WO2006020706A2 - Tie complex binding proteins - Google Patents

Tie complex binding proteins Download PDF

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WO2006020706A2
WO2006020706A2 PCT/US2005/028413 US2005028413W WO2006020706A2 WO 2006020706 A2 WO2006020706 A2 WO 2006020706A2 US 2005028413 W US2005028413 W US 2005028413W WO 2006020706 A2 WO2006020706 A2 WO 2006020706A2
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tiel
protein
antibody
variable domain
binding
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PCT/US2005/028413
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WO2006020706A3 (en
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Clive R. Wood
Daniel T. Dransfield
Henk Pieters
Rene Hoet
Simon E. Hufton
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Dyax Corp.
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Priority claimed from PCT/US2004/026116 external-priority patent/WO2005019267A2/en
Priority claimed from US11/049,536 external-priority patent/US7871610B2/en
Application filed by Dyax Corp. filed Critical Dyax Corp.
Priority to CA002576886A priority Critical patent/CA2576886A1/en
Priority to EP05784935A priority patent/EP1789451A4/en
Priority to AU2005272848A priority patent/AU2005272848A1/en
Priority to JP2007525760A priority patent/JP2008532476A/en
Publication of WO2006020706A2 publication Critical patent/WO2006020706A2/en
Publication of WO2006020706A3 publication Critical patent/WO2006020706A3/en

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Abstract

Tie1 and Tie2 are receptor tyrosine kinase proteins that include a transmembrane domain. Tie1 and Tie2 are present on endothelial cells. This disclosure describes agents, such as antibodies, that bind to Tie1, Tie2, and Ang, including ones that inhibit endothelial cell activity and angiogenesis. The agents can be used to treat angiogenesis-associated disorders.

Description

TIE COMPLEX BINDING PROTEINS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Application Serial
No. 11/049,536, filed February 2, 2005, which is a continuation-in-part of U.S. Application Serial No. 10/916,840, filed August 12, 2004, which claims priority to U.S. Application Serial No. 60/494,713, filed on August 12, 2003. This application is also a continuation-in-part of PCT/US2004/ 026116, filed August 12, 2004, which claims priority to U.S. Application Serial No. 60/494,713, filed on August 12, 2003. The contents of each of the foregoing applications are hereby incorporated by reference in their entirety.
BACKGROUND
[0002] The oxygen and nutrients supplied by the blood vessels are crucial for tissue development and function. Indeed, the cardiovascular system is the first organ system to develop in embryos. During organogenesis and the development of tissues or tumors, the proximity of the growing cells to the circulatory system is ensured by the coordinated growth of blood vessels and organ parenchyma. It may be possible to prevent or treat diseases by modulating blood vessel development or angiogenesis.
[0003] Blood vessels are composed of an inner layer of endothelial cells and an outer layer of pericytes or smooth muscle cells. The first tubular structures are formed by endothelial cells that subsequently recruit pericytes and smooth muscle cells to ensheath them. The de novo formation of blood vessels from a dispersed population of mesodermally derived endothelial precursor cells is termed vasculogenesis. This primitive network undergoes successive morphogenetic events including sprouting, splitting, and remodeling to generate the hierarchical vascular network from large to branched small vessels. These successive morphogenetic events are collectively called angiogenesis. Previous studies have identified a number of endothelial cell specific receptor tyrosine kinases (RTKs) and their cognate ligands, which mediate the vasculogenic and angiogenic development of blood vessels. Members of the vascular endothelial growth factor (VEGF) family and their receptors function during the formation of the initial embryonic vascular plexus, whereas angiopoietins (Angs) and their receptor, Tie2, as well as ephrins and their Eph receptors are implicated in the subsequent remodeling processes. See, e.g., Jones et al. (2001) Nat. Rev. Molec. Cell Biol. 2:257 for a review of receptors involved in angiogenic and lyrnphangiogenic responses.
[0004] Tiel and Tie2 are RTKs that are expressed almost exclusively in endothelial cells and hematopoietic precursor cells. These two receptors are required for the normal development of vascular structures during embryogenesis. The two Tie receptors form a RTK subfamily since, unlike other RTK family members, they include extracellular EGF-homology domains. See, e.g., Partanen (1992) MoI. Cell Biol. 12:1698 and WO 93/14124. Targeted disruption of the Tiel gene in mice results in a lethal phenotype characterized by extensive hemorrhage and defective microvessel integrity. See, e.g., Puri et al. (1995) EMBO J. 14:5884. Tie2 null embryos have defects in vascular remodeling and maturation, resulting from improper recruitment of periendothelial supporting cells. Angiopoietins (Ang, e.g., Angl, Ang2, Ang3, and Ang4) are proteins that interact with Tie2.
SUMMARY
[0005] In one aspect, the invention features a method of modulating Tie complex formation, or interactions between Tie complex components, in a subject. The method includes administering, to a subject, an agent that binds to Tiel. For example, the agent promotes Tiel self-association (e.g., homodimerization) or antagonizes an association between at least two of the following: Tiel, Tie2, and an angiopoietin (Ang; such as Angl, Ang2, Ang3, or Ang4). In one embodiment, the agent antagonizes formation of a heteromeric complex of Tiel , Tie2, and Ang. In another embodiment, the binding of the agent can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
[0006] In one embodiment, the agent binds to Tiel . In one embodiment, the agent antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang. In another embodiment, the binding of the agent can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang. In another embodiment, the agent enhances Tiel self-association, e.g., homodimerization, and thereby associates Tiel with Tiel and prevents association of Tiel with Tie2 and/or Ang. The agent can include at least two valencies for binding to Tiel. hi one embodiment, the agent increases phosphorylation of Tiel, e.g., Tiel "aύϊόphosphorylation. This increase can, but need not, depend on Tiel self- association.
[0007] In one embodiment, the agent includes a protein, such as an antibody, that binds to the extracellular domain of human Tiel . For example, the antibody can be one or more of the following: human, humanized, non-immunogenic, isolated, monoclonal, and recombinant. In one embodiment, the antibody can bind to the first Ig-like C2-type domain (Ig 1) or to the second Ig-like C2-type domain (Ig 2) of Tiel. In one embodiment, the antibody binds to an EGF-like domain of Tiel (e.g., first, second, or third EGF-like domain). In one embodiment, the antibody binds to the fibronectin type III repeats region of Tiel . In one embodiment, the antibody binds to amino acid residues 24-124, 74-174, 124-224, 174-274, 224-324, 274-374, 324-424, 374-474, 424-524, 474-574, 524-624, 574-674, 624-724, 674-759, or 724-759 of SEQ ID NO:2.
[0008] In one embodiment, the agent includes a protein that binds to a Tiel ectodomain and includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence. The protein can further include one or more of the following properties: (1) at least one of the variable domain sequences includes at least one CDR of the E3 or E3b antibody (e.g., one, two, or three CDRs of the E3 or E3b antibody); (2) at least one of the variable domain sequences includes CDR sequences at least 85% identical, in sum, to the CDRs of the corresponding variable domain of the E3 or E3b antibody, (3) at least one of the variable domains is at least 85% identical to the corresponding immunoglobulin variable domains of the E3 or E3b antibody, and (4) the protein competes with E3 or E3b for binding to Tiel or binds to an epitope that overlaps the epitope bound by E3 or E3b on Tiel . Example of antibodies that include an antigen binding site that competes with E3 for binding to Tiel include M0044B08, M0056G08, M0045B03, M0053F04, M0055E10, M0060H01, M0054H10, M0058F03, and related antibodies.
[0009] In one embodiment, the agent includes the HC and/or LC variable domain of the E3 or E3b antibody, or a sequence at least 70, 80, 85, 90, 95, 98, 99% identical to the HC and/or LC variable domains of the E3 or E3b antibody. In one embodiment, the amino acid sequences of the HC variable domain sequence include CDRl, CDR2, and CDR3 sequences from the E3 or E3b clone and the LC variable domain sequence includes CDRl, CDR2, and CDR3 sequences from the E3 or E3b clone. In one embodiment, the agent comprises the E3 or E3b antibody. The LC variable domain sequence can include SEQ ID NO: 116. The HC variable domain sequence can include SEQ ID NO: 114. In one embodiment, the HC and LC framework regions are human. In one embodiment, that agent includes SEQ ID NO:723 and SEQ ID NO:724.
[0010] In one embodiment, the agent binds to Tie2. In one embodiment, the agent antagonizes formation of a heteromeric complex of Tie 1, Tie2, and Ang. In another embodiment, the binding of the agent can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang. In another embodiment, the agent enhances Tie2 self-association, e.g., homodimerization, and, thereby associates Tie2 with Tie2 and prevents association of Tie2 with Tiel and/or Ang. In one embodiment, the agent includes a protein, e.g., an antibody that binds to the extracellular domain of human Tie2. For example, the antibody can be one or more of the following: human, humanized, non-immunogenic, isolated, monoclonal, and recombinant. In one embodiment, the antibody can bind to the first Ig-like C2- type domain (Ig 1) or to the second Ig-like C2-type domain (Ig 2) of Tie2. In one embodiment, the antibody binds to an EGF-like domain of Tie2 (e.g., first, second, or third EGF-like domain). In one embodiment, the antibody binds to the fibronectin type III repeats region of Tie2. hi one embodiment, the antibody binds to amino acid residues 19-119, 69-169, 119-229, 169-269, 229-329, 269-369, 329-429, 369-469, 429-529, 469-569, 529-629, 569-669, 629-729, 669-745, 729-745 of SEQ ID NO:162.
[0011] In one embodiment, the agent binds to Ang (e.g., Angl, Ang2, Ang3, or Ang4). In one embodiment, the agent antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang. In another embodiment, the binding of the agent can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang. In one embodiment, the agent includes a protein, e.g., an antibody that binds to Ang. For example, the antibody can be one or more of the following: human, humanized, non-immunogenic, isolated, monoclonal, and recombinant. In one embodiment, the antibody binds to the N-terminal domain of Angl (e.g., the N- terminal 50 amino acids of Angl). hi one embodiment, the antibody binds to the coiled-coil domain of Angl. In one embodiment, the antibody binds to the fibrinogen-like domain of Angl. In one embodiment, the antibody binds to amino acid residues 1-100, 50-150, 100-200, 150-250, 200-300, 250-350, 300-400, 350-450, 400-497, or 450-497 of SEQ ID NO: 163.
[0012] In one embodiment, the agent includes a protein that contains a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence, hi one embodiment, the HC and LC framework regions are human, hi one embodiment, the agent also includes an Fc domain. In one embodiment, the agent includes the constant domains of a human IgGl, IgG2, IgG3, or IgG4. hi one embodiment, the constant domains of the heavy chain are f allotype, (a,z) allotype, or any other allotype.
[0013] hi one embodiment, the agent is administered in an amount effective to decrease vascular development or angiogenesis. In one embodiment, the subject has an angiogenesis-related disorder, hi other embodiments, the subject has for example: a neoplastic disorder, metastatic cancer, an angiogenesis-dependent cancer or tumor, an inflammatory disorder, rheumatoid arthritis, or psoriasis, hi one embodiment, the protein is delivered systemically.
[0014] hi another embodiment, the protein is administered in an amount effective to reduce one or more of the following activities: sprouting, splitting, remodeling of blood vessels, vasculogenesis, and tubule formation. The method can include other features described herein.
[0015] hi one aspect, the invention includes a method of decreasing or inhibiting endothelial cell activity in the subject, the method includes administering an agent that decreases or inhibits Tie complex formation in an amount effective to decrease or inhibit endothelial cell activity in the subject. The method can include other features described herein.
[0016] In one aspect, the invention includes a method of decreasing endothelial cell activity by administering an agent that causes Tiel phosphorylation, hi one embodiment, the phosphorylation decreases endothelial cell differentiation, e.g., sprouting, splitting, and tube formation.
[0017] In another aspect, the invention includes a method of decreasing endothelial cell activity, the method by administering an agent that activates a signaling pathway. In one embodiment, the signaling pathway decreases endothelial cell differentiation, e.g., sprouting, splitting, and tube formation. For example, the agent increases Tiel autophosphorylation. The method can include other features described herein.
[0018] In one aspect, the invention includes an antibody for modulating Tie complex formation in a subject, wherein the antibody antagonizes an association between at least two of the following: Tiel, Tie2, and an angiopoietin (Ang). In one embodiment, the antibody binds to a Tie complex component or to one or more of Tiel, Tie2, and an Ang. In one embodiment, the antibody antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang. In another embodiment, the antibody can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
[0019] In one embodiment, the antibody binds to Tiel. In one embodiment, the antibody antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang. In another embodiment, the binding of the antibody can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang. In another embodiment, the antibody enhances Tiel self-association, e.g., homodimerization, and thereby associates Tiel with Tiel and prevents association of Tiel with Tie2 or Ang. In another embodiment, the antibody increases Tiel phosphorylation and/or prevents association of Tie 1 with Tie2 or Ang. In one embodiment, the antibody includes an antibody that binds to the extracellular domain of human Tiel. For example, the antibody can be one or more of the following: human, humanized, non- immunogenic, isolated, monoclonal, and recombinant. In one embodiment, the antibody can bind to the first Ig-like C2-type domain (Ig 1) or to the second Ig-like C2-type domain (Ig 2) of Tiel. In one embodiment, the antibody binds to an EGF- like domain of Tiel (e.g., first, second, or third EGF-like domain). In one embodiment, the antibody binds to the fibronectin type III repeats region of Tiel. In one embodiment, the antibody binds to amino acid residues 24-124, 1 A-Il A, 124-224, 174-274, 224-324, 274-374, 324-424, 374-474, 424-524, 474-574, 524-624, 574-674, 624-724, 674-759, or 724-759 of SEQ ID NO:2.
[0020] In one embodiment, the antibody binds to a Tiel ectodomain and includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence, the protein further includes one or more of the following properties: (1) at least one of the variable domain sequences includes at least one CDR of the E3 or E3b antibody; (2) at least one of the variable domain sequences includes CDR sequences at least 85% identical, in sum, to the CDRs of the corresponding variable domain of the E3 or E3b antibody; (3) at least one of the variable domains is at least 85% identical to the corresponding immunoglobulin variable domains of the E3 or E3b antibody, and (4) the protein competes with E3 or E3b for binding to Tiel or binds to an epitope that overlaps the epitope bound by E3 or E3b on Tiel. For example, the antibody is at least bivalent, e.g., with at least two antigen binding sites that bind to Tiel. In one embodiment, the antibody comprises the E3, E3b (e.g., DX-2220), or DX-2240.
[0021] In one embodiment, the antibody includes one or more variable domains from the E3 or E3b antibody or a variable domain sequence that is at least 70, 75, 80, 85, 90, 95, 98, or 995 identical to such a variable domain. In one embodiment, the amino acid sequences of the HC variable domain sequence include CDRl, CDR2, and CDR3 sequences from the E3 or E3b clone, and the LC variable domain sequence includes CDRl, CDR2, and CDR3 sequences from the E3 or E3b clone. In one embodiment, the LC variable domain sequence includes SEQ ID NO:116. In one embodiment, the HC variable domain sequence includes SEQ ID NO:114. In one embodiment, the HC and LC framework regions are human.
[0022] In one embodiment, the antibody binds to Tie2. In one embodiment, the antibody antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang. In another embodiment, the binding of the antibody can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang. In another embodiment, the antibody enhances Tie2 self-association, e.g., homodimerization, and thereby associates Tie2 with Tie2 and prevents association of Tie2 with Tiel or Ang. In one embodiment, the antibody causes Tiel phosphorylation. In one embodiment, the antibody prevents association of Tiel with Tie2 or Ang. In one embodiment, the antibody includes an antibody that binds to the extracellular domain of human Tie2. The antibody may have one or more of these properties, e.g., the antibody may cause Tiel phosphorylation and prevent association of Tiel with Tie2 or Ang, etc.
[0023] For example, the antibody can be one or more of the following: human, humanized, non-immunogenic, isolated, monoclonal, and recombinant. In one embodiment, the antibody can bind to the first Ig-like C2-type domain (Ig 1) or to the second Ig-like C2-type domain (Ig 2) of Tie2. In one embodiment, the antibody binds to an EGF-like domain of Tie2 (e.g., first, second, or third EGF-like domain). In one embodiment, the antibody binds to the fibronectin type III repeats region of Tie2. In one embodiment, the antibody binds to amino acid residues 19-119, 69-169, 119-229, 169-269, 229-329, 269-369, 329-429, 369-469, 429-529, 469-569, 529-629, 569-669, 629-729, 669-745, 729-745 of SEQ ID NO: 162.
[0024] In one embodiment, the antibody binds to Ang. In one embodiment, the antibody antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang. In another embodiment, the binding of the antibody can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang. For example, the antibody can be one or more of the following: human, humanized, non- immunogenic, isolated, monoclonal, and recombinant, hi one embodiment, the antibody binds to the N-terminal domain of Angl (i.e., the N-teraiinal 50 amino acids of Angl). Li one embodiment, the antibody binds to the coiled-coil domain of Angl. hi one embodiment, the antibody binds to the fibrinogen-like domain of Angl. hi one embodiment, the antibody binds to amino acid residues 1-100, 50-150, 100-200, 150- 250, 200-300, 250-350, 300-400, 350-450, 400-497, or 450-497 of SEQ ID NO.163.
[0025] hi one embodiment, the antibody includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence.
[0026] hi one embodiment, the HC and LC framework regions are human, hi one embodiment, the antibody also includes an Fc domain, hi one embodiment, the antibody includes the constant domains of a human IgGl, IgG2, IgG3, or IgG4.
[0027] hi one embodiment, the antibody is administered in an amount effective to decrease vascular development and angiogenesis. hi one embodiment, the antibody is delivered systemically. In one embodiment, antibody is administered in an amount effective to reduce one or more of the following activities: sprouting, splitting, remodeling of blood vessels, vasculogenesis, and tubule formation.
[0028] In one aspect, the invention includes an isolated protein that includes one or more variable domains of an antibody described herein.
[0029] In one aspect, the invention includes a nucleic acid that includes a coding sequence that encodes a polypeptide that includes a variable domain of an antibody described herein. [0030] In one aspect, the invention includes a pharmaceutical composition that includes an antibody described herein. The composition and antibody can include other features described herein.
[0031] hi one aspect, the invention includes an antibody described herein for treatment of an angiogenesis-related disorder. The antibody and treatment can include other features described herein.
[0032] hi one aspect, the invention includes an antibody described herein for the manufacture of a medicament for treating an angiogenesis-related disorder. The medicament and antibody can include other features described herein.
[0033] hi one aspect, the invention includes a method of providing a first therapy that includes administering a first agent in combination with a second therapy, e.g., an anti-cancer therapy. The first agent is an agent that decreases Tie complex formation or an agent that increases Tiel homodimerization. For example, the first agent is a Tiel binding protein, hi one embodiment, the second therapy includes radiation therapy or surgery. In one embodiment, the second therapy includes administering a second agent. For example, the second agent antagonizes or decreases Tie complex formation or increases Tiel homodimerization. hi one embodiment, the second agent is an agent that antagonizes signaling through a VEGF pathway, e.g., a VEGF antagonist antibody, e.g., bevacizumab; VEGF-Receptor tyrosine kinase inhibitor, or another agent that antagonizes VEGF pathway signalling. See also "Combination Therapies" below.
[0034] hi another aspect, the invention includes a composition that includes an agent that decreases Tie complex formation and an anti-cancer agent. For example, the anti-cancer agent can be a second agent that antagonizes Tie complex formation or a second agent that antagonizes a VEGF pathway.
[0035] hi one aspect, the invention features an antibody that decreases endothelial cell activity by causing Tiel phosphorylation. For example, the antibody may decrease endothelial cell differentiation, e.g., sprouting, splitting, and tube formation.
[0036] hi one aspect, the invention features a protein (e.g., an isolated protein) that includes a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence and binds to Tiel ectodomain. The binding protein binds to Tiel ectodomain. For example, the protein binds with an affinity K0 of less than 10"8 M, 5-10"9 M, 10"9 M5 10"10 M, 10"11 M, or 1(T12 M.
[0037] In one embodiment, one or more of the CDRs of the heavy and/or light chain variable domain sequence are human, primate, non-rodent (e.g., non-mouse or non-rat), or synthetic. In one embodiment, one or more of the framework regions of the heavy and/or light chain variable domain sequence are human, primate, or non- rodent (e.g., non-mouse or non-rat).
[0038] hi one embodiment, the heavy chain variable domain sequence includes one or more of the following properties: i) a HC CDRl that includes an amino acid sequence as follows:
(AGSR)-Y-(GVK)-M-(GSVF), (SEQ ID NO: 117)
(AGSIMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO: 118), or
(AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO: 119); ii) a HC CDR2 that includes an amino acid sequence as follows:
X-I-Y-P-S-G-G-X-T-X-Y-A-D-S-V-K-G (SEQ ID NO: 120), wherein X is any amino acid,
(GSV)-I-(SY)-P-S-G-G-(WQ)-T-(GY) (SEQ ID NO:121),
(GSV)-I-(SY)-P-S-G-G-(WNQ)-T-(GY) (SEQ ID NO:160)
(GSV)-I-(SY)-P-S-G-G-(WQ)-T-(GY)-Y-A-D-S-V-K-G (SEQ ID NO: 122),
(GSVW)-I-(SY)-P-S-G-G-(AGVMYWPQH)-T-(AGSTLVMYFKH)
(SEQ ID NO: 123), or
X-I-Y-P-S-G-G-(WPS)-T-(YVH)-Y-A-D (SEQ ID NO : 722), wherein X is any amino acid; iii) a HC CDR3 that includes an amino acid sequence as follows:
V-(four or five residues)-F-D-(]7Y) (SEQ ID NO: 124),
G-Y-G-P-I-A-P-G-L-D-Y (SEQ ID NO: 125),
(GV)-N-Y-Y-(GYD)-S-(SD)-G-Y-G-P-I-A-P-G-L-D-Y (SEQ ID NO: 126),
(GVD)-(AGLN)-(LYR)-(GSTLYH)-(GYD)-(AGSYFP)-(SFD)-(AGYD)- (IY)-(GFD)-(YDP)-(IP)-A-P-G-L-D-Y (SEQ ID NO: 127),
A-P-R-G-Y-S-Y-G-Y-Y-Y (SEQ ID NO:727),
VNYYDSSGYGPIAPGLDY (SEQ ID NO:128), or
G-X-X-G-(AY)-F-D-(YI) (SEQ ID NO:705), wherein X is any amino acid. [0039] In one embodiment, the light chain variable domain sequence includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence as follows: R-A-S-Q-S-(IV)-S-(SR)-Xl-Y-L-(AN) (SEQ ID NO: 129), R-A-S-Q-S-(IV)-S-S-(YS)-L-(ALN) (SEQ ID NO:706), T-G-T-(SN)-S-D-V-G-(GS)-Y (SEQ ID NO:707),
(SGQ)-(GS)-(DS)-(NS)-(IL)-(GR)-S-(YKN)-(YS)-(VA) (SEQ ID NO:708), R-A-S-Q-S-V-S-S-X-L (SEQ ID NO: 130), R-A-S-Q-S-(IV)-S-(SR)-(SY)-(LY)-(ALN) (SEQ ID NO: 131), or R-A-S-(REQ)-(GSTRN)-(IV)-(GSTIRN)-(STIRH)-XI-(SYWNH)-(LV)-
(ASN) (SEQ ID NO: 132), wherein Xl can be serine or absent; ii) a LC CDR2 that includes an amino acid sequence as follows: X-A-S-X-R-A-T (SEQ ID NO: 133), wherein X can be any amino acid, (AGD)-A-S-(STN)-R-A-T (SEQ ID NO: 134), (DG)-(AV)-S-N-(RL)-(AP)-ST) (SEQ ID NO:709), (AGD)-A-S-(STN)-(LR)-(AEQ)-(ST) (SEQ ID NO:135), or (AGTKDEH)-A-S-(STN)-(LR)-(AVEQ)-(ST) (SEQ ID NO: 136); and iii) a LC CDR3 that includes an amino acid sequence as follows: Q-Q-(SYFR)-(GSYN)-S-(STYW)-(RP)-(LWRH)-(TIY) (SEQ ID NO:161), Q-Q-(SYFR)-(GSYN)-S-(STYW)-(RP)-(LWR)-(TIY)-T (SEQ ID NO: 137), (LQ)-Q-(SYFR)-(GSYN)-(SKN)-(STYW)-(RP)-(LWR)-(TIY)-T (SEQ ID
NO: 138),
Q-Q-X-S-(SN)-(WS)-P-X-T-F (SEQ ID NO:710), wherein X is any amino acid,
Y-(TG)-(SG)-S-(PGS)-(TN)-X-(VT) (SEQ ID NO:711), wherein X is any amino acid,
Q-Q-(YR)-(GS)-S-(SW)-P-R-Xl-T (SEQ ID NO:139), wherein Xl is any amino acid or absent,
Q-Q-F-N-S-Y-P-H (SEQ ID NO:728), (LQ)-(LQ)-(SYFRD)-(GSYN)-(STRKN)-(STYWF)-(RP)-(ILMWRH)-(TIY)-
(TI) (SEQ ID NO: 140), or
(LQ)-(LRQ)-(SYFRD)-(GSYN)-(ASTRKN)-(STYWF)-(SVRP)-
(STILMWRH)-(TIY)-(STI) (SEQ ID NO:141). [0040] In one embodiment, the light chain variable domain sequence includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence as follows:
S-X-(ND)-(IV)-(AG)-Xl -X2-X3 (SEQ ID NO: 142), or
T-(GR)-(ST)-S-X5-(ND)-(IV)-(AG)-X1-X2-X3-Y-X4-S (SEQ ID NO: 143), wherein Xl is any amino acid (e.g., G or R), X2 is any amino acid (e.g., Y or N), X3 is any amino acid (e.g., F, N, or K), X4 is any amino acid (e.g., aliphatic, e.g., V or A); ii) a LC CDR2 that includes an amino acid sequence as follows:
(DE)-V-N-N-R-P-S (SEQ ID NO:144)
(DE)-(VD)-(STDN)-(YRDN)-R-P-S (SEQ ID NO: 145); iii) a LC CDR3 that includes an amino acid sequence as follows:
(SQ)-S-(SY)-(ASID)-(GSR)-(ST)-(STRN)-(STYR)-(ATLY)-(SVWQ) (SEQ ID NO:146).
[0041] In one embodiment, the HC CDR2 includes an amino acid sequence as follows: (GSVW)-I-(SY)-P-SG-G-(AGVMYWPQH)-T-(AGSTLVMYFKH)-Y-(AT)- D-S-V-K-G (SEQ ID NO: 147) or (GSV)-I-(SY)-P-SG-G-(WQ)-T-(GY)-Y-(AT)-D-S- V-K-G (SEQ ID NO:148).
[0042] In one embodiment, the protein includes HC CDRl and HC CDR2 sequences that are related to the corresponding CDR sequences of p-F3, E3 or E3b. For example, the protein includes the sequence MYGM (SEQ ID NO: 149), at a position corresponding to HC CDRl. The sequence can be followed by a small amino acid, e.g., glycine, alanine, valine, or serine. In another example, the protein the sequence VISPSGGX1TX2YADSAVKG (SEQ ID NO: 150), at a position corresponding to HC CDR2. For example, X1 can be a hydrophilic amino acid, e.g., glutamine or asparagine. For example, X2 can be a small amino acid, e.g., glycine, alanine, valine, or serine.
[0043] In one embodiment, the heavy chain variable domain sequence can have one or more of the following features: the amino acid residue at Kabat position 31 is A, H, K, N, Q, R, S, or T, e.g., H, N, R, or S; the amino acid residue at Kabat position 32 is Y; the amino acid residue at Kabat position 33 is G, K, P, R, or V, e.g., K or V; the amino acid residue at Kabat position 34 is M; the amino acid residue at Kabat position 35 is A, G, H, I, L, M, S, or V, e.g., G, H, M, or V; the amino acid residue at Kabat position 50 is G, R, S, or V, e.g., S or V; the amino acid residue at Kabat position 51 is I; the amino acid residue at Kabat position 52 is S or Y, e.g., Y; the amino acid residue at Kabat position 52a is P or S, e.g., P; the amino acid residue at Kabat position 53 is S; the amino acid residue at Kabat position 54 is G; the amino acid residue at Kabat position 55 is G; the amino acid residue at Kabat position 56 is A, F, H, I, Q, W, or Y, e.g., A, W or Y; the amino acid residue at Kabat position 57 is T; the amino acid residue at Kabat position 58 is R, S, T, or Y, e.g., Y. In one embodiment, the length of CDR3 is between 8-18 amino acids, e.g., between 8-12, 8-10, or 15-17 ammo acids.
[0044] hi one embodiment, two or three of the CDRs of the HC variable domain sequence match motifs that also match a HC variable domain of an antibody described herein. Similarly, in one embodiment, two or three of the CDRs of the LC variable domain sequence match motifs that also match a LC variable domain of an antibody described herein. In still another embodiment, the matched motifs for the CDRs are based on a HC and a LC that are paired in an antibody described herein.
[0045] In one embodiment, the Hl and H2 hypervariable loops have the same canonical structure as an antibody described herein, hi one embodiment, the Ll and L2 hypervariable loops have the same canonical structure as an antibody described herein.
[0046] hi one embodiment, the HC CDRl amino acid sequences have a length of at least 5 amino acids of which at least 3, 4, or 5 amino acids are identical to the CDRl sequence of the HC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, ρ-C6, p- D12, p-F3, ρ-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s- El 1, s-GlO, s-H4, or another antibody described herein, hi one embodiment, the HC CDR2 amino acid sequences have a length of at least 15, 16, or 17 amino acids of which at least 10, 12, 14, 15, 16, or 17 amino acids are identical to the CDR2 sequence of the HC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p- F3, ρ-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s- GlO, s-H4, or another antibody described herein. In one embodiment, the HC CDR2 amino acid sequences have a length of at least 17 amino acids of which at least 14, 15, 16, or 17 amino acids are identical to the CDR2 sequence of the HC of clone E3, E3b, G2, p-Al, p-AlO, p-Bl, p-B3, ρ-C6, p-D12, p-F3, p-F4, ρ-G3, s-AlO, s-Hl, s-A2, s- B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, S-GlO, s-H4, or another antibody described herein. In one embodiment, the HC CDR3 amino acid sequences have a length of at least of at least 7 or 8 amino acids of which at least 5, 6, 7, or 8 amino acids are identical to the CDR3 sequence of the HC of clone E3, E3b, G2, p-Al, p- AlO, p-Bl, p-B3, p-C6, ρ-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s- ClO, s-C2, s-C7, s-Dll, s-El 1, s-GlO, s-H4, or another antibody described herein.
[0047] In one embodiment, two or three of the CDRs of the HC variable domain sequence match motifs described herein such that the motifs are a set of motifs that match a HC variable domain of a clone described herein, e.g., E3, E3b, G2, p-Al, p-AlO, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s- B2, s-B9, s-ClO, s-C2, s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein. For example, the protein may include SEQ ID NO:118 and SEQ ID NO: 160, e.g., motifs that match the E3 HC variable domain.
[0048] In one embodiment, the LC CDRl amino acid sequences have a length of at least 10, 11, or 12 amino acids of which at least 7, 8, 9, 10, or 11 amino acids are identical to the CDRl sequence of the LC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, ρ-D12, p-F3, ρ-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s- C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein. In one embodiment, the LC CDR2 amino acid sequences have a length of at least 6 or 7 amino acids of which at least 5, 6, or 7 amino acids are identical to the CDR2 sequence of the LC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p- F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dl l, s-El 1, s- GlO, s-H4, or another antibody described herein. In one embodiment, the LC CDR3 amino acid sequences have a length of at least of at least 8, 9, or 10 amino acids of which at least 7, 8, 9, or 10 amino acids are identical to the CDR3 sequence of the LC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, ρ-F3, p-F4, p-G3, s- AlO, s-Hl, S-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein.
[0049] In one embodiment, two or three of the CDRs of the LC variable domain sequence match motifs described herein such that the motifs are a set of motifs that match a LC variable domain of a clone described herein, e.g., E3, E3b, G2, p-Al, p-AlO, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s- B9, s-CIO, s-C2, s-C7, s-Dl 1, s-El 1, s-GlO, s-H4, or another antibody described herein. For example, the protein may include SEQ ID NO:132, SEQ ID NO:136, and SEQ ID NO:161, e.g., motifs that match the E3 LC variable domain.
[0050] In one embodiment, the amino acid sequence of the HC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the HC variable domain of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s- C2, s-C7, s-Dl 1, s-El 1, S-GlO, s-H4, or another antibody described herein.
[0051] In one embodiment, the amino acid sequence of the LC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the LC variable domain of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B35 p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s- C2, s-C7, s-Dll, s-El 1, S-GlO, s-H4, or another antibody described herein.
[0052] In one embodiment, the amino acid sequences of the HC and LC variable domain sequences are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequences of the HC and LC variable domains of a clone selected from the group consisting of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p- D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s- EIl, s-GlO, s-H4, and any other antibody described herein.
[0053] In one embodiment, the amino acid sequences of one or more framework regions (e.g., FRl, FR2, FR3, and/or FR4) of the HC and/or LC variable domain are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to corresponding framework regions of the HC and LC variable domains of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s- A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s-G10, s-H4, or another antibody described herein.
[0054] In one embodiment, the amino acid sequences of the HC and LC variable domain sequences comprise a sequence encoded by a nucleic acid that hybridizes (e.g., under high stringency) to a nucleic acid encoding a variable domain of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s- Hl, S-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-El I5 s-GlO, s-H4, or another antibody described herein. [0055] In one embodiment, the light chain variable domain sequence is human or non-immunogenic in a human. In one embodiment, the heavy chain variable domain sequence is human or non-immunogenic in a human.
[0056] The protein can bind to cells that express Tiel, e.g., endothelial cells.
In one embodiment, the protein does not substantially bind (e.g., does not detectably bind) to platelets (e.g., resting and/or activated platelets).
[0057] In one embodiment, the protein inhibits tube formation by HUVECs in vitro. For example, the E3 antibody inhibits tube formation by HUVECs in vitro (e.g., under conditions described in Example 18). In one embodiment, the protein inhibits angiogenesis in an in vivo MATRIGEL™ plug assay. For example, the E3 antibody can inhibit angiogenesis in an exemplary assay (see, e.g., an exemplary assay described in Example 21).
[0058] In one embodiment, the protein recognizes melanoma-associated structures in a histological section, e.g., not only melanoma tissue, but antigen in surrounding structures. In one embodiment, the protein does not stain blood vessels in normal skin in a histological section.
[0059] In one embodiment, the protein specifically binds to Tiel, e.g., it binds with at least a 10, 50, 100, 103, or 104 fold preference for Tiel relative to another human protein, e.g., Tie2, a natural protein other than Tiel that has a Ig-like domain, an EGF-like domain, or fibronectin Type III repeat, or human serum albumin. In one embodiment, the protein binds to a domain of Tiel described herein.
[0060] In another aspect, the invention features a protein (e.g., an isolated protein) that modulates activity of Tiel, e.g., the Tiel receptor. For example, the protein is not naturally occurring. In one embodiment, the protein includes a HC and LC immunoglobulin variable domain sequence. In one embodiment, one or more of the CDRs of the heavy and/or light chain variable domain sequence are human, primate, non-rodent (e.g., non-mouse or non-rat), or synthetic. In one embodiment, one or more of the framework regions of the heavy and/or light chain variable domain sequence are human, primate, or non-rodent (e.g., non-mouse or non-rat). In another embodiment, the protein is substantially free of an immunoglobulin variable domain, e.g., the protein includes a peptide that independently interacts with Tiel or a polypeptide that does not include a immunoglobulin variable domain. [0061] In one embodiment, the protein activates an activity of the Tiel protein, e.g., an activity in the Tiel/EpoR chimeric BaF3 cell assay described in Example 2. A protein that activates in this assay can behave as antagonists in other conditions, for example, in vivo.
[0062] ■ hi one embodiment, the protein includes the HC and LC immunoglobulin variable domains of the E3, E3b, or other antibody, HC and/or LC immunoglobulin variable domain sequences that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical in the CDR regions to the respective CDRs of the E3, E3b or other antibody described herein. In one embodiment, the protein competes with E3, E3b, or other antibody described herein for binding to Tiel or binds to an epitope that overlaps an epitope that is recognized by E3, E3b, or other antibody described herein, or that has at least one, two or three residues in common with an epitope that is recognized by E3, E3b, or other antibody described herein.
[0063] In one embodiment, the activating protein enables IL-3 dependent cells that express a chimeric receptor including the Tiel extracellular domain and the EpoR intracellular domain to survive in the absence of IL-3.
[0064] In one embodiment, the protein can cause dimerization of Tiel . hi one embodiment, the protein can cause auto-phosphorylation of the RTK domain of Tiel.
[0065] In one embodiment, the protein synergizes with the E3 or E3b antibody to activate an activity of Tie, e.g., in the Tiel/EpoR chimeric BaF3 cell assay. In one embodiment, the protein includes the HC and LC immunoglobulin variable domains of the G2 or C7 antibody or domains that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical in the CDR regions. In one embodiment, the protein competes with G2 or C7 for binding to Tiel or binds to an epitope that overlaps an epitope that is recognized by G2 or C7 or that has at least one, two or three residues in common with an epitope that is recognized by G2 or C7.
[0066] hi another embodiment, the protein antagonizes an activity of the Tiel protein. For example, the protein can at least partially inhibit the ability of the E3 or E3b antibody to agonize the Tie protein. In one embodiment, the protein can at least partially inhibit the ability of the E3 or E3b antibody to enable IL-3 dependent cells that express a chimeric receptor including the Tiel extracellular domain and the EpoR intracellular domain to survive in the absence of IL-3. [0067] In one embodiment, the HC and LC immunoglobulin variable domain sequences of the protein include the amino acid sequences that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequences of respective immunoglobulin variable domains of B2 or Dl 1.
[0068] In one embodiment, the Tiel binding protein includes the HC and LC immunoglobulin variable domains of an antibody selected from the group consisting of: B2, Dl 1, A2, AlO, P-Bl, P-B3, and P-C6 or immunoglobulin domains that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical in the CDR regions to the CDR regions of the respective antibodies. For example, the protein binds with an affinity KD of less than 10"8 M, 5-10"9 M, 10"9 M, 10'10 M, 10'π M, or 10'12 M.
[0069] In one embodiment, the protein can at least partially inhibit the ability of a naturally occurring Tiel binding protein from interacting with the Tie protein.
[0070] The protein can include other features described herein.
[0071] In another aspect, the invention features an antibody (e.g., an isolated antibody) that binds to the Tiel ectodomain, but does not substantially bind to platelets, e.g., as detected by fluorescence activated cell sorting. For example, the antibody does not substantially bind to activated platelets and/or resting platelets. In one embodiment, the antibody binds to endothelial cells, hi one embodiment, the protein is a monoclonal antibody. The antibody can be provided in a preparation that is free of other Tiel-binding antibodies that have other specificities, e.g., free of Tiel binding antibodies that bind to platelets. The antibody can include other features described herein.
[0072] In another aspect, the invention features a protein (e.g., an isolated protein) that preferentially binds to a Tiel protein in a conformation stabilized by the E3 or E3b antibody relative to an endogenous Tiel protein in an unstimulated state, hi one embodiment, the protein includes immunoglobulin HC and LC domains, hi another embodiment, the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tiel. For example, the peptide can include one, two, or three disulfide bonds. The protein can include other features described herein. [0073] In another aspect, the invention features a protein (e.g., an isolated protein) that preferentially binds to a Tiel protein in a dimeric conformation relative to a monomelic Tiel protein. In one embodiment, the protein includes immunoglobulin HC and LC domains. In another embodiment, the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tiel . For example, the peptide can include one, two, or three disulfide bonds. The protein can include other features described herein.
[0074] In another aspect, the invention features a protein (e.g., an isolated protein) that preferentially binds to a Tie2 protein in a conformation that is biased against interaction with Ang or Tiel . In one embodiment, the protein includes immunoglobulin HC and LC domains, m another embodiment, the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tie2. For example, the peptide can include one, two, or three disulfide bonds. The protein can include other features described herein. The invention also features nucleic acid aptamers that have one or more of these properties.
[0075] In another aspect, the invention features a protein (e.g., an isolated protein) that preferentially binds to an Ang protein, and modulates (e.g., inhibits) interaction with Tiel and Tie2. hi one embodiment, the protein includes immunoglobulin HC and LC domains, hi another embodiment, the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Ang. For example, the peptide can include one, two, or three disulfide bonds. The protein can include other features described herein. The invention also features nucleic acid aptamers that have one or more of these properties.
[0076] In another aspect, the invention features a protein (e.g., an isolated protein) that binds to an epitope of Tiel ectodomain with a KD of less than 2 X 10"7 M. The epitope overlaps, is within, or includes an epitope bound by E3, E3b, G2, p- Al, p-AlO, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s- B9, s-ClO, s-C2, s-C7, s-Dll, s-Ell, s-G10, s-H4, or another antibody described herein or that includes at least one, two, or three residues in common. For example, the protein binds with an affinity KD of less than 10"8 M, 5-10"9 M, 10"9 M, 10'10 M, 10"n M, or 10"12 M. In one embodiment, the protein includes immunoglobulin HC and LC domains. In another embodiment, the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tiel . For example, the peptide can include one, two, or three disulfide bonds. The protein can include other features described herein. The invention also features nucleic acid aptamers that have one or more of these properties.
[0077] hi another aspect, the invention features a protein (e.g., an isolated protein) that competitively inhibits binding of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s- Dl 1, s-El 1, s-GlO, s-H4, or another antibody described herein to a Tiel ectodomain. In one embodiment, the protein includes immunoglobulin HC and LC domains, hi another embodiment, the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tiel. For example, the peptide can include one, two, or three disulfide bonds. The protein can include other features described herein.
[0078] In another aspect, the invention features a protein (e.g., an isolated protein) that includes a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence and that antagonizes an activity of the Tiel ectodomain. In one embodiment, CDRl of the light chain variable domain sequence includes: Q-S-X-S-S (SEQ ID NO:151) or R-A-S-Q-S-X- S-S-Y-L-A (SEQ ID NO: 152), wherein X is any amino acid or optionally aliphatic, e.g., isoleucine or valine. In one embodiment, CDR2 of the light chain variable domain sequence includes: A-S-X1-R-X2-T (SEQ ID NO:153) or D-A-S-X1-R-X2-T (SEQ ID NO: 154) , wherein X1 is any amino acid or optionally a hydrophilic amino acid, e.g., serine or asparagine, and X2 is any amino acid or optionally aliphatic or small aliphatic, e.g., alanine or valine. In one embodiment, CDR3 of the light chain variable domain sequence includes: Q-R-S-X2-W-P-R (SEQ ID NO: 155) or X1-Q-R- S-X2-W-P-R-T (SEQ ID NO: 156), wherein X1 is any amino acid or optionally leucine or glutamine, and X2 is any amino acid or optionally lysine or serine.
[0079] In one embodiment, the protein competes with the B2 and/or DIl antibody for binding to Tiel or competitively inhibits binding of B2 and/or Dl 1 to Tiel. [0080] In one embodiment, the protein antagonizes a Tiel activity that is stimulated by the E3 or E3b antibody. In one embodiment, the protein inhibits dimerization of Tiel. The protein can include other features described herein.
[0081] In another aspect, the invention features an isolated, mono-specific protein including a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to Tiel ectodomain and includes a human or non-mouse constant domain (e.g., a human IgGl, IgG2, IgG3, or IgG4 constant domain). The protein can include other features described herein.
[0082] hi another aspect, the invention features an isolated, human antibody that binds to a Tiel ectodomain. The protein can include other features described herein.
[0083] In another aspect, the invention features an isolated antibody (e.g., an isolated antibody) that binds to a Tiel ectodomain and contains less than 5, 4, 3, or 2 peptides (of between 6-9 amino acid length) that are non-human in origin or less than 5, 4, 3, or 2 peptides that are potential human T cell epitopes. Li one embodiment, the antibody contains no peptide (of 6-9 amino acid length) that is non-human in origin or that is a potential human T cell epitope.
[0084] In one embodiment, the antibody is obtained by a method that includes deimmunization. For example, the antibody is deimmunized, e.g., completely deimmunized. The protein can include other features described herein.
[0085] In another aspect, the invention features an isolated antibody that binds to a Tiel ectodomain and that includes a modified Fc domain, e.g., a modified human Fc domain. For example, antibodies may include modifications, e.g., that alter Fc function. For example, the human IgGl constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237, e.g., according to the number in US 5,648,260. Other exemplary modifications include those described in US 5,648,260. The protein can include other features described herein.
[0086] In another aspect, the invention features an isolated protein that binds to the Tiel receptor with an affinity K0 of less than 10"7 M, 10"8 M, 5-10"9 M, 10"9 M, 10"10 M, 10"11 M, or 10'12 M. The protein can include other features described herein. [0087] In another aspect, the invention features an isolated protein including a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to Tiel ectodomain and, for example, includes at least one or more CDRs that are a non- primate CDR (e.g., a non-mouse or non-rabbit CDR) or a synthetic CDR. The protein can include other features described herein.
[0088] In another aspect, the invention features an isolated nucleic acid including a coding sequence that encodes a polypeptide including an immunoglobulin HC variable domain of an antigen binding protein that binds to Tiel . The nucleic acid or polypeptide can include one or more other features described herein. The nucleic acid can include one or more altered codons. In one embodiment, the nucleic acid includes SEQ ID NOs:725 and/or 726. Also featured is a mammalian expression vector that includes SEQ ID NOs:725 and/or 726.
[0089] In one embodiment, the nucleic acid further includes a second coding sequence that encodes a polypeptide including an immunoglobulin HC variable domain, e.g., an HC domain described herein. In one embodiment, the nucleic acid further includes a promoter operably linked to the coding sequence.
[0090] In another aspect, the invention features a nucleic acid that includes one or more coding sequence that encodes one or more polypeptide chains that collectively include an immunoglobulin HC or LC variable domain of an antigen binding protein that binds to Tiel. hi one embodiment, the nucleic acid segment encoding at least one of the variable domains hybridizes to a nucleic acid described herein, e.g., under stringent conditions (e.g., high stringency conditions), e.g., it hybridizes to a region encoding a variable domain and is at least 80, 85, 90, 95, or 98% of the length of such a region. The nucleic acid can include other features described herein.
[0091] In another aspect, the invention features a host cell that contains a first nucleic acid sequence encoding a polypeptide including a HC variable domain of an antigen binding protein and a second nucleic acid sequence encoding a polypeptide including a LC variable domain of the antigen binding protein, wherein the antigen binding protein binds to Tiel with a KD of less than 2 x 10"7 M. hi one embodiment, the HC or LC variable domain includes at least one human CDR. The antigen binding protein can include other features described herein.
[0092] In another aspect, the invention features a host cell that contains a first nucleic acid encoding a polypeptide including a HC variable region and a second nucleic acid encoding a polypeptide including a LC variable region, wherein the HC and the LC variable regions each include at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to respective amino acid sequences of the HC and LC variable domains of a clone selected from the group consisting of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, ρ-C6, ρ-D12, ρ-F3, ρ-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s- C2, s-C7, s-Dl 1, s-El 1, s-GlO, and s-H4. The antigen binding protein can include other features described herein.
[0093] In another aspect, the invention features a pharmaceutical composition including a protein described herein that interacts with Tiel and a pharmaceutically acceptable carrier.
[0094] In another aspect, the invention features a therapeutic composition including a protein described herein that interacts with Tiel wherein the composition is sterile and suitable for administration to a subject.
[0095] In another aspect, the invention features a method that includes: providing a signal-dependent or signal-responsive cell that expresses a chimeric receptor including the Tiel extracellular domain and a heterologous intracellular sequence that can produce a signal; contacting a candidate compound to the cell; and evaluating a property of the cell that is dependent on the signal. In one embodiment, the intracellular sequence includes at least a region of an intracellular sequence of the EpoR protein. The method can be used, e.g., to evaluate activity of a candidate compound, or a plurality of compounds.
[0096] In another aspect, the invention features a method that includes: providing an IL-3 dependent cell that expresses a chimeric receptor including the Tiel extracellular domain and the EpoR intracellular domain; contacting a candidate compound to the cell under conditions in which the concentration of IL-3 is not sufficient to sustain viability of the cell; and evaluating a property of the cell. The method can be used, e.g., to evaluate activity of a candidate compound, or a plurality of compounds. In one embodiment, the property is viability. In one embodiment, the evaluating includes an MTT assay. In one embodiment, the method further includes administering the candidate compound to a subject. For example, the candidate compound includes a protein, e.g., a protein that includes an immunoglobulin variable domain.
[0097] hi another aspect, the invention features method of identifying a compound that modulates Tiel activity. The method includes: providing a plurality of candidate compounds; and evaluating each compound of the plurality using a method described herein.
[0098] hi another aspect, the invention features a culture cell that expresses a chimeric transmembrane protein including a region of the Tiel extracellular domain and a heterologous intracellular sequence. In one embodiment, the intracellular sequence includes a region of the EpoR intracellular domain. In one embodiment, the cell requires IL-3 or Tiel for viability. For example, the cell is IL-3 dependent in the absence of the chimeric transmembrane protein, but is viable in the presence of the E3 or E3b antibody and the absence of IL-3.
[0099] In another aspect, the invention features a preparation that includes the isolated mammalian cells (e.g., cells that expresses a chimeric transmembrane protein including a region of the Tiel extracellular domain and a heterologous intracellular sequence) and a Tiel -binding protein, wherein the Tiel -binding protein is necessary to sustain viability of the cells.
[0100] In another aspect, the invention features a kit including: a Tiel -binding protein and a culture cell that expresses a chimeric transmembrane protein including a region of the Tiel extracellular domain and a heterologous intracellular sequence.
[0101] In another aspect, the invention features a method of evaluating a candidate compound. The method includes: providing a preparation that includes (i) a cell or membrane fraction that contains (a) an insoluble protein that includes a region of the Tiel extracellular domain and a kinase domain and (b) ATP; (ii) a ligand that alters activity of the kinase domain; and (iii) the candidate compound; and evaluating the phosphorylation state of the insoluble protein.
[0102] In another aspect, the invention features a method of evaluating a candidate compound. The method includes: providing a preparation that includes (i) a cell or membrane fraction that includes a Tiel protein or a transmembrane protein that includes at least a region of the Tiel extracellular domain and ATP; (ii) a ligand that causes autophosphorylation of Tiel or the transmembrane protein; and (iii) the candidate compound; and evaluating phosphorylation state of the Tiel protein.
[0103] hi one embodiment, the ligand is an antibody. In one embodiment, the ligand includes the HC and LC immunoglobulin variable domains of the E3 or E3b antibody or domains that are at least 90% identical in the CDR regions. In one embodiment, the method further includes administering the candidate compound to a subject.
[0104] hi another aspect, the invention features a method that includes: providing a preparation that includes (i) a cell or membrane fraction that includes a transmembrane protein that includes at least a region of the Tiel extracellular domain and ATP; and (ii) a ligand that causes autophosphorylation of Tiel or the transmembrane protein; and evaluating phosphorylation state of the transmembrane protein.
[0105] hi another aspect, the invention features a method that includes: contacting a mammalian cell with a ligand that (i) can agonize Tiel autophosphorylation and/or (ii) can enable an IL-3 dependent cell that expresses a chimeric receptor including the Tiel extracellular domain and the EpoR intracellular domain to remain viable under conditions in which the concentration of IL-3 is not sufficient to sustain viability of the cell; and evaluating the mammalian cell, hi one embodiment, the cell expresses an endogenous Tiel protein. In one embodiment, the cell is an endothelial cell, hi one embodiment, the method further includes contacting the mammalian cell with a test compound, other than the ligand. For example, the ligand is an antibody. For example, the ligand includes the HC and LC immunoglobulin variable domains of the E3 or E3b antibody or domains that are at least 90% identical in the CDR regions.
[0106] hi another aspect, the invention features a method that includes: contacting a mammalian cell or fraction thereof with an agent that can modulate the activity of Tiel; and evaluating the mammalian cell or fraction thereof. In one embodiment, the agent is contacted to the cell while the cell is living, and the evaluating includes isolating a fraction of the cell, hi one embodiment, the agent is a protein, e.g., an antibody or a peptide. In one embodiment, the agent includes the HC and LC immunoglobulin variable domains of the E3 or E3b antibody or domains that are at least 90% identical in the CDR regions to the E3 or E3b antibody. In one embodiment, the agent includes the HC and LC immunoglobulin variable domains of the B2 or Dl 1 antibody or domains that are at least 90% identical in the CDR regions to the B2 or Dl 1 antibody. In one embodiment, the agent includes the HC and LC immunoglobulin variable domains of the A2, AlO, P-Bl, P-B3, or P-C6 antibody or domains that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical% identical in the CDR regions to the A2, AlO, P-Bl, P-B3, or P-C6 antibody. In one embodiment, the agent includes the HC and LC immunoglobulin variable domains of the G2 or C7 antibody or domains that are at least 90% identical in the CDR regions to the G2, or C7 antibody. The agent can include other features described herein.
[0107] In another aspect, the invention features a method of evaluating a test compound. The method includes evaluating interaction between an agent that can modulate the activity of Tiel and a protein that includes at least a region of the Tiel extracellular domain in the presence of the test compound. In one embodiment, the agent is a test compound is a small organic compound with molecular weight less than 8000, 7000, 6000, 5000, or 3000 Daltons. For example, the evaluating includes contacting cells that include the protein that includes at least a region of the Tiel extracellular domain with the agent in the presence of the test compound. In another example, the evaluating includes forming a cell-free preparation that includes the protein that includes at least a region of the Tiel extracellular domain, the agent, and the test compound.
[0108] hi another aspect, the invention features an artificial protein complex that includes (i) a protein that includes a Tiel extracellular domain and (ii) a Tiel binding protein that can modulate (e.g., agonize or antagonize) an activity of Tiel. In one embodiment, the ligand is an antibody (e.g., an antibody described herein). For example, the ligand includes the HC and LC immunoglobulin variable domains of an antibody selected from the group consisting of: E3, E3b, B2, Dl 1, A2, AlO, P-Bl, P- B3, P-C6, G2 and C7, or immunoglobulin domains that are at least 90% identical in the CDR regions to the CDR regions of the respective antibody. In one embodiment, the complex is present in a membrane fraction, on a mammalian cell, and/or in a subject. [0109] In another aspect the invention features a method that includes: administering a composition that includes a protein that interacts with Tiel, Tie2, or Ang (e.g., a protein described herein) to a subject in an amount effective to reduce angiogenesis in the subject or otherwise treat or prevent a disorder in a subject. For example, the protein binds to Tiel, Tie2, or Ang with an affinity KD of less than 10" M, 5-10-9 M, 10"9 M3 10"10 M, 10"n M, or 10"12 M.
[0110] In one embodiment, the protein is a Tiel binding protein. The protein can have at least two valencies, each of which binds to Tiel . For example, at least one, two, or all of the valencies can be binding sites that competes with E3 for binding to Tiel . In one embodiment, the protein competes with E3 for binding to Tiel or binds to an epitope that overlaps the epitope bound by E3 on Tiel.
[0111] In one embodiment, the protein comprises a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence. The protein further includes one or more of the following properties: (1) at least one of the variable domain sequences comprising at least one CDR of the E3 antibody; (2) at least one of the variable domain sequences comprising CDR sequences at least 85% identical, in sum, to the CDRs of the corresponding variable domain of the E3 antibody; (3) at least one of the variable domains is at least 85% identical to the corresponding immunoglobulin variable domains of the E3 antibody, and (4) the protein competes with E3 for binding to Tiel or binds to an epitope that overlaps the epitope bound by E3 on Tiel.
[0112] In one embodiment, one or more of the CDRs of the heavy and/or light chain variable domain sequence are human, primate, non-rodent (e.g., non-mouse or non-rat), or synthetic. In one embodiment, one or more of the framework regions of the heavy and/or light chain variable domain sequence are human, primate, or non- rodent (e.g., non-mouse or non-rat).
[0113] In one embodiment, the heavy chain includes one or more of the following properties: i) a HC CDRl that includes an amino acid sequence as follows: (AGSR)-Y-(GVK)-M-(GSVF), (SEQ ID NO: 117) (AGSIMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO: 118), or (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO: 119); ii) a HC CDR2 that includes an amino acid sequence as follows:
X-I-Y-P-S-G-G-X-T-X-Y-A-D-S-V-K-G (SEQ ID NO: 120), wherein X is any amino acid,
(GSV)-I-(SY)-P-S-G-G-(WQ)-T-(GY) (SEQ ID NO:121),
(GSV)-I-(SY)-P-S-G-G-(WQ)-T-(GY)-Y-A-D-S-V-K-G (SEQ ID NO: 122),
(GSVW)-I-(SY)-P-S-G-G-(AGVMYWPQH)-T-(AGSTLVMYFKH) (SEQ ID NO: 123); or
X-I-Y-P-S-G-G-(WPS)-T-(YVH)-Y-A-D (SEQ ID NO:704), wherein X is any amino acid; iii) a HC CDR3 that includes an amino acid sequence as follows:
V-(four or five residues)-F-D-(I/Y) (SEQ ID NO: 124),
G-Y-G-P-I-A-P-G-L-D-Y (SEQ ID NO:125),
(GV)-N-Y-Y-(GYD)-S-(SD)-G-Y-G-P-I-A-P-G-L-D-Y (SEQ ID NO: 126),
(GVD)-(AGLN)-(LYR)-(GSTLYH)-(GYD)-(AGSYFP)-(SFD)-(AGYD)- (IY)-(GFD)-(YDP)-(IP)-A-P-G-L-D-Y (SEQ ID NO: 127),
VNYYDSSGYGPIAPGLDY (SEQ ID NO: 128), or
G-X-X-G-(AY)-F-D-(YI) (SEQ ID NO:705), wherein X is any amino acid. [0114] hi one embodiment, the light chain includes one or more of the following properties: i) a light chain cdrl that includes an amino acid sequence as follows:
R-A-S-Q-S-(IV)-S-(SR)-Xl-Y-L-(AN) (SEQ ID NO: 129), R-A-S-Q-S-(IV)-S-S-(YS)-L-(ALN) (SEQ ID NO:706), T-G-T-(SN)-S-D-V-G-(GS)-Y (SEQ ID NO:707),
(SGQ)-(GS)-(DS)-(NS)-(IL)-(GR)-S-(YKN)-(YS)-(VA) (SEQ ID NO:708), R-A-S-Q-S-V-S-S-X-L (SEQ ID NO: 130), R-A-S-Q-S-(IV)-S-(SR)-(SY)-(LY)-(ALN) (SEQ ID NO:131), OR R-A-S-(REQ)-(GSTRN)-(IV)-(GSTIRN)-(STIRH)-XI-(SYWNH)-(LV)- (ASN) (SEQ ID NO: 132), wherein Xl can be serine or absent; ii) a LC CDR2 that includes an amino acid sequence as follows: X-A-S-X-R-A-T (SEQ ID NO:133), wherein X can be any amino acid, (AGD)-A-S-(STN)-R-A-T (SEQ ID NO: 134), (U<αMAV>S-IN-(KL)-(AP)-ST) (SEQ ID NO:709), (AGD)-A-S-(STN)-(LR)-(AEQ)-(ST) (SEQ ID NO-.135), OR (AGTKDEH)-A-S-(STN)-(LR)-(AVEQ)-(ST) (SEQ ID NO: 136); AND iii) a LC CDR3 that includes an amino acid sequence as follows: Q-Q-(SYFR)-(GSYN)-S-(STYW)-(RP)-(LWR)-(TIY)-T (SEQ ID NO: 137), (LQ)-Q-(SYFR)-(GSYN)-(SKN)-(STYW)-(RP)-(LWR)-(TIY)-T (SEQ ID
NO:138),
Q-Q-X-S-(SN)-(WS)-P-X-T-F (SEQ ID NO:710), wherein x is any amino acid,
Y-(TG)-(SG)-S-(PGS)-(TN)-X-(VT) (SEQ ID N0:711), wherein x is any amino acid,
Q-Q-(YR)-(GS)-S-(SW)-P-R-Xl-T (SEQ ID NO: 139), wherein Xl is any amino acid or absent,
(LQ)-(LQ)-(SYFRD)-(GSYN)-(STRKN)-(STYWF)-(RP)-(ILMWRH)-(TIY)-
(TI) (SEQ ID NO: 140), or
(LQ)-(LRQ)-(SYFRD)-(GSYN)-(ASTRKN)-(STYWF)-(SVRP)-
(STDLMWRH)-(TIY)-(STI) (SEQ ID NO: 141).
[0115] In one embodiment, the heavy chain includes one or more of the following properties: i) a HC CDRl that includes an amino acid sequence as follows: (AGSIMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO: 118), or (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID
NO: 119); ii) a HC CDR2 that includes an amino acid sequence as follows: (GSV)-I-(SY)-P-S-G-G-(NWQ)-T-(GY) (SEQ ID NO: 160), (GSV)-I-(SY)-P-S-G-G-(NWQ)-T-(GY)-Y-A-D-S-V-K-G (SEQ ID NO:122), or
(GSVW)-I-(SY)-P-S-G-G-(AGVMYWPQH)-T-(AGSTLVMYFKH) (SEQ ID
NO: 123); iii) a HC CDR3 that includes an amino acid sequence as follows: APRGYSYGYYY (SEQ ID NO:712).
[0116] In one embodiment, the light chain includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence as follows: R-A-S-(REQ)-(GSTRISO-(IV)-(GSTIRN)-(STIRH)-XI-(SYWNH)-(LV)-(ASN) (SEQ ID NO: 132), wherein Xl can be serine or absent; ii) a LC CDR2 that includes an amino acid sequence as follows: (TAGD)-A-S-(STN)-(LR)-(AEQ)-(ST) (SEQ ID NO:713), or (AGTKDEH)-A-S-(STN)-(LR)-(AVEQ)-(ST) (SEQ ID NO:136); and iii) a LC CDR3 that includes an amino acid sequence as follows: Q-Q-(SYFR)- (GSYN)-S-(STYW)-(RP)-(LHWR)-(TIY) (SEQ ID NO:714), (LQ)-Q-(SYFR)- (GSYN)-(SKN)-(STYW)-(RP)-(LHWR)-(TIY) (SEQ ID NO.715), or (LQ)-(LQ)- (SYFRD)-(GSYN)-(STRKN)-(STYWF)-(RP)-(ILMWRH)-(TrY) (SEQ ID NO:716).
[0117] In one embodiment, the light chain includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence as follows: S-X-(ND)-(IV)-(AG)-Xl -X2-X3 (SEQ ID NO:142), or T-(GR)-(ST)-S-X5-(ND)- (rV)-(AG)-Xl-X2-X3-Y-X4-S (SEQ ID NO: 143), wherein Xl is any amino acid (e.g., G or R), X2 is any amino acid (e.g., Y or N), X3 is any amino acid (e.g., F, N, or K), X4 is any amino acid (e.g., aliphatic, e.g., V or A); iii) a LC CDR2 that includes an amino acid sequence as follows: (DE)-V-N-N-R-P-S (SEQ ID NO: 144); (DE)- (VD)-(STDN)-(YRDN)-R-P-S (SEQ ID NO: 145); v) a LC CDR3 that includes an amino acid sequence as follows: (SQ)-S-(SY)-(ASID)-(GSR)-(ST)-(STRN)-(STYR)- (ATLY)-(SVWQ) (SEQ ID NO:146).
[0118] In one embodiment, the HC CDR2 includes an amino acid sequence as follows: (GSVW)-I-(SY)-P-SG-G-(AGVMYWPQH)-T-(AGSTLVMYFKH)-Y-(AT)- D-S-V-K-G (SEQ ID NO:147)or (GSV)-I-(SY)-P-SG-G-(WQ)-T-(GY)-Y-(AT)-D-S- V-K-G (SEQ ID NO: 148).
[0119] In one embodiment, the HC CDRl amino acid sequences have a length of at least 5 amino acids of which at least 3, 4, or 5 amino acids are identical to the CDRl sequence of the HC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, ρ-C6, p- D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s- EIl, S-GlO, s-H4, or another antibody described herein, hi one embodiment, the HC CDR2 amino acid sequences have a length of at least 15, 16, or 17 amino acids of which at least 10, 12, 14, 15, 16, or 17 amino acids are identical to the CDR2 sequence of the HC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, ρ-D12, p- F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s- GlO, s-H4, or another antibody described herein. In one embodiment, the HC CDR2 amino acid sequences have a length of at least 17 amino acids of which at least 14, 15, 16, or 17 amino acids are identical to the CDR2 sequence of the HC of clone E3, E3b, G2, p-Al, p-AlO, p-Bl, p-B3, ρ-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s- B2, s-B9, s-ClO, s-C2, s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein. In one embodiment, the HC CDR3 amino acid sequences have a length of at least of at least 7 or 8 amino acids of which at least 5, 6, 7, or 8 amino acids are identical to the CDR3 sequence of the HC of clone E3, E3b, G2, p-Al, p- AlO, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s- ClO, s-C2, s-C7, S-DIl5 S-EIl, S-GlO5 s-H4, or another antibody described herein.
[0120] In one embodiment, the LC CDRl amino acid sequences have a length of at least 10, 11, or 12 amino acids of which at least 7, 8, 9, 10, or 11 amino acids are identical to the CDRl sequence of the LC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s- C7, s-Dl 1, s-El 1, S-GlO5 s-H4, or another antibody described herein. In one embodiment, the LC CDR2 amino acid sequences have a length of at least 6 or 7 amino acids of which at least 5, 6, or 7 amino acids are identical to the CDR2 sequence of the LC of clone E3, E3b, G2, p-Al, ρ-A10, p-Bl, p-B3, ρ-C6, p-D12, p- F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s- GlO5 s-H4, or another antibody described herein. In one embodiment, the LC CDR3 amino acid sequences have a length of at least of at least 8, 9, or 10 amino acids of which at least 7, 8, 9, or 10 amino acids are identical to the CDR3 sequence of the LC of clone E3, E3b, G2, p-Al5 ρ-A10, p-Bl, ρ-B3, ρ-C6, ρ-D12, ρ-F3, ρ-F4, p-G3, s- AlO, s-Hl, s- A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s-G10, s-H4, or another antibody described herein.
[0121] In one embodiment, the amino acid sequence of the HC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the HC variable domain of clone E3, E3b, G2, p-Al, p-A10, p-Bl, ρ-B3, p-C6, p-D12, ρ-F3, ρ-F4, ρ-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s- C25 s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein.
[0122] In one embodiment, the amino acid sequence of the LC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the LC variable domain of clone E3, E3b5 G2, p-Al, p-A105 p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B95 s-CIO, s- C2, s-C7, s-Dll, s-Ell, S-GlO, s-H4, or another antibody described herein. [0123] In one embodiment, the amino acid sequences of the HC and LC variable domain sequences are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequences of the HC and LC variable domains of a clone selected from the group consisting of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p- D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s- EIl, s-GlO, and s-H4.
[0124] In one embodiment, the amino acid sequences of one or more framework regions (e.g., FRl, FR2, FR3, and/or FR4) of the HC and/or LC variable domain are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to corresponding framework regions of the HC and LC variable domains of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s- A2, S-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein.
[0125] In one embodiment, the light chain variable domain sequence is human or non-immunogenic in a human. In one embodiment, the heavy chain variable domain sequence is human or non-immunogenic in a human.
[0126] The protein can bind to cells that express Tiel, e.g., endothelial cells.
In one embodiment, the protein does not substantially bind (e.g., does not detectably bind) to platelets.
[0127] In one embodiment, the protein specifically binds to Tiel , e.g., it binds with at least a 10, 50, 100, 103, or 104 fold preference for Tiel relative to another human protein, e.g., Tie2, a natural protein other than Tiel that has a Ig-like domain, an EGF-like domain, or fibronectin Type III repeat, or human serum albumin. In one embodiment, the protein binds to a domain of Tiel described herein.
[0128] In one embodiment, the protein is delivered locally. In one embodiment, the protein is delivered systemically.
[0129] In one embodiment, the subject is in need of reduced angiogenesis, or identified as such. For example, the subject has an angiogenesis-related disorder. In another example, the subject has a neoplastic disorder, e.g., a metastatic cancer. For example, the subject has an angiogenesis-dependent cancer or tumor. The tumor can be a solid tumor, e.g., a tumor at least 1, 2, 3, 5, 8 or 10 mm in diameter. In one embodiment, the solid tumor has a hypoxic core. The method can further include administering an anti-metabolite (e.g., 5-FU, with leucovorin), irinotecan, (or other topoisomerase inhibitor), doxorubicin, bevacizumab, or all of these agents. The method can include, prior to administering the antagonist, evaluating the subject and detecting a solid tumor in the subject.
[0130] In another embodiment, the subject has an inflammatory disorder, e.g., rheumatoid arthritis, psoriasis, rheumatoid or rheumatic inflammatory disease, or other chronic inflammatory disorders, such as chronic asthma, arterial or post- transplantational atherosclerosis, and endometriosis.. Other disorders that can be treated include those that have deregulated or undesired angiogenesis, such as ocular neovascularization, e.g., retinopathies (including diabetic retinopathy and age-related macular degeneration) hemangioblastoma, hemangioma, and arteriosclerosis.
[0131] In one embodiment, the protein is administered in an amount effective to reduce one or more of the following activities: sprouting, splitting and remodeling of blood vessels. In one embodiment, the protein is administered in an amount effective to reduce vasculogenesis or tubule formation.
[0132] In one embodiment, the method further includes, prior to the administering, identifying the subject as a subject in need of reduced angiogenesis. In one embodiment, the method further includes administering the protein continuously or in separate boluses. In one embodiment, the method further includes monitoring the subject during the course of administration. For example, the monitoring includes imaging blood vessels (locally or throughout) the subject. In another example, the monitoring include evaluating tumor size or tumor load in the subject.
[0133] In another aspect the invention features a method that includes: administering a composition that includes a protein described herein (e.g., a protein that reduces a Tiel activity) to a subject in an amount effective to reduce a Tiel activity in the subject. The method can include other features described herein.
[0134] In another aspect the invention features a method that includes: administering a composition that includes a protein described herein (e.g., a protein that can modulate an activity of Tiel) to a subject in an amount effective to modulate endothelial cell activity in the subject. In one embodiment, the protein is delivered into the circulation. [0135] In one embodiment, the composition is effective for sensitizing endothelial cells to a treatment, and providing a treatment to the subject that inhibits, kills, ablates, or otherwise arrests the sensitized endothelial cells.
[0136] In another aspect the invention features a method that includes: (i) contacting the sample (and optionally, a reference, e.g., control, sample) with a protein that binds to Tiel, e.g., a protein described herein, under conditions that allow interaction of the Tiel -binding protein and the Tiel protein to occur; and (ii) detecting formation of a complex between the Tiel -binding protein, and the sample (and optionally, the reference, e.g., control, sample).
[0137] In another aspect the invention features a method that includes: (i) administering to a subject (and optionally a control subject) a Tiel -binding protein (e.g., an antibody or antigen binding fragment thereof), under conditions that allow interaction of the Tiel -binding protein and the Tiel protein to occur; and (ii) detecting formation of a complex between the Tiel -binding protein and a Tiel molecule of the subject or detecting distribution of Tiel -binding protein or at least one location of the Tiel-binding protein in the subject. In one embodiment, the Tiel-binding protein does not modulate the activity of Tiel. The Tiel-binding protein can be a protein described herein. In one embodiment, the ligand detects activated Tiel.
[0138] An antibody that binds to Tiel is preferably monospecific, e.g., a monoclonal antibody, or antigen-binding fragment thereof. For example, the antibody can recognize Tiel on a living cell, e.g., an endogenous Tiel molecule or a Tiel molecule that is expressed from a heterologous nucleic acid. In one embodiment, the Tiel-binding protein interacts with primary endothelial cells. The term "monospecific antibody" refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody" which refers to an antibody that is produced as a single molecular species, e.g., from a population of homogenous isolated cells. A "monoclonal antibody composition" refers to a preparation of antibodies or fragments thereof of in a composition that includes a single molecular species of antibody. In one embodiment, a monoclonal antibody is produced by a mammalian cell. One or more monoclonal antibody species may be combined. [0139] The Tiel-binding antibodies can be foil-length (e.g., an IgG (e.g., an
IgGl, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgAl, IgA2), IgD, and IgE) or can include only an antigen-binding fragment (e.g., a Fab, F(ab')2 or scFv fragment), e.g., it does not include an Fc domain or a CH2, CH3, or CH4 sequence. The antibody can include two heavy chain immunoglobulins and two light chain immunoglobulins, or can be a single chain antibody. The antibodies can, optionally, include a constant region chosen from a kappa, lambda, alpha, gamma, delta, epsilon or a mu constant region gene. A Tiel-binding antibody can include a heavy and light chain constant region substantially from a human antibody, e.g., a human IgGl constant region or a portion thereof.
[0140] In one embodiment, the antibody (or fragment thereof) is a recombinant or modified antibody, e.g., a chimeric, a humanized, a deimmunized, or an in vitro generated antibody. The term "recombinant" or "modified" human antibody, as used herein, is intended to include all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include humanized, CDR grafted, chimeric, deimmunized, in vitro generated antibodies, and may optionally include constant regions derived from human germline immunoglobulin sequences.
[0141] In one embodiment, the antibody binds to an epitope distinct from an epitope bound by known monoclonal antibodies that bind to Tiel, e.g., an antibody described in WO 95/26364, e.g., 3C4C7G6 and 10Fl 1G6. In other embodiments, the antibody does not compete with known monoclonal antibodies that bind to Tiel, e.g., 3C4C7G6 and 10F11G6. In still other embodiments, the antibody does not compete with ligand described herein, e.g., the E3 antibody.
[0142] Also within the scope of the invention are antibodies or other agents
(e.g., protein or non-protein agents) that bind overlapping epitopes of, or competitively inhibit the binding of the proteins disclosed herein, e.g., proteins that bind to Tiel, Tie2, or Ang. For example, the antibodies or other agents bind overlapping epitopes of or competitively inhibit the binding of monospecific antibodies, e.g., E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, ρ-F3, p-F4, p- G3, S-AlO, s-Hl, S-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dl l, s-Ell, S-GlO, s-H4, or another antibody described herein to Tiel, or vice versa (e.g., the monospecific antibodies competitively inhibiting binding of the ligands). Overlapping epitopes can include at least one amino acid in common. Agents that competitively inhibit binding of one another do not necessarily bind to overlapping epitopes. For example, they may inhibit binding by steric interference or by altering the conformation of Tiel.
[0143] Any combination of binding proteins is within the scope of the invention, e.g., two or more antibodies that bind to different regions of Tiel, Tie2, or Ang, e.g., antibodies that bind to two different epitopes on the extracellular domain of Tiel, Tie2, or Ang, e.g., abispecific antibody.
[0144] hi one embodiment, the Tiel-binding antibody or antigen-binding fragment thereof includes at least one light or heavy chain immunoglobulin (or preferably, at least one light chain immunoglobulin and at least one heavy chain immunoglobulin). Preferably, each immunoglobulin includes a light or a heavy chain variable region having at least one, two and, preferably, three complementarity determining regions (CDRs) substantially identical to a CDR from an anti-Tiel light or heavy chain variable region, respectively, i.e., from a variable region of an antibody described herein, e.g., E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, ρ-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s- GlO, s-H4, or another antibody described herein.
[0145] In one aspect, the invention features an agent (e.g., an antibody) that decreases endothelial cell activity by increasing Tiel phosphorylation, hi one embodiment, the agent decreases endothelial cell differentiation, e.g., sprouting, splitting, and tube formation.
[0146] In one aspect, the invention features an agent (e.g., an antibody) that decreases endothelial cell activity by activating a signaling pathway. In one embodiment, the antibody decreases endothelial cell differentiation, e.g., sprouting, splitting, and tube formation. This agent-induced effect can be independent or dependent of Tiel self-association. [0147] In one aspect, the invention features an isolated protein that includes a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to Tiel ectodomain and the heavy chain immunoglobulin variable domain sequence includes one or more of the following properties: i) a HC CDRl that includes an amino acid sequence of a clone from the group consisting of: M0044-A06; M0044-A11; M0044- B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06; and M0062-H01, or a sequence that is at least 70, 75, 80, 85, or 90% identical to such a sequence; ii) a HC CDR2 that includes an amino acid sequence of a clone from the group consisting of: M0044- A06; M0044-A11; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; MOO58-GO3; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06; and M0062-H01, or a sequence that is at least 70, 75, 80, 85, or 90% identical to such a sequence; iii) a HC CDR3 that includes an amino acid sequence of a clone from the group consisting of: M0044-A06; M0044-A11; M0044-B04; M0044-B05; M0044- B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06; and M0062-H01, or a sequence that is at least 70, 75, 80, 85, or 90% identical to such a sequence.
[0148] In one embodiment, the protein also includes the light chain immunoglobulin variable domain sequence which includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence of a clone from the group consisting of: M0044-A06; M0044-A11; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02; MOO53-AO3; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11 ; M0062-F10; M0062-G06; and M0062-H01 , or a sequence that is at least 70, 75, 80, 85, or 90% identical to such a sequence; ii) a LC CDR2 that includes an amino acid sequence of a clone from the group consisting of: M0044-A06; M0044- AIl; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06; and M0062-H01, or a sequence that is at least 70, 75, 80, 85, or 90% identical to such a sequence; iii) a LC CDR3 that includes an amino acid sequence of a clone from the group consisting of: M0044-A06; M0044-A11; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11 ; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06; and M0062-H01, or a sequence that is at least 70, 75, 80, 85, or 90% identical to such a sequence.
[0149] In one embodiment, the protein includes the amino acid sequence of the HC variable domain sequence which is at least 85, 90, 95, 98, or 99% identical to the amino acid sequence of the HC variable domain of clone M0044-A06; M0044- Al 1; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06; or M0062-H01.
[0150] In one embodiment, the protein includes the amino acid sequence of the LC variable domain sequence which is at least 85, 90, 95, 98, or 99% identical to the amino acid sequence of the LC variable domain of clone M0044-A06; M0044- AIl; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03; M0058-H01; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06; or M0062-H01.
[0151] An antibody or other binding protein (e.g., a Tiel-binding protein,
Tie2-binding protein, or Ang binding protein) described herein can be administered to a subject or used in vitro in non-derivatized or unconjugated forms. In other embodiments, the binding protein can be derivatized, modified or linked to another functional molecule, e.g., another protein (e.g., HSA, an Fc domain, etc.), a polymer (e.g., PEG) isotope, cell, or insoluble support. For example, the binding protein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as an antibody (e.g., if the protein is an antibody to form a bispecific or a multi-specific antibody), a toxin, a radioisotope, a therapeutic (e.g., a cytotoxic or cytostatic) agent or moiety, among others. For example, the binding protein can be coupled to a radioactive ion (e.g., an α-, γ-, or β-emitter), e.g., iodine (131I or 125I), yttrium (90Y), lutetium (177Lu), actinium (225Ac), rhenium (186Re), or bismuth (212Bi or 213Bi).
[0152] In another aspect, the invention features a nucleic acid that includes a coding sequence that encodes a polypeptide comprising an immunoglobulin heavy or light chain variable domain that binds to Tiel, e.g., an immunoglobulin heavy or light chain variable domain described herein. For example, the nucleic acid can include a particular nucleic acid sequence described herein, a nucleic acid that is at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to a nucleic acid sequence described herein (e.g., a particular nucleic acid sequence), or a nucleic acid that specifically hybridizes (e.g., under conditions described herein, e.g., high stringency conditions) to a nucleic acid sequence described herein (e.g., a particular nucleic acid sequence, e.g., a nucleic acid encoding one or more variable domains of M0044-A06; M0044-A11; M0044- B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-C12; M0045-D01; M0045-D07; M0045-G01; M0045-G10; M0046-A11; M0046-B06; M0046-B10; M0046-G12; M0046-H03; M0046-H10; M0046-H11; M0047-B03; M0047-D01; M0047-D03; M0047-E10; M0047-G09; M0053-A02; M0053-A03; M0053-A05; M0053-A09; M0053-B09; M0053-B11; M0053-D03; M0053-D06; M0053-D12; M0053-E03; M0053-E04; M0053-E08; M0053-F04; M0053-F05; M0053-F06; M0053-F08; M0053-G04; M0053-G05; M0054-A08; M0054-B06; M0054-B08; M0054-C03; M0054-C07; M0054-E04; M0054-G01; M0054-G05; M0054-H10; M0055-A09; M0055-B11; M0055-B12; M0055-C05; M0055-C07; M0055-D03; M0055-D06; M0055-D12; M0055-E04; M0055-E06; M0055-E10; M0055-E12; M0055-F10; M0055-G02; M0055-G03; M0055-H04; M0056-A01; M0056-A06; M0056-B08; M0056-B09; M0056-C03; M0056-C04; M0056-E08; M0056-F01; M0056-F02; M0056-F10; M0056-F11; M0056-G03; M0056-G04; M0056-G08; M0056-G12; M0056-H04; M0056-H12; M0057-B05; M0057-H07; M0058-A09; M0058-D04; M0058-E09; M0058-F03; M0058-G03; MOO58-HO1; M0059-A02; M0059-A06; M0060-B02; M0060-H01; M0061-A03; M0061-C05; M0061-C06; M0061-F07; M0061-G12; M0061-H09; M0062-A12; M0062-B05; M0062-B07; M0062-C08; M0062-D04; M0062-E02; M0062-E03; M0062-E11; M0062-F10; M0062-G06; or M0062-H01), or fragments thereof (e.g., CDR-coding fragments).
[0153] A nucleic acid described herein can further include a promoter operably linked to the coding sequence. A nucleic acid can include a first and second coding sequence, e.g., wherein the first coding sequence encodes a polypeptide that includes an immunoglobulin heavy chain variable domain and the second coding sequence encodes a polypeptide that includes an immunoglobulin light chain variable domain.
[0154] In another aspect, the invention features a host cell that contains a first nucleic acid encoding a polypeptide comprising a heavy chain variable region and a second nucleic acid encoding a polypeptide comprising a light chain variable region. The heavy chain variable region and the light chain variable region can associate to form a Tiel binding protein. These variable regions can have one or more properties described herein, e.g., at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to a sequence described herein. The invention also includes a method of providing a Tiel- binding antibody. The method can include providing a host cell described herein; and expressing said first and second nucleic acids in the host cell under conditions that allow assembly of said light and heavy chain variable regions to form an antigen binding protein that interacts with Tiel . [0155] In another aspect, the invention provides compositions, e.g., pharmaceutical compositions, which include a pharmaceutically acceptable carrier, excipient or stabilizer, and at least one of the Tiel -binding proteins (e.g., antibodies or fragments thereof) described herein. In one embodiment, the compositions, e.g., the pharmaceutical compositions, include a combination of two or more of the aforesaid Tiel -binding proteins.
[0156] In another aspect, the invention features a kit that includes a Tiel- binding antibody (or fragment thereof), e.g., a Tiel-binding antibody (or fragment thereof) as described herein, for use alone or in combination with other therapeutic modalities, e.g., a cytotoxic or labeling agent, e.g., a cytotoxic or labeling agent as described herein, along with instructions on how to use the Tiel antibody or the combination of such agents to treat, prevent or detect a Tiel-related disorder, e.g., an endothelial cell related disorder, e.g., rheumatoid arthritis or metastatic cancer.
[0157] In another aspect, the binding protein that binds to Tiel is a polypeptide that is not an immunoglobulin. For example, the polypeptide can be of variable length, e.g., 4 to 100 amino acid residues in length, preferably 5 to 75, 6 to 50, or 7 to 40 amino acid residues in length, or more preferably 8 to 30 or 10 to 25 amino acid residues in length, hi some embodiments, the polypeptide includes non- standard or synthetic amino acid residues, e.g., norleucine, selenocysteine, pyrrolysine, etc. In some embodiments, the polypeptide includes cross-linking groups, e.g., two cysteine residues that can form a disulfide bond or some other type of chemical cross-linking moieties that can be used to cyclize the peptide. In other preferred embodiments, the polypeptide can be modified, e.g., using polyethylene glycol or fusion to a soluble protein, e.g., to increase the solubility or circulatory half- life of the polypeptide.
[0158] The target-binding protein can be physically associated with (e.g., fused to) another protein, e.g., a protein that does not bind to the target, e.g., to the amino or carboxy terminus. For example, the target-binding protein can be associated with (e.g., fused to) a protein that increases serum residence or alters stability, e.g., an albumin, e.g., a serum albumin, e.g., HSA (human serum albumin). In another example, the target binding protein is physically associated with (e.g., fused to) a moiety that facilitates purification, e.g., a purification tag such as His, PEG, or to a functional moiety, e.g., Fc. [0159] In another aspect, the invention features a method of identifying a protein that specifically binds to Tiel . In preferred embodiments, the invention includes: providing a Tiel antigen; providing a display library (e.g., a phage display library member); identifying a member present in the library, wherein the member expresses a protein that specifically binds to the Tiel antigen. The term "Tiel antigen" refers to any antigenic fragment of Tiel that is at least 8 amino acids in length. For example, a Tiel antigen can include a fragment of the Tiel ectodomain, e.g., a fragment that includes a folded protein domain such as a fragment described herein. In some embodiments, the Tiel antigen is of human origin and includes, e.g., the extracellular domain of human Tiel or a fragment thereof (e.g., a fragment described herein. The Tiel antigen can be a recombinant polypeptide optionally fused to another polypeptide, e.g., a Fc domain, or it can be a cell that expresses Tiel on its surface (e.g., an endothelial cell). In other preferred embodiments, the Tiel antigen has an activated conformation, e.g., the Tiel antigen is a dimeric conformation or a conformation stabilized by the E3 or E3b antibody described herein.
[0160] The methods described here are, for example, applicable to libraries that are based on bacteriophage with a substantially complete genome (e.g., including a modified gene III) and to libraries that are based on bacteriophage particles that include a phagemid nucleic acid. The terms "bacteriophage library member" and "phage" encompass members of both types of libraries. The term "bacteriophage particle" refers to a particle formed of bacteriophage coat proteins that packages a nucleic acid. The packaged nucleic acid can be a modified bacteriophage genome or a phagemid, e.g., a nucleic acid that includes a bacteriophage origin of replication but lacks essential phage genes and cannot propagate in E. coli without help from "helper phage" or phage genes supplied in trans.
[0161] In other embodiments, the invention features a method of identifying a protein that specifically binds to Tiel . The method includes: providing a Tiel antigen (e.g., an region of the Tiel ectodomain); immunizing a non-human animal with the Tiel antigen; and isolating a cell that produces a immunoglobulin that interacts with Tiel . For example, the method can include producing hybridoma cells from the spleen of the animal (e.g., an immunized mouse); and identifying individual hybridoma cell lines expressing an antibody that specifically binds to the Tiel antigen. For example, the
[0162] In preferred embodiments, the Tiel antigen is of human origin and includes, e.g., the extracellular domain of human Tiel or some fragment thereof, e.g., the HA binding domain of Tiel . The Tiel antigen can be a recombinant polypeptide optionally fused to another polypeptide, e.g., a purification handle, or it can be a cell that expresses Tiel (e.g., an endothelial cell) on its surface. In other preferred embodiments, the Tiel antigen has an activated conformation, e.g., dimerized.
[0163] In preferred embodiments, the methods further include isolating a nucleic acid molecule from the identified phage or hybridoma, wherein the nucleic acid molecule encodes the polypeptide or antibody that specifically binds to the Tiel antigen. The isolated nucleic acid molecules can be used to produce therapeutic agents, as described herein.
[0164] In another aspect, the invention features nucleic acids that encode proteins identified by the methods described herein. In preferred embodiments, the nucleic acids include sequences encoding a heavy and light chain immunoglobulin or immunoglobulin fragment described herein. For example, the invention features, a first and second nucleic acid encoding a heavy and light chain variable region, respectively, of a Tiel -binding antibody molecule as described herein. Sequences encoding a heavy and light chain that function together can be present on separate nucleic acid molecules or on the same nucleic acid molecule. In another aspect, the invention features host cells and vectors containing a nucleic acid described herein.
[0165] In yet another aspect, the invention features a method of producing a
Tiel -binding antibody, or antigen-binding fragment thereof. The method includes: providing a host cell that contains a first nucleic acid encoding a polypeptide comprising a heavy chain variable region, e.g., a heavy chain variable region as described herein; providing a second nucleic acid encoding a polypeptide comprising a light chain variable region, e.g., a light chain variable region as described herein; and expressing said first and second nucleic acids in the host cell under conditions that allow assembly of said light and heavy chain variable regions to form an antigen binding protein that interacts with Tiel . The first and second nucleic acids can be linked or unlinked, e.g., expressed on the same or different vector, respectively. The πrst ana second nucleic acids can be components of the same molecule or can reside on different molecules (e.g., different chromosomes or plasmids).
[0166] The host cell can be a eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli. For example, the mammalian cell can be a cultured cell or a cell line. Exemplary mammalian cells include lymphocytic cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, HEK294, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell. For example, nucleic acids encoding the antibodies described herein can be expressed in a transgenic animal. In one embodiment, the nucleic acids are placed under the control of a tissue-specific promoter (e.g., a mammary specific promoter) and the antibody is produced in the transgenic animal. For example, the antibody molecule is secreted into the milk of the transgenic animal, such as a transgenic cow, pig, horse, sheep, goat or rodent. To produce a single chain antibody, the nucleic acid is configured to encode a single polypeptide that comprises both the heavy and light chain variable domains.
[0167] Tiel has been found to be overexpressed in association with a wide range of cancers. Targeting Tiel on the tumor vasculature with Tiel-binding proteins (e.g., antibodies) can be used to inhibit, destroy, or otherwise antagonize the vasculature so that tumor growth and metastasis is reduced. The proteins can be, for example, associated with a toxic payload or can mediate direct functional inhibition. Proteins (e.g., proteins that have an Fc domain) that can cause ADCC can also be used.
[0168] hi another aspect, the invention features a method of inhibiting an activity of a cell, e.g., an endothelial cell, e.g., proliferation, adhesion, growth or survival of a cell, e.g., an endothelial cell, e.g., an endothelial cell in the vicinity of a cancer, e.g., a tumor. Exemplary methods include contacting the cell with a Tiel binding protein, in an amount sufficient to inhibit the adhesion, migration, growth or proliferation of the cell. Methods of administering a Tiel binding protein can be used, for example, to treat or prevent a disorder, e.g., an inflammatory disorder (e.g., rheumatoid arthritis, lupus, restenosis, psoriasis, graft v. host response, or multiple sclerosis), or a cancerous disorder (e.g., a malignant or metastatic disorder), by administering to a subject (e.g., an experimental animal or a human patient) a Tiel- binding protein in an amount effective to treat or prevent such disorder. [0169] A Tiel-binding protein can be used to treat or prevent angiogenesis- related disorders, particularly angiogenesis-dependent cancers and tumors. Angiogenesis-related disorders include, but are not limited to, solid tumors; tumor metastasis; benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis); psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neo vascular glaucoma, retrolental fibroplasia, rubeosis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation.
[0170] "Angiogenesis-dependent cancers and tumors" are cancers tumors that require, for their growth (expansion in volume and/or mass), an increase in the number and density of the blood vessels supplying then with blood, hi one embodiment a Tiel-binding protein causes regression of such cancers and tumors. "Regression" refers to the reduction of tumor mass and size, e.g., a reduction of at least 2, 5, 10, or 25%.
[0171] In addition, Tiel and Tie2 are also expressed in hematopoietic cells.
(Kufck et al (1997) Br. J. Haematol. 98: 195; Iwama et al (1993) Biochem. Biophys. Res. Commun. 195: 301). Accordingly, in another embodiment, a Tiel-binding protein is used to treat hematopoietic conditions, e.g., hematopoietic cancers. Examples of hematopoietic cancers include: cancers derived from hyperplastic/neoplastic cells of hematopoietic origin, e.g., cells arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Exemplary cancers include acute promyeloid leukemia (APML), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM), non-Hodgkin's lymphoma, peripheral T-cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T- cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), B cell chronic lymphocytic leukemia, myelodysplastic syndrome, and Hodgkin's disease.
[0172] In another aspect, the invention features a method of contacting a cell
(in vitro, ex vivo, or in vivo), e.g., an endothelial cell, e.g., an endothelial cell in the vicinity of a cancer, e.g., a tumor. The method can include providing an agent (e.g., a protein) that interacts with Tiel, e.g., a protein described herein, and contacting the cell with the protein, in an amount sufficient to form at least one detectable ligand-cell complex. The protein can include, for example, a label or cytotoxic entity, e.g., an immunoglobulin Fc domain or a cytotoxic drug.
[0173] In another aspect, the invention features administering the agent described herein as an adjuvant therapy, e.g., to a subject. The adjuvant therapy can be a post-operative therapy that is administered to the subject after the subject has undergone surgery to remove all or part of a tumor (e.g., after surgery to treat glioblastoma or colorectal, breast, or lung cancer). For example, the agent is a protein that inhibits Tie complex formation, promotes Tiel homodimerization, or increases Tiel phosphorylation. For example, the agent is a protein that binds Tiel (e.g., an anti-Tiel antibody). In one embodiment, the agent is administered within 6, 12, 24, 48, or 100 hours of surgery. The agent can be administered before as well as after surgery.
[0174] An exemplary agent is a Tiel binding agent that includes (a) a heavy chain variable domain sequence that is at least 85, 90, 95, 98, 99%, or 100% identical to the heavy chain variable domain of the E3 antibody and a light chain variable domain sequence that is at least 85, 90, 95, 98, 99%, or 100% identical to the light chain variable domain of the E3 antibody; (b) a heavy chain variable domain sequence and a light chain variable domain sequence that form an antigen binding site that competes with E3 for binding to Tiel; or (c) one, two, or three, of the CDRs of the heavy chain variable domain of the E3 antibody, and one, two, or three of the CDRs of the light chain variable domain of the E3 antibody. Other Tiel binding agents described herein can also be used, e.g., a Tiel binding agent that includes a heavy chain variable domain sequence that is at least 85, 90, 95, 98, 99%, or 100% identical to the heavy chain variable domain of M0059A02, M0045A02*, M0054G05, M0053F05, M0053G05, M0061C06, M0045B01, M0046G12, M0046H11, M0053A02, M0053A05, M0046B06, M0044B10, M0044B08, M0056G08, M0045B03, M0053F04, M0055E10, M0060H01, M0054H10, or M0058F03, and a light chain variable domain sequence that is at least 85, 90, 95, 98, 99%, or 100% identical to the light chain variable domain of M0059A02, M0045A02*, M0054G05, M0053F05, M0053G05, M0061C06, M0045B01, M0046G12, M0046H11, M0053A02, M0053A05, M0046B06, M0044B10, M0044B08, M0056G08, M0045B03, M0053F04, M0055E10, M0060H01, M0054H10, orM0058F03.
[0175] In another aspect, the invention features a method of treating, e.g., inhibiting, ablating or killing, a cell or impairing at least one activity of the cell. The method includes providing a Tiel -binding protein, e.g. a ligand described herein, and contacting the cell with the protein, in an amount sufficient to impair at least one activity of the cell, inhibit, ablate or kill the cell. The contacting can be in vitro or in vivo. For example, the cell can be, e.g., an endothelial cell, e.g., an endothelial cell in the vicinity of a cancer, e.g., a tumor. The protein can include a cytotoxic entity. Methods of administering a Tiel binding protein or other agent described herein can be used, for example, to treat or prevent a disorder, e.g., a endothelial cell-based disorder, a blood vessel disorder, wound healing, or a cancerous disorder (e.g., a malignant or metastatic disorder), by administering to a subject (e.g., an experimental animal or a human patient) a Tiel -binding protein in an amount effective to treat or prevent such disorder.
[0176] A Tiel binding protein or other agent described herein can be used on cells in culture, e.g. in vitro or ex vivo. For example, an endothelial cell, e.g., an endothelial cell in cancer biopsy, can be cultured in vitro in culture medium and the contacting step can be effected by adding the Tiel -binding protein to the culture medium. The method can be performed on cells (e.g., cancerous or metastatic cells) present in a subject, as part of an in vivo (e.g., therapeutic or prophylactic) protocol. For in vivo embodiments, the contacting step is effected in a subject and includes administering the Tiel -binding protein to the subject under conditions effective to permit both binding of the protein to the cell, and the inhibition of adhesion, migration, growth or proliferation of the cell.
[0177] A Tiel binding protein or other agent described herein can be used to treat or prevent cancerous disorders, e.g., including hematopoietic cancers, solid tumors, soft tissue tumors, and metastatic lesions, particularly tumors that require a blood supply or angiogenesis. Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract (e.g., renal, urothelial cells), pharynx, as well as adenocarcinomas which include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having, or at risk of, a disorder described herein, e.g., an endothelial cell-based disorder, e.g., cancer).
[0178] The Tiel-binding antibody or fragment thereof, e.g., a Tiel-binding antibody or fragment thereof as described herein, can be administered to the subject systemically (e.g., orally, parenterally, subcutaiieously, intravenously, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation), topically, or by application to mucous membranes, such as the nose, throat and bronchial tubes.
[0179] The methods can further include the step of monitoring the subject, e.g., for a reduction in one or more of: a reduction in tumor size; reduction in cancer markers, e.g., levels of cancer specific antigen; reduction in the appearance of new lesions, e.g., in a bone scan; a reduction in the appearance of new disease-related symptoms; or decreased or stabilization of size of soft tissue mass; or any parameter related to improvement in clinical outcome. The subject can be monitored in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Monitoring can be used to evaluate the need for further treatment with the same Tiel-binding protein or for additional treatment with additional agents. Generally, a decrease in one or more of the parameters described above is indicative of the improved condition of the subject. Information about the monitoring can be recorded, e.g., in electronic or digital form.
[0180] The Tiel-binding protein can be used alone in unconjugated form to thereby inhibit adhesion, migration, or extravasation or the Tiel -expressing cells, or ablate or kill the Tiel -expressing cells. If the Tiel-binding protein is an antibody, the ablation or killing can be mediated, e.g., by an antibody-dependent cell killing mechanisms such as complement-mediated cell lysis and/or effector cell-mediated cell killing. In other embodiments, the Tiel-binding protein can be bound (e.g., physically associated, either directly or indirectly, covalently or non-covalently) to a substance, e.g., a cytotoxic agent or moiety, effective to kill or ablate the Tiel- expressing cells. For example, the Tiel-binding protein can be coupled to a radioactive ion (e.g., an α-, γ-, or β-emitter), e.g., iodine (131I or 125I), yttrium (90Y), lutetium (177Lu), actinium (225Ac), or bismuth (212Bi or 213Bi).
[0181] The methods and compositions described herein can be used in combination with other therapeutic modalities. In one embodiment, the methods include administering to the subject a Tiel-binding protein, e.g., a Tiel-binding antibody or fragment thereof, in combination with a cytotoxic agent, in an amount effective to treat or prevent the disorder. The Tiel-binding protein and the cytotoxic agent can be administered simultaneously or sequentially. In other embodiments, a Tiel binding protein or other agent described herein is used in combination with surgical and/or radiation procedures.
[0182] hi another aspect, the invention features methods for detecting the presence of a Tiel protein or a cell expressing Tiel (e.g., an endothelial cell) in a sample, in vitro (e.g., a biological sample, a tissue biopsy, e.g., a cancerous lesion). The subject method can be used to evaluate, e.g., diagnose or stage a disorder described herein, e.g., a cancerous disorder. The method includes: (i) contacting the sample (and optionally, a reference, e.g., control sample) with a Tiel-binding protein, as described herein, under conditions that allow interaction of the Tiel-binding protein and the Tiel protein to occur; and (ii) detecting formation of a complex between the Tiel-binding protein, and the sample (and optionally, the reference, e.g., control, sample). Formation of the complex is indicative of the presence of Tiel protein (e.g., activated Tiel protein), and can indicate the suitability or need for a treatment described herein. For example, a statistically significant change in the formation of the complex in the sample relative to the reference sample, e.g., the control sample, is indicative of the presence of Tiel (e.g., activated Tiel) in the sample.
[0183] In yet another aspect, the invention provides a method for detecting the presence of Tiel (e.g., activated Tiel) in vivo (e.g., in vivo imaging in a subject). The subject method can be used to evaluate, e.g., diagnose, localize, or stage a disorder described herein, e.g., a cancerous disorder. The method includes: (i) administering to a subject (and optionally a control subject) a Tiel-binding protein (e.g., an antibody or antigen binding fragment thereof), under conditions that allow interaction of the Tiel-binding protein and the Tiel protein to occur; and (ii) detecting formation of a complex between the Tiel-binding protein and Tiel, wherein a statistically significant change in the formation of the complex in the subject relative to the reference, e.g., the control subject or subject's baseline, is indicative of the presence of the Tiel. The presence of Tiel in particular locations within a subject can be indicative of an endothelial-cell related disorder, e.g., an angiogenesis-related disorder, e.g., a cancer, e.g., metastatic cancer, or other angiogenesis-related disorder described herein.
[0184] Tumor cells can express Tiel . In one aspect, the invention features a method of providing a sample from a subject and evaluating the Tiel expression in cells in the sample. In one embodiment, the result of evaluating Tiel expression levels is compared to a reference, e.g., a reference value or reference quality. For example, the Tiel expression on the evaluated sample may have the same, less than, or greater than the reference value. A reference value or quality can be determined using a control sample, a statistical value (e.g., an average, median, etc.) or an arbitrary value. For example, the control sample can be a normal sample, e.g., a sample devoid of tumor cells from the same or different subject. A change (e.g., an increase) relative to the reference can indicate that the sample includes tumor cells, e.g., the subject may be indicated as having a tumor.
[0185] In other embodiments, a method of diagnosing or staging a disorder as described herein (e.g., an inflammatory or cancerous disorder), is provided. The method includes: (i) identifying a subject having, or at risk of having, the disorder; (ii) obtaining a sample of a tissue or cell affected with the disorder; (iii) contacting said sample or a control sample with a Tiel -binding protein, under conditions that allow interaction of the binding agent and the Tiel protein to occur, and (iv) detecting formation of a complex. A statistically significant increase in the formation of the complex between the Tiel -binding protein with respect to a reference sample, e.g., a control sample, is indicative of the disorder or the stage of the disorder. For example, the finding of activated Tiel on tumor cells located in a solid tumor can indicate that the tumor is progressing into a metastatic tumor.
[0186] Preferably, the Tiel-binding protein used in the in vivo and in vitro diagnostic methods is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound binding agent. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. In one embodiment, the Tiel- binding protein is coupled to a radioactive ion, e.g., indium (111Li), iodine (131I or 125I), yttrium (90Y), actinium (225Ac), bismuth (212Bi or 213Bi), sulfur (35S), carbon (14C), tritium (3H), rhodium (188Rh), or phosphorous (32P). In another embodiment, the Tiel -binding protein is labeled with an NMR contrast agent.
[0187] hi one aspect, the invention features a method of imaging tumor vasculature, the method includes: providing a protein that binds to Tiel, Tie2, or Ang, e.g., a protein described herein, wherein the protein is physically associated to an imaging agent; administering the protein to a patient, e.g., with a tumor; and imaging the patient, e.g., to detect tumor vasculature.
[0188] In one aspect, the invention features a method of treating a subject with a blood born neoplastic disorder, the method includes administering a protein that binds to Tiel, Tie2, or Ang, e.g., a protein described herein, to a subject with a blood born neoplastic disorder (e.g., a proliferative disorder of hematopoietic cells, e.g., leukemia), thereby treating the disorder.
[0189] In one aspect, the invention features a method of diagnosing and treating a subject, the method includes evaluating a parameter associated with Tiel, Tie2, or Ang in a subject; and, if the parameter is altered relative to a reference, administering a protein described herein to the subject, thereby treating the subject. In one embodiment, the parameter includes a value indicative of protein or mRNA levels, e.g., in a tissue of a subject, hi one embodiment, the reference includes a value determined for a reference subject, e.g., an age/gender matched subject, e.g., a control or normal subject.
[0190] hi one aspect, the invention features a method of treating a subject, the method includes: administering a protein described herein to a subject that has elevated Tiel, Tie2, or Ang biomolecules or activity relative to a reference. The method can include evaluating the subject, e.g., to determine if the subject has elevated Tiel, Tie2, or Ang biomolecules or activity relative to a reference, hi one embodiment, the subject has elevated Tiel protein or mRNA levels.
[0191] The invention also provides polypeptides and nucleic acids that encompass a range of amino acid and nucleic acid sequences, e.g., sequences described herein or sequences related to those described herein. For example, the invention features nucleic acids that encodes each of the polypeptides described herein. The nucleic acid can include the cognate codons or any set of codons that can be translated to produce the respective polypeptide. Such polypeptides include individual subunits of a multi-chain protein, e.g., an antibody that includes a plurality of different polypeptide chains. The nucleic acid may also be a nucleic acid fragment or vector that is not expressed, but includes a sequence encoding at least a part of an immunoglobulin variable region (e.g., including a CDR described herein) or a complement thereof. Such nucleic acids can be used to prepare useful constructs, cells, and proteins. In addition, the invention features a host cell that includes a nucleic acid described herein. The cell can express a protein described herein, e.g., on its surface. The invention also includes are proteins that include an amino acid sequence encoded by a nucleic acid described herein or that hybridize to a nucleic acid described herein.
[0192] As used herein, the term "antibody" refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab')2, a Fd fragment, a Fv fragments, and dAb fragments) as well as complete antibodies.
[0193] The VH and VL 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). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. MoI. Biol. 196:901-917). Kabat definitions are used herein. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
[0194] An "immunoglobulin domain" refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two β-sheets formed of about seven β-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay 1988 Ann. Rev Immunol. 6:381- 405). The canonical structures of hypervariable loops of an immunoglobulin variable can be inferred from its sequence, as described in Chothia et al. (1992) J. MoI. Biol. 227:799-817; Tomlinson et al. (1992) J. MoI. Biol. 227:776-798); and Tomlinson et al. (1995) EMBO J. 14(18):4628-38.
[0195] As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain. For example, the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations. In one embodiment, a polypeptide that includes immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form a target binding structure (or "antigen binding site"), e.g., a structure that interacts with Tiel, e.g., binds to or inhibits Tiel.
[0196] The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region includes three domains, CHl, CH2 and CH3. The light chain constant region includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system. The term "antibody" includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). The light chains of the immunoglobulin may be of types kappa or lambda, hi one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.
[0197] One or more regions of an antibody can be human or effectively human. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs can be human, e.g., HC CDRl, HC CDR2, HC CDR3, LC CDKl, LC CDR2, and LC CDR3. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FRl, FR2, FR3, and FR4 of the HC or LC. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non- hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. One or more of the constant regions can be human or effectively human. In another embodiment, at least 70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FRl, FR2, and FR3, collectively, or FRl, FR2, FR3, and FR4, collectively) or the entire antibody can be human or effectively human. For example, FRl, FR2, and FR3 collectively can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical to a human sequence encoded by a human germline V segment of a locus encoding a light or heavy chain sequence.
[0198] All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin light chains (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2 -terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus. Full-length immunoglobulin heavy chains (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). A light chain refers to any polypeptide that includes a light chain variable domain. A heavy chain refers to any polypeptide that a heavy chain variable domain.
[0199] The term "antigen-binding fragment" of a full-length antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) aF(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, 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 VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., Bird et al. (1988) Science 2<M-A2?>-426; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883.
[0200] Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those with skill in the art. The term "monospecific antibody" refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody" or "monoclonal antibody composition," which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition. As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.
[0201 ] In one embodiment, the HC or LC of an antibody includes sequences that correspond to an amino acid sequence encoded by a human germline sequence, e.g., the framework regions and/or in the CDRs. For example, the antibody can include sequences from the human DP47 antibody. In one embodiment, one or more codons for the antibody are altered relative to the germline nucleic acid sequence, but are chosen to encode the same amino acid sequence. Codons can be selected, e.g., to optimize expression in a particular system, create restriction enzyme sites, create a silent fingerprint, etc.
[0202] In one embodiment, CDR2 of the antibody HC includes at least 11, 12,
13, 14, or 15 amino acid positions that are identical to the amino acids found in CDR2 ofDP47.
[0203] A "humanized" immunoglobulin variable region is an immunoglobulin variable region that includes sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. Descriptions of "humanized" immunoglobulins include, for example, US 6,407,213 and US 5,693,762.
[0204] An "effectively human" immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An "effectively human" antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
[0205] As used herein, "Tie complex" refers to a heteromeric complex that includes Tiel, Tie2, and an angiopoietin (Ang). The Tie complex is formed in part by association of the extracellular domains of Tiel and Tie2 and also includes Ang. As used herein, "complex members" refers to the proteins that are included in a heteromeric Tie complex. Accordingly, Tiel, Tie2, and Ang are all complex members. The term "Ang" includes all angiopoietins, such as Angl, Ang2, Ang3, and Ang4. The heteromeric Tie complex can include other proteins in addition to Tiel, Tie2, and Ang. A protein or ligand that antagonizes complex formation inhibits or decreases the association of Tiel, Tie2, or Ang with at least one other member of the complex and thereby decreases Tie2 signaling and downstream effects such as angiogenesis. Angiogenesis includes all stages of vessel development (e.g., blood or lymphatic vessel development), including initial vessel formation and later vessel remodeling and morphological changes.
[0206] As used herein, the terms "agonist" and "antagonist" describe properties in context of a particular activity or effect. For example, the E3 or E3b antibody can be an agonist in the context of promoting Tiel self-association (e.g., homodimerization), yet an antagonist in the context of decreasing or inhibiting Tie complex formation and tube formation by HUVECs. Likewise, an agent that is an agonist in the context of a Tiel signaling pathway can be an antagonist in the context of endothelial cell sprouting, splitting, and tube formation.
[0207] The term "Tiel ectodomain" refers to an extracellular region of a Tiel protein, e.g., a region that includes about amino acids 25-759 of SEQ DD NO:2. Other exemplary regions are regions that include one or more EGF-like domains (e.g., 214- 256, 258-303, 303-345, 214-303, 258-345, or 214-345 of SEQ ID NO:2); one or more Ig-Like C2-type domains (e.g., 43-105, 43-426, 372-426); one or more Fibronectin Type HI repeats (e.g., 446-540, 543-639, 643-744, 446-639, 543-744, or 446-744 of SEQ ID NO:2); and combinations thereof. The terms "first Ig-like C2-type domain" and "Ig 1" refer to the immunoglobulin-like domain in Tiel or Tie2 that is located closest to the amino terminus of the protein relative to the other Ig-like C2-type domain (the second such domain). For example, for Tiel, the first Immunoglobulin- like C2-type domain is located at about residue 43 to about residue 105 and the second Ig-like C2-type domain is located at about residue 372 to about residue 426.
[0208] As used herein, "binding affinity" refers to the apparent association constant or Ka. The Ka is the reciprocal of the dissociation constant (Kd). A ligand may, for example, have a binding affinity of at least 105, 106, 107 or 10s M"1 for a particular target molecule. Higher affinity binding of a ligand to a first target relative to a second target can be indicated by a higher Ka (or a smaller numerical value Kd) for binding the first target than the K3 (or numerical value Kd) for binding the second target. In such cases the ligand has specificity for the first target relative to the second target. Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 5, 10, 50, 100, or 1000-fold. For example, a Tiel-binding protein may preferentially bind to Tiel at least 1.5, 2, 5, 10, 50, 100, or 1000-fold better than to another antigen, e.g., Tie2, EGF, fibronectin, or human serum albumin. A Tiel- binding protein may also be species-specific or species-general (e.g., can bind to a Tiel protein from more than one species).
[0209] Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). These techniques can be used to measure the concentration of bound and free ligand as a function of ligand (or target) concentration. The concentration of bound ligand ([Bound]) is related to the concentration of free ligand ([Free]) and the concentration of binding sites for the ligand on the target where (N) is the number of binding sites per target molecule by the following equation:
[Bound] =N • [Free}/((1/Ka) + [Free]) [0210] Althouglα quantitative measurements of Ka are routine, it is not always necessary to make an exact determination of K3, though, since sometimes it is sufficient to obtain a qualitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to Ka, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2, 5, 10, 20, or 50 fold higher than a reference. Binding affinity is typically evaluated in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 niM EDTA and 0.005 % (v/v) surfactant P20.
[0211 ] An "isolated composition" refers to a composition that is removed from at least 90% of at least one component of a natural sample from which the isolated composition can be obtained. Compositions produced artificially or naturally can be "compositions of at least" a certain degree of purity if the species or population of species of interests is at least 5, 10, 25, 50, 75, 80, 90, 95, 98, or 99% pure on a weight-weight basis.
[0212] An "epitope" refers to the site on a target compound that is bound by a ligand, e.g., an antigen-binding protein (e.g., a Fab or antibody). In the case where the target compound is a protein, for example, an epitope may refer to the amino acids that are bound by the ligand. Overlapping epitopes include at least one common amino acid residue.
[0213] As used herein, the term "substantially identical" (or "substantially homologous") is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities, hi the case of antibodies, the second antibody has the same specificity and has at least 50% of the affinity of the same.
[0214] Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of this application. In some embodiment, the sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
[0215] Calculations of "homology" or "sequence identity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
[0216] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J MoI. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgaρdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation described herein) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0217] As used herein, the term "homologous" is synonymous with
"similarity" and means that a sequence of interest differs from a reference sequence by the presence of one or more amino acid substitutions (although modest amino acid insertions or deletions) may also be present. Presently preferred means of calculating degrees of homology or similarity to a reference sequence are through the use of BLAST algorithms (available from the National Center of Biotechnology Information (NCBI), National Institutes of Health, Bethesda MD), in each case, using the algorithm default or recommended parameters for determining significance of calculated sequence relatedness. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
[0218] As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 500C (the temperature of the washes can be increased to 550C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 450C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 600C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 650C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.
[0219] It is understood that the proteins described herein may have mutations relative to a particular protein described herein (e.g., a conservative or non-essential ammo acid substitutions), which do not have a substantial effect on function. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect desired biological properties, such as binding activity can be determined as described in Bowie, et al. (1990) Science 247:1306-1310. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is possible, for example, for framework and CDR amino acid residues to include one or more conservative substitutions.
[0220] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the binding agent, e.g., the antibody, without abolishing or more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change.
[0221] Generally, where "X" is used to represent an amino acid residue, any amino acid (e.g., any of the twenty naturally occurring amino acids) can be used at that position, or at least a subset thereof (e.g., any of the nineteen non-cysteine amino acids).
[0222] The terms "polypeptide" or "peptide" (which may be used interchangeably) refer to a polymer of three or more amino acids linked by a peptide bond, e.g., between 3 and 30, 12 and 60, or 30 and 300, or over 300 amino acids in length. The polypeptide may include one or more unnatural amino acids. Typically, the polypeptide includes only natural amino acids. A "protein" can include one or more polypeptide chains. Accordingly, the term "protein" encompasses polypeptides. A protein or polypeptide can also include one or more modifications, e.g., a glycosylation, amidation, phosphorylation, and so forth. The term "small peptide" can be used to describe a polypeptide that is between 3 and 30 amino acids in length, e.g., between 8 and 24 amino acids in length. [0223] Statistical significance can be determined by any art known method.
Exemplary statistical tests include: the Students T-test, Mann Whitney U non- parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05, or 0.02. Particular ligands may show a difference, e.g., in specificity or binding, that are statistically significant (e.g., P value < 0.05 or 0.02).
[0224] Other features and advantages of the instant invention will become more apparent from the following detailed description and claims. Embodiments of the invention can include any combination of features described herein. In no case does the term "embodiment" necessarily exclude one or more other features disclosed herein, e.g., in another embodiment. The contents of all references, patent applications and patents, cited throughout this application are hereby expressly incorporated by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0225] FIG. 1 illustrates a bivariant FACS plot showing labelling with the platelet specific marker CD42 with Tiel and labelling with the E3 antibody. Only a background number of CD42 positive cells are labeled by the E3 antibody.
[0226] FIGs. 2 A, 2B, 2C, and 2D are plots of the number of branching points versus antibody concentration comparing germlined E3 (2C and 2D) with parental E3 (2A and 2B).
[0227] FIG. 3 depicts a graph of blood vessel density in matrigels that were stained with fluorescein-lectin from an in vivo assay using MATRIGEL™ and evaluating the germlined E3 antibody.
[0228] FIG. 4 depicts results of tube formation in HUVECs using the parental
E3 and E3b (germlined) proteins.
[0229] FIG. 5 depicts graphically the results from animal studies in which nu/nu mice were implanted with SW-480 colorectal cancer cells and treated with DX- 2220 (10 mg/kg), cisplatin (4 mg/kg), or a control. Control conditions were: no treatment, PBS vehicle alone, or a non-specific, isotype-matched IgGl antibody (A2- SV) (10 mg/kg). Tumor weight is plotted on the y axis; days after tumor cell injection is plotted on the x axis. [0230] FIG. 6 depicts graphically the results from animal studies in which nu/nu mice were implanted with LNM35 lung cancer cells and treated with DX-2220 (20 mg/kg) or a non-specific, isotype-matched IgGl antibody (A2-SV) (20 mg/kg). Tumor volume (mm3) is plotted on the y axis; days after tumor cell injection is plotted on the x axis.
[0231] FIG 7A and 7B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-Al, respectively.
[0232] FIG 8A and 8B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-A5, respectively.
[0233] FIG 9 A and 9B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-A6, respectively.
[0234] FIG 1OA and 1OB list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-A10, respectively.
[0235] FIG 11 A and HB list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-Bl, respectively.
[0236] FIG 12A and 12B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-B3, respectively.
[0237] FIG 13A and 13B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-C6, respectively.
[0238] FIG 14A and 14B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-D6, respectively.
[0239] FIG 15A and 15B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-D10, respectively.
[0240] FIG 16A and 16B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-D12, respectively.
[0241] FIG 17A and 17B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-F3, respectively.
[0242] FIG 18A and 18B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-F4, respectively. IUZ4JJ *1G 19A and 19B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-G3, respectively.
[0244] FIG 2OA and 2OB list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-A2, respectively.
[0245] FIG 21 A and 21B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone S-AlO, respectively.
[0246] FIG 22 A and 22B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-B2, respectively.
[0247] FIG 23 A and 23B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-B9, respectively.
[0248] FIG 24A and 24B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-C2, respectively.
[0249] FIG 25A and 25B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-C7, respectively.
[0250] FIG 26A and 26B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-CIO, respectively.
[0251] FIG 27A and 27B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-Dll, respectively.
[0252] FIG 28 A and 28B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-Ell, respectively.
[0253] FIG 29A and 29B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-G4, respectively.
[0254] FIG 30 lists the amino acid sequence of the light chain variable domain of clone s-G9.
[0255] FIG 31 A and 31B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-G10, respectively.
[0256] FIG 32 A and 32B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-Hl, respectively. [0257] FIG 33 A and 33B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-H4, respectively.
[0258] FIG 34 A and 34B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone G2, respectively.
[0259] FIG 35 and 36 list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-Al, respectively.
[0260] FIG 37 provides Table 5, a summary of heavy chain sequences.
[0261] FIG 38 provides Table 6, a summary of light chain sequences.
[0262] FIG 39 provides Table 9, characteristics of some exemplary Tiel binding antibodies.
DETAILED DESCRIPTION
[0263] This disclosure provides, inter alia, agents (also referred to as binding proteins and ligands) that bind to components of a Tie complex, e.g.,Tiel, Tie2, and Ang. Examples of such agents include proteins, for example, a small peptide (e.g., a cyclic or linear peptide, e.g., of between 7 and 25 amino acids), a polypeptide (e.g., a polypeptide of at least 20 amino acids), or a multi-chain protein (e.g., including at least two peptides or polypeptides). An example of a multi-chain protein is an IgG full-length antibody that has separate heavy and light chains. An example of a polypeptide is a single chain antibody.
[0264] Agents can be selected that have particular properties, e.g., ability to antagonize Tiel/Tie2/Ang complex formation, ability to promote Tiel homodimerization, and ability to promote Tiel phosphorylation. For example, agents that bind to Tiel, Tie2, or Ang can be tested for their ability to antagonize formation of heteromeric Tie complexes. Antagonism of this complex decreases Tie2 signaling and its downstream effects, such as promoting angiogenesis.
[0265] Tiel is a receptor tyrosine kinase protein that includes a transmembrane domain. Tiel is present almost exclusively on endothelial cells. Accordingly, a Tiel-binding protein can be used, e.g., to specifically recognize or target an endothelial cell. Some Tiel-binding proteins can also be used to agonize or antagonize enαotneliai cells, in some embodiments, these Tiel-binding proteins have an affinity for particular structural features (e.g., a feature listed below), a combination of features listed below, and/or an epitope that includes at least one amino acid in a structural feature listed below (The sequence is relative to the amino acid sequence provided in SEQ DD NO:2, Example 1, below):
Key From T Too LengthDescription
SIGNAL 1 24 24 POTENTIAL .
CHAIN 25 1138 1114 TYROSINE-PROTEIN KINASE RECEPTOR TIEl.
DOMAIN 25 759 735 EXTRACELLULAR (POT-JJNrTXAL) .
TRANSMEM 760 784 25 POTENTIAL.
DOMAIN 785 1138 354 CYTOPLASMIC (POTENTIAL) .
DOMAIN 43 105 63 IG-LIKE C2-TYPE 1.
DOMAIN 214 256 43 EGF-LIKE 1.
DOMAIN 258 303 46 EGF-LIKE 2.
DOMAIN 305 345 41 EGF-LIKE 3.
DOMAIN 372 426 55 IG-LIKE C2-TYPE 2.
DOMAIN 446 540 95 FIBRONECTIN TYPE-III 1.
DOMAIN 543 639 91 FIBRONECTIN TYPE-III 2.
DOMAIN 643 744 102 FIBRONECTIN TYPE-III 3.
DOMAIN 839 1118 280 PROTEIN KINASE.
NP_BIND 845 853 9 ATP (BY SIMILARITY) .
BINDING 870 870 ATP (BY SIMILARITY) .
ACTJSITE 979 979 BY SIMILARITY.
CARBOHYD 83 83 N-LINKED (GLCNAC...) (POTENTIAL) .
CARBOHYD 161 161 N-LINKED (GLCNAC...) (POTENTIAL) .
CARBOHYD 503 503 N-LINKED (GLCNAC... ) (POTENTIAL) .
CARBOHYD 596 596 N-LINKED (GLCNAC...) (POTENTIAL) .
CARBOHYD 709 709 N-LINKED (GLCNAC... ) (POTENTIAL) .
MOD RES 1007 1007 PHOSPHORYLATION (AUTO-) (BY
SIMILARITY) .
[0266] Tie2 is a receptor tyrosine kinase protein that includes a transmembrane domain. Tie2 is present almost exclusively on endothelial cells. Accordingly, a Tie2-binding protein can be used, e.g., to specifically recognize or target an endothelial cell. Some Tie2-binding proteins can also be used to modulate (e.g., agonize or antagonize) an activity of an endothelial cell. In some embodiments, these Tie2-binding proteins have an affinity for particular structural features, a combination of features, and/or an epitope that includes at least one amino acid in a structural feature. Exemplary structural features of Tie2 include: two Ig-like domains, three EGF-like domains, and three fibronectin type III domains.
[0267] The angiopoietins are a family of ligands that bind to Tie2. Some
Ang-binding proteins (e.g., antibodies or artificial Ang-binding proteins) can be used to agonize or antagonize endothelial cells. In some embodiments, these Ang-binding proteins have an affinity for particular structural features, a combination of features, and/or an epitope that includes at least one amino acid in a structural feature. Exemplary structural features include: the N-terminal region of about 50 amino acids, the coiled-coil domain, or the fibrinogen-like domain.
[0268] Examples of Ang-binding proteins include proteins that inhibit Ang multimerization (e.g., ability of Ang proteins to form tetramers), proteins that inhibit Ang-Tie2 interactions, and proteins that inhibit a ternary complex of Tiel-Tie2-Ang. Inhibitory proteins can function by disrupting existing interactions or by preventing interactions from occurring.
[0269] Tiel and Tie2 can associate through their extracellular domains and form a heteromeric complex with an angiopoietin (Ang), such as Angl, Ang2, Ang3, and Ang4. This heteromeric complex activates the intracellular signaling cascade mediated by Tie2. Thus, antagonizing formation of this heteromeric complex provides a novel approach to inhibiting Tie2 signaling and its downstream effects, such as angiogenesis. Complex formation can be antagonized by proteins that bind to the extracellular domains of Tiel or Tie2 or that bind to Ang so as to prevent its recruitment into the complex or to prevent its multimerization.
[0270] One method for identifying proteins that bind to Tiel includes: providing a library and selecting from the library one or more members that encode a protein that binds to the Tiel antigen or a fragment thereof (e.g., the extracellular domain, an EGF domain, a fibronectin repeat, or an Ig-superfamily domain (e.g., a Ig- like C2-type 2 domain)). The selection can be performed in a number of ways. For example, the library can be a display library. The Tiel can be tagged and recombinantly expressed. The Tiel is purified and attached to a support, e.g., to affinity beads, or paramagnetic beads or other magnetically responsive particles. The Tiel can also be expressed on the surface of a cell. Members of the display library that specifically bind to the cell, e.g., only if the Tiel is activated, can be selected. Analogous procedures can be performed to identify proteins that bind to Tie2 or a fragment thereof (e.g., the extracellular domain, an EGF domain, a fibronectin repeat, or an Ig-superfamily domain (e.g., a Ig-like C2-type 2 domain)). Analogous procedures can also be performed to identify proteins that bind to Ang or a fragment thereof (e.g., the N-terminal domain, the coiled-coil domain, or the fibrinogen-like domain).
[0271] Proteins identified as being capable of binding a Tie complex member can be tested for their ability to antagonize heteromeric complex formation, ability to promote Tiel phosphorylatoin, and/or ability to promote Tiel homodimerization, as described in the examples below. Proteins identified as antagonizing formation of the heteromeric complex can be used in pharmaceutical compositions to treat a subject in need of such treatment, for example, a subject with an angiogenesis-dependent cancer or tumor or other angiogenesis-related disorders.
[02721 Exemplary Tiel Modulators
[0273] In one embodiment, a Tiel-binding protein can modulate a Tiel activity. For example, a Tiel-binding protein can function as a Tiel agonist or antagonist in the Tiel/EpoR chimeric BaF3 cell assay described in Example 2. Tiel agonists in this Tiel/EpoR chimeric BaF3 cell assay can stimulate certain activity of an endothelial cell under particular conditions, e.g., the conditions of the Tiel/EpoR chimeric BaF3 cell assay.
[0274] Some Tiel binding proteins increase phosphatidyl inositol 3-kinase
(PI3 kinase) activity in an endothelial cell and/or Akt kinase activity. Kontos et al. suggest that the cytoplasmic domain of Tiel can associate with the p85 subunit of PI3 kinase and activate PD kinase activity. Kontos et al. (2002) MoI. Cell Biol. 22:1704- 1713. The Tiel cytoplasmic domain may also associate with a protein tyrosine phosphatase Shρ2. See, e.g., Marron et al. (2000) Adv. Exp. Med. Biol. 476:35-46.
[0275] Some Tie binding proteins may increase dimerization, and/or tyrosine phosphorylation (e.g., as a result of auto-phosphorylation) of the Tiel cytoplasmic domain, e.g., the tyrosine in the motif YVN at about amino acid 1117. [0276] Tiel-binding protein can be evaluated in a cell assay (e.g., in the
Tiel/EpoR chimeric BaF3 cell assay as described below in Example 2). An exemplary cell assay uses a growth factor dependent cell in which a chimeric receptor that includes the Tiel ectodomain fused to the intracellular domain of the growth factor receptor is expressed. Cells are evaluated for ability to grow in the absence of the essential growth factor, but in the presence of a test compound, e.g., a Tiel- binding protein. If the Tiel-binding protein agonizes Tiel in the Tiel/EpoR chimeric BaF3 cell assay, a signalling activity of the Tiel chimera can substitute for stimulation by the required growth factor thorough its cognate receptor. Thus, survival of the cell in the absence of the required growth factor can be used as an indication that the Tiel-binding protein interacts with the Tiel ectodomain.
[0277] Tiel agonists in the Tiel/EpoR chimeric BaF3 cell assay may behave as inhibitors of Tiel activity under other conditions, e.g., in vivo, and, irrespective of in vitro properties, may be useful as inhibitors of angiogenesis in vivo.
[0278] Tiel binding proteins can be used, e.g., to reduce an activity of an endothelial cell. For example, some Tiel binding proteins can be used to decrease phosphatidyl inositol 3-kinase (PI3 kinase) activity in an endothelial cell, Shp2 activity, and/or Akt kinase activity. Some Tiel binding proteins may also reduce dimerization, and/or tyrosine phosphorylation (e.g., as a result of auto- phosphorylation) of the Tiel cytoplasmic domain, e.g., the tyrosine in the motif YVN at about amino acid 1117.
[0279] Tiel-binding protein can be evaluated for activity in a cell assay. For example, the binding protein can be assayed for ability to prevent another ligand, e.g., the E3 antibody, from modulating a Tiel activity in a cell assay described herein (e.g., the Tiel/EpoR chimeric BaF3 cell assay as described below in Example 2).
[0280] Display Libraries
[0281 ] A number of methods can be used to identify proteins that bind to
Tiel, Tie2, Ang, fragments thereof, complexes that include one or more of these proteins or fragments thereof . In one embodiment, a display library is used to identify such proteins. A display library is a collection of entities; each entity includes an accessible protein component and a recoverable component that encodes or identifies the protein component. The protein component can be of any length, e.g. from three amino acids to over 300 amino acids. In a selection, the protein component of each member of the library is probed with a target, e.g., Tiel protein, and if the protein component binds to the target, the display library member is identified, e.g., by retention on a support. The method can be adapted for other targets, such as Tie2, Ang, fragments thereof, complexes that include one or more of these proteins or fragments thereof.
[0282] Retained display library members are recovered from the support and analyzed. The analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated. The analysis can also include determining the amino acid sequence of the protein component and purification of the protein component for detailed characterization. A variety of formats can be used for display libraries. Examples include the following.
[0283] Phage Display. One format utilizes viruses, particularly bacteriophages. This format is termed "phage display." The protein component is typically covalently linked to a bacteriophage coat protein. The linkage results form translation of a nucleic acid encoding the protein component fused to the coat protein. The linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon. Phage display is described, for example, in Ladner et al, U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBOJ 12:725- 734; Hawkins et al. (1992) J MoI Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods Enzymol. 267:129-49; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.
[0284] Phage display systems have been developed for filamentous phage
(phage fl, fd, and Ml 3) as well as other bacteriophage (e.g. T7 bacteriophage and lambdoid phages; see, e.g., Santini (1998) J. MoI. Biol. 282:125-135; Rosenberg et al. (1996) Innovations 6:1-6; Houshmet al. (1999) Anal Biochem 268:363-370). The filamentous phage display systems typically use fusions to a minor coat protein, such as gene III protein, and gene VIII protein, a major coat protein, but fusions to other coat proteins such as gene VI protein, gene VII protein, gene IX protein, or domains thereof can also been used (see, e.g., WO 00/71694). In one embodiment, the fusion is to a domain of the gene III protein, e.g., the anchor domain or "stump," (see, e.g., U.S. Patent No. 5,658,727 for a description of the gene III protein anchor domain). It is also possible to physically associate the protein being displayed to the coat using a non-peptide linkage, e.g., a non-covalent bond or a non-peptide covalent bond. For example, a disulfide bond and/or c-fos and c-jun coiled-coils can be used for physical associations (see, e.g., Crameri et al. (1993) Gene 137:69 and WO 01/05950).
[0285] Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g., PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected protein components, by infecting cells using the selected phages. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.
[0286] Cell-based Display. In still another format the library is a cell-display library. Proteins are displayed on the surface of a cell, e.g., a eukaryotic or prokaryotic cell. Exemplary prokaryotic cells include E. coli cells, B. subtilis cells, and spores (see, e.g., Lu et al. (1995) Biotechnology 13:366). Exemplary eukaryotic cells include yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hanseula, or Pichia pastoris). Yeast surface display is described, e.g., in Boder and Wittrup (1997) Nat. Biotechnol. 15:553-557 and WO 03/029456, which describes a yeast display system that can be used to display immunoglobulin proteins such as Fab fragments and the use of mating to generate combinations of heavy and light chains.
[0287] Ribosome Display. RNA and the polypeptide encoded by the RNA can be physically associated by stabilizing ribosomes that are translating the RNA and have the nascent polypeptide still attached. Typically, high divalent Mg2+ concentrations and low temperature are used. See, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sd. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; and Schaffitzel et al. (1999) J Immunol Methods. 231(l-2):119-35. [0288] Polypeptide-Nucleic Acid Fusions. Another format utilizes polypeptide-nucleic acid fusions. Polypeptide-nucleic acid fusions can be generated by the in vitro translation of mRNA that include a covalently attached puromycin group, e.g., as described in Roberts and Szostak (1997) Proc. Natl. Acad. ScL USA 94:12297-12302, and U.S. Patent No. 6,207,446. The mRNA can then be reverse transcribed into DNA and crosslinked to the polypeptide.
[0289] Other Display Formats. Yet another display format is a non- biological display in which the protein component is attached to a non-nucleic acid tag that identifies the polypeptide. For example, the tag can be a chemical tag attached to a bead that displays the polypeptide or a radiofrequency tag (see, e.g., U.S. Patent No. 5,874,214).
[0290] Display technology can also be used to obtain binding proteins, e.g., antibodies that interact with particular epitopes of a target. This can be done, for example, by using competing non-target molecules that lack the particular epitope or are mutated within the epitope, e.g., with alanine. Such non-target molecules can be used in a negative selection procedure as described below, as competing molecules when binding a display library to the target, or as a pre-elution agent, e.g., to capture in a wash solution dissociating display library members that are not specific to the target.
[0291] Iterative Selection. In one preferred embodiment, display library technology is used in an iterative mode. A first display library is used to identify one or more binding proteins for a target. These proteins are then varied, e.g., using a mutagenesis method, to form a second display library. Higher affinity binding proteins are then selected from the second library, e.g., by using higher stringency or more competitive binding and washing conditions.
[0292] In some implementations, the mutagenesis is targeted to regions known or likely to be at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs, e.g., framework regions, particular within ten, five, or three amino acids of a CDR junction. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make precise step-wise improvements.
[0293] Some exemplary mutagenesis techniques include: error-prone PCR
(Leung et al. (1989) Technique 1:11-15), recombination (see, e.g., USSN 10/279,633), DNA shuffling using random cleavage (Stemmer (1994) Nature 389- 391; termed "nucleic acid shuffling"), RACHITT™ (Coco et al. (2001) Nature Biotech. 19:354), site-directed mutagenesis (Zoller et al. (1987) Nucl Acids Res 10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) Methods Enzymol. 208:564-586) and incorporation of degenerate oligonucleotides (Griffiths et al. (1994) EMBO J 13:3245).
[0294] In one example of iterative selection, the methods described herein are used to first identify a binding protein from a display library that binds a Tiel with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of greater than 1 nM, 10 nM, or 100 nM. The nucleic acid sequence encoding the initial identified binding protein is used as a template nucleic acid for the introduction of variations, e.g., to identify a second binding protein that has enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial binding protein.
[0295] Off-Rate Selection. Since a slow dissociation rate can be predictive of high affinity, particularly with respect to interactions between polypeptides and their targets, the methods described herein can be used to isolate binding proteins with a desired kinetic dissociation rate (i.e. reduced) for a binding interaction to a target.
[0296] To select for slow dissociating binding proteins from a display library, the library is contacted to an immobilized target. The immobilized target is then washed with a first solution that removes non-specifically or weakly bound biomolecules. Then the immobilized target is eluted with a second solution that includes a saturation amount of free target, i.e., replicates of the target that are not attached to the particle. The free target binds to biomolecules that dissociate from the target. Rebinding is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilized target.
[0297] The second solution can have solution conditions that are substantially physiological or that are stringent. Typically, the solution conditions of the second solution are identical to the solution conditions of the first solution. Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include biomolecules that dissociate at a slower rate from the target than biomolecules in the early fractions. It is also possible to recover display library members that remain bound to the target even after extended incubation. These can either be dissociated using chaotropic conditions or can be amplified while attached to the target. For example, phage bound to the target can be contacted to bacterial cells.
[0298] Selecting and Screening for Specificity. "Selection" refers to a process in which many members of a display library are allowed to contact the target and those that bind are recovered and propagated. The selection can be from a library having numerous members, e.g., more than 1010 members. "Screening" refers to a process in which isolated members of the library are tested singly for binding to the target. Through automation, thousands of candidates may be screened in a highly parallel process. The display library selection methods described herein can include a selection process that discards display library members that bind to a non-target molecule. Examples of non-target molecules include, e.g., extracellular domains of molecules that include an immunoglobulin super-family domain or an EGF domain and receptor tyrosine kinases other than Tiel, e.g., Tie2, or other than Tie2, e.g., Tiel, or other than Tiel and Tie2. In one implementation, a so-called "negative selection" step is used to discriminate between the target and related non-target molecule and a related, but distinct non-target molecules. The display library or a pool thereof is contacted to the non-target molecule. Members of the sample that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections. The negative selection step can be prior to or after selecting library members that bind to the target molecule.
[0299] In another implementation, a screening step is used. After display library members are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target molecule (e.g., a non-target listed above). For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target. The non-target and target binding data are compared (e.g., using a computer and software) to identify library members that etal
specifically bind to Tiel, Tie2, Ang, fragments thereof, or a complex comprising one or more such components.
[0300] The display library selection and screening methods described herein can include a selection or screening process that selects for display library members that bind to specific sites on the target molecule. For example, elution with high concentration of an antibody described herein can be used to select for phage that bind to an epitope that is near or overlaps with the epitope bound by the antibody used for elution. Accordingly, one can screen for a phage that binds to the E3-binding site of Tiel by performing ELISAs with and without E3 antibody in the buffer.
[0301] The following description provides one exemplary method for identifying antibodies that bind to Tiel using a phagemid Fab library. For example, three rounds of selection can be performed with decreasing amounts of target protein (e.g., 100, 50 and 50 μg for first, second, and third rounds, respectively). The target is immobilized on streptavidin coated magnetic beads (Dynal). The library is depleted against streptavidin coated magnetic beads prior to each round of selection and optionally against an unrelated protein which may include a common purification handle. For example, if the target is produced as a fusion to a Fc domain, the library can be depleted against soluble Trail-Fc (a commercially available Fc fusion protein). The depletion process removes Fc binders.
[0302] Each round of selection can include, e.g., two cycles of streptavidin magnetic bead depletion, a cycle of binding of phage to Tiel -coated beads, ten cycles of washes, elution of bound phage, and propagation of enriched phage for the next round. Phage bound to Tiel -coated beads after ten washes can be directly amplified or eluted before amplification. After three rounds of selection, individual clones may be grown in 96-well microtiter plates and individually screened for Tiel binding activity by phage ELISA. ELISAs can include evaluations of binding to Tiel, specificity controls, and unrelated controls. Isolates can be DNA fingerprinted to determine the diversity emerging from the selection process. For example, positive isolates can be PCR amplified with the oligonucleotide primers M13-reverse and genelll-forward (see, e.g., Marks (1991), J. MoI. Biol. 222:581). The products can be analyzed by BstNI fingerprinting. [0303J An exemplary method for performing ELISA's with phage that display a binding protein is as follows. Individual clones can be grown and rescued as described previously (Marks et al. (1991), J. MoI. Biol. 222:581). For ELISAs, 96- well Immulon 2 HB plates (Thermo Labsystems) are coated with 1 μg/well ImmunoPure™ streptavidin (Pierce) in PBS and incubated overnight at 4°C. After three washes with PBS, 100 μL of biotinylated Tiel protein is allowed to bind to the immobilized streptavidin for 30-60 minutes at room temperature. Then, Tiel -coated wells are blocked with 300 μL of 2% milk/lx PBS/0.05% Tween (2% MPBST) for two hours at 37°C. The wells are incubated with 100 μL of phage culture supernatant that had been blocked with 2% MPBST for one hour at room temperature. The wells are washed five times with lxPBS/Tween 0.1% (PBST), and incubated with 100 μL of anti-M13-HRP secondary antibody at a 1 :5,000 dilution for one hour at room temperature. The wells are washed five times with PBST before developing with TMB-solution and read at 630 nm.
[0304] For the cell ELISAs, cells are washed once in PBS and resuspended at a concentration of 1 x 106 to 2 x 106 cells/niL of PBS. A final concentration of 1-2 x 105 cells per well of a 96-well tissue culture plate (Falcon, VWR) can be used. The cells are fixed by adding an equal volume of 0.2% glutaraldehyde (Sigma- Aldrich) and incubating at 370C for 12 minutes. They are then washed three times with PBS using an automated plate washer (Bio-Tek Instruments, Inc.) and blocked with 200 μL of 2% MPBST for one hour at room temperature. The rest of the ELISA procedure can be performed as described above except that lxPBS/Tween 0.05% is used for the washes and incubations.
[0305] Germlining Antibodies
[0306] It is possible to modify an antibody that binds Tiel , Tie2, or Ang, e.g., an antibody described herein, in order to make the variable regions of the antibody more similar to one or more germline sequences. For example, an antibody can include one, two, three or more amino acid substitutions, e.g., in a framework or CDR region, to make it more similar to a reference germline sequence. One exemplary germlining method can include: identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Then mutations (at the amino acid level) can be made in the isolated antibody, either incrementally, in combination, or both. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.
[0307] In one embodiment, mutagenesis is used to substitute or insert one or more germline residues into a CDR region. For example, the germline CDR residue can be from a germline sequence that is similar (e.g., most similar) to the variable region being modified. After mutagenesis, activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine if the germline residue or residues are tolerated. Similar mutagenesis can be performed in the framework regions.
[0308] Selecting a germline sequence can be performed in different ways. For example, a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The selection can be performed using at least 2, 3, 5, or 10 germline sequences. In the case of CDRl and CDR2, identifying a similar germline sequence can include selecting one such sequence. In the case of CDR3, identifying a similar germline sequence can include selecting one such sequence, but may including using two germline sequences that separately contribute to the amino-terminal portion and the carboxy-terminal portion. In other implementations more than one or two germline sequences are used, e.g., to form a consensus sequence.
[0309] In one embodiment, with respect to a particular reference variable domain sequence, e.g., a sequence described herein, a related variable domain sequence has at at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% of the CDR amino acid positions that are not identical to residues in the reference CDR sequences, residues that are identical to residues at corresponding positions in a human germline sequence (i.e., an amino acid sequence encoded by a human germline nucleic acid).
[0310] hi one embodiment, with respect to a particular reference variable domain sequence, e.g., a sequence described herein, a related variable domain sequence has at at least 30, 50, 60, 70, 80, 90 or 100% of the FR regions are identical to FR sequence from a human germline sequence, e.g., a germline sequence related to the reference variable domain sequence.
[0311 ] Accordingly, it is possible to isolate an antibody which has similar activity to a given antibody of interest, but is more similar to one or more germline sequences, particularly one or more human germline sequences. For example, an antibody can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% identical to a germline sequence in a region outside the CDRs (e.g., framework regions). Further an antibody can include at least 1, 2, 3, 4, or 5 germline residues in a CDR region, the germline residue being from a germline sequence of similar (e.g., most similar) to the variable region being modified. Germline sequences of primary interest are human germline sequences. The activity of the antibody (e.g., the binding activity) can be within a factor or 100, 10, 5, 2, 0.5, 0.1, and 0.001 of the original antibody.
[0312] Exemplary germline reference sequences for Vkappa include:
012/02, 018/08, A20, A30, L14, Ll, L15, L4/18a, L5/L19, L8, L23, L9 ,L24, LIl, L12, Oll/Ol, A17, Al, A18, A2, A19/A3, A23, A27, All, L2/L16, L6, L20, L25, B3, B2, A26/A10, and A14. See, e.g., Tomlinson et al. (1995) EMBO J. 14(18):4628-
3.
[0313] A germline reference sequence for the HC variable domain can be based on a sequence that has particular canonical structures, e.g., 1-3 structures in the Hl and H2 hypervariable loops. The canonical structures of hypervariable loops of an immunoglobulin variable domain can be inferred from its sequence, as described in Chothia et al. (1992) J. MoI. Biol. 227:799-817; Tomlinson et al. (1992) J. MoI. Biol. 227:776-798); and Tomlinson et al. (1995) EMBO J. 14(18):4628-38. Exemplary sequences with a 1-3 structure include: DP-I, DP-8, DP-12, DP-2, DP-25, DP-15, DP- 7, DP-4, DP-31, DP-32, DP-33, DP-35, DP-40, 7-2, hv3005, hv3OO5f3, DP-46, DP- 47, DP-58, DP-49, DP-50, DP-51, DP-53, and DP-54.
[03141 Diversity
[0315] Display libraries and other libraries include variation at one or more positions in the displayed polypeptide. The variation at a given position can be synthetic or natural. For some libraries, both synthetic and natural diversity are included. [0316] Synthetic Diversity. Libraries can include regions of diverse nucleic acid sequence that originate from artificially synthesized sequences. Typically, these are formed from degenerate oligonucleotide populations that include a distribution of nucleotides at each given position. The inclusion of a given sequence is random with respect to the distribution. One example of a degenerate source of synthetic diversity is an oligonucleotide that includes NNN wherein N is any of the four nucleotides in equal proportion.
[0317] Synthetic diversity can also be more constrained, e.g., to limit the number of codons in a nucleic acid sequence at a given trinucleotide to a distribution that is smaller than NNN. For example, such a distribution can be constructed using less than four nucleotides at some positions of the codon. In addition, trinucleotide addition technology can be used to further constrain the distribution. So-called "trinucleotide addition technology" is described, e.g., in Wells et al. (1985) Gene 34:315-323, US 4,760,025 and US 5,869,644.
[0318] Natural Diversity. Libraries can include regions of diverse nucleic acid sequence that originate (or are synthesized based on) from different naturally- occurring sequences. An example of natural diversity that can be included in a display library is the sequence diversity present in immune cells (see also below). Nucleic acids are prepared from these immune cells and are manipulated into a format for polypeptide display.
[0319] Antibody Display Libraries
[0320] In one embodiment, the display library presents a diverse pool of proteins, each of which includes an immunoglobulin domain, e.g., an immunoglobulin variable domain. Display libraries are particular useful, for example for identifying human or "humanized" antibodies that recognize human antigens. Such antibodies can be used as therapeutics to treat human disorders such as endothelial-related disorders, e.g., metastatic cancer. Since the constant and framework regions of the antibody are human, these therapeutic antibodies may avoid themselves being recognized and targeted as antigens. The constant regions are also optimized to recruit effector functions of the human immune system. The in vitro display selection process surmounts the inability of a normal human immune system to generate antibodies against self-antigens. [0321 ] A typical antibody display library displays a polypeptide that includes a VH domain and a VL domain. An "immunoglobulin domain" refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two β-sheets formed of about seven β-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay 1988 Ann. Rev Immunol. 6:381-405). The canonical structures of hypervariable loops of an immunoglobulin variable can be inferred from its sequence, as described in Chothia et al. (1992) J. MoI. Biol. 227:799-817; Tomlinson et al. (1992) J. MoI. Biol 227:776- 798); and Tomlinson et al. (1995) EMBO J. 14(18):4628-38. The display library can display the antibody as a Fab fragment (e.g., using two polypeptide chains) or a single chain Fv (e.g., using a single polypeptide chain). Other formats can also be used.
[0322] As in the case of the Fab and other formats, the displayed antibody can include a constant region as part of a light or heavy chain. In one embodiment, each chain includes one constant region, e.g., as in the case of a Fab. In other embodiments, additional constant regions are displayed.
[0323] Antibody libraries can be constructed by a number of processes (see, e.g., de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20. and Hoogenboom et al. (2000) Immunol Today 21:371-8). Further, elements of each process can be combined with those of other processes. The processes can be used such that variation is introduced into a single immunoglobulin domain (e.g., VH or VL) or into multiple immunoglobulin domains (e.g., VH and VL). The variation can be introduced into an immunoglobulin variable domain, e.g., in the region of one or more of CDRl, CDR2, CDR3, FRl, FR2, FR3, and FR4, referring to such regions of either and both of heavy and light chain variable domains. In one embodiment, variation is introduced into all three CDRs of a given variable domain. In another preferred embodiment, the variation is introduced into CDRl and CDR2, e.g., of a heavy chain variable domain. Any combination is feasible. [0324] In one process, antibody libraries are constructed by inserting diverse oligonucleotides that encode CDRs into the corresponding regions of the nucleic acid. The oligonucleotides can be synthesized using monomelic nucleotides or trinucleotides. For example, Knappik et al. (2000) J. MoI. Biol. 296:57-86 describe a method for constructing CDR encoding oligonucleotides using trinucleotide synthesis and a template with engineered restriction sites for accepting the oligonucleotides. [0325] In another process, an animal, e.g., a non-human animal, e.g., a rodent, is immunized with the Tiel . The animal is optionally boosted with the antigen to further stimulate the response. Then spleen cells are isolated from the animal, and nucleic acid encoding VH and/or VL domains is amplified and cloned for expression in the display library. The non-human animal can include one or more human immunoglobulin gene sequences. For example, the animal can include a complete human immunoglobulin locus. The animal may also have an inactivated endogenous immunoglobulin locus.
[0326] hi yet another process, antibody libraries are constructed from nucleic acid amplified from naϊve germline immunoglobulin genes (e.g., human genes). The amplified nucleic acid includes nucleic acid encoding the VH and/or VL domain. Sources of immunoglobulin-encoding nucleic acids are described below. Amplification can include PCR, e.g., with primers that anneal to the conserved constant region, or another amplification method.
[0327] Nucleic acid encoding immunoglobulin domains or fragments thereof can be obtained from the immune cells of, e.g., a human, a primate, mouse, rabbit, camel, or rodent. In one example, the cells are selected for a particular property. B cells at various stages of maturity can be selected. In another example, the B cells are naϊve.
[0328] In one embodiment, fluorescent-activated cell sorting (FACS) is used to sort B cells that express surface-bound IgM, IgD, or IgG molecules. Further, B cells expressing different isotypes of IgG can be isolated, hi another preferred embodiment, the B or T cell is cultured in vitro. The cells can be stimulated in vitro, e.g., by culturing with feeder cells or by adding mitogens or other modulatory reagents, such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin or pokeweed mitogen.
[0329] In still another embodiment, the cells are isolated from a subject that has an immunological disorder, e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, vasculitis, Sjogren syndrome, systemic sclerosis, or anti-phospholipid syndrome. The subject can be a human, or an animal, e.g., an animal model for the human disease, or an animal having an analogous disorder. In yet another eniDOQiment, me cells are isolated from a transgenic non-human animal that includes a human immunoglobulin locus.
[0330] In one preferred embodiment, the cells have activated a program of somatic hypermutation. Cells can be stimulated to undergo somatic mutagenesis of immunoglobulin genes, for example, by treatment with anti-immunoglobulin, anti- CD40, and anti-CD38 antibodies (see, e.g., Bergthorsdottir et al (2001) J Immunol. 166:2228). In another embodiment, the cells are naϊve.
[0331 ] The nucleic acid encoding an immunoglobulin variable domain can be isolated from a natural repertoire by the following exemplary method. First, RNA is isolated from the immune cell. Full length (i.e., capped) mRNAs are separated (e.g. by dephosphorylating uncapped RNAs with calf intestinal phosphatase). The cap is then removed with tobacco acid pyrophosphatase and reverse transcription is used to produce the cDNAs.
[0332] The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., de Haard et al. (1999) J. Biol. Chem 21 A: 18218-30. The primer binding region can be constant among different immunoglobulins, e.g., in order to reverse transcribe different isotypes of immunoglobulin. The primer binding region can also be specific to a particular isotype of immunoglobulin. Typically, the primer is specific for a region that is 3' to a sequence encoding at least one CDR. hi another embodiment, poly-dT primers may be used (and may be preferred for the heavy-chain genes).
[0333] A synthetic sequence can be ligated to the 3' end of the reverse transcribed strand. The synthetic sequence can be used as a primer binding site for binding of the forward primer during PCR amplification after reverse transcription. The use of the synthetic sequence can obviate the need to use a pool of different forward primers to fully capture the available diversity.
[0334] The variable domain-encoding gene is then amplified, e.g., using one or more rounds. If multiple rounds are used, nested primers can be used for increased fidelity. The amplified nucleic acid is then cloned into a display library vector.
[0335] Any method for amplifying nucleic acid sequences may be used for amplification. Methods that maximize and do not bias diversity are preferred. A variety of techniques can be used for nucleic acid amplification. The polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,195 and 4,683,202, Saiki, et al. (1985) Science 230, 1350-1354) utilizes cycles of varying temperature to drive rounds of nucleic acid synthesis. Transcription-based methods utilize RNA synthesis by RNA polymerases to amplify nucleic acid (U.S. Pat. No 6,066,457; U.S. Pat. No 6,132,997; U.S. Pat. No 5,716,785; Sarkar et. al, Science (1989) 244: 331-34 ; Stofler et al, Science (1988) 239: 491). NASBA (U.S. Patent Nos. 5,130,238; 5,409,818; and 5,554,517) utilizes cycles of transcription, reverse-transcription, and RNaseH-based degradation to amplify a DNA sample. Still other amplification methods include rolling circle amplification (RCA; U.S. Patent Nos. 5,854,033 and 6,143,495) and strand displacement amplification (SDA; U.S. Patent Nos. 5,455,166 and 5,624,825).
[0336] Secondary Screening Methods
[0337] After selecting candidate display library members that bind to a target, each candidate display library member can be further analyzed, e.g., to further characterize its binding properties for the target. Similarly candidate binding proteins (e.g., by immunization, etc.) obtained by other methods can also be analyzed. Each candidate binding protein can be subjected to one or more secondary screening assays. The assay can be for a binding property, a catalytic property, a physiological property (e.g., cytotoxicity, renal clearance, irnmunogenicity), a structural property (e.g., stability, conformation, oligomerization state) or another functional property. The same assay can be used repeatedly, but with varying conditions, e.g., to determine pH, ionic, or thermal sensitivities.
[0338] As appropriate, the assays can use the display library member directly, a recombinant polypeptide produced from the nucleic acid encoding a displayed polypeptide, or a synthetic peptide synthesized based on the sequence of a displayed polypeptide. Exemplary assays for binding properties include the following.
[0339] ELISA. Proteins encoded by a display library can also be screened for a binding property using an ELISA assay. For example, each protein is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non-specifically bound polypeptides. Then the amount of the protein bound to the plate is determined by probing the plate with an antibody that can recognize the polypeptide, e.g., a tag or constant portion of the polypeptide. The antibody is linked to an enzyme such as alkaline phosphatase, which produces a colorimetric product when appropriate substrates are provided. The protein can be purified from cells or assayed in a display library format, e.g., as a fusion to a filamentous bacteriophage coat. Alternatively, cells (e.g., live or fixed) that express the target molecule, e.g., Tiel, Tie2, or Ang, can be plated in a microtitre plate and used to test the affinity of the peptides/antibodies present in the display library or obtained by selection from the display library.
[0340] Cell Binding Assays. Binding proteins (e.g., Tiel, Tie2, or Ang binding proteins) can be evaluated for their ability to interact with one or more cell types, e.g., endothelial cells or platelets. Fluorescent activated cell sorting (FACS) is one exemplary method for testing an interaction between a protein and a cell. The binding protein is labeled directly or indirectly with a fluorophore, before or after, binding to the cells, and then cells are counted in a FACS sorter.
[0341] For example, the following method can be used to evaluate whether a
Tiel binding protein interacts with platelets or other cell types.
[0342] Isolation of Platelets. Human blood can be obtained from informed healthy volunteers. For example, venous blood is collected into one-sixth volume of ACD (2.5 g of sodium citrate, 1.5 g citric acid, and 2.5 g glucose in 100 ml dH2O). The blood is centrifuged at 800 Xg for 15 min at room temperature and the platelet- rich plasma is removed and incubated for 60 min at 37°C. in the presence of 1 mM acetylsalicylic acid followed by centrifugation at 1000 x g for 10 min at room temperature. The platelet pellet can be resuspended at a density of 2 x 108 cells/ml with HEPES-buffered Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 3 mM NaH2PO4, 5 mM glucose, 10 mM HEPES pH 7.4, 0.2% bovine serum albumin, and 0.05 U/mL apyrase). See also, e.g., Kornecki et at. (1990) J Biol Chem. 265:10,042-10,048 and Naik et al. (1995) Biochem J. 310:155-162).
[0343] FACS. For example, for FACS analysis of platelets, cells can be resuspended in 0.1% BSA/PBS (4 x 105 cells/sample) in the presence of PGEl (1 mg/mL) and incubated with a candidate Tiel binding protein (e.g., at about 5 μg/mL) or with a control. After a 1-hour incubation at 22°C, the cells are washed with 0.1% BSA/PBS, treated with 50 μL 1/100 diluted FITC-labeled secondary antibody , incubated for 30 minutes on ice, washed, and resuspended in 0.1% BSA/PBS. The samples are analyzed using an Immunocytometry Systems flow cytometer (FACSORT™, Becton Dickinson, San Jose, CA). See also, e.g., Malgorzata et al. (2000) Blood, Vol. 95 No. 8 (April 15 pp. 2600-2609.
[0344] In addition, it is possible to evaluate platelets by Westerns analysis of
SDS-page separated proteins from isolated platelets and by immunoprecipitation. Still other methods involve binding cells to surfaces to which the Tie 1 -binding protein is attached (e.g., coated to).
[0345] Other cell types can be prepared for FACS by methods known in the art.
[0346] Homogeneous Binding Assays. The binding interaction of candidate polypeptide with a target can be analyzed using a homogenous assay, i.e., after all components of the assay are added, additional fluid manipulations are not required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, for example, Lakowicz et al, U.S. Patent No. 5,631,169; Stavrianopoulos, et al, U.S. Patent No. 4,868,103). A fiuorophore label on the first molecule (e.g., the molecule identified in the fraction) is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule. The fluorescent label on the second molecule fluoresces when it absorbs to the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. A binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a flύorimeter). By titrating the amount of the first or second binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.
[0347] Surface Plasmon Resonance (SPR). The binding interaction of a molecule isolated from a display library and a target can be analyzed using SPR. SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Patent No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcore International AB (Uppsala, Sweden).
[0348] Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (Ka), and kinetic parameters, including K0n and Koff, for the binding of a biomolecule to a target. Such data can be used to compare different biomolecules. For example, different proteins can be compared to identify individuals that have high affinity for the target or that have a slow Koff. This information can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow Koff. This information can be combined with structural modeling (e.g., using homology modeling, energy minimization, or structure determination by crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be formulated and used to guide other design processes.
[0349] Protein Arrays. Proteins identified from the display library can be immobilized on a solid support, for example, on a bead or an array. For a protein array, each of the polypeptides is immobilized at a unique address on a support. Typically, the address is a two-dimensional address. Protein arrays are described below (see, e.g., Diagnostics). It is also possible to use a protein array to evaluate any plurality of proteins, e.g., for interaction with Tiel, Tie2, or Ang.
[0350] Cellular Assays. Candidate proteins can be selected from a library by transforming the library into a host cell; the library could have been previously identified from a display library. For example, the library can include vector nucleic acid sequences that include segments that encode the polypeptides and that direct expression, e.g., such that the proteins are produced within the cell, secreted from the cell, or attached to the cell surface. The cells can be screened or selected for proteins that bind to the Tiel, Tie2, or Ang, e.g., as detected by a change in a cellular phenotype or a cell-mediated activity. For example, in the case of an antibody that binds to Tiel, the activity maybe autophosphorylation, activation of PB Kinase, activation of AKT, or a change in endothelial cell activity (e.g., proliferation).
[0351 ] hi another embodiment, the library of cells is in the form of a cellular array. The cellular array can likewise be screened for any appropriate detectable activity. In other embodiments, competition binding assays are used to identify proteins that are compete with a reference protein for binding to Tiel . Similarly, epitope mapping can be used to identify proteins that bind to a particular epitope of Tie. Fragments and mutants of Tiel can be also be used in the binding protein- identification process, e.g., in one or more of characterization, screening, or immunization.
[0352] Methods for Obtaining Target-Binding Antibodies
[0353] In addition to the use of display libraries, other methods can be used to obtain a target-binding antibody or in combination with the use of display libraries. For example, the Tiel ectodomain or a region thereof can be used as an antigen in a non-human animal, e.g., a rodent. Similarly, Tie2 or Ang, or a region thereof can be used as an antigen in a non-human animal, e.g., a rodent.
[0354] hi one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific Mabs derived from the genes with the desired specificity may be produced and selected. See, e.g., XenoMouse™, Green et al. Nature Genetics 7:13-21 (1994), U.S. 20030070185, WO 96/34096, published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996.
[0355] In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized or deimmunized. Winter describes a CDR-grafting method that may be used to prepare the humanized antibodies (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539. AU of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-Human UDKS. it is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.
[0356] Humanized antibodies can be generated by replacing sequences of the
Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a predetermined target, as described above. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.
[0357] A target-binding antibody may also be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317, the contents of which are specifically incorporated by reference herein. Briefly, the heavy and light chain variable regions of an antibody can be analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable regions, or preferably, by single amino acid substitutions. As far as possible conservative substitutions are made, often but not exclusively, an amino acid common at this position in human germline antibody sequences may be used. Human germline sequences are disclosed in Tomlinson, LA. et al. (1992) J. MoI. Biol. 227:776-798; Cook, G. P. et al. (1995) Immunol. Today Vol. 16 (5): 237- 242; Chothia, D. et al. (1992) J. MoI. Bio. 227:799-817. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK). After the deimmunizing changes are identified, nucleic acids encoding VH and VL can be constructed by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, and so forth). Mutagenized variable sequence can, optionally, be fused to a human constant region, e.g., human IgGl or K constant regions.
[0358] In some cases a potential T cell epitope will include residues which are known or predicted to be important for antibody function. For example, potential T cell epitopes are usually biased towards the CDRs. In addition, potential T cell epitopes can occur in framework residues important for antibody structure and binding. Changes to eliminate these potential epitopes will in some cases require more scrutiny, e.g., by making and testing chains with and without the change. Where possible, potential T cell epitopes that overlap the CDRs were eliminated by substitutions outside the CDRs. hi some cases, an alteration within a CDR is the only option, and thus variants with and without this substitution should be tested, hi other cases, the substitution required to remove a potential T cell epitope is at a residue position within the framework that might be critical for antibody binding. In these cases, variants with and without this substitution should be tested. Thus, in some cases several variant deimmunized heavy and light chain variable regions were designed and various heavy/light chain combinations tested in order to identify the optimal deimmunized antibody. The choice of the final deimmunized antibody can then be made by considering the binding affinity of the different variants in conjunction with the extent of deimmunization, i.e., the number of potential T cell epitopes remaining in the variable region. Deimmunization can be used to modify an antibody that includes a non-human sequence, e.g., a murine antibody or other non- human monoclonal antibody. Deimmunization can be used to modify an antibody isolated from a display library.
[03591 Endothelial Cell Assays
[0360] A target-binding protein or a candidate binding protein can be characterized using a cellular assay, e.g., to evaluate a change in a cellular phenotype or other activity when the binding protein is contacted to the cell. Typically the cell is expresses a protein that includes at least part of the ectodomain of Tie. In some embodiments, the cell expresses liel, e.g., a full-length, mature Tiel protein, Tie2, and/or is contacted with Ang..
[0361] Endothelial cell proliferation. A candidate target-binding protein can be tested for endothelial proliferation inhibiting activity using a biological activity assay such as the bovine capillary endothelial cell proliferation assay, the chick CAM assay, the mouse corneal assay, and evaluating the effect of the binding protein on implanted tumors. The chick CAM assay is described, e.g., by O'Reilly, et al. in "Angiogenic Regulation of Metastatic Growth" Cell, vol. 79 (2), Oct. 21, 1994, pp. 315-328. Briefly, three day old chicken embryos with intact yolks are separated from the egg and placed in a petri dish. After three days of incubation a methylcellulose disc containing the protein to be tested is applied to the CAM of individual embryos. After 48 hours of incubation, the embryos and CAMs are observed to determine whether endothelial growth has been inhibited. The mouse corneal assay involves implanting a growth factor-containing pellet, along with another pellet containing the suspected endothelial growth inhibitor, in the cornea of a mouse and observing the pattern of capillaries that are elaborated in the cornea.
[0362] Angiogenesis. Angiogenesis may be assayed , e.g., using various human endothelial cell systems, such as umbilical vein, coronary artery, or dermal cells. Suitable assays include Alamar Blue based assays (available from Biosource International) to measure proliferation; migration assays using fluorescent molecules, such as the use of Becton Dickinson Falcon HTS FLUOROBLOCK™ cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors; and tubule formation assays based on the formation of tubular structures by endothelial cells on MATRIGEL™ (Becton Dickinson) or collagen I.
[0363] Cell adhesion. Cell adhesion assays measure adhesion of cells to purified adhesion proteins or adhesion of cells to each other, in presence or absence of candidate target-binding proteins. Cell-protein adhesion assays measure the ability of agents to modulate the adhesion of cells to purified proteins. For example, recombinant proteins are produced, diluted to 2.5 g/mL in PBS, and used to coat the wells of a microtiter plate. The wells used for negative control are not coated. Coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2 x final test concentration and added to the blocked, coated wells. Cells are then added to the wells, and the unbound cells are washed off. Retained cells are labeled directly on the plate by adding a membrane-permeable fluorescent dye, such as calcein-AM, and the signal is quantified in a fluorescent microplate reader.
[0364] Cell-cell adhesion assays can be used to measure the ability of candidate target-binding proteins to modulate binding of cells to each other. These assays can use cells that naturally or recombinantly express an adhesion protein of choice, hi an exemplary assay, cells expressing the cell adhesion protein are plated in wells of a multiwell plate together with other cells (either more of the same cell type, or another type of cell to which the cells adhere). The cells that can adhere are labeled with a membrane-permeable fluorescent dye, such as BCECF, and allowed to adhere to the monolayers in the presence of candidate binding proteins. Unbound cells are washed off, and bound cells are detected using a fluorescence plate reader. High-throughput cell adhesion assays have also been described. See, e.g., Falsey J R et al., Bioconjug Chem. May-June 2001;12(3):346-53.
[0365] Tubulogenesis. Tubulogenesis assays can be used to monitor the ability of cultured cells, generally endothelial cells, to form tubular structures on a matrix substrate, which generally simulates the environment of the extracellular matrix. Exemplary substrates include MATRIGEL™ (Becton Dickinson), an extract of basement membrane proteins containing laminin, collagen IV, and heparin sulfate proteoglycan, which is liquid at 4°C. and forms a solid gel at 37°C. Other suitable matrices comprise extracellular components such as collagen, fibronectin, and/or fibrin. Cells are stimulated with a pro-angiogenic stimulant, and their ability to form tubules is detected by imaging. Tubules can generally be detected after an overnight incubation with stimuli, but longer or shorter time frames may also be used. Tube formation assays are well known in the art (e.g., Jones M K et al., 1999, Nature Medicine 5:1418-1423). These assays have traditionally involved stimulation with serum or with the growth factors FGF or VEGF. In one embodiment, the assay is performed with cells cultured in serum free medium. In one embodiment, the assay is performed in the presence of one or more pro-angiogenic agents, e.g., inflammatory angiogenic factors such as TNF-α, or FGF, VEGF, phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.
[0366] Cell Migration. An exemplary assay for endothelial cell migration is the human microvascular endothelial (HMVEC) migration assay. See, e.g., Tolsma et al. (1993) J. Cell Biol 122, 497-511. Migration assays are known in the art (e.g., Paik J H et al., 2001, J Biol Chem 276:11830-11837). In one example, cultured endothelial cells are seeded onto a matrix-coated porous lamina, with pore sizes generally smaller than typical cell size. The lamina is typically a membrane, such as the transwell polycarbonate membrane (Corning Costar Corporation, Cambridge, Mass.), and is generally part of an upper chamber that is in fluid contact with a lower chamber containing pro-angiogenic stimuli. Migration is generally assayed after an overnight incubation with stimuli, but longer or shorter time frames may also be used. Migration is assessed as the number of cells that crossed the lamina, and may be detected by staining cells with hemotoxylin solution (VWR Scientific), or by any other method for determining cell number. In another exemplary set up, cells are fluorescently labeled and migration is detected using fluorescent readings, for instance using the Falcon HTS FLUOROBLOK™ (Becton Dickinson). While some migration is observed in the absence of stimulus, migration is greatly increased in response to pro- angiogenic factors. The assay can be used to test the effect of a target-binding protein on endothelial cell migration.
[0367] Sprouting assay. An exemplary sprouting assay is a three-dimensional in vitro angiogenesis assay that uses a cell-number defined spheroid aggregation of endothelial cells ("spheroid"), embedded in a collagen gel-based matrix. The spheroid can serve as a starting point for the sprouting of capillary-like structures by invasion into the extracellular matrix (termed "cell sprouting") and the subsequent formation of complex anastomosing networks (Korff and Augustin, 1999, J Cell Sci 112:3249-58). In an exemplary experimental set-up, spheroids are prepared by pipetting 400 human umbilical vein endothelial cells into individual wells of a nonadhesive 96-well plates to allow overnight spheroidal aggregation (Korff and Augustin: J Cell Biol 143: 1341- 52, 1998). Spheroids are harvested and seeded in 900 μl of methocel-collagen solution and pipetted into individual wells of a 24 well plate to allow collagen gel polymerization. Test agents are added after 30 min by pipetting 100 μl of 10-fold concentrated working dilution of the test substances on top of the gel. Plates are incubated at 370C for 24 h. Dishes are fixed at the end of the experimental incubation period by addition of paraformaldehyde. Sprouting intensity of endothelial cells can be quantitated by an automated image analysis system to determine the cumulative sprout length per spheroid. [0368] In some embodiments, a target-binding protein has a statistically significant effect on an assay described herein, e.g., a cellular assay desrϊbed herein.
[0369] Protein Production
[0370] Standard recombinant nucleic acid methods can be used to express a binding proteinthat binds to Tiel, Tie2, or Ang. See, for example, the techniques described in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, N.Y. (2001) and Ausubel et al, Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley Interscience, N.Y. (1989). Generally, a nucleic acid sequence encoding the binding proteinis cloned into a nucleic acid expression vector. If the protein includes multiple polypeptide chains, each chain can be cloned into an expression vector, e.g., the same or different vectors, that are expressed in the same or different cells. Methods for producing antibodies are also provided below.
[0371] Antibody Production. Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g., E. coli cells. For example, if the Fab is encoded by sequences in a phage display vector that includes a suppressible stop codon between the display entity and a bacteriophage protein (or fragment thereof), the vector nucleic acid can be shuffled into a bacterial cell that cannot suppress a stop codon. In this case, the Fab is not fused to the gene III protein and is secreted into the media.
[0372] Antibodies can also be produced in eukaryotic cells. In one embodiment, the antibodies (e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g., Powers et al. (2001) J Immunol Methods. 251:123-35), Hanseula, or Saccharomyces.
[0373] In one embodiment, antibodies are produced in mammalian cells.
Preferred mammalian host cells for expressing the clone antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. ScL USA 77:4216- 4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) MoI. Biol. 159:601-621), lymphocytic cell lines, e.g., NSO myeloma cells, SP2 cells, COS cells, HEK 293T cells, and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell. [0374] In addition to the nucleic acid sequence encoding the immunoglobulin domain, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhff host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). Another exemplary expression system is the glutamine synthase (GS) vector system available from Lonza Group Ltd. CH (see, e.g., Clark et al. (2004) BioProcess International 2(4):48-52; Barnes et al. (2002) Biotech Bioeng. 81(6):631-639).
[0375] hi an exemplary system for recombinant expression of an antibody, or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/ AdMLP promoter regulatory element or an SV40 enhancer/ AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
[0376] The codon usage can adapted to the codon bias of the host cell, e.g., for
CHO cells it can be adapted for the codon bias Cricetulus griseus genes. In addition, regions of very high (> 80%) or very low (< 30%) GC content can be avoid avoided where possible. During the optimization process following cis-acting sequence motifs were avoided: internal TATA-boxes; chi-sites and ribosomal entry sites; AT-rich or GC-rich sequence stretches; ARE, BSfS, CRS sequence elements; repeat sequences and RNA secondary structures; and (cryptic) splice donor and acceptor sites, branch points. Two STOP codons can be used to ensure efficient termination. The codon optimization of the sequence can be evaluated according to Sharp, P.M., Li, W.H., Nucleic Acids Res. 15 (3), 1987). The standard codon adaptation index (CAI) can be used. Rare codons include those with a quality class between 0-40.
[0377] The invention features isolated nucleic acid molecules that are altered relative to a sequence described herein, e.g., to include improved codons or sequence features, include an isolated nucleic acid molecule that comprises a heavy or light chain coding sequence. For example, at least 30, 40, 45, 50, 60, 65, 70, 75, or 80% of the codons in the heavy or light chain coding sequence are non-rare or frequent codons in a mammalian cell or the heavy or light chain coding sequence includes fewer than 50, 45, 40, 35, 30, 25, 20, 15, 10% rare codons in a mammalian cell, e.g., a Chinese hamster cell {Cricetulus griseus). In one embodiment, the codon adaptation index is greater than 0.6, 0.7, 0.8, 0.85, 0.90, 0.92, 0.94, 0.95, 0.96, 0.97, or 0.98.
[0378] In one embodiment, the heavy chain coding sequence encodes (i) a polypeptide comprising an antibody heavy chain described herein (e.g., an E3 heavy chain as set forth in SEQ ID NO:723), (ii) a polypeptide at least 85, 90, 95, 96, 97, 98, or 99% identical to an antibody heavy chain coding sequence described herein (e.g., SEQ ID NO:723), or (iii) a polypeptide that comprises a heavy chain variable domain sequence having the CDRs of an antibody heavy chain variable domain described herein (e.g., an E3 heavy chain variable domain). In one embodiment, the heavy chain coding sequence differs from SEQ E) NO:703 at at least 2, 3, 5, 6, 8, 9, 10, or 15 codons.
[0379] In one embodiment, the light chain coding sequence encodes (i) a polypeptide comprising an antibody light chain described herein (e.g., an E3 light chain as set forth in SEQ ID NO:724), (ii) a polypeptide at least 85, 90, 95, 96, 97, 98, or 99% identical to an antibody light chain coding sequence described herein (e.g., SEQ ID NO:724), or (iii) a polypeptide that comprises a light chain variable domain sequence having the CDRs of an antibody light chain variable domain described herein (e.g., an E3 light chain variable domain). In one embodiment, the light chain coding sequence differs from SEQ ID NO:702 at at least 3, 5, 6, 8, 9, 10, or 15 codons.
[0380] In one embodiment, for example, one or more of the ala-GCG codons can be changed to GCC; one or more of the arg-CGT codons are changed to CGC; one or more of the pro-CCG codons are changed to CCC, CCT, or CCA; one or more of the ser-TCG codons are changed to TCC; and/or one or more of the thr-ACG codons are changed to ACC.
[0381 ] Codon-altered (e.g., codon-optimized) sequences can be used to produce an antibody. An exemplary method includes providing a mammalian cell that includes an antibody-coding nucleic acid and expressing the nucleic acid in the cell, e.g., maintaining the cell under conditions in which the protein is expressed. The antibody-coding nucleic acid can be providing in a mammalian expression vector, e.g., a vector that is introduced into the cell. The cell can be a non-human mammalian cell, e.g., a CHO cell.
[0382] For antibodies that include an Fc domain, the antibody production system preferably synthesizes antibodies in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. This asparagine is the site for modification with biantennary-type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by Fcγ receptors and complement CIq (Burton and Woof (1992) Adv. Immunol. 51:1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76). In a preferred embodiment, the Fc domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fc domain can also include other eukaryotic post-translational modifications.
[0383] Antibodies can also be produced by a transgenic animal. For example,
U.S 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest. The antibody can be purified from the milk, or for some applications, used directly. [0384] It is also possible to produce antibodies that bind to Tiel , Tie2, or Ang by immunization, e.g., using an animal, e.g., with natural, human, or partially human immunoglobulin loci. Such an antibody can be of any allotype, e.g., a,z allotype, f allotype, or non-A allotype. Non-human antibodies can also be modified to include substitutions for human immunoglobulin sequences, e.g., consensus human amino acid residues at particular positions, e.g., at one or more of the following positions (preferably at least five, ten, twelve, or all): (in the FR of the variable domain of the light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 7OL, 71L, 73L, 85L, 87L, 98L, and/or (in the FR of the variable domain of the heavy chain) 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 6OH, 67H, 68H, 69H, 7OH, 73H, 74H, 75H, 78H, 91H, 92H, 93H, and/or 103H (according to the Kabat numbering). See, e.g., U.S. 6,407,213.
[0385] Tiel production. Methods for producing Tiel ectodomain protein,
Tiel protein, or Tiel liposomes are known in the art. See, e.g., WO 93/14124. Methods for producing Tie2 and Ang are similarly known. See e.g., U.S. Patent Nos. 6,521,424, 6,376,653; WO 96/11269; WO 96/31598 .
[0386] Biotinylation Methods. A variety of methods are available to biotinylate proteins, e.g., an immunoglobulin protein or a target protein. For example, the protein can be incubated with a 5-fold molar excess of sulfo-NHS-SS-biotin in 50 mM HEPES, pH 8.0, 100 mM NaCl overnight at 40C. Free biotin is removed by buffer exchange into PBS, 0.01% Tween 20, e.g., using a BIOMAX® device with a 10 kDa molecular weight cut-off membrane or by dialysis. The number of biotin molecules incorporated per mole of protein can be determined using the HABA assay as described by the manufacturer (Pierce).
[0387] Pharmaceutical Compositions
[0388] In another aspect, the invention provides compositions, e.g., pharmaceutically acceptable compositions, which include an agent that binds to Tiel, Tie2, or Ang, e.g., an antibody molecule, other polypeptide or peptide identified as binding to Tiel, Tie2, or Ang, or described herein, formulated with a pharmaceutically acceptable carrier. Pharmaceutical compositions encompass labeled binding proteins (e.g., for in vivo imaging) as well as therapeutic compositions. [0389] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the binding protein, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
[0390] A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., etal. (1977) J. Pharm. ScL 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
[0391] Compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of humans with antibodies. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular), hi a preferred embodiment, the target-binding protein is administered by intravenous infusion or injection. In another preferred embodiment, the target-binding protein is administered by intramuscular or subcutaneous injection. [0392] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
[0393] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the binding protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
[0394] The binding proteins described herein can be administered by a variety of methods known in the art, although for many applications, the preferred route/mode of administration is intravenous injection or infusion. For example, for therapeutic applications, the target-binding protein can be administered by intravenous infusion, e.g., at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m2 or 7 to 25 mg/m2. The route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[0395] In certain embodiments, the binding protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound described herein by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
[0396] Pharmaceutical compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a pharmaceutical can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4.,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi- chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Of course, many other such implants, delivery systems, and modules are also known.
[0397] In certain embodiments, a binding protein described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic protein crosses the BBB (if desired), it can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes may include one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. CHn. Pharmacol 29:685).
[0398] Dosage regimens are adjusted to provide the optimum desired response
(e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
[0399] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody described herein is 0.1-20 mg/kg, more preferably 1-10 mg/kg. The target-binding antibody can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m2 or about 5 to 30 mg/m2. For binding proteins smaller in molecular weight than an antibody, appropriate amounts can be proportionally less. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. [0400] The pharmaceutical compositions may be prepared using a
"therapeutically effective amount" or a "prophylactically effective amount" of an target-binding protein described herein. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the binding protein to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective dosage" preferably inhibits a measurable parameter, e.g., inflammation or tumor growth rate by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit a measurable parameter, e.g., cancer, can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
[0401] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
[0402] Also within the scope of the invention are kits including the binding protein that binds to Tiel, Tie2, or Ang and instructions for use, e.g., treatment, prophylactic, or diagnostic use. In one embodiment, the instructions for diagnostic applications include the use of the target-binding protein (e.g., antibody or antigen- binding fragment thereof, or other polypeptide or peptide) to detect Tiel, Tie2, or Ang, in vitro, e.g., in a sample, e.g., a biopsy or cells from a patient having an inflammatory disorder or a cancer or neoplastic disorder, or in vivo. In another embodiment, the instructions for therapeutic applications include suggested dosages and/or modes of administration in a patient with a cancer or neoplastic disorder. The kit can further contain at least one additional reagent, such as a diagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agent as described herein, and/or one or more additional target-binding proteins, formulated as appropriate, in one or more separate pharmaceutical preparations.
[0403] In one embodiment, target binding proteins (such as the Tiel antibodies described herein) can be produced from gene-based vectors, such as transgenes or via adenoviral delivery.
[0404] Stabilization and Retention
[0405] In one embodiment, a target-binding agent (e.g., a Tiel -binding protein, polypeptide, antibody, or aptamer described herein) is physically associated with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, lymph, or other tissues.
[0406] For example, a target-binding agent can be associated with a polymer, e.g., a substantially non-antigenic polymers, such as polyalkylene oxides or polyethylene oxides. Suitable polymers will vary substantially by weight. Exemplary polymers include polymers having molecular number average weights ranging from about 200 to about 35,000, from about 1,000 to about 15,000, and 2,000 to about 12,500.
[0407] For example, an target-binding agent can be conjugated to a water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine, D- galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparon.
[0408] Other compounds can also be attached to the same polymer, e.g., a cytotoxin, a label, or another targeting agent, e.g., another target-binding agent or an unrelated agent. Mono-activated, alkoxy-terminated polyalkylene oxides (PAO's), e.g., monomethoxy-terminated polyethylene glycols (mPEG's); C1-4 alkyl-terminated polymers; and bis-activated polyethylene oxides (glycols) can be used for crosslinking. See, e.g., U.S. 5,951,974.
[0409] In its most common form poly(ethylene glycol), PEG, is a linear or branched polyether terminated with hydroxyl groups and having the general structure:
HO(CH2CH2O)n-CH2CH2-OH
PEG can be synthesized by anionic ring opening polymerization of ethylene oxide initiated by nucleophilic attack of a hydroxide ion on the epoxide ring. Particularly useful for polypeptide modification is monomethoxy PEG, mPEG, having the general structure:
CH3O-(CH2CH2O)n-CH2CH2-OH
For further description, see, e.g., Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476.
[0410] In one embodiment, the polymer prior to cross-linking need not be, but preferably is, water soluble. Generally, after crosslinking, the product is water soluble, e.g., exhibits a water solubility of at least about 0.01 mg/ml, and more preferably at least about 0.1 mg/ml, and still more preferably at least about 1 mg/ml. In addition, the polymer should not be highly immunogenic in the conjugate form, nor should it possess viscosity that is incompatible with intravenous infusion or injection if the conjugate is intended to be administered by such routes. [0411] In one embodiment, the polymer contains only a single group which is reactive. This helps to avoid cross-linking of protein molecules. However, it is within the scope herein to maximize reaction conditions to reduce cross-linking, or to purify the reaction products through gel filtration or ion exchange chromatography to recover substantially homogenous derivatives. In other embodiments, the polymer contains two or more reactive groups for the purpose of linking multiple agents to the polymer backbone. Again, gel filtration or ion exchange chromatography can be used to recover the desired derivative in substantially homogeneous form. [0412] The molecular weight of the polymer can range up to about 500,000 D, and preferably is at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. The molecular weight chosen can depend upon the effective size of the conjugate to be achieved, the nature (e.g. structure, such as linear or branched) of the polymer, and the degree of derivatization.
[0413] The covalent crosslink can be used to attach a target-binding agent
(e.g., a protein) to a polymer, for example, crosslinking to the N-terminal amino group and epsilon amino groups found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups. The polymer may be covalently bonded directly to the target-binding protein without the use of a multifunctional (ordinarily bifunctional) crosslinking agent. Covalent binding to amino groups is accomplished by known chemistries based upon cyanuric chloride, carbonyl diimidazole, aldehyde reactive groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, activated succinimidyl esters, activated dithiocarbonate PEG, 2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate activated PEG.) Carboxyl groups can be derivatized by coupling PEG-amine using carbodiimide. Sulfhydryl groups can be derivatized by coupling to maleimido- substituted PEG (e.g. alkoxy-PEG amine plus sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate) WO 97/10847 or PEG-maleimide commercially available from Shearwater Polymers, Inc., Huntsville, Ala.). Alternatively, free amino groups on the binding protein (e.g. epsilon amino groups on lysine residues) can be thiolated with 2-imino-thiolane (Traut's reagent) and then coupled to maleimide-containing derivatives of PEG, e.g., as described in Pedley et al., Br. J. Cancer, 70: 1126-1130 (1994).
[0414] Functionalized PEG polymers that can be attached to a target-binding agent (e.g., protein) are available, e.g., from Shearwater Polymers, Inc. (Huntsville, Ala.). Such commercially available PEG derivatives include, e.g., amino-PEG, PEG amino acid esters, PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propionic acid, PEG amino acids, PEG succinimidyl succinate, PEG succinimidyl propionate, succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidyl esters of amino acid PEGs, PEG- oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether, PEG-aldehyde, PEG vinylsulfone, PEG-maleimide, PEG-orthopyridyl- disulfide, heterofiinctioiial PEGs, PEG vinyl derivatives, PEG silanes, and PEG phospholides. The reaction conditions for coupling these PEG derivatives may vary depending on the Tie 1 -binding protein, the desired degree of PEGylation, and the PEG derivative utilized. Some factors involved in the choice of PEG derivatives include: the desired point of attachment (such as lysine or cysteine R-groups), hydrolytic stability and reactivity of the derivatives, stability, toxicity and antigenicity of the linkage, suitability for analysis, etc. Specific instructions for the use of any particular derivative are available from the manufacturer.
[0415] The conjugates of an target-binding agent (e.g., a Tiel binding protein) and a polymer can be separated from the unreacted starting materials, e.g., by gel filtration or ion exchange chromatography, e.g., HPLC. Heterologous species of the conjugates are purified from one another in the same fashion. Resolution of different species (e.g., containing one or two PEG residues) is also possible, e.g., due to the difference in the ionic properties of unreacted amino acids. See, e.g., WO 96/34015.
[0416] A target binding protein can also be physically associated with a protein that provides a stabilizing or retention function, e.g., an albumin, e.g., human serum albumin. US 20040171794 describes exemplary methods for physically associating a protein with serum albumin. For exemplary, human albumin sequences or fragments thereof, see EP 201 239, EP 322 094 WO 97/24445, WO95/23857 especially the mature form of human albumin as shown in SEQ ID NO:18 of US 20040171794 and WO 01/79480 or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof. Other exemplary human serum albumin proteins can include one or both of the following sets of point mutations Leu-407 to Ala, Leu-408 to VaI, Val-409 to Ala, and Arg-410 to Ala; or Arg-410 to Ala, Lys-413 to GIn, and Lys-414 to GIn (see, e.g., International Publication No. WO95/23857, with reference to SEQ ID NO:18 of US 20040171794).
[04171 Aptamers
[0418] In one embodiment, the invention also features target protein-binding agents such as aptamers. The term nucleic acid "aptamer," as used herein, refers to a nucleic acid molecule which has a conformation that includes an internal non-duplex nucleic acid structure of at least 5 nucleotides. An aptamer can be a single-stranded nucleic acid molecule which has regions of self-complementarity. Exemplary aptamers include nucleic acid molecules that bind to a target molecule other than a nucleic acid, e.g., to Tiel, Tie2, or Ang. Particular aptamers may also modulate formation of a Tie complex or have one or more properties of a target binding agent described herein and can be used in place of a target binding protein.
[0419] Aptamers can be screened in vitro since a selected aptamer can be recovered by standard nucleic acid amplification procedures. The method can be enhanced, e.g., in later rounds of selection, by splitting selected aptamers into pools and modifying each aptamer in the pool with a detectable label such as a fluorophore. Pools having aptamers that functionally alter the properties of the label can be identified. Such pools can be repeatedly split and reanalyzed to identify the individual aptamers with the desired properties (see, e.g., Jhaveri et al. Nature Biotechnol. 18:1293).
[0420] hi addition, aptamers can be screened for activity in vivo. For example, shuffled nucleic acids can be cloned into an expression vector that is introduced into cells. RNA aptamers resulting from the expressed shuffled nucleic acids can be screened for a biological activity. Cells having the activity can be isolated and the expression vector for the selected RNA aptamer recovered.
[0421] An important feature of therapeutic oligomers (e.g., aptamers) is the design of the backbone of the administered oligomer. In some embodiments, the backbone contains internucleoside linkages that are stable in vivo and is structured such that the oligomer is resistant to endogenous nucleases, such as nucleases that attack the phosphodiester linkage. At the same time, the oligomer retains its ability to hybridize to the target DNA or RNA (Agarwal, K. L. et al. (1979) Nucleic Acids Res. 6:3009; Agarwal, S. et al. (1988) Proc. Natl Acad. Sd USA 85:7079). Modified oligonucleotides can be constructed using alternate internucleoside linkages. Several of these exemplary linkages are described in Uhhnann, E. and Peyman, A. (1990) Chemical Reviews 90:543-584. Among these are methylphosphonates (wherein one of the phosphorus-linked oxygens has been replaced by methyl); phosphorothioates (wherein sulphur replaces one of these oxygens) and various amidates (wherein NH2 or an organic amine derivative, such as morpholidates or piperazidates, replace an oxygen). These substitutions confer enhanced stability. WO 91/15500 teaches various oligonucleotide analogs in which one or more of the internucleotide linkages are replaced by a sulfur based linkage, typically sulfamate diesters, which are isosteric and isoelectric with the phosphodiester. WO 89/12060 similarly discloses linkages containing sulfides, sulfoxides, and sulfones. WO 86/05518 suggests a variant of stereoregular polymeric 3',5'linkages. U.S. Pat. No. 5,079,151 discloses a msDNA molecule of branched RNA linked to a single strand DNA via a 2',5' phosphodiester linkage. U.S. Pat. No. 5,264,562 describes modified linkages of the formula -Y'CX'2 Y'~ wherein Y1 is independently O or S and wherein each X' is a stabilizing substituent and independently chosen. Morpholino-type internucleotide linkages are described in U.S. Pat. No. 5,034,506 and in some cases give rise to an increased affinity of the oligomer for complementary target sequences. U.S. Pat. Nos. 5,264,562 5,596,086 disclose modified oligonucleotides having modified nucleoside linkages which are capable of strong hybridization to target RNA and DNA.
[04221 Treatments
[0423] Binding agents that bind to Tiel , Tie2, or Ang have therapeutic and prophylactic utilities. For example, these binding agents can be administered to cells in culture, e.g. in vitro or ex vivo, or can be administered to a subject, e.g., in vivo, to treat, prevent, and/or diagnose a variety of disorders, such as endothelial cell disorders, blood vessel development disorders, wound healing, inflammatory diseases and cancers, particularly metastatic cancers. The term "treat" or "treatment" refers to the application or administration of an agent, alone or in combination with one or more other agents (e.g., a second agent) to a subject, e.g., a patient, e.g., a patient who has a disorder (e.g., a disorder as described herein), a symptom of a disorder or a predisposition for a disorder, e.g., to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder. Treating a cell refers to a reduction in an activity of a cell, e.g., ability of an endothelial cell to form tubes or vessels. A reduction does not necessarily require a total elimination of activity, but a reduction, e.g., a statistically significant reduction, in the activity or the number of the cell.
[0424] As used herein, an amount of a target binding agent effective to treat a disorder, or a "therapeutically effective amount" refers to an amount of the binding agent which is effective, upon single or multiple-dose administration to a subject, in treating a cell, e.g., an endothelial cell (e.g., a Tiel -expressing endothelial cell) or cancer cell (particularly a metastatic cell thereof), or in prolonging curing, alleviating, relieving or improving a subject with a disorder as described herein beyond that expected in the absence of such treatment, hi some cases, a therapeutically effective amount can be ascertained by evaluating the ability of the binding agent to reduce tumor size of a xenograft in a nude mouse model relative to an untreated control mouse. As used herein, "inhibiting the growth" of a tumor or other neoplasm refers to slowing, interrupting, arresting or stopping its growth and metastases and does not necessarily indicate a total elimination of the neoplastic growth.
[0425] As used herein, an amount of an target-binding agent effective to prevent a disorder, or a "a prophylactically effective amount" of the binding agent refers to an amount of a target binding agent, e.g., a Tiel-binding protein, e.g., a Tiel- binding antibody described herein, which is effective, upon single- or multiple-dose administration to the subject, for preventing or delaying the occurrence of the onset or recurrence of a disorder, e.g., an endothelial cell-related disorder, a blood vessel development disorder, an inflammatory disease or a cancer.
[0426] Subjects that can be treated include human and non-human animals.
For example, the human can be a human patient having a disorder characterized by abnormal cell proliferation or cell differentiation. The term "non-human animals" includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, sheep, dog, cow, pig, etc.
[0427] A binding agent described herein can be used to reduce angiogenesis in a subject, e.g., to treat a cancer (e.g., a solid tumor) or an angiogenesis-associated disorder. The method includes administering the binding to the subject, e.g., in an amount effective to modulate angiogenesis, a symptom of the disorder, or progression of the disorder. The agent (e.g., a Tiel-binding protein, e.g., an anti-Tiel antibody, e.g., E3) may be administered multiple times (e.g., at least two, three, five, or ten times) before a therapeutically effective amount is attained.
[0428] The binding agent, e.g., a Tiel binding protein, can be used to treat or prevent cancer, hi one embodiment, reduction in Tiel activity by a Tiel-binding protein can reduce or prevent angiogenesis near and around the tumor, thereby reducing or preventing tumor growth, hi another embodiment, the neoplasia includes endothelial or hematopoietic cells that are proliferating abnormally. A Tiel-binding protein can be used to modulate the cells of a cancer themselves, e.g., to kill or ablate a neoplastic cell that expresses Tiel. For example, the cell is a hematopoietic cell.
[0429] Examples of cancers that can be treated include, but are not limited to, solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract (e.g., renal, urothelial cells), pharynx, prostate, ovary as well as adenocarcinomas which include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and so forth. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the Tiel binding proteins and other agents described herein.
[0430] Still further examples of solid tumors that can be treated include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastrointestinal system carcinomas, colon carcinoma, pancreatic cancer, breast cancer, genitourinary system carcinomas, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, endocrine system carcinomas, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastonia, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
[0431] A Tiel-binding protein can also be used to inhibit the proliferation of hyperplastic/neoplastic cells of hematopoietic origin, e.g., cells arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof, particularly such cells that express Tiel . For instance, the binding proteins described herein can be used for the treatment of various myeloid disorders including, but not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) CritRev. in Oncol./Hemotol. 11 :261-91). Lymphoid malignancies which may be treated include, but are not limited to acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include non-Hodgkin's lymphoma and variants thereof, peripheral T-cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin's disease. As Tiel has been shown to be upregulated in acute myelogenous leukemia and myelodysplastic syndrome (Verstovsek et al., 2001, Leuk, Lymphoma), B cell chronic lymphocytic leukemia (Aguayo et al, 2001. Leukemia Research 25(4):279-85.)5 binding proteins that interact with Tiel can be used to detect, treat, or prevent these diseases.
[0432] Accordingly, a subject having or at risk for a hematopoietic disorder, e.g., a hematopoietic cancer, can be treated by administering a Tiel binding protein, e.g. a Tiel binding protein that increases Tiel homodimerization, or a binding protein that antagonizes Tie complex formation. For example, the Tiel binding protein can be an anti-Tiel antibody, e.g., an antibody described herein. The administration of the binding protein can include multiple administrations, e.g., to achieve a therapeutic concentration using more than one dose. For example, the administrations can be about once a week, every second or third day, etc.
[0433 ] Methods of administering Tie 1 -binding proteins and other agents are also described in "Pharmaceutical Compositions". Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. The binding proteins can be used as competitive agents to inhibit, reduce an undesirable interaction, e.g., between a natural or pathological agent and the Tiel.
[0434] In one embodiment, the Tiel-binding proteins are used to inhibit (e.g., inhibit at least one activity of, reduce proliferation, migration, growth or viability) of a cell, e.g., an endothelial cell in vivo. The binding proteins can be used by themselves or conjugated to an agent, e.g., a cytotoxic drug, cytotoxin enzyme, or radioisotope. This method includes: administering the binding protein alone or attached to a cytotoxic drug, to a subject requiring such treatment. [0435] Since the Tiel-binding proteins recognize Tie 1 -expressing endothelial cells and can bind to endothelial cells that are associated with (e.g., in proximity of or intermingled with) cancer cells, e.g., cancerous lung, liver, colon, breast, ovarian, epidermal, laryngeal, and cartilage cells, and particularly metastatic cells thereof, Tiel-binding proteins can be used to inhibit (e.g., inhibit at least one activity, reduce growth and proliferation, or kill) any such cells and inhibit angiogenesis. Reducing endothelial cell activity near a cancer can indirectly inhibit (e.g., inhibit at least one activity, reduce growth and proliferation, or kill) the cancer cells which may be dependent on the endothelial cells for nutrients, growth signals and so forth.
[0436] Alternatively, the binding proteins bind to cells in the vicinity of the cancerous cells, but are sufficiently close to the cancerous cells to directly or indirectly inhibit (e.g., inhibit at least one activity, reduce growth and proliferation, or kill) the cancers cells. Thus, the Tiel-binding proteins (e.g., modified with a toxin, e.g., a cytotoxin) can be used to selectively inhibit (e.g., kill or ablate cells in cancerous tissue (including the cancerous cells themselves and endothelial cells associated with or invading the cancer).
[0437] The binding proteins may be used to deliver a variety of cytotoxic drugs including therapeutic drugs, a compound emitting radiation, molecules of plants, fungal, or bacterial origin, biological proteins, and mixtures thereof. The cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as toxins short-range radiation emitters, e.g., short-range, high-energy α-emitters.
[0438] To kill or ablate normal, benign hyperplastic, or cancerous cells, a first binding protein is conjugated with a prodrug which is activated only when in close proximity with a prodrug activator. The prodrug activator is conjugated with a second binding protein, preferably one which binds to a non-competing site on the target molecule. Whether two binding proteins bind to competing or non-competing binding sites can be determined by conventional competitive binding assays. Exemplary drug-prodrug pairs are described in Blakely et al., (1996) Cancer Research, 56:3287-3292.
[0439] The Tiel-binding proteins can be used directly in vivo to eliminate antigen-expressing cells via natural complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). The binding proteins described herein can include complement binding effector domain, such as the Fc portions from IgGl , -2, or -3 or corresponding portions of IgM which bind complement. In one embodiment, a population of target cells is ex vivo treated with a binding agent described herein and appropriate effector cells. The treatment can be supplemented by the addition of complement or serum containing complement. Further, phagocytosis of target cells coated with a binding protein described herein can be improved by binding of complement proteins. In another embodiment target, cells coated with the binding protein which includes a complement binding effector domain are lysed by complement.
[0440] Use of the therapeutic methods described herein to treat cancers has a number of benefits. Tiel expression may be induced in response to hypoxic signals that can arise within the interior of a tumor to stimulate changes in vasculature, including blood and lymphatic vessels so as to increase nutrient and oxygen supply to the tumor. Certain Tiel -binding antibodies (e.g., E3 and related antibodies) may be particularly effective because they can inhibit changes to tumor vasculature and may cause a decrease in intra-tumor pressure. These agents may also be well suited as therapeutics in situations in which conventional agents have difficulty in penetrating into a tumor. Furthermore, Tiel binding proteins may leave hematopoiesis unaffected. Treatment can be effectively monitored with clinical parameters. Alternatively, these parameters can be used to indicate when such treatment should be employed.
[0441] A Tiel binding protein, e.g. a Tiel binding protein that increases Tiel homodimerization, or a binding protein that antagonizes Tie complex formation can be administered to a subject to treat or prevent an inflammatory disorder, e.g., psoriasis or rheumatoid arthritis.
[0442] Psoriasis. Psoriasis is a chronic skin disease, characterized by scaling and inflammation. When psoriasis develops, typically patches of skin thicken, redden, and become covered with silvery scales, referred to as plaques. Psoriasis most often occurs on the elbows, knees, scalp, lower back, face, palms, and soles of the feet. The disease also may affect the fingernails, toenails, and the soft tissues inside the mouth and genitalia. About 10 percent of people with psoriasis have joint inflammation that produces symptoms of arthritis. Patients can be evaluated using a static Physician Global Assessment (sPGA), and receive a category score ranging from six categories between clear and very severe. The score is based on plaque, scaling, and erythema. The therapeutic methods herein can be used to achieve an improvement for at least one of these indicia.
[0443] Rheumatoid arthritis ("RA") is a chronic inflammatory disease that causes pain, swelling, stiffness, and loss of function, primarily the joints. RA frequently begins in the synovium, the membrane that surrounds a joint creating a protective sac. In many individuals suffering from RA, leukocytes infiltrate from the circulation into the synovium causing continuous abnormal inflammation (e.g., synovitis). Consequently, the synovium becomes inflamed, causing warmth, redness, swelling, and pain. The collagen in the cartilage is gradually destroyed, narrowing the joint space and eventually damaging bone. The inflammation causes erosive bone damage in the affected area. During this process, the cells of the synovium grow and divide abnormally, making the normally thin synovium thick and resulting in a joint that is swollen and puffy to the touch. RA can be assessed by a variety of clinical measures. Some exemplary indicia include the total Sharp score (TSS), Sharp erosion score, and the HAQ disability index. The therapeutic methods herein can be used to achieve an improvement for at least one of these indicia.
[0444] A Tiel binding protein (e.g. a Tiel binding protein that increases Tiel homodimerization) or a binding protein that antagonizes Tie complex formation can be administered to a subject to treat or prevent a retinal disorder, e.g., a proliferative retinopathy, such as diabetic retinopathy, ischemic retinopathy, or retinopathy of prematurity; choroidal neovascularization; lens neovasculation; corneal neovascularization; iridial neovascularization; or conjunctival neovascularization. The binding protein can be used to reduce the risk of retinal detachment associated with pathological ocular neovascularization, hi some cases, the binding protein is administered by subconjunctival administration.
[0445] Combination Therapies
[0446] Binding proteins described herein can be administered in combination with one or more of the other therapies for treating cancers, including, but not limited to: surgery; radiation therapy, and chemotherapy. For example, proteins that antagonize Tie complex formation or that modulate Tie signalling activity (including, e.g., proteins that promote Tiel homodimerization and/or phosphorylation) can also be used in combination with other anti-cancer therapies, such as radiation therapy, chemotherapy, surgery, or administration of a second agent. For example, the second agent can be one that targets or negatively regulates the VEGF signaling pathway. Examples of this latter class include VEGF antagonists (e.g., anti-VEGF antibodies such as bevacizumab) and VEGF receptor antagonists (e.g., anti-VEGF receptor antibodies). One particularly combination includes bevacizumab. The combination can further include 5 -FU and leucovorin, and/or irinotecan.
[0447] The term "combination" refers to the use of the two or more agents or therapies to treat the same patient, wherein the use or action of the agents or therapies overlap in time. The agents or therapies can be administered at the same time (e.g., as a single formulation that is administered to a patient or as two separate formulations administered concurrently) or sequentially in any order. Sequential administrations are administrations that are given at different times. The time between administration of the one agent and another agent can be minutes, hours, days, or weeks. The use of a Tiel binding protein described herein can also be used to reduce the dosage of another therapy, e.g., to reduce the side-effects associated with another agent that is being administered, e.g., to reduce the side-effects of an anti-VEGF antibody such as bevacizumab. Accordingly, a combination can include administering a second agent at a dosage at least 10, 20, 30, or 50% lower than would be used in the absence of the Tiel binding protein.
[0448] In addition, a subject can be treated for an angiogenesis-associated disorder by administering to the subject a first and second agent. For example, the first agent modulates early stage angiogenesis and the second agent modulates a subsequent stage of angiogenesis or also modulates early stage angiogenesis. The first and second agents can be administered using a single pharmaceutical composition or can be administered separately. In one embodiment, the first agent is a VEGF pathway antagonist (e.g., an inhibitor of a VEGF (e.g., VEGF-A, -B, or -C) or a VEGF receptor (e.g., KDR or VEGF receptor III (Flt4)) or a bFGF pathway antagonist (e.g., an antibody that binds to bFGF or a bFGF receptor). Other VEGF pathway antagonists are also described, herein and elsewhere. In one embodiment, the second agent inhibits or decreases assembly and stabilization of the blood vessels, disrupts maintenance of blood or lymphatic vessels, or alters distribution of lymphatic vessels in tumors. For example, the second agent comprises inhibits a Tie complex formation or promotes Tiel homodimerization. For example, the second agent is a Tiel binding protein described herein.
[0449] Once a tumor reaches a certain size (e.g., ~ 1-2 mm), the tumor requires new vasculature prior to increasing its mass. An early stage of tumor angiogenesis can include a signal from the tumor, e.g., secretion of VEGF, to stimulate the growth of new blood vessels from the host and infiltration of the tumor by the vessels. VEGF can, for example, stimulate proliferation of endothelial cells that are then assembled into blood vessels. A late stage of tumor angiogenesis can include a signal that leads to the assembly and stabilization of the blood vessels. This assembly and stabilization may involve interaction between the endothelial cells and the pericytes that surround the endothelial cells of the vessels. Tiel, for example, may play a role in the assembly and stabilization of the vessels and in maintaining the association between the pericytes and endothelial cells. Thus, an effective therapy to treat angiogenesis-related disorders can involve a combination of an agent that modulates an early stage angiogenesis (e.g., VEGF pathway antagonists, e.g., anti- VEGF (e.g., bevacizumab) or anti-VEGF receptor (e.g., anti-KDR) antibodies; or antagonists of other pro-angiogenic pathways, e.g., anti-bFGF antibodies or anti- bFGF receptor (e.g., anti-bFGF receptor-1, -2, -3) antibodies) and an agent that modulates a late stage of tumor angiogenesis (e.g., antagonists of Tiel (e.g., anti-Tiel antibodies (e.g., an antibody disclosed herein, e.g., an E3 antibody)), of Tie2 (e.g., anti-Tie2 antibodies), or of Angs (e.g., anti-Ang antibodies (e.g., anti-Ang2 antibodies) or anti-Ang2 peptides (e.g., inhibitory Ang2 peptides)). One or more of these agents can be used in combination. One or more of these agents may also be used in combination with other anti-cancer therapies, such as radiation therapy or chemotherapy.
[0450] Exemplary VEGF receptor antagonists include inhibitors of VEGF receptor tyrosine kinase activity. 4-[4-(l-Amino-l-methylethyl)phenyl]-2-[4-(2- moφholin-4-yl-ethyl)phenylamino]pyrimidine-5-carbonitrile (JNJ-17029259) is one of a structural class of 5-cyanopyrimidines that are orally available, selective, nanomolar inhibitors of the vascular endothelial growth factor receptor-2 (VEGF-R2). Additional examples include: PTK-787/ZK222584( Astra-Zeneca), SU5416, SUl 1248 (Pfizer), and ZD6474 ([N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(l- methylpiperidm-4-yl)methoxy]quinazohn-4-amme]). Still other agents that can be used in combination with Tiel -binding proteins are broad specificity tyrosine kinase inhibitors, e.g., SU6668. See, e.g., Bergers, B. et al. (2003) J. Clin. Invest. Ill, 1287-1295.
[0451] The second agent or therapy can also be another anti-cancer agent or therapy. Nonlimiting examples of anti-cancer agents include, e.g., anti-microtubule agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis, radiation, and antibodies against other tumor- associated antigens (including naked antibodies, immunotoxins and radioconjugates). Examples of the particular classes of anti-cancer agents are provided in detail as follows: antitubulin/antimicrotubule, e.g., paclitaxel, vincristine, vinblastine, vindesine, vinorelbin, taxotere; topoisomerase I inhibitors, e.g., irinotecan, topotecan, camptothecin, doxorubicin, etoposide, mitoxantrone, daunorubicin, idarubicin, teniposide, amsacrine, epirubicin, merbarone, piroxantrone hydrochloride; antimetabolites, e.g., 5-fluorouracil (5-FU), methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, cytarabine/Ara-C, trimetrexate, gemcitabine, acivicin, alanosine, pyrazofurin, N-Phosphoracetyl-L-Asparate=PALA, pentostatin, 5-azacitidine, 5-Aza 2'-deoxycytidine, ara-A, cladribine, 5 - fluorouridine, FUDR, tiazofurin, N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yhnethyl)-N- methylamino]-2-thenoyl]-L-glutamic acid; alkylating agents, e.g., cisplatin, carboplatin, mitomycin C, BCNU=Carmustme, melphalan, thiotepa, busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen mustard, uracil mustard, pipobroman, 4-iρomeanol; agents acting via other mechanisms of action, e.g., dihydrolenperone, spiromustine, and desipeptide; biological response modifiers, e.g., to enhance anti-tumor responses, such as interferon; apoptotic agents, such as actinomycin D; and anti-hormones, for example anti-estrogens such as tamoxifen or, for example antiandrogens such as 4'-cyano-3-(4- fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl) propionanilide.
[0452] A combination therapy can include administering an agent that reduces the side effects of other therapies. The agent can be an agent that reduces the side effects of anti-cancer treatments. For example, the agent can be leucovorin.
[0453] Combination therapies that include administering a Tiel binding protein or other binding protein described herein can also be used to treat a subject having or at risk for another angiogenesis related disorder (e.g., a disorder other than cancer, e.g., disorders that include undesired endothelial cell proliferation or undesirable inflammation, e.g., rheumatoid arthritis.
F0454] Diagnostic Uses
[0455] Binding proteins that bind to Tiel (e.g., antibodies, e.g., an antibody described herein) have in vitro and in vivo diagnostic, therapeutic and prophylactic utilities.
[0456] In one aspect, the invention provides a diagnostic method for detecting the presence of a Tiel, in vitro (e.g., a biological sample, such as tissue, biopsy, e.g., a cancerous tissue) or in vivo (e.g., in vivo imaging in a subject). The method includes: (i) contacting a sample with Tiel -binding protein; and (ii) detecting formation of a complex between the Tiel -binding protein and the sample. The method can also include contacting a reference sample (e.g., a control sample) with the binding protein, and determining the extent of formation of the complex between the binding protein and the sample relative to the same for the reference sample. A change, e.g., a statistically significant change, in the formation of the complex in the sample or subject relative to the control sample or subject can be indicative of the presence of Tiel in the sample. The Tiel -binding protein can be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
[0457] Complex formation between the Tiel -binding protein and Tiel can be detected by measuring or visualizing either the binding protein bound to the Tiel or unbound binding protein. Conventional detection assays can be used, e.g., an enzyme-linked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry. Further to labeling the Tiel -binding protein, the presence of Tiel can be assayed in a sample by a competition immunoassay utilizing standards labeled with a detectable substance and an unlabeled Tiel -binding protein. In one example of this assay, the biological sample, the labeled standards and the Tiel binding agent are combined and the amount of labeled standard bound to the unlabeled binding protein is determined. The amount of Tiel in the sample is inversely proportional to the amount of labeled standard bound to the Tiel binding agent.
[0458] Fluorophore and chromophore labeled binding proteins can be prepared. Since antibodies and other proteins absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm and preferably above 400 nm. A variety of suitable fluorescers and chromophores are described by Stryer (1968) Science, 162:526 and Brand, L. et al. (1972) Annual Review of Biochemistry, 41:843-868. The binding proteins can be labeled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Patent Nos. 3,940,475, 4,289,747, and 4,376,110. One group of fluorescers having a number of the desirable properties described above is the xanthene dyes, which include the fluoresceins and rhodamines. Another group of fluorescent compounds are the naphthylamines. Once labeled with a fluorophore or chromophore, the binding protein can be used to detect the presence or localization of the Tiel in a sample, e.g., using fluorescent microscopy (such as confocal or deconvolution microscopy).
[0459] Histological Analysis. Immunohistochemistry can be performed using the binding proteins described herein. For example, in the case of an antibody, the antibody can synthesized with a label (such as a purification or epitope tag), or can be detectably labeled, e.g., by conjugating a label or label-binding group. For example, a chelator can be attached to the antibody. The antibody is then contacted to a histological preparation, e.g., a fixed section of tissue that is on a microscope slide. After an incubation for binding, the preparation is washed to remove unbound antibody. The preparation is then analyzed, e.g., using microscopy, to identify if the antibody bound to the preparation. The method can be used to evaluate an endothelial cell or tissue formed by endothelial cells, e.g., blood vessels. The antibody (or other polypeptide or peptide) can be unlabeled at the time of binding. After binding and washing, the antibody is labeled in order to render it detectable.
[0460] Protein Arrays. The Tiel -binding protein can also be immobilized on a protein array. The protein array can be used as a diagnostic tool, e.g., to screen medical samples (such as isolated cells, blood, sera, biopsies, and the like). Of course, the protein array can also include other binding proteins, e.g., that bind to Tiel or to other target molecules, such as hyaluronic acid. [0461] Methods of producing polypeptide arrays are described, e.g., in De
Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103-111; Ge (2000) Nucleic Acids Res. 28, e3, 1-VII; MacBeath and Schreiber (2000) Science 289:1760-1763; WO 01/40803 and WO 99/51773A1. Polypeptides for the array can be spotted at high speed, e.g., using commercially available robotic apparati. The array substrate can be, for example, nitrocellulose, plastic, glass, e.g., surface-modified glass. The array can also include a porous matrix, e.g., acrylamide, agarose, or another polymer.
[0462] For example, the array can be an array of antibodies, e.g., as described in De Wildt, supra. Cells that produce the binding proteins can be grown on a filter in an arrayed format. Polypeptide production is induced, and the expressed polypeptides are immobilized to the filter at the location of the cell. A protein array can be contacted with a labeled target to determine the extent of binding of the target to each immobilized polypeptide. If the target is unlabeled, a sandwich method can be used, e.g., using a labeled probed, to detect binding of the unlabeled target. Information about the extent of binding at each address of the array can be stored as a profile, e.g., in a computer database. The protein array can be produced in replicates and used to compare binding profiles, e.g., of a target and a non-target.
[0463] FACS. (Fluorescent Activated Cell Sorting). The target-binding protein can be used to label cells, e.g., cells in a sample (e.g., a patient sample). The binding protein can also be attached (or attachable) to a fluorescent compound. The cells can then be sorted using fluorescent activated cell sorted (e.g., using a sorter available from Becton Dickinson Immunocytometry Systems, San Jose CA; see also U.S. 5,627,037; 5,030,002; and 5,137,809). As cells pass through the sorter, a laser beam excites the fluorescent compound while a detector counts cells that pass through and determines whether a fluorescent compound is attached to the cell by detecting fluorescence. The amount of label bound to each cell can be quantified and analyzed to characterize the sample.
[0464] The sorter can also deflect the cell and separate cells bound by the binding protein from those cells not bound. The separated cells can be cultured and/or characterized. [0465] In Vivo Imaging. In still another embodiment, the invention provides a method for detecting the presence of a Tiel-expressing cancerous tissues in vivo. The method includes: administering the Tie 1 -binding protein to a subject; and detecting the Tie 1 -binding protein in the subject. The detecting can include determining location or time of formation of the complex. The method can include scanning or otherwise imaging the subject, e.g., a region of the subject's body. Another method includes (i) administering to a subject (e.g., a patient having a cancer or neoplastic disorder) a Tiel-binding antibody, conjugated to a detectable marker; (ii) exposing the subject to a means for detecting said detectable marker to the Tiel- expressing tissues or cells. For example, the method can be used visualize blood vessels or the location of endothelial cells, e.g., Tiel-expressing endothelial cells. The subject can be imaged, e.g., by NMR or other tomographic means.
[0466] Examples of labels useful for diagnostic imaging include radiolabels such as 1311, 111In, 1231, 99mTc, 32P, 1251, 3H, 14C, and 188Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography ("PET") scanner, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. Short-range radiation emitters, such as isotopes detectable by short-range detector probes can also be employed. The binding protein can be labeled with such reagents using known techniques. For example, see Wensel and Meares (1983) Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York for techniques relating to the radiolabeling of antibodies and D. Colcher et al. (1986) Meth. Enzymol. 121: 802-816.
[0467] A radiolabeled binding protein can also be used for in vitro diagnostic tests. The specific activity of an isotopically-labeled protein depends upon the half-life, the isotopic purity of the radioactive label, and how the label is incorporated into the protein.
[0468] Effective imaging agents for tumor-associated neo-vasculature are needed. Tiel is up regulated on tumor-associated vasculature. The binding proteins described herein can be used to image such vasculature.
The binding proteins described herein can be used for imaging in several ways. A binding protein can be physically associated, e.g., coupled to a chelator for imaging agents such as 99mTc, 186Re, or 188Re. 99mTc and 188Re emit gamma rays suitable for single photon emission computer tomography (SPECT) imaging. Radioactive fluorine (18F), indium (111In), iodine (1231, 131I), gallium (68Ga, 67Ga), carbon (11C), thallium (201Tl), and other elements may be used as imaging agents.
[0469] The binding proteins can also be attached, covalently or non- covalently, to a particle, e.g., a nano-particle, that includes a radionuclide or spin labels suitable for use as an imaging agent. The binding proteins can be linked to a spin label that would allow imaging through MRI. Botnar et al. {Circulation. (2004) 109:2023-2029.) describe MRI imaging using an exemplary gadolinium-labeled peptide. The binding proteins described herein can be similarly labeled for imaging.
[0470] Chen et al. (J. Nucl. Med., (2004) 45:1776-1783) showed that coupling a small PEG molecule (average molecular weight 3.4 KDa) improved that pharmacodynamics of an ovft-binding peptide. Binding proteins (e.g., Tiel, Tie2, or Ang binding proteins) can be coupled to PEG molecules to adjust the clearance rate and pathway.
[0471 ] Positron Emission Tomography (PET) can be used with imaging agents such as positron emitters such as 64Cu and 18F. These isotopes are becoming more readily available. 6 Cu can be captured in the chelator DOTA. DOTA derivatives can be covalently linked to proteins. In one embodiment, one or more DOTA derivatives are attached to a binding protein (e.g., a Fab) through a lysine side group.
[0472] Fabs are useful binding agents for imaging because they: a) clear from the system fairly raipdly, allowing imaging within a few hours of injection, and b) penetrate tumors efficiently.
[0473] Fabs that bind to Tiel , Tie2, or Ang can be produced, e.g., in E. coli or in eukaryotic cells. The Fabs can be purified by chromatography over protein A. Ion exchange chromatography can also be used. For use in imaging, covalent attachment of a chelating group suitable to the desired radionuclide or other imaging agent allows the Fab to be labeled at the time of use. The Fabs can also have spin labels attached to allow MRI imaging. Fabs can also be attached to particles (e.g., nano-particles) that include a radionuclide or spin label suitable for imaging. In particular embodiments, Fabs may be coupled to PEG molecules to adjust the rate and pathway of clearance. In other embodiments, the Fabs are not coupled to PEG, e.g., to maintain their rapid clearance properties.
[0474] Procedures for labeling polypeptides with the radioactive isotopes
(such as 14C, 3H, 35S, 1251, 32P, 131I) are generally known. For example, tritium labeling procedures are described in U.S. Patent No. 4,302,438. Iodinating, tritium labeling, and 35S labeling procedures, e.g., as adapted for murine monoclonal antibodies, are described, e.g., by Goding, J. W. (Monoclonal antibodies : principles and practice production and application of monoclonal antibodies in cell biology, biochemistry, and immunology 2nd ed. London ; Orlando : Academic Press, 1986. pp 124-126) and the references cited therein. Other procedures for iodinating polypeptides, such as antibodies, are described by Hunter and Greenwood (1962) Nature 144:945, David et al. (1974) Biochemistry 13:1014-1021, and U.S. Patent Nos. 3,867,517 and 4,376,110. Radiolabeling elements which are useful in imaging include 1231, 131I5 111In, and 99mTc, for example. Procedures for iodinating antibodies are described by Greenwood, F. et al. (1963) Biochem. J. 89:114-123; Marchalonis, J. (1969) Biochem. J. 113:299-305; and Morrison, M. et al. (1971) Immunochemistry 289-297. Procedures for 99mTc-labeling are described by Rhodes, B. et al. in Burchiel, S. et al. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer, New York: Masson 111-123 (1982) and the references cited therein. Procedures suitable for n 'in-labeling antibodies are described by Hnatowich, DJ. et al. (1983) J. Immul. Methods, 65:147-157, Hnatowich, D. et al. (1984) J. Applied Radiation, 35:554-557, and Buckley, R. G. et al. (1984) F.E.B.S. 166:202-204.
[0475] In the case of a radiolabeled binding protein, the binding protein is administered to the patient, is localized to the tumor bearing the antigen with which the binding protein reacts, and is detected or "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. See e.g., A.R. Bradwell et al., "Developments in Antibody Imaging", Monoclonal Antibodies for Cancer Detection and Therapy, R.W. Baldwin et al., (eds.), pp 65-85 (Academic Press 1985). Alternatively, a positron emission transaxial tomography scanner, such as designated Pet VI located at Brookhaven National Laboratory, can be used where the radiolabel emits positrons (e.g., 1C, F, 5O, and 13N). [0476] MRI Contrast Agents. Magnetic Resonance Imaging (MRI) uses
NMR to visualize internal features of living subject, and is useful for prognosis, diagnosis, treatment, and surgery. MRI can be used without radioactive tracer compounds for obvious benefit. Some MRI techniques are summarized in EP-A-O 502 814. Generally, the differences related to relaxation time constants Tl and T2 of water protons in different environments are used to generate an image. However, these differences can be insufficient to provide sharp high resolution images.
[0477] The differences in these relaxation time constants can be enhanced by contrast agents. Examples of such contrast agents include a number of magnetic agents paramagnetic agents (which primarily alter Tl) and ferromagnetic or superparamagnetic (which primarily alter T2 response). Chelates (e.g., EDTA, DTPA and NTA chelates) can be used to attach (and reduce toxicity) of some paramagnetic substances (e.g., Fe+3, Mn+2, Gd+3). Other agents can be in the form of particles, e.g., less than 10 μm to about 10 nM in diameter). Particles can have ferromagnetic, antiferromagnetic or superparamagnetic properties. Particles can include, e.g., magnetite (Fe3O4), γ-Fe2O3, ferrites, and other magnetic mineral compounds of transition elements. Magnetic particles may include: one or more magnetic crystals with and without nonmagnetic material. The nonmagnetic material can include synthetic or natural polymers (such as sepharose, dextran, dextrin, starch and the like
[0478] The target-binding proteins can also be labeled with an indicating group containing of the NMR-active 19F atom, or a plurality of such atoms inasmuch as (i) substantially all of naturally abundant fluorine atoms are the 19F isotope and, thus, substantially all fluorine-containing compounds are NMR-active; (ii) many chemically active polyfluorinated compounds such as trifluoracetic anhydride are commercially available at relatively low cost, and (iii) many fluorinated compounds have been found medically acceptable for use in humans such as the perfluorinated polyethers utilized to carry oxygen as hemoglobin replacements. After permitting such time for incubation, a whole body MRI is carried out using an apparatus such as one of those described by Pykett (1982) Scientific American, 246:78-88 to locate and image cancerous tissues.
[0479] Information obtained from evaluating an target-binding protein, e.g., a binding protein described herein, can be recorded on machine-compatible media, e.g., computer readable or computer accessible media. The information can be stored as a computer representation, e.g., in a database (e.g., in the case of imaging using a binding protein, a database of images for one or a plurality of subjects). The term "computer representation" refers to information which is in a form that can be manipulated by a computer. The act of storing a computer representation refers to the act of placing the information in a form suitable for manipulation by a computer.
[0480] Also within the scope of the invention are kits including the binding protein that binds to Tiel and instructions for diagnostic use, e.g., the use of the target-binding protein (e.g., antibody or antigen-binding fragment thereof, or other polypeptide or peptide) to detect Tiel, in vitro, e.g., in a sample, e.g., a biopsy or cells from a patient having a cancer or neoplastic disorder, or in vivo, e.g., by imaging a subject. The kit can further contain a least one additional reagent, such as a label or additional diagnostic agent. For in vivo use the binding protein can be formulated as a pharmaceutical composition.
[0481] The following examples are not to be construed as limiting.
EXAMPLES
[04821 Example 1 ; Tiel Sequences
[0483] An exemplary Tiel amino acid sequence (SEQ ID NO:2) is as follows:
MVWRVPPFLLPILFLASHVGAAVDLTLLANLRLTDPQRFFLTCVSGEAGAGRGSDAWGPP LLLEKDDRIVRTPPGPPLRLARNGSHQVTLRGFSKPSDLVGVFSCVGGAGARRTRVIYVH NSPGAHLLPDKVTHTVNKGDTAVLSARVHKEKQTDVIWKSNGSYFYTLDWHEAQDGRFLL QLPNVQPPSSGIYSATYLEASPLGSAFFRLIVRGCGAGRWGPGCTKECPGCLHGGVCHDH DGECVCPPGFTGTRCEQACREGRFGQSCQEQCPGISGCRGLTFCLPDPYGCSCGSGWRGS QCQEACAPGHFGADCRLQCQCQNGGTCDRFSGCVCPSGWHGVHCEKSDRIPQILNMASEL EFNLETMPRINCAAAGNPFPVRGSIELRKPDGTVLLSTKAIVEPEKTTAEFEVPRLVLAD SGFWECRVSTSGGQDSRRFKVNVKVPPVPLAAPRLLTKQSRQLWSPLVSFSGDGPISTV RLHYRPQDSTMDWSTIWDPSENVTLMNLRPKTGYSVRVQLSRPGEGGEGAWGPPTLMTT DCPEPLLQPWLEGWHVEGTDRLRVSWSLPLVPGPLVGDGFLLRLWDGTRGQERRENVSSP QARTALLTGLTPGTHYQLDVQLYHCTLLGPASPPAHVLLPPSGPPAPRHLHAQALSDSEI QLTWKHPEALPGPISKYWEVQVAGGAGDPLWIDVDRPEETSTIIRGLNASTRYLFRMRA SIQGLGDWSNTVEESTLGNGLQAEGPVQESRAAEEGLDQQLILAWGSVSATCLTILAAL LTLVCIRRSCLHRRRTFTYQSGSGEETILQFSSGTLTLTRRPKLQPEPLSYPVLEWEDIT FEDLIGEGNFGQVIRAMIKKDGLKMNAAIKMLKEYASENDHRDFAGELEVLCKLGHHPNI INLLGACKNRGYLYIAIEYAPYGNLLDFLRKSRVLETDPAFAREHGTASTLSSRQLLRFA SDAANGMQYLSEKQFIHRDLAARNVLVGENLASKIADFGLSRGEEVYVKKTMGRLPVRWM AIESLNYSVYTTKSDVWSFGVLLWEIVSLGGTPYCGMTCAELYEKLPQGYRMEQPRNCDD EVYELMRQCWRDRPYERPPFAQIALQLGRMLEARKAYVNMSLFENFTYAGIDATAEEA
[0484] Anexemplarynucleicacidsequence(SEQIDNO:1) thatencodesTiel isasfollows: atggtctggc gggtgccccc tttcttgctc cccatcctct tcttggcttc tcatgtgggc 60 gcggcggtgg acctgacgct gctggccaac ctgcggctca cggaccccca gcgcttcttc 120 ctgacttgcg tgtctgggga ggccggggcg gggaggggct cggacgcctg gggcccgccc 180 ctgctgctgg agaaggacga ccgtatcgtg cgcaccccgc ccgggccacc cctgcgcctg 240 gcgcgcaacg gttcgcacca ggtcacgctt cgcggcttct ccaagccctc ggacctcgtg 300 ggcgtcttct cctgcgtggg cggtgctggg gcgcggcgca cgcgcgtcat ctacgtgcac 360 aacagccctg gagcccacct gcttccagac aaggtcacac acactgtgaa caaaggtgac 420 accgctgtac tttctgcacg tgtgcacaag gagaagcaga cagacgtgat ctggaagagc 480 aacggatcct acttctacac cctggactgg catgaagccc aggatgggcg gttcctgctg 540 cagctcccaa atgtgcagcc accatcgagc ggcatctaca gtgccactta cctggaagcc 600 agccccctgg gcagcgcctt ctttcggctc atcgtgcggg gttgtggggc tgggcgctgg 660 gggccaggct gtaccaagga gtgcccaggt tgcctacatg gaggtgtctg ccacgaccat 720 gacggcgaat gtgtatgccc ccctggcttc actggcaccc gctgtgaaca ggcctgcaga 780 gagggccgtt ttgggcagag ctgccaggag cagtgcccag gcatatcagg ctgccggggc 840 ctcaccttct gcctcccaga cccctatggc tgctcttgtg gatctggctg gagaggaagc 900 cagtgccaag aagcttgtgc ccctggtcat tttggggctg attgccgact ccagtgccag 960 tgtcagaatg gtggcacttg tgaccggttc agtggttgtg tctgcccctc tgggtggcat 1020 ggagtgcact gtgagaagtc agaccggatc ccccagatcc tcaacatggc ctcagaactg 1080 gagttcaact tagagacgat gccccggatc aactgtgcag ctgcagggaa CCCCttCCCG 1140 gtgcggggca gcatagagct acgcaagcca gacggcactg tgctcctgtc caccaaggcc 1200 attgtggagc cagagaagac cacagctgag ttcgaggtgc cccgcttggt tcttgcggac 1260 agtgggttct gggagtgccg tgtgtccaca tctggcggcc aagacagccg gcgcttcaag 1320 gtcaatgtga aagtgccccc cgtgcccctg gctgcacctc ggctcctgac caagcagagc 1380 cgccagcttg tggtctcccc gctggtctcg ttctctgggg atggacccat ctccactgtc 1440 cgcctgcact accggcccca ggacagtacc atggactggt cgaccattgt ggtggacccc 1500 agtgagaacg tgacgttaat gaacctgagg ccaaagacag gatacagtgt tcgtgtgcag 1560 ctgagccggc caggggaagg aggagagggg gcctgggggc ctcccaccct catgaccaca 1620 gactgtcctg agcctttgtt gcagccgtgg ttggagggct ggcatgtgga aggcactgac 1680 cggctgcgag tgagctggtc cttgcccttg gtgcccgggc cactggtggg cgacggtttc 1740 ctgctgcgcc tgtgggacgg gacacggggg caggagcggc gggagaacgt ctcatccccc 1800 caggcccgca ctgccctcct gacgggactc acgcctggca cccactacca gctggatgtg 1860 cagctctacc actgcaccct cctgggcccg gcctcgcccc ctgcacacgt gcttctgccc 1920 cccagtgggc ctccagcccc ccgacacctc cacgcccagg ccctctcaga ctccgagatc 1980 cagctgacat ggaagcaccc ggaggctctg cctgggccaa tatccaagta cgttgtggag 2040 gtgcaggtgg ctgggggtgc aggagaccca ctgtggatag acgtggacag gcctgaggag 2100 acaagcacca tcatccgtgg cctcaacgcc agcacgcgct acctcttccg catgcgggcc 2160 agcattcagg ggctcgggga ctggagcaac acagtagaag agtccaccct gggcaacggg 2220 Gtgcaggctg agggcccagt ccaagagagc cgggcagctg aagagggcct ggatcagcag 2280 ctgatcctgg cggtggtggg ctccgtgtct gccacctgcc tcaccatcct ggccgccctt 2340 ttaaccctgg tgtgcatccg cagaagctgc ctgcatcgga gacgcacctt cacctaccag 2400 tcaggctcgg gcgaggagac catcctgcag ttcagctcag ggaccttgac acttacccgg 2460 cggccaaaac tgcagcccga gcccctgagc tacccagtgc tagagtggga ggacatcacc 2520 tttgaggacc tcatcgggga ggggaacttc ggccaggtca tccgggccat gatcaagaag 2580 gacgggctga agatgaacgc agccatcaaa atgctgaaag agtatgcctc tgaaaatgac 2640 catcgtgact ttgcgggaga actggaagtt ctgtgcaaat tggggcatca ccocaacatc 2700 atcaacctcc tgggggcctg taagaaccga ggttacttgt atatcgctat tgaatatgcc 2760 ccctacggga acctgctaga ttttctgcgg aaaagccggg tcctagagac tgacccagct 2820 tttgctcgag agcatgggac agcctctacG cttagctccc ggcagctgct gcgtttcgcc 2880 agtgatgcgg ccaatggcat gcagtacctg agtgagaagc agttcatcca cagggacctg 2940 gctgcccgga atgtgctggt cggagagaac ctagcctcca agattgcaga cttcggcctt 3000 tctcggggag aggaggttta tgtgaagaag acgatggggc gtctccctgt gcgctggatg 3060 gccattgagt ccctgaacta cagtgtctat accaccaaga gtgatgtctg gtcctttgga 3120 gtccttcttt gggagatagt gagccttgga ggtacaccct actgtggcat gacctgtgcc 3180 gagctctatg aaaagctgcc ccagggctac cgcatggagc agcctcgaaa ctgtgacgat 3240 gaagtgtacg agctgatgcg tcagtgctgg cgggaccgtc cctatgagcg accccccttt 3300 gcccagattg cgctacagct aggccgcatg ctggaagcca ggaaggccta tgtgaacatg 3360 tcgctgtttg agaacttcac ttacgcgggo attgatgcca cagctgagga ggcctga 3417
[04851 Example 2 : Selection and Primary Screening
[0486] We have used phage display to select Tiel-specific antibodies from a very large phage library that displays immunoglobulins as Fab fragments. To isolate antibodies specific to Tiel, a phage displayed Fab antibody library was selected against the Tiel extracellular domain fused to human Fc or to a histidine purification tag.
[0487] Selection in solution was done using biotin labelled antigen which was captured on streptavidin coated magnetic beads (M-280-DYNAL®). Selection on cells expressing Tiel was performed using a KINGFISHER™ automated magnetic bead capture device. Selection on immobilized antigen was performed using Tiel -Fc coated onto immunotubes. Several selection strategies were used :
[0488] Strategy 1 : Round 1 (50OmM biotin labelled Tiel/magnetic beads),
Round 2 (I x IO7 Tiel expressing cells/Kingfisher), Round 3 (I x IO7 Tiel expressing cells/Kingfisher) Strategy 2 : Round 1 (50OmM biotin labelled Tie 1 /magnetic beads), Round 2 (1 x 107 Tiel expressing cells/KINGFISHER™), (30OmM biotin labelled Tiel/magnetic beads)
[0489] Strategy 3 : Round 1 (Tiel Fc coated immunotubes at 5μg/ml), Round
2 (Tiel-Fc coated immunotubes), Round 3 (Tiel Fc coated immunotubes plus depletion with human IgG).
[0490] Library members recovered from the selection strategies were tested for antigen binding in phage ELISA. Each isolate was tested for binding to coated Tiel Fc. Strategy 1 did not identify any binding clones whereas strategy 2 identified 13 positive clones (« = 95). Strategy 3 identified 86 binding clones (n = 95).
[0491] Sequence analysis of the selected clones were grouped on the basis of the CDR3 selected of the heavy chain and resulted in 23 different antibodies with unique VH-CDR3 sequences.
[0492] We reformatted the selected Fabs as completely human antibodies by recloning the VH and VL coding sequences from the display library vector into two vectors of a mammalian expression vector system. These vectors contain the human kappa constant domain and the human gamma- 1 heavy chain constant region. The vectors were co-transfected into mammalian CHO-Kl cells for expression and production of the corresponding complete IgGs. These antibodies were characterized using several assays as described below, including: 1. Western blotting and immunoprecipitation of Tiel transfected cells and primary human endothelial cells; 2. Immunofluorescence of Tiel transfected cells and primary human endothelial cells; 3. Stimulation and inhibition of Tiel in BaF3 cells and primary human endothelial cells; and 4. Immunostaining of human tissues.
[0493] We identified 23 antibodies that interact with Tiel . See FIGs. 7-36.
After sequence confirmation of the reformatted clones they were used in a transient transfection of HEK293T cells. After growth the IgG was purified from culture supernatants using a protein A column. The quality of purified IgGl was determined using SDS-PAGE.
[0494] The specificity of the Tiel specific IgG's can be determined in a whole cell ELISA on mouse lung microvascular endothelial cells (LEII) and LEII-Tiel cells transfected with a Tiel expression construct. Cells are seeded into 96 well plates at a density of 10,000 cells/well and were fixed using 4% paraformaldehyde. Staining and detection of binding of IgGl to LEII cells are detected using standard labelling with a HRP conjugated rabbit anti human HRP and TMB staining. Binding of purified IgGl to LEII-Tiel transfected cells can also be corrected for Tiel protein that is expressed endogenously. Alternatively cells that have little or no endogenous Tiel can be used for the analysis.
[0495] At least one of the binding antibodies - E3 - functions as a Tiel activating antibody in the BaF3 cell bioassay. We studied Tiel phosphorylation in response to E3 IgG treatment in transiently transfected COSl cells and human primary endothelial cells. Our results indicate that E3 IgG activates the Tiel receptor. The BaF3 cell bioassay (also referred to as the "Tiel/EpoR chimericBAF cell assay" may provide an indication of a ligand's ability to cross-link the Tiel receptor. Because the assay is artificial, crosslinking of the non-naturally occurring Tie-Epo fusion proteins may or may not be predictive of a ligand's ability to modulate in vivo function.
[0496] E3 can be used, instead of possible natural ligands to characterize several functions of Tiel in vitro and in vivo. The region of Tiel which interacts with E3 can be the target for small molecular weight compounds for Tiel activation or inhibition.
[0497] Although E3 functions in one particular Tiel activating assay, E3 and other positives in this assay may also have inhibitory effect as to other functions or in other contexts. For example, E3 can inhibit tube formation by HUVEC cells. See below.
[0498] In addition, we found two antibodies that inhibit the survival effect conferred by E3 in the BaF3 cell bioassay. These two antibodies may inhibit dimerization of Tiel induced by E3 in the BaF3 assay. Two antibodies, B2 and DIl, completely blocked the viability of Tiel/EpoR cells when used in combination with E3.
rO4991 Methods
[0500] Cell culture. COSl cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), glutamine and antibiotics. The murine BaF3 pre-B lymphocytes were cultured in DMEM supplemented with 10% FCS, glutamine, antibiotics and 2 ng/ml interleukin-3 (Calbiochem). Human dermal microvascular endothelial cells (HDMVECs), obtained from PromoCell (Heidelberg, Germany) were cultured in endothelial cell medium provided by the supplier and used at passages 4-7.
[0501] Western blotting and immunoprecipitation. COSl cells were transfected with pcDNA3-Tiel-V5 (1 μg DNA per 10 cm cell culture plate) using FUGENE 6 (Roche) according to manufacturer's instruction and incubated for 48 h before stimulation. For immunoprecipitation, Tiel transfected cells and HMVEC cells were lysed in DOC-RIPA lysis buffer (5OmM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton-X-100, 0.1% SDS, 1% DOC, 1OmM EDTA) supplemented with aprotinin, leupeptin, PMSF and sodium vanadate. Immunoprecipitation was carried out from equal amount of cell lysates by incubating with polyclonal anti-human Tiel antibodies (R&D), monoclonal anti-V5 antibodies (Invitrogen) or altogether 23 anti- Tie 1 antibodies (1 μg/ml) for 1 to 2 h followed by incubation with protein G- Sepharose (Amersham Pharmacia Biotech AB) for 1 h. The imniunoprecipitates were washed twice with PBS-T and twice with PBS, followed by elution with the Laemmli buffer and separation in 8% SDS-PAGE. The blots were probed with the 23 anti-Tiel antibodies (5 μg/ml) and subsequently anti-human Fc antibodies conjugated with HRP.
[0502] Immunofluorescence staining. COSl cells on the glass coverslips were transiently transfected with pcDNA3-Tiel-V5 (the V5-epitope was added to the 3' terminus of pcDNA3-Tiel) (1 μg DNA per 10 cm cell culture plate) using FUGENE™ 6 (Roche) according to manufacturer's instruction and incubated for 48 h before staining. Cells were fixed in 4% paraformaldehyde for 10 min at 4°C. If required, the cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min. Unspecific binding sites were blocked by incubation with 1% BSA in PBS for 30 min. The cells were then stained with anti-Tiel antibodies (5 μg/ml) and anti-V5 antibodies for 1 h at room temperature, followed by incubation with FITC- conjugated anti-human antibodies (DAKO, 40 μg/ml) and TRITC-conjugated anti-mouse antibodies (DAKO, 15 μg/ml) for 30 min. Hoechst 33258 fluorochrome (Sigma, 0.5 μg/ml) was used for the staining of the nuclei. [0503] BaF3 bioassay. To generate Tiel-EpoR expressing BaF3 cells for the bioassay, BaF3 pre-B cells were stably transfected with a nucleic acid that expresses chimeric receptor containing the extracellular domain of human Tiel fused with the transmembrane and cytoplasmic domains of the mouse erythropoietin receptor. The nucleic acid used was a Tiel-EpoR chimeric cDNA in a pEF-BOS expression vector. The nucleic acid encoding the chimeric receptor was constructed by cloning the PCR amplified extracellular part of human Tiel (bp 37-2316 of X60975) as EcoRI-BglH fragment into mEpoR-pcDNA vector. The cDNA encoding for the chimeric receptor consisting of the extracellular part of Tiel fused with the transmembrane and intracellular domains of EpoR was subcloned into the pEF-BOS expression vector. Vector was linearized and co-transfected into BaF3 cells with pcDNA3.1(+) Zeo vector (Invitrogen). Stable cell pools were generated by selection with 250 μg/ml Zeocin. The expression of Tiel/EpoR fusion protein in several clones was analyzed by Western blotting with an antibody against EpoR.
[0504] To perform the assays, BaF3 cells expressing the Tiel -EpoR chimera were split in 96- well microtiter plates at 50 000 cells/well in the presence of the indicated concentrations of anti-Tie 1 antibodies. The E3 antibody used in this study was the germ-lined E3 antibody (DX-2220). As controls, Zeocin resistant pools not expressing the Tiel -EpoR were used. After 48 h, the viability of the cells was determined by adding MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma), 0.5 mg/ml), followed by further 2 h of culture, addition of an equal volume of cell lysis solution (10% SDS, 10 niM HCl) and incubation overnight at 37°C. Absorbance was measured at 540 nm.
[0505] Tiel phosphorylation assay. COSl cells were transfected with pcDNA3-Tiel-V5. After 24 h of transfection, the cells were serum starved for 8 h and then treated with E3 IgG. For the Tiel phosphorylation assay, HDMVECs were cultured on 10 cm dishes to near confluence, starved (8-16 h) in serum free medium and stimulated as indicated. After the stimulations, the cells were lysed in lysis buffer (RIPA-DOC: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton-X-100, 0.1% SDS, 0.5% DOC, 10 mM EDTA, supplemented with aprotinin, leupeptin, PMSF and sodium vanadate). Clarified lysates from transfected COSl cells or HDMVECs were immunoprecipitated with anti-V5 or anti-Tiel B9, respectively. Proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted using the anti-phosphotyrosine and anti-Tiel (R&D systems) antibodies.
[0506] Immunostaining of human tissues. To evaluate reactivity of anti-Tiel antibodies in immunohistochemistry, 5 μm cryosections of human kidney and lung were dried at room temperature for 30 min and fixed with cold acetone for 10 min. Slides were washed with PBS and treated with 0.03 % H2O2 in PBS for 15 min to reduce endogenous peroxidase activity. TNB (30 min at room temperature) was used to block non-specific binding and sections were incubated with Tiel antibodies at concentration of 10 μg/ml overnight at + 4°C. After several washings with PBS, biotinylated anti human antibody (1 :300, Zymed) was added to the tissues. Signal was amplified by using a TSA kit and detected with AEC staining.
IO5071 Results
[0508] Western blotting, immunoprecipitation and immunofluorescence of Tiel transfected cells and primary human endothelial cells (see Table 1).
Figure imgf000138_0001
Figure imgf000139_0001
[0509] To confirm the binding ability of the 23 selected anti-Tie 1 antibodies, we first performed western blotting and immunoprecipitation using COSl cells transfected with ρcDNA3-Tiel-V5 (V5 tagged) and primary endothelial cells. Next, to find out if the anti-Tiel antibodies recognize Tiel in living cells, those cells were studied by immunofluorescence staining. All the antibodies analyzed recognized both transfected and endogenous Tiel, although differences were detected in the binding affinity as shown in Table 1.
[0510] Stimulation and inhibition of Tiel in Tiel-EpoR transfected BaF3 cells and hisϊHa.1 primary endothelial cells. Although no ligand for Tiel has been identified, we used the following efficient screening method for Tiel-binding proteins. Interlenkm-3 depends pεe ■B-lynvplioeyte (BaF3) cells were transfected with a construct that expresses a ϊitKRpoR fusion protein. Since BaF3 cells are IL-^ - dependent, they die unless IL-3 is provided. However, Tie-EpoR receptor expressing BaF3 cells can survive and proliferate if the medium contains a Tiel-binding protein, either a natural ligand or an artificial mimetic. Cell survival can be quantitated, e.g., by colorimetric MTT-assay, which measures mitochondrial activity.
[0511] The results from the BaF3 cell assays indicated that, of the 23 different monoclonal antibodies tested, only E3 IgG was able to promote survival of Tiel - EpoR cells whereas the viability of EpoR BaF3 cells used as a control was not affected by E3 IgG. The IgG part of the immunoglobulin molecule was needed for the survival effect of E3 IgG, as the E3 Fab fragment had no effect on the viability of Tiel-EpoR cells. A concentration of 50 ng/ml of E3 IgG gave almost maximal viability in Tiel-EpoR cell survival assays and the viability was dose dependent.
[0512] To test if the E3 IgG binding to the extracellular region of Tiel induces autophosphorylation of Tiel, the Tiel receptor phosphorylation level in response to E3 IgG treatment was studied in transiently transfected COSl cells and human primary endothelial cells. COSl cells were transfected with an expression vector containing a V5-tagged full length Tiel cDNA, and, after serum starvation, the cells were treated with E3 IgG (200 ng/ml). Cell lysates were extracted at several time points and Tiel was immunoprecipitated with anti-V5 followed by western blotting using anti-phosphotyrosine and anti-Tiel antibodies. The results indicated that Tiel is tyrosine phosphorylated after 10 to 30 min of E3 IgG stimulation. To determine if E3 IgG induces Tiel phosphorylation in primary endothelial cells, HDMVEC cells were serum starved and stimulated with several concentrations of E3 for 60 min. Tiel was then immunoprecipitated from cell lysates and subjected to anti-phosphotyrosine blotting analysis, which showed receptor phosphorylation following E3 IgG stimulation at 50-200 ng/ml. Also higher concentrations of E3 (500-1000 ng/ml) induced Tiel phosphorylation but the response was more rapid and was most prominent after 5 min of stimulation.
[0513] To study the kinetics of E3 IgG induced Tiel activation, cells were stimulated with E3 IgG (200 ng/ml) and receptor phosphorylation was studied at various time points. Tiel phosphorylation was highest 15-30 min after E3 IgG treatment but phosphorylation persisted for up to 1 h.
[0514] To determine if any of the other monoclonal antibodies tested inhibit u the survival effect of E3 IgG in Tiel-EpoR BaF3 assay, antibodies were studied in combination with E3 IgG. A concentration of 100 ng/ml of E3 IgG together with 100 (1:1) or 500 (1:5) ng/ml of the other antibodies were used and the viability of Tiel- EpoR cells was measured. The results from both combinations of E3 IgG and the test antibody (in 1:1 and 1:5 ratios) were similar and indicated that two of the 23 antibodies (B2 and Dl 1) blocked completely the survival effect of E3 IgG. Several antibodies (A2, AlO, P-Bl, P-B3 and P-C6) inhibited the viability effect of E3 IgG to some extent and two of the antibodies (G2 and C7) promoted the survival of Tiel/EpoR BaF3 cells in combination with E3 IgG.
[0515] Immunostaining of human tissues. The anti-Tiel antibodies react with human Tiel in cultured cells. It is also possible to determine whether they could stain human tissue samples from lung and kidney as well as from tumors by using biotinylated anti-Tiel antibodies and detecting bound antibodies using labeled streptavidin or avidin. [0516] Example 3 : Exemplary Sequences
[0517] Sequences of exemplary immunoglobulin variable domains are shown in FIGs. 7-36.
rO518] Example 4 : Inhibition of Tube Formation bv HUVEC Cells Using
Anti Tiel E3-IgG
[0519] To demonstrate the ability of E3 to inhibit angiogenesis in vitro, purified E3 was tested for its ability to inhibit tube formation by human umbilical cord endothelial cells (HUVECS). Human Umbilical vein endothelial cells (HUVEC) were obtained by treating fresh human umbilical cord veins with Trypsin-EDTA (Ix) (Gibco/Invitrogen) for 20-25 minutes at 37°C. The cells were cultured in a T-25 flask coated with attachment factor (AF), (Cascade Biologies) in RPMI 1640 medium supplemented with 10 % FCS, 0.4 % BBE, 1% 1-glutamin, 1% penicillin/streptomycin. Primary cultures were detached with warm Trypsin-EDTA and used when confluent at the second or third passage. The cells were,maintained in a proliferative state by culturing them in a split ratio 1 :2 at an approximate density of the monolayer of about 60-80%. To dissociate the cells, HUVEC monolayers were treated with trypsin/EDTA (500 μl/dish) at 37°C for 3 min. Trypsin activity was stopped by adding 3 volumes of complete RPMI medium. The cells were carefully scraped, separated by repeated pipetting, and finally washed with PBS.
[0520] After 2 passages HUVECs were seeded in their culture medium
(40X 103/ 50 μl/well of a 96-well plate) on a collagen gel (50 μl of collagen I 1.5 mg/ml) prepared by mixing 7.5 volumes of 2mg/ml collagen (Collagen R; Serva, Heidelberg, Germany), 1 volume of 1OX MEM, 1.5 volume OfNaHCO3 (15.6 mg/ml) and ~ 1 volume of NaOH to adjust the pH to 7.4. After 1.5h, the culture medium was then discarded and the cells were covered with a new layer of collagen (1.5 mg/ml, new preparation, 50 μl/well). After polymerization of the gel, culture medium was added to each well in presence or in absence of E3 antibody (1 ng/ml to 10 μg/ml). The assay was performed with a streptavidin antibody used as a control (from 1 ng/ml to 10 μg/ml). The total length of the tube network on the culture surface was quantified at 40x magnification by the METAVUE™ Software (Universal Imaging Corporation). Results from triplicate wells were expressed as mean vessel area per field ± SEM (relative units). Each assay was performed at least three times.
[0521 ] E3 is a potent inhibitor of tube formation by HUVECS even at a concentration of 10 ng/ml. The control anti-streptavidin has no effect on the ability of HUVECS to form tubes. This results indicates that E3 can inhibit at least one aspect of angiogenesis.
[0522] Example 5: Immunohistochemical Analysis of E3 Binding to Matched
Tumor and Normal Tissue Sections
[0523] To evaluate the binding of E3 to Tiel in primary tumor and normal tissue the antibody was produced as an IgG and biotin labeled. The E3 antibody and two other anti Tiel antibodies B2 and Dl Lwere reformatted as full length IgG molecules. Nucleic acids encoding these IgGs were transiently transfected into HEK293T cells. Plasmid preparations for transient cell transfections were performed using the HP-GENELUTE™ MIDI prep kit (Sigma, cat. no. NA0200). HEK293T cells (GenHunter Corp. cat. no. Q401) were seeded 24 hours before transfection; 6 x 106 cells were plated per 10-cm culture dish. Transfections were carried out using LIPOFECTAMINE™ 2000 reagent (Invitrogen, cat. no. 11668019) following the manufacturer's instructions. Five micrograms of plasmid DNA was used per 10-cm dish. Cells were cultured in DMEM (Invitrogen, cat. no. 31966021) supplemented with 10 % "ultra-low IgG" fetal calf serum (Invitrogen, cat. no. 16250078), at 37°C, 5 % CO2, in a water saturated atmosphere. Conditioned media were harvested 72 hours and 144 hours after transfection, pooled and sterile filtered.
[0524] One hundred microliters of Protein A beads (rProtein A Sepharose 4
Fast Flow, Amersham Biosciences, cat. no. 17-1279-01) equilibrated in PBS were added to the cell culture supernatants, and these were rotated overnight at 4°C, e.g., in 50 ml tubes. The beads were collected by centrifugation, transferred to a 96-well filter plate (UNI-FILTER 800 GF/B, Whatman, cat. no. 7700-2803) and washed extensively with PBS using a vacuum manifold (Macherey Nagel, cat. no. 760681). Elution of the antibodies was achieved by resuspending the beads in 400 μl of 12.5 mM citric acid. After a 30 to 60 second incubation, the bead eluates were collected, using the vacuum manifold, into the wells of a 96-well collection plate (UNIPLATE 750, Whatman, cat. no. 7701-5750). Each well of the collection plate contained 60 μl of 1 M HEPES pH 7.5 buffer to immediately neutralize the eluted fractions. The elution step was performed twice to maximize antibody recovery. The eluted samples were then dialyzed against PBS using dialysis cassettes (Slide- A-Lyser Dialysis Cassettes, MWCO 10,000, Pierce, cat. no. 66380) and protein concentration was determined from the absorbance at 280 nm assuming that a 1 mg/ml solution has an absorbance of 1.35. The quality of the preparations was analyzed by reducing and non-reducing SDS-PAGE.
[0525] The Tiel antibodies were biotinylated using the EZ-link Sulfo-NHS-
SS-Biotin (Pierce, Cat. 21331). For Tiel/Fc and Tiel-His, the reaction was performed for 2 hours on ice in 50 mM sodium carbonate buffer, pH 9.6, in the presence of a 5- fold molar excess of biotinylating agent. For the antibodies, the reaction was performed for 2 hours on ice in PBS, in the presence of a 15-fold molar excess of EZ- link Sulfo-NHS-SS-Biotin. The reaction was stopped by the addition of Tris-HCl, pH 7.5 (50 mM final concentration) followed by a 1-hour incubation on ice. Samples were then dialyzed against PBS.
[0526] Various normal and tumor tissue sections were stained with biotinylated antibodies. A mouse monoclonal anti-Tiel antibody (7e8) (Alitalo laboratory, University of Helsinki) was used as a positive control. Sections without primary antibody served as negative control. All samples were fresh frozen tissues and staining was performed with the TSA-kit (Perkin-Elmer Life Sciences). After acetone fixation (10-20 min, -200C) the slides were treated with 0.73% H2O2 for 10 min to reduce endogenous peroxidase activity followed by blocking for 30 min with TNB buffer. Sections (5-10 mm thick) were incubated with primary antibodies (10 μg/ml) overnight at 4°C. Sections with the mouse monoclonal anti-tiel antibody (7e8) were treated with biotinylated anti-mouse antibodies (VectaStain) before the addition of streptavidin-HRP. Signal was amplified by using a TSA kit and the visualized by AEC (235 ml NaAc, 15 ml AEC (stock solution: 1600 mg 3-amino-9- ethyl-carbazole and 480 ml N-dimethylformamide), 250 μl H2O2).
[0527] hi general, Tiel expression was upregulated in tumor tissue when compared with matching normal tissue. However, in the tumor tissues the anti Tiel antibodies stained other structures in addition to the vessels. Furthermore, some tissue specificity in the expression of certain epitopes was observed. For example, the E3 antibody stained vessels in the lung and kidney but not in the skin while the B2 antibody stained vessels very faintly in other normal tissues than in the breast. Shedding of the ectodomain of Tie 1 into the tumor tissues can explain observed differences.
[0528] In skin tissue, the E3, B2, and Dl 1 antibodies stained blood vessels very faintly whereas the murine 7e8 control antibody gave a clear staining in the normal skin. In melanoma tissue, the 7e8 antibody stained vessels only but the E3, B2, and DIl antibodies also stained other surrounding structures. The staining pattern was similar with all three of the E3, B2, and DIl antibodies.
[0529] In lung tissue, we observed that the E3 antibody stained especially clearly the large veins in the lung, whereas DIl and 7e8 gave a faint staining. B2 did not stain the same veins. The expression of Tiel was dramatically upregulated in lung carcinoma and all the antibodies stained vessels more strongly in samples with lung carcinoma than in samples from normal lung. In the lung tumors, the E3, B2, and DIl antibodies stained structures other than vessels.
[0530] In kidney, the E3 and DIl antibodies stained kidney tubules in addition to the vessels. B2 gave only very faint staining of either tubules or vessels while 7e8 stained only vessels. In hypernephroma tissue, only the E3 antibody gave a clear staining.
[0531] In breast, E3 gave the brightest staining in the veins and capillaries of the mammary tissue, B2 and 7e8 gave a similar staining while DIl stained those structures rather faintly. In breast carcinoma the Tiel expression was substantially upregulated, and the E3, B2, and DIl antibodies stained also other structures in addition to vessels.
[0532] Example 6 : Binding to Mouse Endothelial Cell Lines of Anti Tiel E3-
IgG Using Flow Cytometry
[0533] We evaluated if E3 cross reacts with mouse Tiel in situ and thus if we can evaluate E3 activity in mouse tumor xenograft models binding to mouse endothelial cells was tested and compared with human and transfected cell lines.
[0534] Specific binding of the Tiel antibodies and of control Mabs to mouse endothelial cells was measured by flow cytometry analysis (FACSscan, Becton Dickinson, Oxnard, Epics, Coulter). Mouse endothelial cell lines MSl, Le-2, Bend3, SVEC (ATCC, Rockville) and Tiel transfected Le-2 cells were stained. Cell staining was modified from existing protocols. About 200,000 cells were used in each experiment: after trypsinization, cells were washed one time in PBS and resuspended PBS, 10% heat inactivated human serum (incubation buffer). To test specificity, antibodies were incubated at different dilutions for 1 h at room temperature. Cells were spun down by centrifugation for 3 min at 611 g. Between incubations cells were washed twice with PBS. Then relevant biotinylated antibodies (A2 against streptavidin, E3 against Tiel, were added and incubated for 1 h at room temperature). The E3 DX-2210 antibody, in which the light chain has been germlined, was used for these studies. This was followed by incubation with Strepatvidin- R-phycoerythrin (Dako, Glostrup, Denmark) for 1 hour at room temperature in incubation buffer. After the final incubation step bound antibodies were detected by means of flow cytometry on a FACSCan and Epics Altra (Becton Dickinson, Oxnard, Coulter) and results analyzed.
[0535] Intracellular Tiel was measured as described above, except for the addition of Saponin to the incubation buffer to a final concentration of 0.1% during incubations. The anti-Tie 1 antibody E3 binds to mouse endothelial cell lines indicating a cross reactivity of E3 with mouse and human Tiel in situ. The binding pattern in mouse cell lines detected by flow cytometry is different from the binding pattern in HUVEC in that in mouse cells there is a greater cell surface staining than that compared to primary human endothelial cell lines. DX-2210 stained positively both mouse endothelial cell lines as well as the HUVEC control cells. There was a shift in the fluorescent signal when the cells were treated with saponin, indicating a significant intracellular pool of sequestered Tiel.
[05361 Example 7 : Determination of anti Tiel E3-IaG Binding to Human
Platelets Using Flow Cytometry
[0537] Binding experiments with a purified polyclonal goat antiserum against
Tiel (R&D systems) had showed binding to human platelets in a previous study (Tsiamis et al., (2000) J. Vase. Res. 31 r:437-42). The conclusion form this study was that platelets represent a large pool of Tiel immunoreactivity which could present a problem for development of Tie 1 as a therapeutic target. To determine if the antibody E3 binds to platelets we performed flow cytometric analysis on both activated and inactivated platelets and compared the staining pattern with the purified anti Tiel polyclonal serum.
[0538] To avoid platelet activation, human platelets were isolated from plasma of healthy donors using the platelet GelSep kit (Biocytex, Marseille, France) kit according to the guidelines of the manufacturer. Platelets were activated by the addition of thrombin to a final concentration of 0.8 U/ml. To distinguish activated from non-activated platelets double staining was performed with Tiel antibodies/control antibodies and antibody CD42 (total platelets) or CD62 (activated platelets).
[0539] After preparation, platelets were resuspended in buffer 2 of the GelSep kit, 10% heat inactivated human serum (incubation buffer) and incubated for 1 hour. To test specificity, biotinylated antibodies human anti-Tiel(E3), human anti- streptavidin (A2-SV, an antibody that does not bind Tiel), human anti-FITC and goat anti-Tie (R&D systems) were incubated with 500 000 platelets per test for 1 hour at different dilutions (2 μg/ml, 10 μg/ml) for 1 h at room temperature. Platelets were spun down by centrifugation for 10 min at 611 g. Between incubations platelets were washed twice with Buffer 1 of the GelSep kit. Then, Strepatvidin- R-phycoerythrin together with anti-CD42-PercP or anti-CD62-PercP were incubated for 30 minutes at room temperature in incubation buffer After the last incubation and washing detection of bound antibodies was performed by means of flow cytometry on a FACSscan and Epics Altra (Becton Dickinson, Oxnard, Coulter, ) and results analyzed. Cells were gated on SSC and anti-CD42-PercP for the total platelets in case non-activated platelets were used and on SSC and anti-CD62-PercP for the activated platelets.
[0540] The polyclonal goat anti-Tiel antibody indeed binds to platelets under the conditions tested. This binding is lower when platelets are activated. In contrast, the human anti-Tiel antibody E3 shows no significant binding to total platelets, nor to activated platelets (FIG. 1).
[0541] Example 10 ; Assessment of Tiel Immunoreactivity in Human
Platelets Using Immunoprecipitation with Anti Tiel E3-IgG [0542] A previous study with a purified polyclonal goat antiserum against
Tiel (R&D Systems) had showed binding to human platelets (Tsiamis et al., 2000). The conclusion from this study was that platelets represent a large pool of Tiel immunoreactivity which could present a problem for development of Tiel as a therapeutic target . To exclude the possibility that the antibody E3 binds to platelets immunoprecipitation of lysates prepared from platelets and HUVECS were performed. Both activated and inactivated platelets were tested.[0543]
Anti-Tiel antibodies B2, DIl, E3, the goat polyclonal AF619 (R&D) and negative control antibodies anti-FITC and anti-Streptavidin were used. HUVECS were retrieved from culture dishes by trypsinization and platelets were prepared with the platelet GelSep kit (Biocytex, Marseille, France) kit according to the guidelines of the manufacturer. Per immunoprecipitation experiment 3-5 x 106 and 3 x 108CeIIs platelets were used for each antibody tested. Platelets and cells were washed with PBS and spun down at 1400 rpm for 4 minutes and supernatant was removed. Then cells were lysed in 1 ml lysis buffer containing 5OmM Tris HCL pH 7.5, 150 mM NaCl, 0.5% Deoxycholic acid (DOC) and 0.5% NP-40 for 5 minutes. The lysed cells were spin down for 10 minutes at 14.000 rpm and 5 μg/ml antibody was added to the supernatant and incubated at 40C on a rotator. 100 μl/sample protein A beads (Uppsala, Sweden) were washed 3 times with lysis buffer (centrifugation speed: 15 seconds, 2000 rpm) then cell lysates incubated with antibody were added for 30 minutes 4°C. Then beads were washed three times with washing buffer containing 5OmM Tris HCL pH 7.5, 400 mM NaCl, 0.5% DOC, 0.5% NP-40. Finally, beads are spun down and the pellets was resuspended in an equal amount in sample buffer to perform SDS-page and Western blotting. In Western blotting Tiel was detected with the polyclonal goat anti-Tiel antibody. The conclusions of this study are that E3 is able to immunoprecipitate Tiel in HUVEC but not in platelets.
[05441 Example 8 : Distribution of Tiel in HUVEC Cells Determined by
Staining with Anti Tiel E3-IeG
[0545] We analyzed the staining pattern of E3 in HUVECS using confocal microscopy. HUVEC were trypsinised, washed with PBS and spotted at a density of 60 000 cells on a gelatine coated microscope slide and incubated for 24 hours in a humidified incubator at 370C. Cells were air dried and fixed with 4% paraformaldehyde for 20 minutes at room temperature. The slides were washed with PBS. The slides were incubated with 10% Heat inactivated human serum (incubation buffer).
[0546] For measuring specific binding to Tiel, biotinylated antibody E3 and biotinylated negative control antibody A2 were used at a concentration of 10 μg/ml and incubated for 1 hour at room temperature. Slides were washed twice with PBS. Then, Strepatvidin- R-phycoerythrin (Dako, Glostrup, Denmark) was added and incubated for 1 hour at room temperature. After the last incubation and washing detection of bound antibodies was performed by means of confocal microscopy.
[0547] E3 binds specifically to HUVEC as detected by confocal microscopy.
The staining is pre-dominantly located inside of the cell which suggests a large intracellular pool of Tiel relative to a smaller pool of cell surface localized Tiel . The localization of E3 was consistent with co-localization of Tiel with a cytoskeletal protein.
[0548] Example 9 : Conversion of Somatic Mutations Positioned in the
Framework Region of Anti Tiel E3 to Germline Residues
[0549] To reduce potential immunogenicity of E3 in humans, all non germline amino acid residues in the LC framework regions were corrected back to germline. An initial analysis was performed which aligned the LC of E3 with a database containing all kappa and lambda light chain germline genes. The LC of E3 was shown to have closest homology to DPK4 and three substitutions in E3 relative to the germline framework regions were identified.
[0550] We constructed a germlined version of E3 in which the LC framework regions were altered to include sequences identical to the DPK4 germline framework regions. The germlined E3 antibody was constructed by engineering a nucleic acid encoding the desired sequence. Changes to nucleic acids encoding the E3 LC variable domain were made by PCR and other standard molecular biological techniques and verified by nucleic acid sequencing. [0551] An exemplary germlined light chain variable domain E3 sequence includes:
DIQMTQSPSSLSASVGDRVTITCRASQGIGHYLAWYQQKPGKVPKLLIYTASTLQSG VPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQFNSYPHTFGQGTRLEIK ( SEQ ID NO : 159 ) . The altered positions are underscored.
[0552] We produced the germlined version of the E3 antibody as both a soluble Fab and as an IgG. The Fab cassette of the positive sFAB-expressing clone was PCR amplified with oligonucleotides, ligated into a mammalian expression vector containing the human IgG4 Fc region and electroporated into XLl Blue MRF' cells. The prokaryotic ribosomal binding sequence and gene three leader sequence were replaced with a mammalian internal ribosomal entry and heavy chain leader sequences. Reformatted antibody clones were sequenced to confirm accuracy following the cloning procedure. Endotoxin-firee DNA was prepared and used for transient transfection studies.
IO553] Example 10 : Production and Testing of Germlined Anti Tiel E3 - Fab for Binding to Recombinant Tie 1 -Fc in ELISA
[0554] To evaluate if the conversion of any of the somatic mutations in the framework of E3 back to germline residues had any effect on binding activity the soluble Fabs were produced. The soluble expression vector containing the parental E3 Fab and the germlined E3 Fab construct were grown overnight at 30°C in 2xTY broth containing 100 μg/ml ampicillin and 2% glucose and use 4 ml of this overnight culture to inoculate 400 ml of 2 x TY broth containing 100 μg/ml ampicillin and 0.1% glucose. Cells were grow at 370C until an OD600 of 0.8-1.0, ImM IPTG was added and the culture was maintained at 3O0C for 4 hours. The cultures were spun down at 4,000 rpm for 15 min at 4 C. The superaatants were discarded and resuspend the pellets resuspended in 4.8 ml of ice cold TES buffer (0.2 M Tris-HCl, 0.5 niM EDTA, 0.5 M sucrose, pH 8.0) containing proteases inhibitors (protease inhibitor cocktail tablets [Roche]: dissolve 1 tablet in 1 ml of water and dilute 50-times in TES buffer). Transfer to 50 ml Falcon tubes and place on ice for 5-10 min. During this incubation, wash the centrifugation bottles with 5.25 ml TES:H2O (1:3) containing proteases inhibitors and add this to the cells. Incubate for 20 more min on ice. Spin at 3000 g for 15 min at 40C and transfer the supernatants into new centrifugation tubes. Resuspend the cell pellets in 6 ml TES containing 15 niM MgSO4 and proteases inhibitors and incubate on ice for 15 min. Centrifuge at 3000 g for 15 min at 4°C. Transfer the supernatants into the centrifugation tubes and spin at 8000 g for 20 min at 4°C. Collect the supernatants and dialyze against PBS. The Fabs were purified by metal chelate chromatography. Incubate the dialyzed periplasmic extracts with 1 ml of TALON™ Metal Affinity Resin (Clontech) and rotate at room temperature for 2 hours. Transfer the beads into empty gravity column (Poly-Prep chromatography columns, Bio-Rad, Cat. 731-1550). Wash the beads with 5 mM imidazole in PBS and elute the Fabs with 150 mM imidazole in PBS. Dialyze against PBS using dialysis cassettes (SLIDE- A-LYSER™ Dialysis Cassettes, MWCO 10,000, Pierce, cat. no. 66380) and determine the protein concentration from the absorbance at 280 nm assuming that a 1 mg/ml solution has an absorbance of 0.86. The quality of the preparations can be analyzed by reducing and non-reducing SDS-PAGE.
[0555] Wells of an IMMULON™ 2 HB plate coated overnight with 500 ng or
50 ng of purified recombinant human Tiel-Fc target antigen per 100 microliters 0.1 M sodium bicarbonate buffer, pH 8.5. Parental E3, E3 germlined (E3g) or a negative control soluble Fab were loaded into wells at either 5 micrograms or 1 microgram per 100 microliters of PBST. Recombinant human Tiel-Fc target antigen is dissolved in an appropriate amount of acetic acid and subsequently diluted into 0.1 M sodium bicarbonate buffer, pH 9.6 at final concentrations of 500 ng and 50 ng per 100 microliters. After addition of the target antigen to the wells the microtitre plate is incubated overnight at 4°C. The plate is subsequently washed 5 times with PBST and blocked with 1% BSA in PBS at 37°C for 2 hours. The plate is again washed plate times with wash buffer, PBST and 100 microliters per well of purified Fab at 5 or 1 micrograms per 100 microliter PBST was added followed by incubation at room temperature for 1 hour. After washing plate 7 times with PBST 100 microliters of a 1:5000 dilution of anti-sFab-HRP in PBST was added (Pierce Product #31414). After washing the wells seven times 100 microliters TMB-H2O2 solution was added to each well and the plate read at 630 nm in an ELISA. Both E3 and germlined E3 bound to the recombinant human Tiel-Fc target antigen by this assay. [0556] Example 11 : Production and Testing of Germlined Anti Tiel - E3 -
Fab for Binding to Recombinant Human Tiel in BIAcore
[0557] Recombinant purified human Tiel-Fc antigen (Stock 2.45 mg/ml) was biotinylated using the EZ-link Sulfo-NHS-SS-Biotin (Pierce, Cat. 21331). The reaction was performed for 2 hours on ice in 50 mM sodium carbonate buffer, pH 9.6, in the presence of a 5-fold molar excess of biotinylating agent and was stopped by the addition of Tris-HCl, pH 7.5 (50 mM final concentration) followed by a 1-hour incubation on ice. Samples were then dialyzed against PBS. The antigen was then diluted 1/100 fold in HBS and was then captured onto a streptavidin chip. This was coated to a density of 830RU (resonance units). All analysis was performed in HBS buffer. The parental Fab E3 and germlined E3 Fab were prepared as described above. A stock solution of 0.587 mg/ml (1174OnM) was diluted 1/587 in HBS + BSA to obtain a stock of 2OnM and the germlined Fab E3 0.025 mg/ml (500 nM) was diluted 1/25 in HBS + BSA to obtain a stock of 2OnM. Serial dilutions were made of each Fab preparation to obtain 1OnM, 5nM, 2.5nM, and 1.25nM solutions. For the association phase samples were injected at 30μl/min for 4 minutes using kinject program. This was followed by a 10 minutes dissociation phase, any remaining sample was stripped from the Tiel Fc surface at a flow of 50 μl/min with a single injection of 5 mM NaOH + IM NaCl for 18 seconds. AU samples were run and analyzed in duplicate.
[0558] Sensorgrams were analyzed using the simultaneous ka/kd fitting program with 1:1 model in the BIAEV ALUATION™ software 3.1. From the analysis we can see that the germlining of the E3 antibody has had minimal effect on the binding activity of the antibody.
Table 2: Comparison of the binding affinity of parental and germlined E3 Fab
E3 Fab Tiel Fc koπ (1/Ms) koff (1/s) KD(1) nM parental Human 3.00E+05 6.10E-04 2.0 germlined Human 3.00E+05 1.02E-03 3.4 [0559] Example 12 : Comparison of Affinity of Germlined Anti Tiel E3 -IgG to Parental Anti Tiel E3 for Binding to Recombinant Human Tiel Using BIAcore [0560] In order to evaluate if the binding behavior had been affected in any way by the conversion of the somatic mutations back to germline residues, the germlined antibody was produced and tested as an IgG. The germlined E3-IgG construct used to transiently transfect HEK293T cells and purified.
[0561] The germlined E3 IgGl stock solution 0.63 mg/ml was diluted 1/50 in a buffer of pH4.5 and the parental E3 IgGl stock solution 0.56 mg/ml (2143-001) was diluted 1/50 in a buffer of pH 4.5. The IgG were directly coated onto a CM5 chip. The surface of the chips was activated with a 7 minute pulse of 0.05M NHS/0.2M EDC and the IgG was flowed over until 780RU germlined E3-IgG and 728 non germlined E3 IgG was coated onto the surface. AU flow cells were subsequently deactivated with a 7 minute pulse of IM ethanolamine hydrochloride pH 8.5. All analysis was performed in HBS buffer. Purified recombinant human Tiel Fc was diluted 1/28.7 in HBS to obtain a 400 nM stock solution. Serial dilutions were made to obtain 20OnM3 10OnM, 5OnM and 25 nM Tiel Fc stocks. For analysis of the association phase samples were injected at 30 μl/min for 8.3 minutes using kinject program. This was followed by a 40 minutes dissociation phase. Any antigen remaining associated to the surface was stripped from the IgG surface at a flow of 50 μl/min with two injections of 1OmM glycine pH 1.5 for 30secods. AU samples were run and analyzed in duplicate
[0562] Sensorgrams were analyzed using the simultaneous ka/kd fitting program with 1 : 1 model in the BIAEV ALUATION™ software 3.1. Germlining had minimal impact on the binding activity of the E3 IgG with respect to human Tiel Fc.
Table 3: Comparison of the binding affinity of parental and germlined E3 IgG
E3 lgG Tiel Fc kon (1/Ms) koff (1/s) KD(1) nM aarental Human 6.19E+03 3.61 E-05 5.83 germlined Human 7.09E+03 3.67E-05 5.17 1O5631 Example 13 : Production and Testing of Germlined Anti Tiel - E3 -
Fab for Binding to Recombinant Mouse Tiel in BIAcore
[0564] Mouse Tie 1-Fc antigen (0.5 mg/ml stock) was biotinylated using established procedures and after dilution 1/100 fold in HBS this was then used for capturing to a streptavidin chip. This was coated to a resonance value of 740RU. All analysis was performed in HBS buffer. The parental Fab E3 0.587 mg/ml (11740 nM) was diluted 1/587 in HBS + BSA to obtain a stock of 20 nM and the germlined Fab E3 0.025 mg/ml (500 nM) was diluted 1/25 in HBS + BSA to obtain a stock of 20 nM. Serial dilutions were made of each Fab preparation to obtain 10 nM, 5 nM, 2.5nM, and 1.25nM. For the association phase samples were injected at 30μl/min for 4 minutes using kinject program. This was followed by a 10 minutes dissociation phase, any remaining sample was stripped from the Tiel Fc surface at a flow of 50 μl/min with a single injection of 50 mM NaOH + 1 M NaCl for 18 seconds. All samples were run and analyzed in duplicate.
[0565] Sensorgrams were analyzed using the simultaneous ka/kd fitting program with 1:1 model in the BIAEV ALUATION™ software 3.1. The germlining of the E3 antibody has had minimal effect on the binding activity of the antibody.
Table 4: Comparison of the binding affinity of parental and germlined E3 Fab
Figure imgf000153_0001
[0566] Example 14 : Comparison of Affinity of Germlined Anti Tiel E3 -IgG to Parental Anti Tiel E3 for Binding to Recombinant Mouse Tiel Using BIAcore
[0567] In order to evaluate if the binding behavior had been affected in any way by the conversion of the somatic mutations back to germline, the germlined antibody was produced and tested as an IgG. The germlined E3 was reformatted to an IgG as described. This was then used to transiently transfect HEK293T cells using established procedures. The IgG was purified from the culture supernatant using protein A column chromatography using established procedures and the subsequent IgG was then tested for binding activity using surface plasmon resonance (BIAcore). The germlined E3 IgGl stock solution 0,63 mg/ml (2146-002) was diluted 1/50 in a buffer of pH 4.5 and the parental E3 IgGl stock solution 0,56 mg/ml (2143-001) was diluted 1/50 in a buffer of pH 4.5. The IgG were directly coated via onto a CM5 chip. The surface of the chips was activated with a 7 minute pulse of 0.05M NHS/0.2M EDC and the IgG was flowed over until 780RU germlined E3-IgG and 728 non germlined E3 IgG was coated onto the surface. AU flow cells were subsequently deactivated with a 7 minute pulse of IM ethanolamine hydrochloride pH8,5. AU analysis was performed in HBS buffer. Purified recombinant mouse Tiel Fc was diluted 1/6,5 in HBS to obtain a 40OnM stock solution. Serial dilutions were made to obtain 20OnM, 10OnM, 5OnM and 25 nM Tiel Fc stocks. For analysis of the association phase samples were injected at 30μl/min for 8,3 minutes using kinject program. This was followed by a 40 minutes dissociation phase. Any antigen remaining associated to the surface was stripped from the IgG surface at a flow of 50 μl/min with two injections of 1OmM glycine pHl,5 for 30seconds. AU samples were run and analyzed in duplicate
[0568] Sensorgrams were analyzed using the simultaneous ka/kd fitting program with 1 : 1 model in the BIAEV ALUATION™ software 3.1. The germlining process had minimal impact on the binding activity of the E3 IgG with respect to mouse Tiel -Fc.
Table 5: Comparison of the binding affinity of parental and germlined E3 IgG
E3 lgG Tiel Fc kon (1/Ms) koff (1/s) KD(1) nM parental Mouse 6.17E+03 9.20E-05 14.9 germlined Mouse 6.00E+03 8.99E-05 15
["0569] Example 15 : Comparison of IC^o of Germlined Anti Tiel - E3 and
Parental Anti Tiel - E3 in Tube Formation Assays using HUVEC Cells
[0570] Germlined E3 (DX-2220) and its parental antibody (DX-2200) were evaluated in the tube formation assay in a collagen type-I matrix. Human Umbilical vein endothelial cells (HUVEC) (freshly isolated) were obtained by treating human umbilical cord veins with Trypsin-EDTA (Ix) (Gibco/Invitrogen) for 20-25 minutes at 37°C. The cells were then cultured in a T-25 flask coated with attachment factor (AF), (Cascade Biologies) in RPMI 1640 medium supplemented with 10 % FCS, 0.4 % BBE, 1% 1-glutamin, 1% penicillin/streptomycin. Primary cultures were detached with warm Trypsin-EDTA and used when confluent at the second or third passage. During culturing, the cells were kept in a proliferative state by culturing them in a split ratio 1:2 at an approximate density of the monolayer of about 60-80%. HUVEC monolayers were treated with trypsin/EDTA (500μl/dish) at 370C for 3 min. Trypsin activity was stopped by adding 3 volumes of complete RPMI medium. The cells were carefully scraped, separated by repeated pipetting, and finally washed with PBS. HUVECs (passage 2) were seeded in their culture medium (40 x 103/50μl/well of a 96-well plate) on a collagen gel (50 μl of collagen 1 1.5mg/ml) prepared by mixing 7.5 volumes of 2mg/ml collagen (Collagen R; Serva, Heidelberg, Germany), 1 volume of 1OX MEM, 1.5 volume of NaHCO3 (15.6 mg/ml) and ~ 1 volume of NaOH to adjust the pH to 7.4. After Ih 30 min., the culture medium was then discarded and the cells were covered with a new layer of collagen (1.5 mg/ml, new preparation, 50μl/well). After polymerization of the gel, culture medium was added to each well in presence or in absence of E3 antibody (DX-2200) or germlined E3 antibody (DX-2220) (0.1 ng/ml to 100 ng/ml). The total length of the tube network on the culture surface was quantified at 40x magnification by the METAVUE™ Software (Universal Imaging Corporation). Results from triplicate wells were expressed as mean vessel area per field ±SEM (relative units). Each assay was performed at least three times. The conclusions are that conversion of the three somatic mutations to germline amino acids in E3 has had little effect on the potency of E3. Both parental E3 (FIGS. 2A and 2B) and germlined E3 (FIGS. 2C and 2D) inhibit tube formation in vitro with an IC50 less than 10 ng/ml, i.e. 66 pM.
[0571] Preliminary studies demonstrated that a monovalent Fab version of
DX-2240 Fab was unable to inhibit tube formation in HUVECs. Thus, in this assay, bivalency is required to elicit an effect in a cell-based assay. [05721 Example 16 : Analysis of Germlined Anti Tiel - E3 in Tube Formation
Assays With Mouse Endothelial Cells
[0573] hi order to assess mouse Tiel cross-reactivity and biological activity on mouse Tiel, both E3 and germlined E3 were evaluated for their ability to inhibit tube formation in vitro using mouse endothelial cell line (LEII).
[0574] LEII lung mouse endothelial cell line (ATCC) was cultured in a T-25 flask in MEM medium with GLUTAMAX™ (Life Technologies Ltd., Paisley, Scotland) supplemented with 10 % FCS, and 1% penicillin/streptomycin. During culturing, the cells were kept in a proliferative state by culturing them in a split ratio 1:5 at an approximate density of the monolayer of about 80%. LEII monolayers were treated with trypsin/EDTA (500 μl/dish) at 37°C for 3 min. Trypsin activity was stopped by adding 3 volumes of complete MEM medium. The cells were carefully scraped, separated by repeated pipetting, and finally washed with PBS. LEII cells were seeded in their culture medium (20-40 X 103/50μl/well of a 96-well plate) on a basement membrane (BIOCOAT™ Angiogenesis System; Becton Dickinson). After polymerization of the MATRIGEL™ (30 min at 370C, 5% CO2 environment) the endothelial cell suspension resuspended in complete culture medium in the presence of the desired molecules (4.105 cells/ml; 50μl/well) was added to each well. The angiogenesis assay plate was then incubated for 16 to 18 hours at 37°C, 5% CO2 atmosphere. The total length of the tube network was then quantified at 40 X magnification by the METAVUE™ Software (Universal Imaging Corporation). Results from triplicate wells were expressed as mean vessel area per field ± SEM (relative units). Each assay was performed at least two times. Germlined E3 is a potent inhibitor of tube formation in mouse endothelial cells.
[0575] Example 17 : Immunohistochemical Analysis of Mouse Tumor Tissue
Sections Using Anti Tiel - E3 IgG
[0576] We determined if antibody E3 binds to mouse endothelial cells in mouse xenographs. Immunohistochemistry was performed with biotinylated E3 IgGl (a,z allotype) antibody and control antibodies anti-CD31 (endothelial cell specific marker) and anti-PCNA (proliferating cell nuclear antigen). Formalin-fixed tumor tissues from a mouse-xenograph containing SW480 cells (ATCC) were tested for the binding pattern of the human anti-Tiel antibody E3. 5 μm sections of paraffin embedded tissues were deparaffinized, rehydrated and pretreated with warm the citrate buffer (0.01 M sodium citrate, pH6 at 95°C) for 45 min. The slides were cooled down in fresh citrate buffer for 20 min and rinsed with distilled water. The slides were hydrogen peroxide treated, (0.3 % H2O2 in PBS), and preincubated with PBS, 5 % FCS, 5% heat inactivated human serum (HS) for 1 hour. Between antibody incubations slides were washed 3 times 5 minutes in PBS. Biotinylated antibodies E3 and A2-SV were diluted to a concentration of 10 μg/ml in PBS5 10 % HS and incubated for 1 hour at RT. Slides were then incubated with an avidin-HRP (Dako) for 30 minutes at room temperature. Staining was detected by AEC (Vector Laboratories, Burlingame) and H2O2. The peroxidase reaction was stopped with water and slides were counter-stained with haematoxylin. The tissues were evaluated for their binding reactivity. The staining pattern was consistent with staining of mouse endothelial cell Tiel and also with Tiel expressed by the E3 binds to Tiel expressed by SW480 tumor cells in a mouse xenograft.
IO5771 Example 18 : E3 Activity in a MATRIGEL™ Plug Assay
[0578] The germlined variant of the E3 IgG antibody was evaluated in an in vivo assay for angiogenesis induced by bFGF in MATRIGEL™ plugs. Growth factor reduced MATRIGEL™ (BD Biosciences, catalog # 354230) was supplemented with 80 ng/ml of bFGF (R&D Systems, catalog #234-FSE). The A2-SV or an IgG4 E3 antibody (10 μg/ml) or PBS was injected subcutaneously into the abdominal area of NMRI nu/nu mice (150 μl of Matri gel/plug).
[0579] In the first assay, two mice were injected with MATRIGEL™ supplemented with bFGF and soluble VEGFR-I (10 μg/ml) as a positive control for an angiogenic inhibitor. At day 7 post-implantation mice were anesthetized and perfused through heart with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). MATRIGEL™ plugs and a piece of liver were removed and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin (H&E).
[0580] The staining revealed modification of MATRIGEL™ and formation of vessel-like structures in the PBS and A2-SV antibody treated plugs. Even though the E3 antibody and soluble VEGFRl supplemented plugs contained single cells, there were neither modification of the matrix nor organization of the cells observed in these plugs. This results indicates that the germlined E3 antibody inhibits angiogenesis in MATRIGEL™ in vivo.
[0581] In a second assay, mice were treated as described above, and then anesthetized eight days post-implantation and injected with fluorescein-conjugated tomato (lycopersicon esculentum) lectin (100 μg in 200 μl of PBS; Vector, catalog #FL-1171) into the tail vein. After five min circulation the animals were perfused through the heart with 10 ml of PBS followed by 10 ml of 4% PFA in PBS. MATRIGEL™ plugs and pieces of kidney and liver were removed and frozen in OCT (Tissue-Tek). Nuclei were visualized on sections by using VECTASHIELD® mounting medium containing DAPI (Vector) and analyzed under fluorescence microscopy.
[0582] Staining of the MATRIGEL™ with fluorescein lectin revealed stain- positive material for the PBS and control antibody (A2-SV) containing plugs, but no staining could be detected in the E3 antibody containing plugs. As a control, the blood vessels of the kidney and liver from the same mice showed nice staining with the fluorescent lectin.
[0583] Results from these two experiments suggest that the anti-Tiel antibody
E3 can inhibit bFGF-induced angiogenesis in vivo.
[0584] To assess further the potential anti-angiogenic activity of the E3 antibody, a third assay examining the effect of DX-2210 (E3 antibody with germlined light chain) on bFGF-induced endothelial cell tube formation in MATRIGEL™ plugs was performed. Growth factor reduced MATRIGEL™ supplemented with HUVECs, bFGF, and DX-2210, A2-SV (negative control IgG), or PBS were injected subcutaneously into the abdominal area of Balb/c nu/nu mice (150 μl MATRIGEL™/plug). At day 8 post-implantation, mice were anesthetized and injected with fluorescein-conjugated tomato (Lycopercicon esculentum) lectin into the tail vein. After a five minute circulation period, the MATRIGEL™ plugs and liver and kidneys were removed and frozen in OCT media. In order to quantitate the amount of blood vessels in the MATRIGEL™ plugs, sections were cut and either stained for endothelial cell content using an anti-CD-31 antibody or analyzed under fluorescence microscopy to assess the amount of functional blood vessels (tomato lectin staining) (data not shown). In addition, the amount of blood vessels per unit area was quantitated. These results demonstrated that DX-2210 inhibits bFGF- induced angiogenesis by 70% in the MATRIGEL™ assay (FIG. 3).
[0585] Example 19 : Evaluating Effects of Ligands on Complex Formation
[0586] A candidate protein (for example, E3 or E3b antibody) that binds a complex member, such as Tiel, Tie2, or an angiopoietin is tested for its ability to antagonize formation of a heteromeric complex that includes Tiel, Tie2, and Ang, by inhibiting its formation or disrupting the heteromeric complex once it forms.
[0587] To test the ability of a candidate protein to disrupt complex formation, cells expressing Tiel and Tie2 are treated with Ang for a period of time sufficient to allow binding of Ang to Tiel and/or Tie2. The cells are contacted with the candidate protein for a period of time sufficient to allow disruption of the complex. The cells are treated with a membrane non-permeable cross-linker, such as DTSSP, to chemically cross-link the proteins. Cell lysates are prepared and subjected to immunoprecipitation with an antibody specific to a complex member. The immunoprecipitated proteins are separated by SDS-PAGE electrophoresis and immunoblotted with antibodies specific to the complex members. A positive control immunoprecipitation-immunoblot is also performed in which cells expressing Tiel and Tie2 are treated with Ang but not with the candidate protein or are treated with a nonspecific protein. If treatment with the candidate protein decreases the amount of a complex member- that is not bound by the immunoprecipitating antibody- associated with the immunoprecipitated member as compared to the positive control, the candidate protein is an antagonist of complex formation.
[0588] To determine if a candidate protein inhibits complex formation, a similar experiment is performed, except that the cells expressing Tiel and Tie2 are treated with the candidate protein prior to treatment of the cells with Ang. The cells are incubated for a period of time sufficient to allow complex formation in the absence of candidate protein. As described above, a positive control in which the cells are not treated with a candidate protein or are treated with a nonspecific protein is performed. The treated cells are then lysed and immunoprecipitations and immunoblots are performed as described above.
[0589] Candidate proteins that antagonize complex formation, by inhibiting complex formation or by disrupting complexes, are then tested for their effects on angiogenesis in an assay described herein.
[0590] Example 20 : Tie2 Amino Acid Sequence
[0591] An exemplary Tie2 amino acid sequence is as follows:
MDSLASLVLC GVSLLLSGTV EGAMDLILIN SLPLVSDAET SLTCIASGWR 50
PHEPITIGRD FEALMNQHQD PLEVTQDVTR EWAKKWWKR EKASKINGAY 100
FCEGRVRGEA IRIRTMKMRQ QASFLPATLT MTVDKGDNVN ISFKKVLIKE 150
EDAVIYKNGS FIHSVPRHEV PDILEVHLPH AQPQDAGVYS ARYIGGNLFT 200
SAFTRLIVRR CEAQKWGPEC NHLCTACMNN GVCHEDTGEC ICPPGFMGRT 250
CEKACELHTF GRTCKERCSG QEGCKSYVFC LPDPYGCSCA TGWKGLQCNE 300
ACHPGFYGPD CKLRCSCNNG EMCDRFQGCL CSPGWQGLQC EREGIPRMTP 350
KiVDLPDHiE VNSGKFNPIC KASGWPLPTN EEMTLVKPDG TVLHPKDFNH 400
TDHFSVAIFT IHRILPPDSG VWVCSVNTVA GMVEKPFNIS VKVLPKPLNA 450
PNVIDTGHNF AVINISSEPY FGDGPIKSKK LLYKPVNHYE AWQHIQVTNE 500
IVTLNYLEPR TEYELCVQLV RRGEGGEGHP GPVRRFTTAS IGLPPPRGLN 550
LLPKSQTTLN LTWQPIFPSS EDDFYVEVER RSVQKSDQQN IKVPGNLTSV 600
LLNNLHPREQ YWRARVNTK AQGEWSEDLT AWTLSDILPP QPENIKISNI 650
THSSAVISWT ILDGYSISSI TIRYKVQGKN EDQHVDVKIK NATIIQYQLK 700
GLEPETAYQV DIFAENNIGS SNPAFSHELV TLPESQAPAD LGGGKMLLIA 750
ILGSAGMTCL TVLLAFLIIL QLKRANVQRR MAQAFQNVRE EPAVQFNSGT 800
LALNRKVKNN PDPTIYPVLD WNDIKFQDVI GEGNFGQVLK ARIKKDGLRM 850
DAAIKRMKEY ASKDDHRDFA GELEVLCKLG HHPNIINLLG ACEHRGYLYL 900
AIEYAPHGNL LDFLRKSRVL ETDPAFAIAN STASTLSSQQ LLHFAADVAR 950
GMDYLSQKQF IHRDLAARNI LVGENYVAKI ADFGLSRGQE VYVKKTMGRL 1000
PVRWMAIESL NYSVYTTNSD VWSYGVLLWE IVSLGGTPYC GMTCAELYEK 1050
LPQGYRLEKP LNCDDEVYDL MRQCWREKPY ERPSFAQILV SLNRMLEERK 1100
TYVNTTLYEK FTYAGIDCSA EEAA 1124
(SEQ ID NO: 162) SWISS PROT ACCESSION NUMBER: Q02763
[0592] Example 21 : Angl Amino Acid Sequence
[0593] An exemplary Angl amino acid sequence is as follows:
1 MTVFLSFAFL AAILTHIGCS NQRRSPENSG RRYNRIQHGQ CAYTFILPEH DGNCRESTTD 61 QYNTNALQRD APHVEPDFSS QKLQHLEHVM ENYTQWLQKL ENYIVENMKS EMAQIQQNAV 121 QNHTATMLEI GTSLLSQTAE QTRKLTDVΞT QVLNQTSRLE IQLLENSLST YKLEKQLLQQ 181 TNEILKIHEK NSLLEHKILE MEGKHKEELD TLKEEKENLQ GLVTRQTYII QELEKQLNRA 241 TTNNSVLQKQ QLELMDTVHN LVNLCTKEVL LKGGKREEEK PFRDCADVYQ AGFNKSGIYT 301 IYINNMPEPK KVFCNMDVNG GGWTVIQHRE DGSLDFQRGW KEYKMGFGNP SGEYWLGNEF 361 IFAITSQRQY MLRIELMDWE GNRAYSQYDR FHIGNΞKQNY RLYLKGHTGT AGKQSSLILH 421 GADPSTKDAD NDNCMCKCAL MLTGGWWFDA CGPSNLNGMF YTAGQNHGKL NGIKWHYFKG 481 PSYSLRSTTM MIRPLDF
(SEQ ID NO:163) NCBI ACCESSION NUMBER: AAM92271; gi: 22203641
[05941 Example 22 : Conversion of a Mutation Positioned in the Framework 3
Region of Anti-Tie 1 E3 Heavy Chain to Germline Residue
[0595] hi order to limit the risk of potential immunogenicity of the E3 antibody after administration to patients, all non-germline mutations in framework regions were corrected back to germline amino acid residues. The anti-Tie 1 E03 antibody was isolated from Dyax Fab 200 library, hi this library, the HC framework regions are unique and correspond to the DP47 germline segment (V3-23). Since the construction of the synthetic HC-CDR1-CDR2 sublibrary was made through the assembling of overlapping oligonucleotides, followed by some PCR cycles, mutations may have been introduced by one of those 2 steps. Therefore, an analysis was performed which aligned the HC of E03 antibody with the DP47 germline gene sequence. One non-germline mutation positioned in framework region number 3 was identified where a methionine residue has been replaced by a valine residue.
[0596] A strategy was designed to repair this mutation. The introduction of the germlined residue was facilitated by the presence of internal restriction sites in the framework flanking regions of the CDRs. Indeed, the design of the HC-CDRl -CDR2 sublibrary, present in FAB 310 library, was made in such a way that the shuffling of every CDR is allowed by the presence of unique restriction sites in the framework flanking regions. Since the valine residue to be corrected is located in FR3 region, 3' near the Xbal site, a primer was designed containing both the Xbal sequence and the corrected methionine germline residue. The changes were introduced by PCR using the Top Xbal -M forward primer combined with a 3' reverse primer (CJ-lift Nhe REV) annealing in the CHl region. A PCR fragment of ~180bp was then generated. The germlining PCR primers were:
Top Xbal -M primer:
5' TTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGaTGAAC 3' (SEQID
NO:717) F T I S R D N S K N T L Y L Q M N (SEQ ID
NO:718)
CJ- lift Nhe REV:
5' GGAGGGTGCTAGCGGGAAGACCG 3' (SEQ ID NO:719)
[05971 Example 23 : Cloning of Germlined Heavy Chain Tiel E3
[0598] In order to determine if the germlined residue introduced into E3 heavy chain had affected the biological activity of the antibody, a soluble Fab expression vector containing the germlined E3 antibody (termed "E3b" or DX-2220) was constructed. The PCR fragment from Example 22 was digested overnight with 50 U/μgXbal restriction enzyme, followed by a 5 hours digestion with 25 U/μg BsiER. The cleaved PCR product was then purified on a 1% TAE-agarose preparative gel. Ligation into the similarly-digested phagemid expression vector (pMIDl) containing the Tiel E3 germlined light chain sequence was performed for three hours at room temperature. Five nanograms of the newly-ligated material were electroporated into TGl bacterial cells. Verification of the correction of the mutation was performed by sequence determination of the heavy chain of 20 randomly picked isolates. The resulting coding construct contained sequences that encode a germlined HC and a germlined LC sequence in a Fab format (termed Fab E3b).
[05991 Example 24 : Production and Purification of E3b (OX-2220)
[0600] The E3b Fab antibody was reformatted to a human IgGl . This construct was then used to transiently transfect HEK293T cells. Plasmid preparations for transient cell transfections were obtained using the Qiagen filter Plasmid Maxi kit (Qiagen, cat. no. 12263). HEK293T cells (GenHunter Corp., cat. no. Q401) were seeded 24 hours before transfection; 220 x 106 cells were plated per CELLSTACK® culture vessel (CeIlST ACK®- 10 Chamber, Corning, cat. no. 3271). Transfections were carried out using the GeneJuice® reagent (VWR, cat. no. novg70967-3) following the manufacturer's instructions. 650 micrograms of plasmid DNA was used per CELLSTACK®. Cells were cultured in DMEM (Invitrogen, cat. no. 31966021) supplemented with 10 % "ultra-low IgG" fetal calf serum (Invitrogen, cat. no. 16250078), at 370C, 5 % CO2, in a water saturated atmosphere. Conditioned media were harvested 72, 144 and 216 hours after transfection, pooled and sterile filtered.
[0601] Cell culture supernatants were loaded on a 25-ml rProteinA FF column
(GE Healthcare, cat. no. 17-1279-02) equilibrated against PBS containing 0.5 M NaCl. The column was washed with PBS containing 0.5 M NaCl, then with 0.1 M sodium acetate pH 5.0 to remove bovine IgGs and the antibody was eluted with 12.5 mM citric acid. Fractions (5 ml) containing the antibody were neutralized by addition of 150 μl of 1 M Tris-HCl, pH 9.0.
[0602] The E3b antibody was further purified by cation exchange.
The antibody was dialyzed against 50 mM sodium citrate, pH 5.0, and loaded on a HiLoad 26/10 SP Sepharose HP column (GE Healthcare, cat. no. 17-1138-01) equilibrated in the same buffer. The antibody was eluted with 50 mM sodium citrate, pH 5.0, containing 1 M NaCl (linear gradient on 10 column volumes). Fractions containing the antibody were pooled and dialyzed against PBS. Antibody concentration was calculated from the absorbance at 280 nm, assuming that a protein concentration of 1 mg/ml has an absorbance of 1.36.
[06031 Example 25 : Testing of E3b - IgGl Binding to TIE-l/Fc in BIAcore
[0604] The germlined E3b IgGl stock solution (0.56 mg/ml) and the parental
E3 IgGl stock solution (0.41 mg/ml) were diluted 50-fold in 10 mM sodium acetate, pH 4.5. The IgGs were directly coated on a CM5 chip. The surface of the chip was activated with a 7-minute pulse of 0.05 M NHS / 0.2 M EDC and the IgG was run over the chip until 823 RU of germlined E3b and 788 RU of parental E3 were coated on the surface. AU flow cells were subsequently deactivated with a 7-minute pulse of 1 M ethanolamine hydrochloride, pH 8.5. AU further experiments were performed in HBS buffer.
[0605] Purified recombinant human Tiel/Fc was diluted in HBS to final concentrations of 200, 100, 50, 25 and 12.5 nM. Samples were injected at 30 μl/min for 8.3 minutes using the kinject program. This was followed by a 50-minute dissociation phase. Any remaining antigen was stripped from the surface with two 30-sec injections of 10 mM glycine, pH 1.5. [0606] The sensorgrams obtained with this approach are shown below. Visual analysis shows that the dissociation (kOff) is extremely slow (only a very small fraction of Tiel/Fc dissociated despite the long dissociation time), which suggests a very tight interaction. Interestingly, the dissociation rates for the IgGs as measured here are much slower that those of the corresponding Fabs (see Example 29 below), indicating that there is a significant increase in the affinity when going from the monovalent Fab to the bivalent IgG (avidity).
[06071 Example 26 : Testing of E3b - Fab Binding to Tiel/Fc in BIAcore
[0608] To evaluate if the binding behaviour had been affected in any way by the conversion of the somatic mutations back to germline residues, the parental and the germlined E3b antibodies were produced and tested in Biacore as Fab fragments. Here, by contrast to what was done for the IgGs (see Example 28), and in order to measure a monovalent interaction, the Fabs were run over the antigen directly coated onto the surface.
[0609] Recombinant human Tiel/Fc was coated on a CM5 chip. The surface of the chip was first activated with a 7-minute pulse of 0.05 M NHS / 0.2 M EDC, then Tiel/Fc (2 μg/ml in 10 mM sodium acetate, pH 4.0) was run over the chip surface until 750 RUs were coated on the surface. All flow cells were subsequently deactivated with a 7-minute pulse of 1 M ethanolamine hydrochloride, pH 8.5. All further experiments were performed in HBS buffer.
[0610] The parental and the germlined E3b Fabs were prepared. A series of dilution (50, 25, 12.5, 6.25 and 3.125 nM) was prepared in HBS buffer. Samples were injected at 30 μl/min for 5.3 minutes using the KINJECT™ program. This was followed by a 10-minute dissociation phase, and the remaining Fab was stripped from the surface with a single 18-sec injection of 50 mM NaOH / I M NaCl.
[0611] Sensorgrams were analyzed using the simultaneous ka/kd fitting program from the BIAEV ALUATION™ software 3.1 assuming a 1:1 model. This analysis proved that the germlining of the E3 antibody has little or no effect on the affinity against Tiel/Fc :
ES Fab Tiel Fc kon (I/Ms) koff(l/s) KD (nM) Parental Human 8,81e4 l,05e-03 12 germlined (E3b) Human 1.36e5 l,01e-03 7
[06121 Example 27 : Testing of E3b - IeG for Biological Activity in Tube
Formation Assays
[0613] The purpose of this study was to determine if the correction of HC mutation back to germline in the parental E3 has any effect on the biological activity. Human umbilical vein endothelial cells (HUVEC) (freshly isolated) were obtained by treating human umbilical cord veins with Trypsin-EDTA (Ix) (Gibco/fnvitrogen) for 20-25 minutes at 37°C. The cells were then cultured in a T-25 flask coated with attachment factor (AF), (Cascade Biologies) in RPMI 1640 medium supplemented with 10 % FCS, 0.4 % BBE, 1% 1-glutamin, 1% penicillin/streptomycin. Primary cultures were detached with warm Trypsin-EDTA and used when confluent at the second or third passage. During culturing, the cells were kept in a proliferative state by culturing them in a split ratio 1:2 at an approximate density of the monolayer of about 60-80%. HUVEC monolayers were treated with trypsin/EDTA (500 μl/dish) at 37°C for 3 min. Trypsin activity was stopped by adding 3 volumes of complete RPMI medium. The cells were carefully scraped, separated by repeated pipetting, and finally washed with PBS. HUVECs (passage 3) were seeded in their culture medium (40 x 103/50 μl/well of a 96-well plate) on a collagen gel (50μl of collagen I 1.5mg/ml) prepared by mixing 7.5 volumes of 2mg/ml collagen (Collagen R; Serva, Heidelberg, Germany), 1 volume of 1OX MEM, 1.5 volume OfNaHCO3 (15.6 mg/ml) and ~ 1 volume of NaOH to adjust the pH to 7.4. After Ih 30 min, the culture medium was then discarded and the cells were covered with a new layer of collagen (1.5mg/ml, new preparation, 50μl/well). After polymerization of the gel, culture medium was added to each well in presence or in absence of E3b-IgGl or parental E3 antibody (1 pg/ml to 10 ng/ml). Endothelial tube formation was assessed with an inverted photomicroscope. Microphoto graphs of the centre of each well at low power (x40) were taken with a Nikon camera with the aid of imaging-capture software. Tube formation in the microphotographs was quantitatively analysed (total tube length) with META VUE® software (data not shown)- Tube formation by untreated HUVECs in full endothelial cell growth medium was used as control. Results from triplicate wells were expressed as mean vessel area per field ± SEM (relative units) (FIG. 4). The conclusions are that E3b-IgGl inhibits tube formation. Correction of HC mutation had no significant effect on biological activity.
[0614] Example 28 : Exemplary Tiel Binding Sequences
[0615] The following are exemplary sequences of immunoglobulin light chain and heavy chain variable domains:
1. 806C-M0044-A06
L-Variable (AA):
QSΞLTQPPSASGTPGQRVTISCSGSSSSIGLNPVNWYQQLPGTAPKWIHSNDQRPS GVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPAFGGGTKLTVL (SEQ ID NO:164)
L-Variable (DNA):
CAGAGCGAATTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACC ATCTCTTGTTCTGGAAGCAGCTCCAGCATCGGACTTAATCCTGTAAACTGGTACCAGCAGCT CCCAGGAACGGCCCCCAAAGTAGTCATCCATAGTAATGATCAGCGGCCCTCAGGGGTCCCTG ACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGACTCCAGTCT GAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTCCGGCATTCGG CGGAGGGACCAAGCTGACCGTCCTAG (SEQ ID NO:165)
H-Variable (AA) :
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYVMMWRQAPGKGLEWVSRIYPSGGITQYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDVYRAFDIWGQGTMVTVSS (SEQ ID NO : 166 )
H-Variable (DNA) :
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACGTTATGATGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCATTACTCAGTATGCTGAC
TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAΆCAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGATGTCTACAGGGCTT TTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:167)
2. 806C-M0044-A11
L-Variable (AA) :
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPGGTFGQGTKVEIK (SEQ ID NO: 168)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC CTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGAC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGA AGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCCGGGGGGAACGTTCGGCC AAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:169) H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMHWVRQAPGKGLEWVSSIYPSGGYTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSHHFHFWGDYYFLEYWGQGTLVTVS S (SEQ ID NO:170)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGCATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTATACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTATATTACTGTGCGAGAGATAGCCATCATTTCC ATTTTTGGGGTGACTATTATTTTCTAGAATACTGGGGCCAGGGAACCCTGGTCACCGTCTCA AGC (SEQ ID NO:171)
3. 806C-M0044-B04 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGGGTKVEIK (SEQ ID NO:172)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC CATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAΆGTGGGGTCCCATCAAGG TTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGA TTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCCACTTTCGGCGGAGGGACCA AGGTGGAGATCAAA (SEQ ID NO:173)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYLMFWVRQAPGKGLEWVSYIYPSGGWTMYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQNYYDSSGYYYRGFDYWGQGTLVTVS S (SEQ ID NO:174)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACCTTATGTTTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTATATCTATCCTTCTGGTGGCTGGACTATGTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAΆCTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGGCAAAATTACTATGATA GTAGTGGTTATTACTATCGTGGCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCA AGC (SEQ ID NO:175)
4. 806C-M0044-B05 L-Variable (AA):
DIHMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPGITFGGGTKVEIK (SEQ ID NO:176
)
L-Variable (DNA) :
GACATCCATATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAΆCCTGGCC AGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTC AGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTT TGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGGGGATCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAA. (SEQ ID NO:177)
H-Variable (AA) :
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMGWVRQAPGKGLEWVSSIYPSGGWTHYAD SVKGRFTISRDNSKNTLYLQMNSLRAΞDTAVYYCARVLLHYFDYWGQGTLVTVSS (SEQ ID NO:178)
H-Variablθ (DNA) :
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGT
CTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGGGTTGGGTTCGCCAΆGC
TCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTCATTATG CTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTG CAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCAAGAGTACTACTACA CTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:179)
5. 806C-M0044-B08 L-Variable (AA):
QDIQMTQSPSFLSASVGDRVTISCRASQYISIYLNWYQQRPGEAPKLLINAASSLQSGDPSR FSGSGSGTDFTLTINSLQPDDFATYYCQQYKSYPLTFGEGTKVEIK (SEQ ID NO:180)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCCGCATCTGTAGGAGACAGAGTCAC CATCTCTTGCCGGGCAΆGTCAGTACATCAGCATATATTTGAATTGGTATCAGCAGAGACCAG GGGAAGCCCCTAAACTCCTGATCAATGCTGCATCCAGTTTGCAAAGTGGGGACCCATCAAGG TTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGATGA TTTTGCAACTTATTACTGCCAACAGTATAAGAGTTACCCCCTCACTTTCGGCGAGGGGACCA AGGTGGAGATCAAA (SEQ ID NO:181)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYGMGWVRQAPGKGLEWVSVISPSGGQTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCAGGDRYGPLHYWGQGTLVTVSS (SEQ ID NO:182)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACGGTATGGGTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCTCTCCTTCTGGTGGCCAGACTTCTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACCGCCTTGTATTACTGTGCGGGAGGGGACAGGTATGGAC CCTTGCACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:183)
6. 806C-M0044-B09 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYHASNLETGVPSR FSGSGSGTDFTLTISSLQPΞDFATYYCLQYKSYPRLFGQGTKVEVK (SEQ ID NO:184)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC CATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAG GGAAΆGCCCCTAAGCTCCTGATCTACCATGCATCCAATTTGGAAACAGGGGTCCCATCAAGG TTCAGTGGAAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGA TTTTGCAACTTATTACTGTCTTCAGTATAΆAAGTTACCCTCGATTGTTCGGCCAAGGGACCA AGGTGGAAGTCAAA (SEQ ID NO:185)
H-Variable (AA):
ΞVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMNWVRQAPGKGLEWVSVIYPSGGWTYYAD SVKGRFTISRDNSKNTL YLQMNSLRAEDTAWYCASGYYDSSGYSRFDYWGQGTLVTVSS (SEQ ID NO : 186 )
H-Variablβ (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGAATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGAC
TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT
GAACAGCTTAAGGGCTGAGGACACGGCTGTGTATTACTGTGCGAGTGGTTACTATGATAGTA
GTGGTTACTCCCGATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ
ID NO:187)
7. 806C-M0044-B10 L-Variable (AA):
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPQLMIYEGSKRPSGLSN RFSGSKSDNTASLTISGLQAEDEADYYCCSYAGSSTLVFGGGTKLTVL (SEQ ID NO : 188 )
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC CTGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAACAACACC CAGGCAAAGCCCCCCAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGACTTTCTAAT CGCTTCTCTGGCTCCAAGTCTGACAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGA GGACGAGGCTGATTATTACTGCTGCTCATATGCAGGTAGTAGCACTTTAGTATTCGGCGGAG
GGACCAAGCTGACCGTCCTA (SEQIDNO:189)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMGWVRQAPGKGLEWVSSIYPSGGPTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARSEVGAPDYWGQGTLVTVSS (SEQ ID NO:190)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGGGTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCCCTACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACAGCCATGTATTACTGTGCGAGAAGCGAAGTGGGAGCCC CCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:191)
8. 806C-M0044-B12 L-Variable (AA):
QDIQMTQSPSTLSASVGDTVTMTCRASQSISGWLAWYQQKPGKAPNLLIFKASTLKSGVPSR FRGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSQTFGQGTKVEIK (SEQ ID NO:192) L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTTTCTGCATCTGTAGGAGACACCGTCAC CATGACTTGCCGGGCCAGTCAGAGTATTAGTGGGTGGTTGGCCTGGTATCAGCAGAAACCAG GGAAAGCCCCTAACCTCCTGATCTTTAAGGCGTCTACTTTAAAAAGTGGGGTCCCGTCAAGG TTTCGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGA TTTTGCAACTTATTACTGCCAACAATATAATAGTTATTCTCAGACGTTCGGCCAAGGGACCA AGGTGGAAATCAAA (SEQ ID NO:193)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYKMHWVRQAPGKGLEWVSSIYPSGGYTVYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATDRWSSGGYGVDFWGQGTLVTVSS (SEQ ID NO:194)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTATGTACAAGATGCATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTATACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCCACAGACCGG
TGGAGCAGTGGCGGGTACGGTGTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAG
C (SEQ ID NO:195)
9. 806C-M0044-C07 L-Variable (AA):
QDIQMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPQFGQGTKVEIK (SEQ ID NO: 196)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTC CATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGT ACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCC GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAG AGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCAGTTCG GCCAAGGGACCAAGGTGGAAATCAAG (SEQ ID NO:197)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYDMSWVRQAPGKGLEWVSYIYPSGGPTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDWASRFATWGQGTTVTVSS (SEQ ID NO: 198)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACGATATGTCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTATATCTATCCTTCTGGTGGCCCTACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGGCGATTGGGCTTCTC GTTTTGCCACCTGGGGCCAGGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:199)
10.806C-M0044-D01 L- Variable (AA):
QYELTQPPSVSVAPGQTARITCGGIrølGIKSVNWYQQKPGQAPVLWYDDSGRPSGIPQRFS GSNSGNTATLTINRVEAGDEADYYCQVWDSGSDHWVFGGGTKLTVL ( SEQ ID NO : 200 )
L-Variable (DNA):
CAGTACGAATTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTAC
CTGTGGGGGAAACAACATTGGAATTAAAAGTGTGAACTGGTACCAGCAGAAGCCAGGCCAGG
CCCCTGTGCTGGTCGTCTATGATGAT
AGTGGCCGGCCCTCAGGGATCCCTCAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCAC
CCTGACCATCAACAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATA
GTGGTAGTGATCATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO:
201)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMGWVRQAPGKGLEWVSSIYPSGGFTRYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNFVESSHYYHDYWGQGTLVTVSS (SEQ ID NO:202)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACAAGATGGGTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTTTACTCGTTATGCTGAC
TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT
GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCCAGAAATTTCGTTGAAAGTA
GTCATTATTACCATGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:203)
11.806C-M0044-E03 L- Variable (AA):
QSELTQPPSVSVAPGQTAVITCGGSNIGGKSVHWYQQKSGQAPVLWFDDRDRPSGIPERFS GSNSGNTATLTITRVEVGDEADYYCQVWDSGTDHRVFGGGTRLTAL (SEQ ID NO:204)
L-Variable (DNA):
CAGAGCGAATTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGACGGCCGTGATTAC CTGTGGGGGGAGCAACATTGGAGGTAΆAAGTGTACACTGGTACCAGCAGAAGTCAGGCCAGG CCCCTGTGCTGGTCGTCTTTGATGATCGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCT GGCTCCAACTCCGGGAACACGGCCACCCTGACCATCACCAGGGTCGAΆGTCGGGGATGAGGC CGACTATTACTGTCAGGTGTGGGATAGTGGAACTGATCATCGGGTGTTCGGCGGAGGGACCA
GGCTGACCGCCCTA (SEQ ID NO:205) H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMFWVRQAPGKGLEWVSGIYPSGGHTRYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGSGGYFDYWGQGTLVTVSS (SEQ ID NO:206)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGTTTTGGGTTCGCCAΆGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCCATACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGACGAGGC TCGGGGGGCTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:207)
12.S06C-M0044-F03 L-Variable (AA):
QSALTQDPAVSVALGQTVRITCRGDRLRSYYSSWYQQKPRQAPVLVMFGRNNRPSGIPDRFS GSTSGSTASLTITATQADDEADYFCSSRDGSGNFLFGGGTKLTVL (SEQ ID NO:208)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGGCAGACAGTCAGGATCAC ATGCCGAGGAGACAGACTCAGAAGTTATTATTCAAGTTGGTACCAGCAGAAGCCACGACAGG CCCCTGTTCTTGTCATGTTTGGTAGAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCT GGCTCCACCTCAGGAΆGCACAGCTTCCTTGACCATCACTGCGACTCAGGCGGACGATGAGGC TGACTATTTCTGTAGTTCCCGGGACGGCAGTGGTAATTTCCTCTTCGGCGGAGGGACCAAAC TGACCGTCCTT (SEQ ID NO:209)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMIWVRQAPGKGLEWVSSIYPSGGTTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAWYCARSDLGSGWYSAEYFQHWGQGTLVTVSS (SEQ ID NO : 210 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGATTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCACTACTTCTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCTGTGTATTACTGTGCGAGAAGCGACCTAGGCAGTG GCTGGTATAGCGCTGAATACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC
(SEQ ID NO:211)
13.806C-M0044-F06 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSGNLLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTITRLEPEDFAVYFCQQYGGSPPVTFGGGTKVEIK (SEQ ID NO: 212)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCGGCAACCTCTTAGCCTGGTATCAGCAGAAAC
CTGGCCAGGCTCCCAGACTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCACCAGACTGGAGCCTGAAGATTTTGCAGTGTATTTCTGTCAGC
AGTATGGTGGCTCACCTCCGGTCACT
TTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:213)
H-Variable (AA):
ΞVQLLESGGGLVQPGGSLRLSCAASGFTFSSYLMIWVRQAPGKGLEWVSRIYPSGGGTEYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVTYYYDSSGYQPAFDIWGQGTMVTVS S (SEQ ID NO:214)
H-Variable (DNA): GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACCTTATGATTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCGGTACTGAGTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAΆTACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTCACGTATTACTATG ATAGTAGTGGTTATCAACCCGCTTTTGATATCTGGGGCCAΆGGGACAATGGTCACCGTCTCA AGC (SEQ ID NO:215)
14.806C-M0044-F09 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLIISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK (SEQ ID NO:216)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG GCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCATCATCAGCAGCCTAGAGCCTGAAGA TTTTGCAGTTTATTATTGTCAGCAGCGTAGCAACTGGCCTCGAACGTTCGGCCAAGGGACCA AGGTGGAAATCAAA (SEQ ID NO:217)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYGMTWVRQAPGKGLEWVSVIGPSGGNTMYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVWGAFDIWGQGTMVTVSS (SEQ ID NO:218)
H-Varϊable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACGGTATGACTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCGGTCCTTCTGGTGGCAATACTATGTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAΆGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTATGGGGTGCTTTTG
ATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:219)
15.806C-M0044-G06 L-Variable (AA):
QDIQMTQSPATLSVSPGERATLSCRASQSVYNNLAWYQQKPGQAPRLLIYDASTTATGIPAR FSGSGSGTDFTLTITSLEPEDFAVYYCQQRSNWPSLTFGGGTKVEIK (SEQ ID NO:220
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAACGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTTACAACAACTTAGCCTGGTACCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCACCACGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCACCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGC
GTAGCAACTGGCCCTCGCTCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:221) H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMGWVRQAPGKGLEWVSSIYPSGGWTHYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVLLHYFDYWGQGTLVTVSS (SEQ ID NO:222)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGGGTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTCATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCAAGAGTACTACTACACTACT TTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:223)
16.806C-M0044-G07 L-Variable (AA):
QDIQMTQSPSFLSASLGDRVTITCRATQGIGTFLAWYQQKAGRAPKLLIYGASTLQSGVPSR FSGSGSGTEFTLTISSLQPEDFATYYCQQPNSFFGQGTKLEIK (SEQ ID NO:224)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTTTAGGAGACAGAGTCAC CATCACTTGTCGGGCCACTCAGGGCATCGGCACTTTTTTAGCCTGGTATCAGCAAAAAGCAG GGAGAGCCCCTAAACTCCTGATCTATGGTGCTTCCACTTTGCAGAGTGGGGTCCCATCAAGG TTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATAAGCAGCCTGCAGCCTGAAGA TTTTGCAACTTATTACTGTCAACAGCCTAATAGTTTTTTTGGGCAGGGGACCAAGCTGGAGA TCAAA (SEQ ID NO:225)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMGWRQAPGKGLEWVSSIYPSGGWTHYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVLLHYFDYWGQGTLVTVSS ( SEQ ID NO : 226 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGGGTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTCATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTG
CAGTCTACTATTGTGCAAGAGTACTA
CTACACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:
227)
17.806C-M0044-G11 L-Variable (AA):
QDIQMTQSPSSVSASVGDRVTITCRASQDISSWLVWYQQKPGKAPKLLIHDASNLQSGVPSR FSGSGSGTDFTLTINSLQPEDFATYYCQQANSFPVTFGGGTKVEIK (SEQ ID NO:228)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCAC CATTACTTGTCGGGCGAGTCAGGATATTAGCAGTTGGTTAGTCTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGCTCCTGATCCATGATGCATCCAATTTGCAAAGTGGGGTCCCATCAAGG TTCAGCGGCAGTGGGTCTGGGACAGATTTTACTCTCACCATCAACAGCCTGCAGCCTGAAGA TTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGGTCACTTTCGGCGGAGGGACCA AGGTGGAGATCAAA (SEQ ID NO:229)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYPMLWVRQAPGKGLEWVSSISPSGGATAYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSYSDYGVFESWGQGTLVTVSS (SEQ ID NO:230)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACCCTATGCTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTCTCCTTCTGGTGGCGCTACTGCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAΆCTCTAAGAΆTACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAAAGGCTCA
TACAGTGATTACGGGGTCTTTGAGTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC
(SEQ ID NO:231)
18.806C-M0044-H03 L- Variable (AA):
QRVLTQPPSASGTPGQRVTISCSGSSSNVGSNNVNWYQQLPGQAPKLLIDSNNHRPSGVPDR FSGSKSGTSASLALSGLQSEDEADYYCATWDDNLIAPVFGGGTKLTVL (SEQ ID NO: 232)
L-Variable (DNA):
CAGAGGGTCTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTC CTGTTCTGGAAGCAGCTCCAATGTCGGAAGTAATAATGTAAACTGGTATCAGCAGCTCCCAG GACAGGCCCCCAAACTCCTCATCGATAGTAATAATCACCGGCCCTCAGGGGTCCCTGACCGA TTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCCTCAGTGGGCTCCAGTCTGAGGA TGAGGCTGATTATTATTGTGCGACATGGGACGACAACCTGATTGCCCCGGTATTCGGCGGAG GGACCAAGCTGACCGTCCTA (SEQ ID NO:233)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMSWVRQAPGKGLEWVSGIVPSGGWTTYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNYYDFWSGYYISRFGMDVWGQGTTV TVSS (SEQ ID NO:234)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGTCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGGTATCGTTCCTTCTGGTGGCTGGACTACTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGATAACTATTACGATT TTTGGAGTGGTTATTATATTTCTCGATTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTC
ACCGTCTCAAGC (SEQ ID NO:235)
19.806C-M0044-H05 L- Variable (AA):
QYELTQPASVSGSPGQSITISCTGSSSDVSGYNYVSWYQHHPGKAPKLMLYDVSNRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTWVFGGGTKLTVL (SEQ ID NO: 236) L-Variable (DNA):
CAGTACGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC CTGCACTGGATCCAGCAGTGACGTTAGTGGTTATAACTATGTCTCCTGGTACCAACACCACC CAGGCAAAGCCCCCAAACTCATGCTTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAAT CGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGA GGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCACTTGGGTGTTCGGCGGAG GGACCAAGCTGACCGTCCTA (SEQ ID NO:237)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYMMFWVRQAPGKGLEWVSRIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAWYCARVTVPLDSGSYYFDYWGQGTLVTVSS (SEQ ID NO : 238 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACATGATGTTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGAC
TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT
GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTTACGGTACCCCTTG
ATAGTGGGAGCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC
(SEQ IDNO:239)
20.806C-M0044-H07 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPWTFGQGTNVEIK (SEQ ID NO:240)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAG
GGAAAGCCCCTAAGCTCCTGATCTAT
GCTGCATCCAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGCACAGA
TTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAAG
ATTACAATTACCCGTGGACGTTCGGC
CAAGGGACCAATGTGGAAATCAAA (SEQ ID NO:241)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYLMTWVRQAPGKGLEWVSSIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAREMYYDFWSGYYRGFDIWGQGTTVTVS S (SEQ ID NO:242)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACCTTATGACTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACAGCCACATATTACTGTGCGAGAGAGATGTATTACGATT TTTGGAGTGGTTATTATCGAGGTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCA
AGC (SEQ ID NO:243)
21.806C-M0044-H09 L-Variable (AA): QDIQMTQSPSTLSASIGDRVTITCRASQRVSTWVAWYQQKPGRAPKLLIYMASRLESGVPSR FSGSGSGTEFTLTISSLQPDDFATYWCQQYNFYPRTFGQGTKVDIK (SEQ ID NO:244)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATAGGAGACAGAGTCAC CATCACTTGCCGGGCCAGTCAGCGTGTTAGTACTTGGGTGGCCTGGTATCAGCAGAAACCAG GGAGAGCCCCAAAACTCTTGATCTATATGGCGTCTAGGTTAGAAAGTGGGGTCCCATCAAGG TTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATAAGCAGCCTGCAGCCTGATGA TTTTGCTACTTATTGGTGCCAACAATATAATTTTTATCCTCGGACGTTCGGCCAAGGGACCA AGGTGGACATCAAA (SEQ ID NO:245)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYGMNWVRQAPGKGLEWVSSISPSGGQTPYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLGGAYIPDSWGQGTLVTVSS (SEQ ID NO:246)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTGGTACGGTATGAATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTCTCCTTCTGGTGGCCAGACTCCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAΆTACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGCGAGATCTC
GGTGGGGCCTACATACCTGACTCCTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC (SEQ
ID NO:247)
22.806C-M0045-A02 L- Variable (AA):
QDIQMTQSPSFLSASVGDRVTITCRASQGISNYLAWYQQEPGKAPKLLIYSASTLQTGVPSR FSGSGSGTEFTLTISSLQPEDFATYYCQQFNSYPRTFGHGTKVEFK (SEQ ID NO:248)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCTTCCTTCCTGTCTGCATCTGTGGGAGACAGAGTCAC CATCACTTGCCGGGCCAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAΆGAACCAG GGAAAGCCCCTAAGCTCCTCATCTATTCTGCGTCCACTTTGCAAACTGGAGTCCCATCAAGG TTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACAATCAGCAGCCTGCAGCCTGAGGA TTTTGCAACTTATTACTGTCAACAGTTTAACAGTTACCCTCGAACGTTCGGCCACGGGACCA AGGTGGAATTCAAA (SEQ ID NO:249)
H-Variable (AA):
EVQLLΞSGGGLVQPGGSLRLSCAASGFTFSTYPMMWVRQAPGKGLEWVSVISPSGGQTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGGRLNAFDIWGQGTMVTVSS (SEQ ID NO:250)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTACTTACCCTATGATGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCTCTCCTTCTGGTGGCCAGACTTCTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACAGCCGTGTATTACTGTACGAGAGGGGGGAGGCTGAATG CTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:251) 23.S06C-M0045-A04 L-Variable (AA):
QSALTQDPAVSVALGQTVRFTCQGDSLRNYHPSWYQQKPGQAPVLVIYGKNNRPSGIPDRFS GSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVFGTGTKVTVL (SEQ ID NO:252)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGTTCAC TTGCCAAGGAGACAGCCTCAGAAATTATCATCCAAGCTGGTACCAGCAGAAGCCAGGACAGG CCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCT GGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGC TGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTCTTCGGAACTGGGACCAAGG TCACCGTCCTA (SEQ ID NO:253)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYQMGWVRQAPGKGLEWVSRIYPSGGVTKYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDFGPGDLWSGYYDAFDIWGQGTMVTV SS (SEQ ID NO:254)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTATTTACCAGATGGGTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCGTTACTAAGTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCTGTGTATTACTGTGCCAGAGATTTCGGTCCGGGCG ATTTATGGAGTGGTTATTATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTC TCAAGC (SEQ ID NO:255)
24.806C-M0045-B01 L-Variable (AA):
QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGVPD RFSGSKSATTASLTVSGLQAEDEADYYCSSYAGSNNLIFGGGTKVTVL (SEQ ID NO: 256)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCTGCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTC
CTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACC
CAGGCAAAGCCCCCAAACTCATGATT
TATGAGGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGCCAC
CACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCT
CATATGCAGGCAGCAACAATTTGATATTCGGCGGGGGGACCAAGGTGACCGTCCTA (SEQ
ID NO:257)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYQMQWVRQAPGKGLEWVSVIYPGGYTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLQFYGSSAAFDIWGQGTMVTVSS (SEQ ID NO:258)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACCAGATGCAGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCTATCCTGGTGGCTATACTTATTATGCTGACTCC GTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAA CAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGACTCCAGTTCTACGGTTCCT CTGCTGCTTTTGACATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO: 259)
25.806C-M0045-B03 L-Variable (AA):
QDIQMTQSPDTLSLSPGERATLSCRASQSISRYLAWYQQRPGQAPSLLIYDASERAAGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRGNWPLTFGGGTKVDIR (SEQ ID NO:260)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGACACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTATTAGTAGATACTTAGCCTGGTACCAACAAAGACCTG
GCCAGGCTCCCAGCCTCCTCATCTAT
GATGCATCCGAAAGGGCCGCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAAC
GTGGCAACTGGCCGCTCACTTTCGGC
GGAGGGACCAAGGTGGACATCAGA (SEQ ID NO:261)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYPMIWVRQAPGKGLΞWVSVISPSGGHTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIQYYGGAFDIWGQGKMVTVSS (SEQ ID NO:262)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACCCTATGATTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCTCTCCTTCTGGTGGCCATACTTCTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAATCCAGTACTACGGTG GGGCTTTTGATATCTGGGGCCAAGGGAAAATGGTCACCGTCTCAAGC (SEQ ID NO:263)
26.806C-M0045-B11 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPHTFGGGTKVEIK (SEQ ID NO:264)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG GCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAΆCAGGGCCACTGGCATCCCAGCCAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCACACTTTCGGCGGAGGGACCA
AGGTGGAGATCAAA (SEQ ID NO:265)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYGMLWVRQAPGKGLEWVSVISPSGGQTFYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLGAEKGMDVWGQGTTVTVSS (SEQ ID NO:266)
H-Variable (DNA): GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCCTTACGGTATGCTTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCTCTCCTTCTGGTGGCCAGACTTTTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAΆCTCTAAGAATACTCTCTACTTGCAGAT GAΆCAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGGCTAGGTGCGGAAΆAAG GTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:267)
27.806C-M0045-C02 L-Variable (AA):
QDIQMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQRPGQAPRLLIYGASSRATGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGQGTKVEIK (SEQ ID NO:268)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAGACCTG GCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGA TTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCGGACGTTCGGCCAΆGGGACCA AGGTGGAAATCAAA (SEQ ID NO:269)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMGWVRQAPGKGLEWVSSIYPSGGYTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPHCSGGSCYGGYYYYGMDVWGQGT TVTVSS (SEQ ID NO:270)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGGGTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAAAGATTCC
CCGCATTGTAGTGGTGGTAGCTGCTACGGGGGCTACTACTACTACGGTATGGACGTCTGGGG
CCAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:271)
28.806C-M0045-C11 L- Variable (AA):
QSELTQPASVSGSPGQSITISCTGTNRDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSN RFSGSKSGNTASLTISGLQADDEAEYYCSSYTSSGTRVFGTGTKVTVL (SEQ ID NO: 272)
L-Variable (DNA):
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC CTGCACTGGAACCAACAGAGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACC CAGGCAAΆGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAAT CGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGA CGACGAGGCTGAGTATTACTGCAGCTCATATACAAGCAGCGGCACTCGAGTCTTCGGAACTG GGACCAAGGTCACCGTCCTA (SEQ ID NO:273)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMVWVRQAPGKGLEWVSSIYPSGGVTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDVAGALDYWGQGTLVTVSS (SEQ ID NO : 274 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACATTATGGTTTGGGTTCGCCAAGCTCCTG
GTAAΆGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGTTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGATGTT
GCCGGAGCTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:275)
29.806C-M0045-C12 L-Variable (AA):
QYELTQPASVSGSPGQSITISCTGTSTDVGGYNYVSWYQKHPGKAPKLMIYDVSNRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCSSYTNTITWFGGGTKLTVL (SEQ ID NO: 276)
L-Variable (DNA):
CAGTACGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC CTGCACTGGAACCAGCACTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAAAAACACC CAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAACCGGCCCTCTGGGGTTTCTAAT CGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGA GGACGAGGCTGACTATTACTGCAGCTCATATACAAACACCATCACCGTGGTGTTCGGCGGAG GGACCAAGCTGACCGTCCTA (SEQ ID NO:277)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYWMHWRQAPGKGLEWSSIYSSGGRTHYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCAHTDSSTWYRWYFDLWGRGTLVTVSS ( SEQ ID NO : 278 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACTGGATGCATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATTCTTCTGGTGGCCGTACTCATTATGCTGAC
TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT
GAACAGCCTAAGGGCTGAGGACACCGCCATGTATTACTGTGCACACACTGATAGCAGCACCT
GGTACCGGTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC (SEQ
ID NO:279)
30.806C-M0045-D01 L-Variable (AA):
QDIQMTQSPSTLSSSVGDRVTITCRASQSVSNWLAWYQQKPGKAPKVLIYKASTLESGVPSR FSGSGSGTEFTLTISSLQPDDFATYYCQHYHRYSRTFGQGTKVEIK (SEQ ID NO:280)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTTCATCTGTAGGAGACAGAGTCAC CATCACTTGCCGGGCCAGTCAGAGTGTTAGTAACTGGTTGGCCTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGGTCCTAATCTATAAGGCGTCTACTTTAGAAAGTGGGGTCCCGTCAAGG TTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGA TTTTGCAACTTATTACTGCCAACATTATCATCGTTATTCTCGAACGTTCGGCCAAGGGACCA AGGTGGAAATCAAA (SEQ ID NO:281) H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYKMTWVRQAPGKGLEWVSSIYPSGGWTWYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNWQGGAFDIWGQGTMVTVSS (SEQ ID NO:282)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACAAGATGACTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTGGTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATAACTGGCAGGGCG
GTGCTTTTGACATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:283)
31.806C-M0045-D07 L- Variable (AA):
QDIQMTQSPGTLSLSPGΞRATLSCRASQSVNSNQLAWYQQKPGQAPRLLIYGASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNFWTFGQGTKVEIK (SEQ ID NO:284)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAACAGCAACCAGTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTCTATTACTGTCAGC
AGCGTAGCAACTTTTGGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:285)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYLMMWVRQAPGKGLEWVSSIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARVAPYDSSGSVNYAFDPWGQGTLVTVS S (SEQ ID NO:286)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCTTATGATGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACCGCCATGTATTACTGTGCCAGAGTCGCCCCCTATGATA GTAGTGGTTCGGTAΆATTACGCGTTCGACCCCTGGGGCCAGGGCACCCTGGTCACCGTCTCA AGC (SEQ ID NO:287)
32.806C-M0045-G01 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTITCRASQNINIYLNWYQQKPGRAPSLLIYTQSNLRSGVPSR FSGSGYGTDFTLTISGLQPEDFATYYCQQSHSAPRTFGQGTRVEIK (SEQ ID NO:288)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC CATCACTTGCCGGGCAAGTCAGAACATTAACATCTATTTGAATTGGTATCAGCAGAAGCCAG GGAGAGCCCCTAGCCTCCTGATTTAT ACTCAATCCAATTTGCGAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATATGGCACAGA TTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGA
GTCACAGTGCCCCCCGGACGTTCGGC
CAGGGGACCAGGGTGGAAATCAAA (SEQ ID NO:289)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMVWVRQAPGKGLEWVSVIYPSGGWTRYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREMIDTISPGWHFDLWGRGTLVTVSS (SEQ ID NO:290)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACAAGATGGTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTGGACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCAAGAGAAATG
ATTGACACTATTTCGCCCGGCTGGCACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGT
CTCAAGC (SEQ ID NO : 29l) 33.806C-M0045-G10
L-Variable (AA):
QSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVMYGKNNRPSGIPDRFS GSSSGNTASLTITGAQAEDEADYYCQSRGSSSGNHYVFGTGTKVTVL (SEQ ID NO : 292
L-Variable (DNA):
CAGAGCGAATTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCAC
ATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGG
CCCCTGTACTTGTCATGTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCT
GGCTCCAGTTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGC
TGACTATTACTGTCAGTCCCGGGGCAGCAGCAGTGGTAACCATTATGTCTTC
GGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO:293)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYQMMWVRQAPGKGLEWVSSIYPSGGFTRYAD SVKGRFTISRDNSKNILYLQMNSLRAEDTAVYYCAKSYYYGSGTYHYSYYGMDVWGQGTTVT VSS (SEQ ID NO:294)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCAGATGATGTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTTTACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATATTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTATATTACTGTGCGAAATCATAT
TACTATGGGTCGGGGACCTATCATTACTCTTACTACGGTATGGACGTCTGGGGCCAAGGGAC
CACGGTCACCGTCTCAAGC (SEQ ID NO:295)
34.806C-M0046-A11
L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATGIPD RFTGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPLTFGGGTKVEIK (SEQ ID NO:296
) L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCTTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGTAGCACCTACTTAGCCTGGTACCAGCAGAAAC CTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGAC AGGTTCACTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGA AGATTTTGCAGTGTATTACTGTCAGCACTATGGTAGCTCACCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAA (SEQ ID NO:297)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMDWVRQAPGKGLEWVSGIYPSGGHTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARLYLWGSYPTQVAFDIWGQGTMVTVSS (SEQ ID NO:298)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGGATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGGTATCTATCCTTCTGGTGGCCATACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACAGCCACGTATTACTGTGCGAGACTTTACCTTTGGGGGA GTTATCCCACCCAGGTTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:299)
35.806C-M0046-B06 L-Variable (AA):
QDIQMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK (SEQ ID NO:300)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAΆCCTG GCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCA AGGTGGAGATCAAA (SEQ ID NO:301)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYPMLWVRQAPGKGLEWVSSIYPSGGMTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQGYYDSSGWTFDYWGQGTLVTVSS (SEQ ID NO:302)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTATGTACCCTATGCTTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCATGACTTATTATGCTGAC TCCGTTAΆAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGACAAGGTTACTATGATA GTAGTGGGTGGACCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ
ID NO:303)
36.806C-M0046-B10 L-Variable (AA): QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK (SEQ ID NO:304)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGC
GTAGCAACTGGCCGCTCACTTTCGGC
GGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:305)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYVMNWVRQAPGKGLEWSGIYSSGGYIYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARRHFNGVGFDLWGQGTMVTVSS (SEQ ID NO:306)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACGTTATGAΆTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGGTATCTATTCTTCTGGTGGCTATATTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACAGCCACATATTACTGTGCGAGAAGACATTTCAACGGGG TTGGTTTTGATCTCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:307)
37.806C-M0046-G12 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSNLAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQLYKTFGGGTKVEIK (SEQ ID NO:308)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCAACTTAGCCTGGTACCAGCAGAAAC CTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGA AGATTTTGCAGTTTATTACTGTCAGCTGTATAAGACTTTCGGCGGAGGGACCAAGGTGGAGA TCAAA (SEQ ID NO:309)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMNWVRQAPGKGLEWVSVIYPSGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGYSSGWFLFYGMDVWGQGTTVTVSS (SEQ ID NO:310)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGAATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGGTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CTGTGTATTACTGTGCGAGAGTCGGG
TATAGCAGTGGCTGGTTTCTCTTTTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCAC
CGTCTCAAGC (SEQ ID NO:311) 38.806C-M0046-H03 L- Variable (AA):
QSALTQPRSVSGSPGQSVTISCTGSNTDVGRYNFVSWYQQKPGKAPKLIIYDVYKRPSGVPD RFSGSKSGNTASLTISGLQADDEADYYCCSYARASTFSYVFGIGTEVTVL (SEQ ID NO: 312)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTC CTGCACTGGATCCAATACTGATGTTGGTCGATACAATTTTGTTTCCTGGTACCAACAAAAGC CAGGCAAAGCCCCCAAACTCATAATTTATGATGTCTATAAGCGGCCCTCAGGGGTCCCTGAT CGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGA CGATGAGGCTGATTATTACTGCTGCTCATATGCTCGCGCCTCCACTTTCTCTTATGTCTTCG GAATTGGGACCGAAGTCACCGTCCTT (SEQ ID NO:313)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMVWVRQAPGKGLEWVSSIYPSGGHTPYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQTGGYAHFDYWGQGTLVTVSS (SEQ ID NO:314)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACATTATGGTTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCCATACTCCTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGACAGACGGGTGGCTACG
CCCACTTTGATTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:315)
39.806C-M0046-H10 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTMTCRASQGIGTYLAWYQQKPGKVPKLLIYAASTLQSGVPSR FSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPRPFGQGTQVΞIK (SEQ ID NO:316)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCGTCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC CATGACTTGCCGGGCGAGTCAGGGCATTGGCACTTATTTAGCCTGGTATCAGCAGAAACCAG GGAAAGTTCCTAAGCTCCTGATCTATGCTGCGTCCACTTTGCAATCAGGGGTCCCATCTCGG TTCAGTGGCAGTGGATCTGGGACGGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGA TGTTGCAΆCTTATTACTGTCAAAAGTATAACAGTGCCCCTCGTCCGTTCGGCCAAGGGACCC AGGTGGAAATCAAA (SEQ ID NO:317)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYVMHWVRQAPGKGLEWVSSIYPSGGWTLYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAVGPFDYWGQGTLVTVSS (SEQ ID NO:318)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACGTTATGCATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCT ATCTATCCTTCTGGTGGCTGGACTCTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAΆCAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGCAGTG GGACCTTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC (SEQ ID NO-.319 )
40.8O6C-MOO46-H11 L-Variable (AA):
QYELIQPPSVSGIPGQRVTISCSGMTSNFGSNTVTWYQQLPGTAPKLLIYSDSRRPSGVPDR FSGSRSDTSASLAISGLQSEDEAEYHCAAWDDSLNGVFGGGTKLTVL (SEQ ID NO:320 )
L-Variable (DNA):
CAGTACGAATTGATTCAGCCACCCTCAGTGTCTGGGATCCCCGGACAGAGGGTCACCATCTC
TTGTTCTGGAAACAACTCCAACTTCGGAAGTAATACTGTAACCTGGTACCAGCAGCTCCCAG
GAACGGCCCCCAAACTCCTCATCTAT
AGTGATAGTCGGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAGGTCTGACACCTC
AGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGAGTATCACTGTGCAGCAT
GGGATGACAGCCTAAΆTGGGGTGTTC
GGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO:321)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMEWVRQAPGKGLEWVSVIYPSGGHTNYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYYDILTGYYKYYFDYWGQGTLVTV SS (SEQ ID NO:322)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGGAGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCCATACTAATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGAGGCTATTACGATA TTTTGACTGGTTATTATAAGTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCAAGC (SEQ ID NO:323)
41.806C-M0047-B03 L-Variable (AA):
QDIQMTQSPSPLSASVGDSVTITCRASQRIGSYLNWYQQNPGKAPKLLIYGASNLESGVPSR FSGRGSGTEFTLTITSLQPEDFATYFCQQTSSVSPLTFGQGTRLDIK (SEQ ID NO:324
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCCCCCTGTCTGCATCTGTAGGAGACAGTGTCAC
CATCACTTGTCGGGCAAGTCAGAGGATTGGCAGCTACTTGAATTGGTATCAGCAGAATCCAG
GCAAAGCCCCAAΆACTCCTGATCTAC
GGTGCATCCAATTTGGAAAGTGGGGTCCCATCAAGGTTCAGTGGCCGTGGATCTGGGACAGA
GTTCACACTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTATTTCTGTCAACAGA
CCTCCAGTGTCTCCCCGCTCACCTTC
GGCCAAGGGACACGACTGGACATTAAA (SEQ ID NO:325)
H-Varϊable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMSWVRQAPGKGLEWVSVIYPSGGWTWYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMMYYYDSSGYLRADAFDIWGQGTMVT VSS (SEQ ID NO:326) H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGTCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCTGGACTTGGTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAATGATGTATTACTATG ATAGTAGTGGTTACCTAAGGGCTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACC GTCTCAAGC (SEQ ID NO:327)
42.806C-M0047-D01 L-Variable (AA):
QDIQMTQSPGTLSTSIGDRVTITCRASQSINEWLAWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPALTFGGGTKVEIK (SEQ ID NO:328
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTCTCTACATCTATAGGAGACAGAGTCAC CATCACTTGCCGGGCCAGTCAGAGTATTAATGAGTGGTTGGCCTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGG TTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGA TTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCCGCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAA (SEQ ID NO:329)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYKMMWVRQAPGKGLEWVSSIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCARSMGYGDAFDIWGQGTMVTVSS (SEQ ID NO:33O)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACAAGATGATGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGTTTAAGGGCTGAGGACACCGCCTTGTATTACTGTGCGAGATCAATGGGCTATGGTG ATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:331)
43.806C-M0047-D03 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTITCRASQTIRSYLNWYQQKPGKAPKLLIYAASNLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSMSSWTFGQGTNLEIK (SEQ ID NO:332
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC AATCACTTGCCGGGCAAGTCAGACCATTAGAAGCTATTTAAATTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAΆGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGA TTTTGCAACTTACTACTGTCAACAGAGTTACAGTATGTCGTCGTGGACTTTTGGCCAGGGGA CCAACCTGGAGATCAAA (SEQ ID NO:333)
H-Variable (AA): EVQLLESGGGLVQPGGSLRLSCAASGFTFSVYPMAWVRQAPGKGLEWVSWISPGGKTYYM)S VKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARGSRHYDKFDYWGQGTLVTVSS (SEQ ID NO:334)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGTTTACCCTATGGCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTGGATCTCTCCTGGTGGCAAGACTTATTATGCTGACTCC GTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAA CAGCTTAAGGGCTGAGGACACAGCCACGTATTACTGTGCGAGAGGGAGCCGCCACTATGATA AGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:335)
44.806C-M0047-E10 L-Variable (AA):
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKVMIYDVSNRPSGVSN RFSGSKSGNTASLTISGLLAEDEADYYCSSYTSTATYVLGTGTRVTW (SEQ ID NO: 336)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCAGCAGTGACGTTGGTGGTTACAACTATGTCTCCTGGTACCAACAACACC
CAGGCAAAGCCCCCAAAGTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAAT
CGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCGGGGCTCCTGGCTGA
GGACGAAGCTGATTATTACTGCAGCTCATATACAAGTACAGCCACCTATGTC
CTCGGAACTGGGACCAGGGTCACCGTCGTA (SEQ ID NO:337)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMAWVRQAPGKGLEWVSVIYPSGGATYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALPGGYFDYWGQGTLVTVSS (SEQ ID NO:338)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACAΆGATGGCTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAΆGGGCTGAGGACACGG
CCGTGTATTACTGTGCAAGGGCCTTA
CCGGGGGGCTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:339)
45.806C-M0047-G09 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLACRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPD RFSGSGSDTDFTLKISRVEAEDVGTYYCMQATFWPYAFGQGTKLEIK (SEQ ID NO:340
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCGCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAACAGGGCCACTGGCATCCCAGACAGATTCAGCGGCAGTGGGTCAGACAC
TGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGACTTATTACTGCATGC AAGCTACATTCTGGCCGTACGCTTTT GGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO:341)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYRMVWVRQAPGKGLEWVSGIYPSGGFTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYYYDSSGYYFRGGFDPWGQGTLVTV SS (SEQ ID NO:342)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTGGTACCGTATGGTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCTTTACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGTGTAT
TACTATGATAGTAGTGGTTATTATTTCCGTGGGGGGTTCGACCCCTGGGGCCAGGGCACCCT
GGTCACCGTCTCAAGC (SEQ ID NO:343)
46.806C-M0053-A02 L-Variable (AA):
QSVLTQPPSVSGIPGQRVTISCSGNNSNFGSNTVTWYQQLPGTAPKLLIYSDSRRPSGVPDR FSGSRSDTSASLAISGLQSEDEAEYHCAAWDDSLNGVFGGGTKLTVL (SEQ ID NO:344
)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGCCACCCTCAGTGTCTGGGATCCCCGGACAGAGGGTCACCATCTC
TTGTTCTGGAAACAACTCCAACTTCGGAAGTAATACTGTAACCTGGTACCAGCAGCTCCCAG
GAΆCGGCCCCCAAACTCCTCATCTAT
AGTGATAGTCGGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAGGTCTGACACCTC
AGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGAGTATCACTGTGCAGCAT
GGGATGACAGCCTAAATGGGGTGTTC
GGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO:345)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYLMQWVRQAPGKGLEWVSSIYPSGGATYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATRKDGYSRSAFDIWGQGTMVTVSS (SEQ ID NO:346)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACCTTATGCAGTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCAACAAGGAAG
GATGGCTACAGTCGAAGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAG
C (SEQ ID NO:347)
47.806C-M0053-A03 L-Variable (AA):
QDIQMTQSPGTLSLSPGΞRATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQRGNWPRTFGQGTKVEIK (SEQ ID N0:348 L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAΆAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGC
AGCGTGGCAACTGGCCCCGGACGTTC
GGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:349)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMWWVRQAPGKGLEWVSGIYPSGWTVYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLGGTRAFDYWGQGTLVTVSS (SEQ ID NO:350)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACGTTATGTGGTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTTGGACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCG
TGTATTACTGTGCGAAAGATCTGGGG
GGGACCCGTGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:351)
48.806C-M0053-A05 L- Variable (AA):
QSELTQPASVSGSPGQSITISCTGTSSDDVGGYNYVSWYQQHPGKAPKLLIYDVSDRPSGVS NRFSGSKSGNTASLTISGLLAEDΞADYYCGSYRVTSVSRSYVFGTETK (SEQ ID NO: 352)
L-Variable (DNA):
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCAGCAGTGACGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAAC
ACCCAGGCAAAGCCCCCAAACTCCTG
ATTTATGATGTCAGTGATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGG
CAACACGGCCTCCCTGACCATCTCTGGGCTCCTGGCTGAGGACGAGGCTGATTATTATTGCG
GCTCATATCGCGTCACCAGCGTCAGC
AGATCCTATGTCTTCGGAACTGAGACCAAG (SEQ ID NO:353)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYPMTWVRQAPGKGLEWVSRIYPSGGYTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRIAALDYWGQGTLVTVSS (SEQ ID NO:354)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAATTACCCTATGACTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGGGGTCGT ATAGCAGCTCTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC ( SEQ ID NO : 355 )
49.806C-M0053-A09 L-Variable (AA):
QSALTQGPTVSVALGQTWITCQGDTLRYFSASWYQQKPGQAPVLVIFGANNRPSGIPDRFS GSRSGVTASLTITGAQAEDEAEYYCNSRDGSGNWLFGGGTKLSVL ( SEQ ID NO : 356)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGGGCCCTACTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCAC
ATGTCAAGGAGACACCCTCAGATACTTTTCTGCAAGTTGGTACCAGCAGAAGCCGGGACAGG
CCCCTGTCCTTGTCATCTTTGGGGCA
AACAATCGGCCCTCAGGGATCCCAGACCGGTTCTCTGGCTCCAGGTCAGGAGTCACCGCTTC
CTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGAGTATTACTGTAACTCCCGGGACG
GCAGTGGTAATTGGCTGTTCGGCGGA
GGGACCAAGCTGTCCGTCCTC (SEQ ID NO:357)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMHWRQAPGKGLEWVSVIYPSGGATLYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAWYCARGQYSSGWYTEGWFDPWGQGTLVTVSS (SEQ ID NO:358)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTCTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGGCCAG
TATAGCAGTGGCTGGTACACGGAGGGCTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTCAC
CGTCTCAAGC (SEQ ID NO:359)
50.806C-M0053-B09 L-Variable (AA):
QYELTQPPSASGTPGQRVTISCSGSSSNIGSNNVNWYQQLPGTAPKLLIYSNDQRPSGVPDR FSGSKSATSASLAISGLQSEDEADYHCAAWDDSLNGPVFGGGTKLTVL (SEQ ID NO: 360)
L-Variable (DNA):
CAGTACGAATTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTC
TTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATAATGTCAACTGGTACCAGCAACTCCCAG
GAACGGCCCCCAAACTCCTCATCTAC
AGTAATGATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAΆGTCTGCCACCTC
AGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATCACTGTGCAGCAT
GGGATGACAGCCTGAATGGTCCGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO:361)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMQWVRQAPGKGLEWVSSIYPSGGITYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRGTTRAFDYWGQGTLVTVSS (SEQ ID NO:362) H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGCAGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCATTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGGACGA
GGAACGACGCGGGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:363)
51.806C-M0053-B11 L- Variable (AA):
QYELTQPPSVSVAPGQTAKILCGGNDIGRKFVHWYQQKPGQAPVLWFDDSDRPSGIPERFS GSNSGSTATLTISGVEAGDEADYFCQVWDLSSDHWVFGGGTKLTVL (SEQ ID NO:364)
L-Variable (DNA):
CAGTACGAATTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAAGATTCT
CTGTGGGGGAAACGACATTGGAAGAAAGTTTGTTCACTGGTACCAGCAGAAGCCAGGCCAGG
CCCCTGTGCTGGTCGTCTTTGATGAT
AGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAATTCTGGGAGCACGGCCAC
CCTGACCATCAGCGGGGTCGAAGCCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGATC
TTAGTAGTGATCATTGGGTGTTCGGC
GGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO:365)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMHWRQAPGKGLEWVSRIGSSGGHTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCATDYYYDSSGYYYPAFDIWGQGTMVTVS S ( SEQ ID NO : 366 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTGATTACGCTATGCATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTCGT
ATCGGTTCTTCTGGTGGCCATACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCG
CCATGTATTACTGTGCGACTGACTAT
TACTATGATAGTAGTGGTTATTACTACCCTGCTTTTGATATCTGGGGCCAAGGGACAATGGT
CACCGTCTCAAGC (SEQ ID NO:367)
52.806C-M0053-D03 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLFGGGTKVEIK (SEQ ID NO:368)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGC AGTATGGTAGCTCACCTCTGTTCGGC GGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:369)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMMWVRQAPGKGLEWVSSIYPSGGSTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVQGGAGAFDIWGQGTMVTVSS (SEQ ID NO:370)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGCTATGATGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGTACAG
GGGGGGGCGGGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:371)
53.8O6C-M0053-D06 L- Variable (AA):
QDIQMTQSPSSLSASVGDRVTITCRASQSINTYLNWYQHKPGKAPELLISAASSLQSGVPSR FSGSGSGTDFTLTISSLRPEDFATYYCQQSHSISTFTFGPGTKVDVK (SEQ ID NO:372
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGACAGAGTCAC
CATCACTTGCCGGGCAAGTCAGAGCATTAACACCTATTTAAATTGGTATCAGCACAAACCAG
GGAΆGGCCCCTGAGCTCCTGATCTCT
GCTGCATCTAGCTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGA
TTTCACTCTCACCATCAGCAGTCTGCGACCTGAAGATTTTGCGACTTACTACTGTCAACAGA
GTCACAGTATATCCACATTCACTTTC
GGCCCTGGGACCAAAGTGGATGTCAAG (SEQ ID NO:373)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMHWVRQAPGKGFEWVSSIVPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMYYYDSSGYYVGRFDIWGQGTTVTV SS (SEQ ID NO:374)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGCATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTTGAGTGGGTTTCTTCT
ATCGTTCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGACAAATG
TATTACTATGATAGTAGTGGTTATTATGTCGGGCGTTTTGATATCTGGGGCCAAGGGACCAC
GGTCACCGTCTCAAGC (SEQ ID NO:375)
54.806C-M0053-D12 L- Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPRITFGGGTKVEIK (SEQ ID NO: 376)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAΆCAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGC
GTAGCAACTGGCCTCCCCGGATCACT
TTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:377)
H-Variable (AA):
ΞVQLLESGGGLVQPGGSLRLSCAASGFTFSSYMMFWVRQAPGKGLEWVSRIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVTVPLDSGSYYFDYWGQGTLVTVSS (SEQ ID NO:378)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACATGATGTTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGTTACG
GTACCCCTTGATAGTGGGAGCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT
CTCAAGC (SEQ ID NO:379)
55.806C-M0053-E03 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPQLTFGGGTKVEIK (SEQ ID NO: 380)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGC
AGTATGGTAGCTCACCCCAGCTCACT
TTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:381)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMWWVRQAPGKGLEWVSSIYPSGGWTQYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDVGGGGFDYWGQGTLVTVSS (SEQ ID NO:382)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAΆTTACAAGATGTGGTGGGTTCGCCAΆGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTCAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTG CCGTGTATTACTGTGCGAAAGATGTT
GGGGGGGGTGGCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:383)
56.806C-M0053-E04 L-Variable (AA):
QDIQMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPAR FSGSGSGTEFTLTISSLQSEDFAVYYCLTRVTFGGGTKVELK (SEQ ID NO:384)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
ATTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCTAACAC
GAGTCACTTTCGGCGGAGGGACCAAG
GTTGAGCTCAAG (SEQ ID NO:385)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMGWVRQAPGKGLEWVSSIYPSGGWTTYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAWYCARDSPLWPAAIKSGAYYYGMDVWGQGT TVTVSS ( SEQ ID NO : 386 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACAAGATGGGTTGGGTTCGCCAAGCTCCTG
GTAAΆGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTACTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAΆCTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGATTCC
CCCCTAGTAGTACCAGCTGCTATTAAGAGCGGGGCCTACTACTACGGTATGGACGTCTGGGG
CCAAGGGACCACGGTCACCGTCTCAAGC (SEQID NO:387)
57.806C-M0053-E08 L-Variable (AA):
QSVLTQPPSASGTPGQRVSISCSGSSYNIGVYDVYWYQQLPGTAPKLLIYTNNQRPSGVPDR FSGSKSGTSASLSISGLRSEDEADYYCAAWDDSLAGWVFGGGTKVTVL (SEQ ID NO: 388)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAGTATCTC
TTGTTCTGGAΆGCAGCTACAΆCATCGGAGTTTATGATGTATACTGGTACCAGCAGCTCCCAG
GAACGGCCCCCAAΆCTCCTCATCTAT
ACCAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTC
AGCCTCCCTGTCCATCAGTGGGCTCCGGTCCGAGGATGΆGGCTGATTATTACTGTGCAGCCT
GGGATGACAGCCTGGCTGGTTGGGTG
TTCGGCGGAGGGACCAAGGTGACCGTCCTA (SEQ ID NO:389)
H-Variable (AA): ΞVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMLWVRQAPGKGLEWVSVIYPSGGYTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVLRAFDIWGQGTMVTVSS (SEQ ID NO:390)
H-Variable (DNA):
GAΆGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAΆGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGGGGTA
CTAAGAGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID
NO:391)
58.806C-M0053-F04 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK (SEQ ID NO:392)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCGGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATACATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGTCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGC
GTAGCAACTGGCCGATCACCTTCGGC
CAAGGGACACGACTGGAGATTAAA (SEQ ID NO:393)
H-Variable (AA):
ΞVQLLESGGGLVQPGGSLRLSCAASGFTFSGYGMYWVRQAPGKGLEWVSVISPSGGYTHYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAYSSGWYLDYWGQGTLVTVSS (SEQ ID NO:394)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGGTTACGGTATGTATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCTATACTCATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGCGTAT
AGCAGTGGCTGGTACCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:395)
59.806C-M0053-F05 L-Variable (AA):
QSVLTQPPSLSVSPGQTARIACSGDNLGSRYISWYQQKSGQSPVWLYQDYRRPSGIPERIS GSNSGNTATLTISGTQAVDEADYYCQAWDRSTAVFGGGTRLTVL (SEQ ID NO:396)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGCCACCCTCACTGTCCGTGTCCCCAGGGCAGACAGCCCGCATCGC CTGCTCTGGAGATAATTTGGGGAGTAGATATATTTCCTGGTATCAGCAGAAGTCAGGCCAGT CTCCTGTGGTGGTCCTCTATCAAGAC TACAGACGGCCCTCAGGGATCCCTGAGCGAATCTCTGGCTCCAACTCTGGGAACACAGCCAC TCTGACCATCAGCGGGACTCAGGCTGTGGATGAGGCGGACTATTATTGTCAGGCGTGGGACA GAAGCACTGCGGTGTTCGGCGGAGGG ACCAGGCTGACCGTCCTA (SEQ ID NO:397)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYMMFWVRQAPGKGLEWVSRIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVTVPLDSGSYYFDYWGQGTLVTVSS (SEQ ID NO:398)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACATGATGTTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGTTACG
GTACCCCTTGATAGTGGGAGCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT
CTCAAGC (SEQ ID NO:399)
60.806C-M0053-F06 L- Variable (AA):
QDIQMTQSPDTLSLSPGERATLSCRASHSVTNNRLAWYQQKPGQSPRLLIYGASNRAAGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSHWLYTFGQGTKLEIK (SEQ ID NO:400
)
L-Vaπable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGACACCCTGTCTTTGTCTCCAGGAGAAAGAGCCAC
CCTCTCATGCAGGGCCAGTCACAGTGTTACTAACAACCGCTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGTCTCCCAGGCTCCTCATC
TATGGTGCATCCAACAGGGCCGCTGGCATCCCTGCCAGGTTCAGTGGCAGTGGCTCTGGGAC
AGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAAC
AGCGTAGCCACTGGCTTTACACTTTT
GGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO:401)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMIWVRQAPGKGLEWVSSIYPSGGQTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDMAVYYCARKNGYNNVFDVWGQGTMVTVSS (SEQ ID NO:402)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACATTATGATTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCCAGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACATGG
CTGTGTATTACTGTGCAAGAΆAAAAT
GGCTACAATAATGTATTTGATGTCTGGGGCCAΆGGGACAATGGTCACCGTCTCAAGC (SEQ
ID NO:403)
61.806C-M0053-F08 L-Variable (AA): QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSSTYVFGTGTKVTVL (SEQ ID NO: 404)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAACAGCACC
CAGGCAAAGCCCCCAAACTCATGATT
TATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAA
CACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTGCT
CATATGCAGGTAGTAGCACTTATGTC
TTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO:405)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYPMLWVRQAPGKGLEWVSSIYPSGGWTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTPTHNWNDDPDAFDIWGQGTTVTVSS (SEQ ID NO:406)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCCTATGCTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACAG
CCGTGTATTACTGTACCACCCCTACC
CACAACTGGAACGATGACCCTGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGT
CTCAAGC (SEQ ID NO:407) 62.806C-M0053-G04 L-Variable (AA):
QSVLTQPPSVSVAPGQTATITCGGNNIGTKSVHWYQQKPGQAPVFVYDDNDRPSGIPERFSG SNSGNTATMTISRVEAGDEADYYCQVWDPTGDQYVFGSGTKVTVL (SEQ ID NO:408)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCACGATTAC
CTGTGGGGGAAACAACATTGGAACTAAAAGTGTACACTGGTACCAGCAGAAGCCAGGCCAGG
CCCCTGTCTTCGTCTATGATGATAAT
GACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCCGGGAACACGGCCACCAT
GACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATCCTA
CTGGTGATCAGTATGTCTTCGGAAGT
GGGACCAAGGTCACCGTCCTA (SEQ ID NO:409)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMLWVRQAPGKGLEWVSVIYPSGGYTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAWYCARVVVPAFYYYYYMDVWGKGTTVTVSS (SEQ ID NO : 410 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGCTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTATACTTACTATGCTGACTCCGTTAAΆGGTCGCTTCACTATCTC
TAGAGACAΆCTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGTAGTA
GTACCAGCTTTCTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGT CTCAAGC (SEQ ID NO:411)
63.806C-M0053-G05 L-Variable (AA):
QSELTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLGGVFGGGTKLTVL (SEQ ID NO: 412)
L-Variable (DNA):
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACC
CAGGCAAAGCCCCCAAACTCATGATT
TATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAA
CACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCT
CATATACAAGCAGCAGCACTCTCGGG
GGGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO:413)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMDWVRQAPGKGLEWSSIYPSGGFTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKMATMDYWGQGTLVTVSS (SEQ ID NO:414)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGGATTGGGTTCGCCAAGCTCCTG
GTAAΆGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTTTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCAAGAGAGAAG
ATGGCTACAATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:415)
64.S06C-M0054-A08 L-Variable (AA):
QYELTQPASVSGSPGQSITISCTGTSSDVGGCNYVSWYQQHPGKAPQLLIYDVSYRPSGVSN RFSGSKSGNTASLTISGLQADDEADYYCSSCTSSSTLFGTGTKVTVL (SEQ ID NO:416
)
L-Variable (DNA):
CAGTACGAATTGACTCAACCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCAGCAGTGACGTTGGTGGTTGTAACTATGTCTCCTGGTACCAACAACACC
CAGGCAAAGCCCCCCAACTCTTGATT
TATGATGTCAGTTATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAA
CACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGACGACGAGGCTGATTACTACTGCAGCT
CATGTACAAGTAGCAGCACTCTCTTC
GGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO:417)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMHWVRQAPGKGLEWVSRIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVAGESNGMDVWGQGTTVTVSS (SEQ ID NO:418)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGTGGCT
GGGGAGTCGAACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:419)
65.806C-M0054-B06 L- Variable (AA):
QDIQMTQSPSSLSASIGDRVTVTCRTSQSIDTYLNWYQQKPGQAPNLLIYGASSLESGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTSYTFGRGTTLEIQ (SEQ ID NO:420)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCAGCATCTATAGGAGACAGAGTCAC
CGTCACTTGCCGGACAAGTCAGAGCATTGACACCTATTTAAATTGGTATCAGCAAAAACCAG
GGCAAGCCCCTAACCTCCTGATCTAT
GGTGCATCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGA
TTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGA
GTTACACTACCTCCTACACTTTTGGC
CGGGGGACCACGCTGGAGATCCAA (SEQ ID NO:421)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYKMQWVRQAPGKGLEWVSSIYPSGGATYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQTYYYDSSGYFRNAFDIWGQGTMVTV SS (SEQ ID NO:422)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTATTTACAAGATGCAGTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAΆTACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGACAAACG
TATTACTATGATAGTAGTGGTTATTTCCGCAATGCTTTTGATATCTGGGGCCAAGGGACAAT
GGTCACCGTCTCAAGC (SEQ ID NO:423)
66.806C-M0054-B08 L-Variable (AA):
QSVLTQAASVSGSPGQSITLSCTGATRDVSWYQQHPGKAPKLVLYEVNSRPSDVSDRFSGSM SGNTASLTISGLQAEDEADYYCSSTTSRAPRVIFGGGTKLTVL (SEQ ID NO:424)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGGCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCCTCTC
CTGCACTGGAGCCACCAGGGACGTCTCCTGGTACCAACAΆCACCCAGGCAAGGCCCCCAAAC
TCGTCCTTTATGAAGTCAATAGTCGC
CCCTCAGACGTTTCCGATCGCTTCTCTGGCTCCATGTCTGGCAACACGGCCTCCCTGACCAT
CTCTGGACTCCAGGCTGAAGACGAGGCTGATTATTACTGCTCCTCAACCACAΆGTCGCGCCC CTCGCGTGATTTTCGGCGGAGGGACC AAACTGACCGTCCTA (SEQ ID NO:425)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMVWVRQAPGKGLEWVSWIYPSGGWTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSNYYDSAATLDIWGQGTMVTVSS
(SEQ ID NO:426)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGGTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTGG
ATCTATCCTTCTGGTGGCTGGACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGGTCAAAT
TACTATGATAGTGCTGCGACTCTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAG
C (SEQ ID NO:427)
67.806C-M0054-C03 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTITCRASQTISSYLNWYQQKPGKAPKLLISAASTLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPSFGQGTKVEIK (SEQ ID NO:428)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGCCGGGCAAGTCAGACCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAG
GGAAAGCCCCTAAGCTCCTGATCTCT
GCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGA
TTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGA
GTTACAGTACCCCCTCGTTCGGCCAA
GGGACCAAGGTGGAAATCAAA (SEQ ID NO:429)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYQMLWVRQAPGKGLEWVSSIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGYSSGWYALTSKTFDYWGQGTLVTV SS (SEQ ID NO:430)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACCAGATGCTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGTGGGG
TATAGCAGTGGCTGGTACGCGTTGACTTCAAAGACTTTTGACTACTGGGGCCAGGGAACCCT
GGTCACCGTCTCAAGC (SEQ 'ID NO:431)
68.806C-M0054-C07 L-Variable (AA):
QDIQMTQSPATLSLSPGDRAILSCRASHNIDNFL1AWYQQKPGQAPRLLIYDASHRATGIPPR FSGSGSGTDFTLTISSLEPEDFAVYFCQQRTNWLFGGGTKVEIK (SEQ ID NO:432) L-Variable CDNAj:
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCGGGGGΆTCGAGCCΆT
CCTCTCCTGTAGGGCCAGTCACAATATTGACAACTTCTTAGCCTGGTATCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCTCATAGGGCCACTGGCATCCCCCCCCGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAΆCCTGAAGATTTTGCTGTGTATTTCTGTCAACAAC
GGACCAACTGGCTTTTCGGCGGAGGG
ACCAAGGTGGAGATCAAA (SEQ ID NO:433)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYPM]SIWVRQAPGKGLEWVSRIWPSGGSTVYAD SVKGRFTISRDNSKNTLYLQMNSLRAΞDTAVYYCARDSSRYFDVWGRGTLVTVSS ( SEQ ID NO : 434 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCCTATGAATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTCGT
ATCTGGCCTTCTGGTGGCTCTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGATTCT
TCTCGATACTTCGATGTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:435)
69.806C-M0054-E04 L-Variable (AA):
QDIQMTQSPATLSVSPGERATLSCRASQSISSNLAWYQQKPGQAPRLLIYGTSTRATGIPAR FSGSGSGTΞFTLTISSLQSEDFWYYCQQYKDWPLTFGGGTTVEIK ( SEQ ID NO : 436 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGTAATTTAGCCTGGTACCAACAAAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GGTACATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACCGA
GTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGTAGTTTATTACTGTCAGCAGT
ATAAAGACTGGCCTCTCACTTTCGGC
GGAGGGACCACGGTGGAGATCAAG (SEQ ID NO:437)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMHWVRQAPGKGLEWVSVIYPSGGVTEYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAWYCARDQYSGHDYWGQGTLVTVSS (SEQ ID NO : 438 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGCATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTT ATCTATCCTTCTGGTGGCGTTACTGAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGATCAA TACAGTGGCCATGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAΆGC (SEQ ID
NO:439) 70.806C-M0054-G01 L- Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRYSWPLTFGGGTKVEIK (SEQ ID NO:440)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAΆGATTTTGCAGTTTATTACTGTCAGCAGC
GTTACAGCTGGCCTCTCACTTTCGGC
GGAGGGACCAAGGTGGAGATCAAG (SEQ IDNO:441)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYQMIWVRQAPGKGLEWVSYIVPSGGFTAYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVNYYGMDVWGQGTTVTVSS (SEQ ID NO:442)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGAGTACCAGATGATTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTATATCGTTCCTTCTGGTGGCTTTACTGCTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTGAACTACTACGGTA TGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:443)
71.806C-M0054-G05 L-Variable (AA):
QSALTQPASVSGSPGQSISISCTGTNTDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTWVFGGGTKLTVL (SEQ ID NO: 444)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCAGCATCTC CTGCACTGGAACCAACACTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACC CAGGCAAΆGCCCCCAΆACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAAT CGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGA GGACGAGGCTGATTATTACTGCAGCTCATATACAΆGTAGTAGCACTTGGGTGTTCGGCGGAG GGACCAAGCTGACCGTCCTA (SEQ ID NO:445)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYLMEWVRQAPGKGLEWVSGIYPSGGKTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVNVISVAGTGYYYYGMDVWGQGTTVT VSS (SEQ ID NO:446)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACCTTATGGAGTGGGTTCGCCAAGCTCCTG GTAAΆGGTTTGGAGTGGGTTTCTGGT ATCTATCCTTCTGGTGGCAAGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGTGAAC
GTTATATCAGTGGCTGGTACTGGCTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGAC CACGGTCACCGTCTCAAGC (SEQ ID NO:447)
72.806C-M0054-H10 L- Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRATDIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSWPITFGLGTRLEIK (SEQ ID NO:448)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCATCTACTTAGCCTGGTACCAACAGAAACCTG
GTCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGACATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAAC
GTAGCAGCTGGCCGATCACCTTCGGC
CTTGGGACACGACTGGAGATTAAA (SEQ ID NO:449)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYPMIWRQAPGKGLEWVSVISPSGGHTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIQYYGGAFDIWGQGKMVTVSS (SEQ ID NO : 450 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACCCTATGATTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCCATACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAATCCAG
TACTACGGTGGGGCTTTTGATATCTGGGGCCAAGGGAAAATGGTCACCGTCTCAAGC (SEQ ID N0:451)
73.806C-M0055-A09 L-Variable (AA):
QDIQMTQSPSSLSASVGDGVTITCRASQSINNHLNWYQQKPGKAPKVLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK (SEQ ID NO:452)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACGGAGTCAC CATCACTTGCCGGGCAAGTCAGAGCATTAACAACCATTTAAATTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGGTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGG TTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGA TTTTGCAΆCTTACTACTGTCAACAGAGTTACAGTACCCCGTGGACGTTCGGCCAAGGGACCA AGGTGGAAATCAAA (SEQ ID NO:453)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYRMSWVRQAPGKGLEWVSGIYPSGGGTTYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPTYYYDSSGYYYSGPIDYWGQGTLVT VSS (SEQ ID NO:454) H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACCGTATGTCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGGTATCTATCCTTCTGGTGGCGGTACTACTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGACCCACGTATTACTATG ATAGTAGTGGTTATTACTACTCGGGGCCTATTGACTACTGGGGCCAGGGAACCCTGGTCACC GTCTCAAGC (SEQ ID NO:455)
74.806C-M0055-B11 L-Variable (AA):
QYELTQPASVSGSPGQSITISCTGTNTDVGGYNLVSWYQQHPGKAPKLIIYEVSNRPSGVSN RFSGSKSGNTASLTISGLQAEDEVDYYCGSYTSSSTHVFGSGTKVTVL (SEQ ID NO: 456)
L-Variable (DNA):
CAGTACGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCAACACTGACGTTGGTGGTTATAACCTTGTCTCCTGGTACCAACAGCACC
CAGGCAAAGCCCCCAAACTCATAATT
TATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAA
CACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGTTGATTATTATTGCGGCT
CATATACAAGCAGCAGTACTCATGTC
TTCGGAAGTGGGACCAAGGTCACCGTCCTA (SEQ ID NO:457)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYKMHWVRQAPGKGLEWVSVIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTAGWFDPWGQGTLVTVSS (SEQ ID NO:458)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACAAGATGCATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCAAGAGGGACT
GCAGGGTGGTTCGACCCTTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:459)
75.806C-M0055-B12 L-Variable (AA):
QSELTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSSTYVFGTGTKVTVL (SEQ ID NO: 460)
L-Variable (DNA):
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC CTGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAΆCAGCACC CAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAAT CGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGA GGACGAGGCTGATTATTACTGCTGCTCATATGCAGGTAGTAGCACTTATGTCTTCGGAACTG GGACCAAGGTCACCGTCCTA (SEQ ID NO:461)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMTWVRQAPGKGLEWVSSIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARQEDGGYGTWGQGTLVTVSS (SEQ ID NO:462)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGACTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCG
CCATGTATTACTGTGCGAGACAGGAG
GATGGTGGCTACGGGACTTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:
463)
76.806C-M0055-C05 L- Variable (AA):
QSVLTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPVLVIYGENNRPSGIPDRFS GSSSGNTGSLTITGAQAEDEADYYCNSRDTSGSHLLFGGGTKLTVL (SEQ ID NO:464)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCAC
ATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAACCTGGTACCAΆCAGAΆGCCAGGACAGG
CCCCTGTACTTGTCATCTATGGTGAA
AACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGTTCAGGAAACACAGGTTC
CTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACA
CCAGTGGTAGTCATCTATTATTCGGC
GGAGGGACCAAGCTGACCGTCCTG (SEQ ID NO:465)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYKMLWVRQAPGKGLEWVSSIYPSGGWTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASYYDSGGYYRENFQFWGQGTLVTVS S (SEQ ID NO:466)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACAAGATGCTTTGGGTTCGCCAΆGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTCTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGCCTCTTACTATGATA GTGGAGGTTATTACCGAGAAAACTTCCAGTTTTGGGGCCAGGGCACCCTGGTCACCGTCTCA AGC (SEQ ID NO:467)
77.806C-M0055-C07 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTIICRASQSISIYLNWYQQKPGKAPKVLIYDASSLQSGVPSR FSGSGSGTDFSLTITSLQPEDFATYYCQQSYSTPPMYTFGQGTKLEIK (SEQ ID NO: 468) L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC CATCATTTGCCGGGCAAGTCAGAGCATCAGCATCTATTTAAATTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGGTCCTGATATATGATGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGG TTCAGTGGCAGTGGATCTGGGACAGATTTCAGTCTCACCATCACCAGTCTGCAACCTGAAGA TTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCCATGTACACTTTTGGCCAGG GGACCAAGCTGGAGATCAAA (SEQ ID NO:469)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMHWVRQAPGKGLEWVSVIYPSGGATYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAKGLDFWSGPDYWGQGTLVTVSS (SEQ ID NO:470)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGCATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCGCTACTTATTATGCTGAC TCTGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACAGCCACATATTACTGTGCAAAAGGGCTCGATTTTTGGA GTGGCCCGGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC (SEQ ID NO:471)
78.806C-M0055-D03 L- Variable (AA):
QDIQMTQSPSSLSASVGDRVTITCWASQDIRTSLAWYQQKPGKPPKLLIFAASTLQGGVPSR FSGSGSGTEFTLTISGLQPEDFATYYCQHLNGYPLTFGDGTKVEIR (SEQ ID NO:472)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGCTGGGCCAGTCAGGATATTCGCACTTCTTTAGCCTGGTATCAGCAGAAACCAG
GGAAACCCCCTAAACTCCTCATCTTTGCTGCGTCTACTTTGCAAGGTGGGGTCCCATCAAGG
TTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCTCCGGCCTGCAGCCTGAGGA
TTTTGCGACTTATTACTGTCAGCACCTTAATGGTTACCCGCTCACTTTCGGC
GATGGGACCAAGGTGGAGATCAGA (SEQ ID NO:473)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYVMQWVRQAPGKGLEWVSVIYPSGGMTNYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARIRGDTRAFDIWGQGTMVTVSS (SEQ ID NO:474)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTAATTACGTTATGCAGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCATGACTAATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACAG CCACGTATTACTGTGCACGGATACGC
GGTGACACCAGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:475)
79.806C-M0055-D06 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDLAVYYCQLFGSSPRITFGQGTRLEIK (SEQ ID NO: 476)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAΆΆGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC CTGGCCAGGCTCCCAGGCTCCTCATC TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTGGCAGTATATTACTGTCAGC
TGTTTGGAAGCTCTCCTCGGATCACC
TTCGGCCAGGGGACGCGGCTGGAAATTAAA (SEQ ID NO:477)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMWWVRQAPGKGLEWVSVIYPSGGATYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSLGCSSTSCYDAFDIWGQGTMVTVS S (SEQ ID NO:478)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGTGGTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAΆAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGGTCTTCT
CTAGGGTGTAGTAGTACCAGCTGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGT
CACCGTCTCAAGC (SEQ ID NO:479)
80.806C-M0055-D12 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSR FSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPWTFGQGTKVEIK (SEQ ID NO:480)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAG
GGAAAGTTCCTAAGCTCCTGATCTAT
GCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGA
TTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGT
ATAACAGTGCCCCCTGGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:481)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMWWVRQAPGKGLEWVSSISSGGSTVYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLTTVTGNYFDYWGQGTLVTVSS (SEQ ID NO:482)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTACTTACGGTATGTGGTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTCTTCTGGTGGCTCTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAG
AGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCG
TGTATTACTGTGCGAGAGATCTGACT ACGGTGACGGGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC ( SEQ ID NO : 483 )
81.806C-M0055-E04 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSQLAWYQHKRGQPPRLLIYGASSRATGIPD RPSGSGSGTDYILTISRLEPEDFAVYYCQHFGSSPPATFGQGTKVEIK (SEQ ID NO: 484)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTATCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTTCCAGCAGCCAGTTAGCCTGGTACCAGCATAAAC
GTGGCCAGCCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTACATTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGC
ATTTTGGTAGTTCACCTCCGGCGACG
TTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:485)
H-Variable (AA):
EVQLLΞSGGGLVQPGGSLRLSCAASGFTFSSYKMVWVRQAPGKGLEWVSSIYPSGGVTIYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGSSSGWYNPRRAFDYWGQGTLVTVS S (SEQ ID NO:486)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGGTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGTTACTATTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAΆTACTCTCTACTTGCAGATGAΆCAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGATGGA
AGTAGCAGTGGCTGGTACAATCCCCGTAGGGCCTTTGACTACTGGGGCCAGGGAACCCTGGT
CACCGTCTCAAGC
(SEQ IDNO:487)
82.806C-M0055-E06 L-Variable (AA):
QYΞLTQPPSLSVSPGQTVKITCSAEKLSEKYVAWYQQRPGQSPVMVIYQDSRRPSGIPERFS GSNSGNTATLTISGTQPMDΞADYYCQAWFSDSLPFGSGTKVTVL (SEQ ID NO:488)
L-Variable (DNA):
CAGTACGAATTGACTCAGCCACCCTCTCTGTCCGTGTCCCCAGGACAGACAGTCAAGATCAC
CTGCTCTGCAGAGAAGTTGAGTGAGAAATATGTTGCTTGGTATCAACAGAGGCCGGGCCAGT
CCCCTGTCATGGTCATCTATCAAGAT
AGTAGGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCAC
TCTGACCATCAGCGGGACCCAGCCCATGGATGAGGCTGACTACTATTGTCAGGCGTGGTTTA
GCGACAGTCTCCCCTTTGGAAGTGGG
ACCAAGGTCACCGTCCTA (SEQ ID NO:489)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMIWVRQAPGKGLEWVSSIYPSGGHTIYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCAREGGGATSFDYWGQGTLVTVSS (SEQ ID NO:490) H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGATCTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCCATACTATTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCG CCATGTATTACTGTGCGAGAGAGGGC
GGGGGAGCTACCTCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:49l)
83.S06C-M0055-E10 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVRTYLGWYQQKHGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK (SEQ ID NO:492)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGGACCTATTTAGGCTGGTACCAACAGAAACATG GCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCA AGGTGGAGATCAAA (SEQ ID NO:493)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYPMFWVRQAPGKGLEWVSVISPSGGQTSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSFSGLAALDFWGQGTLVTVSS (SEQ ID NO:494)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACCCTATGTTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCCAGACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAΆCTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAAATCATTC
TCAGGCTTAGCAGCTCTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ
ID NO:495)
84.806C-M0055-E12 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQTVSSGSLAWYQQKPGLAPRLLIYGASRRGTGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSTLPLTFGGGTKVEIK (SEQ ID NO: 496)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGACAGTGAGCAGCGGCTCCTTAGCCTGGTACCAGCAGAAAC
CTGGCCTGGCTCCCAGGCTCCTCATC
TATGGTGCATCCCGTAGGGGCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGTCAGC AGTATGGTAGTACACTCCCGCTCACT TTCGGCGGAGGGACCAAGGTCGAGATCAAA (SEQ ID NO:497)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYTMYWVRQAPGKGLEWVSSIYPSGGWTNYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDMAVYYCARGRGGSKAFDIWGQGTMVTVSS (SEQ ID NO:498)
H-Variable(DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACACTATGTATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTAATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACATGG CTGTGTATTACTGTGCGAGAGGCCGT
GGTGGTAGCAAAGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:499)
S5.806C-M0055-F10 L-Variable (AA):
QSELTQPASVSGSPGQSITISCTGTTSDVGGYNYVSWYQQDPGKVPKLIIYEVYNRPSGVSN RFSGSKSGNTASLTISGLRAEDEADYYCSSKTSSVTYVFGTGTKVTVL (SEQ ID NO: 500)
L-Variable (DNA):
CAGAGCGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCACCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTATCAACAGGACC
CAGGCAAAGTCCCCAAACTCATAATT
TATGAGGTCTATAΆTCGGCCCTCAGGGGTTTCAAATCGCTTCTCTGGCTCCAAGTCTGGCAA
CACGGCCTCCCTGACCATCTCTGGGCTCCGGGCTGAGGACGAGGCTGATTATTACTGCAGCT
CAAAAACAAGCAGCGTCACTTATGTC
TTTGGAACTGGGACCAAGGTCACCGTCCTA (SEQID NO:50L)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYVMSWVRQAPGKGLEWVSRIYPSGGGTRYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEAGGSYFLDYWGQGTLVTVSS (SEQ ID NO:502)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACGTTATGTCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCGGTACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAAAGAGGCG
GGTGGGAGCTACTTCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:503)
S6.806C-M0055-G02 L-Variable (AA): QSELTQPRSVSGSLGQSVTISCTGTTSDVGRYNFVSWYQQYPGRAPKLIIHDVTRRPSGVSD RFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSFYVFGSGTQVTVL (SEQ ID NO:504 )
L-Variable (DNA):
CAGAGCGAATTGACTCAGCCTCGCTCAGTGTCCGGGTCTCTTGGACAGTCAGTCACCATCTC
CTGCACTGGAACCACCAGTGATGTTGGTCGTTATAACTTTGTCTCCTGGTACCAACAGTATC
CAGGCAGAGCCCCCAAACTCATCATT
CATGATGTCACTCGGCGGCCCTCCGGGGTATCTGATCGCTTCTCTGGCTCCAAGTCCGGCAA
CACGGCCTCCCTGACCATCTCTGGTCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCT
CATATGCAGGCAGCTTTTATGTCTTC
GGATCTGGGACCCAGGTCACCGTCTTG (SEQ ID NO:505)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMIWVRQAPGKGLEWVSGIYPSGGATGYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARDGGDIWPDYWGQGTLVTVSS (SEQ ID NO:506)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAΆGATGATTTGGGTTCGCCAΆGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCGCTACTGGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACAG
CCACGTATTACTGTGCGAGAGATGGG
GGGGATATTGTAGTGCCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ
ID NO:507)
S7.806C-M0055-G03 L-Variable (AA):
QYELTQPPSASGTPGQRVTISCSGSSSNIGTNTVYWYQQLPGTAPKLLIYTNVQRPSGVPDR FSGSKSGTSASLAISGLQSEDEADYYCQSYDGSLSSAVFGGGTQLTVL (SEQ ID NO: 508)
L-Variable (DNA):
CAGTACGAATTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTC
TTGTTCTGGAAGCAGCTCCAACATCGGAACTAATACTGTATACTGGTACCAGCAGCTCCCAG
GAACGGCCCCCAAACTCCTCATCTAT
ACTAATGTCCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTC
AGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGCCAGTCCT
ATGACGGCAGCCTGAGTTCTGCTGTG
TTCGGAGGAGGCACCCAGCTGACCGTCCTC (SEQ ID NO:509)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYHMGWVRQAPGKGLEWVSSIYSSGGITQYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRVGGWSLFNWFDPWGQGTLVTVSS (SEQ ID NO:510)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACCATATGGGTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCT ATCTATTUTTUTUUTUUUATTACTCAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCAAGAGGCCGA
GTCGGTGGCTGGTCCCTTTTTAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTCACCGT CTCAAGC (SEQ ID NO:511)
88.806C-M0055-H04 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSYIAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK (SEQ ID NO:512)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGC
GTAGCAACTGGCCTCGGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:513)
H-Variable (AA):
ΞVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMYWVRQAPGKGLEWVSRIVPSGGWTNYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDKGDWYFDLWGRGTLVTVSS (SEQ ID NO:514)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACCCTATGTATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTCGT
ATCGTTCCTTCTGGTGGCTGGACTAACTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGATAAG
GGGGACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:515)
89.806C-M0056-A01 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDTSNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAIYYCQQRSNWPPALTFGGGTKVEIK (SEQ ID NO: 516)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTCTGTCTCCAGGGGAGAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGGTACTTAGCCTGGTATCAACAAAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATACATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAATTTATTACTGTCAGCAGC
GTAGCAACTGGCCTCCGGCGCTCACT
TTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQIDNO:517)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMGWVRQAPGKGLEWVSWIYPSGGITSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARITYFDTSVIDYWGQGTLVTVSS (SEQ ID NO:518)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGCTATGGGTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTGG
ATCTATCCTTCTGGTGGCATTACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCG
CCATGTATTACTGTGCACGGATTACG
TATTTTGATACCAGCGTTATTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC
(SEQ ID NO:519)
90.806C-M0056-A06 L-Variable CAA):
QSVLTQPASVSGSPGQSITISCTGTSSNVGNYNLVSWYQQHPGKAPKLMIYEDNKRPSGVSN RFSVSKSGNTASLTISGLQTEDEAEYYCCSYAGSGTWCFGRRGTRVTV (SEQ ID NO: 520)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAΆCCAGCAGTAATGTTGGGAATTATAACCTTGTCTCCTGGTACCAGCAGCACC
CAGGCAAAGCCCCCAAACTCATGATT
TATGAGGACAATAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGTGTCCAAGTCTGGCAA
CACGGCCTCCCTGACAATCTCTGGGCTCCAGACTGAGGACGAGGCTGAATATTACTGCTGCT
CATATGCAGGTAGTGGCACTTGGTGT
TTCGGGCGGAGGGGAACCAGAGTGACCGTC (SEQ ID NO:521)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYPMEWVRQAPGKGLEWVSRIVPSGGWTTYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASRWTTYLDYFDYWGQGTLVTVSS ( SEQ ID NO : 522 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACCCTATGGAGTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTCGT
ATCGTTCCTTCTGGTGGCTGGACTACTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGTCGGGTG
GTAACTACGTACTTAGACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAG
C (SEQ ID NO:523)
91.806C-M0056-B08
H-Variable (AA) :
EVQLLESGGGLVQPGGSLRLSCAASGFTFSVYVMSWVRQAPGKGLEWVSSIYPSGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARRKAAAGYLDYWGQGTLVTVSS (SEQ ID NO:524)
H-Variable (DNA) :
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGTTTACGTTATGTCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCGGTACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACAGCCACATATTACTGTGCGAGACGAAAAGCAGCAGCAG GTTACCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:525
)
L-Variable (AA):
QSALTQPASVSGSPGQSITISCTGTSSDIGAYKHVSWYQQHPGKAPKLMIYEVTNRPSGISN RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSRNTWVFGGGTKLTVL (SEQ ID NO: 526)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATTTC
CTGCACTGGAACTAGCAGTGACATTGGTGCTTATAAACATGTCTCCTGGTATCAACAACACC
CAGGCAAAGCCCCCAAACTCATGATT
TATGAGGTCACTAATCGGCCCTCAGGGATTTCTAATCGTTTCTCTGGCTCCAAGTCTGGCAA
CACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGTT
CATATACAAGCCGTAACACTTGGGTA
TTTGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO:527)
92.806C-M0056-B09 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGRSPSFGPGTKVDIK (SEQ ID NO:528)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGATGCATCCAGTAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGC
AGTATGGTAGGTCACCCTCTTTCGGC
CCTGGGACCAAAGTGGATATCAAA (SEQ ID NO:529)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYKMSWVRQAPGKGLEWVSSIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDMAVYYCARDRPGAFDVWGQGTMVTVSS (SEQ ID N0:530)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACAAGATGTCTTGGGTTCGCCAΆGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACATGG
CTGTGTATTACTGTGCAAGAGATCGG
CCTGGAGCTTTTGATGTTTGGGGCCAΆGGGACAATGGTCACCGTCTCAAGC (SEQ ID
NO:531)
93.806C-M0056-C03 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPDDSATYYCQQYNSYPITFGQGTRLEIK (SEQ ID NO-.532
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGATGATTCTGCAACCTATTACTGCCAAC
AATATAATAGTTATCCGATCACCTTC
GGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO:533)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYKMWWVRQAPGKGLE1WVSVIYPSGGATYYAD SVKGRFTISRDNSKNTLYLQMNSLRAΞDTAVYYCARGIGAVGGFDSWGQGTLVTVSS (SEQ ID NO:534)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACAAGATGTGGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGGGATC
GGAGCAGTGGGCGGGTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:535)
94.806C-M0056-C04 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVTIACRASHDISDNLNWYQQKPGRAPKWISDAFNLEAGVPSR FSGSRSGTYFTFTINSLQPEDVATYYCQQFNNVPYTFGQGTKLEIK (SEQ ID NO:536)
L-Variablθ (DNA) :
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC CATCGCTTGCCGGGCGAGTCACGACATTAGTGACAATTTAAATTGGTATCAGCAAAAACCAG GGAGAGCCCCTAAGGTCGTGATCTCCGATGCATTCAATTTGGAAGCAGGGGTCCCATCAAGG TTCAGTGGAAGTAGATCTGGGACATATTTTACTTTCACCATCAACAGCCTGCAGCCTGAAGA TGTTGCAACATATTACTGTCAACAΆTTTAATAATGTCCCGTACACTTTTGGCCAGGGGACCA AGCTGGAGATCAAA (SEQ ID NO:537)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMAWVRQAPGKGLEWVSRIYPSGGKTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQGGGGRAFDIWGQGTMVTVSS (SEQ ID NO:538)
H-Variable (DNA) :
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACATTATGGCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCAAGACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGACAGGGTGGTGGTGGGC GTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:539
) 95.806C-M0056-E08 L-Variable (AA):
QSALTQDPAVSVALGQTVKITCQGDSLRNYYASWYQQKPGQAPIWIYGKNNRPSGIPDRFS GSRSGSTASLTITGAQAVDEADYYCSSRDTTNYRMEFGGGTKLTVL (SEQ ID NO:540)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAAGATCAC
ATGCCAAGGAGACAGTCTCAGAAATTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGG
CCCCTATAGTTGTCATCTATGGTAAA
AACAACCGGCCCTCAGGGATCCCAGACCGTTTCTCTGGCTCCAGGTCAGGAAGCACAGCTTC
CTTGACCATCACTGGGGCTCAGGCGGTAGATGAGGCTGACTATTACTGTAGTTCCCGGGACA
CTACTAATTACCGCATGGAATTCGGC
GGAGGGACCAAGCTGACTGTCCTA (SEQ ID NO:541)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMAWVRQAPGKGLEWVSGIYPSGGFTTYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIAGGAYHLDYWGQGTLVTVSS (SEQ ID NO:542)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACATTATGGCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCTTTACTACTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CTGTGTATTACTGTGCGAAAATTGCA
GGGGGAGCCTACCACCTTGATTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:543)
96.806C-M0056-F01 L-Variable (AA):
QDIQMIQSPATLSLSPGERATLSCRASQSVSSYL1AWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK (SEQ ID NO: 544)
L-Variable (DNA):
CAAGACATCCAGATGATCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG GCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGGCCCTCACTTTCGGCGGAG GGACCAAGGTGGAGATCAAA (SEQ ID NO:545)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMEWVRQAPGKGLEWVSSIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGSGRYFDYWGQGTLVTVSS (SEQ ID NO:546)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGGTATGGAGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGACGGGGTAGTGGCCGGT ACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC (SEQ ID NO:547)
97.806C-M0056-F02 L-Variable (AA):
QSELTQPPSASGSPGQSVTITCTGTSSDVGYYNYVSWYQQHPGKAPKLMIFEVSNRPSGVPD RFSGSKSGNTASLTVSGLQAEDEAHYYCSSYAGSDNFVFGSGTKVTVL (SEQ ID NO: 548)
L-Variable (DNA):
CAGAGCGAATTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCAC CTGCACTGGAACCAGCAGTGACGTTGGTTATTATAΆCTATGTCTCCTGGTATCAACAACACC CAGGCAAAGCCCCCAAACTCATGATTTTTGAGGTCAGTAATCGGCCCTCAGGGGTCCCTGAT CGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGA GGATGAGGCTCATTATTACTGCAGCTCATATGCAGGCAGCGACAATTTTGTCTTCGGAAGTG GGACCAAGGTCACCGTCTTA (SEQ ID NO:549)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYVMGWVRQAPGKGLEWVSSIYPSGGYTWYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQGGGGRAFDIWGQGTTVTVSS (SEQ ID NO:550)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTATTTACGTTATGGGTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTATACTTGGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGACAGGGA
GGAGGCGGTCGTGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:551)
9S.806C-M0056-F10 L-Variable (AA):
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLFYVFGTGTKVTVL (SEQ ID NO: 552)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC CTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACC CAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAAT CGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGA GGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCACTCTCTTTTATGTCTTCG GAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO:553)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMMWVRQAPGKGLEWVSYIVPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVDYYDFWSGYWWSGGYGMDVWGQGTT VTVSS (SEQ ID NO:554) H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGATGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTATATCGTTCCTTCTGGTGGCTGGACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTTGACTATTACGATT TTTGGAGTGGTTATTGGTGGTCGGGGGGGTACGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCAAGC (SEQ ID NO:555)
99.806C-M0056-F11 L-Variable (AA):
QDIQMTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIYATSTLQSGVPSR FSGSGSGTEFTLAISTLQPEDFATYYCQQLNSYPITFGQGTRLEIK (SEQ ID NO:556)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGCCGGGCCAGTCAGGGCATAAGCACTTATTTAGCCTGGTATCAGCAΆAAGCCAG
GGAAAGCCCCTAAGCTCTTGATCTAT
GCTACATCCACTTTGCAAAGTGGAGTCCCATCAAGGTTCAGCGGCAGTGGGTCTGGGACAGA
ATTCACTCTCGCAATCAGCACCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAAC
TCAATAGTTACCCGATCACTTTCGGC
CAAGGGACGCGACTGGAGATTAAA (SEQ ID NO:557)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMLWVRQAPGKGLEWVSVIYPSGGYTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVLRAFDIWGQGTMVTVSS (SEQ ID NO:558)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGGGGTA
CTAAGAGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID
NO:559)
100. 806C-M0056-G03
L-Variable (AA):
QNIQMTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQVPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPRTFGQGTKVEIK (SEQ ID NO:560)
L-Variable (DNA):
CAAAACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAGAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGTTACTTAGCCTGGTATCAACAGAAACCTG
GCCAGGTTCCCAGGCTCCTCATCTAT
GATGCATCCAATAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGT ATGGTAGTTTACCTCGGACGTTCGGC CAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:561)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMHWVRQAPGKGLEWVSVIYPSGGKTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARΞMGGSGWYDYWGQGTLVTVSS (SEQ ID NO:562)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGCATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCATCTGGTGGCAAGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGAAATG
GGTGGTAGTGGCTGGTACGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:563)
101. 806C-M0056-G04 L-Variable (AA):
QDIQMTQSPATLSLSPGARATLSCRASQSVSSYLAWYQQRPGQTPRLLIYGASSRATGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSRHTFGQGTKLEIK (SEQ ID NO:564)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGCAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAΆCAGAGACCTG
GCCAGACTCCCAGGCTCCTCATCTAT
GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGT
ATGGTAGCTCACGACACACTTTTGGC
CAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO:565)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYVMRWVRQAPGKGLEWVSGIYPSGGWTTYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATVAAAAGAFDIWGQGTMVTVSS (SEQ ID NO:566)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACGTTATGCGTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCTGGACTACTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAΆTACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCAACAGTGGCA
GCAGCTGCGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ
ID NO : 567 )
102. 806C-M0056-G08 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSISSSYLAWYQQKPGQAPRLLLYGTSNRATGIPD RFSGSGSGTDFTLTISRLEPEDFALYYCQQRYKWPLTFGPGTKVDFK ( SEQ ID NO : 568
) L-Variable(DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCCGGCTCCTCCTC
TATGGTACATCCAACAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGACTTTGCACTTTATTACTGTCAGC
AGCGTTACAAGTGGCCTCTCACTTTC
GGCCCTGGGACCAAGGTGGATTTCAAA (SEQ ID NO:569)
H-Variable(AA):
EVQLLESGGGLVQPGGSLRLSCAASGPTPSHYGMWWVRQAPGKGLEWVSVISPSGGQTNYAD SVKGRPTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGQIHGGNLASWGQGTLVTVSS (SEQ ID NO:570)
H-Variable(DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACGGTATGTGGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCCAGACTAATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTG CCGTGTATTACTGTGCCAAAGGGCAA
ATCCACGGTGGTAATCTTGCCTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:571)
103. 806C-M0056-G12
L-Variable (AA):
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMISDVSNRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGTGTKVTVL (SEQ ID NO: 572)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACTAGCAGCGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACC
CAGGCAAAGCCCCCAAACTCATGATT
TCTGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAA
CACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCT
CATATACAAGCAGCAGCACTCTGTAT
GTCTTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO:573)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTPSNYKMNWVRQAPGKGLEWVSVIYPSGGATYYAD SVKGRPTISRDNSKNTLYLQMNSLRAΞDTAMYYCARVGYSSSWDPHFDYWGQGTLVTVSS (SEQ ID NO:574)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGAATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCG
CCATGTATTACTGTGCGAGAGTCGGG TATAGCAGCAGCTGGGACCCCCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC AAGC ( SEQ ID NO : 575)
104. 806C-M0056-H04 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTEFTLTISSLQSEDFGVYYCQQYKDWPRTFGQGTKVEIK (SEQ ID NO:576 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGGAGTTTATTATTGTCAGC
AGTATAAGGACTGGCCTCGAACGTTC
GGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:577)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYRMVWVRQAPGKGLEWVSSIYPSGGPTRYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWSYYYDSSGYYPVSGPFDIWGQGTMV TVSS (SEQ ID NO:578)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACCGTATGGTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCCCTACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGATGGTCG
TATTACTATGATAGTAGTGGTTATTACCCCGTGAGTGGGCCTTTTGATATCTGGGGCCAAGG
GACAATGGTCACCGTCTCAAGC (SEQ ID NO:579)
105. 806C-M0056-H12 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQGVRSTYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSQGFTFGPGTKVDIK (SEQ ID NO: 580)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGGGTGTTAGAAGTACCTACTTAGCCTGGTACCAGCAGAΆAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGC
AGTATGGTAGCTCACAGGGTTTCACT
TTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID NO:581)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYKMHWVRQAPGKGLEWVSVIYPSGGITAYAD SVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCTRΞVMGPSDYWGQGTLVTVSS (SEQ ID NO:582) H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTATGTACAAGATGCATTGGGTTCGCCAAGCTCCTG
GTAAΆGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCATTACTGCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGATGACACAG
CCGTGTATTACTGTACTAGAGAGGTT
ATGGGACCATCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID
NO : 583 )
106. 806C-M0057-B05 L-Variable (AA):
QD I QMTQS PATLSVS PGERATLS CRS SQSLSNNLAWYQQKPGQAPRLLI YGASTRATGI PAR FSGSGSGTEFTLTISSLQSEDFATYYCQQANSFPRTFGQGTKLEIK ( SEQ ID NO : 584 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGTCCAGTCAGAGTCTTAGCAACAACTTAGCCTGGTACCAGCAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GGTGCATCCACCAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
GTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAACTTACTATTGTCAACAGG
CTAACAGTTTCCCTCGAACTTTTGGC
CAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO:585)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYVMHWVRQAPGKGLEWVSSIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATSTTYSSRPFDYWGQGTLVTVSS (SEQ ID NO:586)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACGTTATGCATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAΆGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGACCTCTACG
ACTTATAGCAGCAGGCCCTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC
(SEQ ID NO : 587 )
107. 806C-M0057-H07 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQHKPGKAPKLLIYAAS KLEDGVPSR FSGSGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK ( SEQ ID NO : 588
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGC
CATCACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCACAAACCAG
GGAAAGCCCCTAAACTCCTGATCTAT
GCTGCATCCAΆGTTGGAAGACGGGGTCCCATCAAGATTCAGTGGCAGTGGAΆCTGGGACAGA
TTTCACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAGTTATTTCTGTCAACAGA GCTACTCTAGTCCAGGGATCACTTTC GGCCCTGGGACCAAGGTGGAGATCAAA (SEQ ID NO:589)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYPMMWVRQAPGKGLEWVSVIYSSGGYTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSRGIYYAMDVWGQGTTVTVSS (SEQ ID NO:590)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCCTATGATGTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATTCTTCTGGTGGCTATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAΆCAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGTATCT
CGCGGGATCTACTACGCTATGGACGTCTGGGGCCAΆGGGACCACGGTCACCGTCTCAAGC
(SEQ ID NO : 591)
108. 806C-M0058-A09 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFWYYCQQYGRSRYTFGQGTKLEIK ( SEQ ID NO : 592
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGTAGTGTATTACTGTCAGC
AGTATGGTAGGTCACGGTACACTTTT
GGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO:593)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYKMHWVRQAPGKGLEWVSSIYPSGGPTHYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGYSSGWYIHWYFDLWGRGTLVTVSS (SEQ ID NO:594)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTAATTACAAGATGCATTGGGTTCGCCAAGCTCCTG GTAAΆGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCCCTACTCATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAΆCTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGGGTATAGCAGTG GCTGGTACATTCACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC
(SEQ ID NO : 595)
109. 806C-M0058-D04 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQQKPGKAPKLLIYDASNLETGVPSR FSGSGSGTHFTFTISSLQPEDFATYYCQQADSFPITFGQGTRLEIK ( SEQ ID NO : 596 ) L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGC
CATCACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCAGAAACCAG
GGAAAGCCCCTAAGCTCCTGATCTAC
GATGCATCCAΆTTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACACA
CTTTACCTTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGG
CTGACAGTTTCCCGATCACCTTCGGC
CAAGGGACACGACTGGAGATTAAA (SEQ ID NO:597)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYFMTWVRQAPGKGLEWVSGISPSGGITSYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSYSDYGVFNSWGQGTLVTVSS (SEQ ID NO:598)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACTTTATGACTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTCTCCTTCTGGTGGCATTACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAAAGGCTCA
TACAGTGATTACGGGGTCTTTAATTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:599)
110. 806C-M0058-E09
L-Variable (AA):
QDIQMTQSPATLSVSPGERATLSCRASQSISSSLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK (SEQ ID NO:600)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCAGCTTAGCCTGGTACCAGCAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGC
GTAGCAACTGGCCGCTCACTTTCGGC
GGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:601)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYVMAWVRQAPGKGLEWVSVIYPSGGATYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRLAVTHFDYWGQGTLVTVSS (SEQ ID NO:602)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTAATTACGTTATGGCTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGCTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTACGAGACTGGCG
GTTACTCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:603) 111. 806C-M0058-F03 L-Variable (AA):
QDIQMTQSPSTLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYGASNLQSGVSSR FSGSGSATDFTLTISSLQPEDPATYYCQQFNSYPLTFGGGTKVEIK (SEQ ID NO : 604)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAACAGAAACCAG
GGAAAGTTCCTAAACTCCTGATCTAT
GGTGCATCTAATTTGCAGTCAGGGGTCTCATCGCGGTTCAGTGGCAGTGGATCTGCGACAGA
TTTCACCCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGT
TTAATAGTTACCCTCTGACTTTCGGC
GGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:605)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSVISPSGGQTAYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATVRWFGAFDYWGQGTLVTVSS (SEQ ID NO:606)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTGATTACGGTATGGCTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCCAGACTGCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CTGTGTATTACTGTGCCACAGTTAGA
TGGTTCGGGGCATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:607)
112. 806C-M0058-G03 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSVTSSFLSWYQHRPGQAPRLLIYATSTRATGIPD RFSGSGSGTDFTLTISRLΞPΞDFAVYYCQHYHTSPPTYTFGQGTKLEIK (SEQ ID NO : 608 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACGCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAAAGTGTGACCAGCAGCTTCTTATCCTGGTACCAGCACAGAC CTGGCCAGGCTCCCAGGCTCCTCATCTATGCTACATCCACCAGGGCCACAGGCATCCCAGAC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACTATCAGCAGACTGGAGCCTGA AGATTTTGCAGTGTATTACTGTCAGCACTATCATACCTCACCTCCCACTTACACTTTTGGCC AGGGGACCAAGCTGGAGATCAAA (SEQ ID NO:609)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSLYLMYWVRQAPGKGLEWVSVIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARGYYYGMDVWGQGTTVTVSS (SEQ ID NO:610)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCTTTACCTTATGTATTGGGTTCGCCAAGCTCCTG ATCTATCCTTCTGGTGGCTGGACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCG CCATGTATTACTGTGCGAGAGGCTAC
TACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:611)
113. S06C-M0058-H01 L-Variable (AA):
QSALTQPPSVSVAPGETAEITCGGENIGSKSVHWYQQKPGQAPVLVIYYDNDRPSGIPERFS GSNFGSTATLTISRVEAGDEADYYCQVWDSGSEHYVFGTETKVTVLGQ (SEQ ID NO: 612)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCACCCTCAGTCTCAGTGGCCCCAGGGGAGACGGCCGAAATTAC CTGTGGGGGCGAGAACATTGGAAGTAAAAGTGTCCATTGGTACCAGCAGAΆGCCAGGCCAGG CCCCAGTGCTGGTCATCTATTATGATAACGACCGCCCCTCAGGGATCCCTGAGCGATTCTCT GGCTCCAACTTTGGGAGCACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGC CGACTATTACTGTCAGGTCTGGGATAGTGGCAGTGAGCACTATGTCTTCGGAACTGAGACCA AGGTCACCGTCCTAGGTCAG (SEQ ID NO:613)
H-Variable (AA):
ΞVQLLΞSGGGLVQPGGSLRLSCAASGFTFSGYIMMWVRQAPGKGLΞWVSSIYPSGGHTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWYYGMDVWGQGTTVTVSS (SEQ ID NO:614)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTGGTTACATTATGATGTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCCATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGATGGTAT
TACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:
615)
114. 806C-M0059-A02 L-Variable (AA):
QSALTQPASVSGSPGQSITISCTGTNSDVGGYNYVSWYQQHPGKAPKLI IFDVTNRPSGVSN RFSGSKAGNTASLTISGLQAEDEADYYCSSYSSTSPRFGGGTKLTVL (SEQ ID NO : 616
)
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCTGCCTCCGTGTCAGGGTCTCCTGGACAGTCGATCACCATTTC CTGCACTGGAACCAACAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACC CAGGCAΆAGCCCCCAAACTCATAATTTTTGATGTCACTAATCGGCCCTCAGGGGTTTCTAAT CGCTTCTCTGGCTCCAAGGCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGA GGACGAGGCTGATTATTACTGCAGCTCATATTCAAGTACCAGCCCTCGCTTCGGCGGAGGGA CCAAGCTGACCGTCCTG (SEQ ID NO:617)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYQMQWVRQAPGKGLEWVSRIYPSGGWTVYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRITYDSSGYYDYWGQGTLVTVSS (SEQ ID NO:618)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTATGTACCAGATGCAGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCTGGACTGTTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT
GAΆCAGCTTAAGGGCTGAGGACACAGCCGTGTATTACTGTACACGGATCACGTATGATAGTA GTGGTTATTACGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:
619 )
115. 806C-M0059-A06 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQHKPGKAPKLLIYAASKLEDGVPSR FSGSGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK ( SEQ ID NO : 620
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGC CATCACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCACAAACCAG GGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGA TTCAGTGGCAGTGGAACTGGGACAGATTTCACTCTCACCATCAGAAGTCTGCAACCTGAAGA TTTTGCAAGTTATTTCTGTCAACAGAGCTACTCTAGTCCAGGGATCACTTTCGGCCCTGGGA CCAAGGTGGAGATCAAA (SEQ ID NO:621)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYKMIWVRQAPGKGLEWVSGIYPSGGWTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARLLPALRGAVMDVWGQGTTVTVSS (SEQ ID NO:622)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCCTTACAAGATGATTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGGTATCTATCCTTCTGGTGGCTGGACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACCGCCATGTATTACTGTGCGAGACTGTTACCAGCCTTGC
GGGGAGCCGTGATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:623)
116. 806C-M0060-B02 L-Variable (AA):
QSVLTQDPTVSVALGQTVRITCRGDRLRSYYSSWYQQKPRQAPVLVMFGRNNRPSGIPDRFS GSTSGSTASLTITATQADDEADYFCSSRDGSGNFLFGGGTKLTVL (SEQ ID NO:624)
L-Variable (DNA):
CAGAGCGTCTTGACTCAGGACCCTACTGTGTCTGTGGCCTTGGGGCAGACAGTCAGGATCAC ATGCCGAGGAGACAGACTCAGAAGTTATTATTCAAGTTGGTACCAGCAGAAGCCACGACAGG CCCCTGTTCTTGTCATGTTTGGTAGAΆACAACCGGCCCTCAGGGATCCCAGACCGATTCTCT GGCTCCACCTCAGGAAGCACAGCTTCCTTGACCATCACTGCGACTCAGGCGGACGATGAGGC TGACTATTTCTGTAGTTCCCGGGACGGCAGTGGTAATTTCCTCTTCGGCGGAGGGACCAAAC TGACCGTCCTT (SEQ ID NO:625) H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCΆASGFTFSIYPMHWVRQAPGKGLEWVSSIYPSGGITRΎAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCARQRGSGWHDSWGQGTLVTVSS (SEQ
ID NO : 626)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTATTTACCCTATGCATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCATTACTCGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCG CCTTGTATTACTGTGCGAGACAACGG
GGCAGTGGCTGGCATGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:627)
117. 806C-M0060-H01 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYL1AWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPVTFGQGTRLEIK (SEQ ID NO:628)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGC
GTAGCAACTGGCCGGTCACCTTCGGC
CAAGGGACACGACTGGAGATTAAA (SEQ ID NO:629)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYPMVWVRQAPGKGLEWVSVIVPSGGFTAYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARKRPGNAFDIWGQGTMVTVSS (SEQ ID NO:630)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTATTACCCTATGGTTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCGTTCCTTCTGGTGGCTTTACTGCTTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACCGCCATGTATTACTGTGCGAGAAAGCGACCTGGAAATG CTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:631)
118. S06C-M0061-A03 L-Variable (AA):
QDIQMTQSPSFLSASVGDSVAITCRASQDISRFLAWYQQRPGKAPKLLIFSASTLQSGVPSR FSGSGSGTEFTLTINALQPEDFATYYCQQLSRYSTFGQGTKLEIK (SEQ ID NO:632)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGTGTCGC CATCACTTGCCGGGCCAGTCAGGACATTAGTCGTTTTTTAGCCTGGTATCAGCAAAGACCAG GGAAAGCCCCTAAΆCTCCTGATTTTT TCTGCTTCCACTTTACAAAGTGGGGTCCCATCCAGGTTCAGCGGCAGTGGATCTGGGACAGA ATTTACTCTCACAATCAACGCCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAAC TTAGTCGTTATTCGACGTTCGGCCAAGGCACCAAACTGGAAATCAAA (SEQ ID NO:633
)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYKMWWVRQAPGKGLEWVSSISPGGWTHYADS VKGRPTISRDNSKNTLYLQMNSLRAEDTAVYYCARGPVSSGGDYWGQGTLVTVSS (SEQ ID NO:634)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTTATTACAAGATGTGGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTCTCCTGGTGGCTGGACTCATTATGCTGACTCC GTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAA CAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCTAGAGGCCCTGTCAGTAGTGGTG GGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:635)
119. 806C-M0061-C05
L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPΞDFAVYYCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO:636
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAΆCAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGC
GTAGCAACTGGCCTCCGCTCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:637)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYVMMWVRQAPGKGLEWVSSIYPSGGQTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIAGGAYHLDYWGQGTLVTVSS (SEQ ID N0:638)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACGTTATGATGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCCAGACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAΆCTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAAΆATTGCAGGGGGAGCCT ACCACCTTGATTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:639
120. 806C-M0061-C06 L-Variable (AA):
QYELTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLTIFDVTKRPSGVSD RFSGSKSDNTASLTISGLQAEDEADYYCGSYTSSGSRVFGTGTKVTVL (SEQ ID NO: 640) L-Variable (DNA):
CAGTACGAATTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACC
CAGGCAAAGCCCCCAAACTCACGATT
TTTGATGTCACTAAACGGCCCTCAGGGGTTTCTGATCGCTTCTCTGGCTCCAAGTCTGACAA
TACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAAGACGAAGCTGATTATTACTGCGGCT
CATATACAAGCAGCGGCTCTCGGGTC
TTCGGAACTGGGACCAΆGGTCACCGTCCTC (SEQ ID NO:641)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMGWVRQAPGKGLEWVSRIYPSGGFTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAΞDTAVYYCTRIREGYFDYWGQGTLVTVSS (SEQ ID NO:642)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGGGTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTCGTATCTATCCTTCTGGTGGCTTTACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAΆCTCTAAGAATACTCTCTACTTGCAGAT GAΆCAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTACGAGGATAAGGGAAGGGTACT TTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:643)
121. 806C-M0061-F07 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQQKPGKAPKLLIYAASKLEDGVPSR FSGSGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK (SEQ ID NO:644
)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGC CATCACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGA TTCAGTGGCAGTGGAACTGGGACAGATTTCACTCTCACCATCAGAΆGTCTGCAACCTGAAGA TTTTGCAAGTTATTTCTGTCAACAGAGCTACTCTAGTCCAGGGATCACTTTCGGCCCTGGGA CCAAGGTGGAGATCAAA (SEQ ID NO:645)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMTWVRQAPGKGLEWVSSIYPSGGFTAYAD SVTGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCAKSTYYYEGSGYYRAFDIWGQGTMVTVS S (SEQ ID NO:646)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACGTTATGACTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCTTTACTGCTTATGCTGAC
TCCGTTACAGGTCGCTTCACTATCTCTAGAGACAΆCTCTAAGAATACTCTCTACTTGCAGAT
GAACAGCTTAAGGGCTGAGGACACCGCCATGTATTACTGTGCGAAATCGACT
TATTACTATGAGGGTAGTGGTTATTACCGCGCTTTTGATATCTGGGGCCAAGGGACAATGGT
CACCGTCTCAAGC (SEQ ID NO:647)
122. 806C-M0061-G12 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPA RFSGSGSGTDFTLTISGLEPEDFWYYCQKYGSSSLTFGGGTKVEIK ( SEQ ID NO : 648 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTCTATCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAAC
CTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGAC
AGACTTCACTCTCACCATCAGTGGCCTGGAGCCTGAAGATTTTGTAGTGTATTACTGTCAGA
AGTATGGTAGTTCATCGCTCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO:649)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYKMWWVRQAPGKGLEWVSVIYPSGGVTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAISYSPVGAFDIWGQGTMVTVSS (SEQ ID NO:650)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACAAGATGTGGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCGTTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGATCTCGTAT
AGTCCCGTGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:651)
123. 806C-M0061-H09
L-Variable (AA):
QSALTQPPSVSGSPGQSVTI SCTGTSSDVGSYNRVSWYRQPPGTAPKVI IYDINNRPSGVPD RFSGSRSGDTAYLTISGLQVEDEADYYCSSFTSSSTYIFGTGTKVTVL (SEQ ID NO : 652 )
L-Variable (DNA):
CAGAGCGCTTTGACTCAGCCTCCCTCCGTGTCCGGGTCTCCTGGACAGTCAGTCACCATTTC
CTGCACTGGAACCAGCAGTGACGTTGGTAGTTATAACCGTGTCTCCTGGTACCGGCAGCCCC
CAGGCACAGCCCCCAAAGTCATCATT
TATGACATCAATAATCGGCCCTCAGGTGTCCCTGATCGCTTCTCTGGGTCCAGGTCTGGCGA
CACGGCCTACCTGACCATCTCTGGGCTCCAGGTGGAGGACGAGGCTGATTATTACTGTAGCT
CATTTACAAGCAGCAGCACCTATATC
TTCGGAACTGGGACCAAGGTCACCGTCCTG (SEQ ID NO:653)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSVYKMYWVRQAPGKGLEWVSVIYPSGGYTDYAD SVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARQLPMSYFDYWGQGTLVTVSS ( SEQ ID NO : 654 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTGTTTACAAGATGTATTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCTATACTGATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGCGGCAGCTGCCCATGTCGT ACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:655)
124. 806C-M0062-A12 L-Variable (AA):
QDIQMTQSPLSLPVTPGEPASMSCRSSQSLLQSNGYNYLDWYLQKPGQSPQLLIYLGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTWTFGQGTKVEIK (SEQ ID NO: 656)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTC CATGTCCTGCAGGTCTAGTCAGAGCCTCCTGCAAAGTAATGGATACAACTATTTGGATTGGT ACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCC GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAG AGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTTGGACGTTCG GCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:657)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMVWVRQAPGKGLEWVSRIYPSGGFTNYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDKTAHMDVWGKGTTVTVSS (SEQ ID NO:658)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGGTTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCGCGTATCTATCCTTCTGGTGGCTTTACTAATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATAAGACAGCCCACA TGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCAAGC (SEQ ID NO:659)
125. 806C-M0062-B05 L-Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSWPPLTFGGGTKVEIK (SEQ ID NO:660
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG GCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAGCTGGCCTCCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAG (SEQ ID NO:661)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMNWVRQAPGKGLEWVSSIYPSGGWTNYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRYGDYVRHWGQGTLVTVSS (SEQ ID NO:662) H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGAATTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCTGGACTAATTATGCTGACTCCGTTAAΆGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACΆGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCCAGAGGGGGG AGATACGGTGACTACGTGCGTCACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ
ID NO : 663)
126. 806C-M0062-B07 L-Variable (AA):
QDIQMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQHKPGQAPRLLIYGASIRATGIPAR FSGSGSGTEFTLTISSLQSEDFGVYyCQQYKDWPRTFGQGTKVEIK ( SEQ ID NO : 664 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCAC
TCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCACAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GGTGCATCCATCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
GTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGGAGTTTATTATTGTCAGCAGT
ATAAGGACTGGCCTCGAACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO:665)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMAWVRQAPGKGLEWVSSIYPSGGVTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSIAAAGTAYWGQGTLVTVSS (SEQ ID NO:666)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGGCTTGGGTTCGCCAΆGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCGTTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAΆCTCTAAGAΆTACTCTCTACTTGCAGATGAΆCAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCAAGAGATCTT
AGTATAGCAGCAGCTGGTACTGCCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC
(SEQ ID NO : 667 )
127. 806C-M0062-C08 L-Variable (AA):
QDIQMTQSPGTLSLSPGERATLSCRASQSFVGSRNLAWYQQKPGQPPRLLIYGAFNRATGIP GRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTSPRTFGGGTKVEIK (SEQ ID NO : 668 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGGCACGCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTTTTGTCGGCAGCAGAAACTTAGCCTGGTACCAGCAAA AACCTGGCCAGCCTCCCAGGCTCCTCATCTATGGTGCATTCAACAGGGCCACTGGCATCCCA GGCAGGTTTAGTGGCAGTGGCTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCC TGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTACGTCACCTCGGACTTTCGGCGGAG GGACCAAAGTGGAGATCAAA (SEQ ID NO:669) H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMQWVRQAPGKGLEWFSSIYPSGGATIYAD SVKGRPTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGIPGYFDSWGQGTLVTVSS (SEQ ID NO:670)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGCAGTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGTTTTCTTCT
ATCTATCCTTCTGGTGGCGCTACTATTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAAGGGGA
ATTCCGGGCTACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO : 671)
128. S06C-M0062-D04 L- Variable (AA):
QDIQMTQSPLSLSASIGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYATSTLQSGVPSR FSGSGSGTEFILTISGLQPEDFATYYCQQFNFYPLTLGGGTRVEIKRT ( SEQ ID NO : 672 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCACTCTCCCTGTCTGCATCTATAGGAGACAGAGTCAC
CATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGCAGAAGCCAG
GGAAAGCCCCTAAACTCCTGATCTAT
GCAACTTCCACTTTACAGAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGA
ATTCATTCTCACAATCAGCGGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGT
TTAATTTTTATCCTCTCACTCTCGGC
GGAGGGACCAGGGTGGAGATCAAACGAACT (SEQ ID NO:673)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMVWVRQAPGKGLEWVSSISPSGGNTGYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGNGGFDSWGQGTLVTVSS (SEQ ID N0:674)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACGGTATGGTTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTCTCCTTCTGGTGGCAATACTGGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCAAGAGGAAAT
GGTGGCTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID NO:
675)
129. 806C-M0062-E02 L- Variable (AA):
QSVLTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSSTYVFGTGTKVTVL ( SEQ ID NO : 676 ) L-Variable (DNA):
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAACAGCACC
CAGGCAAAGCCCCCAΆACTCATGATT
TATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAA
CACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTGCT
CATATGCAGGTAGTAGCACTTATGTC
TTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO:677)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMSWRQAPGKGLEWSVIYPSGGWTGYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVATTSFDYWGQGTLVTVSS ( SEQ ID NO : 678 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCATTACGTTATGTCTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCTGGACTGGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGGGGTG
GCAACTACTAGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:679)
130. 806C-M0062-E03 L- Variable (AA):
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRSITFGQGTRLEIK (SEQ ID NO: 680)
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA
CTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGC
GTAGCAACTGGCCTCGATCGATCACC
TTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO:681)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYLMRWVRQAPGKGLEWVSGIYPSGGITAYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASGSYYNYYFDYWGQGTLVTVSS (SEQ ID NO:682)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCTTATGCGTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTATCCTTCTGGTGGCATTACTGCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAΆTACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGCTTCG
GGGAGTTATTATAΆTTACTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCAAG
C (SEQ ID NO:683) 131. 806C-M0062-E11 L-Variable (AA):
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQHKPGKAPKLLIYAASKLEDGVPSR FSGSGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK (SEQ ID NO:684 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGC
CATCACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCACAAACCAG
GGAAAGCCCCTAAACTCCTGATCTAT
GCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGATTCAGTGGCAGTGGAACTGGGACAGA
TTTCACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAGTTATTTCTGTCAACAGA
GCTACTCTAGTCCAGGGATCACTTTC
GGCCCTGGGACCAAGGTGGAGATCAAA (SEQ ID NO:685)
H-Variable (AA):
ΞVQLLESGGGLVQPGGSLRLSCAASGFTFSAYVMHWVRQAPGKGLEWVSRIYPSGGITYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGILTGPNYWGQGTLVTVSS (SEQ ID NO:686)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACGTTATGCATTGGGTTCGCCAAGCTCCTG
GTAΆAGGTTTGGAGTGGGTTTCTCGT
ATCTATCCTTCTGGTGGCATTACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC
TAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGG
CCGTGTATTACTGTGCGAGAGGGATT ^
TTGACTGGCCCAAACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID
NO : 687 )
132. 806C-M0062-F10 L-Variable (AA):
QSALTQSPSASASLGASVKLTCSLSSGHSSYAIAWHQQQPEKGPQYLMKVNSDGSHTKGDGI PDRFSGSSSGAERYLTISSLQSEDEADYYCQTWGTGSWVFGGGTKLTVL ( SEQ ID NO : 688 )
L-Variable (DNA):
CAGAGCGCTTTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGCTCAC CTGCAGTCTGAGCAGTGGGCACAGCAGCTACGCCATCGCATGGCATCAGCAGCAGCCAGAGA AGGGCCCCCAGTACTTAATGAAGGTTAACAGTGATGGCAGCCACACCAAGGGGGACGGGATC CCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGCTACCTCACCATCTCCAGCCTCCA GTCTGAGGATGAGGCTGACTATTACTGTCAGACCTGGGGCACTGGCTCTTGGGTGTTCGGCG GAGGGACCAAGCTGACCGTCCTA (SEQ ID NO:689)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYKMSWVRQAPGKGLEWVSYIYPSGGHTΞYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREREGTPDYWGQGTLVTVSS (SEQ ID NO:690)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGTCTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTTAT
ATCTATCCTTCTGGTGGCCATACTGAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTC TAGAGACAACTCTAAGAATACTCTCTACTTGCAGΆTGAACAGCTTAAGGGCTGAGGACACGG CCGTGTATTACTGTGCGAGAGAAAGG GAAGGGACCCCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC (SEQ ID
NO : 691 )
133. 806C-M0062-G06 L- Variable (AA):
QSVLTQPASVSGSPGQSITISCTGTSSDDVGGYNYVSWYQQHPGKAPKLLIYDVINRPSGVS NRFSGSKSGNTASLTISGLQAEDEADYYCSSYASSGARVFGTGTKVTVL (SEQ ID NO : 692 )
L-Variable (DNA):
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTC
CTGCACTGGAACCAGCAGTGACGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAAC
ACCCAGGCAAΆGCCCCCAAACTCCTG
ATTTATGATGTCATTAATCGGCCCTCAGGAGTTTCTAATCGCTTCTCTGGGTCCAAGTCTGG
CAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCA
GCTCATATGCAAGCAGCGGCGCTCGA
GTCTTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO:693)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMIWVRQAPGKGLEWVSVIYPSGGHTRYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRRVYSSGSAYFDLWGRGTLVTVSS (SEQ ID NO:694)
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC
TTGCGCTGCTTCCGGATTCACTTTCTCTATTTACCCTATGATTTGGGTTCGCCAAGCTCCTG
GTAAAGGTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCCATACTCGTTATGCTGAC
TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT
GAACAGCTTAAGGGCTGAGGACACAGCCGTGTATTACTGTACGAGACGGGTATATAGTAGTG
GTTCTGCGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC (SEQ ID
NO:695)
134. 806C-M0062-H01 L- Variable (AA):
QDIQMTQSPSTLSASVGDRVTITCRASQSVAGLLAWFQQKPGKAPKLLISKASILETGVPSR FSGSGSGTEFTLTITSLQPDDFATYYCQQYSFNSGTFGQGTRVEMK ( SEQ ID NO : 696 )
L-Variable (DNA):
CAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTGGGAGACAGAGTCAC CATCACCTGCCGGGCCAGCCAGAGTGTTGCTGGCTTGTTGGCCTGGTTTCAGCAGAAACCGG GCAAAGCCCCTAΆACTCCTCATCTCTAΆGGCGTCTATTTTAGAGACTGGGGTCCCATCAAGG TTCAGCGGCAGTGGATCTGGGACAGAΆTTCACTCTCACCATCACCAGCCTGCAGCCTGATGA TTTCGCAACTTATTACTGCCAACAATATAGTTTCAATTCTGGGACATTCGGCCAΆGGGACCA GGGTGGAAATGAAA (SEQ ID NO:697)
H-Variable (AA):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYKMAWVRQAPGKGLEWVSYIYPSGGYTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCARVRDSAFDIWGQGTMVTVSS ( SEQ ID NO : 698 )
H-Variable (DNA):
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTC TTGCGCTGCTTCCGGATTCACTTTCTCTATGTACAAGATGGCTTGGGTTCGCCAAGCTCCTG GTAAAGGTTTGGAGTGGGTTTCTTATATCTATCCTTCTGGTGGCTATACTTATTATGCTGAC TCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGAT GAACAGCTTAAGGGCTGAGGACACCGCCTTGTATTACTGTGCGAGAGTAAGGGATTCCGCTT TTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC (SEQ ID NO:699)
[0616] Example 29 : Exemplary Tiel Antibodies
[0617] Tables 5 (FIG. 37) and 6 (Fig. 38) list CDR regions of exemplary light and heavy chain variable regions which are listed herein. FIG. 39 (Table 9) list properties of some of the exemplary antibodies
[0618] Some antibodies described herein include related variable domains.
The same variable domain can function with a different partner variable domain. For example, M0044-G06 and M0044-B05 share a HC variable domain, but have different LC variable domains, as do M0044-G07 and M0044-B05. Other antibodies that have the same HC variable domain include: HC 54(M0053-D12) and 19(M0044- H05); HC 59(M0053-F05) and 19(M0044-H05); HC 72QVI0054-H10) and 25(M0045- B03); and HC 98(MOO56-F11) and 57(M0053-E08). Some antibodies that have the same LC variable domain include: LC 114(M0059-A06) and 106(M0057-H07); LC 130(M0062-Ell) and 106(M0057-H07); and LC 115(M0060-B02) and 12(M0044- F03). Some antibodies have the same CDR3. For example, the CDR3 sequence, QGGGGRAFDI, is present in M0056-C04 and M0056-F02. The CDR3 sequence IAGGAYHLDY is present in M0056-E08 and M0061-C05.
[0619] In some cases, an antibody can include a non-germline residue. One or more of such non-germline residues can be modified, e.g., to restore the germline residue. Exemplary non-germline residues include: L45F (see, e.g., M0053-D06); V48F (see, e.g., M0062-C08); delta S53 (see, e.g.,M0045-B01; M0047-D03; M0055- D12; M0061-A03); delta G54 (see, e.g.,M0053-A03); T57I (see, e.g.,M0046-B10); E85D (see, e.g.,M0056-H12); T87M (see, e.g.,M0053-F06; M0055-E12; M0056- B08); V89L (see, e.g.,M0044-B08; M0047-D01; M0060-B02; M0062-H01); V89M (see, e.g.,M0044-B10; M0045-C12; M0045-D07; M0053-B11; M0055-B12; M0055- E06; M0056-A01; M0056-G12; M0058-G03; M0059-A06; M0060-H01; M0061- F07); V89T (see, e.g.,M0044-H07; M0046-A11; M0046-B10; M0047-D03; M0055- C07; M0055-D03; M0055-G02); A93T (see, e.g.,M0045-A02; M0053-F08; M0056- H12; M0058-E09; M0059-A02; M0061-C06; M0062-G06); and T107K (see, e.g.,M0045-B03).
[0620] Example 30 : Sequence of DX-2220 Antibody
[0621] DX-2220 is a full length, IgGl, germlined human anti-Tiel antibody
E3b. The sequence of DX-2220 is as follows:
DX-2220 Light Chain Amino Acid Sequence:
DIQMTQSPSSLSASVGDRVTITCRASQGIGHYLAWYQQKPGKVPKLLI YTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQFNSYPHT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:700)
DX-2220 Heavy Chain Amino Acid Sequence:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYGMVWVRQAPGKGLE
WVSVISPSGGNTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCARAPRGYSYGYYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:701)
An exemplary DX-2220 Light Chain Nucleotide Sequence: ggcgtgcactctgacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtca ccatcacttgccgggcgagtcagggcattggccattatttagcctggtatcagcagaaaccagggaaagt tcctaagctcctgatctatactgcatccactttgcaatcaggggtcccatctcggttcagtggcagtgga tctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaac agtttaatagttaccctcacaccttcggccaagggacacgactggagattaaacgaactgtggctgcacc atctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctg aataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactccc aggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaa agcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcaca aagagcttcaacaggggagagtgttaataa (SEQ ID NO:702)
An exemplary DX-2220 Heavy Chain Nucleotide Sequence: gaagttcaattgttagagtctggtggcggtcttgttcagcctggtggttctttacgtcttt cttgcgctgcttccggattcactttctctatgtacggtatggtttgggttcgccaagctcctggtaaagg tttggagtgggtttctgttatctctccttctggtggcaatactggttatgctgactccgttaaaggtcgc ttcactatctctagagacaactctaagaatactctctacttgcagatgaacagcttaagggctgaggaca ctgcagtctactattgtgcgagagccccacgtggatacagctatggttactactactggggccagggaac cctggtcaccgtctcaagcgcctccaccaagggcccatcggtcttcccgctagcaccctcctccaagagc acctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgt ggaactcaggcgccctgaccagcggcgtccacaccttcccggctgtcctacagtcctccggactctactc cctcagcagcgtagtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcac aagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccac cgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccct catgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaag ttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaaca gcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtg caaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcc tggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta caagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgc agaagagcctctccctgtctccgggtaaatga (SEQ ID NO:703) [0622] Example 31 : DX-2220 Slows Colorectal Cancer Xenograft Tumor
Progression in Nude Mice
[0623] Mice (nu/nu) were implanted subcutaneously with 5 X lO6 SW-480
(colorectal cancer) cells. After 12 days, when rumors reached approximately 100-200 mg, the mice were separated into 5 groups and treated with the following agents (or left untreated):
1- Untreated
2- Vehicle (PBS)
3- Cisplatin (4mg/kg/, IV, q2d X5 times)
4- A2-SV (negative control antibody @ 10 mg/kg, IP, q2d X 14 times)
5- DX-2220 (anti-Tie-1 antibody @ 10 mg/kg, IP, q2d x 14 times)
[0624] Throughout the study, the length (L) and width (W) of any tumors that developed were measured in millimeters using calibrated vernier calipers, where L is the longer of the two dimensions. When applicable, tumor weight (M) in milligrams was calculated by using the formula associated with a prolate ellipsoid: M = (L x W2)/2- Table 7 shows the average weights (in mg) of the tumors for each of the groups. A2-SV is an isotype matched (IgGl) negative control antibody that binds strepavidin.
Table 7: Tumor Weight (mg)
Figure imgf000243_0001
[0625] The results from the animal study shown in Table 7 are depicted graphically in FIG. 5. DX-2220 slowed tumor progression by 44% when compared to vehicle (PBS)-treated control animals. In addition, DX-2220 was as efficacious as the chemotherapeutic control (cisplatin).
IO626] Example 32: Production and Testing of Germlined Anti Tiel E3 Fab and IgG for Binding to Human and Mouse Tiel in BIACore
[0627] Expression and Purification. Fabs were produced in the E. coli strain,
TGl, using an expression vector containing a PeIB leader sequence for secretion into the periplasm. Under the conditions used for induction (overnight incubation at 3O0C in the presence of 1 mM IPTG), the majority of the secreted Fab was localized in the culture medium rather than the periplasm. The secreted Fab was recovered by adding protein A resin to the clarified culture medium. This protein A resin was then packed into a column to facilitate washing, with PBS, and elution with 50 mM sodium phosphate, 150 mM NaCl, pH 2.5. The pH was brought to approximately neutral by addition of one half volume of 1 M HEPES before buffer exchange into PBS The concentration of the purified germlined E3 (DX-2220) Fab was determined using OD280 1.4 = 1.0 mg/ml.
[0628] The IgG was produced transiently in HEK293T cells using either
LΓPOFECTAMINE™ 2OOO or GENEJUICE™ as the transfection reagent. Antibody could be produced from cells harvested at 72, 144, and 216 hours post transfection. Purification of the IgG from the conditioned culture media essentially followed the same protocol outlined above for the Fab purification. The concentration of the purified IgG was determined using OD280 1.4 = 1.0 mg/ml.
[0629] For preclinical animal studies, IgG were purified using a two-step purification procedure, initially with protein A chromatography subsequently followed by ion exchange chromatography (IEX). Purified IgGs were subjected to biochemical analyses to assess endotoxin levels, leached protein A, DNA content, and host cell proteins.
[0630] Biochemical Analysis
[0631 ] Affinity analysis of the Fab and IgGs was performed using surface plasmon resonance using a BIAcore 3000 instrument. For this analysis both dimeric (Tiel -Fc fusion protein) and monomelic (Tiel -HIS) versions of the extracellular domain of the Tiel were used. The sensor chips used in these experiments were CM5 (dextran-coated) which allow immobilization of proteins to the chip via standard amine coupling chemistries. The concentration of flowed antibody was determined using a surface plasmon resonance based method. Using a high-density protein A sensor chip, under mass transport limited conditions, the response signal is dependent only on the concentration of the antibody in the sample under test. This approach allows a precise determination of the antibody concentration, a parameter important for accurate determination of the K^.
[0632] 470 RUs of Tiel HIS protein were coated on a CM5 sensor chip and 5,
20, 100, and 500 nM of the Fab flowed over the chip at four different flow rates (10, 30, 50 and 80 μl/s), rates in which the system is not mass transport limited. Kinetic data was typically determined using a range of analyte concentrations and three different chip coating densities. The data from the lowest coating density that gave a good signal was typically chosen, this was often in a range from 50-100 RUs. Using such low coating densities allowed the sensor curves to be fit using BIAEVALUATION™ 3.0 software to a 1:1 model, often even when using a bivalent analyte (IgG or Tiel -Fc fusion protein). For bivalent analytes, when fit to a 1 :1 model was not possible, the curves were fit using a 2:1 model. The generated data is shown in Table 8.
Table 8: Kinetic data for binding of DX-2220 Fab and IgG to human Tiel-Fc fusion protein
Fab Human Tiel-Fc
K0n KoffO/s) KD (nM)
(I/Ms)
Fab Parental 8.26E+03 4.47E-05 5.4
Fab Germlined 9.30E+03 4.41E-05 4.7
IgG Parental 6.19E+03 3.61E-05 5.8
IgG Germlined 7.09E+03 3.67E-05 5.2
[0633] The anti-Tiel antibodies described here bind to both human and mouse
Tiel molecules. The binding of the anti-Tiel Fab to mouse Tiel-Fc fusion protein was compared with the binding of the anti-Tiel Fab to human Tiel-Fc fusion protein. In both of these experiments the Tiel-Fc fusion protein was immobilized on the sensor chip and the anti Tie-1 Fab served as the analyte. Under these experimental conditions the anti-Tiel Fab has very similar KΌ (~3 nM) values for both the human and mouse Tiel-Fc fusion proteins (Table 9). Under conditions that are likely to mimic those used in the animal efficacy experiments, i.e. Tiel-Fc immobilized to the CM5 sensor chip and anti-Tiel used as the analyte, the measured KQ was 0.2 nM. This greater than 10 fold increase in affinity represents an avidity effect that results from a bivalent molecule (anti-Tiel) binding to a multivalent surface (immobilized Tiel-Fc).
[0634] Example 33: Sequence of DX-2240: Germlined F AUotvoed E3
Antibody
[0635] DX-2240 (Light, heavy - variable, constant). Variable region:
DIQMTQSPSSLSASVGDRVTITCRASQGIGHYLAWYQQKPGKVPKLLIYTAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQFNSYPHTFGQGTRLEIK
Light constant :
RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO : 724 light chain (variable + constant)
[O636 ] DX-2240 Heavy variable :
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYGMVWVRQAPGKGLEWVSVIS PSGGNTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAPRGYSYGYYY WGQGTLVTVSS
Heavy constant (CHl , Hinge , CH2 , CH3 ) :
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I SKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:723 heavy chain (variable + constant) [0637] The light chain can optionally further include the following signal sequence: Light signal sequence: MGWSCIILFLVATATGVHS (SEQ ID NO:729). The heavy chain can optionally further include the following signal sequence MGWSCHLFLVATATGAHS (SEQ ID NO:730)
IO6381 Example 34: Characterization of DX-2240 from a GS-CHO Cell Line
[0639] The anti-Tie 1 antibody DX-2240 (light and heavy chain germlined and f-allotype) antibodies was produced in HEK293T cells. A stable CHO cell line expressing DX-2240 was generated. Using standard molecular biology cloning techniques, the light and heavy chains from DX-2240 was inserted into glutamine synthase (GS) vector system available from Lonza Group Ltd. CH (see, e.g., Clark et al. (2004) BioProcess International 2(4):48-52; Barnes et al. (2002) Biotech Bioeng. 81(6): 631-639). The single vector constructs containing the light and heavy chains respectively, were then combined to create a single, double gene vector. This DNA construct was then used to generate stable CHO cell lines, grown under MSX selection pressure. One of these clones was then expanded and a single 4OL stirred bioreactor seeded and run over the course of 12 days. Following the completion of this run, 36 liters of clarified CHO culture supernatant was loaded onto a 200ml Protein A XK50 column. The column was first washed with PBS pH 7.4, followed by a PBS+0.4M NaCl pH7.4 wash, and then with a final wash of PBS pH 7.4 prior to the low pH elutions. DX-2240 IgGl was eluted first with 0.1M NaCitrate pH 3.5 followed by the same buffer at pH 3.0. A sharp protein peak eluted at pH 3.0. The pH 3.0 elution contained a predominant peak representing DX-2240, with a low level of contaminants eluting shortly thereafter. A high degree of purity (>95%) of DX-2240 was obtained.
[0640] Example 35: Sequence Optimization of Nucleic Acid Encoding DX- 2240 Antibody
[0641] To improve expression of DX-2240 in CHO cells, a synthetic gene with optimized codons and sequences was engineered. The strategies include codon optimization, CpG island and splice site analysis. An optimized DX-2240 sequence nas been syntnesizeα ana reiormatted into the glutamine synthase (GS) vector system. The exemplary codon optimized sequence is as follows:
[0642] DX-2240-heavy chain (SEQ ID NO:725)
Signal sequence
ATGGGCTGGTCCTGTATCATCCTGTTTCTGGTGGCCACCGCCACCGGCGCTCACTCT GAGGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCTCTGAGA CTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCC ATGTACGGCATGGTG TGGGTGAGGCAG GCCCCTGGCAAGGGCCTGGAGTGGGTGTCC GTGATCTCTCCTTCTGGCGGCAATACCGGC TACGCCGACTCTGTGAAGGGC CGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTG
TACCTGCAGATGAACTCCCTGAGAGCCGAGGΆTACCGCCGTGTACTACTGTGCCAGA GCC
CCTAGAGGCTACTCCTACGGCTACTACTAC TGGGGCCAGGGCACCCTGGTGACCGTGTCC TCTGCTTCTACCAAGGGCCCTTCCGTGTTTCCTCT GGCCCCTTCCTCCAAGTCTACCTCT
GGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCCGTGACAGTG
TCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCTGTGCTGCAGTCC
TCCGGCCTGTACTCTCTGTCCTCCGTGGTGACAGTGCCTTCCTCTTCTCTGGGCACCCAG
ACCTACATCTGTAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAG
CCTAAGTCCTGTGACAAGACCCACACCTGCCCTCCTTGTCCTGCCCCTGAGCTGCTGGGC
GGACCTTCTGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCTCCAGGACC
CCTGAGGTGACCTGTGTGGTGGTGGACGTGTCTCACGAGGATCCCGAGGTGAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGGGAGGAGCAGTAC
AACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC
AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAAAAGACCATC
TCCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCAGGGAG
GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGAT
ATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCT
GTGCTGGACTCTGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGA
TGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC
ACCCAGAAGTCCCTGTCTCTGTCCCCCGGCAΆGTGATGAGAATTC
DX-2240Lightchain(SEQIDNO:726):
Signal sequence ATGGGCTGGTCCTGTATCATCCTGTTTCTGGTGGCCACCGCCACCGGCGTGCACTCT
GACATCCAGATGACCCAGTCCCCTTCCTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCΆTC ACCTGTAGAGCCTCTCAGGGCATCGGCCACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGTGCCC
AAGCTGCTGATCTACACCGCCTCCACCCTGCAGTCTGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCT GGCACCGATTTCACCCTGACCATCTCCTCCCTGCAGCCTGAGGATGTGGCCACCTACTACTGC CAGCAGTTCAACTCCTACCCCCACACC TTCGGCCAGGGCACCAGACTGGAGATCAAG
AGAACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCCCCTTCCGΆCGAGCAGCTGAAGTCTGGCACCGCC TCTGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCC CTGCAGTCCGGCAΆTTCCCAGGAGTCTGTGACCGAGCAGGACTCCΆAGGACAGCACCTACTCCCTGTCC TCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAG GGCCTGTCCTCTCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGΆTGAGAATTC
[0643] Example 36: Pharmacokinetic and Biodistribution Studies in Mice
[0644] The in vivo pharmacokinetics and stability of DX-2240 (produced in
HEK293T cells) was determined by iodinating the protein on available tyrosine residues and measuring plasma clearance and stability in mice after a single intravenous dose. Samples were radio-iodinated by the indirect method using the IODO-GEN™ reagent (method from Pierce, and described by Chizzonite et al. ((199I) J. Immunol. 147:1548; (1992) J. Immunol. 148: 3117). Samples were incubated with the 125I-NaI solution for 9 min at which time tyrosine (10 mg/mL, a saturated solution) was added to quench the reaction. After about 15 min a 5 μl aliquot was removed as a standard for counting. For each labeling reaction, the 125I-labeled material (approx. 0.6 mL) was purified using a single 5 mL D-salt 1800 polyacrylamide column (Pierce). Columns were washed with 25 mM Tris, 0.4 M NaCl, pH 7.5 containing 2.5% HSA to block nonspecific sites then extensively with the same buffer minus the HSA. Samples were applied in and columns were eluted with a series of 0.3 mL aliquots. Recovery of applied activity in all protein fractions was > 75% and the total recovery of applied activity was > 90%. The fractions containing peak levels of labeled protein were pooled for animal injections. To prepare the injectate, the pool was diluted with Tris buffer (pH 7.5) so that the 100 μl injection volume contained about 10 μg of labeled material.
[0645] Solutions containing the radio-labeled compounds were administered to all mice by injection into the tail vein. At predetermined times post-administration animals were sacrificed and blood samples were taken for analyses. Time points tested after injection of radio-labeled compounds were: approximately 0, 7, 15, 30 and 90 minutes, 4h, 8h, 16h, 24h, 48h and 72h after injection. Four animals were sacrificed for each time point. At sacrifice, 0.5 mL aliquots of blood were collected into anticoagulant (0.02 mL EDTA) tubes. Plasma was separated from cells by centrifugation and the plasma fraction was divided into two aliquots, one frozen and one stored at 40C for immediate analysis. [0646] Analyses included gamma counting of all samples. In this single dose i.v. study, DX-2240 exhibited a relatively short-half life in mice of less than 5 hours. Analysis of the biodistribution of DX- 2240 in these mice revealed some accumulation of this antibody, at 30 minutes, in the lungs (12.85% ID/g), spleen (7.44% ID/g), kidney (8.34% ID/g), liver (5.42% ID/g) and heart (4.04% ID/g). DX-2240, due to its interaction with Tiel on the surface of endothelial cells, may accumulate in areas of high vascularization such as the lung. Therefore, multiple administrations of DX-2240 may be required to achieve an effective steady-state level of this antibody in the serum of mice. ELISA on ocular bleeds following three every other day dosings, as well as terminal bleed samples from tumor-bearing mice treated with DX-2220, were performed. In each case, levels of DX-2220 in the serum averaged 500 μg/ml, suggesting that despite a short serum half-life in mice, an effective steady-state level of this antibody can be achieved following just three doses of DX-2220.
[0647] In addition, SEC-HPLC analysis of plasma samples to assess the in vivo stability of DX-2240 was performed. Instability of DX-2240 in mouse could have contributed to the fast clearance of this compound from the serum. SEC-HPLC analysis was performed for two plasma samples at time points at 0 min, 30 min, 90 min, 24h and 72h. The analysis of the radio-labeled DX-2240 showed that this compound is stable in vivo both to degradation and to interactions with plasma components. Therefore, the relatively rapid half-life of DX-2240 in mice is not due to degradation of this compound.
IO648] Example 37 : DX-2220 Slows Lung Cancer Xenograft Tumor
Progression in Nude Mice
[0649] The effect of DX-2220 on tumor growth in mice bearing human
LNM35 lung cancer xenografts was tested. For these studies, LNM35 cells were injected subcutaneously in the lateral thorax of athymic nude mice. Four days after tumor cell implantation, treatment was initiated with DX-2220 or A2-SV (negative control antibody) at a dosage of 20 mg/kg, three times a week. Tumor sizes were measured at day 6, 8 and 10 post antibody treatment. LUODUJ Ά.S snown in figure t>, DX-2220 significantly slowed (-60%) tumor progression in this mouse xenograft model (p=0.037). In addition, mice treated with DX-2220 did not exhibit any significant loss in body weight. These data coupled demonstrate that DX-2220 possesses significant tumor growth inhibitory activity in vivo.
IO6511 Example 38: DX-2220 Slows Tumor Progression in Nude Mice
In addition to the in vivo studies presented above, four additional mouse xenografts studies were performed. These studies were conducted either according to the protocols used in the SW-480 or LNM35 study. The results from these studies are listed below.
LLC (mouse lung carcinoma) 20% inhibition @ day 14 after start of treatment
PC-3 (human prostate cancer) 24% inhibition @ day 28 after start of treatment
L1SM35 #3 (human lung carcinoma) 30% inhibition @ day 21 after start of treatment
Colo205 (human colorectal cancer) no effect
[0652] These results suggest that the E3 anti-Tiel antibody has an effect on a variety of tumor types, indicating broad therapeutic applicability.
[0653] Example 39: Immunohistochemical Analysis of Normal Tissues
[0654] A series of immunohistochemical analyses on a series of non- malignant normal human tissues to assess potential areas of immunoreactivity of the E3 anti-Tiel antibody was performed. Antibody titration experiments were conducted on both cryostat and paraformaldehyde fixed sections of select normal human tissues with biotinylated DX-2220 and an IgG isotype control antibody to determine the preferred tissue preservation conditions as well as optimal concentration of the antibody that would result in minimal background and maximal detection of signal. A concentration of 20 μg/ml for the primary antibody was selected for the study with biotinylated DX-2220 and the biotinylated IgG isotype control antibody used as the primary antibodies, and the principal detection system consisting of Streptavidin HRP with DAB as the chromagen. Tissues also were stained with the positive control antibodies (anti-CD31 and anti-vimentin) to ensure that the tissue antigens were preserved and accessible for immunohistochemical analysis. Only tissues that were positive for CD31 and vimentin staining were selected for the remainder of the study. The negative control consisted of performing the entire immunohistochemical procedure on adjacent sections in the absence of primary antibody. Slides were imaged with a DVC 1310C digital camera coupled to a Nikon microscope.
[0655] The negative control (no primary antibody) slides showed occasional faint background staining within renal tubular epithelium and occasional granulocytes, but was uniformly negative in all other cell types, including the positive control cell line (HMEC, Human Microvascular Endothelial Cells) and positive control colon cancer. The IgG isotype control antibody showed faint background staining of granulocytes, macrophages, adrenal cortex, renal tubular epithelium, fallopian tube epithelium, hepatocytes, Leydig cells, and thyroid. The positive control cell line (HMEC) and positive control colon cancer sample showed either no staining or background staining.
[0656] DX-2220 demonstrated moderate membrane staining within the
HMEC cell line, and staining of macrophages, some carcinoma cells, and endothelial cells within the colon cancer positive control samples. Within normal tissues, the antibody showed faint to moderate staining of macrophages, microglia in the brain, squamous epithelium of the cervix, faint staining of skeletal muscle, islets of Langerhans, and placental endothelium. These observations are consistent with low level expression of Tie 1 in some endothelial, hematopoietic and epithelial tissues, as anticipated from previous reports. The islets of Langerhans staining were unexpected and should be investigated further. Most other faint staining was similar to that seen with the IgG isotype control. If the background from the IgG isotype control is subtracted from the analysis of DX-2220, the majority of tissues were negative, including adrenal, bladder, blood, bone marrow, neurons, breast, colon, endothelium, eye, fallopian tube, heart, kidney, liver, lung, lymphocytes, ovary, exocrine pancreas, pituitary, prostate, skin, spinal cord, spleen, seminiferous epithelium of the testis, thymic lymphocytes, ureter, and uterus.
rO657] Example 40: Platelet Studies
[0658] It has been reported that Tiel is expressed on platelets (Tsiamis et al.
(200O) J Vase. Res. 37(6):437). The possibility of platelet immunoreactivity with the E3 anti-Tiel antibody was investigated by FACs analysis and immunoprecipitation studies. DX-2200 did not show significant binding to platelets, nor did it immunoprecipitate Tiel from platelet extracts. In addition, the effect of DX-2200 and DX-2210 on platelet agglutination and aggregation was investigated. Ristocetin, a cofactor that induces platelet agglutination by mediating the binding of von Willebrand factor (vWF) to platelet membrane glycoprotein GPIb (CD42), was used as a positive control for platelet agglutination. Antibodies to CD9 were used as a positive control to activate platelets and induce platelet aggregation, with kinetics and extent comparable to physiological agonists such as thrombin (reference). Neither the DX-2200 nor its light chain germlined variant DX-2210 induced platelet agglutination or aggregation.
[06591 Example 41: Chord Blood Stem Cell Studies
[0660] To evaluate the binding characteristics to blood progenitor cells (stem cells), FACS analysis with the anti-Tiel antibody (or the appropriate negative control antibody) on G-CSF mobilized peripheral blood cells and with bone marrow cells was performed. Briefly, cells were blocked with 10% heat-inactivated human AB serum/ 2% mouse serum. Binding was initiated with biotinylated DX-2220 or biotinylated A2 negative control antibody. After washing the cells, primary antibodies were detected using FITC-labeled streptavidin. Following an additional 30 minute incubation period, remaining erythrocytes were lysed, and the resulting cell pellet after centrifugation was resuspended in PBS prior to FACS analysis. Data acquisition was performed on a FACSCanto™ (Becton-Dickinson) using FacsDiva™ software. Active gating on SSC/CD45 was used. Progenitor cells were gated on CD45+ CD34+ cells and were acquired automatically with at least 100,000 CD45+ CD34+ counted. [0661] While the expression of Tie 1 has been reported on certain hematopoietic malignancies, this experiment demonstrated that neither the negative control IgG A02, nor the E3 anti-Tie 1 antibody DX-2220, positively stained CD45+ CD34+ blood progenitor cells. This finding supports the hypothesis that targeting Tiel with E3 should have no deleterious effects on stem cells, unlike certain chemotherapeutic agents.
[0662] Example 42: In Vitro Hematopoiesis Studies:
[0663] The effect of anti-Tiel antibodies (DX-2220 and DX-2240) on human myeloid and erythroid progenitors was evaluated using methylcellulose-based in vitro colony assays and megakaryocyte progenitors using collagen-based in vitro assays. Neither DX-2220 nor DX-2240 inhibited colony formation in the particular conditions of this in vitro assay at concentrations up to 100 μg/ml .
[0664] The effect of the E3 anti-Tiel antibody DX-2240 on the recovery of the mouse hematopoietic system using an in vivo myeloablation model was evaluated. Mice were injected with 5-FU on day 0 and then received either DX-2240 or a negative control antibody. At various time points following injection (days 2, 4, 6, 8, 10, 12 and 14), 4 mice were sacrificed from control and treated groups and peripheral blood and femurs were harvested. The peripheral blood and femoral cells were analyzed to determine the following:
• Total nucleated cells per femur
• Frequency of bone marrow colony forming cells for both myeloid and erythroid progenitors
• Total hematopoietic CFC per femur
• Total megakaryocytic CFC per femur
• Total white blood cell count and differential analysis of mature cells
[0665] DX-2240 had no effect on the recovery of the mouse hematopoietic system following 5-FU administration. This supports the in vitro findings that DX-2240 possesses no hematological toxicities under these assay conditions. Thus, it is particularly useful as a therapeutic as it will not interfere with nuimαi iicuiaiupuicLic iuiiuiums required to maintain red cell and lymphocyte production.
[0666] The anti-Tie 1 antibody was also evaluated for its effect on implanted tumors. Tumor cells were injected subcutaneously into the abdominal region of mice (Balb/C nu/nu female mice, 5-6 weeks old). The following tumor cells were tested: a lung carcinoma (mouse syngeneic Lewis lung carcinoma (LLC)); human lung carcinoma LNM35; an aggressive human colon carcinoma clone (SW480R). Treatments with anti-Tie 1 antibody were initiated 4-6 days post-implantation. The anti-Tie 1 antibodies or a control antibody (the A2 anti-streptavidin antibody) were administered intraperitoneally at 20 mg/kg every second day. Tumor volume was measured every second day and calculated as 0.5 x height x width x depth.
[0667] The E3 anti-Tie 1 antibody (also termed DX-2240) inhibits primary tumor growth of LLC (about a 20% effect, p = 0.078; ANOVA, single factor test) and LNM35. In one study, this anti-Tie 1 antibody inhibited primary tumor growth by 60% (most responsive). (Doubling of the antibody dose with administration only twice a week rather than every second day resulted in only a modest effect on primary tumor growth). The SW480R tumor was not responsive to the antibody treatment under these conditions. As described in Example 34, the antibody was effective for inhibiting tumor growth of SW480 cells (i.e., rather than the derivative SW480R cells).
[0668] Histological analysis of tumor sections from the experiment in which
60% inhibition was observed indicated that blood vessels in anti-Tiel antibody treated tumors have a distinct morphology even though blood vessel density may not be altered, hi anti-Tie antibody treated tumors, the vessels form septa-like structures in between lobuli of tumor cells. These tumors also have more necrosis than control antibody treated tumors. The distribution of smooth muscle cells (as detected by anti- smooth muscle actin antibody staining) was also altered. The lymphatic vessels in anti-Tiel antibody treated tumors were also more dispersed, somewhat dilated and in several instances composed of adjacent lumina clustered together in a string. These observations indicate that this anti-Tie antibody has a distinctive effect on tumor necrosis and vessel organization within the tumor. [0669] Example 43: Evaluating Combination Therapies
[0670] An animal model can be used to evaluate combination therapies. For example, the combinations (provided intraperitoneally) can be tested for ability to modulate tumor growth in female NCr nu/nu mice with xenografts of HT29, COLO205, or PC3. The following are some exemplary test regimes:
Figure imgf000256_0001
[0671] Another regime is as follows:
Figure imgf000256_0002
[0672] A2-SV is the control anti-streptavidin antibody. Twelve animals can be used in each group. Clinical signs, mean group weights are evaluated every day. Individual body weights and tumor burden are evaluated twice weekly. At study termination, tissue samples can be obtained from tumors, liver, lung, spleen, heart, axial node, kidney, and uterus.
Other embodiments are within the following claims:

Claims

WHAT IS CLAIMED:
1. An isolated protein comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to Tiel ectodomain and
(A) the heavy chain immunoglobulin variable domain sequence comprises one or more of the following properties: i) a HC CDRl that includes (AGSMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO: 118); ii) a HC CDR2 that includes (GSV)-I-(SY)-P-S-G-G-(WNQ)-T-(GY) (SEQ ID NO: 160); and iii) a HC CDR3 that includes A-P-R-G-Y-S-Y-G-Y-Y-Y (SEQ ID NO:727); and/or
(B) the light chain immunoglobulin variable domain sequence comprises one or more of the following properties: i) a LC CDRl that includes R-A-S-(REQ)-(GSTRN)-(IV)-(GSTIRN)- (STIRH)-X1-(SYWNH)-(LV)-(ASN) (SEQ ID NO: 132), wherein Xl can be serine or absent; ii) a LC CDR2 that includes Q-Q-(SYFR)-(GSYN)-S-(STYW)-(RP)- (LWRH)-(TIY) (SEQ ID NO:161); and iii) a LC CDR3 that includes Q-Q-F-N-S-Y-P-H (SEQ ID NO:728).
2. The protein of claim 1 wherein the heavy chain immunoglobulin variable domain sequence comprises i) a HC CDRl that includes (AGSIMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO: 118); ii) a HC CDR2 that includes (GSV)-I-(SY)-P-S-G-G-(WNQ)-T-(GY) (SEQ ID NO: 160); and iii) a HC CDR3 that includes A-P-R-G-Y-S-Y-G-Y-Y-Y (SEQ ID NO:727).
3. The protein of claim 1 wherein the light chain immunoglobulin variable domain sequence comprises: i) a LC CDRl that includes R-A-S-(REQ)-(GSTRN)-(rV)-(GSTIRN)- (STIRH)-X1-(SYWNH)-(LV)-(ASN) (SEQ ID NO-.132), wherein Xl can be serine or absent; ii) a LC CDR2 that includes Q-Q-(SYFR)-(GSYN)-S-(STYW)-(RP)- (LWRH)-(TIY) (SEQ ID NO:161); and iii) a LC CDR3 that includes Q-Q-F-N-S-Y-P-H (SEQ ID NO.728).
4. The protein of claim 1 wherein the amino acid sequences of the HC variable domain sequence comprises CDRl, CDR2, and CDR3 sequences from the E3 clone, and the LC variable domain sequence comprises CDRl, CDR2, and CDR3 sequences from the E3 clone.
5. The protein of claim 1 that comprises (i) the HC and/or LC immunoglobulin variable domains of the E3 antibody , (ii) HC and/or LC immunoglobulin variable domain sequences that are at least 85% identical to the HC and LC immunoglobulin variable domains of the E3 antibody, respectively, or (iii) HC and/or LC immunoglobulin variable domain sequences that are encoded by a nucleic acid that hybridizes with high stringency to a nucleic acid encoding a HC or LC variable domain of E3, respectively.
6. The protein of any preceding claim that inhibits tube formation by HUVEC cells in vitro.
7. The protein of any preceding claim wherein the protein is a Fab.
8. The protein of any preceding claim wherein the protein is an IgG.
9. An isolated protein comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to a Tiel ectodomain and competes with E3, G2, p-Al, p- AlO, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s- ClO, s-C2, s-C7, s-Dll, s-Ell, S-GlO, or s-H4 for binding to Tiel or binds to an epitope that overlaps an epitope that is recognized by E3, G2, p-Al, p-A10, p-Bl, p- B3, p-C6, P-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-El 1, s-GlO, or s-H4or that has at least one, two or three residues in common with an epitope that is recognized by E3, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, ρ-F3, p-F4, ρ-G3, S-AlO5 s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s- G10, or s-H4.
10. A pharmaceutical composition comprising a protein of any preceding claim and a pharmaceutically acceptable carrier.
11. A method of inhibiting vascular development in a subject, the method comprising administering, to a subject having or at risk for a disorder requiring inhibition of vascular development, a Tiel binding protein according to any of claims- 1-9.
12. The method of claim 11 wherein the Tiel binding protein one or more of the following properties:
(1) at least one of the variable domain sequences comprising at least two CDR of the E3 antibody;
(2) at least one of the variable domain sequences comprising CDR sequences at least 85% identical, in sum, to the CDRs of the corresponding variable domain of the E3 antibody,
(3) at least one of the variable domains is at least 85% identical to the corresponding immunoglobulin variable domains of the E3 antibody,
(4) the protein competes with E3 for binding to Tiel or binds to an epitope that overlaps the epitope bound by E3 on Tiel, and (5) the protein comprises a domain that is encoded by a nucleic acid that hybridizes with high stringency to a nucleic acid encoding a HC or LC variable domain of E3 or E3b.
13. The method of claim 11 wherein the antibody comprises the variable domains of E3.
14. The method of any of claims 11-13 wherein the agent is administered in an amount and/or a time effective to decrease vascular development in a subject.
15. The method of any of claims 11-14 wherein the subj ect has an vasculature-dependent cancer or tumor.
16. The method of claim 15 wherein the tumor is a solid tumor.
17. The method of any of claims 11-16 further comprising providing a second therapy that is an anti-cancer therapy.
18. The method of claim 17 wherein the second therapy is a chemotherapeutic.
19. The method of claim 17 wherein the second therapy comprises administering an agent that antagonizes signaling through a VEGF pathway.
20. The method of claim 17 wherein the second therapy comprises administering bevacizumab.
21. The method of claim 17 wherein the second therapy comprises administering 5 -FU, leucovorin, and/or irinotecan.
22. A method of providing a post-operative adjuvant therapy, the method comprising administering a Tiel binding protein according to claim 1, to a subject who has had surgery to remove a tumor.
23. The method of claim 22 wherein the Tiel binding protein comprises (a) a heavy chain variable domain sequence that is at least 90% identical to the heavy chain variable domain of the E3 antibody and a light chain variable domain sequence that is at least 90% identical to the light chain variable domain of the E3 antibody;
(b) a heavy chain variable domain sequence and a light chain variable domain sequence that form an antigen binding site that competes with E3 for binding to Tiel; or
(c) one, two, or three, of the CDRs of the heavy chain variable domain of the E3 antibody, and one, two, or three of the CDRs of the light chain variable domain of the E3 antibody.
24. The method of claim 22 wherein the Tiel binding protein is administered within 48 hours of surgery.
25. The method of claim 22 wherein the Tiel binding protein is administered before and after surgery.
26. An isolated protein comprising SEQ ID NO:723 and SEQ ID NO:724.
27. The protein of claim 26 that comprises two antigen binding sites, each of which binds to Tiel .
28. An isolated nucleic acid encoding the heavy chain immunoglobulin variable domain sequence or the light chain immunoglobulin variable domain sequence of the protein of claim 1.
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