HUMAN SERUM ALBUMIN LINKERS AND CONJUGATES THEREOF

Abstract

Disclosed is a human serum albumin (HSA) linker and HSA linker with binding, diagnostic, and therapeutic agents conjugated thereto. Also disclosed is a conjugate in which the HSA linker is covalently bonded to amino and carboxy terminal binding moieties that are first and second single-chain Fv molecules (scFvs). Exemplified conjugates are useful, e.g., in reducing tumor cell proliferation, e.g., for therapeutic therapeutic applications. Also disclosed are methods and kits for the diagnostic and therapeutic application of an HSA linker conjugate.

Description

FIELD OF THE INVENTION

[0001] Provided are a human serum albumin (HSA) linker conjugates and binding, diagnostic, and therapeutic conjugates thereof. In one embodiment, the HSA linker includes two amino acid substitutions. Another embodiment is a conjugate in which the HSA linker is covalently bonded to amino and carboxy terminal binding moieties that are first and second single-chain Fv molecules (scFvs). Exemplified conjugates are useful, e.g., in reducing tumor cell proliferation, e.g., for therapeutic applications. Further provided are methods for the manufacture and administration of diagnostic and therapeutic HSA linker conjugates.

BACKGROUND OF THE INVENTION

[0002] Antibody-like binding moieties (including those in intact antibodies, antibody fragments, and scFvs) are often used for therapeutic applications. Antibody fragments and scFvs generally exhibit shorter serum half lives than intact antibodies, and in some therapeutic applications increased in vivo half lives would be desirable for therapeutic agents possessing the functionality of such fragments and scFvs.

[0003] Human serum albumin (HSA) is a protein of about 66,500 kD and is comprised of 585 amino acids including at least 17 disulphide bridges. As with many of the members of the albumin family, human serum albumin plays an important role in human physiology and is located in virtually every human tissue and bodily secretion. HSA has the ability to bind and transport a wide spectrum of ligands throughout the circulatory system, including the long-chain fatty acids, which are otherwise insoluble in circulating plasma.

[0004] The serum albumins belong to a family of proteins that includes alpha-fetoprotein and human group-specific component, also known as vitamin-D binding protein. The serum albumins are the major soluble proteins of the circulatory system and contribute to many vital physiological processes. Serum albumin generally comprises about 50% of the total blood component by dry weight. The 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 variety of chemically diverse molecules, such as fatty acids, amino acids, steroids, calcium, metals such as copper and zinc, and various pharmaceutical agents. The albumin family of molecules is generally thought to facilitate transfer of many of these ligands across organ-circulatory interfaces, such as the liver, intestines, kidneys, and the brain. The albumins are thus involved in a wide range of circulatory and metabolic functions.

SUMMARY OF THE INVENTION

[0005] In a first aspect, the invention provides an HSA linker conjugate that includes a human serum albumin (HSA) linker that comprises an amino acid sequence set forth in any one of SEQ ID NOS:6-15 and first and second binding moieties selected from antibodies, single-chain Fv molecules, bispecific single chain Fv ((scFv')₂) molecules, domain antibodies, diabodies, triabodies, hormones, Fab fragments, F(ab')₂ molecules, tandem scFv (taFv) fragments, receptors (e.g., cell surface receptors), ligands, aptamers, and biologically-active fragments thereof, in which the first binding moiety is bonded to the amino terminus of the HSA linker and the second binding moiety is bonded to the carboxy terminus of the HSA linker. In one embodiment, the first binding moiety specifically binds ErbB3 and the second binding moiety specifically binds ErbB2. In other embodiments, the HSA linker comprises an amino acid sequence set forth in SEQ ID NO:1, 9, 10, 14 or 15.

[0006] In a second aspect, three or more binding moieties (e.g., 4, 5, 6, 7, 8, 9, 10, or more) can be included in the agent; these additional binding moieties can be added to the agent, e.g., in tandem (e.g., 2, 3, 4, or 5 or more in tandem) with the first or second binding moiety.

[0007] In a third aspect, the invention provides an HSA linker that comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:1, and a serine residue at position 34 and a glutamine residue at position 503 of the amino acid sequence set forth in SEQ ID NO:1. In one embodiment, the amino acid sequence has at least 95% sequence identity to the sequence set forth in SEQ ID NO:1. In another embodiment, the HSA linker comprises the amino acid sequence set forth in SEQ ID NO:1. In yet another embodiment, the HSA linker has the amino acid sequence set forth in SEQ ID NO:1.

[0008] In a fourth aspect, the invention provides an HSA linker conjugate that includes an HSA linker having at least 90% amino acid sequence identity to the sequence set forth in SEQ ID NO:1 and at least a first binding moiety. In one embodiment, the HSA linker conjugate includes a first peptide connector that binds the first binding moiety to the HSA linker.

[0009] In a fifth aspect, the invention features an HSA linker conjugate that includes an HSA linker having an amino acid sequence set forth in any one of SEQ ID NOs:11-15, or a fragment or variant of any one of these sequences, and at least a first binding moiety.

[0010] In certain embodiments of either the fourth or fifth aspect of the invention, the HSA linker conjugate further includes 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 an embodiment, the first connector covalently binds the first binding moiety to the HSA linker.

[0011] In certain embodiments of either the fourth or fifth aspect of the invention, the HSA linker conjugate further includes at least a second binding moiety. In an embodiment, the HSA linker conjugate further includes a second peptide connector (e.g., AAS, AAQ, or AAAL (SEQ ID NO:5)) 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 a further embodiment, the second connector covalently binds the second binding moiety to the HSA linker. In other embodiments, the HSA linker conjugate further includes three or more binding moieties which are included in tandem with the first or second binding moiety; the three or more binding moieties can further include a connector sequence that joins the three or more binding moieties to the first or second binding moiety and to each other.

[0012] In certain embodiments of either the fourth or fifth aspect of the invention, the HSA linker conjugate includes a first peptide connector that covalently binds a first binding moiety to the amino terminus of the HSA linker and a second peptide connector that covalently binds a second binding moiety to the carboxy terminus of the HSA linker. In one embodiment, the first connector has the amino acid sequence AAS or AAQ and the second connector has the amino acid sequence set forth in SEQ ID NO:5.

[0013] In certain embodiments of either the fourth or fifth aspect of the invention, the first or second binding moiety (or third or more binding moiety) is an antibody, single-chain Fv molecule, bispecific single chain Fv ((scFv')₂) molecule, domain antibody, diabody, triabody, hormone, Fab fragment, F(ab')₂ molecule, tandem scFv (taFv) fragment, receptor (e.g., cell surface receptor), ligand, aptamer, or biologically-active fragment thereof. In other embodiments, the HSA linker conjugates provided herein include combinations 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.

[0014] In an 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 an insulin-like growth factor 1 receptor (IGF1R), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transition 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), heregulin, 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-α), TNF-β, folate receptor (FOLR), folate, transferrin receptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, an integrin (e.g., an α4 integrin or a β-1 integrin), P-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronate 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 cell 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, MART-1, gp100, MAGE-1, ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen (CEA), Lewis^Y, MUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG-72 antigen, and fragments thereof. In a further 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 erythroblastic leukemia viral oncogene homolog (ErbB) receptor (e.g., ErbB1 receptor; ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor). In 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 alpha-fetoprotein (AFP) or an interferon, or a biologically-active fragment thereof. In a further embodiment, one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab, certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab, or anakinra.

[0015] In any one of the first, second, third, fourth, or fifth aspects of the invention, the HSA linker conjugate is conjoined to a diagnostic, a therapeutic agent, or both. In one embodiment, the diagnostic agent is a detectable label, such as a radioactive, fluorescent, or heavy metal label. In another embodiment, the therapeutic agent is a cytotoxic, cytostatic, or immunomodulatory agent. Cytotoxic agents include alkylating agents, antibiotics, antineoplastic agents, antiproliferative agents, antimetabolites, tubulin inhibitors, topoisomerase I or II inhibitors, hormonal agonists or antagonists, immunomodulators, DNA minor groove binders, and radioactive agents, or any agent capable of binding to and killing a tumor cell or inhibiting tumor cell proliferation. Antineoplastic agents include cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and their derivatives.

[0016] In any one 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 exhibits an in vivo half-life of between 6 hours and 7 days. In another embodiment, the agent exhibits an in vivo half-life greater than 8 hours.

[0017] In a sixth aspect, the invention features a method for treating a mammal having a disease or disorder by administering any one of the HSA linker conjugates described herein. In one embodiment, the disease or disorder is associated with cellular signaling through a cell surface receptor. In another embodiment, the mammal is a human. In a further embodiment, the disease or disorder is a proliferative or autoimmune disease. Proliferative diseases include such cancers 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 combination with one or more therapeutic agents, such as an antineoplastic agent.

[0018] In a seventh aspect, the invention features a method for making an HSA linker conjugate by bonding at least a first binding moiety to the amino terminus and a second binding moiety to the carboxy terminus of an HSA linker having the amino acid sequence set forth in any one of SEQ ID NOS:1, 3, or 6-15, or a sequence having at least 90%, 95%, 97%, or 100% sequence identity to a sequence set forth in any one of SEQ ID NOS:1, 3, or 6-15. In one embodiment, the first or second binding moiety is covalently joined 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 joined in tandem with the first or second binding moiety to the amino or carboxy terminus of the HSA linker. 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, single-chain Fv molecule, bispecific single chain Fv ((scFv')₂) molecule, domain antibody, diabody, triabody, hormone, Fab fragment, F(ab')₂ molecule, tandem scFv (taFv) fragment, receptor (e.g., cell surface receptor), ligand, or 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 transition 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), heregulin, 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-α), TNF-β, folate receptor (FOLR), folate, transferrin receptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, an integrin (e.g., an α4 integrin or a β-1 integrin), P-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronate 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 cell 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, MART-1, gp100, MAGE-1, ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen (CEA), Lewis^Y, MUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG-72 antigen, and fragments thereof. In a further 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 erythroblastic leukemia viral oncogene homolog (ErbB) receptor (e.g., ErbB1 receptor; ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor). In 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 alpha-fetoprotein (AFP) or an interferon, or a biologically-active fragment thereof. In a further embodiment, one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab, certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab, or anakinra. In another embodiment, the agent is conjoined to a diagnostic or therapeutic agent. In one embodiment, the diagnostic agent is a detectable label, such as a radioactive, bioluminescent, fluorescent, heavy metal, or epitope tag. In another embodiment, the therapeutic agent is a cytotoxic agent, cytostatic, or immunomodulatory agent. Cytotoxic agents include alkylating agents, antibiotics, antineoplastic agents, antiproliferative agents, antimetabolites, tubulin inhibitors, topoisomerase I and II inhibitors, hormonal agonists or antagonists, immunomodulators, DNA minor groove binders, and radioactive agents, or any agent capable of binding to and killing a tumor cell or inhibiting tumor cell proliferation. Antineoplastic agents include cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and their derivatives. In a further embodiment, the agent is admixed with a pharmaceutically acceptable carrier, excipient, or diluent.

[0019] In a eighth aspect, the invention features a method for making an HSA linker by substituting one or more surface-exposed amino acid residues in the amino acid sequences set forth in any one of SEQ ID NOS:1, 3, and 6-15 with a substitute amino acid capable of chemical modification that allows conjugation of a diagnostic or therapeutic agent. In one embodiment, the substitute amino acid is cysteine and the surface exposed amino acid residues are serine or threonine. In another embodiment, the chemical modification results in a covalent bond between the substitute amino acid and the diagnostic or therapeutic agent. In a further embodiment, the surface-exposed amino acid residues 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.

[0020] In a ninth aspect, the invention features a method for making an HSA linker by substituting one or more of the residues in the amino acid sequences set forth in any one of SEQ ID NOS:1, 3, and 6-15 with an asparagine, serine, or threonine, thereby incorporating a glycosylation site within the HSA agent.

[0021] In a tenth aspect, the invention features a method for making an HSA linker by substituting one or more of the asparagine, serine, or threonine residues in the amino acid sequences set forth in any one of SEQ ID NOS:1, 3, and 6-15 with any amino acid other than asparagine, serine, or threonine, thereby removing a glycosylation site from the HSA agent.

[0022] In an eleventh aspect, the invention features an HSA linker that comprises a sequence that has at least 90% sequence identity to one of the amino acid sequences set forth in SEQ ID NOS:16-25. In one embodiment, the HSA linker has at least 95% sequence identity to one of the amino acid sequences set forth in SEQ ID NOS:16-25. In another embodiment, the HSA linker comprises one of the amino acid sequences set forth in SEQ ID NOS:16-25. In yet another embodiment, the HSA linker has one of the amino acid sequences set forth in SEQ ID NOS:16-25.

[0023] In a further embodiment, the HSA linker or HSA linker conjugate is conjoined to a diagnostic or therapeutic agent. Diagnostic agents include detectable labels, such as a radioactive, bioluminescent, fluorescent, or heavy metal labels, or epitope tags. Fluorescent molecules that can serve 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 a further embodiment, the therapeutic agent is a cytotoxic polypeptide such as cytochrome c, caspase 1-10, granzyme A or B, tumor necrosis factor-alpha (TNF-α), TNF-β, Fas, Fas ligand, Fas-associated death doman-like IL-1β converting enzyme (FLICE), TRAIL/APO2L, TWEAK/APO3L, Bax, Bid, Bik, Bad, Bak, RICK, vascular apoptosis inducing proteins 1 and 2 (VAP1 and VAP2), pierisin, apoptosis-inducing protein (AIP), IL-1α propiece polypeptide, apoptin, apoptin-associated protein 1 (AAP-1), endostatin, angiostatin, and biologically-active fragments thereof. An HSA linker or HSA linker conjugate can be combined with (e.g., conjoined to or mixed with in a pharmaceutical composition) one or more therapeutic agents such as cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and derivatives thereof.

[0024] In an embodiment of any aspect described herein, the first and second binding moieties (and, if present, one or more of the third or further binding moiety) specifically bind the same target molecule. In another embodiment of any aspect, the first and second binding moieties (and, if present, one or more of the third or further binding moiety) specifically bind different target molecules. In a further embodiment of any aspect, the first and second binding moieties (and, if present, one or more of the third or further binding moiety) specifically bind different epitopes on the same target molecule.

[0025] In a twelfth aspect, the invention features an HSA linker that comprises one or both of amino acid residues 25-44 and 494-513 of the amino acid sequence set forth in SEQ ID NO:1. In one embodiment, the HSA linker comprises amino acid residues 25-70 and 450-513 of the amino acid sequence set forth in SEQ ID NO:1. In another embodiment, the HSA linker comprises amino acid residues 15-100 and 400-520 of the amino acid sequence set forth in SEQ ID NO:1. In a further embodiment, the HSA linker comprises amino acid residues 10-200 and 300-575 of the amino acid sequence set forth in SEQ ID NO:1. In another embodiment, the HSA linker comprises amino acid residues 5-250 and 275-580 of the amino acid sequence set forth in SEQ ID NO:1.

[0026] In the twelfth aspect of the invention, the HSA linker is conjoined to at least a first binding moiety, for form an HSA linker conjugate. In one embodiment, the HSA linker conjugate includes 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 a further embodiment, the HSA linker includes a second binding moiety. In one embodiment, the HSA linker includes 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 a further embodiment, the second connector covalently binds the second binding moiety to the HSA linker. In other embodiments, the HSA linker includes 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 moiety. In yet other embodiments, a peptide connector (e.g., AAS, AAQ, or AAAL (SEQ ID NO:5)) separates one or more of these additional binding moities from each other, the first or second binding moiety, or the HSA linker.

[0027] In the twelfth aspect of the invention, the HSA linker includes a first peptide connector that covalently binds a first binding moiety to the amino terminus of the polypeptide linker and a second peptide connector that covalently binds a second binding moiety to the carboxy terminus of the HSA linker. In one embodiment, the first connector has the amino acid sequence AAS or AAQ and the second connector has the amino acid sequence set forth in SEQ ID NO:5.

[0028] In the 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, single-chain Fv molecule, bispecific single chain Fv ((scFv')₂) molecule, domain antibody, diabody, triabody, hormone, Fab fragment, F(ab')₂ molecule, tandem scFv (taFv) fragment, receptor (e.g., cell surface receptor), ligand, aptamer, or biologically-active fragment thereof. In one embodiment, one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is a human or humanized single-chain Fv molecule.

[0029] In the 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 an insulin-like growth factor 1 receptor (IGF1R), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transition 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), heregulin, 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-α), TNF-β, folate receptor (FOLR), folate, transferrin receptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, an integrin (e.g., an α4 integrin or a β-1 integrin), P-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronate 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 cell 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, MART-1, gp100, MAGE-1, ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen (CEA), Lewis^Y, MUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG-72 antigen, and fragments thereof. In a further embodiment, the first or second binding moiety is or specifically binds to erythroblastic leukemia viral oncogene homolog (ErbB) receptor (e.g., ErbB1 receptor; ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor). In 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 alpha-fetoprotein (AFP) or an interferon, or a biologically-active fragment thereof. In a further embodiment, one or more of the first or second binding moiety (or, if present, the third or further binding moiety) is natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab, certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, panitumumab, or anakinra.

[0030] In the twelfth aspect of the invention, the HSA linker is conjoined to a diagnostic agent, a therapeutic agent, or both. In one embodiment, the diagnostic agent is a detectable label, such as a radioactive, fluorescent, or heavy metal label. In another embodiment, the therapeutic agent is a cytotoxic agent, cytostatic, or immunomodulatory agent. Cytotoxic agents include alkylating agents, antibiotics, antineoplastic agents, antiproliferative agents, antimetabolites, tubulin inhibitors, topoisomerase I or II inhibitors, hormonal agonists or antagonists, immunomodulators, DNA minor groove binders, and radioactive agents, or any agent capable of binding to and killing a tumor cell or inhibiting tumor cell proliferation. Antineoplastic agents include cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and their derivatives. In one embodiment, the conjoined HSA linker is admixed with a pharmaceutically acceptable carrier, excipient, or diluent. In another embodiment, the HSA linker exhibits an in vivo half-life of between 6 hours and 7 days. In a further embodiment, the HSA linker exhibits an in vivo half-life greater than 8 hours.

[0031] A thirteenth aspect of the invention features an agent of any of the prior aspects of the invention (one through twelve), in which the HSA linker is replaced by another polypeptide linker. For example, the polypeptide linker sequence could be a mammalian, non-human serum albumin polypeptide sequence, such as, e.g., a bovine, murine, feline, and canine serum albumin (BSA) polypeptide sequence. In other embodiments this polypeptide linker sequence is between 5 and 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 this range. In other embodiments, the polypeptide linker sequence includes a single amino acid (including, but not limited to, e.g., glycine, alanine, serine, glutamine, leucine, and valine), or combinations of amino acids.

[0032] In another embodiment, the HSA linker is replaced by an alpha-fetoprotein (AFP) polypeptide, e.g., mammalian AFP polypeptide, such as a human, murine, bovine, or canine AFP polypeptide. In an embodiment, the AFP linker corresponds to the full-length human AFP polypeptide sequence (a.a. 1-609; SEQ ID NO:58), the mature human AFP polypeptide sequence lacking amino acids 1-18 of the signal sequence (a.a., 19-609 of SEQ ID NO:58), or fragments thereof. In other embodiments, the AFP polypeptide linker contains at least 5 to 8 contiguous amino acids, 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 of SEQ ID NO:58, or has at least 90% sequence identity (e.g., at least 95%, 97%, 99%, or more sequence identity) to a contiguous polypeptide sequence of SEQ ID NO:58 having one or more of these lengths. For example, an AFP polypeptide linker sequence having 90% sequence identity to a 34-mer human AFP peptide corresponding to amino acids 446-479 of SEQ ID NO:58 (LSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGV; SEQ ID NO:59) may contain up to 3 amino acids altered from the 446-479 segment of SEQ ID NO:58. One such example of sequence deviation in biologically active human AFP fragments is found in, e.g., U.S. Pat. No. 5,707,963 (incorporated by reference herein), which discloses a 34-amino acid fragment of human AFP (SEQ ID NO:59) with flexibility at two amino acid residues (amino acid 9 and 22 of SEQ ID NO:59). Other examples of AFP polypeptide linker sequences include, e.g., amino acids 19-198 of SEQ ID NO:58 (human AFP Domain I), amino acids 217-408 of SEQ ID NO:58 (human AFP Domain II), amino acids 409-609 of SEQ ID NO:58 (human AFP Domain III), amino acids 19-408 of SEQ ID NO:58 (human AFP Domain I+II), amino acids 217-609 of SEQ ID NO:58 (human AFP Domain II+III), and amino acids 285-609 of SEQ ID NO:58 (human AFP Fragment I). In another embodiment, the human AFP polypeptide linker sequence is an 8-amino acid sequence that includes amino acids 489-496 (i.e., EMTPVNPG) of SEQ ID NO:58.

[0033] A fourteenth aspect of the invention features kits that include any of the HSA linkers, HSA linker conjugates, or any other agents described in the first, second, third, fourth, fifth, eleventh, twelfth, and thirteenth aspects discussed above. The kits further include instructions to allow a practitioner (e.g., a physician, nurse, or patient) to administer the compositions and agents contained therein. In an embodiment, the kits include multiple packages of a single- or multi-dose pharmaceutical composition containing an effective amount of an agent, e.g., HSA linker conjugate as described herein or an HSA linker that includes, e.g., one or more binding moieties (e.g., antibodies or antibody fragments (e.g., scFv)), diagnostic agents (e.g., radionuclide or chelating agents), and/or therapeutic agents (e.g., cytotoxic or immunomodulatory agents). Optionally, instruments or devices necessary for administering the pharmaceutical composition(s) may be included in the kits. For instance, a kit may provide one or more pre-filled syringes containing an effective amount of an HSA linker conjugate or HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto. Furthermore, the kits may also include additional components such as instructions or administration schedules for a patient suffering from a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease) to use the pharmaceutical composition(s) containing, e.g., an HSA linker conjugate or HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto.

DEFINITIONS

[0034] The term "antibody" as used interchangeably herein, includes whole antibodies or immunoglobulins and any antigen-binding fragment or single chains thereof. Antibodies, as used herein, can be mammalian (e.g., human or mouse), humanized, chimeric, recombinant, synthetically produced, or naturally isolated. In most mammals, including humans, whole antibodies have at least two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region consists of three domains, C_H1, C_H2, and C_H3 and a hinge region between C_H1 and C_H2. Each light chain consists 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. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies of the present invention include all known forms of antibodies and other protein scaffolds 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 also can be a Fab, Fab'2, scFv, SMIP, diabody, nanobody, aptamers, or a domain antibody. The antibody can have any of the following isotypes: IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, and IgAsec), IgD, or IgE. Antibodies that can be used as binding moieties, as defined herein, in combination with an HSA linker include, but are not limited to, natalizumab, infliximab, adalimumab, rituximab, alemtuzumab, bevacizumab, daclizumab, efalizumab, golimumab, certolizumab, trastuzumab, abatacept, etanercept, pertuzumab, cetuximab, and panitumumab.

[0035] The term "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., ErbB2). The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L, and C_H1 domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and C_H1 domains; (iv) a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (v) a dAb including V_H and V_L domains; (vi) a dAb fragment (Ward et al., Nature 341:544-546 (1989)), which consists of a V_H domain; (vii) a dAb which consists of a V_H or a V_L domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antibody fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0036] By "autoimmune disease" is meant a disease in which an immune system response is generated against self epitopes or antigens. 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, celiac 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, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin dependent diabetes, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, 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, Stiff-Man syndrome, systemic lupus erythematosus (SLE), Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis (e.g., birdshot retinochoroidopathy uveitis and sarcoid uveitis), vasculitis, vitiligo, Wegener's granulomatosis, and myasthenia gravis.

[0037] By "binding moiety" is meant 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 antibodies), antibody fragments (e.g., Fab fragments, Fab'2, scFv antibodies, SMIP, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and domain antibodies), receptors, ligands, aptamers, and other molecules having a known binding partner.

[0038] By "biologically-active" is meant that a molecule, including biological molecules, such as nucleic acids, peptides, polypeptides, and proteins, exerts a physical or chemical activity on itself or other molecule. For example, a "biologically-active" molecule may possess, e.g., enzymatic activity, protein binding activity (e.g., antibody interactions), or cytotoxic activities are "biologically-active."

[0039] The term "chimeric antibody" refers to an immunoglobulin or antibody whose variable regions derive from a first species and whose constant regions derive from a second species. Chimeric antibodies can be constructed, for example, by genetic engineering, from immunoglobulin gene segments belonging to different species (e.g., from a mouse and a human).

[0040] By "connector" or "peptide connector" is meant an amino acid sequence of 2 to 20 residues in length that is covalently attached to one or both of the amino or carboxy termini of an HSA linker, or is covalently attached to one or more residues of an HSA linker (e.g., a residue between the amino and carboxy terminal residues). In preferred embodiments, the peptide connector attached to the amino terminus of an HSA linker has the amino acid sequence AAS or AAQ and the connector attached to the carboxy terminus has the amino acid sequence "AAAL" (SEQ ID NO:5).

[0041] The terms "effective amount" or "amount effective to" or "therapeutically effective amount" means an amount of an agent (e.g., an HSA linker bonded with one or more binding moieties or diagnostic or therapeutic agents with or without a connector sequence) sufficient to produce a desired result, for example, killing a cancer cell, reducing tumor cell proliferation, reducing inflammation in a diseased tissue or organ, or labeling a specific population of cells in a tissue, organ, or organism (e.g., a human).

[0042] The term "human antibody," as used herein, is intended to include antibodies, or fragments thereof, having variable regions in which both the 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, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies may include amino acid residues 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, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., a humanized antibody or antibody fragment).

[0043] The term "humanized antibody" refers to any antibody or antibody fragment that includes at least one immunoglobulin domain having a variable region that includes a variable framework region substantially derived from a human immunoglobulin or antibody and complementarity determining regions (e.g., at least one CDR) substantially derived from a non-human immunoglobulin or antibody.

[0044] As used herein, an "inflammatory signaling inhibitor" or "ISI" is an agent that decreases the binding between a pro-inflammatory cytokine (e.g., TNF-alpha, TNF-beta, or IL-1) and its receptor (e.g., TNF receptor 1 or 2, or IL-1 receptor, respectively); decreases the binding of activating molecules to pro-inflammatory cell surface signaling molecules (e.g., CD20, CD25, CTLA-4, CD80/CD86, or CD28); or decreases the downstream activation of, or activity of, intracellular signaling molecules that are activated following the binding of pro-inflammatory cytokines to their receptors or the binding of activating molecules to pro-inflammatory cell surface signaling molecules (e.g., an agent that decreases the activation of, or activity of, signaling molecules in the p38 MAPK signaling pathway). The decrease mediated by an ISI may be a decrease in binding between a pro-inflammatory cytokine and its receptor, a decrease in binding of an activating molecule to a pro-inflammatory cell surface signaling molecule, or a decrease in intracellular signaling which occurs following the binding of pro-inflammatory cytokines to their receptors or activating molecules to pro-inflammatory cell surface signaling molecules. Preferably, such a decrease mediated by an ISI is a decrease 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%). An ISI may act by reducing the amount of pro-inflammatory cytokine (e.g., TNF-alpha, TNF-beta, or IL-1) freely available to bind the receptor. For example, an ISI may be a soluble pro-inflammatory cytokine receptor protein (e.g., a soluble TNF receptor fusion protein such as etanercept (ENBREL®) or lenercept), or a soluble pro-inflammatory cell surface signaling molecule (e.g., a soluble CTLA-4 (abatacept)), or an antibody directed against 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; an anti-CD20 antibody, such as rituximab; or TRU-015 (Trubion Pharmaceuticals)). In addition, an ISI may act by disrupting the ability of the endogenous wild-type pro-inflammatory cytokine or the pro-inflammatory cell surface signaling molecule to bind to its receptor (e.g., TNF receptor 1 or 2, IL-1 receptor--e.g., anakinra, or CD11a--e.g., efalizumab (RAPTIVA®, Genentech)). Examples of dominant-negative TNF-alpha variants are XENP345 (a pegylated version of TNF variant A145R/I97T) and Xpro®1595, and further variants disclosed in U.S. Patent Application Publication Nos. 20030166559 and 20050265962, herein incorporated by reference. An example of a dominant negative IL-1 variant is anakinra (KINERET®), which is a soluble form of IL-1 that binds to the IL-1 receptor without activating intracellular signaling pathways. Inflammatory signaling inhibitors, which can be used in the present invention, are also small molecules which inhibit or reduce the signaling pathways downstream of pro-inflammatory cytokine or pro-inflammatory cell surface signaling molecules (e.g., DE 096). Examples of ISIs of this kind include inhibitors of p38 MAP kinase, e.g., 5-amino-2-carbonylthiopene derivatives (as described in WO 04/089929, herein incorporated); ARRY-797; BIRB 796 BS, (1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-2(morpholin-4-yl-ethoxy)-- naphtalen-1-yl]-urea); CHR-3620; CNI-1493; FR-167653 (Fujisawa Pharmaceutical, Osaka, Japan); ISIS 101757 (Isis Pharmaceuticals); ML3404; NPC31145; PD169316; PHZ1112; RJW67657, (4-(4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-y- l)-3-butyn-1-ol; SCIO-469; SB202190; SB203580, (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole)- ; SB239063, trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl-methoxypyridimidin-4-yl)i- midazole; SB242235; SD-282; SKF-86002; TAK 715; VX702; and VX745. Furthermore, an ISI may interfere with the processing of a pro-inflammatory cytokine (e.g., TNF-alpha and TNF-beta) from its membrane bound form to its soluble form. Inhibitors of TACE are ISIs of this class. Examples of inhibitors of TACE include BB-1101, BB-3103, BMS-561392, butynyloxyphenyl β-sulfone piperidine hydroxomates, CH4474, DPC333, DPH-067517, GM6001, GW3333, Ro 32-7315, TAPI-1, TAPI-2, and TMI 005. Additional examples of ISIs include short peptides derived from the E. coli heat shock proteins engineered for disease-specific immunomodulatory activity (e.g., dnaJP1).

[0045] By "integrin antagonist" is meant any agent that reduces or inhibits the biological activity of an integrin molecule (e.g., a reduction or inhibition of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more relative to the biological activity in the absence of the integrin antagonist), such as the α4 subunit of an integrin molecule. The agent may act directly or indirectly on the α4 integrin subunit (NCBI Accession No. P13612; Takada et al., EMBO J. 8:1361-1368 (1989)) by inhibiting the activity or expression of the α4 integrin subunit, or may act on a target to which the intact integrin containing an α4 subunit binds. For example, an antibody or blocking peptide that binds to vascular cell adhesion molecule-1 (VCAM-1), thus preventing the binding of α4β1 integrin to VCAM-1 is considered an integrin antagonist for purposes of the present invention. Non-limiting exemplary integrin antagonists suitable for use with the present invention may include proteins, blocking peptides, antibodies, such as natalizumab (TYSABRI®), and small molecule inhibitors. Examples of α4 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 by reference herein), oMEPUPA-V (Biogen; U.S. Pat. No. 6,495,525; incorporated by reference herein), alefacept, CDP-323 (Celltech); firategrast (SB-68399; GlaxoSmithKline); TR-9109 (Pfizer); ISIS-107248 (Antisense Therapeutics); R-1295 (Roche); and TBC-4746 (Schering-Plough). Additional non-limiting examples of α4 integrin antagonists include the small molecules described in U.S. Pat. Nos. 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 herein incorporated by reference); in 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 herein incorporated by reference); in European Patent Nos. EP 0842943; EP 0842944; EP 0842945; EP 0903353; and EP 0918059; and in PCT Publication Nos. WO 95/15973; WO 96/06108; WO 96/40781; WO 98/04247; WO 98/04913; WO 98/42656; WO 98/53814; WO 98/53817; WO 98/53818; WO 98/54207; WO 98/58902; WO 99/06390; WO 99/06431; WO 99/06432; WO 99/06433; WO 99/06434; WO 99/06435; WO 99/06436; WO 99/06437; WO 99/10312; WO 99/10313; WO 99/20272; WO 99/23063; WO 99/24398; WO 99/25685; WO 99/26615; WO 99/26921; WO 99/26922; WO 99/26923; WO 99/35163; WO 99/36393; WO 99/37605; WO 99/37618; WO 99/43642; WO 01/42215; and WO 02/28830; all of which are incorporated by reference herein. Additional examples of α4 integrin antagonists include the phenylalanine derivatives described in: U.S. Pat. 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 herein incorporated by reference). Additional amino acid derivatives that are α4 integrin antagonists include those described in, e.g., U.S. Patent Application Publication Nos. 2004/0229859 and 2006/0211630 (herein incorporated by reference), and PCT Publication Nos. WO 01/36376; WO 01/47868; and WO 01/70670; all of which are incorporated by reference herein. Other examples of α4 integrin antagonists include the peptides, and the peptide and semi-peptide compounds described in, e.g., PCT Publication Nos. 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. An additional example of an α4 integrin antagonist is the pegylated molecule described in U.S. Patent Application Publication No. 2007/066533 (herein incorporated by reference). Examples of antibodies that are α4 integrin antagonists include those described in, e.g., PCT Publication Nos. WO 93/13798; WO 93/15764; WO 94/16094; and WO 95/19790. Additional examples of α4 integrin antagonists are described herein.

[0046] By "interferon" is meant a mammalian (e.g., a human) interferon-alpha, -beta, -gamma, or -tau polypeptide, or biologically-active fragment thereof, e.g., IFN-α (e.g., IFN-α-1a; see U.S. Patent Application No. 20070274950, incorporated herein by reference), IFN-α-1b, IFN-α-2a (see PCT Application No. WO 07/044083, herein incorporated by reference), and IFN-α-2b), IFN-β (e.g., described in U.S. Pat. No. 7,238,344, incorporated by reference; IFN-b-1a (AVONEX® and REBIF®), as described in U.S. Pat. No. 6,962,978, incorporated by reference, and IFN-β-1b (BETASERON®, as described in U.S. Pat. Nos. 4,588,585; 4,959,314; 4,737,462; and 4,450,103; incorporated by reference in their entirety), 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; incorporated by reference).

[0047] By "HSA linker conjugate" is meant a human serum albumin (HSA) linker in combination with (preferably covalently linked to) one or more binding moieties, peptide connectors, diagnostic agents, or therapeutic agents.

[0048] The term "monoclonal antibody" as used herein 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, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies can be prepared using any art recognized technique and those described herein such as, for example, a hybridoma method, as described by Kohler et al., Nature 256:495 (1975), a transgenic animal (e.g., Lonberg et al., Nature 368(6474):856-859 (1994)), recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567), or using phage, yeast, or synthetic scaffold antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0049] By "pharmaceutically acceptable carrier" is meant a carrier which is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.

[0050] 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 breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, bilecyst, bile duct, small intestine, urinary system (including the kidney, bladder, and epithelium of the urinary tract), female genital system (including the uterine neck, uterus, ovary, chorioma, and gestational trophoblast), male genital system (including the prostate, seminal vesicle, and testicles), endocrine glands (including the thyroid gland, adrenal gland, and pituitary body), skin (including angioma, melanoma, sarcoma originating from bone or soft tissue, and Kaposi's sarcoma), brain and meninges (including astrocytoma, neuroastrocytoma, spongioblastoma, retinoblastoma, neuroma, neuroblastoma, neurinoma and neuroblastoma), nerves, eyes, hemopoietic system (including chloroleukemia, plasmacytoma and dermal T lymphoma/leukemia), and immune system (including lymphoma, e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma). An example of a non-solid tumor proliferative disease is leukemia (e.g., acute lymphoblastic leukemia).

[0051] The term "recombinant antibody," refers to an antibody prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes (e.g., human immunoglobulin genes) or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library (e.g., containing human antibody sequences) using phage, yeast, or synthetic scaffold display, and (d) antibodies prepared, expressed, created, or isolated by any other means that involve splicing of immunoglobulin gene sequences (e.g., human immunoglobulin genes) to other DNA sequences.

[0052] By "specifically bind" is meant the preferential association of a binding moiety (e.g., an antibody, antibody fragment, receptor, ligand, or small molecule portion of an agent as described herein) to a target molecule (e.g., a secreted target molecule, such as a cytokine, chemokine, hormone, receptor, or ligand) or to a cell or tissue bearing the target molecule (e.g., a cell surface antigen, such as a receptor or ligand) and not to non-target cells or tissues lacking the target molecule. It is recognized that a certain degree of non-specific interaction may occur between a binding moiety and a non-target molecule (present alone or in combination with a cell or tissue). Nevertheless, specific binding may be distinguished as mediated through specific recognition of the target molecule. Specific binding results in a stronger association between the binding moiety (e.g., an antibody) and e.g., cells bearing the target molecule (e.g., an antigen) than between the binding moiety and e.g., cells lacking the target molecule. Specific binding typically results in greater than 2-fold, preferably greater than 5-fold, more preferably greater than 10-fold and most preferably greater than 100-fold increase in amount of bound binding moiety (per unit time) to e.g., a cell or tissue bearing the target molecule or marker as compared to a cell or tissue lacking that target molecule or marker. Binding moieties bind to the target molecule or marker 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^-15M. Specific binding to a protein under such conditions requires a binding moiety that is selected for its specificity for that particular protein. A variety of assay formats are appropriate for selecting binding moieties (e.g., antibodies) capable of specifically binding to a particular target molecule. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0053] By "sequence identity" is meant (in the context of comparing a polynucleotide or polypeptide sequence to a reference sequence) that the polynucleotide or polypeptide sequence is the same as the reference sequence or has a specified percentage of nucleotides or amino acid residues that are the same at the corresponding locations within the reference sequence when the two sequences are optimally aligned.

[0054] By "surface-exposed amino acid residue" or "surface-exposed" is meant an amino acid residue that is present on the exterior face of the folded and conformationally-correct tertiary structure of a HSA polypeptide. Such residues can be substituted with e.g., other, chemically-reactive, amino acids (e.g., cysteine) to allow for site-specific conjugation of diagnostic or therapeutic agents. Additionally, surface-exposed amino acid residues can be substituted to allow (e.g., by addition of serine, threonine, or asparagine residues, or glycosylation motifs) or prevent (e.g., by removal of 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 (amino acid numbering is relative to e.g., the sequence of the HSA linker set forth in SEQ ID NO:1). Other surface-exposed residues can be identified by the skilled artisan using the HSA crystal structure (Sugio et al., "Crystal structure of human serum albumin at 2.5 A resolution," Protein Eng. 12:439-446 (1999)). A "subject" refers to a human patient or a nude mouse xenograft model comprising human tumor cells.

[0055] A "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), or an HSA conjugate that contains one or more binding moieties (e.g., an HSA linker bonded to one or more antibodies or antibody fragments) can specifically bind. Preferred target molecules are exposed on the exterior of a target cell (e.g., a cell-surface or secreted protein) but target molecules may alternately or also be present in the interior of a target cell.

[0056] "Treating" preferably provides a reduction (e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100%) in the progression or severity of a human disease or disorder (e.g., an autoimmune or proliferative disease), or in the progression, severity, or frequency of one or more symptoms of the human disease or disorder in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 is an illustration showing the schematic representation of an exemplary HSA linker conjugate. The connector between the amino terminal binding moiety and the HSA linker has a sequence of alanine, alanine and serine. The connector between the HSA linker and the carboxy terminal binding moiety has a sequence of alanine, alanine, alanine, leucine (SEQ ID NO:5).

[0058] FIG. 2 is a graph showing that B2B3 variants inhibit HRG-induced pErbB3 in ZR75-1 breast cancer cells.

[0059] FIG. 3A is a graph showing the inhibition of phosphorylated ErbB3 in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B1D2-2 (A5-HSA-B1D2. Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0060] FIG. 3B is a graph showing the inhibition of phosphorylated ErbB3 in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B1D2-1 (H3-HSA-B1D2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0061] FIG. 3C is a graph showing the inhibition of phosphorylated ErbB3 in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B2B3-10 (H3-HSA-F5B6H2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0062] FIG. 3D is a graph showing the inhibition of phosphorylated ErbB3 in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B2B3-8 (F4-HSA-F5B6H2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0063] FIG. 4A is a graph showing the inhibition of phosphorylated AKT in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B1D2-2 (A5-HSA-B1D2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0064] FIG. 4B is a graph showing the inhibition of phosphorylated AKT in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B1D2-1 (H3-HSA-B1D2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0065] FIG. 4C is a graph showing the inhibition of phosphorylated AKT in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B2B3-10 (H3-HSA-F5B6H2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0066] FIG. 4D is a graph showing the inhibition of phosphorylated AKT in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B2B3-8 (F4-HSA-F5B6H2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0067] FIG. 5A is a graph showing that inhibition of phosphorylated ERK in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B1D2-2 (A5-HSA-B1D2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0068] FIG. 5B is a graph showing that inhibition of phosphorylated ERK in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B1D2-1 (H3-HSA-B1D2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0069] FIG. 5C is a graph showing that inhibition of phosphorylated ERK in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B2B3-10 (H3-HSA-F5B6H2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0070] FIG. 5D is a graph showing that inhibition of phosphorylated ERK in BT474 breast cancer cells following 24 hour pre-treatment with the HSA linker conjugate B2B3-8 (F4-HSA-F5B6H2). Further details regarding this HSA linker conjugate is set forth below, e.g., in Table 6.

[0071] FIG. 6 is a graph showing that treatment of BT474 breast cancer cells with B2B3-1 variants causes G1 arrest and a decrease in the number of cells in S phase.

[0072] FIG. 7 is a flow cytometry histogram showing that pre-incubation of BT-474-M3 cells with 1 μM B2B3-1 substantially blocks binding of HRG.

[0073] FIG. 8A is a graph showing that B2B3-1 inhibits ErbB3 phosphorylation in B-T474-M3 cells. Breast cancer cells BT-474-M3 were pre-treated with a dose titration of B2B3-1 for 24 hours and then stimulated for 10 minutes with 5 nM of HRG 1βEGF domain. The phosphorylation status of ErbB3 was then examined using an ELISA assay.

[0074] FIG. 8B is a graph showing that B2B3-1 inhibits ErbB3 phosphorylation in ZR75-3 0 cells. Breast cancer cells ZR75-3 0 (FIGS. 8B and 8D) were pre-treated with a dose titration of B2B3-1 for 24 hours and then stimulated for 10 minutes with 5 nM of HRG 1β EGF domain. The phosphorylation status of ErbB3 was then examined using an ELISA assay.

[0075] FIG. 8C is a graph showing that B2B3-1 inhibits AKT phosphorylation in B-T474-M3 cells. Breast cancer cells BT-474-M3 were pre-treated with a dose titration of B2B3-1 for 24 hours and then stimulated for 10 minutes with 5 nM of HRG 1βEGF domain. The phosphorylation status of AKT was then examined using an ELISA assay.

[0076] FIG. 8D is a graph showing that B2B3-1 inhibits AKT phosphorylation in ZR75-3 0 cells. Breast cancer cells ZR75-3 0 were pre-treated with a dose titration of B2B3-1 for 24 hours and then stimulated for 10 minutes with 5 nM of HRG 1βEGF domain. The phosphorylation status of AKT was then examined using an ELISA assay.

[0077] FIG. 9 is a photograph of a Western blot that shows the effect of treatment with increasing concentrations of B2B3-1 on signaling proteins in BT474 breast cancer cells. "p-" indicates the tyrosine-posphorylated form of the signaling protein. Beta tubulin (not a signaling protein in this context) provides a loading control. Beta tubulin (not a signaling protein in this context) provides a loading control.

[0078] FIG. 10 is a photograph of a Western blot that shows the immunoprecipitation of B2B3-1 treated BT474 breast cancer cells. Beta tubulin provides a control for levels of cellular proteins input into the immunoprecipitation reactions.

[0079] FIG. 11A is a graph showing B2B3-1 treatment of BT-474 cell line causes G1 arrest and a decrease in the population of cells in S phase (FIG. 11A).

[0080] FIG. 11B is a graph showing B2B3-1 treatment of BT-474 cell line inhibits colony formation in both BT-474 and SKBr3 cells compared to untreated cells.

[0081] FIG. 11C is a graph showing B2B3-1 inhibits proliferation of BT-474-M3 cells in a cell impedance assay.

[0082] FIG. 12 is a graph showing that B2B3-1 does not stimulate ErbB3 phosphorylation in ZR75-1 cells.

[0083] FIG. 13A is a graph showing that B2B3-1 binds specifically to ErbB3.

[0084] FIG. 13B is a graph showing that B2B3-1 binds specifically to ErbB2.

[0085] FIG. 14 is a graph showing that avidity binding of B2B3-1 to MALME-3 cells results in a significant increase in apparent binding affinity compared to ErbB2-only binding variant, SKO-3, and ErbB3-only binding variant, SKO-2.

[0086] FIG. 15A is a graph showing the stability of B2B3-1 in mouse serum. 100 nM B2B3-1 was incubated in mouse serum at 37° C. for a period of 120 hours. Samples were removed at 0, 24, 48, 72, 96 and 120 hours and the ability of B2B3-1 to bind both ErbB2 and ErbB3 was measured by ELISA (RLU=relative light units).

[0087] FIG. 15B is a graph showing the stability of B2B3-1 in Cynomolgus monkey serum. 100 nM B2B3-1 was incubated in Cynomolgus monkey serum at 37° C. for a period of 120 hours. Samples were removed at 0, 24, 48, 72, 96 and 120 hours and the ability of B2B3-1 to bind both ErbB2 and ErbB3 was measured by ELISA (RLU=relative light units).

[0088] FIG. 15C is a graph showing the stability of B2B3-1 in human serum. 100 nM B2B3-1 was incubated in human serum at 37° C. for a period of 120 hours. Samples were removed at 0, 24, 48, 72, 96 and 120 hours and the ability of B2B3-1 to bind both ErbB2 and ErbB3 was measured by ELISA (RLU=relative light units).

[0089] FIG. 16 is a graph showing B2B3-1 dose response in a BT-474-M3 breast cancer xenograft model. The relationship of B2B3-1 dose on tumor response was assessed in the BT-474-M3 breast tumor line at the doses indicated. HSA was given at 52.5 mg/kg, which is an equimolar dose to the 90 mg/kg B2B3-1 dose.

[0090] FIG. 17A is a graph showing that B2B3-1 is effective in a Calu-3 (human lung adenocarcinoma) xenograft model in an ErbB2 dependent manner. Mice were treated with 30 mg/kg of B2B3-1 every 3 days or HSA control at an equimolar dose to B2B3-1.

[0091] FIG. 17B is a graph showing that B2B3-1 is effective in a SKOV-3 (human ovarian adenocarcinoma) xenograft model in an ErbB2 dependent manner. Mice were treated with 30 mg/kg of B2B3-1 every 3 days or HSA control at an equimolar dose to B2B3-1.

[0092] FIG. 17C is a graph showing that B2B3-1 is effective in a NCI-N87 (human gastric carcinoma) xenograft model in an ErbB2 dependent manner. Mice were treated with 30 mg/kg of B2B3-1 every 3 days or HSA control at an equimolar dose to B2B3-1.

[0093] FIG. 17D is a graph showing that B2B3-1 is effective in a ACHN (human kidney adenocarcinoma) xenograft model in an ErbB2 dependent manner. Mice were treated with 30 mg/kg of B2B3-1 every 3 days or HSA control at an equimolar dose to B2B3-1.

[0094] FIG. 17E is a graph showing that B2B3-1 is effective in a MDA-MB-361 (human breast adenocarcinoma) xenograft model in an ErbB2 dependent manner. Mice were treated with 30 mg/kg of B2B3-1 every 3 days or HSA control at an equimolar dose to B2B3-1.

[0095] FIG. 18A is a graph showing that the over-expression of ErbB2 converts B2B3-1 non-responder ADRr breast cancer xenograft model into a responder. ErbB2 was over-expressed in wild type ADRr xenografts using a retroviral expression system.

[0096] FIG. 18B is a graph showing that the over-expression of ErbB2 converts B2B3-1 non-responder ADRr breast cancer xenograft model into a responder. ErbB2 was over-expressed in ADRr-E2 xenografts using a retroviral expression system.

[0097] FIG. 19A is a graph showing that B2B3-1 activity correlates positively with ErbB2 expression levels in vitro.

[0098] FIG. 19B is a graph showing that B2B3-1 activity correlates positively with ErbB2 expression levels in vivo.

[0099] FIG. 20A shows that B2B3-1 treatment modifies tumor cell cycling. FIG. 20A includes fluorescent micrographs showing that B2B3-1 treatment of BT474-M3 breast tumor cells for 6 hours results in translocation of cell cycle inhibitor p27.sup.kip1 to the nucleus. Hoechst stain was used to identify the nucleus.

[0100] 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 levels of the cell cycle regulator Cyclin D1. The cytoskeleton protein vinculin was probed as a protein loading control in this experiment.

[0101] FIG. 21A is a micrograph showing that B2B3-1 treatment of BT474 breast tumor xenografts results in translocation of p27.sup.kip1 to the nucleus. BT474 breast tumor xenografts were treated with B2B3-1 at a dose of 30 mg/kg every 3 days for a total of 4 doses and stained for p27.sup.kip1.

[0102] FIG. 21B is a micrograph showing the effect of an equimolar dose of HSA on BT474 breast tumor xenografts treated every 3 days for a total of 4 doses and stained for p27.sup.kip1.

[0103] FIG. 22A is a fluorescent micrograph showing that B2B3-1 treatment results in a reduction of the proliferation marker Ki67 in BT474-M3 breast cancer xenograft. BT474-M3 breast tumor xenografts were treated with B2B3-1 at a dose of 30 mg/kg every 3 days for a total of 4 doses.

[0104] FIG. 22B is a fluorescent micrograph showing the effect of an equimolar dose of HSA on BT474-M3 breast cancer xenograft treated every 3 days for a total of 4 doses.

[0105] FIG. 23A is a fluorescent micrograph showing that B2B3-1 treatment results in a reduction of vessel density in BT474-M3 breast cancer xenograft tumors (as determined by a reduction in CD31 staining). BT474-M3 breast tumor xenografts were treated with B2B3-1 at a dose of 30 mg/kg every 3 days for a total of 4 doses.

[0106] FIG. 23B is a fluorescent micrograph showing the effect of an equimolar dose of HSA on vessel density in BT474-M3 breast cancer xenograft tumors (as determined by a reduction in CD31 staining) treated every 3 days for a total of 4 doses.

[0107] FIG. 24A is a graph showing that B2B3-1 inhibits phosphorylation of ErbB3 in vivo. Lysates from individual BT-474-M3 xenograft tumors treated with B2B3-1 (M1-M5) or control HSA (H1-H2) were subjected to SDS-PAGE and probed for pErbB3 and beta tubulin using Western blot analysis.

[0108] FIG. 24B is a graph showing that normalization of the mean pErbB3 signal to the mean beta tubulin signal demonstrated that B2B3-1 treated tumors contained significantly less pErbB3 than HSA tumors.

[0109] FIGS. 25A and B are graphs showing the in vivo activity of B2B3-1 in BT-474-M3 shPTEN and shControl xenografts. Cultured BT-474-M3 tumor cells were transfected with a control vector (FIG. 25A) or with a retroviral vector expressing shPTEN (FIG. 25B), which knocks out PTEN activity. BT-474-M3 breast cancer cells thus engineered to knock out PTEN activity were injected into the right flank of mice, while cells transfected with control vector were injected into the left flank of the same mouse. Mice were treated with 30 mg/kg B2B3-1 every 3 days or 10 mg/kg Herceptin every week and HSA was injected as a control at an equimolar dose to B2B3-1. B2B3-1 and Herceptin promoted a reduction in the size of tumors formed by control BT-474-M3 breast cancer cells (FIG. 25A), whereas only B2B3-1 (and not Herceptin) promoted a reduction in the size of tumors formed by BT-474-M3 breast cancer cells lacking expression of PTEN (FIG. 25B).

[0110] FIGS. 26A-B show that B2B3-1 inhibits phosphorylation of AKT in BT-474-M3 xenografts that have reduced PTEN activity. Tumors were lysed following the completion of treatment (q3d×11) and tested for PTEN, pErbB3, and pAKT expression levels by Western blot analysis (FIG. 26A). Densitometry on the band intensity for pAKT normalized to total AKT and total protein demonstrate that B2B3-1 was able to inhibit phosphorylation of this protein, when Herceptin did not (FIG. 26B).

[0111] FIG. 27A is a graph showing the single dose pharmacokinetic properties of 5 mg/kg bolus dose of B2B3-1 in nu/nu mice. B2B3-1 serum concentrations are comparable measured by the HSA assay or ErbB2/ErbB3 assay, indicating that the antigen binding activity of B2B3-1 is retained in circulation.

[0112] FIG. 27B is a graph showing the single dose pharmacokinetic properties of 15 mg/kg bolus dose of B2B3-1 in nu/nu mice. B2B3-1 serum concentrations are comparable measured by the HSA assay or ErbB2/ErbB3 assay, indicating that the antigen binding activity of B2B3-1 is retained in circulation.

[0113] FIG. 27C is a graph showing the single dose pharmacokinetic properties of 30 mg/kg bolus dose of B2B3-1 in nu/nu mice. B2B3-1 serum concentrations are comparable measured by the HSA assay or ErbB2/ErbB3 assay, indicating that the antigen binding activity of B2B3-1 is retained in circulation.

[0114] FIG. 27D is a graph showing the single dose pharmacokinetic properties of 45 mg/kg bolus dose of B2B3-1 in nu/nu mice. B2B3-1 serum concentrations are comparable measured by the HSA assay or ErbB2/ErbB3 assay, indicating that the antigen binding activity of B2B3-1 is retained in circulation.

[0115] FIG. 28 is a graph showing the dose-exposure relationship for 5, 15, 30, and 45 mg/kg bolus doses of B2B3-1 in nude mice. Increases in dose result in a linear increase in overall exposure to B2B3-1.

[0116] FIG. 29 is a graph showing the B2B3-1 serum concentrations measured in Cynomolgus monkeys dosed every three days for 4 doses with 4 mg/kg (n=2), 20 mg/kg (n=2) and 200 mg/kg (up to 336 hour n=4, for 384, 552 and 672 hour time points n=2).

[0117] FIG. 30 is an illustration of the B2B3-1 expression plasmid pMP10k4H3-mHSA-B1D2.

[0118] FIG. 31 is an illustration of the neomycin resistance plasmid pSV2-neo.

[0119] FIG. 32 is an illustration of the hygromycin resistance plasmid pTK-Hyg.

[0120] FIG. 33 shows data demonstrating that B2B3-1 dosed q7d shows equivalent efficacy to q3d dosing.

[0121] FIG. 34 shows western blot data demonstrating that B2B3-1 and trastuzumab exhibit different mechanisms of ErbB3 inhibition.

[0122] FIG. 35 A-C shows the results of the experiments detailed in Example 43 in which B2B3-1 combination treatment with trastuzumab was studied in spheroids of various human breast cancer cell lines, which serve as a model for human breast tumors.

[0123] 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 is one third that of each indicated concentration of B2B3-1.

[0124] FIG. 36 show the results of the in vivo tumor xenograft experiments detailed in Example 44. "Days" on the X-axis indicates days post tumor implant. Error bars for each data point represent the response for at least two independent xenografts.

[0125] FIG. 37 shows data obtained from a xenograft model essentially as described in Example 44, except that the tumor cells used are N-87 gastric tumor cells which may be obtained from the US National Cancer Institute.

[0126] FIG. 38 shows a subset of the data presented in FIG. 36.

DETAILED DESCRIPTION

[0127] This invention provides human serum albumin (HSA) linkers, as well as HSA linker conjugates (e.g., binding, diagnostic, or therapeutic agents) that comprise an HSA linker and one or more additional moieties such as binding moieties. Such HSA linker conjugates have desirable properties such as, for example, an increased in vivo half-life of between 6 hours and 7 days, and do not induce significant humoral or cell-mediated immune responses when administered in vivo to a mammal (e.g., a human). In one aspect, the invention provides a mutated HSA linker that has 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 an HSA linker bonded to one or more binding moieties (e.g., antibodies, antibody fragments, receptor/ligands, or small molecules) for diagnostic or therapeutic applications in a mammal (e.g., a human) in vivo or for use in vitro in connection with mammalian cells, tissues, or organs. In a further aspect, the HSA linker may be coupled to one or more immunomodulatory agents, cytotoxic or cytostatic agents, detectable labels, or radioactive agents for diagnostic or therapeutic applications in a mammal (or in connection with a mammalian cell, tissue, or organ). An HSA linker conjugate, which includes the HSA linker, can be optionally combined with one or more pharmaceutically acceptable carriers or excipients and can be formulated to be administered intravenously, intramuscularly, orally, by inhalation, parenterally, intraperitoneally, intraarterially, transdermally, sublingually, nasally, through use of suppositories, transbuccally, liposomally, adiposally, opthalmically, intraocularly, subcutaneously, intrathecally, topically, or locally. An HSA linker conjugate can, but need not, be combined or coadministered with one or more biologically-active agents (e.g., biological or chemical agents, such as chemotherapeutics and antineoplastic agents). In a further aspect, the invention provides a kit, with instructions, for the conjugation of binding moieties (e.g., antibodies, antibody fragments, receptors or ligands), immunomodulatory agents, cytotoxic or cytostatic agents, detectable labels, or radioactive agents to the HSA linker to prepare HSA linker conjugates that can be used for diagnostic or therapeutic applications.

Human Serum Albumin (HSA) Linkers

[0128] An HSA linker may comprise a wild-type HSA amino acid sequence, as set forth in SEQ ID NO:3. Alternatively, the HSA linker may comprise an altered, or mutated, sequence. One mutated HSA linker contains two amino acid mutations, at positions 34 and 503, relative to the wild-type HSA amino acid sequence set forth in SEQ ID NO:3. 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 the amino acid and corresponding nucleotide sequence set forth in SEQ ID NOS:1 and 2, respectively. This mutated HSA linker contains two amino acid substitutions (i.e., serine for cysteine at amino acid residue 34 ("C34S") and glutamine for asparagine at amino acid residue 503 ("N503Q")). The HSA linker, when bonded to one or more binding moieties (e.g., antibodies, antibody fragments (e.g., single chain antibodies), or other targeting or biologically active agents (e.g., receptors and ligands)), confers several beneficial pharmacologic properties to those conjugates and to additional diagnostic or therapeutic agents also conjoined (e.g., immunomodulatory agents, cytotoxic or cytostatic agents, detectable labels, or radioactive agents)) relative to the pharmacologic properties of these agents in the absence of the HSA linker. These benefits can include decreased immunogenicity (e.g., decreased host antibody neutralization of linker-antibody conjugates), increased detection of HSA linker conjugates (e.g., by mass spectroscopy) and increased pharmacologic 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, the asparagine at amino acid residue 503 is sensitive to deamination, likely resulting in reduced pharmacologic half-life. The substitution of glutamine for asparagine at amino acid residue 503 can result in increased pharmacologic half-life of the HSA linker, and correspondingly, of conjugate agents that include the HSA linker when administered to a mammal (e.g., a human) or cells, tissues, or organs thereof.

[0129] In other embodiments, the mutated HSA linker includes domain I of HSA (SEQ ID NO:53; residues 1-197 of SEQ ID NO:1), domain III of HSA (SEQ ID NO:55; residues 381-585 of SEQ ID NO:1), combination of domains I and III of HSA, or a combination of domain I or III of HSA with domain II of HSA (SEQ ID NO:54; residues 189-385 of SEQ ID NO:1). For example, an HSA linker can include domains I and II, I and III, or II and III. In addition, the cysteine residue at position 34 (i.e., C34) of domain I (SEQ ID NO:53) 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) of domain III (SEQ ID NO:55) can be mutated to any amino acid residue other than asparagine (e.g., glutamine, serine, histidine, or alanine). These HSA linkers can be incorporated into an HSA linker conjugate, which includes one or more of a peptide connector, a binding moiety, and therapeutic or diagnostic agents, each of which is described in detail below.

Peptide Connectors

[0130] To facilitate the conjugation of binding moieties, as defined herein, to the HSA linker, short (e.g., 2-20 amino acids in length) peptide connectors that can be bonded (e.g, covalently (e.g., a peptidic bond), ionically, or hydrophobically bonded, or via a high-affinity protein-protein binding interaction (e.g., biotin and avidin)) to the amino or carboxy termini of an HSA linker. These connectors provide flexible tethers to which any of the binding moieties described herein can be attached. A peptide connector 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 connector is a sequence of, e.g., glycine, alanine, serine, glutamine, leucine, or valine residues. Although not specifically enumerated herein, the connector can be solely glycine, alanine, serine, glutamine, leucine, or valine residues, or it may be any combination of these residues up to about 20 amino acids in length. In a preferred embodiment, the connector attached to the amino terminus of an HSA linker has the amino acid sequence AAS or AAQ and the connector attached to the carboxy terminus has the amino acid sequence "AAAL" (SEQ ID NO:5). The connector can be covalently bound to the amino or carboxy terminal residue of the HSA linker, to an amino acid residue within the HSA linker, or can be included between one or more binding moieties, if present.

HSA Linker Manufacture

[0131] The HSA linker, with or without one or more peptide connectors described above, one or more of the binding moieties described below, polypeptide-based detectable labels, and other polypeptide-based therapeutic agents, can be produced recombinantly. For example, a nucleotide sequence encoding the HSA linker (and one or more of the optional elements) may be expressed (e.g., in a plasmid, viral vector, or transgenically) in a bacterial (e.g., E. coli), insect, yeast, or mammalian cell (e.g., a CHO cell), or a mammalian tissue, organ, or organism (e.g., a transgenic rodent, ungulate (e.g., a goat), or non-human primate). After expression of the HSA linker in the host cell, tissue, or organ, the skilled artisan may isolate and purify the HSA linker using standard protein purification methods (e.g., FPLC or affinity chromatography). A recombinant expression system for the production of an HSA linker in combination with two binding moieties is illustrated in FIG. 1.

[0132] Alternatively, the HSA linker, with or without one or more of the optional elements described above, can be synthetically produced. For example, the HSA linker or HSA linker conjugate can be prepared by techniques generally established in the art of peptide synthesis, such as the solid-phase approach. Solid-phase synthesis involves the stepwise addition of amino acid residues to a growing peptide chain that is linked to an insoluble support or matrix, such as polystyrene. The C-terminus residue of the peptide is first anchored to a commercially available support with its amino group protected with an N-protecting agent such as a t-butyloxycarbonyl group (tBoc) or a fluorenylmethoxycarbonyl (FMOC) group. The amino-protecting group is removed with suitable deprotecting agents such as TFA in the case of tBOC or piperidine for FMOC and the next amino acid residue (in N-protected form) is added with a coupling agent such as dicyclocarbodiimide (DCC). Upon formation of a peptide bond, the reagents are washed from the support. After addition of the final residue, the agent is cleaved from the support 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 manufactured in one, two, three, or more segments, which can then be ligated to form the whole HSA linker construct.

Binding Moieties

[0133] HSA linker conjugates may include one or more binding moieties, such as antibodies, antibody fragments (as defined herein, e.g., a single chain Fv (scFv)) or receptor/ligands (i.e., protein or glycoprotein ligands or receptors)) that allow selective and specific binding of the HSA linker conjugate to a target cell, tissue, or organ. The binding moieties can be bonded to the HSA linker (e.g., via a covalent (e.g., a peptide bond), ionic, or hydrophobic bond, or via a high-affinity protein-protein binding interaction (e.g., biotin and avidin)).

[0134] One or more binding moieties can be bonded to an HSA linker. In one embodiment, two or more identical binding moieties (i.e., moieties having the same structure and binding affinities) are bonded to an HSA linker, one or more (e.g., in tandem) each at the amino and carboxy termini, the HSA linker thereby affording improved avidity of the binding moieties for their target antigen. Alternatively, two or more different binding moieties (e.g., an antibody, such as a scFv, with binding affinities for two or more different target molecules, or scFv with binding affinities for two or more different epitopes on the same target molecule) can be bonded to an HSA linker (e.g., a bispecific HSA linker conjugate) to allow multiple target antigens or epitopes to be bound by the HSA linker conjugate. In another embodiment, different species of binding moieties can also be bonded to an HSA linker to bestow, for example, two or more different binding specificities or agonistic/antagonistic biological properties on the linker conjugate. Useful combinations of binding moiety pairs for use in the preparation of bispecific HSA linker conjugates are disclosed in, e.g., International Patent Application Publications WO 2006/091209 and WO 2005/117973, herein incorporated by reference. In other embodiments, more than two binding moieties (e.g., the same or different binding moieties) can be bonded to an HSA linker to form an HSA linker conjugate.

[0135] The invention features an HSA linker conjugate having at least first and second binding moieties, each of which can be bound at either the amino or carboxy terminus of the HSA linker, or to peptide connectors, as defined herein, present at either or both termini FIG. 1 illustrates an exemplary mutated HSA linker in which two binding moieties ("arm 1" and "arm 2") are bonded to the mutated HSA linker by the amino terminal peptide connector AAS and carboxy terminal peptide connector AAAL (SEQ ID NO:5). Binding moieties (e.g., an antibody or scFv) can also be bound to other loci (e.g., internal amino acid residues of the HSA linker), for example, 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 the HSA linker.

[0136] Binding moieties that can be included in an HSA linker conjugate include antibodies, antibody fragments, receptors, and ligands. Binding moieties bound to an HSA linker may be recombinant (e.g., human, murine, chimeric, or humanized), synthetic, or natural. Representative binding moieties include, for example, complete antibodies, domain antibodies, diabodies, triabodies, bi-specific antibodies, antibody fragments, Fab fragments, F(ab')2 molecules, single chain Fv (scFv) molecules, bispecific single chain Fv ((scFv')₂) molecules, tandem scFv fragments, antibody fusion proteins, hormones, receptors, ligands, and aptamers, and biologically-active fragments thereof.

Antibodies

[0137] Antibodies include the IgG, IgA, IgM, IgD, and IgE isotypes. Antibodies or antibody fragments thereof, as used herein, contain one or more complementarity determining regions (CDR) or binding peptides that bind to target proteins, glycoproteins, or epitopes present on the exterior or in the interior of target cells.

[0138] Many of the antibodies, or fragments thereof, described herein can undergo non-critical amino-acid substitutions, additions or deletions in both the variable and constant regions without loss of binding specificity or effector functions, or intolerable reduction of binding affinity (e.g., below about 10^-7 M). Usually, an antibody or antibody fragment incorporating such alterations exhibits substantial sequence identity to a reference antibody or antibody fragment from which it is derived. Occasionally, a mutated antibody or antibody fragment can be selected having the same specificity and increased affinity compared with a reference antibody or antibody fragment from which it was derived. Phage-display technology offers powerful techniques for selecting such antibodies. See, e.g., Dower et al., WO 91/17271 McCafferty et al., WO 92/01047; and Huse, WO 92/06204, incorporated by reference herein.

[0139] The HSA linker can also be bonded to one or more fragments of an antibody that retain the ability to bind with specificity to a target antigen. Antibody fragments include separate variable heavy chains, variable light chains, Fab, Fab', F(ab')₂, Fabc, and scFv. Fragments can be produced by enzymatic or chemical separation of intact immunoglobulins. For example, a F(ab')₂ fragment can be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Pubs., N.Y. (1988). Fab fragments may be obtained from F(ab')₂ fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents. Fragments can also be produced by recombinant DNA techniques. Segments of nucleic acids encoding selected fragments are produced by digestion of full-length coding sequences with restriction enzymes, or by de novo synthesis. Often fragments are expressed in the form of phage-coat fusion proteins. This manner of expression is advantageous for affinity-sharpening of antibodies.

Humanized Antibodies

[0140] Humanized antibodies may also be used in combination with the HSA linker, in which one or more of the antibody CDRs are derived from a non-human antibody sequence, and one or more, but preferably all, of the CDRs bind specifically to an antigen (e.g., a protein, glycoprotein, or other suitable epitope).

[0141] A humanized antibody contains constant framework regions derived substantially from a human antibody (termed an acceptor antibody), as well as, in some instances, a majority of the variable region derived from a human antibody. One or more of the CDRs (all or a portion thereof, as well as discreet amino acids surrounding one or more of the CDRs) are provided from a non-human antibody, such as a mouse antibody. The constant region(s) of the antibody, may or may not be present.

[0142] The substitution of one or more mouse CDRs into a human variable domain framework is most likely to result in retention of their 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 originated. This is achieved by obtaining the human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence and structural identity with the murine variable framework domains from which the CDRs were derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies, consensus sequences of several human antibodies, or can be human germline variable domain sequences. See, e.g., Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).

[0143] Suitable human antibody sequences are identified by alignments of the amino acid sequences of the mouse variable regions with the sequences of known human antibodies. The comparison is performed separately for heavy and light chains but the principles are similar for each.

[0144] Methods of preparing chimeric and humanized antibodies and antibody fragments are described in, e.g., U.S. Pat. Nos. 4,816,567, 5,530,101, 5,622,701, 5,800,815, 5,874,540, 5,914,110, 5,928,904, 6,210,670, 6,677,436, and 7,067,313 and U.S. Patent Application Nos. 2002/0031508, 2004/0265311, and 2005/0226876. Preparation of antibody or fragments thereof is further described in, e.g., U.S. Pat. Nos. 6,331,415, 6,818,216, and 7,067,313.

Receptors and Ligands

[0145] Within certain HSA linker conjugates, protein or glycoprotein receptors or ligands are bound to an HSA linker. HSA linkers bonded with a receptor or ligand can be used, for example, to specifically target a secreted protein, a cell (e.g., a cancer cell), tissue, or organ. Furthermore, the specific binding of the HSA linker-receptor or -ligand conjugate to cognate target receptors or ligands can cause agonistic or antagonistic biological activity in intracellular or intercellular signaling pathways. As with the other binding moieties described herein, receptors and ligands, or fragments thereof, can be conjoined to the amino and/or carboxy termini of an HSA linker, to a peptide connector linked to the HSA linker or to an amino acid residue within the HSA linker.

[0146] Exemplary receptors and ligands that can be joined to an HSA linker include, but are not limited to, insulin-like growth factor 1 receptor (IGF1R), IGF2R, insulin-like growth factor (IGF), mesenchymal epithelial transition 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), heregulin, 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-α), TNF-β, folate receptor (FOLR), folate, transferrin receptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, α4 integrin, P-selectin, sphingosine-1-phosphate receptor-1 (S1PR), hyaluronate 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 cell 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^Y, MUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG-72 antigen, and biologically-active fragments thereof.

[0147] Receptors and ligands can be expressed, isolated, or joined to an HSA linker using any of the methods described supra.

Diagnostic Agents

[0148] The HSA linker, or any binding moiety conjugated thereto (e.g., antibody, antibody fragment, receptor, or ligand), can be coupled to a chelating agent or to a detectable label to form a diagnostic agent. Also contemplated are HSA linker conjugates that include a detectable label, as described herein, as well as one or more of the therapeutic agents or binding moieties described herein.

[0149] The HSA linker (or HSA linker conjugate) and chelator components can be coupled by reacting the free amino group of a threonine residue of the HSA linker (or HSA linker conjugate) with an appropriate functional group of the chelator, such as a carboxyl group or activated ester. For example, a such coupling may be achieved by incorporating the chelator ethylenediaminetetraacetic acid (EDTA), which is common in the art of coordination chemistry, when functionalized with a carboxyl substituent on the ethylene chain. Synthesis of EDTA derivatives of this type are 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 is free to react with the amino group of a peptide portion of the agent.

[0150] An HSA linker or an HSA linker conjugate may incorporate a metal chelator component that is peptidic, i.e., compatible with solid-phase peptide synthesis. In this case, the chelator may be coupled in the same manner as EDTA described above or, more conveniently, the chelator and HSA linker or HSA linker conjugate are synthesized in toto starting from the C-terminal residue of the HSA linker or HSA linker conjugate and ending with the N-terminal residue of the chelator.

[0151] An HSA linker or an HSA linker conjugate may further incorporate a linking group component that serves to couple the HSA linker to the chelator while not adversely affecting the biological properties of the HSA linker, the targeting function of the binding moiety portion(s) 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 reactive groups for coupling to the HSA linker or the HSA linker conjugate and to the chelator. An amino acid chain is the preferred linking group when the chelator is peptidic so that the HSA linker or HSA linker conjugate can be synthesized in toto by solid-phase techniques. An alkyl chain-linking group may be incorporated in the HSA linker or HSA linker conjugate by reacting the amino group of a threonine residue of a peptide portion of an HSA linker with a first functional group on the alkyl chain, such as a carboxyl group or an activated ester. Subsequently the chelator is attached to the alkyl chain to complete the formation of the HSA linker or HSA linker conjugate by reacting a second functional group on the alkyl chain with an appropriate group on the chelator. The second functional group on the alkyl chain is selected from substituents that are reactive with a functional group on the chelator while not being reactive with a threonine residue of the mutated HSA linker. For example, when the chelator incorporates a functional group such as a carboxyl group or an activated ester, the second functional group of the alkyl chain-linking group can be an amino group. It will be appreciated that formation of the HSA linker or HSA linker conjugate may require protection and deprotection of the functional groups present in order to avoid formation of undesired products. Protection and deprotection are accomplished using protecting groups, reagents, and protocols common in the art of organic synthesis. Particularly, protection and deprotection techniques employed in solid phase peptide synthesis described above may be used.

[0152] An alternative chemical linking group to an alkyl chain is polyethylene glycol (PEG), which is functionalized in the same manner as the alkyl chain described above for incorporation in the HSA linker or HSA linker conjugate. It will be appreciated that linking groups may alternatively be coupled first to the chelator and then to the HSA linker or HSA linker conjugate.

[0153] In one aspect, an HSA linker or HSA linker conjugate is coupled to a diagnostically useful metal capable of forming a complex. Suitable metals include, e.g., radionuclides, such as technetium and rhenium in their various forms (e.g., ⁹⁹ mTcO³+, ⁹⁹ mTcO₂.sup.+, ReO³+, and ReO₂.sup.+). Incorporation of the metal within the HSA linker or HSA linker conjugate can be achieved by various methods common in the art of coordination chemistry. When the metal is technetium-99 m, the following general procedure may be used to form a technetium complex. An HSA linker-chelator conjugate solution is formed initially by dissolving the HSA linker or HSA linker conjugate in aqueous alcohol such as ethanol. The solution is then degassed to remove oxygen then thiol protecting groups are removed with a suitable reagent, for example, with 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 the conjugate with an amount of a reducing agent such as stannous chloride sufficient to reduce technetium and heated. The labeled HSA linker or HSA linker conjugate may be separated from contaminants ⁹⁹ mTcO₄^- and colloidal ⁹⁹ mTcO₂ chromatographically, for example, with a C-18 Sep Pak cartridge.

[0154] In an alternative method, labeling of an HSA linker can be accomplished by a transchelation reaction. The technetium source is a solution of technetium complexed with labile ligands facilitating ligand exchange with the selected chelator. Suitable ligands for transchelation include tartarate, citrate, and heptagluconate. In this instance the preferred reducing reagent is sodium dithionite. It will be appreciated that the HSA linker or HSA linker conjugate may be labeled using the techniques described above, or alternatively the chelator itself may be labeled and subsequently coupled to an HSA linker to form an HSA linker-chelator conjugate; a process referred to as the "prelabeled ligand" method.

[0155] Another approach for labeling an HSA linker, or any agent conjugated thereto, involves immobilizing the HSA linker-chelator conjugate on a solid-phase support through a linkage that is cleaved upon metal chelation. This is achieved when the chelator is coupled to a functional group of the support by one of the complexing atoms. Preferably, a complexing sulfur atom is coupled to the support which is functionalized with a sulfur protecting group such as maleimide.

[0156] When labeled with a diagnostically useful metal, an agent that includes an HSA linker-chelator conjugate can be used to detect tissue at risk of developing cancer (e.g., lung cancer, breast cancer, colon cancer, and prostate cancer), age-related diseases (e.g., cardiovascular disease, cerebrovascular disease, or Alzheimer's disease), tobacco-related diseases (e.g., emphysema, aortic aneurysms, esophageal cancer, or squamous cell cancer of the head and neck) by procedures established in the art of diagnostic imaging. An agent that incorporates an HSA linker labeled with a radionuclide metal, such as technetium-99 m, may be administered to a mammal (e.g., a human) by intravenous injection in a pharmaceutically acceptable solution, such as isotonic saline, or by other methods described herein. The amount of a labeled agent appropriate for administration is dependent upon the distribution profile of the chosen HSA linker or HSA linker conjugate in the sense that an agent that incorporates a rapidly cleared HSA linker or HSA linker conjugate may be administered at higher doses than an agent that incorporates an HSA linker or HSA linker conjugate that clears less rapidly. Unit doses acceptable for imaging tissues are in the range of about 5-40 mCi for a 70 kg individual. The in vivo distribution and localization of an agent that incorporates a labeled HSA linker or HSA linker conjugate can be tracked by standard techniques described herein at an appropriate time subsequent to administration, typically between 30 minutes and 180 minutes and up to about 5 days depending upon the rate of accumulation at the target site with respect to the rate of clearance at non-target tissue.

[0157] An HSA linker, or any molecule or moiety conjugated thereto, can also be modified or labeled to facilitate diagnostic or therapeutic uses. Detectable labels such as a radioactive, fluorescent, heavy metal, or other molecules may be bound to any of the agents. Single, dual, or multiple labeling of an agent may be advantageous. For example, dual labeling with radioactive iodination of one or more residues combined with the additional coupling of, for example, ⁹⁰Y via a chelating group to amine-containing side or reactive groups, would allow combination labeling. This may be useful for specialized diagnostic needs such as identification of widely dispersed small neoplastic cell masses.

[0158] An HSA linker, or any molecule or moiety conjugated thereto, can also be modified, for example, by halogenation of the tyrosine residues of the peptide component. Halogens include fluorine, chlorine, bromine, iodine, and astatine. Such halogenated agents may be detectably labeled, e.g., if the halogen is a radioisotope, such as, for example, ¹⁸F, ⁷⁵Br, ⁷⁷Br, ¹²² I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, or ²¹¹At. Halogenated agents contain a halogen covalently bound to at least one amino acid, and preferably to D-Tyr residues in each agent molecule. Other suitable detectable modifications include binding of other compounds (e.g., a fluorochrome such as fluorescein) to a lysine residue of the agent, or analog, particularly an agent or analog having a linker including lysines.

[0159] Radioisotopes for radiolabeling an HSA linker, or any molecule or moiety conjugated thereto, include any radioisotope that can be covalently bound to a residue of the peptide component of the agent or analog thereof. The radioisotopes can also be selected from radioisotopes that emit either beta or gamma radiation, or alternatively, any of the agents can be modified to contain chelating groups that, for example, can be covalently bonded to lysine residue(s) of the HSA linker or any peptidic agent conjugated thereto. The chelating groups can then be modified to contain any of a variety of radioisotopes, such as gallium, indium, technetium, ytterbium, rhenium, or thallium (e.g., ¹²⁵I, ⁶⁷Ga, ¹¹¹In, ⁹⁹mTc, ¹⁶⁹Yb, ¹⁸⁶Re).

[0160] An HSA linker, or any molecule or moiety conjugated thereto, can be modified by attachment of a radioisotope. Preferable radioisotopes are those having a radioactive half-life corresponding to, or 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 preferably ⁷⁵Br, ⁷⁷Br, ⁷⁶Br, ¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, or ²¹¹At.

[0161] An agent that incorporates an HSA linker, or any molecule or moiety conjugated thereto, can be coupled to radioactive metals and used in 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, ²³Bi and ²14Bi. The choice of metal is determined based on the desired therapeutic or diagnostic application.

[0162] An HSA linker, or any molecule or moiety conjugated thereto, can be coupled to a metal component, to produce a diagnostic or therapeutic agent. A detectable label may be a metal ion from heavy elements or rare earth ions, such as Gd³+, Fe³+, Mn³+, or Cr²+. Agents that incorporate an HSA linker having paramagnetic or superparamagnetic metals conjoined thereto are useful as diagnostic agents in MRI imaging applications. Paramagnetic metals include, but are not limited to, chromium (III), manganese (II), iron (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).

[0163] Chelating groups may be used to indirectly couple detectable labels or other molecules to an HSA linker or to an agent conjugated thereto. Chelating groups can link agents with radiolabels, such as a bifunctional stable chelator, or can be linked to one or more terminal or internal amino acid reactive groups. An HSA linker, or any molecule or moeity conjugated thereto, can be linked via an isothiocyanate β-Ala or appropriate non-α-amino acid linker which prevents Edman degradation. Examples of chelators known in the art include, for example, the ininocarboxylic and polyaminopolycarboxylic reactive groups, ininocarboxylic and polyaminopolycarboxylic reactive groups, diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0164] An HSA linker, when expressed recombinantly, can be joined to a peptidic detectable label or diagnostic agent. Peptides and proteins that can be used as a detectable label with an 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 can also be incorporated into an HSA linker conjugate that also includes a therapeutic, cytotoxic, or cytostatic agent.

[0165] Fluorescent proteins or fluorochromes, 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 label joined to an HSA linker. Fluorescent proteins can be recombinantly expressed in a cell (e.g., a blood cell, such as a lymphocyte) following transfection or transduction of the cell with an expression vector that encodes the nucleotide sequence of the fluorescent protein. Upon exposure of the fluorescent protein to a stimulating frequency of light, the fluorescent protein will emit light at a low, medium, or high intensity that can be observed by eye under a microscope or by an optical imaging device. Exemplary fluorescent proteins suitable for use as the diagnostic sequence in agents are described in, e.g., U.S. Pat. Nos. 7,417,131 and 7,413,874, each of which is herein incorporated by reference.

[0166] Bioluminescent proteins can also be used as a detectable label incorporated into an HSA linker. Bioluminescent proteins, such as luciferase (e.g., firefly (SEQ ID NO:51), Renilla (SEQ ID NO:52), and Omphalotus luciferase) and aequorin, emit light as part of a chemical reaction with a substrate (e.g., luciferin and coelenterazine). In one embodiment, a vector encoding a luciferase gene provides for the in vivo, in vitro, or ex vivo 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 a diagnostic sequence and methods for their use are described in, e.g., U.S. Pat. Nos. 5,292,658, 5,670,356, 6,171,809, and 7,183,092, each of which is herein incorporated by reference.

[0167] Epitope tags are short amino acid sequences, e.g., 5-20 amino acid residues in length, that can be incorporated into an HSA linker conjugate as a detectable label to facilitate detection once expressed in a cell, secreted from the cell, or bound to a target cell. An agent that incorporates an epitope tag as a diagnostic sequence can be detected by virtue of its interaction with an antibody, antibody fragment, or other binding molecule specific for the epitope tag. Nucleotide sequences encoding the epitope tag are produced either by cloning appropriate portions of natural genes or by synthesizing a polynucleotide that encodes the epitope tag. An antibody, antibody fragment, or other binding molecule that binds an epitope tag can directly incorporate a detectable label (e.g., a fluorochrome, radiolabel, heavy metal, or enzyme such as horseradish peroxidase) or serve itself as a target for a secondary antibody, antibody fragment, or other binding molecule that incorporates such a label. Exemplary epitope tags that can be used as a diagnostic sequence include c-myc (SEQ ID NO:33), hemagglutinin (HA; SEQ ID NO:34), and histidine tag (His₆; SEQ ID NO:35). Furthermore, fluorescent (e.g., GFP) and bioluminescent proteins can also serve as epitope tags, as antibodies, antibody fragments, and other binding molecules are commercially available for the detection of these proteins.

[0168] The in vivo, in vitro, or ex vivo detection, imaging, or tracking of an HSA linker conjugate that incorporates a diagnostic sequence (e.g., a fluorescent protein, bioluminescent protein, or epitope tag) or any cell expressing or bound thereto can be accomplished using a microscope, flow cytometer, luminometer, or other state of the art optical imaging device, such as an IVIS® Imaging System (Caliper LifeSciences, Hopkinton, Mass.).

Therapeutic or Cytotoxic Agents Coupled to the HSA Linker

[0169] An HSA linker, or any molecule or moiety conjugated thereto, can be coupled to any known cytotoxic or therapeutic moiety to form an agent (an HSA linker conjugate) that can be administered to treat, inhibit, reduce, or ameliorate disease (e.g., a cancer, autoimmune disease, or cardiovascular disease) or one or more symptoms of disease. Examples include but are not limited to antineoplastic agents such as: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; adriamycin; aldesleukin; altretamine; ambomycin; a. metantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; camptothecin; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; combretestatin a-4; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daca (n-[2-(dimethyl-amino)ethyl]acridine-4-carboxamide); dactinomycin; daunorubicin hydrochloride; daunomycin; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; dolasatins; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; ellipticine; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; ethiodized oil i 131; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; 5-fdump; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; gold au 198; homocamptothecin; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta-i a; interferon gamma-i b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peploycinsulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; rhizoxin; rhizoxin d; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; strontium chloride sr 89; sulofenur; talisomycin; taxane; taxoid; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; thymitaq; tiazofurin; tirapazamine; tomudex; top53; topotecan hydrochloride; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine; vinblastine sulfate; vincristine; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride; 2-chlorodeoxyadenosine; 2' deoxyformycin; 9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid; 2chloro-2'-arabino-fluoro-2'-deoxyadenosine; 2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlor ethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-Nnitrosourea (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); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; cisplatin; carboplatin; ormaplatin; oxaliplatin; C1-973; DWA 2114R; JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6-mercaptopurine; 6-thioguanine; hypoxanthine; teniposide 9-amino camptothecin; topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14-hydroxy-retro-retinol; all-trans retinoic acid; 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).

[0170] Other therapeutic compounds include, but are not limited to, 20-pi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bleomycin A2; bleomycin B2; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin); canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2'deoxycoformycin (DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; discodermolide; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epothilones (A, R=H; B, R=Me); epithilones; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide; etoposide 4'-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maytansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; rnerbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; ifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mithracin; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; podophyllotoxin; porfimer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

[0171] HSA linker conjugates can also include site-specifically conjugated molecules and moieties. Site-specific conjugation allows for the controlled stoichiometric attachment to specific residues in the HSA linker of cytotoxic, immunomodulatory, or cytostatic agents including, e.g., anti tubulin agents, DNA minor groove binders, DNA replication inhibitors, alkylating agents, anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy or radiation sensitizer, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, steroids, taxanes, topoisomerase inhibitors, and vinca alkaloids or any other molecules or moieties described herein.

[0172] Techniques for conjugating therapeutic agents to proteins, and in particular to antibodies, are well-known (e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting 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 Drug Delivery," in Controlled Drug Delivery (Robinson et al., eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al., eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al., eds., Academic Press, 1985); Thorpe et al., Immunol. Rev. 62:119-58 (1982); and Doronina et al., "Development of potent monoclonal antibody auristatin conjugates for cancer therapy," Nature Biotech. 21:(7)778-784 (2003)). See also, e.g., PCT publication WO 89/12624.

[0173] An HSA linker, or any molecule or moiety conjugated thereto, can also be coupled to a lytic peptide. Such lytic peptides induce cell death and include, but are not limited to, streptolysin O; stoichactis toxin; phallolysin; staphylococcus alpha toxin; holothurin A; digitonin; melittin; lysolecithin; cardiotoxin; and cerebratulus A toxin (Kem et al., J. Biol. Chem. 253(16):5752-5757, 1978). An HSA linker, or any molecule or moiety conjugated thereto (e.g., antibody or antibody fragment conjugates), may be coupled to a synthetic peptide that shares some sequence homology or chemical characteristics with any of the naturally occurring peptide lysins; such characteristics include, but are not limited to, linearity, positive charge, amphipathicity, and formation of alpha-helical structures in a hydrophobic environment (Leuschner et al., Biology of Reproduction 73:860-865, 2005). An HSA linker, or any molecule or moiety conjugated thereto, can also be coupled to an agent that induces complement-mediated cell lysis such as, for example, the immunoglobulin F_c subunit. An HSA linker, or any molecule or moiety conjugated thereto, can also be coupled to any member of the phospholipase family of enzymes (including phospholipase A, phospholipase B, phospholipase C, or phospholipase D) or to a catalytically-active subunit thereof.

[0174] An HSA linker, or any molecule or moiety conjugated thereto, can also be coupled to a radioactive agent to form an agent that can be used for detection or therapeutic applications. Radioactive agents that can be used include but are not limited to Fibrinogen ¹²⁵I; Fludeoxyglucose ¹⁸F; Fluorodopa ¹⁸F; Insulin ¹²⁵I; Insulin ¹³¹I; lobenguane ¹²³I; Iodipamide Sodium ¹³¹I; Iodoantipyrine ¹³¹I; Iodocholesterol ¹³¹I; lodohippurate Sodium ¹²³I; Iodohippurate Sodium ¹²⁵I; Iodohippurate Sodium ¹³¹I; Iodopyracet ¹²⁵I; Iodopyracet ¹³¹I; lofetamine Hydrochloride ¹²³I; Iomethin ¹²⁵I; Iomethin ¹³¹I; ¹³¹I; Iothalamate Sodium ¹²⁵I; Iothalamate Sodium ¹³¹I tyrosine ¹³¹I; Liothyronine ¹²⁵I; Liothyronine ¹³¹I; Merisoprol Acetate ¹⁹⁷Hg; Merisoprol Acetate ²⁰³Hg; Merisoprol ¹⁹⁷Hg; Selenomethionine ⁷⁵Se; Technetium ⁹⁹mTc Antimony Trisulfide Colloid; Technetium ⁹⁹mTc Bicisate; Technetium ⁹⁹mTc Disofenin; Technetium ⁹⁹mTc Etidronate; Technetium ⁹⁹mTc Exametazime; Technetium ⁹⁹mTc Furifosmin; Technetium ⁹⁹mTc Gluceptate; Technetium ⁹⁹mTc Lidofenin; Technetium ⁹⁹mTc Mebrofenin; Technetium ⁹⁹mTc Medronate; Technetium ⁹⁹mTc Medronate Disodium; Technetium ⁹⁹mTc Mertiatide; Technetium ⁹⁹mTc Oxidronate; Technetium ⁹⁹mTc Pentetate; Technetium ⁹⁹mTc Pentetate Calcium Trisodium; Technetium ⁹⁹mTc Sestamibi; Technetium ⁹⁹mTc Siboroxime; Technetium ⁹⁹mTc; Succimer; Technetium ⁹⁹mTc Sulfur Colloid; Technetium ⁹⁹mTc Teboroxime; Technetium ⁹⁹mTc Tetrofosmin; Technetium ⁹⁹mTc Tiatide; Thyroxine ¹²⁵I; Thyroxine ¹³¹I; Tolpovidone ¹³¹I; Triolein ¹²⁵I; or Triolein ¹³¹I.

[0175] Additionally, a radioisotope can be site-specifically conjoined to an HSA linker or HSA linker conjugate. The available reactive groups could be used to conjugate site-specific bifunctional chelating agents for labeling of radioisotopes, including ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ⁹⁹ mTc, ¹¹¹In, ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁹⁰Y, ⁷⁷As, ⁷²As, ⁸⁶Y, ⁸⁹Zr, ²¹¹At, ²12Bi, ²¹³Bi, or ²25Ac.

[0176] Therapeutic or cytotoxic agents incorporated into or coupled with an HSA linker or an HSA linker conjugate may further include, for example, anti-cancer Supplementary Potentiating Agents, including, but not limited to: tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine, and maprotiline); non-tricyclic anti-depressant 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; Triparanol analogs (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g., buthionine and sulfoximine) and Multiple Drug Resistance reducing agents such as Cremaphor EL.

[0177] An agent that includes an HSA linker, or any molecule or moiety conjugated thereto, can also be coupled to or administered with one or more cytokines (e.g., granulocyte colony stimulating factor, interferon-alpha, and tumor necrosis factor-alpha). An HSA linker, or any molecule or moiety conjugated thereto, can also be coupled to an antimetabolic agent. Antimetabolic agents include, but are not limited to, the following compounds and their derivatives: azathioprine, cladribine, cytarabine, dacarbazine, fludarabine phosphate, fluorouracil, gencitabine chlorhydrate, mercaptopurine, methotrexate, mitobronitol, mitotane, proguanil chlorohydrate, pyrimethamine, raltitrexed, trimetrexate glucuronate, urethane, vinblastine sulfate, vincristine sulfate, etc. More preferably, an HSA linker or conjugate can be coupled to a folic acid-type antimetabolite, a class of agents that includes, for example, methotrexate, proguanil chlorhydrate, pyrimethanime, trimethoprime, or trimetrexate glucuronate, or derivatives of these compounds.

[0178] An HSA linker, or any molecule or moiety conjugated thereto, can also be coupled to a member of the anthracycline family of neoplastic agents, including but not limited to aclarubicine chlorhydrate, daunorubicine chlorhydrate, doxorubicine chlorhydrate, epirubicine chlorhydrate, idarubicine chlorhydrate, pirarubicine, or zorubicine chlorhydrate; a camptothecin, or its derivatives or related compounds, such as 10, 11 methylenedioxycamptothecin; or a member of the maytansinoid family of compounds, which includes a variety of structurally-related compounds, e.g., ansamitocin P3, maytansine, 2'-N-demethylmaytanbutine, and maytanbicyclinol.

[0179] An HSA linker, or any molecule or moiety conjugated thereto, can be coupled directly to a cytotoxic or therapeutic agent using known chemical methods, or coupled indirectly to a cytotoxic or therapeutic agent via an indirect linkage. For example, an HSA linker can be attached to a chelating group that is attached to the cytotoxic or therapeutic agent. Chelating groups include, but are not limited to, ininocarboxylic and polyaminopolycarboxylic 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. Pat. No. 5,753,627; and WO 91/01144 (each of which are hereby incorporated by reference).

[0180] An HSA linker conjugate that includes, e.g., an HSA linker, one or more binding moieties (with or without intervening peptide connectors, as defined herein), and a therapeutic or cytotoxic agent, can be specifically targeted by the binding moiety (e.g., antibody, antibody fragment, or receptor/ligand) to a cell or tissue, thereby allowing selective destruction of the target cell or tissue to which the binding moiety is directed. For example, an HSA linker conjugate can be used to target and destroy cancer cells of the lung, breast, prostate, and colon in order to prevent, stabilize, inhibit the progression of, or treat cancers originating in these organs when the HSA linker conjugate includes a binding moiety that specifically binds to the cancer cells in these organs. Also, for example, an HSA linker conjugate can be used to target and destroy cells of the vasculature, brain, liver, kidney, heart, lung, prostate, colon, nasopharynx, oropharynx, larynx, bronchus, and skin in order to prevent, stabilize, inhibit the progression of, or treat age-related, tobacco-related, or autoimmune diseases or conditions relating to these organs by targeting, in the case of autoimmune disease for example, autoreactive T cells (e.g., by binding to and agonizing tumor necrosis factor receptor 2 (TNFR2) present on the autoreactive T cells).

[0181] An HSA linker, when expressed recombinantly, can be joined to a cytotoxic polypeptide. Cytotoxic polypeptides, when brought into contact with a target cell (e.g., a cancer cell), exerts cytotoxic or cytostatic effects on the cell. For example, a cytotoxic polypeptide, when joined with an HSA linker, can induce events in a target cell upon binding of the target cell that leads to cell death through, for example, apoptosis, necrosis, or senescence. Alternatively, a cytotoxic polypeptide joined with an HSA linker can interfere or inhibit normal cellular biological activities, such as division, metabolism, and growth, or abnormal cellular biological activities, such as metastasis.

[0182] For example, an HSA linker joined to caspase 3 will bind a target cell (e.g., a cancer cell) and undergoe endocytosis. Once internalized by the target cell, the caspase portion of the HSA linker conjugate can initiate the pro-apoptotic caspase cascade, ultimately resulting in the apoptosis of the target cell.

[0183] In a preferred embodiment, an HSA linker conjugate includes a cytotoxic polypeptide capable of killing a cancer cell. In another embodiment, the cytotoxic polypeptide inhibits the growth or metastasis of a cancer cell. The cytotoxic polypeptide joined with an HSA linker can also be used to kill or inhibit the growth of cells associated with, necessary for, or beneficial to cancer growth, such as endothelial cells that form blood vessels that perfuse solid tumors.

[0184] In an embodiment, an HSA linker conjugate can include two or more cytotoxic polypeptides so as to modulate (e.g., increase) the specificity, intensity, or duration of the cytotoxic or cytostatic effect on a target cell (e.g., a cancer cell).

[0185] In another embodiment, the HSA linker is joined to an activatable form of cytotoxic polypeptide (e.g., a biologically-inactive pro-agent that is capable of activation upon cleavage by an enzyme or drug). In this embodiment, texposure (e.g., in vivo) of the cytotoxic polypeptide pro-agent to an enzyme or drug capable of cleaving the cytotoxic polypeptide, renders the cytotoxic polypeptide 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 procaspase (e.g., procaspase 8 or 3). For example, the procaspase 8 domain of an HSA linker can be cleaved by TRAIL or FasL upon internalization by a target cell (e.g., a cancer cell). Once cleaved, the biologically active caspase 8 can promote apoptosis of the target cell.

[0186] In one embodiment, the cytotoxic polypeptide joined to an HSA linker can include a full-length peptide, polypeptide, or protein, or biologically-active fragment thereof (e.g., a "death domain"), known to have cytotoxic or cytostatic properties. Peptides, polypeptides, or proteins with cytotoxic or cytostatic properties can be altered (e.g., by making amino acid substitutions, mutations, truncations, or additions) to facilitate incorporation of the cytotoxic sequence into an agent as described herein. Desirable alterations include, for example, changes to the amino acid sequence that facilitate protein expression, longevity, cell secretion, and target cell toxicity.

[0187] The present invention also provides a nucleic acid molecule encoding a cytotoxic polypeptide as a fusion protein with an HSA linker, optionally including binding moieities and peptide connectors. The nucleic acid molecule can be incorporated into a vector (e.g., an expression vector), such that, upon expression of the HSA linker in a cell transfected or transduced with the vector, the cytotoxic polypeptide, HSA linker, and binding moieties, if present, are operably linked (e.g., fused, contiguously-joined, or tethered together). Examples of peptides, polypeptides, and proteins that can be used as a 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-alpha (TNFα; SEQ ID NO:40)), TNF-beta, Fas (SEQ ID NO:41) and Fas ligand; Fas-associated death domain-like IL-1β converting enzyme (FLICE); TRAIL/APO2L (SEQ ID NO:45) and TWEAK/APO3L (see, e.g., U.S. Patent Application Publication No. 2005/0187177, herein incorporated 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, herein incorporate by reference); vascular apoptosis inducing proteins 1 and 2 (VAP1 and VAP2; 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-1α propiece polypeptide (see, e.g., U.S. Pat. No. 6,191,269, herein incorporated by reference); apoptin and apoptin-associated proteins such as AAP-1 (see, e.g., European Patent Application Publication No. EP 1083224, herein incorporated 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 herein incorporated by reference: U.S. 2003/0054994, U.S. 2003/0086919, U.S. 2007/0031423, WO 2004/078112, WO 2007/012430, and WO 2006/0125001 (intracellular domain of delta 1 and jagged 1).

Wild-Type HSA Linker Conjugates

[0188] The present invention also encompasses a naturally-occurring wild-type HSA linker, the amino acid and nucleotide sequences of which are provided in SEQ ID NOS:3 and 4, respectively, in the formation of binding, diagnostic, or therapeutic agents. In all embodiments utilizing an HSA linker with the amino acid sequence listed in SEQ ID NO:3, one or more peptide connectors, as described above, are covalently attached to the amino and/or carboxy termini of the HSA linker, or to an amino acid residue within the HSA linker sequence, to facilitate conjugation of one or more binding moieties.

Truncations

[0189] The invention further provides an HSA linker conjugate that is formed using a truncated wild-type HSA polypeptide, optionally combined with one or more peptide connectors or binding moieties. 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) can be conjoined to any of the binding moieties or diagnostic or therapeutic agents described herein. Truncations can occur at one or both ends of the HSA linker, or can include a deletion of internal residues. Truncation of more than one amino acid residue need not be linear (i.e., consecutive). Examples of wild-type HSA linkers include those having, in combination with one or more peptide connectors or binding moieties, 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 189-385 of SEQ ID NO:3), or domain III (SEQ ID NO:57; residues 381-585 of SEQ ID NO:3), or combinations thereof, e.g., domains I and II, I and III, and II and III.

[0190] Serum clearance rates of a conjugate (e.g., a bispecific HSA-drug or radioisotope-containing agent), can be optimized by testing conjugates containing a truncated wild-type HSA linker, as described above.

Additional HSA Linker Modifications

[0191] HSA linkers may, but need not, be modified by site-specific chemical modification of amino acid residues in the HSA linker. The correctly-folded tertiary structure of HSA displays certain amino acid residues on the external face of the protein. Chemically-reactive amino acid residues (e.g., cysteine) can be substituted for these surface-exposed residues to allow site-specific conjugation of a diagnostic or therapeutic agent.

[0192] Alternatively, or in addition, HSA linkers may optionally be modified by the addition or removal of asparagine, serine, or threonine residues from an HSA linker sequence to alter glycosylation of these amino acid residues. Glycosylation sites added to an HSA linker are preferably surface-exposed, as discussed herein. Glycosyl or other carbohydrate moieties introduced to an HSA linker can be directly conjugated to diagnostic, therapeutic, or cytotoic agents.

Cysteine (Thiol) Conjugation

[0193] Surface-exposed amino acid residues of the HSA linker may be substituted with cysteine residues to allow for chemical conjugation of diagnostic, therapeutic, or cytotoxic agents. Cysteine residues exposed on the surface of the HSA linker (when folded into its native tertiary structure) allow the specific conjugation of a diagnostic, therapeutic, or cytotoxic agent to a thiol reactive group such as maleimide or haloacetyl. The nucleophilic reactivity of the thiol functionality of a cysteine residue to a maleimide group is about 1000 times higher compared to any other amino acid functionality in a protein, such as the amino group of a lysine residue or the N-terminal amino group. Thiol specific functionality in iodoacetyl and maleimide reagents may react with amine groups, but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London). The amount of free thiol in a protein may be estimated using the standard Ellman's assay. In some instances, reduction of the disulfide bonds with a reagent such as dithiothreitol (DTT) or selenol (Singh et al., Anal. Biochem. 304:147-156 (2002)) is required to generate the reactive free thiol.

[0194] Sites for cysteine substitution can be identified by analysis of surface accessibility of the HSA linker (e.g., the identification of serine and threonine residues as suitable for substitution are described in Example 1 below). The surface accessibility can be expressed as the surface area (e.g., square angstroms) that can be contacted by a solvent molecule, e.g., water. The occupied space of water is approximated as a sphere with a 1.4 angstrom radius. Software for calculating the surface accessibility of each amino acid of a protein is freely available or licensable. For example, the CCP4 Suite of crystallography programs which employ algorithms to calculate the surface accessibility of each amino acid of a protein with known x-ray crystallography derived coordinates ("The CCP4 Suite: Programs for Protein Crystallography" Acta. Cryst. D50:760-763 (1994); www.ccp4.ac.uk/dist/html/INDEX.html). Solvent accessibility may also be assessed using the free software DeepView Swiss PDB Viewer downloaded from the Swiss Institute of Bioinformatics. The substitution of cysteines at surface-exposed sites allows for conjugation of the reactive cysteine to a thiol reactive group linked to the diagnostic or therapeutic agent.

Glycosylation

[0195] In addition, altered serum clearance rates can be achieved by engineering glycosylation sites into the HSA linker. In certain embodiments, an HSA linker is glycosylated. Glycosylation of polypeptides is typically either 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 the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0196] Addition or deletion of glycosylation sites to the HSA linker is conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) is created. The alteration 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 O-linked glycosylation sites). The resulting carbohydrate structures on HSA can also be used for site-specific conjugation of cytotoxic, immunomodulatory or cytostatic agents as described above.

HSA Linker Conjugates in Combination with Other Therapeutic Agents

[0197] HSA linker conjugates described herein may be administered with one or more of the therapeutic, cytotoxic, or cytostatic agents described herein. For example, a patient suffering from breast cancer can be administered an HSA linker containing ErbB2 and ErbB3 scFvs (e.g., B2B3-1) can be co-administered with, e.g., doxorubicin, cyclophosphamide, and paclitaxel, a common chemotherapeutic regimen for the treatment of breast cancer. A preferred therapeutic agent for use in this regard is trastuzumab. Data regarding this combination are set forth in Examples 42-44 below. Additional biological and chemical agents useful for the treatment of cancer are set forth herein, e.g., in Appendix 2.

HSA Linker Conjugates in Combination with Radiotherapy or Surgery

[0198] HSA linker conjugates may be administered prior to, concurrent with, or following radiotherapy or surgery. For example, a patient suffering from a proliferative disorder (e.g., breast cancer) can receive an HSA linker conjugate, alone or in combination with other therapeutic, cytotoxic, or cytotoxic agents as described herein concurrent with targeted radiotherapy or surgical intervention (e.g., lumpectomy or mastectomy) at the site of the cancerous tissue. Radiotherapies suitable for use in combination with HSA linker conjugates include brachytherapy and targeted intraoperative radiotherapy (TARGIT).

Pharmaceutical Compositions

[0199] Pharmaceutical compositions provided herein contain a therapeutically or diagnostically effective amount of an HSA linker conjugate that includes one or more of a binding moiety (e.g., antibodies or antibody fragments), a diagnostic agent (e.g., radionuclide or chelating agents), or a therapeutic agent (e.g., cytotoxic or immunomodulatory agents) agent. The active ingredients, an HSA linker conjugate (prepared with one or more of a binding moiety, diagnostic agent, or therapeutic agent) can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the compositions for proper formulation. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer Science 249:1527-1533 (1990).

[0200] The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment. Commonly, the pharmaceutical compositions are administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application. Thus, compositions for parenteral administration may include an HSA linker, with or without one or more binding, diagnostic, and/or therapeutic agent conjugated 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 delivery, which may contain inert ingredients such as binders or fillers for the formulation of a tablet, a capsule, and the like. Furthermore, this invention provides compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, and the like.

[0201] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) in a sealed package of tablets or capsules, for example. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

[0202] The compositions containing an effective amount of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) can be administered to a mammal (e.g., a human) for prophylactic and/or therapeutic treatments. In prophylactic applications, compositions containing an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) are administered to a patient susceptible to or otherwise at risk of developing a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease). Such an amount is defined to be a "prophylactically effective dose." In this use, the precise amounts again depend on the patient's state of health, but generally range from about 0.5 mg to about 400 mg of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) per dose (e.g., 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, or 400 mg or more per dose) and from about 0.1 μg to about 300 mg of one or more immunomodulatory agents per dose (e.g., 10 μg, 30 μg, 50 μg, 0.1 mg, 10 mg, 50 mg, 100 mg, or 200 mg per dose). A dose of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) 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 commonly, a single dose per week of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) is administered.

[0203] In therapeutic applications, a dose of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) is administered to a mammal (e.g., a human) already suffering from a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease) in an amount sufficient to cure or at least partially arrest or alleviate one or more of the symptoms of the disease or condition and its complications. An amount adequate to accomplish this purpose is defined as a "therapeutically effective dose." Amounts effective for this use may depend on the severity of the disease or condition and general state of the patient, but generally range from about 0.5 mg to about 400 mg of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) per dose (e.g., 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, or 400 mg or more per dose). A dose of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) can be administered therapeutically 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 commonly, a single dose per week of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) is administered.

[0204] In several embodiments, the patient may receive an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) in the range of about 0.5 to about 400 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more per week), preferably about 5 mg to about 300 mg per dose one or more times per week, and even more preferably about 5 mg to about 200 mg per dose one or more times per week. The patient may also receive a biweekly dose of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) in the range of about 50 mg to about 800 mg or a monthly dose of an HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto, in the range of about 50 mg to about 1,200 mg.

[0205] In other embodiments, an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may be administered to a patient in a typical dosage range of about 0.5 mg per week to about 2000 mg per week, about 1.0 mg per week to about 1000 mg per week, about 5 mg per week to about 500 mg per week, about 10 mg per week to about 100 mg per week, about 20 mg per week to about 80 mg per week, about 100 mg per week to about 300 mg per week, or about 100 mg per week to about 200 mg per week. In another aspect, the dosages for administration to a 70 kg patient can range from, for example, about 1 μg to about 5000 mg, about 2 μg to about 4500 mg, about 3 μg to about 4000 mg, about 4 μg to about 3,500 mg, about 5 μg to about 3000 mg, about 6 μg to about 2500 mg, about 7 μg to about 2000 mg, about μg to about 1900 mg, about 9 μg to about 1,800 mg, about 10 μg to about 1,700 mg, about 15 μg to about 1,600 mg, about 20 μg to about 1,575 mg, about 30 μg to about 1,550 mg, about 40 μg to about 1,500 mg, about 50 μg to about 1,475 mg, about 100 μg to about 1,450 mg, about 200 μg to about 1,425 mg, about 300 μg to about 1,000 mg, about 400 μg to about 975 mg, about 500 μg to about 650 mg, about 0.5 mg to about 625 mg, about 1 mg to about 600 mg, about 1.25 mg to about 575 mg, about 1.5 mg to about 550 mg, about 2.0 mg to about 525 mg, about 2.5 mg to about 500 mg, about 3.0 mg to about 475 mg, about 3.5 mg to about 450 mg, about 4.0 mg to about 425 mg, about 4.5 mg to about 400 mg, about 5 mg to about 375 mg, about 10 mg to about 350 mg, about 20 mg to about 325 mg, about 30 mg to about 300 mg, about 40 mg to about 275 mg, about 50 mg to about 250 mg, about 100 mg to about 225 mg, about 90 mg to about 200 mg, about 80 mg to about 175 mg, about 70 mg to about 150 mg, or about 60 mg to about 125 mg, of an HSA linker conjugate provided herein. Dosage regimen may be adjusted to provide the optimum therapeutic response. In another aspect, an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may be administered in the range of about 0.5 mg every other day to about 500 mg every other day, preferably about 5 mg every other day to about 75 mg every other day, more preferably about 10 mg every other day to about 50 mg every other day, and even more preferably 20 mg every other day to about 40 mg every other day. An HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may also be administered in the range of about 0.5 mg three times per week to about 100 mg three times per week, preferably about 5 mg three times per week to about 75 mg three times per week, more preferably about 10 mg three times per week to about 50 mg three times per week, and even more preferably about 20 mg three times per week to about 40 mg three times per week.

[0206] In non-limiting embodiments of the methods of the present invention, an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) is administered to a mammal (e.g., a human) continuously 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; biweekly; 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; bimonthly; 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 biannually. An HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may be administered at different frequencies during a therapeutic regime. In additional embodiments, an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) may be administered to a patient at the same frequency or at a different frequency.

[0207] The amount of one or more diagnostic or therapeutic agents and an HSA linker, or any agent conjugated thereto, required to achieve the desired therapeutic effect depends on a number of factors, such as the specific diagnostic or therapeutic agent(s) chosen, the mode of administration, and clinical condition of the recipient. A skilled artisan will be able to determine the appropriate dosages of one or more diagnostic or therapeutic agents and an HSA linker, or any agent conjugated thereto, to achieve the desired results.

[0208] Single or multiple administrations of the compositions comprising an effective amount of an HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) can be carried out with dose levels and pattern being selected by the treating physician. The dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in a mammal (e.g., a human), which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.

[0209] An HSA linker conjugate (prepared with one or more of a binding, diagnostic, and/or therapeutic agent) can be administered to a mammalian subject, such as a human, directly or in combination with any pharmaceutically acceptable carrier or salt known in the art. Pharmaceutically acceptable salts may include non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.

Diagnostic and Therapeutic Applications

[0210] HSA linker conjugates can be used for diagnostic and therapeutic applications in a human, including, for example, the diagnosis or treatment of proliferative diseases (e.g., cancers, such as melanoma, clear cell sarcoma, and renal cancer) and autoimmune diseases (e.g., multiple sclerosis, rheumatoid arthritis, and uveitis). The following discussion of human proliferative and autoimmune diseases is meant to provide the skilled practitioner with a general understanding of how HSA linker conjugates can be applied in diagnostic and therapeutic applications and is not meant to limit the scope of the present invention.

Proliferative Diseases (Cancer)

[0211] An 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, gastric cancer, and ovarian cancer. Binding moieties to be conjoined with an HSA linker for diagnostic or therapeutic application in a patient suspected of having or suffering from a proliferative disease may be chosen based on their ability to specifically bind, agonize, activate, antagonize, or inhibit target molecules (e.g., cell surface receptors such as tyrosine kinase receptors) associated with a proliferative disease. Binding moieties that target, for example, insulin-like growth factor receptor (IGFR, 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 transition factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)) can be conjoined to an HSA linker to diagnose or treat a proliferative disease. Specific binding of a cancer cell by an HSA linker conjugate can allow for detection (e.g., an HSA linker conjoined to a detectable label, as defined herein) or destruction (e.g., an HSA linker conjoined to a cytotoxic agent) of the bound cancer cell. Specific application of HSA linker conjugates for the treatment of breast and renal cancer is described below.

Breast Cancer

[0212] Common forms of breast cancer include invasive ductal carcinoma, a malignant cancer in the breast's ducts, and invasive lobular carcinoma, a malignant cancer in the breast's lobules. Some types of breast cancer cells are known to express high levels of epidermal growth factor receptors, especially ErbB2 (i.e., HER2/neu). Aberrant signaling or unregulated activation of EGFRs has been linked to the development and progression of many cancers, including breast cancer. Uncontrolled cellular proliferation mediated via dysfunctional EGFR pathways can be found in a wide variety of solid tumors of epithelial origin and data have linked tumor EGFR expression, overexpression, and dysregulation to advanced disease, metastatic phenotype, resistance to chemotherapy, and an overall poorer prognosis.

[0213] An HSA linker conjoined to one or more binding moieties specific for an EGFR (e.g., anti-ErbB2; trastuzumab) can be used with a diagnostic (e.g., a detectable label) or cytotoxic, cytostatic, or therapeutic agent, as described herein, to diagnose or treat breast cancer. Alternatively, a bispecific HSA linker conjugate that comprises binding moieties specific for ErbB2 and ErbB3, such as "B2B3-1," described further herein, can be employed to diagnose or treat cancers, e.g., breast, kidney, ovarian, and lung cancers.

[0214] As described above, an HSA linker conjugate used to treat breast cancer can be administered prior to (e.g., neoadjuvant chemotherapy), concurrent with, or following (e.g., adjuvant chemotherapy) radiotherapy or surgical intervention. An HSA linker conjugate can also be co-administered with other compounds (e.g., antineoplastic agents, such as biological or chemical therapeutics) useful for the treatment of breast cancer. For example, the antineoplastic agents listed in Table 1, including mitotic inhibitors (e.g., taxanes), topoisomerase inhibitors, alkylating agents (including, e.g., platinum-based agents), selective estrogen modulators (SERM), aromatase inhibitors, antimetabolites, antitumor antibiotics (e.g., anthracycline antibiotics), anti-VEGF agents, anti-ErbB2 (HER2/neu) agents, and anti-ErbB3 agents, are known to be particularly useful for the treatment of breast cancer. An HSA linker conjugate can be administered by a clinician in combination with any compound, including those listed in Appendix 2, known or thought to be beneficial for the treatment of breast cancer.

TABLE-US-00001 TABLE 1 Exemplary antineoplastic agents for treatment of breast cancer in combination with HSA linker conjugates. Exemplary Agent Therapeutic Class (Generic/Tradename) Exemplary Dose Mitotic Inhibitors paclitaxel (TAXOL ®; 175 mg/m² ABRAXANE ®) docetaxel (TAXOTERE ®) 60-100 mg/m² Topoisomerase Inhibitors camptothecin topotecan hydrochloride (HYCAMTIN ®) etoposide (EPOSIN ®) Alkylating Agents cyclophosphamide (CYTOXAN ®) 600 mg/m² Platinum-Based Agents Cisplatin 20-100 mg/m² carboplatin (PARAPLATIN ®) 300 mg/m² nedaplatin (AQUPLA ®) oxaliplatin (ELOXATIN ®) 65-85 mg/m² satraplatin (SPERA ®) triplatin tetranitrate Selective Estrogen Modulators tamoxifen (NOLVADEX ®) 20-40 mg/day (SERM) raloxifene (EVISTA ®) 60 mg/day toremifene (FARESTON ®) Antimetabolites methotrexate 40 mg/m² Fluorouracil (5-FU) 500 mg/m² Raltitrexed Antitumor Antibiotics Doxorubicin (ADRIAMYCIN ®) 40-75 mg/m² epirubicin (ELLENCE ®) 60-120 mg/m² Aromatase Inhibitors aminoglutethimide (CYTADREN ® 250-2000 mg/day anastrozole (ARIMIDEX ®) 1 mg/day letrozole (FEMARA ®) 2.5 mg/day Vorozole exemestane (AROMASIN ®) 25-50 mg/day Testolactone fadrozole (AFEMA ®) Anti-VEGF Agents bevacizumab (AVASTIN ®) 10 mg/kg Anti-ErbB2 (HER2/neu) Agents trastuzumab (HERCEPTIN ®) 2-8 mg/kg Pertuzumab (OMNITARG ®) Anti-ErbB3 (HER3) Agents U3-1287 (AMG 888)

Renal Cancer

[0215] Kidney cancers, such as renal cell carcinoma, are particularly resistant to traditional radiological and chemical therapies. As such, the application of biological therapeutics, conjoined with an HSA linker, represents an attractive option for patients suffering from these cancers. For example, an HSA linker conjoined with binding moieties that agonize type I interferon or interleukin 2 receptors can be used to treat a renal cancer. As a solid tumor, binding moieties that target and inhibit tumor vascularization (e.g., anti-vascular endothelial growth factor (VEGF) antibodies such as bevacizumab) can also be used for therapeutic effect.

Autoimmune Diseases

[0216] An HSA linker conjugate can be used to diagnose, treat, prevent, or stabilize autoimmune diseases and disorders in e.g., a human patient, such as, e.g., multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), uveitis, Sjogren's syndrome, Grave's disease, psoriasis, and myasthenia gravis. Autoimmune diseases and disorders are caused by self-reactive elements of the immune system (e.g., T cells, B cells, and self-reactive antibodies). As such, binding moieties that inhibit, block, antagonize, or deplete (e.g., anti-lymphocyte or anti-thymocyte globulins; basiliximab, daclizumab, or muromonab-CD3 monoclonal antibodies) self-reactive immune cells and antibodies can be conjoined with an HSA linker for therapeutic use. Binding moieties that function as inflammatory signaling inhibitors (ISI), as defined herein, can be conjoined to an HSA linker for the treatment of autoimmunity. In addition, binding moieties that inhibit or antagonize integrin function (e.g., an integrin antagonist, as defined herein) can ameliorate or halt disease progression.

[0217] In other embodiments, the binding moiety is a soluble TNF receptor, such as etanercept or lenercept; an antibody directed against a pro-inflammatory cytokine or a pro-inflammatory cell surface signaling molecule, such as adalimumab, certolizumab, inflixamab, golimumab, and rituxan; a dominant-negative pro-inflammatory cytokine variant, such as XENP345, XPRO®1595, anakinra, and variants disclosed in U.S. Patent Application Publication Nos. 20030166559 and 20050265962; an inhibitor of the signaling pathways downstream of pro-inflammatory cytokine or pro-inflammatory cell surface signaling molecules, such as DE 096, 5-amino-2-carbonylthiopene derivatives (as described in WO2004089929), ARRY-797, BIRB 796 BS, (1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-2(morpholin-4-yl-ethoxy)-- naphtalen-1-yl]-urea, CHR-3620, CNI-1493, FR-167653 (Fujisawa Pharmaceutical, Osaka, Japan), ISIS 101757 (Isis Pharmaceuticals), ML3404, NPC31145, PD169316, PHZ1112, RJW67657, 4-(4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl- )-3-butyn-1-ol, SCIO-469, SB202190, SB203580, (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole)- , SB239063, trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl-methoxypyridimidin-4-yl)i- midazole, SB242235, SD-282, SKF-86002, TAK 715, VX702, and VX745; or an inhibitor of TNF-alpha converting enzyme (TACE), such as BB-1101, BB-3103, BMS-561392, butynyloxyphenyl β-sulfone piperidine hydroxomates, CH4474, DPC333, DPH-067517, GM6001, GW3333, Ro 32-7315, TAPI-1, TAPI-2, and TMI 005); or an anti-idiotypic agent, such as monoclonal antibodies, LJP 394 (abetimus, RIQUENT®, La Jolla Pharmaceuticals).

[0218] In other embodiments, the binding moiety is an interferon, as described herein. Binding moieties that can be conjoined to an HSA linker include, e.g., interferon-beta (REBIF® (IFN-β-1a), AVONEX® (IFN-β-1a), and BETASERON® (IFN-β-1b)), interferon-t (TAUFERON®), interferon-alpha (e.g., ROFERON-A® (IFN-α-2a), INTRON-A® (IFN-α-2b), REBETRON® (IFN-α-2b), ALFERON-N® (IFN-α-n3), PEG-INTRON® (IFN-α-2b covalently conjugated with monomethoxy polyethylene glycol), INFERGEN® (a non-naturally occurring type 1 interferon with 88% homology to IFN-α-2b), or PEGASYS® (pegylated IFN-α-1a)), and ACTIMMUNE® (IFN-g-1b).

[0219] The present invention further provides HSA linker conjugates with binding moieties that antagonize these pro-inflammatory molecules or their specific receptors to treat autoimmunity. Specific application of HSA linker conjugates for the diagnosis and treatment of MS and RA are described below.

Multiple Sclerosis

[0220] Multiple sclerosis (MS) is a neurological disease characterized by irreversible degeneration of the nerves of the central nervous system (CNS). Although the underlying cause is unclear, the neurodegeneration in MS is the direct result of demyelination, or the stripping of myelin, a protein that normally lines the outer layer and insulates the nerves. T cells play a key role in the development of MS. Inflamed MS lesions, but not normal white matter, can have infiltrating CD4.sup.+ T cells that respond to self antigens presented by MHC class II-linked molecules such as human HLA-DR2. The infiltrating CD4 T cells (T_H1 cells) produce the pro-inflammatory cytokines IL-2, IFN-γ, and TNF-α that activate antigen-presenting cells (APCs) such as macrophages to produce additional pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-8, and IL-12. IL-12 induces further IFN-γ synthesis. The result is progressive demyelination of neuronal sheaths, leading to human disease.

[0221] HSA linker conjugates can be used to aid in the diagnosis of MS. Diagnostic HSA linker conjugates that include binding moieties that specifically target one or more (e.g., a bispecific HSA linker conjugate) immune cell activation markers (e.g., CD69, CD28, HLA-DR, and CD45). An imbalance of one or more of these pro-inflammatory or immune cell activation mediators relative to other factors or cells may be measured using an HSA linker conjugate conjoined with a diagnostic agent (e.g., a radioisotope or fluorochrome).

[0222] An HSA linker conjugate can be used to treat a person at risk of developing or suffering from MS or to prevent, ameliorate, or cure the symptoms of the disease. For example, binding moieties that specifically target and antagonize α4 integrin (e.g., natalizumab), CD52 (e.g., alemtuzumab), CD80, P-selectin, sphingosine-1-phosphate receptor-1 (S1PR1), hyaluronate receptor, leukocyte function antigen-1 (LFA-1), CD11 (e.g., efalizumab), CD18, CD20 (e.g., rituximab), CD85, ICOS ligand, CCR2, CXCR3, or CCR5 can be useful when conjoined to an HSA linker for therapeutic use in a patient suffering from MS. Similarly, binding moieties that neutralize type I interferons (e.g., interferons-alpha and -beta) or that antagonize type I interferon receptors (e.g., IFNαR1) can also be conjoined to an HSA linker for therapeutic application.

Rheumatoid Arthritis

[0223] Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. It is a disabling and painful inflammatory condition, which can lead to substantial loss of mobility due to pain and joint destruction. RA is a systemic disease, often affecting extra-articular tissues throughout the body including the skin, blood vessels, heart, lungs, and muscles.

[0224] Patients suffering from RA frequently have an increase in cellular expression of the HLA-DR4/DR1 cluster. HSA linker conjugates specific for one or both of these cell surface molecules are useful for the diagnosis of RA.

[0225] An HSA linker conjugate can be used to treat a person at risk of developing of suffering from RA to prevent, ameliorate, or cure the symptoms of the disease. For example, binding moieties, as defined herein, that specifically target and antagonize TNF-α (e.g., etanercept, infliximab, and adalimumab), IL-1 (e.g., anakinra), or CTLA-4 (e.g., abatacept). Binding moieties that specifically target and deplete B cells (e.g., an anti-CD20 antibody, such as rituximab) can also be conjoined to the HSA linker described herein to treat or prevent RA.

Uveitis

[0226] Uveitis specifically refers to inflammation of the middle layer of the eye, but may refer to any inflammatory process involving the interior of the eye. Uveitis may be autoimmune or idiopathic in origin

[0227] An HSA linker conjugate can be used to treat a person at risk of developing of suffering from autoimmune uveitis to prevent, ameliorate, or cure the symptoms of the disease. For example, alpha-fetoprotein (e.g., human AFP; NCBI Accession No. NM_--001134), or biologically-active fragments thereof, can be conjoined to an HSA linker to reduce or eliminate inflammation associated with autoimmune or idiopathic uveitis.

Kits

[0228] The present invention further provides kits that include a pharmaceutical composition containing an HSA linker, and one or more of a binding moiety (e.g., antibodies or antibody fragments), a diagnostic agent (e.g., radionuclide or chelating agents), and a therapeutic agent (e.g., cytotoxic or immunomodulatory agents) with reagents that can be used to conjugate them to the HSA linker, if necessary, and including a pharmaceutically-acceptable carrier, in a therapeutically effective amount for treating a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease). The kits include instructions to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein.

[0229] Preferably, the kits include multiple packages of the single-dose pharmaceutical composition(s) containing an effective amount of an HSA linker, or any binding (e.g., antibodies or antibody fragments (e.g., scFv)), diagnostic (e.g., radionuclide or chelating agents), and/or therapeutic (e.g., cytotoxic or immunomodulatory agents) conjugate thereof. Optionally, instruments or devices necessary for administering the pharmaceutical composition(s) may be included in the kits. For instance, a kit may provide one or more pre-filled syringes containing an effective amount of an HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto. Furthermore, the kits may also include additional components such as instructions or administration schedules for a patient suffering from a disease or condition (e.g., a cancer, autoimmune disease, or cardiovascular disease) to use the pharmaceutical composition(s) containing an HSA linker, or any binding, diagnostic, and/or therapeutic agent conjugated thereto.

[0230] 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

[0231] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

Example 1

Methods to Identify Residues in the HSA Linker for Site-Specific Conjugation of Cytotoxic or Cytostatic Drugs

[0232] To identify sites for specific conjugation of drug to HSA the crystal structure is studied and surface exposed serine and threonine residues are identified. These particular surface-exposed amino acids can then be mutated to cysteine allowing drug conjugation to the substituted cysteine using a thiol-specific conjugating agent such as maleimide. Mild reduction may be required prior to drug conjugation. The number of drugs conjugated is controlled by the number of surface exposed cysteine residues introduced into HSA. Serine and threonine are selected as the most suitable residues for mutation as they share the most structural identity with cysteine, however, other surface exposed residues may also be mutated to cysteine and successfully conjugated to cytostatic or cytotoxic drugs.

[0233] The crystal structure of HSA is deposited in the RSCB Protein Data Bank (1bm0-Sugio et al., "Crystal structure of human serum albumin at 2.5 A resolution," Protein Eng. 12:439-446 (1999)). This structure is analyzed using the DeepView Swiss PDB Viewer downloaded from the Swiss Institute of Bioinformatics. Serine and threonine residues with 50%, 40%, and 30% surface exposure were identified as the most suitable for mutation to cysteine (Table 2). Mutations can be introduced using standard molecular biology procedures. Conjugation of a thiol reactive drug or chelating agent to introduced cysteines can be tested using standard protein chemistry techniques.

TABLE-US-00002 TABLE 2 % Surface Exposure Residue 50 T496 40 S58 30 T76, T79, T83, T125, T236, S270, S273, S304, S435, T478, T50 T508 indicates data missing or illegible when filed

Example 2

Methods to Identify Residues in the HSA Linker for Introduction of Asparagine-Linked Glycosylation Sites

[0234] To identify regions for introduction of asparagine-linked glycosylation sites in HSA, the crystal structure is studied to identify surface exposed (>30%) asparagine, serine and threonine residues that would be suitable for mutation. Glycosylation occurs on asparagine residues when the consensus sequence asparagine-x-serine/threonine is present, where x cannot be a proline. Table 2 lists possible mutation sites in HSA for the introduction of asparagine-linked glycosylation.

TABLE-US-00003 TABLE 3 Residue Proposed Mutation 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*

[0235] *These mutations have also been reported to occur very rarely in HSA (Carlson et al., "Alloalbuminemia in Sweden: Structural study and phenotypic distribution of nine albumin variants," Proc. Nat. Acad. Sci. USA 89:8225-8229 (1992); Madison et al., "Genetic variants of human serum albumin in Italy: point mutants and a carboxyl-terminal variant," Proc. Nat. Acad. Sci. USA 91:6476-6480 (1994); Hutchinson et al., "The N-terminal sequence of albumin Redhill, a variant of human serum albumin," FEBS Lett. 193:211-212 (1985); Brennan et al., "Albumin Redhill (-1 Arg, 320 Ala-Thr): a glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site," Proc. Nat. Acad. Sci. USA 87:26-30 (1990); Minchiotti et al., "Structural characterization of four genetic variants of human serum albumin associated with alloalbuminemia in Italy," Eur. J. Biochem. 247:476-482 (1997); Peach et al., "Structural characterization of a glycoprotein variant of human serum albumin albumin Casebrook (494 Asp-Asn)," Biochim. Biophys. Acta 1097:49-54 (1991)).

Example 3

[0236] B2B3-1 is a bispecific scFv antibody fusion molecule comprising B1D2, a human anti-ErbB2 scFv antibody (SEQ ID NO:27) and H3, a human anti-ErbB3 scFv (SEQ ID NO:26). The two scFvs are joined by a modified human serum albumin (HSA) linker. The anti-ErbB3 scFv, H3, is recombinantly fused to the amino terminus of the HSA linker incorporating a short connector polypeptide and the anti-ErbB2 scFv, B1D2, is recombinantly fused to the carboxy terminus of the modified HSA linker incorporating an additional short connector polypeptide. Each connector polypeptide is selected based on protease resistance properties. The modified HSA linker contains two amino acid substitutions. A cysteine residue at position 34 of native HSA is mutated to serine in order to reduce potential protein heterogeneity due to oxidation at this site. An asparagine residue at amino acid 503 of native HSA, which in native HSA may be sensitive to deamidation which can result in decreased pharmacologic half-life, is mutated to glutamine. It is believed that B2B3-1 selectively binds ErbB2 over-expressing tumors by virtue of its high affinity anti-ErbB2 scFv binding moiety, which has a kD in the range of 10.0 nM to 0.01 nM and more preferably a kD of about 0.3 nM. Subsequent binding of ErbB3 by the anti-ErbB3 scFv, which has a kD in the range of 50 to 1 nM and more preferably about 16 nM, inhibits HRG induced phosphorylation of ErbB3. The modified HSA linker confers an extended circulating half-life on the bispecific molecule. B2B3-1 has a molecular weight of 119.6 kDa and is preferably not glycosylated.

[0237] B2B3-1 inhibits ligand-induced phosphorylation of ErbB3 with sub-nanomolar potency; this activity is believed to be due, at least in part, to the abundant expression of its dimerization partner, ErbB2, which facilitates specific targeting of cancer cells that express both receptors.

Example 4

[0238] As shown in FIG. 2, B2B3-1 variants inhibit HRG-induced pErbB3 in ZR75-1 breast cancer cells. ZR75-1 breast cancer cells are treated with a dose range of B2B3-1 variants for 24 hours followed by HRG stimulation. pErbB3 levels are measured in cell lysates by ELISA and IC₅₀ values are calculated together with the percent of inhibition. Shown are the mean IC₅₀ values (Y axis) with error bars representing replicate experiments. Percent inhibition values are shown above the corresponding bar.

ELISA Assays

[0239] Except as noted, ELISA reagents for total and phospho-ErbB3 ELISAs are purchased from R&D Systems as DUOSET kits. 96-well NUNC MAXISORB plates are coated with 50 μl of an antibody and incubated overnight at room temperature. Next morning, plates are washed 3 times with 1000 μl/well in a BIOTEK plate washer with Dulbecco's phosphate buffered saline without calcium or magnesium (PBS) with added Tween detergent (PBST) (0.05% Tween-20). Plates are subsequently blocked for about 1 hr at room temperature with 2% BSA in PBS. The plates are washed 3 times with 1000 μl/well in the BIOTEK plate washer with PBST. Cells are grown at 37° C. and 5% carbon dioxide, washed with cold PBS, then harvested with mammalian protein extract (MPER) lysis buffer (Pierce, 78505) to which 150 mM NaCl, 5 mM sodium pyrophosphate, 10 uM bpV (phen), 50 uM phenylarsine, 1 mM sodium orthovanadate, and protease inhibitor cocktail (Sigma, P2714) is added. 50 μL of cell lysates and standards diluted in 50% Lysis buffer and 1% BSA are used in duplicates for further processing. Samples are incubated for 2 hrs at 4° C. on a plate shaker and washed as before. About 50 μl of a detection antibody diluted in 2% BSA, PBST is added and incubated for about 1-2 hrs at room temperature. For phospho-ErbB3, the detection antibody is directly conjugated to horseradish peroxidase (HRP), while for total ErbB3 and biotinylated mouse anti-human ErbB3 secondary detection antibody is used. The plate is washed as before. For total ErbB3 about 50 μl of Streptavidin-HRP is added and incubation is for 30 min and the plates washed as before. About 50 μL of SUPERSIGNAL ELISA Pico (Thermo Scientific) substrate is added and the plate is read using a FUSION plate reader. Duplicate samples are averaged and, where shown, error bars represent the standard deviation between the two replicates.

Example 5

[0240] Inhibition of phosphorylated ErbB3 (FIGS. 3A-D), AKT (FIGS. 4A-D), and ERK (FIGS. 5A-D) following 24 hour pre-treatment with B2B3-1 variants A5-HSA-B1D2 (panel A of FIGS. 3-5), H3-HSA-B1D2 (panel B of FIGS. 3-5), H3-HSA-F5B6H2 (panel C of FIGS. 3-5), and F4-HSA-F5B6H2 (panel D of FIGS. 3-5) is measured. BT474 breast cancer cells are treated with a dose range of B2B3-1 variants for 24 hours followed by HRG stimulation. pErbB3, pAKT, and pERK levels are measured in cell lysates by ELISA and IC₅₀ values are calculated together with the percent of inhibition. These results demonstrate that B1B2-1 is the only HSA linker conjugate of those tested that provides greater than 50% inhibition of HRG-induced phosphorylation of AKT, ERK, and ErbB3 at a concentration of 10^-8 molar and above.

Example 6

[0241] As shown in FIG. 6, treatment of BT474 breast tumor cells with B2B3-1 variants causes G1 cell cycle arrest and a decrease in the population of cells in S phase. BT474 cells are treated with 1 μM of B2B3-1 variants and controls for 72 hours. After the end of treatment, cells are trypsinized, gently resuspended in hypotonic solution containing propidium iodide and single cells are analyzed by flow cytometry. Cell cycle distribution in G1 and S phases is measured using curve-fitting algorithms designed for cell cycle analysis (FlowJo software cell cycle platform).

Example 7

[0242] B2B3-1 (SEQ ID NO:16) inhibits ErbB3 activation, utilizing the abundant expression of its dimerization partner, ErbB2, to target tumor cells. A high affinity anti-ErbB2 scFv antibody, B1D2, facilitates targeting of B2B3-1 to tumor cells over-expressing ErbB2. B1D2 is connected by a modified HSA linker to a lower affinity anti-ErbB3 scFv antibody, H3, which blocks binding of ErbB3's ligand, HRG. It is believed that the inhibition of ErbB3 phosphorylation and downstream AKT signaling mediated by B2B3-1 is due to this blockade. The ErbB2 binding scFv, B1D2, is derived from parent scFv C6.5, which possesses neither agonistic nor antagonistic activity at ErbB2. B1D2, therefore, likely functions solely as a targeting agent. The lower affinity binding of the ErbB3 binding scFv is believed to prevent strong binding of B2B3-1 to normal, non-cancerous tissues which express ErbB3 but little or no ErbB2, thereby reducing the potential for non-specific toxicity. In tumor cells expressing both ErbB2 and ErbB3, there is an avidity effect of bispecific B2B3-1 binding to both receptors that overcomes the low affinity of the ErbB3 scFv allowing strong inhibition of HRG interaction with ErbB3 receptor complexes.

[0243] The ability of B2B3-1 to inhibit HRG binding to ErbB3 is investigated using flow cytometry (FACS). Cells of the breast cancer cell line human (a variant of BT-474 that over-express ErbB2), are pretreated with 1 μM B2B3-1 then incubated with 10 nM biotinylated HRG 1β EGF domain. After extensive washing, binding is assessed using streptavidin-AlexaFluor 647 conjugate. All incubations are performed at 4° C. FIG. 7 shows that B2B3-1 is capable of blocking the binding of HRG to ErbB3. and appears to provide 100% blockade at a concentration of 1 μM.

Example 8

[0244] After demonstrating B2B3-1's ability to block HRG binding to ErbB3, the effect of B2B3-1 on ErbB3 signaling in vitro on two cell lines that express ErbB3 and over-express ErbB2 is investigated. Human breast cancer cell lines BT-474-M3 (described in, e.g., Drummond et al. (2005) Clin. Cancer Res. 11:3392; Park et al. (2002) Clin. Cancer Res. 8:1172; Kirpotin et al. (2006) Cancer Res. 66:6732) and ZR7530 (obtainable from the US NIH Lawrence Berkeley National Laboratory breast cancer cell collection) are serum starved overnight, pre-treated with a dose titration of B2B3-1 for 24 hours and then stimulated for 10 minutes with 5 nM of HRG 1β EGF domain. The phosphorylation status of ErbB3 and AKT is then examined using ELISA assays generally as described above. The results show that B2B3-1 inhibits HRG induced activation of both ErbB3 and AKT phosphorylation in a dose-dependent manner and with potent IC₅₀s in both cell lines (FIGS. 8A-D).

Example 9

[0245] FIG. 9 shows the effect of B2B3-1 treatment on signaling proteins in BT474 breast cancer cells. Cells are treated with a dose range of B2B3-1 for 24 hours, followed by heregulin stimulation. Levels of pErbB2, pErbB3, pErk and pAKT and their corresponding total protein levels are determined on cell lysates by Western blot analysis. Results indicate that levels of at least pErbB2 and pErbB3 are reduced in a dose-dependent manner by B2B3-1 treatment.

Example 10

[0246] FIG. 10 shows the immunoprecipitation-Western blot analysis of B2B3-1 treated BT474 breast cancer cells. Cells are treated with a dose range of B2B3-1 for 24 hours, followed by heregulin stimulation. ErbB2-associated complexes are isolated from cell lysates using an anti-ErbB2 antibody followed by Western blot analysis to detect pErbB2 and pErbB3 and the corresponding total protein levels. The results show that B2B3-1 crosslinks ErbB2 to ErbB3, so that substantially more ErbB3 and phospho-ErbB3 is precipitated by the anti-ErbB2 antibody.

Example 11

[0247] The anti-tumor activity of B2B3-1 is investigated in vitro using a number of 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 decrease in S phase of the cell cycle is examined Briefly, cells are treated with 1 μM B2B3-1 or PBS vehicle for 72 hours. After the end of treatment, cells are trypsinized, gently resuspended in hypotonic solution containing propidium iodide and single cells are analyzed by flow cytometry. Cell cycle distribution in G1 and S phases is measured using curve-fitting algorithms designed for cell cycle analysis (FlowJo software cell cycle platform, Tree Star, Inc.). B2B3-1 was found to modestly decrease the percentage of cells in S phase and increase the population of cells in G1 phase (FIG. 11A). In a second experiment, the number of cell colonies formed following treatment with B2B3-1 is studied. BT-474 and SKBR3 breast cancer cells are plated in the presence of 1 μM B2B3-1 and compared to cells plated in media only. Media only or media including treatment is replenished every 3 days. After 14 days the number of colonies is counted and compared to untreated cells. FIG. 11B illustrates the 40-45% decrease in the number of colonies formed when cells are treated with B2B3-1 compared to control cells. Finally, the ability of B2B3-1 to inhibit cell proliferation is assessed in a cell impedance assay using a Real-Time Cell Electronic Sensing System (RT-CES: ACEA Biosciences). BT-474 cells are seeded on plates integrated with microelectronic sensor arrays and treated with a dose titration of B2B3-1 or media only for 72 hours. Data reflecting the generation of cell-electrode impedance response are collected every hour for 72 hours and IC₅₀ values are calculated 68 hours after treatment. FIG. 11C illustrates that B2B3-1 was able to inhibit impedance of BT-474 cells with an IC₅₀ of 33 nM.

Example 12

[0248] We also investigated whether B2B3-1 exhibits agonistic activity based on its ability to simultaneously bind and cross-link ErbB2 and ErbB3 receptors. Serum starved ZR75-1 breast cancer cells are incubated with 1 μM B2B3-1 or PBS vehicle for 24 hours. Cells are also treated with B2B3-1 or PBS vehicle for 24 hours followed by a 10-minute stimulation with 5 nM HRG 1β EGF domain. Cells are lysed and the pErbB3 content of the lysates is assessed by ELISA generally as described above. FIG. 12 shows that cells treated with B2B3-1 alone contained levels of phosphorylated ErbB3 that were comparable to the levels in untreated cells, indicating that B2B3-1 does not act as an agonist promoting ErbB3 phosphorylation.

Example 13

[0249] The ability of B2B3-1 to bind with specificity to ErbB2 and ErbB3 and not to related ErbB family members, EGFR and ErbB4, is investigated by ELISA. Plates are coated with the recombinant extracellular domain of either ErbB2 or ErbB3. Plates are blocked and incubated with a half-maximal binding concentration of B2B3-1 in the presence of a dilution series of competing recombinant extracellular domains of EGFR, ErbB2, ErbB3 or ErbB4. The results show 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 the anti-ErbB2 arm B1D2 for ErbB2, and of the anti-ErbB3 arm H3 for ErbB3. The increased signal observed when soluble ErbB2 extracellular domain was incubated with B2B3-1 on the ErbB3 coated plate is assumed to be due to formation of an ErbB2, ErbB3, B2B3-1 complex on the plate.

Example 14

[0250] The ability of B2B3-1 to bind to tumor cells expressing both ErbB2 and ErbB3 is studied using monospecific variants of B3B3-1. 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.

[0251] SKO-2 and SKO-3 are constructed using the QUIKCHANGE Site Directed Mutagenesis kit (STRATAGENE) which uses oligonucleotide primer pairs, each complementary to opposite strands of the template vector. These are extended during temperature cycling generating a mutated plasmid containing staggered nicks. Following temperature cycling, the product is treated with Dpn I, which is used to digest the parental DNA template. The nicked vector DNA containing the desired mutations is then transformed into XL1-BLUE supercompetent cells STRATAGENE) to propogate plasmid DNA.

[0252] To create SKO-2, 5 nucleotides in the VH CDR3 loop of the anti-ErbB2 scFv, B1D2, are mutated to create the following amino acid substitutions; H95A, W100hA, and E100jA. Mutations at these positions have been demonstrated to knock-out binding of the B1D2 parent scFv, C6.5, to ErbB2 (Schier et al, 1996 JMB). Mutations are introduced to the B2B3-1 plasmid pMP9043 (SEQ ID NO:60) in a stepwise manner. First, mutations c295g and a296c are 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 GGC ACA AAA GTA C-3' (SEQ ID NO:62) and temperature cycling of 95° C. for 1 minute followed by 30 cycles of 95° C. for 1 minute, 55° C. for minute, and 65° C. for 17.2 minutes. Mutations are confirmed by DNA sequencing of plasmid DNA. To introduce mutations at t334g, g335c, and a341c, a second round of site-directed mutagenesis is performed on the plasmid with sequence-confirmed mutations at c295g and a296c using primers 5'-GAC ATG TGC CAA GGC CCC CGC GTG GCT GGG AGT G-3' (SEQ ID NO:63) and 5'-CAC TCC CAG CCA CGC GGG GGC CTT GGC ACA TGT C-3' (SEQ ID NO:64) and temperature cycling of 95° C. for 30 seconds and 18 cycles of 95° C. for 30 seconds, 55° C. for 1 minute and 68° C. for 17.2 minutes. Mutations are confirmed by DNA sequencing of resulting plasmid DNA.

[0253] To create SKO-3, the mutated B1D2 scFv is subcloned into the original B2B3-1 plasmid to replace the anti-ErbB3 scFv, H3. Primers annealing to SKO-2, 5'ACAGTGGCGGCCGCCACCATGGGCTGGTCTCTGATCCTGCTGTTCCTGGTGG CCGTGGCCACGCGTGTGCTGTCCCAGGTGCAGCTCGTCCAGAGCGGCGC (SEQ ID NO:65) and 5'GGAGGCGGCGCCCAGGACTGTCAGCTTGGTGCCACCGCCG (SEQ ID NO:66) are used to isolate the mutated B1D2 scFv from SKO-2 and to introduce Kas I and Not I restriction sites for subcloning into Kas I/Not I restriction digested B2B3-1 plasmid. PCR is performed as follows: 94° C. for 1 minute followed by 30 cycles of 94° C. for 30 seconds, 58° C. for 1 minute, 72° C. for 1 minute, followed by one cycle of 72° C. for 5 minutes to amplify the mutant B1D2. Successful cloning is monitored by DNA sequencing. SKO-2 and SKO-3 plasmids are stably expressed from CHO-K1 cells in shake flasks or 10 L WAVE bags and purified from conditioned media using Blue SEPHAROSE and cation exchange chromatography.

[0254] MALME-3M melanoma cells, which express approximately equal numbers of ErbB2 and ErbB3 receptors, are incubated with a dilution series of B2B3-1, SKO-2, or SKO-3 in the presence of saturating concentrations of a goat anti-HSA Alexafluor-647 conjugated antibody. Cell binding is assessed by flow cytometry and apparent binding affinities are determined for each molecule. Control cells are incubated with secondary antibody alone. No measurable cell binding is observed for SKO-2, which retains only the low affinity binding to ErbB3 mediated by H3 and lacks ErbB2 binding activity. SKO-3, which retains a functional, high affinity ErbB2 binding B1D2 scFv but lacks the ability to bind ErbB3 had a K_D of 6.7 nM. B2B3-1 bound cells with a K_D of 0.2 nM, demonstrating the increased binding mediated by the bispecific design of this molecule (FIG. 14).

Example 15

[0255] The stability of B2B3-1 under physiological conditions is assessed by incubating 100 nM B2B3-1 in human, Cynomolgus monkey, or mouse serum at 37° C. for a period of 120 hours. Samples are removed at 0, 24, 48, 72, 96 and 120 hours and the ability of B2B3-1 to bind both ErbB2 and ErbB3 is measured by ELISA. This ELISA involves coating a 96-well plate with recombinant ErbB2 extracellular domain overnight followed by a blocking step and then incubation with a dilution series of B2B3-1. Plates are then incubated with an Fc-ErbB3 extracellular domain fusion protein followed by a goat antihuman-Fc-HRP conjugate. Plates are developed by addition of supersignal chemiluminescense substrate. FIGS. 15A-C show that B2B3-1 remains stable in serum from all three species under physiological conditions, retaining comparable ability to bind both ErbB2 and ErbB3 at all time points measured.

Example 16

B2B3-1 Dose Response in BT-474-M3 Human Breast Cancer In Vivo Xenograft Model

[0256] The efficacy of B2B3-1 in vivo is assessed using nude mice bearing human BT-474-M3 xenografts. Ten mice per group are treated with 12 doses of 0.3, 1, 3, 10, 30 or 90 mg/kg of B2B3-1 every 3 days. Control groups are administered PBS vehicle or HSA at an equimolar dose to the 90 mg/kg B2B3-1 dose. All doses are administered interperitoneally (i.p.). Tumor size is measured twice a week and the corresponding tumor volume is calculated. The results show that B2B3-1 treatment leads to significant reduction in BT-474-M3 tumor size as compared to the control group (FIG. 16). Complete regressions were observed in each of the B2B3-1 treatment groups except mice treated with the lowest dose of B2B3-1 (0.1 mg/kg).

Example 17

[0257] As shown in FIGS. 17A-E, B2B3-1 reduces tumor size in multiple xenograft models in an ErbB2 dependent manner. B2B3-1 was efficacious in the Calu-3 (FIG. 17A), SKOV-3 (FIG. 17B), NCI-N87 (FIG. 17C), and MDA-MB-361 (FIG. 17E) xenograft models expressing ErbB2 at >1×10⁵ receptors/cell but worked less well in the ACHN (FIG. 17D) xenograft model which expresses 4.5×10⁴ ErbB2 receptors/cell. Mice were treated with 30 mg/kg of B2B3-1 every 3 days.

Example 18

[0258] Over-expression of ErbB2 converts B2B3-1 non-responder ADRr breast cancer xenograft model into a responder to B2B3-1 (FIGS. 18A and 18B). ErbB2 is over-expressed in ADRr breast cancer cells using a retroviral expression system. Transfected cells expressing high levels of ErbB2 (ADRr-E2) are selected using FACS and subsequently injected subcutaneously into nude mice to establish xenograft tumors. Mice are treated with 30 mg/kg of B2B3-1 every 3 days. While no response to B2B3-1 was observed in wild type ADRr xenografts (FIG. 18A), ADRr-E2 xenografts (FIG. 18B) responded to B2B3-1.

Example 19

[0259] As shown in FIGS. 19A-B, B2B3-1 activity correlates positively with ErbB2 expression levels in vitro (FIG. 19A) and in vivo (FIG. 19B). B2B3-1 inhibition of ErbB3 phosphorylation is determined in 9 tumor cell lines with expression levels of ErbB2 ranging from 5×10⁴ receptors/cell to 2.4×10⁶ receptors/cell using an ELISA assay. The extent of B2B3-1's ability to inhibit ErbB3 phosphorylation to basal levels (% pErbB3 inhibition) was found to correlate positively with ErbB2 expression levels. Similarly, B2B3-1 activity is assessed in 10 tumor xenograft models expressing low to high levels of ErbB2. Xenograft response also correlated positively with ErbB2 expression levels.

Example 20

[0260] B2B3-1 treatment of BT474-M3 breast tumor cells results in translocation of p27.sup.kip1 to the nucleus (FIG. 20A). BT474-M3 cells are treated with 1 μM of B2B3-1 for 6 hours. The sub-cellular location of p27.sup.kip1 is assessed using immunofluorescence techniques. In cells treated with B2B3-1, p27.sup.kip1 translocated to the nucleus, which has been shown to result in inhibition of cell proliferation. p27.sup.kip1 remained in the cytoplasm of untreated cells.

[0261] To further study the effect of B2B3-1 on the cell cycle, BT-474-M3 cells treated with B2B3-1 for 72 hours are probed for the cell cycle regulator Cyclin D1 using Western blot analysis (FIG. 20B). The cytoskeleton protein vinculin is used as a protein loading control in this experiment. B2B3-1 treatment resulted in a decrease in the levels of Cyclin D1 compared to untreated cells.

Example 21

[0262] As shown in FIGS. 21A-B, B2B3-1 treatment of BT474-M3 breast tumor xenografts results in translocation of p27.sup.kip1 to the nucleus. BT474 breast tumor xenografts are treated with B2B3-1 (FIGS. 21A) at a dose of 30 mg/kg or an equimolar dose of HSA (FIGS. 21B) every 3 days for a total of 4 doses. Increased nuclear staining for p27.sup.kip1 was observed in B2B3-1 treated tumors compared to HSA control tumors indicating an anti-proliferative effect of B2B3-1 in vivo.

Example 22

[0263] B2B3-1 treatment results in a reduction of the proliferation marker Ki67 in BT474 breast cancer xenograft tumors. BT474-M3 breast tumor xenografts are treated with B2B3-1 (FIG. 22A) at a dose of 30 mg/kg or an equimolar dose of HSA (FIG. 22B) every 3 days for a total of 4 doses. Subsequent staining of tumor sections for Ki67 demonstrated a reduced expression pattern for B2B3-1 treated tumors compared to HSA treated tumors.

Example 23

[0264] B2B3-1 treatment results in a reduction of vessel density in BT474-M3 breast cancer xenograft tumors, as determined by assaying for CD31 expression (FIGS. 23A-B). BT474 breast tumor xenografts are treated with B2B3-1 (FIG. 23A) at a dose of 30 mg/kg or an equimolar dose of HSA (FIG. 23B) every 3 days for a total of 4 doses. Tumors are stained for the presence of vascular marker CD31. Tumors treated with B2B3-1 show a marked decrease in vessel density compared to control tumors treated with HSA.

Example 24

B2B3-1 Inhibits Phosphorylation of ErbB3 In Vivo

[0265] BT-474-M3 xenograft tumors are treated with 30 mg/kg B2B3-1 or 17.5 mg/kg HSA every 3 days for a total of 4 doses and tumors are harvested 24 hours after the final dose. Tumors are lysed and subjected to SDS-PAGE followed by Western analysis to assess relative levels of phosphorylation of B2B3-1's target ErbB3. Equal quantities of protein are loaded in each lane and total protein levels are controlled by probing for beta tubulin. Western blot analysis using antibodies specific for phosphorylated ErbB3 show that B2B3-1 treated tumors contain less pErbB3 than HSA treated tumors (FIG. 24A). Densitometry of the western blot analysis followed by normalization of the mean pErbB3 integral band intensity to the mean beta tubulin integral band intensity demonstrated that B2B3-1 treated tumors contained significantly less pErbB3 than control HSA treated tumors (FIG. 24B). These data confirmed that B2B3-1 inhibits phosphorylation of its target ErbB3 in vivo.

Example 25

In Vivo Activity of B2B3-1 in BT-474-M3 Xenografts which have Reduced PTEN Activity

[0266] ShRNA technology is applied to knock out the activity of the tumor suppressor gene phosphatase and tensin homolog (PTEN) in BT-474-M3 breast cancer cells. Briefly, cultured BT-474-M3 cells are transfected with shPTEN or shControl RNA by retroviral transfection. Transfected cells with reduced PTEN are selected using FACS and subsequently injected subcutaneously into the right flank of nude mice to establish xenograft tumors. Cells transfected with a control vector are injected into the left flank of the same mouse. Mice are treated with 30 mg/kg B2B3-1 every 3 days or 10 mg/kg trastuzumab every week. HSA is injected as a control at an equimolar dose to B2B3-1. All injections are done i.p.

[0267] B2B3-1 and trastuzumab promoted a reduction in the size of tumors formed by control BT-474-M3 breast cancer cells (FIG. 25A), whereas only B2B3-1 (and 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 Having Reduced PTEN Activity

[0268] The ability of B2B3-1 to inhibit phosphorylation of ErbB3 signaling in tumor xenografts is studied using the PTEN deficient BT-474-M3 model described above. Xenograft tumors of the engineered cell line or control cell line are treated with 30 mg/kg B2B3-1, 17.5 mg/kg HSA every 3 days or 10 mg/kg trastuzumab weekly and tumors are harvested 24 hours after the final dose. Tumors are lysed and subjected to SDS-PAGE followed by Western analysis to assess relative levels of phosphorylation of B2B3-1's target ErbB3, AKT and total PTEN levels. Equal quantities of protein are loaded in each lane and total protein levels are controlled by probing for PCNA. Western blot analysis using antibodies specific for phosphorylated ErbB3 shows that B2B3-1 treated tumors contain less pAKT than HSA treated or Herceptin treated tumors (FIG. 26A). Densitometry of the western blot analysis followed by normalization of the mean pAKT integral band intensity to the mean PCNA integral band intensity demonstrated that B2B3-1 treated tumors contained significantly less pAKT than control HSA treated and Herceptin-treated tumors (FIG. 26B).

Example 27

[0269] The pharmacokinetic parameters for B2B3-1 are investigated in nu/nu mice. Animals are randomized into groups and administered intravenous (IV) with a single dose of 5, 15, 30, or 45 mg/kg B2B3-1 (FIGS. 27A-D, respectively). Blood is collected pre-dose and at 0.5, 4, 8, 24, 48, 72, and 120 hours post dose. Three mice are used for each time point. Serum levels of B2B3-1 are measured using two ELISA methods. The first method requires functional binding of B2B3-1 to both ErbB2 and ErbB3 while the second method measures only the HSA component of B2B3-1 in the serum. The HSA ELISA utilizes a polyclonal-anti HSA capture antibody and a HRP-conjugated polyclonal anti-HSA detection antibody. A reduction in B2B3-1 serum concentration measured using the ErbB2/ErbB3 binding method versus the HSA method would indicate a loss in functional B2B3-1. FIGS. 27A-D show that the pharmacokinetic properties of B2B3-1 are comparable when assessed using either ELISA method, indicating that B2B3-1 is stable in circulation in mice.

Example 28

[0270] B2B3-1 serum concentrations are fit using a two-compartment, biexponential model and show biphasic disposition. Terminal half-lives were calculated to be 17, 16, 23, and 18 hrs for the 5, 15, 30, or 45 mg/kg doses, respectively, and are shown in Table 4. Increases in B2B3-1 dose resulted in a linear increase in exposure (FIG. 28).

TABLE-US-00004 TABLE 4 Pharmacokinetic properties of B2B3-1 in mice and Cynomolgus monkeys. Dose T1/2β AUC Clearance (mg/kg) Species N (hrs) (hr-μg/ml) (ml/hr/kg) 5 Mouse (single dose) 3 16.9 1.58E+03 3.19 15 Mouse (single dose) 3 16.2 6.10E+03 2.47 30 Mouse (single dose) 3 22.6 1.18E+04 2.54 45 Mouse (single dose) 3 17.5 1.84E+04 2.46 4 Cynomolgus 2 39.1 3.44E+03 1.17 monkey (1st dose) 4 Cynomolgus 2 44.9 7.20E+03 0.60 monkey (4th dose) 20 Cynomolgus 2 33.1 2.29E+04 0.88 monkey (1st dose) 20 Cynomolgus 2 122.5 8.20E+04 0.25 monkey (4th dose) 200 Cynomolgus 4 68.8 3.18E+05 0.64 monkey (1st dose) 200 Cynomolgus 2 69.7 5.72E+05 0.35 monkey (4th dose) 200 Cynomolgus 2 66.6 5.99E+05 0.34 monkey (4th dose)* *recovery animals

Example 29

[0271] Blood samples for pharmacokinetic analysis are also obtained from a dose range-finding toxicology study in female Cynomolgus monkeys. In this study, animals are infused with 4, 20 or 200 mg/kg of B2B3-1 administered every 3 days for 4 doses. Sampling occurred prior to and 5 minutes after dosing on each dosing day (study days 1, 4, 7 and 10) to provide pre-dose and peak/trough concentrations, and at 1, 2, 4, 8, 24 and 48 hours after the end of the first infusion on day 1 and at 1, 2, 4, 8, 24, 48, 72 and 120 hours after the last infusion on day 10. For recovery animals dosed at 200 mg/kg serum samples are also collected at 168, 336 and 456 hours after the last infusion.

[0272] Cynomolgus monkey serum samples are assayed using the ErbB2/ErbB3 ELISA method described previously. Serum concentrations for each dose over the time course are shown in FIG. 29. The analysis shows that mean concentration-time profiles for serum B2B3-1 after dosing on days 1 and 10 were qualitatively similar with concentrations generally declining with time from Cmax. Mean half-life estimates ranged from 38.3-67.2 hours on day 1 and 45.0 to 121.0 hours on day 10 (Table 4).

Example 30

[0273] The plasmid encoding the B2B3-1 bispecific scFv antibody fusion protein is created combining gene sequences of a unique human anti-ErbB3 scFv (designated "H3"), a human anti-ErbB2 scFv (designated "B1D2"), and a modified human serum albumin (HSA) linker. The anti-ErbB3 scFv, H3, is recombinantly linked to the amino terminus of the HSA linker via a connecting peptide (Ala-Ala-Ser) and the anti-ErbB2 scFv, B1D2, is genetically linked the carboxy terminus of the HSA linker via a connecting peptide (Ala-Ala-Ala-Leu--SEQ ID NO:5). The peptide connectors are formed through the introduction of restriction sites during construction of the mammalian expression vector and are synthesized with optimized codon usage for mammalian expression together with the single chain antibody fragments and HSA linker.

[0274] The B1D2 scFv is selected from a combinatorial phage display library created by mutagenesis of the ErbB2-binding scFv C6.5, which is selected from a non-immune phage display library. The H3 scFv is selected from a non-immune phage display library originally made by Sheets et al. The gene sequences encoding the B1D2 and H3 single chain antibody fragments are optimized for CHO cell codon preferences and synthesized for subsequent construction of the B2B3-1 encoding plasmid.

[0275] The modified HSA linker contains two amino acid substitutions. A cysteine residue at position 34 is mutated to serine in order to reduce potential protein heterogeneity due to oxidation at this site. An asparagine residue at amino acid 503 is mutated to glutamine, which in wild type HSA is sensitive to deamination and can result in decreased pharmacologic half-life.

[0276] The gene sequence encoding the modified HSA linker is synthesized with optimized codon usage for mammalian expression for subsequent construction of the B2B3-1 encoding plasmid.

Example 31

[0277] The B2B3-1 coding sequence is cloned into pMP10k base vector using standard molecular biology techniques to create plasmid pMP10k4H3-mHSA-B1D2, shown in FIG. 30. For the most part this construct employs commonly used genetic elements. B2B3-1 expression is driven by the human GAPD promoter. This vector utilizes genetic elements referred to as Matrix Attachment Regions or MAR elements. The MAR genetic elements control the dynamic organization of chromatin, and insulate nearby genes from the effect of surrounding chromatin thereby increasing copy number dependent, position-independent, expression of genes. MAR elements have been shown to improve the probability of isolating a clone exhibiting the desired level of expression for the production of a recombinant protein and to increase the stability of production. The MAR elements used in the B2B3-1 constructs are non-coding human MAR elements. In addition to the B2B3-1 plasmid, a neomycin antibiotic resistance plasmid (FIG. 31) and a hygromycin resistance plasmid (FIG. 32) are also used to select for stable transformants.

Example 32

First Round of Gene Transfection

[0278] Chinese Hamster Ovary CHO-K1 cells are purchased from ATCC (ATCC # CCL-61). The CHO-K1 cell line is a serum and proline dependent adherent sub-clone of the parental CHO cell line created by T. T. Puck. The CHO-K1 cells used for B2B3-1 transfection are pre-adapted for suspension growth in serum free media prior to transfection. An iterative transfection procedure is used to develop the B2B3-1 cell line. Twenty-four hours before transfection, CHO-K1 cells are sub passaged to 1.0×10⁶ cells/mL in SFM4CHO (Serum Free) medium (HyClone, Logan, Utah) supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine. On the day of transfection, cells are resuspended in OptiMEM medium (Invitrogen Corp, Carlsbad, Calif.) and 40,000 cells are placed in each well of a twenty-four well plate. In the first transfection, the B2B3-1 expression plasmid (pMP10k4H3-mHSA-B1D2) and the neomycin resistance plasmid (FIG. 30; pSV2-neo (Selexis, Inc., Marlborough, Mass.) are mixed together using a molar plasmid ratio of 75:1 (B2B3-1:neomycin resistance). The plasmid mixture is subsequently mixed with a cationic lipid transfection reagent (Lipofectamine LTX, Invitrogen Corp, Carlsbad, Calif.) and lipid/DNA complexes are allowed to form for thirty minutes. The DNA/Lipid complex is then added to the CHO-K1 cells and the 24-well plates are placed in a 37° C., 5% CO₂ incubator.

Example 33

Selection and Screening for High Producers

[0279] The contents of each transfection well are washed with PBS, trypsinized and distributed across two, ninety-six well plates. The growth media used consists of DMEM/F12 (Invitrogen Corp, Carlsbad, Calif.) with 10% FBS (Invitrogen Corp, Carlsbad, Calif.) and 500 mg/L of geneticin (G418; Invitrogen Corp, Carlsbad, Calif.). Media in the 96-well plates is changed on day 4 to SFM4CHO medium supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, 0.016 mM thymidine, and 500 mg/L geneticin. Following an additional two weeks of culture in selection medium, surviving cells have formed well-defined colonies. The clones are evaluated using quantitative spot blot techniques. The top producing colonies are trypsinized, and expanded to a single well of a 24-well plate.

[0280] A seven day productivity assay is used to screen for high B2B3-1 producing colonies. Upon expansion the cells in 24-well plates are allowed to proliferate for seven days in SFM4CHO medium supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine. The B2B3-1 concentration in the spent media is determined Top clones from the 24-well scale are expanded into 125 mL baffled shake flasks. A seven day study in the shake flask in SFM4CHO medium supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine is used to screen the cell pools for growth and B2B3-1 production.

Example 34

Second Round of Gene Transfection

[0281] The highest producing cell pool determined from the first round of transfection (supra) is transfected a second time to increase production. Twenty-four hours before transfection, the cell pool is sub passaged to 1.0×10⁶ cells/mL in SFM4CHO (Serum Free) medium supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine. On the day of transfection, cells are resuspended in OptiMEM medium (Invitrogen Corp, Carlsbad, Calif.) and 40,000 cells re placed in each well of a twenty-four well plate. In the first transfection, the B2B3-1 and hygromycin resistance plasmid (FIG. 32; pTK-Hyg (Clontech, Mountain View, Calif.)) are mixed together using a molar plasmid ratio of 50:1 (B2B3-1:hygromycin resistance). The plasmid mixture is subsequently mixed with a cationic lipid transfection reagent (Lipofectamine LTX, Invitrogen Corp) and lipid/DNA complexes are allowed to form for thirty minutes. The DNA/Lipid complex is then added to the cell pool and the 24-well plates are placed in a 37° C., 5% CO₂ incubator.

Example 35

Selection and Screening for High Producers from Second Transfection

[0282] The contents of each transfection well are washed with PBS, trypsinized and distributed across two, 96-well plates. The growth media used consists of DMEM/F12 supplemented with 10% FBS and 400 mg/L of hygromycin B (Invitrogen Corp). Media in the 96-well plates is changed on day 4 to Hyclone SFM4CHO medium supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, 0.016 mM thymidine, and 400 mg/L of hygromycin B. After an additional two weeks of selection, surviving cells have formed well-defined colonies. The clones are evaluated using quantitative spot blot techniques. The top producing colonies are trypsinized, and expanded to a single well of a 24-well plate.

[0283] A seven day productivity assay is used to screen for high B2B3-1 producing colonies.

Upon expansion the cells are allowed to proliferate for seven days, and the B2B3-1 concentration in the spent media is determined.

[0284] Top clones from the 24-well plates are expanded into 125 mL baffled shaker flasks in the Hyclone SFM4CHO medium supplemented with 8 mM L-glutamine, 0.1 mM sodium hypoxanthine, and 0.016 mM thymidine. A seven day study in shake flask is used to screen the cell pools for growth and B2B3-1 production. The spent media is quantitated using Protein Aresin and an HPLC instrument.

Example 36

Limiting Dilution Cloning

[0285] The best growing and highest B2B3-1-producing colony identified by the productivity assay is transferred from the 125 mL shaker flask and plated in five 96-well plates at a cell concentration calculated to give one cell/well. The 96-well plates are placed in an incubator at 37° C. and 5% CO₂. The wells are examined bi-weekly to track formation of colonies. Colonies arising from a single cell are identified based on the symmetrical shape of the colony. Wells containing such colonies are marked for further screening by 24-well 7-day assessment, and 125 mL shaker flask 7-day assessment.

[0286] The second round of limiting dilution cloning is performed in a similar manner to the first round. An additional 100 mL fed batch evaluation is performed to confirm clone choice. A pre-seed bank is cryopreserved.

Example 37

Formulation of B2B3-1

[0287] B2B3-1 is administered once a week via intravenous infusion over a period of 60 or 90 minutes, depending on patient tolerability. B2B3-1 is formulated in a sterile 20 mM L-histidine hydrochloride, 150 mM sodium chloride, pH 6.5 solution at a concentration of 25 mg/mL for administration to a patient (e.g., a human).

Example 38

Treatment of Breast Cancer

[0288] If a patient's cancer is believed to be expressing high levels of epidermal growth factor receptors, including ErbB2 (HER2/neu), then treatment with an HSA linker conjoined to an ErbB2 biding moiety, such as B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9, or B3B3-10 (see Table 6, below) would be indicated. Such would be the case where genotypic or histologic screens of cancer biopsies reveals increased expression of ErbB2 in the patient's tumor.

[0289] A B2B3 HSA linker conjugate (e.g., B2B3-1, SEQ ID NO:16) is administered to a patient diagnosed with breast cancer once a week or twice a week via intravenous infusion over a period of, e.g., 60 or 90 minutes, depending on patient tolerability, at a dose no higher than 30 mg/kg. The B2B3 HSA linker conjugate is formulated in a sterile 20 mM L-histidine hydrochloride, 150 mM sodium chloride, pH 6.5 solution at a concentration of 25 mg/mL for administration to the patient. A clinician supervising the administration of the B2B3 HSA linker conjugate follows common formulation and dosing practices to determine the proper course of therapy for the patient.

[0290] The clinician may further co-administer one or more therapies with the B2B3 HSA linker conjugate. E.g., one or more therapeutic drugs or compounds may be administered in combination with the B2B3 HSA linker conjugate, such as the common chemotherapeutic regimen for the treatment of breast cancer, which includes doxorubicin, cyclophosphamide, and paclitaxel. Alternatively, a clinician can administer the B2B3 HSA linker conjugate in combination with surgical or radiation therapy to treat breast cancer the patient.

Example 39

Treatment of Ovarian Cancer

[0291] If a patient's cancer is believed to be expressing high levels of epidermal growth factor receptors, including ErbB2 (HER2/neu), then treatment with an HSA linker conjoined to an ErbB2 biding moiety, such as B2B3-1, B2B3-2, v-3, B2B3-4, B2B3-5, B2B3-6, B2B3-7, B2B3-8, B2B3-9, or B3B3-10 (see Table 6, below) would be indicated. Such would be the case where genotypic or histologic screens of cancer biopsies reveals increased expression of ErbB2 in the patient's tumor.

[0292] A B2B3 HSA linker conjugate (e.g., B2B3-1, SEQ ID NO:16) is administered to the patient diagnosed with ovarian cancer alone or in combination with one or more other therapies essentially as described in the preceding Example.

Example 40

Additional HSA Linker Conjugates

[0293] HSA linker conjugates are constructed using one or more of the elements (groups A-E) listed in Table 5 below. In particular, an HSA linker conjugate, which is shown as Group C in Table 5 below, incorporates one or more binding moieties selected from groups A and E shown in Table 5. In addition, the HSA linker conjugates can also include one or more peptide connectors, which are selected from groups B and D in Table 5, at each of the amino and carboxy terminal ends of the HSA linker. Peptide connectors can be repeated or truncated to increase or decrease the length of the connector sequence.

Example 41

In Vivo, B2B3-1 Dosed q7d Shows Equivalent Efficacy as B2B3-1 Dosed q3d

[0294] B2B3-1 efficacy using a q7d (once every 7 days) dosing regimen is determined in in female athymic nude mice (nu/nu) from Charles River Labs, 5-6 weeks of age bearing xenograft tumors of the human breast cancer cell line BT-474-M3 (FIG. 33). Mice receive a subcutaneous estrogen-releasing implant in the opposite flank (0.72 mg pellet, 60 day, slow-release, Innovative Research of America, Sarasota, Fla.) 24 h prior to the injection of 20×10⁶ human BT-474-M3 cells in PBS. Dosing is initiated when tumor growth is established (tumor volumes of approximately 400 mm3) and B2B3-1 is administered to 10 mice per group either once every 3 days (q3d) at 30 mg/kg for the course of the study or once every 7 days at 22 mg/kg, 66 mg/kg, 132 mg/kg or 198 mg/kg by intraperitoneal injection. Tumors are measured twice a week using digital calipers. Tumor volume is calculated using the formula: π/6×(W²×L), where W is the short diameter and L is the long diameter. Pharmacokinetic calculations suggest that a 66 mg/kg dose q7d should give a similar exposure of B2B3-1 to the xenograft tumor as a 30 mg/kg dose q3d. PBS vehicle is used as a negative control. B2B3-1 efficacy was equivalent for the 30 mg/kg, q3d dose and the 3 highest doses of B2B3-1 administered q7d, indicating a q7d dosing schedule for B2B3-1 is suitable in this model.

Example 42

B2B3-1 and Trastuzumab have Different Mechanisms of ErbB3 Inhibition

[0295] The ability of B2B3-1 to inhibit heregulin induced ErbB3 activity is tested using Western blot analysis. Monolayers of serum-starved BT-474-M3 cells are treated for 24 hours with 100 nM B2B3-1 or trastuzumab and then stimulated with 5 nM HRG 1β EGF for 10 minutes. Cells are also treated with 10nM and 100 nM B2B3-1 or 10nM and 100 nM trastuzumab and left unstimulated. Lysates are subjected to immunoblot analysis for ErbB3, pErbB3, AKT, and pAKT. Western blot analysis (FIG. 34) demonstrated that B2B3-1 treatment results in inhibition of pErbB3 and pAKT in a ligand dependent manner, whereas inhibition of pErbB3 and pAKT by trastuzumab was only seen in the absence of ligand.

Example 43

B2B3-1 has an Additive Effect when Administered with Trastuzumab In Vitro

[0296] The effects of B2B3-1, trastuzumab and the combination of both drugs on the growth of cancer cell spheroids was examined using four different breast cancer cell lines. 2,000 cells of BT-474-M3, SKBR3 (ATCC), or MDA-MB-361 (ATCC) human breast cancer cells were seeded in round-bottom low adherence 96-well plates (Corning® 96 Well Clear Round Bottom Ultra Low Attachment Microplate-Product #7007) and the following day the spheroids were measured and treated with a dose range of B2B3-1, trastuzumab or a combination of both at a ratio of 3 fold molar excess B2B3-1 to trastuzumab. After 12 days of growth the surface area of the spheroids was measured and compared to untreated cells. As can be seen in FIGS. 35A-C, the combination of B2B3-1 with trastuzumab over a range of concentrations resulted in greater inhibition of spheroid growth compared to the single agents in all cell lines tested at all but the lowest concentrations of drug(s). These results also indicate that B2B3-1 does not compete with trastuzumab for binding to ErbB2 (HER2).

Example 44

B2B3-1 has an Additive Effect when Administered with Trastuzumab In Vivo

[0297] The effects of B2B3-1 when co-administered with trastuzumab in vivo is studied in female athymic nude mice (nu/nu) from Charles River Labs, 5-6 weeks of age, using a BT-474-M3 xenograft model. Mice receive a subcutaneous estrogen-releasing implant in the opposite flank (0.72 mg pellet, 60 day, slow-release, Innovative Research of America, Sarasota, Fla.) 24 h prior to the injection of 20×10⁶ human BT-474-M3 cells in PBS. Dosing is initiated when tumor growth is established (tumor volumes of approximately 400 mm³) Tumors are measured twice a week using digital calipers. Tumor volume is calculated using the formula: π/6×(W²×L), where W is the short diameter and L is the long diameter. Ten mice per group are administered B2B3-1 at 3 mg/kg or 10 mg/kg q3d, trastuzumab at 1 mg/kg or 0.1 mg/kg q7d or the combination of both drugs for the course of the study by intraperitoneal injection. All combinations of B2B3-1 and trastuzumab (10 mg/kg B2B3-1+1 mg/kg trastuzumab, 10 mg/kg B2B3-1+0.1 mg/kg trastuzumab, 3 mg/kg B2B3-1+1 mg/kg trastuzumab, 3 mg/kg B2B3-1+0.1 mg/kg trastuzumab) are dosed as for the corresponding single agent.

[0298] As shown in FIG. 36, substantially greater efficacy was seen for all the combinations compared to the single agents and significant efficacy was observed from at least as early as day 20 onward for the combination groups dosed with 10 mg/kg B2B3-1 and both doses of trastuzumab and with 3 mg/kg B2B3-1 and 1 mg/kg trastuzumab. In the 10 mg/kg B2B3-1+1 mg/kg trastuzumab combination group, 5 out of the 10 mice had completely regressed tumors compared to 0 out of 10 for the single agent groups given equivalent doses as the combination. In the 3 mg/kg B2B3-1+1 mg/kg trastuzumab combination group, 7 out of 10 mice had completely regressed tumors compared to 0 out of 10 for the single agent groups given equivalent doses as the combination. These results indicate that B2B3-1 does not compete with trastuzumab for binding to ErbB2 (HER2). These results also demonstrate that treatment with a combination of at least 3 mg/kg of B2B3-1 and at least 0.1 mg/kg trastuzumab is more effective than treatment with 3 mg/kg or 10 mg/kg of B2B3-1 alone or with 0.1 mg/kg or 1 mg/kg of trastuzumab alone. In particular, the combination of at least 3 mg/kg of B2B3-1 and 1 mg/kg trastuzumab induces essentially complete tumor regression in at least about 50% of nude mice carrying human breast tumor cell xenografts, while the same concentrations of either B2B3-1 or trastuzumab alone do not provide complete regression in even 10% of such mice.

[0299] Further provided are specific embodiments of the HSA linkers, peptide connectors, and binding moieties discussed above. Table 6, below, lists ten HSA linker conjugates with varying ErbB2-specific or ErbB3-specific binding moieties, as well as peptide connectors, at the amino and carboxy termini of an HSA linker.

[0300] Those skilled in the art will recognize, and will be able to ascertain and implement using no more than routine experimentation, many equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combinations of the embodiments disclosed in the dependent claims are contemplated to be within the scope of the disclosure.

[0301] The disclosure of each and every US and foreign patent and pending patent application and publication referred to herein is hereby incorporated herein by reference in its entirety.

TABLE-US-00005 TABLE 6 HSA Linker Amino Terminal N-Terminal C-Terminal Carboxy Terminal Conjugate Binding Moiety Connector HSA Connector Binding Moiety B2B3-1 H3 (SEQ ID NO: 26) AAS mHSA (SEQ ID NO: 1) AAAL B1D2 (SEQ ID NO: 27) (SEQ ID NO: 16) (SEQ ID NO: 5) B2B3-2 A5 (SEQ ID NO: 28) AAS mHSA (SEQ ID NO: 1) AAAL B1D2 (SEQ ID NO: 27) (SEQ ID NO: 17) (SEQ ID NO: 5) B2B3-3 A5 (SEQ ID NO: 28) AAS mHSA (SEQ ID NO: 1) AAAL F5B6H2 (SEQ ID NO: 32) (SEQ ID NO: 18) (SEQ ID NO: 5) B2B3-4 A5 (SEQ ID NO: 28) AAS mHSA (SEQ ID NO: 1) AAAL ML3.9 (SEQ ID NO: 29) (SEQ ID NO: 19) (SEQ ID NO: 5) B2B3-5 B12 (SEQ ID NO: 30) AAS mHSA (SEQ ID NO: 1) AAAL B1D2 (SEQ ID NO: 27) (SEQ ID NO: 20) (SEQ ID NO: 5) B2B3-6 B12 (SEQ ID NO: 30) AAS mHSA (SEQ ID NO: 1) AAAL F5B6H2 (SEQ ID NO: 32) (SEQ ID NO: 21) (SEQ ID NO: 5) B2B3-7 F4 (SEQ ID NO: 31) AAS mHSA (SEQ ID NO: 1) AAAL B1D2 (SEQ ID NO: 27) (SEQ ID NO: 22) (SEQ ID NO: 5) B2B3-8 F4 (SEQ ID NO: 31) AAS mHSA (SEQ ID NO: 1) AAAL F5B6H2 (SEQ ID NO: 32) (SEQ ID NO: 23) (SEQ ID NO: 5) B2B3-9 H3 (SEQ ID NO: 26) AAS HSA (SEQ ID NO: 3) AAAL B1D2 (SEQ ID NO: 27) (SEQ ID NO: 24) (SEQ ID NO: 5) B2B3-10 H3 (SEQ ID NO: 26) AAS mHSA (SEQ ID NO: 1) AAAL F5B6H2 (SEQ ID NO: 32) (SEQ ID NO: 25) (SEQ ID NO: 5)

TABLE-US-00006 APPENDIX 1 SEQUENCES, SEQUENCE ANNOTATIONS AND SEQUENCE ALIGNMENTS pMP9043 SEQ ID NO: 60 gtgccgacgatagagcagacctcgctaaatatatctgcgagaatcaggattccattagctctaagctgaaagaa- tgttgcgagaagccc ctcctggaaaagagtcattgtatcgccgaggtggaaaacgacgagatgccagcagatctgccatcactcgctgc- cgactttgtggaat ccaaagatgtctgcaagaattacgcagaggctaaagacgtgttcctggggatgtttctgtatgagtacgcccgg- cgtcaccccgattat agcgtcgtgctcctgctccgactggcaaagacctacgaaacaactctggagaaatgttgcgctgccgcagaccc- tcatgaatgttatgc taaggtgttcgatgagtttaagccactcgtcgaagagccccagaacctgattaaacagaattgcgaactgttcg- agcagctcggtgaat acaagtttcagaacgccctgctcgtgcgttataccaaaaaggtccctcaggtgtctacaccaactctggtggag- gtcagtaggaatctg ggcaaagtgggatcaaagtgttgcaaacaccccgaggcaaagagaatgccttgtgctgaagattacctctccgt- cgtgctgaaccagc tctgcgtgctgcatgaaaagaccccagtcagcgatcgggtgacaaaatgttgcaccgaatctctggtcaatcgc- cgaccctgtttcagt gccctcgaagtggacgaaacttatgtgcctaaggagtttcaggctgaaacattcacctttcacgccgatatctg- cactctgtccgagaaa gaaaggcagattaagaaacagacagcactggtcgagctcgtgaagcataaaccaaaggctaccaaggagcagct- gaaagccgtca tggacgatttcgcagcttttgtggaaaagtgttgcaaagccgacgataaggagacttgtttcgcagaagagggg- aaaaagctcgtggc tgccagccaggcagctctgggtctggccgcagctctgcaggtgcagctcgtccagagcggcgctgaggtgaaga- agccaggcgag tccctgaagatctcctgtaagggctccggctacagcttcacctcctactggatcgcttgggtgaggcagatgcc- aggaaagggactgg agtacatgggcctgatctaccctggcgactccgacaccaagtactccccatccttccagggccaggtgaccatc- agcgtggacaagtc cgtgtctaccgcctacctgcaatggtcctccctgaagccttctgactctgccgtgtacttttgtgcccggcacg- atgtgggctactgcacc gaccggacatgtgccaagtggcccgagtggctgggagtgtggggacagggaacactggtgacagtgagttctgg- cggtggcggct cttccggcggtggctctggtggcggcggatctcagagcgtgctgacacagccacctagcgtgtccgctgcccct- ggccagaaggtg acaatcagctgctccggcagctcttccaacatcggcaacaactacgtgtcttggtatcagcagctgcccggaac- agctccaaaactgct gatctatgaccacaccaatcggcctgccggcgtgccagatcggttctctggctctaagagcggcacctccgcca- gcctggctatctct ggcttcagatctgaggatgaggctgactactattgtgcctcctgggactacaccctgtctggctgggtgttcgg- cggtggcaccaagct gacagtcctgggatgatgactcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttcta- gttgccagccatctgt tgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgagg- aaattgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagca- ggcatgctggg gatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctg- tagcggcgcatta agcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctccUtcgct- ttcttcccttccttt ctcgccacgttcgccggctttccccgtcaagctctaaatcgggggtccctttagggttccgatttagtgcttta- cggcacctcgaccccaa aaaacttgattagggtgatggttcacgtacctagaagttcctattccgaagttcctattctctagaaagtatag- gaacttccttggccaaaa agcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcag- ctctcggagggcga agaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtt- tctacaaagatcgttat gtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaattcagcgagagcct- gacctattgcatctcc cgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcagccggtcgcgga- ggccatggatgc gatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacacta- catggcgtgatttc atatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgc- gcaggctctcgatga gctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcc- tgacggacaatgg ccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatcttct- tctggaggccgtgg ttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccg- ggcgtatatgctc cgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcg- atgcgacgcaatcgt ccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtg- tagaagtactc gccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaatagcacgtactacgagatttcgattc- caccgccgccttc tatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgct- ggagttcttcgccca ccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcat- ttttttcactgcattctagtt gtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaa- tcatggtcatagctgtttcct gtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgc- ctaatgagtgagcta actcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaa- tcggccaacgcgcgg ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctg- cggcgagcggtatca gctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaagg- ccagcaaaagg ccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaat- cgacgctcaagtca gaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctg- ttccgaccctgccg cttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatct- cagttcggtgtaggtcgt tcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtc- ttgagtccaacccgg taagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgct- acagagttcttgaa gtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcg- gaaaaagagttggtag ctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaa- aaaaaggatctcaag aagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatg- agattatcaaaaaggatc ttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctga- cagttaccaatgcttaatcag tgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataacta- cgatacgggagggcttac catctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccag- ccagccggaagg gccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagt- aagtagttcgccagtt aatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcatt- cagctccggttcccaacg atcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtca- gaagtaagttggccgc agtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctg- tgactggtgagtactcaacc aagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgcc- acatagcagaacttt aaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagtt- cgatgtaacccactcgt gcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgc- cgcaaaaaagggaat aagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttatt- gtctcatgagcggatacata tttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcga- cggatcgggagatct cccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgc- ttgtgtgttggaggtcgc tgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgctta- gggttaggcgttttg cgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaatt- acggggtcattagttcata gcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgc- ccattgacgtcaata atgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac- tgcccacttggcagtac atcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcc- cagtacatgaccttat gggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtac- atcaatgggcgtggatag cggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatca- acgggactttccaaaat gtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagct- ctctggctaactag agaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttctagaattcgct- gtctgcgagggccagc tgttggggtgagtactccctctcaaaagcgggcatgacttctgcgctaagattgtcagtttccaaaaacgagga- ggatttgatattcacct ggcccgcggtgatgcctttgagggtggccgcgtccatctggtcagaaaagacaatctttttgttgtcaagcttg- aggtgtggcaggcttg agatctggccatacacttgagtgacaatgacatccactttgcctttctctccacaggtgtccactcccaggtcc- aactgcagatatccagc acagtggcggccgccaccatgggctggtctctgatcctgctgttcctggtggccgtggccacgcgtgtgctgtc- ccaggtgcagctgc aggagtctggcggcggactggtgaagcctggcggctccctgcggctgtcctgcgccgcctccggcttcaccttc- tcctcctactggat gtcctgggtgcggcaggcccctggcaagggcctggagtgggtggccaacatcaaccgggacggctccgcctcct- actacgtggact

ccgtgaagggccggttcaccatctcccgggacgacgccaagaactccctgtacctgcagatgaactccctgcgg- gccgaggacacc gccgtgtactactgcgccagggaccggggcgtgggctacttcgacctgtggggcaggggcaccctggtgaccgt- gtcctccgctag tactggcggcggaggatctggcggaggagggagcgggggcggtggatcccagtccgccctgacccagcctgcct- ccgtgtccgg ctcccctggccagtccatcaccatcagctgcaccggcacctcctccgacgtgggcggctacaacttcgtgtcct- ggtatcagcagcac cccggcaaggcccctaagctgatgatctacgacgtgtccgaccggccttccggcgtgtccgacaggttctccgg- ctccaagtccggc aacaccgcctccctgatcatcagcggcctgcaggcagacgacgaggccgactactactgctcctcctacggctc- ctcctccacccac gtgatctttggcggcggaacaaaggtgaccgtgctgggcgccgcctccgacgctcacaagagcgaagtggcaca- taggttcaaaga tctgggcgaagagaactttaaggccctcgtcctgatcgctttcgcacagtacctccagcagtctccctttgaag- atcacgtgaaactggt caatgaggtgaccgaatttgccaagacatgcgtggctgatgagagtgcagaaaactgtgacaaatcactgcata- ctctctttggagata agctgtgcaccgtcgccacactcagagagacttatggggaaatggctgactgttgcgcaaaacaggagcctgaa- cggaatgagtgttt cctccagcacaaggatgacaacccaaatctgccccgcctcgtgcgacctgaggtcgatgtgatgtgcaccgcct- ttcatgacaacgaa gagacattcctgaagaaatacctgtatgaaattgctcgtaggcacccatacttttatgcccccgagctcctgtt- ctttgcaaagagataca aagctgccttcactgaatgttgccaggcagctgataaggccgcatgtctcctgcctaaactggacgagctccgg- gatgaaggtaaggc ttccagcgccaaacagcgcctgaagtgcgcttctctccagaagtttggcgagcgagcattcaaagcctgggctg- tggcccgtctcagt cagaggtttccaaaggcagaatttgctgaggtctcaaaactggtgaccgacctcacaaaggtccatactgagtg- ttgccacggagatct gctggaat SEQ ID NO: 67 QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDS DTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARADVGYCTDRTCAKAP AWLGVWGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGS SSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSED EADYYCASWDYTLSGWVFGGGTKLTVLGAASDAHKSEVAHRFKDLGEENFKALVL IAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLR ETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA KQRLKCAvSLQKFGERAFKAWAVARLvSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAAD FVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTP TLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCC TESLVNRRPCFSALEVDETYVPKEFQAETFTFHADICTLSEKERQIKKQTALVELVKH KPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQ VQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSD TKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAKWP EWLGVWGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSS SNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDE ADYYCASWDYTLSGWVFGGGTKLTVLG SEQ ID NO: 68 QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDG SASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGR GTLVTVSSASTGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGY NFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYY CSSYGSSSTHVIFGGGTKVTVLGAASDAHKSEVAHRFKDLGEENFKALVLIAFAQYL QQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEM ADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECAD DRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESK DVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHE CYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVE VSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL VNRRPCFSALEVDETYVPKEFQAETFTFHADICTLSEKERQIKKQTALVELVKHKPKA TKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLV QSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSP SFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARADVGYCTDRTCAKAPAWLGV WGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGN NYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYY CASWDYTLSGWVFGGGTKLTVLG

B2B3-1 (H3-mHSA-B1D2)

TABLE-US-00007 1 51 101 151 201 251 DAHKSEVAH 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 QAALGL QVQLVQSGAE 851 901 951 1001 1051

CDR loops are highlighted within H3 (underlined 1-248 with bold italicized CDRs) and B1D2 (underlined 841-1095 with bold italicized CDRs). Connectors to modified HSA are dotted-underlined. Sequence Alignments for Various HSA Linker Conjugates Comprising B2B3 and mHSA

TABLE-US-00008 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--

TABLE-US-00009 APPENDIX 2 ANTICANCER AGENTS Anticancer agents for combination with B2B3-1 Manufacturer/Proprietor Brand Name(s) Anti-IGF1R Antibodies AMG 479 (fully humanized mAb) Amgen IMCA12 (fully humanized mAb) ImClone NSC-742460 Dyax 19D12 (fully humanized mAb) CP751-871 (fully humanized mAb) Pfizer H7C10 (humanized mAb) alphaIR3 (mouse) scFV/FC (mouse/human chimera) EM/164 (mouse) MK-0646, F50035 Pierre Fabre Medicament, Merck Small Molecules Targeting IGF1R NVP-AEW541 Novartis BMS-536,924 (1H-benzoimidazol-2-yl)- Bristol-Myers Squibb 1H-pyridin-2-one) BMS-554,417 Bristol-Myers Squibb Cycloligan TAE226 PQ401 Anti-EGFR Monoclonal Antibodies INCB7839 Incyte Bevacizumab Avastin ® Genentech Cetuximab Erbitux ® IMCLONE mAb 806 Matuzumab (EMD72000) Nimotuzumab (TheraCIM) Panitumumab Vectibix ® Amgen Anti-ErbB3 Therapeutics -- -- U3-1287/AMG888 U3 Pharma/Amgen MM-121 Merrimack Pharmaceuticals Anti-ErbB2 Therapeutics -- -- trastuzumab Herceptin ® Genentech HKI-272 - neratinib Wyeth KOS-953 - tanespimycin Kosan Biosciences Her/ErbB Dimerization Inhibitors 2C4, R1273 - Pertuzumab Omnitarg ® Genentech, Roche Small Molecules Targeting EGFR CI-1033 (PD 183805) Pzifer, Inc. EKB-569 Gefitinib IRESSA ® AstraZeneca Lapatinib (GW572016) GlaxoSmithKline Lapatinib Ditosylate Tykerb ® SmithKline Beecham Erlotinib HCl (OSI-774) Tarceva ® OSI Pharms PD158780 PKI-166 Novartis Tyrphostin AG 1478 (4-(3-Chloroanillino)- 6,7-dimethoxyquinazoline) Anti-cmet Antibody Therapies AVEO (AV299) AVEO AMG102 Amgen 5D5 (OA-5D5) Genentech Small Molecules Targeting cmet PHA665752 ARQ-650RP ArQule ARQ 197 ArQule Alkylating Agents BCNU→ 1,3-bis t2-chloroethyl)- nitrosourea Bendamustine Busulfan Myleran GlaxoSmithKline Carboplatin Paraplatin Bristol-Myers Squibb Carboquone Carmustine CCNU→ 1,-(2-chloroethyl)-3-cyclohexyl- 1-nitrosourea (methyl CCNU) Chlorambucil Leukeran ® Smithkline Beecham Chlormethine Cisplatin (Cisplatinum, CDDP) Platinol Bristol-Myers Cyclophosphamide Cytoxan Bristol-Myers Squibb Neosar Teva Parenteral Dacarbazine (DTIC) Fotemustine Hexamethylmelamine (Altretamine, HMM) Hexalen ® MGI Pharma, Inc. Ifosfamide Mitoxana ® ASTA Medica Lomustine Mannosulfan Melphalan Alkeran ® GlaxoSmithKline Nedaplatin Nimustine Oxaliplatin Eloxatin ® Sanofi-Aventis US Prednimustine, Matulane Sigma-Tau Pharmaceuticals, Inc. Procarbazine HCL Ribonucleotide Reductase Inhibitor (RNR) Ranimustine Satraplatin Semustine Streptozocin Temozolomide Treosulfan Triaziquone Triethylene Melamine ThioTEPA Bedford, Abraxis, Teva Triplatin tetranitrate Trofosfamide Uramustine Antimetabolites 5-azacytidine Flourouracil (5-FU)/Capecitabine 6-mercaptopurine (Mercaptopurine, 6-MP) 6-Thioguanine (6-TG) Purinethol ® Teva Cytosine Arabinoside (Cytarabine, Thioguanine ® GlaxoSmithKline Ara-C) Azathioprine Azasan ® AAIPHARMA LLC Capecitabine XELODA ® HLR (Roche) Cladribine (2-CdA, 2- Leustatin ® Ortho Biotech chlorodeoxyadenosine) 5-Trifluoromethyl-2'-deoxyuridine Fludarabine phosphate Fludara ® Bayer Health Care Floxuridine (5-fluoro-2) FUDR ® Hospira, Inc. Methotrexate sodium Trexall Barr Pemetrexed Alimta ® Lilly Pentostatin Nipent ® Hospira, Inc. Raltitrexed Tomudex ® AstraZeneca Tegafur Aromatose Inhibitor Ketoconazole Glucocorticoids Dexamethasone Decadron ® Dexasone, Wyeth, Inc. Diodex, Hexadrol, Maxidex Prednisolone Prednisone Deltasone, Orasone, Liquid Pred, Sterapred ® Immunotherapeutics Alpha interferon Angiogenesis Inhibitor Avastin ® Genentech IL-12→ Interleukin 12 IL-2→ Interleukin 2 (Aldesleukin) Proleukin ® Chiron Kinase Inhibitors AMG 386 Amgen Axitinib ((AG-013736) Pfizer, Inc Bosutinib (SKI-606) Wyeth Brivanib alalinate (BMS-582664) BMS Cediranib (AZD2171) Recentin AstraVeneca Dasatinib (BMS-354825) Sprycel ® Bristol-Myers Squibb Imatinib mesylate Gleevec Novartis Lestaurtinib (CEP-701) Cephalon Motesanib diphosphate (AMG-706) Amgen/Takeda Nilotinib hydrochloride monohydrate Tasigna ® Novartis Pazopanib HCL (GW786034) Armala GSK Semaxanib (SU5416) Pharmacia, Sorafenib tosylate Nexavar ® Bayer Sunitinib malate Sutent ® Pfizer, Inc. Vandetanib (AZD647) Zactima AstraZeneca Vatalanib; PTK-787 Novartis; Bayer Schering Pharma XL184, NSC718781 Exelixis, GSK Microtubule-Targeting Agents Colchicine Docetaxel Taxotere ® Sanofi-Aventis US Ixabepilone IXEMPRA ® Bristol-Myers Squibb Larotaxel Sanofi-aventis Ortataxel Spectrum Pharmaceuticals Nanoparticle paclitaxel (ABI-007) Abraxane ® Abraxis BioScience, Inc. Paclitaxel Taxol ® Bristol-Myers Squibb Tesetaxel Genta Vinblastine sulfate Velban ® Lilly Vincristine Oncovin ® Lilly Vindesine sulphate Eldisine ® Lilly Vinflunine Pierre Fabre Vinorelbine tartrate Navelbine ® Pierre Fabre mTOR Inhibitors Deforolimus (AP23573, MK 8669) ARIAD Pharmaceuticals, Inc Everolimus (RAD001, RAD001C) Certican ®, Afinitor Novartis Sirolimus (Rapamycin) Rapamune ® Wyeth Pharama Temsirolimus (CCI-779) Torisel ® Wyeth Pharama Protein Synthesis Inhibitor L-asparaginase Elspar ® Merck & Co. Somatostatin Analogue Octreotide acetate Sandostatin ® Novartis Topoisomerase Inhibitors Actinomycin D Camptothecin (CPT) Belotecan Daunorubicin citrate Daunoxome ® Gilead Doxorubicin hydrochloride Doxil ® Alza Vepesid ® Bristol-Myers Squibb Etoposide Etopophos Hospira, Bedford, Teva Parenteral, Etc. Irinotecan HCL (CPT-11) Camptosar ® Pharmacia & Upjohn Mitoxantrone HCL Novantrone EMD Serono Rubitecan Teniposide (VM-26) Vumon ® Bristol-Myers Squibb Topotecan HCL Hycamtin ® GlaxoSmithKline Chemotherapeutic Agents Adriamycin, 5-Fluorouracil, Cytoxin, Bleomycin, Mitomycin C, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins Clofarabine, Mercaptopurine, Pentostatin, Thioguanine, Cytarabine, Decitabine, Floxuridine, Gemcitabine (Gemzar), Enocitabine, Sapacitabine Hormonal Therapies Abarelix Plenaxis ® Amgen Abiraterone acetate CB7630 BTG plc Afimoxifene TamoGel Ascend Therapeutics, Inc. Anastrazole Arimidex ® AstraZeneca Aromatase inhibitor Atamestane plus toremifene Intarcia Therapeutics, Inc. Arzoxifene Eli Lilly & Co. Asentar; DN 101 Novartis; Oregon Health & Science Univ. Bicalutamide Casodex ® AstraZeneca Buserelin Suprefact ® Sanofi Aventis Cetrorelix Cetrotide ® EMD Serono Exemestane Aromasin ® Pfizer Exemestane Xtane Natco Pharma, Ltd. Fadrozole (CGS 16949A) Flutamide Eulexin ® Schering Flutamide Prostacur Laboratorios Almirall, S.A. Fulvestrant Faslodex ® AstraZeneca Goserelin acetate Zoladex ® AstraZeneca Letrozole Femara ® Novartis Letrozole (CGS20267) Femara Chugai Pharmaceutical Co., Ltd. Letrozole Estrochek Jagsonpal Pharmaceuticals, Ltd. Letrozole Letrozole Indchemie Health Specialities Leuprolide acetate Eligard ® Sanofi Aventis Leuprolide acetate Leopril VHB Life Sciences, Inc. Leuprolide acetate Lupron ®/Lupron Depot TAP Pharma Leuprolide acetate Viador Bayer AG Megestrol acetate Megace ® Bristol-Myers Squibb Magestrol acetate Estradiol Valerate Jagsonpal Pharmaceuticals, Ltd. (Delestrogen) Medroxyprogesterone acetate Veraplex Combiphar MT206 Medisyn Technologies, Inc. Nafarelin Nandrolone decanoate Zestabolin Mankind Pharma, Ltd. Nilutamide Nilandron ® Aventis Pharmaceuticals Raloxifene HCL Evista ® Lilly Tamoxifen Taxifen Yung Shin Pharmaceutical Tamoxifen Tomifen Alkem Laboratories, Ltd. Tamoxifen citrate Nolvadex AstraZeneca Tamoxifen citrate Soltamox EUSA Pharma, Inc. Tamoxifen citrate Tamoxifen citrate Sopharma JSCo. SOPHARMA Toremifene citrate Fareston ® GTX, Inc. Triptorelin pamoate Trelstar ® Watson Labs Triptorelin pamoate Trelstar Depot Paladin Labs, Inc. Protein Kinase B (PKB) Inhibitors Akt Inhibitor ASTEX Astex Therapeutics Akt Inhibitors NERVIANO Nerviano Medical Sciences AKT Kinase Inhibitor TELIK Telik, Inc. AKT DECIPHERA Deciphera Pharmaceuticals, LLC

Perifosine (KRX0401, D-21266) Keryx Biopharmaceuticals, Inc., AEterna Zentaris, Inc. Perifosine with Docetaxel Keryx Biopharmaceuticals, Inc., AEterna Zentaris, Inc. Perifosine with Gemcitabine AEterna Zentaris, Inc. Perifosine with Paclitaxel Keryx Biopharmaceuticals, Inc, AEterna Zentaris, Inc. Protein Kinase-B inhibitor DEVELOGEN DeveloGen AG PX316 Oncothyreon, Inc. RX0183 Rexahn Pharmaceuticals, Inc. RX0201 Rexahn Pharmaceuticals, Inc. VQD002 VioQuest Pharmaceuticals, Inc. XL418 Exelixis, Inc. ZEN027 AEterna Zentaris, Inc. Phosphatidylinositol 3-Kinase (PI3K) Inhibitors BEZ235 Novartis AG BGT226 Novartis AG CAL101 Calistoga Pharmaceuticals, Inc. CHR4432 Chroma Therapeutics, Ltd. Erk/PI3K Inhibitors ETERNA AEterna Zentaris, Inc. GDC0941 Genentech Inc./Piramed Limited/Roche Holdings, Ltd. Enzastaurin HCL (LY317615) Enzastaurin Eli Lilly LY294002/Wortmannin PI3K Inhibitors SEMAFORE Semafore Pharmaceuticals PX866 Oncothyreon, Inc. SF1126 Semafore Pharmaceuticals VMD-8000 VM Discovery, Inc. XL147 Exelixis, Inc. XL147 with XL647 Exelixis, Inc. XL765 Exelixis, Inc. PI-103 Roche/Piramed Cyclin-dependent kinase inhibitors CYC200, r-roscovitine Seliciclib Cyclacel Pharma NSC-649890, L86-8275, HMR-1275 Alvocidib NCI TLr9, CD289 IMOxine Merck KGaA HYB2055 Idera IMO-2055 Isis Pharma 1018 ISS Dynavax Technologies/UCSF PF-3512676 Pfizer Enzyme Inhibitor Lonafarnib (SCH66336) Sarasar SuperGen, U Arizona Anti-TRAIL AMG-655 Aeterna Zentaris, Keryx Biopharma Apo2L/TRAIL, AMG951 Genentech, Amgen Apomab (fully humanized mAb Genentech Target Other Imprime PGG Biothera CHR-2797 AminopeptidaseM1 Chroma Therapeutics E7820, NSC 719239 Integrin-alpha2 Eisai INCB007839 ADAM 17, TACE Incyte CNF2024, BIIB021 Hsp90 Biogen Idec MP470, HPK-56 Kit/Met/Ret Shering-Plough SNDX-275/MS-275 HDAC Syndax Zarnestra, Tipifarnib, R115777 Ras Janssen Pharma Volociximab; Eos 200-4, M200 alpha581 integrin Biogen Idec; Eli Lilly/UCSF/PDL BioPharma Apricoxib (TP2001) COX-2 Inhibitor Daiichi Sankyo; Tragara Pharma indicates data missing or illegible when filed

Other Embodiments

[0302] While the invention has been described in connection with specific embodiments thereof, it will be understood 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 that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0303] All patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Sequence CWU 1

1

681585PRTArtificial SequenceSynthetic construct 1Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 21755DNAArtificial SequenceSynthetic construct 2gacgctcaca agagcgaagt ggcacatagg ttcaaagatc tgggcgaaga gaactttaag 60gccctcgtcc tgatcgcttt cgcacagtac ctccagcagt ctccctttga agatcacgtg 120aaactggtca atgaggtgac cgaatttgcc aagacatgcg tggctgatga gagtgcagaa 180aactgtgaca aatcactgca tactctcttt ggagataagc tgtgcaccgt cgccacactc 240agagagactt atggggaaat ggctgactgt tgcgcaaaac aggagcctga acggaatgag 300tgtttcctcc agcacaagga tgacaaccca aatctgcccc gcctcgtgcg acctgaggtc 360gatgtgatgt gcaccgcctt tcatgacaac gaagagacat tcctgaagaa atacctgtat 420gaaattgctc gtaggcaccc atacttttat gcccccgagc tcctgttctt tgcaaagaga 480tacaaagctg ccttcactga atgttgccag gcagctgata aggccgcatg tctcctgcct 540aaactggacg agctccggga tgaaggtaag gcttccagcg ccaaacagcg cctgaagtgc 600gcttctctcc agaagtttgg cgagcgagca ttcaaagcct gggctgtggc ccgtctcagt 660cagaggtttc caaaggcaga atttgctgag gtctcaaaac tggtgaccga cctcacaaag 720gtccatactg agtgttgcca cggagatctg ctggaatgtg ccgacgatag agcagacctc 780gctaaatata tctgcgagaa tcaggattcc attagctcta agctgaaaga atgttgcgag 840aagcccctcc tggaaaagag tcattgtatc gccgaggtgg aaaacgacga gatgccagca 900gatctgccat cactcgctgc cgactttgtg gaatccaaag atgtctgcaa gaattacgca 960gaggctaaag acgtgttcct ggggatgttt ctgtatgagt acgcccggcg tcaccccgat 1020tatagcgtcg tgctcctgct ccgactggca aagacctacg aaacaactct ggagaaatgt 1080tgcgctgccg cagaccctca tgaatgttat gctaaggtgt tcgatgagtt taagccactc 1140gtcgaagagc cccagaacct gattaaacag aattgcgaac tgttcgagca gctcggtgaa 1200tacaagtttc agaacgccct gctcgtgcgt tataccaaaa aggtccctca ggtgtctaca 1260ccaactctgg tggaggtcag taggaatctg ggcaaagtgg gatcaaagtg ttgcaaacac 1320cccgaggcaa agagaatgcc ttgtgctgaa gattacctct ccgtcgtgct gaaccagctc 1380tgcgtgctgc atgaaaagac cccagtcagc gatcgggtga caaaatgttg caccgaatct 1440ctggtcaatc gccgaccctg tttcagtgcc ctcgaagtgg acgaaactta tgtgcctaag 1500gagtttcagg ctgaaacatt cacctttcac gccgatatct gcactctgtc cgagaaagaa 1560aggcagatta agaaacagac agcactggtc gagctcgtga agcataaacc aaaggctacc 1620aaggagcagc tgaaagccgt catggacgat ttcgcagctt ttgtggaaaa gtgttgcaaa 1680gccgacgata aggagacttg tttcgcagaa gaggggaaaa agctcgtggc tgccagccag 1740gcagctctgg gtctg 17553585PRTHomo sapiens 3Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 41755DNAHomo sapiens 4gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg tagctgatga gtcagctgaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaaatgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtggc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaggattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aactgtgagc tttttaagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca caaaatgctg cacagagtcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagcttgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gctta 175554PRTArtificial sequenceSynthetic Construct 5Ala Ala Ala Leu 1 6588PRTArtificial sequenceSynthetic Construct 6Ala Ala Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 1 5 10 15 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 20 25 30 Tyr Leu Gln Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 35 40 45 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 50 55 60 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 65 70 75 80 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 85 90 95 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 100 105 110 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 115 120 125 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 130 135 140 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 145 150 155 160 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 165 170 175 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 180 185 190 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 195 200 205 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 210 215 220 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 225 230 235 240 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 245 250 255 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 260 265 270 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 275 280 285 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 290 295 300 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 305 310 315 320 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 325 330 335 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 340 345 350 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 355 360 365 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 370 375 380 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 385 390 395 400 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 405 410 415 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 420 425 430 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 435 440 445 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 450 455 460 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 465 470 475 480 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 485 490 495 Asp Glu Thr Tyr Val Pro Lys Glu Phe Gln Ala Glu Thr Phe Thr Phe 500 505 510 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 515 520 525 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 530 535 540 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala

Ala Phe Val Glu Lys 545 550 555 560 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 565 570 575 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 7588PRTArtificial SequenceSynthetic Construct 7Ala Ala Gln Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 1 5 10 15 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 20 25 30 Tyr Leu Gln Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 35 40 45 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 50 55 60 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 65 70 75 80 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 85 90 95 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 100 105 110 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 115 120 125 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 130 135 140 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 145 150 155 160 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 165 170 175 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 180 185 190 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 195 200 205 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 210 215 220 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 225 230 235 240 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 245 250 255 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 260 265 270 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 275 280 285 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 290 295 300 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 305 310 315 320 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 325 330 335 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 340 345 350 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 355 360 365 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 370 375 380 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 385 390 395 400 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 405 410 415 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 420 425 430 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 435 440 445 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 450 455 460 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 465 470 475 480 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 485 490 495 Asp Glu Thr Tyr Val Pro Lys Glu Phe Gln Ala Glu Thr Phe Thr Phe 500 505 510 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 515 520 525 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 530 535 540 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 545 550 555 560 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 565 570 575 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 8589PRTArtificial SequenceSynthetic Construct 8Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala Ala Ala Leu 580 585 9592PRTArtificial SequenceSynthetic Construct 9Ala Ala Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 1 5 10 15 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 20 25 30 Tyr Leu Gln Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 35 40 45 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 50 55 60 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 65 70 75 80 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 85 90 95 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 100 105 110 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 115 120 125 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 130 135 140 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 145 150 155 160 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 165 170 175 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 180 185 190 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 195 200 205 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 210 215 220 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 225 230 235 240 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 245 250 255 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 260 265 270 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 275 280 285 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 290 295 300 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 305 310 315 320 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 325 330 335 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 340 345 350 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 355 360 365 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 370 375 380 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 385 390 395 400 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 405 410 415 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 420 425 430 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 435 440 445 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 450 455 460 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 465 470 475 480 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 485 490 495 Asp Glu Thr Tyr Val Pro Lys Glu Phe Gln Ala Glu Thr Phe Thr Phe 500 505 510 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 515 520 525 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 530 535 540 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 545 550 555 560 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 565 570 575 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala Ala Ala Leu 580 585 590 10592PRTArtificial SequenceSynthetic Construct 10Ala Ala Gln Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 1 5 10 15 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 20 25 30 Tyr Leu Gln Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 35 40 45 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 50 55 60 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 65 70 75 80 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 85 90 95 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 100 105 110 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 115 120 125 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 130 135 140 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 145 150 155 160 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 165 170 175 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 180 185 190 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 195 200 205 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 210 215 220 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 225 230 235 240 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 245 250 255 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 260 265 270 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 275 280 285 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 290 295 300 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 305 310 315 320 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 325 330 335 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 340 345 350 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 355 360 365 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 370 375 380 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 385 390 395 400 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 405 410 415 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 420 425 430 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 435 440 445 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 450 455 460 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 465

470 475 480 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 485 490 495 Asp Glu Thr Tyr Val Pro Lys Glu Phe Gln Ala Glu Thr Phe Thr Phe 500 505 510 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 515 520 525 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 530 535 540 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 545 550 555 560 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 565 570 575 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala Ala Ala Leu 580 585 590 11588PRTArtificial SequenceSynthetic Construct 11Ala Ala Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 1 5 10 15 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 20 25 30 Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 35 40 45 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 50 55 60 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 65 70 75 80 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 85 90 95 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 100 105 110 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 115 120 125 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 130 135 140 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 145 150 155 160 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 165 170 175 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 180 185 190 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 195 200 205 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 210 215 220 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 225 230 235 240 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 245 250 255 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 260 265 270 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 275 280 285 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 290 295 300 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 305 310 315 320 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 325 330 335 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 340 345 350 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 355 360 365 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 370 375 380 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 385 390 395 400 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 405 410 415 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 420 425 430 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 435 440 445 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 450 455 460 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 465 470 475 480 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 485 490 495 Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe 500 505 510 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 515 520 525 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 530 535 540 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 545 550 555 560 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 565 570 575 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 12588PRTArtificial SequenceSynthetic Construct 12Ala Ala Gln Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 1 5 10 15 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 20 25 30 Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 35 40 45 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 50 55 60 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 65 70 75 80 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 85 90 95 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 100 105 110 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 115 120 125 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 130 135 140 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 145 150 155 160 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 165 170 175 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 180 185 190 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 195 200 205 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 210 215 220 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 225 230 235 240 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 245 250 255 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 260 265 270 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 275 280 285 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 290 295 300 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 305 310 315 320 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 325 330 335 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 340 345 350 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 355 360 365 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 370 375 380 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 385 390 395 400 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 405 410 415 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 420 425 430 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 435 440 445 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 450 455 460 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 465 470 475 480 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 485 490 495 Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe 500 505 510 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 515 520 525 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 530 535 540 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 545 550 555 560 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 565 570 575 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 13589PRTArtificial SequenceSynthetic Construct 13Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala Ala Ala Leu 580 585 14592PRTArtificial SequenceSynthetic Construct 14Ala Ala Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 1 5 10 15 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 20 25 30 Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 35 40 45 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 50 55 60 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 65 70 75 80 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 85 90 95 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 100 105 110 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 115 120 125 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 130 135 140 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 145 150 155 160 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 165 170 175 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 180 185 190 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 195 200 205 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 210 215 220 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 225 230 235 240 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 245 250 255 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 260 265 270 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 275 280 285 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 290 295 300 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 305 310 315 320 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 325 330 335 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 340 345 350 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 355 360 365 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 370 375 380 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 385 390

395 400 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 405 410 415 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 420 425 430 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 435 440 445 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 450 455 460 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 465 470 475 480 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 485 490 495 Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe 500 505 510 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 515 520 525 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 530 535 540 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 545 550 555 560 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 565 570 575 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala Ala Ala Leu 580 585 590 15592PRTArtificial SequenceSynthethic Construct 15Ala Ala Gln Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 1 5 10 15 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 20 25 30 Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 35 40 45 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 50 55 60 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 65 70 75 80 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 85 90 95 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 100 105 110 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 115 120 125 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 130 135 140 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 145 150 155 160 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 165 170 175 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 180 185 190 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 195 200 205 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 210 215 220 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 225 230 235 240 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 245 250 255 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 260 265 270 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 275 280 285 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 290 295 300 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 305 310 315 320 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 325 330 335 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 340 345 350 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 355 360 365 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 370 375 380 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 385 390 395 400 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 405 410 415 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 420 425 430 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 435 440 445 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 450 455 460 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 465 470 475 480 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 485 490 495 Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe 500 505 510 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 515 520 525 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 530 535 540 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 545 550 555 560 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 565 570 575 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala Ala Ala Leu 580 585 590 161095PRTArtificial SequenceSynthetic Construct 16Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Asn Arg Asp Gly Ser Ala Ser Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Val Gly Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala 130 135 140 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly 145 150 155 160 Thr Ser Ser Asp Val Gly Gly Tyr Asn Phe Val Ser Trp Tyr Gln Gln 165 170 175 His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asp Arg 180 185 190 Pro Ser Gly Val Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr 195 200 205 Ala Ser Leu Ile Ile Ser Gly Leu Gln Ala Asp Asp Glu Ala Asp Tyr 210 215 220 Tyr Cys Ser Ser Tyr Gly Ser Ser Ser Thr His Val Ile Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Thr Val Leu Gly Ala Ala Ser Asp Ala His Lys Ser 245 250 255 Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala 260 265 270 Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu 275 280 285 Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys 290 295 300 Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu 305 310 315 320 Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly 325 330 335 Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys 340 345 350 Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg 355 360 365 Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr 370 375 380 Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe 385 390 395 400 Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 405 410 415 Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys 420 425 430 Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg 435 440 445 Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala 450 455 460 Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala 465 470 475 480 Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys 485 490 495 Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala 500 505 510 Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 515 520 525 Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val 530 535 540 Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe 545 550 555 560 Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val 565 570 575 Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr 580 585 590 Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu 595 600 605 Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val 610 615 620 Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys 625 630 635 640 Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn 645 650 655 Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro 660 665 670 Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys 675 680 685 Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu 690 695 700 Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val 705 710 715 720 Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg 725 730 735 Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu 740 745 750 Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser 755 760 765 Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val 770 775 780 Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp 785 790 795 800 Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu 805 810 815 Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala 820 825 830 Ala Leu Gly Leu Ala Ala Ala Leu Gln Val Gln Leu Val Gln Ser Gly 835 840 845 Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly 850 855 860 Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Ala Trp Val Arg Gln Met 865 870 875 880 Pro Gly Lys Gly Leu Glu Tyr Met Gly Leu Ile Tyr Pro Gly Asp Ser 885 890 895 Asp Thr Lys Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Val 900 905 910 Asp Lys Ser Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro 915 920 925 Ser Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Val Gly Tyr Cys 930 935 940 Thr Asp Arg Thr Cys Ala Lys Trp Pro Glu Trp Leu Gly Val Trp Gly 945 950 955 960 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser Gly 965 970 975 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro 980 985 990 Ser Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly 995 1000 1005 Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln 1010 1015 1020 Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp His Thr Asn 1025 1030 1035 Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly 1040 1045 1050 Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg Ser Glu Asp Glu 1055 1060 1065 Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr Thr Leu Ser Gly Trp 1070 1075 1080 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 1085 1090 1095 171092PRTArtificial SequenceSynthetic Construct 17Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Thr Tyr 20 25 30 Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Val Ala Thr Thr Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val 130 135 140 Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser 145 150 155 160 Ser Asn Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro 165 170 175 Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser 180 185 190 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 195 200 205 Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 210 215 220 Gln Ser Tyr Asp Ser Ser Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys 225 230 235 240 Leu Thr Val Leu Gly Ala Ala Ser Asp Ala His Lys Ser Glu Val Ala 245 250 255 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 260 265 270 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu Asp His Val 275 280 285 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 290 295 300 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 305 310 315 320 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 325 330 335 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 340 345 350 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 355 360 365 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 370 375 380 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 385 390 395 400 Glu Leu Leu

Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 405 410 415 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 420 425 430 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 435 440 445 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 450 455 460 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 465 470 475 480 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 485 490 495 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 500 505 510 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 515 520 525 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 530 535 540 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 545 550 555 560 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 565 570 575 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 580 585 590 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 595 600 605 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 610 615 620 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 625 630 635 640 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 645 650 655 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 660 665 670 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 675 680 685 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 690 695 700 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 705 710 715 720 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 725 730 735 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Gln Ala 740 745 750 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 755 760 765 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 770 775 780 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 785 790 795 800 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 805 810 815 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 820 825 830 Leu Ala Ala Ala Leu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 835 840 845 Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr 850 855 860 Ser Phe Thr Ser Tyr Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys 865 870 875 880 Gly Leu Glu Tyr Met Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys 885 890 895 Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser 900 905 910 Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser 915 920 925 Ala Val Tyr Phe Cys Ala Arg His Asp Val Gly Tyr Cys Thr Asp Arg 930 935 940 Thr Cys Ala Lys Trp Pro Glu Trp Leu Gly Val Trp Gly Gln Gly Thr 945 950 955 960 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser 965 970 975 Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser 980 985 990 Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser 995 1000 1005 Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly 1010 1015 1020 Thr Ala Pro Lys Leu Leu Ile Tyr Asp His Thr Asn Arg Pro Ala 1025 1030 1035 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala 1040 1045 1050 Ser Leu Ala Ile Ser Gly Phe Arg Ser Glu Asp Glu Ala Asp Tyr 1055 1060 1065 Tyr Cys Ala Ser Trp Asp Tyr Thr Leu Ser Gly Trp Val Phe Gly 1070 1075 1080 Gly Gly Thr Lys Leu Thr Val Leu Gly 1085 1090 181083PRTArtificial SequenceSynthetic Construct 18Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Thr Tyr 20 25 30 Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Val Ala Thr Thr Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val 130 135 140 Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser 145 150 155 160 Ser Asn Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro 165 170 175 Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser 180 185 190 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 195 200 205 Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 210 215 220 Gln Ser Tyr Asp Ser Ser Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys 225 230 235 240 Leu Thr Val Leu Gly Ala Ala Ser Asp Ala His Lys Ser Glu Val Ala 245 250 255 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 260 265 270 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu Asp His Val 275 280 285 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 290 295 300 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 305 310 315 320 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 325 330 335 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 340 345 350 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 355 360 365 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 370 375 380 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 385 390 395 400 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 405 410 415 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 420 425 430 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 435 440 445 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 450 455 460 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 465 470 475 480 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 485 490 495 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 500 505 510 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 515 520 525 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 530 535 540 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 545 550 555 560 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 565 570 575 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 580 585 590 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 595 600 605 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 610 615 620 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 625 630 635 640 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 645 650 655 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 660 665 670 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 675 680 685 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 690 695 700 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 705 710 715 720 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 725 730 735 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Gln Ala 740 745 750 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 755 760 765 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 770 775 780 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 785 790 795 800 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 805 810 815 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 820 825 830 Leu Ala Ala Ala Leu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu 835 840 845 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 850 855 860 Thr Phe Arg Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys 865 870 875 880 Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Arg Gly Asp Asn Thr Tyr 885 890 895 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 900 905 910 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 915 920 925 Ala Val Tyr Tyr Cys Ala Lys Met Thr Ser Asn Ala Val Gly Phe Asp 930 935 940 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 945 950 955 960 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gln Ser Val Leu Thr 965 970 975 Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser 980 985 990 Cys Thr Gly Arg His Ser Asn Ile Gly Leu Gly Tyr Gly Val His Trp 995 1000 1005 Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly 1010 1015 1020 Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Phe 1025 1030 1035 Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala 1040 1045 1050 Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Arg Thr 1055 1060 1065 Pro Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 1070 1075 1080 191092PRTArtificial SequenceSynthetic Construct 19Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Thr Tyr 20 25 30 Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Val Ala Thr Thr Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val 130 135 140 Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser 145 150 155 160 Ser Asn Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro 165 170 175 Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser 180 185 190 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 195 200 205 Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 210 215 220 Gln Ser Tyr Asp Ser Ser Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys 225 230 235 240 Leu Thr Val Leu Gly Ala Ala Ser Asp Ala His Lys Ser Glu Val Ala 245 250 255 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 260 265 270 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu Asp His Val 275 280 285 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 290 295 300 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 305 310 315 320 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 325 330 335 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 340 345 350 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 355 360 365 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 370 375 380 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 385 390 395 400 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 405 410 415 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 420 425 430 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 435 440 445 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 450 455 460 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 465 470 475 480 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 485 490 495 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 500 505

510 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 515 520 525 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 530 535 540 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 545 550 555 560 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 565 570 575 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 580 585 590 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 595 600 605 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 610 615 620 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 625 630 635 640 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 645 650 655 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 660 665 670 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 675 680 685 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 690 695 700 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 705 710 715 720 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 725 730 735 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Gln Ala 740 745 750 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 755 760 765 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 770 775 780 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 785 790 795 800 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 805 810 815 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 820 825 830 Leu Ala Ala Ala Leu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 835 840 845 Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr 850 855 860 Ser Phe Thr Ser Tyr Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys 865 870 875 880 Gly Leu Glu Tyr Met Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys 885 890 895 Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser 900 905 910 Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser 915 920 925 Ala Val Tyr Phe Cys Ala Arg His Asp Val Gly Tyr Cys Ser Ser Ser 930 935 940 Asn Cys Ala Lys Trp Pro Glu Tyr Phe Gln His Trp Gly Gln Gly Thr 945 950 955 960 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser 965 970 975 Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser 980 985 990 Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser 995 1000 1005 Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly 1010 1015 1020 Thr Ala Pro Lys Leu Leu Ile Tyr Asp His Thr Asn Arg Pro Ala 1025 1030 1035 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala 1040 1045 1050 Ser Leu Ala Ile Ser Gly Phe Arg Ser Glu Asp Glu Ala Asp Tyr 1055 1060 1065 Tyr Cys Ala Ser Trp Asp Tyr Thr Leu Ser Gly Trp Val Phe Gly 1070 1075 1080 Gly Gly Thr Lys Leu Thr Val Leu Gly 1085 1090 201096PRTArtificial SequenceSynthetic Construct 20Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Gly Ala Lys Gln Trp Leu Glu Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu 130 135 140 Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile 145 150 155 160 Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn 180 185 190 Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Thr Ser Gly Asn 195 200 205 Ser Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp 210 215 220 Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Val Thr Val Leu Gly Ala Ala Ser Asp Ala His Lys 245 250 255 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 260 265 270 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe 275 280 285 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 290 295 300 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 305 310 315 320 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 325 330 335 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 340 345 350 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 355 360 365 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 370 375 380 Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 385 390 395 400 Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 405 410 415 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 420 425 430 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 435 440 445 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 450 455 460 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 465 470 475 480 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 485 490 495 Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 500 505 510 Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 515 520 525 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 530 535 540 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 545 550 555 560 Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 565 570 575 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 580 585 590 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 595 600 605 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 610 615 620 Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 625 630 635 640 Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln 645 650 655 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 660 665 670 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 675 680 685 Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 690 695 700 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 705 710 715 720 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 725 730 735 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 740 745 750 Glu Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu 755 760 765 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 770 775 780 Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 785 790 795 800 Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 805 810 815 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 820 825 830 Ala Ala Leu Gly Leu Ala Ala Ala Leu Gln Val Gln Leu Val Gln Ser 835 840 845 Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys 850 855 860 Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Ala Trp Val Arg Gln 865 870 875 880 Met Pro Gly Lys Gly Leu Glu Tyr Met Gly Leu Ile Tyr Pro Gly Asp 885 890 895 Ser Asp Thr Lys Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser 900 905 910 Val Asp Lys Ser Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys 915 920 925 Pro Ser Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Val Gly Tyr 930 935 940 Cys Thr Asp Arg Thr Cys Ala Lys Trp Pro Glu Trp Leu Gly Val Trp 945 950 955 960 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser 965 970 975 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro 980 985 990 Pro Ser Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser 995 1000 1005 Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln 1010 1015 1020 Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp His Thr 1025 1030 1035 Asn Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 1040 1045 1050 Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg Ser Glu Asp 1055 1060 1065 Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr Thr Leu Ser Gly 1070 1075 1080 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 1085 1090 1095 211086PRTArtificial SequenceSynthetic Construct 21Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Gly Ala Lys Gln Trp Leu Glu Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu 130 135 140 Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile 145 150 155 160 Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn 180 185 190 Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Thr Ser Gly Asn 195 200 205 Ser Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp 210 215 220 Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Val Thr Val Leu Gly Ala Ala Ser Asp Ala His Lys 245 250 255 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 260 265 270 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe 275 280 285 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 290 295 300 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 305 310 315 320 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 325 330 335 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 340 345 350 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 355 360 365 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 370 375 380 Thr Phe Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe 385 390 395 400 Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 405 410 415 Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys 420 425 430 Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg 435 440 445 Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala 450 455 460 Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala 465 470 475 480 Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys 485 490 495 Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala 500 505 510 Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 515 520 525 Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val 530 535 540 Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe 545 550 555 560 Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val 565 570 575 Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr 580 585 590 Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu 595 600

605 Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val 610 615 620 Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys 625 630 635 640 Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn 645 650 655 Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro 660 665 670 Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys 675 680 685 Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu 690 695 700 Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val 705 710 715 720 Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg 725 730 735 Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu 740 745 750 Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser 755 760 765 Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val 770 775 780 Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp 785 790 795 800 Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu 805 810 815 Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala 820 825 830 Ala Leu Gly Leu Ala Ala Ala Leu Gln Val Gln Leu Val Glu Ser Gly 835 840 845 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 850 855 860 Ser Gly Phe Thr Phe Arg Ser Tyr Ala Met Ser Trp Val Arg Gln Ala 865 870 875 880 Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Arg Gly Asp 885 890 895 Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 900 905 910 Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 915 920 925 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Met Thr Ser Asn Ala Val 930 935 940 Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 945 950 955 960 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gln Ser 965 970 975 Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val 980 985 990 Thr Ile Ser Cys Thr Gly Arg His Ser Asn Ile Gly Leu Gly Tyr Gly 995 1000 1005 Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 1010 1015 1020 Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 1025 1030 1035 Ser Gly Phe Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly 1040 1045 1050 Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp 1055 1060 1065 Arg Arg Thr Pro Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr 1070 1075 1080 Val Leu Gly 1085 221096PRTArtificial SequenceSynthetic Construct 22Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Tyr Ser Ser Ser Trp Ser Glu Val Ala Ser Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Val Met 130 135 140 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 145 150 155 160 Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr 165 170 175 Gln Gln Lys Ala Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser 180 185 190 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 210 215 220 Thr Tyr Phe Cys Gln Gln Ala His Ser Phe Pro Pro Thr Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Glu Ile Lys Arg Gly Ala Ala Ser Asp Ala His Lys 245 250 255 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 260 265 270 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe 275 280 285 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 290 295 300 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 305 310 315 320 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 325 330 335 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 340 345 350 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 355 360 365 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 370 375 380 Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 385 390 395 400 Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 405 410 415 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 420 425 430 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 435 440 445 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 450 455 460 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 465 470 475 480 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 485 490 495 Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 500 505 510 Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 515 520 525 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 530 535 540 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 545 550 555 560 Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 565 570 575 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 580 585 590 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 595 600 605 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 610 615 620 Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 625 630 635 640 Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln 645 650 655 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 660 665 670 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 675 680 685 Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 690 695 700 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 705 710 715 720 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 725 730 735 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 740 745 750 Glu Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu 755 760 765 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 770 775 780 Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 785 790 795 800 Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 805 810 815 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 820 825 830 Ala Ala Leu Gly Leu Ala Ala Ala Leu Gln Val Gln Leu Val Gln Ser 835 840 845 Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys 850 855 860 Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Ala Trp Val Arg Gln 865 870 875 880 Met Pro Gly Lys Gly Leu Glu Tyr Met Gly Leu Ile Tyr Pro Gly Asp 885 890 895 Ser Asp Thr Lys Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser 900 905 910 Val Asp Lys Ser Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys 915 920 925 Pro Ser Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Val Gly Tyr 930 935 940 Cys Thr Asp Arg Thr Cys Ala Lys Trp Pro Glu Trp Leu Gly Val Trp 945 950 955 960 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser 965 970 975 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro 980 985 990 Pro Ser Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser 995 1000 1005 Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln 1010 1015 1020 Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp His Thr 1025 1030 1035 Asn Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 1040 1045 1050 Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg Ser Glu Asp 1055 1060 1065 Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr Thr Leu Ser Gly 1070 1075 1080 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 1085 1090 1095 231087PRTArtificial SequenceSynthethic Construct 23Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Tyr Ser Ser Ser Trp Ser Glu Val Ala Ser Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Val Met 130 135 140 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 145 150 155 160 Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr 165 170 175 Gln Gln Lys Ala Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser 180 185 190 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 210 215 220 Thr Tyr Phe Cys Gln Gln Ala His Ser Phe Pro Pro Thr Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Glu Ile Lys Arg Gly Ala Ala Ser Asp Ala His Lys 245 250 255 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 260 265 270 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe 275 280 285 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 290 295 300 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 305 310 315 320 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 325 330 335 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu 340 345 350 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 355 360 365 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 370 375 380 Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 385 390 395 400 Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 405 410 415 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 420 425 430 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 435 440 445 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 450 455 460 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 465 470 475 480 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 485 490 495 Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 500 505 510 Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 515 520 525 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 530 535 540 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 545 550 555 560 Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 565 570 575 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 580 585 590 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 595 600 605 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 610 615 620 Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 625 630 635 640 Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln 645 650 655 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 660 665 670 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 675 680 685 Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 690 695 700

Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 705 710 715 720 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 725 730 735 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 740 745 750 Glu Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu 755 760 765 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 770 775 780 Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 785 790 795 800 Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 805 810 815 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 820 825 830 Ala Ala Leu Gly Leu Ala Ala Ala Leu Gln Val Gln Leu Val Glu Ser 835 840 845 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 850 855 860 Ala Ser Gly Phe Thr Phe Arg Ser Tyr Ala Met Ser Trp Val Arg Gln 865 870 875 880 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Arg Gly 885 890 895 Asp Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 900 905 910 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 915 920 925 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Met Thr Ser Asn Ala 930 935 940 Val Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 945 950 955 960 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gln 965 970 975 Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg 980 985 990 Val Thr Ile Ser Cys Thr Gly Arg His Ser Asn Ile Gly Leu Gly Tyr 995 1000 1005 Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 1010 1015 1020 Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg 1025 1030 1035 Phe Ser Gly Phe Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr 1040 1045 1050 Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr 1055 1060 1065 Asp Arg Arg Thr Pro Gly Trp Val Phe Gly Gly Gly Thr Lys Leu 1070 1075 1080 Thr Val Leu Gly 1085 241095PRTArtificial SequenceSynthetic Construct 24Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Asn Arg Asp Gly Ser Ala Ser Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Val Gly Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala 130 135 140 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly 145 150 155 160 Thr Ser Ser Asp Val Gly Gly Tyr Asn Phe Val Ser Trp Tyr Gln Gln 165 170 175 His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asp Arg 180 185 190 Pro Ser Gly Val Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr 195 200 205 Ala Ser Leu Ile Ile Ser Gly Leu Gln Ala Asp Asp Glu Ala Asp Tyr 210 215 220 Tyr Cys Ser Ser Tyr Gly Ser Ser Ser Thr His Val Ile Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Thr Val Leu Gly Ala Ala Ser Asp Ala His Lys Ser 245 250 255 Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala 260 265 270 Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu 275 280 285 Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys 290 295 300 Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu 305 310 315 320 Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly 325 330 335 Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys 340 345 350 Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg 355 360 365 Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr 370 375 380 Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe 385 390 395 400 Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 405 410 415 Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys 420 425 430 Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg 435 440 445 Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala 450 455 460 Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala 465 470 475 480 Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys 485 490 495 Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala 500 505 510 Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 515 520 525 Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val 530 535 540 Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe 545 550 555 560 Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val 565 570 575 Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr 580 585 590 Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu 595 600 605 Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val 610 615 620 Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys 625 630 635 640 Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn 645 650 655 Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro 660 665 670 Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys 675 680 685 Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu 690 695 700 Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val 705 710 715 720 Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg 725 730 735 Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu 740 745 750 Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser 755 760 765 Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val 770 775 780 Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp 785 790 795 800 Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu 805 810 815 Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala 820 825 830 Ala Leu Gly Leu Ala Ala Ala Leu Gln Val Gln Leu Val Gln Ser Gly 835 840 845 Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly 850 855 860 Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Ala Trp Val Arg Gln Met 865 870 875 880 Pro Gly Lys Gly Leu Glu Tyr Met Gly Leu Ile Tyr Pro Gly Asp Ser 885 890 895 Asp Thr Lys Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Val 900 905 910 Asp Lys Ser Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro 915 920 925 Ser Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Val Gly Tyr Cys 930 935 940 Thr Asp Arg Thr Cys Ala Lys Trp Pro Glu Trp Leu Gly Val Trp Gly 945 950 955 960 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser Gly 965 970 975 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro 980 985 990 Ser Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly 995 1000 1005 Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln 1010 1015 1020 Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp His Thr Asn 1025 1030 1035 Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly 1040 1045 1050 Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg Ser Glu Asp Glu 1055 1060 1065 Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr Thr Leu Ser Gly Trp 1070 1075 1080 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 1085 1090 1095 251086PRTArtificial SequenceSynthetic Construct 25Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Asn Arg Asp Gly Ser Ala Ser Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Val Gly Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala 130 135 140 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly 145 150 155 160 Thr Ser Ser Asp Val Gly Gly Tyr Asn Phe Val Ser Trp Tyr Gln Gln 165 170 175 His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asp Arg 180 185 190 Pro Ser Gly Val Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr 195 200 205 Ala Ser Leu Ile Ile Ser Gly Leu Gln Ala Asp Asp Glu Ala Asp Tyr 210 215 220 Tyr Cys Ser Ser Tyr Gly Ser Ser Ser Thr His Val Ile Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Thr Val Leu Gly Ala Ala Ser Asp Ala His Lys Ser 245 250 255 Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala 260 265 270 Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu 275 280 285 Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys 290 295 300 Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu 305 310 315 320 Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly 325 330 335 Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys 340 345 350 Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg 355 360 365 Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr 370 375 380 Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe 385 390 395 400 Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 405 410 415 Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys 420 425 430 Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg 435 440 445 Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala 450 455 460 Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala 465 470 475 480 Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys 485 490 495 Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala 500 505 510 Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 515 520 525 Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val 530 535 540 Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe 545 550 555 560 Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val 565 570 575 Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr 580 585 590 Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu 595 600 605 Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val 610 615 620 Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys 625 630 635 640 Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn 645 650 655 Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro 660 665 670 Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys 675 680 685 Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu 690 695 700 Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val 705 710 715 720 Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg 725 730 735 Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu 740 745 750 Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser 755 760 765 Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val 770 775 780 Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp 785 790 795 800 Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

Ala Asp Asp Lys Glu 805 810 815 Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala 820 825 830 Ala Leu Gly Leu Ala Ala Ala Leu Gln Val Gln Leu Val Glu Ser Gly 835 840 845 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 850 855 860 Ser Gly Phe Thr Phe Arg Ser Tyr Ala Met Ser Trp Val Arg Gln Ala 865 870 875 880 Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Arg Gly Asp 885 890 895 Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 900 905 910 Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 915 920 925 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Met Thr Ser Asn Ala Val 930 935 940 Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 945 950 955 960 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gln Ser 965 970 975 Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val 980 985 990 Thr Ile Ser Cys Thr Gly Arg His Ser Asn Ile Gly Leu Gly Tyr Gly 995 1000 1005 Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 1010 1015 1020 Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 1025 1030 1035 Ser Gly Phe Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly 1040 1045 1050 Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp 1055 1060 1065 Arg Arg Thr Pro Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr 1070 1075 1080 Val Leu Gly 1085 26248PRTArtificial SequenceSynthetic construct 26Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Asn Arg Asp Gly Ser Ala Ser Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Val Gly Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala 130 135 140 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly 145 150 155 160 Thr Ser Ser Asp Val Gly Gly Tyr Asn Phe Val Ser Trp Tyr Gln Gln 165 170 175 His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asp Arg 180 185 190 Pro Ser Gly Val Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr 195 200 205 Ala Ser Leu Ile Ile Ser Gly Leu Gln Ala Asp Asp Glu Ala Asp Tyr 210 215 220 Tyr Cys Ser Ser Tyr Gly Ser Ser Ser Thr His Val Ile Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Thr Val Leu Gly 245 27255PRTArtificial SequenceSynthetic Construct 27Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Tyr Met 35 40 45 Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg His Asp Val Gly Tyr Cys Thr Asp Arg Thr Cys Ala Lys Trp 100 105 110 Pro Glu Trp Leu Gly Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 145 150 155 160 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 165 170 175 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 180 185 190 Ile Tyr Asp His Thr Asn Arg Pro Ala Gly Val Pro Asp Arg Phe Ser 195 200 205 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg 210 215 220 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr Thr Leu 225 230 235 240 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 245 250 255 28245PRTArtificial SequenceSynthetic Construct 28Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Thr Tyr 20 25 30 Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Val Ala Thr Thr Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val 130 135 140 Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser 145 150 155 160 Ser Asn Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro 165 170 175 Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser 180 185 190 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 195 200 205 Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 210 215 220 Gln Ser Tyr Asp Ser Ser Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys 225 230 235 240 Leu Thr Val Leu Gly 245 29255PRTArtificial SequenceSynthetic Construct 29Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Tyr Met 35 40 45 Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg His Asp Val Gly Tyr Cys Ser Ser Ser Asn Cys Ala Lys Trp 100 105 110 Pro Glu Tyr Phe Gln His Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 145 150 155 160 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 165 170 175 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 180 185 190 Ile Tyr Asp His Thr Asn Arg Pro Ala Gly Val Pro Asp Arg Phe Ser 195 200 205 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg 210 215 220 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr Thr Leu 225 230 235 240 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 245 250 255 30249PRTArtificial SequenceSynthetic Construct 30Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Gly Ala Lys Gln Trp Leu Glu Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu 130 135 140 Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile 145 150 155 160 Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn 180 185 190 Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Thr Ser Gly Asn 195 200 205 Ser Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp 210 215 220 Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Val Thr Val Leu Gly 245 31249PRTArtificial SequenceSynthetic construct 31Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Tyr Ser Ser Ser Trp Ser Glu Val Ala Ser Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Val Met 130 135 140 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 145 150 155 160 Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr 165 170 175 Gln Gln Lys Ala Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser 180 185 190 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 210 215 220 Thr Tyr Phe Cys Gln Gln Ala His Ser Phe Pro Pro Thr Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Glu Ile Lys Arg Gly 245 32246PRTArtificial SequenceSynthetic Construct 32Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Arg Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Met Thr Ser Asn Ala Val Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gln Ser Val Leu Thr Gln Pro Pro Ser Val 130 135 140 Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Arg His 145 150 155 160 Ser Asn Ile Gly Leu Gly Tyr Gly Val His Trp Tyr Gln Gln Leu Pro 165 170 175 Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser 180 185 190 Gly Val Pro Asp Arg Phe Ser Gly Phe Lys Ser Gly Thr Ser Ala Ser 195 200 205 Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 210 215 220 Gln Ser Tyr Asp Arg Arg Thr Pro Gly Trp Val Phe Gly Gly Gly Thr 225 230 235 240 Lys Leu Thr Val Leu Gly 245 3310PRTHomo sapiens 33Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 349PRTOrthomxyoviridue 34Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 356PRTArtificial SequenceSynthetic Construct 35His His His His His His 1 5 36277PRTHomo sapiens 36Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Met Ser 20 25 30 Trp Asp Thr Gly Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser 165 170 175 Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Asp Ala Asp

180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr Phe Tyr His 275 37479PRTHomo sapiens 37Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser 1 5 10 15 Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln 20 25 30 Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Met Leu Phe Gln Arg Leu 35 40 45 Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu 50 55 60 Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Leu Ile Thr Tyr Leu Asn 65 70 75 80 Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gln Thr Pro Gly Arg Ala 85 90 95 Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val 100 105 110 Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile 115 120 125 Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile 130 135 140 Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu 145 150 155 160 Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile Ile Asn 165 170 175 Asp Tyr Glu Glu Phe Ser Lys Glu Arg Ser Ser Ser Leu Glu Gly Ser 180 185 190 Pro Asp Glu Phe Ser Asn Gly Glu Glu Leu Cys Gly Val Met Thr Ile 195 200 205 Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Ser Gln Thr Leu Asp Lys 210 215 220 Val Tyr Gln Met Lys Ser Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn 225 230 235 240 Asn His Asn Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Leu His Ser 245 250 255 Ile Arg Asp Arg Asn Gly Thr His Leu Asp Ala Gly Ala Leu Thr Thr 260 265 270 Thr Phe Glu Glu Leu His Phe Glu Ile Lys Pro His Asp Asp Cys Thr 275 280 285 Val Glu Gln Ile Tyr Asp Ile Leu Lys Ile Tyr Gln Leu Met Asp His 290 295 300 Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Leu Ser His Gly Asp Lys 305 310 315 320 Gly Ile Ile Tyr Gly Thr Asp Gly Gln Glu Pro Pro Ile Tyr Glu Leu 325 330 335 Thr Ser Gln Phe Thr Gly Leu Lys Cys Pro Ser Leu Ala Gly Lys Pro 340 345 350 Lys Val Phe Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln Lys Gly 355 360 365 Ile Pro Val Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu Glu Met Asp 370 375 380 Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala Asp Phe Leu 385 390 395 400 Leu Gly Met Ala Thr Val Asn Asn Cys Val Ser Tyr Arg Asn Pro Ala 405 410 415 Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gln Ser Leu Arg Glu Arg 420 425 430 Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Leu Thr Glu Val Asn Tyr 435 440 445 Glu Val Ser Asn Lys Asp Asp Lys Lys Asn Met Gly Lys Gln Met Pro 450 455 460 Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu Val Phe Pro Ser Asp 465 470 475 38247PRTHomo sapiens 38Met Gln Pro Ile Leu Leu Leu Leu Ala Phe Leu Leu Leu Pro Arg Ala 1 5 10 15 Asp Ala Gly Glu Ile Ile Gly Gly His Glu Ala Lys Pro His Ser Arg 20 25 30 Pro Tyr Met Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser Leu Lys Arg 35 40 45 Cys Gly Gly Phe Leu Ile Gln Asp Asp Phe Val Leu Thr Ala Ala His 50 55 60 Cys Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His Asn Ile Lys 65 70 75 80 Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg Ala Ile Pro 85 90 95 His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp Ile Met Leu Leu 100 105 110 Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln Pro Leu Arg 115 120 125 Leu Pro Ser Asn Lys Ala Gln Val Lys Pro Gly Gln Thr Cys Ser Val 130 135 140 Ala Gly Trp Gly Gln Thr Ala Pro Leu Gly Lys His Ser His Thr Leu 145 150 155 160 Gln Glu Val Lys Met Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp 165 170 175 Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro 180 185 190 Glu Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val 195 200 205 Cys Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly 210 215 220 Met Pro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe Val His Trp Ile 225 230 235 240 Lys Lys Thr Met Lys Arg Tyr 245 39105PRTHomo sapiens 39Met Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser 1 5 10 15 Gln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn 20 25 30 Leu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser 35 40 45 Tyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr 50 55 60 Leu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys 65 70 75 80 Met Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile 85 90 95 Ala Tyr Leu Lys Lys Ala Thr Asn Glu 100 105 40233PRTHomo sapiens 40Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 41314PRTHomo sapiens 41Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala 1 5 10 15 Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser 20 25 30 Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn 35 40 45 Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro 50 55 60 Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro 65 70 75 80 Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95 Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110 Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120 125 Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 130 135 140 Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr 145 150 155 160 Ser Asn Thr Lys Cys Lys Glu Glu Val Lys Arg Lys Glu Val Gln Lys 165 170 175 Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly Ser His Glu Ser Pro 180 185 190 Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu Ser Asp Val Asp Leu 195 200 205 Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met Thr Leu Ser Gln Val 210 215 220 Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu Ala Lys Ile Asp Glu 225 230 235 240 Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu Gln Lys Val Gln Leu 245 250 255 Leu Arg Asn Trp His Gln Leu His Gly Lys Lys Glu Ala Tyr Asp Thr 260 265 270 Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys Thr Leu Ala Glu Lys 275 280 285 Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser Asp Ser Glu Asn Ser 290 295 300 Asn Phe Arg Asn Glu Ile Gln Ser Leu Val 305 310 42168PRTHomo sapiens 42Met Phe Gln Ile Pro Glu Phe Glu Pro Ser Glu Gln Glu Asp Ser Ser 1 5 10 15 Ser Ala Glu Arg Gly Leu Gly Pro Ser Pro Ala Gly Asp Gly Pro Ser 20 25 30 Gly Ser Gly Lys His His Arg Gln Ala Pro Gly Leu Leu Trp Asp Ala 35 40 45 Ser His Gln Gln Glu Gln Pro Thr Ser Ser Ser His His Gly Gly Ala 50 55 60 Gly Ala Val Glu Ile Arg Ser Arg His Ser Ser Tyr Pro Ala Gly Thr 65 70 75 80 Glu Asp Asp Glu Gly Met Gly Glu Glu Pro Ser Pro Phe Arg Gly Arg 85 90 95 Ser Arg Ser Ala Pro Pro Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg 100 105 110 Glu Leu Arg Arg Met Ser Asp Glu Phe Val Asp Ser Phe Lys Lys Gly 115 120 125 Leu Pro Arg Pro Lys Ser Ala Gly Thr Ala Thr Gln Met Arg Gln Ser 130 135 140 Ser Ser Trp Thr Arg Val Phe Gln Ser Trp Trp Asp Arg Asn Leu Gly 145 150 155 160 Arg Gly Ser Ser Ala Pro Ser Gln 165 43279PRTHomo sapiens 43Met Val Asp His Leu Ala Asn Thr Glu Ile Asn Ser Gln Arg Ile Ala 1 5 10 15 Ala Val Glu Ser Cys Phe Gly Ala Ser Gly Gln Pro Leu Ala Leu Pro 20 25 30 Gly Arg Val Leu Leu Gly Glu Gly Val Leu Thr Lys Glu Cys Arg Lys 35 40 45 Lys Ala Lys Pro Arg Ile Phe Phe Leu Phe Asn Asp Ile Leu Val Tyr 50 55 60 Gly Ser Ile Val Leu Asn Lys Arg Lys Tyr Arg Ser Gln His Ile Ile 65 70 75 80 Pro Leu Glu Glu Val Thr Leu Glu Leu Leu Pro Glu Thr Leu Gln Ala 85 90 95 Lys Asn Arg Trp Met Ile Lys Thr Ala Lys Lys Ser Phe Val Val Ser 100 105 110 Ala Ala Ser Ala Thr Glu Arg Gln Glu Trp Ile Ser His Ile Glu Glu 115 120 125 Cys Val Arg Arg Gln Leu Lys Ala Thr Gly Arg Pro Pro Ser Thr Glu 130 135 140 His Ala Ala Pro Trp Ile Pro Asp Lys Ala Thr Asp Ile Cys Met Arg 145 150 155 160 Cys Thr Gln Thr Arg Phe Ser Ala Leu Thr Arg Arg His His Cys Arg 165 170 175 Lys Cys Gly Phe Val Val Cys Ala Glu Cys Ser Arg Gln Arg Phe Leu 180 185 190 Leu Pro Arg Leu Ser Pro Lys Pro Val Arg Val Cys Ser Leu Cys Tyr 195 200 205 Arg Glu Leu Ala Ala Gln Gln Arg Gln Glu Glu Ala Glu Glu Gln Gly 210 215 220 Ala Gly Ser Pro Arg Gln Pro Ala His Leu Ala Arg Pro Ile Cys Gly 225 230 235 240 Ala Ser Ser Gly Asp Asp Asp Asp Ser Asp Glu Asp Lys Glu Gly Ser 245 250 255 Arg Asp Gly Asp Trp Pro Ser Ser Val Glu Phe Tyr Ala Ser Gly Val 260 265 270 Ala Trp Ser Ala Phe His Ser 275 44850PRTPieris rapae 44Met Ala Asp Arg Gln Pro Tyr Met Thr Asn Gly Ile Gln Ala Ala Val 1 5 10 15 Val Glu Trp Ile Arg Ala Leu Asp Leu Glu Ile Ile Ser Leu Leu Leu 20 25 30 Ser Arg Ala Trp Pro Met Ala Leu Leu Ala Thr Ser Glu Leu Arg Trp 35 40 45 Arg Pro Thr Val Leu Thr Asp Thr Asp Asn Val Val Arg Leu Asp Arg 50 55 60 Arg Gln Arg Leu Val Arg Trp Asp Arg Arg Pro Pro Asn Glu Ile Phe 65 70 75 80 Leu Asp Gly Phe Val Pro Ile Val Thr Arg Glu Asn Pro Asp Trp Glu 85 90 95 Glu Thr Asp Leu Tyr Gly Phe Ala Lys Asn Asn His Pro Ser Ile Phe 100 105 110 Val Ser Thr Thr Lys Thr Gln Arg Asn Lys Lys Lys Tyr Val Trp Thr 115 120 125 Pro Arg Asn Ala Asn Arg Gly Ile Val Tyr Gln Tyr Glu Ile Tyr Ala 130 135 140 Pro Gly Gly Val Asp Val Asn Asp Ser Phe Ser Asp Ala Ser Pro Trp 145 150 155 160 Pro Asn Gln Met Glu Val Ala Phe Pro Gly Gly Ile Gln Asn Ile Tyr 165 170 175 Ile Arg Ser Ala Arg Glu Leu His Asn Gly Arg Ile Gln Arg Ile Trp 180 185 190 Ile Asn Pro Asn Phe Leu Asp Pro Gly Asp Leu Glu Pro Ile Val Ser 195 200 205 Ser Ser Arg Thr Pro Gln Val Ile Trp Arg Met Asn His Pro Asp Gly 210 215 220 Gly His Arg Asp Gln Arg Ser Glu Arg Ser Ala Ser Ser Tyr Asp Asp 225 230 235 240 Leu Met Tyr Gly Gly Thr Gly Asn Val Gln Glu Asp Thr Phe Gly Asp 245 250 255 Glu Pro Asn Asn Pro Lys Pro Ile Ala Ala Gly Glu Phe Met Ile Glu 260 265 270 Ser Ile Lys Asp Lys Asn Ser Phe Leu Asp Leu Ser Lys Asn Val Asn 275 280 285 Gly Gly Val Ile His Ser Asn Leu Tyr Ser Gly Gly Asp Asn Gln Ile 290 295 300 Trp Val Phe Ser Tyr Asp Asp Asn Lys Lys Ala Tyr Arg Ile Gln Ser 305 310 315 320 Tyr Gln Asn Ser Tyr Leu Tyr Leu Ser Trp Asp Ser Asn Ala Ser Ser 325 330 335 Lys Glu Met Ile Leu Arg Gly Tyr Thr Asn Ser Gly Ser Asn Asn Gln 340 345 350 Tyr Trp Gln Ile Glu Gln Thr Gly Lys Asn Tyr Arg Leu Arg Asn Leu 355

360 365 Leu Asn Leu Asp Met Ile Ile Thr Ala Gln Asp Lys Pro Ser Ala Phe 370 375 380 Gly Gly Lys Glu Val Ile Val Asn Thr Glu Ile Ser Asn Ser Asn Thr 385 390 395 400 Lys Ile Ser Gln Glu Trp Lys Met Ile Pro Phe Asp Phe Arg Pro Ile 405 410 415 Ile Asp Gly Asp Tyr Asn Ile Phe Asn Val Asp Leu Ser Asn Gln Val 420 425 430 Val Asp Phe Ser Asn Gln Pro Asp Leu Leu Val His Gly His Ile Phe 435 440 445 Cys Asp Asn Glu Asn Gln Thr Trp His Phe Thr Tyr Asn Ser Thr Tyr 450 455 460 His Ala Tyr Lys Ile Trp Ser Gly Arg Lys Ser Asn Leu Leu Leu Thr 465 470 475 480 Trp Asp Ser Asn Ala Ala Ser Lys Glu Met Val Val Arg Ala Tyr Thr 485 490 495 Glu Ser Arg Ser Lys Asn Gln Tyr Trp Arg Ile Glu Gln Thr Gly Ser 500 505 510 Lys Ser Tyr Lys Val Arg Asn Leu Glu Asn Ser Ser Met Ile Leu Gly 515 520 525 Leu Thr Arg Val Ser Thr Pro Tyr Gly Gly Leu Asn Leu Met Val Glu 530 535 540 Asp Asp Ser Asp Gly His Ser Asp Leu His Ser Asp Trp Asp Ile Lys 545 550 555 560 Pro Ile Phe Tyr Gln Asp Ile Pro Asp Gly Asp Tyr Asn Ile Phe Asn 565 570 575 Asp Asn Phe Pro Asn Ile Ala Ile Asp Phe Thr Asn Gln Glu Gly Ser 580 585 590 Leu Ile His Gly His Asn Phe Cys Ser Asn Asn Asn Gln Lys Trp Ser 595 600 605 Phe Val Phe Asp Gly Lys Arg Lys Ala Tyr Arg Ile Lys Ser Gly Val 610 615 620 Arg Ser Asn Leu Trp Leu Ser Trp Asp Ser Asn Ala Ser Ser Lys Glu 625 630 635 640 Met Val Leu Arg Ala Tyr Thr Glu Ser Gly Ser Ser Asn Gln Tyr Trp 645 650 655 Arg Leu Asp Glu Ala Asn Asp Gly Ser Tyr Arg Ile Arg Asn Leu Gln 660 665 670 Asp Tyr Tyr Lys Leu Ile Ala Leu Thr Asn Lys Asn Thr Pro Tyr Gly 675 680 685 Gly Lys Glu Leu Ile Val Ser Asp Asn Lys Glu Ser Gly Asn Thr Trp 690 695 700 Tyr Leu Lys Lys Leu Gly Glu Val Pro Leu Pro Asn Arg Lys Phe Arg 705 710 715 720 Ile Ala Thr Lys Leu Asn Tyr Lys Lys Val Ile Asp Ser Ser Thr Ser 725 730 735 Tyr Asn Leu Ile Ile Thr His Asp Leu Asn Phe Ala Ser Ser Ile Trp 740 745 750 Glu Leu Val Tyr Asp Ser Ser Lys Lys Ala Tyr Asn Ile Tyr Ser Ser 755 760 765 Asp Ile Asn Asn Leu Gly Trp Ile Tyr Gln Asn Lys Asn Phe Phe Val 770 775 780 Lys Leu Gly Asn Ile Asp Gly Pro Asp His Gly Asp Leu Arg Tyr Phe 785 790 795 800 Trp Thr Ile Glu Tyr Ser Met Gln Thr Gly Cys Tyr Leu Ile Arg Ser 805 810 815 Leu His Asp Pro Ala Asn Ala Val Gly Tyr Thr Asp Ser Glu Ser Val 820 825 830 Ile Thr Asp Thr Ser Thr Tyr Ser Asp Asn Gln Leu Phe His Phe Ile 835 840 845 Leu Met 850 45281PRTHomo sapiens 45Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys 1 5 10 15 Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30 Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45 Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60 Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val 65 70 75 80 Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95 Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105 110 Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly 115 120 125 Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu 130 135 140 Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly 145 150 155 160 His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile 165 170 175 His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190 Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200 205 Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220 Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr 225 230 235 240 Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile 245 250 255 Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala 260 265 270 Ser Phe Phe Gly Ala Phe Leu Val Gly 275 280 46192PRTHomo sapiens 46Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gly Gly Pro Thr Ser Ser 1 5 10 15 Glu Gln Ile Met Lys Thr Gly Ala Leu Leu Leu Gln Gly Phe Ile Gln 20 25 30 Asp Arg Ala Gly Arg Met Gly Gly Glu Ala Pro Glu Leu Ala Leu Asp 35 40 45 Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys 50 55 60 Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile 65 70 75 80 Ala Ala Val Asp Thr Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala 85 90 95 Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala 100 105 110 Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala Leu Cys Thr Lys 115 120 125 Val Pro Glu Leu Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe Leu 130 135 140 Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly Trp Asp Gly 145 150 155 160 Leu Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gln Thr Val Thr Ile Phe 165 170 175 Val Ala Gly Val Leu Thr Ala Ser Leu Thr Ile Trp Lys Lys Met Gly 180 185 190 47238PRTAequorea victoria 47Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 48194PRTAliivibrio fischeri 48Met Phe Lys Gly Ile Val Glu Gly Ile Gly Ile Ile Glu Lys Ile Asp 1 5 10 15 Ile Tyr Thr Asp Leu Asp Lys Tyr Ala Ile Arg Phe Pro Glu Asn Met 20 25 30 Leu Asn Gly Ile Lys Lys Glu Ser Ser Ile Met Phe Asn Gly Cys Phe 35 40 45 Leu Thr Val Thr Ser Val Asn Ser Asn Ile Val Trp Phe Asp Ile Phe 50 55 60 Glu Lys Glu Ala Arg Lys Leu Asp Thr Phe Arg Glu Tyr Lys Val Gly 65 70 75 80 Asp Arg Val Asn Leu Gly Thr Phe Pro Lys Phe Gly Ala Ala Ser Gly 85 90 95 Gly His Ile Leu Ser Ala Arg Ile Ser Cys Val Ala Ser Ile Ile Glu 100 105 110 Ile Ile Glu Asn Glu Asp Tyr Gln Gln Met Trp Ile Gln Ile Pro Glu 115 120 125 Asn Phe Thr Glu Phe Leu Ile Asp Lys Asp Tyr Ile Ala Val Asp Gly 130 135 140 Ile Ser Leu Thr Ile Asp Thr Ile Lys Asn Asn Gln Phe Phe Ile Ser 145 150 155 160 Leu Pro Leu Lys Ile Ala Gln Asn Thr Asn Met Lys Trp Arg Lys Lys 165 170 175 Gly Asp Lys Val Asn Val Glu Leu Ser Asn Lys Ile Asn Ala Asn Gln 180 185 190 Cys Trp 49225PRTMontastraea cavernosa 49Met Ser Val Ile Lys Ser Val Met Lys Ile Lys Leu Arg Met Asp Gly 1 5 10 15 Ile Val Asn Gly His Lys Phe Met Ile Thr Gly Glu Gly Glu Gly Lys 20 25 30 Pro Phe Glu Gly Thr His Thr Ile Ile Leu Lys Val Lys Glu Gly Gly 35 40 45 Pro Leu Pro Phe Ala Tyr Asp Ile Leu Thr Thr Ala Phe Gln Tyr Gly 50 55 60 Asn Arg Val Phe Thr Lys Tyr Pro Lys Asp Ile Pro Asp Tyr Phe Lys 65 70 75 80 Gln Ser Phe Pro Glu Gly Tyr Ser Trp Glu Arg Ser Met Thr Phe Glu 85 90 95 Asp Gln Gly Val Cys Thr Val Thr Ser Asp Ile Lys Leu Glu Gly Asp 100 105 110 Cys Phe Phe Tyr Glu Ile Arg Phe Tyr Gly Val Asn Phe Pro Ser Ser 115 120 125 Gly Pro Val Met Gln Lys Lys Thr Leu Lys Trp Glu Pro Ser Thr Glu 130 135 140 Asn Met Tyr Val Arg Asp Gly Val Leu Leu Gly Asp Val Ser Arg Thr 145 150 155 160 Leu Leu Leu Glu Gly Asp Lys His His Arg Cys Asn Phe Arg Ser Thr 165 170 175 Tyr Gly Ala Lys Lys Gly Val Val Leu Pro Glu Tyr His Phe Val Asp 180 185 190 His Arg Ile Glu Ile Leu Ser His Asp Lys Asp Tyr Asn Thr Val Glu 195 200 205 Val Tyr Glu Asn Ala Val Ala Arg Pro Ser Met Leu Pro Val Lys Ala 210 215 220 Lys 225 50230PRTDiscosoma sp. 50Met Ser Cys Ser Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val 1 5 10 15 Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile Lys Gly Glu 20 25 30 Gly Glu Gly Arg Pro Tyr Glu Gly His Cys Ser Val Lys Leu Met Val 35 40 45 Thr Lys Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ser Pro Gln 50 55 60 Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro 65 70 75 80 Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val 85 90 95 Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Ser Gln Asp Ser Ser 100 105 110 Leu Lys Asp Gly Cys Phe Ile Tyr Glu Val Lys Phe Ile Gly Val Asn 115 120 125 Phe Pro Ser Asp Gly Pro Val Met Gln Arg Arg Thr Arg Gly Trp Glu 130 135 140 Ala Ser Ser Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Asp 145 150 155 160 Ile His Met Ala Leu Arg Leu Glu Gly Gly Gly His Tyr Leu Val Glu 165 170 175 Phe Lys Ser Ile Tyr Met Val Lys Lys Pro Ser Val Gln Leu Pro Gly 180 185 190 Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Met Thr Ser His Asn Glu Asp 195 200 205 Tyr Thr Val Val Glu Gln Tyr Glu Lys Thr Gln Gly Arg His His Pro 210 215 220 Phe Ile Lys Pro Leu Gln 225 230 51550PRTPhotinus pyralis 51Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 10 15 Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg 20 25 30 Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu 35 40 45 Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 55 60 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val 65 70 75 80 Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu 85 90 95 Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg 100 105 110 Glu Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val Phe Val 115 120 125 Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro 130 135 140 Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly 145 150 155 160 Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile 180 185 190 Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Ser Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp 210 215 220 Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val 225 230 235 240 Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 260 265 270 Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val 275 280 285 Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr 290 295 300 Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315 320 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile 325 330 335 Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr 340 345 350 Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe 355 360 365 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val 370 375 380 Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly 385 390 395 400 Tyr Val Asn Asp Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly 405 410 415 Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe 420 425 430 Phe

Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Cys Gln 435 440 445 Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile 450 455 460 Phe Asp Ala Gly Val Ala Gly Leu Pro Gly Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu 500 505 510 Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly 515 520 525 Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys 530 535 540 Gly Gly Lys Ser Lys Leu 545 550 52311PRTRenilla reniformis 52Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr 1 5 10 15 Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser 20 25 30 Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile 35 40 45 Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val 50 55 60 Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly 65 70 75 80 Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp 85 90 95 His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys 100 105 110 Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His 115 120 125 Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu 130 135 140 Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu 145 150 155 160 Glu Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu 165 170 175 Glu Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys Ile Met Arg 180 185 190 Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 195 200 205 Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro 210 215 220 Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr 225 230 235 240 Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe Ile Glu 245 250 255 Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys 260 265 270 Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gln 275 280 285 Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu 290 295 300 Arg Val Leu Lys Asn Glu Gln 305 310 53197PRTArtificial SequenceSynthetic Construct 53Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg 195 54197PRTHomo sapiens 54Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln 1 5 10 15 Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser 20 25 30 Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr 35 40 45 Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu 50 55 60 Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln 65 70 75 80 Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu 85 90 95 Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala 100 105 110 Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys 115 120 125 Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr 130 135 140 Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg 145 150 155 160 Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala 165 170 175 Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu 180 185 190 Val Glu Glu Pro Gln 195 55205PRTArtificial sequenceSynthetic construct 55Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu 1 5 10 15 Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr 20 25 30 Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 35 40 45 Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys 50 55 60 Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu 65 70 75 80 Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys 85 90 95 Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu 100 105 110 Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Gln Ala Glu Thr Phe Thr 115 120 125 Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys 130 135 140 Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr 145 150 155 160 Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu 165 170 175 Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly 180 185 190 Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 195 200 205 56197PRTHomo sapiens 56Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg 195 57205PRTHomo sapiens 57Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu 1 5 10 15 Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr 20 25 30 Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 35 40 45 Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys 50 55 60 Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu 65 70 75 80 Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys 85 90 95 Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu 100 105 110 Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr 115 120 125 Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys 130 135 140 Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr 145 150 155 160 Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu 165 170 175 Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly 180 185 190 Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 195 200 205 58609PRTHomo sapiens 58Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr 1 5 10 15 Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu 20 25 30 Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr 35 40 45 Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser 50 55 60 Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp 65 70 75 80 Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu 85 90 95 Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp 100 105 110 Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His 115 120 125 Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro 130 135 140 Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn 145 150 155 160 Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro 165 170 175 Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys 180 185 190 Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr 195 200 205 Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys 210 215 220 Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val 225 230 235 240 Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln 245 250 255 Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly 260 265 270 Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile 275 280 285 Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys 290 295 300 Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp 305 310 315 320 Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp 325 330 335 Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala 340 345 350 Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser 355 360 365 Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys 370 375 380 Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu 385 390 395 400 Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys 405 410 415 Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu 420 425 430 Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met 435 440 445 Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu 450 455 460 Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile 465 470 475 480 Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly 485 490 495 Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp 515 520 525 Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala 530 535 540 Leu Gln Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys 545 550 555 560 Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser 565 570 575 Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe 580 585 590 Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly 595 600 605 Val 5934PRTHuman 59Leu Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile 1 5 10 15 Ile Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro 20 25 30 Gly Val 608645DNAArtificial SequenceSynthetic Construct 60gtgccgacga tagagcagac ctcgctaaat atatctgcga gaatcaggat tccattagct 60ctaagctgaa agaatgttgc gagaagcccc tcctggaaaa gagtcattgt atcgccgagg 120tggaaaacga cgagatgcca gcagatctgc catcactcgc tgccgacttt gtggaatcca 180aagatgtctg caagaattac gcagaggcta aagacgtgtt cctggggatg tttctgtatg 240agtacgcccg gcgtcacccc gattatagcg tcgtgctcct gctccgactg gcaaagacct 300acgaaacaac tctggagaaa tgttgcgctg ccgcagaccc tcatgaatgt tatgctaagg 360tgttcgatga gtttaagcca ctcgtcgaag agccccagaa cctgattaaa cagaattgcg 420aactgttcga gcagctcggt gaatacaagt ttcagaacgc cctgctcgtg cgttatacca 480aaaaggtccc tcaggtgtct acaccaactc tggtggaggt cagtaggaat ctgggcaaag 540tgggatcaaa gtgttgcaaa caccccgagg caaagagaat gccttgtgct gaagattacc 600tctccgtcgt gctgaaccag ctctgcgtgc tgcatgaaaa gaccccagtc agcgatcggg 660tgacaaaatg ttgcaccgaa tctctggtca atcgccgacc ctgtttcagt gccctcgaag 720tggacgaaac ttatgtgcct aaggagtttc aggctgaaac attcaccttt cacgccgata 780tctgcactct gtccgagaaa gaaaggcaga ttaagaaaca gacagcactg gtcgagctcg 840tgaagcataa accaaaggct accaaggagc agctgaaagc cgtcatggac gatttcgcag 900cttttgtgga aaagtgttgc aaagccgacg ataaggagac ttgtttcgca gaagagggga 960aaaagctcgt ggctgccagc caggcagctc tgggtctggc cgcagctctg caggtgcagc 1020tcgtccagag cggcgctgag gtgaagaagc caggcgagtc cctgaagatc tcctgtaagg 1080gctccggcta cagcttcacc tcctactgga tcgcttgggt gaggcagatg ccaggaaagg 1140gactggagta catgggcctg atctaccctg gcgactccga caccaagtac tccccatcct 1200tccagggcca ggtgaccatc agcgtggaca agtccgtgtc taccgcctac ctgcaatggt 1260cctccctgaa gccttctgac tctgccgtgt acttttgtgc

ccggcacgat gtgggctact 1320gcaccgaccg gacatgtgcc aagtggcccg agtggctggg agtgtgggga cagggaacac 1380tggtgacagt gagttctggc ggtggcggct cttccggcgg tggctctggt ggcggcggat 1440ctcagagcgt gctgacacag ccacctagcg tgtccgctgc ccctggccag aaggtgacaa 1500tcagctgctc cggcagctct tccaacatcg gcaacaacta cgtgtcttgg tatcagcagc 1560tgcccggaac agctccaaaa ctgctgatct atgaccacac caatcggcct gccggcgtgc 1620cagatcggtt ctctggctct aagagcggca cctccgccag cctggctatc tctggcttca 1680gatctgagga tgaggctgac tactattgtg cctcctggga ctacaccctg tctggctggg 1740tgttcggcgg tggcaccaag ctgacagtcc tgggatgatg actcgagtct agagggcccg 1800tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 1860cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 1920atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 1980ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 2040gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc 2100cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2160ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2220ccggctttcc ccgtcaagct ctaaatcggg ggtcccttta gggttccgat ttagtgcttt 2280acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct 2340attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact 2400caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat 2460gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata 2520tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca 2580ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag 2640cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac 2700cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga 2760tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac 2820atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat 2880ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga 2940ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac 3000ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca 3060atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac 3120gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat 3180gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc 3240agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg 3300tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc 3360cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg 3420agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga 3480cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa 3540cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 3600taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta 3660tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt 3720tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 3780gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 3840gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 3900ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 3960ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 4020cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 4080gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 4140tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 4200ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 4260atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 4320gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 4380tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 4440cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 4500cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 4560tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 4620cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 4680cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 4740gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 4800gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 4860gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 4920ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 4980atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 5040agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 5100ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 5160tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 5220ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 5280caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 5340gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 5400atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 5460accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 5520aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 5580gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 5640tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 5700aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 5760ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 5820aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga 5880tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 5940cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa 6000gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt 6060tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt 6120tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 6180acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 6240tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 6300gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 6360acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 6420accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 6480gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 6540ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 6600tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 6660tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt 6720atcgaaatta atacgactca ctatagggag acccaagctt ctagaattcg ctgtctgcga 6780gggccagctg ttggggtgag tactccctct caaaagcggg catgacttct gcgctaagat 6840tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgcggtg atgcctttga 6900gggtggccgc gtccatctgg tcagaaaaga caatcttttt gttgtcaagc ttgaggtgtg 6960gcaggcttga gatctggcca tacacttgag tgacaatgac atccactttg cctttctctc 7020cacaggtgtc cactcccagg tccaactgca gatatccagc acagtggcgg ccgccaccat 7080gggctggtct ctgatcctgc tgttcctggt ggccgtggcc acgcgtgtgc tgtcccaggt 7140gcagctgcag gagtctggcg gcggactggt gaagcctggc ggctccctgc ggctgtcctg 7200cgccgcctcc ggcttcacct tctcctccta ctggatgtcc tgggtgcggc aggcccctgg 7260caagggcctg gagtgggtgg ccaacatcaa ccgggacggc tccgcctcct actacgtgga 7320ctccgtgaag ggccggttca ccatctcccg ggacgacgcc aagaactccc tgtacctgca 7380gatgaactcc ctgcgggccg aggacaccgc cgtgtactac tgcgccaggg accggggcgt 7440gggctacttc gacctgtggg gcaggggcac cctggtgacc gtgtcctccg ctagtactgg 7500cggcggagga tctggcggag gagggagcgg gggcggtgga tcccagtccg ccctgaccca 7560gcctgcctcc gtgtccggct cccctggcca gtccatcacc atcagctgca ccggcacctc 7620ctccgacgtg ggcggctaca acttcgtgtc ctggtatcag cagcaccccg gcaaggcccc 7680taagctgatg atctacgacg tgtccgaccg gccttccggc gtgtccgaca ggttctccgg 7740ctccaagtcc ggcaacaccg cctccctgat catcagcggc ctgcaggcag acgacgaggc 7800cgactactac tgctcctcct acggctcctc ctccacccac gtgatctttg gcggcggaac 7860aaaggtgacc gtgctgggcg ccgcctccga cgctcacaag agcgaagtgg cacataggtt 7920caaagatctg ggcgaagaga actttaaggc cctcgtcctg atcgctttcg cacagtacct 7980ccagcagtct ccctttgaag atcacgtgaa actggtcaat gaggtgaccg aatttgccaa 8040gacatgcgtg gctgatgaga gtgcagaaaa ctgtgacaaa tcactgcata ctctctttgg 8100agataagctg tgcaccgtcg ccacactcag agagacttat ggggaaatgg ctgactgttg 8160cgcaaaacag gagcctgaac ggaatgagtg tttcctccag cacaaggatg acaacccaaa 8220tctgccccgc ctcgtgcgac ctgaggtcga tgtgatgtgc accgcctttc atgacaacga 8280agagacattc ctgaagaaat acctgtatga aattgctcgt aggcacccat acttttatgc 8340ccccgagctc ctgttctttg caaagagata caaagctgcc ttcactgaat gttgccaggc 8400agctgataag gccgcatgtc tcctgcctaa actggacgag ctccgggatg aaggtaaggc 8460ttccagcgcc aaacagcgcc tgaagtgcgc ttctctccag aagtttggcg agcgagcatt 8520caaagcctgg gctgtggccc gtctcagtca gaggtttcca aaggcagaat ttgctgaggt 8580ctcaaaactg gtgaccgacc tcacaaaggt ccatactgag tgttgccacg gagatctgct 8640ggaat 86456134DNAArtificial SequenceSynthetic Construct 61gtacttttgt gcccgggccg atgtgggcta ctgc 346234DNAArtificial SequenceSynthetic Construct 62gcagtagccc acatcggccc gggcacaaaa gtac 346334DNAArtificial SequenceSynthetic Construct 63gacatgtgcc aaggcccccg cgtggctggg agtg 346434DNAArtificial SequenceSynthetic Construct 64cactcccagc cacgcggggg ccttggcaca tgtc 3465101DNAArtificial SequenceSynthetic Construct 65acagtggcgg ccgccaccat gggctggtct ctgatcctgc tgttcctggt ggccgtggcc 60acgcgtgtgc tgtcccaggt gcagctcgtc cagagcggcg c 1016640DNAArtificial SequenceSynthetic Construct 66ggaggcggcg cccaggactg tcagcttggt gccaccgccg 40671102PRTArtificial SequenceSynthetic Construct 67Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Tyr Met 35 40 45 Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ala Asp Val Gly Tyr Cys Thr Asp Arg Thr Cys Ala Lys Ala 100 105 110 Pro Ala Trp Leu Gly Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 145 150 155 160 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 165 170 175 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 180 185 190 Ile Tyr Asp His Thr Asn Arg Pro Ala Gly Val Pro Asp Arg Phe Ser 195 200 205 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg 210 215 220 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr Thr Leu 225 230 235 240 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ala 245 250 255 Ala Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu 260 265 270 Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr 275 280 285 Leu Gln Gln Ser Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val 290 295 300 Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys 305 310 315 320 Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala 325 330 335 Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln 340 345 350 Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro 355 360 365 Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala 370 375 380 Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile 385 390 395 400 Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala 405 410 415 Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys 420 425 430 Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys 435 440 445 Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe 450 455 460 Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg 465 470 475 480 Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu 485 490 495 Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala 500 505 510 Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser 515 520 525 Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys 530 535 540 Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu 545 550 555 560 Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn 565 570 575 Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr 580 585 590 Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala 595 600 605 Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro 610 615 620 His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu 625 630 635 640 Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu 645 650 655 Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys 660 665 670 Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu 675 680 685 Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met 690 695 700 Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val 705 710 715 720 Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr 725 730 735 Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp 740 745 750 Glu Thr Tyr Val Pro Lys Glu Phe Gln Ala Glu Thr Phe Thr Phe His 755 760 765 Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln 770 775 780 Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu 785 790 795 800 Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys 805 810 815 Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys 820 825 830 Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala Ala Ala Leu Gln 835 840 845 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser 850 855 860 Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp 865 870 875 880 Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Tyr Met Gly 885 890 895 Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe Gln 900 905 910 Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val Ser Thr Ala Tyr Leu 915 920 925 Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser Ala Val Tyr Phe Cys Ala 930 935 940 Arg His Asp Val Gly Tyr Cys Thr Asp Arg Thr Cys Ala Lys Trp Pro 945 950 955 960 Glu Trp Leu Gly Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 965 970 975 Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 980 985 990 Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln Lys 995 1000 1005 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 1010 1015 1020 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 1025 1030 1035 Leu Ile Tyr Asp His Thr Asn Arg Pro Ala Gly Val Pro Asp Arg 1040 1045 1050 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser

1055 1060 1065 Gly Phe Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp 1070 1075 1080 Asp Tyr Thr Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu 1085 1090 1095 Thr Val Leu Gly 1100 681095PRTArtificial SequenceSynthetic Construct 68Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Asn Arg Asp Gly Ser Ala Ser Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Val Gly Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala 130 135 140 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly 145 150 155 160 Thr Ser Ser Asp Val Gly Gly Tyr Asn Phe Val Ser Trp Tyr Gln Gln 165 170 175 His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asp Arg 180 185 190 Pro Ser Gly Val Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr 195 200 205 Ala Ser Leu Ile Ile Ser Gly Leu Gln Ala Asp Asp Glu Ala Asp Tyr 210 215 220 Tyr Cys Ser Ser Tyr Gly Ser Ser Ser Thr His Val Ile Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Thr Val Leu Gly Ala Ala Ser Asp Ala His Lys Ser 245 250 255 Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala 260 265 270 Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu 275 280 285 Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys 290 295 300 Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu 305 310 315 320 Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly 325 330 335 Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys 340 345 350 Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg 355 360 365 Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr 370 375 380 Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe 385 390 395 400 Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 405 410 415 Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys 420 425 430 Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg 435 440 445 Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala 450 455 460 Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala 465 470 475 480 Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys 485 490 495 Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala 500 505 510 Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 515 520 525 Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val 530 535 540 Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe 545 550 555 560 Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val 565 570 575 Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr 580 585 590 Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu 595 600 605 Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val 610 615 620 Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys 625 630 635 640 Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn 645 650 655 Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro 660 665 670 Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys 675 680 685 Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu 690 695 700 Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val 705 710 715 720 Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg 725 730 735 Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu 740 745 750 Phe Gln Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser 755 760 765 Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val 770 775 780 Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp 785 790 795 800 Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu 805 810 815 Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala 820 825 830 Ala Leu Gly Leu Ala Ala Ala Leu Gln Val Gln Leu Val Gln Ser Gly 835 840 845 Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly 850 855 860 Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Ala Trp Val Arg Gln Met 865 870 875 880 Pro Gly Lys Gly Leu Glu Tyr Met Gly Leu Ile Tyr Pro Gly Asp Ser 885 890 895 Asp Thr Lys Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Val 900 905 910 Asp Lys Ser Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro 915 920 925 Ser Asp Ser Ala Val Tyr Phe Cys Ala Arg Ala Asp Val Gly Tyr Cys 930 935 940 Thr Asp Arg Thr Cys Ala Lys Ala Pro Ala Trp Leu Gly Val Trp Gly 945 950 955 960 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser Gly 965 970 975 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro 980 985 990 Ser Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly 995 1000 1005 Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln 1010 1015 1020 Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp His Thr Asn 1025 1030 1035 Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly 1040 1045 1050 Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg Ser Glu Asp Glu 1055 1060 1065 Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr Thr Leu Ser Gly Trp 1070 1075 1080 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 1085 1090 1095

Claims

1.-36. (canceled)

37. A conjugate comprising a human serum albumin (HSA) linker having an amino terminus and a carboxy terminus, said HSA linker comprising an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 1 and comprising a first binding moiety bonded to the amino terminus of said HSA linker and a second binding moiety bonded to the carboxy terminus of said HSA linker, wherein said first binding moiety specifically binds ErbB3 with a dissociation constant (K_d) of about 16 nM, as determined by surface plasmon resonance, and said second binding moiety specifically binds ErbB2 with a K_d of about 0.3 nM, as determined by surface plasmon resonance.

38. The conjugate of claim 37, wherein the HSA linker further comprises a peptide connector comprising 2 to 20 amino acid residues at the amino or carboxy terminus of said HSA linker, wherein said peptide connector covalently joins said HSA linker to said first or second binding moiety.

39. A composition comprising the conjugate of claim 37 admixed with a pharmaceutically acceptable carrier, excipient, or diluent.

40. The composition of claim 39 in a solution comprising 25 mg/mL of the conjugate.

41. The conjugate of claim 37, wherein said first binding moiety is a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 26 and said second binding moiety is an scFv comprising the amino acid sequence of SEQ ID NO: 27.

42. The conjugate of claim 38, wherein said HSA linker comprises a peptide connector of 2 to 20 amino acid residues between said first binding moiety and said HSA linker and a peptide connector of 2 to 20 amino acid residues between said second binding moiety and said HSA linker.

43. The conjugate of claim 37, wherein said HSA linker comprises a serine residue at position 34 of SEQ ID NO: 1 and a glutamine residue at position 503 of SEQ ID NO: 1.

44. The conjugate of claim 37, wherein said HSA linker comprises the amino acid sequence of SEQ ID NO: 1.

45. The conjugate of claim 37, wherein each of said first and second binding moieties is an scFv.

46. The conjugate of claim 45, wherein each said scFv is human or humanized.

47. The conjugate of claim 37, wherein said conjugate exhibits an in vivo half-life of greater than 8 hours.

48. The conjugate of claim 37, wherein said HSA linker comprises amino acid residues 25-44 and amino acid residues 494-513 of SEQ ID NO:1.

49. The conjugate of claim 48, wherein said HSA linker comprises amino acid residues 25-70 and amino acid residues 450-513 of SEQ ID NO:1.

50. The conjugate of claim 49, wherein said HSA linker comprises amino acid residues 15-100 and amino acid residues 400-520 of SEQ ID NO:1.

51. The conjugate of claim 50, wherein said HSA linker comprises amino acid residues 10-200 and amino acid residues 300-575 of SEQ ID NO:1.

52. The conjugate of claim 51, wherein said HSA linker comprises amino acid residues 5-250 and amino acid residues 275-580 of SEQ ID NO:1.

53. The conjugate of claim 37, wherein said conjugate exhibits the ability to bind both ErbB2 and ErbB3 before and after incubation in human serum at 37.degree. C. for 120 hours as measured by enzyme-linked immunosorbent assay (ELISA).

54. A kit comprising the conjugate of claim 37 in a package with instructions for administering said conjugate to a patient.

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