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WO2025077833A1 - Anticorps ciblant tim-3 et pd-1 et leurs utilisations - Google Patents

Anticorps ciblant tim-3 et pd-1 et leurs utilisations Download PDF

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Publication number
WO2025077833A1
WO2025077833A1 PCT/CN2024/124165 CN2024124165W WO2025077833A1 WO 2025077833 A1 WO2025077833 A1 WO 2025077833A1 CN 2024124165 W CN2024124165 W CN 2024124165W WO 2025077833 A1 WO2025077833 A1 WO 2025077833A1
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seq
region
terminus
amino acid
acid sequence
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Xin Dong
Yu Zhang
Jinyu DONG
Binbin Wang
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Suzhou Neologics Bioscience Co Ltd
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Suzhou Neologics Bioscience Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • bispecific antibodies comprising (i) a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1) , wherein the VL1/VH1 pair specifically binds to human Tim-3, and wherein the VL1 comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 101, 102, and 103, respectively, and wherein the VH1 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 104, 105, and 106, respectively; or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VL CDRs and VH CDRs; and (ii) a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) , wherein the VL2/VH2 pair specifically binds to human PD-1; and wherein the VL2 comprises VL CDR1, VL2/VH
  • VL1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84; VH1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 85-91; and the VL2 and VH2 have the amino acid sequences of SEQ ID NOs: 1 and 2, respectively. In some embodiments, the VL1 and VH1 have the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.
  • the bispecific antibodies provided herein comprise: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a light chain constant (CL) region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant (CH) region and a single chain variable fragment (scFv) , wherein the scFv comprises, from N-terminus to C-terminus, VL2, a linker (L) , and VH2.
  • the CL region is C ⁇ (SEQ ID NO: 7) or C ⁇ (SEQ ID NO: 8) , or a variant thereof having up to ten amino acids substitutions; or (2) the CH region is human IgG1 CH region (SEQ ID NO: 9) , IgG2 CH region (SEQ ID NO: 10) , IgG3 CH region (SEQ ID NO: 11) , or IgG4 CH region (SEQ ID NO: 12) , or a variant thereof having up to ten amino acids substitutions; or both (1) and (2) .
  • the CL region is C ⁇ (SEQ ID NO: 7) , and the CH region is human IgG1 CH region with L234A, L235A and P329G substitutions (SEQ ID NO: 25) ; or (2) the CL region is C ⁇ (SEQ ID NO: 7) , and the CH region is human IgG1 CH region with L234A, L235A, P329G and K447A substitutions (SEQ ID NO: 27) .
  • the bispecific antibodies provided herein comprise: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2, VL1, and a CL region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, VH1, and a CH region.
  • the VL1 and VL2 are connected by a first linker, and the VH1 and VH2 are connected by a second linker.
  • the first linker consists of amino acids 1 to m of the CL region
  • the second linker consists of amino acids 1 to n of the CH region; wherein m and n are each independently an integer between 4 and 20.
  • the CL region is C ⁇ (SEQ ID NO: 7) , and the CH region is human IgG1 CH region with L234A, L235A and P329G substitutions (SEQ ID NO: 25) ; or (2) the CL region is C ⁇ (SEQ ID NO: 7) , and the CH region is human IgG1 CH region with L234A, L235A, P329G and K447A substitutions (SEQ ID NO: 27) .
  • the first linker has the amino acid sequence of SEQ ID NO: 54; and the second linker has the amino acid sequence of SEQ ID NO: 55.
  • C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 67
  • C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 68.
  • C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 69
  • C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 70.
  • the bispecific antibodies provided herein comprise: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant domain 1 (CH1 domain) , and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Knob-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VL2, a linker (L) , and VH2.
  • the bispecific antibodies provided herein comprise: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a CH1 domain, and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Knob-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VH2, a linker (L) , and VL2.
  • the bispecific antibodies provided herein comprise: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a CH1 domain, and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Hole-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VH2, a linker (L) , and VL2.
  • the bispecific antibodies provided herein comprise: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH1 domain, and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Knob-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VL1, a linker (L) , and VH1.
  • the bispecific antibodies provided herein comprise: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH1 domain, and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Hole-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VH1, a linker (L) , and VL1.
  • the linker is a GS linker having the amino acid sequence of SEQ ID NO: 44 or 46, or a TF linker having the amino acid sequence of SEQ ID NO: 50.
  • the CL region is kappa CL (C ⁇ ; SEQ ID NO: 7) or lambda CL (C ⁇ , SEQ ID NO: 8) , or a variant thereof having up to ten amino acids substitutions;
  • the CH1 domain is human IgG1 CH1 domain (SEQ ID NO: 13) or a variant thereof having up to ten amino acids substitutions; or
  • the Knob-Fc region and the Hole-Fc region have the amino acid sequences of (i) SEQ ID NOs: 29 and 30, respectively; (ii) SEQ ID NOs: 31 and 32, respectively; (iii) SEQ ID NOs: 33 and 34, respectively; (iv) SEQ ID NOs: 35 and 36, respectively; (v) SEQ ID NOs: 37 and 38, respectively; or (vi) SEQ ID NOs: 39 and 40, respectively; or any combination of (1) - (3) .
  • vectors comprising the polynucleotide described herein.
  • provided herein are methods of inducing or stimulating immune cell activation and/or proliferation, comprising contacting an immune cell with an effective amount of the bispecific antibody described herein.
  • provided herein are methods of reducing PD-1-mediated or Tim-3-mediated suppression of an immune cell comprising contacting the immune cell with an effective amount of the bispecific antibody described herein.
  • the immune cell is a T cell, an NK cell, an NKT cell, or a myeloid cell.
  • the immune cell is a T cell.
  • the immune cell is a NK cell.
  • the immune cell is a myeloid cell, wherein the myeloid cell is a macrophage or a dendritic cell.
  • the subject is a human.
  • provided herein are uses of the bispecific antibody described herein in cancer treatment. In some embodiments, provided herein are uses of the bispecific antibody described herein for the preparation of a medicament for the treatment of cancer.
  • the cancer is a hematological cancer or a solid tumor. In some embodiments, the cancer has a high degree of microsatellite instability. In some embodiments, the cancer is a Tim-3 expressing cancer. In some embodiments, the cancer is a PD-L1 expressing cancer. In some embodiments, the cancer is resistant to anti-PD-1 treatment.
  • FIGs. 1A-1C provide diagrams illustrating the three different types of bispecific antibodies disclosed herein.
  • N means the N-terminus
  • C means the C-terminus
  • L means a linker.
  • FIG. 1A illustrates the “IgG-scFv” format, which includes two pairs of two distinct peptides, a first peptide chain (C1) and a second peptide chain (C2) , with the configurations shown below:
  • FIG. 1B illustrates the “DVD-Ig” format, which includes two pairs of two distinct peptides, a first peptide chain (C1) and a second peptide chain (C2) , with the configurations shown below:
  • FIG. 1C illustrates the “KIH” format, which includes three distinct peptides, a first peptide chain (C1) , a second peptide chain (C2) , and a third peptide chain (C3) , with the configurations shown below:
  • FIG. 3 provides the results of Mini PDX tumor animal models. As shown, 3E6 inhibited patient tissue derived tumor cell growth. T/C: treated to control value. na means the treatment group wasn’ t set up for this patient.
  • FIG. 5 provides the results of a PBMC stimulation assay showing that the candidate antibody NBP-4 significantly augmented the secretion of IFN- ⁇ by activated T cells, surpassing the effect of both anti-PD-1 antibody (nivolumab) and anti-Tim-3 antibody (3E6) , alone or in combination.
  • FIG. 6 provides the results of a cytokine production assay in a MLR showing that the candidate antibody NBP-4 significantly increased IL-2 release by T cells in MLR, surpassing the effect of both anti-PD-1 antibody (nivolumab) and anti-Tim-3 antibody (3E6) , alone or in combination.
  • the present disclosure provides bispecific antibodies that specifically bind to human Tim-3 and human PD-1.
  • Pharmaceutical compositions comprising a therapeutically effective amount of such bispecific antibodies, methods of uses of the bispecific antibodies or the pharmaceutical compositions disclosed herein for treating cancer (e.g., Tim-3 and/or PD-1-expressing cancer) are also provided.
  • Tim-3 expression is also important in regulating the activities of many innate immune cells (e.g., macrophages, monocytes, dendritic cells, mast cells, and natural killer cells) .
  • innate immune cells e.g., macrophages, monocytes, dendritic cells, mast cells, and natural killer cells
  • Tim-3 is considered a promising candidate for cancer immunotherapy, in part, because it is upregulated in immune cell populations that constitute immunosuppression in tumor environment of many human cancers.
  • Tim-3 is found to suppress antitumor T-cell responses by T-cell extrinsic mechanisms involving myeloid cells and different Tim-3/ligand interactions.
  • PD-1 Programmed Cell Death 1 protein
  • DCD1 Programmed Cell Death 1 protein
  • HPD-1 Protein PD-1
  • CD279 Antigen HPD-L
  • HSLE1 SLEB2
  • PD-1 functioning as an immune checkpoint, plays an important role in down-regulating the immune system by inhibiting the activation of T-cells, which in turn reduces autoimmunity and promotes self-tolerance.
  • PD-L1 is expressed by a variety of cancer cells and the expression thereof is thought to be at least in part responsible for a dampening of an immune response against the cancer cell.
  • a number of antibodies that block the activity of PD-1 or PD-L1 activate the immune system to attack tumors and are therefore used with success to treat some types of cancer.
  • PD-1 includes any variants or isoforms of PD-1 which are naturally expressed by cells.
  • An exemplary sequence is provided below (SEQ ID NO: 98; UniProt: Q15116) .
  • Human PD- consists of 288 amino acids, including an extracellular region (amino acid residues 24-170) , a transmembrane domain (amino acid residues 171-191) , and a cytoplasmic domain (amino acid residues 192-288) .
  • the sequence can be further processed into a mature form.
  • Exemplary sequences of various isoforms of human PD-1 can be found with accession numbers, e.g., ENSG00000188389; OMIM: 600244; and NCBI: NP_005009.2.
  • Tim-3 the inhibitory receptor PD-1.
  • many tumor-specific T cells express both PD-1 and Tim-3, and these T cells have been shown to be more dysfunctional compared to T cells that express only PD-1 or Tim-3 (Fourcade et al., 2010, J. Exp. Med. 207, 2175-2186) .
  • Provided herein are bispecific antibodies or that bind both PD-1 and Tim-3, which provide surprising and synergist anti-tumor response in mouse models, even compared to the combination of anti-PD-1 antibody and anti-Tim-3 antibody.
  • an entity refers to one or more of that entity; for example, “an antibody, ” is understood to represent one or more antibodies.
  • polynucleotide, ” “nucleic acid, ” and their grammatical equivalents as used interchangeably herein mean polymers of nucleotides of any length and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • binding affinity generally refers to the strength of the sum total of noncovalent interactions between a binding moiety and a target molecule (e.g., antigen) .
  • the binding of a binding moiety and a target molecule is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (K D ) .
  • K D is the ratio of a dissociation rate (k off or k d ) to the association rate (k on or k a ) .
  • K D is the ratio of a dissociation rate (k off or k d ) to the association rate (k on or k a ) .
  • the lower the K D of a binding pair the higher the affinity.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following.
  • the “K D ” or “K D value” can be measured by assays known in the art, for example by a binding assay.
  • the K D may be measured in a radiolabeled antigen binding assay (RIA) (Chen, et al., (1999) J. Mol Biol 293: 865-881) .
  • the K D or K D value can also be measured by using biolayer interferometry (BLI) using, for example, the Gator system (Probe Life) , or the Octet-96 system (Sartorius AG) .
  • the K D or K D value can also be measured by using surface plasmon resonance assays (SPR) by Biacore, using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 BIAcore, Inc., Piscataway, NJ) .
  • SPR surface plasmon resonance assays
  • identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between.
  • identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
  • a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • treat and its grammatical equivalents as used herein in connection with a disease or a condition, or a subject having a disease or a condition refer to an action that suppresses, eliminates, reduces, and/or ameliorates a symptom, the severity of the symptom, and/or the frequency of the symptom associated with the disease or disorder being treated.
  • administer and its grammatical equivalents as used herein refer to the act of delivering, or causing to be delivered, a therapeutic or a pharmaceutical composition to the body of a subject by a method described herein or otherwise known in the art.
  • the therapeutic can be a compound, a polypeptide, an antibody, a cell, or a population of cells.
  • Administering a therapeutic or a pharmaceutical composition includes prescribing a therapeutic or a pharmaceutical composition to be delivered into the body of a subject.
  • an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to the subject.
  • the therapeutically effective amount can be ascertained by measuring relevant physiological effects. The exact amount required varies from subject to subject, depending on the age, weight, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. An appropriate “effective amount” in any individual case can be determined by one of ordinary skill in the art using routine experimentation.
  • a subject can have a particular disease or condition.
  • a subject can be at risk of having a particular disease or condition.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • GenBank numbers GI numbers and/or SEQ ID NOS. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to Uniprot (https: //www. uniprot. org/) , GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .
  • bispecific antibodies that specifically bind both human Tim-3 and human PD-1.
  • the bispecific antibodies provided herein are monoclonal antibodies.
  • the bispecific antibodies provided herein are isolated.
  • the bispecific antibodies provided herein are substantially pure.
  • an “antibody” is an immunoglobulin molecule that recognizes and specifically binds a target (e.g., a protein) through at least one antigen-binding fragment which is typically within the variable region of the immunoglobulin molecule.
  • An “antibody” can be of many different types and structures. For example, antibodies can be polyclonal antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, or any other modified immunoglobulin molecule comprising an antigen-binding site. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
  • An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) , based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the term “antibody” as used herein include “antigen-binding fragment” of intact antibodies.
  • the term “antigen-binding fragment” as used herein refers to a portion or fragment of an intact antibody that is the antigenic determining variable region of an intact antibody.
  • immunoglobulins have been well characterized (see, e.g., FUNDAMENTAL IMMUNOLOGY Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989) ) .
  • immunoglobulins comprise two pairs of polypeptide chains, one pair of light (L; low molecular weight) chains and one pair of heavy (H; high molecular weight) chains, all four inter-connected by disulfide bonds.
  • Each light chain of an immunoglobulin typically includes a light chain variable region ( “VL region” ) and a light chain constant region ( “CL region” ) .
  • VL region variable region
  • CL region light chain constant region
  • ⁇ kappa
  • ⁇ lambda
  • the amino acid sequences of the CL regions are well known in the art.
  • the CH region of immunoglobulins comprise more than one domain.
  • the CH region of an IgG antibody is comprised of three domains, heavy chain constant domain 1 (CH1) , heavy chain constant domain 2 (CH2) , and heavy chain constant domain 3 (CH3) .
  • CH1 and CH2 domains The highly flexible region between the CH1 and CH2 domains is referred to as the “hinge region. ” Disulfide bonds in the hinge region are part of the interactions between two heavy chains in an immunoglobulin.
  • the “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • Table 1B Exemplary variants of human IgG constant regions/domains.
  • the VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops) , also termed complementarity determining regions (CDRs) , interspersed with regions that are more conserved, termed framework regions (FRs) .
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR) .
  • the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen.
  • the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with (1) Q1S and Y3V substitutions, (2) Q1S and N4Q substitutions, (3) Q1S and S5P substitutions, (4) Q1S and P7N substitutions, (5) Y3V and N4Q substitutions, (6) Y3V and S5P substitutions, (7) Y3V and P7N substitutions, (8) N4Q and S5P substitutions, (9) N4Q and P7N substitutions, (10) S5P and P7N substitutions, (11) Q1S, Y3V and N4Q substitutions, (12) Q1S, Y3V and S5P substitutions, (13) Q1S, Y3V and P7N substitutions, (14) Q1S, N4Q and S5P substitutions, (15) Q1S, N4Q and P7N substitutions, (16) Q1S, S5P and P7N substitutions, (17) Y3
  • the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1S substitution. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1E substitution. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Y3V substitution. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with N4Q substitution. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with S5P substitution.
  • the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with P7N substitution. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1S and Y3V substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1S and N4Q substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1S and S5P substitutions.
  • the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with N4Q, S5P and P7N substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1S, Y3V, N4Q and S5P substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1S, Y3V, N4Q, and P7N substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1S, Y3V, S5P and P7N substitutions.
  • the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1E and N4Q substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1E and S5P substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1E and P7N substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1E, Y3V and N4Q substitutions.
  • the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1E, Y3V and S5P substitutions. In some embodiments, the VL CDR3 o fVL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1E, Y3V and P7N substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1E, N4Q and S5P substitutions. In some embodiments, the VL CDR3 of VL1 can have the amino acid sequence of SEQ ID NO: 103 with Q1E, N4Q and P7N substitutions.
  • the VL1/VH1 pair comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 101, 102, 137, 104, 105, and 106 respectively.
  • the VL1/VH1pair comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 101, 102, 138, 104, 105, and 106 respectively.
  • the VL1/VH1 pair comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 101, 102, 139, 104, 105, and 106 respectively.
  • the VL1/VH1 pair comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 101, 102, 140, 104, 105, and 106respectively.
  • the VL1/VH1 pair comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 101, 102, 143, 104, 105, and 106 respectively.
  • bispecific antibodies provided herein comprise a VL/VH pair of anti-Tim-3 antibody h3E6, which is a humanized antibody based on chimeric antibody 3E6 is disclosed in PCT Application Nos: PCT/CN2022/088770 and PCT/CN2022/127052, which are hereby incorporated by reference in their entireties. Some exemplary VLs and VHs are provided below. As used herein, a VL/VH pair of h3E6 can comprise the VL/VH of any humanized 3E6.
  • the VL1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the VL1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%identical to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the VL1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 80.
  • the VL1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the VL1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, the VL1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the VL1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 84.
  • the VH1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 85.
  • the VH1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the VH1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the VH1 is at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 88.
  • the VH1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the VH1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the VH1 is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 91.
  • Table 2A Exemplary sequences of humanized 3E6 (h3E6) .
  • the VL1/VH1 pair of the bispecific antibodies disclosed herein that specific binds human Tim-3 can be any combination of the specific sequences provided in Table 2A, or variants thereof that have at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity with the exemplified sequences.
  • the VL1/VH1 pair that specifically binds human Tim-3 can have a VL1 having the amino acid of SEQ ID NO: 3, and a VH1 having an amino acid selected from the group consisting of SEQ ID NOs: 4 and 85-91.
  • the VL1 has the amino acid sequence of SEQ ID NO: 76, and the VH1 has an amino acid selected from the group consisting of SEQ ID NOs: 4 and 85-91. In some embodiments, the VL1 has the amino acid sequence of SEQ ID NO: 77, and the VH1 has an amino acid selected from the group consisting of SEQ ID NOs: 4 and 85-91. In some embodiments, the VL1 has the amino acid sequence of SEQ ID NO: 78, and the VH1 has an amino acid selected from the group consisting of SEQ ID NOs: 4 and 85-91.
  • the VL1 has the amino acid sequence of SEQ ID NO: 82, and the VH1 has an amino acid selected from the group consisting of SEQ ID NOs: 4 and 85-91. In some embodiments, the VL1 has the amino acid sequence of SEQ ID NO: 83, and the VH1 has an amino acid selected from the group consisting of SEQ ID NOs: 4 and 85-91. In some embodiments, the VL1 has the amino acid sequence of SEQ ID NO: 84, and the VH1 has an amino acid selected from the group consisting of SEQ ID NOs: 4 and 85-91.
  • the VL1/VH1 pair that specifically binds human Tim-3 can have a VL1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84, and a VH1 having the amino acid sequence of SEQ ID NO: 4.
  • the VL1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84, and a VH1 having the amino acid sequence of SEQ ID NO: 85.
  • the VL1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84, and a VH1 having the amino acid sequence of SEQ ID NO: 86.
  • the VL1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84, and a VH1 having the amino acid sequence of SEQ ID NO: 90. In some embodiments, the VL1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84, and a VH1 having the amino acid sequence of SEQ ID NO: 91.
  • the VL2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to SEQ ID NO: 1.
  • the VH2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to SEQ ID NO: 2, respectively.
  • the VL2 and VH2 have amino acid sequences of SEQ ID NOs: 1 and 2, respectively.
  • the VL2 and VH2 that specifically bind to human PD-1 have the VL and VH of zimberelimab. In some embodiments, the VL2 and VH2 that specifically bind to human PD-1 have the amino acid sequences of SEQ ID NO: 119 and SEQ ID NO: 120, respectively. In some embodiments of the bispecific antibodies disclosed herein, the VL2 and VH2 that specifically bind to human PD-1 have the VL and VH of toripalimab. In some embodiments, the VL2 and VH2 that specifically bind to human PD-1 have the amino acid sequences of SEQ ID NO: 121 and SEQ ID NO: 122, respectively.
  • the VL2 and VH2 that specifically bind to human PD-1 have the VL and VH of pucotenlimab. In some embodiments, the VL2 and VH2 that specifically bind to human PD-1 have the amino acid sequences of SEQ ID NO: 131 and SEQ ID NO: 132, respectively.
  • the bispecific antibodies provided herein have a VL1/VH1 pair that specifically binds to human Tim-3 and a VL2/VH2 pair that specifically binds to human PD-1.
  • the VL1/VH1 pair and the VL2/VH2 pair can be any of the VL/VH pairs disclosed herein.
  • the VL1/VH1 pair that specifically binds to human Tim-3 can be those disclosed in Table 2A.
  • the VL2/VH2 pair that specifically binds to human PD-1 can be those disclosed in Table 2B.
  • VL1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84; VH1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 85-91; and the VL2 and VH2 have the amino acid sequences of SEQ ID NOs: 1 and 2, respectively. In some embodiments, the VL1 and VH1 have the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.
  • bispecific antibodies provided herein have (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a CL region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a CH region and a single chain variable fragment (scFv) , wherein the scFv comprises, from N-terminus to C-terminus, VL2, a linker (L) , and VH2.
  • bispecific antibodies provided herein have (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a CL region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a CH region and an scFv, wherein the scFv comprises, from N-terminus to C-terminus, VH2, a linker (L) , and VL2.
  • bispecific antibodies provided herein have (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH region, and an scFv, wherein the scFv comprises, from N-terminus to C-terminus, VL1, a linker (L) , and VH1.
  • the bispecific antibodies provided herein comprises two identical pairs of C1/C2, forming two binding sites for Tim-3 and two binding sites for PD-1.
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH region is human IgG1 CH region with L234A, L235A and P329G substitutions (SEQ ID NO: 25)
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH region is human IgG1 CH region with L234A and L235A substitutions (SEQ ID NO: 26) .
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH region is human IgG1 CH region with L234A, L235A, P329G and K447A substitutions (SEQ ID NO: 27)
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH region is human IgG4 CH region with S228P substitution (SEQ ID NO: 28) .
  • the CL region is C ⁇ (SEQ ID NO: 8) , and the CH region is human IgG1 CH region with L234A, L235A and P329G substitutions (SEQ ID NO: 25) .
  • the CL region is C ⁇ (SEQ ID NO: 8) , and the CH region is human IgG1 CH region with L234A and L235A substitutions (SEQ ID NO: 26) .
  • the GS linker can have the amino acid sequence of SEQ ID NO: 47.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 48.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 49.
  • the linker is a TF linker having the amino acid sequence of SEQ ID NO: 50.
  • the linker has the amino acid sequence selected from the group consisting of SEQ ID NOs: 51-53.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 42.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 45.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 47.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 48.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 49.
  • the second linker is a TF linker having the amino acid sequence of SEQ ID NO: 50.
  • the second linker has the amino acid sequence selected from the group consisting of SEQ ID NOs: 51-53. z
  • Ab No. 10 has two peptide chains, C1 and C2, wherein (1) C1 comprises, from N-terminus to C-terminus, VL of h3E6 (e.g., SEQ ID NO: 3) and a C ⁇ region (e.g., SEQ ID NO: 7) ; and (2) C2 comprising, from N-terminus to C-terminus, a VH of h3E6 (e.g., SEQ ID NO: 4) , an CH region with L234A, L235A, P329G and K447A substitutions (e.g., SEQ ID NO: 27) , a linker (e.g., SEQ ID NO: 49) , and an scFv comprising, from N-terminus to C-terminus, VH of
  • Ab No. 12 has two peptide chains, C1 and C2, wherein (1) C1 comprises, from N-terminus to C-terminus, VL of nivolumab (e.g., SEQ ID NO: 1) and a C ⁇ region (e.g., SEQ ID NO: 7) ; and (2) C2 comprising, from N-terminus to C-terminus, a VH of nivolumab (e.g., SEQ ID NO: 2) , an CH region with L234A, L235A, P329G and K447A substitutions (e.g., SEQ ID NO: 27) , a linker (e.g., SEQ ID NO: 49) , and an scFv comprising, from N-terminus to C-terminus, VH of h3E6 (e.g., SEQ ID NO: 4) , a linker (e.g., SEQ ID NO: 49) and VL of h3E6 (e
  • the VL of h3E6 can be the VL of any humanized 3E6. In some embodiments, the VL of h3E6 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84.
  • the VH of h3E6 can be the VH of any humanized 3E6. In some embodiments, the VH of h3E6 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 85-91.
  • Table 3A Exemplary Bispecific Antibodies (IgG-scFv)
  • C1 has an amino acid sequence that is at least 85%identical to SEQ ID NO: 63.
  • C1 can have an amino acid sequence that is at least 90%identical to SEQ ID NO: 63.
  • C1 can have an amino acid sequence that is at least 95%identical to SEQ ID NO: 63.
  • C1 can have an amino acid sequence that is at least 98%identical to SEQ ID NO: 63.
  • C1 can have an amino acid sequence that is at least 99%identical to SEQ ID NO: 63.
  • C1 can have the amino acid sequence of SEQ ID NO: 63.
  • C2 has an amino acid sequence that is at least 85%identical to SEQ ID NO: 64.
  • bispecific antibody that specifically bind to human Tim-3 and human PD-1, wherein the bispecific antibody has a first peptide chain (C1) , and a second peptide chain (C2) , wherein C1 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 65, and C2 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 66.
  • C1 has an amino acid sequence that is
  • the bispecific antibodies provided herein have a VL1/VH1 pair that specifically binds to human Tim-3 and a VL2/VH2 pair that specifically binds to human PD-1.
  • the VL1/VH1 pair and the VL2/VH2 pair can be any of the VL/VH pairs disclosed herein.
  • the VL1/VH1 pair that specifically binds to human Tim-3 can be those disclosed in Table 2A.
  • the VL2/VH2 pair that specifically binds to human PD-1 can be those disclosed in Table 2B.
  • the bispecific antibodies provided herein have (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1, VL2, and a CL region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, VH2, and a CH region.
  • the bispecific antibodies provided herein comprises two identical pairs of C1/C2, forming two binding sites for Tim-3 and two binding sites for PD-1.
  • the bispecific antibodies provided herein in DVD-Ig format also comprise a CL region and a CH region.
  • the amino acid sequences of the CL region and the CH region of the bispecific antibodies disclosed herein can be derived from any appropriate source, e.g., a constant region of an antibody such as an IgG1, IgG2, IgG3, or IgG4.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG1.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG2.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG3.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG4.
  • the amino acid sequences of the CL region and the CH of the bispecific antibodies disclosed herein can comprise one or more amino acid substitutions that differ from the wildtype immunoglobulin, e.g., one or more amino acid substitutions in a wild type IgG1 or IgG4. Such substitutions are known in the art (see, e.g., US7704497, US7083784, US6821505, US 8323962, US6737056, and US7416727) .
  • the CH region of the bispecific antibodies provided herein is human IgG1 CH region (SEQ ID NO: 9) . In some embodiments, the CH region of the bispecific antibodies provided herein is human IgG1 CH region with L234A, L235A and P329G substitutions (SEQ ID NO: 25) . In some embodiments, the CH region of the bispecific antibodies provided herein is human IgG1 CH region with L234A and L235A substitutions (SEQ ID NO: 26) . In some embodiments, the CH region of the bispecific antibodies provided herein is human IgG1 CH region with L234A, L235A, P329G and K447A substitutions (SEQ ID NO: 27) . In some embodiments, the CH region is human IgG4 CH region (SEQ ID NO: 12) . In some embodiments, the CH region is human IgG4 CH region with S228P substitution (SEQ ID NO: 28) .
  • the CL region of the bispecific antibodies provided herein can be kappa CL (C ⁇ ; SEQ ID NO: 7) . In some embodiments, the CL region of the bispecific antibodies provided herein can be lambda CL (C ⁇ ; SEQ ID NO: 8) .
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH region is human IgG1 CH region with L234A, L235A and P329G substitutions (SEQ ID NO: 25)
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH region is human IgG1 CH region with L234A and L235A substitutions (SEQ ID NO: 26) .
  • the CL region is C ⁇ (SEQ ID NO: 8) , and the CH region is human IgG1 CH region with L234A, L235A and P329G substitutions (SEQ ID NO: 25) .
  • the CL region is C ⁇ (SEQ ID NO: 8) , and the CH region is human IgG1 CH region with L234A and L235A substitutions (SEQ ID NO: 26) .
  • VL1 and VL2 can be connected by a first linker, and the VH1 and VH2 can be connected by a second linker.
  • the first linker and second linker can independently be any suitable linker disclosed herein or otherwise known in the art.
  • the first linker can be selected from those identified in Table 1C.
  • the first linker is a GS linker having the amino acid sequence of SEQ ID NO: 44 or 46, or a TF linker having the amino acid sequence of SEQ ID NO: 50.
  • the second linker is a GS linker having the amino acid sequence of SEQ ID NO: 44 or 46, or a TF linker having the amino acid sequence of SEQ ID NO: 50.
  • the first linker consists of amino acids 1 to m of the CL region and the second linker consists of amino acids 1 to n of the CH region; wherein m and n are each independently an integer between 4 and 20. In some embodiments, the first linker consists of amino acids 1 to m of the CL region, wherein m is an integer between 4 and 20.
  • the CL region can be kappa CL (C ⁇ ; SEQ ID NO: 7) .
  • the CL region can be lambda CL (C ⁇ ; SEQ ID NO: 8) .
  • m can be an integer between 8 and 16. In some embodiments, m can be an integer between 10 and 14. In some embodiments, m is 8. In some embodiments, m is 12.
  • the VL of h3E6 can be the VL of any humanized 3E6. In some embodiments, the VL of h3E6 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84.
  • the VH of h3E6 can be the VH of any humanized 3E6. In some embodiments, the VH of h3E6 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 85-91.
  • Ab No. 4 has two peptide chains, C1 and C2, wherein (1) C1 comprises, from N-terminus to C-terminus, VL of nivolumab (e.g., SEQ ID NO: 1) , a first linker (e.g., SEQ ID NO: 54) , VL of h3E6 (e.g., SEQ ID NO: 3) , and a C ⁇ region (e.g., SEQ ID NO: 7) ; and (2) C2 comprising, from N-terminus to C-terminus, a VH of nivolumab (e.g., SEQ ID NO: 2) , a second linker (e.g., SEQ ID NO: 55) , VH of h3E6 (e.g., SEQ ID NO: 4) , and an IgG1 CH region with L234A, L235A, and P329G substitutions (e.g., SEQ ID NO: 25)
  • C1 comprises, from
  • Table 4A Exemplary Bispecific Antibodies (DVD-Ig)
  • C2 can have an amino acid sequence that is at least 90%identical to SEQ ID NO: 70.
  • C2 can have an amino acid sequence that is at least 95%identical to SEQ ID NO: 70.
  • C2 can have an amino acid sequence that is at least 98%identical to SEQ ID NO: 70.
  • C2 can have an amino acid sequence that is at least 99%identical to SEQ ID NO: 70.
  • C2 can have the amino acid sequence of SEQ ID NO: 70.
  • provided herein are bispecific antibody that specifically bind to human Tim-3 and human PD-1 having a first peptide chain (C1) and a second peptide chain (C2) , wherein C1 and C2 have amino acid sequences of SEQ ID NOs: 69 and 70, respectively.
  • the bispecific antibodies have four peptide chains, including two identical C1 and two identical C2.
  • the bispecific antibodies provided herein have the “knobs-into-holes” or “KIH” structure depicted in FIG. 1C.
  • the “KIH” model promotes formation of heterodimers of the engineered bispecific antibody instead of heavy chain homodimers.
  • the modification promoting the association of a pair of Fc domains in a bispecific antibody includes the so-called “knob-into-hole” modification, comprising a “knob” modification in one Fc domain and a “hole” modification in the other one.
  • the knob-into-hole technology is described e.g., in US 5,731,168; US 7,695,936; Ridgway et al., Prot. Eng. 9, 617-621 (1996) and Carter, J Immunol. Meth. 248, 7-15 (2001) .
  • the method involves introducing a protuberance ( “knob” ) at the interface of a first Fc (the “Knob-Fc” ) and a corresponding cavity ( “hole” ) in the interface of a second Fc (the “Hole-Fc” ) , such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan) .
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) .
  • a “Knob-Fc region” and a “Hole-Fc region” are designed to form heterodimer pair.
  • the Knob-Fc region refers to the Fc region in which an amino acid of the CH3 domain is replaced with an amino acid residue having a larger side chain volume, generating a protuberance within the CH3 domain positionable in a cavity within the CH3 domain of the Hole-Fc region, in which an amino acid residue of the CH3 domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain within which the protuberance within the CH3 domain of the first subunit is positionable.
  • the C2 and C3 pair of the bispecific antibodies can adopts a KIH design, wherein C2 includes a Hole-Fc region and the C3 includes a Knob-Fc region.
  • the C2 includes a Knob-Fc region and the C3 includes a Hole-Fc region.
  • the bispecific antibodies provided herein have the KIH structure and have a VL1/VH1 pair that specifically binds to human Tim-3 and a VL2/VH2 pair that specifically binds to human PD-1.
  • the VL1/VH1 pair and the VL2/VH2 pair can be any of the VL/VH pairs disclosed herein.
  • the VL1/VH1 pair that specifically binds to human Tim-3 can be those disclosed in Table 2A.
  • the VL2/VH2 pair that specifically binds to human PD-1 can be those disclosed in Table 2B.
  • VL1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84; VH1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 85-91; and the VL2 and VH2 have the amino acid sequences of SEQ ID NOs: 1 and 2, respectively. In some embodiments, the VL1 and VH1 have the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.
  • the bispecific antibodies provided herein can have (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a CH1 domain, and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Hole-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VH2, a linker (L) , and VL2.
  • the bispecific antibodies provided herein can have (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH1 domain, and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Knob-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VH1, a linker (L) , and VL1.
  • the bispecific antibodies provided herein can have (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH1 domain, and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Hole-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VL1, a linker (L) , and VH1.
  • the bispecific antibodies provided herein can have (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH1 domain, and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Hole-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VH1, a linker (L) , and VL1.
  • the bispecific antibodies provided herein in KIH format also comprise a CL region, a CH1 domain, a Hole-Fc and a Knob-Fc region.
  • the amino acid sequences of the CH1 domain, the CL region, and the Fc regions of the bispecific antibodies disclosed herein can be derived from any appropriate source, e.g., a constant region of an antibody such as an IgG1, IgG2, IgG3, or IgG4.
  • Antibody heavy and light chain constant regions amino acid sequences are well known in the art, e.g., those provided in the IMGT database (www. imgt. org) or at www. vbase2. org/vbstat. php., both of which are incorporated by reference herein.
  • the CH1 domain of the bispecific antibodies provided herein can be selected from the group consisting of a human IgG1 CH1 domain (SEQ ID NO: 13) , a human IgG2 CH1 domain (SEQ ID NO: 14) , a human IgG3 CH1 domain (SEQ ID NO: 15) , and a human IgG4 CH1 domain (SEQ ID NO: 16) .
  • the CH1 domain is human IgG1 CH1 domain (SEQ ID NO: 13) .
  • the Knob-Fc region is a human IgG1 Fc region variant having up to ten amino acids substitutions, including a T366W substitution; and the Hole-Fc region is a human IgG1 Fc region variant having up to ten amino acids substitutions, including a Y407V substitution.
  • the Hole-Fc region is human IgG1 Fc having a Y407T substitution.
  • the Hole-Fc region can further include T366S and L368A substitutions.
  • the Knob-Fc and Hole-Fc regions can further include S354C and Y349C substitutions, respectively.
  • the Knob-Fc and Hole-Fc regions can further include E356C and Y349C substitutions, respectively.
  • the Fc region further have L234A, L235A and P329G substitutions.
  • the Fc region further have L234A and L235A substitutions. All amino acid residues are numbered according to the EU Index.
  • the Knob-Fc region can have an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, 35, 37, and 39.
  • the Hole-Fc region can have an amino acid sequence selected from group consisting of SEQ ID NOs: 30, 32, 34, 36, 38, and 40.
  • the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 29 and 30, respectively. In some embodiments, the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 31 and 32, respectively. In some embodiments, the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 33 and 34, respectively. In some embodiments, the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 35 and 36, respectively.
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH1 region is human IgG1 CH1 region (SEQ ID NO: 13)
  • the Knob-Fc and Hole-Fc have the amino acid sequences of SEQ ID NOs: 29 and 30, respectively.
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH1 region is human IgG1 CH1 region (SEQ ID NO: 13)
  • the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 31 and 32, respectively.
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH1 region is human IgG1 CH1 region (SEQ ID NO: 13)
  • the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 33 and 34, respectively.
  • the CL region is C ⁇ (SEQ ID NO: 7)
  • the CH1 region is human IgG1 CH1 region (SEQ ID NO: 13)
  • the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 35 and 36, respectively.
  • the VL and the VH are connected by a linker.
  • the linker can be any suitable linker disclosed herein or otherwise known in the art.
  • the linker can be selected from those identified in Table 1C.
  • the linker is a GS linker having the amino acid sequence of SEQ ID NO: 44 or 46, or a TF linker having the amino acid sequence of SEQ ID NO: 50.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 44.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 46.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 42.
  • the GS linker can have the amino acid sequence of SEQ ID NO: 45.
  • h3E6L a VL of h3E6
  • h3E6H a VH of h3E6
  • NivL the VL of nivolumab (SEQ ID NO: 1)
  • NivH refers: the VH of Nivolumab (SEQ ID NO: 2)
  • C ⁇ kappa CL
  • C ⁇ lambda CL
  • IgG1 CH1 IgG1 CH1 domain (SEQ ID NO: 13)
  • (L) linker
  • IgG1 Fc AA-C-SAV IgG1 with L234A, L235A, Y349C, T366S, L368A, Y407V
  • IgG1 Fc AAG-C-SAV IgG1 with L234A, L235A, P329G, Y349C, T366S, L368A, Y407V
  • IgG1 Fc AA-C-W IgG1 with L2
  • bispecific antibody that specifically bind to human Tim-3 and human PD-1, wherein the bispecific antibody has a first peptide chain (C1) , a second peptide chain (C2) and a third peptide chain (C3) , wherein C1 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 57, C2 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 57, C
  • C2 can have an amino acid sequence that is at least 90%identical to SEQ ID NO: 58.
  • C2 can have an amino acid sequence that is at least 95%identical to SEQ ID NO: 58.
  • C2 can have an amino acid sequence that is at least 98%identical to SEQ ID NO: 58.
  • C2 can have an amino acid sequence that is at least 99%identical to SEQ ID NO: 58.
  • C2 can have the amino acid sequence of SEQ ID NO: 58.
  • C3 has an amino acid sequence that is at least 85%identical to SEQ ID NO: 59.
  • C3 can have an amino acid sequence that is at least 90%identical to SEQ ID NO: 59.
  • C3 can have an amino acid sequence that is at least 95%identical to SEQ ID NO: 59.
  • C3 can have an amino acid sequence that is at least 98%identical to SEQ ID NO: 59.
  • C3 can have an amino acid sequence that is at least 99%identical to SEQ ID NO: 59.
  • C3 can have the amino acid sequence of SEQ ID NO: 59.
  • bispecific antibody that specifically bind to human Tim-3 and human PD-1 having a first peptide chain (C1) , a second peptide chain (C2) , and a third peptide chain (C3) , wherein C1, C2, and C3 have amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively.
  • C1 has an amino acid sequence that is at least 85%identical to SEQ ID NO: 60.
  • C1 can have an amino acid sequence that is at least 90%identical to SEQ ID NO: 60.
  • C1 can have an amino acid sequence that is at least 95%identical to SEQ ID NO: 60.
  • C1 can have an amino acid sequence that is at least 98%identical to SEQ ID NO: 60.
  • C1 can have an amino acid sequence that is at least 99%identical to SEQ ID NO: 60.
  • C1 can have the amino acid sequence of SEQ ID NO: 60.
  • C2 has an amino acid sequence that is at least 85%identical to SEQ ID NO: 61.
  • C2 can have an amino acid sequence that is at least 90%identical to SEQ ID NO: 61.
  • bispecific antibody that specifically bind to human Tim-3 and human PD-1 having a first peptide chain (C1) , a second peptide chain (C2) , and a third peptide chain (C3) , wherein C1, C2, and C3 have amino acid sequences of SEQ ID NOs: 60, 61, and 62, respectively.
  • the present disclosure further contemplates additional variants and equivalents that are substantially homologous to the bispecific antibodies described herein.
  • it is desirable to modulate biological properties of the antibody including but not limited to, specificity, thermostability, expression level, effector function (s) , glycosylation, immunogenicity, and/or solubility.
  • amino acid changes may alter post-translational processes of an antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.
  • Antibodies comprising functional variants of the heavy chain, light chains, VL regions, VH regions, or one or more CDRs of the antibodies of the examples as also provided herein.
  • a functional variant of a heavy chain, a light chain, VL, VH, or CDRs used in the context of an antibody still allows the antibody to retain at least a substantial proportion (at least about 90%, 95%or more) of functional features of the “reference” and/or “parent” antibody, including affinity and/or the specificity/selectivity, Fc inertness and PK parameters such as half-life, Tmax, Cmax.
  • Such functional variants typically retain significant sequence identity to the parent antibody and/or have substantially similar length of heavy and light chains.
  • Exemplary variants include those which differ from heavy and/or light chains, VH and/or VL, and/or CDR regions of the parent antibody sequences mainly by conservative substitutions, e.g., 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant may be conservative amino acid residue replacements.
  • a variant of a bispecific antibody disclosed herein comprises the amino acid sequence of the parent antibody with one or more non-conservative amino acid substitutions. In some embodiments, a variant of a bispecific antibody disclosed herein comprises the amino acid sequence of the parent binding antibody with one or more non-conservative amino acid substitution, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant. In certain embodiments, the one or more conservative amino acid substitutions and/or the one or more non-conservative amino acid substitutions can enhance a biological activity of the variant, such that the biological activity of the functional variant is increased as compared to the parent antibody.
  • Variations can be a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native antibody or polypeptide sequence.
  • amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • Insertions or deletions can be in the range of about 1 to 5 amino acids.
  • a variant comprises one to 30 amino acid substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to 25 amino acid substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to 20 substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to 15 substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment.
  • a variant comprises one to 10 substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to five amino acid substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to three amino acid substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the amino acid substitution (s) is in a CDR of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitution (s) is not in a CDR of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitution (s) is in a framework region of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions, additions, and/or deletions are conservative amino acid substitutions.
  • variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.
  • Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) TECHNIQUES IN MOLECULAR BIOLOGY (MacMillan Publishing Company, New York) ; Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985) ; Kunkel et al., Methods Enzymol. 54: 367-382 (1987) ; Sambrook et al.
  • an antibody does not have one or more effector functions (e.g., “effectorless” antibodies) .
  • the antibody has no ADCC activity and/or no CDC activity.
  • the antibody does not bind an Fc receptor and/or complement factors.
  • the antibody has no effector function (s) .
  • the constant region modifications increase or enhance ADCC and/or CDC of the antibody.
  • the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. In some embodiments, the constant region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites.
  • the effector function can be complement dependent cytotoxicity (CDC) , antibody-dependent cell-mediated cytotoxicity (ADCC) , antibody-dependent cellular phagocytosis (ADCP) , cytokine secretion, or any combination thereof.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cytokine secretion or any combination thereof.
  • the effector function is ADCC.
  • the same one or more amino acid substitution is present in each of the two subunits of the Fc region.
  • the one or more amino acid substitution reduces the binding affinity of the Fc region to an Fc receptor.
  • the one or more amino acid substitution reduces the binding affinity of the Fc region to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.
  • Variants with reduced effector functions are known in the art and can be incorporated in the antibodies disclosed herein. For example, amino acid substitutions are known to reduce effector function.
  • hIgG1 L235A/G237A/E318A antibody is unable to bind to human cell lines expressing Fc ⁇ Rs, resulting in reduced ADCC.
  • hIgG1 and hIgG4 antibodies with L234A/L235A Fc domains have no detectable binding to the low affinity Fc ⁇ Rs and C1q and significantly reduced ADCC and CDC.
  • Mutations at specific residues in hIgG1 known to interact with both Fc ⁇ Rs and C1q can reduce binding to the low affinity Fc ⁇ Rs and result in no detectable binding to Fc ⁇ RI.
  • the G236R/L328R mutation pair reduces or completely abrogates binding to the Fc ⁇ Rs.
  • S267E substitution also reduces binding for all low affinity hFc ⁇ Rs.
  • the combination ofL234F/L235E/D265A potently silences the Fc region, resulting in no detectable binding to FcyRI, reduced binding to the low affinity FcyRs and reduced binding to C1q.
  • the S298G/T299A mutations are found to abolish or significantly reduce binding to C1q and most Fc ⁇ Rs except for Fc ⁇ RIIA-R131 and Fc ⁇ RIIB.
  • hIgG4 which has low affinity for all Fc ⁇ R
  • the serine at position 228 plays a pivotal role in F (ab) arm exchange.
  • the S228P substitution can provide homogeneous hIgG4, and is commonly introduced in therapeutic hIgG4 antibodies.
  • the human ⁇ 4 constant region can be used in Fc-silencing approaches. For example, exchanging the human ⁇ 1 region with that of human ⁇ 4 can reduce effector functionality.
  • Murine IgG2b isotype which also has low Fc ⁇ R binding activity, differs from hIgG4 at position 235.
  • Incorporating the mouse IgG2b residue (glutamic acid) into the hIgG4 antibody at this position can further minimize Fc effector function, resulting in an antibody (with the S228P/L235E mutations) with substantially reduced, if any, binding to all Fc ⁇ Rs and C1q, and no measurable ADCC. Additionally, rather than replacing the whole constant region of hIgG1 with hIgG4, specific amino acids from human ⁇ 4 can be introduced into antibodies of other IgG isotypes.
  • a combination of amino acid mutations-H268Q/V309L/A330S/P331S (IgG2m4) , when introduced into a hIgG2 backbone, can lead to no detectable binding to hFc ⁇ RI, hFc ⁇ RIIIA or C1q, reduced binding to hFc ⁇ RIIB and no change in binding to Fc ⁇ RIIA-H131 when compared to the WT hIgG2 antibody.
  • V234A/G237A/P238S/H268A/V309L/A330S/P331S (IgG2c4d) mutations can result in no detectable binding to any Fc ⁇ Rs or C1q and no measurable ADCC, ADCP or CDC when compared to the WT hIgG2 counterpart.
  • the bispecific antibodies disclosed herein comprise the NA mutation. In some embodiments, the bispecific antibodies disclosed herein comprise the AAA mutations. In some embodiments, the bispecific antibodies disclosed herein comprise the LALA mutations. In some embodiments, the bispecific antibodies disclosed herein comprise the RR mutations. In some embodiments, the bispecific antibodies disclosed herein comprise the GA mutations. In some embodiments, the bispecific antibodies disclosed herein comprise the FES mutations.
  • the bispecific antibodies disclosed herein comprise the LALAPG mutations. In some embodiments, the bispecific antibodies disclosed herein comprise the FEA mutations. In some embodiments, the bispecific antibodies disclosed herein comprise the IgG2m4 mutations. In some embodiments, the bispecific antibodies disclosed herein comprise the IgG2-PE mutations. In some embodiments, the bispecific antibodies disclosed herein comprise the IgG2c4d mutations. As a person of ordinary skill in the art would understand, the bispecific antibodies disclosed herein are not limited by specific Fc modifications, and any combination and permutations of the Fc modifications disclosed herein or otherwise known in the art that reduce the effector function or binding affinity to Fc ⁇ R can be adopted in bispecific antibodies disclosed herein.
  • the Fc region of the bispecific antibodies provided herein can include an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, P329 and K447. In some embodiments, the Fc region includes an amino acid substitution at a position selected from the group of L234, L235, P329 and K447. In some embodiments, the Fc region includes an amino acid substitution at a position selected from the group of L234, L235 and P329. In some embodiments, the Fc region includes the amino acid substitutions L234A and L235A. In some embodiments, the Fc region is an IgG1 Fc region, particularly a human IgG1 Fc region.
  • the Fc region comprises the amino acid mutations L234A, L235A, P329G and K447A. In some embodiments, the Fc region includes amino acid substitutions at positions P329, L234 and L235. In some embodiments, the Fc region comprises the amino acid mutations L234A, L235A and P329G. In some embodiments, the Fc region is an IgG4 Fc region, particularly a human IgG4 Fc region. In some embodiments, the Fc region includes amino acid substitution at position S228. In some embodiments, the Fc region comprises amino acid substitution S228P. All amino acid residues are numbered according to the EU index.
  • variants can include addition of amino acid residues at the amino-and/or carboxyl-terminal end of the antibody.
  • the length of additional amino acids residues can range from one residue to a hundred or more residues.
  • a variant comprises an N-terminal methionyl residue.
  • a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g., a fluorescent tag or an enzyme) .
  • bispecific antibodies disclosed herein can be chemically modified naturally or by intervention.
  • the bispecific antibodies are chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications can be carried out by known techniques.
  • the bispecific antibodies provided herein can comprise one or more analogs of an amino acid (including, for example, unnatural amino acids) , as well as other modifications known in the art.
  • bispecific antibodies of the present disclosure can be analyzed for their physical, chemical and/or biological properties by various methods known in the art.
  • abispecific antibody provided herein is tested for its ability to bind human Tim-3 and/or human PD-1.
  • Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore) , ELISA, and FACS.
  • antibodies can be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.
  • the anti-PD-1/Tim-3 bispecific antibodies disclosed herein can be attached to a solid support.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • an immobilized bispecific antibody is used in an immunoassay.
  • an immobilized bispecific antibody is used in purification.
  • polynucleotides encoding at least one peptide chain of the bispecific antibodies disclosed herein.
  • the polynucleotides provided herein encode one peptide.
  • the polynucleotides provided herein encode more than one peptide.
  • the polynucleotides provided herein can encode, for example, both peptide chains of a bispecific antibody provided herein (e.g., the bispecific antibodies in IgG-scFv or DVD-Ig format) .
  • the polynucleotides provided herein can encode two or three of the peptide chains of a bispecific antibody provided herein (e.g., the bispecific antibodies in KIH format) .
  • a bispecific antibody provided herein e.g., the bispecific antibodies in KIH format
  • polynucleotides encoding the C1, C2, C3 or any combination thereof of the anti-Tim-3/PD-1 bispecific antibodies disclosed herein in the KIH format are provided herein are polynucleotides encoding the C1, C2, C3 or any combination thereof of the bispecific antibodies exemplified in Table 6A.
  • polynucleotides encoding the C1, C2, or both of the anti-Tim-3/PD-1 bispecific antibodies disclosed herein in the IgG-scFv format are polynucleotides encoding the C1, C2, or both of the bispecific antibodies exemplified in Table 3A.
  • polynucleotides encoding the C1, C2, or both of the anti-Tim-3/PD-1bispecific antibodies disclosed herein in the DVD-Ig format for example, provided herein are polynucleotides encoding the C1, C2, or both of the bispecific antibodies exemplified in Table 4A.
  • Cistrons can be separated by, for example, an internal ribosomal entry site (IRES) or 2A element.
  • IRES refers to nucleotide sequences in an expression cassette which when transcribed into mRNA, can recruit ribosomes directly, without a previous scanning of untranslated region of mRNA by the ribosomes.
  • a 2A element as understood in the art, encoding self-cleaving short peptides (about 20 amino acids) that provide a mechanism for subsequent separation of equimolarly produced polypeptides of interest.
  • Illustrative 2A self-cleaving peptides include P2A, E2A, F2A, and T2A.
  • the term “encode” and its grammatical equivalents refer to the inherent property of specific sequences of nucleotides in a polynucleotide or a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.
  • polynucleotide that encodes a polypeptide encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences.
  • the polynucleotides of the disclosure can be in the form of RNA or in the form of DNA.
  • DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. Single stranded DNA can be the coding strand or non-coding (anti-sense) strand.
  • the polynucleotides of the disclosure can be mRNA.
  • a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence up to 5%of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted into the reference sequence.
  • These mutations of the reference sequence can occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both.
  • a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide.
  • a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code) .
  • Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. coli) .
  • a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.
  • a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • a polynucleotide is isolated. In some embodiments, a polynucleotide is substantially pure.
  • vectors comprising a polynucleotide disclosed herein.
  • vehicle and its grammatical equivalents as used herein refer to a vehicle that is used to carry genetic material (e.g., a polynucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • polynucleotides into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotides are expressed in a sufficient amount to produce a desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • vectors provided herein can be expression vectors.
  • vectors provided herein comprise a polynucleotide encoding at least one peptide chain of the bispecific antibodies described herein.
  • recombinant expression vectors which can be used to amplify and express a polynucleotide encoding at least one peptide chain of the bispecific antibodies described herein.
  • structural elements intended for use in certain expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • a polypeptide in situations where recombinant protein is expressed without a leader or transport sequence, can include an N-terminal methionine residue.
  • vectors are plasmid, autonomously replicating sequences, and transposable elements.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.
  • Exemplary transposon systems such as Sleeping Beauty and PiggyBac can be used, which can be stably integrated into the genome (e.g., Ivics et al., Cell, 91 (4) : 501–510 (1997) ; et al., (2007) Nucleic Acids Research. 35 (12) : e87) .
  • the vector is an episomal vector or a vector that is maintained extrachromosomally.
  • episomal vector refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
  • the vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV.
  • the lymphotrophic herpes virus may be Epstein Barr virus (EBV) , Kaposi's sarcoma herpes virus (KSHV) , Herpes virus saimiri (HS) , or Marek's disease virus (MDV) .
  • Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus.
  • the host cell comprises the viral replication transactivator protein that activates the replication.
  • “Expression control sequences, ” “control elements, ” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector-origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions-which interact with host cellular proteins to carry out transcription and translation.
  • Such elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including ubiquitous promoters and inducible promoters can be used.
  • Illustrative ubiquitous expression control sequences that can be used in present disclosure include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus40 (SV40) promoter (e.g., early or late) , a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1) , ferritin H (FerH) , ferritin L (FerL) , Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , eukaryotic translation initiation factor 4A1 (EIF4A1) , heat shock 70kDa protein 5 (H
  • inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone) , metallothionine promoter (inducible by treatment with various heavy metals) , MX-1 promoter (inducible by interferon) , the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323: 67) , the cumate inducible gene switch (WO 2002/088346) , tetracycline-dependent regulatory systems, etc.
  • steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone)
  • metallothionine promoter inducible by treatment with various heavy metals
  • MX-1 promoter inducible by interfer
  • bispecific antibodies described herein can be produced by any method known in the art, including chemical synthesis and recombinant expression techniques.
  • the practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art.
  • cells comprising the polynucleotides disclosed herein that encode at least one peptide chain of the bispecific antibodies described herein.
  • cells provided herein comprise a polynucleotide that encodes the C1, C2 and C3 of the bispecific antibodies disclosed herein in the KIH format.
  • cells provided herein comprise a plurality of the polynucleotides that collectively encode the C1, C2 and C3 of the bispecific antibodies disclosed herein in the KIH format.
  • cells provided herein comprise a polynucleotide that encodes both the C1 and C2 of the bispecific antibodies disclosed herein in the IgG-scFv format or the DVD-Ig format. In some embodiments, cells provided herein comprise a first polynucleotide that encodes C1 and a second polynucleotide that encodes C2 of the bispecific antibodies disclosed herein in the IgG-scFv format or the DVD-Ig format.
  • host cells comprising vectors disclosed herein are also contemplated.
  • host cells comprising a vector comprising a polynucleotide disclosed herein.
  • host cells provided herein comprise a vector or multiple vectors that collectively comprise the polynucleotides encoding the polypeptide chains of the bispecific antibodies described herein.
  • host cells provided herein produce the bispecific antibodies described herein.
  • suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived) , L-929 (murine fibroblast-derived) , C127 (murine mammary tumor-derived) , 3T3 (murine fibroblast-derived) , CHO (Chinese hamster ovary-derived) , HeLa (human cervical cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.
  • COS-7 monkey kidney-derived
  • L-929 murine fibroblast-derived
  • C127 murine mammary tumor-derived
  • 3T3 murine fibroblast-derived
  • CHO Choinese hamster ovary-derived
  • HeLa human cervical cancer-derived
  • BHK hamster kidney fibroblast-derived
  • HEK-293 human embryonic kidney-derived
  • Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • expression of recombinant proteins in insect cell culture systems e.g., baculovirus
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • the bispecific antibodies disclosed herein are comprised of more than one polypeptide chain, which can be produced separately or together.
  • methods provided herein produce at least one polypeptide chain of the bispecific antibodies disclosed herein.
  • methods provided herein produce all polypeptide chains of the bispecific antibodies disclosed herein.
  • bispecific antibodies or polypeptides described herein can be produced and isolated using methods known in the art.
  • Polyeptides can be synthesized, in whole or in part, using chemical methods (see, e.g., Caruthers (1980) . Nucleic Acids Res. Symp. Ser. 215; Horn (1980) ; and Banga, A.K., THERAPEUTIC PEPTIDES AND PROTEINS, FORMULATION, PROCESSING AND DELIVERY SYSTEMS (1995) Technomic Publishing Co., Lancaster, PA) .
  • Peptide synthesis can be performed using various solid phase techniques (see, e.g., Roberge, Science 269: 202 (1995) ; Merrifield, Methods. Enzymol.
  • host-expression vector systems can be utilized to recombinantly express the bispecific antibodies described herein or one or more of their polypeptide chains.
  • Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well-known in the art.
  • Such host-expression systems represent vehicles by which the coding sequences of the bispecific antibodies described herein can be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate polynucleotide coding sequences, express the bispecific antibodies described herein in situ.
  • These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for the compounds described herein; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding the compounds described herein; insect cell systems infected with recombinant virus expression vectors (e.g., baclovirus) containing the sequences encoding the compounds described herein; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding the molecules compounds described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S.
  • mammalian cell systems e.
  • Per C. 6 cells human retinal cells developed by Crucell harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • vectors can be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of the bispecific antibodies described herein, vectors which direct the expression of high levels of protein products that are readily purified can be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., (1983) , EMBO J. 2: 1791-1794) ; pIN vectors (Inouye et al., (1985) , Nucleic Acids Res. 13: 3101-3110; Van Heeke et al., (1989) , J. Biol. Chem.
  • pGEX vectors can also be used to express polypeptides as fusion proteins with glutathione S-transferase (GST) .
  • GST glutathione S-transferase
  • proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. In mammalian host cells, a number of viral-based expression systems can be utilized.
  • suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived) , L-929 (murine fibroblast-derived) , C127 (murine mammary tumor-derived) , 3T3 (murine fibroblast-derived) , CHO (Chinese hamster ovary-derived) , HeLa (human cervical cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.
  • COS-7 monkey kidney-derived
  • L-929 murine fibroblast-derived
  • C127 murine mammary tumor-derived
  • 3T3 murine fibroblast-derived
  • CHO Choinese hamster ovary-derived
  • HeLa human cervical cancer-derived
  • BHK hamster kidney fibroblast-derived
  • HEK-293 human embryonic kidney-derived
  • Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • Expression of recombinant proteins in insect cell culture systems e.g., baculovirus
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • modifications e.g., glycosylation
  • processing e.g., cleavage
  • the antibodies described herein can be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a polyprotein precursor) , requiring proteolytic cleavage by native or recombinant cellular mechanisms to form separate polypeptides of the bispecific antibodies described herein.
  • the disclosure thus encompasses engineering a nucleic acid sequence to encode a polyprotein precursor molecule comprising the polypeptides of the bispecific antibodies described herein, which includes coding sequences capable of directing post translational cleavage of said polyprotein precursor.
  • Post-translational cleavage of the polyprotein precursor results in the polypeptides of the bispecific antibodies described herein.
  • the post translational cleavage of the precursor molecule comprising the polypeptides of the compounds described herein can occur in vivo (i.e., within the host cell by native or recombinant cell systems/mechanisms, e.g. furin cleavage at an appropriate site) or can occur in vitro (e.g.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
  • cell lines which stably express compounds described herein can be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc. ) , and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method can advantageously be used to engineer cell lines which express the compounds described herein. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the compounds described herein.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., (1977) , Cell 11: 223-232) , hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., (1992) Bioessays 14: 495-500) , and adenine phosphoribosyltransferase (Lowy et al., (1980) , Cell 22: 817-823) genes can be employed in tk-, hgprt-or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., (1980) PNAS 77: 3567-3570; O' Hare et al., (1981) PNAS, 78: 1527-1531) ; gpt, which confers resistance to mycophenolic acid (Mulligan et al., (1981) PNAS, 78: 2072-2076) ; neo, which confers resistance to the aminoglycoside G-418 (Tolstoshev (1993) , Ann. Rev. Pharmacol. Toxicol.
  • bispecific antibodies described herein or their polypeptide chains can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987) .
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987) .
  • a marker in the vector system described herein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of a protein of interest, production of the protein of interest will also increase (Crouse et al., (1983) Mol. Cell. Biol
  • the host cell can be co-transfected with more than one expression vectors, each encoding a polypeptide chain of a bispecific antibody described herein.
  • the vectors can contain identical selectable markers which enable equal expression of all polypeptides.
  • a single vector can be used which encodes two or more polypeptides.
  • the coding sequences for the polypeptides of compounds described herein can comprise cDNA or genomic DNA.
  • a bispecific antibody described herein or polypeptide described herein can be purified by any method known in the art for purification of polypeptides, polyproteins or antibodies (e.g., analogous to antibody purification schemes based on antigen selectivity) for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the compound comprises an Fc domain (or portion thereof) ) , and sizing column chromatography) , centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the compound comprises an Fc domain (or portion thereof)
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies.
  • bispecific antibodies described herein or a polypeptide chain of a bispecific antibody described herein comprising obtaining a cell described herein and expressing the polynucleotide described herein in said cell.
  • the method further comprises isolating and purifying a bispecific antibody or polypeptide chain described herein.
  • Antibodies can be further tested by flow cytometry for binding to a cell line expressing human Tim3 and/or PD-1, but not to a control cell line that does not express the target antigen. Briefly, the binding of antibodies can be assessed by incubating Tim3 and/or PD-1 expressing CHO cells with the bispecific antibody provided herein. The cells can be washed, and binding can be detected with an anti-human IgG Ab. Flow cytometric analyses can be performed using a FACS can flow cytometry (Becton Dickinson, San Jose, CA) .
  • bispecific antibodies provided herein can be further tested for reactivity with the target antigen (s) by Western blotting, and other methods known in the art for analyzing binding affinity, cross-reactivity, and binding kinetics of various bispecific antibodies described herein include, for example, biolayer interferometry (BLI) using, for example, Gator system (Probe Life) or the Octet-96 system (Sartorius AG) , or BIACORE TM surface plasmon resonance (SPR) analysis using a BIACORE TM 2000 SPR instrument (Biacore AB, Uppsala, Sweden) .
  • BLI biolayer interferometry
  • Gator system Probe Life
  • Octet-96 system Sesartorius AG
  • SPR BIACORE TM surface plasmon resonance
  • T cell activation assays such as assays using purified T cells obtained from PBMCs of human donors. Assays can be conducted with total T cells or subpopulations thereof, e.g., Th1 cells, T cytotoxic cells, Treg cells, CD4+T cells, CD8+T cells, provided that they express TIM-3. Activation may be measured by determining the level of secretion of certain cytokines, e.g., interferon-g or IL-2 or the level of proliferation of the T cells.
  • cytokines e.g., interferon-g or IL-2
  • binding of anti-TIM-3 antibodies to TIM-3 on T cells may prevent binding of TIM-3 to a TIM-3 ligand (TIM-3 putative ligands include Galectin-9, HMGB 1, Semaphorin-4A, CEACAM-1, ILT-4and phosphatidylserine) and thereby prevent TIM-3 mediated signaling in the T cell thereby preventing negatively regulation of T cells by TIM-3.
  • TIM-3 putative ligands include Galectin-9, HMGB 1, Semaphorin-4A, CEACAM-1, ILT-4and phosphatidylserine
  • Exemplary assays including Th1 assays, TIL assays and mixed lymphocyte reactions (MLRs) are well known in the art; (2) assays measuring stimulation of macrophages, e.g., M1 or M2 macrophage; (3) assays measuring secretion of myeloid-associated cytokines, e.g., TNF ⁇ , IL-I ⁇ , GM-CSF, IL-6, IL-2, IL-10, CCL2, CCL3, CCL4 or CCL5 from TIM-3 positive myeloid cells.
  • any method for testing the biological activity of an agent that affects immune responses can be used to characterize the biological activity of the antibodies disclosed herein.
  • compositions comprising the bispecific antibodies disclosed herein.
  • the pharmaceutical composition comprises a therapeutically effective amount of the bispecific antibodies disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions are useful in immunotherapy.
  • the pharmaceutical compositions are useful in immuno-oncology.
  • the pharmaceutical compositions are useful in inhibiting tumor growth in a subject (e.g., a human patient) .
  • the pharmaceutical compositions are useful in treating cancer in a subject (e.g., a human patient) .
  • the pharmaceutical compositions provided herein comprise bispecific antibodies provided herein.
  • Pharmaceutically acceptable carriers that can be used in compositions provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) .
  • the active ingredient i.e., the bispecific antibodies
  • the bispecific antibodies can be coated in a material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.
  • kits for preparation of pharmaceutical compositions having the bispecific antibodies disclosed herein comprising the bispecific antibodies disclosed herein and a pharmaceutically acceptable carrier in one or more containers.
  • the kits can comprise bispecific antibodies disclosed herein for administration to a subject.
  • the kits comprise instructions regarding the preparation and/or administration of the bispecific antibodies disclosed herein.
  • the pharmaceutical composition or formulation disclosed herein comprises: (a) bispecific antibodies disclosed herein disclosed herein; (b) a buffering agent; (c) a stabilizing agent; (d) a salt; (e) a bulking agent; and/or (f) a surfactant.
  • the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5years or more.
  • the pharmaceutical composition or formulation is stable when stored at 4°C, 25°C, or 40°C.
  • Stabilizing agents are added to a pharmaceutical product to stabilize that product. Such agents can stabilize proteins in different ways. Common stabilizing agents include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine, carbohydrates such as glucose, sucrose, trehalose, rafftnose, or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrins or destrans of any kind and molecular weight, or PEG. In some embodiments, the stabilizing agent is chosen to maximize the stability of FIX polypeptide in lyophilized preparations. In certain embodiments, the stabilizing agent is sucrose and/or arginine.
  • Bulking agents can be added to a pharmaceutical composition or formulation to add volume and mass to the product, thereby facilitating precise metering and handling thereof.
  • Common bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.
  • Surfactants are amphipathic substances with lyophilic and lyophobic groups.
  • a surfactant can be anionic, cationic, zwitterionic, or nonionic.
  • nonionic surfactants include, but are not limited to, alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyl dimethylamine oxide.
  • the surfactant is polysorbate 20 or polysorbate 80.
  • the pharmaceutical composition is an aqueous formulation.
  • aqueous formulation is typically a solution or a suspension, but can also include colloids, dispersions, emulsions, and multi-phase materials.
  • aqueous formulation is defined as a formulation comprising at least50%w/w water.
  • aqueous solution is defined as a solution comprising at least50%w/w water
  • aqueous suspension is defined as a suspension comprising at least50%w/w water.
  • compositions disclosed herein can also include a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA) , butylated hydroxytoluene (BHT) , lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA) , sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butyl
  • compositions or formulations typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein.
  • some methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions disclosed herein can be prepared with carriers that protect the active ingredient against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and poly lactic acid.
  • Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See. e.g., SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • the bispecific antibodies described herein can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of diseases. Accordingly, provided herein are methods of modifying an immune response in a subject comprising administering to the subject a bispecific antibody or pharmaceutical described herein such that the immune response in the subject is modified. In some embodiments, the response is enhanced, stimulated or up-regulated.
  • provided herein are methods of inhibiting the interaction between TIM-3 and a TIM-3 ligand (e.g., phosphatidylserine) on an immune cell comprising contacting the immune cell with an effective amount of a bispecific antibody described herein.
  • a TIM-3 ligand e.g., phosphatidylserine
  • methods of reducing PD-1 mediated suppression of an immune cell proliferation and/or activation comprising contacting an immune cell with an effective amount a bispecific antibody described herein.
  • provided herein are methods of inhibiting the interaction between PD-1 and a PD-1 ligand (e.g., PD-L1) on an immune cell comprising contacting the immune cell with an effective amount of a bispecific antibody described herein.
  • cytokine e.g., IFN- ⁇
  • methods of increasing cytokine comprising contacting an immune cell with an effective amount of a bispecific antibody described herein.
  • Subjects suitable for the present methods include human patients in whom enhancement of an immune response would be desirable.
  • the methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting an immune response (e.g., a T-cell mediated immune response, e.g., an antigen specific T cell response) .
  • the methods are particularly suitable for treatment of cancer in vivo.
  • a bispecific antibody described herein can be administered together with an antigen of interest or the antigen can already be present in the subject to be treated (e.g., a tumor-bearing or virus-bearing subject) .
  • the two can be administered separately or simultaneously.
  • bispecific antibodies disclosed herein to stimulate or co-stimulate T cell responses, e.g., antigen-specific T cell responses, such as by inhibiting negative effects of TIM-3 and/or PD-1, provided herein are in vitro and in vivo methods of using the bispecific antibodies disclosed herein to stimulate, enhance or upregulate antigen-specific T cell responses, e.g., anti-tumor T cell responses.
  • methods for inhibiting growth of tumor cells in a subject comprising administering to the subject a bispecific antibody described herein such that growth of the tumor is inhibited in the subject.
  • methods of treating a viral infection in a subject comprising administering to the subject a bispecific antibody described herein such that the viral infection is treated in the subject.
  • treatment of a subject with bispecific antibodies disclosed herein does not result in overstimulation of the immune system to the extent that the subject’s immune system then attacks the subject itself (e.g., autoimmune response) or results in, e.g., anaphylaxis.
  • the bispecific antibodies provided herein do not cause anaphylaxis.
  • treatment of a subject with bispecific antibodies described herein does not cause significant inflammatory reactions, e.g., immune-mediated pneumonitis, immune-mediated colitis, immune mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypophysitis, immune-mediated hypothyroidism and hyperthyroidism, or other immune-mediated adverse reactions.
  • inflammatory reactions e.g., immune-mediated pneumonitis, immune-mediated colitis, immune mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypophysitis, immune-mediated hypothyroidism and hyperthyroidism, or other immune-mediated adverse reactions.
  • bispecific antibodies provided herein cause limited inflammatory reactions, e.g., immune-mediated pneumonitis, immune-mediated colitis, immune mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypophysitis, immune-mediated hypothyroidism and hyperthyroidism, anaphylaxis or other immune-mediated adverse reactions.
  • treatment of a subject with bispecific antibodies disclosed herein does not cause significant cardiac disorders, e.g., ventricular arrhythmia; eye disorders, e.g., iridocyclitis; infusion-related reactions; increased amylase, increased lipase; nervous system disorders, e.g., dizziness, peripheral and sensory neuropathy; skin and subcutaneous tissue disorders, e.g., rash, pruritus, exfoliative dermatitis, erythema multiforme, vitiligo or psoriasis; respiratory, thoracic and mediastinal disorders, e.g., cough; fatigue; nausea; decreased appetite; constipation; arthralgia; or diarrhea.
  • cardiac disorders e.g., ventricular arrhythmia
  • eye disorders e.g., iridocyclitis
  • infusion-related reactions e.g., increased amylase, increased lipase
  • nervous system disorders e.g., dizziness, peripheral and sensory
  • the active ingredients i.e., the bispecific antibodies provided herein
  • dosage levels of the active ingredients can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • bispecific antibodies or pharmaceutical compositions provided herein can be administered to a subject by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes of administration, for example by injection or infusion, or direct administration to the thymus.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.
  • subcutaneous administration is adopted.
  • intravenous administration is adopted.
  • oral administration is adopted.
  • the antibodies or antigen-binding fragments or cells provided herein can be delivered regionally to a tumor using well known methods, including but not limited to, hepatic or aortic pump; limb, lung or liver perfusion; in the portal vein; through a venous shunt; in a cavity or in a vein that is nearby a tumor, and the like.
  • the bispecific antibodies provided herein can be administered systemically.
  • the bispecific antibodies are administered regionally at the site of a tumor.
  • the bispecific antibodies can also be administered intratumorally, for example, by direct injection of the cells at the site of a tumor and/or into the tumor vasculature.
  • administration is preferably by intrapleural administration (see Adusumilli et al., Science Translational Medicine 6 (261) : 261 ra 151 (2014) ) .
  • One skilled in the art can select a suitable mode of administration based on the type of cancer and/or location of a tumor to be treated.
  • the bispecific antibodies can be introduced by injection or catheter.
  • the bispecific antibodies are pleurally administered to the subject in need, for example, using an intrapleural catheter.
  • Cancers or tumors to be treated using the bispecific antibodies or pharmaceutical compositions provided herein comprise those typically responsive to immunotherapy and those that are not typically responsive to immunotherapy. Cancers that can be treated also include TIM-3 positive cancers. Cancers that can be treated also include PD-L1 positive cancers. In some embodiments, the cancer has a high degree of microsatellite instability.
  • cancers or tumors that can be treated with the bispecific antibodies or pharmaceutical compositions disclosed herein are hematological cancers. In some embodiments, cancers or tumors that can be treated with the bispecific antibodies or pharmaceutical compositions disclosed herein disclosed herein are solid tumors. In some embodiments, cancers or tumors that can be treated with the bispecific antibodies or pharmaceutical compositions disclosed herein disclosed herein are breast cancers.
  • an anti-tumor effect can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An anti-tumor effect can also be manifested by the ability of the antibodies or antigen binding fragments, or pharmaceutical compositions provided herein in prevention of the occurrence of tumor in the first place.
  • an “anti-tumor effect” can be manifested by the reduction in cancer-induced immunosuppression.
  • Clinical improvement comprises decreased risk or rate of progression or reduction in pathological consequences of the cancer or tumor.
  • a method of treating cancer can include any effect that ameliorates a sign or symptom associated with cancer.
  • signs or symptoms include, but are not limited to, reducing tumor burden, including inhibiting growth of a tumor, slowing the growth rate of a tumor, reducing the size of a tumor, reducing the number of tumors, eliminating a tumor, all of which can be measured using routine tumor imaging techniques well known in the art.
  • Other signs or symptoms associated with cancer include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers.
  • the methods or uses provided herein can reduce tumor burden.
  • administration of the bispecific antibodies or pharmaceutical compositions disclosed herein can reduce the number of tumor cells, reduce tumor size, and/or eradicate the tumor in the subject.
  • Methods for monitoring patient response to administration of a pharmaceutical composition disclosed herein are known in the art and can be employed in accordance with methods disclosed herein.
  • treatment of a subject having cancer with a bispecific antibody disclosed herein can result in, e.g., stable disease, partial response, increased overall survival, increased disease-free survival, or enhanced progression free survival.
  • an anti-tumor effect is observed in a subject having a tumor or cancer who has been administered with a bispecific antibody described herein as a single therapy, namely, not in combination with another therapeutic.
  • tumor burden is reduced in a subject having a tumor or cancer who has been administered with a bispecific antibody described herein as a single therapy, namely, not in combination with another therapeutic.
  • a therapeutically effective amount of the bispecific antibodies or pharmaceutical compositions disclosed herein are administered to a subject in need of cancer treatment.
  • the subject can be a mammal.
  • the subject is a human.
  • these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases.
  • This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts and are suitably defined for different types of cancers.
  • Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.
  • a method of treating cancer in a subject comprises first determining whether the subject is TIM-3 positive, e.g., has tumor cells or TILs that express TIM-3, and if the subject has TIM-3 positive cancer or TIL cells, then administering to the subject the bispecific antibodies or pharmaceutical compositions described herein.
  • a method of treating a subject having cancer with the bispecific antibodies or pharmaceutical compositions described herein can comprise administering to a subject who has cancer cells or TIL cells that express TIM-3, a therapeutically effective amount of the bispecific antibodies or pharmaceutical compositions described herein.
  • a method of treating cancer in a subject comprises first determining whether the subject is PD-L1 or PD-1 positive, e.g., has tumor cells or TILs that express PD-L1 or PD-1, and if the subject has PD-L1 or PD-1 positive cancer or TIL cells, then administering to the subject the bispecific antibodies or pharmaceutical compositions described herein.
  • a method of treating a subject having cancer with the bispecific antibodies or pharmaceutical compositions described herein can comprise administering to a subject who has cancer cells or TIL cells that express PD-L1 or PD-1, a therapeutically effective amount of the bispecific antibodies or pharmaceutical compositions described herein.
  • the cancer to be treated by the bispecific antibodies disclosed herein are Tim-3 expressing cancers (i.e., Tim-3 positive cancers) .
  • the cancer to be treated by the bispecific antibodies disclosed herein are PD-L1 expressing cancers (i.e., PD-L1 positive cancers) .
  • the cancer to be treated by the bispecific antibodies disclosed herein are resistant to anti-PD-1 treatment.
  • the bispecific antibodies or pharmaceutical compositions described herein can be administered with a standard of care treatment.
  • the bispecific antibodies or pharmaceutical compositions described herein can be administered as a maintenance therapy, e.g., a therapy that is intended to prevent the occurrence or recurrence of tumors.
  • the bispecific antibodies or pharmaceutical compositions described herein can be administered with another treatment, e.g., radiation, surgery, or chemotherapy.
  • the bispecific antibodies or pharmaceutical compositions described herein can be administered as an adjunctive therapy when there is a risk that micrometastases can be present and/or in order to reduce the risk of a relapse.
  • the additional therapy can be administered prior to, concurrently with, or subsequent to administration of the bispecific antibodies or pharmaceutical compositions described herein.
  • Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • a person skilled in the art can readily determine appropriate regimens for administering a pharmaceutical composition described herein and an additional therapy in combination, including the timing and dosing of an additional agent to be used in a combination therapy, based on the needs of the subject being treated.
  • Embodiment 1 A bispecific antibody comprising (i) a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1) , wherein the VL1/VH1 pair specifically binds to human Tim-3, and wherein the VL1 comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 101, 102, and 103, respectively, and wherein the VH1 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 104, 105, and 106, respectively; or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VL CDRs and VH CDRs; and (ii) a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) , wherein the VL2/VH2 pair specifically binds to human PD-1; and wherein the VL2 comprises VL CDR
  • Embodiment 3 The bispecific antibody of Embodiment 1 or Embodiment 2, wherein VL1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 76-84; VH1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 85-91; and the VL2 and VH2 have the amino acid sequences of SEQ ID NOs: 1 and 2, respectively.
  • Embodiment 4 The bispecific antibody of Embodiment 3, wherein the VL1 and VH1 have the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.
  • Embodiment 5 The bispecific antibody of any one of Embodiments 1 to 4, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a light chain constant (CL) region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant (CH) region and a single chain variable fragment (scFv) , wherein the scFv comprises, from N-terminus to C-terminus, VL2, a linker (L) , and VH2.
  • Embodiment 6 The bispecific antibody of any one of Embodiments 1 to 4, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a CL region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a CH region and an scFv, wherein the scFv comprises, from N-terminus to C-terminus, VH2, a linker (L) , and VL2.
  • Embodiment 7 The bispecific antibody of any one of Embodiments 1 to 4, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH region, and an scFv, wherein the scFv comprises, from N-terminus to C-terminus, VL1, a linker (L) , and VH1.
  • Embodiment 8 The bispecific antibody of any one of Embodiments 1 to 4, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH region, and an scFv, wherein the scFv comprises, from N-terminus to C-terminus, VH1, a linker (L) , and VL1.
  • Embodiment 9 The bispecific antibody of any one of Embodiments 5 to 8, wherein the linker is a GS linker having the amino acid sequence of SEQ ID NO: 44 or 46, or a TF linker having the amino acid sequence of SEQ ID NO: 50.
  • Embodiment 10 The bispecific antibody of any one of Embodiments 5 to 9, wherein the scFv is connected to the CH region via a second linker.
  • Embodiment 11 The bispecific antibody of Embodiment 10, wherein the second linker is a GS linker having the amino acid sequence of SEQ ID NO: 44 or 46, or a TF linker having the amino acid sequence of SEQ ID NO: 50.
  • Embodiment 14 The bispecific antibody of Embodiment 6, wherein C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 63, and C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 64.
  • Embodiment 17 The bispecific antibody of any one of Embodiments 1 to 4, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2, VL1, and a CL region; and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, VH1, and a CH region.
  • C1 a first peptide chain
  • C2 a second peptide chain comprising, from N-terminus to C-terminus, VH2, VH1, and a CH region.
  • Embodiment 20 The bispecific antibody of any one of Embodiments 16 to 19, wherein (1) the CL region is C ⁇ (SEQ ID NO: 7) or C ⁇ (SEQ ID NO: 8) , or a variant thereof having up to ten amino acids substitutions; or (2) the CH region is human IgG1 CH region (SEQ ID NO: 9) , IgG2 CH region (SEQ ID NO: 10) , IgG3 CH region (SEQ ID NO: 11) , or IgG4 CH region (SEQ ID NO: 12) , or a variant thereof having up to ten amino acids substitutions; or both (1) and (2) .
  • the CL region is C ⁇ (SEQ ID NO: 7) or C ⁇ (SEQ ID NO: 8) , or a variant thereof having up to ten amino acids substitutions
  • the CH region is human IgG1 CH region (SEQ ID NO: 9) , IgG2 CH region (SEQ ID NO: 10) , IgG3 CH region (SEQ ID NO: 11
  • Embodiment 21 The bispecific antibody of Embodiment 20, wherein (1) the CL region is C ⁇ (SEQ ID NO: 7) , and the CH region is human IgG1 CH region with L234A, L235A and P329G substitutions (SEQ ID NO: 25) ; or (2) the CL region is C ⁇ (SEQ ID NO: 7) , and the CH region is human IgG1 CH region with L234A, L235A, P329G and K447A substitutions (SEQ ID NO: 27) .
  • Embodiment 24 The bispecific antibody of Embodiment 17, wherein C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 69, and C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 70.
  • Embodiment 25 The bispecific antibody of any one of Embodiments 1 to 4, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant domain1 (CH1 domain) , and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Knob-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VL2, a linker (L) , and VH2.
  • Embodiment 26 The bispecific antibody of any one of Embodiments 1 to 4, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a CH1 domain, and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Knob-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VH2, a linker (L) , and VL2.
  • Embodiment 29 The bispecific antibody of any one of Embodiments 1 to 4, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH1 domain, and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Knob-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VL1, a linker (L) , and VH1.
  • Embodiment 32 The bispecific antibody of any one of Embodiments 1 to 4, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a CL region; (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a CH1 domain, and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, an scFv, and a Hole-Fc region; wherein the scFv comprises, from N-terminus to C-terminus, VH1, a linker (L) , and VL1.
  • Embodiment 35 The bispecific antibody of Embodiment 34, wherein the Hole-Fc region further comprises T366S and L368A substitutions.
  • Embodiment 36 The bispecific antibody of Embodiment 34 or 35, wherein the Knob-Fc region further comprises S354C substitution, and the Hole-Fc region further comprises Y349C substitution.
  • Embodiment 37 The bispecific antibody of Embodiment 34 or 35, wherein the Hole-Fc region further comprises S354C substitution, and the Knob-Fc region further comprises Y349C substitution.
  • Embodiment 39 The bispecific antibody of Embodiment 38, wherein the Knob-Fc region and the Hole-Fc region further comprise the P329G substitution.
  • Embodiment 42 The bispecific antibody of Embodiment 26, wherein C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 57; C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 58; and C3 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 59.
  • Embodiment 43 The bispecific antibody of Embodiment 30, wherein C1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 60; C2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61; and C3 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 62.
  • Embodiment 49 A cell comprising the polynucleotide or plurality of polynucleotides of any one of Embodiments 45 to 47, or the vector of Embodiment 48.
  • Embodiment 52 The method of Embodiment 50 or 51, wherein the immune cell is a T cell, an NK cell, an NKT cell, or a myeloid cell.
  • Embodiment 53 The method of Embodiment 52, wherein the immune cell is a T cell.
  • Embodiment 55 The method of Embodiment 52, wherein the immune cell is a myeloid cell, wherein the myeloid cell is a macrophage or a dendritic cell.
  • Embodiment 58 The method of Embodiment 56 or 57, further comprising administering an additional therapy to the subject.
  • Embodiment 59 The method of any one of Embodiments 56 to 58, wherein the subject is a human.
  • Embodiment 60 Use of the bispecific antibody of any one of Embodiments 1 to 43 in cancer treatment.
  • Embodiment 62 The method or use of any one of Embodiments 56 to 61, wherein the cancer is a hematological cancer.
  • Embodiment 63 The method or use of any one of Embodiments 56 to 61, wherein the cancer is a solid tumor.
  • Embodiment 65 The method or use of any one of Embodiments 56 to 64, wherein the cancer is a Tim-3 expressing cancer.
  • the antibody-coupled HFC sensor was balanced in the SD buffer solution for 1 minute, and then incubated in the buffer solution containing different concentrations of TIM3-his or PD1-his for 200s to measure the association of the protein to captured antibody. Finally, the sensor combining the antigen and antibody was placed in SD buffer and waited for 300s to measure dissociation of protein from the antibodies captured on sensor. Data were fitted using a 1: 1 model, and the association rate (k on ) and the dissociation rate (k off ) were calculated. The equilibrium dissociation constant (K D ) was calculated by the ratio k off /k on .
  • 96-well microplates were coated with 60 ⁇ L/well 2 ⁇ g/ml PD1-ECD-hFc and incubated at 4°C over night. After washing (3x250 ⁇ L/well with PBST-buffer) , 60 ⁇ L serially diluted candidate antibodies were added and incubated for 1h at room temperatures (RT) . Following another round of washing, 60 ⁇ L/well 0.3 ⁇ g/ml Biotin-TIM-3-ECD-hFc was added and incubated for 1 h at RT. Then, 60 ⁇ L/well strep-HRP was added in 1: 1000 dilution and incubated at RT for 30min. After washing (3x250 ⁇ L/well with PBST-buffer) , 60 ⁇ L/well TMB substrate was added and incubated until OD 2-3. Measurement took place at 450nm.
  • FIGs. 4A-4C genes associated with antigen processing-cross presentation were up-regulated after 3E6 treatment.
  • FIG. 4A shows the gene ontology analysis on upregulated genes of 3E6 treatment versus medium control.
  • the heatmap in FIG. 4B provides visualization of the expression of the enriched genes.
  • FIG. 4C provides results of the Gene Set Enrichment Analysis (GSEA) .
  • GSEA Gene Set Enrichment Analysis
  • PBMC derived T-cells were used to explore the functional activities of candidate antibodies.
  • 1 ⁇ g/ml OKT3 (anti-CD3 mAb) was used to coat plates by incubating overnight at 4°C. On the following day, coating solution containing OKT3 was discarded, and PBMCs were added to OKT3-coated plates.
  • activated PBMC-derived T cells were mixed with stable transfected cells 293T-PD1/OKT3 [293T cells with stable expression of PD-1 and OKT3 (anti-CD3) scFv on cell membrane] at a ratio of 5: 1 (T: 293T-OKT3) , followed by the addition of serial diluted antibodies. After overnight incubation, IFN- ⁇ secretion in supernatant was detected by ELISA.
  • NBP-4 as a novel humanized Fc-silent IgG1 ⁇ antibody, specifically bound to human TIM-3 and PD-1 with nM level affinity and simultaneously bound to TIM-3 and PD-1 receptors through a trans-manner; demonstrated its potent capacity to enhance the interaction between T cells and dendritic cells (DCs) .
  • NBP-4 was bridged the innate and acquired immunity in tumor immunity and optimized the therapeutic response of anti-PD1 therapy by maximizing TIM-3 blockade on antigen-experienced T cells, enhancing T cells activation and killing activity of NK cells, restoring DCs function, and promoting antigen presentation of antigen-presenting cells (APC) .

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Abstract

L'invention concerne des anticorps bispécifiques ciblant Tim-3 et PD-1 humains. L'invention concerne également des polynucléotides codant pour les anticorps bispécifiques, des compositions pharmaceutiques comprenant les anticorps bispécifiques, et des procédés de production des anticorps bispécifiques. L'invention concerne par ailleurs des utilisations médicales des anticorps bispécifiques selon l'invention.
PCT/CN2024/124165 2023-10-11 2024-10-11 Anticorps ciblant tim-3 et pd-1 et leurs utilisations Pending WO2025077833A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016166360A1 (fr) * 2015-04-17 2016-10-20 Bayer Pharma Aktiengesellschaft Constructions d'anticorps bispécifiques pour cdh3 et cd3
WO2017021356A1 (fr) * 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps bispécifiques se liant à la mésothéline et à cd3
WO2017055404A1 (fr) * 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anticorps bispécifiques spécifiques de pd1 et tim3
WO2018009507A1 (fr) * 2016-07-06 2018-01-11 Bristol-Myers Squibb Company Combinaison d'un antagoniste tim -4 et procédés d'utilisation
WO2021057930A1 (fr) * 2019-09-26 2021-04-01 Wuxi Biologics (Shanghai) Co., Ltd. Nouvel anticorps bispécifique anti-pd-l1/anti-lag-3 et ses utilisations
WO2022223048A1 (fr) * 2021-04-23 2022-10-27 Suzhou Neologics Bioscience Co. , Ltd. Anticorps ciblant tim-3 et leurs utilisations

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016166360A1 (fr) * 2015-04-17 2016-10-20 Bayer Pharma Aktiengesellschaft Constructions d'anticorps bispécifiques pour cdh3 et cd3
WO2017021356A1 (fr) * 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps bispécifiques se liant à la mésothéline et à cd3
WO2017055404A1 (fr) * 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anticorps bispécifiques spécifiques de pd1 et tim3
WO2018009507A1 (fr) * 2016-07-06 2018-01-11 Bristol-Myers Squibb Company Combinaison d'un antagoniste tim -4 et procédés d'utilisation
WO2021057930A1 (fr) * 2019-09-26 2021-04-01 Wuxi Biologics (Shanghai) Co., Ltd. Nouvel anticorps bispécifique anti-pd-l1/anti-lag-3 et ses utilisations
WO2022223048A1 (fr) * 2021-04-23 2022-10-27 Suzhou Neologics Bioscience Co. , Ltd. Anticorps ciblant tim-3 et leurs utilisations

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