RU2847298C1 - Biologically activated drugs based on cytokines and methods for use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to biologically activated polypeptide medicinal structures based on cytokines, and can be used in medicine for treating colon cancer and inhibiting its metastasis. Disclosed is a platform of biologically activated medicinal structure D1-D2-D3 based on cytokines ("VitoKine"), containing, in the direction from the N end to the C end, connected to each other by peptide linkers: domain D1 of group with double function – antagonist humanised antibody to PD1 (programmed cell death 1 protein); D2 domain of a biologically activated moiety – polypeptide of IL-2 variant (interleukin-2); and domain D3 of masking moiety – cognate receptor/binding partner for IL-2, which is IL-2RαSushi. Function of D1 consists in the direction and retention of the biologically activated group in the intended place of therapy, D3 is capable of masking the functional activity of D2 before activation in the intended place of therapy.

EFFECT: invention enables reducing systemic toxicity in treating colon cancer and inhibiting its metastasis.

7 cl, 46 dwg, 21 tbl, 25 ex

Description

Many cytokines have been studied as potential therapeutic agents for disease treatment. However, their associated excessive systemic stimulation or excessive suppression of the body's immune system have significantly limited their development and clinical application.

Interleukin-2 (IL-2) and interleukin-15 (IL-15) share receptor components (γc and IL-2Rβ) and signaling pathways and perform a number of similar functions. Both cytokines stimulate T-cell proliferation, induce the development of cytotoxic T lymphocytes (CTLs), promote B-cell proliferation and immunoglobulin synthesis, and induce the development and persistence of natural killer cells (NK cells). Based on numerous preclinical studies and numerous clinical trials, both cytokines are considered potentially valuable therapeutic agents in cancer, autoimmune disorders, inflammatory disorders, transplantation, and various other disorders. Recombinant IL-2 has been approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. IL-15 is currently undergoing several clinical trials in oncology, but its use has not yet been approved. Furthermore, both IL-2 and IL-15 have a third, unique, non-signaling receptor α-subunit: IL-2Ra (also known as CD25) or IL-15Ra, respectively, which may contribute to differences in their receptor specificity and biological functions.

Recombinant human IL-2 is an effective immunotherapy used in metastatic melanoma and renal cell carcinoma, with durable responses in approximately 10% of patients. However, optimal dosing of IL-2 is limited by its short half-life and significant toxicity. Furthermore, IL-2 also binds to its heterotrimeric receptor IL-2Rαβγ with higher affinity, leading to a preferential expansion of immunosuppressive regulatory T cells (Treg) with high levels of constitutive IL-2Rα expression. Treg expansion may be an undesirable effect of IL-2 in cancer immunotherapy. However, the ability of IL-2 to stimulate Treg cells, even at low doses, may be exploited to treat autoimmune and chronic inflammatory disorders. Recently, it has been discovered that IL-2 can be modified to selectively stimulate cytotoxic effector T cells, or Treg cells. Various techniques have made it possible to obtain IL-2 variants with improved and selective immunomodulatory activity.

Both IL-2 and IL-15 are potent agonists of effector immune cells, and it is crucial that cytotoxic immune cells be fully activated only when they are at or near the site of disease, such as at a cancer site for the specific destruction of tumor cells or at an inflammatory site for local action against autoimmune or chronic inflammatory disorders. Improving target specificity and selectivity while leaving healthy cells and tissues intact and undamaged is of great interest for all cytokines, chemokines, and growth factors.

DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a biologically activatable cytokine drug platform ("VitoKine") aimed at reducing mechanism-based systemic toxicity and enabling broader therapeutic utility of cytokines, chemokines, hormones, and growth factors such as IL-15 and IL-2 for the treatment of cancer, autoimmune disorders, inflammatory disorders, and various other disorders. The VitoKine platform is defined by the constructs shown in FIG. 1 and the proposed activation methods shown in FIG. 2. Referring to FIG. 1, the novel VitoKine constructs of the present invention comprise 3 domains: (1) a D1 domain ("D1") selected from the group consisting of a tissue-targeting domain, a half-life-enhancing domain, or a dual-function moiety domain; (2) a D2 domain ("D2"), which is an "active moiety domain"; and (3) the D3 domain (“D3”), which is a “masking moiety domain.” Importantly, the D2 domain of the VitoKine construct remains nearly inert or minimally active until locally activated by proteases upregulated in diseased tissues or by hydrolysis at disease sites, which will limit the binding of the active moiety to its receptors at the periphery or on the surface of non-disease-affected cells or normal tissues, preventing excessive activation of its pathway and reducing unwanted toxicity due to off-target tissue binding and unwanted clearance due to on-target binding.

In various embodiments, the VitoKine constructs of the present invention comprise D1, which is a targeting moiety, such as an antibody or antibody fragment, that binds to a tumor-associated antigen (TAA), or an immune checkpoint modulator, or a tissue-specific antigen, a cell surface molecule, or an extracellular matrix protein, or a protease(s), or any residue(s) subjected to post-translational modification. In various embodiments, the VitoKine constructs of the present invention comprise D1, which is a targeting moiety, such as a protein or peptide, that exhibits binding affinity for a cell or tissue affected by a disease. Examples of antibodies contemplated for use as D1 in the VitoKine constructs of the present invention include various PD-1 (programmed death protein 1) antagonist antibodies, the PD-L1 (PD-1 ligand) blocking antibody Tecentriq, the anti-CTLA4 (cytotoxic T lymphocyte antigen 4) antibody ipilimumab, the CD40 agonist antibody RO7009789, antibodies directed to tumor antigens including L19 directed against the extra domain of fibronectin, rituximab directed against CD20, Herceptin directed against Her-2, cetuximab directed against EGFR (epidermal growth factor receptor), an anti-FAP (fibroblast activation protein) antibody for tumor targeting and retention, and the anti-inflammatory antibodies vedolizumab against _{integrin α4β7} and Humira against TNFα (tumor necrosis factor a).

In various embodiments, D1 is an antibody that is an antagonist antibody or an antagonist antibody fragment to fibroblast activation protein (FAP). In various embodiments, the antibody is a humanized anti-FAP antibody comprising the amino acid sequences set forth in SEQ ID NOs: 193 and 194. In various embodiments, D1 is an antibody or an antibody fragment to an immune checkpoint modulator. In various embodiments, the antibody is an antagonist antibody or an antagonist antibody fragment to PD-1. In various embodiments, the antibody is an antagonist humanized PD-1 antibody comprising the amino acid sequences set forth in SEQ ID NOs: 195 and 196. In various embodiments, the antibody is an antagonist humanized PD-1 antibody comprising the amino acid sequences set forth in SEQ ID NOs: 197 and 198. In various embodiments, the antibody is an antagonist humanized PD-1 antibody comprising the amino acid sequences set forth in SEQ ID NOs: 275 and 276. In various embodiments, the antibody is an antagonist human PD-1 antibody comprising the amino acid sequences set forth in SEQ ID NOs: 277 and 278. In various embodiments, the antibody is an antagonist PD-L1 antibody or antagonist antibody fragment. In various embodiments, the antibody is an antagonist antibody to human PD-L1 comprising the amino acid sequences set forth in SEQ ID NOs: 279 and 280. In various embodiments, the VitoKine constructs comprise the amino acid sequences set forth in SEQ ID NOs: 128-142, 180-181, 281-286, 296-297, and 303-306.

In various embodiments, the VitoKine constructs of the present invention comprise a D1 that is a modified protein or peptide, such as a glycan-modified protein or peptide, that exhibits binding affinity for a particular receptor, such as a type c lectin receptor, expressed on a cell or tissue affected by a disease. In various embodiments, the VitoKine constructs of the present invention comprise a D1 that functions to retain the cytokine at a tissue site. In various embodiments, the VitoKine constructs of the present invention comprise a D1 that is bifunctional, such as tissue-targeted and tissue-retaining. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is a polymer. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is a half-life-enhancing moiety. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is an Fc domain (or a functional fragment thereof).

"Fc domain" refers to a dimer of two Fc domain monomers that typically contains all or a portion of a hinge region. In various embodiments, the Fc domain is selected from the group consisting of the human immunoglobulin G1 (IgG1) Fc domain, the human IgG2 Fc domain, the human IgG3 Fc domain, the human IgG4 Fc domain, the IgA Fc domain, the IgD Fc domain, the IgE Fc domain, the IgG Fc domain, and the IgM Fc domain, or any combination thereof. In various embodiments, the Fc domain comprises an amino acid change resulting in an Fc domain having altered complement or Fc receptor binding properties. Amino acid changes resulting in an Fc domain with altered complement or Fc receptor binding properties are known in the art. In various embodiments, the Fc domain sequence used to produce the VitoKine constructs is the human IgG1 Fc domain sequence set forth in SEQ ID NO: 13. In various embodiments, the Fc domain sequence used to produce the VitoKine constructs is the sequence set forth in SEQ ID NO: 14, comprising amino acid substitutions that abolish binding to FcγR and C1q. In various embodiments, the Fc domain comprises amino acid changes that result in a further increase in half-life in vivo. Amino acid changes that result in an Fc domain with a further increased half-life are known in the art. In various embodiments, the Fc domain sequence used to generate the VitoKine constructs is the sequence set forth in SEQ ID NO: 156 or 166, both of which contain amino acid substitutions that abolish binding to FcγR and C1q and increase half-life in vivo. In various embodiments, the heterodimeric Fc domain sequence used to produce the VitoKine constructs is derived from the Knob Fc domain sequence set forth in SEQ ID NO: 15. In various embodiments, the heterodimeric Fc domain sequence used to produce the VitoKine constructs is derived from the Hole Fc domain sequence set forth in SEQ ID NO: 16. In various embodiments, the heterodimeric Fc domain sequence used to produce the VitoKine constructs is derived from the Knob Fc domain sequence with an increased in vivo half-life set forth in SEQ ID NO: 167. In various embodiments, the heterodimeric Fc domain sequence used to produce the VitoKine constructs is derived from the Hole Fc domain sequence with an increased in vivo half-life set forth in SEQ ID NO: 168.

In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain that is a protein. In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain that is a cytokine selected from the group consisting of, but not limited to, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-15, IL-23, and transforming growth factor β (TGFβ) superfamily ligands, such as TGFβ (SEQ ID NO: 24). In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain that is IL-15. In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain that is a variant (or mutant) of IL-15 that comprises one or more amino acid substitutions, deletions, or insertions in the IL-15 polypeptide. In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain that is an IL-2. In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain that is a variant (or mutant) of IL-2 that contains one or more amino acid substitutions, deletions, or insertions compared to the IL-2 polypeptide.

In various embodiments, the D2 domain of the VitoKine construct is an IL-15 domain comprising the mature human IL-15 polypeptide sequence (also referred to herein as huIL-15 or wild-type (wt) IL-15) set forth in SEQ ID NO: 2. In various embodiments, the IL-15 domain will be a variant (or mutant) of IL-15 comprising a sequence derived from the mature human IL-15 polypeptide sequence set forth in SEQ ID NO: 2. In various embodiments, the IL-15 domain will be a variant (or mutant) of IL-15 comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% homologous to SEQ ID NO: 2. IL-15 variants (or mutants) are named herein using the native amino acid, its position in the mature sequence, and the amino acid variant. For example, huIL-15 "S58D" refers to a human IL-15 containing an S to D substitution at position 58 of SEQ ID NO: 2. In various embodiments, the IL-15 variant functions as an IL-15 agonist, as demonstrated, for example, by increased binding activity to IL-15Rβγc receptors compared to a native IL-15 polypeptide. In various embodiments, the IL-15 variant functions as an IL-15 antagonist, as demonstrated, for example, by reduced binding activity to IL-15Rβγc receptors or similar or increased binding activity to IL-15Rβγc receptors, but reduced signaling activity compared to a native IL-15 polypeptide or its elimination. In various embodiments, the IL-15 variant has an increased binding affinity or decreased binding activity for IL-15Rβγc receptors compared to a native IL-15 polypeptide. In various embodiments, the IL-15 variant sequence has at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid change compared to a native IL-15 sequence. The amino acid change may include one or more amino acid substitutions, deletions, or insertions in the IL-15 polypeptide, such as a substitution, deletion, or insertion in the IL-15Rβ and/or IL-15Rβγc interacting domain of IL-15. In various embodiments, the amino acid change is one or more amino acid substitutions at position 30, 31, 32, 58, 62, 63, 67, 68, or 108 of SEQ ID NO: 2. In various embodiments, the amino acid change is a substitution of D to T at position 30, V to Y at position 31, H to E at position 32, S to D, or G, or H, or R, or Q, or I, or P at position 58, T to D at position 61, V to F, or A, or K, or R at position 63, I to V at position 67, I to F, or H, or D, or K, or Q, or G at position 68, or Q to A, or M, or S, or E, or K at position 108 of the mature human IL-15 sequence, or any combination of these substitutions. In various embodiments, the amino acid change is a deletion of 1, or 2, or 3, or 4, 5, or 6 amino acids from the N-terminus of SEQ ID NO: 2. In various embodiments, the amino acid change is a deletion of 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acids from the C-terminus of SEQ ID NO: 2. In various embodiments, the amino acid change is an insertion of the amino acids "GS", "GSGS" (SEQ ID NO: 116), or "GGSGG" (SEQ ID NO: 119), or "GSSGGSGGS" (SEQ ID NO: 110) after position N95 of SEQ ID NO: 2. In various embodiments, the IL-15 domain has any combination of amino acid substitutions, deletions, and insertions. In various embodiments, the VitoKine construct will utilize an IL-15 variant with optimally attenuated activity, resulting in reduced intrinsic basal activity of the corresponding VitoKine construct. In various embodiments, the IL-15 variant comprises the amino acid sequence set forth in SEQ ID NOs: 3, 182-192, and 199-215.

In various embodiments, the D2 domain of the VitoKine constructs of the present invention comprises an IL-2 polypeptide. In various embodiments, the VitoKine constructs of the present invention comprise a D2 domain that is a variant (or mutant) of IL-2 comprising one or more amino acid substitutions, deletions, or insertions. In various embodiments, the VitoKine construct comprises a D2 domain, wherein the IL-2 domain comprises the mature human IL-2 polypeptide sequence (also referred to herein as huIL-2 or wild-type (wt) IL-2) set forth in SEQ ID NO: 8. In various embodiments, the IL-2 domain will be a variant (or mutant) of IL-2 comprising a sequence derived from the mature human IL-2 polypeptide sequence set forth in SEQ ID NO: 8. In various embodiments, the IL-2 domain will be a variant (or mutant) of IL-2 comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% homologous to SEQ ID NO: 8. In various embodiments, the IL-2 variant functions as an IL-2 agonist. In various embodiments, the IL-2 variant functions as an IL-2 antagonist. In various embodiments, the amino acid change is one or more amino acid substitutions at position 19, 20, 38, 41, 42, 44, 62, 65, 68, 88, 107, 125, or 126 of SEQ ID NO: 8. In various embodiments, the amino acid change is a substitution of L to D or H or N or P or Q or R or S or Y at position 19, D to E or I or N or Q or S or T or Y at position 20, R to E or A at position 38, T to A or G or V at position 41, F to A at position 42, F to G or V at position 44, E to A or F or H or L at position 62, P to G or E or H or R or A or K, or N, or Q at position 65, E to A, or F, or H, or L, or P at position 68, N to D, E, or G, or I, or M, or Q, or T, or R at position 88, Y to G, or H, or L, or V at position 107, S to E, or H, or K, or I, or L, or V, or W at position 125, Q to D, or E, or K, or L, or M, or N at position 126 of the mature human IL-2 sequence, or any combination of these substitutions. In various embodiments, the VitoKine construct will comprise an IL-2 moiety designed to reduce/eliminate binding to IL-2Ra. In various embodiments, the IL-2 variant has reduced binding activity to IL-2Rβγc receptors compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant has both reduced/aborted binding to IL-2Rα and altered binding activity to IL-2Rβγc receptors compared to a native IL-2 polypeptide. In various embodiments, the IL-2 variant with reduced/aborted binding to IL-2Rα comprises the amino acid sequence set forth in SEQ ID NOs: 232-247. In various embodiments, VitoKine constructs containing IL-2 were designed to selectively increase the number of Teff cells, including constructs comprising the amino acid sequences set forth in SEQ ID NOs: 59-61, 271-274, and 286-291. In various embodiments, the IL-2 variant in the VitoKine construct allows for fine-tuning of the intrinsic basal activity of the VitoKine IL-2 construct to ensure an optimal balance between desired antitumor efficacy and undesirable systemic toxicity.

In various embodiments, the VitoKine constructs of the present invention comprise a "masking moiety domain" (D3) that is a cognate receptor/binding partner or any binding partner identified for the D2 protein or cytokine. In various embodiments, the D3 domain is a variant of a cognate receptor/binding partner for the D2 domain. In various embodiments, the D3 domain has enhanced binding to the D2 domain compared to the wild-type cognate receptor/binding partner. In various embodiments, the D3 domain has reduced binding to the D2 domain compared to the wild-type cognate receptor/binding partner or its binding to the D2 domain is abolished. In various embodiments, the D3 domain is a protein or peptide or antibody or antibody fragment capable of masking D2 activity. In various embodiments, the D3 domain is DNA, an RNA fragment, or a polymer such as PEG (polyethylene glycol). In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the extracellular domain of IL-15Rα or a functional fragment thereof. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the IL-15RαSushi domain. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the extracellular domain of IL-2Rα or a functional fragment thereof. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the IL-15RαSushi domains. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is a variant (mutant) of the IL-15RαSushi domains. In various embodiments, the IL-2RαSushi domain variant (or mutant) comprises amino acid changes in a sequence derived from the sequence set forth in SEQ ID NO: 10. In various embodiments, the amino acid change is one or more amino acid substitutions at position 36, 38, 42, or 43 of SEQ ID NO: 10. In various embodiments, the amino acid change is a substitution of R to A at position 36, K to E at position 38, L to G at position 42, Y to A at position 43. In various embodiments, the D3 domain is capable of masking the functional activity of D2 prior to activation at the intended site of therapy. In various embodiments, the D3 domain is designed to facilitate dissociation and diffusion following proteolytic cleavage and activation.

In various embodiments, the D1, D2, and D3 domains of the VitoKine construct are connected by a protease-cleavable polypeptide linker sequence. In various embodiments, the D1, D2, and D3 domains of the VitoKine construct are connected by a non-cleavable polypeptide linker sequence. In various embodiments, both L1 and L2 of the VitoKine constructs of the present invention are protease-cleavable peptide linkers. In various embodiments, L1 of the VitoKine constructs of the present invention is a protease-cleavable peptide linker and L2 is a non-cleavable peptide linker. In various embodiments, L1 of the VitoKine constructs of the present invention is a non-cleavable peptide linker and L2 is a protease-cleavable peptide linker. In various embodiments, both L1 and L2 of the VitoKine constructs of the present invention are non-cleavable linkers. In various embodiments, the non-cleavable linker has a high G/S content (e.g., at least about 60%, 70%, 80%, 90%, or more of the amino acids of the linker are G or S). The sequence of each peptide linker can be independently selected. In various embodiments, the protease-cleavable linker is selected from the group of sequences set forth in SEQ ID NOs: 71-96 and 157-161. In various embodiments, the protease-cleavable linker may have an additional peptide spacer of variable length at the N-terminus of the cleavable linker, or at the C-terminus of the cleavable linker, or at both ends of the cleavable linker. In various embodiments, the non-cleavable linker is selected from the group of sequences set forth in SEQ ID NO: 107-127. In various embodiments, the linker is a flexible or rigid linker of variable length.

In various embodiments, the D2 and D3 domains of the VitoKine construct are located at the N-terminus of the D1 domain, as shown in FIG. 1A. In various embodiments, the D2 and D3 domains of the VitoKine construct are located at the C-terminus of the D1 domain, as shown in FIG. 1B.

In various embodiments, the D1, D2, and D3 domains of the VitoKine construct may be monomers or dimers, or a combination of a dimer and a monomer, such as a combination wherein D1 is a dimer and D2 and D3 are monomers.

In another aspect, the present invention provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma, or any cancer.

In another aspect, the present invention provides a method of treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention in combination with a second therapy selected from the group consisting of cytotoxic chemotherapy, immunotherapy, targeted therapy with small molecule kinase inhibitors, surgery, radiation therapy, stem cell transplantation, cellular therapy including CAR-T, CAR-NK, iPS-induced CAR-T or iPS-induced CAR-NK cells, and vaccines such as bacillus Calmette-Guerin (BCG). In various embodiments, combination therapy may comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but not limited to: treatment using depleting antibodies to certain tumor antigens; treatment using antibody-drug conjugates; Treatment with agonist, antagonist, or blocking antibodies to costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec-7, Siglec-8, Siglec-9, Siglec-15, and VISTA Treatment with bispecific T-cell engager (BiTE®) antibodies such as blinatumomab Treatment including administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ Treatment with therapeutic vaccines such as sipuleucel-T Treatment with dendritic cell vaccines or tumor antigen peptide vaccines therapy using chimeric antigen receptor T cells (CAR-T cells); therapy using CAR-NK cells; therapy using tumor-infiltrating lymphocytes (TILs); therapy using adoptively transferred anti-tumor T cells (ex vivo cultured and/or TCR transgenic); therapy using TALL-104 cells; and therapy using immunostimulatory agents such as Toll-like receptor (TLR) CpG agonists and imiquimod; and therapy using a vaccine such as BCG; where the combination therapy provides enhanced killing of tumor cells by effector cells, i.e., synergy occurs when VitoKine constructs and immunotherapy are used together.

In another aspect, the present invention provides a method for treating a viral infection in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to the subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the virus is HIV (human immunodeficiency virus).

In another aspect, the present invention provides a method of treating a viral infection in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention in combination with a second therapy, including, but not limited to, acyclovir, epclusa, mavyret, zidovudine and enfuvirtide.

In another aspect, the present invention provides a method for treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenic purpura, Graves' disease, Sjogren's disease, dermatomyositis, Hashimoto's disease, polymyositis, inflammatory bowel disease, multiple sclerosis (MS), diabetes mellitus, rheumatoid arthritis, and scleroderma.

In another aspect, the present invention provides a method for treating an inflammatory disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to the subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the inflammatory disease is selected from the group consisting of Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, inflammatory bowel disease, Behcet's syndrome, and colitis not otherwise specified.

In various embodiments, the inflammatory disease is selected from the group consisting of other autoimmune and inflammatory diseases such as: achalasia, adult Still's disease, agammaglobulinemia, amyloidosis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy and gangliopathy, autoimmune encephalomyelitis, autoimmune inner ear disease, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy, Balo's disease, Behcet's disease, benign mucous membrane pemphigoid, Castleman's disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal Osteomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, Coxsackie myocarditis, CREST syndrome, dermatitis herpetiformis, Devic disease/neuromyelitis optica, discoid lupus, Dressler syndrome, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, fibrosing alveolitis, giant cell arteritis, giant cell myocarditis, Henoch-Schonlein purpura, herpes gestationis or pemphigoid gestationis, IgA nephropathy, IgG4-associated sclerosing disease, immune-mediated adverse events, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile myositis, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease, chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Moren ulcer, Mucha-Habermann disease, multifocal motor neuropathy, optic neuritis, palindromic rheumatism, PANDAS, paraneoplastic cerebellar degeneration, Parry-Romberg syndrome, intermediate uveitis, Parsonage-Turner syndrome, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polyglandular syndromes, polymyalgia rheumatica, postinfarction syndrome, postpericardiotomy syndrome, primary sclerosing cholangitis, progesterone dermatitis, Psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reflex sympathetic dystrophy, relapsing polychondritis, retroperitoneal fibrosis, scleritis, autoimmune diseases of seminal fluid and testicles, stiff-person syndrome, subacute bacterial endocarditis, Susak syndrome, sympathetic ophthalmia, Takayasu arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, undifferentiated connective tissue disease, Vogt-Koyanagi-Harada disease.

In another aspect, the invention provides the use of VitoKine constructs for the manufacture of a medicament for the treatment of cancer.

In another aspect, the invention provides the use of VitoKine constructs for the manufacture of a medicament for the treatment of a viral infection.

In another aspect, the invention provides the use of VitoKine constructs for the manufacture of a medicament for the treatment of an autoimmune disease.

In another aspect, the invention provides the use of VitoKine constructs for the manufacture of a medicament for the treatment of inflammation.

In another aspect, the invention provides the use of the VitoKine constructs of the invention in combination with a second therapeutic agent or cell therapy for the treatment of cancer, a viral infection, or an autoimmune disease, or inflammation.

In another aspect, the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a VitoKine construct of the present invention. In another aspect, the present invention provides vectors comprising the nucleic acids described herein. In various embodiments, the vector is an expression vector. In another aspect, the present invention provides isolated cells comprising the nucleic acids of the invention. In various embodiments, the cell is a host cell comprising an expression vector of the invention. In another aspect, methods for producing VitoKine constructs are provided, comprising culturing the host cells under conditions conducive to the expression of proteins or polypeptides.

In another aspect, the present invention provides a pharmaceutical composition comprising isolated VitoKine constructs in admixture with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

FIG. 1 shows exemplary formats of VitoKine constructs of the present invention. FIG. 1A shows a VitoKine construct with the D2 and D3 domains located at the C-terminus of the D1 domain. FIG. 1B shows a VitoKine construct with the D2 and D3 domains located at the N-terminus of the D1 domain. FIGS. 1C-1E further show a VitoKine construct with the D2 and D3 domains located at the C-terminus of the D1 domain, where the D1 domain is an antibody; (1C) a VitoKine construct with the D2 and D3 domains located at the C-terminus of a heterodimeric antibody heavy chain; (1D) a VitoKine construct with the D2 and D3 domains located at the C-terminus of homodimeric antibody heavy chains; (1E) a VitoKine construct with the D2 and D3 domains located at the C-terminus of an antibody light chain. Similarly, the D2 and D3 domains can be located at the N-terminus of the heavy chain (both heterodimeric and homodimeric) and light chain of an antibody.

FIG. 2 shows a proposed mechanism for the activation of VitoKine constructs. A typical VitoKine construct contains two protease-cleavable linkers; activation by protease 1 via cleavage of linker L1 results in the formation of active form 1; activation by protease 2 via cleavage of linker L2 results in the formation of active form 2; activation by both proteases via cleavage of linkers L1 and L2 results in the formation of active form 3. FIG. 2A shows an example of the retention of the D3 domain in a non-covalent complex with D2 after proteolysis; FIG. 2B shows an example of the release and diffusion of D3 from D2 after protease cleavage.

FIG. 3 shows the protein profile by (A) SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) in the absence and presence of reducing agent and (B) size exclusion chromatography of a representative VitoKine construct with IL-15 and Fcβ-0315 after protein A purification.

FIG. 4 shows the binding and functional activity of the VitoKine construct to IL-15 P-0172 compared to the highly active IL-15 fusion protein P-0198.

(A) Binding activity to IL-2Rβ measured by ELISA (enzyme-linked immunosorbent assay); (B-C) induction of CD69 expression on human CD8+ T cells (B) and NK cells (C) from fresh human PBMCs (peripheral blood mononuclear cells) by FACS (fluorescence-activated cell sorting).

FIG. 5 shows the functional activity of the VitoKine monomeric construct with IL-15 and Fc P-0170 compared to the highly active IL-15 fusion protein P-0166. Induction of CD69 expression on human CD8+ T cells from fresh human PMBCs was measured and analyzed by FACS.

FIG. 6 shows the induction of CD69 expression on (A) CD8+ T cells and

(B) NK cells (CD56+) of human PMBCs were exposed to representative VitoKine IL-15 and Fc constructs (P-0204, P-0205, and P-0206) with different linker lengths compared to the highly active IL-15/IL-15Rα and Fc fusion protein P-0165.

FIG. 7 shows the proliferation of human RMBC NK cells (CD56+) in response to representative VitoKine constructs with different L1 and L2 linkers (P-0202, P-0203, and P-0204) compared to fully active IL-15/IL-15Rα and Fc fusion proteins P-0207 and P-0217.

FIG. 8 shows the proliferation of (A) CD8+ T cells and (B) NK cells (CD56+) of human PMBCs by representative VitoKine IL-15 and Fc constructs (P-0351, P-0488, and P-0489) with different L2 linker sequence compositions, measured by FACS, compared to the IL-15/IL-15Rα and Fc fusion protein P-0156.

FIG. 9 shows in vitro proteolysis analysis of the VitoKine construct with IL-15 and Fcβ-0315 by SDS-PAGE using varying amounts of MMP-2 (matrix metalloproteinase-2).

FIG. 10 shows SDS-PAGE analysis of in vitro proteolysis of the VitoKine construct with IL-15 and Fcβ-0203 using uPA under various conditions to determine suitable interaction conditions for complete cleavage.

FIG. 11 shows: (A) SDS-PAGE analysis of the VitoKine IL-15-Fc P-0203 construct before and after in vitro proteolysis with uPA; (B) protein profile of the activated VitoKine P-0203 construct after uPA cleavage and protein A purification to remove Fc-containing fragments.

FIG. 12 shows: (A) SDS-PAGE analysis of the VitoKine IL-15-Fcβ P-0315 construct before and after in vitro MMP-2 proteolysis, the gel also showing the profile of MMP-2-cleaved P-0315 purified on Protein A; (B) the protein profile of the active form 2 of the VitoKine P-0315 construct obtained by MMP-2 cleavage and subsequent Protein A purification; (C) the protein profile of the active form 3 of the VitoKine P-0315 construct obtained by dual proteolysis with both MMP-2 and uPA and subsequent Protein A purification in flow-through mode.

FIG. 13 shows the activity of the VitoKine construct with IL-15 and Fc protease-activated protein (uPA) P-0203 by assaying the induction of the activation marker CD69 on (A) CD56+ NK cells and (B) CD8+ T cells. P-0165, a highly active IL-15 fusion protein, was included as a positive control.

FIG. 14 shows the evaluation of the activity of two forms of the VitoKine IL-15/Fc protease-activated P-0315 construct by assaying the induction of the activation marker CD69 on (A) CD56+ NK cells and (B) CD8+ T cells. The active form 2 of P-0315 was generated by cleavage with MMP-2, and the active form 3 of P-0315 was generated by dual proteolysis with both MMP-2 and uPA. P-0313, a highly active IL-15 fusion protein structurally similar to the active form 2 of P-0315, was included as a positive control.

FIG. 15 shows the activity assessment of the VitoKine construct with IL-15 and Fc P-0315 activated by MMP-2 (active form 2) by assaying the induction of the proliferation marker Ki67 on (A) CD56+ NK cells and (B) CD8+ T cells. P-0351, where both linkers, L1 and L2, are non-cleavable and the length of linker L2 is the same as in P-0315, was included for comparison.

FIG. 16 shows the dose- and time-dependent effects of the cleavable VitoKine construct with IL-15 and Fcββ-0315 and the non-cleavable VitoKine construct with IL-15 and Fcββ-0351 on the increase in (A) CD8+ T cells, (B) NK cells, and (C) leukocytes in peripheral blood after a single injection in Balb/C mice. The fully active IL-15 and Fcββ-0313 fusion protein was included for comparison. Blood was collected for lymphocyte phenotyping by FACS analysis at days -1, 3, 5, and 7. Data are presented as the mean plus/minus SEM. Statistical analysis was performed by two-way ANOVA followed by Tukey's post hoc test. ****p less than 0.0001, ***p less than 0.001, *p less than 0.05 compared with the PBS (phosphate-buffered saline) group at the corresponding time point.

FIG. 17 shows the inhibition of pulmonary metastatic nodules in the CT26 mouse pulmonary metastasis model one day after 4-fold administration of P-0315, P-0351, P-0313, or control PBS once every 5 days (Q5D). The first administration was performed one day after CT26 cell injection. Unless otherwise stated, all comparisons are against the PBS group; ****p less than 0.0001; **p less than 0.01; *p less than 0.05.

FIG. 18 shows (A) the % of CD8+ T cells and (B) the % of NK cells among total blood lymphocytes in mice with CT26 metastases. Cell numbers were determined by flow cytometry 4 days after three intraperitoneal injections of P-0315, P-0351, P-0313, or control PBS, Q5D. All comparisons are versus the PBS group; ****p less than 0.0001; **p less than 0.01; *p less than 0.05.

FIG. 19 shows the antitumor efficacy of the VitoKine IL-15-Fcβββ construct compared to the fully active IL-15-Fcβββ fusion protein in the CT26 mouse established colorectal cancer model. The growth curve of subcutaneous CT26 tumors in individual mice after two treatments, Q5D, is shown for (A) the PBS vehicle group, (B) the 0.1 mg/kg P-0315 group, and (C) the 0.1 mg/kg P-0313 group; (D) the mean tumor volume plus/minus SEM (standard error of the mean) over time for each treatment group. All comparisons are versus vehicle; n is 11 per group; ****p less than 0.0001.

FIG. 20 shows immunopharmacodynamic profiling of mouse peripheral blood following administration of VitoKine with IL-15 and Fcβ-0315 or a highly active IL-15 and Fcβ-0313 fusion protein in the CT26 mouse colorectal cancer model. Following two administrations, Q5D, starting at day 11 after tumor implantation, the percentage increase in the proliferation marker Ki67 in (A) NK cells and (B) CD8+ T cells at day 19 was determined using flow cytometry. ****p less than 0.0001 vs. PBS.

FIG. 21 shows immunopharmacodynamic profiling of mouse peripheral blood following administration of P-0315 or P-0313 in the CT26 mouse colorectal cancer model. Following two Q5D administrations, beginning 11 days after tumor implantation, increases in total circulating leukocytes (A), NK cells (B), and CD8+ T cells (C) per µL of whole blood were determined at day 19 using flow cytometry. ****p less than 0.0001 vs. PBS.

FIG. 22 shows immunopharmacodynamic profiling of the spleen following administration of P-0315 or P-0313 in the CT26 mouse colorectal cancer model. Following two Q5D administrations, beginning 11 days after tumor implantation, increases in total leukocytes (A), NK cells (B), and CD8+ T cells (C) in the spleen were determined at day 19 using flow cytometry. ****p less than 0.0001, *p less than 0.05, vs. PBS.

FIG. 23 shows the activity of various IL-15/IL-15Rα and Fc fusion proteins containing one or two amino acid substitutions at positions V63, I68, and Q108 by assaying the induction of the proliferation marker Ki67 on (A) CD8+ T cells and (B) CD56+ NK cells. P-0313, a highly active IL-15/IL-15Rα and Fc fusion protein, was included for comparison.

FIG. 24 shows the activity assessment of P-0764, an IL-15 Q108S/IL-15Rα Fc fusion protein, and its corresponding VitoKine Fc construct, designated P-0682, by assaying the induction of the proliferation marker Ki67 on (A) CD8+ T cells and (B) CD56+ NK cells. P-0313, a highly active IL-15/IL-15Rα Fc fusion protein, was included for comparison.

FIG. 25 shows a comparison of the activity of the non-cleavable VitoKine construct with IL-15 and Fcβ-0351 and the reference molecule (Benchmark) by assaying the induction of the proliferation marker Ki67 on (A) CD56+ NK cells and (B) CD8+ T cells.

FIG. 26 shows the protein profile by (A) SDS-PAGE, in the absence and presence of reducing agent, and (B) size exclusion chromatography of a representative VitoKine construct with IL-2 P-0320 after purification on protein A.

FIG. 27 shows the activity of two VitoKine IL-2 and Fc constructs, P-0320 (IL-2 fused to the C-terminus of Fc) and P-0329 (IL-2 fused to the N-terminus of Fc), by analyzing pStat5 levels in (A) CD4+Foxp3+/CD25high ^Treg cell subsets and (B) CD4+Foxp3-/ ^D25low CD4 conventional T cell subsets of fresh human PMBC. P-0250, a highly active IL-2 and Fc fusion protein, was included as a positive control.

FIG. 28 shows: (A) SDS-PAGE analysis of the VitoKine IL-2 construct P-0382 and its activation by MMP-2 cleavage followed by Ni-Excel purification; and (B) protein profile of P-0382 activated by MMP-2 and purified on protein A in bind-and-elute mode.

FIG. 29 shows the activity of the VitoKine IL-2 and Fc protease-activated P-0382 construct by analyzing pStat5 levels in (A) CD4+Foxp3+/CD25 ^high Treg and (B) CD4+Foxp3-/D25 ^low CD4 conventional T cell (Tconv) subsets of fresh human PBMCs. The two activated samples were purified either on Ni-Excel resin with the protease removed (P-0382 activ. 1) or on protein A with both the protease and the IL-2RαSushi domain cleaved by proteolysis removed (P-0382 activ. 2). P-0250, a high-activity IL-2 and Fc fusion protein, was included as a positive control.

FIG. 30 shows the activity of the VitoKine IL-2-Fc construct P-0398 before and after proteolysis by MMP-2 by analyzing pStat5 levels in (A) CD4+Foxp3+/CD25high ^Treg and (B) CD4+Foxp3-/ ^D25low CD4 Tconv cell subsets of fresh human PMBC. P-0382, which differs from P-0398 only in the length of the L2 linker, and P-0250, a high-activity IL-2-Fc fusion protein, were included for comparison.

FIG. 31 shows the binding of IL-2 and Fc variant fusion proteins P-0704, P-0707, P-0708, and P-0709, containing different amino acid substitutions at position P65, in an ELISA with IL-2Rα. P-0689, an equivalent protein with wild-type IL-2, was included for comparison.

FIG. 32 shows the activity of P-0704 and P-0689 using the Ki67 proliferation marker induction assay on fresh human PMBC CD8+ T cells. P-0704 is a fusion protein of IL-2 P65R and Fc that has lost binding activity to IL-2Rα, while P-0689 is the equivalent protein with wild-type IL-2.

FIG. 33 shows the binding of different IL-2RαSushi variants P-0751, P-0752, and P-0753 in an ELISA with the monovalent fusion protein wild-type IL-2 and Fc P-0689. P-0757 contains wild-type IL-2RαSushi and was included for comparison.

FIG. 34 shows the activity of VitoKine IL-2-Fc constructs containing either wild-type IL-2RαSushi (P-0701) or one of the IL-2RαSushi variants (P-0754, P-0755, and P-0756) as the D3 domain. P-0704, an IL-2 P65R-Fc fusion protein that has lost IL-2Rα binding activity but fully retains IL-2Rβγ activity, was included as a control. Activity was assessed by analyzing the induction of the proliferation marker Ki67 on (A) CD8+ T cells and (B) CD56+ NK cells, determined by flow cytometry.

FIG. 35 shows the activity of P-0704, an IL-2 P65R Fc fusion protein, and its corresponding VitoKine Fc constructs containing either wild-type IL-2RαSushi (P-0745) or one of the IL-2RαSushi variants (P-0807, P-0808, and P-0809) as the D3 domain. Activity was assessed by analyzing the induction of the proliferation marker Ki67 on (A) CD8+ T cells and (B) CD56+ NK cells, determined by flow cytometry.

FIG. 36 shows the activity of P-0755, a VitoKine IL-2 Fc construct containing the IL-2RαSushi L42G variant as the D3 domain, before and after in vitro protease activation. P-0704, an IL-2 P65R Fc fusion protein that fully retains IL-2Rβγ activity, was included as a control. Activity was assessed by analyzing the induction of the proliferation marker Ki67 on (A) CD8+ T cells and (B) CD56+ NK cells from fresh human PBMCs.

FIG. 37 shows the evaluation of the activity of the VitoKine IL-15/Fcβ P-0315 construct compared to the VitoKine IL-15/Fcβ P-0485 construct by flow cytometric analysis of the induction of the proliferation marker Ki67 on (A) CD8+ T cells and (B) CD56+ NK cells from fresh human PMBC.

FIG. 38 shows flow cytometric analysis of Ki67 expression on CD8+ T cells induced by various VitoKine IL-15 constructs compared to their corresponding non-VitoKine equivalent fusion proteins. (A) VitoKine IL-15 Fc construct P-0315 compared to the equivalent Fc fusion protein in human PMBC; (B) VitoKine IL-15 P-0875 construct compared to the equivalent P-0870 Fc fusion protein in human PMBC; (C) P-0875 compared to P-0773 in cynomolgus monkey PMBC.

FIG. 39 shows the activity of various VitoKine IL-2/antibody constructs P-0800, P-0830, P-0831, and P-0802 compared to a non-VitoKine IL-2/antibody fusion protein, P-0782, by assaying the induction of the proliferation marker Ki67 on (A) CD8+ T cells and (B) CD56+ NK cells, as determined by flow cytometry. The four VitoKine IL-2/antibody constructs differ only in the IL-2 moiety, which has different levels of binding strength to IL-2Rα.

FIG. 40 shows the cleavage and activation of the VitoKine construct with IL-2 and the P-0872 antibody by protease using (A) SDS-PAGE analysis under reducing conditions and (B) flow cytometric analysis of the dose-dependent induction of Ki67 expression on human PMBC CD8+ T cells. P-0872 contains a monovalent IL-2 moiety and one protease-cleavable linker connecting the D2 and D3 domains.

FIG. 41 shows the cleavage and activation of the VitoKine construct with IL-2 and the antibody P-0929 by protease using (A) SDS-PAGE analysis under reducing conditions and (B) flow cytometric analysis of the dose-dependent induction of Ki67 expression on human PMBC CD8+ T cells. P-0929 contains a bivalent IL-2 moiety and two protease-cleavable linkers.

FIG. 42 shows the binding of IL-2Rβ-based blocking peptides (L01, L02, L03, L04, and L05) to IL-15 in an ELISA format.

FIG. 43 shows the binding of IL-15 fusion proteins (P-0153, P-0159, P-0160, and P-0161) to IL-2Rβ immobilized on a plate. P-0159, P-0160, and P-0161 contain different IL-2Rβ-based blocking peptides.

FIG. 44 shows the size exclusion chromatograms of four VitoKine IL-2 constructs (P-0320, P-0382, P-0362, and P-0379) (FIGS. 40B-40E) compared to their equivalent Fc fusion protein P-0250 (FIG. 44A). P-0531, which differs from P-0250 by a single amino acid substitution, S125I, in IL-2, was included for comparison (FIG. 44F).

FIG. 45 shows SDS-PAGE analysis of VitoKine constructs with IL-15 and Fc P-0389 (A) and P-0315 (B), containing different D3 domains.

FIG. 46 shows the antitumor efficacy of VitoKine constructs with IL-2 and the PD-1 antagonist antibody P-0922A, P-0928A, and P-0929A compared to their non-cleavable equivalent P-0877 in the MC38 mouse colon cancer model. Tumor size is shown for each individual mouse in each group at day 7 after a single administration.

EMBODIMENT(S) OF THE INVENTION

The present invention provides novel "VitoKine" constructs as a technology platform for reducing target binding-mediated systemic toxicity and enhancing the therapeutic index of cytokines intended for use in the treatment of cancer, viral infection, autoimmune diseases, or inflammatory diseases. The VitoKine platform is defined by the constructs shown in FIG. 1 and the proposed activation methods shown in FIG. 2. Referring to FIG. 1, the novel VitoKine constructs of the present invention comprise 3 domains: (1) a D1 domain ("D1") selected from the group consisting of a tissue-targeting domain, a half-life-enhancing domain, an immune checkpoint modulator-targeting domain, or a dual-function moiety domain; (2) a D2 domain ("D2"), which is an "active moiety domain"; and (3) a D3 domain ("D3"), which is a "masking moiety domain". Importantly, the D3 domain has the ability to mask or attenuate the functional activity of D2 prior to its activation at the intended site of therapy.

The three domains are linked using linkers of variable length and rigidity, coupled with protease-cleavable sequences representing peptide substrates of specific protease subtypes whose expression is upregulated or misregulated at disease sites, allowing for the uncovering or release of the functional D2 domain at the disease site. The linker length and composition were optimized to best mask the accessibility of the D2 domain to its receptors, reducing its systemic exposure while maintaining the stability of VitoKine constructs in circulating blood and ensuring their efficient cleavage upon contact with specific proteases at the target disease site. The development of VitoKine also took into account rational considerations based on existing data on the molecular interactions of cytokines with their cognate receptors. Cytokine receptors typically function as oligomeric complexes consisting of two to four receptor subunits. Different subunits perform specialized functions, such as ligand binding or signal transduction. The alpha subunit of cytokine receptors is a binding receptor that confers ligand specificity, enhances ligand interaction with signaling receptors, and converts low-affinity signaling receptors into high-affinity ones. Thus, the D3 domain of the VitoKine construct preferably represents a cognate binding receptor for the D2 domain. After cleavage, dissociation or reassociation of the D3 and D2 domains is possible, with full restoration of the binding and signaling activity of the D2 domain in place. Thus, the D3 domain may play a dual role in regulating the function of the D2 domain. It keeps the D2 domain inactive when the VitoKine construct is inactivated and can participate in D2 function after cleavage and activation of the VitoKine construct. However, the D3 domain can be any protein, peptide, antibody, antibody fragment, polymer, or nucleotides capable of masking D2 activity.

In another aspect, the addition of the D3 domain may also significantly expand the possibilities for designing a VitoKine construct with higher expression yield and less aggregation propensity.

The D1 domain may serve as a half-life-enhancing domain, extending the VitoKine construct's circulating half-life and serving as an additional domain masking the functional activity of the D2 domain. The D1 domain may also serve as a disease- or tissue-targeting motif, specifically directing the VitoKine construct to the site of interest and limiting local activation of the VitoKine construct, further improving the therapeutic index. Therefore, the VitoKine platform enables selective cytokine activation at the target site and offers the advantages of reducing systemic toxicity while simultaneously enhancing the therapeutic effect at disease sites, thereby improving their therapeutic index.

The D2 domain of the VitoKine construct is the active moiety but remains inert until locally activated by proteases upregulated in disease-affected tissues; this will limit the binding of the active moiety to its receptors at the periphery or on the surface of cells or tissues not affected by the disease, preventing excessive activation of its pathway and reducing unwanted toxicity due to target binding outside the target tissue. Furthermore, the inertness of the VitoKine active moiety until activated by proteases will substantially reduce potential clearance due to antigen binding, thereby increasing the in vivo half-life and improving biodistribution, bioavailability, and efficacy at the intended therapeutic sites. Furthermore, based on the present invention, the VitoKine platform may offer expanded protein design options, including, but not limited to, enhancing expression levels and reducing aggregation propensity, such as by using the cognate receptor alpha as the D3 domain.

Although cleavable linkers are preferred for most VitoKine constructs to limit their systemic activation and release the active domain at the intended site after administration, non-cleavable linkers may be desirable to ensure prolonged, low-potency systemic action of the pharmacologically active VitoKine construct and to enhance therapeutic efficacy.

In exemplary embodiments, the VitoKine constructs comprise an active moiety (D2) based on IL-15, an IL-15 variant, IL-2, or an IL-2 variant. For these IL-15 and/or IL-2-based VitoKine constructs, the unique and non-signaling α-subunit of each cytokine receptor is used as the masking moiety (D3) domain subunit via a protease-cleavable linker to reversibly mask the cytokine's activity. Depending on the selective properties of each receptor complex and the varying requirements of different diseases that are putative indications for treatment with VitoKine molecules, it may be preferable for the masking α-subunit to complex with the activated cytokine through non-covalent associations (e.g., in the case of IL-15) or dissociate from it (e.g., in the case of IL-2) following protease cleavage of the linker. As a result, amino acid modifications of the α-receptor that modulate the binding affinity to its cognate cytokine may prove necessary and beneficial.

In typical embodiments, VitoKine constructs comprise an active moiety (D2) based on IL-15, an IL-15 variant, IL-2, or an IL-2 variant. In the case of these IL-15 and/or IL-2-based VitoKine constructs, a common receptor β-subunit or a receptor β-subunit-based blocking peptide is used as the masking moiety (D3) domain via a protease-cleavable linker to reversibly mask cytokine activity.

This concept of combining a cognate receptor, protein, antibody, antibody fragment, or binding peptide with a cytokine via an activatable linker to mask its functional activity prior to activation at the target therapeutic site can be adapted for a variety of cytokines, including, but not limited to, IL-4, IL-7, IL-9, IL-10, IL-12, IL-22, IL-23, and TGFβ, chemokines such as CXCR3, or various growth factors such as the TNF family, TGFα, and TGFβ, and hormones. The same concept can also be applied to other proteins to generate proproteins for enhanced disease targeting and expanded therapeutic applications.

Definitions

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, "peptides," "polypeptides," and "proteins" are chains of amino acids whose alpha carbons are linked by peptide bonds. Thus, the terminal amino acid at one end of the chain (the N-terminal) has a free amino group, while the terminal amino acid at the other end of the chain (the C-terminal) has a free carboxyl group. As used herein, the term "N-terminus" refers to the free α-amino group of the amino acid at the N-terminus of the peptide or to the α-amino group (the amino group when involved in a peptide bond) of the amino acid elsewhere within the peptide. Similarly, the term "C-terminus" refers to the free carboxyl group at the C-terminus of the peptide or the carboxyl group of the amino acid elsewhere within the peptide. Peptides also include virtually any polyamino acid, including, without limitation, peptide mimetics such as amino acids linked by ester rather than amide bonds.

The polypeptides of the invention include polypeptides that have been modified in any manner and for any reason, such as to: (1) reduce susceptibility to proteolysis; (2) reduce susceptibility to oxidation; (3) alter binding affinity for forming protein complexes; (4) alter binding affinity; and (5) impart or modify other physicochemical or functional properties.

As used herein, "amino acid substitution" refers to the replacement of one amino acid of a polypeptide at a particular position in the parent polypeptide sequence with a different amino acid. Amino acid substitutions can be made using genetic or chemical methods well known in the art. For example, a single amino acid substitution or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in a portion of a polypeptide outside the domain(s) forming intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid of a polypeptide with a functionally similar amino acid. Each of the following six groups includes amino acids that are conservative substitutions for each other:

1) alanine (A), serine (S) and threonine (T);

2) aspartic acid (D) and glutamic acid (E);

3) asparagine (N) and glutamine (Q);

4) arginine (R) and lysine (K);

5) isoleucine (I), leucine (L), methionine (M) and valine (V);

6) phenylalanine (F), tyrosine (Y) and tryptophan (W).

A "non-conservative amino acid substitution" refers to the replacement of a member of one of these classes with a member of another class. When making such changes, according to various embodiments, the hydrophobicity index of amino acids can be taken into account. Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge characteristics. It is: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);

cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydrophobicity index of amino acids in imparting biological interaction function to a protein is known in the art (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids can be substituted for other amino acids having a similar hydrophobicity index or value while maintaining similar biological activity. In various embodiments, changes based on the hydrophobicity index include substitutions of amino acids whose hydrophobicity indices differ by no more than 2. In various embodiments, substitutions of amino acids whose hydrophobicity indices differ by no more than 1 are included, and, in various embodiments, substitutions of amino acids whose hydrophobicity indices differ by no more than 0.5 are included.

It is also known in the art that substitution of similar amino acids can be effectively performed based on hydrophilicity, particularly where the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the highest local average hydrophilicity of a protein, determined by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

The following hydrophilicity values were assigned to the listed amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 plus or minus 0.1); glutamate (+3.0 plus or minus 0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 plus or minus 0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In various embodiments, changes based on similar hydrophilicity values include substitutions of amino acids whose hydrophilicity values differ by no more than 2, in various embodiments, substitutions of amino acids whose hydrophilicity values differ by no more than 1 are included, and, in various embodiments, substitutions of amino acids whose hydrophilicity values differ by no more than 0.5 are included.

Examples of amino acid substitutions are given in Table 1.

One skilled in the art will be able to identify suitable variants of the polypeptides described herein using well-known techniques. In various embodiments, one skilled in the art will be able to identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions not believed to be essential for activity. In other embodiments, one skilled in the art will be able to identify residues and portions of the molecule conserved among similar polypeptides. In other embodiments, even regions that may be important for biological activity or structure can be subject to conservative amino acid substitutions without disrupting biological activity or adversely affecting the polypeptide structure.

Furthermore, a specialist in this field will be able to review structure-function studies that identify residues in similar polypeptides that are important for their activity or structure. Based on such comparisons, the specialist will be able to predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues important for the activity or structure of similar polypeptides. A specialist in this field may prefer to replace such predicted important amino acid residues with chemically similar amino acid residues.

A person skilled in the art can also analyze the three-dimensional structure and amino acid sequence in comparison to similar polypeptides. Based on this information, a person skilled in the art can predict the alignment of the polypeptide's amino acid residues in relation to its three-dimensional structure. In various embodiments, a person skilled in the art may avoid radical changes to amino acid residues presumably located on the surface of the polypeptide, as such residues may be involved in important interactions with other molecules. Furthermore, a person skilled in the art can generate test variants containing a single amino acid substitution for each desired amino acid residue. These variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to obtain information about suitable variants. For example, if it is discovered that a change to a particular amino acid residue results in a disruption, an undesirable decrease in activity, or inappropriate activity, variants with such a change can be avoided. In other words, based on the information obtained in such routine experiments, a person skilled in the art can easily identify amino acids where further substitutions, either alone or in combination with other mutations, should be avoided.

As used herein, the terms "polypeptide fragment" and "truncated polypeptide" refer to a polypeptide having an N-terminal and/or C-terminal deletion compared to the corresponding full-length protein. In various embodiments, the fragments can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids long. In various embodiments, the length of the fragments may also be, for example, no more than 1000, no more than 900, no more than 800, no more than 700, no more than 600, no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 50, no more than 25, no more than 10, or no more than 5 amino acids. The fragment may further comprise, at one or both ends thereof, one or more additional amino acids, such as an amino acid sequence from another naturally occurring protein (e.g., an Fc domain or a leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

As used herein, the terms "polypeptide variant," "hybrid polypeptide," and "polypeptide mutant" refer to a polypeptide comprising an amino acid sequence with an insertion, deletion, and/or substitution of one or more amino acid residues of the amino acid sequence relative to the sequence of another polypeptide. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. Hybrids of the present invention include fusion proteins.

A "derivative" of a polypeptide is a polypeptide that has been chemically modified, such as by conjugation with another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

The term "% sequence identity" is used herein interchangeably with the term "% identity" and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity as determined by a given algorithm and means that a given sequence is at least 80% identical to another region of another sequence. In various embodiments, the % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or greater sequence identity to a given sequence. In various embodiments, the % identity ranges from, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

The term "% sequence homology" is used herein interchangeably with the term "% homology" and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same as 80% sequence homology as determined by a given algorithm, and, accordingly, a homologue of a given sequence has more than 80% sequence homology over a region of the given sequence. In various embodiments, the % homology is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence homology to a given sequence. In various embodiments, the % homology ranges from, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

Typical computer programs that can be used to determine the identity of two sequences include, but are not limited to, the BLAST suite of programs, such as BLASTN, BLASTX, TBLASTX, BLASTP, and TBLASTN, which are publicly available online at the NCBI website. See also Altschul et al., J. Mol. Biol. 215:403–10, 1990 (with special reference to the published default settings, i.e., w = 4 and t = 17) and Altschul et al., Nucleic Acids Res., 25:3389–3402, 1997. When evaluating a given amino acid sequence against amino acid sequences from GenBank Protein Sequences and other publicly available databases, sequence searches are typically performed using the BLASTP program. BLASTX is the preferred program for searching nucleic acid sequences translated in all reading frames against amino acid sequences from GenBank Protein Sequences and other publicly available databases. Both BLASTP and BLASTX are run using the default gap opening penalty of 11.0 and gap extension penalty of 1.0, and the BLOSUM-62 matrix.

In addition to calculating percent identity, the BLAST algorithm also allows for statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 90:5873–5787, 1993). One measure of similarity when using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability of a random match between two nucleotide or amino acid sequences. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the analyzed nucleic acid to the reference nucleic acid is, for example, less than about 0.1, less than about 0.01, or less than about 0.001.

As used herein, the term "modification" refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modifications (e.g., glycosylation) of the polypeptide.

As used herein, the term "knob-into-hole modification" refers to a modification of the facing surfaces of two immunoglobulin heavy chains in the CH3 domain. In one embodiment, the knob-into-hole modification comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one antibody heavy chain and the amino acid substitutions T366S, L368A, Y407V, and optionally Y349C in the other antibody heavy chain. Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001).

As used herein, the term "biologically activated drug" or "VitoKine" refers to a drug precursor compound that, upon administration to a subject, releases the drug in vivo through some chemical or physiological process such that the bioactivated drug is converted into a product active in target tissues. A bioactivated drug is any compound that undergoes biological activation before exerting its pharmacological effects. Thus, bioactivated drugs can be thought of as drugs containing specialized non-toxic protecting groups used temporarily to alter or eliminate undesirable properties of the parent molecule.

As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising two or more genes originally encoding separate proteins, wherein the components of the fusion protein are joined to each other by peptide bonds, either directly or through peptide linkers. As used herein, the term "fusion" refers to components joined by peptide bonds, either directly or through one or more peptide linkers.

A "linker" refers to a molecule that connects two other molecules covalently or through ionic, van der Waals, or hydrogen bonds, such as a nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 5' end, thereby joining two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded, cleaved, or otherwise separated to separate the two components joined by the cleavable linker. Cleavable linkers are typically cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers can also be cleaved by environmental factors, such as changes in temperature, pH, salt concentration, and so on.

As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids, typically approximately 1-30 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, ( _G4S ) _n , ( _SG4 ) _n , or _G4 ( _SG4 ) _n peptide linkers. In most cases, "n" is a number from 1 to 10, typically from 2 to 4.

"Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal. The pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to that amount of the agent that is effective in producing the intended pharmacological result. "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, fillers, buffers, and excipients, such as phosphate-buffered saline, 5% aqueous dextrose solution, emulsions such as oil/water or water/oil emulsions, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and compositions are described in Remington's Pharmaceutical Sciences, ^21st Ed. 2005, Mack Publishing Co, Easton. A "pharmaceutically acceptable salt" is a salt that can be incorporated into a compound for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

As used herein, "treatment" (and its grammatical variations, such as "treat") refers to a clinical intervention in an attempt to alter the natural course of a disease in the individual being treated and may be administered prophylactically or during the progression of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of disease development or recurrence, alleviation of symptoms, reduction in any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or temporary alleviation of a painful condition, and remission or improvement of prognosis. As used herein, "alleviation" of a disease, disorder, or condition means a reduction in the severity and/or frequency of symptoms of the disease, disorder, or condition. Furthermore, reference to "treatment" herein includes references to curative, palliative, and prophylactic treatment.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat the specified disorder, condition, or disease, such as ameliorating, alleviating, reducing, and/or delaying one or more of its symptoms. With respect to cancer or other unwanted cell proliferation, an effective amount includes an amount sufficient to: (1) reduce the number of cancer cells; (2) reduce the size of a tumor; (3) inhibit, slow down, somewhat slow down, and preferably stop the infiltration of peripheral organs by cancer cells; (4) inhibit (i.e., somewhat slow down, and preferably stop) tumor metastasis; (5) inhibit tumor growth; (6) prevent or delay the development and/or recurrence of a tumor; and/or (7) somewhat alleviate one or more of the symptoms associated with cancer. An effective amount may be administered in one or more administrations.

The phrase "administration" or "actions leading to administration" refers to actions taken by a healthcare professional (e.g., a physician) or a person supervising the care of a patient who oversees and/or authorizes the administration of the relevant agent(s)/compound(s) to the patient. Actions leading to administration may include diagnosis and/or determination of an appropriate therapeutic regimen and/or prescribing the specific agent(s)/compound(s) to the patient. Such prescribing may include, for example, filling a prescription form, recording it in a patient's medical record, and the like. When describing administration, "actions leading to administration" are also included herein.

The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or non-human primate, but also to domesticated mammals (e.g., cats or dogs), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and farm mammals (e.g., horses, bovines, pigs, sheep). In various embodiments, a patient may be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a child male, or a child female) being cared for by a physician or other health care professional in an inpatient setting, a mental health facility, an outpatient setting, or other clinical context. In various embodiments, the patient may be an immunocompromised patient or a patient with a weakened immune system, including, but not limited to, patients with primary immunodeficiency, AIDS (acquired immunodeficiency syndrome), cancer patients, and transplant patients receiving certain immunosuppressive drugs, and patients with inherited diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient is a patient with an immunogenic cancer, including, but not limited to, bladder cancer, lung cancer, melanoma, and other cancers reported to have an increased mutation rate (Lawrence et al., Nature, 499(7457):214-218, 2013).

The term "immunotherapy" refers to the treatment of cancer, including, but not limited to: treatment using depleting antibodies to certain tumor antigens; treatment using antibody-drug conjugates; treatment using agonist, antagonist, or blocking antibodies to costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1, CD40, OX-40, CD 137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec7, Siglec 8, Siglec 9, Siglec 15 and VISTA, CD276, CD272, TIM-3, B7-H4; treatment using bispecific T-cell engager (BiTE®) antibodies such as blinatumomab; treatment involving the administration of biological response modifiers such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, IL-22, GM-CSF, IFN-α, IFN-β and IFN-γ, a TGF-β antagonist or a TGF-β trap; treatment using therapeutic vaccines such as sipuleucel-T; treatment using therapeutic viruses, including but not limited to oncolytic viruses such as T-vec; treatment using dendritic cell vaccines or vaccines based on tumor antigen peptides or neoantigens; treatment using NK cells; treatment using chimeric antigen receptor T cells (CAR-T cells); treatment using CAR-NK cells; treatment using dendritic cells (DCs) or T cells; treatment using iPS-induced NK cells; treatment using iPS-induced T cells; and treatment using a vaccine such as bacille Calmette-Guérin (BCG); treatment using tumor-infiltrating lymphocytes (TIL); treatment using adoptively transferred anti-tumor T cells (ex vivo cultured and/or TC β R T cells); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9 and imiquimod.

"Resistant or refractory cancer" refers to tumor cells or cancer that have failed to respond to prior anticancer therapy, including, for example, chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells may be resistant or refractory at the start of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that are unresponsive at the start of treatment or that initially respond for a short period but ultimately fail to respond. Refractory tumor cells also include tumors that respond to anticancer therapy but fail to respond to subsequent rounds of therapy. For the purposes of this invention, refractory tumor cells also encompass tumors that are suppressed by anticancer therapy but relapse within five years, sometimes ten years or more, after cessation of therapy. Anti-cancer therapy may involve chemotherapy alone, radiation alone, targeted therapy alone, surgery alone, or combinations thereof. For simplicity and not to be limiting, it should be understood that "refractory tumor cells" are interchangeable with "resistant tumor."

The term "tumor-associated antigen" (TAA) refers, for example, to cell surface antigens selectively expressed by cancer cells or overexpressed in cancer cells compared to most normal cells. As used herein, the terms "TAA variant" and "TAA mutant" refer to a TAA containing an amino acid sequence with an insertion, deletion, and/or substitution of one or more amino acid residues relative to another TAA sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length.

The term "neoantigen" refers, for example, to cell surface antigens that are previously unknown to the immune system, especially antigens that arise from changes in host antigens due to radiation, chemotherapy, viral infection, neoplastic transformation/mutation, drug metabolism, etc., that are selectively expressed by cancer cells or overexpressed in cancer cells compared to most normal cells.

As used herein, the term "antibody" is used in its broadest sense and encompasses various antibody structures (IgG1, 2, 3 or 4, IgM, IgA, IgE), including, without limitation, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or bifunctional antibodies) and antibody fragments, provided that they exhibit the desired antigen-binding activity.

As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies.

As used herein, the term "Fab fragment" refers to an immunoglobulin fragment comprising the VL domain and constant domain (CL) of the light chain with the VH domain and first constant domain (CH1) of the heavy chain.

As used herein, the terms "variable region" or "variable domain" refer to the domain of the heavy or light chain of an immunoglobulin or antibody that is typically involved in binding the immunoglobulin or antibody to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of an immunoglobulin or antibody typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs).

As used herein, "human immunoglobulin" means an immunoglobulin having an amino acid sequence corresponding to the amino acid sequence of an immunoglobulin produced by a human being or a human cell, or derived from a non-human source, that utilizes human immunoglobulin repertoires or other sequences encoding human immunoglobulins. This definition of human immunoglobulin expressly excludes humanized immunoglobulin containing non-human antigen-binding residues.

As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. The Fc region of IgG comprises a CH2 domain of IgG and a CH3 domain of IgG. The CH3 region may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain that has a knob introduced into one chain and a corresponding cavity introduced into the other chain; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical immunoglobulin heavy chains, as described herein. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system.

As used herein, the term "effector functions" refers to the biological activity inherent in the Fc region of an immunoglobulin, which varies among immunoglobulin isotypes. Examples of immunoglobulin effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

When used here, the term "regulatory T cell" or "Treg cell" refers to a specialized type of CD4+ T cell that can suppress the responses of other T cells (effector T cells). Tregs are characterized by the expression of CD4, the IL-2 receptor α subunit (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531–62 (2004)) and play a critical role in inducing and maintaining peripheral self-tolerance to antigens, including tumor-expressed antigens.

When used herein, the term "conventional CD4+ T cells" refers to CD4+ T cells other than regulatory T cells.

When used herein, the term “selective Treg cell activation” refers to the activation of Treg cells with little or no concomitant activation of other T cell subsets (such as CD4+ helper T cells, CD8+ cytotoxic T cells, NK T cells) or natural killer (NK) cells.

As used here, "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an immunoglobulin to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR).

As used herein, the terms "affinity" or "binding affinity" refer to the strength of the total sum of noncovalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can typically be represented as a dissociation constant (K _D ), which is the ratio of the dissociation and association rate constants (k _off and k _on , respectively). A specific method for measuring affinity is surface plasmon resonance (SPR).

As used herein, the term "decreased binding" refers to a decrease in the affinity of the corresponding interaction, measured, for example, by SPR. Conversely, "enhanced binding" refers to an increase in the binding affinity of the corresponding interaction.

As used herein, the term "polymer" generally includes, without limitation: homopolymers; copolymers, such as block copolymers, graft copolymers, random copolymers, and alternating copolymers; and terpolymers; and blends and modifications thereof. Furthermore, unless expressly limited, the term "polymer" includes all possible geometric configurations of the substance. These configurations include, without limitation, isotactic, syndiotactic, and random symmetry.

"Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include those containing bases that are not naturally occurring, nucleotides that form linkages with other nucleotides other than the naturally occurring phosphodiester bond, or those containing bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranephosphonates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNA), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, typically no more than approximately 50 nucleotides long. It should be understood that the representation of a nucleotide sequence as a DNA sequence (i.e., A, T, G, C) also includes an RNA sequence (i.e., A, U, G, C), in which "T" is replaced by "U."

A traditional notation is used to describe polynucleotide sequences: the left end of a single-stranded polynucleotide sequence is the 5' end; the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5'-3' direction of nucleotide addition to synthesized RNA transcripts is referred to as the direction of transcription. The DNA strand that has the same sequence as the mRNA (messenger RNA) is called the "coding strand"; sequences of the DNA strand that have the same sequence as the mRNA transcribed from this DNA and are located 5' from the 5' end of the RNA transcript are called "upstream sequences"; sequences of the DNA strand that have the same sequence as the RNA and are located 5' from the 5' end of the coding RNA transcript are called "downstream sequences".

"Complementarity" refers to the topological compatibility or mutual comparability of the interacting surfaces of two polynucleotides. Thus, two molecules can be described as complementary and, furthermore, the characteristics of their contact surfaces are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide or if the first polynucleotide can hybridize with the second polynucleotide under stringent hybridization conditions.

"Specifically hybridizing to," or "specific hybridization," or "hybridize selectively to," refers to the preferential binding, duplex formation, or hybridization of a nucleic acid molecule to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) of DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target subsequence and, to a lesser extent, to other sequences, or will not hybridize at all to other sequences. "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence-dependent and vary under different environmental conditions. Comprehensive guidelines for nucleic acid hybridization are provided by Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, N.Y.; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd sup. ed., NY; and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

Typically, highly stringent hybridization and wash conditions are selected to be approximately 5°C below the melting temperature (Tm) of a particular sequence at a given ionic strength and pH. Tm is the temperature (at a given ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Highly stringent conditions are selected to be equal to the Tm for the corresponding probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids with more than approximately 100 complementary residues on a Southern or Northern blot filter is 50% formalin with 1 mg heparin at 42°C, with hybridization performed overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent wash conditions is a wash with 0.2x SSC at 65°C for 15 minutes. The SSC buffer is described in Sambrook et al. A wash under high stringency conditions may be preceded by a wash under low stringency conditions to remove background probe signal. A typical medium-stringency wash for a duplex of, for example, more than approximately 100 nucleotides is 1x SSC at 45°C for 15 minutes. A typical low-stringency wash for a duplex of, for example, more than approximately 100 nucleotides is 4-6x SSC at 40°C for 15 minutes. Typically, a signal-to-noise ratio 2-fold (or more) higher than that observed for an unrelated probe in a given hybridization assay indicates detection of specific hybridization.

A "primer" refers to a polynucleotide capable of specifically hybridizing to a designated polynucleotide template and providing the initiation site for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions that trigger synthesis, that is, in the presence of nucleotides, a complementary polynucleotide template, and a polymerization agent such as DNA polymerase. A primer is typically single-stranded but may be double-stranded. Primers are typically deoxyribonucleic acids, but in many cases, a wide variety of synthetic and naturally occurring primers are applicable. A primer is complementary to the template to which it is designed to hybridize, providing the initiation site for synthesis, but it does not necessarily reflect the exact sequence of the template. In such cases, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, for example, chromogenic, radioactive or fluorescent moieties and used as detectable moieties.

When used in connection with a polynucleotide, "probe" refers to a polynucleotide capable of specifically hybridizing to a designated sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide but does not necessarily reflect the exact complementary sequence of the template. In such cases, specific hybridization of the probe to the target polynucleotide depends on the stringency of hybridization conditions. Probes can be labeled, for example, with chromogenic, radioactive, or fluorescent moieties and used as detectable moieties. In cases where the probe provides the initiation point for complementary polynucleotide synthesis, the probe can also serve as a primer.

A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), where additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors containing the bacterial replicator and episomal vectors for mammalian cells). Other vectors (e.g., non-episomal vectors for mammalian cells) undergo integration into the host cell genome upon introduction into the host cell and, thereby, undergo replication along with the host genome. An "expression vector" is a type of vector capable of directing the expression of a selected polynucleotide.

A "regulatory sequence" is a nucleic acid that influences the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence may, for example, exert its effects on the regulated nucleic acid directly or through the action of one or more other molecules (e.g., polypeptides linked to the regulatory sequence and/or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Other examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif, and Baron et al., 1995, Nucleic Acids Res. 23:3605–06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the nucleotide sequence.

A "host cell" is a cell that can be used to express a polynucleotide of the invention. A host cell can be a prokaryote, such as E. coli, or it can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or another fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to refer to a host cell transformed or transfected with a nucleic acid to be expressed. A host cell may also be a cell that contains a nucleic acid but does not express it at the desired level, unless a regulatory sequence has been introduced into the host cell in such a way that it becomes operably linked to that nucleic acid. It should be understood that the term "host cell" refers not only to the specific cell in question but also to the progeny or potential progeny of such a cell. Since certain modifications are possible in subsequent generations, due, for example, to mutations or environmental influences, such progeny may not actually be identical to the original cell, but the scope of the term, when used herein, still includes such progeny.

The term "isolated molecule" (where the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that, by virtue of its source: (1) is not associated with the components with which it is naturally associated in its native state; (2) is substantially free of other molecules of the same species; (3) is expressed by a cell of a different species; or (4) is not naturally occurring. Thus, a chemically synthesized molecule or a molecule expressed in a cellular system other than the cell of its natural source will be "isolated" from the components with which it is naturally associated. A molecule may also be made substantially free of the components with which it is naturally associated by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be analyzed using a number of techniques well known in the art. For example, the purity of a polypeptide sample can be analyzed using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques well known in the art. For certain applications, higher resolution can be achieved using high performance liquid chromatography (HPLC) or other purification methods well known in the art.

A protein or polypeptide is "substantially pure," "substantially uniform," or "substantially purified" when at least about 60% to 75% of the sample is represented by a single polypeptide species. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise approximately 50%, 60%, 70%, 80%, or 90%, w/w, of the protein sample, more typically approximately 95%, and will preferably be greater than 99% pure. The purity or uniformity of a protein can be demonstrated by a number of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample followed by visualization of a single polypeptide band after staining the gel with a dye well known in the art. For certain applications, higher resolution can be achieved using HPLC or other purification methods well known in the art.

As used herein, the terms "label" or "labeled"/"labeled" refer to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as by incorporating a radioactively labeled amino acid or by attaching biotinyl moieties to a polypeptide that can be detected by avidin containing a marker (e.g., streptavidin containing a fluorescent marker or having enzymatic activity that can be detected by optical or calorimetric methods). In another embodiment, the label or marker can be therapeutic, such as a drug conjugate or a toxin. Various methods for labeling polypeptides and glycoproteins known in the art can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope labels), magnetic agents such as gadolinium chelates, toxins such as Pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinatedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and their analogs and homologues. In various embodiments, the labels are attached via spacers of varying lengths to reduce potential steric hindrance.

As used herein, the term "heterologous" refers to a composition or structure that is not native or found in nature, such as a composition or structure that can be obtained by replacing an existing natural composition or structure with one derived from another source. Similarly, expression of a protein in an organism different from the organism that naturally expresses the protein constitutes a heterologous expression system and a heterologous protein.

It should be understood that aspects and embodiments of the invention described herein include aspects and embodiments “consisting of” and/or “consisting essentially of” the described aspects and embodiments.

References to "approximately" in a description of a value or parameter herein include (and describe) variations that directly address that value or parameter. For example, a description that states "approximately X" includes the description "X."

Unless the context clearly dictates otherwise, as used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural. It is understood that aspects and variants of the invention described herein include aspects and variants "consisting of" and/or "consisting essentially of" the described aspects and variants.

Description of the VitoKine platform

According to the present invention, a biologically activatable cytokine-based drug platform ("VitoKine") is provided, aimed at reducing systemic toxicity based on the mechanism of action and providing a broader therapeutic application of proteins, such as cytokines. With reference to FIG. 1, the novel VitoKine constructs of the present invention comprise a D1 domain, which is a targeting domain, an immune checkpoint modulator targeting domain, a half-life-extending domain, or a dual-function or multifunctional moiety domain, an "active moiety domain" (D2), and a "masking moiety domain" (D3). The proposed methods for activating the D2 domain of the VitoKine construct are shown in FIG. 2. Importantly, maintaining the inertness or reduced activity of the D2 moiety of the VitoKine construct prior to its local activation by proteases upregulated in disease-affected tissues will limit binding of the active moiety to its receptors at the periphery or on the surface of unaffected cells, preventing excessive activation of its pathway and reducing unwanted toxicity associated with target binding outside the target tissue. Furthermore, the inertness of the active moiety of the VitoKine construct prior to its activation by proteases will significantly reduce potential clearance due to antigen or target binding, thereby increasing the in vivo half-life and improving biodistribution and bioavailability at the intended therapeutic sites.

D1 domain (targeting domain, half-life-extending domain, or dual-function or multifunctional moiety domain)

In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is a targeting moiety in the form of an antibody, or an antibody fragment, or a protein, or a peptide to a tumor-associated antigen. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is an antibody, an antibody fragment, a protein, or a peptide to an immune checkpoint modulator. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain that is an antibody, or an antibody fragment, or a protein, or a peptide as an autoimmune modulator. In various embodiments, the VitoKine constructs of the present invention comprise a D1 whose function is to retain the D2 domain in a tissue focus, such as the tumor microenvironment (TME) or tissue inflammatory foci. In various embodiments, the VitoKine constructs of the present invention comprise a D1 that is bifunctional, for example, tissue-targeted and provides tissue retention. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain, which is a polymer. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain, which is a half-life-enhancing moiety. In various embodiments, the VitoKine constructs of the present invention comprise a D1 domain, which is an Fc domain.

Fc domains

IgG immunoglobulins are among the most abundant proteins in human blood. Their circulating half-life can be as long as 21 days. Fusion proteins containing combinations of IgG Fc regions with domains of other proteins, such as various cytokines and receptors, have been reported (see, e.g., Capon et al., Nature, 337:525–531, 1989; Chamow et al., Trends Biotechnol., 14:52–60, 1996; Patent Nos. 5,116,964 and 5,541,087). The prototype of such a fusion protein is a homodimeric protein linked through cysteine residues in the hinge Fc region of IgG to produce an IgG-like molecule lacking the variable domain and the CH1 domain of the heavy chain and light chains. The dimeric nature of Fc-domain-containing fusion proteins may be advantageous for enabling higher-order interactions (i.e., bivalent or bispecific binding) with other molecules. Due to structural homology, the in vivo pharmacokinetic profile of Fc-fusion proteins is comparable to that of human IgG of a similar isotype.

The term "Fc" refers to a molecule or sequence comprising the sequence of a fragment of a whole antibody other than an antigen-binding fragment, in monomeric or multimeric form. The immunoglobulin that is the source of native Fc is preferably of human origin and can be any of the immunoglobulins, but IgG1 and IgG2 are preferred. Native Fc are composed of monomeric polypeptides that can be linked to form dimeric or multimeric forms by covalent (i.e., disulfide) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules varies from 1 to 4 depending on the class (e.g., IgG, IgM, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2). One example of a native Fc is the disulfide-linked dimer produced by papain digestion of IgG (see Ellison et al. (1982) Nucleic Acids Res. 10: 4071–9). When used here, the term "native Fc" is a generic term for monomeric, dimeric, and multimeric forms. Fc domains contain binding sites for protein A, protein G, various Fc receptors, and complement proteins.

In various embodiments, the term "Fc variant" refers to a molecule or sequence modified relative to a native Fc but still containing a binding site for the salvage receptor FcRn. Exemplary Fc variants, as well as interactions with the salvage receptor, are described in International Applications WO 97/34631 (published September 25, 1997) and WO 96/32478, incorporated herein by reference. Furthermore, a native Fc contains sites that may be deleted because they impart structural characteristics or biological activity that are not essential for the fusion molecules of the present invention. Thus, in various embodiments, the term "Fc variant" includes a molecule or sequence that lacks one or more sites or residues of a native Fc that affect or are involved in (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N- or C-terminal heterogeneity when expressed in the selected host cell, (4) glycosylation, (5) interaction with complement such as CDC, (6) binding to a Pc receptor other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether produced by cleavage of a whole antibody, recombinant gene expression, or otherwise. In various embodiments, an "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO: 13), which typically contains all or a portion of a hinge region. In various embodiments, mutations of the Fc domain are possible to eliminate effector functions. In various embodiments, each of the Fc domain monomers in the Fc domain comprises amino acid substitutions in the CH2 constant domain of the antibody to reduce interaction or binding between the Fc domain and the Fc receptor. In various embodiments, each Fc domain subunit comprises two amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function, wherein said amino acid substitutions are L234A and L235A. In various embodiments, each Fc domain subunit comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function, wherein said amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO: 14). In various embodiments, each Fc domain subunit comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function, wherein said amino acid substitutions are L234A, L235A, and P329A.

In various embodiments, mutations of the Fc domain are possible to further increase the half-life in vivo. In various embodiments, each Fc domain subunit comprises three amino acid substitutions that enhance binding to human FcRn, wherein said amino acid substitutions are M252Y, S254T, and T256E, disclosed in U.S. Patent Publication No. 7,658,921 (SEQ ID NO: 156). In various embodiments, each Fc domain subunit comprises one amino acid substitution that enhances binding to human FcRn, wherein said amino acid substitution is N434A (SEQ ID NO: 166), disclosed in U.S. Patent Publication No. 7,371,826. In various embodiments, each Fc domain subunit comprises one amino acid substitution that enhances binding to human FcRn, wherein said amino acid substitutions are M428L and N434S, disclosed in U.S. Patent Publication No. 8,546,543. In various embodiments, combinations of mutations that increase half-life with amino acid substitutions that reduce binding to the activating Fc receptor and/or effector function are possible.

In various embodiments, each of the two Fc domain monomers in the Fc domain contains amino acid substitutions that promote heterodimerization of the two monomers. In various embodiments, heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions into the two Fc domain monomers, such as knob-into-hole residue pairs. The knob-into-hole technique is also disclosed in U.S. Patent Publication No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob mutation T366W and the other Fc domain monomer contains the hole mutations T366S, L358A, and Y407V. In various embodiments, two Cys residues (S354C on the knob side and Y349C on the trough side) are introduced to form a stabilizing disulfide bond (SEQ ID NOs: 15 and 16). Use of a heterodimeric Fc can result in a monovalent VitoKine construct.

Antibodies and proteins/peptides that bind to disease-associated targets or tumor-associated antigens

In various embodiments, D1 may be a targeting moiety in the form of an antibody to a tumor-associated antigen (TAA) or another protein or peptide exhibiting binding affinity for a cell affected by a disease or tissue affected by a disease. A TAA may be any molecule, macromolecule, combination of molecules, etc., against which an immune response is desired. A TAA may be a protein comprising more than one polypeptide subunit. For example, the protein may be a dimer, trimer, or higher-order multimer. In various embodiments, two or more protein subunits may be joined by a covalent bond, such as, for example, a disulfide bond. In various embodiments, the protein subunits may be held together by non-covalent interactions. Thus, the TAA may be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, against which one skilled in the art desires to induce an immune response. In various embodiments, the TAA is a peptide comprising about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 amino acids. In various embodiments, the peptide, polypeptide, or protein is a molecule typically administered to subjects by injection. In various embodiments, following administration, the tumor-specific antibody or binding protein acts as a targeting moiety, directing the VitoKine construct to the site of disease, such as a cancer lesion, where the active domain can be released and interact with its cognate receptors on disease-affected cells or tissue.

Any of the above-mentioned markers can be used as a disease-associated target or TAA target for the VitoKine constructs of the present invention. In various embodiments, one or more disease-associated targets, or variants thereof, or TAA, TAA variants, or TAA mutants intended for use in the VitoKine constructs and methods of the present invention are selected from the list presented in Table 2 or are derived from the antigens presented in this list.

Other examples of tumor-associated antigens include TRP-1, TRP-2, MAG-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-BSO (LAGE), SCP-1, Hom/Mel-40, H-Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) E6 and E7 antigens, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, Numa, K-ras, β-catenin, CDK4, Muni-1, p16, TAGE, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA 15-3 (CA 27.29/BCAA), CA 195, CA242, CA-50, CAM43, CD68/KF1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac binding protein-2/cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.

Immune checkpoint modulators

Various immune cells have been reported to express a number of immune checkpoint protein antigens, including, for example, CD152 (expressed by activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed on tumor-infiltrating lymphocytes, expressed by activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, anergic T cells, monocytes, dendritic cells), CD274 (expressed on T cells, B cells, dendritic cells, vascular endothelial cells, pancreatic islet cells), and CD223 (expressed by activated T cells, regulatory T cells, anergic T cells, NK cells, NKT cells, and plasmacytoid dendritic cells) (see, e.g., Pardoll, D., Nature Reviews Cancer, 12:252-264, 2012). Antibodies that bind to an antigen identified as an immune checkpoint protein are known to those skilled in the art. For example, various anti-CD276 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20120294796 (Johnson et al.) and references cited therein); various anti-CD272 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20140017255 (Mataraza et al.) and references cited therein); Various anti-CD152/CTLA-4 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20130136749 (Korman et al.) and references cited therein); Various anti-LAG-3/CD223 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20110150892 (Thudium et al.) and references cited therein); Various anti-CD279/PD-1 antibodies have been described in the art (see, e.g., U.S. Patent No. 7,488,802 (Collins et al.) and references cited therein); Various anti-PD-L1 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20130122014 (Korman et al.) and references cited therein); Various anti-TIM-3 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20140044728 (Takayanagi et al.) and references cited therein); and various anti-B7-H4 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20110085970 (Terrett et al.) and references cited therein). Each of these references is hereby incorporated by reference in its entirety with respect to the particular antibodies and sequences disclosed therein.

In various embodiments, D1 may comprise an antibody, an antibody fragment, or a protein or peptide that exhibits binding to an immune checkpoint protein antigen present on the surface of an immune cell. In various embodiments, the immune checkpoint protein antigen is selected from the group consisting of, but not limited to, CD276, CD272, CD152, CD223, CD279, CD274, CD40, SIRPα, CD47, OX-40, GITR, ICOS, CD27, 4-1BB, TIM-3, B7-H4, Siglec-7, Siglec-8, Siglec-9, Siglec-15, and VISTA.

In various embodiments, D1 may comprise an antibody to an immune checkpoint protein antigen present on the surface of a tumor cell, selected from the group consisting of, but not limited to, PD-L1, B7-H3, and B7-H4.

In various embodiments, D1 is an antibody that is an antagonist antibody or an antagonist antibody fragment to fibroblast activation protein (FAP). In various embodiments, the antibody is a humanized anti-FAP antibody comprising the amino acid sequences set forth in SEQ ID NOs: 193 and 194. In various embodiments, D1 is an antibody or an antibody fragment to an immune checkpoint modulator. In various embodiments, the antibody is an antagonist antibody or an antagonist antibody fragment to PD-1. In various embodiments, the antibody is an antagonist humanized PD-1 antibody comprising the amino acid sequences set forth in SEQ ID NOs: 195 and 196. In various embodiments, the antibody is an antagonist humanized PD-1 antibody comprising the amino acid sequences set forth in SEQ ID NOs: 197 and 198. In various embodiments, the antibody is an antagonist humanized PD-1 antibody comprising the amino acid sequences set forth in SEQ ID NOs: 275 and 276. In various embodiments, the antibody is an antagonist human PD-1 antibody comprising the amino acid sequences set forth in SEQ ID NOs: 277 and 278. In various embodiments, the antibody is an antagonist PD-L1 antibody or antagonist antibody fragment. In various embodiments, the antibody is an antagonist antibody to human PD-L1 comprising the amino acid sequences set forth in SEQ ID NOs: 279 and 280. In various embodiments, VitoKine antibody constructs comprise the amino acid sequences set forth in SEQ ID NOs: 128-142, 180-181, 281-286, 296-297, and 303-306.

Modulators of autoimmune and inflammatory disorders

Any of the above-mentioned proteins, the expression of which is highly expressed in various inflammatory tissues or on immune cells, can be used as a target associated with autoimmune/inflammatory diseases for the VitoKine constructs of the present invention. In various embodiments, one or more targets associated with autoimmune/inflammatory diseases, their variants, or their mutants/isoforms, which are intended for use in the VitoKine constructs and methods of the present invention, are selected from the list presented in Table 3 or are derived from the targets presented in this list. These targets can also be used for targeting cancer.

In various embodiments, the D1 targeting moiety may be an antibody, antibody fragment, other protein, or peptide specific for inflamed tissue that exhibits binding to a cell affected by the disease or the disease microenvironment, such as _TNF , TNFR, integrin _A4β7 , IL-6Rα, BLYS, TSLP. In various embodiments, the VitoKine antibody constructs comprise the amino acid sequences set forth in SEQ ID NOs: 143-146.

Polymers

In various embodiments, D1 may be a polymer, such as polyethylene glycol (PEG). In various embodiments, the polymer, such as PEG, may be covalently attached to the N- or C-terminus or at an internal position using conventional chemistry, such as chemical conjugation. In various embodiments, the polymer, such as PEG, may be covalently attached to the N-terminus of the D2 domain by site-specific conjugation or by replacing the cytokine amino acids with other amino acids or specially designed amino acids. Half-life enhancing moieties

In various embodiments, other half-life-enhancing moieties may be used as D1 domains in the present invention to increase the serum half-life of VitoKine constructs. Half-life-enhancing moieties include, but are not limited to, an Fc domain, an Fc variant, an antibody fragment (Fab, scFv), EXTEN (Schellenberger et al., Nat. Biotechnol. 27:1 186-1 192, 2009), and human serum albumin.

Domain D2 (active grouping domain)

D2 is the active moiety of the VitoKine construct, whose activity is reversibly masked within the construct and can be restored upon cleavage by proteases at the site of disease. This active moiety can be any protein, including, without limitation, a polypeptide of any native or variant interleukin or cytokine. Importantly, maintaining the inertness or attenuated activity of the "active moiety" of the VitoKine construct prior to its local activation by proteases upregulated in disease-affected tissues will limit the active moiety's binding to its receptors at the periphery or on the surface of non-disease-affected cells, preventing excessive activation of its pathway and mitigating unwanted toxicity associated with target binding outside the target tissue. Furthermore, the inertness of the active moiety of the VitoKine construct prior to its activation by proteases will significantly reduce the potential clearance due to antigen or target binding, and thus increase the in vivo half-life and improve biodistribution and action at the intended sites of therapy.

IL-15

Interleukin-15 (IL-15) is a cytokine identified by two independent groups based on its ability to stimulate proliferation of the IL-2-dependent T cell line CTLL-2 in the presence of neutralizing anti-IL-2 antibodies (Steel et al., Trends in Pharmacological Sciences, 33(1):35–41, 2012). IL-15 and interleukin-2 (IL-2) have similar biological properties in vitro consistent with their shared receptor (R) signaling components (IL-2/15Rβγ _c ). However, specificity for IL-15, unlike IL-2, is provided by unique self-receptors consisting of α-chains that terminate the heterotrimeric high-affinity receptor complexes IL-15Rαβγ and IL-2Rαβγ, thus allowing cells to respond differentially depending on the ligand and the high-affinity receptor expressed. Notably, both IL-15 and IL-15Rα transcripts are much more widely distributed in tissues than IL-2/IL-2Rα. Furthermore, tight control of IL-15 expression is mediated by a multitude of complex post-transcriptional regulatory mechanisms. Thus, based on the complex regulation and differences in expression patterns of IL-15 and IL-15Rα, it is likely that the critical functions of this receptor/ligand pair in vivo differ from those of IL-2 and IL-2Rα. Studies to date on IL-15 biology have identified several key, non-exhaustive functions, such as the importance of IL-15 in the development and function of natural killer (NK) cells, NK T cells, and intestinal intraepithelial lymphocytes. The role of IL-15 in autoimmune processes such as rheumatoid arthritis and malignancies such as adult T-cell lymphoma suggests that dysregulation of IL-15 may have deleterious effects on the host (Fehniger et al., Blood, 97:14-32, 2001).

When used herein in the context of proteins or polypeptides, the terms "native IL-15" and "native interleukin-15" refer to any naturally occurring amino acid sequence of mammalian interleukin-15, including both immature or precursor forms and mature forms. Non-limiting examples of amino acid sequence accession numbers of various native mammalian interleukin-15 species in GenBank include NP 032383 (Mu musculus, immature form), AAB60398 (Macaca mulatta, immature form), NP000576 (human, immature form), CAA62616 (human, immature form), AAI00964 (human, immature form), and AAH18149 (human). In various embodiments of the present invention, native IL-15 is an immature form or a precursor form of naturally occurring mammalian IL-15. In other embodiments, native IL-15 is a mature form of naturally occurring mammalian IL-15. In various embodiments, native IL-15 is a precursor form of naturally occurring human IL-15. In various embodiments, native IL-15 is a mature form of naturally occurring human IL-15. In various embodiments, the native IL-15 protein/polypeptide is isolated or purified. In various embodiments, the IL-15-based D2 domain is derived from the amino acid sequence of the human IL-15 precursor set forth in SEQ ID NO: 1:

In various embodiments, the IL-15-based D2 domain comprises the amino acid sequence of the mature form of human IL-15 set forth in SEQ ID NO: 2:

In various embodiments, the IL-15-based D2 domain will be an IL-15 variant (or mutant) comprising a sequence derived from the mature human IL-15 polypeptide sequence set forth in SEQ ID NO: 2. IL-15 variants (or mutants) are named herein using the native amino acid, its position in the mature sequence, and the amino acid of the variant. For example, "huIL-15 S58D" refers to human IL-15 comprising an S to D substitution at position 58 of SEQ ID NO: 2. In various embodiments, the D2 domain of the present invention comprises an IL-15 domain that is an IL-15 variant (also referred to herein as an IL-15 mutant domain). In various embodiments, the IL-15 variant comprises an amino acid sequence that is different from the native IL-15 protein (or wild-type IL-15 protein). In various embodiments, an IL-15 variant binds to an IL-15Rα polypeptide and functions as an IL-15 agonist or antagonist. In various embodiments, IL-15 variants with agonist activity have superagonist activity. In various embodiments, an IL-15 variant can function as an IL-15 agonist or antagonist independent of its binding to IL-15Rα. IL-15 agonists are characterized by comparable or increased biological activity compared to wild-type IL-15. IL-15 antagonists are characterized by decreased biological activity compared to wild-type IL-15 or the ability to inhibit IL-15-mediated responses. In various embodiments, an IL-15 variant binds to IL-15Rβγc receptors with increased or decreased activity. In various embodiments, the IL-15 variant sequence has at least one amino acid change, such as a substitution or deletion, compared to the sequence of native IL-15, wherein such changes result in agonistic or antagonistic activity relative to IL-15. In various embodiments, the amino acid substitutions/deletions are present in the IL-15Rβ and/or _γc interacting domains of IL-15. In various embodiments, the amino acid substitutions/deletions do not affect binding to the IL-15Rα polypeptide or the ability to produce the IL-15 variant. Suitable amino acid substitutions/deletions for producing IL-15 variants can be determined based on known IL-15 structures, comparisons of IL-15 with homologous molecules, such as IL-2, of known structure, through rational or random mutagenesis and functional assays, as provided herein, or other empirical methods. In addition, suitable amino acid substitutions may include conservative or non-conservative changes and insertions of additional amino acids. In various embodiments, the IL-15 variants of the invention comprise one or more amino acid deletions or one or more amino acid substitutions at position 30, 31, 32, 58, 62, 63, 67, 68 or 108 of the mature human IL-15 sequence set forth in SEQ ID NO: 2. In various embodiments, the substitutions D30T ("D30" refers to the amino acid and residue position in the native mature human IL-15 sequence and "T" refers to the substitution amino acid residue at the indicated position of the IL-15 variant), V31Y, H32E, S58H, S58I, S58P, S58R, S58Q, D62T, V63A, V63F, V63K, V63R, I67V, I68H, I68FJ68Q, I68G, I68K, I68D, Q108A, Q108S, Q108E, Q108K, or Q108M result in IL-15 variants with antagonist activity, and the S58D substitution results in IL-15 variants with agonist activity. In various embodiments, the IL-15 variant comprises a deletion of 1, or 2, or 3, or 4, 5, or 6 amino acids from the N-terminus of SEQ ID NO: 2. In various embodiments, the IL-15 variant comprises a deletion of 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acids from the C-terminus of SEQ ID NO: 2. In various embodiments, the IL-15 variant comprises an insertion of the amino acids "GS" (SEQ ID NO: 12) or "GGSGG" (SEQ ID NO: 153) or "GSSGGSGGS" (SEQ ID NO: 154) after position N95 of SEQ ID NO: 2. In various embodiments, the IL-15 variant comprises the amino acid sequence set forth in SEQ ID NOs: 3, 182-192, and 199-215.

Typical VitoKine constructs with IL-15 and Fc are shown in Table 4.

In various embodiments of the VitoKine IL-15 and antibody construct or IL-15 and Fc fusion molecules, the IL-15 and Fc fusion molecules will comprise two or more than two heterodimeric chains as set forth in Table 5.

In various embodiments, the IL-15-based D2 domain will comprise an IL-15 construct comprising IL-2RJ3 as set forth in SEQ ID NO: 12 or an IL-2Rβ-based blocking peptide selected from constructs having the amino acid sequences set forth in SEQ ID NO: 66-70.

In various embodiments, the IL-15-based D2 domain will comprise an IL-15 construct comprising an IL-2Rβ-based blocking peptide and having two or more heterodimeric chains as set forth in Table 6.

IL-2

Interleukin-2 (IL-2), a classic Th1 cytokine, is produced by T cells following activation via the T-cell antigen receptor and the costimulatory molecule CD28. IL-2 is regulated through the activation of signaling pathways and transcription factors acting on the IL-2 promoter, leading to new gene transcription, but also includes modulation of IL-2 mRNA stability. IL-2 binds to a receptor containing multiple chains, including the highly regulated α chain and the β and γ chains that mediate signaling through the Jak-STAT pathway. IL-2 transmits activation, growth, and differentiation signals to T cells, B cells, and NK cells. IL-2 is also important for mediating activation-induced T-cell death, a function that is a critical mechanism for terminating immune responses. A commercially available preparation of nonglycosylated human recombinant IL-2, aldesleukin (available under the trade name Proleukin® as desalanyl-1-serine-125 human interleukin-2 from Prometheus Laboratories Inc., San Diego, California), is approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been proposed for administration to patients infected with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, optimal dosing of IL-2 is limited by a short half-life and significant toxicity.

When used herein in the context of proteins or polypeptides, the terms "native IL-2" and "native interleukin-2" refer to any naturally occurring amino acid sequences of mammalian interleukin-2, including both immature or precursor forms and mature forms. Non-limiting examples of amino acid sequence accession numbers of various native mammalian interleukin-2 species in GenBank include NP 032392.1 (Mus muscuius, immature form), NP 001040595.1 (Macaca mulatta, immature form), NP 000577.2 (human, precursor form), CAA01199.1 (human, immature form), AAD48509.1 (human, immature form), and AAB20900.1 (human). In various embodiments of the present invention, native IL-2 is an immature form or a precursor form of naturally occurring mammalian IL-2. In other embodiments, native IL-2 is a mature form of naturally occurring mammalian IL-2. In various embodiments, native IL-2 is a precursor form of naturally occurring human IL-2. In various embodiments, native IL-2 is a mature form of naturally occurring human IL-2. In various embodiments, the IL-2-based D2 domain is derived from the amino acid sequence of the human IL-2 precursor set forth in SEQ ID NO: 6:

In various embodiments, the IL-2-based D2 domain comprises the amino acid sequence of the mature form of wild-type human IL-2 set forth in SEQ ID NO: 8, containing a substitution of cysteine at position 125 to serine, but does not alter binding to the IL-2 receptor compared to naturally occurring IL-2:

In various embodiments, the IL-2-based D2 domain will be a variant (or mutant) of IL-2 comprising a sequence derived from the mature human IL-2 polypeptide sequence set forth in SEQ ID NO: 8. In various embodiments, the IL-2 variant comprises an amino acid sequence different from the native IL-2 protein (or wild-type IL-15 protein). In various embodiments, the IL-2 variant binds to the IL-2Rα polypeptide and functions as an IL-2 agonist or antagonist. In various embodiments, IL-2 variants with agonist activity have superagonist activity. In various embodiments, an IL-2 variant can function as an IL-2 agonist or antagonist independent of its binding to IL-2Rα. IL-2 agonists are characterized by comparable or enhanced biological activity compared to wild-type IL-2. IL-2 antagonists are characterized by reduced biological activity compared to wild-type IL-2 or the ability to inhibit IL-2-mediated responses. In various embodiments, the IL-2 variant sequence has at least one amino acid change, such as a substitution or deletion, compared to the sequence of native IL-2, wherein such changes result in agonistic or antagonistic activity relative to IL-2. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO: 8, with reduced/aborted binding to IL-2Rα for selective activation and proliferation of effector T cells (Teff) for the treatment of cancer; exemplary amino acid substitutions are provided in Table 7. In various embodiments, the IL-2 variant with reduced/aborted binding to IL-2Rα comprises the amino acid sequence set forth in SEQ ID NO: 232-247. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO: 8, with reduced binding to IL-2Rβ and/or γβ and increased selectivity for activation and proliferation of regulatory T cells (Treg) for the treatment of autoimmune diseases; exemplary amino acid substitutions are provided in Table 7. As will be understood by those skilled in the art, all mutations can be optionally and independently combined in any manner to achieve optimal affinity and modulation of activity.

In various embodiments, the active moiety is selected from the group of sequences consisting of, but not limited to, the amino acid sequences of interleukin-4 (IL-4) (SEQ ID NO: 17), interleukin-7 (IL-7) (SEQ ID NO: 18), interleukin-9 (IL-9) (SEQ ID NO: 19), interleukin-10 (IL-10) (SEQ ID NO: 20), interleukin-12 alpha (IL-12α) (SEQ ID NO: 21), interleukin-12 beta (IL-12β) (SEQ ID NO: 22), interleukin-23 alpha (IL-23α) (SEQ ID NO: 23) and TGFβ (SEQ ID NO: 24). In various embodiments, the active moiety is a heterodimeric human cytokine IL-12 comprising SEQ ID NO: 21 as chain 1 and SEQ ID NO: 22 as chain 2. In various embodiments, the active moiety is a heterodimeric human cytokine IL-23 comprising SEQ ID NO: 23 as chain 1 and SEQ ID NO: 22 as chain 2.

Domain D3 (masking grouping domain)

The D3 domain is a "masking moiety domain" and is primarily used to reversibly mask the activity of the D2 domain in a particular VitoKine construct. The D3 domain is capable of masking the functional activity of D2 prior to activation at the intended site of therapy. In various embodiments, the VitoKine constructs of the present invention comprise a "masking moiety domain" (D3) that is a cognate receptor/binding partner for the D2 protein or cytokine. In various embodiments, the D3 domain is a variant of the cognate receptor/binding partner or a specific binding agent, such as a peptide or antibody fragment, for the D2 domain. In various embodiments, the D3 domain has enhanced binding to the D2 domain compared to the wild-type cognate receptor/binding partner. In various embodiments, the D3 domain has reduced binding to the D2 domain compared to the wild-type cognate receptor/binding partner or its binding to the D2 domain is abolished. In various embodiments, the D3 domain is a protein or peptide or antibody or antibody fragment capable of masking D2 activity. In various embodiments, the D3 domain is DNA, an RNA fragment, or a polymer such as PEG via a cleavable linker. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the extracellular domain of IL-15Rα or a functional fragment or variant thereof. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the IL-15RαSushi domain (amino acids 1-65 of SEQ ID NO: 5). In various preferred embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the IL-15RαSushi+ domain containing 1-30 additional IL-15Rα residues at the C-terminus of the Sushi domain (e.g., SEQ ID NO: 5). In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the extracellular domain of IL-2Rα or a functional fragment thereof. In various preferred embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the IL-2RαSushi domain. In various preferred embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is a variant of the IL-2RαSushi domain. In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is the extracellular domain of IL-2Rβ or a blocking peptide derived from IL-2Rβ. In various embodiments, the D3 domain is capable of masking the functional activity of D2 prior to activation at the intended site of therapy.

IL-15 alpha receptor

The IL-15 receptor is a type 1 cytokine receptor consisting of beta (β) and gamma (γ) subunits, which are common to it and the IL-2 receptor, and an alpha (α) subunit that binds IL-15 with high affinity. Full-length human IL-15Rα is a type 1 transmembrane protein with a 32-amino acid signal peptide, a 173-amino acid extracellular domain, a 21-amino acid transmembrane domain, a 37-amino acid cytoplasmic tail, and multiple N- or O-glycosylation sites (Anderson et al., J. Biol Chem., 270:29862–29869, 1995). It has previously been demonstrated that the natural soluble form of the IL-15R alpha chain, corresponding to the full-length extracellular domain of IL-15R alpha, acts as a high-affinity antagonist of IL-15. However, in stark contrast to these data, it has been demonstrated that the recombinant soluble sushi domain of IL-15R alpha, which possesses the majority of binding affinity for IL-15, acts as a potent IL-15 agonist, enhancing its binding to the IL-15R beta/gamma heterodimer and the biological effects (proliferation and protection from apoptosis) mediated through it, while not affecting the binding of the IL-15R alpha/beta/gamma tripartite membrane receptor to IL-15 and its function. These results suggest that in vivo such soluble sushi domains may be involved in the transpresentation mechanism of IL-15 (Mortier et al., J. Biol Chem, 281(3): 1612–1619, 2006).

When used herein in the context of proteins or polypeptides, the terms "native IL-15Rα" and "native interleukin-15 receptor alpha" refer to any naturally occurring amino acid sequence of mammalian interleukin-15 receptor alpha ("IL-15Rα"), including both immature or precursor forms and mature forms, and naturally occurring isoforms. Non-limiting examples of amino acid sequence accession numbers of various native mammalian IL-15Rα in GenBank include NP_002180 (human), ABK41438 (Macaca mulatto), NP_032384 (Mus musculus), Q60819 (Mus musculus), CA141082 (human). In various embodiments, the native IL-15Rα is an immature form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is a mature form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is a form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is a full-length form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is an immature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is a mature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the native IL-15Rα is the full-length form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the native IL-15Rα protein or polypeptide is isolated or purified. In various embodiments, the IL-15Rα domain is derived from the amino acid sequence of human IL-15Rα set forth in SEQ ID NO: 4:

In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is an IL-15RαSushi+ domain comprising the amino acid sequence of a mature human IL-15Rα polypeptide as set forth in SEQ ID NO: 5:

In various embodiments, IL-15RαSushi+ (SEQ ID NO: 5), a truncated IL-15 cognate coreceptor that recapitulates most of the binding affinity of full-length IL-15Rα (SEQ ID NO: 4), was used as the D3 domain to mask IL-15 activity by selecting a cleavable or non-cleavable linker connecting EL-15 and IL-15RαShushi+ to produce the VitoKine IL-15 construct. One skilled in the art will appreciate that the length of the D3 domain may differ from the sequence set forth in SEQ ID NO: 5, so long as it can recapitulate most of the binding activity of full-length IL-15Rα (SEQ ID NO: 4), i.e., is a functional fragment thereof. The unique design of VitoKine with IL-15 is its ability to fully utilize the unique properties of the IL-15 pathway, including the exceptionally high affinity between IL-15 and IL-15Rα (30 pM) and the fact that complex formation with IL-15Rα enhances IL-15 activity in vivo. Following cleavage of the linker connecting IL-15 and IL-15aSushi+ by proteases upregulated at the disease site, IL-15RαShushi+ or any functional fragment derived from IL-15RαECD is expected to maintain a non-covalent association with IL-15 and enhance IL-15 activity.

IL-2 receptor

The IL-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes, that binds to and responds to a cytokine called IL-2. IL-2R has three subunits: α (CD25), β (CD 122), and _γ (CD 132, a common chain shared with five other cytokine receptors: IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R). The alpha chain (also called Tas antigen or p55) of the human receptor is encoded on chromosome 10p14-15 by the IL-2RA gene. The human β-chain gene (IL-2RB, CD122) of this receptor is located on chromosome 22q11.2-12, while the gene encoding the common IL2Rγ chain _c (IL2RG) is located on chromosome Xq13. Assembly of all three receptor subunits is important for signal transduction in B and T cells. IL-2R is present (transiently or permanently) on the surface of almost all hematopoietic cells, including lymphoid T, B, and NK cell lineages, as well as myeloid lineages such as macrophages, monocytes, and neutrophils. Signal transduction in cells occurs through the Janus kinases Jak1 and Jak3. Phosphorylation of the intracytosolic portion of the receptor β-chain leads to the possibility of forming homodimers of STAT-3 and STAT-5 factors. STAT-3 and STAT-5 homodimers exhibit increased affinity for the nucleus, where they bind to specific DNA elements that enhance transcription of IL-2-dependent genes.

When used herein in the context of proteins or polypeptides, the terms "native IL-2Rα" and "native interleukin-2 receptor alpha" refer to any naturally occurring amino acid sequence of mammalian interleukin-2 receptor alpha ("IL-2Rα"), including both immature or precursor forms and mature forms, and naturally occurring isoforms. Non-limiting examples of amino acid sequence accession numbers of various native mammalian IL-2Rα include NP_032393.3 (Mus musculus), CAK26553.1 (human), and NP_000408.1 (human). In various embodiments, the native IL-2Rα is an immature form of a naturally occurring mammalian IL-2Rα polypeptide. In various embodiments, native IL-2Rα is a mature form of a naturally occurring mammalian IL-2Rα polypeptide. In various embodiments, native IL-2Rα is a form of a naturally occurring mammalian IL-2Rα polypeptide. In various embodiments, native IL-2Rα is a full-length form of a naturally occurring mammalian IL-2Rα polypeptide. In various embodiments, native IL-2Rα is an immature form of a naturally occurring human IL-2Rα polypeptide. In various embodiments, native IL-2Rα is a mature form of a naturally occurring human IL-2Rα polypeptide. In various embodiments, native IL-2Rα is a full-length form of a naturally occurring human IL-2Rα polypeptide. In various embodiments, the native IL-2Rα protein or polypeptide is isolated or purified. In various embodiments, the IL-2Rα domain is derived from the amino acid sequence of human IL-2Rα set forth in SEQ ID NO: 9:

In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is an IL-2RαSushi domain comprising the amino acid sequence of a mature human IL-2Rα polypeptide as set forth in SEQ ID NO: 10:

In various embodiments, IL-2RαSushi (SEQ ID NO: 10) was used to generate VitoKine IL-2 constructs to mask IL-2 activity. Unlike IL-15Rα, which contains a single sushi domain, IL-2Rα contains two sushi domains separated by a linker region. In various embodiments, the VitoKine IL-2 construct comprises an IL-2RαSushi variant containing amino acid substitutions to disrupt the specific non-covalent interactions between IL-2Rα and IL-2, thereby reducing the binding affinity of IL-2Rα for IL-2. Although native IL-2Rα binds IL-2 with moderate affinity of 30 nM, it is possible that IL-2Rα may not dissociate following linker cleavage. Binding of IL-2Rα to IL-2 may reduce IL-2 activity and/or shift the balance of T cell subsets in an undesirable direction. If mutation(s) that reduce affinity are introduced into IL-2RαSushi, such as R36A, K38E, L42G, Y43A, or any combination of these substitutions, there is a high probability of dissociation of the IL-2Rα sushi domains from IL-2 after protease cleavage of the linker.

In various embodiments, the VitoKine constructs of the present invention comprise a D3 domain that is a variant of the IL-2RαSushi domain comprising the amino acid sequence as set forth in SEQ ID NO: 267-270.

Linkers L1 and L2 Cleavable linkers

A cleavable linker or a disease-associated enzyme-sensitive linker may contain a moiety, such as a protein substrate, that can be specifically cleaved by a protease whose levels are elevated in disease sites compared to non-disease tissues. Increased levels of enzymes with known substrates have been reported in various cancer types, such as solid tumors. See, e.g., La Rocca et al., Brit. J. Cancer 90:1414–1421 and Ducry et al., Bioconjug. Chem. 21:5–13, 2010, each of which is incorporated by reference in its entirety. In various embodiments, the protease capable of cleaving the protease-cleavable linker is selected from the group consisting of metalloproteases, such as matrix metalloproteinases (MMPs) 1-28, serine proteases, such as urokinase-type plasminogen activator (uPA) and matriptase, cysteine proteases, such as legumain, aspartic proteases, and cathepsin proteases. The peptide sequences of typical protease substrates are presented in Table 9.

Peptide sequences of typical protease substrates that can be used as protease-cleavable linkers, with or without peptide spacers of varying lengths at the C-terminus, or at the N-terminus, or at both ends of the cleavable linker, are presented in Table 10.

In various embodiments, the protease is MMP-9 or MMP-2. In another specific embodiment, the protease is uRA. In another specific embodiment, the protease is MMP-14. In another specific embodiment, the protease is legumain. In various embodiments, a single VitoKine molecule comprises two different proteases. In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "GPLGMLSQ" (SEQ ID NO: 77). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "LGGSGRSANAILE" (SEQ ID NO: 80). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "SGRSENIRTA" (SEQ ID NO: 157). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "GPTNKVR" (SEQ ID NO: 158). In various embodiments, the linker (e.g., the cleavable linker) can be cleaved by tumor-associated proteases. In various embodiments, for diseases other than cancer, such as inflammatory diseases, the cleavable linker can be cleaved by other disease-specific proteases.

In various embodiments, peptide spacers may be included on either side of the protease-cleavable sequence, or to flank the protease-cleavable sequence on both sides, or as a non-cleavable linker without a protease substrate site. The peptide spacer is used to position the cleavable linker, increasing its accessibility to the enzyme that mediates cleavage. The length of the spacers may be altered or optimized to balance accessibility for enzymatic cleavage and the spatial constraints necessary for reversible masking of the biological activity of the D2 domain. The spacer may contain 1-100 amino acids. Suitable peptide spacers are known in the art and include, but are not limited to, peptide linkers containing flexible amino acid residues such as glycine and serine. In various embodiments, the spacer may comprise from 1 to 12 amino acids, including the motifs G, S, GS, GSGS (SEQ ID NO: 116), GGS (SEQ ID NO: 117), GSGS (SEQ ID NO: 121), GSGSGS (SEQ ID NO: 122), GSGSGSGS (SEQ ID NO: 123), GSGSGSGSGS (SEQ ID NO: 124) or GSGSGSGSGSGS (SEQ ID NO: 125). In other embodiments, the spacer may comprise the motifs GGGGS (SEQ ID NO: 118) _n , where n is an integer from 1 to 10. In other embodiments, the spacer may also comprise amino acids other than glycine and serine.

Typical protease-cleavable linkers with spacer peptides flanking the protease substrate peptide (underlined) are shown in Table 11.

In various embodiments, the cleavable linker may be activated by mechanisms other than proteolysis, including, but not limited to, hydrolysis, such as in the case of a PEGylated polymer that provides release via a controlled mechanism at different pH values.

Non-cleavable linkers

A non-cleavable linker provides covalent attachment and additional structural and/or spatial flexibility between protein domains. As is known in the art, peptide linkers containing flexible amino acid residues, such as glycine and serine, can be used as non-cleavable linkers. In various embodiments, a non-cleavable linker can contain 1-100 amino acids. In various embodiments, the spacer can contain GS, GSGS (SEQ ID NO: 116), GGS (SEQ ID NO: 117), GGGGS (SEQ ID NO: 118), GGSG (SEQ ID NO: 119), or SGGG (SEQ ID NO: 120) motifs. In other embodiments, the linker may comprise motifs (GGGGS)(SEQ IDNO: 118) _n , where n is an integer from 1 to 10. In other embodiments, the linker may also comprise amino acids other than glycine and serine. In another embodiment, the non-cleavable linker may be a simple chemical bond, such as an amide bond (e.g., in PEG chemical conjugation). The non-cleavable linker is stable under physiological conditions, as well as at the site of a disease, such as a cancer lesion or inflammatory disease lesion.

Typical non-cleavable linkers are shown in Table 12.

Combination of cleavable and non-cleavable linkers

In various embodiments, both linkers, L1 and L2, may be cleavable or non-cleavable, or a combination of cleavable and non-cleavable linkers, yielding different forms of the active moiety of the D2 domain to address different therapeutic goals or to provide a benefit-risk balance or different cytokine properties. Exemplary active forms released upon cleavage of the linkers are shown in FIG. 2. Active forms 1 and 3, resulting from cleavage of L1 and both L1 and L2, respectively, are short-acting cytokines with varying degrees of functional activity depending on the D3 conformation. Cleavage and dissociation from the D1 moiety, which increases half-life or targets disease-affected tissue, will increase local concentrations of the activated D2 domain. After local action, the short-acting active forms can be rapidly cleared from systemic circulation to reduce toxicity. In contrast, the active form 2, obtained as a result of L2 cleavage, is a long-acting cytokine with fully restored function and preserved tissue targeting, stably retained at the site of the disease, providing longer-lasting and higher efficacy.

Polynucleotides

In another aspect, the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding IL-15, an IL-15 variant, IL-15Rα, an IL-15Rα variant, IL-2, an IL-2 variant, IL-2Rα, IL-2Rα, Fc, an Fc variant, an antibody directed to a TAA or an immune checkpoint modulator, an antibody fragment, or a VitoKine construct of the present invention. The nucleic acids in question may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. DNA includes, for example, cDNA (complementary DNA), genomic DNA, synthetic DNA, DNA amplified by PCR (polymerase chain reaction), and combinations thereof. Genomic DNA encoding the VitoKine constructs is obtained from genomic libraries available for a number of species. Synthetic DNA can be produced by chemical synthesis of overlapping oligonucleotide fragments, followed by fragment assembly to restore part or all of the coding regions and flanking sequences. RNA can be produced using prokaryotic expression vectors that produce high levels of mRNA synthesis, such as vectors with promoters and T7 RNA polymerase. DNA molecules according to the invention include full-length genes, as well as polynucleotides and fragments thereof. A full-length gene may also contain sequences encoding an N-terminal signal sequence. Such nucleic acids can be used, for example, in methods for producing new VitoKine constructs.

In various embodiments, the isolated nucleic acid molecules comprise the polynucleotides described herein and further comprise a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecules further comprise polynucleotides encoding linkers or hinge linkers described herein.

In various embodiments, the recombinant nucleic acids of the present invention may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory sequences are known in the art and are selected to control expression of the VitoKine construct. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. The present invention contemplates constitutive or inducible promoters, as known in the art. Promoters may be naturally occurring promoters or hybrid promoters that combine elements of more than one promoter. The expression construct may be present in the cell on an episome, such as a plasmid, or the expression construct may be introduced into the chromosome. In various embodiments, the expression vector contains a selectable marker gene, allowing for selection of transformed host cells. Selectable marker genes are well known in the art and will vary depending on the host cell used.

In another aspect of the present invention, the nucleic acid in question is presented in an expression vector comprising a nucleotide sequence encoding the VitoKine construct and operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector for expressing a polypeptide with a polynucleotide sequence. Vectors suitable for expression in host cells are readily available, and nucleic acid molecules are introduced into vectors using standard recombinant DNA techniques. Such vectors may contain a wide range of expression control sequences that, when operably linked to the DNA sequence, control its expression and can be used in these vectors to express DNA sequences encoding the VitoKine construct. Such useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, the adenovirus or cytomegalovirus immediate early promoter, the RSV promoters, the lac system, the trp system, the TAC or TRC system, the T7 promoter expressed by T7 RNA polymerase, the basic operator and promoter regions of the lambda phage, the control regions of the fd coat protein, the 3-phosphoglycerate kinase or other glycolytic enzyme promoters, acid phosphatase promoters such as PhoS, yeast mating factor a-promoters, the baculovirus polyhedrin promoter, and other known sequences that control the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It will be appreciated that the structure of the expression vector may depend on factors such as the choice of host cell to be transformed and/or the type of protein desired to be expressed. Furthermore, the vector copy number, the ability to control this copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, must be considered. Typical expression vectors suitable for expressing VitoKine constructs include pDSRa and its derivatives containing the VitoKine construct polynucleotides, as well as any additional suitable vectors known in the art or described below.

The recombinant nucleic acid of the present invention can be produced by ligating the cloned gene or a portion thereof into a vector suitable for expression in prokaryotic cells and/or eukaryotic cells (yeast, avian, insect, or mammalian cells). Expression vectors for producing the recombinant VitoKine construct include plasmids and other vectors. For example, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as E. coli.

Some mammalian cell expression vectors contain both prokaryotic sequences that facilitate vector replication in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. Examples of mammalian cell expression vectors suitable for transfection of eukaryotic cells include vectors derived from pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, PTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg. Some of these vectors are modified using sequences from bacterial plasmids, such as pBR322, to facilitate replication and selection for drug resistance in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, derived from pREP and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems are given below under the description of delivery systems for gene therapy. The various methods used for producing plasmids and transforming host organisms are well known in the art. Other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant techniques, are given in Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press, 1989), Chapters 16 and 17. In some cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as pBlueBac III, which contains B-gal).

In various embodiments, a vector will be developed for producing the subject VitoKine constructs in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, CA), pcDNA4 vectors (Invitrogen, Carlsbad, CA), and pCI-neo vectors (Promega, Madison, WI). As will be appreciated, the subject gene constructs can be used to express the subject VitoKine constructs in cultured cells, for example, to produce proteins, including fusion proteins or protein variants, for purification.

The present invention also relates to a host cell transfected with a recombinant gene comprising a nucleotide sequence encoding the amino acid sequence of one or more of the contemplated VitoKine constructs. The host cell may be any prokaryotic or eukaryotic cell. For example, the VitoKine construct of the present invention may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast cells, or mammalian cells. Other suitable host cells are known to those skilled in the art, such as Chinese Hamster Ovary (CHO) cells or Human Embryonic Kidney 293 (HEL293) cells.

Accordingly, the present invention also relates to methods for producing the subject VitoKine constructs. For example, a host cell transfected with an expression vector encoding the VitoKine construct can be cultured under suitable conditions allowing expression of the VitoKine construct. The VitoKine construct can be secreted and isolated from a mixture of cells and medium containing the VitoKine construct. Alternatively, the VitoKine construct can remain in the cytoplasmic or membrane fraction, and the cells can be harvested, lysed, and the protein isolated. The cell culture comprises host cells, medium, and other byproducts. Suitable cell culture media are well known in the art.

The polypeptides and proteins of the present invention can be purified using protein purification techniques well known to those skilled in the art. These techniques include, at one level, the separation of crude protein and non-protein fractions. After separation of the peptides and polypeptides from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). As used herein, the term "isolated polypeptide" or "purified polypeptide" is intended to refer to a composition that can be separated from other components, wherein the polypeptide is purified to any extent compared to its natural state. Thus, a purified polypeptide also refers to a polypeptide free from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has been fractionated to remove various other components and substantially retains its expressed biological activity. Where the term "substantially purified" is used, the designation will refer to a peptide or polypeptide composition wherein the polypeptide or peptide is a major component of the composition, comprising about 50%, about 60%, about 70%, about 80%, about 85%, or about 90%, or more of the proteins of the composition.

Various techniques suitable for use in purification will be well known to those skilled in the art. These include, for example: precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), and the like, or by thermal denaturation followed by centrifugation; chromatography such as affinity chromatography (protein A columns), ion exchange chromatography, gel filtration, reverse phase chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques. As is well known in the art, it is contemplated that the order of the various purification steps may be varied, or that certain steps may be omitted, and the method will still be suitable for obtaining a substantially purified polypeptide.

Pharmaceutical compositions

In another aspect, the present invention provides a pharmaceutical composition comprising VitoKine constructs in admixture with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those skilled in the art and are described in numerous references (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990). Pharmaceutically acceptable carriers can be included to modify, maintain, or preserve, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition. Such pharmaceutical compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide. Suitable pharmaceutically acceptable carriers include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); Antimicrobial agents; Antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen sulfite); Buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); Bulking agents (such as mannitol or glycine); Chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); Complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); Fillers; Monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins); Proteins (such as serum albumin, gelatin, or immunoglobulins); Dyes; Flavorings and diluents; Emulsifiers; Hydrophilic polymers (such as polyvinylpyrrolidone); Low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, cyclodextrin, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal); stability-enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol); delivery vehicles; diluents; excipients; and/or pharmaceutical adjuvants.

The primary excipient or carrier in a pharmaceutical composition may be aqueous or non-aqueous. For example, a suitable excipient or carrier may be water for injection, saline, or artificial cerebrospinal fluid, optionally supplemented with other substances frequently used in parenteral compositions. Other typical excipients include neutral buffered saline or saline mixed with serum albumin. Other typical pharmaceutical compositions contain a Tris buffer of approximately pH 7.0-8.5 or an acetate buffer of approximately pH 4.0-5.5, which may additionally contain sorbitol or a suitable substitute. In one embodiment of the present invention, the compositions may be prepared for storage by mixing the selected composition, having the desired degree of purity, with additional agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized mass or an aqueous solution. Additionally, the therapeutic composition can be formulated as a lyophilisate using suitable excipients, such as sucrose. The optimal pharmaceutical composition will be determined by a person skilled in the art based on, for example, the intended route of administration, delivery format, and desired dosage.

When parenteral administration is intended, therapeutic pharmaceutical compositions may be in the form of a pyrogen-free aqueous solution suitable for parenteral administration containing the desired VitoKine construct in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the polypeptide is included in the composition in the form of a sterile isotonic solution with the desired preservative(s). In various embodiments, pharmaceutical compositions suitable for injection may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. In addition, suspensions of the active compounds may be prepared in the form of suitable oily injection suspensions. The suspension may also optionally contain suitable stabilizers or agents to enhance the solubility of the compounds, allowing highly concentrated solutions to be obtained.

In various embodiments, therapeutic pharmaceutical compositions can be formulated for targeted delivery using a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful in the preparation of liposomes include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Representative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Targeting of liposomes based on, for example, their specificity for organs, cells, and organelles is also possible, as is known in the art.

In various embodiments, oral administration of the pharmaceutical compositions is contemplated. The pharmaceutical compositions administered in this manner may be prepared with or without those carriers commonly used in the preparation of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or calcium hydrogen phosphate, and/or any of the following: (1) fillers or excipients, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binding agents, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) agents retarding dissolution, such as paraffin; (6) agents accelerating absorption, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricating agents, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugars, as well as high-molecular-weight polyethylene glycols, and the like. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as aqueous or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular cottonseed, peanut, corn, germ, olive, castor and sesame oil), glycerin, tetrahydrofuryl alcohol, polyethylene glycols, sorbitan esters of fatty acids and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, coloring agents, perfumes and preservatives.

Various embodiments contemplate topical administration of the pharmaceutical compositions to the skin or mucous membranes. Compositions for topical administration may further comprise one or more of a wide range of known agents that effectively enhance penetration through the skin or stratum corneum. Examples include 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Further, additional agents may be included to ensure cosmetic acceptability of the composition. Examples include fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface-active agents. Keratolytic agents, such as those known in the art, may also be included. Examples include salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalation dosage forms. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants that may be necessary. Ointments, pastes, creams, and gels may contain, in addition to the compound of the invention (e.g., VitoKine constructs), excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, gum tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

Additional pharmaceutical compositions contemplated for use herein include those containing polypeptides in sustained- or controlled-delivery formulations. In various embodiments, the pharmaceutical compositions can be formulated as nanoparticles, such as slow-release hydrogels, or incorporated into oncolytic viruses. Such nanoparticle-based methods include, for example, encapsulation in nanoparticles composed of polymers with a hydrophobic backbone and hydrophilic side chains as drug carriers, encapsulation in microparticles, incorporation into liposomes in emulsions, and conjugation with other molecules. Examples of nanoparticles include chitosan and carbopol-coated mucoadhesive nanoparticles (Takeuchi et al., Adv. Drag Deliv. Rev. 47(1):3 9-54, 2001) and nanoparticles containing charged combination polyesters, poly(2-sulfobutylvinyl alcohol) and poly(D,L-lactic-glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50(1):147-160, 2000). Albumin-based nanoparticle compositions have been developed as a drug delivery system for the delivery of hydrophobic drugs such as taxane. See, for example, U.S. Patents 5,916,596, 6,506,405, 6,749,868, 6,537,579, 7,820,788, and 7,923,536. Abraxane®, a nanoparticle formulation of paclitaxel stabilized with albumin, was approved in the United States in 2005 and subsequently in various other countries for the treatment of metastatic breast cancer.

Techniques for preparing a variety of other means for sustained or controlled delivery, such as liposomal carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art.

An effective amount of a pharmaceutical composition for therapeutic use will depend, for example, on the therapeutic context and the objectives being pursued. One skilled in the art will appreciate that appropriate dosage levels for treatment will thus vary depending, in part, on the molecule being delivered, the indication for use of the polypeptide, the route of administration, and the biometric data (body weight, body surface area, or organ size) and condition (age and general health) of the patient. Accordingly, a clinician can adjust the dose and modify the route of administration to achieve the optimal therapeutic effect. A typical dose can range from about 0.0001 mg/kg to about 100 mg/kg or more, depending on the factors noted above. Preferably, injection or administration of polypeptide compositions is performed intravenously. Long-acting pharmaceutical compositions can be administered once every three or four days, once a week, or once every two weeks, depending on the half-life and clearance rate of a particular composition. The frequency of administration will depend on the pharmacokinetic parameters of the polypeptide in the formulation being used. Typically, the composition is administered until a dose that achieves the desired effect is reached. Thus, the composition can be administered single or multiple times (at the same or different concentrations/doses) over time, or as a continuous infusion. Furthermore, titration of the appropriate dose is typically performed. Appropriate doses can be determined using available dose-response data.

The route of administration of the pharmaceutical composition corresponds to known methods, for example, oral administration, administration by intravenous, intraperitoneal, intratumoral, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional injection, intramedullary, intrathecal, intraventricular, intravesical, transdermal, subcutaneous or intraperitoneal administration, as well as administration by the intranasal, enteral, topical, sublingual, urethral, vaginal or rectal route, using sustained-release systems or implantable devices. Where desired, the compositions can be administered by bolus injection or continuous infusion or using an implantable device. Alternatively or additionally, the composition can be administered locally by implanting a membrane, sponge, or other suitable material onto which the desired molecule is absorbed or encapsulated. When using an implantable device, it can be implanted into any suitable tissue or organ, and delivery of the desired molecule can occur via diffusion, time-dependent bolus administration, or continuous administration.

Therapeutic use

According to the present invention, there is provided a method of treating cancer cells in a subject, comprising administering to said subject a therapeutically effective amount (as a monotherapy or in a combination therapy regimen) of the VitoKine construct of the present invention in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and/or proliferation of a cancer cell. Specifically, the VitoKine construct of the present invention is useful in the treatment of disorders characterized as cancer. Such disorders include, but are not limited to, solid tumors such as cancer of the breast, respiratory system, brain, reproductive organs, digestive system, urinary system, eye, liver, skin, head and neck, thyroid, parathyroid glands and their distant metastases, lymphomas, sarcomas, multiple myeloma and leukemia. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ and lobular carcinoma in situ. Examples of respiratory system cancers include, but are not limited to, small cell and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancers include, but are not limited to, brainstem and hypothalamic glioma, cerebellar and cerebellar astrocytoma, neuroblastoma, medulloblastoma, ependymoma, as well as neuroectodermal tumors and pineal gland tumors. Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal and vulvar cancer, and uterine sarcoma. Tumors of the digestive system include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, stomach cancer, liver cancer, breast cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer. Tumors of the urinary system include, but are not limited to, cancer of the bladder, penis, kidney, renal pelvis, ureter, and urethra. Eye cancer includes, but is not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without the fibrolamellar variant), cholangiocarcinoma (cancer of the intrahepatic bile ducts), and mixed hepatocellular cholangiocarcinoma. Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaposi sarcoma, malignant melanoma, Merkel cell skin cancer, and nonmelanoma skin cancer. Head and neck cancer includes, but is not limited to, nasopharyngeal cancer and cancer of the lip and oral cavity. Lymphomas include, but are not limited to, lymphoma, AIDS-associated lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. In various embodiments, the cancer will be a cancer with high expression of a member of the TGF-β family, such as activin A, myostatin, TGF-β and GDF15, such as pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, lung cancer, prostate cancer, brain cancer, bladder cancer and head and neck cancer.

In various embodiments, the VitoKine design can be used as a monotherapy for the treatment of all types of cancer, including, but not limited to, non-small cell lung cancer, small cell lung cancer, melanoma, renal cell carcinoma, urothelial cancer, liver cancer, breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer and sarcoma.

In another aspect, the present invention provides a method of treating an autoimmune disease in a subject, comprising administering to said subject a therapeutically effective amount (as a monotherapy or in a combination therapy regimen) of a VitoKine construct of the present invention in a pharmaceutically acceptable carrier. "Autoimmune disease" refers to a non-malignant disease or disorder originating from and directed against an individual's own tissues. Examples of autoimmune diseases or disorders include, but are not limited to: inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); dermatitis; allergic conditions such as eczema and asthma; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes mellitus (eg, type 1 diabetes mellitus or insulin-dependent diabetes mellitus); multiple sclerosis and juvenile diabetes.

In another aspect, the present invention provides a method for treating an inflammatory disease in a subject, comprising administering to said subject a therapeutically effective amount (as monotherapy or in a combination therapy regimen) of the VitoKine construct of the present invention in a pharmaceutically acceptable carrier. "Inflammatory diseases" include all diseases associated with acute or chronic inflammation. Acute inflammation is the body's initial response to harmful stimuli and results from an increased movement of plasma and leukocytes (such as, for example, granulocytes) from the blood into damaged tissue. A number of biochemical events lead to the spread and maturation of the inflammatory response, affecting the local vascular system, the immune system, and various cells of the damaged tissue. Long-term inflammation is called chronic inflammation, which leads to a progressive change in the cell types present at the site of inflammation and is characterized by the simultaneous destruction and healing of tissue under the influence of the inflammatory process. Examples of inflammatory diseases are well known in the art. In various embodiments, the inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, and bacterial sepsis. As used herein, the term "inflammatory bowel disease" refers to a group of inflammatory conditions of the colon and small intestine, including, for example, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, inflammatory bowel disease, Behcet's syndrome, and colitis not otherwise specified.

In another aspect, the present invention provides a method for treating a viral infection in a subject, comprising administering to said subject a therapeutically effective amount (as monotherapy or in a combination therapy regimen) of the VitoKine construct of the present invention in a pharmaceutically acceptable carrier. In various embodiments, the viral infection to be treated may be caused by infectious agents including, but not limited to, bacteria, fungi, protozoa, and viruses. Viral diseases that can be prevented, treated and/or controlled according to the methods described herein include, but are not limited to, diseases caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, chickenpox, adenovirus, herpes simplex type I (HSY-I), herpes simplex type II (HSY-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackievirus, mumps virus, measles virus, rubella virus, polio virus, smallpox, Epstein-Barr virus, human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2) and the causative agents of viral diseases such as viral meningitis, encephalitis, dengue fever or smallpox.

Bacterial diseases caused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Proteus vulgaris, Staphylococcus viridians, and Pseudomonas aeruginosa) that can be prevented, treated, and/or controlled according to the methods described herein include, but are not limited to, mycobacteria, rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus anthracis (anthrax), tetanus, streptococci, staphylococci, mycobacteria, whooping cough, cholera, plague, diphtheria, chlamydia, S. aureus, and legionella.

Protozoan diseases caused by protozoa that can be prevented, treated and/or controlled according to the methods described herein include, but are not limited to, leishmania, coccidia, trypanosomes, or malaria.

Parasitic diseases caused by parasites that can be prevented, treated and/or controlled according to the methods described herein include, but are not limited to, chlamydia and rickettsia.

A "therapeutically effective amount" or "therapeutically effective dose" refers to that amount of a therapeutic agent administered that will alleviate to some extent one or more symptoms of the disorder being treated.

An initial estimate of the therapeutically effective dose can be made in cell culture assays by determining the IC ₅₀ . A dose can then be adjusted in animal models to achieve a circulating plasma concentration range that includes the IC ₅₀ determined in cell culture. This information can be used to more accurately determine doses suitable for humans. Plasma levels can be measured, for example, by HPLC. The exact composition, route of administration, and dose can be selected by the individual physician based on the patient's condition.

Administration regimens can be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus administration can be administered, several divided doses (multiple, repeat, or maintenance) can be administered over a period of time, and the dose can be proportionally decreased or increased depending on the specific therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages in mammalian subjects to be treated, wherein each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The characteristics of the unit dosage forms of the present invention will be dictated primarily by the unique properties of the antibody and the specific therapeutic or prophylactic effect to be achieved.

Thus, based on the description provided herein, it will be clear to one skilled in the art that the dose and administration schedule are adjusted in accordance with methods well known in the therapeutic field. That is, the maximum tolerated dose can be readily determined, as can the effective amount providing a detectable therapeutic benefit in a subject, as well as the time requirements for administering each agent to provide a detectable therapeutic benefit in a subject. Accordingly, although examples of specific doses and administration schedules are provided herein, these examples in no way limit the dose and administration schedule that can be used in a subject practicing the present invention.

It should be noted that dosage values may vary depending on the type and severity of the condition being treated and may include single or multiple doses. It should also be understood that for any particular subject, specific administration regimens should be adjusted over time in accordance with the individual needs and the professional judgment of the person performing or supervising the administration of the compositions, and that the dosage ranges provided herein are merely examples and are not intended to limit the scope or practical application of the claimed composition. Furthermore, the administration regimen for the compositions of the present invention may be based on a variety of factors, including the type of disease, age, body weight, sex and health status of the subject, the severity of the condition, the route of administration and the particular antibody used. Thus, the administration regimen may vary widely, but can be routinely determined using standard methods. For example, doses can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, such as toxic effects, and/or laboratory parameters. Thus, the present invention encompasses increasing the dose administered to a subject at the discretion of the skilled artisan. Determining appropriate doses and administration schedules is well known in the art and will be readily apparent to those skilled in the art with the information disclosed herein.

Non-limiting examples of daily dose ranges for a therapeutically or prophylactically effective amount of a VitoKine construct or VitoKine variant of the invention may be ranges of from 0.0001 to 100 mg/kg, from 0.0001 to 90 mg/kg, from 0.0001 to 80 mg/kg, from 0.0001 to 70 mg/kg, from 0.0001 to 60 mg/kg, from 0.0001 to 50 mg/kg, from 0.0001 to 40 mg/kg, from 0.0001 to 30 mg/kg, from 0.0001 to 20 mg/kg, from 0.0001 to 10 mg/kg, from 0.0001 to 5 mg/kg, from 0.0001 to 4 mg/kg, from 0.0001 to 3 mg/kg, from 0.0001 to 2 mg/kg, from 0.0001 to 1 mg/kg, from 0.001 to 50 mg/kg, from 0.001 to 40 mg/kg, from 0.001 to 30 mg/kg, from 0.001 to 20 mg/kg, from 0.001 to 10 mg/kg, from 0.001 to 5 mg/kg, from 0.001 to 4 mg/kg, from 0.001 to 3 mg/kg, from 0.001 to 2 mg/kg, from 0.001 to 1 mg/kg, from 0.010 to 50 mg/kg, from 0.010 to 40 mg/kg, from 0.010 to 30 mg/kg, from 0.010 to 20 mg/kg, from 0.010 up to 10 mg/kg, from 0.010 to 5 mg/kg, from 0.010 to 4 mg/kg, from 0.010 to 3 mg/kg, from 0.010 to 2 mg/kg, from 0.010 to 1 mg/kg, from 0.1 to 50 mg/kg, from 0.1 to 40 mg/kg, from 0.1 up to 30 mg/kg, from 0.1 to 20 mg/kg, from 0.1 to 10 mg/kg, from 0.1 to 5 mg/kg, from 0.1 to 4 mg/kg, from 0.1 to 3 mg/kg, from 0.1 to 2 mg/kg, from 0.1 to 1 mg/kg, from 1 to 50 mg/kg, from 1 to 40 mg/kg, from 1 to 30 mg/kg, from 1 to 20 mg/kg, 1 to 10 mg/kg, 1 to 5 mg/kg, 1 to 4 mg/kg, 1 to 3 mg/kg, 1 to 2 mg/kg, or 1 to 1 mg/kg of body weight. It should be noted that dosage values may vary depending on the type and severity of the conditions to be alleviated. It should also be understood that for any particular subject, specific administration regimens should be adjusted over time in accordance with the individual needs and the professional judgment of the person performing or supervising the administration of the compositions, and that the dosage ranges given herein are merely examples and are not intended to limit the scope or practical application of the claimed composition.

The toxicity and therapeutic index of the pharmaceutical compositions of the invention can be determined by standard pharmaceutical techniques in cell cultures or in experimental animals, for example, by methods for determining the LD50 (the dose lethal to 50% of the population) and _ED50 (the dose therapeutically effective for 50% of the population). The ratio of the toxic to therapeutically effective dose is the therapeutic index, and it can be expressed as the ratio _LD50 / _ED50 . Compositions exhibiting large therapeutic indices are generally preferred.

The frequency of administration of the pharmaceutical composition of the VitoKine design depends on the type of therapy and the specific disease being treated. The composition can be administered to the subject at regular intervals, such as weekly or monthly, until the desired therapeutic result is achieved. Examples of administration frequencies include, but are not limited to: once a week without interruption; once a week; once every two weeks; once every 2 weeks; once every 3 weeks; once a week without interruption for 2 weeks, then once a month; once a week without interruption for 3 weeks, then once a month; once a month; once every two months; once every three months; once every four months; once every five months; or once every six months; or once a year.

Combination therapy

As used herein, the terms "co-administration," "co-administered," and "in combination with" referring to the VitoKine construct of the invention and one or more other therapeutic agents are intended to mean and include the following: the simultaneous administration of such combination of the VitoKine construct of the invention and the therapeutic agent(s) to a subject in need of treatment, wherein such components are included together in a single dosage form that releases said components in said subject at substantially the same time; the substantially simultaneous administration of such combination of the VitoKine construct of the invention and the therapeutic agent(s) to a subject in need of treatment, wherein such components are included separately from one another in separate dosage forms that are administered to said subject at substantially the same time, whereby release of said components in said subject occurs at substantially the same time; sequential administration of such a combination of the VitoKine construct of the invention and the therapeutic agent(s) to a subject in need of treatment, wherein such components are included separately from one another in separate dosage forms taken by said subject sequentially with a significant time interval between each administration, as a result of which the release of said components in said subject occurs at substantially different times; and sequential administration of such a combination of the VitoKine construct of the invention and the therapeutic agent(s) to a subject in need of treatment, wherein such components are included together in a single dosage form releasing said components in a controlled manner, as a result of which their simultaneous, sequential and/or overlapping release occurs in said patient, at the same and/or at different times, wherein all parts may be administered by the same route of administration or by different routes of administration.

In another aspect, the present invention provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention in combination with a second therapy, including, but not limited to, immunotherapy, cytotoxic chemotherapy, targeted therapy with small molecule kinase inhibitors, surgery, radiation therapy, and stem cell transplantation. For example, such methods can be used in the prophylactic prevention of cancer, the prevention of cancer recurrence and metastasis after surgery, and as an adjunct to other standard cancer therapy. According to the present invention, the use of the combination methods described herein can enhance the effectiveness of standard cancer therapy (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery).

A wide range of standard compounds have been shown to possess antitumor activity. These compounds are used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or reduce the number of malignant T cells in leukemias or bone marrow malignancies. Despite the effectiveness of chemotherapy in treating various types of malignancies, many antitumor compounds cause undesirable side effects. It has been shown that when two or more different treatments are combined, they can act synergistically, allowing for a reduction in the dose of each, resulting in a reduction in the harmful side effects associated with higher doses of each compound. In other cases, treatment-refractory malignancies may respond to combination therapy of two or more different treatments.

In various embodiments, the patient will be administered a second anti-cancer agent, such as a chemotherapeutic agent. Typical chemotherapeutic agents include, but are not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyurea taxanes (such as paclitaxel and docetaxel) and/or anthracycline antibiotics, as well as combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX. In various embodiments, dosages of such chemotherapeutic agents include, but are not limited to, any of about 10 mg/ ^m2 , 20 mg/ ^m2 , 30 mg/ ^m2 , 40 mg/ ^m2 , 50 mg/ ^m2 , 60 mg/ ^m2 , 75 mg/ ^m2 , 80 mg/ ^m2 , 90 mg/ ^m2 , 100 mg/ ^m2 , 120 mg/ ^m2 , 150 mg/ ^m2 , 175 mg/ ^m2 , 200 mg/ ^m2 , 210 mg/ ^m2 , 220 mg/ ^m2 , 230 mg/ ^m2 , 240 mg/ ^{m2, 250 mg/m2 ,} 260 mg/ ^m2 , and 300 mg/ ^m2 .

In various embodiments, the combination therapy methods of the present invention may further comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but not limited to: treatment using depleting antibodies to certain tumor antigens; treatment using antibody-drug conjugates; treatment using agonist, antagonist, or blocking antibodies to costimulatory or coinhibitory molecules (immune checkpoints), such as, including, but not limited to, antibodies to CTLA-4, PD-1, PD-L1, CD40, OX-40, CD 137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec 7, Siglec 8, Siglec 9, Siglec 15 and VISTA, CD276, CD272, TIM-3, B7-H4; Treatment using bispecific T cell engaging antibodies (BiTE®) such as blinatumomab; Treatment including administration of biological response modifiers such as IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-22, GM-CSF, IFN-α, IFN-β, IFN-γ, a TGF-β antagonist, or a TGF-β trap; Treatment using therapeutic vaccines, including but not limited to oncolytic viruses such as T-vec or therapeutic vaccines such as sipuleucel-T; Treatment using dendritic cell vaccines or vaccines based on tumor antigen peptides or neoantigens; Treatment using chimeric antigen receptor T cells (CAR-T cells); Treatment using CAR-NK cells; treatment using NK cells; treatment using iPS-induced NK cells; treatment using iPS-induced T cells; treatment using iPS-induced CAR-T cells or iPS-induced CAR-NK cells; treatment using tumor-infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo cultured and/or TC ΔR T cells); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9, and vaccines such as bacille Calmette-Guérin (BCG) and imiquimod; where combination therapy provides enhanced destruction of tumor cells by effector cells, that is, with the combined use of VitoKine structures and immunotherapy, synergy occurs.

In various embodiments, the combination therapy methods of the present invention may further comprise administering to the subject a therapeutically effective amount of anti-inflammatory agents for the treatment of autoimmune diseases, inflammatory diseases and other immune disorders, including, but not limited to: treatment using depleting antibodies to certain immune cells; treatment using modulating antibodies (agonist, antagonist or blocking) as targeted immune response modifiers acting on targets (ligands or their receptors) including, but not limited to, IL-1α, IL-1β or IL-1R, IL-4 or IL-4R, IL-5 or IL-5R, IL-6 or IL-6R, IL-8 or IL-8R, IL-7 or IL-7R, IL-10 or IL-10R, IL-11 or IL-11R, IL-12 or IL-12R, IL-17 or IL-17R, IL-18 or IL-18R, IL-21 or IL-18R, IL-22 or IL-22R, IL-23 or IL-23R, MCSF or MCSF-R, GM-CSF or GM-CSFR, IFN-α, IFN-β, IFN-γ, TGF-α, TGF-β or TGF-β, TNF family or their corresponding receptors, integrin family (e.g., α4β7), TSLP, complement component 5 (C5) or C5a, IgE, APRIL, TACI, BCMA, CD20, CD22, CD40/CD40L, B7H1, B7H2, ICOS, BAFF, BCR, BLys, B7RP1, TLR7, TLR8, TLR9; treatment using small molecules (agonist or antagonist) as targeted immune response modifiers acting on targets including, but not limited to, NFkB, Jak1, Jak2, Jak3, Tyk2, Syk, BTK, PIK3, cyclooxygenase-2 and NMDA receptors; where combination therapy increases the effectiveness of immune response modulation, i.e., synergy occurs with the combined use of VitoKine constructs and anti-inflammatory therapy.

In various embodiments, combination therapy comprises administering the VitoKine construct and the second agent composition simultaneously, in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the VitoKine construct composition and the second agent composition are administered sequentially, i.e., the VitoKine construct composition is administered before or after the second agent composition. In various embodiments, the VitoKine construct composition and the second agent composition are administered simultaneously, i.e., the periods of administration of the VitoKine construct composition and the second agent composition overlap. In various embodiments, the VitoKine construct composition and the second agent composition are administered non-simultaneously. For example, in various embodiments, administration of the VitoKine construct composition is discontinued before administration of the second agent composition. In various embodiments, administration of the second agent composition is discontinued before administration of the VitoKine construct composition.

The following examples are provided to more fully explain the invention, but do not limit its scope.

Example 1

Design and production of VitoKine constructs with IL-15 and Fc

The goal was to develop VitoKine constructs with IL-15 that would remain inert until locally activated by proteases upregulated in cancer or disease-associated tissue. Described herein are VitoKine constructs with wild-type IL-15 (SEQ ID NO: 2) or an IL-15 mutein (e.g., SEQ ID NO: 3) as the active moiety, reversibly masked between the Fc domain and IL-15RαSushi+ (SEQ ID NO: 5). These constructs contain one or two cleavable linkers recognized by tumor-specific proteases. In the presence of tumor cells expressing proteases, cleavage of the linker connecting Fc and IL-15 mutein and/or the linker connecting IL-15 and IL-15αSushi+ will occur, thereby restoring IL-15 activity. It should be noted that after proteolysis, IL-15αSushi+ is expected to remain non-covalently associated with IL-15 due to the exceptionally high affinity between IL-15 and IL-15Rα (K _D is 30 pM). VitoKine IL-15 and Fc constructs with various combinations of linkers and peptide spacers were prepared, which are schematically shown in FIG. 1, and their corresponding sequences are provided as SEQ ID NOs: 25-43, 162-165, and 169-174.

All genes synthesized and subcloned into the recipient mammalian cell expression vector (GenScript) were codon optimized for expression in mammalian cells. Protein expression is driven by the CMV promoter, and a synthetic SV40 poly-A signal sequence is present at the 3' end of the CDS. A leader sequence was designed at the N-terminus of the constructs to provide the necessary signals and processing for secretion.

Constructs were generated by cotransfection of HEK293-F cells grown in suspension with mammalian cell expression vectors using polyethyleneimine (PEI, linear, molecular weight 25,000, Polysciences). When two or more expression vectors were present, they were used for transfection at a 1:1 ratio. For transfection, HEK293 cells were cultured in FreeStyle™ 293 serum-free expression medium (ThermoFisher). For production in 1000 ml shake flasks (working volume 330 ml), HEK293 cells were seeded at a density of 0.8 × 10 ⁶ cells/ml 24 h before transfection. Expression vectors, to a total DNA amount of 330 μg, were mixed with 16.7 ml of Opti-MEM medium (ThermoFisher). After adding 0.33 mg PEI diluted in 16.7 ml Opti-MEM medium, the mixture was vortexed for 15 sec and then incubated for 10 min at room temperature. The DNA/PEI solution was then added to the cells and incubated at 37°C in an incubator with an 8% _CO2 atmosphere. On day 4, sodium butyrate (Millipore Sigma) was added to the cell culture at a final concentration of 2 mg/l to promote stable protein expression. After 6 days of culturing, the supernatant was obtained for purification by centrifugation for 20 min at 2200 rpm. The resulting solution was sterilized by filtration (0.22 μm filter, Corning). The secreted protein was purified from the cell culture supernatants using protein A affinity chromatography.

For affinity chromatography, the supernatant was loaded onto a HiTrap MabSelect SuRe Protein A FF column (CV 5 ml, GE Healthcare) equilibrated with 25 ml of phosphate-buffered saline, pH 7.2 (ThermoFisher). Unbound protein was removed by washing with 5 column volumes of PBS, pH 7.2, and the target protein was eluted with 25 mM sodium citrate, 25 mM sodium chloride, pH 3.2. The protein solution was neutralized by adding 3% 1 M Tris, pH 10.2. The target protein was concentrated using an Amicon® Ultra-15 concentrator with a molecular weight cutoff of 10 kDa (Merck Millipore Ltd.).

The purity and molecular weight of the purified constructs were analyzed by SDS-PAGE in the presence or absence of a reducing agent and Coomassie staining (Imperial ^R Stain). The NuPAGE® Pre-Cast gel system (4-12% or 8-16% Bis-Tris, ThermoFisher) was used according to the manufacturer's instructions. The protein concentration of the purified protein samples was determined by measuring the UV absorbance at 280 nm (Nanodrop spectrophotometer, ThermoFisher) divided by the molar extinction coefficient calculated from the amino acid sequence. The aggregate content of the constructs was analyzed on an Agilent 1200 high-performance liquid chromatography (HPLC) system. Samples were applied to an AdvanceBio size-exclusion column (300Å, 4.6×150 mm, 2.7 μm, LC column, Agilent) using 150 μM sodium phosphate, pH 7.0, as the mobile phase at 25°C.

P-0315 is a VitoKine dimeric construct with IL-15 and Fc at the C-terminus, containing uPA and MMP cleavage sequences in the L1 and L2 linkers, respectively. IL-15 is a protein variant with S58D. As an example demonstrating the protein profile of the VitoKine IL-15 and Fc constructs, SDS-PAGE analysis of P-0315 (SEQ ID NO: 33) is shown in FIG. 3A. The size exclusion chromatogram is shown in FIG. 3B.

Example 2

In vitro IL-15 activity was effectively masked in the VitoKine format

VitoKine IL-15 construct P-0172 (SEQ ID NO: 27) contains an IL-15/IL-15RαSushi+ fusion polypeptide linked by a short GS peptide linker connecting the C-terminus of the homodimeric Fc domain via a uPA-cleavable linker in a homodimeric fusion format. P-0198 is a dimeric IL-15 fusion protein with Fc at the C-terminus in a non-covalent complex with IL-15RαSushi. The two molecules have a similar configuration of the fusion moiety between Fc and IL-15 and differ significantly in the way IL-15RαSushi is incorporated. In one, it is fused via a short GS linker (P-0172), while in the other, it is linked non-covalently (P-0198). The binding activity of P-0172 to IL-2Rβ was determined by enzyme-linked immunosorbent assay (ELISA) in comparison with P-0198 (containing SEQ ID NOs: 45, 44, and 5), a highly active IL-15/IL-15Rα-Fc fusion protein.

Briefly, IL-2Rβ-ECD (SEQ ID NO: 12) was coated onto the wells of 96-well Nunc Maxisorp microplates at 1 μg per well. After overnight incubation at 4°C and blocking with superblock (ThermoFisher), 100 μl of 3-fold serial dilutions of IL-15 compounds, starting at 100 nM, were added to each well. After 1 h of incubation at room temperature, 100 μl of goat anti-human IgG Fc antibody conjugated to HRP (horseradish peroxidase) (1:5000 dilution in diluent) were added to each well and incubation was carried out at room temperature for 1 h. After each step, the wells were carefully aspirated and washed three times with PBS/0.05% Tween-20. Finally, 100 µl of TMB substrate (ThermoFisher) was added to each well, the plate was developed at room temperature in the dark for 10 min, and stopping solution (2 N sulfuric acid, Ricca Chemical) was added at 100 µl per well. Optical density was determined at 450 nm, and curves were plotted using Prism software (GraphPad). As shown in FIG. 4A, VitoKine P-0172 bound to IL-2Rβ with significantly less potency than P-0198 (12.2 nM vs. 0.21 nM), likely due to steric constraints resulting from the short covalent binding of IL-15 and IL-15RαSushi, indicating that IL-15RαSushi in the VitoKine platform effectively interfered with the binding of the IL-15 domain to its receptor.

Further evaluation of the functional activity of IL-15 VitoKine P-0172 compared to P-0198 was performed by examining IL-15-mediated induction of CD69 expression on human NK cells and CD8+ T cells from fresh peripheral blood mononuclear cells (PBMCs) using FACS analysis. CD69 is a cell surface glycoprotein induced early in lymphoid activation, including by NK and T cells.

Briefly, human PMBCs were isolated by Ficoll-Hypaque centrifugation from buffy coats purchased from the Oklahoma Blood Institute. Purified human PMBCs were treated with serial dilutions of each IL-15 assay and incubated at 37°C for 48 h. Cells were collected by centrifugation at 300 g and resuspended in FACS buffer. After blocking Fc receptors with Human TraStain FcX (1:50 dilution), cells were stained with anti-human CD56 with FITC, anti-human CD69 with PE, and anti-human CD8 with APC (1:50 dilution). After 30 min of incubation with antibodies at room temperature, cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry. CD69 expression was determined by analyzing CD56+ NK cells and CD8+ T cells, and data are expressed as % of CD69-positive cells in the analyzed population.

As demonstrated in FIGS. 4B and 4C, CD69 activation on CD8+ T and NK cells by VitoKine P-0172 was significantly reduced and measurable only at the highest concentration tested, with activity at least 2-3 logarithms lower than that of P-0198. This indicates effective masking of IL-15 activity in the VitoKine format. This masking effect was more pronounced in the PMBC CD69 activation assay than in the IL-2Rβ binding ELISA, indicating a more pronounced reduction in IL-15 activity under physiological conditions compared to in vitro conditions in ELISA. Due to spatial constraints, the VitoKine design significantly reduces IL-15 binding to the IL-2Rβ/γ complex expressed on lymphocytes, resulting in inefficient pathway activation and significant reduction in activity.

The biological activity of the VitoKine monomeric construct with IL-15 and Fc was also analyzed. P-0170 (SEQ ID NOs: 26 and 15) is a monomeric analogue of P-0172, having the same linkers and fusion configuration. Compared to the highly active IL-15 and Fc fusion protein P-0166, P-0172 showed significantly less ability to activate CD69 on CD8+ T cells (FIG. 5), indicating that the VitoKine monomeric platform also effectively masked the biological activity of IL-15 in the D2 domain.

Example 3

Comparison of masking efficiency in VitoKine constructs with IL-15 and Fc with different lengths and compositions of linkers between IL-15 and IL-15RαSushi+ and between Fc and IL-15

VitoKine IL-15 constructs were constructed by fusing human IL-15 between two different domains, the half-life-enhancing Fc domain and the alpha domain of its cognate high-affinity coreceptor, via linkers L1 and L2, as shown in FIG. 1. The differential effect of the two linkers, connecting IL-15 Fc and IL-15RαSushi domain, as well as the length and composition of these linkers, on the biological activity of IL-15 were analyzed for the desired activity reduction.

To evaluate VitoKine IL-15 constructs with different lengths of the non-cleavable linker between IL-15 and IL-15RαSushi+ (L2), FACS analysis of the activation marker CD69 was performed in subpopulations of fresh human RMBC immune cells. The same protocol was followed as in Example 2.

P-0204 (SEQ ID NO: 30), P-0205 (SEQ ID NO: 31), and P-0206 (SEQ ID NO: 32) are IL-15 and Fc VitoKine constructs that have the same uPA cleavable linker sequence between Fc and IL-15 (L1), but the linkers connecting the IL-15 and IL-15RαSushi+ (L2) domains in these three VitoKine constructs are of different lengths and are (GGGGS) ₃ (SEQ ID NO: 127), (GGGGS) ₂ (SEQ ID NO: 126), and GGGGS (SEQ ID NO: 118), respectively.

As shown in FIG. 6, all VitoKine IL-15 and Fc constructs with different linkers ranging from 5 to 15 amino acids in length between IL-15 and IL-15RαSushi+ resulted in a significant decrease in activity in activating CD8+ T cells (FIG. 6A) or NK cells (FIG. 6B). When comparing the activity of P-0206, P-0205, and P-0204, it was evident that the shorter the length of the linker connecting the IL-15 and IL-15RαSushi+ domains, the more inactive the VitoKine construct became, indicating that the degree of activity reduction can be further adjusted by varying the length of the L2 linker. In conclusion, almost complete abolition of IL-15 activity was demonstrated by masking it between the Fc domain and its cognate high-affinity co-receptor alpha and adjusting the length of the linker between IL-15 and IL-15Rα (L2) to obtain a suitable level of spatial constraint.

The effect of the linker connecting Fc and IL-15 (L1) on the biological activity of the VitoKine constructs was also analyzed, as shown in FIG. 7. P-0204 and P-0203 (SEQ ID NO: 29) have the same flexible 15 amino acid (G ₄ S) ₃ linker (SEQ ID NO: 112) between IL-15 and IL-15Rα (L2), but differ in the length of the L1 linker, while P-0203 contains a longer, 7 GS-rich residues longer peptide spacer than P-0204, flanking the uPA substrate peptide connecting Fc and IL-15. Despite the differences in the length of the L1 linker connecting Fc and IL-15, the biological activities of P-0204 and P-0203 were similar (FIG. 7), indicating that the L1 linker connecting Fc and IL-15 had minimal effect on reducing IL-15 activity when its length ranged from 13 to 35 amino acid residues. However, when the L1 linker length is less than 13 amino acid residues or greater than 35 amino acid residues, or in the context of other cytokines, it may affect the masking of the D2 domain activity. P-0202, which has the same L1 linker connecting Fc and IL-15, but a 13 amino acid shorter L2 linker connecting IL-15 and the IL-15RαSushi domain than P-0203, was also included in the same study. P-0202 showed lower biological activity than P-0203, confirming that linker L2 is more important than linker L1 in masking the activity of the VitoKine construct.

The effect of linker composition or linker peptide sequence on the activity of VitoKine constructs was assessed by measuring Ki67 expression in the nuclei of NK cells and CD8 T cells after treatment with VitoKine constructs plus IL-15. Ki67 is a marker of cell proliferation, and an ex vivo assay was developed with human PMBCs. Briefly, purified human PMBCs were treated with serial dilutions of VitoKine compounds plus IL-15 and incubated at 37°C for 5 days. On day 5, the cells were washed once with FACS buffer (1% FBS/PBS) and initially stained with Fc blocking solution and antibodies to surface markers, including anti-human CD56 with FITC and anti-human CD8 with APC (diluted 1:50). After 30 minutes of incubation and washing, the cell pellets were completely resuspended by adding 1x Foxp3 fixative and permeabilizing working solution (200 μl per well) and incubating for 30 minutes at room temperature in the dark. After centrifugation, 200 μl of 1x permeabilization buffer was added to each well for another wash. The cell pellets were resuspended in permeabilization buffer with anti-human Ki67 antibody with PE (diluted 1:25). After 30 minutes of incubation at room temperature, the cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry. Data are expressed as the percentage of Ki67-positive cells in the analyzed population.

Since the L2 linker has a more significant effect on the activity of the VitoKine IL-15 construct than the L1 linker, the effect of different sequence compositions of the L2 linker on the biological activity of the VitoKine IL-15 constructs was studied. P-0351 (SEQ ID NO: 25), P-0488 (SEQ ID NO: 163), and P-0489 (SEQ ID NO: 164) have the same linker (G ₄ S) ₃ connecting Fc and IL-15 (L1). All linkers connecting IL-15 and IL-15Rα are 10 amino acids long, but have different sequences. This linker is (G ₄ S) ₂ in P-0351, the MMP-14 substrate peptide (SEQ ID NO: 157) in P-0488, or the legumain substrate peptide (SEQ ID NO: 160) in P-0489.

As shown in FIG. 8, all three VitoKine IL-15 constructs had significantly reduced activity in CD8+ T cell (FIG. 8A) or NK cell (FIG. 8B) proliferation compared to the highly active IL-15/IL-15Rα and Fcββ fusion protein (SEQ ID NOs: 175 and 176). The different peptide linker sequences had little effect on the biological activity of the respective VitoKine constructs (FIGS. 8A and 8B), likely due to the structural flexibility of each linker peptide. The more rigid the L2 linker, the more structural constraints it imposes on the VitoKine molecules, which may result in a greater reduction in activity. However, the effect of the L2 linker sequence on VitoKine activity was minor, and these data indicate the possibility of incorporating different cleavable linkers as the L2 linker to effectively mask the activity of the D2 domain, expanding the design and application of VitoKine constructs.

In conclusion, the data obtained, taken together, demonstrated that the L2 linker, which connects the IL-15 (D2) and IL-15RαSushi+ (D3) domains, played a fundamental role in masking D2 activity to ensure the inertness of the VitoKine constructs. The degree of activity reduction can be further adjusted by varying the length and sequence/flexibility of the L2 linker. When selecting the length and sequence of the cleavable L2 linker, the presence of specific proteases at the site of the suspected disease, the accessibility of the substrate peptide to proteases, and the desired rate of proteolysis should be considered.

Example 4

Determination of suitable interaction conditions for complete protease digestion in vitro

Initial in vitro protease cleavage experiments were performed using the VitoKine IL-15 and Fc constructs P-0315 and P-0203 to determine the feasibility of protease cleavage and the optimal conditions for cleavage by MMP-2 and uPA, respectively. P-0315 (SEQ ID NO: 33) contains a uPA-cleavable linker connecting the Fc and IL-15 (L1) domains and an MMP-2/9-cleavable linker connecting the IL-15 and IL-15RαSushi+ (L2) domains. P-0203 (SEQ ID NO: 29) contains a single protease (uPA) cleavable linker connecting the Fc and IL-15 (L1) domains. The linker between the IL-15 and IL-15RαSushi+ domains in P-0203 is a flexible linker ( _G4S ) ₃ . Recombinant human uPA and MMP-2 were purchased from BioLegend. MMP-2 was supplied in a latent form and activated with mercuric n-aminophenylacetate (APMA, Millipore Sigma) according to the manufacturer's instructions.

For proteolytic cleavage by MMP-2, 4 μg of P-0315 were incubated with 30 ng, 100 ng, or 300 ng of APMA-activated MMP-2 in the manufacturer's recommended assay buffer (100 mM Tris, 20 mM _CaCl2 , 300 mM NaCl, 0.1% (w/v) Brij 35, pH 7.5) at 37°C for 3 h. To stop the reaction, SDS-PAGE loading dye was added to the reaction mixture, and the resulting mixture was heated at 95°C for 5 min. To assess cleavage, the digested samples were separated on an SDS-PAGE gel with 4-12% Bis-Tris. Comparison of untreated and treated samples demonstrated complete cleavage of the VitoKine IL-15 constructs after treatment with MMP-2 at all concentrations analyzed. This was indicated by a change in size and the appearance of a distinct band of approximately 9 kDa on an SDS-PAGE gel (FIG. 9), which represented the IL-15RαSushi+ domain cleaved from P-0315.

The ability of uPA to cleave was assessed using P-0203. First, different amounts of uPA were added to 2 μg of P-0203 in 20 μl of PBS buffer, pH 7.2, and the reaction mixture was incubated at 37°C for 2 h. Cleavage was performed using 0, 25 ng, 50 ng, 100 ng, and 300 ng of uPA; the results are shown in FIG. 10A. The three arrows in FIG. 10A refer to non-reduced (NR) samples and indicate the change in the Fc chain upon proteolysis by uPA. In “partial cleavage,” the IL-15/IL-15RαSushi+ fusion polypeptide was cleaved from only one of the two Fc chains, while in “complete cleavage,” the IL-15/IL-15RαSushi+ fusion polypeptide was cleaved from both Fc chains. The smeared band circled in FIG. 10A represented the IL-15/IL-15RαSushi+ fusion polypeptide cleaved from Fc, and its smeared appearance was most likely due to glycosylation. In the recovered samples (R), the upper band represented the Fc chain associated with the IL-15/IL-15RαSushi+ fusion polypeptide, and the distinct lower band represented the Fc chain after cleavage of the IL-15/IL-15RαSushi+ fusion polypeptide.

SDS-PAGE gels clearly demonstrate that with increasing amounts of uPA, there was a gradual increase in the amount of completely cleaved protein in the unreduced samples. Similarly, there was an increase in the amount of cleaved Fc chain in the reduced samples, indicating a higher level of cleavage. However, neither condition resulted in complete cleavage. To achieve complete cleavage, similar uPA cleavage reactions were incubated for longer periods. FIG. 10B shows the cleavage of 2 μg P-0203 using 50 ng, 100 ng, and 300 ng uPA at 37°C for 24 hours. These data indicate that 100 ng uPA resulted in nearly complete cleavage after a 24-hour incubation.

Example 5

Protease cleavage of the VitoKine construct with IL-15 and Fc P-0203 to generate activated IL-15 products

VitoKine P-0203 (SEQ ID NO: 29) contains a uPA substrate peptide linker with spacer peptides flanking both ends (SEQ ID NO: 90) connecting Fc and IL-15, and a second flexible linker of 15 amino acids (GGGGS) ₃ (SEQ ID NO: 127) connecting the IL-15 and IL-15RαSushi+ domains. In vitro protease digestion was performed by incubating 100 μg VitoKine P-0203 with 5 μg recombinant human uPA (BioLegend) in 500 μl PBS buffer, pH 7.2, for 24 hours at 37°C. To stop the interaction, 25 μl of Ni-Excel resin (50% suspension equilibrated in PBS, GE Healthcare) was added to remove 6-His-tagged uPA from the solution. At the same time, 50 μl of MabSelect SuRe Protein A resin (50% suspension equilibrated in PBS, GE Healthcare) was also added to the reaction mixture to remove the fraction of cleaved Fc and uncleaved or incompletely cleaved P-0203. After incubation with both affinity resins at room temperature for 15 min, the resins were removed by centrifugation to obtain an eluate containing protease-activated P-0203, namely the IL-15/IL-15αSushi+ fusion polypeptide (schematically shown as active form 1 in FIG. 2). As shown in FIG. 11A and 11B, the activated fragment of P-0203 moves as a smeared band, which is most likely due to glycosylation.

Example 6

Protease cleavage of the VitoKine construct with IL-15 and Fc P-0315 to produce activated IL-15 products

VitoKine P-0315 (SEQ ID NO: 33) contains a uPA substrate peptide linker (SEQ ID NO: 92) connecting Fc and IL-15, and a second 10-amino acid linker, cleavable by MMP-2/9 (SEQ ID NO: 95), between the IL-15 and IL-15RαSushi+ domains. The IL-15 domain of P-0315 contains an S58D substitution to enhance binding to the receptor β-subunit. Protease cleavage resulted in two activated forms of P-0315.

One activated form of P-0315 (shown schematically as active form 2 in FIG. 2) was generated by in vitro protease cleavage using MMP-2. Briefly, 660 ng of latent MMP-2 (BioLegend) was activated using APMA (Millipore Sigma) following the manufacturer's instructions, buffer exchanged, and added to P-0315 (80 μg) in 0.4 ml of the manufacturer's recommended assay buffer (100 mM Tris, 20 mM _CaCl2 , 300 mM NaCl, 0.1% (w/v) Brij 35, pH 7.5). After incubation at 37°C for 3 hours, 50 μl of MabSelect SuRe Protein A resin (50% suspension equilibrated in PBS, GE Healthcare) was added to the reaction mixture. The desired activated form 1 was eluted using 25 mM sodium citrate, 25 mM sodium chloride, pH 3.2. The protein was neutralized by adding 3% 1 M Tris, pH 10.2. To assess cleavage, samples were separated on an SDS-PAGE gel with 4-12% Bis-Tris (FIG. 12A). P-0315 before MMP-2 cleavage in the presence of a reducing agent is shown in lane 1, while lanes 2 and 3 show unreduced and reduced P-0315 after MMP-2 proteolysis but before Protein A purification. The appearance of a distinct 9 kDa IL-15Rα-sushi+ domain band on the gel confirmed efficient cleavage by MMP-2 at the MMP-2/9 substrate peptide linker. After Protein A purification, the samples (lanes 4 and 5) showed an identical migration pattern. These data indicate that the IL-15RαSushi+ domain, released from covalent binding, remains non-covalently associated with IL-15 Fc-fused, which is shown as active form 2 in FIG. 2; This bond was strong enough to withstand the low pH conditions during elution from protein A. FIG. 12B further shows the two non-covalently bound components of this activated form.

Another activated form of P-0315 (shown schematically as active form 3 in FIG. 2) was generated by protease cleavage of P-0315 using both uPA and MMP-2. Briefly, 100 μg of P-0315 was incubated with 5 μg of uPA in 400 μl of PBS buffer, pH 7.2, for 20 hours. An equal volume of buffer containing 200 mM Tris, 40 mM CaCl2, 450 mM NaCl, 0.2% (w/v) Brij 35, pH 7.5 was then added to the reaction mixture to adjust the buffer to obtain a buffer close to the assay buffer recommended by the manufacturer for MMP-2 (100 mM Tris, 20 mM _CaCl2 , 300 mM NaCl, 0.1% (w/v) Brij 35, pH 7.5). Latent MMP-2 (660 ng) was activated using APMA, the buffer was exchanged with the assay buffer, it was added to the reaction mixture and incubated at 37°C for 3 h. Ni-Excel resin (50 μl of a 50% suspension equilibrated in PBS, GE Healthcare) was added to remove His-tagged MMP-2 and uPA from the solution. At the same time, 100 μl of MabSelect SuRe Protein A resin (50% suspension equilibrated in PBS, GE Healthcare) was added to the reaction mixture to remove the cleaved Fc fraction and remaining uncleaved or incompletely cleaved P-0315. After incubation with both affinity resins at room temperature for 15 min, the resins were removed by centrifugation to obtain an eluate containing the active form 3 of P-0315, schematically shown in FIG. 2. As shown in FIG. 12C, the active form 3 of P-0315 contains a non-covalent IL-15/IL-15αSushi+ complex, as expected after dual proteolysis; IL-15 migrates as a smeared band, while IL-15RαSushi+ is a distinct band of approximately 9 kDa, as seen in active form 2 (FIG. 12B).

Example 7

Evaluation of the activity of VitoKine IL-15 and protease-activated Fc constructs in a human RMBC assay

To assess the activity of the VitoKine IL-15 protease-activated constructs, FACS analysis of the activation marker CD69 was performed in immune cell subsets from fresh human PMBCs as detailed in Example 2. A comparison of P-0203 and its corresponding activated form (P-0203 Activ.; schematically shown as active form 1 in FIG. 2) obtained by uPA cleavage is shown in FIG. 13. The activity of VitoKine before protease cleavage was approximately 3 logs lower than that of the highly active IL-15/IL-15Rα Fc fusion protein P-0165, consistent with the activity of VitoKine described in Example 3. Following uPA cleavage, the activity in activating both CD56+ NK cells (FIG. 13A) and CD8+ T cells (FIG. 13B) was significantly increased, but was still markedly lower than that of P-0165, possibly due to covalent binding of IL-15 and the IL-15Rα domain. Presumably, increasing the length of the flexible linker connecting IL-15 and IL-15Rα would enhance the activity of the activated form. Paradoxically, increasing the length of the flexible linker would likely also reduce the efficiency of activity masking by the D3 domain and, consequently, result in VitoKine constructs with higher basal activity.

The biological activity of another VitoKine IL-15/Fc construct, P-0315, and its two activated forms was assessed by measuring CD69 activation upon activation of fresh human PMBC immune cell subsets. As shown in FIG. 14, the activity of uncleaved P-0315 was nearly undetectable at the concentrations analyzed, confirming the efficient masking of the active moiety in the VitoKine format. Active form 2, P-0315, contains the Fc-fused IL-15 in a non-covalent complex with the IL-15RαSushi+ domain released upon MMP-2-mediated cleavage, as shown in FIG. 2; it is structurally similar to the positive control P-0313, a highly active IL-15, IL-15Rα, and Fc fusion protein. Active form 3 of P-0315 contains the free IL-15 domain cleaved from the Fc domain by uPA and the IL-15RαSushi+ domain released by MMP-2 cleavage, which form non-covalent complexes as shown in FIG. 2. Both activated forms of P-0315 demonstrated complete or near-complete restoration of both CD56+ NK cell (FIG. 14A) and CD8+ T cell (FIG. 14B) activating activity, with active form 3 being slightly more active than active form 2. The absence of the Fc domain in active form 3 may be advantageous when transient activation of a putative pathway in the tumor microenvironment is desired.

The activity of P-0315 before and after proteolysis by MMP-2 was also examined by measuring Ki67 expression in the nuclei of NK cells (FIG. 15A) and CD8+ T cells (FIG. 15B) after treatment. P-0351, which contains two non-cleavable flexible linkers, was included for comparison. The data also demonstrated the inertness of the VitoKine construct and an approximately 3-log increase in activity against both NK cells and CD8+ T cells following in vitro proteolytic activation. The observed identical activity of P-0351 and P-0315 indicates that the two cleavable linkers in P-0315 remained intact during production, expression, and storage and were specific for the corresponding proteases.

In conclusion, cleavage of IL-15 VitoKine P-0315 by MMP-2/9 and/or uPA results in activation of the molecule, and cytokine activity was restored to levels similar to the highly active compound IL-15 P-0313 with an _EC50 in the subnanomolar range.

Example 8

Minimal systemic cytokine effects using VitoKine IL-15 and Fc constructs in healthy mice

The VitoKine technology platform aims to reduce target-mediated systemic toxicity and expand the therapeutic window. The VitoKine design masks the active cytokine in an inert state, preventing it from binding to receptors in the periphery or on the surface of normal cells. Consequently, the VitoKine platform limits excessive cytokine pathway activation and mitigates unwanted toxicity due to target binding outside the target tissue. The VitoKine design is designed for local activation by proteases, which are upregulated in disease-affected tissues. To test this hypothesis, the protease-cleavable VitoKine construct and the non-cleavable VitoKine construct were administered to healthy mice, and their systemic cytokine activity was assessed in comparison with a highly active IL-15-Fc fusion protein.

P-0313 (SEQ ID NOs: 47 and 5) is a fully active IL-15/IL-15Rα Fc fusion molecule used as a positive control. P-0315 (SEQ ID NO: 33) is a VitoKine IL-15 Fc construct containing two protease-cleavable linkers. P-0351 (SEQ ID NO: 25) is a VitoKine IL-15 Fc construct containing two non-cleavable linkers. Vehicle (PBS) was included as a negative control. Compounds were administered by a single i.p. (intraperitoneal) injection to healthy BALB/c mice (8-10 weeks old, n = 6 per group) at doses of 0.1 and 0.3 mg/kg. Blood samples for immunophenotyping were obtained before administration (-1 day) or on days 3, 5, and 7 after administration.

After lysis of red blood cells with BD Pharm lysis buffer, the total number of viable mononuclear blood cells was calculated by exclusion of nonviable cells using trypan blue. After blocking Fc receptors with purified anti-mouse CD16/CD32 antibody (diluted 1:50), cells were stained with anti-mouse CD3 antibody with FITC, anti-mouse CD49b antibody with APC, and anti-mouse CD8 antibody with Percpcy5.5 (diluted 1:50). After 30 minutes of incubation, cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry.

As shown in FIG. 16, P-0313, a fully active IL-15 Fc fusion protein, at both doses tested resulted in a significant dose-dependent increase in the number of cytotoxic CD8+ T cells (FIG. 16A), NK cells (FIG. 16B), and total leukocytes (FIG. 16C) in peripheral blood. Cell numbers increased at day 3, peaked at day 5, and nearly returned to baseline values at day 7. In contrast, both the cleavable VitoKine construct (P-0315) and the non-cleavable VitoKine construct (P-0351) showed no increase in CD8+ T cells throughout the 7-day study. A minor and delayed increase in NK cell numbers was observed in mice receiving the high-dose cleavable VitoKine P-0315. P-0351 and the low dose of P-0315 showed no evidence of an increase in any of the analyzed target cell populations. Overall, compared with the active P-0313 molecule, the two analyzed VitoKine constructs demonstrated minimal systemic activation and an increase in target lymphocyte populations, successfully masking and delaying IL-15 activity in the periphery.

Example 9

Inhibition of pulmonary metastasis of colon cancer cells by VitoKine constructs with IL-15 and Fc in mice

The antimetastatic efficacy and immunological responses of VitoKine molecules with IL-15 and Fc were studied in a CT26 mouse lung metastasis model. Briefly, 1× ¹⁰⁵ mouse colon cancer cells, CT26-WT (ATCC CRL-2638), were administered intravenously to female Balb/C mice (9-11 weeks old). On the following day (day 1), administration of the study constructs was started by intraperitoneal injections four times, once every 5 days (Q5D). Administration groups (6 in total, n = 7 per group) included 0.3 mg/kg P-0315, 0.3 mg/kg P-0351, and 0.1 mg/kg P-0313. P-0315 (SEQ ID NO: 33) is a VitoKine IL-15 Fc construct containing two protease-cleavable linkers. P-0351 (SEQ ID NO: 25) is a non-cleavable VitoKine IL-15 Fc construct. P-0313 (SEQ ID NOS: 47 and 5) is a fully active IL-15/IL-15Rα Fc fusion. Vehicle (PBS) was included as a negative control. On day 17, all mice were sacrificed for tissue collection. Lungs were inflated with 15% India ink and destained in Fekete's solution (10% formaldehyde, 5% glacial acetic acid, and 60% ethanol). Tumor nodules in the lungs were counted and the antimetastatic effect was presented as the different number of tumor nodules in different administration groups and in the vehicle control group.

As shown in FIG. 17, P-0313 exerted significant effects in suppressing the formation and growth of lung metastases. At a dose of 0.1 mg/kg, administration of P-0313 resulted in nearly complete inhibition of lung metastasis. The cleavable VitoKine P-0315 construct demonstrated 70% inhibition of lung nodule development; its antimetastatic efficacy was comparable at all three doses (0.3, 1, or 3 mg/kg). The non-cleavable VitoKine P-0351 construct demonstrated a relatively weak but significant effect in reducing metastasis development, indicating some intrinsic basal activity at the high dose. However, compared with P-0351, P-0315 demonstrated significantly better antimetastatic efficacy (p < 0.05; FIG. 17), indicating that proteolytic cleavage of one or both linkers of P-0315 and subsequent release of the active form of IL-15 likely contributed to the greater in vivo efficacy of P-0315 than that of P-0351. The development of tumor metastases may lead to increased proteolytic activity near the tumor microenvironment.

The immunological response after administration of IL-15 compounds was examined by flow cytometric analysis of the peripheral blood of mice on day 15 (4 days after the third administration). Compared with the control, an increase in the number of CD8+ T cells was noted in mice receiving the active IL-15-Fc fusion protein P-0313, but not the non-cleavable VitoKine P-0315 construct or the non-cleavable VitoKine P-0351 construct, indicating that the antimetastatic efficacy against colon cancer was not accompanied by a systemic increase in CD8+ T cells induced by the VitoKine constructs used (FIGS. 17 and 18A). However, an increase in peripheral blood NK cells was noted in all three IL-15 compound administration groups, which was most pronounced in the non-cleavable VitoKine construct group after multiple administrations (FIG. 18B). The enhanced systemic increase in NK cells, but not CD8+ T cells, in the VitoKine groups suggests that NK cells are more sensitive to IL-15 administration than CD8+ T cells, and the intrinsic basal activity of the VitoKine construct may lead to an increase in NK cell numbers. Therefore, it is critical to adjust the concentration of VitoKine constructs with IL-15 administered to reduce residual systemic effects. The marked increase in NK cells in the P-0351 group also suggests that the low activity of the non-cleavable VitoKine construct may lead to weak but persistent activation of its pathway and prolonged immune responses.

Example 10

VitoKine construct with IL-15 and Fc P-0315 inhibited the growth of established CT26 tumors in mice with minimal systemic cytokine activation.

The antitumor efficacy and immunological responses of IL-15 Fc VitoKine P-0315 were studied in a CT26 murine colorectal cancer model in comparison with a fully active IL-15/IL-15Rα fusion protein and Fc P-0313. Briefly, female Balb/C mice (10–12 weeks old) received a subcutaneous injection of 1 × ¹⁰ CT26 cells in the right flank. On day 11, when the mean tumor volume was approximately 70 mm ³ , mice were randomized into three groups (n = 11 per group) and received an intraperitoneal injection of vehicle (PBS) or 0.1 mg/kg P-0315 or P-0313 on the same day. One additional intraperitoneal injection of the respective study agents was administered on day 16 (2 × Q5D). Tumors were measured three times a week using calipers, and tumor volume was calculated as follows: volume equals 0.5 x (width) x ² x (length). To study the immunological response, non-terminal peripheral blood was collected into heparin-treated tubes on day 19. On day 21, all mice were sacrificed for tissue collection.

As shown in FIG. 19A, mice treated with PBS rapidly developed large subcutaneous tumors, and treatment of mice with P-0315 or P-0313 delayed tumor growth to approximately the same extent (FIGS. 19B and 19C). At 21 days post-tumor cell administration, the mean tumor volume in control-treated mice was approximately 1000 ^mm3 versus approximately 450 ^mm3 in mice treated with P-0315 or P-0313 (****, P less than 0.0001; one-way ANOVA with Tukey's post-hoc test) (FIG. 19D). Note that P-0313 initially showed a greater reduction in tumor burden than P-0315, but this difference diminished as treatment continued. The delayed antitumor effect of P-0315 was likely due to the time required for the protease(s) to accumulate in sufficient quantities to reach and cleave the peptide linkers that are their substrates and activate the VitoKine construct.

The effect of P-0315 on the proliferation of peripheral blood CD8+ T cells and NK cells was then examined in comparison with P-0313 and vehicle using flow cytometry. In addition, the effect of P-0315 on total peripheral and splenic leukocyte populations and lymphocyte subpopulations (CD8+ T and NK cells) was similarly assessed.

Injection of the fully active IL-15/IL-15Rα fusion protein and Fc P-0313 into tumor-bearing mice resulted in marked proliferation and increased lymphocyte numbers in both peripheral blood and the spleen (FIGS. 20-22). Compared with the PBS group, proliferation assessed by Ki67 was increased 4-fold in NK cells (61% vs. 15%; FIG. 20A) and 5.3-fold in CD8+ cells (46% vs. 8.6%; FIG. 20B) after P-0313 administration. Similarly, administration of P-0313 resulted in marked increases in total leukocyte, NK cell, and CD8+ T cell counts in both peripheral blood (FIGS. 21A–21C) and the spleen (FIGS. 22A–22C). For example, total leukocyte counts in the peripheral blood increased 6-fold, CD8+ T cell counts increased 5-fold, and NK cell counts increased dramatically by 85-fold. In the spleen, the most pronounced increases were observed for NK cells (10-fold) and CD8+ T cells, which increased 2.9-fold. Total leukocyte counts in the spleen increased moderately by 1.7-fold. The pronounced activation of cytotoxic CD8+ T cells and NK cells is consistent with the general immunomodulatory properties of IL-15, and robust immune responses were likely the primary factor contributing to the in vivo antitumor activity of P-0313. However, abrupt changes in lymphocyte subsets in the blood may lead to toxicity and reduce the therapeutic index.

In marked contrast to P-0313, administration of the VitoKine construct with IL-15 and Fc P-0315 resulted in minimal changes in the homeostasis of lymphocyte subsets in the blood. These observations are shown in FIG. 20 for peripheral NK and CD8+ T cell proliferation assessed by Ki67, and in FIG. 21 for the increase in total leukocytes, NK cells, and CD8+ T cells in the peripheral blood. The only noticeable pharmacodynamic effect after administration of P-0315 was a 4-fold increase in the number of NK cells in the spleen (FIG. 22B). Because P-0315 and P-0313 inhibited the growth of established CT26 tumors to approximately equal extents (FIGS. 19A-19D), the in vivo antitumor activity of P-0315 likely resulted from proteolysis of the cleavable linker(s) and subsequent activation of the VitoKine construct near the tumor microenvironment. Because the activated VitoKine construct was present only near the tumor, the peripheral lymphocyte response to administration of the inert VitoKine molecule was much less pronounced than that observed with the fully active P-0313.

Overall, the VitoKine construct with IL-15 and Fc, exemplified by P-0315, was able to effectively inhibit tumor growth without significant changes in proliferation and lymphocyte subpopulation numbers in the blood and spleen. Therefore, the VitoKine format prevented excessive pathway activation, unwanted toxicity due to off-target binding, and unwanted clearance due to on-target binding, which typically occur with administration of a fully active cytokine, without compromising the antitumor effect.

Example 11

Reduction of IL-15 group activity to minimize the intrinsic basal activity of the corresponding VitoKine design

Despite a greater than 3 log reduction in activity when comparing P-0313, a fully active bivalent IL-15 S58D/IL-15Rα and Fc fusion protein, and its corresponding VitoKine Fc construct P-0315, P-0315 still possesses intrinsic basal activity that can lead to ex vivo stimulation of effector cells with an _{EC50 of} 50-100 nM (as shown in FIG. 15). At high in vivo doses, the intrinsic basal activity of the VitoKine construct could potentially lead to peripheral receptor stimulation with persistent pharmacodynamic effects in vivo and systemic toxicity. Therefore, the present inventors hypothesized that a reduction in the activity of the IL-15 moiety could minimize the intrinsic basal activity of the corresponding VitoKine construct.

A panel of IL-15 muteins with amino acid substitutions that disrupt interaction with IL-15Rβγ were expressed as IL-15/IL-15Rα Fc fusion proteins (non-covalently) and screened for attenuation of activity in the human PMBC assay as described previously. P-0313 was used as a control molecule. A summary of muteins with representative single amino acid changes at residues V63, 168, and Q108 of IL-15, or combinations thereof, that resulted in attenuation of activity in CD8 T cell proliferation is presented in Table 13. Compared with P-0313, fusion proteins of these IL-15/IL-15Rα Fc variants showed a broad spectrum of attenuation, ranging from 5-fold to approximately 6700-fold. In FIG. 23A and 23B also show the percentage of Ki67 expression on CD8 T cells and NK cells following administration of a number of representative IL-15/IL-15Rα and Fc variant fusion proteins, including P-0736, P-0772, P-0737, P-0768, P-0793, and P-0764. A summary of the IL-15 amino acid changes in the fusion proteins shown in FIG. 23 is presented in Table 13.

Furthermore, IL-15 variants containing an amino acid deletion or insertion, or a combination of an amino acid substitution and a deletion/insertion, as shown in SEQ ID NOs: 182-192, demonstrated varying levels of attenuation of activity when expressed as IL-15/IL-15RαSushi and Fc fusion proteins (data not shown). Such a grouping of IL-15 can be similarly used in the VitoKine IL-15 format to achieve optimal intrinsic basal activity. As will be appreciated by those skilled in the art, all mutations (amino acid substitutions, deletions, and insertions) can, optionally and independently, be combined in any manner to achieve optimal activity modulation.

To determine whether reducing the activity of an IL-15 moiety minimizes the intrinsic basal activity of the corresponding VitoKine construct, IL-15 Q108S, which exhibits significantly reduced activity, was included as the D2 domain in a VitoKine construct designated P-0682. In addition to containing a different IL-15 variant, P-0682 also differs from P-0315 in that it has a non-cleavable flexible L1 linker. However, the two VitoKine constructs are otherwise identical. As shown in FIGS. 24A and 24B, P-0682 completely lost its activity in inducing Ki67 expression on CD8 T cells or NK cells, even at the highest concentration assayed of 1 μM. P-0764 is an Fc-fused analog of P-0682 and is similar to the activated form of P-0682. Taken together, the data of the present invention demonstrate that the VitoKine IL-15 platform typically provides 1,000- to 2,000-fold attenuation of cytokine activity in ex vivo assays, and the _EC50 values calculated for the IL-15 Q108S P-0682 VitoKine construct for CD8 T cell and NK cell induction in these assays are 100 μM and 20 μM, respectively. Thus, the data presented indeed confirm that attenuation of IL-15 activity results in a decrease in the intrinsic basal activity of the corresponding VitoKine construct.

Because the intrinsic basal activity of the VitoKine IL-15 construct is proportional to the activity of the cytokine moiety, it can be adjusted by including an IL-15 moiety with different activity levels, such as the IL-15 variants listed in Table 13. For example, the basal activity of P-0806 (SEQ ID NO: 231), a VitoKine IL-15 Fc construct containing IL-15 V63A/I68H as the D2 domain, is expected to be intermediate between P-0315 and P-0682, with an estimated _EC50 for CD8 T cell induction of 2-3 μM. Importantly, the ability to adjust the intrinsic basal activity of the VitoKine IL-15 construct allows for an optimal balance to be achieved between the inertness of the VitoKine construct before cleavage and the activity after its activation, facilitating the desired antitumor efficacy while minimizing unwanted systemic toxicity.

Example 12

The non-cleavable VitoKine construct as a cytokine form with reduced activity

It is known in the field that a cytokine active in vitro may not elicit the most potent lymphocyte response in vivo. Highly active cytokines are often associated with greater stimulation, internalization, and desensitization of receptors, attenuated signaling, proliferation, and function, as well as increased cell death or clonal exhaustion. Therefore, a cytokine with attenuated activity may be highly desirable for preventing excessive lymphocyte activation and achieving a sustained and enhanced pharmacodynamic effect and antitumor efficacy in vivo.

The VitoKine non-cleavable construct with IL-15 and Fcβ-0351 demonstrated significantly reduced activity compared to fully active IL-15 compounds in vitro; however, it demonstrated antimetastatic efficacy and robust NK cell responses in a CT26 mouse lung metastasis model (Example 8). Thus, non-cleavable VitoKine constructs may be useful as cytokines with attenuated and prolonged activity to optimize pharmacodynamics in vivo.

The activity of P-0351, including NK cell and CD8+ T cell proliferation assessed by Ki67 (FIGS. 25A and 25B), was the same as that of the benchmark molecule (SEQ ID NOs: 177 and 178), which is equivalent to XENP024306 in patent application WO 2018071919 Al. XENP024306 is an IL-15/IL-15Rα and Fc fusion molecule containing amino acid substitutions (D30N/E64Q/N65D) in IL-15 and half-life-increasing mutations in Fc. These three mutations in the IL-15 XENP024306 chain were reported to result in a 200-fold decrease in in vitro activity, but XENP024306 was shown to be more active in vivo, likely due to optimized in vivo pharmacodynamics.

Similarly, P-0351 with attenuated potency is expected to result in a longer duration of action and improved pharmacodynamics (PD) due to the absence or reduction of excessive activation and unwanted target-binding-mediated clearance typically associated with administration of a fully active cytokine. Thus, an extended-half-life analogue of P-0351, P-0651 (SEQ ID NO: 170), will promote a longer half-life and further prolong pharmacodynamic action in vivo.

Example 13

Design and production of VitoKine constructs with IL-2 and Fc for selective expansion of regulatory T cells (Treg)

The challenge is to develop VitoKine IL-2 constructs that will remain inert until locally activated by proteases upregulated at sites of inflammation. Low doses of wild-type IL-2 preferentially stimulate Tregs over effector T cells, and IL-2 muteins with reduced binding affinity to IL-2Rβ have been reported to expand the window of selectivity. These molecules could be developed as therapeutic agents for the prevention of autoimmune diseases. Other mutations that interfere with binding to IL-2Rβ or _γC and do not affect interaction with IL-2Rα may also increase the window of selectivity for preferential activation of Tregs over Teff.

VitoKine IL-2 and Fc constructs were used, containing either wild-type IL-2 or an IL-2 mutein with enhanced selectivity for Treg stimulation over effector T cells, reversibly masked between the Fc domain and IL-2RαSushi (SEQ ID NO: 10) as the active moiety. IL-2Rα (SEQ ID NO: 9) contains two sushi domains separated by a natural peptide linker region. VitoKine IL-2 constructs contain one or two cleavable linkers recognized by proteases, which are reported to be upregulated at sites of inflammatory disorders. While the linker connecting Fc and IL-2/mutein can be either cleavable or non-cleavable, it is preferred that the linker connecting IL-2 and IL-2αSushi be specifically cleavable by a protease.

The activity of the IL-2 mutein for selective stimulation of Tregs is expected to be restored by the release and diffusion of IL-2Rα from IL-2 following protease cleavage. Due to the nanomolar binding affinity of IL-2Rα and IL-2, it is possible that IL-2RαSushi will remain non-covalently bound to IL-2 following linker cleavage, resulting in blocked interaction of IL-2 with IL-2Rα on Tregs. To address this potential issue, IL-2Rα muteins with amino acid substitutions on their IL-2-interacting surface were developed to weaken binding to IL-2. Thus, following protease cleavage of the linker, the IL-2RαSushi mutant will dissociate and diffuse from IL-2; This activation mechanism (shown schematically in FIG. 2B) is somewhat different from that shown in FIG. 2A.

Exemplary amino acid substitutions were made at positions 38 (i.e., K38E) and 43 (i.e., Y43A) of the IL-2Rα domain. Other IL-2Rα variants with substitutions of IL-2-interacting residues that are expected to disrupt IL-2 and IL-2Rα interactions may also be included. As will be apparent to those skilled in the art, all mutations may be optionally and independently combined in any manner to achieve optimal affinity modulation. VitoKine IL-2 molecules containing various combinations of linkers, wild-type IL-2 or IL-2 variants, and wild-type IL-2RαSushi or IL-2RαSushi variants were generated, and their respective sequences are provided as SEQ ID NOs: 49-65.

Gene synthesis, expression vector construction, and protein production, purification, and characterization were performed following the same procedures detailed in Example 1. As an example demonstrating the protein profile of the VitoKine IL-2 constructs, SDS-PAGE analysis of P-0320 is shown in FIG. 26A. The size exclusion chromatogram shown in FIG. 26B demonstrates that after the initial capture step on Protein A without a final purification step, the aggregate content was less than 5%. This low aggregation propensity indicates broad potential for the development of VitoKine IL-2 constructs.

Example 14

In vitro evaluation of the activity of VitoKine constructs with IL-2 and Fc targeting Tregs

The biological activity of VitoKine IL-2 constructs on T cells was determined by measuring phosphorylated STAT5 (pStat5) levels in defined T cell subsets of fresh human PBMCs. Stat5 is known to be involved in downstream intracellular signaling induced by IL-2 binding to the IL- _12RβγC transmembrane complex. pStatS levels were measured by flow cytometry in fixed and permeabilized cells using an antibody to the pStat5 peptide. Briefly, human PBMCs were isolated by Ficoll-Hypaque centrifugation from healthy donor buffy coats purchased from the Oklahoma Blood Institute. PBMCs (2 × ¹⁰⁵ ) were treated with serial dilutions of test compounds for 30 min at 37°C. Cells were then treated with the Foxp3/Transcription Factor Staining Buffer Set (EBIO) according to the manufacturer's instructions. Cells were then fixed with Cytofix, permeabilized with Perm Buffer III (BD Biosciences), and washed. After blocking Fc receptors with Human TruStain FcX (1:50 dilution), cells were stained with a mixture of anti-CD25 with PE, anti-FOXP3 with APC, anti-pSTAT5 with FITC, and anti-CD4 with PerCP-Cy5.5 antibodies at the manufacturer's recommended concentrations for 60 minutes at room temperature. Cells were then harvested and washed, resuspended in FACS buffer, and analyzed by flow cytometry. Flow cytometry data for Treg and CD4+ normal I cell subsets were analyzed as Foxp3+/CD25 ^high and Foxp3-/D25 ^low , respectively. Data are expressed as the percentage of pStat5-positive cells in the analyzed population.

VitoKine IL-2 constructs P-0320 (SEQ ID NO: 49) and P-0329 (SEQ ID NO: 62) were evaluated for pStat5 activation compared to P-0250 (SEQ ID NO: 48). P-0320 contains the wild-type IL-2 domain with its N-terminus fused to the Fc domain via a uPA-cleavable linker and its C-terminus fused to the IL-2RαSushi domain via a flexible linker (GGGGS) ₃ (SEQ ID NO: 127). P-0329 contains the wild-type IL-2 domain with its C-terminus fused to the Fc domain via a uPA-cleavable linker and its N-terminus fused to the IL-2RαSushi domain via a flexible linker (GGGGS) ₃ (SEQ ID NO: 127). P-0250 is a highly active IL-2-Fc fusion protein. The percentage of pStatS-positive cells in the Treg and CD4+ conventional T cell (Tconv) subsets using the test compounds is shown in FIG. 27. It is clearly seen that with both VitoKine IL-2 constructs, pStatS activation of Tregs is significantly lower than with the fully active IL-2 fusion protein, while pStat5 activation of CD4+ Tconv cells was almost undetectable. These data clearly demonstrate the effective masking of IL-2 activity in the VitoKine format.

Example 15

Activation of the VitoKine construct with IL-2 and Fc protease and evaluation of its activity in vitro

The VitoKine IL-2 and Fc construct P-0382 (SEQ ID NO: 51) contains a flexible GGGSGGGS linker (SEQ ID NO: 115) connecting Fc and IL-2, and a 10-amino acid linker cleavable by MMP-2/9 (SEQ ID NO: 77) between the IL-2 and IL-2RαSushi domains. The IL-2RαSushi domain of P-0382 contains an amino acid substitution (K38E) designed to reduce its binding affinity to IL-2 and ensure dissociation and subsequent diffusion from IL-2 after linker cleavage by protease.

P-0382 was activated by in vitro protease cleavage using MMP-2. Briefly, 3.3 μg of latent MMP-2 (BioLegend) was first activated using APMA (Millipore Sigma) following the manufacturer's instructions, then buffer exchanged and added to 120 μg of P-0382 in 0.4 ml of the manufacturer's recommended assay buffer (100 mM Tris, 20 mM _CaCl2 , 300 mM NaCl, 0.1% (w/v) Brij 35, pH 7.5). After incubation at 37°C for 20 hours, half of the reaction mixture was purified on MabSelect SuRe Protein A resin, and the activated VitoKine construct was eluted with 25 mM sodium citrate, 25 mM sodium chloride, pH 3.2. The protein was neutralized by adding 3% 1 M Tris, pH 10.2. The other half of the sample was incubated with Ni-Excel resin to stop the interaction by removing His-tagged MMP-2 protein, and the activated VitoKine construct was obtained by removing the Ni-resin by centrifugation. Protein A purification was performed to confirm the absence of non-covalent binding of the IL-2RαSushi domain to IL-2 after cleavage of the polypeptide chain, as schematically shown in FIG. 2B. Analysis of the samples by SDS-PAGE with 4-12% Bis-Tris is shown in FIG. 28. Despite the higher protease abundance and longer interaction time compared to structurally similar VitoKine constructs with IL-15 (e.g., P-0315), the interaction did not result in complete cleavage. Comparison with MMP-2-treated samples with and without Protein A purification (FIGS. 29A and 29B) confirmed the release of the IL-2RαSushi domain from covalent binding and its absence in the Protein A-purified Fc-IL-2 fusion polypeptide.

Despite incomplete cleavage, these two MMP-2-activated samples, one as a Ni-Excel eluate (Activ. 1) and one as a post-protein A eluate (Activ. 2), were evaluated in the pStat5 activation assay described in Example 14, and the data are shown in FIG. 29. P-0382 activity was very low in Tregs and nearly undetectable in CD4+ Tconv cells, further confirming the efficient masking of the active moiety in the VitoKine IL-2 format. Both activated samples demonstrated nearly complete restoration of activity. The moderate decrease in activity compared to P-0250 was likely due to incomplete proteolysis.

The presence of the MMP-2-cleaved IL-2RαSushi domain in the Activ. 1 sample did not appear to affect the activity of the activated VitoKine IL-2 construct, as Activ. 1 and Activ. 2 had comparable potency in inducing pStat5 phosphorylation in both Treg and Tconv cells (FIGS. 28A and 28B). These data indicate that the MMP-2-cleaved IL-2RαSushi domain was not associated with IL-2 and should not interfere with IL-2 binding to receptor complexes expressed on lymphocytes.

Proteolysis of P-0382 by MMP-2 did not result in complete cleavage, suggesting that increasing the length of the cleavable linker may improve the accessibility of the substrate peptide to the cleavage protease. The 10-amino acid linker (SEQ ID NO: 95) in P-0382 was replaced with a 15-amino acid linker cleavable by MMP-2/9 (SEQ ID NO: 94) containing additional flanking residues, yielding the new VitoKine construct P-0398 (SEQ ID NO: 52). P-0398 was activated by in vitro protease cleavage using MMP-2, following the same protocol detailed above. Three times less MMP-2 (1.5 μg MMP-2 per 180 μg P-0398 versus 3.3 μg MMP-2 per 120 μg P-0382) resulted in complete cleavage of P-0398, as indicated by the presence of only a “complete cleavage” band on the SDS-PAGE gel (data not shown).

The biological activity of activated P-0398 with the IL-2RαSushi domain deleted by protein A purification was determined in the pStat5 assay (FIGS. 29A and 29B). Activated P-0398 is similar to the IL-2-Fc fusion molecule P-0250 in sequence and structure, and they have nearly identical activity in inducing Stat5 phosphorylation in both Treg and Tconv cells. Although both VitoKine constructs, P-0382 and P-0398, had significantly reduced (by 4 logs) biological activity due to covalent linkage to the IL-2RαSushi domain, P-0398 containing the longer L2 linker appeared to have slightly higher activity. Similar to what was observed with the VitoKine IL-15 and Fc constructs, the degree of activity reduction in the VitoKine IL-2 constructs could be further adjusted by varying the length of the L2 linker. Similarly, when selecting the length and sequence of the cleavable L2 linker, the presence of specific proteases at the target disease site, the availability of the substrate peptide for proteases, and the desired rate of proteolysis should be considered.

In conclusion, compared to VitoKine constructs with IL-15, VitoKine constructs with IL-2 required a longer L2 linker to ensure optimal enzyme accessibility and complete proteolysis. Cleavage of typical IL-2 VitoKine constructs P-0382 and P-0398 by MMP-2 resulted in full activation of these molecules. Activated VitoKine constructs with IL-2 achieved biological activity similar to the highly active IL-2-Fc fusion P-0250.

Example 16

Design and production of VitoKine constructs with IL-2 and Fc for selective expansion of effector T cells (Teff)

The challenge is to develop VitoKine IL-2 constructs that will remain inert until locally activated by proteases present or upregulated only in tumor sites. IL-2-induced preferential expansion of Tregs is an undesirable effect of IL-2 for cancer immunotherapy, as Tregs can inhibit effector T cell responses. IL-2-induced preferential expansion of Tregs is due to the high and constitutive expression of IL-2Rα on Tregs, along with the βγ signaling receptor subunits. To overcome these limitations, IL-2 variants designed to eliminate binding to IL-2Rα are expected to avoid preferential activation of Tregs, activating them only at concentrations that also activate CD8+ T and NK cells. Following this concept, a panel of IL-2 variants containing one or more substitutions at amino acid residues that interact with IL-2Rα was developed. Residues R38, T41, F42, F44, E62, P65, E68, and Y107 contact IL-2Rα and form hydrogen/salt bonds or hydrophobic interactions with many IL-2Rα residues (Mathias Rickert, et al. (2005) Science 308, 1477-80). Amino acid substitutions at these sites would be expected to disrupt interaction with IL-2Rα and result in IL-2 variants with reduced or abolished binding to IL-2Rα.

A panel of IL-2 muteins in the form of C-terminal fusion proteins with an Fc homodimer or Fc heterodimer were expressed and screened for binding to IL-2Rα in an enzyme-linked immunosorbent assay (ELISA). Briefly, IL-2Rα-ECD was adsorbed onto wells at 0.1 μg per well. After overnight incubation at 4°C and blocking, 100 μl of serial dilutions of IL-2 and Fc fusion proteins were added to each well. After 1 h of incubation at room temperature, 100 μl of goat anti-human IgG Fc antibody conjugated to HRP (1:5000 dilution in diluent) were added to each well and incubation was carried out at room temperature for 1 h. The plate was developed at room temperature in the dark after the addition of 100 μl of TMB substrate for 10 minutes, and then stop solution was added at 100 μl per well. Optical density was determined at 450 nm, and curves were plotted using Prism software (GraphPad). P-0531 (SEQ ID NO: 248) and P-0689 (SEQ ID NOs: 249 and 168), the S1251 equivalents of wild-type IL-2 and Fc fusion proteins with bivalent and monovalent IL-2 moieties, respectively, were included as controls.

A summary of single or combined amino acid changes in IL-2 that resulted in decreased or abolished binding is presented in Table 14. Binding curves of a number of representative monovalent IL-2 Fc variant fusion proteins, P-0704, P-0707, P-0708, and P-0709, compared to P-0689, to IL-2Rα in ELISA are shown in FIG. 31.

In addition to amino acid substitutions that disrupt interaction with IL-2Rα, all IL-2 variants presented in Table 14 also contain the S1251 mutation, which significantly enhances protein expression and reduces its tendency to aggregate. Those skilled in the art will appreciate that the essence and scope of the present invention encompasses mutants with any other combinations for modulating their affinity for IL-2Rα, regardless of changes in their affinity for the aforementioned IL-2 receptor components.

Furthermore, despite reduced/aborted binding to CD25, these IL-2 variants fully retained binding and functional activity for the dimeric IL-2Rβy receptor and the ability to activate effector cells due to preserved IL-2Rβy signaling. This was demonstrated for P-0704 in a human PMBC assay. The potency of P-0704, a monovalent IL-2 P65R and Fc fusion protein with ablated IL-2Rα binding, in inducing Ki67 expression on CD8 T cells was equal to that of its wild-type IL-2 counterpart P-0689 (FIG. 32). In the following examples, P-0704 and P-0689 will be used interchangeably as controls for full IL-2 activity against Teff.

VitoKine IL-2 and Fc constructs targeting Teff contain an IL-2 variant with reduced/aborted binding to IL-2Rα as the active moiety, reversibly masked between the Fc domain and IL-2RαSushi (SEQ ID NO: 10). These constructs contain one or two cleavable linkers recognized by proteases reported to be upregulated in various cancer types, such as solid tumors. While the linker connecting the Fc and IL-2 mutein can be either cleavable or non-cleavable, the linker connecting IL-2 and IL-2RαSushi is preferably susceptible to specific protease cleavage. Because the IL-2 moiety in the VitoKine construct is designed to eliminate binding to IL-2Rα, the D3 mask moiety of the VitoKine IL-2Rα construct is likely to diffuse after proteolytic cleavage in vivo (FIG. 28), resulting in restoration of IL-2 mutein activity. VitoKine IL-2 molecules containing various IL-2 muteins as the active moiety are schematically shown in FIG. 1. Representative VitoKine IL-2 Fc molecules were designed and produced to selectively increase the number of Teff cells, and their respective sequences are provided as SEQ ID NOs: 59-61 and 271-274.

Gene synthesis, construction of expression vectors, and protein production, purification, and characterization were performed following the same procedures as described in detail in Example 1.

Example 17

VitoKine IL-2 constructs containing IL-2Rα variants as a masking moiety D3

IL-2Rα variants were engineered to reduce IL-2 binding by incorporating mutations in IL-2-interacting residues to ensure rapid diffusion of the D3 moiety following in vivo proteolysis. Three representative IL-2RαSushi variants (SEQ ID NOs: 267-269) were expressed as monovalent Fc fusion proteins corresponding to P-0751 (Y43A), P-0752 (L42G), and P-0753 (R36A), respectively. The fusion proteins of the three IL-2RαSushi and Fc variants were assessed for the effect of individual mutations on IL-2 binding in ELISA compared to a similar fusion protein with wild-type IL-2RαSushi, designated P-0757.

Briefly, monovalent fusion proteins of IL-2RαSushi variants and Fc were adsorbed onto the wells of 96-well Nunc Maxisorp microplates at 1 μg per well. After overnight incubation at 4°C and blocking with 1% BSA, 100 μl of serial dilutions of P-0689, a monovalent fusion protein of IL-2 S125I and Fc, were added to each well. After incubation for 1 hour at room temperature, 100 μl of anti-IL-2 antibody clone B33-2 with biotin (BD Biosiences), 1 μg/ml, were added to each well, and incubation was carried out at room temperature for 1 hour. Then, 100 µl of avidin-HRP (BioLegend) at a 1:500 dilution was added to each well and incubated for 30 min. Finally, 100 µl of TMB substrate was added to each well, the plate was developed at room temperature, and stopping solution was added, 100 µl per well. Optical density was determined at 450 nm, and curves were plotted using Prism software (GraphPad).

As shown in Table 15 and FIG. 33, the Y43A, L42G, and R36A amino acid substitutions in IL-2Rα resulted in disruption of its interaction with IL-2. Y43A resulted in a moderate decrease in IL-2 binding (8.1-fold), the R36A mutation resulted in a more significant decrease in binding (EC ₅₀ ) of 346-fold, and the L42G mutation resulted in an intermediate decrease in IL-2 binding of 35-fold.

The three IL-2Rα variants mentioned above, as well as wild-type IL-2Rα, were used to construct four Teff-targeted IL-2–Fc VitoKine molecules, all containing monomeric IL-2 S125I (wild-type equivalent) as the D2 domain and a 15-amino acid L2 linker cleavable by MMP-2/9. The masking efficiency of the IL-2Rα variants was then assessed by analyzing their activity in inducing Ki67 expression on CD8 T and NK cells in a human PMBC assay compared to P-0704, an Fc fusion protein with activity equivalent to P-0689, an Fc fusion protein similar to this panel of VitoKine constructs. The resulting data are presented in Table 16 and shown in FIGS. 34A and 34B.

As shown in FIG. 34, wild-type IL-2RαSushi in the VitoKine P-0701 construct as a D3 masking domain resulted in a significant 3-log reduction in CD8+ T and NK cell proliferation induction compared to P-0704, a similar fully active Fc fusion protein. Incorporation of a mutation that disrupts IL-2 binding into IL-2RαSushi was predicted to reduce the masking effect of the D3 domain and thus reduce the inertness of the VitoKine construct. The degree of reduction in masking effect was also expected to correlate with the degree of binding strength reduction between IL-2 and the IL-2RαSushi variants.

As shown in FIG. 34A and Table 16, the IL-2RαSushi variants Y43A and R36A as the D3 domains in the corresponding VitoKine Fc constructs (P-0754 and P-0756) indeed demonstrated a reduced masking effect and, consequently, less inertness of the VitoKine constructs compared to P-0701 in inducing CD8 T cell proliferation. The same trend was observed when the activity against NK cells was analyzed (FIG. 34B). However, the IL-2RαSushi Y43A variant exhibited only a modest reduction in IL-2 binding (8.1-fold), while the R36A substitution resulted in a significantly larger 346-fold reduction in IL-2 binding, yet their corresponding VitoKine constructs exhibited similar reductions in masking, contrary to the prediction that the degree of reduction in masking would correlate with the degree of reduction in binding strength between IL-2 and the IL-2RαSushi variants. More surprisingly, despite a 35-fold reduction in IL-2 binding, the IL-2RαSushi L42G variant did not result in a reduction in masking compared to its wild-type counterpart, and thus its corresponding VitoKine construct, P-0755, retained almost complete inertness (FIGS. 34A and 34B). The experimental observation that the inclusion of mutations that disrupt IL-2 binding in IL-2Rα exhibits varying levels of influence on the masking effect without correlation with the degree of change in binding strength was unexpected but has been confirmed in multiple experiments. This may be due to optimal interaction between the IL-2 and IL-2Rα domains at the spatial level. The IL-2RαSushi L42G variant was selected as the preferred D3 domain for VitoKine IL-2 constructs because it preserves the masking effect, maintaining the inertness of the corresponding VitoKine constructs, but at the same time undergoes rapid diffusion after proteolysis in vivo, ensuring full activation due to its weakened binding to IL-2. At the same time, IL-2Rα variants, such as R36A, can be used as D3 domains when it is desired to adjust the intrinsic basal activity of the VitoKine IL-2 construct to ensure an optimal balance between desired antitumor efficacy and unwanted systemic toxicity.

Additionally, IL-2 Fc VitoKine constructs containing IL-2 P65R as the D2 domain and wild-type IL-2RαSushi (P-0745), IL-2RαSushi Y43A (P-0807), IL-2RαSushi L42G (P-0808), or IL-2RαSushi R36A (P-0809) as the D3 domain were generated and analyzed for their activity in inducing CD8 T and NK cell proliferation. As shown in FIG. 35A and FIG. 35B, all of these VitoKine constructs demonstrated comparable 10- to 20-fold reductions in activity compared to P-0704, an Fc fusion protein similar to this panel of VitoKine constructs. Because the P65R mutation abolished IL-2 binding to IL-2Rα, the modest masking effect was likely due to the spatial correspondence between IL-2 and IL-2RαSushi, and any additional IL-2Rα mutations that disrupted interaction with IL-2 had no additional effect on the masking effect.

In addition, the VitoKine IL-2-Fcβ β-0755 construct, containing IL-2 as the D2 domain and IL-2RαSushi L42G as the D3 domain, was activated by in vitro MMP-2 cleavage following the method detailed in Example 15 and evaluated in a human PMBC assay. After cleavage and diffusion of the D3 domain, the activated β-0755 is similar to the analogous fusion protein Fcβ β-0689 with several additional residues that were part of the protease substrate and remained at the C-terminus of the IL-2 moiety. As shown in FIG. 36A and 36B, the activity of P-0755 in the VitoKine construct was reduced by approximately 3 logs and upon activation could be fully restored in stimulating the proliferation of effector cells, including CD8 T and NK cells.

In conclusion, representative VitoKine IL-2-Fc constructs targeting Teff, containing IL-2RαSushi variants as the D3 masking domain, were constructed and analyzed. The IL-2RαSushi L42G variant was chosen as the preferred D3 domain because it retains the wild-type masking effect in the VitoKine construct and is more diffusible, ensuring full activation after proteolysis due to its weakened binding to IL-2. When higher intrinsic basal activity of the VitoKine IL-2 construct is desired to ensure an optimal balance between desired antitumor efficacy and undesirable systemic toxicity, other IL-2RαSushi mutations with reduced masking capacity can be used.

Example 18

Construction, expression, and purification of VitoKine antibody constructs

The use of recombinant antibody-cytokine fusion proteins (immunocytokines) may enhance the therapeutic index of cytokines by targeting them to the site of disease. However, fusion of a fully active cytokine with an antibody may result in peripheral activation and insufficient tumor targeting. The inertness of VitoKine constructs prior to activation at the intended therapeutic site makes VitoKine antibody constructs a new and innovative form of immunocytokines. In addition to tumor-targeted antibodies, VitoKine antibody constructs can also be produced using antibodies that block immune checkpoints, thereby bypassing the immunosuppressive effects of the tumor microenvironment, or immunostimulatory antibodies to enhance existing responses, which may further enhance the immune system's antitumor activity. Furthermore, VitoKine antibody constructs targeting inflamed tissue may be used to treat autoimmune and chronic inflammatory disorders.

Following this concept, VitoKine proteins were constructed with antibodies containing, as the D2 domain, either wild-type or IL-15 variant IL-15, or wild-type or IL-2 variant IL-2. Examples of antibodies include various PD-1 antagonist antibodies, including various human/humanized PD-1 antagonist antibodies (SEQ ID NOs: 195-198 and 275-278), the PD-L1 blocking antibody atezolizumab (SEQ ID NOs: 279-280), the anti-CTLA4 antibody ipilimumab, the CD40 agonist antibody RO7009789, antibodies directed to tumor antigens, including L19 directed against the extra domain of fibronectin, rituximab directed against CD20, Herceptin directed against Neg-2, cetuximab directed against EGFR, an anti-FAP antibody for tumor targeting and retention (SEQ ID NOs: 193-194), and the anti-inflammatory antibody vedolizumab against integrin _{α4β 7} and Humira against TNFα. Sequences of typical VitoKine antibody constructs are provided as SEQ ID NOs: 128-146, 180-181, 281-286, 296-297, and 303-306.

Gene synthesis, expression vector construction, and protein production, purification, and characterization were performed following the same procedures as detailed in Example 1. Representative VitoKine constructs with IL-15 or IL-2 and antibodies demonstrated expression profiles, such as productivity and aggregation propensity, similar to similar VitoKine constructs with Fc.

Example 19

Functional evaluation of the biological activity of VitoKine constructs with IL-15 and antibodies ex vivo

The biological activity of a representative VitoKine construct with IL-15 and anti-PD-L1 antibody P-0485 (SEQ ID NOs: 180 and 181) was analyzed by measuring Ki67 expression in CD8+ T cells (FIG. 37A) and NK cells (FIG. 37B) following treatment of human PMBCs with VitoKine-IL-15 compounds. P-0485 shares the same L1 and L2 linkers and D2 and D3 domains as its counterpart, the VitoKine Fc construct P-0315. P-0485 had slightly higher activity, which may have been facilitated by lymphocyte activation resulting from PD-L1 blockade.

As shown in Example 11, the intrinsic basal activity of the VitoKine construct with IL-15 and Fc can be adjusted by incorporating IL-15 moieties with different activities. Similarly, a VitoKine construct with IL-15 and the PD-1 antagonist antibody P-0875 (SEQ ID NOs: 196 and 284) was generated with the IL-15 variant V63A/I68H as the D2 domain. P-0875 was analyzed by measuring Ki67 expression in CD8+ T cells after treatment with human PMBCs in comparison with a similar IL-15/IL-15RαSushi fusion protein and P-0870 antibody (SEQ ID NOs: 196, 297, and 5) and a similar IL-15/IL-15RαSushi fusion protein and Fc P-0773 (FIG. 38B). P-0773 and P-0870 were equally potent in inducers of Ki67 expression with an _{EC50 of} 18.5 nM, indicating that the fusion protein format did not affect IL-15 activity. Taking into account all the data of the present invention, according to which the VitoKine platform with IL-15 typically results in approximately 1000-fold reduction in activity, the predicted _EC50 of P-0875 for inducing CD8 T cells is therefore 18 μM. This low predicted activity is consistent with the lack of activity of P-0875 even at the highest concentration assayed of 1 μM (FIG. 38B); however, these data do not allow a reliable determination of the activity of P-0875. P-0875 and P-0773 were then also assayed using cynomolgus monkey PBMSCs, generated in a manner similar to human PBMSCs. Both compounds showed a proportional increase in biological activity compared to human cells, and activity curves allowing reliable determination of _EC50 values were obtained over the concentration range assayed (FIG. 38C). P-0773 and P-0875 induced Ki67 expression in cynomolgus monkey CD8+ T cells with EC50s _{of 0.259} nM and 254 nM, respectively. A 1,000-fold reduction in activity was a signature of the VitoKine IL-15 platform, consistently demonstrated by VitoKine IL-15 and Fc constructs based on the prototype VitoKine IL-15 compound, P-0315, compared to P-0313 ( _{EC50s of} 18.6 pM for P-0313 and 16.9 pM for P-0315 for inducing Ki67 expression in human CD8+ T cells, as shown in FIG. 38A).

In conclusion, VitoKine constructs with IL-15 and antibodies provide the attenuation of cytokine activity characteristic of this platform. Furthermore, reduced activity of the IL-15 moiety may minimize the intrinsic basal activity of the corresponding VitoKine construct.

Example 20

Functional evaluation of the biological activity of VitoKine constructs with IL-2 and an antibody targeting Teff ex vivo

Representative VitoKine IL-2–antibody constructs targeting Teff were generated and analyzed for the masking efficacy of the D3 domain in the context of each IL-2 mutation. All four representative VitoKine IL-2–antibody constructs, P-0800, P-0830, P-0831, and P-0802, contained the anti-mouse PD-1 antibody (SEQ ID NOs: 299 and 302) as the D1 domain and the IL-2RαSushi L42G variant as the D3 domain. The monovalent D2 domain, containing IL-2 P65R in P-0800, IL-2 P65N in P-0830, IL-2 P65Q in P-0831, and the equivalent wild-type IL-2 in P-0802, is fused to the C-terminus of the heterodimeric HC chain (SEQ ID NO: 301) via a non-cleavable linker (G ₄ S) ₃ (SEQ ID NO: 112) and connected to the N-terminus of the D3 domain by a linker cleavable by MMP-2/9 (SEQ ID NO: 94). Each of these four VitoKine IL-2 antibody constructs also contained two additional polypeptides, as set forth in SEQ ID NOs: 300 and 302. The IL-2 moiety in each of these four VitoKine constructs fully retained its activity against Teff, but with varying levels of binding strength to IL-2Rα. As shown in Table 14 of Example 16, the P65R mutation abolished binding to IL-2Rα, while P65N and P65Q reduced binding strength by 8.6-fold and 43-fold, respectively.

The four VitoKine IL-2 antibody constructs were evaluated for their respective activities in inducing dose-dependent Ki67 expression on CD8+ T cells (FIG. 39A) and NK cells (FIG. 39B) of fresh human PMBC. P-0782, containing anti-mouse PD-1 antibody fused to monovalent IL-2 P65R fused to the C-terminus of a heterodimeric heavy chain, was included for comparison. P-0782, P-0800, and P-0802 are antibody fusion proteins similar to P-0704, P-0808, and P-0755, respectively. Taken together, the data shown in FIG. 34, 35, and 39 clearly demonstrated that the format of the D1 domain, whether Fc or antibody, as exemplified here, does not affect the efficiency of masking cytokine activity by the D3 domain. When the D2 domain is wild-type IL-2, the IL-2Rα-based D3 domain provides approximately 3 log reduction in activity; when the D2 domain is an IL-2 variant whose binding to IL-2Rα is abolished, the D3 domain provides only a 10- to 20-fold masking effect.

Interestingly, when IL-2 variants with intermediate binding strength to IL-2Rα, such as P65N and P65Q, were used as the D2 domain, the D3 domain provided masking efficiency similar to that observed with wild-type IL-2. Data on the dose-dependent induction of Ki67 expression on CD8+ T cells are shown in FIG. 39A, and on Ki67 expression on NK cells are shown in FIG. 39B. In addition, the _EC50 values of each compound for inducing Ki67 expression on NK cells are provided in Table 17.

For VitoKine IL-2 constructs targeting Teff, the threshold binding affinity between the D2 and D3 domains and the optimal spatial configuration of their binding surfaces were hypothesized to be important in determining the masking efficiency. Since the IL-2 P65Q variant has significantly lower binding strength to IL-2Rα but can still be effectively masked by IL-2RαSushi L42G while remaining inert in the VitoKine construct form, IL-2 P65Q is expected to exhibit significantly less Treg cell stimulating ability than wild-type IL-2 after in vivo protease cleavage and full restoration of biological activity (data not shown). Therefore, IL-2 P65Q was selected as the preferred D2 domain for VitoKine IL-2 constructs targeting Teff. However, to achieve an optimal balance between desired antitumor efficacy and undesirable systemic toxicity, IL-2 variants with other mutations may be used.

Using various combinations of cytokine valence and linkers, a variety of VitoKine IL-2–antibody constructs were generated, containing the human PD-1 antagonist antibody as the D1 domain, the IL-2 P65Q variant as the D2 domain, and the IL-2RαSushi L42G variant as the D3 domain. Representative VitoKine IL-2–anti-PD-1 antibody constructs are shown in Table 18.

Representative VitoKine IL-2 and anti-PD-1 antibody constructs P-0872 and P-0929 were also evaluated for protease cleavage efficiency in the presence of abundant D1 domain. P-0872 contains a monovalent IL-2 moiety and a single MMP-2/9 cleavable linker (SEQ ID NO: 94) connecting the D2 and D3 domains. P-0872 was cleaved with MMP-2 protease following the protocol detailed in Example 15. The cleaved sample was then purified on protein A in bind-and-elute mode, the eluted sample was analyzed on a reduced SDS-PAGE gel, and its biological function was assessed in an ex vivo functional assay.

As shown in FIG. 40A, the D3 domain of P-0872 was efficiently and completely cleaved to yield the active form 2 (P-0972-Activ.) shown in FIG. 2B, resulting in complete restoration of activity, exemplified by comparable activity to a similar non-VitoKine IL-2/anti-PD-1 antibody fusion protein, P-0879 (SEQ ID NOs: 285 and 296), in inducing dose-dependent Ki67 expression on fresh human PMBC CD8+ T cells (FIG. 40B).

Another exemplary VitoKine IL-2 antibody construct, P-0929, contains a bivalent IL-2 moiety and two protease-cleavable linkers, including an MMP-2/9-cleavable linker connecting the D1 and D3 domains and an MMP-14-cleavable linker (SEQ ID NO: 298) connecting the D2 and D3 domains. P-0929 was cleaved with MMP-14 protease following a protocol similar to MMP-2 cleavage. The cleaved sample was purified using protein A, and both the eluate and eluted sample were analyzed on a reduced SDS-PAGE gel.

The SDS-PAGE gel image shown in FIG. 41A demonstrated that MMP-14 protease can recognize and efficiently cleave both the MMP-2/9 substrate peptide and the MMP-14 substrate peptide, resulting in active form 1 and active form 3 in the absence of active form 2. This observation is consistent with the fact that MMP substrates have low specificity for one member of the MMP family. The presence of the cleaved D3 domain in the sample was due to the purification scheme and did not indicate a lack of diffusion of the D3 domain after cleavage. The sample obtained from the elution of protein A (P-0929-Activ.), containing active forms 1 and 3, was then analyzed using human PMBC. As shown in FIG. 41B, activated P-0929 induces dose-dependent Ki67 expression on CD8+ T cells even more potently than a similar monovalent IL-2–anti-PD-1 fusion protein, which is not a VitoKine construct, P-0879. In conclusion, the biological activity of IL-2 with reduced binding to IL-2Rα targeting Teff can be effectively masked in the VitoKine IL-2–antibody format and rapidly restored by proteolysis. With two protease-cleavable linkers, the sequence and choice of the two cleavable linkers can be further optimized to adapt to different diseases and/or disease stages.

Example 21

In vivo evaluation of the VitoKine construct with an IL-15 variant and antibody

The goal of VitoKine IL-15 constructs containing IL-15 variants with attenuated biological activity as the D2 domain is to modulate the intrinsic basal activity of the VitoKine construct to further minimize target-binding-associated systemic toxicity and undesirable antigen-associated clearance, thereby improving bioavailability and expanding the therapeutic window. To evaluate this hypothesis, an in vivo assay of the VitoKine IL-15 construct and the P-0869 antibody is being conducted. P-0869 contains a surrogate antibody to mouse PD-1 (SEQ ID NOs: 299 and 302) as the D1 domain, the IL-15 V63A/I68H variant (SEQ ID NO: 213) as the D2 domain, non-cleavable linkers, an L2 linker cleavable by MMP-2/9 (SEQ ID NO: 95), and IL-15RαSushi (SEQ ID NO: 5) as the D3 domain. As shown in Example 11 and Table 13, the V63A/I68H amino acid substitutions in IL-15 resulted in a decrease in the activity of inducing Ki67 expression on CD8+ T cells ex vivo by approximately 2 logarithms. Furthermore, P-0875, an anti-human PD-1 analog of P-0869, demonstrated no detectable biological activity at the highest assayed concentration of 1 μM (FIG. 38B) in fresh human PMBC, indicating a significant reduction in the intrinsic basal activity of the VitoKine construct.

P-0869 and vehicle (PBS) as a negative control were administered by a single i.p. injection to healthy BALB/c mice (8-10 weeks old, n = 6 per group) at doses of 1, 3, 5, and 1 mg/kg. P-0773 (SEQ ID NOs: 227 and 5), a fusion protein of the IL-15 V63A/I68H/IL-15RαSushi variant and Fc, was included as a positive control and was administered by a single i.p. injection at a dose of 0.5 mg/kg. Blood samples for immunophenotyping were obtained before administration (-1 day) or on days 3, 5, and 7 after administration. Consistent with expectations based on the VitoKine IL-15 platform, P-0869 exhibits minimal systemic activation and expansion of target lymphocyte populations even at a very high dose due to significantly reduced basal activity.

P-0869 is also being tested in various syngeneic mouse models, including the CT26 mouse lung metastasis model, the CT26 mouse subcutaneous tumor model, and the MC38 mouse subcutaneous colon cancer model. Experimental procedures are similar to those described in Examples 9 and 10. As predicted, the VitoKine IL-15 antibody construct containing the attenuated D2 domain demonstrated tumor growth inhibition with minimal systemic cytokine activation at high doses. The inertness of the VitoKine constructs prior to activation at the target site of therapy makes the VitoKine antibody constructs a novel and innovative form of immunocytokine. Reducing the basal activity of the VitoKine construct by reducing D2 domain activity also facilitates stoichiometric equilibrium between the cytokine and targeting antibody to ensure optimal dosing.

Example 22

In vivo evaluation of the VitoKine construct with IL-2 and an antibody targeting Teff

P-0831, a VitoKine IL-2–antibody construct targeting Teff, was evaluated in vivo in a manner similar to that described in Example 21. P-0831 contains an anti-mouse PD-1 antibody (SEQ ID NOs: 300, 301, and 302) as the D1 domain, an IL-2 variant P65Q/S125I (SEQ ID NO: 240) as the D2 domain, and an IL-2RαSushi variant L42G (SEQ ID NO: 268) as the D3 domain. P-831 also contains a non-cleavable L1 linker (SEQ ID NO: 112) and a 15-amino acid linker cleavable by MMP2/9 (SEQ ID NO: 94). The monomeric IL-2 moiety retains full activity against Teff, but with significantly reduced binding strength to IL-2Rα (43-fold), which is expected to reduce stimulation of the unwanted Treg subset. As described in Example 20, the D3 domain of P-0831 effectively masked IL-2 activity and provided an approximately 1,000-fold reduction in activity (FIG. 39).

P-0831 was also analyzed in various syngeneic mouse models, including the CT26 mouse lung metastasis model, the CT26 mouse subcutaneous tumor model, and the MC38 mouse subcutaneous colon cancer model. The experimental procedures were similar to those described in Examples 9 and 10.

Several VitoKine constructs with IL-2 and the antibody P-0922A, P-0928A, P-0929A, and the similar non-cleavable protein P-0877 were also analyzed in the mouse model of established subcutaneous colon cancer MC38. All four of these VitoKine constructs contain the IL-2 variant P65Q/S125I (SEQ ID NO: 240) as the D2 domain and the IL-2RαSushi variant L42G (SEQ ID NO: 268) as the D3 domain. P-0922A and P-0929A contain a surrogate murine PD-1 antagonist antibody with a homodimeric heavy chain (SEQ ID NOs: 299 and 302) as the D1 domain, while P-0928A and P-0877 contain a surrogate murine PD-1 antagonist antibody with a heterodimeric heavy chain (SEQ ID NOs: 300, 301, and 302) as the D1 domain. The L1 and L2 linkers in P-0922A are a non-cleavable linker (G ₄ S) ₃ (SEQ ID NO: 112) and a cleavable linker (SEQ ID NO: 94), respectively. Both linkers L1 and L2 in P-0928A are cleavable with SEQ ID NO: 298 and SEQ ID NO: 94, respectively. Both linkers L1 and L2 in P-0929A are cleavable with SEQ ID NO: 94 and SEQ ID NO: 298, respectively. P-0877 contains two non-cleavable linkers (G ₄ S) ₃ (SEQ ID NO: 112). Briefly, 7-week-old female C57BL/6 mice were subcutaneously implanted with 5× ¹⁰⁵ murine colorectal cancer MC38 cells. Intraperitoneal administration of vehicle or VitoKine constructs was started when tumors reached a volume of 50-80 mm3. There were 7 mice in each group, and tumor volume was monitored twice a week. In FIG. 46 shows the tumor volume in individual mice in each group at day 7 post-administration. Compared to vehicle, all cleavable VitoKine constructs with IL-2 and antibody P-0922A, P-0928A, and P-0929A demonstrated significant and similar tumor growth inhibition (TGI) of 75-80%, while their non-cleavable counterpart P-0877 demonstrated a relatively weak but still significant tumor growth inhibition effect with a TGI of 47%, likely due to PD-1 antagonist activity. The fact that the VitoKine cleavable constructs demonstrated significantly superior antitumor efficacy compared to P-0877 suggests that proteolytic cleavage of one or both linkers in the VitoKine cleavable IL-2 antibody constructs and subsequent release of the active form of IL-2 into or around the tumor or tumor microenvironment likely contributed to the superior in vivo efficacy of the VitoKine cleavable constructs compared to their non-cleavable counterpart.

The inertness of VitoKine constructs until activated at the intended therapy site makes VitoKine antibody constructs a new and innovative form of immunocytokine and facilitates the establishment of a stoichiometric equilibrium between the cytokine and targeting antibody to ensure optimal dosing.

P-0831 was also analyzed in various syngeneic mouse models, including the CT26 mouse lung metastasis model, the CT26 mouse subcutaneous tumor model, and the MC38 mouse subcutaneous colon cancer model. The experimental procedures were similar to those described in Examples 9 and 10. As predicted, the VitoKine antibody-IL-2 construct demonstrated tumor growth inhibition with minimal systemic cytokine activation at high doses. The inertness of the VitoKine antibody constructs prior to activation at the target site makes the VitoKine antibody constructs a novel and innovative form of immunocytokine and facilitates stoichiometric equilibrium between the cytokine and targeting antibody for optimal dosing.

Example 23

IL-2Rβ-based blocking peptides for the resulting inert IL-15 or IL-2 protease-activated fusion proteins

Another method for producing inert protease-activated IL-15 or IL-2 fusion proteins is the genetic fusion of blocking peptides (e.g., a blocking peptide based on IL-2Rβ) to IL-15 or IL-2 via a cleavable linker. The blocking peptides studied are based on two loops of IL-2Rβ (SEQ ID NOs: 97 and 98) containing key residues that directly contact IL-15 and IL-2. The peptides listed in Table 19 are based on the sequences of these two loops.

Five peptides, L01-L05 (SEQ ID NOs: 97-101) from Table 19, were synthesized and analyzed for their binding to IL-15 in an ELISA format. Briefly, P-0153 (SEQ ID NOs: 44 and 46), an IL-15/IL-15RαSushi+ Fc fusion protein, was adsorbed onto the wells of 96-well Nunc Maxisorp microplates, 1 μg per well, and 3-fold serial dilutions of the biotinylated peptides starting at 100 μM were added to each well. Streptavidin-HRP complex was added at the concentration recommended by the manufacturer, and the signal was developed with TMB substrate. As shown in FIG. 42, specific binding of L03 (SEQ ID NO: 99), which represented the cyclized loop 2 (SEQ ID NO: 98), was noted.

The loop 2-based sequence was used as the blocking peptides and incorporated into the IL-15 fusion protein. Representative sequences of fusion proteins containing the IL-2Rβ-based blocking peptide fused to IL-15 via a cleavable linker and peptide spacers (SEQ ID NOs: 102-106) are shown in Table 19, where the IL-15Rβ-based blocking peptide is shown in bold, the cleavable linker is shown in wavy underlining, and the spacer peptides are shown in straight underlining. IL-15RαSushi+ (SEQ ID NO: 5) was coexpressed and a non-covalent complex was formed with the IL-15 fusion protein containing the blocking peptide.

Gene synthesis, expression vector construction, and protein production, purification, and characterization were performed following the same procedures as detailed in Example 1. These IL-15 fusion proteins containing blocking peptides were first tested in an ELISA assay to assess their binding ability to IL-2Rβ. As shown in FIG. 43, compared to P-0153, the binding affinity was moderately reduced due to association with various blocking peptides. However, an PMBC assay assessing the activation of immune cells, including CD56+ NK cells or CD8+ T cells, by these IL-15 fusion proteins containing blocking peptides did not show a significant decrease in activity (data not shown), indicating insufficient masking efficiency of the analyzed blocking peptides. Blocking peptides of varying lengths, including the full-length extracellular domain of IL-2Rβ, are being studied and are expected to have greater efficacy in masking activity. A similar approach could be applied to IL-2.

Example 24

VitoKine Format Expands Possibilities for Developing Fusion Proteins

It is known in the art that the naturally occurring IL-2 protein tends to be not very stable and is prone to aggregation. In experiments conducted by the inventors, it was demonstrated that a fusion protein of wild-type IL-2 and Fc (P-0250), expressed at a low level (approximately 3 mg/L, transiently, in HEK-293F cells), has a high propensity to aggregate, as exemplified by the SEC chromatogram shown in FIG. 44A. Four VitoKine IL-2 molecules, P-0320, P-0382, P-0362, and P-0379, were compared with P-0250. P-0320 (SEQ ID NO: 49) contains the wild-type IL-2 domain, the N-terminus of which is fused to the Fc domain, and the C-terminus is fused to the IL-2RαSushi domain. The L1 linker connecting Fc and IL-2 is a cleavable linker containing the uPA substrate peptide and flanking spacer peptides (SEQ ID NO: 92), and the L2 linker between IL-2 and IL-2RαSushi is a flexible linker (GGGGS) ₃ (SEQ ID NO: 127). P-0382 (SEQ ID NO: 51) differs from P-0320 only in the linker sequences; the L1 linker of P-0382 is a flexible linker (G ₃ S) ₂ (SEQ ID NO: 115), and the L2 linker is a linker cleavable by MMP-2/9 (SEQ ID NO: 95). P-0362 (SEQ ID NO:) and P-0379 (SEQ ID NO:59) differ from P-382 by one point mutation. P-0362 contains a K38E mutation in the IL-2RαSushi domain, while P-0379 contains an F42A substitution in the IL-2 domain. P-0250 (SEQ ID NO:48) is an IL-2 and Fc fusion protein, where IL-2 is fused to the C-terminus of Fc using a flexible linker ( _G3S ) ₂ (SEQ ID NO:115).

The exclusion diagrams of these five molecules are shown in FIGS. 33A–33E. The chromatograms show that all four VitoKine IL-2 constructs have significantly better purity profiles than the IL-2–Fc fusion protein. P-0250 contains more than 25% of unwanted high-molecular-weight species. In contrast, all four VitoKine molecules exhibit sharp monomer peaks with monomer contents greater than 96%. Linker changes, mutations in IL-2, or IL-2RαSushi did not affect quality. This significant increase in protein quality was likely due to the fusion of the IL-2αSushi domain to the VitoKine construct.

In addition to protein quality, the expression level of VitoKine IL-2 constructs was also increased, particularly in the VitoKine format with a GS linker between Fc and IL-2 and a 10-amino acid linker activated by MMP-2/9 between IL-2 and IL-2RαSushi. Although protein expression levels may vary between batches due to cell growth conditions, it is clear that the expression level of VitoKine constructs consistently exceeds the expression level of the IL-2 and Fc fusion protein by several times. Table 20 lists protein expression titers in mg/L and the percentage of protein monomer.

Furthermore, the inventors' efforts to develop IL-2 constructs also led to the discovery of a single amino acid substitution of serine to isoleucine at position 125, which provided a versatile expansion of the design possibilities for IL-2-Fc fusion constructs with full preservation of biological activity. The substitution of isoleucine at position 125 in wild-type IL-2 and IL-2 variants with different mutational contexts in the Fc fusion format in all cases resulted in a 4- to 11-fold increase in expression levels and a similarly low aggregation propensity. Summary data on the expression level in mg/L and the purity of the protein A-purified substance, assessed by SEC chromatography, as a percentage of aggregation of typical molecules are presented in Table 21. Two molecules in the same row of Table 21 have the same other amino acid substitution(s) and differ only in residue 125, which is serine or isoleucine. As an example, FIG. 44F also shows an SEC chromatogram of P-0531, a P-250 analog molecule with IL-2 S125I.

In conclusion, the VitoKine platform significantly expanded protein development capabilities, as demonstrated by increased protein expression and a significant reduction in the aggregation propensity of VitoKine IL-2 and Fc constructs. Furthermore, VitoKine IL-2 constructs (wild-type or variants) containing the beneficial S125I amino acid in IL-2 will provide even broader development options.

Example 25

The choice of the D3 domain of the VitoKine construct can greatly influence protein expression

The VitoKine platform was also studied with D3 domains, which are variants of the cognate receptor D2 domain or a domain from an unrelated protein. Based on crystallographic analysis (Wang et al., Science 310: 1159–1163, 2005), sushi domains 1 and 2 of IL-2Rα are involved in chain exchange, resulting in residues 1–19 of IL-2Rα being part of sushi domain 2 and residues 102–122 being part of sushi domain 1. This structural arrangement was reproduced in the IL-2RαSushi variant (SEQ ID NO: 147), which contains residues 102–122 of IL-2Rα (SEQ ID NO: 10) at the N-terminus and residues 20–68 of IL2Rα at the C-terminus. This IL-2RαSushi variant contains most of the residues that interact with IL-2 and is expected to exhibit most of the activity while maintaining structural integrity. Replacing the IL-2RαSushi domain in P-0320 (SEQ ID NO: 49) with the IL-2RαSushi variant resulted in the IL-2-containing VitoKine construct P-0321 (SEQ ID NO: 179). Surprisingly, P-0321, which contained the IL-2RαSushi variant as the D3 domain, was not expressible at all or was expressed at such a low level that its isolation and purification were not possible.

Similarly, the IL-15αSushi+ domain in the VitoKine P-0315 construct (SEQ ID NO: 33) was replaced with IL-2RαSushi (SEQ ID NO: 10), and the resulting protein was P-0389 (SEQ ID NO: 42). The expression level of P-0389 was significantly lower than the expression level of P-0315. More notably, P-0389 was mainly represented by high molecular weight aggregates, as demonstrated by the SDS-PAGE gel image shown in FIG. 45A. For comparison, an SDS-PAGE gel image with a similar molecule of P-0315 is shown in FIG. Furthermore, purified P-0389 was resistant to MMP-2 cleavage despite the presence of the MMP-2/9 substrate peptide in its sequence, suggesting misfolding of the molecule or limited accessibility to the protease due to aggregation.

In conclusion, D3 is a critical component of VitoKine constructs. Besides acting as a masking moiety, it can have a profound impact on protein design, both positively and negatively.

In light of the present invention, all articles and methods disclosed and claimed herein can be made and carried out without undue experimentation. Although the articles and methods of the present invention have been described by means of preferred embodiments, it will be clear to those skilled in the art that said articles and methods can be modified without departing from the spirit and scope of the invention. It is intended that all such modified variants and equivalents, obvious to those skilled in the art, whether existing at the time of filing of this application or later developed, are included within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The invention illustratively described herein can be suitably practiced in the absence of any element(s) not specifically disclosed herein. It should therefore be understood that, although the present invention has been particularly disclosed through preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may occur to those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

List of sequences

The nucleic acid sequences and amino acid sequences shown in the appended sequence listing are shown using standard nucleotide base abbreviations and three-letter amino acid codes as defined in 37 C.F.R. 1.822.

SEQ ID NO: 1 is the amino acid sequence of the human IL-15 precursor.

SEQ ID NO: 2 is the amino acid sequence of the mature form of human IL-15.

SEQ ID NO: 3 is the amino acid sequence of the IL-15 variant polypeptide.

SEQ ID NO: 4 is the amino acid sequence of human IL-15R.

SEQ ID NO: 5 is the amino acid sequence of the sushi+ domain of human IL-15R.

SEQ ID NO: 6 is the amino acid sequence of the human IL-2 precursor.

SEQ ID NO: 7 is the naturally occurring amino acid sequence of the mature form of human IL-2.

SEQ ID NO: 8 is the amino acid sequence of the mature form of wild-type human IL-2.

SEQ ID NO: 9 is the amino acid sequence of human IL-2R precursor (CD25).

SEQ ID NO: 10 is the amino acid sequence of the sushi domain of human IL-2R.

SEQ ID NO: ll is the amino acid sequence of human IL-2R S1251.

SEQ ID NO: 12 is the amino acid sequence of the extracellular domain of human IL-2Rβ.

SEQ ID NO: 13 is the amino acid sequence of Fc of human IgG1.

SEQ ID NO: 14 is the Fc sequence of human IgG1 with reduced/abstracted effector function. SEQ ID NO: 15 is the amino acid sequence of Knob-Fc.

SEQ ID NO: 16 is the amino acid sequence of Hole-Fc.

SEQ ID NO: 17 is the amino acid sequence of the mature form of human IL-4.

SEQ ID NO: 18 is the amino acid sequence of the mature form of human IL-7.

SEQ ID NO: 19 is the amino acid sequence of the mature form of human IL-9.

SEQ ID NO: 20 is the amino acid sequence of the mature form of human IL-10.

SEQ ID NO: 21 is the sequence of the mature form of the alpha subunit of human IL-12.

SEQ ID NO: 22 is the sequence of the mature form of the beta subunit of human IL-12.

SEQ ID NO: 23 is the sequence of the mature form of the alpha subunit of human IL-23.

SEQ ID NO: 24 is the sequence of the mature form of the beta subunit of human IL-27.

SEQ ID NOs: 25-43 are the amino acid sequences of various VitoKine constructs with IL-15 and Fc.

SEQ ID NO: 44 is the amino acid sequence of the Hole-Fc-IL-15 fusion protein.

SEQ ID NO: 45 is the amino acid sequence of the Knob-Fc-IL-15 fusion protein.

SEQ ID NO: 46 is the amino acid sequence of the Knob-Fc-IL-15RSushi+ fusion protein.

SEQ ID NO: 47 is the amino acid sequence of the Fc-IL-15 S58D fusion protein.

SEQ ID NO: 48 is the amino acid sequence of the IL-2 fusion protein.

SEQ ID NOs: 49-65 are the amino acid sequences of various VitoKine constructs with IL-2 and Fc.

SEQ ID NO: 66-70 are the amino acid sequences of various IL-15 constructs containing the blocking peptide.

SEQ ID NOs: 71-87 and 157-159 are the amino acid sequences of various protease substrate peptides.

SEQ ID NOs: 88-96, 160-161 and 298 are the amino acid sequences of various protease cleavable linkers containing various spacer peptides flanking protease substrate peptides.

SEQ ID NOs: 97-106 are the amino acid sequences of various blocking peptide sequences.

SEQ ID NOs: 107-127 are the amino acid sequences of various non-cleavable linker sequences.

SEQ ID NOs: 128-146 are the amino acid sequences of various VitoKine antibody constructs.

SEQ ID NO: 147 is the sequence of a human IL-2R variant.

SEQ ID NOs: 148-149 are the amino acid sequences of the Hole-Fc-IL-15 fusion constructs.

SEQ ID NO: 150-155 are the amino acid sequences of various VitoKine constructs with IL-2 and Fc.

SEQ ID NO: 156 is the Fc sequence of human IgG1 with reduced/abstracted effector function and increased half-life.

SEQ ID NOs: 162-165 are the amino acid sequences of various VitoKine constructs with IL-15 and Fc.

SEQ ID NO: 166 is a human IgG1 Fc sequence with reduced/abstracted effector function and increased half-life.

SEQ ID NO: 167 is the amino acid sequence of Knob-Fc with extended half-life.

SEQ ID NO: 168 is the amino acid sequence of Hole-Fc with extended half-life.

SEQ ID NOs: 169-174 are the amino acid sequences of various VitoKine constructs with IL-15 and Fc.

SEQ ID NOs: 175-178 are the amino acid sequences of various IL-15 Fc fusion constructs.

SEQ ID NO: 179 is the amino acid sequence of the VitoKine IL-2 Fc construct.

SEQ ID NO: 180-181 are the amino acid sequences of various VitoKine antibody constructs with IL-15.

SEQ ID NO: 182-192 represent the amino acid sequences of polypeptides of various IL-15 variants containing amino acid deletions, insertions and/or substitutions.

SEQ ID NO: 193-194 are the amino acid sequences of the heavy chain and light chain of the humanized anti-FAP antibody.

SEQ ID NO: 195-196 are the amino acid sequences of the heavy chain and light chain of the humanized PD-1 antagonist antibody.

SEQ ID NOs: 197-198 are the amino acid sequences of the heavy chain and light chain of the human PD-1 antagonist antibody.

SEQ ID NOs: 199-215 are the amino acid sequences of polypeptides of various IL-15 variants.

SEQ ID NOs: 216-229 are the amino acid sequences of fusion constructs of various IL-15 and Fc variants.

SEQ ID NO: 230-231 are the amino acid sequences of various VitoKine constructs with IL-15 and Fc.

SEQ ID NO: 232-247 are the amino acid sequences of polypeptides of various IL-2 variants.

SEQ ID NO: 248-266 are the amino acid sequences of fusion constructs of various IL-2 and Fc variants.

SEQ ID NOs: 267-270 are the amino acid sequences of polypeptides of various variants of the sushi domain of human IL-2R.

SEQ ID NOs: 271-274 and 292-295 are the amino acid sequences of various VitoKine constructs with IL-2 and Fc.

SEQ ID NO: 275-276 are the amino acid sequences of the heavy chain and light chain of the PD-1 antagonist antibody.

SEQ ID NO: 277-278 are the amino acid sequences of the heavy chain and light chain of the PD-1 antagonist antibody.

SEQ ID NO: 279-280 are the amino acid sequences of the heavy chain and light chain of the PD-L1 antagonist antibody.

SEQ ID NOs: 281-291 are the amino acid sequences of various VitoKine IL-15/IL-2 antibody constructs.

SEQ ID NO: 296 is the amino acid sequence of the Knob HC chain of the PD-1 antagonist antibody fused to an IL-2 variant

SEQ ID NO: 297 is the amino acid sequence of the PD-1 antagonist antibody HC fused to an IL-15 variant.

SEQ ID NO: 299-302 are the amino acid sequences of the anti-mouse PD-1 surrogate antibody with homodimeric or heterodimeric heavy chains.

SEQ ID NO: 303-306 are the amino acid sequences of various VitoKine IL-15 antibody constructs.

SEQUENCE LISTING Sequence of the human IL-15 precursor

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SEQUENCE LIST

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85 90 95

Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro

100 105 110

Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro

115 120 125

Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val

130 135 140

Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His

145 150 155 160

Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg

165 170 175

Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln

180 185 190

Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu

195 200 205

Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr

210 215 220

Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln

225 230 235 240

Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu

245 250 255

Ser Gly Leu Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile

260 265 270

<210> 10

<211> 165

<212> PRT

<213> Homo sapiens

<400> 10

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly

165

<210> 11

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 S125I sequence

<400> 11

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 12

<211> 214

<212> PRT

<213> Homo sapiens

<400> 12

Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala

1 5 10 15

Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser

20 25 30

Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys

35 40 45

Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu

50 55 60

Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu

65 70 75 80

Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln

85 90 95

Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu

100 105 110

Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile

115 120 125

Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg

130 135 140

Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu

145 150 155 160

Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr

165 170 175

Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr

180 185 190

Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala

195 200 205

Ala Leu Gly Lys Asp Thr

210

<210> 13

<211> 226

<212> PRT

<213> Homo sapiens

<400> 13

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 14

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Human IgG1 Fc with reduced/eliminated effector

function

<400> 14

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 15

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Knob-Fc human IgG1 with reduced/eliminated effector

function

<400> 15

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 16

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc human IgG1 with reduced/eliminated effector

function

<400> 16

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 17

<211> 129

<212> PRT

<213> Homo sapiens

<400> 17

His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser

1 5 10 15

Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile

20 25 30

Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala

35 40 45

Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg

50 55 60

Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile

65 70 75 80

Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu

85 90 95

Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe

100 105 110

Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser

115 120 125

Ser

<210> 18

<211> 152

<212> PRT

<213> Homo sapiens

<400> 18

Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu

1 5 10 15

Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

115 120 125

Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135 140

Ile Leu Met Gly Thr Lys Glu His

145 150

<210> 19

<211> 126

<212> PRT

<213> Homo sapiens

<400> 19

Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile Asn Phe Leu Ile

1 5 10 15

Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys Ser Ala Asn

20 25 30

Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp Asn Cys Thr Arg

35 40 45

Pro Cys Phe Ser Glu Arg Leu Ser Gln Met Thr Asn Thr Thr Met Gln

50 55 60

Thr Arg Tyr Pro Leu Ile Phe Ser Arg Val Lys Lys Ser Val Glu Val

65 70 75 80

Leu Lys Asn Asn Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro Cys Asn

85 90 95

Gln Thr Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu

100 105 110

Ile Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile

115 120 125

<210> 20

<211> 160

<212> PRT

<213> Homo sapiens

<400> 20

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

1 5 10 15

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

65 70 75 80

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

85 90 95

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

145 150 155 160

<210> 21

<211> 197

<212> PRT

<213> Homo sapiens

<400> 21

Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu

1 5 10 15

His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys

20 25 30

Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp

35 40 45

His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu

50 55 60

Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr

65 70 75 80

Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe

85 90 95

Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr

100 105 110

Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys

115 120 125

Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu

130 135 140

Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser

145 150 155 160

Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu

165 170 175

Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser

180 185 190

Tyr Leu Asn Ala Ser

195

<210> 22

<211> 306

<212> PRT

<213> Homo sapiens

<400> 22

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

Cys Ser

305

<210> 23

<211> 170

<212> PRT

<213> Homo sapiens

<400> 23

Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gln Cys Gln Gln

1 5 10 15

Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His Pro Leu Val

20 25 30

Gly His Met Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr Thr Asn Asp

35 40 45

Val Pro His Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln Gly Leu Arg

50 55 60

Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly Leu Ile Phe

65 70 75 80

Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu Pro Ser Leu

85 90 95

Leu Pro Asp Ser Pro Val Gly Gln Leu His Ala Ser Leu Leu Gly Leu

100 105 110

Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu Thr Gln Gln Ile

115 120 125

Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu Leu Arg Phe

130 135 140

Lys Ile Leu Arg Ser Leu Gln Ala Phe Val Ala Val Ala Ala Arg Val

145 150 155 160

Phe Ala His Gly Ala Ala Thr Leu Ser Pro

165 170

<210> 24

<211> 112

<212> PRT

<213> Homo sapiens

<400> 24

Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys

1 5 10 15

Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp

20 25 30

Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys

35 40 45

Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu

50 55 60

Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro

65 70 75 80

Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro

85 90 95

Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser

100 105 110

<210> 25

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0351 (VitoKine construct with IL-15 and Fc)

<400> 25

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 26

<211> 432

<212> PRT

<213> Artificial

<220>

<223> Hole chain P-0170 (VitoKine constructs with IL-15 and Fc)

<400> 26

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu Asn

225 230 235 240

Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln

245 250 255

Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro

260 265 270

Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val

275 280 285

Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn

290 295 300

Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr

305 310 315 320

Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys

325 330 335

Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr

340 345 350

Ser Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp

355 360 365

Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys

370 375 380

Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys

385 390 395 400

Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu

405 410 415

Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

420 425 430

<210> 27

<211> 432

<212> PRT

<213> Artificial

<220>

<223> P-0172 (VitoKine construct with IL-15 and Fc)

<400> 27

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu Asn

225 230 235 240

Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln

245 250 255

Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro

260 265 270

Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val

275 280 285

Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn

290 295 300

Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr

305 310 315 320

Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys

325 330 335

Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr

340 345 350

Ser Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp

355 360 365

Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys

370 375 380

Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys

385 390 395 400

Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu

405 410 415

Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

420 425 430

<210> 28

<211> 450

<212> PRT

<213> Artificial

<220>

<223> P-0202 (VitoKine construct with IL-15 and Fc)

<400> 28

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Leu Ser Gly Arg Ser Asp Asn His Gly Gly Ser Gly Gly Gly Gly

245 250 255

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

260 265 270

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

275 280 285

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

290 295 300

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

305 310 315 320

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

325 330 335

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

340 345 350

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

355 360 365

Asn Thr Ser Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His

370 375 380

Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr

385 390 395 400

Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr

405 410 415

Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro

420 425 430

Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala

435 440 445

Pro Pro

450

<210> 29

<211> 467

<212> PRT

<213> Artificial

<220>

<223> P-0203 (VitoKine construct with IL-15 and Fc)

<400> 29

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser

225 230 235 240

Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys

260 265 270

Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr

275 280 285

Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe

290 295 300

Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile

305 310 315 320

His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser

325 330 335

Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu

340 345 350

Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val

355 360 365

Gln Met Phe Ile Asn Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu

385 390 395 400

His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg

405 410 415

Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu

420 425 430

Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr

435 440 445

Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro

450 455 460

Ala Pro Pro

465

<210> 30

<211> 460

<212> PRT

<213> Artificial

<220>

<223> P-0204 (VitoKine construct with IL-15 and Fc)

<400> 30

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gly Gly Ser

225 230 235 240

Gly Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly Gly Gly Ser Asn Trp

245 250 255

Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser

260 265 270

Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser

275 280 285

Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile

290 295 300

Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu

305 310 315 320

Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu

325 330 335

Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu

340 345 350

Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

355 360 365

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile

370 375 380

Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys

385 390 395 400

Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe

405 410 415

Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys

420 425 430

Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

435 440 445

Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

450 455 460

<210> 31

<211> 455

<212> PRT

<213> Artificial

<220>

<223> P-0205 (VitoKine construct with IL-15 and Fc)

<400> 31

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gly Gly Ser

225 230 235 240

Gly Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly Gly Gly Ser Asn Trp

245 250 255

Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser

260 265 270

Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser

275 280 285

Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile

290 295 300

Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu

305 310 315 320

Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu

325 330 335

Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu

340 345 350

Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

355 360 365

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro

370 375 380

Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr

385 390 395 400

Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly

405 410 415

Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala

420 425 430

His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val

435 440 445

His Gln Arg Pro Ala Pro Pro

450 455

<210> 32

<211> 450

<212> PRT

<213> Artificial

<220>

<223> P-0206 (VitoKine construct with IL-15 and Fc)

<400> 32

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gly Gly Ser

225 230 235 240

Gly Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly Gly Gly Ser Asn Trp

245 250 255

Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser

260 265 270

Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser

275 280 285

Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile

290 295 300

Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu

305 310 315 320

Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu

325 330 335

Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu

340 345 350

Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

355 360 365

Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His

370 375 380

Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr

385 390 395 400

Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr

405 410 415

Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro

420 425 430

Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala

435 440 445

Pro Pro

450

<210> 33

<211> 448

<212> PRT

<213> Artificial

<220>

<223> P-0315 (VitoKine construct with IL-15 and Fc)

<400> 33

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu

245 250 255

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

260 265 270

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

275 280 285

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

290 295 300

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

305 310 315 320

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

325 330 335

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

340 345 350

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu

355 360 365

Ser Gln Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp

370 375 380

Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys

385 390 395 400

Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys

405 410 415

Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu

420 425 430

Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440 445

<210> 34

<211> 451

<212> PRT

<213> Artificial

<220>

<223> P-0316 (VitoKine construct with IL-15 and Fc)

<400> 34

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu

245 250 255

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

260 265 270

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

275 280 285

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

290 295 300

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

305 310 315 320

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

325 330 335

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

340 345 350

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Gly Pro Leu Gly Met

355 360 365

Leu Ser Gln Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu

370 375 380

His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg

385 390 395 400

Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu

405 410 415

Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr

420 425 430

Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro

435 440 445

Ala Pro Pro

450

<210> 35

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0350 (VitoKine construct with IL-15 and Fc)

<400> 35

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 36

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0354 (VitoKine construct with IL-15 and Fc)

<400> 36

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 37

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0355 (VitoKine construct with IL-15 and Fc)

<400> 37

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 38

<211> 446

<212> PRT

<213> Artificial

<220>

<223> P-0385 (VitoKine construct with IL-15 and Fc)

<400> 38

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu

245 250 255

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

260 265 270

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

275 280 285

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

290 295 300

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

305 310 315 320

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

325 330 335

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

340 345 350

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Pro Leu Gly Met Leu Ser

355 360 365

Gln Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp

370 375 380

Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser

385 390 395 400

Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu

405 410 415

Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys

420 425 430

Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440 445

<210> 39

<211> 448

<212> PRT

<213> Artificial

<220>

<223> P-0386 (VitoKine construct with IL-15 and Fc)

<400> 39

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu

245 250 255

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

260 265 270

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

275 280 285

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

290 295 300

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

305 310 315 320

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

325 330 335

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

340 345 350

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu

355 360 365

Ser Gln Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp

370 375 380

Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Ser

385 390 395 400

Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys

405 410 415

Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu

420 425 430

Lys Ser Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440 445

<210> 40

<211> 448

<212> PRT

<213> Artificial

<220>

<223> P-0387 (VitoKine construct with IL-15 and Fc)

<400> 40

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu

245 250 255

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

260 265 270

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

275 280 285

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

290 295 300

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

305 310 315 320

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

325 330 335

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

340 345 350

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu

355 360 365

Ser Gln Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp

370 375 380

Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Glu Tyr Ile Cys

385 390 395 400

Asn Ser Gly Phe Lys Glu Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys

405 410 415

Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu

420 425 430

Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440 445

<210> 41

<211> 433

<212> PRT

<213> Artificial

<220>

<223> P-0388 (VitoKine construct with IL-15 and Fc)

<400> 41

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu

245 250 255

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

260 265 270

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

275 280 285

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

290 295 300

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

305 310 315 320

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

325 330 335

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

340 345 350

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu

355 360 365

Ser Gln Ser Asp Cys Gly Leu Pro Pro Asp Val Pro Asn Ala Gln Pro

370 375 380

Ala Leu Glu Gly Arg Thr Ser Phe Pro Glu Asp Thr Val Ile Thr Tyr

385 390 395 400

Lys Cys Glu Glu Ser Phe Val Lys Ile Pro Gly Glu Lys Asp Ser Val

405 410 415

Ile Cys Leu Lys Gly Ser Gln Trp Ser Asp Ile Glu Glu Phe Cys Asn

420 425 430

Arg

<210> 42

<211> 536

<212> PRT

<213> Artificial

<220>

<223> P-0389 (VitoKine construct with IL-15 and Fc)

<400> 42

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu

245 250 255

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

260 265 270

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

275 280 285

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

290 295 300

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

305 310 315 320

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

325 330 335

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

340 345 350

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu

355 360 365

Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala

370 375 380

Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu

385 390 395 400

Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu

405 410 415

Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys

420 425 430

Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro

435 440 445

Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln

450 455 460

Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro

465 470 475 480

Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln

485 490 495

Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly

500 505 510

Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr

515 520 525

Gln Pro Gln Leu Ile Cys Thr Gly

530 535

<210> 43

<211> 443

<212> PRT

<213> Artificial

<220>

<223> P-0397 (VitoKine construct with IL-15 and Fc)

<400> 43

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Gly Gly Pro

65 70 75 80

Leu Gly Met Leu Ser Gln Ser Asn Trp Val Asn Val Ile Ser Asp Leu

85 90 95

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

100 105 110

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

115 120 125

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

130 135 140

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

145 150 155 160

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

165 170 175

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

180 185 190

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Gly Gly Ser Leu Gly

195 200 205

Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly Ser Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 44

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc-IL-15

<400> 44

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser

225 230 235 240

Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 45

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Knob-Fc-IL-15

<400> 45

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser

225 230 235 240

Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 46

<211> 318

<212> PRT

<213> Artificial

<220>

<223> Knob-Fc-IL-15RαSushi+

<400> 46

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser

225 230 235 240

Pro Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp

245 250 255

Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser

260 265 270

Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu

275 280 285

Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys

290 295 300

Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

305 310 315

<210> 47

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 S58D

<400> 47

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 48

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0250 (IL-2-Fc fusion protein)

<400> 48

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 49

<211> 560

<212> PRT

<213> Artificial

<220>

<223> P-0320 (VitoKine construct with IL-2 and Fc)

<400> 49

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys

245 250 255

Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu

260 265 270

Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr

275 280 285

Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln

290 295 300

Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala

305 310 315 320

Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile

325 330 335

Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys

340 345 350

Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp

355 360 365

Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Cys Asp Asp

385 390 395 400

Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys

405 410 415

Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile

420 425 430

Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser

435 440 445

Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr

450 455 460

Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr

465 470 475 480

Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro

485 490 495

Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg

500 505 510

Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln

515 520 525

Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met

530 535 540

Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

545 550 555 560

<210> 50

<211> 560

<212> PRT

<213> Artificial

<220>

<223> P-0352 (VitoKine construct with IL-2 and Fc)

<400> 50

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys

245 250 255

Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu

260 265 270

Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr

275 280 285

Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln

290 295 300

Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala

305 310 315 320

Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile

325 330 335

Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys

340 345 350

Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp

355 360 365

Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Cys Asp Asp

385 390 395 400

Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys

405 410 415

Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile

420 425 430

Glu Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser

435 440 445

Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr

450 455 460

Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr

465 470 475 480

Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro

485 490 495

Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg

500 505 510

Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln

515 520 525

Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met

530 535 540

Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

545 550 555 560

<210> 51

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0382 (VitoKine construct with IL-2 and Fc)

<400> 51

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 52

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0398 (VitoKine construct with IL-2 and Fc)

<400> 52

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 53

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0362 (VitoKine construct with IL-2 and Fc)

<400> 53

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 54

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0380 (VitoKine construct with IL-2 and Fc)

<400> 54

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

405 410 415

Gly Ser Leu Ala Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 55

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0384 (VitoKine construct with IL-2 and Fc)

<400> 55

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser

405 410 415

Gly Ser Leu Ala Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 56

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0400 (VitoKine construct with IL-2 and Fc)

<400> 56

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Asn Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 57

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0404 (VitoKine construct with IL-2 and Fc)

<400> 57

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Glu Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 58

<211> 454

<212> PRT

<213> Artificial

<220>

<223> P-0399 (VitoKine construct with IL-2 and Fc)

<400> 58

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys Pro Pro Pro Met

370 375 380

Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser

385 390 395 400

Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr

405 410 415

Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His

420 425 430

Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His

435 440 445

Gln Arg Pro Ala Pro Pro

450

<210> 59

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0379 (VitoKine construct with IL-2 and Fc)

<400> 59

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 60

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0381 (VitoKine construct with IL-2 and Fc)

<400> 60

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Glu

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 61

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0383 (VitoKine construct with IL-2 and Fc)

<400> 61

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Glu

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 62

<211> 555

<212> PRT

<213> Artificial

<220>

<223> P-0329 (VitoKine construct with IL-2 and Fc)

<400> 62

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

165 170 175

Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

180 185 190

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

195 200 205

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

210 215 220

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

225 230 235 240

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

245 250 255

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

260 265 270

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

275 280 285

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

290 295 300

Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Leu Gly

305 310 315 320

Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly Ser Cys Pro

325 330 335

Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe

340 345 350

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

355 360 365

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

370 375 380

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

385 390 395 400

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

405 410 415

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

420 425 430

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

435 440 445

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

450 455 460

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

465 470 475 480

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

485 490 495

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

500 505 510

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

515 520 525

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

530 535 540

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

545 550 555

<210> 63

<211> 549

<212> PRT

<213> Artificial

<220>

<223> P-0401 (VitoKine construct with IL-2 and Fc)

<400> 63

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ala

165 170 175

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

180 185 190

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

195 200 205

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

210 215 220

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

225 230 235 240

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

245 250 255

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

260 265 270

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

275 280 285

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

290 295 300

Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu

325 330 335

Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

340 345 350

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

355 360 365

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

370 375 380

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

385 390 395 400

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

405 410 415

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

420 425 430

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

435 440 445

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

450 455 460

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

465 470 475 480

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

485 490 495

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

500 505 510

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

515 520 525

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

530 535 540

Ser Leu Ser Pro Gly

545

<210> 64

<211> 554

<212> PRT

<213> Artificial

<220>

<223> P-0402 (VitoKine construct with IL-2 and Fc)

<400> 64

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly Gly Gly Ser Gly Pro Leu Gly Met Leu Ser Gln

165 170 175

Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

180 185 190

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

195 200 205

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

210 215 220

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

225 230 235 240

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

245 250 255

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

260 265 270

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

275 280 285

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

290 295 300

Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro

325 330 335

Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro

340 345 350

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

355 360 365

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

370 375 380

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

385 390 395 400

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

405 410 415

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

420 425 430

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

435 440 445

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

450 455 460

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

465 470 475 480

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

485 490 495

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

500 505 510

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

515 520 525

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

530 535 540

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

545 550

<210> 65

<211> 546

<212> PRT

<213> Artificial

<220>

<223> P-0403 (VitoKine construct with IL-2 and Fc)

<400> 65

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ala

165 170 175

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

180 185 190

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

195 200 205

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

210 215 220

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

225 230 235 240

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

245 250 255

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

260 265 270

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

275 280 285

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

290 295 300

Ser Thr Leu Thr Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

305 310 315 320

Ala Ala Ala Lys Ala Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

325 330 335

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

340 345 350

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

355 360 365

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

370 375 380

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

385 390 395 400

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

405 410 415

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

420 425 430

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

435 440 445

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

450 455 460

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

465 470 475 480

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

485 490 495

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

500 505 510

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

515 520 525

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

530 535 540

Pro Gly

545

<210> 66

<211> 405

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc-15p1

<400> 66

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser

225 230 235 240

Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

355 360 365

Ala Ile Leu Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

370 375 380

Ile Tyr Asn Cys Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His

385 390 395 400

Leu Cys Tyr Ser Ile

405

<210> 67

<211> 407

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc-15p2

<400> 67

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser

225 230 235 240

Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

355 360 365

Ala Ile Leu Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

370 375 380

Ile Tyr Asn Cys Glu Leu His Arg Glu Phe Tyr His Ser Ala Gln Ser

385 390 395 400

Ile Glu Trp Cys Tyr Ser Ile

405

<210> 68

<211> 417

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc-15p3

<400> 68

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser

225 230 235 240

Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

355 360 365

Ala Ile Leu Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

370 375 380

Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His

385 390 395 400

Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr Leu Cys Pro Gly

405 410 415

His

<210> 69

<211> 386

<212> PRT

<213> Artificial

<220>

<223> p1'-15-Fc

<400> 69

Gln Gly Gln Ser Gly Gln Cys Glu Ile Ser Gln Ala Ser His Tyr Phe

1 5 10 15

Glu Arg His Leu Cys Tyr Ser Ile Gly Ser Ser Gly Gly Ser Gly Gly

20 25 30

Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser

35 40 45

Gly Thr Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp

50 55 60

Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp

65 70 75 80

Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu

85 90 95

Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr

100 105 110

Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly

115 120 125

Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys

130 135 140

Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe

145 150 155 160

Ile Asn Thr Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

165 170 175

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

180 185 190

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

195 200 205

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

210 215 220

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

225 230 235 240

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

245 250 255

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

260 265 270

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

275 280 285

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

290 295 300

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

305 310 315 320

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

325 330 335

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

340 345 350

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

355 360 365

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

370 375 380

Pro Gly

385

<210> 70

<211> 397

<212> PRT

<213> Artificial

<220>

<223> p3'-15-Fc

<400> 70

Gln Gly Gln Ser Gly Gln Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala

1 5 10 15

Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr Leu Cys

20 25 30

Pro Gly His Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

35 40 45

Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser Gly Thr Asn Trp Val

50 55 60

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

65 70 75 80

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

85 90 95

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

100 105 110

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

115 120 125

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

130 135 140

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

145 150 155 160

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Gly

165 170 175

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

180 185 190

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

195 200 205

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

210 215 220

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

225 230 235 240

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

245 250 255

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

260 265 270

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

275 280 285

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

290 295 300

Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

305 310 315 320

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

325 330 335

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

340 345 350

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

355 360 365

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

370 375 380

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

385 390 395

<210> 71

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 71

Ser Pro Leu Gly Leu Ala Gly Ser

1 5

<210> 72

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 72

Glu Pro Leu Glu Leu Arg Ala Gly

1 5

<210> 73

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 73

Leu Ser Gly Arg Ser Asp Asn His

1 5

<210> 74

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 74

Gly Pro Leu Gly Ile Ala Gly Gln

1 5

<210> 75

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 75

Gly Thr Ala His Leu Met Gly Gly

1 5

<210> 76

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 76

Arg Ile Gly Ser Leu Arg Thr Ala

1 5

<210> 77

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 77

Gly Pro Leu Gly Met Leu Ser Gln

1 5

<210> 78

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 78

Arg Pro Ser Ala Ser Arg Ser Ala

1 5

<210> 79

<211> 6

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 79

Pro Leu Gly Leu Ala Gly

1 5

<210> 80

<211> 13

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 80

Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu

1 5 10

<210> 81

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 81

Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile

1 5 10

<210> 82

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 82

Ser Gly Arg Ser Ala

1 5

<210> 83

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 83

Ala Ala Asn Leu

1

<210> 84

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 84

Gly Phe Phe Tyr

1

<210> 85

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 85

Gly Pro Ile Cys Phe Arg Leu Gly

1 5

<210> 86

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 86

Arg Gln Ala Gly Phe Ser Leu

1 5

<210> 87

<211> 6

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 87

His Ser Ser Lys Leu Gln

1 5

<210> 88

<211> 30

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 88

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Ser

1 5 10 15

Gly Arg Ser Asp Asn His Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 89

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 89

Gly Ser Ser Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala Gly Thr

1 5 10 15

<210> 90

<211> 35

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 90

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu Gly

1 5 10 15

Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser

35

<210> 91

<211> 28

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 91

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg

1 5 10 15

Ser Ala Asn Ala Ile Leu Glu Gly Gly Gly Gly Ser

20 25

<210> 92

<211> 21

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 92

Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile

1 5 10 15

Leu Glu Gly Gly Ser

20

<210> 93

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 93

Gly Gly Gly Ser Gly Pro Thr Asn Lys Val Arg Gly Gly Ser

1 5 10

<210> 94

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 94

Gly Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser

1 5 10 15

<210> 95

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 95

Gly Gly Pro Leu Gly Met Leu Ser Gln Ser

1 5 10

<210> 96

<211> 13

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 96

Gly Gly Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Ser

1 5 10

<210> 97

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 97

Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val

1 5 10 15

<210> 98

<211> 13

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 98

Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu

1 5 10

<210> 99

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 99

Cys Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Cys

1 5 10 15

<210> 100

<211> 36

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 100

Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Gly

1 5 10 15

Gly Gly Gly Gly Gly Gly Gly Glu Ile Ser Gln Ala Ser His Tyr Phe

20 25 30

Glu Arg His Leu

35

<210> 101

<211> 13

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 101

Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser

1 5 10

<210> 102

<211> 50

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 102

Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu

1 5 10 15

Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ile Tyr Asn

20 25 30

Cys Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Cys Tyr

35 40 45

Ser Ile

50

<210> 103

<211> 52

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 103

Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu

1 5 10 15

Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ile Tyr Asn

20 25 30

Cys Glu Leu His Arg Glu Phe Tyr His Ser Ala Gln Ser Ile Glu Trp

35 40 45

Cys Tyr Ser Ile

50

<210> 104

<211> 62

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 104

Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu

1 5 10 15

Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Thr His

20 25 30

Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu

35 40 45

Arg His Leu Glu Phe Glu Ala Arg Thr Leu Cys Pro Gly His

50 55 60

<210> 105

<211> 50

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 105

Gln Gly Gln Ser Gly Gln Cys Glu Ile Ser Gln Ala Ser His Tyr Phe

1 5 10 15

Glu Arg His Leu Cys Tyr Ser Ile Gly Ser Ser Gly Gly Ser Gly Gly

20 25 30

Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser

35 40 45

Gly Thr

50

<210> 106

<211> 61

<212> PRT

<213> Artificial

<220>

<223> Sequence of a blocking peptide based on IL-2Rβ

<400> 106

Gln Gly Gln Ser Gly Gln Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala

1 5 10 15

Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr Leu Cys

20 25 30

Pro Gly His Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

35 40 45

Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser Gly Thr

50 55 60

<210> 107

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 107

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser

1 5 10

<210> 108

<211> 12

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 108

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10

<210> 109

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 109

Gly Gly Gly Ser

1

<210> 110

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 110

Gly Ser Ser Gly Gly Ser Gly Gly Ser

1 5

<210> 111

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 111

Gly Ser Ser Gly Thr

1 5

<210> 112

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 112

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 113

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 113

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

Ala

<210> 114

<211> 20

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 114

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 115

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 115

Gly Gly Gly Ser Gly Gly Gly Ser

1 5

<210> 116

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 116

Gly Ser Gly Ser

1

<210> 117

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 117

Gly Gly Ser Ser

1

<210> 118

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 118

Gly Gly Gly Gly Ser

1 5

<210> 119

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 119

Gly Gly Ser Gly Gly

1 5

<210> 120

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 120

Ser Gly Gly Gly

1

<210> 121

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 121

Gly Ser Gly Ser

1

<210> 122

<211> 6

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 122

Gly Ser Gly Ser Gly Ser

1 5

<210> 123

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 123

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210> 124

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 124

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 125

<211> 12

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 125

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 126

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 126

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 127

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Sequence of the non-cleavable linker

<400> 127

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 128

<211> 676

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-15

<400> 128

Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Lys Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly

450 455 460

Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val

465 470 475 480

Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile

485 490 495

Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val

500 505 510

Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu

515 520 525

Ser Gly Asp Ala Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu

530 535 540

Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys

545 550 555 560

Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln

565 570 575

Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Pro

580 585 590

Leu Gly Met Leu Ser Gln Ser Ile Thr Cys Pro Pro Pro Met Ser Val

595 600 605

Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu

610 615 620

Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser

625 630 635 640

Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr

645 650 655

Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg

660 665 670

Pro Ala Pro Pro

675

<210> 129

<211> 219

<212> PRT

<213> Artificial

<220>

<223> LC of humanized PD-1 antagonist antibody

<400> 129

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 130

<211> 669

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a CTLA-4 and IL-15 antagonist antibody

<400> 130

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

435 440 445

Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu

450 455 460

Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile

465 470 475 480

Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu

485 490 495

Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu

500 505 510

Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His

515 520 525

Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser

530 535 540

Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu

545 550 555 560

Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln

565 570 575

Met Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser

580 585 590

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

595 600 605

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

610 615 620

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

625 630 635 640

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

645 650 655

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

660 665

<210> 131

<211> 215

<212> PRT

<213> Artificial

<220>

<223> CTLA-4 antagonist antibody LC

<400> 131

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 132

<211> 677

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with CD40 agonist antibody and IL-15

<400> 132

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gln Pro Leu Gly Tyr Cys Thr Asn Gly Val Cys Ser Tyr

100 105 110

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

145 150 155 160

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

165 170 175

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

180 185 190

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

195 200 205

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

210 215 220

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

225 230 235 240

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

245 250 255

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

260 265 270

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

275 280 285

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295 300

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305 310 315 320

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

325 330 335

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

340 345 350

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

355 360 365

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

370 375 380

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

385 390 395 400

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

405 410 415

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

420 425 430

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

435 440 445

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser

450 455 460

Gly Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn

465 470 475 480

Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His

485 490 495

Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys

500 505 510

Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu

515 520 525

Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile

530 535 540

Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly

545 550 555 560

Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu

565 570 575

Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly

580 585 590

Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys Pro Pro Pro Met Ser

595 600 605

Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg

610 615 620

Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser

625 630 635 640

Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp

645 650 655

Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln

660 665 670

Arg Pro Ala Pro Pro

675

<210> 133

<211> 214

<212> PRT

<213> Artificial

<220>

<223>CD40 agonist antibody LC

<400> 133

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ile Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 134

<211> 666

<212> PRT

<213> Artificial

<220>

<223> HC VitoKine constructs with humanized antibody against

fibronectin and IL-15

<400> 134

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly

435 440 445

Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu Gly Gly

450 455 460

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

465 470 475 480

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

485 490 495

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

500 505 510

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

515 520 525

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

530 535 540

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

545 550 555 560

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

565 570 575

Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys

580 585 590

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

595 600 605

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

610 615 620

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

625 630 635 640

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

645 650 655

Ala Leu Val His Gln Arg Pro Ala Pro Pro

660 665

<210> 135

<211> 215

<212> PRT

<213> Artificial

<220>

<223> LC humanized anti-fibronectin antibody

<400> 135

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro

85 90 95

Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 136

<211> 766

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with rituximab and IL-2

<400> 136

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

450 455 460

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

465 470 475 480

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

485 490 495

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

500 505 510

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

515 520 525

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

530 535 540

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

545 550 555 560

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

565 570 575

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

580 585 590

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

595 600 605

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

610 615 620

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser

625 630 635 640

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

645 650 655

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

660 665 670

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

675 680 685

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

690 695 700

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

705 710 715 720

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

725 730 735

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

740 745 750

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

755 760 765

<210> 137

<211> 213

<212> PRT

<213> Artificial

<220>

<223> Rituximab LC

<400> 137

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 138

<211> 766

<212> PRT

<213> Artificial

<220>

<223> HC constructs of VitoKine with Herceptin and IL-2

<400> 138

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

450 455 460

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

465 470 475 480

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

485 490 495

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

500 505 510

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

515 520 525

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

530 535 540

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

545 550 555 560

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

565 570 575

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

580 585 590

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

595 600 605

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

610 615 620

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser

625 630 635 640

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

645 650 655

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

660 665 670

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

675 680 685

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

690 695 700

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

705 710 715 720

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

725 730 735

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

740 745 750

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

755 760 765

<210> 139

<211> 214

<212> PRT

<213> Artificial

<220>

<223> LC Herceptin

<400> 139

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 140

<211> 764

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with cetuximab and IL-2

<400> 140

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys

450 455 460

Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile

465 470 475 480

Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu

485 490 495

Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu

500 505 510

Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu

515 520 525

Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn

530 535 540

Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met

545 550 555 560

Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg

565 570 575

Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Pro

580 585 590

Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro Pro Glu

595 600 605

Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met

610 615 620

Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser

625 630 635 640

Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn

645 650 655

Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val

660 665 670

Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln

675 680 685

Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg

690 695 700

Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe

705 710 715 720

Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala

725 730 735

Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys

740 745 750

Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

755 760

<210> 141

<211> 214

<212> PRT

<213> Artificial

<220>

<223> LC cetuximab

<400> 141

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 142

<211> 770

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-15

<400> 142

Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Lys Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro

450 455 460

Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu

465 470 475 480

Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro

485 490 495

Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala

500 505 510

Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu

515 520 525

Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro

530 535 540

Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly

545 550 555 560

Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile

565 570 575

Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser

580 585 590

Thr Leu Thr Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys

595 600 605

Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala

610 615 620

Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg

625 630 635 640

Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser

645 650 655

His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg

660 665 670

Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg

675 680 685

Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser

690 695 700

Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr

705 710 715 720

Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys

725 730 735

Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys

740 745 750

Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys

755 760 765

Thr Gly

770

<210> 143

<211> 766

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with vedolizumab and IL-2

<400> 143

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Ser Glu Ser Asn Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Ile Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Gly Trp Asp Tyr Ala Ile Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly

225 230 235 240

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

450 455 460

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Thr Leu Gln

465 470 475 480

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

485 490 495

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

500 505 510

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

515 520 525

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

530 535 540

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

545 550 555 560

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

565 570 575

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

580 585 590

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

595 600 605

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

610 615 620

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

625 630 635 640

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

645 650 655

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

660 665 670

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

675 680 685

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

690 695 700

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

705 710 715 720

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

725 730 735

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

740 745 750

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

755 760 765

<210> 144

<211> 219

<212> PRT

<213> Artificial

<220>

<223> LC vedolizumab

<400> 144

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ala Lys Ser

20 25 30

Tyr Gly Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Gly

85 90 95

Thr His Gln Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 145

<211> 766

<212> PRT

<213> Artificial

<220>

<223> HC designs of VitoKine with Humira and IL-2

<400> 145

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

450 455 460

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Thr Leu Gln

465 470 475 480

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

485 490 495

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

500 505 510

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

515 520 525

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

530 535 540

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

545 550 555 560

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

565 570 575

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

580 585 590

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

595 600 605

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

610 615 620

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

625 630 635 640

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

645 650 655

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

660 665 670

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

675 680 685

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

690 695 700

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

705 710 715 720

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

725 730 735

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

740 745 750

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

755 760 765

<210> 146

<211> 214

<212> PRT

<213> Artificial

<220>

<223> LC Humira

<400> 146

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 147

<211> 70

<212> PRT

<213> Artificial

<220>

<223> Altered IL-2RαSushi domain

<400> 147

Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg

1 5 10 15

Ile Tyr His Phe Val Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys

20 25 30

Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys

35 40 45

Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr

50 55 60

Ser Ser Ala Thr Arg Asn

65 70

<210> 148

<211> 350

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc-IL-15 2

<400> 148

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn

225 230 235 240

Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His

245 250 255

Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys

260 265 270

Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu

275 280 285

Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile

290 295 300

Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly

305 310 315 320

Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu

325 330 335

Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

340 345 350

<210> 149

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc-IL-15 3

<400> 149

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 150

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0420 (VitoKine design with IL-2 variant acting

mainly on Treg and Fc)

<400> 150

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Arg Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 151

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0421 (VitoKine design with IL-2 variant acting

mainly on Treg and Fc)

<400> 151

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Thr Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 152

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0423 (VitoKine design with IL-2 variant acting

mainly on Treg and Fc)

<400> 152

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 153

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0424 (VitoKine design with IL-2 variant acting

mainly on Treg and Fc)

<400> 153

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Asn Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 154

<211> 542

<212> PRT

<213> Artificial

<220>

<223> P-0425 (VitoKine design with IL-2 variant acting

mainly on Treg and Fc)

<400> 154

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Arg Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Pro Leu Gly Met Leu Ser Gln Ser Glu Leu Cys Asp Asp Asp Pro

370 375 380

Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly

385 390 395 400

Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Glu Ser

405 410 415

Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp

420 425 430

Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys

435 440 445

Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu

450 455 460

Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His

465 470 475 480

Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr

485 490 495

His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr

500 505 510

Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His

515 520 525

Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

530 535 540

<210> 155

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0426 (VitoKine design with IL-2 variant acting

mainly on Treg and Fc)

<400> 155

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu His Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Glu Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Arg Ile Glu Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 156

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Human IgG1 Fc with reduced/eliminated effector

function and increased half-life

<400> 156

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr

20 25 30

Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 157

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 157

Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala

1 5 10

<210> 158

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 158

Gly Pro Thr Asn Lys Val Arg

1 5

<210> 159

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Sequence of the protease substrate peptide

<400> 159

Arg Gln Ala Arg Ala Val Gly Gly

1 5

<210> 160

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 160

Gly Gly Pro Thr Asn Lys Val Arg Gly Ser

1 5 10

<210> 161

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 161

Gly Arg Gln Ala Arg Ala Val Gly Gly Ser

1 5 10

<210> 162

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0660 (VitoKine construct with IL-15 and Fc)

<400> 162

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Ser Ser Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala Gly

225 230 235 240

Thr Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 163

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0488 (VitoKine construct with IL-15 and Fc)

<400> 163

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 164

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0489 (VitoKine construct with IL-15 and Fc)

<400> 164

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Pro Thr Asn Lys Val Arg Gly Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 165

<211> 441

<212> PRT

<213> Artificial

<220>

<223> P-0661 (VitoKine construct with IL-15 and Fc)

<400> 165

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Pro Thr Asn Lys Val Arg Gly Gly Ser

225 230 235 240

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

245 250 255

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

260 265 270

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

275 280 285

Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val Glu

290 295 300

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

305 310 315 320

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

325 330 335

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

340 345 350

Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys Pro

355 360 365

Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser

370 375 380

Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys

385 390 395 400

Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn

405 410 415

Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala

420 425 430

Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 166

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Human IgG1 Fc with reduced/eliminated effector

function and increased half-life in vivo

<400> 166

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 167

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Knob-Fc human IgG1 with extended half-life

in vivo

<400> 167

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 168

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc human IgG1 with extended half-life

in vivo

<400> 168

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 169

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0650 (VitoKine construct with IL-15 and Fc)

<400> 169

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 170

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0651 (VitoKine construct with IL-15 and Fc)

<400> 170

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 171

<211> 448

<212> PRT

<213> Artificial

<220>

<223> Hole chain P-0662 (VitoKine constructs with IL-15 and Fc)

<400> 171

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu

245 250 255

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

260 265 270

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

275 280 285

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

290 295 300

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

305 310 315 320

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

325 330 335

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

340 345 350

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu

355 360 365

Ser Gln Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp

370 375 380

Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys

385 390 395 400

Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys

405 410 415

Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu

420 425 430

Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440 445

<210> 172

<211> 448

<212> PRT

<213> Artificial

<220>

<223> Hole chain P-0663 (VitoKine constructs with IL-15 and Fc) with increased

half-life

<400> 172

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu

245 250 255

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

260 265 270

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

275 280 285

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

290 295 300

Asp Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

305 310 315 320

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

325 330 335

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

340 345 350

Ile Val Gln Met Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu

355 360 365

Ser Gln Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp

370 375 380

Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys

385 390 395 400

Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys

405 410 415

Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu

420 425 430

Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440 445

<210> 173

<211> 442

<212> PRT

<213> Artificial

<220>

<223> Hole chain P-0664 (VitoKine constructs with IL-15 and Fc) with an increased

half-life

<400> 173

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 174

<211> 442

<212> PRT

<213> Artificial

<220>

<223> Hole chain P-0665 (VitoKine constructs with IL-15 and Fc) with increased

half-life

<400> 174

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Ser Ser Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala Gly

225 230 235 240

Thr Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 175

<211> 299

<212> PRT

<213> Artificial

<220>

<223> Chain 1 P-0156

<400> 175

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Gly Cys Pro

65 70 75 80

Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe

85 90 95

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

100 105 110

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

115 120 125

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

130 135 140

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

145 150 155 160

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

165 170 175

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

180 185 190

Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg

195 200 205

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

210 215 220

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

225 230 235 240

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

245 250 255

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

260 265 270

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

275 280 285

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

290 295

<210> 176

<211> 336

<212> PRT

<213> Artificial

<220>

<223> Chain 2 P-0156

<400> 176

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro

115 120 125

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

130 135 140

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

145 150 155 160

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

165 170 175

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

180 185 190

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

195 200 205

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

210 215 220

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

225 230 235 240

Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

245 250 255

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

260 265 270

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

275 280 285

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

290 295 300

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

305 310 315 320

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

325 330 335

<210> 177

<211> 350

<212> PRT

<213> Artificial

<220>

<223> Benchmark, chain 1

<400> 177

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asn Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Gln

50 55 60

Asp Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His

115 120 125

Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Lys His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Glu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Asp Val

260 265 270

Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asp Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Glu Gln Gly Asp Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His

325 330 335

Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 178

<211> 301

<212> PRT

<213> Artificial

<220>

<223> Benchmark, chain 2

<400> 178

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr

65 70 75 80

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

85 90 95

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

100 105 110

Val Thr Cys Val Val Val Asp Val Lys His Glu Asp Pro Glu Val Lys

115 120 125

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

130 135 140

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

145 150 155 160

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

165 170 175

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

180 185 190

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

195 200 205

Arg Glu Gln Met Thr Lys Asn Gln Val Lys Leu Thr Cys Leu Val Lys

210 215 220

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

225 230 235 240

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

245 250 255

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

260 265 270

Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser

275 280 285

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

290 295 300

<210> 179

<211> 465

<212> PRT

<213> Artificial

<220>

<223> P-0321 (VitoKine construct with IL-2 and Fc)

<400> 179

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn

225 230 235 240

Ala Ile Leu Glu Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys

245 250 255

Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu

260 265 270

Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr

275 280 285

Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln

290 295 300

Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala

305 310 315 320

Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile

325 330 335

Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys

340 345 350

Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp

355 360 365

Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly His Cys Arg Glu

385 390 395 400

Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val

405 410 415

Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg

420 425 430

Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser

435 440 445

His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg

450 455 460

Asn

465

<210> 180

<211> 669

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with PD-L1-blocking antibody and IL-15

<400> 180

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

435 440 445

Gly Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu

450 455 460

Glu Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile

465 470 475 480

Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu

485 490 495

Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu

500 505 510

Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Asp Ile His

515 520 525

Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser

530 535 540

Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu

545 550 555 560

Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln

565 570 575

Met Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser

580 585 590

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

595 600 605

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

610 615 620

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

625 630 635 640

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

645 650 655

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

660 665

<210> 181

<211> 214

<212> PRT

<213> Artificial

<220>

<223> LC PD-L1 blocking antibody

<400> 181

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 182

<211> 110

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide with deletion 111-114

<400> 182

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe

100 105 110

<210> 183

<211> 108

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide with deletion 109-114

<400> 183

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln

100 105

<210> 184

<211> 107

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide with deletion 108-114

<400> 184

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val

100 105

<210> 185

<211> 116

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide with a "GS" insertion after N95

<400> 185

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Gly

85 90 95

Ser Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe

100 105 110

Ile Asn Thr Ser

115

<210> 186

<211> 119

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide with "GGSGG" inserted after

N95

<400> 186

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Gly

85 90 95

Gly Ser Gly Gly Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val

100 105 110

Gln Met Phe Ile Asn Thr Ser

115

<210> 187

<211> 113

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant with deletion of 1 amino acid at the N-terminus

<400> 187

Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln

1 5 10 15

Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro

20 25 30

Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val

35 40 45

Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn

50 55 60

Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr

65 70 75 80

Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys

85 90 95

Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr

100 105 110

Ser

<210> 188

<211> 112

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant with deletion of 2 amino acids at the N-terminus

<400> 188

Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser

1 5 10 15

Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser

20 25 30

Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile

35 40 45

Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu

50 55 60

Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu

65 70 75 80

Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu

85 90 95

Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

100 105 110

<210> 189

<211> 111

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant with deletion of 3 amino acids at the N-terminus

<400> 189

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

1 5 10 15

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

20 25 30

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

35 40 45

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

50 55 60

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

65 70 75 80

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

85 90 95

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

100 105 110

<210> 190

<211> 110

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant with a deletion of 4 amino acids at the N-terminus

<400> 190

Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His

1 5 10 15

Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys

20 25 30

Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu

35 40 45

Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile

50 55 60

Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly

65 70 75 80

Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu

85 90 95

Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

100 105 110

<210> 191

<211> 112

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 V63A variant with a deletion of 2 amino acids

N-terminus

<400> 191

Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser

1 5 10 15

Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser

20 25 30

Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile

35 40 45

Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala Glu Asn Leu

50 55 60

Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu

65 70 75 80

Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu

85 90 95

Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

100 105 110

<210> 192

<211> 112

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant I68H with a deletion of 2 amino acids

N-terminus

<400> 192

Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser

1 5 10 15

Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser

20 25 30

Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile

35 40 45

Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu

50 55 60

Ile His Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu

65 70 75 80

Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu

85 90 95

Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

100 105 110

<210> 193

<211> 449

<212> PRT

<213> Artificial

<220>

<223> Heavy chain of humanized anti-FAP antibody

<400> 193

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Asn

20 25 30

Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Phe His Pro Gly Ser Gly Ser Ile Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Gly Gly Thr Gly Arg Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 194

<211> 218

<212> PRT

<213> Artificial

<220>

<223> Humanized anti-FAP antibody kappa light chain

<400> 194

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Ser Thr Ser

20 25 30

Ala Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 195

<211> 446

<212> PRT

<213> Artificial

<220>

<223> Heavy chain humanized PD-1 antagonist antibody

<400> 195

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 196

<211> 214

<212> PRT

<213> Artificial

<220>

<223> Humanized PD-1 antagonist antibody kappa light chain

<400> 196

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Glu Thr Val

20 25 30

Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr Ser Arg Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 197

<211> 448

<212> PRT

<213> Artificial

<220>

<223> Heavy chain of human PD-1 antagonist antibody

<400> 197

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ala Asp Tyr Ser Ser Gly Ser Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 198

<211> 214

<212> PRT

<213> Artificial

<220>

<223> Human PD-1 antagonist antibody lambda light chain

<400> 198

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Ile Tyr

35 40 45

Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Lys Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Asn Ser Ile Thr Tyr

85 90 95

Arg Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 199

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 V63A variant polypeptide

<400> 199

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 200

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 V63K variant polypeptide

<400> 200

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Lys Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 201

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 V63R variant polypeptide

<400> 201

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Arg Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 202

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide I68H

<400> 202

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile His Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 203

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide I68F

<400> 203

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Phe Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 204

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide I68Q

<400> 204

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Gln Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 205

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide I68G

<400> 205

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Gly Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 206

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide I68K

<400> 206

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Lys Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 207

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide I68D

<400> 207

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Asp Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 208

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide Q108S

<400> 208

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Ser Met Phe Ile Asn

100 105 110

Thr Ser

<210> 209

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide Q108A

<400> 209

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Ala Met Phe Ile Asn

100 105 110

Thr Ser

<210> 210

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide Q108E

<400> 210

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Glu Met Phe Ile Asn

100 105 110

Thr Ser

<210> 211

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide Q108K

<400> 211

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Lys Met Phe Ile Asn

100 105 110

Thr Ser

<210> 212

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide Q108M

<400> 212

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Met Met Phe Ile Asn

100 105 110

Thr Ser

<210> 213

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide V63A/I68H

<400> 213

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala Glu

50 55 60

Asn Leu Ile His Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 214

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide V63A/I68Q

<400> 214

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala Glu

50 55 60

Asn Leu Ile Gln Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 215

<211> 114

<212> PRT

<213> Artificial

<220>

<223> Human IL-15 variant polypeptide V63A/I68G

<400> 215

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala Glu

50 55 60

Asn Leu Ile Gly Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 216

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 V63K chain

<400> 216

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Lys

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 217

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 V63R chain

<400> 217

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Arg

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 218

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 V63A chain

<400> 218

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 219

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 I68H chain

<400> 219

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile His Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 220

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 I68F chain

<400> 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Phe Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 221

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 I68Q chain

<400> 221

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Gln Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 222

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 I68G chain

<400> 222

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Gly Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 223

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 I68K chain

<400> 223

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Lys Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 224

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 I68D chain

<400> 224

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Asp Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 225

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Hole chain Fc-IL-15 Q108S

<400> 225

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser

225 230 235 240

Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Ser Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 226

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Hole chain Fc-IL-15 Q108A

<400> 226

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser

225 230 235 240

Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Ala Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 227

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 V63A/I68H chain

<400> 227

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala

290 295 300

Glu Asn Leu Ile His Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 228

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Fc-IL-15 V63A/I68Q chain

<400> 228

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala

290 295 300

Glu Asn Leu Ile Gln Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 229

<211> 355

<212> PRT

<213> Artificial

<220>

<223> Circuit Fc-IL-15 V63A/I68G

<400> 229

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala

290 295 300

Glu Asn Leu Ile Gly Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser

355

<210> 230

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0682 (VitoKine construct with IL-15 and Fc)

<400> 230

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

290 295 300

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Ser Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 231

<211> 442

<212> PRT

<213> Artificial

<220>

<223> P-0806 (VitoKine construct with IL-15 and Fc)

<400> 231

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

245 250 255

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

260 265 270

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

275 280 285

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala

290 295 300

Glu Asn Leu Ile His Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

305 310 315 320

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

325 330 335

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

340 345 350

Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys

355 360 365

Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr

370 375 380

Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg

385 390 395 400

Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr

405 410 415

Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro

420 425 430

Ala Leu Val His Gln Arg Pro Ala Pro Pro

435 440

<210> 232

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 R38A/S125I variant polypeptide

<400> 232

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Ala Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 233

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 F42A/S125I variant polypeptide

<400> 233

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 234

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide E62A/S125I

<400> 234

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 235

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide E62F/S125I

<400> 235

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Phe Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 236

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide E62H/S125I

<400> 236

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu His Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 237

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide E62L/S125I

<400> 237

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Leu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 238

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide P65H/S125I

<400> 238

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

His Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 239

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide P65N/S125I

<400> 239

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Asn Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 240

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide P65Q/S125I

<400> 240

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Gln Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 241

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide P65R/S125I

<400> 241

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 242

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide P65K/S125I

<400> 242

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Lys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 243

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide F42A/E62F/S125I

<400> 243

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Phe Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 244

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide F42A/E62A/S125I

<400> 244

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 245

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide F42A/E62H/S125I

<400> 245

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu His Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 246

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide F42A/P65H/S125I

<400> 246

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

His Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 247

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Human IL-2 variant polypeptide F42A/P65R/S125I

<400> 247

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 248

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0531 (IL-2-Fc fusion protein)

<400> 248

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 249

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0689 (fusion protein of IL-2 and Fc, Knob chain)

<400> 249

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 250

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0602 (IL-2-Fc fusion protein)

<400> 250

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 251

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0613 (IL-2-Fc fusion protein)

<400> 251

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 252

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0624 (IL-2-Fc fusion protein)

<400> 252

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Ala Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 253

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0625 (IL-2-Fc fusion protein)

<400> 253

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Phe Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 254

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0626 (IL-2-Fc fusion protein)

<400> 254

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu His Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 255

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0627 (IL-2-Fc fusion protein)

<400> 255

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Leu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 256

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0634 (IL-2-Fc fusion protein)

<400> 256

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys His Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 257

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0708 (fusion protein of IL-2 and Fc, Knob chain)

<400> 257

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Asn Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 258

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0709 (fusion protein of IL-2 and Fc, Knob chain)

<400> 258

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gln Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 259

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0635 (IL-2-Fc fusion protein)

<400> 259

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 260

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0704 (fusion protein of IL-2 and Fc, Knob chain)

<400> 260

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 261

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0707 (fusion protein of IL-2 and Fc, Knob chain)

<400> 261

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Lys Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 262

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0702 (fusion protein of IL-2 and Fc, Knob chain)

<400> 262

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Phe Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 263

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0766 (fusion protein of IL-2 and Fc, Knob chain)

<400> 263

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Ala Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 264

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0767 (fusion protein of IL-2 and Fc, Knob chain)

<400> 264

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu His Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 265

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0703 (fusion protein of IL-2 and Fc, Knob chain)

<400> 265

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys His Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 266

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0705 (fusion protein of IL-2 and Fc, Knob chain)

<400> 266

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 267

<211> 165

<212> PRT

<213> Artificial

<220>

<223> Human IL-2RαSushi Y43A variant

<400> 267

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Ala Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly

165

<210> 268

<211> 165

<212> PRT

<213> Artificial

<220>

<223> Human IL-2RαSushi L42G variant

<400> 268

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Lys Ser Gly Ser Gly Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly

165

<210> 269

<211> 165

<212> PRT

<213> Artificial

<220>

<223> Human IL-2RαSushi R36A variant

<400> 269

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Ala Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly

165

<210> 270

<211> 165

<212> PRT

<213> Artificial

<220>

<223> Human IL-2Rα variant Sushi K38E

<400> 270

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Glu Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly

165

<210> 271

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0754 (VitoKine construct with IL-2 and Fc, Knob chain)

<400> 271

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Arg Ile Lys Ser Gly Ser Leu Ala Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 272

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0755 (VitoKine construct with IL-2 and Fc, Knob chain)

<400> 272

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Arg Ile Lys Ser Gly Ser Gly Tyr Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 273

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0756 (VitoKine construct with IL-2 and Fc, Knob chain)

<400> 273

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Ala Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 274

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0745 (VitoKine construct with IL-2 and Fc, Knob chain)

<400> 274

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 275

<211> 447

<212> PRT

<213> Artificial

<220>

<223> HC humanized PD-1 antagonist antibody

<400> 275

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 276

<211> 218

<212> PRT

<213> Artificial

<220>

<223> LC of humanized PD-1 antagonist antibody

<400> 276

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 277

<211> 440

<212> PRT

<213> Artificial

<220>

<223> HC antibody antagonist of human PD-1

<400> 277

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 278

<211> 214

<212> PRT

<213> Artificial

<220>

<223> LC antibody antagonist of human PD-1

<400> 278

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 279

<211> 448

<212> PRT

<213> Artificial

<220>

<223> HC humanized PD-L1 antagonist antibody

<400> 279

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 280

<211> 214

<212> PRT

<213> Artificial

<220>

<223> LC of humanized PD-L1 antagonist antibody

<400> 280

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 281

<211> 662

<212> PRT

<213> Artificial

<220>

<223> VitoKine Knob-HC chain with humanized antibody-

PD-1 and IL-15 antagonist

<400> 281

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val

450 455 460

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

465 470 475 480

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

485 490 495

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

500 505 510

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

515 520 525

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

530 535 540

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

545 550 555 560

Leu Gln Ser Phe Val His Ile Val Ser Met Phe Ile Asn Thr Ser Gly

565 570 575

Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys Pro Pro Pro Met

580 585 590

Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser

595 600 605

Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr

610 615 620

Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His

625 630 635 640

Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His

645 650 655

Gln Arg Pro Ala Pro Pro

660

<210> 282

<211> 662

<212> PRT

<213> Artificial

<220>

<223> VitoKine Knob-HC chain with humanized antibody-

PD-1 and IL-15 antagonist

<400> 282

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val

450 455 460

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

465 470 475 480

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

485 490 495

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

500 505 510

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala Glu Asn Leu Ile

515 520 525

Gly Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

530 535 540

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

545 550 555 560

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Gly

565 570 575

Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys Pro Pro Pro Met

580 585 590

Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser

595 600 605

Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr

610 615 620

Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His

625 630 635 640

Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His

645 650 655

Gln Arg Pro Ala Pro Pro

660

<210> 283

<211> 662

<212> PRT

<213> Artificial

<220>

<223> VitoKine Knob-HC chain with humanized antibody-

PD-1 and IL-15 antagonist

<400> 283

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val

450 455 460

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

465 470 475 480

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

485 490 495

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

500 505 510

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

515 520 525

Gly Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

530 535 540

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

545 550 555 560

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Gly

565 570 575

Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys Pro Pro Pro Met

580 585 590

Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser

595 600 605

Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr

610 615 620

Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His

625 630 635 640

Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His

645 650 655

Gln Arg Pro Ala Pro Pro

660

<210> 284

<211> 662

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-15

<400> 284

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val

450 455 460

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

465 470 475 480

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

485 490 495

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

500 505 510

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala Glu Asn Leu Ile

515 520 525

His Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

530 535 540

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

545 550 555 560

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Gly

565 570 575

Gly Pro Leu Gly Met Leu Ser Gln Ser Ile Thr Cys Pro Pro Pro Met

580 585 590

Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser

595 600 605

Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr

610 615 620

Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His

625 630 635 640

Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His

645 650 655

Gln Arg Pro Ala Pro Pro

660

<210> 285

<211> 446

<212> PRT

<213> Artificial

<220>

<223> Hole-HC chain humanized PD-1 antagonist antibody

<400> 285

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 286

<211> 767

<212> PRT

<213> Artificial

<220>

<223> VitoKine Knob-HC chain with humanized antibody-

PD-1 and IL-2 antagonist

<400> 286

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr

450 455 460

Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn

465 470 475 480

Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe

485 490 495

Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys

500 505 510

Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu Asn Leu Ala Gln

515 520 525

Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn

530 535 540

Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu

545 550 555 560

Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile

565 570 575

Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Gly Pro

580 585 590

Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp

595 600 605

Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu

610 615 620

Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys

625 630 635 640

Ser Gly Ser Gly Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser

645 650 655

Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr

660 665 670

Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr

675 680 685

Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly

690 695 700

His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile

705 710 715 720

Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly

725 730 735

Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr

740 745 750

His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

755 760 765

<210> 287

<211> 767

<212> PRT

<213> Artificial

<220>

<223> VitoKine Knob-HC chain with humanized antibody-

PD-1 and IL-2 antagonist

<400> 287

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr

450 455 460

Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn

465 470 475 480

Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe

485 490 495

Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys

500 505 510

Leu Glu Glu Glu Leu Lys Gln Leu Glu Glu Val Leu Asn Leu Ala Gln

515 520 525

Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn

530 535 540

Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu

545 550 555 560

Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile

565 570 575

Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Gly Pro

580 585 590

Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp

595 600 605

Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu

610 615 620

Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Ala Ile Lys

625 630 635 640

Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser

645 650 655

Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr

660 665 670

Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr

675 680 685

Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly

690 695 700

His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile

705 710 715 720

Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly

725 730 735

Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr

740 745 750

His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

755 760 765

<210> 288

<211> 767

<212> PRT

<213> Artificial

<220>

<223> VitoKine Knob-HC chain with humanized antibody-

PD-1 and IL-2 antagonist

<400> 288

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr

450 455 460

Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn

465 470 475 480

Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe

485 490 495

Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys

500 505 510

Leu Glu Glu Glu Leu Lys Gln Leu Glu Glu Val Leu Asn Leu Ala Gln

515 520 525

Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn

530 535 540

Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu

545 550 555 560

Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile

565 570 575

Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Gly Pro

580 585 590

Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp

595 600 605

Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu

610 615 620

Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys

625 630 635 640

Ser Gly Ser Gly Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser

645 650 655

Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr

660 665 670

Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr

675 680 685

Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly

690 695 700

His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile

705 710 715 720

Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly

725 730 735

Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr

740 745 750

His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

755 760 765

<210> 289

<211> 767

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-2

<400> 289

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr

450 455 460

Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn

465 470 475 480

Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe

485 490 495

Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys

500 505 510

Leu Glu Glu Glu Leu Lys Gln Leu Glu Glu Val Leu Asn Leu Ala Gln

515 520 525

Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn

530 535 540

Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu

545 550 555 560

Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile

565 570 575

Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Gly Pro

580 585 590

Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp

595 600 605

Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu

610 615 620

Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys

625 630 635 640

Ser Gly Ser Gly Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser

645 650 655

Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr

660 665 670

Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr

675 680 685

Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly

690 695 700

His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile

705 710 715 720

Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly

725 730 735

Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr

740 745 750

His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly

755 760 765

<210> 290

<211> 774

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-2

<400> 290

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala Gly Gly Ala Pro Thr

450 455 460

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu

465 470 475 480

Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys

485 490 495

Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr

500 505 510

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gln Leu Glu

515 520 525

Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg

530 535 540

Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser

545 550 555 560

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

565 570 575

Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr

580 585 590

Leu Thr Gly Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly

595 600 605

Ser Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe

610 615 620

Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys

625 630 635 640

Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Gly Tyr Met Leu Cys Thr

645 650 655

Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser

660 665 670

Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu

675 680 685

Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val

690 695 700

Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu

705 710 715 720

Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val

725 730 735

Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala

740 745 750

Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro

755 760 765

Gln Leu Ile Cys Thr Gly

770

<210> 291

<211> 774

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-2

<400> 291

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Ala Pro Thr

450 455 460

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu

465 470 475 480

Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys

485 490 495

Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr

500 505 510

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gln Leu Glu

515 520 525

Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg

530 535 540

Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser

545 550 555 560

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

565 570 575

Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr

580 585 590

Leu Thr Gly Gly Gly Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala Gly

595 600 605

Gly Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe

610 615 620

Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys

625 630 635 640

Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Gly Tyr Met Leu Cys Thr

645 650 655

Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser

660 665 670

Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu

675 680 685

Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val

690 695 700

Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu

705 710 715 720

Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val

725 730 735

Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala

740 745 750

Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro

755 760 765

Gln Leu Ile Cys Thr Gly

770

<210> 292

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0701 (VitoKine construct with IL-2 and Fc, Knob chain)

<400> 292

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 293

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0807 (VitoKine construct with IL-2 and Fc, Knob chain)

<400> 293

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Arg Ile Lys Ser Gly Ser Leu Ala Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 294

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0708 (VitoKine construct with IL-2 and Fc, Knob chain)

<400> 294

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Arg Ile Lys Ser Gly Ser Gly Tyr Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 295

<211> 547

<212> PRT

<213> Artificial

<220>

<223> P-0809 (VitoKine construct with IL-2 and Fc, Knob chain)

<400> 295

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr Gly

355 360 365

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Glu Leu

370 375 380

Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met

385 390 395 400

Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe

405 410 415

Arg Ala Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser

420 425 430

Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr

435 440 445

Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu

450 455 460

Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala

465 470 475 480

Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala

485 490 495

Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln

500 505 510

Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val

515 520 525

Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile

530 535 540

Cys Thr Gly

545

<210> 296

<211> 594

<212> PRT

<213> Artificial

<220>

<223> Knob-HC chain of humanized PD-1 antagonist antibody with IL-2

<400> 296

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr

450 455 460

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu

465 470 475 480

Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys

485 490 495

Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr

500 505 510

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gln Leu Glu

515 520 525

Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg

530 535 540

Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser

545 550 555 560

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

565 570 575

Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr

580 585 590

Leu Thr

<210> 297

<211> 575

<212> PRT

<213> Artificial

<220>

<223> HC humanized PD-1 antagonist antibody with IL-15

<400> 297

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val

450 455 460

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

465 470 475 480

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

485 490 495

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

500 505 510

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Ala Glu Asn Leu Ile

515 520 525

His Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

530 535 540

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

545 550 555 560

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

565 570 575

<210> 298

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Protease-cleavable linker sequence

<400> 298

Gly Gly Gly Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala Gly Gly

1 5 10 15

<210> 299

<211> 444

<212> PRT

<213> Artificial

<220>

<223> HC chain of a surrogate antibody antagonist of murine PD-1

<400> 299

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ser

20 25 30

Tyr Arg Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Arg Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Ser Ala Gly Ile Ser Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Arg Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Val Asn Ser Val Thr Thr Glu Asp Ala Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Ser Asp Asn Met Gly Thr Thr Pro Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

180 185 190

Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala

195 200 205

Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

210 215 220

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

245 250 255

Thr Cys Val Val Val Ala Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

260 265 270

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro

275 280 285

Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

290 295 300

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

305 310 315 320

Asn Ser Ala Ala Phe Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro

340 345 350

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

355 360 365

Asn Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

370 375 380

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

385 390 395 400

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

405 410 415

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

420 425 430

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

<210> 300

<211> 443

<212> PRT

<213> Artificial

<220>

<223> Heterodimeric HC chain 1 surrogate antibody antagonist

mouse PD-1

<400> 300

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ser

20 25 30

Tyr Arg Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Arg Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Ser Ala Gly Ile Ser Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Arg Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Val Asn Ser Val Thr Thr Glu Asp Ala Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Ser Asp Asn Met Gly Thr Thr Pro Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

180 185 190

Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala

195 200 205

Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

210 215 220

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

245 250 255

Thr Cys Val Val Val Ala Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

260 265 270

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro

275 280 285

Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

290 295 300

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

305 310 315 320

Asn Ser Ala Ala Phe Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

340 345 350

Lys Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn

355 360 365

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

370 375 380

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Lys Thr Asp Gly Ser

385 390 395 400

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

405 410 415

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

420 425 430

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

<210> 301

<211> 443

<212> PRT

<213> Artificial

<220>

<223> Heterodimeric HC chain 2 surrogate antibody antagonist

mouse PD-1

<400> 301

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ser

20 25 30

Tyr Arg Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Arg Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Ser Ala Gly Ile Ser Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Arg Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Val Asn Ser Val Thr Thr Glu Asp Ala Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Ser Asp Asn Met Gly Thr Thr Pro Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

180 185 190

Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala

195 200 205

Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

210 215 220

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

245 250 255

Thr Cys Val Val Val Ala Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

260 265 270

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro

275 280 285

Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

290 295 300

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

305 310 315 320

Asn Ser Ala Ala Phe Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

340 345 350

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn

355 360 365

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

370 375 380

Ala Glu Asn Tyr Asp Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

385 390 395 400

Tyr Phe Val Tyr Ser Asp Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

405 410 415

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

420 425 430

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

<210> 302

<211> 218

<212> PRT

<213> Artificial

<220>

<223> LC of a surrogate antibody antagonist of murine PD-1

<400> 302

Asp Ile Val Met Thr Gln Gly Thr Leu Pro Asn Pro Val Pro Ser Gly

1 5 10 15

Glu Ser Val Ser Ile Thr Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Gln Gln Gly

85 90 95

Leu Glu Phe Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg

100 105 110

Thr Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

115 120 125

Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln

145 150 155 160

Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

180 185 190

His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

195 200 205

Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 303

<211> 668

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-15

<400> 303

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu

450 455 460

Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

465 470 475 480

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

485 490 495

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

500 505 510

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

515 520 525

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

530 535 540

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

545 550 555 560

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

565 570 575

Phe Ile Asn Thr Ser Gly Gly Pro Leu Gly Met Leu Ser Gln Ser Ile

580 585 590

Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys

595 600 605

Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe

610 615 620

Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys

625 630 635 640

Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

645 650 655

Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

660 665

<210> 304

<211> 673

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-15

<400> 304

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu

450 455 460

Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

465 470 475 480

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

485 490 495

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

500 505 510

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

515 520 525

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

530 535 540

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

545 550 555 560

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

565 570 575

Phe Ile Asn Thr Ser Gly Gly Ser Gly Pro Leu Gly Met Leu Ser Gln

580 585 590

Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala

595 600 605

Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile

610 615 620

Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu

625 630 635 640

Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser

645 650 655

Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro

660 665 670

Pro

<210> 305

<211> 667

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-15

<400> 305

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala Gly Gly Asn Trp Val

450 455 460

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

465 470 475 480

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

485 490 495

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

500 505 510

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

515 520 525

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

530 535 540

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

545 550 555 560

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Gly

565 570 575

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Ile Thr

580 585 590

Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser

595 600 605

Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys

610 615 620

Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala

625 630 635 640

Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp

645 650 655

Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

660 665

<210> 306

<211> 667

<212> PRT

<213> Artificial

<220>

<223> VitoKine HC constructs with a humanized antagonist antibody

PD-1 and IL-15

<400> 306

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val

450 455 460

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

465 470 475 480

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

485 490 495

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

500 505 510

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

515 520 525

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

530 535 540

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

545 550 555 560

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Gly

565 570 575

Gly Ser Gly Pro Leu Gly Met Leu Ser Gln Gly Gly Gly Ser Ile Thr

580 585 590

Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser

595 600 605

Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys

610 615 620

Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala

625 630 635 640

Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp

645 650 655

Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

660 665

<---

Claims (12)

1. A biologically activatable polypeptide drug construct D1-D2-D3 for the treatment of colon cancer and inhibition of colon cancer metastasis, comprising, in the direction from the N-terminus to the C-terminus: (1) a D1 domain of a dual-function moiety; (2) a D2 domain of a biologically activatable moiety; and (3) a D3 domain of a masking moiety; where the function of D1 is to direct and maintain the biologically activated moiety at the intended site of therapy; where D3 is capable of masking the functional activity of D2 prior to activation at the intended site of therapy; and wherein the D2 domain is an IL-2 (interleukin-2) variant polypeptide selected from the group of polypeptides having the amino acid sequence set forth in SEQ ID NO: 232-247, wherein D1 is an antagonist humanized PD-1 (programmed cell death protein 1) antibody selected from an antibody comprising: the heavy chain and light chain amino acid sequences set forth in SEQ ID NOs: 195 and 196; the heavy chain and light chain amino acid sequences set forth in SEQ ID NOs: 197 and 198; the heavy chain and light chain amino acid sequences set forth in SEQ ID NOs: 275 and 276; or the heavy chain and light chain amino acid sequences set forth in SEQ ID NOs: 277 and 278; and. wherein the D3 domain is a cognate receptor/binding partner for IL-2, which is IL-2RαSushi, comprising the amino acid sequence set forth in SEQ ID NO: 10 and 267-270; where D2 is connected to D1 by a peptide linker L1 selected from the group consisting of a protease-cleavable peptide linker and a non-cleavable peptide linker, and D2 is connected to D3 by a peptide linker L2 selected from the group consisting of a protease-cleavable peptide linker and a non-cleavable peptide linker, where the non-cleavable peptide linker is selected from the group of sequences indicated in SEQ ID NO: 107, 108, 110, 112, 115, 123-127. 2. The construct according to claim 1, wherein each of the domains D1, D2 and D3 of the construct has the form of a monomer, the form of a dimer, or they, taken together, have the form of a combination of a dimer and a monomer. 3. The construct according to claim 1, wherein the protease-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NO: 71-96, 157-161 and 298. 4. The construct according to any one of claims 1 to 3, wherein both L1 and L2 are protease-cleavable peptide linkers, or where both L1 and L2 are non-cleavable peptide linkers, or where L1 is a protease-cleavable peptide linker and L2 is a non-cleavable peptide linker, or where L1 is a non-cleavable peptide linker and L2 is a protease-cleavable peptide linker. 5. The design according to any of paragraphs. 1-4, which is selected from a construct comprising the heavy chain amino acid sequence of SEQ ID NO: 286 and the light chain amino acid sequence presented in SEQ ID NO: 196, a construct comprising the heavy chain amino acid sequence of SEQ ID NO: 287 and the light chain amino acid sequence presented in SEQ ID NO: 196, a construct comprising the heavy chain amino acid sequence of SEQ ID NO: 288 and the light chain amino acid sequence presented in SEQ ID NO: 196, a construct comprising the heavy chain amino acid sequence of SEQ ID NO: 289 and the light chain amino acid sequence presented in SEQ ID NO: 196, a construct comprising the heavy chain amino acid sequence of SEQ ID NO: 290 and the light chain amino acid sequence presented in SEQ ID NO: 196, a construct comprising the heavy chain amino acid sequence of SEQ ID NO: 291 and the light chain amino acid sequence presented in SEQ ID NO: 196, and a construct comprising the heavy chain amino acid sequence of SEQ ID NO: 296 and the light chain amino acid sequence presented in SEQ ID NO: 196. 6. A pharmaceutical composition for treating colon cancer and inhibiting colon cancer metastasis, comprising an effective amount of the construct according to any one of claims 1-5 in a mixture with a pharmaceutically acceptable carrier. 7. The use of a pharmaceutical composition according to claim 6 in the treatment of colon cancer or metastasis of colon cancer in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 6.