HK1164903A - Human serum albumin linkers and conjugates thereof - Google Patents

Description

Human serum albumin linkers and conjugates thereof

Technical Field

The present invention provides Human Serum Albumin (HSA) linker conjugates and binding, diagnostic, and therapeutic conjugates thereof. In one embodiment, the HSA linker comprises two amino acid substitutions. Another embodiment is a conjugate wherein the HSA linker is covalently bound to an amino-and carboxy-terminal binding moiety, which are first and second single chain Fv molecules (scfvs). Exemplary conjugates are useful, for example, for reducing tumor cell proliferation, e.g., for therapeutic applications. The invention further provides methods for making and administering diagnostic and therapeutic HSA linker conjugates.

Background

Antibody-like binding moieties (including those of intact antibodies, antibody fragments, and scfvs) are often used for therapeutic applications. Antibody fragments and scfvs typically exhibit a shorter serum half-life than intact antibodies, and in some therapeutic applications, it is desirable to increase the in vivo half-life of therapeutic agents that function with such fragments and scfvs.

Human Serum Albumin (HSA) is an approximately 66,500kD protein, consisting of 585 amino acids including at least 17 disulfide bridges. As with many members of the albumin family, human serum albumin has an important role in human physiology and is found in essentially every human tissue and body secretion. HSA is capable of binding and transporting a wide variety of ligands in the circulatory system, including long chain fatty acids, which are otherwise insoluble in circulating plasma.

Serum albumin belongs to a family of proteins that includes alpha-fetoprotein and human population specific components, also known as vitamin D binding proteins. Serum albumin is a major soluble protein of the circulatory system and contributes to many important physiological processes. Serum albumin typically comprises about 50% total blood components by dry weight. Albumins and their related blood proteins also play an important role in the transport, distribution, and metabolism of many endogenous and exogenous ligands in the human body, including a wide variety of chemically diverse molecules such as fatty acids, amino acids, steroids, calcium, metals such as copper and zinc, and various pharmaceutical agents. It is generally believed that albumin family molecules facilitate the transfer of many of these ligands across organ circulatory interfaces, such as the liver, intestine, kidney, and brain. Albumin is therefore involved in a wide variety of circulatory and metabolic functions.

Disclosure of Invention

In a first aspect, the present invention provides an HSA linker conjugate comprising a Human Serum Albumin (HSA) linker comprising the sequence of seq id NO: 6-15 and a first and second binding moiety selected from the group consisting of an antibody, a single chain Fv molecule, a bispecific single chain Fv ((scFv')₂) Molecules, domain antibodies, diabodiesTriabodies, hormones, Fab fragments, F (ab')₂Molecules, tandem scfv (tafv) fragments, receptors (e.g., cell surface receptors), ligands, aptamers, and biologically active fragments thereof, wherein a first binding moiety is bound to the amino terminus and a second binding moiety is bound to the carboxy terminus of an HSA linker. In one embodiment, the first binding moiety specifically binds ErbB3 and the second binding moiety specifically binds ErbB 2. In other embodiments, the HSA linker comprises the amino acid sequence set forth in SEQ ID NO: 1. 9, 10, 14 or 15.

In a second aspect, three or more binding moieties (e.g., 4,5, 6,7, 8,9, 10, or more) can be included in a medicament; these additional binding moieties may be added to the agent, for example, in tandem (e.g., 2, 3, 4, or 5 or more tandem) with the first or second binding moiety.

In a third aspect, the invention provides an HSA linker comprising a sequence corresponding to the sequence set forth in SEQ ID NO: 1, and an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 1, a serine residue at position 34 and a glutamine residue at position 503. In one embodiment, the amino acid sequence is relative to the amino acid sequence set forth in SEQ ID NO: 1 has at least 95% sequence identity. In another embodiment, the HSA linker comprises the amino acid sequence set forth in SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. In another embodiment, the HSA linker has the sequence set forth in SEQ id no: 1, or a pharmaceutically acceptable salt thereof.

In a fourth aspect, the invention provides an HSA linker conjugate comprising an HSA linker that is complementary to a HSA linker in SEQ ID NO: 1, and at least a first binding moiety, and at least 90% amino acid sequence identity. In one embodiment, the HSA linker conjugate comprises a first peptide connector (connector) that binds a first binding moiety to the HSA linker.

In a fifth aspect, the invention features an HSA linker conjugate that includes an HSA linker having the amino acid sequence set forth in SEQ ID NO: 11-15, or a fragment or variant of any of these sequences, and at least a first binding moiety.

In certain embodiments of the fourth or fifth aspect of the invention, the HSA linker conjugate described above further comprises a first peptide connector (e.g., AAS, AAQ, or AAAL (SEQ ID NO: 5)) that binds the first binding moiety to the amino-or carboxy-terminus of the HSA linker. In one embodiment, the first connector covalently binds the first binding moiety to the HSA linker.

In certain embodiments of the fourth or fifth aspects of the invention, the HSA linker conjugate described above further comprises at least a second binding moiety. In one embodiment, the above HSA linker conjugate further comprises a second peptide connector (e.g., AAS, AAQ, or AAAL (SEQ ID NO: 5)) that binds a second binding moiety to the HSA linker. In other embodiments, the second connector binds the second binding moiety to the amino or carboxy terminus of the HSA linker. In yet another embodiment, the second connector covalently binds the second binding moiety to the HSA linker. In other embodiments, the HSA linker conjugate described above further comprises three or more binding moieties in series with the first or second binding moiety; the three or more binding moieties may further comprise a linker sequence linking the three or more binding moieties to the first or second binding moieties and linking the three or more binding moieties to each other.

In certain embodiments of the fourth or fifth aspect of the present invention, the HSA linker conjugate comprises a first peptide connector covalently binding the first binding moiety to the amino terminus of the HSA linker, and a second peptide connector covalently binding the second binding moiety to the carboxy terminus of the HSA linker. In one embodiment, the first linker has the amino acid sequence AAS or AAQ, and the second linker has the amino acid sequence set forth in SEQ id no: 5, or a pharmaceutically acceptable salt thereof.

In certain embodiments of the fourth or fifth aspect of the invention, the first or second binding moiety (or the third or more binding moieties) is an antibody, a single chain Fv molecule, a bispecific single chain Fv ((scFv')₂) Molecules, domain antibodies, diabodies, triabodies, hormones, Fab fragments, F (ab')₂A molecule, a tandem scfv (tafv) fragment, a receptor (e.g., a cell surface receptor), a ligand, an aptamer, or a biologically active fragment thereof. In other embodiments, the HSA linker conjugates provided herein comprise a combination of these different types of binding moieties. In one embodiment, at least the first or second binding moiety is a human or humanized single chain Fv molecule.

In one embodiment of any one of the first, second, third, fourth or fifth aspects of the invention, one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is or specifically binds to a protein selected from the group consisting of insulin-like growth factor 1 receptor (IGF1R), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transforming factor receptor (c-met; also known as Hepatocyte Growth Factor Receptor (HGFR)), Hepatocyte Growth Factor (HGF), Epidermal Growth Factor Receptor (EGFR), Epidermal Growth Factor (EGF), nerve growth factor, Fibroblast Growth Factor Receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), Vascular Endothelial Growth Factor Receptor (VEGFR), Vascular Endothelial Growth Factor (VEGF), Tumor Necrosis Factor Receptor (TNFR), tumor necrosis factor alpha (TNF-alpha), TNF-beta, folate receptor (FOLR), folate transferrin receptor (TfR), mesothelin, Fc receptor, c-kit, integrin (e.g., α 4 integrin or β -1 integrin), P-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronic acid receptor, leukocyte functional antigen-1 (LFA-1), CD4, CD11, CD18, CD20, CD25, CD27, CD52, CD70, CD80, CD85, CD95(Fas receptor), CD106 (vascular cell adhesion molecule 1(VCAM1), CD166 (activated leukocyte adhesion molecule (am)), CD178 (alc ligand), CD253 (TNF-related apoptosis inducing ligand (TRAIL)), ICOS ligand, CCR2, CXC.R3, CCR5, CXCL12 (stromal cell derived factor 1(SDF-1)), interleukin 1(IL-1), CTLA-4, MART1, gp100, MAGE-1, ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1(MAdCAM-1), carcinoembryonic antigen (CEA), Lewis^YMUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125(CA125), Prostate Specific Membrane Antigen (PSMA), TAG-72 antigen, and fragments thereof. In yet another embodiment, one or more of the first or second binding moieties (or, if present, the third or further binding moieties) is or specifically binds to an erythroblastic leukemia virus (erythroblastic leukemia virus) oncogene homolog (ErbB) receptor (e.g., the ErbB1 receptor; the ErbB2 receptor; the ErbB3 receptor; and the ErbB4 receptor). In another embodiment, one or more of the first or second binding moieties (or, if present, the third or further binding moieties) is or specifically binds to alpha-fetoprotein (AFP) or interferon, or a biologically active fragment thereof. In yet another embodiment, one or more of the first or second binding moieties (or, if present, the third or further binding moieties) is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efletuzumab, golimumab, certolizumab, trastuzumab, abamectin, etanercept, pertuzumab, cetuximab, panitumumab (panitumumab), or anakinra.

In any one of the first, second, third, fourth, or fifth aspects of the invention, the HSA linker conjugate is linked to a diagnostic agent, a therapeutic agent, or both. In one embodiment, the diagnostic agent is a detectable label, such as a radioactive label, a fluorescent label, or a heavy metal label. In another embodiment, the therapeutic agent is a cytotoxic agent, cytostatic agent, or immunomodulatory agent. Cytotoxic agents include alkylating agents, antibiotics, antineoplastic agents, antiproliferative agents, antimetabolites, tubulin inhibitors, topoisomerase I or II inhibitors, hormone agonists or antagonists, immunomodulators, DNA minor groove binding agents, and radioactive agents, or any agent capable of binding to and killing tumor cells or inhibiting tumor cell proliferation. Antineoplastic agents include cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, rhizomycin, dolastatin (dolastatin), ansamitocin p3, maytansinoids (maytansinoids), auristatins, calicheamicin (caleacimicin), methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and derivatives thereof.

In any of the first, second, third, fourth, or fifth aspects of the invention, the HSA linker conjugate is admixed with a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment, the agent has an in vivo half-life of 6 hours to 7 days. In another embodiment, the agent has an in vivo half-life of greater than 8 hours.

In a sixth aspect, the invention features a method for treating a mammal having a disease or disorder, the method comprising administering any one of the HSA linker conjugates described herein. In one embodiment, the disease or disorder is associated with cell signaling through a cell surface receptor. In another embodiment, the mammal is a human. In yet another embodiment, the disease or disorder is a proliferative or autoimmune disease. Proliferative diseases include cancers such as melanoma, clear cell sarcoma, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, renal cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, and brain cancer. Autoimmune diseases include multiple sclerosis, psoriasis, myasthenia gravis, uveitis, systemic lupus erythematosus, and rheumatoid arthritis. In one embodiment, the HSA linker conjugate is administered in conjunction with one or more therapeutic agents, such as an antineoplastic agent.

In a seventh aspect, the invention features a method for making an HSA linker conjugateThe method of (a), the method comprising binding at least a first binding moiety to the amino terminus and a second binding moiety to the carboxy terminus of an HSA linker, respectively, the HSA linker having the amino acid sequence set forth in SEQ ID NO: 1. 3, or 6-15, or a sequence of amino acids set forth in any one of SEQ ID NOs: 1. 3, or 6-15, having at least 90%, 95%, 97%, or 100% sequence identity. In one embodiment, the first or second binding moiety is covalently attached to the amino or carboxy terminus of the HSA linker. In other embodiments, a third or additional binding moiety (e.g., a fourth, fifth, sixth, seventh, eighth, ninth, or tenth binding moiety) is covalently attached to the amino or carboxy terminus of the HSA linker in tandem with the first or second binding moiety. In another embodiment, one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is an antibody, a single chain Fv molecule, a bispecific single chain Fv ((scFv')₂) Molecules, domain antibodies, diabodies, triabodies, hormones, Fab fragments, F (ab')₂A molecule, a tandem scfv (tafv) fragment, a receptor (e.g., a cell surface receptor), a ligand, or an aptamer. In another embodiment, the first or second binding moiety (or, if present, the third or further binding moiety) is a human or humanized single chain Fv molecule. In yet another embodiment, one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is or specifically binds to insulin-like growth factor 1 receptor (IGF1R), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transformation factor receptor (c-met; also known as Hepatocyte Growth Factor Receptor (HGFR)), Hepatocyte Growth Factor (HGF), Epidermal Growth Factor Receptor (EGFR), Epidermal Growth Factor (EGF), nerve growth factor, Fibroblast Growth Factor Receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), Vascular Endothelial Growth Factor Receptor (VEGFR), Vascular Endothelial Growth Factor (VEGF), Tumor Necrosis Factor Receptor (TNFR), tumor necrosis factor alpha (TNF-alpha), TNF-beta, alpha, beta, or combinations thereof, Folate receptor (FOLR), folate transferrin receptor (TfR), mesothelinFc receptor, c-kit, integrin (e.g., α 4 integrin or β -1 integrin), P-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronic acid receptor, leukocyte function antigen-1 (LFA-1), CD4, CD11, CD18, CD20, CD25, CD27, CD52, CD70, CD80, CD85, CD95(Fas receptor), CD106 (vascular cell adhesion molecule 1(VCAM1), CD166 (activated leukocyte adhesion molecule (ALCAM)), CD178(Fas ligand), CD253 (TNF-related apoptosis inducing ligand (TRAIL)), ICOS ligand, Ep 2, CXCR3, 5, CXCL12 (CCR 1(SDF-1)), interleukin 1(IL-1), CTLA-4, MART-1, 100, MAhrin-1, Ephrin-1, and mucosal cell adhesion receptor (dC-1) molecules (dC-1 (DCAM-1)) Carcinoembryonic antigen (CEA), Lewis^YMUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125(CA125), Prostate Specific Membrane Antigen (PSMA), TAG-72 antigen, and fragments thereof. In yet another embodiment, one or more of the first or second binding moieties (or, if present, the third or additional binding moieties) is or specifically binds to an erythroblastic leukemia virus oncogene homolog (ErbB) receptor (e.g., the ErbB1 receptor; the ErbB2 receptor; the ErbB3 receptor; and the ErbB4 receptor). In another embodiment, one or more of the first or second binding moieties (or, if present, the third or further binding moieties) is or specifically binds to alpha-fetoprotein (AFP) or interferon, or a biologically active fragment thereof. In yet another embodiment, one or more of the first or second binding moieties (or, if present, the third or further binding moieties) is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efletuzumab, golimumab, certolizumab, trastuzumab, abamectin, etanercept, pertuzumab, cetuximab, panitumumab (panitumumab), or anakinra. In another embodiment, the agent is linked to a diagnostic or therapeutic agent. In one embodiment, the diagnostic agent is a detectable label, such as a radioactive label, a bioluminescent label, a fluorescent label, a heavy metal label, or an epitope tag. In another embodiment, the therapeutic agent is a drugA cytotoxic agent, cytostatic agent, or immunomodulatory agent. Cytotoxic agents include alkylating agents, antibiotics, antineoplastic agents, antiproliferative agents, antimetabolites, tubulin inhibitors, topoisomerase I and II inhibitors, hormone agonists or antagonists, immunomodulators, DNA minor groove binding agents, and radioactive agents, or any agent capable of binding to and killing tumor cells or inhibiting tumor cell proliferation. Antineoplastic agents include cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, rhizomycin, dolastatin (dolastatin), ansamitocin p3, maytansinoids (maytansinoids), auristatins, calicheamicin (caleachimicin), methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and derivatives thereof. In yet another embodiment, the pharmaceutical agent is mixed with a pharmaceutically acceptable carrier, excipient, or diluent.

In an eighth aspect, the invention features a method for making an HSA linker, the method comprising substituting a substituted amino acid capable of chemical modification for a peptide set forth in SEQ ID NO: 1. 3, and 6-15, wherein the chemical modification facilitates binding of a diagnostic or therapeutic agent. In one embodiment, the substituted amino acid is cysteine and the surface exposed amino acid residue is serine or threonine. In another embodiment, the chemical modification results in covalent binding between the substituted amino acid and the diagnostic or therapeutic agent. In yet another embodiment, the surface exposed amino acid residue is threonine at position 496, serine at position 58, threonine at position 76, threonine at position 79, threonine at position 83, threonine at position 125, threonine at position 236, serine at position 270, serine at position 273, serine at position 304, serine at position 435, threonine at position 478, threonine at position 506, or threonine at position 508.

In a ninth aspect, the invention features a method for making an HSA linker, the method comprising substituting asparagine, serine, or threonine for the amino acid sequence set forth in SEQ ID NO: 1. 3, and 6-15, thereby comprising a glycosylation site in the HSA agent.

In a tenth aspect, the invention features a method for making an HSA linker that includes substituting any amino acid other than asparagine, serine, or threonine in SEQ ID NO: 1. 3, and 6-15, thereby removing glycosylation sites from the HSA agent.

In an eleventh aspect, the invention features an HSA linker comprising an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 16-25, having at least 90% sequence identity to one of the amino acid sequences listed in seq id no. In one embodiment, the HSA linker described above is substituted with respect to the HSA linker set forth in SEQ ID NO: 16-25 has at least 95% sequence identity to one of the amino acid sequences listed. In another embodiment, the HSA linker comprises a sequence having a sequence set forth in SEQ id no: 16-25, or a pharmaceutically acceptable salt thereof. In another embodiment, the HSA linker has the sequence set forth in SEQ ID NO: 16-25, or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the HSA linker or HSA linker conjugate is linked to a diagnostic or therapeutic agent. Diagnostic agents include detectable labels, such as radioactive labels, bioluminescent labels, fluorescent labels, or heavy metal labels, or epitope tags. Fluorescent molecules that can be used as detectable labels include Green Fluorescent Protein (GFP), enhanced GFP (egfp), Yellow Fluorescent Protein (YFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP), and dsRed. In one embodiment, the bioluminescent molecule is luciferase. In another embodiment, the epitope tag is c-myc, hemagglutinin, or a histidine tag. In yet another embodiment, the therapeutic agent is a cytotoxic polypeptide such as cytochrome c, caspase 1-10, granzyme A or B, tumor necrosis factor-alpha (TNF-alpha), TNF-beta, Fas ligand, Fas-related death domain-like IL-1 beta converting enzyme (FLICE), TRAIL/APO2L, TWEAK/APO3L, Bax, Bid, Bik, Bad, Bak, RICK, apoptosis-inducing proteins 1 and 2(VAP1 and VAP2), pirisin, apoptosis-inducing protein (AIP), IL-1 alpha pre-segment polypeptide (IL-1 alpha propeecec polypeptide), apoptotic protein-related protein 1(AAP-1), endostatin, angiostatin, and biologically active fragments thereof. The HSA linker or HSA linker conjugate can be incorporated into (e.g., linked to or mixed with in a pharmaceutical composition) one or more therapeutic agents such as cyclophosphamide, camptothecin, homocamptothecin, colchicine, combretastatin, rhizomycin, dolastatin (dolastatin), ansamitocin p3, maytansinoids (maytansinoids), auristatins, calicheamicin (calechinicin), methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and derivatives thereof.

In one embodiment of any of the aspects described herein, the first and second binding moieties (and, if present, one or more third or further binding moieties) specifically bind to the same target molecule. In another embodiment of any aspect, the first and second binding moieties (and, if present, one or more third or further binding moieties) specifically bind different target molecules. In a further embodiment of any aspect, the first and second binding moieties (and, if present, one or more third or further binding moieties) specifically bind to different epitopes on the same target molecule.

In a twelfth aspect, the invention features an HSA linker comprising the sequence set forth in SEQ ID NO: 1, and one or both of amino acid residues 25-44 and 494-513 of the amino acid sequence listed in seq id No. 1. In one embodiment, the HSA linker is comprised in seq id NO: 1 and amino acid residues 25-70 and 450-513 of the amino acid sequence listed in seq id No. 1. In another embodiment, the HSA linker comprises the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 15-100 and 400-520 of the amino acid sequences listed in 1. In yet another embodiment, the HSA linker comprises the amino acid sequence set forth in SEQ ID NO: 1, amino acid residues 10-200 and 300-575 of the amino acid sequence listed in seq id no. In another embodiment, the HSA linker comprises the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 5-250 and 275-580 of the amino acid sequence listed in 1.

In a twelfth aspect of the invention, the HSA linker is linked to at least a first binding moiety in order to form an HSA linker conjugate. In one embodiment, the HSA linker conjugate comprises at least a first peptide connector that binds the first binding moiety to the amino or carboxy terminus of the HSA linker. In another embodiment, the first peptide connector covalently binds the first binding moiety to the HSA linker. In yet another embodiment, the HSA linker comprises a second binding moiety. In one embodiment, the HSA linker comprises a second peptide connector that binds the second binding moiety to the HSA linker. In other embodiments, the second connector binds the second binding moiety to the amino or carboxy terminus of the HSA linker. In yet another embodiment, the second connector covalently binds the second binding moiety to the HSA linker. In other embodiments, the HSA linker comprises a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth binding moiety. In other embodiments, these additional binding moieties are present in tandem with one or both of the first or second binding moieties. In other embodiments, a peptide linker (e.g., AAS, AAQ, or AAAL (SEQ ID NO: 5)) separates one or more of these additional binding moieties from each other, from the first or second binding moieties, or from the HSA linker.

In a twelfth aspect of the invention, the HSA linker comprises a first polypeptide connector having a first binding moiety covalently bound to the amino terminus of the polypeptide linker and a second polypeptide connector having a second binding moiety covalently bound to the carboxy terminus of the HSA linker. In one embodiment, the first linker has the amino acid sequence AAS or AAQ and the second linker has the amino acid sequence set forth in SEQ ID NO: 5, or a pharmaceutically acceptable salt thereof.

In a twelfth aspect of the invention, one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is an antibody, a single chain Fv molecule, a bispecific single chain Fv ((scFv')₂) Molecules, domain antibodies, diabodies, triabodies, hormones, Fab fragments, F (ab')₂A molecule, a tandem scfv (tafv) fragment, a receptor (e.g., a cell surface receptor), a ligand, an aptamer, or a biologically active fragment thereof. In one embodiment, one or more of the first or second binding moieties (or, if present, the third or further binding moieties) is a human or humanized single chain Fv molecule.

In a twelfth aspect of the invention, one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is or specifically binds to a protein selected from the group consisting of insulin-like growth factor 1 receptor (IGF1R), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transformation factor receptor (c-met; also known as Hepatocyte Growth Factor Receptor (HGFR)), Hepatocyte Growth Factor (HGF), Epidermal Growth Factor Receptor (EGFR), Epidermal Growth Factor (EGF), nerve growth factor, Fibroblast Growth Factor Receptor (FGFR), platelet-derived growth factor receptor (PDGF), Vascular Endothelial Growth Factor Receptor (VEGFR), vascular endothelial growth factor receptor (VEGF), Tumor Necrosis Factor Receptor (TNFR), tumor necrosis factor alpha (TNF-alpha), TNF-beta, folate receptor (FOLR), folate transferrin receptor (TfR), mesothelin, Fc receptor, c-kit, integrin (e.g., alpha 4 integrin or beta-1 integrin), P-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronan receptor, leukocyte functional antigen-1 (LFA-1), CD4, CD11, CD18, CD20, CD25, CD27, CD52, CD70, CD80, CD85, CD95(Fas receptor), CD106 (vascular cell adhesion molecule 1(VCAM1), CD166 (activated leukocyte adhesion molecule (ALCAM)), CD178(Fas ligand), CD253 (TNF-related apoptosis inducing ligand (TRAIL)), ICOS ligand, CCR2, CXCR3, 5, CXCL12 (stromal cell derived factor 12)1(SDF-1)), interleukin 1(IL-1), CTLA-4, MART1, gp100, MAGE-1, ephrin (Eph) receptor, mucosal addrin cell adhesion molecule 1(MAdCAM-1), carcinoembryonic antigen (CEA), Lewis^YMUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125(CA125), Prostate Specific Membrane Antigen (PSMA), TAG-72 antigen, and fragments thereof. In yet another embodiment, the first or second binding moiety is or specifically binds to an erythroblastic leukemia virus oncogene homolog (ErbB) receptor (e.g., the ErbB1 receptor; the ErbB2 receptor; the ErbB3 receptor; and the ErbB4 receptor). In another embodiment, one or more of the first or second binding moieties (or, if present, the third or additional binding moieties) is or specifically binds to alpha-fetoprotein (AFP) or interferon, or a biologically active fragment thereof. In yet another embodiment, one or more portions of the first or second binding moiety (or, if present, the third or further binding moiety) is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efletuzumab, golimumab, certolizumab, trastuzumab, abamectin, etanercept, pertuzumab, cetuximab, panitumumab (panitumumab), or anakinra.

In a twelfth aspect of the invention, the HSA linker is linked to the diagnostic agent, the therapeutic agent, or both. In one embodiment, the diagnostic agent is a detectable label, such as a radioactive label, a fluorescent label, or a heavy metal label. In another embodiment, the therapeutic agent is a cytotoxic agent, cytostatic agent, or immunomodulatory agent. Cytotoxic agents include alkylating agents, antibiotics, antineoplastic agents, antiproliferative agents, antimetabolites, tubulin inhibitors, topoisomerase I or II inhibitors, hormone agonists or antagonists, immunomodulators, DNA minor groove binding agents, and radioactive agents, or any agent capable of binding to and killing tumor cells or inhibiting tumor cell proliferation. Antineoplastic agents include cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, rhizomycin, dolastatin (dolastatin), ansamitocin p3, maytansinoids (maytansinoids), auristatins, calicheamicin (caleachimicin), methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and derivatives thereof. In one embodiment, the bound HSA linker is mixed with a pharmaceutically acceptable carrier, excipient, or diluent. In another embodiment, the HSA linker has an in vivo half-life of 6 hours to 7 days. In yet another embodiment, the HSA linker has an in vivo half-life of greater than 8 hours.

A thirteenth aspect of the invention features the agent of any of the previous aspects (1 to 12) of the invention, wherein the HSA linker is replaced by another polypeptide linker. For example, the polypeptide linker sequence may be a mammalian non-human serum albumin polypeptide sequence, such as, for example, bovine, murine, feline, and canine serum albumin (BSA) polypeptide sequences. In other embodiments, such polypeptide linker sequences are 5 to 1,000 amino acids in length, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 amino acids in length, or any number of amino acids within the above ranges. In other embodiments, the polypeptide linker sequence comprises a single amino acid (including, but not limited to, for example, glycine, alanine, serine, glutamine, leucine, and valine), or a combination of amino acids.

In another embodiment, the HSA linker is substituted with a polypeptide of alpha-fetoprotein (AFP), e.g., a mammalian AFP polypeptide, such as a human, murine, bovine, or canine AFP polypeptide. In one embodiment, the AFP is grafted to a mature human AFP polypeptide sequence corresponding to the full-length human AFP polypeptide sequence (a.a. 1-609; SEQ ID NO: 58), lacking the signal sequence of amino acids 1-18 (a.a., 19-609 of SEQ ID NO: 58), or a fragment thereof. In other embodiments, the AFP polypeptide linker comprises SEQ ID NO: 58, preferably at least 10, 20, or 50 contiguous amino acids, more preferably at least 100 contiguous amino acids, and most preferably at least 200, 300, 400, or more contiguous amino acids, or a sequence that is complementary to a sequence of SEQ ID NO: 58 have at least 90% sequence identity (e.g., at least 95%, 97%, 99%, or more sequence identity) to a contiguous polypeptide sequence of one or more of these lengths. For example, relative to the nucleotide sequence corresponding to SEQ ID NO: the 34-mer (34-mer) human AFP peptide of amino acid 446-479 of 58 (LSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGV; SEQ ID NO: 59) the AFP polypeptide linker sequence having 90% sequence identity may comprise up to 3 amino acid sequences altered from the amino acid sequence of SEQ ID NO: 58, 446 and 479. One such example of sequence variation in a biologically active fragment of human AFP is found, for example, in U.S. Pat. No. 5,707,963 (incorporated herein by reference), which discloses a34 amino acid fragment with flexibility at two amino acid residues (amino acids 9 and 22 of SEQ ID NO: 59) of human AFP (SEQ ID NO: 59). Other examples of AFP polypeptide linker sequences include, for example, SEQ ID NO: 58 (human AFP domain I), amino acids 19-198 of SEQ ID NO: 58 (human AFP domain II), SEQ ID NO: 58 (human AFP domain III), amino acid 409 of SEQ ID NO: 58 (human AFP domain I + II), seq id NO: amino acid 217-609 of SEQ ID NO: amino acid 285-609 of 58 (fragment I of human AFP). In another embodiment, the human AFP polypeptide linker sequence is an 8 amino acid sequence comprising SEQ ID NO: amino acids 489-496 of 58 (i.e., EMTPVNPG).

A fourteenth aspect of the invention features a kit that includes any HSA linker, HSA linker conjugate, or any other agent described in the first, second, third, fourth, fifth, eleventh, twelfth, and thirteenth aspects discussed above. The kit further includes instructions for administration of the composition and the agents contained therein to a user (e.g., a physician, nurse, or patient). In one embodiment, the kit comprises a plurality of packaged single or multi-dose pharmaceutical compositions comprising an effective amount of an agent, e.g., an HSA linker conjugate or HSA linker as described herein, comprising, e.g., one or more binding moieties (e.g., an antibody or antibody fragment (e.g., scFv)), a diagnostic agent (e.g., a radionuclide or chelator), and/or a therapeutic agent (e.g., a cytotoxic agent or an immunomodulatory agent). Optionally, the instruments or devices necessary for administering the pharmaceutical composition may be included in a kit. For example, the kit can provide one or more prefilled syringes comprising an effective amount of HSA linker conjugate or HSA linker, or any binding agent, diagnostic agent, and/or therapeutic agent conjugated thereto. In addition, the kit can also include additional components such as instructions or a dosing schedule for administering a pharmaceutical composition comprising, for example, an HSA linker conjugate or HSA linker, or any binding agent, diagnostic agent, and/or therapeutic agent conjugated thereto, to a patient having a disease or disorder (e.g., cancer, autoimmune disease, or cardiovascular disease).

Definition of

As used interchangeably herein, the term "antibody" includes whole antibodies or immunoglobulins as well as any antigen-binding fragment or single chain thereof. As used herein, an antibody can be a mammalian (e.g., human or mouse) antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a synthetically produced antibody, or a naturally isolated antibody. In most mammals, including humans, whole antibodies have at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V)_H) And a heavy chain constant region. The heavy chain constant region is composed of three domains (C)_H1、C_H2. And C_H3) And in C_H1 and C_H2 between them. Each light chain is composed of a light chain variable region (abbreviated herein as V)_L) And a light chain constant region. The light chain constant region consists of one domain (C)_L) And (4) forming. V_HAnd V_LThe regions may be further subdivided into highly variable regions, so-called Complementarity Determining Regions (CDRs), alternating with more conserved regions, so-called Framework Regions (FRs). Each V_HAnd V_LConsists of three CDRs and four FRs, and is arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. Variable regions of heavy and light chainsComprising a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) as well as the first component of the classical complement system (Clq). The antibodies of the invention include all known forms of antibodies as well as other protein backbones with antibody-like properties. For example, the antibody can be a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, or a protein scaffold with antibody-like properties, such as fibronectin or ankyrin repeats. The antibody can also be a Fab, Fab' 2, scFv, SMIP, diabody, nanobody, aptamer, or domain antibody. The antibody may be of any of the following types: IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, and IgAsec), IgD, or IgE. Antibodies that may be used as binding moieties in combination with an HSA linker, as defined herein, include, but are not limited to, natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efletuzumab, golimumab, certuzumab, trastuzumab, abacatel, etanercept, pertuzumab, cetuximab, and panitumumab.

As used herein, the term "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., ErbB 2). The antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include, but are not limited to: (i) fab fragment from V_L、V_H、C_LAnd C_H1 domain; (ii) f (ab')₂A fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) fd fragment of from V_HAnd C_H1 domain composition; (iv) fv fragment, V from one arm of an antibody_LAnd V_HDomain composition; (v) dAb, comprising V_HAnd V_LA domain; (vi) dAb fragments ((Ward et al, Nature 341: 544-546(1989))) consisting of V_HDomain composition; (vii)dAb of V_HOr V_LDomain composition; (viii) an isolated Complementarity Determining Region (CDR); and (ix) a combination of two or more isolated CDRs, wherein the CDRs may optionally be joined by synthetic linkers. In addition, although two domains of the Fv fragment, V_LAnd V_HEncoded by different genes, but they can be joined by recombinant methods and by synthetic linkers that allow them to be made into single protein chains, where V_LAnd V_HThe regions are paired to form monovalent molecules (known as single chain fv (scFv); see, e.g., Bird et al, Science 242: 423-. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for use in the same manner as intact antibodies. Antibody fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

An "autoimmune disease" refers to a disease in which an immune system response is generated against an autologous epitope or antigen. Examples of autoimmune diseases include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, sprue dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, churg-strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, lupus erythematosus, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves ' disease, Guillain-Barr syndrome, Hashimoto thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile arthritis, Graves's disease, Guillain-Barr syndrome, Lichen planus, lupus, meniere's disease, mixed connective tissue disease, multiple sclerosis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, glandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, raynaud's phenomenon, reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, stelmn's syndrome, Systemic Lupus Erythematosus (SLE), takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis (e.g., shotgun bullet-like retinal choroidopathitis (birdshocrocoto herpetic uveitis) and sarcoidosis uveitis), vasculitis, vitiligo, wegener's granulomatosis, and myasthenia gravis.

"binding moiety" refers to any molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding moieties include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric), antibody fragments (e.g., Fab fragments, Fab' 2, scFv antibodies, SMIPs, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and domain antibodies), receptors, ligands, aptamers, and other molecules with known binding partners.

By "biologically active" is meant that a molecule (including biomolecules such as nucleic acids, peptides, polypeptides, and proteins) exerts physical or chemical activity on itself or on other molecules. For example, a "biologically active" molecule can have, for example, enzymatic activity, protein binding activity (e.g., antibody interaction), or cytotoxic activity.

The term "chimeric antibody" refers to an immunoglobulin or antibody whose variable regions are from a first species and whose constant regions are from a second species. Chimeric antibodies can be constructed, for example, by genetic engineering, from immunoglobulin gene fragments belonging to different species (e.g., from mouse and human).

"linker" or "peptide linker" refers to an amino acid sequence of 2 to 20 residues in length that is covalently attached to one or both amino or carboxy termini of the HSA linker, or to one or more residues of the HSA linker (e.g., residues between the amino and carbonyl terminal residues). In a preferred embodiment, the peptide linker attached to the amino terminus of the HSA linker has the amino acid sequence AAS or AAQ and the linker attached to the carboxy terminus has the amino acid sequence "AAAL" (SEQ ID NO: 5).

The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent (e.g., an HSA linker conjugated with one or more binding moieties or diagnostic or therapeutic agents and with or without a connector sequence) sufficient to produce a desired result, e.g., killing cancer cells, reducing tumor cell proliferation, reducing inflammation in diseased tissues or organs, or labeling a particular cell population in a tissue, organ, or organism (e.g., a human).

As used herein, the term "Human antibody" is intended to include antibodies, or fragments thereof, having variable regions in which both framework and CDR regions are derived from Human germline immunoglobulin Sequences as described, for example, by Kabat et al, (Sequences of proteins of Immunological Interest, fine Edition, u.s.department of Health and Human Services, NIH Publication No.91-3242 (1991)). In addition, if the antibody comprises a constant region, the constant region is also derived from a human germline immunoglobulin sequence. Human antibodies can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences from the germline of another mammalian species (e.g., a mouse) have been grafted onto framework sequences (i.e., humanized antibodies or antibody fragments).

The term "humanized antibody" refers to any antibody or antibody fragment that includes at least one immunoglobulin domain having a variable region that includes variable framework regions substantially from a human immunoglobulin or antibody and complementarity determining regions (e.g., at least one CDR) substantially from a non-human immunoglobulin or antibody.

As used herein, an "inflammatory signaling inhibitor" or "ISI" is an agent that reduces the binding between a proinflammatory cytokine (e.g., TNF- α, TNF- β, or IL-1) and its receptor (e.g., TNF receptor 1 or 2, or IL-1 receptor, respectively); reducing binding of the activating molecule to a proinflammatory cell surface signaling molecule (e.g., CD20, CD25, CTLA-4, CD80/CD86, or CD 28); or reducing downstream activation, or activity, of an intracellular signaling molecule that is activated upon binding of pro-inflammatory cytokines to their receptors or upon binding of an activating molecule to a pro-inflammatory cell surface signaling molecule (e.g., an agent that reduces activation, or activity, of a signaling molecule in the p38MAPK signaling pathway). The ISI-mediated decrease may be a decrease in binding between pro-inflammatory cytokines and their receptors, a decrease in binding of activating molecules to pro-inflammatory cell surface signaling molecules, or a decrease in intramolecular signaling that occurs after pro-inflammatory cytokines bind to their receptors or activating molecules bind to pro-inflammatory cell surface signaling molecules. Preferably, such ISI-mediated reduction is a reduction of at least about 10%, preferably at least 20%, 30%, 40%, or 50%, more preferably at least 60%, 70%, 80%, or 90% (up to 100%). ISI can act by reducing the amount of proinflammatory cytokines (e.g., TNF-alpha, TNF-beta, or IL-1) that are freely available to bind to the receptor. For example, the ISI may be soluble proinflammatory cytokine receptor proteins (e.g., soluble TNF receptor fusion proteins such as etanercept)Or lenacicept), or a soluble pro-inflammatory cell surface signaling molecule (e.g., soluble CTLA-4 (abacavir)), or an antibody that targets a pro-inflammatory cytokine or a pro-inflammatory cell surface signaling molecule (e.g., an anti-TNF antibody such as adalimumab, certolizumab, inflixamab, or golimumab; anti-CD 20 antibodies, such as rituximab; or TRU-015(Trubion Pharmaceuticals)). In addition, ISI may be mediated by disruption of binding of endogenous wild-type proinflammatory cytokines or proinflammatory cell surface signaling molecules to their receptors (e.g., TNF receptor 1 or 2, IL-1 receptors such as anakinra, gamma-,Or CD11a such as efletuzumab (Genentech)) to function. Examples of dominant-negative (negative-negative) TNF-alpha variants are XENP345 (pegylated form of TNF variant a 145R/I97T) and xpro 1595, as well as additional variants disclosed in U.S. patent application publication nos. 20030166559 and 20050265962, which are incorporated herein by reference. An example of a dominant negative IL-1 variant is anakinraIt is a soluble form of IL-1, wherein IL-1 binds to the IL-1 receptor without activating intracellular signaling pathways. Inflammatory signaling inhibitors that may be used in the present invention are also small molecules that inhibit or decrease the signaling pathway downstream of a proinflammatory cytokine or proinflammatory cell surface signaling molecule (e.g., DE 096). Examples of this type of ISI include inhibitors of p38MAP kinase, e.g., 5-amino-2-carbonylthiophene derivatives (5-amino-2-arylthiopen derivatives) (as described in WO04/089929, incorporated herein); ARRY-797; BIRB 796BS, (1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- [4-2 (morpholin-4-yl-ethoxy) -naphthalen-1-yl]-urea); CHR-3620; CNI-1493; FR-167653(Fujisawa Pharmaceutical, Osaka, Japan); ISIS 101757(ISIS Pharmaceuticals); ML 3404; NPC 31145; PD 169383; PHZ 1112; RJW67657, (4- (4- (4-fluorophenyl) -1- (3-phenylpropyl) -5- (4-pyridyl) -1H-imidazol-2-yl) -3-butyn-1-ol, SCIO-469; SB 202190; SB203580, (4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- (4-pyridyl) 1H-imidazole), SB239063, trans-1- (4-hydroxycyclohexyl) -4- (4-fluorophenyl-methoxypyrimidin-4-yl) imidazole (trans-1- (4-hydroxycyclohexyl) -4- (4-fluorophenoxy-4-yl) imidazole), SB242235, SD-282, SKF-86002, TAK 715, VX702, and VX745. further, ISI can interfere with the processing of pro-inflammatory cytokines (e.g., TNF- α and TNF- β) from its membrane-bound form to its soluble form. Inhibitors of TACE are this type of ISI. Examples of inhibitors of TACE include BB-1101, BB-3103, BMS-561392, butaneAlkynyl hydroxyphenyl β -sulfone piperidine hydroxamates (butyloxyphenyl β -sulfone piperidine hydroxamates), CH4474, DPC333, DPH-067517, GM6001, GW3333, Ro 32-7315, TAPI-1, TAPI-2, and TMI 005. Additional examples of ISI include short peptides from heat shock proteins of e.coli that are designed to have disease-specific immunomodulatory activity (e.g., dnaJP 1).

An "integrin antagonist" refers to any agent that reduces or inhibits the biological activity of an integrin molecule (e.g., reduces or inhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more relative to the biological activity in the absence of an integrin antagonist), such as the α 4 subunit of an integrin molecule. The above agents may act directly or indirectly on the α 4 integrin subunit by inhibiting the activity or expression of the α 4 integrin subunit (NCBI Access No. P13612; Takada et al, EMBO J. 8: 1361-1368(1989)), or may act on a target to which an intact integrin comprising the α 4 subunit binds. For example, antibodies or blocking peptides that bind to vascular cell adhesion molecule-1 (VCAM-1) and thus prevent α 4 β 1 integrin binding to VCAM-1 are considered integrin antagonists for the purposes of the present invention. Non-limiting exemplary integrin antagonists suitable for use in the present invention can include proteins, blocking peptides, antibodies, such as natalizumabAnd small molecule inhibitors. Examples of alpha 4 integrin antagonists include, but are not limited to, natalizumab (Elan/Biogen Idec; see, e.g., U.S. Pat. nos. 5,840,299, 6,033,665, 6,602,503, 5,168,062, 5,385,839, and 5,730,978, incorporated herein by reference), oemupa-V (Biogen; U.S. Pat. No. 6,495,525, incorporated herein by reference), alefacept, CDP-323 (Celltech); firataegras (SB-68399; GlaxoSmithKline); TR-9109 (Pfizer); ISIS-107248 (Antisene therapeutics); r-1295 (Roche); and TBC-4746 (Schering-PloUgh). Additional non-limiting examples of α 4 integrin antagonists include small molecules, see: united states patentNumbers 5,821,231, 5,869,448, 5,936,065, 6,265,572, 6,288,267, 6,365,619, 6,423,728, 6,426,348, 6,458,844, 6,479,666, 6,482,849, 6,596,752, 6,667,331, 6,668,527, 6,685,617, 6,903,128, and 7,015,216 (each incorporated herein by reference); U.S. patent application publication nos. 2002/0049236, 2003/0004196, 2003/0018016, 2003/0078249, 2003/0083267, 2003/0100585, 2004/0039040, 2004/0053907, 2004/0087574, 2004/0102496, 2004/0132809, 2004/0229858, 2006/0014966, 2006/0030553, 2006/0166866, 2006/0166961, 2006/0241132, 2007/0054909, and 2007/0232601 (each incorporated herein by reference); european patent nos. EP 0842943, EP 0842944, EP 0842945, EP 0903353, and EP 0918059; and PCT publication Nos. WO 95/15973, WO 96/06108, WO 96/40781, WO 98/04247, WO 98/04913, WO98/42656, WO 98/53814, WO 98/53817, WO 98/53818, WO98/54207, WO 98/58902, WO 99/06390, WO 99/06431, WO99/06432, WO 99/06433, WO 99/06434, WO 99/06435, WO99/06436, WO 99/06437, WO 99/10312, WO 99/10313, WO99/20272, WO 99/23063, WO 99/24398, WO 99/25685, WO99/26615, WO 99/26921, WO 99/26922, WO 99/26923, WO99/35163, WO 99/36393, WO 99/37605, WO 99/37618, WO99/43642, WO 01/42215, and WO 02/28830, all of which are incorporated herein by reference. Additional examples of alpha 4 integrin antagonists include phenylalanine derivatives, see U.S. patent nos. 6,197,794, 6,229,011, 6,329,372, 6,388,084, 6,348,463, 6,362,204, 6,380,387, 6,445,550, 6,806,365, 6,835,738, 6,855,706, 6,872,719, 6,878,718, 6,911,451, 6,916,933, 7,105,520, 7,153,963, 7,160,874, 7,193,108, 7,250,516, and 7,291,645 (each incorporated herein by reference). Additional amino acid derivatives that are antagonists of α 4 integrins include those derivatives. Which are described, for example, in U.S. patent application publication nos. 2004/0229859 and 2006/0211630 (incorporated herein by reference), and PCT publication nos. WO 01/36376, WO 01/47868, and WO 01/70670, all of which are incorporated herein by reference. Other examples of alpha 4 integrin antagonists include peptides, andand peptide and semi-peptide compounds, see, for example, PCT publications WO 94/15958, WO 95/15973, WO 96/00581, WO 96/06108, WO 96/22966(Leu-Asp-Val tripeptide; Biogen, Inc.), WO 97/02289, WO 97/03094, and WO 97/49731. Further examples of alpha 4 integrin antagonists are the pegylated molecules described in U.S. patent application publication No. 2007/066533 (incorporated herein by reference). Examples of antibodies that are alpha 4 integrin antagonists include those described in, for example, PCT publication nos. WO 93/13798, WO 93/15764, WO 94/16094, and WO 95/19790. Additional examples of alpha 4 integrin antagonists are described herein.

"Interferon" refers to mammalian (e.g., human) interferon-alpha, -beta, -gamma, or-tau polypeptides, or biologically active fragments thereof, such as IFN-alpha (e.g., IFN-alpha-1 a, see U.S. patent application No. 20070274950, incorporated herein by reference), IFN-alpha-1 b, IFN-alpha-2 a (see PCT application No. WO 07/044083, incorporated herein by reference), and IFN-alpha-2 b), IFN-beta (e.g., described in U.S. patent No. 7,238,344, incorporated herein by reference; IFN-b-1a (And) As described in U.S. Pat. No. 6,962,978, which is incorporated herein by reference, and IFN- β -1b (As described in U.S. Pat. Nos. 4,588,585, 4,959,314, 4,737,462, and 4,450,103, which are incorporated herein by reference), IFN-g, and IFN-t (as described in U.S. Pat. No. 5,738,845 and U.S. patent application publication Nos. 20040247565 and 20070243163, which are incorporated herein by reference).

By "HSA linker conjugate" is meant a Human Serum Albumin (HSA) linker along with (preferably covalently bound to) one or more binding moieties, peptide connectors, diagnostic agents, or therapeutic agents.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, targeting single antigenic sites. In addition, unlike conventional (polyclonal) antibody agents, which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. Monoclonal antibodies can be prepared using any art-recognized technique and those herein, such as, for example. Hybridoma methods, such as those described by Kohler et al, Nature 256: 495(1975), transgenic animals (e.g., Lonberg et al, Nature 368 (6474): 856-859(1994)), recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567), or recombinant DNA methods using phage, yeast, or synthetic scaffold antibody libraries and methods described in, e.g., Clackson et al, Nature 352: 624-: 581-597 (1991).

By "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to the mammal to be treated while retaining the therapeutic properties of the compound with which it is administered. One typical pharmaceutical carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's pharmaceutical Sciences, (18)^thedition), ed.a. gennaro, 1990, mack publishing Company, Easton, PA.

By "proliferative disease" or "cancer" is meant any condition characterized by abnormal or unregulated cell growth. Examples of proliferative diseases include, for example, solid tumors such as: sarcomas (e.g., clear cell sarcoma), carcinomas (e.g., renal cell carcinoma), and lymphomas; tumors of the following tissues, organs, or systems: chest, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder, bile ducts, small intestine, urinary system (including the epithelium of the kidney, bladder, and urinary tract), female reproductive system (including the cervix, uterus, ovary, chorioma, and gestational trophoblasts), male reproductive system (including the prostate, seminal vesicle, and testis), endocrine glands (including the thyroid, adrenal, and pituitary), skin (including hemangioma, melanoma, sarcomas from bone or soft tissue, and kaposi's sarcoma), brain and meninges (including astrocytoma, neuroastrocytoma, spongioblastoma, neuroma, neuroblastoma, schwanoma, and neuroblastoma), nerve, eye, hematopoiesis system (including chloromycetic leukemia, plasmacytoma, and dermal T lymphoma/leukemia), And the immune system (including lymphomas, e.g., hodgkin lymphoma and non-hodgkin lymphoma). An example of a non-solid tumor proliferative disease is leukemia (e.g., acute lymphoblastic leukemia).

The term "recombinant antibody" refers to an antibody that has been prepared, expressed, produced, or isolated by recombinant means, such as (a) an antibody isolated from an animal (e.g., a mouse), which is transgenic or transchromosomal for an immunoglobulin gene (e.g., a human immunoglobulin gene) or a hybridoma prepared therefrom, (b) isolated from a host cell transformed to express the antibody, e.g., antibodies isolated from transfectomas, (c) antibodies isolated from recombinant, combinatorial antibody libraries (e.g., comprising human antibody sequences), wherein phage, yeast, or synthetic scaffold (synthetic scaffold) display is utilized, and (d) an antibody prepared, expressed, produced, or isolated by any other means, wherein any of the other above-described means involves splicing of immunoglobulin gene sequences (e.g., human immunoglobulin genes) into other DNA sequences.

By "specifically binds" is meant that the binding moiety (e.g., an antibody, antibody fragment, receptor, ligand, or small molecule portion of an agent described herein) preferentially binds to a target molecule (e.g., a secreted target molecule such as a cytokine, chemokine, hormone, receptor, or ligand) or preferentially binds to a molecule containing a target molecule(e.g., a cell surface antigen such as a receptor or ligand) and does not preferentially bind to non-target cells or tissues lacking the target molecule. It is generally recognized that a degree of non-specific interaction can occur between the binding moiety and the non-target molecule (alone or in conjunction with a cell or tissue). However, specific binding may be said to be mediated by specific recognition of the target molecule. Specific binding results in stronger binding between the binding moiety (e.g., an antibody) and, for example, a cell containing the target molecule (e.g., an antigen) than between the binding moiety and, for example, a cell lacking the target molecule. Specific binding typically results in a greater than 2-fold increase (per unit time) in the amount of bound binding moiety compared to, for example, cells or tissues containing the target molecule or label, preferably greater than 5-fold, more preferably greater than 10-fold and most preferably greater than 100-fold, compared to cells or tissues lacking the target molecule or label. The binding moiety binds to a target molecule or label with a dissociation constant of, e.g., less than 10^-6M, more preferably less than 10^-7M、10^-8M、10^-9M、10^-10M、10^-11M, or 10^-12M, and most preferably less than 10^-13M、10^-14M, or 10^-15And M. Specific binding to proteins under the above conditions requires a binding moiety that is selected for its specificity for a particular protein. Various assay formats are suitable for selecting binding moieties (e.g., antibodies) that are capable of specifically binding to a particular target molecule. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specific for immunological activity with proteins. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Harlow&Lane，Antibodies，ALaboratory Manual，Cold Spring Harbor Publications，New York(1988)。

The term "sequence identity" means (when comparing a polynucleotide or polypeptide sequence to a reference sequence) that the polynucleotide or polypeptide sequence is identical to the reference sequence or that a specified percentage of nucleotides or amino acid residues are identical to the corresponding positions in the reference sequence when the two sequences are optimally aligned.

By "surface exposed amino acid residues" or "surface exposed" is meant amino acid residues that are present on the outer surface of the folded and conformationally correct tertiary structure of the HSA polypeptide. The above residues may be substituted, for example, with other chemically active amino acids (e.g., cysteine) to facilitate site-specific binding of diagnostic or therapeutic agents. In addition, surface exposed amino acid residues can be substituted to facilitate (e.g., by adding serine, threonine, asparagine residues, or glycosylation motifs) or prevent (e.g., by removing serine, threonine, or asparagine residues, or glycosylation motifs) glycosylation. Surface exposed amino acid residues include, but are not limited to, threonine at position 496, serine at position 58, threonine at position 76, threonine at position 79, threonine at position 83, threonine at position 125, threonine at position 236, serine at position 270, serine at position 273, serine at position 304, serine at position 435, threonine at position 478, threonine at position 506, and threonine at position 508 (the amino acid numbering is relative to, for example, the sequence of the HSA linker listed in SEQ ID NO: 1). Using the HSA crystal structure, one skilled in the art can identify other surface-exposed residues (Sugio et al, "crystallization of human serum album at 2.5A resolution," Protein Eng.12: 439-446 (1999)). By "subject" is meant a human patient or a nude mouse xenograft model comprising human tumor cells.

By "target molecule" or "target cell" is meant a molecule (e.g., a protein, epitope, antigen, receptor, or ligand) or cell to which a binding moiety (e.g., an antibody) is capable of binding, or an HSA conjugate comprising one or more binding moieties (e.g., an HSA linker bound to one or more antibodies or antibody fragments). Preferred target molecules are exposed to the exterior of the target cell (e.g., cell surface or secreted proteins) but the target molecules may alternatively or also be present in the interior of the target cell.

"treating" preferably provides reducing (e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100%) the progression or severity of a human disease or disorder (e.g., an autoimmune or proliferative disease) or reducing the progression, severity, or frequency of one or more symptoms of a human disease or disorder in a subject.

Drawings

Figure 1 is a schematic of a typical HSA linker conjugate. The connector between the amino terminal binding moiety and the HSA linker has the sequence of alanine, and serine. The linker between the HSA linker and the carboxy-terminal binding portion has the sequence of alanine, leucine (SEQ ID NO: 5).

Fig. 2 is a graph showing that B2B3 variant inhibits HRG-induced pErbB3 in ZR75-1 breast cancer cells.

Fig. 3A-D are graphs showing inhibition of phosphorylated ErbB3 in BT474 breast cancer cells after 24 hours of pretreatment with B2B3HSA linker conjugate B1D2-2(a5-HSA-B1D2, fig. 3A), B1D2-1 (H3-HSA-B1D2, fig. 3B), B2B3-10(H3-HSA-F5B6H2, fig. 3C), and B2B3-8(F4-HSA-F5B6H2, fig. 3D). Further details regarding these HSA linker conjugates are set forth below (e.g., in table 6).

Fig. 4A-D are graphs showing inhibition of phosphorylated AKT in BT474 breast cancer cells after 24 hours of pretreatment with B2B3HSA linker conjugate B1D2-2(a5-HSA-B1D2, fig. 4A), B1D2-1 (H3-HSA-B1D2, fig. 4B), B2B3-10(H3-HSA-F5B6H2, fig. 4C), and B2B3-8(F4-HSA-F5B6H2, fig. 4D). Further details regarding these HSA linker conjugates are set forth below (e.g., in table 6).

FIGS. 5A-D are graphs showing linker binding B1D2-2(A5-HSA-B1D2, FIG. 5A), B1D2-1 with B2B3HSA in BT474 breast cancer cells

Inhibition of phosphorylated ERK after 24 hours of pretreatment (H3-HSA-B1D2, FIG. 5B), B2B3-10(H3-HSA-F5B6H2, FIG. 5C), and B2B3-8(F4-HSA-F5B6H2, FIG. 5D). Further details regarding these HSA linker conjugates are set forth below (e.g., in table 6).

Fig. 6 is a graph showing that treatment of BT474 breast cancer cells with B2B3-1 variant resulted in G1 arrest and a reduction in the number of cells in S phase.

Figure 7 is a flow cytometry bar graph showing that preincubation of BT-474-M3 cells with 1 μ M B2B3-1 significantly blocked HRG binding.

FIGS. 8A-D are graphs showing that B2B3-1 inhibits ErbB3 and AKT phosphorylation in B-T474-M3 and ZR75-30 cell lines. The breast cancer cell lines BT-474-M3 (FIGS. 8A and 8C) and ZR75-30 (FIGS. 8B and 8D) were pretreated with dose-titrated B2B3-1 for 24 hours, followed by stimulation with 5nM of HRG 1 β EGF domain for 10 min. The phosphorylation status of ErbB3 and AKT was then examined using an ELISA assay.

FIG. 9 is a Western blot photograph showing the effect of treatment with increasing concentrations of B2B3-1 on signaling proteins in BT474 breast cancer cells. "p-" represents the tyrosine phosphorylated form of the signal protein. Beta tubulin (not a signal protein herein) provides a loading control. Beta tubulin (not a signal protein herein) provides a loading control.

Fig. 10 is a western blot photograph showing immunoprecipitation of BT474 breast cancer cells treated with B2B 3-1. Beta tubulin provides a control for the level of cellular protein input into the immunoprecipitation reaction.

11A-C show that B2B3-1 treatment of BT-474 cell line resulted in G1 arrest and a reduction in cell population in S phase (FIG. 11A), and inhibited colony formation in BT-474 and SKBr3 cells compared to untreated cells (FIG. 11B). In addition, B2B3-1 inhibited the proliferation of BT-474-M3 cells in a cell impedance assay (FIG. 11C).

FIG. 12 is a graph showing that B2B3-1 does not stimulate ErbB3 phosphorylation in ZR75-1 cells.

Figures 13A-B are graphs showing that B2B3-1 specifically binds to ErbB3 (figure 13A) and ErbB2 (figure 13B).

Figure 14 is a graph showing that affinity binding of B2B3-1 to MALME-3 cells results in a significant increase in apparent binding affinity compared to the binding variant SKO-3 unique to ErbB2, and SKO-2 unique to ErbB 3.

Fig. 15A-C are graphs showing the stability of B2B3-1 in mouse, cynomolgus monkey, and human serum. In mouse (FIG. 15A), cynomolgus monkey (FIG. 15B), or human (FIG. 15C) serum and at 37 ℃ temperature in 100nM B2B3-1 for 120 hours. Samples were removed at 0, 24, 48, 72, 96 and 120 hours and the ability of B2B3-1 to bind ErbB2 and ErbB3 was measured by ELISA (RLU relative light units).

FIG. 16 is a graph showing B2B3-1 dose response in a BT-474-M3 breast cancer xenograft model. Dose-to-tumor response relationship of B2B3-1 evaluated in BT-474-M3 breast tumor lines and at the indicated doses. HSA was administered at 52.5mg/kg, which is an equimolar dose to the 90mg/kg B2B3-1 dose.

Figures 17A-E are graphs showing that B2B3-1 is effective in a variety of xenograft models in an ErbB2 dependent manner. Calu-3 (human lung adenocarcinoma; FIG. 17A), SKOV-3 (human ovarian adenocarcinoma; FIG. 17B), NCI-N87 (human gastric carcinoma; FIG. 17C), ACHN (human renal adenocarcinoma; FIG. 17D), and MDA-MB-361 (human thymus carcinoma; FIG. 17E) xenograft models are shown. Mice were treated every 3 days with 30mg/kg of B2B3-1 or with an HSA control at an equimolar dose to B2B 3-1.

Figures 18A-B are graphs showing that overexpression of ErbB2 converted B2B3-1 non-responder ADRr breast cancer xenograft models to responders. ErbB2 was overexpressed in wild-type ADRr xenografts (fig. 18A) and in ADRr-E2 xenografts (fig. 18B) using a retroviral expression system.

FIGS. 19A-B show that B2B3-1 activity was positively correlated with levels of ErbB2 expression in vitro (FIG. 19A) and in vivo (FIG. 19B).

FIGS. 20A-B show that B2B3-1 treatment altered the tumor cell cycle. FIG. 20A includes fluorescence micrographs showing that treatment of BT474-M3 breast tumor cells with B2B3-1 for 6 hours results in the cell cycle inhibitor p27^kip1Translocate to the nucleus. Hoechst staining was used to identify nuclei. FIG. 20B is a Western blot of BT-474-M3 cells treated with B2B3-1 for 72 hours, which resulted in a decrease in the level of the cell cycle regulator cyclin D1. In this experiment, the cytoskeletal protein neunin was used as a protein loading control.

FIGS. 21A-B are photomicrographs showing that treatment of BT474 breast tumor xenografts with B2B3-1 resulted in p27^kip1Translocate to the nucleus. BT474 breast tumor xenografts were treated every 3 days with B2B3-1 (FIG. 21A) at a dose of 30mg/kg or with an equimolar dose of HSA (FIG. 21B), for a total of 4 doses, followed by p27^kip1And (6) dyeing.

FIGS. 22A-B are fluorescence micrographs showing that B2B3-1 treatment resulted in a decrease in the proliferation marker Ki67 in BT474-M3 breast cancer xenografts. BT474-M3 breast tumor xenografts were treated every 3 days with B2B3-1 at a dose of 30mg/kg (FIG. 22A) or with an equimolar dose of HSA (FIG. 22B), for a total of 4 doses.

Fig. 23A-B are fluorescence micrographs showing that B2B3-1 treatment resulted in a decrease in vascular density (as determined by a decrease in CD31 staining) in BT474-M3 breast cancer xenograft tumors. BT474-M3 breast tumor xenografts were treated every 3 days with B2B3-1 at a dose of 30mg/kg (FIG. 23A) or with an equimolar dose of HSA (FIG. 23B), for a total of 4 doses.

FIGS. 24A-B show that B2B3-1 inhibits phosphorylation of ErbB3 in vivo. Lysates from BT-474-M3 xenograft tumors alone and treated with B2B3-1(M1-M5) or control HSA (H1-H2) were subjected to SDS-PAGE and detected for pErbB3 and beta tubulin using Western blot analysis (FIG. 24A). Normalization of the mean pErbB3 signal relative to the mean beta tubulin signal indicated that B2B3-1 treated tumors contained much less pErbB3 than HSA tumors (fig. 24B).

Fig. 25A and B are graphs showing the in vivo activity of B2B3-1 in BT-474-M3shPTEN and sh control xenografts. Cultured BT-474-M3 tumor cells were transfected with control vectors (fig. 25A) or with retroviral vectors expressing shPTEN (fig. 25B), which knockdown PTEN activity. Thus, genetically engineered BT-474-M3 breast cancer cells knock-out for PTEN activity were injected into the right flank of mice, while cells transfected with control vectors were injected into the left flank of the same mice. Mice were treated every 3 days with 30mg/kg B2B3-1 or weekly with 10mg/kg trastuzumab, and injected with an equimolar dose of HSA relative to B2B3-1 as a control. B2B3-1 and trastuzumab promoted a reduction in tumor size formed by control BT-474-M3 breast cancer cells (fig. 25A), while B2B3-1 alone (and not trastuzumab) promoted a reduction in tumor size formed by BT-474-M3 breast cancer cells lacking PTEN expression (fig. 25B).

FIGS. 26A-B show that B2B3-1 inhibits phosphorylation of AKT in BT-474-M3 xenografts with decreased PTEN activity. After completion of treatment (q3dx11), tumors were lysed and tested for PTEN, pErbB3, and pAKT expression levels by western blot analysis (fig. 26A). Densitometry of the band intensities for pAKT (normalized to total AKT and total protein) showed that B2B3-1 was able to inhibit phosphorylation of this protein when trastuzumab was not able (fig. 26B).

Figures 27A-D are graphs showing single dose pharmacokinetic performance of 5 (figure 27A), 15 (figure 27B), 30 (figure 27C), and 45 (figure 27D) mg/kg bolus doses of B2B3-1 in nu/nu mice. Serum concentrations of B2B3-1 measured by the HSA assay or the ErbB2/ErbB3 assay were comparable, indicating that the antigen binding activity of B2B3-1 was retained in the circulation.

FIG. 28 shows dose exposure relationships for 5, 15, 30, and 45mg/kg bolus doses of B2B3-1 in nude mice. An increase in dose resulted in a linear increase in total exposure to B2B 3-1.

Fig. 29 shows B2B3-1 serum concentrations measured in cynomolgus monkeys dosed a total of 4 doses every 3 days, with doses of 4mg/kg (n-2), 20mg/kg (n-2), and 200mg/kg (up to 336 hours, n-4, for 384, 552, and 672 hour time points, n-2).

FIG. 30 shows the B2B3-1 expression plasmid pMP10k4H3-mHSA-B1D 2.

FIG. 31 shows the neomycin resistance plasmid pSV 2-neo.

FIG. 32 shows the hygromycin-resistant plasmid pTK-Hyg.

Figure 33 shows data representative of q7d administration of B2B3-1, equivalent to the efficacy of q3d administration.

Figure 34 shows western blot data representing that B2B3-1 and trastuzumab exhibit different mechanisms of ErbB3 inhibition.

FIGS. 35A-C show the detailed experimental results in example 43, in which B2B3-1 was studied in combination with trastuzumab in spheroid cells of various human breast cancer cell lines as a model of human breast cancer. FIG. 35A shows data obtained using BT-474-M3 cells, FIG. 35B shows data obtained using SKBR3 cells, and FIG. 35C shows data obtained using MDA-MB-361 cells. The molar concentration of B2B3-1, alone or in combination, is given along the X-axis. The molar concentration of trastuzumab, alone or in combination, was one-third of the concentration of each of the indicated B2B 3-1.

Figure 36 shows the results of in vivo tumor xenograft experiments performed specifically in example 44. "days" on the X-axis indicate days after tumor implantation. The error bars for each data point represent the response to at least two independent biological xenografts.

FIG. 37 shows data obtained from a xenograft model substantially as described in example 44, except that the tumor cells used were N-87 gastric tumor cells, which were obtained from the National Cancer Institute (US National Cancer Institute).

FIG. 38 shows data for the subgroups shown in FIG. 36.

Detailed Description

The invention provides Human Serum Albumin (HSA) linkers, as well as HSA linker conjugates (e.g., binding, diagnostic, or therapeutic agents) comprising an HSA linker and one or more additional moieties (e.g., binding moieties). Such HSA linker conjugates have desirable properties, e.g., increased in vivo half-life of 6 hours to 7 days, and do not induce a significant humoral or cell-mediated immune response when administered in vivo to a mammal (e.g., a human). In one aspect, the invention provides a mutant HSA linker having two defined amino acid substitutions (i.e., the "C34S" and "N503Q" substitutions, as set forth in SEQ ID NO: 1). In another aspect, the invention provides HSA linkers that bind to one or more binding moieties (e.g., antibodies, antibody fragments, receptors/ligands, or small molecules), for diagnostic or therapeutic use in vivo in a mammal (e.g., a human), or for use in vitro in conjunction with a mammalian cell, tissue, or organ. In yet another aspect, the HSA linker can be linked to one or more immunomodulators, cytotoxic or cytostatic agents, detectable labels, or radioactive agents for diagnostic or therapeutic use in a mammal (or in conjunction with a mammalian cell, tissue, or organ). The HSA linker conjugate including the HSA linker can optionally be combined with one or more pharmaceutically acceptable carriers or excipients, and can be formulated for administration by intravenous route, intramuscular route, oral, inhalation route, parenteral route, intraperitoneal route, intraarterial route, transdermal route, sublingual route, nasal route, by using suppositories, buccal route, liposomal route, fat route, ocular route, intraocular route, subcutaneous route, intrathecal route, topical route, or topical route. The HSA linker conjugate can, but need not, be combined with or administered with one or more bioactive agents (e.g., biological or chemical agents, such as chemotherapeutic and antineoplastic agents). In yet another aspect, the invention provides kits with instructions for binding a binding moiety (e.g., an antibody, antibody fragment, receptor, or ligand), an immunomodulator, a cytotoxic or cytostatic agent, a detectable label, or a radioactive agent to an HSA linker to prepare an HSA linker conjugate useful for diagnostic or therapeutic use.

Human Serum Albumin (HSA) linker

The HSA linker can comprise various types of HSA amino acid sequences, such as SEQ id no: 3, and (c) is listed in (3). Alternatively, the HSA linker may comprise an altered, or mutated, sequence. A mutant HSA linker comprises a sequence corresponding to the sequence set forth in SEQ ID NO: 3 at positions 34 and 503. The cysteine residue at position 34 (i.e., C34) can be mutated to any amino acid residue other than cysteine (e.g., serine, threonine, or alanine). Likewise, the asparagine residue at position 503 (i.e., N503) can be mutated to any amino acid residue other than asparagine (e.g., glutamine, serine, histidine, or alanine). In one embodiment, the HSA linker has the amino acid sequences set forth in SEQ ID NOs: 1 and 2 and the corresponding nucleotide sequences. This mutant HSA linker comprises two amino acid substitutions (i.e., a serine substitution for cysteine at amino acid residue 34 ("C34S") and a glutamine substitution for asparagine at amino acid residue 503 ("N503Q")). The HSA linker, when conjugated to one or more binding moieties (e.g., antibodies, antibody fragments (e.g., single chain antibodies), or other targeting or bioactive agents (e.g., receptors and ligands)) can confer several advantageous pharmacological properties to those conjugates and to linked additional diagnostic or therapeutic agents (e.g., immunomodulators, cytotoxic or cytostatic agents, detectable labels, or radioactive agents) relative to the pharmacological properties of these agents in the absence of the HSA linker. These benefits can include reduced immunogenicity (e.g., reduced host antibody neutralization of the linker-antibody conjugate), increased detection of the HSA linker conjugate (e.g., by mass spectrometry), and increased pharmacological half-life (e.g., a half-life greater than 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) when administered to a mammal (e.g., a human). Specifically, the substitution of serine for cysteine at amino acid residue 34 results in reduced oxidation and protein heterogeneity of the HSA linker. In wild-type HSA, asparagine at amino acid residue 503 is susceptible to deamination, again resulting in a reduced pharmacological half-life. The substitution of glutamine for asparagine at amino acid residue 503 can result in an extended pharmacological half-life of the HSA linker, thus, when administered to a mammal (e.g., a human) or a cell, tissue, or organ thereof, correspondingly increasing the pharmacological half-life of a binding agent comprising the HSA linker.

In other embodiments, the mutant HSA linker comprises domain I of HSA (SEQ ID NO: 53; residues 1-197 of SEQ ID NO: 1), domain III of HSA (SEQ ID NO: 55; residue 381-585 of SEQ ID NO: 1), a combination of domains I and III of HSA, or a combination of domain I or III of HSA and domain II of HSA (SEQ ID NO: 54; residue 189-385 of SEQ ID NO: 1). For example, the HSA linker can include domains I and II, I and III, or II and III. Furthermore, the cysteine residue at position 34 of domain I (SEQ ID NO: 53) (i.e., C34) can be mutated to any amino acid residue other than cysteine (e.g., serine, threonine, or alanine). Likewise, the asparagine residue at position 503 of Domain III (SEQ ID NO: 55) (i.e., N503) can be mutated to any amino acid residue other than asparagine (e.g., glutamine, serine, histidine, or alanine). These HSA linkers can be added to HSA linker conjugates that include one or more peptide connectors, binding moieties, and therapeutic or diagnostic agents, each of which is described in detail below.

Peptide linker

To facilitate binding of the binding moiety as defined herein to the HSA linker, a short (e.g., 2-20 amino acids in length) peptide linker (e.g., covalent (e.g., peptide bond), ionic, or hydrophobic binding, or via high affinity protein-protein binding interactions (e.g., biotin and avidin)) can be bound to the amino or carboxy terminus of the HSA linker. These linkers provide a flexible chain to which any of the binding moieties described herein can be attached. The peptide linker may be 2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length. In one embodiment, the linker is, for example, a sequence of glycine, alanine, serine, glutamine, leucine, or valine residues. Although not specifically enumerated herein, the linker may be simply a glycine, alanine, serine, glutamine, leucine, or valine residue, and may be any combination of these residues (up to about 20 amino acids in length). In a preferred embodiment, the linker attached to the amino terminus of the HSA linker has the amino acid sequence AAS or AAQ and the linker attached to the carboxy terminus has the amino acid sequence "AAAL" (SEQ ID NO: 5). The connector may be covalently bound to an amino or carbonyl terminal residue of the HSA linker, to an amino acid residue in the HSA linker, or may be included between one or more binding moieties (if present).

HSA linker preparation

The HSA linker, with or without one or more peptide connectors, one or more binding moieties described below, detectable polypeptide-based labels, and other polypeptide-based therapeutic agents, can be recombinantly produced. For example, the nucleotide sequence encoding the HSA linker (and one or more optional elements) can be expressed (e.g., in a plasmid, viral vector, or transgenically) in a bacterium (e.g., e.coli), insect, yeast, or mammalian cell (e.g., CHO cell), or in a mammalian tissue, organ, or organism (e.g., a transgenic rodent, ungulate (e.g., goat), or non-human primate). After expression of the HSA linker in the host cell, tissue, or organ, the HSA linker can be isolated and purified by one skilled in the art using standard protein purification methods (e.g., FPLC or affinity chromatography). The recombinant expression system for the production of the HSA linker together with the two binding moieties is shown in figure 1.

Alternatively, the HSA linker with or without one or more of the optional elements described above may be produced synthetically. For example, the HSA linker or HSA linker conjugate can be prepared by techniques commonly established in the art of peptide synthesis, such as solid phase means. Solid phase synthesis involves the stepwise addition of amino acid residues to a growing peptide chain attached to an insoluble support or matrix, such as polystyrene. The carboxy-terminal residue of the peptide is first anchored to a commercial carrier in which its amino group is protected by an N-protecting agent, such as a t-butoxycarbonyl group (tBoc) or Fluorenylmethoxycarbonyl (FMOC) group. The amino protecting group is removed with a suitable deprotecting agent such as TFA in the case of tBOC or piperidine (for FMOC) followed by addition of the next amino acid residue (in N-protected form) together with a coupling agent such as bicyclic carbodiimide (DCC). After peptide bond formation, the reagent is washed from the carrier. After addition of the final residue, the agent is cleaved from the carrier with a suitable reagent, such as trifluoroacetic acid (TFA) or Hydrogen Fluoride (HF). If desired, the HSA linker, with or without one or more of the optional elements described above, can be prepared in one, two, three, or more segments, which can then be joined to form the entire HSA linker construct.

Binding moieties

The HSA linker conjugate can include one or more binding moieties, such as an antibody, antibody fragment (e.g., single chain fv (scfv)), or receptor/ligand (i.e., protein or glycoprotein ligand or receptor), as defined herein, which facilitates selective and specific binding of the HSA linker conjugate to a target cell, tissue, or organ. The binding moiety can be bound to the HSA linker (e.g., via a covalent (e.g., peptide bond), ionic, or hydrophobic bond, or via a high affinity protein-protein binding interaction (e.g., biotin and avidin)).

One or more binding moieties can be bound to the HSA linker. In one embodiment, two or more identical binding moieties (i.e., moieties having the same structure and binding affinity) can be bound to the HSA linker, one or more (e.g., in tandem) each at the amino and carboxy termini, such that the HSA linker provides greater affinity of the binding moieties to their target antigens. Alternatively, two or more different binding moieties (e.g., antibodies, such as scfvs having binding affinity for two or more different target molecules, or scfvs having binding affinity for two or more different epitopes on the same target molecule) can be conjugated to an HSA linker (e.g., a bispecific HSA linker conjugate) to facilitate binding of the HSA linker conjugate to multiple target antigens or epitopes. In another embodiment, binding moieties of different species may also be bound to the HSA linker to confer a linker binding, e.g., two or more different binding specificities or agonist/antagonist properties. Advantageous combinations of binding moiety pairs for making bispecific HSA linker conjugates are disclosed, for example, in international patent application publications WO2006/091209 and WO 2005/117973, which are incorporated herein by reference. In other embodiments, two or more binding moieties (e.g., the same or different binding moieties) can be conjugated to the HSA linker to form an HSA linker conjugate.

The invention features an HSA linker conjugate having at least first and second binding moieties, each of which can be conjugated to the amino or carboxy terminus of the HSA linker, or to a peptide linker (as defined herein) present at either or both termini. FIG. 1 shows a typical mutant HSA linker, wherein two binding moieties ("arm 1" and "arm 2") are bound to the mutant HSA linker via an amino-terminal peptide linker AAS and a carboxy-terminal peptide linker AAAL (SEQ ID NO: 5). Binding moieties (e.g., antibodies or scfvs) can also be bound to other positions (e.g., internal amino acid residues of an HSA linker), e.g., covalently or ionically, e.g., using biotin-avidin interactions. Biotinylation of amine (e.g., lysine residues) and sulfhydryl (e.g., cysteine residues) amino acid side chains is known in the art and can be used to attach binding moieties to HSA linkers.

Binding moieties that may be included in an HSA linker conjugate include antibodies, antibody fragments, receptors, and ligands. The binding moiety that binds to the HSA linker can be recombinant (e.g., human,Murine, chimeric, or humanized), synthetic, or natural. Typical binding moieties include, for example, a full antibody, a domain antibody, a diabody, a triabody, a bispecific antibody, an antibody fragment, a Fab fragment, a F (ab ') 2 molecule, a single chain Fv (scFv) molecule, a bispecific single chain Fv ((scFv')₂) Molecules, tandem scFv fragments, antibody fusion proteins, hormones, receptors, ligands, and aptamers, as well as biologically active fragments thereof.

Antibodies

Antibodies include IgG, IgA, IgM, IgD, and IgE classes. As used herein, their antibodies or antibody fragments comprise one or more Complementarity Determining Regions (CDRs) or binding peptides that bind to a target protein, glycoprotein, or epitope present outside or inside a target cell.

Many of the antibodies, or fragments thereof, described herein can be subjected to non-critical amino acid substitutions, additions or deletions in the variable and constant regions without loss of binding specificity or effector function, or excessive reduction in binding affinity (e.g., less than about 10)^-7M). Typically, an antibody or antibody fragment comprising the above-described alterations has significant sequence identity with respect to the reference antibody or antibody fragment from which it is derived. Sometimes, a mutant antibody or antibody fragment can be selected that has the same specificity and increased affinity as compared to the reference antibody or antibody fragment from which it was derived. Phage display technology provides a powerful technique for selecting such antibodies. See, e.g., Dower et al, WO 91/17271 McCafferty et al, WO 92/01047; and hue, WO 92/06204, which is incorporated herein by reference.

The HSA linker can also be bound to one or more fragments of the antibody that retain the ability to specifically bind to the target antigen. Antibody fragments include isolated heavy chain variable regions, light chain variable regions, Fab ', F (ab')₂Fabc, and scFv. Fragments may be generated by enzymatic or chemical isolation of intact immunoglobulins. For example, by performing the protein with pepsin at pH3.0-3.5Hydrolytic digestion and digestion using standard methods such as those described in Harlow and Lane, Antibodies: standard methods such as those described in A Laboratory Manual, Cold spring Harbor Pubs, N.Y. (1988), F (ab')₂Fragments may be obtained from IgG molecules. The Fab fragments may be obtained from the F (ab') 2 fragment by limited reduction, or from the whole antibody by digestion with papain in the presence of a reducing agent. Fragments may also be generated by recombinant DNA techniques. Segments of nucleic acid encoding the selected fragments are generated by digestion of the full-length coding sequence with restriction endonucleases, or by de novo synthesis. Fragments are often expressed as phage coat fusion proteins. This expression pattern is advantageous for enhancing the affinity of the antibody.

Humanized antibodies

Humanized antibodies can be used in combination with an HSA linker, wherein one or more antibody CDRs are from a non-human antibody sequence and one or more, but preferably all, CDRs specifically bind to an antigen (e.g., a protein, glycoprotein, or other suitable epitope).

Humanized antibodies comprise constant framework regions (so-called acceptor antibodies) essentially derived from human antibodies, and, in some cases, a majority of the variable regions are derived from human antibodies. One or more CDRs (all or portions thereof, and discrete amino acids surrounding the one or more CDRs) are provided from a non-human antibody, such as a mouse antibody. The constant region of the antibody may or may not be present.

Substitution of one or more mouse CDRs into a human variable domain framework is more likely to cause them to retain the correct spatial orientation if the human variable domain framework adopts the same or a similar conformation as the mouse variable framework from which the CDRs are derived. This is achieved by obtaining human variable domains from human antibodies. Wherein the framework sequences of the human antibody have a high degree of sequence and structural identity to the murine variable framework domain from which the CDRs are derived. The heavy and light chain variable framework regions may be from the same or different human antibody sequences. The human antibody sequence may be the sequence of a naturally occurring human antibody, a consensus sequence of several human antibodies, or may be a human germline variable domain sequence. See, e.g., keyborough et al, Protein Engineering 4: 773 (1991); kolbinger et al, Protein Engineering 6: 971(1993).

Suitable human antibody sequences are identified by sequence comparison of the amino acid sequence of the mouse variable region to the sequence of a known human antibody. The heavy and light chains were compared separately, but the principle was similar.

Methods for making chimeric and humanized antibodies and antibody fragments are described, for example, in U.S. Pat. Nos. 4,816,567, 5,530,101, 5,622,701, 5,800,815, 5,874,540, 5,914,110,http://patft.uspto.gov/netacgi/- hOhttp://patft.uspto.gov/netacgi/-h25,928,904, 6,210,670, 6,677,436, and 7,067,313, and U.S. patent application nos. 2002/0031508, 2004/0265311, and 2005/0226876. The preparation of antibodies or fragments thereof is further described, for example, in U.S. Pat. nos. 6,331,415, 6,818,216, and 7,067,313.

Receptors and ligands

In certain HSA linker conjugates, a protein or glycoprotein receptor or ligand is conjugated to the HSA linker. An HSA linker bound to a receptor or ligand can be used, for example, to specifically target a secreted protein, a cell (e.g., a cancer cell), a tissue, or an organ. In addition, specific binding of the HSA linker-receptor or-ligand conjugate to the target receptor or ligand may be associated with eliciting agonistic or antagonistic biological activity in intracellular or intercellular signaling pathways. As with the other binding moieties described herein, the receptors and ligands, or fragments thereof, may be linked to the amino and/or carboxy terminus of the HSA linker, or to a peptide connector linked to the HSA linker or to amino acid residues in the HSA linker.

Typical receptors and ligands that may be attached to the HSA linker include, but are not limited to, insulin-like growth factor 1 receptor (IGF1R), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transformation factor receptor (c-met; also known as Hepatocyte Growth Factor Receptor (HGFR)), Hepatocyte Growth Factor (HGF)Epidermal Growth Factor Receptor (EGFR), Epidermal Growth Factor (EGF), nerve growth factor, Fibroblast Growth Factor Receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), Vascular Endothelial Growth Factor Receptor (VEGFR), Vascular Endothelial Growth Factor (VEGF), Tumor Necrosis Factor Receptor (TNFR), tumor necrosis factor alpha (TNF-alpha), TNF-beta, folate receptor (FOLR), folate transferrin receptor (TfR), mesothelin, Fc receptor, c-kit, alpha 4 integrin, P-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronic acid receptor, leukocyte functional antigen-1 (LFA-1), CD4, CD11, CD18, CD20, CD25, CD27, CD52, CD70, alpha 4, alpha-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronic acid receptor, leukocyte functional antigen-1 (LFA-1), CD4, CD11, CD, CD80, CD85, CD95(Fas receptor), CD106 (vascular cell adhesion molecule 1(VCAM1), CD166 (activated leukocyte adhesion molecule (ALCAM)), CD178(Fas ligand), CD253 (TNF-related apoptosis inducing ligand (TRAIL)), ICOS ligand, CCR2, CXCR3, CCR5, CXCL12 (stromal cell derived factor 1(SDF-1)), interleukin 1(IL-1), CTLA-4, receptors alpha and beta, MART-1, gp100, MAGE-1, ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1(MADCAM-1), carcinoembryonic antigen (CEA), Lewis^YMUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125(CA125), Prostate Specific Membrane Antigen (PSMA), TAG-72 antigen, and biologically active fragments thereof.

The receptor and ligand may be expressed, isolated, or linked to the HSA linker using any of the methods described above.

Diagnostic agent

The HSA linker, or any binding moiety bound thereto (e.g., an antibody, antibody fragment, receptor, or ligand), can be linked to a chelator or to a detectable label to form a diagnostic agent. Also contemplated are HSA linker conjugates comprising a detectable label (as described herein), and one or more therapeutic agents or binding moieties described herein.

The HSA linker (or HSA linker conjugate) and the chelator component may be conjugated by reacting the free amino group of the threonine residue of the HSA linker (or HSA linker conjugate) and an appropriate functional group of the chelator, such as a carboxyl group or an activated ester. For example, when functionalized with carboxyl substituents on the ethylene chain, such binding can be achieved by the addition of the chelating agent ethylenediaminetetraacetic acid (EDTA), which is common in the field of coordination chemistry. The synthesis of this type of EDTA derivative is reported in Arya et al (Bioconjugate Chemistry 2: 323(1991)), which describes blocking each of the four coordinating carboxyl groups with a t-butyl group, while the carboxyl substituent on the ethylene chain can react freely with the amino group of the peptide portion of the agent.

The HSA linker or HSA linker conjugate may be conjugated to a metal chelator component, the metal chelator component being a peptide, i.e. compatible with solid phase peptide synthesis. In this case, the chelator may be conjugated in the same manner as EDTA described above, or more conveniently, the chelator and HSA linker or HSA linker conjugate may be synthesized: beginning entirely at the carboxy-terminal residue of the HSA linker or HSA linker conjugate and ending at the amino-terminal residue of the chelator.

The HSA linker or HSA linker conjugate can further incorporate a linker component that serves to link the HSA linker to the chelator without adversely affecting the biological properties of the HSA linker, the targeting function of the binding portion of the HSA linker conjugate, or the metal binding function of the chelator. Suitable linking groups include amino acid chains and alkyl chains functionalized with an active group to facilitate coupling to the HSA linker or HSA linker conjugate and to the chelator. When the chelator is a peptide, the amino acid chain is the preferred linking group so that the HSA linker or HSA linker conjugate can be synthesized entirely by solid phase techniques. An alkyl chain-linking group can be added to the HSA linker or HSA linker conjugate by reacting the amino group of the threonine residue of the peptide portion of the HSA linker with a first functional group on the alkyl chain, such as a carboxyl group or an activated ester. Subsequently, a chelator is attached to the alkyl chain by reacting a second functional group on the alkyl chain with an appropriate group on the chelator to complete formation of the HSA linker or HSA linker conjugate. The second functional group on the alkyl chain is selected from substituents that are reactive with the functional group on the chelator but not with the threonine residue of the mutant HSA linker. For example, when the chelating agent binds a functional group such as a carboxyl group or an activated ester, the second functional group of the alkyl chain-linking group may be an amino group. It will be appreciated that the formation of an HSA linker or HSA linker conjugate may require protection and deprotection of functional groups present to avoid the formation of undesirable products. Protection and deprotection are accomplished using protecting groups, reagents, and methods common to the art of organic synthesis. In particular, the protection and deprotection techniques described above for solid phase peptide synthesis may be used.

An alternative chemical linking group for the alkyl chain is polyethylene glycol (PEG), which is functionalized in the same manner as the alkyl chain described above to add an HSA linker or HSA linker conjugate. It will be appreciated that the linking group may alternatively be conjugated first to the chelator and then to the HSA linker or HSA linker conjugate.

In one aspect, the HSA linker or HSA linker conjugate is conjugated to a diagnostically useful metal capable of forming a complex. Suitable metals include, for example, radionuclides such as technetium and rhenium in their different forms (e.g.,^99mTcO³⁺、^99mTcO₂ ⁺、ReO³⁺and ReO₂ ⁺). Metals can be added to the HSA linker or HSA linker conjugate by various methods common in the art of coordination chemistry. When the metal is technetium-99 m, the following general procedure can be used to form technetium complexes. The HSA linker-chelator conjugate solution is initially formed by dissolving the HSA linker or HSA linker conjugate in an aqueous alcohol, such as ethanol. The solution is then degassed to remove oxygen, followed by removal of thiol protecting groups with a suitable reagent, e.g., sodium hydroxide, and then neutralized with an organic acid, such as acetic acid (ph 6.0-6.5). In the labeling step, a stoichiometric excess of sodium pertechnetate obtained from a molybdenum generator is added to a solution of conjugate in which a reducing agent such as stannous chloride is present in an amount sufficient to reduce technetium, and heated. The labeled HSA linker or HSA linker conjugate can be separated from contaminants by chromatography, e.g., with the aid of a C-18Sep Pak cartridge^99mTcO₄ ^-And colloidal state^99mTcO₂。

In an alternative method, the HSA linker may be labeled by transchelation reaction (transchelation). The technetium source is a solution of technetium complexed to a labile ligand to facilitate exchange of the ligand with the selected chelator. Suitable ligands for transchelation include tartrate, citrate, and heptogluconate (heptagluconate). In this case, the preferred reducing agent is sodium dithionate (sodium dithionite). It will be appreciated that the HSA linker or HSA linker conjugate can be labeled using the techniques described above, or alternatively, the chelator itself can be labeled and then linked to the HSA linker to form an HSA linker-chelator conjugate; a process known as the "pre-label ligand" method.

Another means for labeling the HSA linker, or any agent bound thereto, involves the immunoaffinity of the HSA linker-chelator conjugate on a solid support through a linkage that is cleaved under metal chelation. This is achieved when the chelating agent is attached to the functional group of the support via a complexing atom. Preferably, the complexing sulfur atom is attached to a support that is functionalized with a sulfur protecting group such as maleimide.

Agents comprising HSA linker-chelator conjugates can be used to detect tissues that are at risk of developing the following diseases, when labeled with diagnostically useful metals, by procedures established in the field of diagnostic imaging: cancers (e.g., lung, breast, colon, and prostate), age-related diseases (e.g., cardiovascular, cerebrovascular, or alzheimer's), tobacco-related diseases (e.g., emphysema, aortic aneurysm, esophageal cancer, or squamous cell carcinoma of the head and neck). The agent incorporating the HSA linker labeled with a radionuclide metal, such as technetium-99 m, can be administered to a mammal (e.g., a human) by intravenous injection and with a pharmaceutical solution, such as isotonic saline, or by other methods described herein. The amount of labeling agent suitable for administration depends on the distribution characteristics of the HSA linker or HSA linker conjugate selected, which means that: agents that bind a faster clearing HSA linker or HSA linker conjugate can be administered at higher doses than agents that bind a slower clearing HSA linker or HSA linker conjugate. For a 70kg individual, an acceptable unit dose for imaging tissue is about 5-40 mCi. At an appropriate time after administration, typically between 30 minutes and 180 minutes and up to about 5 days (which depends on the rate of accumulation at the target site relative to clearance at non-target tissues), the in vivo distribution and localization of the agent conjugated to the labeled HSA linker or HSA linker conjugate can be traced by standard techniques described herein.

The HSA linker, or any molecule or moiety bound thereto, may also be modified or labeled to facilitate diagnostic or therapeutic applications. A detectable label such as a radioactive label, a fluorescent label, a heavy metal label, or other molecular label can be conjugated to any agent. Single, double, or multiple labeling of agents may be advantageous. For example, double labeling by radioiodination of one or more residues, together with additional, for example,⁹⁰y is bound to the amine-containing side or the reactive group via a chelating group, which will facilitate combinatorial labeling. This may be advantageous for specific diagnostic needs such as identification of widely distributed small tumor cell mass.

The HSA linker, or any molecule or moiety bound thereto, may also be modified, for example, by halogenating tyrosine residues of the peptide component. Halogens include fluorine, chlorine, bromine, iodine, and astatine. The above-mentioned halogenating agents may be detectably labeled, for example, if the halogen is a radioisotope, such as, for example,¹⁸F、⁷⁵Br、⁷⁷Br、¹²²I、¹²³I、¹²⁴I、¹²⁵I、¹²⁹I、¹³¹I. or²¹¹At. The halogenating agent comprises a halogen covalently bound to at least one amino acid, and preferably to a D-Tyr residue in each agent molecule. Other suitable detectable modifications include binding of other compounds (e.g., fluorochromes such as fluorescein) to lysine residues of the agent, or the like, particularly agents or the like having linkers that include lysine.

The radioisotope used to radiolabel the HSA linker, or any molecule or moiety bound thereto, includes any radioisotope which may be covalently bound to a residue of a peptide component of an agent or analogue thereof. The radioisotope may also be selected from beta or gamma radiation emitting radioisotopes, or alternatively, any agent may be modified to include a chelating group, for example, a lysine residue which may be covalently bound to the HSA linker or any peptide agent bound thereto. The chelating group can then be modified to include any of a variety of radioisotopes, such as gallium, indium, technetium, ytterbium, rhenium, or thallium (e.g.,¹²⁵I、⁶⁷Ga、¹¹¹In、⁹⁹mTc、¹⁶⁹Yb、¹⁸⁶Re)。

the HSA linker, or any molecule or moiety bound thereto, may be modified by the attachment of a radioisotope. Preferred radioisotopes are those whose radioactive half-life corresponds to, or is longer than, the biological half-life of the HSA conjugate used. More preferably, the radioisotope is a radioisotope of a halogen atom (e.g., a radioisotope of fluorine, chlorine, bromine, iodine, and astatine), even more preferably⁷⁵Br、⁷⁷Br、⁷⁶Br、¹²²I、¹²³I、¹²⁴I、¹²⁵I、¹²⁹I、¹³¹I. Or²¹¹At。

An agent that binds to the HSA linker, or any molecule or moiety bound thereto, can be linked to a radiometal and used for radiographic imaging or radiotherapy. Preferred radioisotopes also include^99mTc、⁵¹Cr、⁶⁷Ga、⁶⁸Ga、¹¹¹In、¹⁶⁸Yb、¹⁴⁰La、⁹⁰Y、⁸⁸Y、¹⁵³Sm、¹⁵⁶Ho、¹⁶⁵Dy、⁶⁴Cu、⁹⁷Ru、¹⁰³Ru、¹⁸⁶Re、¹⁸⁸Re、²⁰³Pb、²¹¹Bi、²¹²Bi、²¹³Bi. And²¹⁴and (4) Bi. The choice of metal depends on the desired treatmentTherapeutic or diagnostic use.

The HSA linker, or any molecule or moiety bound thereto, can be attached to the metal component to produce a diagnostic or therapeutic agent. The detectable label may be a metal ion from a heavy element or a rare earth ion, such as Gd³⁺、Fe³⁺、Mn³⁺Or Cr²⁺. Agents that incorporate HSA linkers with paramagnetic or superparamagnetic metals attached thereto can be used as diagnostic agents in MRI imaging applications. Paramagnetic metals include, but are not limited to, chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), and ytterbium (III).

The chelating group can be used to indirectly attach a detectable label or other molecule to the HSA linker or an agent bound thereto. The chelating group may be attached to a pharmaceutical agent and a radiolabel, such as a bifunctional stable chelating agent, or may be attached to one or more terminal or internal amino acid reactive groups. The HSA linker, or any molecule or moiety bound thereto, may be connected via an isothiocyanate beta-Ala or a suitable non-alpha-amino acid linker which prevents idenemen degradation. Examples of chelating agents known in the art include, for example, iminocarboxylic acid (iminocarboxylic acid) and polyaminopolycarboxylic acid (polyaminopolycarboxylic) reactive groups, iminocarboxylic acid and polyaminopolycarboxylic acid reactive groups, diethylenetriaminepentaacetic acid (DTPA), and 1,4, 7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA).

The HSA linker, when recombinantly expressed, may be linked to a peptide detectable label or diagnostic agent. Peptides and proteins that can be used as detectable labels for the HSA linker include, but are not limited to, fluorescent proteins, bioluminescent proteins, and epitope tags, each of which is discussed in detail below. One or more of these detectable labels may also be added to the HSA linker conjugate, which also includes a therapeutic agent, cytotoxic agent, or cytostatic agent.

Fluorescent proteins or pigments, such as green fluorescent protein (GFP; SEQ ID NO: 47), enhanced GFP (eGFP), yellow fluorescent protein (SEQ ID NO: 48; YFP), cyan fluorescent protein (SEQ ID NO: 49; CFP), and red fluorescent protein (SEQ ID NO: 50; RFP or DsRed) can be used as detectable labels attached to the HSA linker. The fluorescent protein can be recombinantly expressed in a cell (e.g., a blood cell, such as a lymphocyte) after transfection or transduction of the cell with an expression vector encoding a nucleotide sequence for the fluorescent protein. After exposure of the fluorescent protein to the stimulus frequency of light, the fluorescent protein will emit light at low, medium, or high intensity, which can be observed under a microscope with the eye or by an optical imaging device. Typical fluorescent proteins suitable for use as diagnostic sequences for a pharmaceutical agent are described, for example, in U.S. patent nos. 7,417,131 and 7,413,874, each of which is incorporated herein by reference.

The bioluminescent protein may also be used as a detectable label for the addition of the HSA linker. Bioluminescent proteins, such as luciferases (e.g., firefly (SEQ ID NO: 51), Renilla (SEQ ID NO: 52), and Omphalatus luciferase) and aequorin, emit light as part of a chemical reaction with a substance (e.g., luciferin and coelenterazine). In one embodiment, the vector encoding the luciferase gene provides for in vivo, in vitro, or in vitro detection of cells (e.g., blood cells, such as lymphocytes) that have been transduced or transfected according to standard methods such as those described herein. Exemplary bioluminescent proteins suitable for use as diagnostic sequences and methods for their use are described, for example, in U.S. Pat. Nos. 5,292,658, 5,670,356, 6,171,809, and 7,183,092, each of which is incorporated herein by reference.

Epitope tags are short amino acid sequences, e.g., 5-20 amino acid residues in length, that can be added as a detectable label to the HSA linker conjugate to facilitate detection after expression in a cell, secretion from a cell, or binding to a target cell. Agents that bind to an epitope tag as a diagnostic sequence can be detected by interaction with an antibody, antibody fragment, or other binding molecule that is specific for the epitope tag. By cloning of natural genesThe nucleotide sequence encoding the epitope tag is generated in suitable portions or by synthesizing the polynucleotide encoding the epitope tag. An antibody, antibody fragment, or other binding molecule that binds an epitope tag can be directly bound to a detectable label (e.g., a fluorescent dye, a radiolabel, a heavy metal, or an enzyme such as horseradish peroxidase) or can itself serve as the target for a secondary antibody, antibody fragment, or other binding molecule that is bound to the label. Typical epitope tags that can be used as diagnostic sequences include c-myc (SEQ ID NO: 33), hemagglutinin (HA; SEQ ID NO: 34), and histidine tag (His)₆(ii) a SEQ ID NO: 35). In addition, fluorescent (e.g., GFP) and bioluminescent proteins can also be used as epitope tags, as antibodies, antibody fragments, and other binding molecules are commercially available to detect these proteins.

Microscopes, flow cytometers, photometers, or other prior art optical imaging devices, e.g.Imaging System (Caliper life sciences, Hopkinton, MA), detects, images, or tracks HSA linker conjugates in vivo, in vitro, or in vitro, the HSA linker conjugates being conjugated to a diagnostic sequence (e.g., a fluorescent protein, a bioluminescent protein, or an epitope tag) or any cell expressing or conjugated to an HSA linker conjugate.

Therapeutic or cytotoxic agents linked to an HSA linker

The HSA linker, or any molecule or moiety bound thereto, can be linked to any known cytotoxic or therapeutic moiety to form an agent (HSA linker conjugate) that can be administered to treat, inhibit, reduce, or ameliorate a disease (e.g., cancer, autoimmune disease, or cardiovascular disease) or one or more symptoms of a disease. Examples include, but are not limited to, antineoplastic agents such as: acivicin; aclarubicin; (ii) aristozole hydrochloride; (ii) abelmoscine; (ii) Alexanox; doxorubicin; aldesleukin; altretamine; an apramycin; amenthraquinone acetate; aminoglutethimide; amsacrine; anastrozole; an atramycin; asparaginyl-hexazyme; a triptyline; azacitidine; azatepa; (ii) azomycin; batimastat; benzotepa; bicalutamide; bisantrene hydrochloride; bisnefad mesylate; bizelesin; bleomycin sulfate; brequinar sodium; briprimine; busulfan; actinomycin c; (ii) carpoterone; camptothecin; a carbimide; a carbapenem; carboplatin; carmustine; a doxorubicin hydrochloride; folding to get new; cediogo; chlorambucil; a sirolimus; cisplatin; cladribine; combretastatin a-4 (combretastatin a-4); cllinaltol mesylate; cyclophosphamide; cytarabine; dacarbazine; daca (n- [2- (dimethylamino) ethyl ] acridine-4-imidazole carboxamide); actinomycin d; daunorubicin hydrochloride; daunomycin; decitabine; (ii) dexomaplatin; 2, dizagutanin; 1, dizagutinine mesylate; diazaquinone; docetaxel; dolastatins (dolastatins); doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; drotandrosterone propionate; daptomycin; edatrexae; eflornithine hydrochloride; ellipticine; elsamitrucin; enloplatin; an enpu urethane; epinastine; epirubicin hydrochloride; (ii) ebuzole; isosbacin hydrochloride; estramustine; estramustine sodium sulfate; etanidazole; ethiodized oil i 131; etoposide; etoposide phosphate; chlorphenethyl pyrimethanil; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; 5-fdump; (iii) flucitabine; a phosphorus quinolone; fostrexasin sodium; gemcitabine; gemcitabine hydrochloride; gold au 198; homocamptothecin; a hydroxyurea; idarubicin hydrochloride; ifosfamide; ilofovir dipivoxil; interferon alpha-2 a; interferon alpha-2 b; interferon alpha-nl; interferon alpha-n 3; interferon beta-ia; interferon gamma-ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liazole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; (ii) maxolone; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; (ii) a melanoril; mercaptopurine; methotrexate; methotrexate sodium; chlorpheniramine; meltupipide; mitodomide; mitokacin; mitorubin; mitoxantrone; mitomacin; mitomycin; mitospirane culturing; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; a noramycin; ormaplatin; oshuzuren; paclitaxel; a pemetrexed; a pelithromycin; nemadectin; pellomycin sulfate; cultivating phosphoramide; pipobroman; piposulfan; piroxantrone hydrochloride; (ii) a plicamycin; pramipexole; porfimer sodium; a podomycin; deltemustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazolofuran; rhizomycin; rhizomycin d; (ii) lybodenosine; ludwimine; safrog; safrog hydrochloride; semustine; octreozine; sodium phosphono-aspartate; a sparamycin; germanospiramine hydrochloride; spiromustine; spiroplatinum; streptomycin; a streptozocin; strontium chloride sr 89; a sulfochlorophenylurea; a talithromycin; a taxane; taxoid; sodium tegafur; tegafur; tiloxanthraquinone hydrochloride; temoporfin; (ii) teniposide; a tiroxiron; a testosterone ester; thiamiprine; thioguanine; thiotepa; thymotaq; thiazolfurin; tirapazamine; carrying out seeking; top 53; topotecan hydrochloride; toremifene citrate; triton acetate; triciribine phosphate; trimetrexate; tritrosa glucuronide; triptorelin; tobramzole hydrochloride; uramustine; uretipi; vapreotide; verteporfin; vinblastine; vinblastine sulfate; vincristine; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinblastine sulfate; vinorelbine tartrate; vinblastine sulfate; vinzolidine sulfate; (ii) vorozole; zeniplatin; 1, neat setastine; zorubicin hydrochloride; 2-chlorodeoxyadenosine; 2' desoxymetrythromycin; 9-aminocamptothecin; raltitrexed; n-propargyl-5, 8-dideazafolic acid (N-propargyl-5, 8-dideazafolic acid); 2chloro-2 '-arabino-fluoro-2' -deoxyadenosine (2chloro-2 '-arabino-fluoro-2' -deoxyadenosine); 2-chloro-2' -deoxyadenosine; anisomycin; trichostatin a; hPRL-G129R; CEP-751; linoamine; sulfur mustard gas; a nitrogen mustard; cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-N nitrosourea (MNU); n, N' -bis (2-chloroethyl) -N-nitrosourea (BCNU); n- (2-chloroethyl) -N' cyclohexyl-N-nitrosourea (CCNU); n- (2-chloroethyl) -N '- (trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU), N- (2-chloroethyl) -N' - (diethyl) ethylphosphonate-N-nitrosourea (fotemustine), streptozocin (streptozocin), Diaccabazine (DTIC), mitozolamide, temozolomide, thiotepa, mitomycin C, AZQ, adolesin, cisplatin, carboplatin, ormaplatin, oxaliplatin, C1-973, DWA 2114R, JM216, JM335, bis (platinum), topotecan, azacitidine, cytarabine, gemcitabine, 6-mercaptopurine, 6-thioguanine, hypoxanthine, teniposide 9-aminocamptothecin, topotecan, CPT-11, doxorubicin, daunomycin, epirubicin, mitoxantrone, actinomycin D, acridine, pyrazosine (pyrazoline), tranzoxazole, tramadol, mitoxantrone, doxycycline, and tramadol An alcohol; 14-hydroxy-retro-retinol; all-trans retinoic acid; n- (4-Hydroxyphenyl) retinamide (N- (4-Hydroxyphenyl) retinamide); 13-cis retinoic acid; 3-methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); or 2-chlorodeoxyadenosine (2-Cda).

Other therapeutic compounds include, but are not limited to, 20-pi-1, 25 dihydroxy vitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; an acylfulvene; adecyenol; (ii) Alexanox; aldesleukin; ALL-TK antagonist; altretamine; an ammonia lamp; (ii) amidox; amifostine; 5-aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; an angiogenesis inhibitor; an antagonist D; an antagonist G; antarelix; anti-dorsal morphogenetic protein-1 (anti-dorsallizing morphogenetic protein-1); anti-androgens, prostate cancer; anti-estrogen agents; an antineoplastic ketone; an antisense oligonucleotide; aphidicolin aminoacetate; an apoptosis gene modulator; a modulator of apoptosis; (ii) an allopurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestan; amoxicillin; axinatatin 1; axinatatin 2; axinatatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; a BCR/ABL antagonist; benzo chlorin; benzoyl staurosporine; beta lactam derivatives; beta-alethine; betacylomycin B; betulinic acid; a bFGF inhibitor; bicalutamide; a bisantrene group; bis-aziridinyl spermine; (ii) bisnefarde; bistetralene A; bizelesin; brefflate; bleomycin a 2; bleomycin B2; briprimine; titanium is distributed; buthionine esulfoximine; calcipotriol; calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin); canarypox IL-2; capecitabine; imidazole carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; an inhibitor derived from cartilage; folding to get new; casein kinase Inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide (chloroquinoxaline sulfonamide); (ii) cicaprost; a cis-porphyrin; cladribine; clomiphene analogs; clotrimazole; colismycin A; colismycin B; combretastatin a 4; combretastatin analogs; a concanagen; crambescidin 816; clinatot; cryptophycin 8; cryptophycinA derivatives; curve A; cyclopentanthanthrones (cyclopentanthraquinones); cycloplatam; cypemycin; cytarabine ocfosfate; a cytolytic factor; cytostatins (cytostatins); daclizumab; decitabine; dehydrogenatedmin B; 2' deoxy syndiomycin (DCF); deslorelin; dexfosfamide; dexrazoxane; (ii) verapamil; diazaquinone; didemnin B; didox; diethylnorpermine; dihydro-5-azacytidine; dihydro taxol, 9-; a dioxamycin; diphenylspiromustine; discodermolide; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; etokomustine; edifulin; epirubicin; eflornithine; elemene; ethirimuron fluoride; epirubicin; epothilones (A, R ═ H; B, R ═ Me); epithilones; epristeride; an estramustine analogue; an estrogen agonist; an estrogen antagonist; etanidazole; etoposide; etoposide 4' -phosphate (pirimid); exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flutemastine; a flashterone; fludarabine; fluoroaurourigenin hydrochloride; fowler; 2, fulvestrant; fostrexasin sodium; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; (ii) a gelatinase inhibitor; gemcitabine; a glutathione inhibitor; hepsulfam; a nerve growth factor; hexamethylene bisamide; homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin; idoxifene; iloperidone; ilofovir dipivoxil; ilomastat; an imidazocridinone; imiquimod; immunostimulatory peptides; insulin-like growth factor-1 receptor inhibitors; an interferon agonist; an interferon; an interleukin; iodobenzylguanidine; doxorubicin iodoxide; epoetin, 4-; irinotecan; iprop; isradine; isobengazole; isohomohalicondrin B; itasetron; j asplexolide; kahalalide F; lamellarin-N triacetic acid; lanreotide; leinamycin; leguminous kiosks; sulfuric acid lentinan; leptin statin; letrozole; leukemia inhibitory factor; leukocyte interferon-alpha; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liazole; a linear polyamine analog; a lipophilic glycopeptide; a lipophilic platinum compound; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxanthraquinone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; a lytic peptide; maytansine; mannostatin A; marimastat; (ii) maxolone; maspin; matrilysin inhibitors; an interstitial metalloprotease inhibitor; (ii) a melanoril; rnerbarone; 1, meperiline; methioninase; metoclopramide; an inhibitor of MIF; an ifepisetone; miltefosine; a Millisetil; mismatched double-stranded RNA; mithramycin; mitoguazone; dibromodulcitol; mitomycin analogs; mitonaphthylamine; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofagotine; moraxest; monoclonal antibodies, human chorionic gonadotropin; monophosphoryl lipid a + myobacterium cell wall sk; mopidanol; anti-multidrug gene inhibitors; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; a mycobacterial cell wall extract; myriaporone; n-acetyldinaline; an N-substituted benzamide; nafarelin; spraying naretide; naloxone + pentazocine; napavin; naphterpin; a nartostim; nedaplatin; nemorubicin; neridronic acid; neutral peptide chain wind-cleaving enzyme; nilutamide; nisamycin; a nitric oxide modulator; a nitroxide antioxidant; nitrulyn; 06-benzylguanine; octreotide; okicenone; an oligonucleotide; onapristone; ondansetron; ondansetron; oracin; an oral cytokine inducer; ormaplatin; an oxateclone; oxaliplatin; oxanonomycin; a paclitaxel analog; a paclitaxel derivative; palauamine; palmitoyl rhizomycin; pamidronic acid; panaxatriol; panomifen; a parabencin; pazeliptin; a pemetrexed; pedunculing; sodium pentapolythionate; pentostatin; (ii) pentazole; perfluorobromoalkane; cultivating phosphoramide; perillyl alcohol; phenazinomomycin; phenyl acetate; inhibitors of phosphatidase; a hemolytic streptococcal agent; pilocarpine hydrochloride; pirarubicin; pirtroxine; placetin A; placetin B; a plasminogen activation inhibitor; a platinum complex; a platinum compound; a platinum-triamine complex; (ii) a podophyllotoxin; porfimer sodium; a podomycin; propylbisacridone; prostaglandin J2; a proteasome inhibitor; protein a-based immunomodulators; inhibitors of protein kinase C; protein kinase C inhibitors, microelgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurin; pyrazole acridine; pyridoxalated hemoglobin polyoxyethylene conjugates; a raf antagonist; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; (ii) a ras inhibitor; ras-GAP inhibitors; demethylated reteplatin; rhenium (Re) 186 etidronate; rhizomycin; a ribozyme; RII retinoamide; ludwimine; rohitukine; romurtide; loquimex; rubiginoneB 1; ruboxyl; safrog; saintopin; SarCNU; sarcophylol A; sargrastim; a Sdi 1 mimetic; semustine; an aging-derived inhibitor 1; a sense oligonucleotide; a signal transduction inhibitor; a signal transduction modulator; a single-chain antigen-binding protein; a texaphyrin; sobuconazole; sodium boron carbonate; sodium phenylacetate; solverol; a growth regulator binding protein; sonaming; phosphono-winteric acid; spicamycin D; spiromustine; spleenetin; spongistatin 1; squalamine; a stem cell inhibitor; inhibitors of stem cell division; stiiamide; a matrilysin inhibitor; sulfinosine; a superactive vasoactive intestinal peptide antagonist; (ii) surfasta; suramin; swainsonine triol octahydro; synthesizing glycosaminoglycan; tamustine; tamoxifen methyl iodide; taulomustine; tazarotene; sodium tegafur; tegafur; telluropyrylium; a telomerase inhibitor; temoporfin; temozolomide; (ii) teniposide; tetrachlorodecaoxide; tetrazomine; (ii) a thioablistatin; thalidomide; thiocoraline; thrombopoietin; a thrombopoietin mimetic; thymalfasin (Thymalfasin); a thymosin receptor agonist; thymotreonam; thyroid stimulating hormone; the rhodopin ethyl ester tin; tirapazamine; titanocene dichloride; topotecan; topstein; toremifene; a totipotent stem cell factor; a translation inhibitor; tretinoin; triacetyl uridine; (iii) triciribine; trimetrexate; triptorelin; tropisetron; toleromide; tyrosine kinase inhibitors; tyrphostins; an UBC inhibitor; ubenimex; growth inhibitory factor derived from the urogenital sinuses; a urokinase receptor antagonist; vapreotide; variolin B; vector systems, erythrocyte gene therapy; vilareol; veramine; verdins; verteporfin; vinorelbine; vinxaline; vitaxin; (ii) vorozole; zanoteron; zeniplatin; benzal vitamin C; and neat stastatin ester.

HSA linker conjugates can also include site-specific binding molecules and moieties. Site-specific binding facilitates controlled stoichiometric attachment to specific residues in the HSA linker of a cytotoxic, immunomodulatory, or cytostatic agent, including, for example, antimicrotubulin agents, DNA minor groove binders, DNA replication inhibitors, alkylating agents, anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapeutic or radiosensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, lexins, nitrosoureas, bikrebs, purine antimetabolites, puromycin, steroids, taxanes, topoisomerase inhibitors, and vinca alkaloids or any other molecule or moiety described herein.

Techniques For binding Therapeutic Agents to proteins, particularly Antibodies, are well known (e.g., Arnon et al, "Monoclonal Antibodies against proteins Of Drugs In Cancer Therapy," In Monoclonal Antibodies And Cancer Therapy "(Reisfeld et al, eds., Alan R.Liss, Inc., 1985), Hellstrom et al," Antibodies For Drugs Delivery, "In controlled Drug Delivery (Robinson et al, feeds, Markel Delivery, Inc., 2nd, 1987), Thoro," Antibodies Of Cytotoxic Agents Of Drugs In Cancer Therapy: A video, "In Antibody Therapy, vaccine, "Development of cell monoclonal antibodies against microorganisms for cancer therapy," Nature Biotech.21: (7)778-784(2003)). See also, for example, PCT publication WO 89/12624.

The HSA linker, or any molecule or moiety bound thereto, may also be linked to the cleavage peptide. The above lytic peptides induce cell death and include, but are not limited to, streptolysin O; the stoichactin toxin; a goose cream peptide; staphylococcal alpha toxin; holothurin A; digitonin; melittin; lysolecithin; a cardiotoxin; and cerebratulus A toxin (Kemet al., J. biol. chem.253 (16): 5752-. The HSA linker, or any molecule or moiety bound thereto (e.g., an antibody or antibody fragment conjugate) can be conjugated to a synthetic peptide that shares some sequence homology or chemical properties with any naturally occurring peptide lysin to form the agents of the present invention; such properties include, but are not limited to, linearity, positive charge, amphiphilicity, and formation of alpha-helical structures in a hydrophobic environment (Leuschner et al, Biology of Reproduction 73: 860-865, 2005). The HSA linker, or any molecule or portion bound thereto, can also be linked to an agent that induces complement-mediated cell lysis such as, for example, an immunoglobulin Fc subunit. The HSA linker, or any molecule or portion that binds thereto, can also be linked to any member of the phospholipase family (including phospholipase a, phospholipase B, phospholipase C, or phospholipase D) or to a catalytically active subunit thereof.

The HSA linker, or any molecule or moiety bound thereto, can also be linked to a radioactive agent to form an agent that can be used for detection or therapeutic use. Useful radioactive agents include, but are not limited to, iodofibrinogen¹²⁵I; fluorodeoxyglucose¹⁸F; fludopa¹⁸F; insulin¹²⁵I; insulin¹³¹I; iodobenzylguanidine (IBM)¹²³I; sodium cholate¹³¹I; iodine antipyrine¹³¹I; iodine cholesterol¹³¹I; iodine hippurate sodium¹²³I; iodine hippurate sodium¹²⁵I; iodine hippurate sodium¹³¹I; iodoxolone¹²⁵I; iodoxolone¹³¹I; iodobutamine hydrochloride¹²³I; iodometin¹²⁵I; iodometin¹³¹I; sodium Iolatate¹²⁵I; sodium Iolatate¹³¹I; tyrosine¹³¹I; liothyronine (I) and its preparation method¹²⁵I; liothyronine (I) and its preparation method¹³¹I; mercury acetate propanol¹⁹⁷Hg; mercury acetate propanol²⁰³Hg; mercury propanol¹⁹⁷Hg; selenomethionine⁷⁵Se; antimony trisulfide technetium^99mTc colloid; technetium bicincetate^99mTc; desoxybenzine technetium^99mTc; technetium etidronate^99mTc; technetium Yimeishaoxie^99mTc; technetium^99mTc-fosfomycin; technetium glucoheptonate^99mTc; lituophenine technetium^99mTc; technetium bromobenzine^99mTc; technetium methylenephosphonate^99mTc; technetium^99mTc disodium methylenephosphonate; technetium^99mTc thiotepide; oxyphosphonic acid technetium^99mTc; technetium pentetate^99mTc; technetium^99mTc sodium calcium pentetate; setibetium^99mTc; technetium boroxime^99mTc; technetium dimercaptosuccinate^99mTc; technetium^99mTc sulphur colloid; teximetium^99mTc; tetrofosete technetium^99mTc; technetium^99mTc Tiatide; thyroxine preparation¹²⁵I; thyroxine preparation¹³¹I; iodophorone¹³¹I; triolein iodine¹²⁵I; or triolein iodine¹³¹I。

In addition, the radioisotope can be site-specifically linked to the HSA linker or HSA linker conjugate. The available reactive groups can be used to bind to site-specific bifunctional chelating agents for the labeling of radioisotopes, including¹²³I、¹²⁴I、¹²⁵I、¹²¹I、^99mTc、¹¹¹In、⁶⁴Cu、⁶⁷Cu、¹⁸⁶Re、¹⁸⁸Re、¹⁷⁷Lu、⁹⁰Y、⁷⁷As、⁷²As、⁸⁶Y、⁸⁹Zr、²¹¹At、²¹²Bi、²¹³Bi. Or²²⁵Ac。

The therapeutic or cytotoxic agent added or linked to the HSA linker or HSA linker conjugate may further include, for example, an anti-cancer co-enhancer including, but not limited to: tricyclic depressants (e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine, and maprotiline); non-tricyclic depressive drugs (e.g., sertraline, trazodone, and citalopram); ca²⁺Antagonists (e.g., verapamil, nifedipine, nitrendipine, and caroverine); calmodulin inhibitors (e.g., prenylamine, trifluoroperazine, and clomipramine); amphotericin B; tripareol analogs (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive agents (e.g., reserpine); thiol scavengers (thioldeples) (e.g., buthionine and sulfoximine); and multidrug resistant reducing agents such as polyoxyethylene castor oil.

Agents comprising the HSA linker, or any molecule or moiety bound thereto, may also be linked to or administered in conjunction with one or more cytokines (e.g., granulocyte colony stimulating factor, interferon- α, and tumor necrosis factor- α). The HSA linker, or any molecule or moiety that binds thereto, can also be attached to an antimetabolite agent. Antimetabolites include, but are not limited to, the following compounds and their derivatives: azathioprine, cladribine, cytarabine, dacarbazine, fludarabine phosphate, fluorouracil, genitabine chlorohydrate, mercaptopurine, methotrexate, dibromomannitol, mitotane, proguanil hydrochloride, pyrimethamine, raltitrexed, trimetrexate glucuronate, urethane, vinblastine sulfate, vincristine sulfate, and the like. More preferably, the HSA linker or conjugate can be attached to the folate antimetabolite, a class of agents that includes, for example, methotrexate, proguanil hydrochloride, pyrimethamine, trimethoprim, or trimetrexate glucuronate, or derivatives of these compounds.

The HSA linker, or any molecule or moiety bound thereto, can also be attached to a member of the anthracycline family of neoplasia agents, which includes, but is not limited to, aclarubicin hydrochloride, daunorubicin hydrochloride, doxorubicin hydrochloride, epirubicin hydrochloride, idarubicin hydrochloride, pirarubicin, or zorubicin hydrochloride; camptothecin, or a derivative or related compound thereof, such as 10, 11-methylenedioxycamptothecin; or members of the maytansinoid (maytansinoid) family of compounds, which include various structurally related compounds, for example, ansamitocin P3, maytansine, 2' -N-demethylmaytansinoid, and maytansbicylinol.

The HSA linker, or any molecule or moiety bound thereto, can be directly connected to the cytotoxic or therapeutic agent using known chemistry, or indirectly connected to the cytotoxic or therapeutic agent via an indirect connection. For example, the HSA linker can be attached to a chelating group that is attached to a cytotoxic or therapeutic agent. Chelating groups include, but are not limited to, iminocarboxylic acid and polyaminopolycarboxylic acid reactive groups, diethylenetriaminepentaacetic acid (DTPA), and 1,4, 7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA). For general methods, see, e.g., Liu et al, Bioconjugate chem.12 (4): 653, 2001; cheng et al, WO 89/12631; kieffer et al, WO 93/12112; albert et al, u.s.p.n.5,753, 627; and WO 91/01144 (each of which is incorporated herein by reference).

HSA linker conjugates include, for example, an HSA linker, one or more binding moieties (with or without a spacer peptide linker, as defined herein), and a therapeutic or cytotoxic agent that can be specifically targeted to a cell or tissue via the binding moiety (e.g., an antibody, antibody fragment, or receptor/ligand), thereby facilitating selective disruption of the target cell or tissue to which the binding moiety is directed. For example, when the HSA linker conjugate includes a binding moiety that specifically binds to cancer cells in an organ, the HSA linker conjugate can be used to target and destroy cancer cells of the lung, breast, prostate, and colon, to prevent, stabilize, inhibit progression of, or treat cancer derived from the above-mentioned organ. In addition, the HSA linker conjugates can be used to target and destroy cells of the vasculature, brain, liver, kidney, heart, lung, prostate, colon, nasopharynx, oropharynx, larynx, bronchi, and skin, for example, by targeting, in the case of, for example, autoimmune diseases, autoreactive T cells (e.g., by binding to and agonizing tumor necrosis factor receptor 2(TNFR2)) to prevent, stabilize, inhibit progression of age-related, tobacco-related, or autoimmune diseases or conditions associated with these organs, or to treat them.

The HSA linker, when recombinantly expressed, may be linked to the cytotoxic polypeptide. A cytotoxic polypeptide, which, when contacted with a target cell (e.g., a cancer cell), exerts a cytotoxic or cytostatic effect on the cell. For example, a cytotoxic polypeptide, when linked to an HSA linker, can be induced in a target cell upon binding to the target cell, which results in cell death (by, e.g., apoptosis, necrosis, or senescence). Alternatively, a cytotoxic polypeptide attached to the HSA linker can interfere with or inhibit normal cellular biological activities, such as division, metabolism, and growth, or abnormal cellular biological activities, such as metastasis.

For example, an HSA linker attached to caspase 3 will bind to a target cell (e.g., a cancer cell) and be endocytosed. Upon internalization by the target cell, the caspase moiety of the HSA linker conjugate can initiate a pro-apoptotic caspase cascade, ultimately leading to apoptosis of the target cell.

In a preferred embodiment, the HSA linker conjugate comprises a cytotoxic polypeptide capable of killing cancer cells. In another embodiment, the cytotoxic polypeptide inhibits growth or metastasis of cancer cells. The cytotoxic polypeptide attached to the HSA linker can also be used to kill or inhibit cell growth associated with, necessary for, or beneficial for cancer growth, such as endothelial cells forming blood vessels throughout a solid tumor.

In one embodiment, the HSA linker conjugate can include two or more cytotoxic polypeptides to modulate (e.g., increase) the specificity, intensity, or duration of cytotoxic or cytostatic effects on target cells (e.g., cancer cells).

In another embodiment, the HSA linker is linked to an activatable form of the cytotoxic polypeptide (e.g., a biologically inactive prodrug that is capable of being activated upon cleavage by an enzyme or drug). In such embodiments, exposure (e.g., in vivo) of the cytotoxic polypeptide prodrug to an enzyme or drug capable of cleaving the cytotoxic polypeptide can cause the cytotoxic polypeptide to become biologically active (e.g., cytotoxic or cytostatic). An example of a biologically inactive cytotoxic polypeptide that can be converted to a biologically active form for use with an HSA linker is a pro-caspase (e.g., pro-caspase 8 or 3). For example, following internalization of a target cell (e.g., a cancer cell), the pre-caspase 8 domain of the HSA linker can be cleaved by TRAIL or FasL. After cleavage, biologically active caspase 8 may promote apoptosis of the target cell.

In one embodiment, the cytotoxic polypeptide attached to the HSA linker can include a full-length peptide, polypeptide, or protein, or a biologically active fragment thereof (e.g., a "death domain"), known to have cytotoxic or cytostatic properties. Peptides, polypeptides, or proteins having cytotoxic or cytostatic properties can be altered (e.g., by making amino acid substitutions, mutations, truncations, or additions) to facilitate the addition of cytotoxic sequences to the agents described herein. Desirable changes include, for example, changes to amino acid sequences that can facilitate protein expression, prolong life, cellular secretion, and target cytotoxicity.

The invention also provides nucleic acid molecules encoding cytotoxic polypeptides as fusion proteins with an HSA linker, optionally including a binding moiety and a peptide linker. The nucleic acid molecule can be added to a vector (e.g., an expressible vector) such that, after expression of the HSA linker in a cell transfected or transduced with the vector, the cytotoxic polypeptide, HSA linker, and binding moiety, if present, can be operably linked (e.g., fused, adjacently linked, or defined together). Examples of peptides, polypeptides, and proteins that may be used as the cytotoxic polypeptide of the present invention include, but are not limited to, apoptosis-inducing proteins such as cytochrome c (SEQ ID NO: 39); caspases (e.g., caspase 3(SEQ ID NO: 36) and caspase 8(SEQ ID NO: 37)); procaspases, granzymes (e.g., granzymes A and B (SEQ ID NO: 38)); tumor Necrosis Factor (TNF) and TNF receptor family members, including TNF- α (TNF α; SEQ ID NO: 40)), TNF- β, Fas (SEQ ID NO: 41) and a Fas ligand; fas-associated death domain-like IL-1 β converting enzyme (FLICE); TRAIL/APO2L (SEQ ID NO: 45) and TWEAK/APO3L (see, e.g., U.S. patent application publication No. 2005/0187177, incorporated herein by reference); pro-apoptotic members of the Bcl-2 family, including Bax (SEQ ID NO: 46), Bid, Bik, Bad (SEQ ID NO: 42), Bak, and RICK (see, e.g., U.S. patent application publication No. 2004/0224389, incorporated herein by reference); apoptosis-inducing proteins 1 and 2(VAP1 and VAP 2; Masuda et al, biochem. Biophys. Res. Commun.278: 197-204 (2000)); pierisin (SEQ ID NO: 44; Watanabe et al, Biochemistry 96: 10608-10613 (1999)); apoptosis-inducing protein (SEQ ID NO: 43; AIP; Murawaka et al, Nature 8: 298-307 (2001)); IL-1. alpha. preprotein (see, e.g., U.S. Pat. No. 6,191,269, incorporated herein by reference); apoptotic and apoptotic-protein-related proteins such as AAP-1 (see, e.g., European patent application publication No. EP 1083224, incorporated herein by reference); anti-angiogenic factors such as endostatin and angiostatin; and other apoptosis-inducing proteins, including those described in the following international and U.S. patent application publications, each incorporated herein by reference: U.S.2003/0054994, U.S.2003/0086919, U.S.2007/0031423, WO 2004/078112, WO2007/012430, and WO 2006/0125001(δ 1 and notched 1 intracellular domain).

Wild type HSA linker conjugates

In forming a binding, diagnostic, or therapeutic agent, the invention also includes a naturally occurring wild-type HSA linker whose amino acid and nucleotide sequences are provided in SEQ ID NO: 3 and 4. In the use of a polypeptide having the sequence set forth in SEQ ID NO: 3, one or more peptide connectors (as described above) are covalently attached to the amino and/or carboxy terminus of the HSA linker, or to amino acid residues in the HSA linker sequence, to facilitate binding of the one or more binding moieties.

Cutting to length

The invention also provides HSA linker conjugates formed using truncated wild-type HSA polypeptides, which are optionally conjugated to one or more peptide connectors or binding portions. It is possible to link a wild-type HSA polypeptide lacking 1,2, 3, 4,5, 10, 15, 20, 50, 100, 200 or more amino acids of the full length wild-type HSA amino acid sequence (i.e., SEQ ID NO: 3) to any of the binding moieties or diagnostic or therapeutic agents described herein. Truncation may occur at one or both ends of the HSA linker, or may include deletion of internal residues. Truncation of more than one amino acid residue need not be linear (i.e., contiguous). Examples of wild type HSA linkers include those having one or more of domain I (SEQ ID NO: 56; residues 1-197 of SEQ ID NO: 3), domain II (SEQ ID NO: 54; residues 189 and 385 of SEQ ID NO: 3), or domain III (SEQ ID NO: 57; residues 381 and 585 of SEQ ID NO: 3), or combinations thereof, e.g., domains I and II, I and III, and II and III, along with one or more peptide linkers or binding moieties.

Serum clearance of a conjugate (e.g., a bispecific HSA-drug or a radioisotope-containing agent) can be optimized by testing a conjugate comprising a truncated wild-type HSA linker (as described above).

Additional HSA linker modifications

The HSA linker can be modified by site-specific chemical modification of amino acid residues in the HSA linker, but is not required. The properly folded tertiary structure of HSA presents certain amino acid residues on the outside of the protein. Chemically active amino acid residues (e.g., cysteine) may be substituted for these surface exposed residues to allow for site-specific binding of diagnostic or therapeutic agents.

Alternatively or additionally, the HSA linker may be optionally modified by adding or removing asparagine, serine, or threonine residues from the HSA linker sequence to alter glycosylation of these amino acid residues. The glycosylation site to which the HSA linker is added is preferably surface exposed (as discussed herein). The carbohydrate moiety or other carbohydrate moieties introduced into the HSA linker can be directly conjugated to a diagnostic, therapeutic, or cytotoxic agent.

Cysteine (thiol) conjugates

The surface exposed amino acid residues of the HSA linker may be substituted with cysteine residues to facilitate chemical binding of diagnostic, therapeutic, or cytotoxic agents. Cysteine residues exposed on the surface of the HSA linker (when folded into its native tertiary structure) facilitate specific binding of diagnostic, therapeutic, or cytotoxic agents to thiol-reactive groups such as maleimide or haloacetyl groups. The nucleophilic reactivity of the thiol functional group of a cysteine residue with a maleimide group is about 1000 times higher than any other amino acid functional group in the protein (e.g., the amino group or amino-terminal amino group of a lysine residue). Thiol-specific functional groups in iodoacetyl and maleimide reagents can react with amine groups but require higher pH (> 9.0) and longer reaction times (Garman, 1997, Non-Radioactive laboratory: analytical Press, London). The amount of free thiol groups in the protein can be estimated using standard elman assays. In some cases, reagents such as Dithiothreitol (DTT) or selenol are required to reduce the disulfide bond (Singh et al, anal. biochem. 304: 147-156(2002)) to generate a reactive free thiol group.

Sites for cysteine substitutions can be identified by analyzing the surface accessibility of the HSA linker (e.g., identification of serine and threonine residues suitable for substitution as described in example 1 below). Surface accessibility may be expressed as the surface area (e.g., square angstroms) that may be contacted by solvent molecules such as water. The space occupied by the water is approximately a sphere with a radius of 1.4 angstroms. The software for calculating the surface accessibility of each amino acid of a protein is freely available or privileged. For example, CCP4Suite, a Crystallography program that employs algorithms to calculate surface accessibility for each amino acid of a protein with known coordinates from X-ray Crystallography ("The CCP4 Suite: Programs for protein Crystallography" acta. Crystal. D50: 760-763 (1994);www.ccp4.ac.uk/dist/html/INDEX.html). Solvent accessibility can also be assessed using the freeware DeepView Swiss PDB Viewer downloaded from SwissInstitute of B Ioinformatics. Substitution of the cysteine at the surface exposed site facilitates the binding of the reactive cysteine to the thiol-reactive group attached to the diagnostic or therapeutic agent.

Glycosylation

In addition, serum clearance can be altered by designing glycosylation sites in the HSA linker. In certain embodiments, the HSA linker is glycosylated. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X represents any amino acid except proline, are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition or deletion of glycosylation sites of the HSA linker is conveniently accomplished by altering the amino acid sequence so as to produce one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Such alterations may also be made by the addition, deletion, or substitution of one or more serine or threonine residues to the sequence of the original HSA linker (for the O-linked glycosylation site). The resulting carbohydrate structures on HSA can also be used for site-specific binding of cytotoxic, immunomodulatory or cytostatic agents as described above.

HSA linker conjugates in combination with other therapeutic agents

The HSA linker conjugates described herein can be administered in combination with one or more of the therapeutic, cytotoxic, or cytostatic agents described herein. For example, a breast cancer patient can be administered an HSA linker comprising ErbB2, and ErbB3scFv (e.g., B2B3-1), a common chemotherapeutic regimen for treating breast cancer, can be co-administered in combination with, for example, doxorubicin, cyclophosphamide, and paclitaxel. One preferred therapeutic agent for this use is trastuzumab. Data on this combination is set forth in examples 42-44 below. Additional biological and chemical agents useful in the treatment of cancer are listed herein, for example in appendix 2.

HSA linker conjugates in combination with radiotherapy or surgery

The HSA linker conjugate can be administered before, during, or after radiation therapy or surgery. For example, a patient with a proliferative disease (e.g., breast cancer) may receive an HSA linker conjugate alone or together with other therapeutic agents, cytotoxic agents, or a cytotoxic agent and an HSA linker conjugate as described herein while undergoing targeted radiation therapy or surgical intervention (e.g., focal resection or mastectomy) at a cancerous tissue site. Radiation therapies suitable for use in combination with HSA linker conjugates include brachytherapy and targeted intraoperative radiation therapy (TARGIT).

Pharmaceutical composition

The pharmaceutical compositions provided herein comprise a therapeutically or diagnostically effective amount of an HSA linker conjugate comprising one or more of a binding moiety (e.g., an antibody or antibody fragment), a diagnostic agent (e.g., a radionuclide or chelator), or a therapeutic agent (e.g., a cytotoxic agent or immunomodulatory agent). The active ingredient, the HSA linker conjugate (prepared with one or more binding moieties, diagnostic agents, or therapeutic agents) can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for proper formulation. Suitable dosage forms for use in the present invention are described in Remington's pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of drug delivery methods, see langer science 249: 1527-1533(1990). The pharmaceutical compositions are for parenteral administration, intranasal administration, topical administration, oral administration, or topical administration, such as by transdermal means, for prophylactic and/or therapeutic treatment. Typically, the pharmaceutical composition is administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral administration, or by topical application. Thus, compositions for parenteral administration can include an HSA linker, with or without one or more binding, diagnostic, and/or therapeutic agents bound thereto, dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, PBS, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. The invention also provides compositions for oral administration which may contain inert ingredients such as binders or fillers used in the formulation of tablets, capsules and the like. In addition, the present invention provides compositions for topical administration which may contain inert components such as solvents or emulsifiers for formulating creams, ointments and the like.

These compositions may be sterilized by conventional sterilization techniques, or they may be sterile filtered. The aqueous solution obtained may be packaged for use as is, or lyophilized, the lyophilized preparation being incorporated in a sterile aqueous carrier prior to administration. The pH of the agent is typically from 3 to 11, more preferably from 5 to 9 or from 6 to 8, and most preferably from 7 to 8, such as from 7 to 7.5. The resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) in a sealed package, such as a tablet or capsule. The compositions in solid form may also be packaged in containers having variable amounts, such as in squeezable tubes designed for topical application of creams or ointments. Compositions comprising an effective amount of an HSA linker conjugate formulated with one or more binding, diagnostic, and/or therapeutic agents can be administered to a mammal (e.g., a human) for prophylactic and/or therapeutic treatment. In prophylactic applications, a composition comprising an HSA linker conjugate formulated with one or more binding, diagnostic, and/or therapeutic agents is administered to a patient susceptible to or having a developing disease or disorder (e.g., cancer, autoimmune disease, or cardiovascular disease). The above amount is defined as a "prophylactically effective dose". In such applications, the precise amount again depends on the health of the patient, but typically is about 0.5mg to about 400mg of HSA linker conjugate (prepared with one or more binding, diagnostic, and/or therapeutic agents) (e.g., 10mg, 50mg, 100mg, 200mg, 300mg, or 400mg or more per dose) per administration and about 0.1 μ g to about 300mg of one or more immunomodulators (e.g., 10 μ g, 30 μ g, 50 μ g, 0.1mg, 10mg, 50mg, 100mg, or 200mg per dose). The dosage of HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) can be administered prophylactically to a patient one or more times per hour, day, week, month, or year (e.g., 2, 4,5, 6,7, 8,9, 10, 11, or 12 times per hour, day, week, month, or year). More typically, a single dose of HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) is administered weekly.

In therapeutic use, a dose of HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) is administered to a mammal (e.g., a human) already suffering from a disease or disorder (e.g., cancer, autoimmune disease, or cardiovascular disease), wherein the amount administered is sufficient to cure or at least partially arrest or alleviate one or more symptoms of the disease or disorder, as well as its complications. An amount suitable for achieving the above purpose is defined as a "therapeutically effective dose". The amount effective for such use may depend on the severity of the disease or disorder and the general condition of the patient, but is typically from about 0.5mg to about 400mg of HSA linker conjugate (prepared with one or more binding, diagnostic, and/or therapeutic agents) per administration (e.g., 10mg, 50mg, 100mg, 200mg, 300mg, or 400mg or more per dose). A dose of HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) can be administered to a patient therapeutically one or more times per hour, day, week, month, or year (e.g., 2, 4,5, 6,7, 8,9, 10, 11, or 12 times per hour, day, week, month, or year). More typically, a single dose of HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) is administered weekly.

In several embodiments, the patient may receive from about 0.5 to about 400mg per dose of HSA linker conjugate (prepared with one or more binding, diagnostic, and/or therapeutic agents) once or more times per week (e.g., 2, 3, 4,5, 6, or 7 or more times per week), preferably once or more times per week, with from about 5mg to about 300mg per administration, and even more preferably once or more times per week, with from about 5mg to about 200mg per administration. The patient may also receive a biweekly dose of HSA linker conjugate (prepared with one or more binding, diagnostic, and/or therapeutic agents) (about 50mg to about 800mg) or a monthly dose of HSA linker, or any binding, diagnostic, or/and therapeutic agent conjugated thereto (about 50mg to about 1,200 mg).

In other embodiments, HSA linker conjugates (formulated with one or more binding, diagnostic, and/or therapeutic agents) can be administered to a patient in a typical dosage range: about 0.5 mg/week to about 2000 mg/week, about 1.0 mg/week to about 1000 mg/week, about 5 mg/week to about 500 mg/week, about 10 mg/week to about 100 mg/week, about 20 mg/week to about 80 mg/week, about 100 mg/week to about 300 mg/week, or about 100 mg/week to about 200 mg/week. In another aspect, the dosage range of an HSA linker conjugate described herein for administration to a 70kg patient may be, for example, from about 1 μ g to about 5000mg, from about 2 μ g to about 4500mg, from about 3 μ g to about 4000mg, from about 4 μ g to about 3,500mg, from about 5 μ g to about 3000mg, from about 6 μ g to about 2500mg, from about 7 μ g to about 2000mg, from about μ g to about 1900mg, from about 9 μ g to about 1,800mg, from about 10 μ g to about 1,700mg, from about 15 μ g to about 1,600mg, from about 20 μ g to about 1,575mg, from about 30 μ g to about 1,550mg, from about 40 μ g to about 1,500mg, from about 50 μ g to about 1,475mg, from about 100 μ g to about 1,450mg, from about 200 μ g to about 1,425mg, from about 300 μ g to about 1,000mg, from about 400 μ g to about 975mg, from about 500 μ g to about 650mg, from about 1.5mg, from about 1,525 mg to about 2.525 mg, from about 1.500 mg, from about 1mg, about 2mg to about 2.525 mg, About 3.0mg to about 475mg, about 3.5mg to about 450mg, about 4.0mg to about 425mg, about 4.5mg to about 400mg, about 5mg to about 375mg, about 10mg to about 350mg, about 20mg to about 325mg, about 30mg to about 300mg, about 40mg to about 275mg, about 50mg to about 250mg, about 100mg to about 225mg, about 90mg to about 200mg, about 80mg to about 175mg, about 70mg to about 150mg, or about 60mg to about 125 mg. The dosage regimen may be adjusted to provide the optimal therapeutic response. In another aspect, the HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) can be administered at about 0.5mg every other day to about 500mg every other day, preferably at about 5mg every other day to about 75mg every other day, more preferably at about 10mg every other day to about 50mg every other day, and even more preferably at about 20mg every other day to about 40mg every other day. The HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) can also be administered from about 0.5mg three times per week to about 100mg three times per week, preferably from about 5mg three times per week to about 75mg three times per week, more preferably from about 10mg three times per week to about 50mg three times per week, and even more preferably from about 20mg three times per week to about 40mg three times per week.

In non-limiting embodiments of the methods of the invention, the HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) can be administered to a mammal (e.g., a human) for 1,2, 3, or 4 hours; 1,2, 3, or 4 times a day; every other day or every third, fourth, fifth, or sixth day; 1,2, 3, 4,5, 6,7, 8,9, or 10 times a week; every two weeks; 1,2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times a month; every two months; 1,2, 3, 4,5, 6,7, 8,9, or 10 times every six months; 1,2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times a year; or every two years. The HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) can be administered at different frequencies during a treatment regimen. In additional embodiments, the HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) can be administered to the patient at the same frequency or at different frequencies.

The amount of the one or more diagnostic or therapeutic agents and the HSA linker, or any agent bound thereto, needed to achieve the desired therapeutic effect depends on several factors, such as the particular diagnostic or therapeutic agent selected, the mode of administration, and the clinical condition of the recipient. The skilled artisan will be able to determine the appropriate dosages of one or more diagnostic or therapeutic agents and the HSA linker, or any agent conjugated thereto, to achieve the desired result.

Compositions comprising an effective amount of an HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) can be administered in single or multiple administrations, where the dose level and pattern are selected by the treating physician. Based on the severity of the disease or condition in the mammal (e.g., human), dosages and schedules for administration may be determined and adjusted, and the clinician or those described herein may monitor the severity throughout the course of treatment according to commonly practiced methods.

The HSA linker conjugate (formulated with one or more binding, diagnostic, and/or therapeutic agents) can be administered to a mammalian subject, such as a human, either directly or in conjunction with any pharmaceutically acceptable carrier or salt known in the art. Pharmaceutically acceptable salts may include non-toxic salts commonly used in the pharmaceutical industryAcid addition salts or metal complexes. Examples of the acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid, or the like; high molecular acids such as tannic acid, carboxymethyl cellulose, etc.; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like. The metal complex includes zinc, iron, etc. One typical pharmaceutical carrier is physiological saline. Other physiologically acceptable carriers and their dosage forms are known to the person skilled in the art and are described, for example, inRemington’s Pharmaceutical Sciences，(18^thedition), ed.a. gennaro, 1990, Mack publishing company, Easton, PA.

Diagnostic and therapeutic applications

HSA linker conjugates can be used for diagnostic and therapeutic applications in humans, including, for example, in the diagnosis or treatment of proliferative diseases (e.g., cancers such as melanoma, clear cell sarcoma, and kidney cancer) and autoimmune diseases (e.g., multiple sclerosis, rheumatoid arthritis, and uveitis). The following discussion of human proliferative and autoimmune diseases is intended to provide the skilled artisan with a general understanding of how HSA linker conjugates can be used for diagnostic and therapeutic applications, and is not intended to limit the scope of the invention.

Proliferative diseases (cancer)

The HSA linker conjugate can be used to diagnose, treat, prevent, or eliminate proliferative diseases such as, but not limited to, breast cancer, melanoma, clear cell sarcoma, renal cancer (e.g., renal cell carcinoma), prostate cancer, lung cancer, stomach cancer, and ovarian cancer. The selection of binding moieties to be linked to an HSA linker and used in the diagnosis or treatment of a patient suspected of having or having a proliferative disease may be based on their ability to specifically bind to, agonize, activate, antagonize, or inhibit a target molecule associated with a proliferative disease (e.g., a cell surface receptor such as a tyrosine kinase receptor). Binding moieties may be linked to the HSA linker to diagnose or treat proliferative diseases, wherein the binding moieties target, for example, insulin-like growth factor receptors (IGFRs, e.g., IGF1R and IGF2R), Fibroblast Growth Factor Receptor (FGFR), platelet-derived growth factor receptor (PDGFR), Vascular Endothelial Growth Factor Receptor (VEGFR), Tumor Necrosis Factor Receptor (TNFR), epidermal growth factor receptor (EGFR, e.g., ErbB2(HER2/neu)), Fc receptor, c-kit receptor, or mesenchymal epithelial transforming factor receptor (c-met; also known as Hepatocyte Growth Factor Receptor (HGFR)). Specific binding of the HSA linker conjugate to the cancer cell can facilitate detection (e.g., an HSA linker linked to a detectable label, as defined herein) or disruption (e.g., an HSA linker linked to a cytotoxic agent) of the bound cancer cell. Specific applications of HSA linker conjugates in the treatment of breast and kidney cancer are described below.

Breast cancer

Common forms of breast cancer include invasive ductal carcinoma, malignant cancer in the thoracic duct, and invasive lobular carcinoma, malignant cancer in the thoracic lobules. Some types of breast cancer cells are known to express high levels of epidermal growth factor receptor, particularly ErbB2 (i.e., HER 2/neu). Aberrant signaling or unregulated activation of EGFR has been associated with the development and progression of many cancers, including breast cancer. Uncontrolled cell proliferation mediated via the dysfunctional EGFR pathway can be found in various solid tumors of epithelial origin, and data indicate that tumor EGFR expression, overexpression, and dysregulation are associated with progressive disease, metastatic phenotype, resistance to chemotherapy, and overall poor prognosis.

An HSA linker linked to one or more binding moieties specific for EGFR (e.g., anti-ErbB 2; trastuzumab) can be used in conjunction with a diagnostic agent (e.g., a detectable label) or cytotoxic, cytostatic, or therapeutic agent (as described herein) to diagnose or treat breast cancer. Alternatively, bispecific HSA linker conjugates comprising a binding moiety specific for ErbB2 and ErbB3, such as "B2B 3-1" (described further below), can be used to diagnose or treat cancer, e.g., breast, kidney, ovarian, and lung cancer.

As described above, the HSA linker conjugate used to treat breast cancer can be administered prior to radiation therapy or surgical intervention (e.g., neoadjuvant chemotherapy), concurrently with, or after (e.g., adjuvant chemotherapy). The HSA linker conjugate can also be administered in combination with other compounds useful in the treatment of breast cancer (e.g., antineoplastic agents, such as biological or chemotherapeutic agents). For example, the antineoplastic agents listed in table 1, which include mitotic inhibitors (e.g., taxanes), topoisomerase inhibitors, alkylating agents (including, e.g., platinum-based agents), selective estrogen modulators (SERMs), aromatase inhibitors, antimetabolites, antitumor antibiotics (e.g., anthracycline antibiotics), anti-VEGF agents, anti-ErbB 2(HER2/neu) agents, and anti-ErbB 3 agents, are known to be particularly advantageous for the treatment of breast cancer. The HSA linker conjugate can be administered by the clinician in combination with any compound, including those listed in appendix 2, which are known or believed to be beneficial in the treatment of breast cancer.

Table 1: along with HSA linker conjugates are typical antineoplastic agents used to treat breast cancer.

Renal cancer

Renal cancers, such as renal cell carcinoma, are particularly resistant to traditional radiation and chemotherapy. Thus, the use of a biotherapeutic agent in conjunction with the HSA linker is a good choice for patients with these cancers. For example, an HSA linker linked to a binding moiety that agonizes the type I interferon or interleukin 2 receptor may be used to treat kidney cancer. As solid tumors, binding moieties that target and inhibit tumor vascularization (e.g., anti-Vascular Endothelial Growth Factor (VEGF) antibodies such as bevacizumab) may also be used for therapeutic effect.

Autoimmune diseases

The HSA linker conjugates can be used to diagnose, treat, prevent, or stabilize, for example, autoimmune diseases and disorders in patients, such as, for example, Multiple Sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), Rheumatoid Arthritis (RA), uveitis, sjogren's syndrome, graves' disease, psoriasis, and myasthenia gravis. Autoimmune diseases and disorders are caused by self-reactive components of the immune system (e.g., T cells, B cells, and self-reactive antibodies). Thus, binding moieties that inhibit, block, antagonize, or deplete self-reactive immune cells and antibodies (e.g., anti-lymphocyte or anti-thymocyte globulin; basiliximab, daclizumab, or moluzumab-CD 3 monoclonal antibodies) may be attached to the HSA linker for therapeutic use. A binding moiety that is an Inflammatory Signal Inhibitor (ISI) (as defined herein) may be linked to the HSA linker for use in the treatment of autoimmunity. In addition, binding moieties that inhibit or antagonize integrin function (e.g., integrin antagonists, as defined herein) can ameliorate or stop disease progression.

In other embodiments, the binding moiety is a soluble TNF receptor, such as etanercept or lenacicept; antibodies that target proinflammatory cytokines or proinflammatory cell surface signal molecules, such as adalimumab, certolizumab, infliximab, golimumab, and rituxan; dominant negative pro-inflammatory cytokine variants, such as XENP345, XPROTM1595, anakinra, and variants disclosed in U.S. patent application publication nos. 20030166559 and 20050265962; proinflammatory cytokines or inhibitors of the signaling pathway downstream of proinflammatory cell surface signaling molecules, e.g. DE 096, 5-amino-2-carbonylthiophene derivatives (as described in WO 2004089929), ARRY-797, BIRB 796BS, (1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- [4-2 (morpholin-4-yl-Ethoxy) -naphthalen-1-yl]-urea, CHR-3620, CNI-1493, FR-167653(Fujisawa Pharmaceutical, Osaka, Japan), ISIS 101757(Isis pharmaceuticals), ML3404, NPC31145, PD 169383 16, PHZ1112, RJW67657, 4- (4- (4-fluorophenyl) -1- (3-phenylpropyl) -5- (4-pyridyl) -1H-imidazol-2-yl) -3-butyn-1-ol, SCIO-469, SB 190, SB203580, (4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- (4-pyridyl) 1H-imidazole), SB239063, trans-1- (4-hydroxycyclohexyl) -4- (4-fluorophenyl-methoxypyrimidin-4-yl) imidazole, and, SB242235, SD-282, SKF-86002, TAK 715, VX702, and VX 745; or inhibitors of TNF-alpha converting enzyme (TACE), such as BB-1101, BB-3103, BMS-561392, butynylphenylene beta-sulfone piperidine hydroxymates, CH4474, DPC333, DPH-067517, GM6001, GW3333, Ro 32-7315, TAPI-1, TAPI-2, and TMI 005); or anti-idiotypic agents, such as monoclonal antibodies, LJP 394 (abelimus,La Jolla Pharmaceuticals)。

in other embodiments, the binding moiety is an interferon (as described herein). Binding moieties that can be linked to the HSA linker include, for example, interferon-beta (C.)(IFN-β-1a)、(IFN-. beta. -1a), and(IFN-. beta. -1b)), interferon-t (Tauferotm), interferon-. alpha.e.ROFERON-(IFN-α-2a)、INTRON-(IFN-α-2b)、(IFN-α-2b)、(IFN-α-n3)、(IFN-. alpha. -2b, covalently bound to monomethoxypolyethylene glycol),(non-naturally occurring type 1 interferon with 88% homology to IFN-. alpha. -2b), or(pegylated IFN-. alpha. -1a)), and(IFN-g-1b)。

the invention further provides HSA linker conjugates having a binding moiety that antagonizes these pro-inflammatory molecules or their specific receptors to treat autoimmunity. Specific applications of HSA linker conjugates in the diagnosis and treatment of MS and RA are described below.

Multiple sclerosis

Multiple Sclerosis (MS) is a neurological disease characterized by irreversible degeneration of nerves of the Central Nervous System (CNS). Although the underlying cause is unclear, neurodegeneration in MS is a direct result of demyelination, or the exfoliation of myelin, a protein that is usually located in the outer layers and insulates nerves. T cells play a key role in the development of MS. Inflammatory MS lesions, not normal proteins, may have infiltrative CD4⁺T cells that respond to self-antigens presented by MHC class II-linked molecules such as human HLA-DR 2. Infiltrating CD4T cells (T)_H1 cell) produces the proinflammatory cytokines IL-2, IFN-gamma, and TNF-alpha, which activate Antigen Presenting Cells (APCs) such as megakaryocytesPhagocytosis to produce additional proinflammatory cytokines (e.g., IL-1 β, IL-6, IL-8, and IL-12). IL-12 will further induce IFN- γ synthesis. The result is a gradual demyelination of the myelin sheath of neurons, leading to human disease.

HSA linker conjugates can be used to aid in the diagnosis of MS. Diagnostic HSA linker conjugates including a binding moiety will specifically target one or more (e.g., bispecific HSA linker conjugates) immune cell activation markers (e.g., CD69, CD28, HLA-DR, and CD 45). The imbalance of one or more of these pro-inflammatory or immune cell activation mediators relative to other factors or cells can be measured by using an HSA linker conjugate linked to a diagnostic agent (e.g., a radioisotope or a fluorescent pigment).

The HSA linker conjugate can be used to treat a human at risk of developing or suffering from MS, or to prevent, ameliorate, or cure the symptoms of the above-mentioned disease. For example, binding moieties that specifically target and antagonize α 4 integrin (e.g., natalizumab), CD52 (e.g., alemtuzumab), CD80, P-selectin, sphingosine-1-phosphate receptor-1 (S1PR1), hyaluronic acid receptor, leukocyte functional antigen-1 (LFA-1), CD11 (e.g., efavirenzumab), CD18, CD20 (e.g., rituximab), CD85, ICOS ligand, CCR2, CXCR3, or CCR5, when linked to an HSA linker, can be used to treat MS patients. Similarly, binding moieties that neutralize type I interferons (e.g., interferon- α and- β) or antagonize type I interferon receptors (e.g., IFN α R1) may also be linked to an HSA linker for therapeutic use.

Rheumatoid arthritis

Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune disease that causes the immune system to attack the joints. It is a disabling and painful inflammatory condition that can lead to substantial loss of mobility due to pain and joint destruction. RA is a systemic disease that often affects the extraarticular tissues of the entire body, including the skin, blood vessels, heart, lungs, and muscles.

RA patients often have increased cellular expression of the HLA-DR4/DR1 cluster. HSA linker conjugates specific for one or both of these cell surface molecules can be used for the diagnosis of RA.

The HSA linker conjugate can be used to treat a human at risk of developing or suffering from RA to prevent, ameliorate, or cure the symptoms of the above-mentioned disease. For example, a binding moiety (as defined herein) that specifically targets and antagonizes TNF-a (e.g., etanercept, infliximab, and adalimumab), IL-1 (e.g., anakinra), or CTLA-4 (e.g., abamectin). Binding moieties that specifically target and deplete B cells (e.g., anti-CD 20 antibodies, such as rituximab) can also be linked to the HSA linker described herein to treat or prevent RA.

Uveitis

Uveitis is specific to inflammation in the middle layer of the eye, but may refer to any inflammatory process involved in the interior of the eye. Uveitis may be autoimmune or idiopathic in origin.

The HSA linker conjugate can be used to treat a person at risk of developing or suffering from autoimmune uveitis to prevent, ameliorate, or cure the symptoms of the above-mentioned disease. For example, an alpha-fetoprotein (e.g., human AFP; NCBIAccess No. NM 001134), or a biologically active fragment thereof, can be linked to an HSA linker to reduce or eliminate inflammation associated with autoimmune or idiopathic uveitis.

Reagent kit

The invention also provides kits comprising a pharmaceutical composition comprising an HSA linker, and one or more binding moieties (e.g., an antibody or antibody fragment), a diagnostic agent (e.g., a radionuclide or chelator), and a therapeutic agent (e.g., a cytotoxic agent or immunomodulatory agent), and agents that can be used to bind them to the HSA linker, including a pharmaceutically acceptable carrier if necessary, and in a therapeutically effective amount to treat a disease or disorder (e.g., cancer, autoimmune disease, or cardiovascular disease). The kit includes instructions for administration of the composition contained therein by a user (e.g., a physician, nurse, or patient).

Preferably, the kit comprises a plurality of packaged single dose pharmaceutical compositions comprising an effective amount of an HSA linker, or any binding (e.g., antibody or antibody fragment (e.g., scFv)), diagnostic (e.g., radionuclide or chelator), and/or therapeutic (e.g., cytotoxic or immunomodulatory agent) conjugate thereof. Optionally, the instruments or devices necessary for administering the pharmaceutical composition may be included in a kit. For example, the kit can provide one or more prefilled syringes comprising an effective amount of the HSA linker, or any binding, diagnostic, and/or therapeutic agent bound thereto. In addition, the kit can also include additional components such as instructions or an administration schedule for administration of a pharmaceutical composition comprising an HSA linker, or any binding, diagnostic, and/or therapeutic agent associated therewith, to a patient with a disease or disorder (e.g., cancer, autoimmune disease, or cardiovascular disease).

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, methods, and kits of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Examples

The following examples are illustrative and not limiting. Those skilled in the art will readily appreciate that various non-critical parameters may be varied or modified to produce substantially the same or similar results.

Example 1: methods for identifying residues in an HSA linker for site-specific binding of a cytotoxic or cytostatic drug.

To identify sites where drugs specifically bind to HSA, the crystal structure was studied and surface exposed serine and threonine residues were identified. These specific surface exposed amino acids can then be mutated to cysteines to facilitate drug binding to the substituted cysteines using thiol-specific binding agents such as maleimides. Mild reduction may be required before drug binding. The number of drugs bound is controlled by the number of surface exposed cysteine residues introduced into HSA. Serine and threonine are chosen as the most suitable residues for mutation because they share the greatest structural identity with cysteine, however, other surface exposed residues can also be mutated to cysteine and successfully bind to cytostatic or cytotoxic drugs.

The Crystal structure of HSA is stored in RSCB Protein Data Bank (1bm0-Sugioet al, "Crystal structure of human serum album at 2.5A resolution," Protein Eng.12: 439 446 (1999)). The above structure was analyzed using a DeepView Swiss PDB Viewer downloaded from Swiss Institute of Bioinformatics. Serine and threonine residues with 50%, 40%, and 30% surface exposure were identified as the most suitable mutations to cysteine (table 2). Mutations can be introduced using standard molecular biology procedures. The binding of thiol-reactive drugs or chelators to the introduced cysteine can be tested using standard protein chemistry techniques.

TABLE 2

Surface exposed%	Residue of
		50	T496
40	S58
		30	T76、T79、T83、T125、T236、S270、S273
	S304、S435、T478、T506、T508

Example 2: methods for identifying residues for introducing asparagine-linked glycosylation sites in the HSA linker.

To identify the regions for introducing asparagine-linked glycosylation sites in HSA, the crystal structure was investigated to identify surface exposed (> 30%) asparagine, serine, and threonine residues that could be suitable for mutation. When the consensus asparagine-x-serine/threonine is present, where x cannot be proline, glycosylation occurs at the asparagine residue. Table 2 lists possible mutation sites for introducing asparagine-linked glycosylation in HSA.

TABLE 3

Residue of	Proposed mutations
		Gln32	Asn
Val46	Ser/Thr
		Asp56	Asn
Asp63	Ser/Thr*
		Glu231	Asn
Asp237	Asn
		Gln268	Asn
Asp269	Ser/Thr
		Glu285	Asn
Ala320	Ser/Thr*
		Asp340	Asn
Glu354	Asn
		Gln437	Asn
Glu425	Asn
		Glu465	Asn
Asp494	Asn*

These mutations have also been reported to occur very rarely in HSA (Carlson et al, "Allobulinemia in Sweden: Structural study and phenotypic distribution of peptide album variants," Proc. Nat. Acad. Sci. USA 89: 8225 and 8229 (1992); Madison et al, "Genetic variants of human serum in metals: poromtans and a carboxy-tertiary variants," Proc. Nat. Acad. Sci. USA 91: 6476. 6480 (1994); Hutchinson et al, "the N-tertiary sequence of albumin Redhailin, a serum of serum album, BSP. Leu. 193: Kleine.1985. 1997; Kluyn. xanth. Ala. 19826. 1987. xanth. Ala. 1987. xanth. 19832. Ala. 12. aspartic.247. 12. Biochemical of polysaccharide IV. xanth et al. (Biochemical) No. 320. xanth et al., Ala. xanth 7. xanth et al., Ala. 1987. xanth. 1987. "Structural characterization of aggregate protein variant of human serum album: albumin Casebrook (494Asp-Asn), "biocim.biophysis.acta 1097: 49-54(1991)).

Example 3

B2B3-1 is a bispecific scFv antibody fusion molecule comprising B1D2, a human anti-ErbB 2scFv antibody (SEQ ID NO: 27), and H3, a human anti-ErbB 3scFv (SEQ ID NO: 26). The two scfvs were linked by modifying the Human Serum Albumin (HSA) linker. The anti-ErbB 3scFv, H3 was recombinantly fused to the amino terminus of the HSA linker conjugated to the short connector polypeptide, and the anti-ErbB 2scFv, B1D2 was recombinantly fused to the carboxy terminus of the modified HSA linker conjugated to the additional short connector polypeptide. The selection of each linker polypeptide is based on protease resistance properties. The modified HSA linker comprises two amino acid substitutions. The cysteine residue at position 34 of native HSA was mutated to serine to reduce potential protein heterogeneity (due to oxidation at this site). The asparagine residue at amino acid 503 of native HSA, which in native HSA may be susceptible to deamidation resulting in a decrease in pharmacological half-life, is mutated to glutamine. It is believed that B2B3-1 selectively binds to ErbB2 overexpressing tumors with a kD of 10.0nM to 0.01nM, and more preferably about 0.3nM, by virtue of a high affinity anti-ErbB 2scFv binding moiety. Subsequent binding of anti-ErbB 3scFv to ErbB3, with a kD of 50 to 1nM and more preferably about 16nM, inhibits HRG-induced phosphorylation of ErbB 3. The modified HSA linker confers the bispecific molecule an extended circulating half-life. B2B3-1 has a molecular weight of 119.6kDa and is preferably not glycosylated.

B2B3-1 inhibits ligand-induced phosphorylation of ErbB3 with subnanomolar potency; this activity is believed to be due, at least in part, to the high abundant expression of its dimerization partner ErbB2, which facilitates the specific targeting of cancer cells expressing both receptors.

Example 4

As shown in fig. 2, the B2B3-1 variant inhibited HRG-induced pErbB3 in ZR75-1 breast cancer cells. ZR75-1 breast cancer cells were treated with a range of doses of the B2B3-1 variant for 24 hours, followed by HRG stimulation. Measurement of pErbB3 levels in cell lysates by ELISA and calculation of IC together with percent inhibition₅₀The value is obtained. Shown is the average IC₅₀Values (Y-axis), where error bars represent duplicate experiments. The inhibition percentage values are shown above the corresponding bars.

ELISA assay

Except as indicated, ELISA reagents for total ELISA and phospho-ErbB 3ELISA were purchased from R & D Systems in the form of DUOSET kits. 96-well NUNC MAXISORB plates were coated with 50. mu.l of antibody and incubated overnight at room temperature. The next morning, the plates were washed 3 times in a BIOTEK plate washer with 1000 μ l/well of calcium or magnesium free dubiaceae Phosphate Buffered Saline (PBS) supplemented with Tween detergent (PBST) (0.05% Tween-20). The plates were then blocked with 2% BSA in PBS for about 1 hour at room temperature. Plates were washed 3 times with 1000. mu.l/well PBST in a BIOTEK plate washer. Cells were grown at 37 ℃ and 5% carbon dioxide, washed with cold PBS, and then enriched with Mammalian Protein Extract (MPER) lysis buffer (Pierce, 78505) supplemented with 150mM NaCl, 5mM sodium pyrophosphate, 10. mu.M bpV (phen), 50uM phenylarsine, 1mM sodium orthovanadate, and protease inhibitor cocktail (Sigma, P2714). 50 μ L of cell lysate and standard diluted in 50% lysis buffer and 1% BSA were performed in duplicate for further processing. The samples were incubated at 4 ℃ for 2 hours on a plate shaker and washed as before. Approximately 50. mu.l of detection antibody was diluted in 2% BSA, PBST was added and incubated at room temperature for about 1-2 hours. For phospho-ErbB 3, the detection antibody was directly conjugated to horseradish peroxidase (HRP), while for total ErbB3 and biotinylated murine anti-human ErbB3, a second detection antibody was used. The plates were cleaned as described previously. For total ErbB3, approximately 50. mu.l of streptavidin-HRP was added and incubated for 30 minutes, and the plates were washed as before. Approximately 50. mu.L of SUPERSIGNALAELISA Pico (Thermo Scientific) substrate was added and plate reads were performed using a FUSION plate reader. Duplicate samples were averaged and the error bars shown represent the standard deviation between the two samples.

Example 5

Inhibition of phosphorylated ErbB3 (fig. 3A-D), AKT (fig. 4A-D), and ERK (fig. 5A-D) was measured after 24 hours of pretreatment with B2B3-1 variant a5-HSA-B1D2 (fig. 3-5 panel a), H3-HSA-B1D2 (fig. 3-5 panel B), H3-HSA-F5B6H2 (fig. 3-5 panel C), and F4-HSA-F5B6H2 (fig. 3-5 panel D). Treatment of BT474 breast cancer with a dose range of B2B3-1 variantsCells were stimulated for 24 hours, followed by HRG. By ELISA in cell lysate in pErbB3, pAKT, and pERK levels, and together with the percentage of inhibition calculated IC₅₀The value is obtained. These results show that 10^-8Or higher, B1B2-1 was the only HSA linker connector tested to provide greater than 50% inhibition of HRG-induced phosphorylation of AKT, ERK and ErbB 3.

Example 6

As shown in fig. 6, treatment of BT474 breast tumor cells with B2B3-1 variant resulted in G1 cell cycle arrest and a reduction in S phase cell population. BT474 cells were treated with 1. mu.M of the B2B3-1 variant and control for 72 hours. After the end of the treatment, the cells were trypsinized, gently resuspended in a hypotonic solution containing propidium iodide, and then analyzed for single cells by flow cytometry. Cell cycle distribution in G1 and S phases was measured using a curve fitting algorithm designed for cell cycle analysis (FlowJo software cell cycle platform).

Example 7

By utilizing the dimerization partner, the high-abundance expression of ErbB2, B2B3-1(SEQ ID NO: 16) can inhibit ErbB3 activation, thereby targeting tumor cells. High affinity anti-ErbB 2scFv antibody, B1D2, facilitates targeting of B2B3-1 to tumor cells overexpressing ErbB 2. B1D2 was linked to the low affinity anti-ErbB 3scFv antibody H3 by a modified HSA linker, which blocks binding of the ligand HRG of ErbB 3. Inhibition of ErbB3 phosphorylation and downstream AKT signaling mediated by B2B3-1 is thought to be due to this blockade. ErbB2, B1D2, that binds to scFv, derived from the parent scFv C6.5, has neither agonistic nor antagonistic activity against ErbB 2. Thus, B1D2 also acts only as a targeting agent. Low affinity binding of ErbB3 to scFv is thought to prevent strong binding of B2B3-1 to normal, non-cancerous tissues that express ErbB3 but little or no ErbB2, thereby reducing the efficacy of non-specific toxicity. In tumor cells expressing ErbB2 and ErbB3, the affinity effect of the presence of bispecific B2B3-1 binding to both receptors would overcome the low affinity of ErbB3scFv, allowing strong inhibition of the interaction of HRG with the ErbB3 receptor complex.

The ability of B2B3-1 to inhibit HRG binding to ErbB3 was studied using flow cytometry (FACS). Cells of a human breast cancer cell line (variant of BT-474, which overexpresses ErbB2) were pretreated with 1 μ M B2B3-1 and then incubated with 10nM biotinylated HRG 1 β EGF domain. After extensive washing, the binding was assessed using streptavidin-AlexaFluor 647 conjugate. All incubations were performed at 4 ℃. Figure 7 shows that B2B3-1 was able to block HRG binding to ErbB3 and provided 100% blocking at a concentration of 1 μ M.

Example 8

After demonstrating the ability of B2B3-1 to block HRG binding to ErbB3, the effect of two cell lines B2B3-1 expressing ErbB3 and overexpressing ErbB2 on ErbB3 signaling in vitro was investigated. Human breast cancer cell line BT-474-M3 (in, e.g., Drummondet al (2005) clin.11：3392；Park et al.(2002)Clin.CancerRes.8：1172；Kirpotin et al.(2006)Cancer Res.66: 6732) and ZR7530 (available from US NIH Lawrence Berkeley National Laboratory Breast cancer cell Collection) were serum starved overnight, pretreated with dose-titrated B2B3-1 for 24 hours, and then stimulated with 5nM HRG 1 β EGF domain for 10 minutes. Phosphorylation status of ErbB3 and AKT was then examined using an ELISA assay essentially as described previously. The results indicate that B2B3-1 inhibited HRG-induced activation of ErbB3 and AKT phosphorylation in a dose-dependent manner in both cell lines with potent IC₅₀(FIGS. 8A-D).

Example 9

FIG. 9 shows the effect of B2B3-1 treatment on signaling proteins in BT474 breast cancer cells. Cells were treated with a range of doses of B2B3-1 for 24 hours, followed by nerve growth factor stimulation. By western blot analysis, cell lysates were determined for pErbB2, pErbB3, pErk, and pAKT levels, as well as their corresponding total protein levels. The results show that at least the levels of pErbB2 and pErbB3 were reduced in a dose-dependent manner by B2B3-1 treatment.

Example 10

Fig. 10 shows immunoprecipitation-western blot analysis of B2B3-1 treated BT474 breast cancer cells. Cells were treated with a range of doses of B2B3-1 for 24 hours, followed by nerve growth factor stimulation. The ErbB2 binding complex was isolated from cell lysates using anti-ErbB 2 antibody, followed by western blot analysis to detect pErbB2 and pErbB3 and corresponding total protein levels. The results indicate that B2B3-1 cross-linked ErbB2 with ErbB3 so that substantially more ErbB3 and phosphorus-ErbB 3 could be precipitated by anti-ErbB 2 antibodies.

Example 11

The anti-tumor activity of B2B3-1 was studied in vitro using several assays. In the first assay, the effect of B2B3-1 on the accumulation of BT-474 or SKBR3 cells in G1 phase and the concomitant reduction in cells in S phase of the cell cycle was examined. Briefly, cells were treated with 1 μ M B2B3-1 or PBS carrier for 72 hours. After the end of the treatment, the cells were trypsinized, gently resuspended in a hypotonic solution containing propidium iodide, and then analyzed for single cells by flow cytometry. Cell cycle distribution in G1 and S phases was measured using a curve fitting algorithm designed for cell cycle analysis (FlowJo software cell cycle platform, Tree Star, Inc.). It was found that B2B3-1 mildly decreased the percentage of S phase cells and increased the population of G1 phase cells (fig. 11A). In a second experiment, the number of cell colonies formed after treatment with B2B3-1 was investigated. BT-474 and SKBR3 breast cancer cells were plated in the presence of 1. mu.MB 2B3-1, and cells plated only in medium were compared. Media was replenished only or media including treatments every 3 days. After 14 days, the number of colonies was counted and compared to untreated cells. FIG. 11B shows that the number of colonies formed was reduced by 40-45% when cells were treated with B2B3-1 compared to control cells. Finally, the ability of B2B3-1 to inhibit cell proliferation was assessed using a cell impedance assay and using a real-time cell electronic sensing system (RT-CES: ACEA Biosciences). BT-474 cells were seeded on a plate incorporating an array of microelectronic sensors and then dosedTitrated B2B3-1 or media only treatment for 72 hours. Data reflecting the occurrence of cell-electrode impedance reactions were collected every hour for 72 hours, and IC was then calculated 68 hours after treatment₅₀The value is obtained. FIG. 11C shows that B2B3-1 was able to inhibit the impedance of BT-474 cells and IC₅₀Was 33 nM.

Example 12

We also investigated whether B2B3-1 has agonist activity based on its ability to bind and cross-link ErbB2 and ErbB3 receptors simultaneously. Serum-starved ZR75-1 breast cancer cells were incubated with 1 μ M B2B3-1 or PBS vehicle for 24 hours. Cells were also treated with B2B3-1 or PBS vehicle for 24 hours followed by 10 minutes of stimulation with 5nM HRG 1. beta. EGF domain. The cells were lysed and lysates were assessed for pErbB3 content by ELISA essentially as described above. Figure 12 shows that cells treated with B2B3-1 alone contain levels of phosphorylated ErbB3 comparable to those in untreated cells, suggesting that B2B3-1 does not act as an agonist promoting ErbB3 phosphorylation.

Example 13

The ability of B2B3-1 to specifically bind to ErbB2 and ErbB3 and not to the relevant ErbB family members EGFR and ErbB4 was studied by ELISA. The plates were coated with recombinant extracellular domains of ErbB2 or ErbB 3. The plates were blocked and incubated with B2B3-1 at half maximal binding concentration in the presence of serial dilutions of the competing recombinant extracellular domain of EGFR, ErbB2, ErbB3, or ErbB 4. The results showed that only soluble ErbB2 extracellular domain blocked B2B3-1 binding to ErbB2 coated plates (fig. 13A). Likewise, only soluble ErbB3 extracellular domain blocked B2B3-1 binding to ErbB3 coated plates (fig. 13B). These results are believed to demonstrate the specificity of anti-ErbB 2 arm B1D2 for ErbB2 and anti-ErbB 3 arm H3 for ErbB 3. The increased signal observed when the soluble ErbB2 ectodomain was incubated with B2B3-1 on ErbB3 coated plates was thought to be due to the formation of ErbB2, ErbB3, B2B3-1 complexes on the plates.

Example 14

The ability of B2B3-1 to bind to tumor cells expressing ErbB2 and ErbB3 was studied using monospecific variants of B3B 3-1. SKO-2(SEQ ID NO: 67) and SKO-3(SEQ ID NO: 68) are variants of B2B3-1 that lack the ability to interact with ErbB2 or ErbB3, respectively.

SKO-2 and SKO-3 were constructed using the QUIKCHANE Site directed mutagenesis kit (STRATAGENE) using oligonucleotide primer pairs each complementary to opposite strands of the template vector. These primers were extended during temperature cycling to generate mutant plasmids containing staggered nicks. After temperature cycling, the product was treated with DpnI, which was used to digest the parental DNA template. The nicked vector DNA containing the desired mutation was then transported into XL1-BLUE super competent cells (STRATAGENE) to amplify the plasmid DNA.

To prepare SKO-2, anti-ErbB 2scFv, 5 nucleotides in the VH CDR3 loop of B1D2 were mutated to generate the following amino acid substitutions; H95A, W100hA, and E100 jA. Mutations at these positions have been shown to knock out the B1D2 parent scFv, C6.5 binding to ErbB2 (Schier et al, 1996 JMB). Mutations were introduced into the B2B3-1 plasmid pMP9043(SEQ ID NO: 60) in a stepwise manner. First, mutations c295g and a296c were generated using primers 5'-GTA CTT TTG TGC CCG GGC CGA TGT GGG CTA CTG C-3' (SEQ ID NO: 61) and 5'-GCA GTA GCC CAC ATC GGC CCG GGCACA AAA GTA C-3' (SEQ ID NO: 62) and cycling at 95 ℃ for 1 minute, followed by 95 ℃ for 1 minute 30 times, 55 ℃ for 1 minute, and at 65 ℃ for 17.2 minutes. Mutations were confirmed by DNA sequencing of plasmid DNA. To introduce mutations at t334g, g335c, and a341c, a second round of site-directed mutagenesis was performed at c295g and a296c on a plasmid with a mutation of confirmed sequence using primers 5'-GAC ATG TGC CAA GGC CCC CGC GTG GCTGGG AGT G-3' (SEQ ID NO: 63) and 5'-CAC TCC CAG CCA CGCGGG GGC CTT GGC ACA TGT C-3' (SEQ ID NO: 64) and a temperature cycle of 95 ℃ for 30 seconds and 95 ℃ for 30 seconds 18 times, 55 ℃ for 1 minute and 68 ℃ for 17.2 minutes. The mutation was confirmed by DNA sequencing of the resulting plasmid DNA.

To generate SKO-3, the mutated B1D2scFv was subcloned into the original B2B3-1 plasmid in place of the anti-ErbB 3scFv, H3. The primers were annealed to SKO-2, the mutated B1D2scFv was isolated from SKO-2 using 5 'ACAGTGGCGGCCGCCACCATGGGCTGGTCTCTGATCCTGCTGTTCCTGGTGGCCGTGGCCACGCGTGTGCTGTCCCAGGTGCAGCTCGTCCAGAGCGGCGC (SEQ ID NO: 65) and 5' GGAGGCGGCGCCCAGGACTGTCAGCTTGGTGCCACCGCCG (SEQ ID NO: 66) and the Kas I and Not I restriction sites for subcloning were introduced into the Kas I/Not I restriction digested B2B3-1 plasmid. PCR was performed as follows: the mutant B1D2 was amplified by cycling 30 times at 94 ℃ for 1 minute followed by 94 ℃ for 30 seconds, 1 minute at 58 ℃,1 minute at 72 ℃, and once at 72 ℃ for 5 minutes. Sequential clones were monitored by DNA sequencing. The SKO-2 and SKO-3 plasmids were stably expressed from CHO-K1 in shake flasks or 10L WAVE bags and purified from the conditioned medium using Blue SEPHAROSE and cation exchange chromatography.

MALME-3M melanoma cells expressing approximately equal numbers of ErbB2 and ErbB3 receptors were incubated with serial dilutions of B2B3-1, SKO-2, or SKO-3 in the presence of saturating concentrations of goat anti-HSA Alexafluor-647 binding antibody. Cell binding was assessed by flow cytometry and the apparent binding affinity of each molecule was determined. Control cells were incubated with secondary antibody only. No measurable cellular binding of SKO-2 was observed, and SKO-2 retained only low affinity binding to ErbB3 mediated by H3 and lacked binding activity to ErbB 2. SKO-3, which retains functional, high affinity ErbB2 binding to B1D2scFv but lacks the ability to bind ErbB3, has a K of 6.7nM_D. B2B3-1 binding cell, K_DAt 0.2nM, indicating increased binding mediated by the bispecific design of this molecule (fig. 14).

Example 15

The stability of B2B3-1 under physiological conditions was assessed by incubation of 100nMB2B3-1 in human, cynomolgus, or mouse serum at 37 ℃ for 120 hours. Samples were taken at 0, 24, 48, 72, 96 and 120 hours and measured for the ability of B2B3-1 to bind ErbB2 and ErbB3 by ELISA. The ELISA described above involved coating 96-well plates with recombinant ErbB2 extracellular domain overnight, followed by a blocking step, followed by incubation with serial dilutions of B2B 3-1. The plates were then incubated with Fc-ErbB3 extracellular domain fusion protein followed by goat anti-human Fc-HRP conjugate. The plate was developed by adding a supersignal chemiluminescent substrate. Figures 15A-C show that B2B3-1 was still stable in serum from all three species under physiological conditions and retained comparable binding capacity for ErbB2 and ErbB3 at all time points measured.

Example 16: B2B3-1 dose response in an in vivo xenograft model of BT-474-M3 human breast cancer.

The in vivo efficacy of B2B3-1 was evaluated in nude mice carrying human BT-474-M3 xenografts. Each group of 10 mice was given B2B3-1 in an amount of 0.3, 1, 3,10, 30, or 90mg/kg 12 times every 3 days. Control groups were administered PBS carrier or HSA at equimolar doses relative to the 90mg/kg B2B3-1 dose. All doses were given intraperitoneally (i.p.). Tumor size was measured twice weekly and the corresponding tumor volume was calculated. The results indicate that B2B3-1 treatment resulted in a significant reduction in BT-474-M3 tumor volume compared to the control group (fig. 16). Complete regression was observed in each B2B3-1 treated group except mice treated with the lowest dose of B2B3-1(0.1 mg/kg).

Example 17

As shown in fig. 17A-E, B2B3-1 reduced tumor size in an ErbB 2-dependent manner in multiple xenograft models. In expressing ErbB2 > 1x10⁵Calu-3 (FIG. 17A), SKOV-3 (FIG. 17B), NCI-N87 (FIG. 17C), and MDA-MB-361 (FIG. 17E) xenograft models of recipients/cells B2B3-1 was effective, but expressed 4.5X10⁴The ErbB2 receptor/cell ACHN (figure 17D) xenograft model was less effective. Mice were treated every 3 days with 30mg/kg of B2B 3-1.

Example 18

Overexpression of ErbB2 transformed the B2B3-1 non-responder ADRr breast cancer xenograft model into responders to B2B3-1 (FIGS. 18A and 18B). ErbB2 was overexpressed in ADRr breast cancer cells using a retroviral expression system. Transfected cells expressing high levels of ErbB2 (ADRr-E2) were selected using FACS, followed by subcutaneous injection into nude mice to establish xenograft tumors. Mice were treated every 3 days with 30mg/kg of B2B 3-1. While no response to B2B3-1 was observed in wild-type ADRr xenografts (fig. 18A), ADRr-E2 xenografts (fig. 18B) responded to B2B 3-1.

Example 19

As shown in fig. 19A-B, B2B3-1 activity was positively correlated with levels of ErbB2 expression in vitro (fig. 19A) and in vivo (fig. 19B). Expression levels in ErbB2 were 5x10 as determined by ELISA⁴Receptor/cell to 2.4x10⁶Inhibition of phosphorylation of ErbB3 by B2B3-1 was determined in 9 tumor cell lines of receptors/cell. The extent of the ability of B2B3-1 to inhibit ErbB3 phosphorylation relative to basal levels (pErbB3 inhibition%) was found to be positively correlated with ErbB2 expression levels. Similarly, B2B3-1 activity was evaluated in 10 tumor xenograft models expressing low to high levels of ErbB 2. Xenograft responses were also positively correlated with levels of ErbB2 expression.

Example 20

B2B3-1 treatment of BT474-M3 breast tumor cells resulted in p27^kip1Translocated to the nucleus (FIG. 20A). BT474-M3 cells were treated with 1. mu.M of B2B3-1 for 6 hours. P27 was evaluated using immunofluorescence techniques^kip1The subcellular location of (a). In B2B3-1 treated cells, p27^kip1Translocation to the nucleus has been shown to result in inhibition of cell proliferation. p27^kip1Still in the cytoplasm of the untreated cells.

To further investigate the effect of B2B3-1 on cell cycle, cell cycle regulator cyclin D1 was examined for BT-474-M3 cells treated with B2B3-1 for 72 hours using Western blot analysis (FIG. 20B). The cytoskeletal protein neunin was used as a protein loading control in this experiment. Treatment with B2B3-1 resulted in a reduction in cyclin D1 levels compared to untreated cells.

Example 21

As shown in FIGS. 21A-B, B2B3-1 treatment of BT474-M3 breast tumor xenografts resulted in p27^kip1Translocate to the nucleus. BT474 breast tumor xenografts were treated with B2B3-1 at a dose of 30mg/kg (FIG. 21A) or with an equimolar dose of HSA (FIG. 21B) every 3 days for a total of 4 doses. P27 was observed in B2B3-1 treated tumors compared to HSA control tumors^kip1Indicating an antiproliferative effect of B2B3-1 in vivo.

Example 22

B2B3-1 treatment resulted in a decrease in the proliferation marker Ki67 in BT474 breast cancer xenograft tumors. BT474-M3 breast tumor xenografts were treated with B2B3-1 at a dose of 30mg/kg (FIG. 22A) or with an equimolar dose of HSA (FIG. 22B) every 3 days for a total of 4 doses. Subsequent Ki67 staining of tumor sections indicated that B2B3-1 treated tumors had a reduced expression pattern compared to HSA treated tumors.

Example 23

B2B3-1 treatment resulted in a decrease in vascular density in BT474-M3 breast cancer xenograft tumors as determined by measuring CD31 expression (fig. 23A-B). BT474 breast tumor xenografts were treated with B2B3-1 at a dose of 30mg/kg (FIG. 23A) or with an equimolar dose of HSA (FIG. 23B) every 3 days for a total of 4 doses. Tumor staining to check for the presence of the vascular marker CD 31. Tumors treated with B2B3-1 showed significantly reduced vascular density compared to control tumors treated with HSA.

Example 24: B2B3-1 inhibits phosphorylation of ErbB3 in vivo.

BT-474-M3 xenograft tumors were treated with 30mg/kg of B2B3-1 or 17.5mg/kg of HSA every 3 days for a total of 4 doses and tumors were harvested 24 hours after the last dose. Tumors were lysed and subjected to SDS-PAGE followed by protein analysis to assess the relative level of phosphorylation of target ErbB3 of B2B 3-1. Equal amounts of protein were loaded in each lane and total protein levels were controlled by examining beta tubulin. Western blot analysis with an antibody specific for phosphorylated ErbB3 showed that B2B3-1 treated tumors contained less pErbB3 than HSA treated tumors (fig. 24A). Densitometry of western blot analysis and subsequent normalization of the average pErbB3 integrated band intensity to the average beta tubulin integrated band intensity demonstrated that B2B3-1 treated tumors contained significantly less pErbB3 compared to control HSA treated tumors (fig. 24B). These data demonstrate that B2B3-1 inhibits phosphorylation of its target ErbB3 in vivo.

Example 25: in vivo activity of B2B3-1 in BT-474-M3 xenografts with reduced PTEN activity.

ShRNA technology was used to knock out the activity of the tumor suppressor gene phosphotriesterase and tensin homolog (PTEN) in BT-474-M3 breast cancer cells. Briefly, cultured BT-474-M3 cells were transfected with shPTEN or shControl RNA by retroviral transfection. Transfected cells with reduced PTEN were selected using FACS and then injected subcutaneously into the right flank of nude mice to establish xenograft tumors. Cells transfected with the control vector were injected into the left flank of the same mouse. Mice were treated with 30mg/kg of B2B3-1 every 3 days or 10mg/kg trastuzumab weekly. HSA was injected as a control at an equimolar dose relative to B2B 3-1. All were injected into the abdominal cavity.

B2B3-1 and trastuzumab promoted a reduction in the size of tumors formed by control BT-474-M3 breast cancer cells (fig. 25A), while B2B3-1 alone (but not trastuzumab) promoted a reduction in the size of tumors formed by BT-474-M3 human breast cancer cells lacking expression of PTEN (fig. 25B).

Example 26: B2B3-1 inhibits ErbB3 signaling in BT-474-M3 breast cancer cells with reduced PTEN activity.

The ability of B2B3-1 to inhibit phosphorylation of ErbB3 signals in tumor xenografts was investigated using the PTEN deficient BT-474-M3 model described above. Xenograft tumors of engineered or control cell lines were treated every 3 days with 30mg/kg B2B3-1, 17.5mg/kg HSA, or weekly with 10mg/kg trastuzumab, and tumors were harvested 24 hours after the last dose. Tumors were lysed and subjected to SDS-PAGE followed by protein analysis to assess the target ErbB3 of B2B3-1, the relative level of phosphorylation of AKT, and the total PTEN level. Equal amounts of protein were loaded in each lane and total protein levels were controlled by examining PCNA. Western blot analysis with an antibody specific for phosphorylated ErbB3 showed that B2B3-1 treated tumors contained less pAKT than HSA-treated or trastuzumab-treated tumors (fig. 26A). Densitometry of western blot analysis and subsequent normalization of the average pAKT integrated band intensity to the average PCNA integrated band intensity demonstrated that B2B3-1 treated tumors contained significantly less pAKT than control HSA-treated and trastuzumab-treated tumors (fig. 26B).

Example 27

Pharmacokinetic parameters of B2B3-1 were studied in nu/nu mice. Animals were randomized and given a single Intravenous (IV) administration of 5, 15, 30, or 45mg/kg of B2B3-1 (FIGS. 27A-D, respectively). Blood was collected before dosing and 0.5, 4,8, 24, 48, 72, and 120 hours post-dosing. Three mice were used for each time point. Serum levels of B2B3-1 were measured using two ELISA methods. The first method required functional binding of B2B3-1 to ErbB2 and ErbB3, while the second method only measured the HSA component of B2B3-1 in serum. The HSA ELISA employed a polyclonal anti-HSA capture antibody and an HRP-conjugated polyclonal anti-HSA detection antibody. A decrease in serum concentration of B2B3-1, measured using the ErbB2/ErbB3 binding method, compared to the HSA method would indicate a loss of functional B2B 3-1. FIGS. 27A-D show that the pharmacokinetic performance of B2B3-1 was comparable when evaluated using either ELISA method, indicating that B2B3-1 was stable in the circulation of mice.

Example 28

A two-compartment, two-exponential model was used to fit B2B3-1 serum concentrations and to show a biphasic profile. The final half-lives were calculated as 17, 16, 23, and 18 hours for the 5, 15, 30, or 45mg/kg doses, respectively, and are listed in table 4. An increase in the dose of B2B3-1 resulted in a linear increase in exposure (fig. 28).

Table 4: pharmacokinetic Properties of B2B3-1 in mice and cynomolgus monkeys

Example 29

Blood samples for pharmacokinetic analysis were also obtained from dose-range-finding toxicology studies performed on female cynomolgus monkeys. In this study, animals were infused with 4, 20, or 200mg/kg of B2B3-1 given every 3 days for a total of 4 doses. On each dosing day, (study days 1,4, 7, and 10) samples were taken before and 5 minutes after dosing to provide pre-dose and peak/trough concentrations, and samples were taken 1,2, 4,8, 24, and 48 hours after the end of the first infusion on day 1 and 1,2, 4,8, 24, 48, 72, and 120 hours after the last infusion on day 10. Serum samples were also collected 168, 336 and 456 hours after the last infusion for recovery animals dosed at 200 mg/kg.

Cynomolgus monkey serum samples were assayed using the previously described ErbB2/ErbB3ELISA method. Serum concentrations over time for each dose are shown in figure 29. The analysis showed that the mean concentration-time distribution of serum B2B3-1 after dose administration on day 1 and day 10 was qualitatively similar: the concentration generally decreases from a maximum concentration over time. The mean half-life estimates were 38.3-67.2 hours on day 1 and 45.0 to 121.0 hours on day 10 (table 4).

Example 30

Plasmids encoding the B2B3-1 bispecific scFv antibody fusion proteins were generated that incorporate unique gene sequences for human anti-ErbB 3scFv (designated "H3"), human anti-ErbB 2scFv (designated "B1D 2"), and a modified Human Serum Albumin (HSA) linker. The anti-ErbB 3scFv, H3, was recombinantly linked to the amino terminus of the HSA linker by means of a linker peptide (Ala-Ala-Ser), and the anti-ErbB 2scFv, B1D2, was genetically linked to the carboxy terminus of the HSA linker by means of a linker peptide (Ala-Ala-Ala-Leu-SEQ ID NO: 5). Peptide linkers are formed by introducing restriction sites during construction of mammalian expression vectors and are synthesized along with single chain antibody fragments and HSA linkers to codons preferentially used for mammalian expression.

The B1D2scFv was selected from a combinatorial phage display library generated by mutagenesis of ErbB2 binding to scFv C6.5, which was selected from a non-immune phage display library. The H3scFv was selected from a non-immune phage display library originally prepared by Sheets et al. The gene sequences encoding the B1D2 and H3 single chain antibody fragments were optimized according to CHO cell codon bias and synthesized for subsequent construction of the B2B3-1 encoding plasmid.

The modified HSA linker comprises two amino acid substitutions. The cysteine residue at position 34 was mutated to serine to reduce potential protein heterogeneity (due to oxidation at this site). The asparagine residue at amino acid 503 is mutated to glutamine, which is susceptible to deamination in wild-type HSA and results in a shortened pharmacological half-life.

The gene sequence encoding the modified HSA linker was synthesized with optimized codon usage for mammalian expression for subsequent construction of a B2B3-1 encoding plasmid.

Example 31

The B2B3-1 coding sequence was cloned into the pMP10k base vector using standard molecular biology techniques to generate the plasmid pMP10k4H3-mHSA-B1D2 shown in FIG. 30. For the most part, such constructs employ common genetic elements. B2B3-1 expression was driven by the human GAPD promoter. Such vectors employ genetic elements known as interstitial junction regions or MAR elements. MAR gene elements control the dynamic organization of chromatin and sequester nearby genes from the effects of surrounding chromatin, thereby increasing copy number-dependent, location-independent gene expression. MAR elements have been shown to improve the probability of isolating clones exhibiting a desired level of expression for the production of recombinant proteins, as well as to increase the stability of production. The MAR element used in the B2B3-1 construct was a non-coding human MAR element. In addition to the B2B3-1 plasmid, the neomycin antibiotic resistance plasmid (FIG. 31) and the hygromycin resistance plasmid (FIG. 32) were used to select stable transformants.

Example 32: first round of Gene transfection

Chinese hamster ovary CHO-K1 cells were purchased from ATCC (ATCC # CCL-61). The CHO-K1 cell line is a serum and proline dependent adherent subclone of the parental CHO cell line produced by t.t. Prior to transfection, CHO-K1 cells used for B2B3-1 transfection were previously adapted to growth in suspension in serum-free media. The transfection procedure was repeated to grow the B2B3-1 cell line. CHO-K1 cells were sub-passaged to 1.0X10 in SFM4CHO (serum free) medium (HyClone, Logan, UT) supplemented with 8mM L-glutamine, 0.1mM sodium hypoxanthine, and 0.016mM thymidine 24 hours prior to transfection⁶cells/mL. On the day of transfection, cells were resuspended in OptiMEM media (Invitrogen Corp, Carlsbad, CA) and 40,000 cells were placed in each well of a 24-well plate. In the first transfection, 75: 1(B2B 3-1: neomycin resistance) B2B3-1 expression plasmid (pMP10K4H3-mHSA-B1D2) and neomycin resistance plasmid (FIG. 30; pSV2-neo (Selexis, Inc., Marlborough, Mass.) were mixed together the plasmid mixture was then mixed with cationic Lipofectamine LTX, Invitrogen Corp, Carlsbad, CA) and the lipid/DNA complex was allowed to form for 30 minutes, then DNA/lipid complex was added to CHO-K1 cells and the 24-well plates were placed at 37 ℃ with 5% CO₂In a thermostat of (1).

Example 33: high Producer selection and screening

The contents of each transfection well were washed with PBS, trypsinized and distributed in two, 96-well plates. The growth medium used included DMEM/F12(Invitrogen Corp, Carlsbad, CA) containing 10% FBS (Invitrogen Corp, Carlsbad, CA) and 500mg/L neomycin (G418; Invitrogen Corp, Carlsbad, CA). The medium in the 96-well plate was changed to SFM4CHO medium supplemented with 8mM L-glutamine, 0.1mM sodium hypoxanthine, 0.016mM thymidine, and 500mg/L neomycin on day 4. After two more weeks of culture in selection medium, surviving cells had formed well defined colonies. Clones were evaluated using quantitative dot blot techniques. The top-generated colonies were trypsinized and spread to a single well of a 24-well plate.

The 7 day productivity assay was used to screen high B2B3-1 producing colonies. After expansion, cells in 24-well plates can be propagated in SFM4CHO medium supplemented with 8mM L-glutamine, 0.1mM sodium hypoxanthine, and 0.016mM thymidine for 7 days. The concentration of B2B3-1 in the spent medium was determined. Top clones from the 24-well scale were expanded to 125mL baffled shake flasks. A7 day study in SFM4CHO medium supplemented with 8mM L-glutamine, 0.1mM sodium hypoxanthine, and 0.016mM thymidine in shake flasks was used to screen cell populations for growth and B2B3-1 production.

Example 34: second round of Gene transfection

The highest producing cell population determined from the first round of transfection (above) was transfected a second time to increase production. 24 hours prior to transfection, the cell population was sub-passaged to 1.0X10 in SFM4CHO (serum-free) medium supplemented with 8mM L-glutamine, 0.1mM sodium hypoxanthine, and 0.016mM thymidine⁶cells/mL. On the day of transfection, cells were resuspended in OptiMEM medium (Invitrogen Corp, Carlsbad, CA) and 40,000 cells were placed in each well of a 24-well plate. In the first transfection, the ratio between 50: 1(B2B 3-1: hygromycin resistance) B2B3-1 and the hygromycin resistance plasmid (FIG. 32; pTK-Hyg (Clontech, Mountain View, CA)) were mixed together. The plasmid mixture was then mixed with a cationic lipofection reagent (Lipofectamine LTX, Invitrogen Corp) and the lipid/DNA complex allowed to form for 30 minutes. The DNA/lipid complexes were then added to the cell population and the 24-well plates were placed at 37 ℃ in 5% CO₂In a thermostat of (1).

Example 35: selection and screening of high Producers from second transfection

The contents of each transfection well were washed with PBS, trypsinized, and distributed to two 96-well plates. The growth medium used included DMEM/F12 supplemented with 10% FBS and 400mg/L hygromycin B (Invitrogen Corp). On day 4, the medium in the 96-well plate was changed to HycloneSFM4CHO medium supplemented with 8mM L-glutamine, 0.1mM sodium hypoxanthine, 0.016mM thymidine, and 400mg/L hygromycin B. After two weeks of continued selection, surviving cells had formed well defined colonies. Clones were evaluated using quantitative dot blot techniques. The top-generated colonies were trypsinized and spread to a single well of a 24-well plate.

The 7 day productivity assay was used to screen high B2B3-1 producing colonies. After expansion, the cells were allowed to proliferate for 7 days, and then the concentration of B2B3-1 in the spent medium was determined.

Top clones from 24-well plates were expanded into 125mL baffled shake flasks containing Hyclone SFM4CHO medium supplemented with 8mM L-glutamine, 0.1mM sodium hypoxanthine, and 0.016mM thymidine. A7-day study in shake flasks was used to screen the cell population for growth and B2B3-1 production. The spent media was quantified using a Protein Aresin and HPLC instrument.

Example 36: limiting dilution cloning

Colonies identified by the productivity assay as best growing and highest B2B3-1 yield were transferred from 125mL shake flasks and plated in 596 well plates at the cell concentration used to generate one cell/well. Place 96-well plate at 37 ℃ and 5% CO₂In a thermostat of (1). The wells were examined every two weeks to track the formation of colonies. Based on the symmetric shape of the colonies, colonies from single cells were identified. Wells containing the above colonies were labeled for further screening by 24 well 7 day evaluation, and 125mL shake flask 7 day evaluation.

A second round of limiting dilution cloning was performed in a similar manner to the first round. An additional 100mL of the supplied material was evaluated to confirm clonal selection. The pre-inoculation chamber is refrigerated.

Example 37: preparation of B2B3-1

B2B3-1 was administered once a week via intravenous infusion for 60 or 90 minutes depending on the patient's tolerance.

B2B3-1 can be formulated in a sterile 20mM L-histidine hydrochloride, 150mM sodium chloride, pH6.5 solution at a concentration of 25mg/mL for administration to a patient (e.g., a human).

Example 38: treatment of breast cancer

If the patient's cancer is considered to express high levels of epidermal growth factor receptor, including ErbB2(HER2/neu), then treated with an HSA linker that binds to an ErbB binding moiety, B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9, or B3B3-10 are shown, for example (see Table 6 below). This is also the case when genotypic or histological screening of cancer biopsies reveals increased expression of ErbB2 in patient tumors.

The B2B3HSA linker conjugate (e.g., B2B3-1, SEQ ID NO: 6) can be administered to a patient diagnosed with breast cancer at a dose of no greater than 30mg/kg by intravenous infusion once a week or twice a week for a period of time, e.g., 60 or 90 minutes, depending on the patient's tolerance. The B2B3HSA linker conjugate can be formulated in a sterile 20mM L-histidine hydrochloride, 150mM sodium chloride, pH6.5 solution at a concentration of 25mg/mL for administration to a patient. The clinician supervising the administration of the B2B3HSA linker conjugate follows normal formulation and administration practices to determine the appropriate course of treatment for the patient.

The clinician may also administer one or more therapeutic agents in combination with the B2B3HSA linker conjugate. For example, one or more therapeutic drugs or compounds may be administered in combination with a B2B3HSA linker conjugate, such as the usual chemotherapeutic regimen for the treatment of breast cancer, including doxorubicin, cyclophosphamide, and paclitaxel. Alternatively, a clinician may administer the B2B3HSA linker conjugate in conjunction with surgery or radiation therapy to treat breast cancer in a patient in need thereof.

EXAMPLE 39 treatment of ovarian cancer

If the patient's cancer is considered to exhibit high levels of epidermal growth factor receptors, including ErbB2(HER2/neu), treatment with an HSA linker linked to a selected portion of ErbB2 (biding motif), such as B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9, or B3B3-10 listed (see Table 6 below). This is also the case when genotypic or histological screening of cancer biopsies reveals increased expression of ErbB2 in patient tumors.

The B2B3HSA linker conjugate (e.g., B2B3-1, SEQ ID NO: 16) is administered alone or in combination with one or more other therapeutic agents substantially as described in the previous example to a patient diagnosed with ovarian cancer.

Example 40: other HSA linker conjugates

HSA linker conjugates can be constructed using one or more of the components (groups a-E) listed in table 5 below. In particular, HSA linker conjugates, shown as group C in table 5 below, bind to one or more binding moieties selected from groups a and E shown in table 5. In addition, the HSA linker conjugate can further comprise one or more peptide connectors at the amino or carboxy terminus of the HSA linker selected from groups B and D in table 5. The peptide linker may be repeated or truncated to increase or decrease the length of the linker sequence.

Example 41: in vivo administration of q7d to B2B3-1 showed equivalent efficacy to that of q3d to B2B3-1

B2B3-1 efficacy using a q7d (once every 7 days) dose regimen was determined in 5-6 week old female athymic nude mice (nu/nu) from Charles River Labs, with xenograft tumors of the human breast cancer cell line BT-474-M3 (fig. 33). Mice were injected with 20X 10 in PBS⁶Human BT-474-M3 cells received a subcutaneous estrogen-releasing transplant in the opposite flank 24 hours prior to implantation (0.72mg pellet, 60 days, slow release, Innovative Research of America, Sarasota, FL). When the tumor starts to grow (tumor volume is about 400 mm)³) Dosing was started and B2B3-1 was administered to 10 mice per group by intraperitoneal injection over a study period of 30mg/kg once every 3 days (q3d) or 22mg/kg, 66mg/kg, 132mg/kg or 198mg/kg once every 7 days. Tumors were measured bilaterally weekly using a digital caliper. Utilize the followingTumor volume was calculated by formula: pi/6 (W)²X L) where W is the short diameter and L is the long diameter. Pharmacokinetic calculations indicate that 66mg/kg of q7d administered should give a similar B2B3-1 exposure as a xenograft tumor administered 30mg/kg of q3d administered. PBS vector was used as negative control. The three highest doses of B2B3-1, B2B3-1, given at 30mg/kg for q3d and q7d were equally efficacious, indicating that the q7d dosing regimen for B2B3-1 was appropriate in this model.

Example 42: B2B3-1 and trastuzumab have different ErbB inhibition mechanisms

The ability of B2B3-1 to inhibit nerve growth factor inducing ErbB3 activity was tested using western blot analysis. Serum-free BT-474-M3 cell monolayers were treated with 100nM B2B3-1 or trastuzumab for 24 h, followed by 10 min stimulation with 5nM HRG 1. beta. EGF. Cells were then treated with trastuzumab at 10nM and 100nM B2B3-1 or 10nM and 100nM and left unstimulated. Lysates were subjected to immunoblot analysis for ErbB3, pErbB3, AKT, and pAKT. Western blot analysis (fig. 34) showed that B2B3-1 treatment resulted in inhibition of pErbB3 and pAKT in a ligand-dependent manner, whereas inhibition of pErbB3 and pAKT was only observed with trastuzumab in the absence of ligand.

Example 43: B2B3-1 has additional effects when administered in vitro with trastuzumab

The effect of B2B3-1, trastuzumab, and the combination of the two drugs on cancer cell spheroid growth was studied using four different breast cancer cell lines. 2000 human breast cancer cells of BT-474-M3, SKBR3(ATCC), or MDA-MB-361(ATCC) were seeded in round bottom low adhesion 96well plates (96Well clean round Bottom Ultra Low Attachment Microplate-product #7007) and the spheroids were measured and treated the next day with the dose range of B2B3-1, trastuzumab, and the combination of both at a ratio of B2B3-1 to a 3-fold molar excess of trastuzumab. After 12 days of growth, the surface area of spheroids was measured and compared with untreated cellsA comparison is made. As shown in fig. 35A-C, the combination of B2B3-1 with trastuzumab resulted in greater inhibition of spheroid growth than the single agent over a range of concentrations in all cell lines tested, with the lowest concentration of drug. These results also indicate that B2B3-1 does not compete with trastuzumab for binding to ErbB2(HER 2).

Example 44: B2B3-1 has an additive effect when administered in vivo with trastuzumab

The effect of B2B3-1 when administered in vivo with trastuzumab was studied in 5-6 week old female athymic nude mice (nu/nu) from Charles River Labs using the BT-474-M3 xenograft model. 20X 10 in injected PBS⁶Mice received estrogen-releasing grafts (0.72mg pellets, 60 days, slow release, Innovative Research of america, Sarasota, FL) subcutaneously in the contralateral flank 24 hours prior to human BT-474-M3 cells. When tumor growth was established (tumor volume approximately 400 mm)³) Administration is initiated. Tumors were measured twice weekly using a digital caliper. Tumor volume was calculated using the following formula: pi/6 x (W)²x L) where W is the short diameter and L is the long diameter. During the study, 10 mice/group were given B2B3-1 at 3mg/kg or 10mg/kg q3d, trastuzumab at 1mg/kg or 0.1mg/kgq7d, or a combination of both drugs by intraperitoneal injection. All combinations of B2B3-1 and trastuzumab were administered for the corresponding single agents (10mg/kg B2B3-1+1mg/kg trastuzumab, 10mg/kgB2B3-1+0.1mg/kg trastuzumab, 3mg/kg B2B3-1+1mg/kg trastuzumab, 3mg/kg B2B3-1+0.1mg/kg trastuzumab).

As shown in figure 36, significantly greater efficacy was observed for all combinations compared to the single agent, and significant efficacy was observed for at least 20 days prior to administration of 10mg/kg B2B3-1 and the combination of trastuzumab at the same time with 3mg/kg B2B3-1 and 1mg/kg trastuzumab. In the 10mg/kg B2B3-1+1mg/kg trastuzumab combination group, 5 of 10 mice had completely regressed tumors, in contrast to 10 mice in the single agent group given the same dose as in the combination, which did not have completely regressed tumors. In the 3mg/kg B2B3-1+1mg/kg trastuzumab combination group, 7 of 10 mice had completely regressed tumors, in contrast to 10 mice in the single agent group given an equivalent dose to the combination, which did not have completely regressed tumors. These results indicate that B2B3-1 does not compete with trastuzumab for binding to ErbB2(HER 2). These results also indicate that treatment with a combination of at least 3mg/kg B2B3-1 and at least 0.1mg/kg trastuzumab is more effective than treatment with 3mg/kg or 10mg/kg B2B3-1 alone or 0.1mg/kg or 1mg/kg trastuzumab alone. In particular, the combination of at least 3mg/kgB2B3-1 and 1mg/kg trastuzumab induced substantially complete tumor regression in at least about 50% of nude mice bearing human breast cancer cell xenografts, whereas administration of the same concentration of B2B3-1 or trastuzumab alone failed to provide 10% complete regression in such mice.

Specific embodiments of the HSA linkers, peptide connectors, and binding moieties described above are also provided. Table 6 below lists 10 HSA linker conjugates with different ErbB 2-specific or ErbB 3-specific binding moieties at the amino and carboxy termini of the HSA linker, as well as peptide connectors.

Those skilled in the art will recognize, and be able to ascertain and practice, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the dependent claims is included within the scope of the disclosure herein.

The disclosure of each of the U.S. and foreign patents and pending patent applications and publications cited herein is hereby incorporated by reference in its entirety.

TABLE 6

Appendix 1

Sequences, sequence annotations, and sequence alignments

SEQ ID NO：60

pMP9043

gtgccgacgatagagcagacctcgctaaatatatctgcgagaatcaggattccattagctctaagctga

aagaatgttgcgagaagcccctcctggaaaagagtcattgtatcgccgaggtggaaaacgacgagat

gccagcagatctgccatcactcgctgccgactttgtggaatccaaagatgtctgcaagaattacgcag

aggctaaagacgtgttcctggggatgtttctgtatgagtacgcccggcgtcaccccgattatagcgtcg

tgctcctgctccgactggcaaagacctacgaaacaactctggagaaatgttgcgctgccgcagaccc

tcatgaatgttatgctaaggtgttcgatgagtttaagccactcgtcgaagagccccagaacctgattaaa

cagaattgcgaactgttcgagcagctcggtgaatacaagtttcagaacgccctgctcgtgcgttatacc

aaaaaggtccctcaggtgtctacaccaactctggtggaggtcagtaggaatctgggcaaagtgggat

caaagtgttgcaaacaccccgaggcaaagagaatgccttgtgctgaagattacctctccgtcgtgctg

aaccagctctgcgtgctgcatgaaaagaccccagtcagcgatcgggtgacaaaatgttgcaccgaat

ctctggtcaatcgccgaccctgtttcagtgccctcgaagtggacgaaacttatgtgcctaaggagtttca

ggctgaaacattcacctttcacgccgatatctgcactctgtccgagaaagaaaggcagattaagaaac

agacagcactggtcgagctcgtgaagcataaaccaaaggctaccaaggagcagctgaaagccgtc

atggacgatttcgcagcttttgtggaaaagtgttgcaaagccgacgataaggagacttgtttcgcagaa

gaggggaaaaagctcgtggctgccagccaggcagctctgggtctggccgcagctctgcaggtgca

gctcgtccagagcggcgctgaggtgaagaagccaggcgagtccctgaagatctcctgtaagggctc

cggctacagcttcacctcctactggatcgcttgggtgaggcagatgccaggaaagggactggagtac

atgggcctgatctaccctggcgactccgacaccaagtactccccatccttccagggccaggtgaccat

cagcgtggacaagtccgtgtctaccgcctacctgcaatggtcctccctgaagccttctgactctgccgt

gtacttttgtgcccggcacgatgtgggctactgcaccgaccggacatgtgccaagtggcccgagtgg

ctgggagtgtggggacagggaacactggtgacagtgagttctggcggtggcggctcttccggcggt

ggctctggtggcggcggatctcagagcgtgctgacacagccacctagcgtgtccgctgcccctggc

cagaaggtgacaatcagctgctccggcagctcttccaacatcggcaacaactacgtgtcttggtatca

gcagctgcccggaacagctccaaaactgctgatctatgaccacaccaatcggcctgccggcgtgcc

agatcggttctctggctctaagagcggcacctccgccagcctggctatctctggcttcagatctgagga

tgaggctgactactattgtgcctcctgggactacaccctgtctggctgggtgttcggcggtggcaccaa

gctgacagtcctgggatgatgactcgagtctagagggcccgtttaaacccgctgatcagcctcgactg

tgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccact

cccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctgggg

ggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggat

gcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcg

ccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgcc

agcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtca

agctctaaatcgggggtccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttg

attagggtgatggttcacgtacctagaagttcctattccgaagttcctattctctagaaagtataggaactt

ccttggccaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagc

gtctccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcg

tggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttg

catcggccgcgctcccgattccggaagtgcttgacattggggaattcagcgagagcctgacctattgc

atctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgca

gccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcc

cattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatcccc

atgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgag

ctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaaca

atgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattc

ccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgct

acttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattg

gtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgat

gcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgc

ggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcg

tccgagggcaaaggaatagcacgtactacgagatttcgattccaccgccgccttctatgaaaggttgg

gcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttc

ttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcac

aaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtat

accgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgc

tcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgag

ctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcat

taatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcac

tgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacgg

ttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccag

gaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaa

aatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctg

gaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttc

gggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa

gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttg

agtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagag

cgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggac

agtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggc

aaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagg

atctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaaggg

attttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaat

ctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcg

atctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggctt

accatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagca

ataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagt

ctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccatt

gctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaa

ggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtca

gaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgcca

tccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgac

cgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctc

atcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgt

aacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaac

aggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttc

ctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaa

aataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcggga

gatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatct

gctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaag

gcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacggg

ccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatag

cccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacc

cccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtca

atgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcc

ccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggacttt

cctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaat

gggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttg

ttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggc

ggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgctta

ctggcttatcgaaattaatacgactcactatagggagacccaagcttctagaattcgctgtctgcgagg

gccagctgttggggtgagtactccctctcaaaagcgggcatgacttctgcgctaagattgtcagtttcc

aaaaacgaggaggatttgatattcacctggcccgcggtgatgcctttgagggtggccgcgtccatctg

gtcagaaaagacaatctttttgttgtcaagcttgaggtgtggcaggcttgagatctggccatacacttga

gtgacaatgacatccactttgcctttctctccacaggtgtccactcccaggtccaactgcagatatccag

cacagtggcggccgccaccatgggctggtctctgatcctgctgttcctggtggccgtggccacgcgt

gtgctgtcccaggtgcagctgcaggagtctggcggcggactggtgaagcctggcggctccctgcgg

ctgtcctgcgccgcctccggcttcaccttctcctcctactggatgtcctgggtgcggcaggcccctggc

aagggcctggagtgggtggccaacatcaaccgggacggctccgcctcctactacgtggactccgtg

aagggccggttcaccatctcccgggacgacgccaagaactccctgtacctgcagatgaactccctgc

gggccgaggacaccgccgtgtactactgcgccagggaccggggcgtgggctacttcgacctgtgg

ggcaggggcaccctggtgaccgtgtcctccgctagtactggcggcggaggatctggcggaggagg

gagcgggggcggtggatcccagtccgccctgacccagcctgcctccgtgtccggctcccctggcca

gtccatcaccatcagctgcaccggcacctcctccgacgtgggcggctacaacttcgtgtcctggtatc

agcagcaccccggcaaggcccctaagctgatgatctacgacgtgtccgaccggccttccggcgtgt

ccgacaggttctccggctccaagtccggcaacaccgcctccctgatcatcagcggcctgcaggcag

acgacgaggccgactactactgctcctcctacggctcctcctccacccacgtgatctttggcggcgga

acaaaggtgaccgtgctgggcgccgcctccgacgctcacaagagcgaagtggcacataggttcaa

agatctgggcgaagagaactttaaggccctcgtcctgatcgctttcgcacagtacctccagcagtctcc

ctttgaagatcacgtgaaactggtcaatgaggtgaccgaatttgccaagacatgcgtggctgatgaga

gtgcagaaaactgtgacaaatcactgcatactctctttggagataagctgtgcaccgtcgccacactca

gagagacttatggggaaatggctgactgttgcgcaaaacaggagcctgaacggaatgagtgtttcct

ccagcacaaggatgacaacccaaatctgccccgcctcgtgcgacctgaggtcgatgtgatgtgcacc

gcctttcatgacaacgaagagacattcctgaagaaatacctgtatgaaattgctcgtaggcacccatac

ttttatgcccccgagctcctgttctttgcaaagagatacaaagctgccttcactgaatgttgccaggcag

ctgataaggccgcatgtctcctgcctaaactggacgagctccgggatgaaggtaaggcttccagcgc

caaacagcgcctgaagtgcgcttctctccagaagtttggcgagcgagcattcaaagcctgggctgtg

gcccgtctcagtcagaggtttccaaaggcagaatttgctgaggtctcaaaactggtgaccgacctcac

aaaggtccatactgagtgttgccacggagatctgctggaat

SEQ ID NO：67

QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGK

GLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLK

PSDSAVYFCARADVGYCTDRTCAKAPAWLGVWGQGTLVTVSS

GGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIG

NNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASL

AISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLGAASDAH

KSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVT

EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC

CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEET

FLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAAC

LLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLS

QRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYI

CENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADF

VESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKT

YETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE

QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCK

HPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLV

NRRPCFSALEVDETYVPKEFQAETFTFHADICTLSEKERQIKKQT

ALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAE

EGKKLVAASQAALGLAAALQVQLVQSGAEVKKPGESLKISCKG

SGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQ

VTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAK

WPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPS

VSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHT

NRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLS

GWVFGGGTKLTVLG

SEQ ID NO：68

QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPG

KGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNS

LRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSASTGGGGSGG

GGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSW

YQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQA

DDEADYYCSSYGSSSTHVIFGGGTKVTVLGAASDAHKSEVAHR

FKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCV

ADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPE

RNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLY

EIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDE

LRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEF

AEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS

SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVC

KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK

CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKF

QNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRM

PCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA

LEVDETYVPKEFQAETFTFHADICTLSEKERQIKKQTALVELVKH

KPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAA

SQAALGLAAALQVQLVQSGAEVKKPGESLKISCKGSGYSFTSY

WIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKS

VSTAYLQWSSLKPSDSAVYFCARADVGYCTDRTCAKAPAWLGV

WGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQK

VTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDR

FSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGT

KLTVLG

B2B3-1(H3-mHSA-B1D2)

1 QVQLQESGGG LVKPGGSLRL SCAASGFTFS WVRQA PGKGLEWVA

51RFTI SRDDAKNSLY LQMNSLRAED TAVYYCAR

101WGR GTLVTVSSAS TGGGGSGGGG SGGGGSQSAL TQPASVSGSP

151 GQSITISC WYQQHPGK APKLMIYDVS GVSDRF

201 SGSKSGNTAS LIISGLQADD EADYYC FGG GTKVTVLGAA

251 SDAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQSPFEDH VKLVNEVTEF

301 AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERN

351 ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF

401 YAPELLFFAK RYKAAFTECC QAADKAACLL PKLDELRDEG KASSAKQRLK

451 CASLQKFGER AFKAWAVARL SQRFPKAEFA EVSKLVTDLT KVHTECCHGD

501 LLECADDRAD LAKYICENQD SISSKLKECC EKPLLEKSHC IAEVENDEMP

551 ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHP DYSVVLLLRL

601 AKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLG

651 EYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA

701 EDYLSVVLNQ LCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP

751 KEFQAETFTF HADICTLSEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD

801 DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGIQVQLVQS GAE

851 VKKPGESLKI SCKGSGYSFT WVRQM PGKGLEYMG

901QVTI SVDKSVSTAY LQWSSLKPSD SAVYFCAR

951WG QGTLVTVSSG GGGSSGGGSG GGGSQSVLTQ PPSVSAAPGQ

1001 KVTISC W YQQLPGTAPK LLIY GVPDRFSGS

1051 KSGTSASLAI SGFRSEDEAD YYC FGGGTK LTVLG

CDR loops are highlighted in H3 (underlined 1-248 with CDRs in bold slant) and B1D2 (underlined 841-1095 with CDRs in bold slant). The linker modifying HSA is underlined.

Alignment of various HAS linker conjugates comprising B2B3 and mHSA

1 45

A5-mHSA-ML3.9 (1)QVQLVQSGGGLVKPGGSLRLSCAASGFSFNTYDMNWVRQAPGKGL

A5-mHSA-B1D2 (1)QVQLVQSGGGLVKPGGSLRLSCAASGFSFNTYDMNWVRQAPGKGL

A5-mHSA-F5B6H2 (1)QVQLVQSGGGLVKPGGSLRLSCAASGFSFNTYDMNWVRQAPGKGL

B12-mHSA-B1D2 (1)QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGL

B12-mHSA-F5B6H2 (1)QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGL

F4-mHSA-B1D2 (1)QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL

F4-mHSA-F5B6H2 (1)QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL

H3-mHSA-B1D2 (1)QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGL

H3-mHSA-F5B6H2 (1)QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGL

46 90

A5-mHSA-ML3.9 (46)EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED

A5-mHSA-B1D2 (46)EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED

A5-mHSA-F5B6H2 (46)EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED

B12-mHSA-B1D2 (46)EWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRPED

B12-mHSA-F5B6H2 (46)EWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRPED

F4-mHSA-B1D2 (46)EWVSTISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED

F4-mHSA-F5B6H2 (46)EWVSTISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED

H3-mHSA-B1D2 (46)EWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAED

H3-mHSA-F5B6H2 (46)EWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAED

91 135

A5-mHSA-ML3.9 (91)TAVYYCARDG---VATTPFDYWGQGTLVTVS---SGGGGSGGGGS

A5-mHSA-B1D2 (91)TAVYYCARDG---VATTPFDYWGQGTLVTVS---SGGGGSGGGGS

A5-mHSA-F5B6H2 (91)TAVYYCARDG---VATTPFDYWGQGTLVTVS---SGGGGSGGGGS

B12-mHSA-B1D2 (91)TAVYYCARDLGAKQWLEGFDYWGQGTLVTVSSASTGGGGSGGGGS

B12-mHSA-F5B6H2 (91)TAVYYCARDLGAKQWLEGFDYWGQGTLVTVSSASTGGGGSGGGGS

F4-mHSA-B1D2 (91)TAVYYCAKGYSSSWSEVASGYWGQGTLVTVSSASTGGGGSGGGGS

F4-mHSA-F5B6H2 (91)TAVYYCAKGYSSSWSEVASGYWGQGTLVTVSSASTGGGGSGGGGS

H3-mHSA-B1D2 (91)TAVYYCARDR----GVGYFDLWGRGTLVTVSSASTGGGGSGGGGS

H3-mHSA-F5B6H2 (91)TAVYYCARDR----GVGYFDLWGRGTLVTVSSASTGGGGSGGGGS

136 180

A5-mHSA-ML3.9 (130)GGGGSQSVLTQPPS-VSGAPGQRVTISCTGSSSNIGAGYDVHWYQ

A5-mHSA-B1D2 (130)GGGGSQSVLTQPPS-VSGAPGQRVTISCTGSSSNIGAGYDVHWYQ

A5-mHSA-F5B6H2 (130)GGGGSQSVLTQPPS-VSGAPGQRVTISCTGSSSNIGAGYDVHWYQ

B12-mHSA-B1D2 (136)GGGGSSYELTQDPA-VSVALGQTVRITCQGDSLRS---YYASWYQ

B12-mHSA-F5B6H2 (136)GGGGSSYELTQDPA-VSVALGQTVRITCQGDSLRS---YYASWYQ

F4-mHSA-B1D2 (136)GGGGSAIVMTQSPSSLSASVGDRVTITCRASQGIR---NDLGWYQ

F4-mHSA-F5B6H2 (136)GGGGSAIVMTQSPSSLSASVGDRVTITCRASQGIR---NDLGWYQ

H3-mHSA-B1D2 (132)GGGGSQSALTQPAS-VSGSPGQSITISCTGTSSDVGGYNFVSWYQ

H3-mHSA-F5B6H2 (132)GGGGSQSALTQPAS-VSGSPGQSITISCTGTSSDVGGYNFVSWYQ

181 225

A5-mHSA-ML3.9 (174)QLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAED

A5-mHSA-B1D2 (174)QLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAED

A5-mHSA-F5B6H2 (174)QLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAED

B12-mHSA-B1D2 (177)QKPGQAPVLVIYGKNNRPSGIPDRFSGSTSGNSASLTITGAQAED

B12-mHSA-F5B6H2 (177)QKPGQAPVLVIYGKNNRPSGIPDRFSGSTSGNSASLTITGAQAED

F4-mHSA-B1D2 (178)QKAGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPDD

F4-mHSA-F5B6H2 (178)QKAGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPDD

H3-mHSA-B1D2 (176)QHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADD

H3-mHSA-F5B6H2 (176)QHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADD

226 270

A5-mHSA-ML3.9 (219)EADYYCQSYDSS-LSALFGGGTKLTVLG-AASDAHKSEVAHRFKD

A5-mHSA-B1D2 (219)EADYYCQSYDSS-LSALFGGGTKLTVLG-AASDAHKSEVAHRFKD

A5-mHSA-F5B6H2 (219)EADYYCQSYDSS-LSALFGGGTKLTVLG-AASDAHKSEVAHRFKD

B12-mHSA-B1D2 (222)EADYYCNSRDSSGNHWVFGGGTKVTVLG-AASDAHKSEVAHRFKD

B12-mHSA-F5B6H2 (222)EADYYCNSRDSSGNHWVFGGGTKVTVLG-AASDAHKSEVAHRFKD

F4-mHSA-B1D2 (223)FATYFCQQAHSF--PPTFGGGTKVEIKRGAASDAHKSEVAHRFKD

F4-mHSA-F5B6H2 (223)FATYFCQQAHSF--PPTFGGGTKVEIKRGAASDAHKSEVAHRFKD

H3-mHSA-B1D2 (221)EADYYCSSYGSSSTHVIFGGGTKVTVLG-AASDAHKSEVAHRFKD

H3-mHSA-F5B6H2 (221)EADYYCSSYGSSSTHVIFGGGTKVTVLG-AASDAHKSEVAHRFKD

271 315

A5-mHSA-ML3.9 (262)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES

A5-mHSA-B1D2 (262)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES

A5-mHSA-F5B6H2 (262)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES

B12-mHSA-B1D2 (266)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES

B12-mHSA-F5B6H2 (266)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES

F4-mHSA-B1D2 (266)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES

F4-mHSA-F5B6H2 (266)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES

H3-mHSA-B1D2 (265)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES

H3-mHSA-F5B6H2 (265)LGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADES

316 360

A5-mHSA-ML3.9 (307)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

A5-mHSA-B1D2 (307)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

A5-mHSA-F5B6H2 (307)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

B12-mHSA-B1D2 (311)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

B12-mHSA-F5B6H2 (311)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

F4-mHSA-B1D2 (311)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

F4-mHSA-F5B6H2 (311)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

H3-mHSA-B1D2 (310)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

H3-mHSA-F5B6H2 (310)AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

361 405

A5-mHSA-ML3.9 (352)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY

A5-mHSA-B1D2 (352)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY

A5-mHSA-F5B6H2 (352)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY

B12-mHSA-B1D2 (356)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY

B12-mHSA-F5B6H2 (356)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY

F4-mHSA-B1D2 (356)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY

F4-mHSA-F5B6H2 (356)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY

H3-mHSA-B1D2 (355)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY

H3-mHSA-F5B6H2 (355)QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY

406 450

A5-mHSA-ML3.9 (397)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS

A5-mHSA-B1D2 (397)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS

A5-mHSA-F5B6H2 (397)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS

B12-mHSA-B1D2 (401)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS

B12-mHSA-F5B6H2 (401)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS

F4-mHSA-B1D2 (401)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS

F4-mHSA-F5B6H2 (401)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS

H3-mHSA-B1D2 (400)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS

H3-mHSA-F5B6H2 (400)FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS

451 495

A5-mHSA-ML3.9 (442)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL

A5-mHSA-B1D2 (442)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL

A5-mHSA-F5B6H2 (442)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL

B12-mHSA-B1D2 (446)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL

B12-mHSA-F5B6H2 (446)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL

F4-mHSA-B1D2 (446)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL

F4-mHSA-F5B6H2 (446)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL

H3-mHSA-B1D2 (445)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL

H3-mHSA-F5B6H2 (445)AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL

496 540

A5-mHSA-ML3.9 (487)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL

A5-mHSA-B1D2 (487)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL

A5-mHSA-F5B6H2 (487)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL

B12-mHSA-B1D2 (491)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL

B12-mHSA-F5B6H2 (491)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL

F4-mHSA-B1D2 (491)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL

F4-mHSA-F5B6H2 (491)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL

H3-mHSA-B1D2 (490)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL

H3-mHSA-F5B6H2 (490)TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL

541 585

A5-mHSA-ML3.9 (532)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG

A5-mHSA-B1D2 (532)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG

A5-mHSA-F5B6H2 (532)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG

B12-mHSA-B1D2 (536)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG

B12-mHSA-F5B6H2 (536)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG

F4-mHSA-B1D2 (536)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG

F4-mHSA-F5B6H2 (536)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG

H3-mHSA-B1D2 (535)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG

H3-mHSA-F5B6H2 (535)LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG

586 630

A5-mHSA-ML3.9 (577)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV

A5-mHSA-B1D2 (577)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV

A5-mHSA-F5B6H2 (577)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV

B12-mHSA-B1D2 (581)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV

B12-mHSA-F5B6H2 (581)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV

F4-mHSA-B1D2 (581)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV

F4-mHSA-F5B6H2 (581)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV

H3-mHSA-B1D2 (580)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV

H3-mHSA-F5B6H2 (580)MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKV

631 675

A5-mHSA-ML3.9 (622)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV

A5-mHSA-B1D2 (622)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV

A5-mHSA-F5B6H2 (622)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV

B12-mHSA-B1D2 (626)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV

B12-mHSA-F5B6H2 (626)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV

F4-mHSA-B1D2 (626)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV

F4-mHSA-F5B6H2 (626)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV

H3-mHSA-B1D2 (625)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV

H3-mHSA-F5B6H2 (625)FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV

676 720

A5-mHSA-ML3.9 (667)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL

A5-mHSA-B1D2 (667)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL

A5-mHSA-F5B6H2 (667)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL

B12-mHSA-B1D2 (671)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL

B12-mHSA-F5B6H2 (671)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL

F4-mHSA-B1D2 (671)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL

F4-mHSA-F5B6H2 (671)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL

H3-mHSA-B1D2 (670)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL

H3-mHSA-F5B6H2 (670)STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVL

721 765

A5-mHSA-ML3.9 (712)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT

A5-mHSA-B1D2 (712)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT

A5-mHSA-F5B6H2 (712)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT

B12-mHSA-B1D2 (716)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT

B12-mHSA-F5B6H2 (716)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT

F4-mHSA-B1D2 (716)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT

F4-mHSA-F5B6H2 (716)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT

H3-mHSA-B1D2 (715)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT

H3-mHSA-F5B6H2 (715)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFT

766 810

A5-mHSA-ML3.9 (757)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

A5-mHSA-B1D2 (757)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

A5-mHSA-F5B6H2 (757)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

B12-mHSA-B1D2 (761)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

B12-mHSA-F5B6H2 (761)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

F4-mHSA-B1D2 (761)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

F4-mHSA-F5B6H2 (761)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

H3-mHSA-B1D2 (760)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

H3-mHSA-F5B6H2 (760)FHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA

811 855

A5-mHSA-ML3.9 (802)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA

A5-mHSA-B1D2 (802)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA

A5-mHSA-F5B6H2 (802)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVESGG

B12-mHSA-B1D2 (806)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA

B12-mHSA-F5B6H2 (806)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVESGG

F4-mHSA-B1D2 (806)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA

F4-mHSA-F5B6H2 (806)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVESGG

H3-mHSA-B1D2 (805)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGA

H3-mHSA-F5B6H2 (805)FVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVESGG

856 900

A5-mHSA-ML3.9 (847)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG

A5-mHSA-B1D2 (847)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG

A5-mHSA-F5B6H2 (847)GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR

B12-mHSA-B1D2 (851)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG

B12-mHSA-F5B6H2 (851)GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR

F4-mHSA-B1D2 (851)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG

F4-mHSA-F5B6H2 (851)GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR

H3-mHSA-B1D2 (850)EVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPG

H3-mHSA-F5B6H2 (850)GLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGR

901 945

A5-mHSA-ML3.9 (892)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH

A5-mHSA-B1D2 (892)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH

A5-mHSA-F5B6H2 (892)GDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKM

B12-mHSA-B1D2 (896)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH

B12-mHSA-F5B6H2 (896)GDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKM

F4-mHSA-B1D2 (896)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH

F4-mHSA-F5B6H2 (896)GDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKM

H3-mHSA-B1D2 (895)DSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARH

H3-mHSA-F5B6H2 (895)GDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKM

946 990

A5-mHSA-ML3.9 (937)DVGYCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSSGGGSGGGGS

A5-mHSA-B1D2 (937)DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGS

A5-mHSA-F5B6H2 (937)TSNAVG----------FDYWGQGTLVTVSSGGGGSGGGSGGGGSG

B12-mHSA-B1D2 (941)DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGS

B12-mHSA-F5B6H2 (941)TSNAVG----------FDYWGQGTLVTVSSGGGGSGGGSGGGGSG

F4-mHSA-B1D2 (941)DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGS

F4-mHSA-F5B6H2 (941)TS----------NAVGFDYWGQGTLVTVSSGGGGSGGGSGGGGSG

H3-mHSA-B1D2 (940)DVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGS

H3-mHSA-F5B6H2 (940)TSNAVG----------FDYWGQGTLVTVSSGGGGSGGGSGGGGSG

991 1035

A5-mHSA-ML3.9 (982)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA

A5-mHSA-B1D2 (982)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA

A5-mHSA-F5B6H2 (972)

QSVLTQPPSVSGAPGQRVTISCTGRHSNIGLGYGVHWYQQLPGTA

B12-mHSA-B1D2 (986)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA

B12-mHSA-F5B6H2 (976)

QSVLTQPPSVSGAPGQRVTISCTGRHSNIGLGYGVHWYQQLPGTA

F4-mHSA-B1D2 (986)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA

F4-mHSA-F5B6H2 (976)

QSVLTQPPSVSGAPGQRVTISCTGRHSNIGLGYGVHWYQQLPGTA

H3-mHSA-B1D2 (985)QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNY-VSWYQQLPGTA

H3-mHSA-F5B6H2 (975)

QSVLTQPPSVSGAPGQRVTISCTGRHSNIGLGYGVHWYQQLPGTA

1036 1080

A5-mHSA-ML3.9 (1026)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC

A5-mHSA-B1D2 (1026)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC

A5-mHSA-F5B6H2 (1017)PKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYC

B12-mHSA-B1D2 (1030)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC

B12-mHSA-F5B6H2 (1021)PKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYC

F4-mHSA-B1D2 (1030)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC

F4-mHSA-F5B6H2 (1021)PKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYC

H3-mHSA-B1D2 (1029)PKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYC

H3-mHSA-F5B6H2 (1020)PKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYC

1081 1104

A5-mHSA-ML3.9 (1071)ASWDYTLSGWVFGGGTKLTVLG--

A5-mHSA-B1D2 (1071)ASWDYTLSGWVFGGGTKLTVLG--

A5-mHSA-F5B6H2 (1062)QSYDRRTPGWVFGGGTKLTVLG--

B12-mHSA-B1D2 (1075)ASWDYTLSGWVFGGGTKLTVLG--

Appendix 2

Anticancer agent

Other embodiments

While the invention has been described in terms of specific embodiments, it will be appreciated that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

All patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims (36)

1. An HSA linker conjugate comprising:

i) comprises the amino acid sequence shown in SEQ ID NO: a Human Serum Albumin (HSA) linker of the amino acid sequence set forth in any one of claims 6-15; and

ii) selected from the group consisting of antibodies, single chain Fv molecules, bispecific single chain Fv ((scFv')₂) Molecules, domain antibodies, diabodies, triabodies, hormones, Fab fragments, F (ab')₂Molecules, first and second binding moieties of the group consisting of tandem scFv (taFv) fragments, receptors, ligands, aptamers, and biologically active fragments thereof, and methods of making and using the sameWherein the first binding moiety is bound to the amino terminus of the HSA linker and the second binding moiety is bound to the carboxy terminus of the HSA linker.

2. The HSA linker conjugate of claim 1, wherein the HSA linker comprises the amino acid sequence set forth in SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

3. The HSA linker conjugate of claim 1, wherein said first binding moiety specifically binds ErbB3 and said second binding moiety specifically binds ErbB 2.

4. Has or comprises SEQ ID NO: 16, HSA linker conjugate B2B 3-1.

5. Has or comprises SEQ ID NO: 17, HSA linker conjugate B2B 3-2.

6. Has or comprises SEQ ID NO: 18, HSA linker conjugate B2B 3-3.

7. Has or comprises SEQ ID NO: 19, HSA linker conjugate B2B 3-4.

8. Has or comprises SEQ ID NO: 20, HSA linker conjugate B2B 3-5.

9. Has or comprises SEQ ID NO: 21, HSA linker conjugate B2B 3-6.

10. Has or comprises SEQ ID NO: 22, HSA linker conjugate B2B 3-7.

11. Has or comprises SEQ ID NO: 23, HSA linker conjugate B2B 3-8.

12. Has or comprises SEQ ID NO: 24, HSA linker conjugate B2B 3-9.

13. Has or comprises SEQ ID NO: 25, HSA linker conjugate B2B 3-10.

14. The HSA linker conjugate of any one of claims 1-13 admixed with a pharmaceutically acceptable carrier, excipient, or diluent.

15. A method for treating a patient having a disease or disorder, wherein the method comprises administering to the mammal an effective amount of the HSA linker conjugate of any one of claims 1-13.

16. A composition comprising the HAS linker conjugate of any one of claims 1-13 in combination with one or more therapeutic agents selected from the group consisting of: cyclophosphamide, camptothecin, homocamptothecin, colchicine, combretastatin, rhizomycin, dolastatin (dolastatin), ansamitocin p3, maytansinoids, auristatin, calicheamicin (caleachimicin), methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and derivatives thereof.

17. The composition of claim 16, wherein the therapeutic agent is trastuzumab.

18. The composition of claim 16, wherein the HSA linker conjugate is a polypeptide having or comprising SEQ ID NO: 16, HSA linker conjugate B2B 3-1.

19. The composition of claim 16, wherein the therapeutic agent is trastuzumab and the HSA linker conjugate is a polypeptide having or comprising SEQ ID NO: 16, HSA linker conjugate B2B 3-1.

20. The composition of claim 19, wherein the combination provides greater efficacy when the therapeutic agent at a first dose and the HSA linker conjugate at a second dose are tested in a nude mouse human xenograft model using human breast cancer cells than the efficacy provided by the therapeutic agent at only the first dose or the HSA linker conjugate at only the second dose in the same model.

21. The composition of claim 20 in a dosage form to be administered to a subject to provide at least 3mg/kg of B2B3-1 and at least 0.1mg/kg trastuzumab.

22. The composition of claim 21 in a dosage form suitable for administration to a subject to provide 3mg/kg of B2B3-1 and 0.1mg/kg trastuzumab.

23. The composition of claim 21 in a dosage form suitable for administration to a subject to provide 10mg/kg of B2B3-1 and 1mg/kg trastuzumab.

24. The composition of claim 22, wherein the administration of 3mg/kg of B2B3-1 and 0.1mg/kg trastuzumab to the subject is more effective than treatment of the matched subject with 3mg/kg of B2B3-1 alone or 0.1mg/kg of trastuzumab alone.

25. The composition of claim 23, wherein administration to a subject to provide 10mg/kg of B2B3-1 and 1mg/kg trastuzumab is more effective than treatment of a matched subject with 10mg/kg of B2B3-1 alone or 1mg/kg of trastuzumab alone.

26. The composition of claim 22, wherein when administered to a plurality of subjects to provide each with 3mg/kg of B2B3-1 and 0.1mg/kg trastuzumab, induces tumor regression in more than 10% of the subjects after 20 days.

27. The composition of claim 23, wherein when administered to a plurality of subjects to provide each with 10mg/kg of B2B3-1 and 1mg/kg trastuzumab, induces tumor regression in at least 50% of the subjects after 20 days.

28. The composition of claim 16, wherein the therapeutic agent is lapatinib.

29. The composition of claim 16, wherein the therapeutic agent is a taxane.

30. The composition of claim 29, wherein the therapeutic agent is docetaxel.

31. The composition of claim 16, wherein the therapeutic agent is letrozole.

32. The HSA linker conjugate of claim 3, wherein said first binding moiety has a K of about 16nM_dBinds ErBb3 and the second binding moiety has a K of about 0.3nM_dBinds ErbB 2.

33. A method of treating a subject in need of tumor treatment comprising administering to the subject an effective amount of the HSA linker conjugate of any one of claims 1-14 and 32.

34. A method of treating a subject in need of tumor treatment, the treatment comprising administering to the subject an effective amount of the composition of any one of claims 16-30.

35. The use of the HSA linker conjugate of any of claims 1-14 and 32 in the manufacture of a medicament for the treatment of a tumor.

36. Use of a composition according to any one of claims 16 to 30 in the manufacture of a medicament for the treatment of a tumour.