HK1136836A - Angiopoietin-2 specific binding agents - Google Patents

Description

Angiopoietin-2 specific binding agents

This is a continuation-in-part application of U.S. patent application 10/269,805 (filed 10/2002) and PCT/US02/32613 (filed 10/11/2002) claiming priority to U.S. provisional patent application 60/328,604 (filed 10/11/2001), all of which are incorporated herein by reference. Priority is also claimed for U.S. provisional patent application 60/620,161 (application 10/19/2004), which is incorporated herein by reference.

Technical Field

The present invention relates to specific binding agents (binding agents) that recognize and bind to angiopoietin-2 (Ang-2). More specifically, the invention relates to the production and diagnostic and therapeutic applications of polyclonal antibodies, monoclonal antibodies and fragments thereof that specifically bind to Ang-2.

Background

Angiogenesis, the formation of new blood vessels from existing blood vessels, is essential for many physiological and pathological processes. Normally, angiogenesis is tightly regulated by pro-and anti-angiogenic factors, but in disease states such as cancer, intra-ocular dermal growth factor disease, arthritis and psoriasis, the process is biased. (Folkman, J., nat. Med., 1: 27-31(1995))

Many diseases are known to be associated with deregulated angiogenesis and undesirable angiogenesis. These diseases include, but are not limited to, ocular neovascular diseases such as retinopathy (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammation such as rheumatoid or rheumatic inflammation, especially arthritis (including rheumatoid arthritis), or other chronic inflammation such as chronic asthma, arteriosclerosis or post-transplant arteriosclerosis, endometriosis, tumors such as so-called solid tumors and liquid (or blood) tumors (such as leukemias and lymphomas). Other diseases associated with deregulated angiogenesis and undesirable angiogenesis will be apparent to those skilled in the art.

Although angiogenesis regulation is involved in many signal transduction systems, one of the most well-identified systems is the endothelial cell selection system, which includes the Tie-2 receptor tyrosine kinase (i.e., Tie-2 or Tie-2R, or ORK; murine Tie-2 is also known as tek) and its ligand, angiopoietin (Gale, N.W. and Yancopoulos, G.D., Genes Dev.13: 1055-1066 (1999)). There are now 4 known angiopoietins: angiopoietin-1 (Ang-1) to angiopoietin-4 (Ang-4). These angiopoietins are also known as "Tie-2 ligands". (Davis, S., et al, Cell, 87: 1161- &Visual Science, 42: 1617-; koblizek, t.i., et al, Current Biology, 8: 529- "532 (1998); lin, p, et al, Proc Natl Acad SciUSA, 95: 8829-8834 (1998); maison pierre, p.c., et al, Science, 277: 55-60 (1997); papapeetropoulos, a., et al, Lab Invest, 79: 213-223 (1999); sato, t.n., et al, Nature, 375: 70-74 (1998); shyu, k.g., et al, Circulation, 98: 2081-2087 (1998); suri, c, et al, Cell, 87: 1171-1180 (1996); suri, c, et al, Science, 282: 468-471 (1998); vallenzuela, D.M., et al, Proceedings of National Academy of Sciences of the USA, 96: 1904-1909 (1999); witzenbichler, b., et al, J Biol Chem, 273: 18514-18521(1998)). Although binding of Ang-1 to Tie-stimulates receptor phosphorylation in cultured endothelial cells, it has been observed that Ang-2 both activates and antagonizes Tie-2 receptor phosphorylation (Davis, S., et al, (1996), supra; Maison pierre, P.C., et al, (1997), supra; Kim, I., J.H.Kim, et al,Oncogene19(39): 4549-4552 (2000); Teichert-Kuliszewska, k., p.c. maison pierre, et al, Cardiovascular Research 49(3)：659-70(2001))。

Mice knocked out for Tie-2 and those knocked out for Ang-1 are phenotypically similar, suggesting that Tie-2 phosphorylation, stimulated by Ang-1, mediates embryonic vascular remodeling and stabilization by maintaining endothelial cell-supporting cell adhesion. (Dumont, D.J., et al, Genes & Development, 8: 1897-. The role of Ang-1 in vascular stabilization is thought to be maintained until adulthood, with its widespread and sustained expression (Hanahan, D., Science, 277: 48-50 (1997); Zagzag, D., et al, Experimental neurology, 159: 391 (1999)). In contrast, expression of Ang-2 is primarily restricted to sites of vascular remodeling, where Ang-2 is thought to repress the function of Ang-1, thereby inducing a plastic state of the blood vessel, favoring angiogenesis (Hanahan, D., (1997), supra; Holash, J., et al, Science, 284: 1994-1998 (1999); Maison pierre, P.C., et al, (1997), supra).

A number of published studies have demonstrated vascular-selective Ang-2 expression in disease states associated with angiogenesis. These pathological conditions include, for example, psoriasis, macular degeneration and Cancer (Bunone, G., et al, American Journal of Pathology, 155: 1967-audio 1976 (1999); Etoh, T., et al, Cancer Research, 61: 2145-audio 2153 (2001); Hangai, M., et al, Investigative opthalmology & Visualscience, 42: 1617-audio 1625 (2001); Holash, J., et al, (1999) supra; Kuroda, K., et al, Journal of Investigative Dermatology, 116: 713-audio 720 (2001); Otani, A., et al, Investigative halmology & Visualscience, 40: 1912-audio 1920 (1999); manatan, A., et al, Journal of Pathology, 153: 1459, 1999, S.103, J., 103, J.201, 103, J., 103, J.103, J.1999), and so on-audio J.103, 103, J.103, J.. Most of these studies have focused attention on cancer, and many tumors show expression of angio Ang-2. Expression of Ang-2 in normal tissues is greatly restricted compared to its expression in pathological angiogenesis (Maison pierre, P.C., et al, (1997), supra; Mezzita, J., et al, Biochemical and biophysical research Communications, 260: 492) -498 (1999)). The three major sites of angiogenesis in normal adults are the ovary, placenta and uterus; these sites are the predominant tissue in normal (i.e., non-cancerous) tissue where Ang-2mRNA is detectable.

Some functional studies suggest that Ang-2 may be involved in tumor angiogenesis. Ahmad et al (Cancer Res., 61: 1255-1259(2001)) describe Ang-2 overexpression in a xenografted mouse model and argue that it is associated with increased tumor growth. Also, see Etoh et al and Tanaka, supra, for reports that Ang-2 overexpression is associated with tumor angiogenesis. In contrast, Yu et al (am.J.Path., 158: 563-570(2001)) reported that overexpression of Ang-2 in Lewis lung and breast cancer cells in mouse transfectants extended the lifespan of mice.

In the past few years, a variety of published reports have indicated that Ang-1, Ang-2 and/or Tie-2 may be targets for anticancer therapy. For example, U.S. Pat. Nos. 6,166,185, 5,650,490, and 5,814,464 all disclose the concept of anti-Tie-2 ligand antibodies and receptor antibodies. Lin et al (Proc. Natl. Acad. Sci USA, 95: 8829-8834(1998)) injected soluble Tie-2 expressing adenovirus into mice; soluble Tie-2 was said to reduce the number and size of mouse tumors. In a related study, Lin et al (J.Clin.invest., 100: 2072-2078(1997)) injected soluble Tie-2 into rats; the compounds are said to reduce tumor size in rats. Siemeister et al (Cancer Res., 59: 3185-3189(1999)) generated a human melanoma cell line expressing the Tie-2 ectodomain, injected this cell into nude mice, and stated that soluble Tie-2 caused "significant inhibition" of tumor growth and tumor angiogenesis. Given this information, and because both Ang-1 and Ang-2 bind to Tie-2, it is not clear from these studies whether Ang-1, Ang-2 or Tie-2 might be attractive targets for anticancer therapy.

Various approaches have been used to fuse specific peptides to stable cytoplasmic proteins (e.g., Ig constant regions) to increase the half-life of the molecule, as described in, e.g., U.S. patent 5,480,981; zheng et al, j.immunol., 154: 5590-; fisher et al, n.engl.j.med., 334: 1697-; van Zee, k, et al, j.immunol., 156: 2221-; us patent 5,808,029, grant date: 9 month 15 of 1998; capon et al, Nature, 337: 525-531, (1989); harvil et al, Immunotech, 1: 95-105, (1995); WO 97/23614, publication date: 3 days 7 month 1997; PCT/US 97/23183, filing date: 11/12/1997; linsley, j.exp.med., 174: 561-; WO 95/21258, publication date: 8/10/1995).

Effective anti-Ang-2 therapy may be beneficial for a large number of cancer patients, since most solid tumors require neovascularization to grow to a diameter greater than 1-2 mm. This therapy may also have broad application in other angiogenesis-related diseases, such as retinopathy, arthritis and psoriasis.

There is a need to fill the gap with new drugs that specifically recognize and bind to Ang-2. These drugs may play a role in diagnostic screening and treatment of diseases associated with Ang-2 activity.

It is therefore an object of the present invention to provide Ang-2 binding agents that specifically bind to Ang-2 and modulate Ang-2 activity.

Summary of The Invention

The invention provides antibodies comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region sequence selected from 526HC (SEQ ID NO: 1); 528HC (SEQ ID NO: 3); 531HC (SEQ ID NO: 5); 533HC (SEQ ID NO: 7); 535HC (SEQ ID NO: 9); 536HC (SEQ ID NO: 11); 537HC (SEQ ID NO: 13); 540HC (SEQID NO: 15); 543HC (SEQ ID NO: 17); 544HC (SEQ ID NO: 19); 545HC (SEQ ID NO: 21); 546HC (SEQ ID NO: 23); 551HC (SEQ ID NO: 25); 553HC (SEQ ID NO: 27); 555HC (SEQ ID NO: 29); 558HC (SEQID NO: 31); 559HC (SEQ ID NO: 33); 565HC (SEQ ID NO: 35); F1-C6HC (SEQ ID NO: 37); FB1-A7HC (SEQ ID NO: 39); FD-B2HC (SEQ ID NO: 41); FE-B7HC (SEQ ID NO: 43); FJ-G11HC (SEQ ID NO: 45); FK-E3HC (SEQ ID NO: 47); G1D4HC (SEQ ID NO: 49); GC1E8HC (SEQ ID NO: 51); H1C12HC (SEQ ID NO: 53); IA1-1E7HC (SEQ ID NO: 55); IF-1C10HC (SEQ ID NO: 57); IK-2E2HC (SEQ ID NO: 59); IP-2C11HC (SEQ ID NO: 61), and antigen-binding fragments of these sequences. The light chain comprises a light chain variable region selected from 526 kappa (SEQ ID NO: 2); 536(THW) κ (SEQ ID NO: 12); 536(LQT) κ (SEQ ID NO: 210); 543 κ (SEQ ID NO: 18); 544 κ (SEQ ID NO: 20); 551 κ (SEQ ID NO: 26); 553 κ (SEQ ID NO: 28); 555 κ (SEQ ID NO: 30); 558 kappa (SEQ ID NO: 32); 565 κ (SEQ ID NO: 36); FE-B7 kappa (SEQ ID NO: 44); FJ-G11 kappa (SEQ ID NO: 46); FK-E3 kappa (SEQ ID NO: 48); IA1-1E7 κ (SEQ ID NO: 56); IP-2C11 kappa (SEQ ID NO: 62); 528 lambda (SEQ ID NO: 4); 531 λ (SEQ ID NO: 6); 533 lambda (SEQ ID NO: 8); 535 λ (SEQ ID NO: 10); 537 λ (SEQ ID NO: 14); 540 λ (SEQ ID NO: 16); 545 lambda (SEQ ID NO: 22); 546 λ (SEQ ID NO: 24); 559 λ (SEQ ID NO: 34); F1-C6 lambda (SEQ ID NO: 38); FB1-A7 λ (SEQ ID NO: 40); FD-B2 lambda (SEQ ID NO: 42); G1D4 λ (SEQ ID NO: 50); GC1E8 λ (SEQ ID NO: 52); H1C12 λ (SEQ ID NO: 54); IF-1C10 λ (SEQ ID NO: 58); IK-2E2 lambda (SEQ ID NO: 60), and antigen-binding fragments of these sequences.

The invention also provides a specific binding agent comprising at least one of the following peptides: SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 43; SEQ ID NO: 45, a first step of; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 51; SEQ ID NO: 53; SEQ ID NO: 55; SEQ ID NO: 57; SEQ ID NO: 59; SEQ ID NO: 61; SEQ ID NO: 2; SEQ ID NO: 12; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30, of a nitrogen-containing gas; SEQ ID NO: 32, a first step of removing the first layer; SEQ ID NO: 36; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 56; SEQ ID NO: 62, a first step of mixing; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 34; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID NO: 50; SEQ ID NO: 52; SEQ ID NO: 54, a first electrode; SEQ ID NO: 58; and SEQ ID NO: 60, and fragments of these sequences.

It will be appreciated that the specific binding agent may be, for example, an antibody, such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody. Such antibodies may also be single chain antibodies. The invention further relates to hybridomas producing a monoclonal antibody of the invention.

The invention also relates to nucleic acid molecules encoding specific binding agents, such as antibodies, vectors containing these nucleic acid molecules and host cells containing these vectors.

In addition, the present invention also provides a method for preparing a specific binding agent, comprising the steps of (a) transforming a host cell with at least one nucleic acid molecule encoding a specific binding agent of claim 1; (b) expressing the nucleic acid molecule in said host cell; and (c) isolating the specific binding agent. The present invention also provides a method for producing an antibody, comprising the steps of: (a) transforming a host cell with at least one nucleic acid molecule encoding an antibody of the invention; (b) expressing the nucleic acid molecule in said host cell; and (c) isolating the specific binding agent.

The invention also relates to a method of inhibiting undesired angiogenesis in a mammal by administering a therapeutically effective amount of a specific binding agent of the invention. The invention also provides methods of treating cancer in a mammal by administering a therapeutic amount of a specific binding agent of the invention.

The invention also relates to methods of inhibiting undesired angiogenesis in a mammal by administering a therapeutically effective amount of an antibody of the invention. The invention also provides methods of treating cancer in a mammal by administering a therapeutic dose of an antibody of the invention.

It will be appreciated that the invention also relates to pharmaceutical compositions comprising a specific binding agent of the invention and a pharmaceutically acceptable excipient (formulation agent). The pharmaceutical composition may comprise an antibody of the invention and a pharmaceutically acceptable excipient.

The invention provides methods of modulating or inhibiting angiopoietin-2 activity by administering one or more specific binding agents described herein. The invention also provides methods of modulating or inhibiting angiopoietin-2 by administering the antibodies of the invention.

The invention also relates to a method of modulating at least one of vascular permeability and plasma leakage in a mammal comprising the step of administering a therapeutically effective amount of a specific binding agent of the invention. The present invention also relates to methods of treating at least one of the following mammalian diseases: ocular neovascular disease, obesity, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, inflammation, atherosclerosis, endometriosis, tumor, bone-related disease or psoriasis, comprising the step of administering a therapeutically effective amount of a specific binding agent of the invention.

The invention also relates to a method of modulating at least one of vascular permeability and plasma leakage in a mammal comprising the step of administering a therapeutically effective amount of an antibody of the invention. The present invention also relates to methods of treating at least one of the following mammalian diseases: ocular neovascular disease, obesity, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, inflammation, atherosclerosis, endometriosis, tumor, bone-related disease or psoriasis, comprising the step of administering a therapeutically effective amount of an antibody of the invention.

The invention also relates to a method of treating cancer in a mammal comprising the step of administering a therapeutically effective amount of a specific binding agent of the invention and a chemotherapeutic agent. It will be understood by those skilled in the art that the specific binding agent and the chemotherapeutic agent need not be administered simultaneously.

The invention also relates to a method of treating cancer in a mammal comprising the step of administering a therapeutically effective amount of an antibody of the invention and a chemotherapeutic agent. The antibody and chemotherapeutic agent need not be administered simultaneously.

The invention also provides a specific binding agent comprising the complementarity determining region 1(CDR1) of any one of the following sequences: 526HC (SEQ ID NO: 1); 528HC (SEQ ID NO: 3); 531HC (SEQ ID NO: 5); 533HC (SEQ ID NO: 7); 535HC (SEQ ID NO: 9); 536HC (SEQ ID NO: 11); 537HC (SEQ ID NO: 13); 540HC (SEQ ID NO: 15); 543HC (SEQ ID NO: 17); 544HC (SEQ ID NO: 19); 545HC (SEQ ID NO: 21); 546HC (SEQ ID NO: 23); 551HC (SEQ ID NO: 25); 553HC (SEQ ID NO: 27); 555HC (SEQ ID NO: 29); 558HC (SEQID NO: 31); 559HC (SEQ ID NO: 33); 565HC (SEQ ID NO: 35); F1-C6HC (SEQ ID NO: 37); FB1-A7HC (SEQ ID NO: 39); FD-B2HC (SEQ ID NO: 41); FE-B7HC (SEQ ID NO: 43); FJ-G11HC (SEQ ID NO: 45); FK-E3HC (SEQ ID NO: 47); G1D4HC (SEQ ID NO: 49); GC1E8HC (SEQ ID NO: 51); H1C12HC (SEQ ID NO: 53); IA1-1E7HC (SEQ ID NO: 55); IF-1C10HC (SEQ ID NO: 57); IK-2E2HC (SEQ ID NO: 59); IP-2C11HC (SEQ ID NO: 61); 526 κ (SEQ ID NO: 2); 536(THW) κ (SEQ ID NO: 12); 536(LQT) κ (SEQ ID NO: 210); 543 κ (SEQ ID NO: 18); 544 κ (SEQ ID NO: 20); 551 κ (SEQ ID NO: 26); 553 κ (SEQ ID NO: 28); 555 κ (SEQ ID NO: 30); 558 kappa (SEQ ID NO: 32); 565 κ (SEQ ID NO: 36); FE-B7 kappa (SEQ ID NO: 44); FJ-G11 kappa (SEQ ID NO: 46); FK-E3 kappa (SEQ ID NO: 48); IA1-1E7 κ (SEQ ID NO: 56); IP-2C11 kappa (SEQ ID NO: 62); 528 lambda (SEQ ID NO: 4); 531 λ (SEQ ID NO: 6); 533 lambda (SEQ ID NO: 8); 535 λ (SEQ ID NO: 10); 537 λ (SEQ ID NO: 14); 540 λ (SEQ ID NO: 16); 545 lambda (SEQ ID NO: 22); 546 λ (SEQ ID NO: 24); 559 λ (SEQ ID NO: 34); F1-C6 lambda (SEQ ID NO: 38); FB1-A7 λ (SEQ ID NO: 40); FD-B2 lambda (SEQ ID NO: 42); G1D4 λ (SEQ ID NO: 50); GC1E8 λ (SEQ ID NO: 52); H1C12 λ (SEQ ID NO: 54); IF-1C10 λ (SEQ ID NO: 58); and IK-2E2 lambda (SEQ ID NO: 60).

The invention also provides a specific binding agent comprising a complementarity determining region 2(CDR2) of any one of the following sequences: 526HC (SEQ ID NO: 1); 528HC (SEQ ID NO: 3); 531HC (SEQ ID NO: 5); 533HC (SEQ ID NO: 7); 535HC (SEQ ID NO: 9); 536HC (SEQ ID NO: 11); 537HC (SEQ ID NO: 13); 540HC (SEQ ID NO: 15); 543HC (SEQ ID NO: 17); 544HC (SEQ ID NO: 19); 545HC (SEQ ID NO: 21); 546HC (SEQ ID NO: 23); 551HC (SEQ ID NO: 25); 553HC (SEQ ID NO: 27); 555HC (SEQ ID NO: 29); 558HC (SEQID NO: 31); 559HC (SEQ ID NO: 33); 565HC (SEQ ID NO: 35); F1-C6HC (SEQ ID NO: 37); FB1-A7HC (SEQ ID NO: 39); FD-B2HC (SEQ ID NO: 41); FE-B7HC (SEQ ID NO: 43); FJ-G11HC (SEQ ID NO: 45); FK-E3HC (SEQ ID NO: 47); G1D4HC (SEQ ID NO: 49); GC1E8HC (SEQ ID NO: 51); H1C12HC (SEQ ID NO: 53); IA1-1E7HC (SEQ ID NO: 55); IF-1C10HC (SEQ ID NO: 57); IK-2E2HC (SEQ ID NO: 59); IP-2C11HC (SEQ ID NO: 61); 526 κ (SEQ ID NO: 2); 536(THW) κ (SEQ ID NO: 12); 536(LQT) κ (SEQ ID NO: 210); 543 κ (SEQ ID NO: 18); 544 κ (SEQ ID NO: 20); 551 κ (SEQ ID NO: 26); 553 κ (SEQ ID NO: 28); 555 κ (SEQ ID NO: 30); 558 kappa (SEQ ID NO: 32); 565 κ (SEQ ID NO: 36); FE-B7 kappa (SEQ ID NO: 44); FJ-G11 kappa (SEQ ID NO: 46); FK-E3 kappa (SEQ ID NO: 48); IA1-1E7 κ (SEQ ID NO: 56); IP-2C11 kappa (SEQ ID NO: 62); 528 lambda (SEQ ID NO: 4); 531 λ (SEQ ID NO: 6); 533 lambda (SEQ ID NO: 8); 535 λ (SEQ ID NO: 10); 537 λ (SEQ ID NO: 14); 540 λ (SEQ ID NO: 16); 545 lambda (SEQ ID NO: 22); 546 λ (SEQ ID NO: 24); 559 λ (SEQ ID NO: 34); F1-C6 lambda (SEQ ID NO: 38); FB1-A7 λ (SEQ ID NO: 40); FD-B2 lambda (SEQ ID NO: 42); G1D4 λ (SEQ ID NO: 50); GC1E8 λ (SEQ ID NO: 52); H1C12 λ (SEQ ID NO: 54); IF-1C10 λ (SEQ ID NO: 58); and IK-2E2 lambda (SEQ ID NO: 60).

The invention also provides a specific binding agent comprising the complementarity determining region 3(CDR3) of any one of the following sequences: 526HC (SEQ ID NO: 1); 528HC (SEQ ID NO: 3); 531HC (SEQ ID NO: 5); 533HC (SEQ ID NO: 7); 535HC (SEQ ID NO: 9); 536HC (SEQ ID NO: 11); 537HC (SEQ ID NO: 13); 540HC (SEQ ID NO: 15); 543HC (SEQ ID NO: 17); 544HC (SEQ ID NO: 19); 545HC (SEQ ID NO: 21); 546HC (SEQ ID NO: 23); 551HC (SEQ ID NO: 25); 553HC (SEQ ID NO: 27); 555HC (SEQ ID NO: 29); 558HC (SEQID NO: 31); 559HC (SEQ ID NO: 33); 565HC (SEQ ID NO: 35); F1-C6HC (SEQ ID NO: 37); FB1-A7HC (SEQ ID NO: 39); FD-B2HC (SEQ ID NO: 41); FE-B7HC (SEQ ID NO: 43); FJ-G11HC (SEQ ID NO: 45); FK-E3HC (SEQ ID NO: 47); G1D4HC (SEQ ID NO: 49); GC1E8HC (SEQ ID NO: 51); H1C12HC (SEQ ID NO: 53); IA1-1E7HC (SEQ ID NO: 55); IF-1C10HC (SEQ ID NO: 57); IK-2E2HC (SEQ ID NO: 59); IP-2C11HC (SEQ ID NO: 61); 526 κ (SEQ ID NO: 2); 536(THW) κ (SEQ ID NO: 12); 536(LQT) kappa (SEQ ID NO: 210)543 kappa (SEQ ID NO: 18); 544 κ (SEQ ID NO: 20); 551 κ (SEQ ID NO: 26); 553 κ (SEQ ID NO: 28); 555 κ (SEQ ID NO: 30); 558 kappa (SEQ ID NO: 32); 565 κ (SEQ ID NO: 36); FE-B7 kappa (SEQ ID NO: 44); FJ-G11 kappa (SEQ ID NO: 46); FK-E3 kappa (SEQ ID NO: 48); IA1-1E7 κ (SEQ ID NO: 56); IP-2C11 kappa (SEQ ID NO: 62); 528 lambda (SEQ ID NO: 4); 531 λ (SEQ ID NO: 6); 533 lambda (SEQ ID NO: 8); 535 λ (SEQ ID NO: 10); 537 λ (SEQ ID NO: 14); 540 λ (SEQ ID NO: 16); 545 lambda (SEQ ID NO: 22); 546 λ (SEQ ID NO: 24); 559 λ (SEQ ID NO: 34); F1-C6 lambda (SEQ ID NO: 38); FB1-A7 λ (SEQ ID NO: 40); FD-B2 lambda (SEQ ID NO: 42); G1D4 λ (SEQ ID NO: 50); GC1E8 λ (SEQ ID NO: 52); H1C12 λ (SEQ ID NO: 54); IF-1C10 λ (SEQ ID NO: 58); and IK-2E2 lambda (SEQ ID NO: 60).

The invention also provides nucleic acid molecules encoding the specific binding agents of the invention.

Furthermore, the present invention also relates to a method for detecting angiopoietin-2 in a biological sample, comprising the steps of (a) contacting a specific binding agent of the present invention with the biological sample; and (b) determining the extent of binding of the specific binding agent to the sample. The present invention also relates to a method for detecting angiopoietin-2 in a biological sample, comprising the steps of (a) contacting the biological sample with an antibody of the present invention; and (b) determining the extent of binding of the antibody to the sample.

The invention also relates to a method of inhibiting undesired angiogenesis in a mammal comprising the step of administering a therapeutically effective amount of a polypeptide or composition described herein. The invention also relates to a method of modulating angiogenesis in a mammal comprising the step of administering a therapeutically effective amount of a polypeptide or composition described herein. The invention also relates to a method of inhibiting a tumor characterized by undesired angiogenesis in a mammal comprising the step of administering a therapeutically effective amount of a polypeptide or composition described herein. In addition, the invention also relates to a method of treating cancer in a mammal comprising the step of administering a therapeutically effective amount of a polypeptide or composition as described herein, and a chemotherapeutic agent. In a preferred embodiment, the chemotherapeutic agent comprises at least 5-Fu, CPT-11 and Taxotere (Taxotere). Of course, other suitable chemotherapeutic agents and other cancer therapies may be used.

Of course, specific binding agents of the invention may be used to treat a variety of diseases associated with deregulated angiogenesis or with undesired angiogenesis. These diseases include, but are not limited to, ocular neovascularisation such as retinopathy (including diabetic retinopathy), psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammation such as rheumatic or rheumatic inflammation, in particular arthritis (including rheumatoid arthritis), or other chronic inflammation such as chronic asthma, arteriosclerosis or post-transplant arteriosclerosis, endometriosis, tumours such as so-called solid tumours and liquid tumours (such as leukaemias). Other diseases that may be treated by administration of a specific binding agent of the invention will be apparent to those skilled in the art. Such diseases include, but are not limited to, obesity, vascular permeability disorders, plasma leakage, bone related disorders including osteoporosis. The invention therefore also relates to methods of treating such diseases associated with deregulated angiogenesis or with undesired angiogenesis.

Other embodiments of the present invention will be readily understood by the description herein.

Brief Description of Drawings

FIG. 1 is a plot of tumor size (Y-axis) versus time (X-axis) in mice treated with an anti-Ang-2 antibody of the present invention (clone 533, 537, or 544), a control antibody, or Phosphate Buffered Saline (PBS). See the examples section for details.

FIGS. 2A, 2B and 2C are epitope mapping data (O.D.370) for the peptibody (TN) of the invention TN8-Con4-C, L1-7-N and 12-9-3-C, as well as for the control peptibody, Tie2-Fc, C2B8 or 5B12, bound to the N-terminus of full-length human Ang-2(hAng-2), hAng-2, and C-terminus of hAng-2, respectively. See the examples for details.

Detailed Description

The headings used in this section are for organizational purposes only and are not meant to be limiting in any way.

Standard procedures can be used for recombinant DNA, recombinant protein, antibody production, tissue culture and cell transformation procedures. The enzymatic reactions and purification processes are carried out essentially according to the instructions for the use of the product or according to methods customary in the art, for example as listed in Sambrook et al (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) or according to the methods listed therein. The terms used herein, analytical chemistry experimentation and techniques, organic synthesis and medicinal chemistry related matters are all well known and commonly used in the related art, unless otherwise specified. Standard methods can be used for chemical synthesis, chemical analysis, preparation, formulation and administration of drugs, and treatment of patients.

Definition of

When the following concepts are used herein, the meanings follow, unless otherwise indicated, in the following text:

"Ang-2" refers to the polypeptide listed in FIG. 6 of U.S. Pat. No.6,166,185 ("Tie-2 ligand-2") or fragments thereof, and also refers to related polypeptides including allelic variants, splice variants, derivatives, substitution/deletion and/or insertion variants, fusion peptides and polypeptides, and intermediate homologues. Depending on the method of preparation, the Ang-2 polypeptide may or may not include additional terminal residues, such as leader, targeting, amino terminal methionine and lysine residues, and/or tag or fusion protein sequences.

When "biologically active" is used to refer to an Ang-2 or Ang-2 specific binding agent, it is meant a peptide or polypeptide having at least one activity characteristic of an Ang-2 or Ang-2 specific binding agent. When referring to at least one biological activity of Ang-2, Ang-2 specific binding agents may have agonist, antagonist, neutralizing or repressing activity.

By "specific binding agent" is meant a molecule, preferably a protein molecule, which binds Ang-2 (and variants and derivatives thereof as defined herein) with a much higher affinity than other angiopoietins. Specific binding agents can be proteins, peptides, nucleic acids, sugars, lipids, or small molecule compounds that preferentially bind to Ang-2. In a preferred embodiment, the specific binding agents of the invention are antibodies, such as monoclonal antibodies, polyclonal antibodies (mabs), chimeric antibodies, CDR-grafted antibodies, multispecific antibodies, bispecific antibodies, catalytic antibodies, humanized antibodies, human antibodies, anti-idiotypic antibodies (anti-ids), and antibodies that can be labeled in soluble or immobilized form, as well as fragments, variants, or derivatives of the above, alone or in combination with other amino acid sequences, as is known in the art. These techniques include, but are not limited to, enzymatic cleavage, chemical cleavage, peptide synthesis, or recombinant techniques. The anti-Ang-2 specific binding agents of the invention are capable of binding to a moiety on Ang-2 that can modulate (e.g., inhibit or promote) Ang-2 biological activity and/or other Ang-2 related activities.

"polyclonal antibody" refers to a mixture of heterologous antibodies that recognize and bind to different epitopes on the same antigen. The polyclonal antibodies can be obtained from natural serum preparations or purified using methods such as antigen affinity chromatography or protein A/G affinity chromatography.

"monoclonal antibody" refers to a collection of antibodies encoded by the same nucleic acid molecule, optionally produced by a single hybridoma or other cell line or transgenic animal, such that each monoclonal antibody will typically recognize the same epitope on an antigen. Here, "mAbs" can be obtained from a variety of different methods and from a variety of different animals, such as mice, rats, etc.

"chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence of an antibody of one animal origin or of one species (or subclass) of antibody, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody of another animal origin or of another species (or subclass) of antibody. Also included are fragments of such antibodies which possess the desired biological activity (i.e., the ability to specifically bind Ang-2). See U.S. Pat. No.4,816,567 and Morrison et al, Proc Natl Acad Sci (USA), 81: 6851-6855(1985).

"CDR-inhibiting antibody" refers to an antibody of one species or isotype whose CDRs are recombinantly inserted into the framework regions of another antibody of the same or different species (or isotype).

By "multispecific antibody" is meant an antibody whose variable region can recognize multiple epitopes on one or more antigens. One subspecies of such antibodies is a "bispecific antibody," which recognizes two different epitopes on the same antigen or on different antigens.

"catalytic antibody" refers to an antibody that is toxic or catalytically active, wherein one or more cytotoxic groups or, more commonly, one or more biologically active groups are attached to a target binding agent.

"humanized antibodies" refer to a particular class of CDR-grafted antibodies in which the framework regions are of human origin, but each CDR is replaced by a CDR of another origin, such as a murine CDR. "CDR" is defined below.

By "fully human" antibody is meant an antibody in which the CDRs and framework regions are derived from one or more human DNA molecules.

"anti-idiotype antibody" refers to any antibody capable of binding to other antibodies that recognize an antigen. Any of the Ang-2 specific binding antibody production methods described herein can be used to produce anti-idiotypic antibodies, except as follows: these antibodies are obtained by immunizing an animal with an Ang-2 specific antibody or Ang-2 binding fragment thereof, rather than using the Ang-2 polypeptide itself or a fragment thereof.

As used herein, "variant" includes binding agent polypeptides in which residue insertions, deletions, and/or substitutions occur in the naturally occurring (or at least in known) amino acid sequence. Variants of the invention include the fusion proteins described below.

"derivatives" include those binding agents that have been chemically modified in a manner different from that of the inserted, deleted or substituted variant.

"specifically binds to Ang-2" refers to the ability of a specific binding agent of the invention (e.g., an antibody or fragment thereof) to recognize and bind to a mature full-length (or partial-length) human Ang-2 polypeptide (or ortholog thereof), with an Affinity (as determined by the Affinity ELISA or bibiacore assay as described herein) or wherein the neutralizing capacity (as determined by the neutralization ELISA or similar methods described herein) is at least 10-fold, or optionally 50-fold, 100-fold, 250-fold, 500-fold, or at least 1000-fold, of the corresponding effect on other angiogenic proteins or other peptides (polypeptides).

An "antigen binding domain" or "antigen binding region" refers to a portion of a specific binding agent: it contains amino acid residues (or other groups) that interact with the antigen and confer specificity and affinity to the antigen on the binding agent. In antibodies, antigen binding agent domains are often referred to as "complementarity determining regions" or "CDRs".

An "epitope" refers to a portion of any molecule that is capable of being recognized and bound by a specific binding agent (e.g., an antibody) in one or more binding agent antigen binding regions. Epitopes are usually composed of chemically active surface groups of molecules, such as amino acids or glycosyl side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. The epitopes referred to herein may be continuous or discontinuous. Furthermore, since it has the same three-dimensional structure as the epitope used to generate the antibody, it is possible to mimic the epitope which does not contain (or contains only some) of the amino acid residues of Ang-2 used to elicit an immune response from the antibody.

"inhibitory and/or neutralizing epitopes" are characterized by: when bound by a specific binding agent (e.g., an antibody) in vivo, in vitro, or in situ, results in the loss (or at least reduction) of biological activity of the molecule, cell, or organism comprising the epitope. Herein, the neutralizing epitope is located within, or associated with, the biologically active region of Ang-2. Alternatively, an "activating epitope" refers to an epitope that: when bound by a specific binding agent of the invention (e.g., an antibody), activate Ang-2, or at least maintain Ang-2 in its biologically active conformation.

An "antibody fragment" refers to a peptide or polypeptide comprising the entire antibody partial sequence. An intact antibody comprises two functionally independent parts or fragments: the antigen binding fragment "Fab" and the C-terminal crystallizable fragment- -the "Fc" fragment. Fab fragments include the first constant regions of the heavy and light chains (CH1 and CL1), as well as the variable regions of the heavy and light chains (binding to a specific antigen). The variable regions of both heavy and light chains comprise threeComplementarity Determining Regions (CDRs) and framework region amino acid residues that separate individual CDRs. The Fc region includes the second and third constant regions of the heavy chain (CH2 and CH3), which are involved in effector functions such as complementation activation and phagocytic attack. Some antibodies have the Fc and Fab regions separated by an antibody "hinge region" which can be attached to the Fab or Fc fragment depending on how the full length antibody is cleaved enzymatically. For example, papain cleavage of an antibody results in the hinge region being attached to the resulting Fc fragment, whereas pepsin cleavage results in the hinge in the resulting fragment being attached to both Fab's simultaneously. Since the two Fab fragments are in fact covalently linked together after pepsin cleavage, the resulting fragment is designated F (ab') ₂And (3) fragment.

The Fc domain has a relatively long serum half-life, while the Fab has a short lifetime. (Capon et al, Nature, 337: 525-31 (1989)). When expressed as part of a fusion protein, the Fc domain may confer a longer lifespan to the fused protein or may confer functions such as Fc receptor binding, protein a binding, complement fixation, and possibly even placental transfer (placental transfer). The Fc region may be a native Fc region or may be Fc modified or improved for certain properties, such as therapeutic properties or circulation time.

"variable region" or "variable domain" refers to a portion of an antibody light chain and/or heavy chain, typically comprising about 120-130 amino acid residues at the amino terminus of the heavy chain, and about 100-110 amino acid residues at the amino terminus of the light chain. Even the variable regions of antibodies of the same class vary greatly in amino acid sequence. The variable regions of an antibody determine the binding characteristics and specificity of each particular antibody for its particular antigen. The variability of the sequence is concentrated within the Complementarity Determining Regions (CDRs), while the highly conserved regions in the variable domains are called Framework Regions (FRs). The CDR regions of the light and heavy chains contain amino acids that are essential for the direct interaction of the antibody with the antigen, although amino acids in the FR regions can also have a significant effect on antigen binding/recognition, as described below.

The "light chain" of an antibody is a generic term for two different types of light chains, called kappa (k) or lambda (l), respectively, depending on the constant region amino acid sequence.

The "heavy chain" of an antibody is a collective term for 5 different types of heavy chains, called α, δ, ε, γ, and μ, respectively, depending on the heavy chain constant region amino acid sequence. The combination of heavy and light chains is known to form 5 antibodies: IgA, IgD, IgE, IgG and IgM, IgG comprising 4 subspecies, called IgG₁、IgG₂、IgG₃And IgG₄。

When referring to biological materials (e.g., nucleic acid molecules, polypeptides, host cells, etc.) by "naturally occurring," it is meant that the materials are in their native form and have not been artificially modified.

When "isolated" is used to modify Ang-2 or a specific binding agent therefor, it is meant that a substance is at least free of one contaminating polypeptide or contaminating compound found in its natural environment, and preferably substantially free of any other contaminating mammalian polypeptide that may interfere with the therapeutic or diagnostic use of the substance.

When "mature" is used to describe Ang-2, anti-Ang-2 antibodies or any other Ang-2 specific binding protein molecule, it means that the peptide or polypeptide has no leader or signal sequence. When a binding agent of the invention is expressed (e.g., in a prokaryotic host cell), the "mature" peptide or polypeptide may also comprise additional amino acid residues (but still not the leader sequence), such as an amino-terminal methionine, or one or more methionine and lysine residues. The peptides or polypeptides prepared in this manner may or may not contain these amino acid residues removed at the time of use.

When an "effective dose" or "therapeutically effective amount" of a specific binding agent for Ang-2 is used, it is meant that the amount of specific binding agent is effective or sufficient to support a measurable change in the level of one or more biological activities of Ang-2. Such alteration in the level of biological activity of Ang-2 may be either enhancement or attenuation. Preferably, the alteration is a decrease in Ang-2 activity.

Specific binding agents and antibodies

As used herein, "specific binding agent" refers to a molecule that specifically recognizes and binds said Ang-2. Suitable specific binding agents include, but are not limited to, antibodies and derivatives thereof, polypeptides, and small molecules. Suitable specific binding agents can be prepared using methods known in the art. The polypeptide Ang-2 specific binding agents of the invention are capable of binding to a specific portion of an Ang-2 polypeptide and preferably modulate the activity or function of the Ang-2 polypeptide.

Specific binding agents, such as antibodies and antibody fragments, that can specifically bind to an Ang-2 polypeptide are included within the scope of the invention. The antibody may be a polyclonal antibody including a monospecific polyclonal antibody, a monoclonal antibody (mAb), a recombinant, a chimeric antibody, a humanized antibody such as a CDR-grafted antibody, a human antibody, a single chain antibody, a catalytic antibody, a multispecific antibody and/or a bispecific antibody and fragments thereof, including variants and/or derivatives thereof.

Multiple antibodies against Ang-2 are typically prepared by multiple subcutaneous or intraperitoneal injections of Ang-2 polypeptide or fragment thereof (with or without an adjuvant) into an animal (e.g., rabbit, hamster, goat, sheep, horse, pig, rat, gerbil, guinea pig, mouse, or any other suitable mammal, and other non-mammals). Such adjuvants include, but are not limited to, Freund's complete/incomplete adjuvant, mineral gums such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions (oil emulsions), keyhole limpetHemocyanin and dinitrophenol. BCG (bacille Calmette-Guerin) and anaerobic Corynebacterium are particularly useful as human adjuvants. Coupling a carrier protein, such as a keyhole limpet, to an antigen, which carrier protein is immunogenic for the animal to be immunized, may be an effective methodHemocyanin, serum albumin, bovine thyroid protein, and soybean insulin inhibitor. Flocculants (a) may also be usedggregating agents) such as alum to enhance the immune response. Blood from the animals is taken after immunization and the sera is tested for anti-Ang-2 polypeptide antibody titers using the methods described in the "examples" section. The polyclonal antibody can be used in combination with serum in which the polyclonal antibody is detected, or can be purified from the serum using methods such as antigen affinity chromatography or protein a or G affinity chromatography.

Monoclonal antibodies against Ang-2 polypeptides can be prepared using, for example, but not limited to, conventional hybridoma methods or newer phage display technologies. For example, reference may be made to Kohler et al, Nature 256: 495 (1975); kosbor et al, immunological Today 4: 72(1983) (human B cell hybridoma technology); cote et al, Proc Natl Acad Sci (USA) 80: 2026-; brodeur et al, Monoclonal Antibody Production technologies and applications, pp.51-63, Marcel Dekker, Inc., New York, (1987)), and Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96, (1985) (EBV hybridoma technology). The invention also provides hybridoma cell lines that produce monoclonal antibodies having Ang-2 polypeptide reactivity.

Myeloma cell lines may be used in hybridoma technology. This cell line produces no antibody, has high fusion efficiency, and lacks some enzymes, making it unable to grow in certain selective media (only the desired fused cells (hybridomas) can grow) and is therefore suitable for use in hybridoma fusion methods. For example, cell lines used in mouse fusion procedures are Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/u, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; the cell lines used in the rat fusion procedure were R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B 210. Other cell lines used in cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC 729-6. Hybridomas and other monoclonal antibody-producing cell lines are contemplated as novel compositions of the present invention.

Any of a variety of monoclonal antibodies can also be prepared using phage display technology. Preferably, the fully human monoclonal antibody is prepared using this technique, wherein a polynucleotide encoding a single Fab or Fv antibody fragment is expressed on the surface of a phage particle. (Hoogenboom et al, J Mol Biol 227: 381 (1991); Marks et al, J Mol Biol 222: 581 (1991); see also U.S. Pat. No.5,885,793)). Each phage can be "screened" using the binding assays described herein to identify antibody fragments with an Ang-2 affinity effect. Thus, these methods mimic immunoselection by displaying a pool of antibody fragments on the filamentous (flamentous) surface of a bacteriophage, and then selecting the bacteriophage for binding to Ang-2. One such method is described in PCT application No. PCT/US98/17364 (Applicant Adams et al) which provides for the purification of antibody fragments with high affinity and antagonistic function for the MPL and msk receptors. In this method, a complete library of human antibody genes can be prepared by cloning naturally rearranged human V genes from peripheral blood lymphocytes as described above (Mullinax et al, Proc Natl Acad Sci (USA) 87: 8095-.

Once a polynucleotide sequence encoding each chain of the full-length mAb of the invention (or encoding the Fab or Fv fragment of the mAb) has been identified, the mAb polynucleotide can be expressed using either eukaryotic or prokaryotic host cells, according to recombinant techniques well known and routinely practiced in the art. Alternatively, a polynucleotide encoding a specific binding agent may be introduced into the genome of a recipient animal in a manner that allows expression of the polynucleotide encoding the monoclonal antibody or other specific binding agent to construct a transgenic animal, e.g., a mouse, rat, goat, or cow. In one aspect, a polynucleotide encoding a monoclonal antibody or other specific binding agent can be linked to the mammary gland-specific regulatory sequence, and the chimeric polynucleotide can be administered to the germline of the target animal. The resulting transgenic animals produce the desired antibody in their milk (Pollock et al, J Immunol Meth 231: 147-157 (1999); Little et al, Immunol Today 8: 364-370 (2000)). Alternatively, plants can be expressed and generate Ang-2 specific binding agents (e.g., polyclonal antibodies) by transfecting the plants with an appropriate polynucleotide encoding a monoclonal antibody or other specific binding agent.

In another embodiment of the invention, a monoclonal or polyclonal antibody (or fragment thereof) of non-human origin may be "humanized" or "chimeric". Procedures for humanizing antibodies of non-human origin are well known in the art. (see U.S. Pat. Nos. 5,859,205, 5,585,089, and 5,693,762). Replacement of at least part of the complementarity determining region (e.g., rodents) with the corresponding region of a human antibody can be performed using methods such as those described in the art (Jones et al, Nature 321: 522-525 (1986); Riechmann et al, Nature, 332: 323-327 (1988); Verhoeyen et al, Science 239: 1534-1536 (1988)). Variants and derivatives of these human antibodies are also provided herein, as discussed herein (and well known in the art).

The invention also relates to fully human antibodies, and fragments, variants and/or derivatives thereof, that bind to Ang-2 polypeptides. These antibodies can be prepared using phage display techniques as described above. Alternatively, transgenic animals (e.g., mice) can be used to produce these antibodies, provided that such transgenic animals are capable of generating a human antibody repertoire in the absence of endogenous immunoglobulin production. This can be achieved by immunizing an animal with an Ang-2 antigen or fragment thereof, when the Ang-2 fragment contains the unique amino acid sequence of Ang-2. These immunogens can optionally be coupled to a carrier. See, e.g., Jakobovits et al, Proc Natl Acad Sci (USA), 90: 2551 2555 (1993); jakobovits et al, Nature 362: 255-258 (1993); bruggermann et al, Year in immuno, 7: 33(1993). In one method of making a transgenic animal, endogenous immunogen heavy and light chain encoding loci are inactivated in the animal, and then the encoding loci for human heavy and light chain proteins are inserted into the genome of the animal. Partially modified animals are those that have not been fully modified, and are crossed to obtain an animal that contains all of the desired immune system modifications. When administered with an immunogen, these transgenic animals are capable of producing antibodies containing human variable regions that comprise human (rather than murine) amino acid sequences with immunological specificity for the desired antigen. See PCT applications PCT/US96/05928 and PCT/US 93/06926. Other methods are described in U.S. Pat. No. 5,545,807, PCT application No. PCT/US91/245, PCT/GB89/01207, EP 546073B1 and EP 546073A 1. Human antibodies can also be produced by expressing recombinant DNA in a host cell or hybridoma, as described herein.

Transgenic manipulation can be accomplished by a variety of different methods. See, e.g., Bruggeman et al, Immunol Today 17: 391-7(1996). In one approach, the miniloci are constructed to artificially bring gene segments in germline configuration into close proximity with each other. Due to the limited length (i.e., typically less than 30kb), the resulting miniloci contain a limited number of different gene fragments, but are still capable of generating a large antibody repertoire. The minilocus contains only human DNA sequences (including promoters and enhancers) and is fully functional in transgenic animals.

When it is desired to contain a large number of gene fragments in the transgenic animal body, Yeast Artificial Chromosomes (YACs) can be used. YACs vary in length from a few hundred kb to 1Mb and are introduced into mice (or other suitable animals) by direct microinjection into eggs, or YACs are transferred into embryonic stem cell lines. YACs are usually administered to ES cells using lipofection with purified DNA, or by yeast protoplast fusion, where the purified DNA is carried by mouse cells, in a manner similar to hybridoma fusion. Selection of desired ES cells following DNA transfer can be performed by any selectable marker known in the art on YACs.

Alternatively, the P1 phage vector was amplified in an E.coli host. Since these vectors typically carry less DNA than that inserted in YACs, sufficient quantities of clones are more readily available for direct microinjection into mouse ova. It has been shown that the use of multiple P1 vectors in combination will result in higher levels of homologous recombination.

Serum levels of circulating antibodies are detected using any method known in the art, such as ELISA, and once a suitable transgenic mouse (or other suitable animal) is identified, the transgenic animal is crossed with a mouse in which the endogenous Ig locus has been disrupted. Substantially all of the B cells of the resulting progeny express human antibodies.

Alternatively, the entire locus of an animal is replaced with a human Ig locus, and the resulting animal expresses only human antibodies. In another method, a portion of the loci of the animal are replaced with corresponding specific human loci. In some cases, the resulting animals of this approach may express chimeric antibodies (as opposed to fully human antibodies) depending on the replacement characteristics of the mouse Ig locus.

Human antibodies can also be prepared by exposing human splenocytes (B or T cells) to antigen in vitro and then reconstituting the exposed cells in immunocompromised mice (e.g., SCID or nod/SCID). See Brams et al, J Immunol, 160: 2051-2058 (1998); carballido et al, NatMed, 6: 103-106(2000). In one approach, administration of human embryonic cells to SCID mice (SCID-hu) results in long-lasting hematopoiesis and human T cell development (McCune et al, Science 241: 1532-. Any humoral immune response in these chimeric animals is entirely dependent on the coordinated development of T cells (Martensson et al, Immunol 83: 1271-179 (1994)). In an alternative approach, human peripheral blood lymphocytes are administered intraperitoneally (membrane) or otherwise into SCID mice (Mosier et al, Nature 335: 256-259 (1988)). Higher levels of B cells were detected when the introduced cells were treated with a neutrophil, such as Staphylococcal Enterotoxin A (SEA) (Martensson et al, Immunol 84: 224-230(1995)) or anti-human CD40 mAb (Murphy et al, Blood 86: 1946-1953 (1995)).

Alternatively, a fully synthetic human heavy chain repertoire was prepared from unrearranged V genes by combining each human VH segment with a D segment of random nucleotides and a human J segment (Hoogenboom et al, JMol Biol 227: 381-388 (1992)). Light chain libraries were also prepared by combining each human V fragment with the J fragment (Griffiths et al, EMBO J13: 3245-. The nucleotides encoding the intact antibody (i.e., the heavy and light chains) are linked to a single chain Fv fragment, and the polynucleotide is linked to nucleotides encoding the filamentous phage microcoating protein. When this fusion protein is expressed on the phage surface, selection is performed using the immobilized antigen to identify polynucleotides encoding the same miniantibody.

In an alternative method, the antibody fragment is assembled into two Fab fragments by fusing one chain to a phage protein and the other secreted into the periplasm of the bacterium (Hoogenboom et al, Nucleacids Res 19: 4133. sup. 4137 (1991); Barbas et al, Proc Natl Acad Sci (USA) 88: 7978. sup. 7982 (1991)).

A typical method for mass production of chimeric antibodies, humanized antibodies, CDR-grafted antibodies and fully human antibodies (or fragments thereof) is recombinant technology. Polynucleotide molecules encoding each of the antibody heavy and light chains (or fragments thereof) can be introduced into a host cell and expressed using the materials and methods described herein. In a preferred embodiment, the antibody is produced in a mammalian host cell, such as a CHO cell. Details of this method will be described below.

Fusion partner of specific binding agents

In another embodiment of the invention, a polypeptide comprising the amino acid sequence of the Ang-2 antibody variable region (e.g., a heavy chain variable region having the amino acid sequence described herein or a light chain variable region having the amino acid sequence described herein) can be fused to one or more domains (N-or C-terminal) of a human IgG Fc region. The Fc domain, when co-formed with a therapeutic protein (e.g., Fab of Ang-2 specific antibodies), may provide the fusion protein with a longer lifespan or provide functions such as Fc receptor binding, protein a binding, complement binding, and possibly even placental transport to the fusion protein. (Capon et al, Nature, 337: 525-531 (1989)).

In one example, the antibody hinge, CH2, and CH3 regions can be fused to the N-terminus or C-terminus of a specific binding agent polypeptide (e.g., an anti-Ang-2 Fab or Fv fragment obtained from a phage display library) using methods known in the art. The resulting fusion protein can be purified using a protein a or protein G affinity chromatography column. Peptides and proteins fused to the Fc region have been found to have a sufficiently longer life span in vivo than the same molecule without fusion. Fusion to the Fc region also allows dimerization/multimerization of the fusion polypeptide. The Fc region may be a native Fc region, or may be modified to improve certain properties, such as therapeutic properties, circulation time, reduce aggregation problems, and the like. Other examples known in the art include the fusion of the Fc region (which may be of human or other origin, or synthetic) to the N-terminus of CD30L for the treatment of Hodgkin's disease, anaplastic lymphoma and T-cell leukemia (U.S. Pat. No.5,480,981), the fusion of the Fc region to the TNF receptor for the treatment of septic shock (Fisher et al, N EnglJ Med, 334: 1697-1702(1996)), and the fusion of the Fc region to the Cd4 receptor for the treatment of AIDS (Capon et al, Nature, 337: 525-31 (1989)).

Catalytic antibodies are another fusion molecule with one or more cytotoxic groups or (more commonly) one or more bioactive groups attached to a specific binding agent (antibody). See, e.g., (Rader et al, Chem Eur J12: 2091-2095 (2000)). Cytotoxic agents of this type may increase antibody-mediated cytotoxicity and comprise portions of such cytokines that directly or indirectly cause cell death, radioisotopes, chemotherapeutic drugs (including prodrugs), bacterial toxins (e.g., pseudomonas exotoxin, diphtheria toxin, etc.), plant toxins (e.g., ricin, poliumulin, etc.), chemical conjugates (e.g., maytansinoid, caclechaemicin, etc.), radioconjugates (conjugates), enzyme conjugates (RNase conjugates), antibody-directed enzyme-prodrug therapy (ADEPT), etc. In one approach, a cytotoxic agent can be linked to a portion of a bispecific or multispecific antibody by binding the cytotoxic agent to one of the antibody's selectable antigen recognition sites. Alternatively, a polynucleotide encoding a toxin can be linked to a polynucleotide encoding a binding agent and expressed, and the protein cytotoxin is expressed as a fusion protein with the specific binding agent. In an alternative approach, the specific binding agent may be covalently modified to incorporate the desired cytotoxin.

Examples of such fusion proteins include immunopeptides, proteins with long circulating half-lives (e.g., immunoglobulin constant regions), marker proteins, proteins or polypeptides that facilitate purification of the desired specific binder polypeptide, and polypeptides that facilitate formation of multimeric proteins (e.g., leucine zipper motifs that facilitate polymer formation and stabilization).

Typically, in such insertional variants, the entire native molecule or a substantial portion (substantaial portion) is linked at its N-terminus or C-terminus to a second polypeptide or portion thereof. For example, fusion proteins often use other kinds of leader sequences to allow expression of the recombinant in a heterologous host. Other useful fusion proteins include the addition of immunoglobulin active domains (e.g., antibody epitopes) to facilitate purification of the purified protein. The inclusion of a cleavage site at or near the fusion junction site may facilitate removal of the foreign polypeptide after purification. Other useful fusions include attachment of functional domains, such as an active site of an enzyme, a glycosylation domain, a cellular target signal, or a transmembrane region.

A variety of commercially available fusion protein expression systems can be used in the present invention. Particularly useful systems include, but are not limited to, the glutathione-S-transferase (GST) system (Pharmacia), the maltose binding protein system (NEB, Beverley, MA), the FLAG system (IBI, New Haven, CT) and the 6XHis system (Qiagen, Chatsworth, CA). These systems enable the production of recombinant polypeptides carrying only a small number of additional amino acids which are unlikely to affect the antigenic activity of the recombinant polypeptide. For example, the FLAG system and the 6xHis system add only short sequences, which are known to be scarcely antigenic and do not affect the folding of the polypeptide into its native conformation. Another efficient fusion contemplated is the fusion of the Met-Lys dipeptide at the N-terminus of the protein or peptide. Such fusions may result in a beneficial increase in protein expression or activity.

The fusion of a specific binding agent to a hapten to enhance the immunogenicity of a specific binding agent fusion construct (which facilitates, for example, the production of an anti-idiotypic antibody of the invention) may be a particularly effective fusion construct. It is known in the art that such immunogenicity enhancing fusions, for example, of specific binding agents to a co-antigen (e.g., hsp70), or to a cytokine (e.g., I1-2), will facilitate the initiation of an immune response. In another embodiment, fusion can be performed to increase the targeting activity of the antigen binding composition to a specific site or cell.

Other contemplated fusion constructs include fusion of heterologous polypeptides (e.g., Ig constant regions) with desired properties to increase serum half-life, or fusion of antibodies (or fragments thereof) to improve target localization activity. Still other fusion systems produce hybrid polypeptides that require cleavage of the fusion partner from the desired polypeptide. In one embodiment, such fusion partners are linked to a recombinant specific binding agent polypeptide, wherein the linker peptide sequence comprises a recognition sequence specific for the protease. Suitable proteases are, for example, tobacco etch virus protease (Life Technologies, Gaithersburg, Md.) or factor Xa (New England Biolabs, Beverley, Mass.).

The present invention also provides a fusion polypeptide comprising all or part of the variable region of an Ang-2 antibody, for example, a heavy (or light) chain variable region having an amino acid sequence as described herein linked to truncated tissue factor (tTF), which is a vascular target molecule consisting of a human coagulation-inducing protein (tumor vascular coagulant) from which a partial sequence has been excised. Fusion of tTF to anti-Ang-2 antibodies (or fragments thereof) may facilitate the transport of anti-Ang-2 to target cells.

Variants of specific binding agents

Variants of the specific binding agents of the invention include insertion, deletion and/or substitution variants. In one aspect of the invention, insertional variants are provided in which one or more amino acid residues are added to the specific binding agent. Insertion may occur at the end (either simultaneously or at only one end) or at an internal sequence of the specific binding agent. Insertion variants with additional residues at both or one end may comprise, for example, fusion proteins and proteins with amino acid tags or labels. Insertion variants include specific binding agent sequences (or fragments thereof) with the addition of one or more residues.

Variant products of the invention also include mature specific binding agent products. The leader or signal sequence of this specific binding agent product is removed, but the resulting protein still has additional terminal residues compared to the wild-type Ang-2 polypeptide. Such additional terminal residues may be from other proteins or may contain one or more residues that cannot be identified as being specific for which protein. Specific binding agent products having an additional methionine residue at position-1 (Met) are also contemplated ^-1-specific binding agent), and a specific binding agent product (Met) with additional methionine and lysine residues at positions-2 and-1^-2-Lys^-1-a specific binding agent). Variants of specific binding agents having additional Met, Met-Lys, Lys (or, generally, one or more basic residues) residues are particularly advantageous for enhancing recombinant protein production in bacterial host cells.

The invention also relates to variants of specific lower binding agents with additional amino acid residues from specific expression systems. For example, using a commercial vector system in which the desired polypeptide is expressed as part of a glutathione-S transferase (GST) fusion product, the resulting product will have an additional glycine residue at amino acid position-1 after removal of the GST component from the desired protein. Variants from other vector system expression are also contemplated, including sequences comprising a poly-his tag in the amino acid sequence, typically at the carboxy terminus or amino terminus of the sequence.

Insertional variants also include fusion proteins as described above in which the amino and/or carboxy terminus of a specific binding agent polypeptide is fused to another polypeptide (or fragment thereof), or to an amino acid sequence not normally recognized as part of any particular protein sequence.

In another aspect, the invention provides deletion variants in which one or more amino acid residues of a specific binding agent polypeptide are deleted. Deletions may occur at one or both ends of a specific binding agent, or within an amino acid sequence. Deletion variants must contain all fragments of a specific binding agent polypeptide.

Antibody fragments include those moieties that bind to an epitope on an antigenic polypeptide. Such fragments include, for example, Fab and F (ab')₂And (3) fragment. Other binding fragments include the products of recombinant DNA technology, such as the expression product of recombinant plasmids containing the antibody variable region encoding sequences. The invention also relates to polypeptide fragments of Ang-2 binding agents that retain the ability to specifically bind Ang-2 polypeptides. This is understood to be a fragment comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more conserved amino acid residues of a peptide or polypeptide of the invention. Preferred polypeptide fragments have specific or specific immunological properties for the antigen binding agents of the invention. Any method known and practiced in the art can be used to prepare the polypeptides of the invention having the immunological properties of interest.

In another aspect, the invention also provides alternative variants of the specific binding agents. Substitution variants are generally considered to be similar or have some sequence identity to the original polypeptide, with one or more residues of the substitution variant removed and replaced with alternative residues. Conservative substitutions are contemplated by the present invention, as well as non-conservative substitutions.

The identity and similarity of related polypeptides can be readily calculated by known methods. These methods include, but are not limited to, those described in the following documents: computationmolecular Biology, Lesk, a.m., ed., Oxford university Press, New York (1988); biocomputing: information and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); computer Analysis of sequence data, Part1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, NewJersey (1994); sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); sequence Analysis Primer, Gribskov, m.anddevereux, j., eds., m.stockton Press, New York (1991); and Carillo et al, sia j. applied math, 48: 1073(1988).

Preferred methods for determining the percent of relatedness or identity between two polypeptides are designed to give the greatest degree of match between the sequences to be sequenced. Methods of determining identity are described in published computer software. Preferred Computer software for determining identity between two sequences include, but are not limited to, The GCG software package, including GAP (Devereux et al, Nucl. acid, Res, 12: 387 (1984)), Genetics Computer Group, univariate of Wisconsin, Madison, WI, BLASTP, BLASTN, and FASTA (Altschul et al, J.mol.biol., 215: 403-.

A certain alignment between two amino acid sequences may result in only a short region of match between the two sequences, which may have a high degree of sequence identity even if there is no significant correlation between the two full-length sequences. Thus, in some embodiments, the selected alignment method (GAP program) will obtain an alignment result that spans at least 10% of the full length of the target polypeptide to be tested, i.e., at least 40 consecutive amino acids are aligned when the polypeptide to be tested is at least 400 amino acids long, at least 30 consecutive amino acids are aligned when the polypeptide to be tested is at least 300-400 amino acids long, at least 20 consecutive amino acids are aligned when the polypeptide to be tested is at least 200-300 amino acids long, and at least 10 consecutive amino acids are aligned when the polypeptide to be tested is at least 100-200 amino acids long.

For example, using the GAP algorithm (Genetics Computer Group, unity of Wisconsin, Madison, Wis.), two polypeptides of which the sequence to be determined is a percentage of the same are aligned to obtain the best match (range of match, as determined by the algorithm) of the two respective amino acids. In certain embodiments, a gap open deduct score (which is typically calculated as a 3X average diagonal; the "average diagonal" is the average diagonal of the alignment matrix used; the "diagonal" is the score or value assigned to each optimal amino acid match by a particular alignment matrix), a gap extended deduct score (typically 1/10 for a gap open deduct score), and an alignment matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. Standard contrast matrices are also used in some embodiments by algorithms (Dayhoff et al Atlas of Protein sequences and structures, 5(3) (1978), PAM 250 contrast matrices; Henikoff et al Proc. Natl. Acad. Sci USA 89: 10915-.

In certain embodiments, the parameters used for polypeptide sequence alignment include:

the algorithm is as follows: needleman et al, j.mol.biol., 48: 443-453 (1970);

comparing the matrixes: BLOSUM 62from Henikoff et al, supra (1992);

Gap buckle value: 12

Gap length score value: 4

Similarity threshold: 0

The above parameters may be valid in the GAP program. In certain embodiments, the aforementioned parameters for polypeptide comparison (end GAP deduct value) using the GAP algorithm are default parameters.

In certain embodiments, the parameters used for polypeptide molecule sequence alignment include:

the algorithm is as follows: needleman et al, supra (1970)

Comparing the matrixes: match +10 and mismatch-0

Gap buckle value: 50

Gap length score value: 3

The above parameters may also be valid in the GAP program. In certain embodiments, the aforementioned parameters for polypeptide comparison are default parameters.

Other exemplary algorithms, gap opening score, gap extension score, contrast matrices, similarity thresholds, etc., may also be used, including those listed in Program Manual, Wisconsin Package, Version 9, September, 1997. It will be apparent to those skilled in the art that the choice will be made in detail depending on the particular comparison to be made (e.g., DNA-DNA, protein-protein, protein-DNA; and additionally depending on whether a comparison is made between a given pair of molecules between sequences (in which case GAP or BedtFit is generally preferred) or between sequences and a large sequence database (in which case FASTA or BLASTA is preferred).

Abbreviations for 20 conventional amino acids are used herein according to conventional usage. See Immunology- -A Synthesis (2nd Edition, E.S. Golub and D.R.Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)) incorporated herein by reference.

Amino acids may have either the L configuration or the D configuration (with the exception of glycine, which has no distinction between the L and D configurations), and the polypeptides and compositions of the invention may comprise different stereoisomers. The L configuration is preferred. The invention also provides inverted molecules in which the amino acid sequence of the carboxyl to amino group is inverted. E.g. normal sequence X₁-X₂-X₃The reverse molecule of (a) is X₃-X₂-X₁. The invention also provides a transformed (retro) -inverted molecule, as described above, in which the amino acid sequence of an amino acid sequence from carboxy to amino is inverted and the residues normally of the L enantiomer are changed to the D configuration.

Stereoisomers of 20 conventional amino acids (e.g.D-amino acids), unnatural amino acids such as alpha-, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids may also be suitable as polypeptide components of the invention. Non-conventional amino acids include, for example, but are not limited to, aminoadipic acid, beta-alanine, beta-aminopropionic acid, aminobutyric acid, pipecolic acid, aminocaproic acid, aminoheptanoic acid, aminoisobutyric acid, aminopimelic acid, diaminobutyric acid, desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine, N-ethylaspartic acid, hydroxylysine, allo-hydroxylysine, hydroxyproline, isoclavine, allo-isoleucine, N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, ornithine, 4-hydroxyproline, gamma-carboxyglutamic acid, epsilon-N, N, N-trimethyllysine, epsilon-N-acetyl lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methylarginine and other similar amino acids (e.g., 4-hydroxyproline).

Similarly, the left-hand end of a single-stranded polynucleotide sequence is the 5' end, if not otherwise specified; the left-hand direction of a double-stranded polynucleotide sequence refers to the 5' direction. The direction of 5 'to 3' addition of the nascent RNA transcript is the direction of transcription; a sequence region on a DNA strand having the same sequence as RNA and located in the 5 'direction of the 5' end of an RNA transcript is an "upstream sequence"; the sequence region on the DNA strand having the same sequence as the RNA and located in the 3 'direction of the 3' end of the RNA transcript is the "downstream sequence".

Conservative amino acid substitutions may include unnatural amino acid residues, which typically occur in chemical rather than biological systems of peptide synthesis. This includes peptide mimetics and other inverted or transformed forms of amino acid moieties.

Natural amino acids can be classified into the following classes according to side chain commonality:

1) hydrophobic amino acids: met, Ala, Val, Leu, Ile

2) Neutral hydrophilic amino acids: cys, Ser, Thr, Asn, Gln

3) Acidic amino acids: asp, Glu

4) Basic amino acids: his, Lys, Arg

5) Chain orientation affecting residues: gly, Pro and

6) aromatic amino acids: trp, Tyr, Phe

For example, a non-conservative substitution may be a substitution of an amino acid of one species with an amino acid of another species. Such replacement residues may be introduced into a region of the human antibody that is homologous to the non-human antibody, or into a region of the molecule that is non-homologous.

In certain embodiments, the hydropathic value of an amino acid can be considered in making such a change. Each amino acid is assigned a hydropathic value according to its hydrophobic and charge characteristics: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tyrosine (-0.9); tryptophan (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); arginine (-4.5).

The importance of amino acid hydropathic values in conferring interactive biological function on proteins is known in the art. Kyte et al, j.mol.biol., 157: 105-131(1982). It is known that certain amino acids may be substituted for other amino acids having similar hydropathic values and still retain similar biological functions. In certain embodiments such changes comprise substitutions of amino acids having a difference in hydropathic index within ± 2. In certain embodiments the alteration comprises a substitution of an amino acid having a difference in hydropathic index within ± 1, and in certain embodiments the alteration comprises a substitution of an amino acid having a difference in hydropathic index within ± 0.5.

It is also understood in the art that substitution of like amino acids can be made effectively based on hydrophobicity, particularly when the biological function of the protein or peptide is to be prepared for immunological use, as in the present invention. In certain embodiments, the maximum local average hydrophobicity of a protein (determined by the hydrophobicity of its adjacent amino acids) is correlated with its immunogenicity and antigenicity, i.e., correlated with the biological properties of the protein.

The hydropathic values of the amino acid residues are assigned as follows: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In certain embodiments, changes based on similar hydropathic values include substitutions of amino acids having hydropathic value differences within + -2, in certain embodiments within + -1, and in certain embodiments within + -0.5. Epitopes can be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions".

Exemplary amino acid substitutions are listed in table 1.

TABLE 1

Amino acid substitutions

Original residues	Alternative examples	Preferred alternatives
			Ala	Val，Leu，Ile	Val
Arg	Lys，Gln，Asn	Lys
			Asn	Gln，Glu，Asp	Gln

Asp	Glu，Gln，Asn	Glu
			Cys	Ser，Ala	Ser
Gln	Asn，Glu，Asp	Asn
			Glu	Asp，Asn，Gln	Asp
Gly	Pro，Ala	Ala
			His	Asn，Gln，Lys，Arg	Arg
Ile	Leu, Val, Met, Ala, Phe, norleucine	Leu
			Leu	Norleucine, Ile, Val, Met, Ala, Phe	Ile
Lys	Arg, 1, 4 diamino-butyric acid, Gln, Asn	Arg
			Met	Leu，Phe，Ile	Leu
Phe	Leu，Val，Ile，Ala，Tyr	Leu
			Pro	Ala	Gly
Ser	Thr，Ala，Cys	Thr
			Thr	Ser	Ser
Trp	Tyr，Phe	Tyr
			Tyr	Trp，Phe，Thr，Ser	Phe
Val	Ile, Met, Leu, Phe, Ala, norleucine	Leu

One skilled in the art can determine suitable variants from the listed polypeptides using well known methods. In certain embodiments, the skilled artisan can identify suitable regions of the molecule that can be replaced without disrupting activity by targeting portions of these regions that are not believed to be important for activity. In certain embodiments, even conservative amino acid substitutions may be made in regions that may be important to biological activity or structure without disrupting biological activity or adversely affecting polypeptide structure.

In addition, one skilled in the art can review structure-function studies in which residues important to activity or structure in similar polypeptides are identified. In making such comparisons, the importance of the amino acid residues in a protein corresponding to those residues in a similar protein that play an important role in activity or structure can be predicted.

One skilled in the art can analyze the three-dimensional structure and amino acid sequence of similar polypeptides. Given this information, one skilled in the art can predict the alignment between the three-dimensional structure of an antibody and its amino acid residues. In certain embodiments, because these residues may be involved in important interactions between the protein and other molecules, one skilled in the art may choose not to make drastic changes in the amino acid residues predicted to be located on the surface of the protein. Furthermore, one skilled in the art can prepare test variants in which substitutions are made at each desired amino acid residue. These variants can then be screened using activity detection methods known in the art. For example, if a particular amino acid residue is found to cause loss of activity, unintended reduction in activity, or inappropriate activity, then variants with such changes are not selected. In other words, from the information gathered from these routine experiments, one skilled in the art can readily determine amino acids that cannot be substituted (whether by themselves or in combination with other mutations).

There is a large body of scientific literature relating to secondary structure prediction. See Moult j, curr op in biotech, 7 (4): 422-: 222-245 (1974); chou et al, Biochemistry, 113 (2): 211-222 (1974); chou et al, adv.enzymol.relat.areas mol.biol., 47: 45-148 (1978); chou et al, ann.rev.biochem., 47: 251, 276and Chou et al, Biophys.J., 26: 367-384(1979). In addition, commercially available computer programs are available that can assist in predicting secondary structure. One method of predicting secondary structure is based on homology modeling (homology modeling). Two polypeptides or proteins have greater than 30% sequence identity or greater than 40% similarity, and the two molecules are generally similar in topology. Recent developments in protein structure databases (PDB) have increased the predictability of secondary structure, including the number of potential folds within a polypeptide or protein structure. See Holm et al, nuclear, Res, 27 (1): 244-247(1999). There is literature (Brenner et al, curr. Op. struct. biol., 7 (3): 369-376(1997)) that the number of folds within a given polypeptide or protein is limited and that the accuracy of structure prediction is greatly improved once the cut-off for the structure is resolved.

Other secondary structure prediction methods include: the "reading" method (Jones, D., curr. Opin. Structure. biol., 7 (3): 377-87 (1997); Sippl et al, Structure, 4 (1): 15-19 (1996)); "Profile analysis" ("profile analysis") (Bowie et al, Science, 253: 164-; "evolutionary linkage" ("evolutionary linkage") (Holm, supra (1999), and Brenner, supra (1997)).

In certain embodiments, antibody variants include glycosylation variants, in which the number and/or type of glycosylation sites is altered compared to the original polypeptide amino acid sequence. In certain embodiments, a protein variant comprises an increased or decreased number of N-linked glycosylation sites as compared to the native protein. The characteristic sequence of the N-linked glycosylation site is: Asn-X-Ser or Asn-X-Thr, wherein X can be any amino acid except proline. The substitution of amino acids to construct this sequence provides a new potential site for the addition of N-linked glycosyl side chains. Alternatively, substitution to remove this sequence will remove the existing N-linked glycosyl side chain. Rearrangement of the N-linked glycosyl side chains is also provided, wherein one or more N-linked glycosylation sites (typically the native sites) are removed or created. Other preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted or replaced with another amino acid (e.g., serine) compared to the original amino acid sequence. Cysteine variants may be effective when the antibody is required to refold into a biologically active conformation (e.g., after separation from soluble endosomes). Cysteine variants generally contain fewer cysteine residues than the native protein, and typically contain an even number of cysteines to minimize interactions caused by unpaired cysteines.

In certain embodiments, the amino acid residue substitutions are for the purpose of: (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for protein complex formation, (4) altered binding affinity, and/or (5) additional or modified functional properties imparted to these polypeptides. In certain embodiments, single or multiple amino acid substitutions (in certain embodiments conservative substitutions) may be made in the native sequence (in certain embodiments, on portions of the sequence outside of the contacts that form the molecule). In certain embodiments, conservative substitutions may typically not result in substantial changes in the structural characteristics of the original sequence (e.g., amino acid substitutions that do not tend to disrupt the helices in the original sequence (or disrupt other characteristic secondary structures of the original sequence)). Methods for identifying secondary and tertiary Structures of polypeptides are described, for example, in protein, structure and Molecular rules "Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); "Protein structural information" Introduction to Protein Structure (C.Branden and J.Tooze, eds., Garland publishing, New York, N.Y. (1991)); and Thornton et at nature 354: 105(1991).

The specific binding agent is a polypeptide or peptide substitution variant, and can contain amino acid substitution proportion as high as 12%. For antibody variants, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid may be substituted in the heavy chain, and 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid may be substituted in the light chain.

Derivatives of specific binding agents

The invention also provides derivatives of specific binding agents. Derivatives include specific binding agents that are modified (except for insertion, deletion, substitution of amino acid residues). These modifications are preferably covalent in nature, including, for example, chemical attachment to polymers, lipids, other organic and inorganic groups. The derivatives of the invention may be prepared for the purpose of increasing the circulating half-life of the specific binding agent polypeptide or may be designed to improve the targeting ability of the polypeptide to a cell, tissue or organ of interest.

The binder derivatives to which the present invention also relates may be covalently modified to include one or more water soluble polymer linkers, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol, see U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, 4,179,337. Other polymers known in the art to be useful include monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate polymers, poly- (N-vinyl pyrrolidone) -polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (such as glycerol) and polyvinyl alcohol, and mixtures thereof. Covalent modification of specific binding agents with Polyethylene (PEG) subunits is particularly preferred. The water-soluble polymer may be attached to a specific location, for example the amino terminus of a specific binding agent product, or randomly attached to one or more side chains of the polypeptide. The use of PEG to improve the therapeutic ability of specific binding agents (particularly for humanised antibodies) is described in us patent 6,133,426 and Gonzales et al, grant date: year 2000, 10 months and 17 days.

Target sites for antibody mutagenesis

Certain measures can be taken to manipulate the intrinsic properties of Ang-2 specific antibodies, such as the affinity of the antibody for its target. These include site-directed or random mutagenesis of the polynucleotide encoding the antibody to obtain antibody variants, which are then screened to recover antibody variants exhibiting the desired change (e.g., increased or decreased affinity).

The most commonly used sites for amino acid residue mutation are residues within the CDRs. As described above, residues that actually interact with Ang-2, as well as residues that affect the spatial rearrangement of these interacting residues, are contained within the CDRs. However, residues within the framework of the variable domains outside the CDR regions have also been found to contribute substantially to the antigen binding properties of the antibody, and these residues may also serve as targets for mutations to manipulate these properties. See Hudson, Curr Opin Biotech, 9: 395-402(1999) and references therein.

Sites within the CDRs corresponding to regions susceptible to "hypermutation" can be restriction randomly mutated or site-directed mutated during somatic (somatic) affinity maturation to yield smaller and more efficient antibody screening libraries. See, Chowdhury and pasta, nature biotech, 17: 568-572(1999) and references therein. The types of DNA elements known to define hypermutation sites in this approach include direct and inverted repeats (direct and inverted repeats), certain consensus sequences, secondary structures, and palindromic structures. Wherein the consensus DNA sequence comprises the four base sequence purine-G-pyrimidine-A/T (i.e., A or G-G-C or T-A or T) and the serine codon AGY (wherein Y can be C or T).

Thus, the objective of using a mutation strategy in one embodiment of the invention is to increase the affinity of the antibody for its target. These strategies include mutations throughout the variable heavy and light chains, mutations only in the CDR regions, mutations at common mutational hot spots within the CDRs, mutations within the framework regions, or a combination of these mutational approaches (where the mutations can be random or site-directed mutations). The structure of the test antibody and antibody-ligand complex can be analyzed using methods known in the art, such as X-ray crystallography techniques, to accurately delineate the CDR regions and identify residues (including binding sites). Based on the analysis and characterization of the crystal structure of such antibodies, a variety of methods are known in the art for approximating (although not exactly) CDRs. Commonly used methods are, for example, Kabat, Chothia, AbM and contact-delineation (contictdefinition).

Kabat delineation (Kabat definition) is the most commonly used CDR region prediction delineation method, which is based on sequence variability. (Johnson and Wu, Nucleic Acids Res, 28: 214-8(2000)) Chothia delineation (Chothia definition) location based on loop structures. (Chothia et al, J Mol Biol, 196: 901-17 (1986); Chothia et al, Nature, 342: 877-83 (1989)). Between the two AbM definitions, a complete set of methods for antibody structural modeling from Oxford Molecular Group (Martin et al, Proc Natl Acad Sci (USA) 86: 9268- ^TMABM (antibody variable region modeling) software^TMA computer program for modifying variable regions of antioxidants, Oxford, UK; oxford Molecular, Ltd.). It uses a database in conjunction with a de novo algorithm to model the primary to tertiary structure of antibodies. The other kind isMethod contact-delineation, based on analysis of commercial crystal structure complexes.

By convention, the CDR regions in the heavy chain are typically referred to as H1, H2, and H3, counted from amino terminus to carboxy terminus. The CDR regions in the light chain are typically designated L1, L2, and L3, counted from amino terminus to carboxy terminus.

CDR-H1 is approximately 10-12 residues in length, depicted by Chothia and AbM, typically starting at 4 residues after Cys, or 5 residues after Cys, depicted by Kabat. Typical H1 is followed by Trp, usually Trp-Val, but also Trp-Ile or Trp-Ala. H1 was approximately 10-12 residues long according to AbM delineation, but Chothia delineation removed the last 4 residues.

CDR-H2 typically starts 15 residues after the H1 terminus, depicted by Kabat and AbM. A typical residue preceding H2 is Leu-Glu-Trp-Ile-Gly, but there are many variations. A typical sequence after H2 is Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. According to the Kabat mapping, H2 is about 16-19 residues long, while the AbM mapping predicts it as typically 9-12 residues long.

CDR-H3 typically begins 33 residues after the end of H2, preceded by the typical sequence Cys-Ala-Arg. H3 is typically followed by Gly. H3 is between 3 and 25 residues in length.

CDR-L1 typically begins at about residue 24, preceded by Cys. Usually the residue after CDR-L1 is Trp, followed by the typical sequence Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln or Trp-Tyr-Leu. CDR-L1 is about 10-17 residues in length. The putative (positive) CDR-L1 of the antibody of the invention strictly follows this pattern, being located after the Cys residue followed by 15 amino acids, and then Trp-Tyr-Gln.

CDR-L2 begins at about 16 residues after the end of L1. The preceding residue is usually Ile-Tyr, Val-Tyr, Ile-Lys or Ile-Phe, which is about 7 residues in length.

CDR-L3 generally begins 33 residues after the end of L2, preceded by Cys, which is the typical residue. L3 is typically followed by a Phe-Gly-XXX-Gly sequence that is about 7 to 11 residues in length.

Various methods of antibody modification are known in the art. For example, U.S. Pat. No. 5,530,101(Queen et al, June 25, 1996) describes methods for generating humanized antibodies in which the sequence of the humanized immunoglobulin heavy chain variable region framework region is 65-95% identical to the corresponding sequence of the source immunoglobulin. Each humanized immunoglobulin chain typically comprises, in addition to the CDRs, residues in the framework region of the source immunoglobulin that are capable of interacting with the CDRs to affect binding affinity, e.g., residues in the source immunoglobulin that are directly adjacent to the CDRs, or those that are within 3 angstroms of the CDRs (as predicted by molecular modeling techniques). When combined into a whole antibody, the humanized immunoglobulin of the invention will be substantially non-immunogenic to humans, but will retain substantially the same affinity for an antigen (e.g., an epitope-containing protein or other compound) as the source immunoglobulin. Related methods are described in U.S. Pat. No. 5,693,761(Queen, et al, issued (issued) at 12 months 2 days 1997, "Polynucleotides encoding immunoglobulins with improved humanization properties" "" Polynucleotides encoding engineered immunoglobulins "; U.S. Pat. No. 5,693,762(Queen, et al, entitled "humanized immunoglobulins" and "Humanized immunoglobulins" on 12 months and 2 days 1997); U.S. Pat. No. 5,585,089(Queen, et al, entitled "Humanized immunoglobulins" and "Humanized Immuno globulins" on 12 months and 17 days 1996).

For example, U.S. Pat. No. 5,565,332(Hoogenboom et al, entitled 10/15, 1996, "preparation of chimeric antibodies-a combinatorial approach". A Production of recombinant antibodies-a combinatorial approach ") describes methods for preparing antibodies (and antibody fragments) that have similar binding activity as the parental antibody, but with reduced human antibody characteristics. Humanized antibodies are obtained by using, for example, phage display technology, chain rearrangement (shuffling), comprising the heavy (or light) chain variable region of a non-human antibody, and a polypeptide specific for the antigen of interest in combination with a library of human complementary (light or heavy) chain variable regions. Hybrid pairs specific for the antigen of interest are identified, and the human strands from the selected hybrid pair are combined with a pool of human complementary (light or heavy) chain variable regions. In another embodiment, CDR components from a non-human antibody are combined with a library of CDR components from a human. Hybrids from the resulting library of antibody polypeptide dimers are selected and used to perform a second round of humanized shuffling procedure. Or not, provided that the human characteristics of the hybrid are sufficient to be treated. Methods of modification to enhance human characteristics are also described in the literature, see Winter, FEBS letters 430: 92-92(1998).

Also for example, U.S. Pat. No. 5,766,886(Studnicka et al, granted at 16.6.1998, "Modified antibody variable regions" identifies residues in antibody variable regions that can be Modified without reducing the natural affinity of the antigen binding domain, while reducing the immunogenicity of the antibody to a xenogeneic animal, and methods for making such Modified antibody variable regions that facilitate introduction of the antibody into a xenogeneic animal. See also us patent 5,869,619(Studnicka, granted on 2/9/1999).

As described herein, the purpose of modifying an antibody using any method known in the art is generally to increase the binding affinity of the antibody to an antigen and/or to reduce the immunogenicity of the antibody to a recipient. In one approach, humanized antibodies can be modified to remove glycosylation sites, thereby increasing the affinity of the antibody for its cognate antigen (Co et al, Mol Immunol 30: 1361. sup. 1367 (1993)). Methods such as remodeling ("rejuvenating"), hypercomplex ("hyperchemization"), and veneering/resurfacing ("tunneling") are used to prepare humanized antibodies with increased therapeutic potential. (Vaswami et al, Annals of Allergy, Asthma, & Immunol 81: 105 (1998); Roguska et al, Prot Engineer 9: 895-. See also U.S. patent 6,072,035(Hardman et al, 6.6.2000) which describes a method of reshaping antibodies. Although these techniques reduce the immunogenicity of antibodies by reducing the number of foreign residues, they do not prevent anti-idiotypic and anti-allotypic antigen reactions that occur after repeated administrations of these antibodies. In addition to these methods, alternative methods for reducing immunogenicity are described in Gilliland et al, J Immunol 62 (6): 3663-71(1999).

In many instances, humanized antibodies cause a loss of antigen binding ability. It is therefore preferred to "back mutate" the humanized antibody to include one or more amino acid residues in the original antibody (most often in rodents) in the humanized antibody in an attempt to restore the binding affinity of the antibody. See, e.g., Saldanha et al, MolImmunol 36: 709-19(1999).

Analogues/mimetics of non-peptide specific binding agents (Protein Mimetic)

In addition, the present invention also contemplates non-peptide analogs of peptide-specific binding agents that provide a stabilized structure or reduced biodegradation. Starting from the selected inhibitory peptide, one or more residues are replaced with non-peptide components to prepare peptidomimetic analogs of specific binding agents. The non-peptide component preferably allows the peptide to retain its native conformation, or alternatively, may stabilize a preferred (e.g., biologically active) conformation that retains the ability to recognize and bind Ang-2. On the one hand the resulting analogue/mimetic has an improved binding affinity for Ang-2. The literature, Nachman et al, Regul Pept 57: 359-370(1995) describe the preparation of non-peptidic mimetic analogs starting from peptide-specific binding agents. The peptide-specific binding agents of the invention may be modified if desired, for example by glycosylation, amidation, carboxylation or phosphorylation, or by formation of acid addition salts, amides, esters, especially C-terminal esters, and N-acyl derivatives of the peptides of the invention. The modification may also be carried out by bringing the peptide-specific binding agent together with other components into a covalent or non-covalent complex. The chemical moiety may be attached to a functional group on a side chain of the peptide-specific binding agent, or to the N-terminus or C-terminus to form a covalent binding complex.

In particular, it is contemplated that the peptide-specific binding agent may be coupled to a receptor group, including, but not limited to, a radiolabel, a fluorescent label, an enzyme (e.g., an enzyme that catalyzes a colorimetric or fluorescent reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). The invention therefore provides a molecule comprising an antibody molecule, which molecule preferably further comprises an acceptor group for an acceptor selected from the group consisting of: radiolabel, fluorescent label, enzyme, substrate, solid matrix or support. These markers are well known to the person skilled in the art and, for example, the use of biotin markers is particularly contemplated. The use of such labels is also well known to those skilled in the art and is described in U.S. Pat. Nos. 3,817,837; U.S. patent nos. 3,850,752; U.S. Pat. Nos. 3,996,345; U.S. Pat. No.4,277,437. Other labels that may be used include, but are not limited to, radioactive labels, fluorescent labels, and chemiluminescent labels. U.S. patents relating to the use of these markers include U.S. Pat. Nos. 3,817,837; U.S. patent nos. 3,850,752; U.S. patent nos. 3,939,350; U.S. Pat. No.3,996,345. Any peptide of the invention may comprise any kind of such labels, the number of which may be one, two or more.

Preparation method of specific binding agent

The protein-specific binding agents of the present invention can be prepared by chemical synthesis according to conventional methods on a solution or solid support. The upper limit of the length of the current solid phase synthesized proteins is about 85 to 100 amino acid residues. Chemical synthesis techniques are often used to combine a series of smaller peptides into a full-length polypeptide. There are a variety of commercial automated synthesis devices and can be used according to known methods. See, for example, Stewart and Young, "Solid Phase peptide Synthesis", Solid Phase peptide Synthesis, 2d.ed., Pierce Chemical Co., 1984; tam et al, J Am Chem Soc, 105: 6442, (1983); merrifield, Science, 232: 341-347, (1986); barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284; barany et al, int.J.peptide Protein Res., 30, 705-739 (1987); U.S. patent No.5,424,398, both of which are incorporated herein by reference.

The solid phase peptide synthesis method uses copoly (styrene-divinylbenzene) containing 0.1-1.0mM amine per gram of polymer. These syntheses use t-butyloxycarbonyl (t-BOC) or 9-Fluorenylmethoxycarbonyl (FMOC)) to protect the alpha-amino group. Both protection methods are stepwise synthesis, adding one amino acid per step starting from the C-terminus of the peptide (see Coligan et al, "Current Protocols in Immunology, Wiley Interscience, 1991, unit 9). After chemical synthesis is complete, the t-BOC or FMOC amino acid blocking group can be removed to deprotect the synthetic peptide and treated with acid at low temperature (e.g., liquid HF-10% anisole at 0 ℃ for about 1h) to cleave the peptide from the polymer. After evaporation of the reagents, the peptide specific binding agent was extracted from the polymer using 1% acetic acid solution to give a crude material. The crude material can be usually purified by gel filtration using 5% acetic acid as a solvent using Sephadex G-15. Lyophilization of an appropriate portion of the column will yield a homogeneous peptide-specific binding agent or peptide derivative, which can then be identified using standard methods such as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility detection, and quantified using solid-phase Edman degradation.

In the chemical synthesis of anti-Ang-2 antibodies, derivatives, variants (and fragments of these molecules) and other protein-based Ang-2 binding agents, the inclusion of unnatural amino acids in the binding agent is permitted.

Recombinant DNA technology is a convenient method for making antibodies and other protein-specific binding agents (and fragments thereof) of the invention. The cDNA molecules encoding the antibodies or fragments thereof can be inserted into an expression vector, which is then inserted into a host cell to produce the antibodies or fragments. Of course, the cDNAs encoding these antibodies can be modified to make them different from the "original" cDNA (translated from the mRNA) to provide codon degeneracy or allow codon preference for use in different host cells.

In general, DNA molecules encoding antibodies can be obtained using the methods described in the examples section herein. When it is desired to obtain relevant Fab fragments or CDRs of the original antibody molecule, the skilled person can use standard methods to screen from an appropriate library (phage display library; lymphocyte library) to identify and clone the relevant Fab/CDR. The probes used for the screening may be full or partial Fab probes encoding the Fab portion of the original antibody, probes directed to one or more CDRs in the Fab portion of the original antibody, or other suitable probes. However, typical hybridization conditions when using DNA fragments as probes are described in Ausubel et al ("eds. Molecular Biology laboratory, Current Protocols in Molecular Biology, Current Protocols Press (1994)). The probe imprints are washed under appropriate stringency conditions based on factors such as probe size, predicted homology between probe and clone, type of library used, and number of clones screened. High stringency screening conditions are for example 50-65 ℃, 0.1X SSC and 0.1% SDS.

A variety of expression vector/host systems can be used to contain and express polynucleotides encoding specific binding agents of the invention. These systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage, plasmid or cosmid DNA expression vectors; yeast transformed with a yeast expression vector; insect cells infected with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, and tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or an animal cell system.

Mammalian cells that can be effectively used in the production of recombinant specific binding agent proteins include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary cell lines (Chineshamster ovary (CHO) cells lines), COS cells (e.g., COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, and 293, and hybridoma cells as described herein. In the preparation of specific binding agents such as antibodies and antibody fragments that are typically glycosylated and require proper refolding, mammalian cells are preferably used. Preferred mammalian cells include CHO cells, hybridoma cells, and bone marrow cells.

Some examples of recombinant expression of specific binding agents are given below.

"expression vector" refers to a plasmid, virus, or vector used to express polynucleotides of DNA (RNA) sequences. Expression vectors may comprise transcription units comprising a combination of (1) genetic elements or elements having a regulatory role in gene expression, such as promoters or enhancers, (2) structural units or sequences encoding specific binding agents that are transcribed into mRNA and translated into protein, (3) appropriate transcription initiation and termination sequences. The building blocks used in yeast or eukaryotic expression systems preferably contain leader sequences so that the host cell can secrete the translated protein extracellularly. Alternatively, when the recombinant specific binding agent protein is expressed without a leader or transport (transport) sequence, the structural unit may comprise the amino acid terminator methionine residue. This residue can then be cleaved from the expressed recombinant protein or retained to provide the final product of the specific binding agent.

For example, specific binders can be recombinantly expressed in yeast using commercially available Expression systems, such as the Pichia Expression System (Invitrogen, San Diego, Calif.) according to the instructions for use. This system relies on the pre-alpha (pre-pro-alpha) sequence for direct secretion, while transcription of the insert is driven by alcohol oxidase (AOX1) under methanol induction.

Secreted peptide-specific binding agents are purified from yeast growth media using methods such as purification of peptides from bacterial and mammalian cell culture supernatants.

Alternatively, the cDNA encoding the peptide-specific binding agent was cloned into the baculovirus expression system pVL1393, (PharMingen, San Diego, CA). The vector can be used to infect Spodoptera frugiperda (Spodoptera frugiperda) cells in sF9 protein-free medium according to the instructions for use to produce recombinant proteins. Specific binding agent proteins can be purified and concentrated from the culture medium using a heparin-sepharose column (Pharmacia).

Alternatively, the peptides can be expressed using an insect system. Insect systems for protein expression are well known to those skilled in the art. In an insect expression system, an exogenous gene can be expressed in Spodoptera frugiperda or Trichoplusia ni (Trichoplusia larvae) using Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. The coding sequence for the peptide-specific binding agent may be cloned into a non-essential region of the virus, e.g., into the polyhedrin gene and placed under the control of, e.g., the polyhedrin promoter. Successful insertion of a peptide-specific binding agent results in inactivation of, for example, the polyhedrin gene, thereby producing a recombinant virus lacking the capsid protein coat. The recombinant virus can be used to infect Spodoptera frugiperda or Trichoplusia ni, and the exogenous peptide is expressed. (Smith et al, J Virol 46: 584 (1983); Engelhard et al, Proc Nat Acad Sci (USA) 91: 3224-7(1994))

In another example, the DNA sequence encoding the peptide-specific binding agent may be amplified using PCR and cloned into a suitable vector, such as pGEX-3X (pharmacia). The pGEX-3X vector encodes glutathione-S-transferase (GST), which produces a fusion protein comprising this enzyme and a specific binder protein encoded by a DNA insert inserted into the cloning site of the vector. The PCR primers may comprise, for example, suitable cleavage sites. When the moiety fused to the specific binding agent is used only to facilitate expression, or is not a linker for its insertion into the protein, the recombinant specific binding agent can be cleaved from the GST moiety in the fusion protein. Coli XL-1Blue cells (Stratagene, La Jolla CA) were transformed with pGEX-3X/specific binding agent peptide, and single transformant diseases were isolated and cultured. Plasmid DNA can be purified from single transformants and partially sequenced using an automated sequencer to confirm that the specific binding agent-encoding nucleic acid is inserted in the correct orientation.

Expression of antibodies encoding anti-Ang-2 antibodies and fragments thereof using the recombinant systems described above may result in antibodies or fragments thereof that require "refolding" (to establish multiple correct disulfide bonds) for biological activity. Typical refolding methods for these antibodies are described in the examples below.

Specific binding agents prepared in bacterial cells can be produced as bacterial insoluble inclusion bodies and purified as follows. Host cells can be processed using centrifugation; washing with 0.15M NaCl, 10mM Tris, pH 8, 1mM EDTA; and treated with 0.1mg/ml lysozyme (Sigma, St. Louis, Mo.) for 15min at room temperature. The lysate can be clarified by sonication and then centrifuged at 12,000Xg for 10min to pellet the cell debris. The pellet containing the specific binding agent was resuspended with 50mM Tris, pH 8, and 10mM EDTA, layered with 50% glycerol, and centrifuged at 6000Xg. for 30 min. With no Mg⁺⁺And Ca⁺⁺Resuspend the pellet in standard phosphate solution (PBS). The specific binding agent can then be further purified by denaturing SDS polyacrylamide gel separation (Sambrook et al, supra). The gel may be soaked with 0.4M KCl to visualize the proteins, the gel may be excised and electrophoresed in gel electrophoresis buffer without SDS. If a GST fusion protein (soluble protein) is produced in bacteria, Purification can be carried out using GST Purification Module (Pharmacia).

The use of mammalian cells for the expression of recombinant proteins is well known to those skilled in the art. Host cell lines may be selected for their specific ability to process the expressed protein or to produce certain post-translational modifications that will facilitate production of protein activity. Such polypeptide modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Various host cells such as CHO, HeLa, MDCK, 293 and hybridoma cell lines have specific cellular and characteristic mechanisms associated with this post-translational modification activity that may be selected to ensure proper modification and processing of the foreign protein administered.

Transformed cells (for recombinant protein production) can be recovered using a variety of selection systems. These selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase genes in tk-, hgprt-, or aprt-cells, respectively. Antimetabolite resistance may also be used for selection, DHFR conferring resistance to methotrexate to cells; gpt confers resistance to mycophenolic acid on cells; neo confers resistance to the aminoglycoside G418 and chlorsulfuron on cells; hygro confers resistance to hygromycin to cells. Other useful selection genes include trpB (which allows cells to use indole instead of tryptophan) or hisD (which allows cells to use histidinol instead of histidine). Markers for visually identifying transformants include anthocyanin, beta-glucosidase and GUS as a substrate thereof, luciferase and luciferin as a substrate thereof.

Purification and refolding of specific binding agents

In some cases, specific binding agents produced using the methods described above may require "refolding" and oxidation to form the correct quaternary structure and to establish disulfide bonds for biological activity. Refolding can be performed using a variety of methods known in the art. These methods include, for example, exposing the solubilized polypeptide to a pH above 7 (typically) in the presence of a chaotropic agent. The selection of chaotropic agents is similar to the selection in terms of solubilization of inclusion bodies, but chaotropic agents are typically used at low concentrations. Chaotropic agents such as guanidine. In most cases, the refolding/oxidation solution also contains a reducing agent and its oxidized form, at concentrations that are maintained at a particular ratio to produce a particular redox potential, so that disulfide shuffling (bisekide shuffling) can occur to form disulfide bonds. Some redox molecule pairs commonly used include cysteine/cystamine, glutathione/dithiogsh, cuprous chloride, dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol (bME)/disulfide-bME. Cosolvents are used in many cases to improve refolding efficiency. Commonly used co-solvents include glycerol, polyethylene glycols of various molecular weights and arginine.

It is desirable to purify the specific binding agent proteins of the invention or variants thereof. Protein purification methods are well known to those skilled in the art. These methods involve, at one level, crude separation of polypeptide and non-polypeptide fragments. After separation of the specific binding agent polypeptide from other proteins, the polypeptide of interest can be purified using chromatographic and electrophoretic methods to achieve partial or complete purification (or purification to homogeneity). Particularly suitable analytical methods for pure specific binding agent peptides are ion exchange chromatography, exclusion chromatography, polyacrylamide electrophoresis, isoelectric focusing. One particularly effective method of peptide purification is rapid protein liquid chromatography or HPLC.

Certain aspects of the invention relate to purification, and in particular embodiments, to substantial purification of specific binding agent proteins or peptides. As used herein, "purified specific binding agent protein or peptide" refers to a composition that is separated from other components, wherein the specific binding agent protein or peptide is purified to any degree relative to its naturally available state. Thus, a purified specific binding agent protein or peptide also refers to a specific binding agent protein or peptide that is free from its environment in which it may naturally occur.

Generally, "purified" refers to a specific binding agent composition that has been separated from various other components and which retains sufficient biological activity that it is expressed. As used herein, "substantially purified" refers to a specific binding agent composition in which the specific binding agent protein or peptide is the major component of the composition, e.g., the specific binding agent protein or peptide is present in a proportion of about 50%, 60%, 70%, 80%, 90%, 95% or more of the composition protein.

One skilled in the art will appreciate a variety of methods for quantifying the degree of purification of a specific binding agent in light of the present disclosure. These methods include, for example, determining the specific binding activity of a partially purified product, or detecting the amount of specific binding agent polypeptide in a portion of the purified product by SDS/PAGE. A preferred method of assessing the degree of purification of a portion of the purified fraction of a specific binding agent is to calculate the binding agent activity of that portion and compare it to the starting extract to calculate the degree of purification, herein denoted as "fold purification". The actual unit used to represent binding activity will, of course, depend on the particular assay chosen after purification and whether the expressed purified binding agent protein or peptide exhibits detectable binding activity.

The skilled artisan is familiar with a variety of methods that can be used for the purification of specific binding agent proteins. These include, for example, ammonium sulfate precipitation, PEG, antibody methods (immunoprecipitation), or centrifugation after heat denaturation, chromatography such as affinity chromatography (e.g., protein A-Sepharose), ion exchange chromatography, reverse phase chromatography, gel filtration, hydroxyapatite and affinity chromatography, isoelectric focusing, gel electrophoresis, and combinations thereof. As is well known in the art, the order of the different purification steps may be varied, or certain steps may be omitted, and still suitable purification methods may be obtained to obtain substantially purified specific binding agents.

It is not generally required that the most pure state of a specific binding agent be always provided. In fact, in particular embodiments it is contemplated to use binding agent products that are not particularly high specificity. The partially purified product can be obtained using fewer purification steps in a combined process or using different forms of the same process of purification by passage. For example, cation exchange column chromatography using an HPLC apparatus generally yields higher purification times than the same procedure using a low pressure chromatography system. Methods with a relatively low degree of purification may be advantageous for the total recovery of specific binder protein product, or for maintaining the binding activity of the expressed specific binder protein.

It is known that polypeptides may migrate differently, sometimes significantly, under different SDS/PAGE conditions (Capaldi et al, Biochem Biophys \ Res Comm, 76: 425 (1977)). Thus, the apparent molecular weight of the purified or partially purified specific binding agent expression product may, of course, vary under different electrophoretic conditions.

Binding experiments

Immunological binding assays typically use capture reagents to specifically bind to and often immobilize a target antigen of an analyte. The capture reagent is a group that specifically binds to the analyte. In one embodiment of the invention, the capture reagent is an antibody or fragment thereof that specifically binds to Ang-2. These immunological binding assays are well known in the art (Asai, ed., "Methods in Cell Biology," Vol.37, "Antibodies in Cell Biology, Academic Press, Inc., New York (1993)).

Immunological binding assays often use labels to indicate the presence of a fixed complex formed by a capture reagent and an antigen. The label may be one of the molecules comprising the immobilized complex, i.e. the label may be a labeled specific binding agent or a labeled antibody directed against a specific binding agent. Alternatively, the label may be a third molecule, typically a third antibody, which binds to the immobilized complex. The label may be, for example, an antibody carrying a label against the specific binding agent. The second antibody is specific for the immobilized complex and may be unlabeled, but it may be immobilized by a fourth molecule specific for the species to which the second antibody belongs. For example, the second antibody may be modified with a detectable group such as biotin, which may be immobilized by a fourth molecule, such as labeled streptavidin. Other proteins that specifically bind to immunoglobulin constant regions, such as protein A or protein G, may also be used as labels. These marker proteins are usually components of the cell wall of streptococci and exhibit strong nonimmunogenic activity with a variety of immunoglobulin constant regions (see generally Akerstrom, J Immunol, 135: 2589-2542 (1985); Chaubert, Mod Pathol, 10: 585-591(1997))

Throughout these experiments, incubation and/or washing steps may be required after each substance reaction. Incubation times may vary from 5s to several hours, preferably from about 5min to about 24 h. However, the incubation time depends on factors such as the assay format, analyte, solution volume, concentration, etc. Typically these measurements are made at ambient temperature, but may be made over a range of temperatures.

A. Non-competitive binding experiments:

the immunological binding assay may be a non-competitive binding assay. There are many analytes captured in these experiments that are measured directly. For example, in a preferred "sandwich" assay, the capture reagent (antibody) may be immobilized directly on a solid substrate. These immobilized antibodies are then captured (bound) by the antigen in the sample to be tested. The protein thus immobilized is then immobilized on a label, for example a second antibody carrying a label. In another preferred "sandwich" assay, the second antibody does not carry a label, but may be immobilized by another antibody carrying a label, which is specific for the species to which the second antibody belongs. The second antibody may also be modified to add a detectable group such as biotin to which a third labeled molecule specifically binds (see Harlow and Lane, "antibody Laboratory Manual," Antibodies, A Laboratory Manual, Ch 14, Cold Spring Harbor Laboratory, NY (1988), incorporated herein by reference).

B. Competitive binding assays

The immunological binding assay may be a competitive binding assay. The amount of analyte present in the sample is indirectly measured by measuring the amount of additional analyte added that is displaced or competed for by the analyte in the sample. In a preferred competitive binding assay, a known amount of analyte (usually labeled) is added to a sample, which is then contacted with an antibody (capture reagent). The amount of label immobilized by the antibody is inversely proportional to the amount of analyte in the sample (See See, Harlowand Lane, "guide for Antibodies", A Laboratory Manual, Ch 14, pp.579-583, supra).

In another preferred competitive binding assay, the antibody is immobilized on a solid substrate. The amount of protein immobilized on the antibody can be determined by measuring the amount of protein in the protein/antibody complex or measuring the amount of protein remaining unbound to the antibody. The amount of protein can be measured by providing a labeled protein (See See, Harlow and Lane, "antibody protocols," Antibodies, A Laboratory Manual, Ch 14, pp.579-583, supra).

Hapten inhibition is used in another preferred competitive binding assay. Here, known analytes are immobilized on a solid matrix. A known amount of antibody is added to the sample, which is then contacted with the immobilized analyte. The amount of antibody captured by the immobilized analyte is inversely proportional to the amount of analyte in the sample. The amount of immobilized antibody can be detected by detecting the amount of immobilized antibody or detecting the amount of antibody remaining in the solution. Direct detection may be performed when the antibody is labeled, or indirect detection may be performed by subsequent addition of a labeled group (which is specific for the antibody, as described above).

C. Use of competitive binding assays

The skilled artisan can determine cross-reactivity using competitive binding assays to determine whether a protein or enzyme complex recognized by a specific binding agent of the invention is a desired protein and is not a cross-reactive molecule, or whether an antibody is specific for an antigen and does not bind to an antigen of non-interest. In this type of experiment, the antigen may be immobilized on a solid support and an unknown mixture of proteins is added to the assay solution, which mixture will competitively bind to the immobilized protein with a specific binding agent. The competitor also binds to one or more antigens unrelated to the antigen of interest. The binding competition between the protein and the specific binding agent antibody for the immobilized antigen is compared to the binding of the same protein immobilized on the solid support to determine the cross-reactivity of the protein mixture.

D. Other binding experiments

The invention also provides a Western blot method to detect or quantify Ang-2 in a sample. The method steps generally involve separating the sample proteins by gel electrophoresis according to molecular weight, transferring the proteins to a suitable solid support, such as a nitrocellulose membrane, a nylon membrane, or a derivatized nylon membrane. The sample is then incubated with an antibody or fragment thereof that specifically binds Ang-2 and the resulting complex is detected. These antibodies may be directly labeled or subsequently detected using a labeled antibody that specifically binds to the first antibody.

Assays for the detection of binding of Ang-2 specific binding agents that disrupt the binding of Ang-2 to its receptor, see the examples section herein.

Diagnostic test

The antibodies or fragments thereof of the invention are useful for the diagnosis of conditions or diseases characterized by the expression of Ang-2 (or subunits thereof), or for the monitoring of patients treated with Ang-2 inducers and fragments thereof, antagonists or inhibitors of Ang-2 activity in assays. The Ang-2 diagnostic assay involves the use of specific binding agents and labels to detect Ang-2 in human body fluids or cell (or tissue) extracts. The specific binding agents of the present invention may be used with or without modification. In a preferred diagnostic assay, the specific binding agent is labeled by attachment of, for example, a label or receptor molecule. A variety of label and receptor molecules are known, some of which have been described herein. The present invention is particularly effective for human diseases.

Various methods are known in the art for measuring Ang-2 protein using polyclonal antibodies or monoclonal antibodies specific for the respective receptor proteins. Including, for example, enzyme-linked immuno-assays (ELISA), Radioimmunoassays (RIA) and Fluorescence Activated Cell Sorting (FACS). Preferably, a two-site, monoclonal antibody-based immunoassay is used in which a monoclonal antibody that reacts with two non-interfering epitopes on Ang-2 is used, although competitive binding assays may be used. These experiments are described, for example, in Maddox et al, J Exp Med.158: 1211(1983).

To provide a basis for diagnosis, normal or standard Ang-2 expression values are typically established in humans. This value can be established by the combined use of a body fluid or cell extract from a normal subject, preferably a human, and a specific binding agent for Ang-2, such as an antibody, under conditions well known in the art to be suitable for complex formation. The effect of standard complex formation can be quantified by comparing the binding of a specific binding agent to a known amount of Ang-2 protein, to the corresponding effects of control and disease samples and quantifying the comparison. The normalized values obtained from normal samples can then be compared to sample values obtained from a subject that may be infected. Deviation between the standard and subject values suggests a role for Ang-2 in the disease state.

In certain embodiments, specific binding agents are typically labeled with a detectable group for diagnostic purposes. Such a detectable group may be any molecule/group that can directly or indirectly generate a detectable signal. For example, a radioactive isotope such as³H、¹⁴C、³²P、³⁵S、¹²⁵I; fluorescent or chemiluminescent compounds such as fluorescent isothiocyanates, rhodamines or luciferin; or an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase (Bayer et al, MethEnz, 184: 138-163, (1990)).

Disease and disorder

The present invention provides specific binding agents that bind to Ang-2 that are effective in treating human diseases and ameliorating symptoms. Substances that modulate Ang-2 binding activity or other cellular activity may be used in combination with other therapeutic agents to enhance their therapeutic effect or reduce possible side effects.

In one aspect, the invention provides medicaments and methods effective in treating diseases or conditions characterized by non-essential or abnormal levels of Ang-2 activity in cells. These diseases include cancer and other hyperproliferative conditions such as hyperplasia, psoriasis, contact dermatitis, immune disorders and infertility.

The invention also provides a method of treating cancer in an animal, including a human, comprising the step of administering to the animal an effective amount of a specific binding agent that inhibits or reduces Ang-2 activity. The invention also relates to methods of inhibiting the growth of cancer cells, processes including cell proliferation, invasion and metastasis in biological systems. These methods comprise using the compounds of the present invention as cancer cell growth inhibitors. Preferably, these methods are used to inhibit or reduce the growth, invasion, metastasis or tumor extent of cancer cells in a living animal, such as a mammal. The methods of the invention may also be conveniently modulated in assay systems, such as assays for cancer cell growth and its properties, and the identification of compounds that affect cancer cell growth.

The cancer treatable using the methods of the invention is preferably a mammalian cancer. Mammals include, for example, humans and other primates, companion or companion animals such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep and cattle.

Tumors or neoplasms include tissue cell growth where cell proliferation is uncontrolled and invasive. Some of these growths are benign, while others are expected to be malignant and may cause death of the organism. Malignant tumors or cancers are distinguished from benign growths in that cancer cells, in addition to exhibiting invasive cell proliferation, may invade surrounding tissues and metastasize. Malignant tumors are also characterized by a greater loss of their ability to differentiate (greater extent of anaplastic development) and a greater decrease in their tissue extent relative to each other and to the tissues surrounding them. This property is called "degenerative development".

Tumors that can be treated using the present disclosure include solid tumors (solid tumors), i.e., carcinomas (carcinomas) and sarcomas (sarcomas). Cancers include those malignancies that originate in epithelial cells that infiltrate (invade) the surrounding tissue and cause metastases. Adenocarcinoma originates in glandular tissue, or from tissue forming recognizable glandular structures. Another large class of carcinomas includes sarcomas, in which the cells of the tumor are embedded in a fibrous or homogeneous body, such as embryonic connective tissue. The invention may also be used to treat cancers of the myeloid or lymphoid systems, including leukemias, lymphomas, and other cancers that typically do not appear as tumor masses but are distributed throughout the vascular or lymphatic vasculature.

The types of cancers or tumors that are effectively treated by the present invention include, for example, ACTH-producing tumors, acute lymphocytic leukemia, acute non-lymphocytic leukemia, adrenocortical carcinoma, bladder cancer, brain cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's tumor, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and non-small cells), malignant effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, non-Hodgkin's tumor, osteosarcoma, ovarian cancer, germ cell ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, eye cancer, skin cancer, soft tissue tumor, squamous cell carcinoma, gastric cancer, testicular cancer, and abdominal carcinoma, Thyroid cancer, trophoblastic tumors, uterine cancer, vaginal cancer, vulvar cancer, and nephroblastoma.

Certain cancers treatable by the present invention are specifically exemplified herein. In these illustrative examples, standard in vitro and in vivo models at the current technological level are used. These methods can be used to identify agents that are predicted to be effective for in vivo therapy. The method of the invention is of course not limited to the treatment of only these types of tumors, but the method of the invention can be used to treat any solid tumor originating from any organ system. Invasion or metastasis of cancers associated with Ang-2 expression (or activity) is particularly susceptible to inhibition by the methods of the present methods, or even to induction for recovery.

The mode of applying the present invention may also be to use a specific binding agent of the present invention such as an antibody. The specific binding agents of the present invention are used in combination with other anti-cancer chemotherapeutic agents, such as any conventional chemotherapeutic agent. The combination of a specific binding agent and a chemotherapeutic agent may enhance the effectiveness of the chemotherapeutic method. One skilled in the art will recognize a number of chemotherapeutic methods that can be incorporated into the methods of the invention. Any chemotherapeutic agent may be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, and natural products. For example, the compounds of the present invention may be administered in combination with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, paclitaxel and natural or synthetic derivatives thereof, and the like. Also for example, in the treatment of mixed tumors such as breast cancer (tumor cells include both gonadotropin-dependent and gonadotropin-independent cells), the compounds of the invention may be administered with leuprolide or goserelin (a synthetic analogue of LH-RH). Other anti-tumor approaches include the use of tetracycline compounds in combination with other therapeutic modalities (e.g., surgery, radiation therapy, etc., also referred to herein as "adjuvant anti-tumor approaches"). The methods of the present invention can therefore be used in combination with these conventional treatments to reduce side effects and improve therapeutic efficacy.

Thus, the present invention provides compositions and methods that are effective in the treatment of a wide variety of cancers, including solid tumors and leukemias. Types of cancers that can be treated include (but are not limited to): breast, prostate and colon cancers; all forms of bronchopulmonary carcinoma; a myeloma cell; melanoma; hepatoma; neuroblastoma; mastoid lymphoma; (iii) an apdd tumor; a granuloma; gill primary tumor; malignant syndrome (malignant carcinoid syndrome); carcinoid heart disease; cancers (e.g., Walker cancer, basal cell carcinoma, basal squamous cell carcinoma, Brown-pearl cancer, ductal carcinoma, Ehrlich tumor, Krebs 2, merkel cell carcinoma, mucinous carcinoma, non-cell lung cancer, oat cell lung cancer, papillary carcinoma, dura mater, bronchial carcinoma, bronchogenic carcinoma, squamous cell carcinoma, transitional cell carcinoma); (ii) a tissue cell disorder; leukemia; malignant histiocytosis; hodgkin's tumor; non-small cell immunoproliferative lung cancer; non-hodgkin's lymphoma; a plasmacytoma; reticuloendotheliosis; melanoma; lipoma; chondroma; chondrosarcoma; fibroids; fibrosarcoma; giant cell tumor; a histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; clonal cell tumors; hamartoma; interstitial tumors; middle kidney tumor; myosarcoma; amelogenic cytomas; cementoma; dental tumors; teratoma; thymoma; trophoblastic tumors (topoblast tumor). Other types of cancers that can be treated are: adenoma; biliary duct tumors; a pearloma; cyclindroma; cystic carcinoma; a cystic tumor; granulosa cell tumors; amphoterial blastoma; hepatoma; sweat gland adenoma; islet cell tumor of pancreas; a stromal cell tumor; mastoid lymphoma; sertoli cell tumor; a follicular membrane cell tumor; leiomyoma; leiomyoma; myoblastoma; a glioma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; an ependymoma; a ganglionic cell tumor; a glioma; medulloblastoma; meningioma; schwannoma; neuroblastoma; epithelial tumors; neurofibromas; neuroma; paragangliomas; non-chromophilous paragangliomas; a glioma; vascular lymphoid hyperplasia with eosinophilia; sclerosing angioma; hemangioma; glomus; vascular endothelioma; hemangioma; vascular endothelial cell tumor; angiosarcoma; lymphangioma; lymphangioleiomyomata; lymphangioleiomyosarcoma; pineal tumor; a carcinosarcoma; chondrosarcoma; phyllocystic sarcoma; fibrosarcoma; angiosarcoma; leiomyosarcoma; white sarcoma; liposarcoma; lymphangioleiomyosarcoma; myosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; a sarcoma; tumors; neurofibromatosis; and cervical abnormalities.

In addition, the materials and methods of the present invention may be used to prevent and/or treat any skin hyperproliferation, including psoriasis and contact dermatitis, as well as other hyperproliferative diseases. Ang-2 activity has been shown to be elevated in patients with psoriasis and contact dermatitis in these disease states (Ogoshi et al, J.Inv.Dermatol., 110: 818-23 (1998)). It is preferred to use specific binding agents specific for Ang-2 in combination with other drugs to treat patients exhibiting these clinical symptoms. Specific binding agents can be administered to a patient using a variety of carriers by the routes described herein and by other routes well known to those skilled in the art.

In addition, the invention includes the treatment of retinopathies involving angiogenesis (including diabetic retinopathy and age-related macular degeneration) as well as female genital tract disorders/diseases such as endometriosis, uterine fibroids and other such conditions associated with dysfunctional vascular hyperproliferation (including endometrial microvascular growth) in the female reproductive cycle.

In addition, the invention relates to the treatment of abnormal vascular growth including cerebral arteriovenous malformations (AVM), gastrointestinal mucosal damage and repair, gastroduodenal mucosal ulceration in patients with a history of digestive organ ulceration, including ischemia due to heart attack, a large category of pulmonary vascular disorders in liver disease and portal hypertension in patients with non-hepatic portal hypertension.

The invention also relates to the use of the compositions and methods of the invention to prevent cancer. These drugs will include specific binding agents for Ang-2.

Pharmaceutical composition

The invention also includes pharmaceutical compositions of Ang-2 specific binding agents. Pharmaceutical compositions comprise antibodies as detailed in U.S. Pat. No. 6,171,586(Lam et al, granted on 9.1.2001). These compositions comprise a therapeutically or prophylactically effective amount of a specific binding agent (e.g., an antibody or antibody fragment, variant, derivative, and fusion protein described herein) in admixture with a pharmaceutically acceptable agent. In a preferred embodiment, the pharmaceutical composition comprises an antagonistic specific binding agent which partially or completely modulates at least one of Ang-2 activity in admixture with a pharmaceutically acceptable agent. Typically, the specific binding agent is sufficiently purified to allow introduction into an animal.

Pharmaceutical compositions may contain formulation materials to modify, maintain or maintain properties of the composition such as pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dispersion or release rate, adsorption or permeation characteristics. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamic acid, aspartic acid, arginine, or lysine); antibacterial agents, antioxidants (e.g., ascorbic acid, sodium sulfite or sodium bisulfite), buffers (e.g., borate buffer, bicarbonate buffer, Tris-HCl, citrate buffer, phosphate buffer, other organic acid buffers), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., EDTA), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), bulking agents, monosaccharides, disaccharides and other carbohydrates (e.g., glucose, mannose, or dextrins), proteins (e.g., serum albumin, gelatin or immunoglobulins), colorants, flavors or diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride), preservatives (e.g., sodium chloride), and the like, Benzoic acid, salicylic acid, mercurial, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, aminobutyric acid, lecithin, cholesterol, tyloxapol), stabilizers (sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides (preferably potassium chloride or potassium, mannitol, sorbitol)), transport vehicles, diluents, excipients, and/or Pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 18 Edition, a.r. geno, ed., Mack Publishing Company, 1990).

The optimal pharmaceutical composition can be determined by one skilled in the art based on, for example, the intended route of administration, the form of delivery and the desired dosage. See Remington's pharmaceutical sciences, supra. Such compositions may affect the physical body, stability, rate of in vivo release, rate of in vivo clearance of the specific binding agent.

The primary vehicle or carrier in the pharmaceutical composition may be naturally aqueous or non-aqueous. For example, a suitable vehicle or carrier may be water for injection, saline, or artificial cerebrospinal fluid, and may be supplemented with materials for administration by injection, as are otherwise commonly used in compositions. The carrier is, for example, a neutral salt buffer or a serum albumin-salt mixture. Other examples of pharmaceutical compositions comprise Tirs buffer (pH about 7.0-8.5) or acetate buffer (pH about 4.0-5.5), and may further comprise sorbitol or a suitable substitute thereof. In one embodiment of the invention, in preparing a binder composition for storage, the selected composition (having the desired degree of purification) may be combined with an excipient (Remington's pharmaceutical Sciences, supra) optionally in the form of a lyophilized cake or an aqueous solution. The binder product can then be made into a lyophilized product using a suitable excipient, such as sucrose.

Alternatively, the pharmaceutical composition may be administered by injection. Or by administration to the respiratory or intestinal tract, for example orally, otically, ocularly (opthlmolicaly), rectally, or intravaginally. The preparation of such pharmaceutically acceptable compositions is known to those skilled in the art.

The concentration of the formulation ingredients should be acceptable for the site of introduction. For example, the composition is maintained at physiological pH or slightly lower pH, typically between about 5 and 8, with a buffer.

When administration by injection is contemplated, the therapeutic compositions used in the present invention may be in the form of pyrogen-free and injectable aqueous solutions comprising the desired specific binding agent and a pharmaceutically acceptable carrier therefor. A particularly suitable carrier for injection is sterile distilled water in which the specific binding agent is prepared as a sterile, sterile ionic solution and stored properly. Another way that can be used is to combine the desired molecule with an agent, such as injectable microspheres, biodegradable particles, polymers (polylactic acid, polyglycolic acid), beads or liposomes, which allow controlled or sustained release of the desired product, and to make the formulation for administration by depot injection. Hyaluronic acid, which has the function of prolonging the duration of the (drug) circulation, may also be used. Other suitable means of administering the desired molecule include implanting a drug delivery device.

Alternatively, pharmaceutical compositions suitable for injectable administration may be in the form of aqueous solutions, preferably in physiologically compatible buffers, such as Hanks 'solution, ringer' solution, or physiological saline buffer. Aqueous suspensions for injection may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol and dextran. Alternatively, suspensions of the active mixture may be prepared in the form of suitable oily suspensions. Suitable lipophilic solvents or vehicles include fats such as sesame oil, synthetic fatty acid esters such as ethyl oleic acid, triglycerides, liposomes. Non-lipid polycationic amino polymers (Non-lipid polymeric amides) may also be used for delivery. The suspension may optionally contain suitable stabilizers or agents capable of enhancing the solubility of the compound to allow for the preparation of highly concentrated solutions.

In another embodiment, the pharmaceutical composition may be formulated for respiratory administration. For example, the specific binding agent is made into inhalable dry powder. The polypeptide or nucleic acid molecule can also be inhaled into solution and made into aerosol with propellant. In another embodiment, the solution may be made into a spray. In addition, pulmonary administration is described in PCT application No. PCT/US94/001875, which describes the pulmonary administration of chemically modified proteins.

Certain orally administrable dosage forms are also contemplated. In one embodiment of the invention, the specific binding agent molecules administered in this manner may or may not be formulated with carriers commonly used in solid dosage forms, such as tablets or capsules. For example, the capsule can be designed such that release of the active portion of the composition occurs in the gastrointestinal tract when bioavailability is maximized and systemic pre-degradation is minimized. Other agents may also be employed to facilitate absorption of the binder molecules. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binding agents may also be used.

The pharmaceutical compositions may also be formulated for oral administration using pharmaceutically acceptable carriers well known in the art for dosage forms suitable for oral administration. These carriers enable the pharmaceutical compositions to be in a form for absorption by the patient, e.g., tablets, pills, dragees, capsules, solutions, gels, syrups, slurries (suspensions), suspensions.

Pharmaceutical preparations for oral administration can be obtained by combining the active compound with solid excipients and by granulating a resulting mixture, optionally after comminution, to give tablets or dragee cores. Suitable auxiliaries can be added if desired. Suitable excipients include carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, and sorbitol; corn starch, wheat starch, rice starch, potato starch, or other plant starch; cellulose such as methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose; the resin comprises acacia gum and tragacanth gum; proteins include gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as cross-linked polyvinylpyrrolidone, agar and alginic acid or a salt thereof, for example sodium alginate.

Dragee cores may be combined with suitable coatings, such as concentrated sugar solutions, which may also contain acacia, talc, polyvinyl pyrrolidone carbomer gel (carbopol gel), polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or coatings to identify the product or to characterize the active compound, i.e., the dosage.

Pharmaceutical preparations for oral use may also comprise push-fit (push-fit) capsules made of gelatin, or soft, sealed capsules made of gelatin and a coating (e.g. glycerol and sorbitol). Push-fit capsules can contain the active ingredient in admixture with fillers or binders such as sucrose or starch, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compound may be dispersed or suspended in a suitable liquid, such as a fat, liquid (liquid) or liquid polyethylene glycol (liquid), with or without stabilizers.

Pharmaceutical preparations may also be prepared by mixing an effective amount of the binding agent with excipients that are non-toxic and suitable for the manufacture of pills. Solutions can be prepared in unit dosage form by dissolving the pill in sterile water or other carrier. Suitable excipients include, but are not limited to, diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, calcium phosphate; or a binder such as starch, gelatin or gum arabic; lubricants such as magnesium stearate, stearic acid or talc.

Other forms of pharmaceutical compositions, including dosage forms that provide sustained or controlled release of the binder molecule, will also be apparent to those skilled in the art. Those skilled in the art are familiar with a variety of formulation methods involving other sustained or controlled release delivery means, such as liposome carriers, biodegradable microparticles, or porous beads. See, e.g., PCT/US93/00829, which describes the controlled release of pharmaceutical compositions delivered through porous polymeric microparticles. Other examples of sustained release include shaped semipermeable polymer matrices, such as films, or microcapsules. Sustained release mechanisms may include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and γ -ethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-Astrona 556(1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al, J Biomed Mater Res, 15: 167-Astrona 277, (1981)) and (Langer et al, Chem Tech, 12: 98-105(1982)), ethylene-vinyl acetate copolymers (Langer et al, supra) or poly-D (-) -3-hydroxybutyric acid (EP 133,988). Sustained release compositions also include liposomes, which can be prepared by several methods known in the art. See, e.g., Eppstein et al, Proc Natl Acad Sci (USA), 82: 3688-3692 (1985); EP36,676; EP 88,046; EP 143,949.

Pharmaceutical compositions for in vivo administration typically must be sterile. This can be achieved by using armless travel membrane filtration. When the composition is in lyophilized form, this sterilization process can be performed before or after lyophilization (after reconstitution). Compositions for injection may be stored in lyophilized form or in solution. In addition, containers for holding injectable compositions typically have sterile valves, for example using an intravenous solution bag or via a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition formulation is complete, it may be contained in sterile vials in the form of solutions, suspensions, gels, emulsions, solids, dehydrated or lyophilized powders. These formulations may be stored in a ready-to-use form or in a form that requires reconstitution (e.g., lyophilization) prior to use.

In a particular embodiment of the invention, a kit is used to obtain a single dosage unit. The kit may contain two containers, a first containing the dried protein and a second containing the aqueous formulation. The present invention also contemplates the use of such kit packages, i.e., containing single or multiple chamber pre-filled syringes (e.g., liquid syringes and liquid sol syringes).

The effective dosage of the pharmaceutical composition to be used depends on, for example, the environment and the object to be treated. One skilled in the art will appreciate that the appropriate therapeutic dosage level will thus vary in height, depending in part on the delivery molecule, the instructions for use of the binder molecule to be used, the route of administration, and the physical constitution (body species, body surface area or organ volume) and condition (age and general health) of the patient. Thus, clinicians may vary the dosage and adjust the route of administration to achieve optimal therapeutic results. Depending on the factors mentioned above, a typical dosage range may be from about 0.1mg/kg to 100 mg/kg. In other embodiments, the dosage range is from about 0.1mg/kg to 100mg/kg, alternatively from about 1mg/kg to 100mg/kg, alternatively from about 5mg/kg to 100 mg/kg.

The therapeutically effective dose of any compound can be estimated initially by cell culture assays or animal models, such as those using mice, rats, rabbits, dogs, or pigs. Animal models can be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine effective dosages and routes of administration for humans.

The precise dosage will be determined by the relevant factors of the subject in need of treatment. The dosage and mode of administration are adjusted to provide a sufficient level of the active compound or to maintain the desired effect. Factors to be taken into consideration may include the severity of the disease state, the overall health status of the subject, age, body weight, sex, timing and frequency of administration, pharmaceutical compound, reaction sensitivity and therapeutic response. The long acting pharmaceutical composition may be administered once every 3d or 4d, once a week, or once every two weeks, depending on the half-life and clearance rate of the particular composition.

The frequency of administration will depend on the pharmacokinetic parameters of the binder in the dosage form used. Typically, the composition is administered until the desired effect of the drug is achieved. Thus the composition may be administered in a single dose, or in multiple doses (at the same or different concentrations/doses) over a period of time, or continuously infused. It would be routine to accurately determine the appropriate dosage. Appropriate dosages can be estimated by using appropriate dosing response data.

The route of administration of the pharmaceutical composition is in accordance with known methods, such as oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, intramedullary, intracerobrospinal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal routes, by sustained release systems or by implantation devices. Intravenous bolus injection or continuous infusion may be used if desired.

Alternatively, topical administration may be achieved by implanting a membrane, sponge or other suitable material which absorbs or encapsulates the desired molecule. Where a graft device is used, the graft may be administered to any suitable combination or organ, and the desired molecule may be delivered by diffusion, timed release bolus, or sustained administration.

In some cases, it may be desirable to use the pharmaceutical composition ex vivo (ex vivo). In this manner, cells, tissues or organs are removed from the patient and then exposed to the pharmaceutical composition and transplanted back into the patient.

In other cases, where the binding agent is a polypeptide molecule, certain cells can be genetically manipulated using methods described herein and then transplanted into a subject to express and secrete the polypeptide. The cell may be an animal cell or a human cell, or may be an autologous cell, a heterologous cell or a heterologous cell. Optionally, immortalized cells may be used. To reduce the chance of an immune response, these cells may be encapsulated to avoid infiltration into surrounding tissues. The capsule material is typically a biocompatible, semi-permeable polymeric shell or envelope that must permit the release of proteins and prevent the destruction of cells by the patient's immune system or other environmentally harmful factors.

Combination therapy

Specific binding agents of the invention may be used in combination with other therapeutic approaches in the treatment of Ang-2 related diseases. These treatments include radiotherapy, chemotherapy, and targeted therapies, as described below. Other combination therapies not specifically listed herein are also contemplated by the present invention.

Thus, the invention encompasses the combined use of one or more specific binding agents of the invention and one or more other suitable drugs for administration to the same patient in a manner suitable for each drug. This includes the simultaneous administration of a specific binding agent of the invention and one or more suitable drugs. As used herein, "concurrently administered" means that one or more specific binding agents of the invention and one or more other suitable drugs are administered substantially simultaneously.

As used herein, "non-concurrently administered" refers to the administration of one or more selective binding agents of the invention with another or more suitable agents at different times, whether overlapping or not, and optionally in a sequential order. This includes, but is not limited to, sequential administration of the combination (e.g., pre-treatment, post-treatment, or overlapping treatment), and alternating administration of the drugs, as well as chronic administration of one component and intermittent administration of the other. The various ingredients may be administered in the same formulation or in separate formulations, and the routes of administration may be the same or different.

In certain embodiments, the combination therapy comprises the use of a specific binding agent capable of binding Ang-2, and at least one other anti-angiopoietin agent. Such agents include, but are not limited to, chemical compositions synthesized in vitro, antibodies, antigen binding regions, radionuclides, and combinations thereof. In certain embodiments, the drug used may inhibit or stimulate its target (e.g., receptor, enzyme activation or inhibition), thereby initiating cell death or disrupting cell growth.

Chemotherapy may use antineoplastic drugs, such as alkyl compounds, including: nitrogen mustards such as dichloromethyl diethylamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas such as nitrosourea nitrogen mustard (BCNU), lomustine (CCNU), semustine (methyl-CCNU); ethyleneimine/methyl melamines such as Triethylenemelamine (TEM), triethylenethiophosphoramide (thiotepa), hexamethylmelamine (HMM, hexamethylmelamine); alkyl sulfonates such as busulfan; triazines such as Dacarbazine (DTIC); antimetabolites include folic acid analogs such as methotrexate and trimetrexate; pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytarabine (AraC, cytarabine), 5-azacytidine, 2' -difluorodeoxycytidine; purine analogs such as 6-mercaptopurine, 6-thioguanine, imidazole mercaptopurine, 2' -deoxysynethamycin (pentostatin), erythro-9- (2-hydroxy-3-nonyl) adenine (EHNA), fludarabine phosphate, 2-chlorodeoxyadenosine (cladribine), 2-CdA; natural products include antimitotic drugs such as paclitaxel (paclitaxel), vinca alkaloids including Vinblastine (VLB), vincristine and vinorelbine (vinorelbine), docetaxel, Estramustine (Estramustine) and Estramustine phosphate; epipodophyllotoxins (ppipodophyllotoxins) such as etoposide and teniposide; antibiotics such as spinosyn D, daunomycin (rubicin), doxorubicin, mitoxantrone, demethoxydaunorubicin, bleomycin, plicamycin (mithramycin), mitomycin C, actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; various drugs include platinum complexes such as cisplatin and cisplatin, anthraquinones (anthraquinones) such as mitoxantrone, substituted ureas such as hydroxyurea, procarbazine derivatives including N-procarbazine (MIH) and procarbazine; adrenocortical suppressants such as mitotane (o, p' -DDD) and aminoglutethimide; hormones and antagonists include adrenocorticosteroids such as prednisone and equivalents, dexamethasone, and aminoglutethimide; progesterone such as progesterone caproate monoester, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; male hormones including testosterone propionate and fluantel methyltestosterone/equivalent; antiandrogens such as flutamide, gonadotropin-releasing hormone analogues and leuprolide; and non-steroidal antiandrogens such as flutamide.

Cancer therapy (which may be by administration of a specific binding agent to Ang-2) also includes, but is not limited to, targeted therapies as described herein. Targeted therapies such as, but not limited to, the use of therapeutic antibodies. Therapeutic antibodies include, for example, but are not limited to, murine antibodies, murine-human chimeric antibodies, CDR-grafted antibodies, humanized antibodies, or fully human antibodies, as well as synthetic antibodies, including, but not limited to, antibodies screened from antibody libraries. Antibodies include, for example, but are not limited to, antibodies that bind to cell surface proteins Her2, CDC20, CDC33, mucin-like glycoproteins (mucin-like glycoproteins), and Epidermal Growth Factor Receptor (EGFR) on tumor cells, and optionally produce cytostatic and/or cytotoxic effects on tumor cells displaying these proteins. Antibodies also include, e.g., HERCEPTIN^TMTrastuzumab (which can be used to treat breast cancer and other cancers), ITUXAN^TM(rituximab)、ZEVALIN^TM(ibritumomab tiuxetan), GLEEVEC^TMAnd LYMPHOCIDE^TM(epratuzumab) (which can be used to treat non-hodgkin's tumors and other cancers). Some examples of antibodies also include ERBITUX^TM(IMC-C225)、IRESSA^TM(ertinolib)、BEXXAR^TM(iodo-131 tositumomab), KDR (kinase domain receptor) inhibitors, anti-VEGF antibodies and antagonists (e.g. AVASTIN) ^TMAnd VEGAF-TRAP), anti-VEGF receptor antibodies and antigen binding regions, anti-Ang-1 antibodies and antigen binding regions, antibodies to Tie-2 and other Ang-1/Ang-2 receptors, Tie-2 ligands, antibodies to Tie-2 kinase inhibitors, and(alemtuzumab). In certain embodiments, the anti-cancer agent is a polypeptide capable of selectively causing apoptosis in tumor cells, including but not limited to a TNF-related polypeptide, such as TRAIL (TNF receptor apoptosis inducing ligand).

In certain embodiments, suitable anti-cancer drugs are known to have anti-angiogenic effects. Some of these drugs include, but are not limited to, IL-8, Campath^TMB-FGF, FGF antagonists, Tek antagonists (Cerretti et al, U.S. publication)No.2003/0162712, Cerretti et al, U.S. Pat. No.6,413,932; cerretti et al, U.S. Pat. No.6,521,424, all incorporated herein by reference), anti-TWEAK drugs (including, but not limited to, antibodies and antigen binding regions), soluble TWEAK receptor antagonists (Wiley, U.S. Pat. No.6,727,225), ADAM disintegrins (or fragments thereof to inhibit the binding of integrins to their ligands, Fanslow et al, U.S. publication No.2002/0042368), anti-eph receptor and anti-hrepsin antibodies (and antigen binding regions) or antagonists (U.S. Pat. Nos. 5,981,245, 5,728,813, 5,969,110, 6,596,852, 6,232,447, 6,057,124 and their patent family members), anti-VEGF drugs (e.g., antibodies and antigen binding regions that specifically bind VEGF, or soluble VEGF receptors or their ligand binding regions) as described herein such as ASAVTIN ^TMOr VEGF-TRAP^TManti-VEGF receptor drugs (e.g., antibodies and antigen binding regions that specifically bind thereto), EGFR inhibitors (e.g., antibodies and antigen binding regions that specifically bind thereto) such as panitumumab, IRESSA^TM(gefitinib)，TARCEVA^TM(erlotinib), anti-Ang-1 and anti-Ang-2 drugs (such as antibodies and antigen binding regions that specifically bind to it or antibodies and antigen binding regions that specifically bind to their receptors (such as Tie-2/TEK)), and anti-Tie-2 kinase inhibitors (antibodies that specifically bind to growth factors and inhibit their activity, such as stem cell growth factor inhibitors (HGF), also known as scatter factor (scatter factor), and antibodies and antigen binding regions that specifically bind to their receptor "c-met"), anti-PDGF-BB antagonists, antibodies and antigen binding regions to PDGF-BB ligands, PDGFR kinase inhibitors.

In certain embodiments, the cancer treatment agent is an angiopoietin inhibitor. Some of these include, but are not limited to, SD-7784(Pfizer, USA); cilengitide (cilengitide) (Merck KGaA, Germany, EPO 770622); pegaptanibocantisdium, (Gilead Sciences, USA); tumor angiostatic peptide (Alphastatin), (BioActa, UK); M-PGA, (Celgene, USA, US 5712291); ilomastat (ilomastat) (ariva, USA, US 5892112); semaxanib, (Pfizer, USA, US 5792783); vatalanib, (Novartis, Switzerland); 2-methoxyestradiol, (entrmed, USA); TLC ELL-12, (Elan, Ireland); anecortave acetate, (Alcon, USA); α -D148Mab, (Amgen, USA); CEP-7055, (Cephalon, USA); anti-VnMab, (Crucell, Netherlands); DAC: anti-angiogenesis, (ConjuChem, Canada); angiocidin, (inkinene Pharmaceutical, USA); KM-2550, (KyowaHakko, Japan); SU-0879, (Pfizer, USA); CGP-79787, (Novartis, Switzerland, EP 970070); ARGENT technology, (Ariad, USA); YIGSR-Stealth, (Johnson & Johnson, USA); fibrinogen-E fragment, (bioatta, UK); angiogenesis inhibitors, (Trigen, UK); TBC-1635, (Encysivepharmaceuticals, USA); SC-236, (Pfizer, USA); ABT-567, (Abbott, USA); metastatin, (entrmed, USA); angiogenesis inhibitors (Tripep, Sweden); maspin, (Sosei, Japan); 2-methoxyestradiol, (Oncology sciences corporation, USA); ER-68203-00, (IVAX, USA); flutolamine (Benefin), (Lane Labs, USA); tz-93, (Tsumura, Japan); TAN-1120, (Takeda, Japan); FR-111142, (Fujisawa, Japan, JP 02233610); platelet fourth factor, (RepliGen, USA, EP 407122); vascular epithelial growth factor inhibitors, (Borean, Denmark); cancer therapy (cancer therapy), (univariate of South Carolina, USA); bevacizumab (pINN), (Genentech, USA); angiogenesis inhibitors, (SUGEN, USA); XL 784, (Exelixis, USA); XL 647, (Exelixis, USA); MAb, α 5 β 3 integrin, second generation, (Applied Molecular Evolution, USA and medimmunee, USA); gene therapy, retinopathy, (Oxford BioMedica, UK); enzastaurin hydrochloride (USAN), (Lilly, USA); CEP 7055, (Cephalon, USA and Sanofi-Synthelabo, France); BC1 (Genoa Institute of Cancer Research, Italy); angiogenesis inhibitors, (Alchemia, Australia); VEGF antagonists, (Regeneron, USA); rBPI 21 and BPI-derived anti-angiogenic agents, (XOMA, USA); PI 88, (Progen, Australia); cilengitide (pINN), (Merck KGaA; Munich technical university, Germany, Scripps clinical and Research Foundation, USA); cetuximab (inn), (Aventis, France); AVE 8062, (Ajinomoto, Japan); AS1404, (Cancer Research Laboratory, New Zealand); SG 292(Telios, USA); endostatin, (Boston Childrens Hospital, USA); ATN 161, (Attenuon, USA); human angiostatin (angiostatin), (Boston Childrens Hospital, USA); 2-methoxyestradiol, (Boston Childrens Hospital, USA); ZD 6474, (AstraZeneca, UK); ZD 6126, (Angiogene Pharmaceuticals, UK); PPI2458, (Praecis, USA); AZD 9935, (AstraZeneca, UK); AZD 2171, (AstraZeneca, UK); vatalanib (pINN), (Novartis, Switzerland and Schering AG, Germany); tissue factor assault inhibitors (entrmed, USA); pegaptanib (Pinn), (Gilead Sciences, USA); xanthorrhizol (xanthorrhizol), (Yonsei unity, South Korea); the genetic vaccine VEGF-2, (Scripps clinical and research Foundation, USA); SPV5.2, (Supratek, Canada); SDX 103, (unity of California at San Diego, USA); PX 478, (ProlX, USA); METAS TATIN, (EntreMed, USA); troponin I (Harvard unitarity, USA); SU 6668, (SUGEN, USA); OXI 4503, (OXiGENE, USA); o-guanidine, (Dimensional Pharmaceuticals, USA); motoporamine C, (british columbia university, Canada); CDP 791, (Celltech Group, UK); atiprimod (pinn), (GlaxoSmithKline, UK); e7820, (Eisai, Japan); CYC381, (Harvard university, USA); AE 941, (Aeterna, Canada); vaccine, angiogenesis (USA); urokinase plasminogen activator inhibitor (Dendreon, USA); oglufanide (pINN), (Melmote, USA); HIF-1alfa inhibotors, (Xenova, UK); CEP 5214, (Cephalon, USA); BAY RES 2622, (Bayer, Germany); angiocidin, (InKine, USA); a6, (angustrom, USA); KR 31372 (Korea Research Institute of chemical technology, South Korea); GW 2286, (GlaxoSmithKline, UK); EHT0101, (exohit, France); CP 868596, (Pfizer, USA); CP 564959, (OSI, USA); CP 547632, (Pfizer, USA); 786034, (GlaxoSmithKline, UK); KRN 633, (Kirin Brewery, Japan); drug delivery system, intraocular, 2-methoxyestradiol (drug delivery system, intraocularly, 2-methoxyestradiol) (EntreMed, USA); angionex, (Maastricht unity, Netherlands, and Minnesotaunity, USA); ABT 510, (Abbott, USA); AAL 993, (Novartis, Switzerland); VEGI, (ProteomTech, USA); tumor necrosis factor alpha inhibitors, (National Institute on Aging, USA); SU 11248, (Pfizer, USA and SUGENUSA); ABT 518, (Abbott, USA); YH16, (Yantai Rongchang, China); s-3APG, (Boston Childrens Hospital, USA and Entremed, USA); MAb, KDR, (immunocone Systems, USA); MAb, α 5 β 1, (Protein Design, USA); KDR kinase inhibitors (Celltech Group, UK, and Johnson & Johnson, USA); GFB 116 (South Florida unity, USA and Yale unity, USA); CS706, (Sankyo, Japan); combretastatin a4 prodrug, (Arizona stateunity, USA); chondroitin AC, (IBEX, Canada); BAY RES 2690, (Bayer, Germany); AGM 1470, (Harvard uneverrity, USA, Takeda, Japan, andTAP, USA); AG 13925, (agoron, USA); ammonium tetrathiomolybdate, (university of michigan, USA); GCS 100, (Wayne State unity, USA) CV 247, (Ivymedical, UK); CKD 732, (Chong Kun Dang, South Korea); MAb, vascular epithelial growth factor (Xenova, UK); irsogladine (inn), (Nippon Shinyaku, Japan); RG 13577, (Aventis, France); WX 360, (Wilex, Germany); squalamine (pINN), (Genaera, USA); RPI 4610, (Sirna, USA); cancercherapey, (Marinova, Australia); heptanase inhibitors, (InSight, Israel); KL 3106, (Kolon, South Korea); honokiol, (organism unity, USA); ZK CDK, (Schering AG, Germany); ZK Angio, (Schering AG, Germany); ZK 229561, (Novartis, Switzerland, and Schering AG, Germany); XMP 300, (XOMA, USA); VGA 1102, (Taisho, Japan); VEGF receptor modulators, (Pharmacopeia, USA); VE-cadherin-2 antagonists, (Imclone Systems, USA); vasostatin, (National Institutes of Health, USA); vaccine, Flk-1, (Imclone Systems, USA); TZ 93, (Tsumura, Japan); tumstatin, (Beth Israel Hospital, USA); partial length soluble FLT 1(vascular endothelial growth factor receiver 1), (Merck & Co, USA); tie-2 ligand, (Regeneron, USA); thrombospondin 1 inhibitors, (allegheny health, edition and Research Foundation, USA); (ii) a 2-benzenesulfonamide, 4- (5- (4-chlorophenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl) -; arriva; and C-met. ave 8062((2S) -2-amino-3-hydroxy-N- [ 2-methoxy-5- [ (1Z) -2- (3, 4, 5-trimethoxyphenyl) vinyl ] phenylpropionamide monohydrochloride); metelilimumab (pINN) (immunoglobulin G4, anti- (human transforming growth factor beta.1 (human monoclonal antibody CAT192. gamma. 4-chain)), human disulfide monoclonal antibody CAT192. kappa. -chain dimer); flt3 ligand; a CD40 ligand; interleukin-2; interleukin-12; 4-1BB ligand; an anti-4-1 BB antibody; TNF antagonists and TNF receptor antagonists, including TNFR/Fc, TWEAK antagonists and TWEAK-R antagonists, including TWEAK-R/Fc; TRAIL; VEGF antagonists include anti-VEGF antibodies; VEGF receptor (including VEGF-R1 and VEGF-R2, also known as Flt1 and Flk1 or KDR) antagonists; CD148 (also known as DEP-1, ECRTP, and PTPRJ, see Takahashi et al, J.Am.Soc.Nephrol.10: 2135-45(1999), incorporated herein by reference) antagonists; an inhibitor of thrombospondin 1, and an inhibitor of Tie-2 or one of the Tie-2 ligands (e.g., Ang-2), or both. Many Ang-2 inhibitors are known in the art, including certain anti-Ang-2 antibodies (U.S. patent application No.20030124129 (corresponding to PCT application No. wo03/030833) and U.S. patent No.6,166,185, the contents of which are incorporated herein by reference). Also known in the art are Ang-2 peptibodies (peptibodies) as can be found in U.S. patent application No.20030229023 (corresponding to PCT application No. wo03/057134) and U.S. patent application No.20030236193, the contents of which are incorporated herein by reference.

Certain cancer therapeutic agents include, but are not limited to, thalidomide and its analogs (N- (2, 6-dioxo-3-piperidyl) phthalimide); sodium sulfated polysaccharides (sulfated polysaccharides); TAN 1120 (8-acetyl-7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-1-methoxy-10- [ [ octahydro-5-hydroxy-2- (2-hydroxypropyl) -4, 10-dimethylpyran [3, 4-d ] -1, 3, 6-dioxazocine (dioxazocin) -8-yl ] oxy ] -5, 12-naphthalenedione); surfista (7, 7' - [ carbonylbis [ imino (1-methyl-1H-pyrrole-4, 2-diyl) carbonylimino ] ] bis-1, 3-naphthalenedisulfonic acid tetrasodium salt); SU 302; SU 301; SU 1498((E) -2-cyano-3- [ 4-hydroxy-3, 5-bis (1-methylethyl) phenyl ] -N- (3-phenylpropyl) -2-propionamide); SU 1433(4- (6, 7-dimethyl-2-quinoxalinyl) -1, 2-benzenediol); ST 1514; SR 25989; soluble Tie-2; SERM derivatives, Pharmos; semaxanib (pINN) (3- [ (3, 5-dimethyl-1H-pyrrol-2-yl) methylene ] -1, 3-dihydro-2H-indol-2-one); s836; RG 8803; RESTIN; r440 (3- (1-methyl-1H-indol-3-yl) -4- (1-methyl-6-nitro-1H-indol-3-yl) -1H-pyrrole-2, 5-dione); r123942 (1- [6- (1, 2, 4-thiadiazol-5-yl) -3-pyridazinyl ] -N- [3- (trifluoromethyl) phenyl ] -4-piperidinamine); prolyl hydroxylase inhibitors; progression accelerated genes (progression accelerated genes); prilinostat (INN) ((S) -2, 2-dimethyl-4- [ [ p- (4-pyridinyloxy) phenyl ] sulfonyl ] -3-thiomorpholinoietic hydroxamic acid); NV 1030; NM 3 (8-hydroxy-6-methoxy- α -methyl-1-oxo-1H-2-benzopyranyl-3 acetic acid); NF 681; NF 050; MIG; METH 2; METH 1; manassantin B (α - [1- [4- [5- [4- [2- (3, 4-dimethoxyphenyl) -2-hydroxy-1-methylethoxy ] -3-methoxyphenyl ] tetrahydro-3, 4-dimethyl-2-furan ] -2-methoxyphenoxy ] ethyl ] -1, 3-benzodioxole-5-methanol); KDR monoclonal antibody; α 5 β 3 integrin monoclonal antibody; LY 290293 (2-amino-4- (3-pyridyl) -4H-naphtho [1, 2-b ] pyran-3-carbonitrile); KP 0201448; KM 2550; an integrin-specific peptide; INGN 401; GYKI 66475; GYKI 66462; greenstatin (101-; gene therapy agents for rheumatoid arthritis, prostate cancer, ovarian cancer, glioma, endostatin, intestinal cancer, ATF BTPI, anti-angiogenic genes, angiogenesis inhibitors or antagonists; gelatinase inhibitor, FR 111142(4, 5-dihydroxy-2-hexenoic acid 5-methoxy-4- [ 2-methyl-3- (3-methyl-2-butenyl) oxiranyl ] -1-oxaspiro [2.5] oct-6-yl ester); forfenimex (pINN) (S) - α -amino-3-hydroxy-4- (hydroxymethyl) phenylacetic acid); fibronectin antagonists (1-acetyl-L-prolyl-L-histidyl-L-seryl-L-cysteinyl-L-asparagine); inhibitors of fibroblast growth factor receptors; (ii) a tissue mother cell growth factor antagonist; FCE 27164(7, 7' - [ carbonylbis [ imino (1-methyl-1H-pyrrole-4, 2-diyl) carbonylimino ] ] bis-1, 3, 5-naphthalenetrisulfonic acid hexasodium salt); FCE 26752(8, 8' - [ carbonylbis [ imino (1-methyl-1H-pyrrole-4, 2-diyl) carbonylimino ] ] bis-1, 3, 6-naphthalenetrisulfonic acid); endothelial monocyte-active polypeptide II; a VEGFR antisense oligonucleotide; anti-angiogenic and liver trophic factors; an ANCHOR antagonist; endostatin; del-1 angiogenic protein; CT 3577; a contortstatin; a CM 101; chondroitinase AC; a CDP 845; CanStatin; BST 2002; BST 2001; BLS 0597; BIBF 1000; ARRESTIN; apomigren (1304-1388-XV collagen-like (human gene COL15A 1. alpha. chain precursor)); angiostatin; aaATIII; a36; 9 α -fluoromethoxyprogesterone acetate ((6- α) -17- (acetoxy) -9-fluoro-6-methyl-pregn-4-ene-3, 20-dione); 2-methyl-2-phthalimido-glutaric acid (2- (1, 3-dihydro-1-oxo-2H-isoindol-2-yl) -2-methylglutaric acid); monoclonal antibody BC-1 marked by Yttrium 90; semaxanib (3- (4, 5-dimethylpyrrol-2-ylmethylene) indol-2-one) (C15H14N 2O); PI 88 (sulfated mannopentose phosphate); alvocidib (4H-1-benzopyran-4-one, 2- (2-chlorophenyl) -5, 7-dihydroxy-8- (3-hydroxy-1-methyl-4-piperazinyl) -cis- (-) - (C21H20C1N O5); e7820; SU 11248(5- [ 3-fluoro-2-oxo-1, 2-indolino- (3Z) -ylidenemethyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) amide) (C22H27F N4O 2); squalamine (cholestane-7, 24-diol, 3- [ [3- [ (4-aminobutyl) aminopropyl ] amino ] -, 24- (hydrogensulfate), (3. beta., 5.a., 7.a.) - (C34H65N3O 5S); eriochrome Black T; AGM 1470 (carbamic acid, (chloroacetyl) -, 5-methoxy-4- [ 2-methyl-3- (3-methyl-2-butenyl) oxiranyl ] -1-oxaspiro [2, 5] oct-6-yl ester [3R- [3 α, α (2R, 3R), 5 β,6 β ] ]) (C19H28C1N O6); AZD 9935; BIBF 1000; AZD 2171; ABT 828; KS-interleukin-2; utoglobin; a6; NSC 639366 fumaric acid (1- [3- (diethylamino) -2-hydroxypropylamino ] -4- (oxapropane (oxoran) -2-ylmethylamino) anthraquinone) (c24h29n3o4. c4h4o4); an ISV 616; anti-ED-B fusion protein; HUI 77; troponin I; BC-1 monoclonal antibody; SPV 5.2; ER 68203; CKD731(3- (3, 4, 5-trimethoxyphenyl) -2(E) -acrylic acid (3R, 4S, 5S, 6R) -4- [2(R) -methyl-3 (R) -3(R) - (3-methyl-2-butenyl) oxapropan-2-yl ] -5-methoxy-1-oxaspiro [ [2.5] oct-6-yl ester) (C28H38O 8); IMC-1C 11; aaATIII; SC 7; a CM 101; angiocol; kringle 5; CKD 732(3- [4- [2- (dimethylamino) ethoxy ] phenyl ] -2(E) -acrylic acid) (C29H41N O6); u995; a Canstatin; SQ 885; CT 2584(1- [11- (dodecylamino) -10-hydroxyundecyl ] -3, 7-dimethylxanthine) (C30H55N5O 3); salmosin; EMAP II; TX 1920(1- (4-methylpiperazino) -2- (2-nitro-1H-1-imidazoloyl) -1-ethanone) (C10H15N5O 3); an alpha-v beta-x inhibitor; CHIR 11509(N- (1-propynyl) glycyl- [ N- (2-naphthyl) ] glycyl- [ N- (carbamoylmethyl) ] glycine bis (4-methoxyphenyl) methylamide) (C36H37N5O 6); BST 2002; BST 2001; b0829; FR 111142; 4, 5-dihydroxy-2 (E) -hexenoic acid (3R, 4S, 5S, 6R) -4- [1(R), 2(R) -epoxy-1, 5-dimethyl-4-hexenyl ] -5-methoxy-1-oxaspiro [2.5] octan-6-yl ester (C22H34O 7); and kinase inhibitors including, but not limited to, N- (4-chlorophenyl) -4- (4-pyridylmethyl) -1-phthalazine amine; 4- [4- [ [ [ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino ] carbonyl ] amino ] phenoxy ] -N-methyl-2-pyridinecarboxamide; n- [2- (diethylamino) ethyl ] -5- [ (5-fluoro-1, 2-dihydro-2-oxo-3H-indol-3-ylidene) methyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxamide; 3- [ (4-bromo-2, 6-difluorophenyl) methoxy ] -5- [ [ [ [4- (1-pyrrolidinyl) butyl ] amino ] carbonyl ] amino ] -4-isothiazolecarboxamide; n- (4-bromo-2-fluorophenyl) -6-methoxy-7- [ (1-methyl-4-piperidinyl) methoxy ] -4-quinazolinamine; 3- [5, 6,7, 13-tetrahydro-9- [ (1-methylethoxy) methyl ] -5-oxo-12H-indeno [2, 1-a ] pyrrolo [3, 4-c ] carbazol-12-yl ] propyl ester N, N-dimethyl-glycine; n- [5- [ [ [5- (1, 1-dimethylethyl) -2-oxazolyl ] methyl ] thio ] -2-thiazolyl ] -4-piperidinecarboxamide; n- [ 3-chloro-4- [ (3-fluorophenyl) methoxy ] phenyl ] -6- [5- [ [ [2- (methylsulfonyl) ethyl ] amino ] methyl ] -2-furyl ] -4-quinazolinamine; 4- [ (4-methyl-1-piperazine) methyl ] -N- [ 4-methyl-3- [ [4- (3-pyridinyl) -2-piperazine ] amino ] -phenyl ] benzamide; n- (3-chloro-4-fluorophenyl) -7-methoxy-6- [3- (4-morpholinyl) propoxy ] -4-quinazolinamine; n- (3-ethylphenyl) -6, 7-bis (2-methoxyethoxy) -4-quinazolinamine; n- (3- ((((2R) -1-methyl-2-pyrrolidinyl) methyl) oxy) -5- (trifluoromethyl) phenyl) -2- ((3- (1, 3-oxazepin-5-yl) phenyl) amino) -3-pyridinecarboxamide; 2- (((4-fluoromethyl) methyl) amino) -N- (3- (((((2R) -1-methyl-2-pyrrolidinyl) methyl) oxy) -5- (trifluoromethyl) phenyl) -3-pyridinecarboxamide; n- [3- (azetidin-3-ylmethoxy) -5-trifluoromethyl) phenyl ] -2- (4-fluoro-benzylamino) -nicotinamide; 6-fluoro-N- (4- (1-methylethyl) phenyl) -2- ((4-picolyl) amino) -3-pyridinecarboxamide; 2- ((4-picolyl) amino) -N- (3- (((2S) -2-pyrrolidinylmethyl) oxy) -5- (trifluoromethyl) phenyl) -3-pyridinecarboxamide; n- (3- (1, 1-dimethylethyl) -1H-pyrazol-5-yl) -2- ((4-picolyl) amino) -3-pyridinecarboxamide; n- (3, 3-dimethyl-2, 3-dihydro-1-benzofuran-6-yl) -2- ((4-picolyl) amino) -3-pyridinecarboxamide; n- (3- ((((2S) -1-methyl-2-pyrrolidinyl) methyl) oxy) -5- (trifluoromethyl) phenyl) -2- ((4-picolyl) amino) -3-pyridinecarboxamide; 2- ((4-picolyl) amino) -N- (3- ((2- (1-pyrrolidinyl) ethyl) oxy) -4- (trifluoromethyl) phenyl) -3-pyridinecarboxamide; n- (3, 3-dimethyl-2, 3-dihydro-1H-indol-6-yl) -2- ((4-picolyl) amino) -3-pyridinecarboxamide; n- (4- (pentafluoroethyl) -3- (((2S) -2-pyrrolidinylmethyl) oxy) phenyl) -2- ((4-pyridylmethyl) amino) -3-pyridinecarboxamide; n- (3- ((3-azetidinylmethyl) oxy) -5- (trifluoromethyl) phenyl) -2- ((4-picolyl) amino) -3-pyridinecarboxamide; n- (3- (4-piperidinyloxy) -5- (trifluoromethyl) phenyl) -2- ((2- (3-pyridine) ethyl) amino) -3-pyridinecarboxamide; n- (4, 4-dimethyl-1, 2, 3, 4-tetrahydro-isoquinolin-7-yl) -2- (1H-indol-6-ylamino) -nicotinamide; 2- (1H-indol-6-ylamino) -N- [3- (1-methylpyridin-2-ylmethoxy) -5-trifluoromethyl-phenyl ] -nicotinamide; n- [1- (2-dimethylamino-acetyl) -3, 3-dimethyl-2, 3-dihydro-1H-indol-6-yl ] -2- (1H-indazol-6-ylamino) -nicotinamide; 2- (1H-indazol-6-ylamino) -N- [3- (pyridin-2-ylmethoxy) -5-trifluoromethyl-phenyl ] -nicotinamide; n- (1-acetyl-3, 3-dimethyl-2, 3-dihydro-1H-indol-6-yl) -2- (1H-indazol-6-ylamino) -nicotinamide; n- (4, 4-dimethyl-1-oxo-1, 2, 3, 4-tetrahydro-isoquinolin-7-yl) -2- (1H-indazol-6-ylamino) -nicotinamide; n- [4- (tert-butyl) -3- (3-piperidinopropyl) phenyl ] [2- (1H-indazol-6-ylamino) (3-pyridyl) ] carboxamide; n- [5- (tert-butyl) isoxazol-3-yl ] [2- (1H-indazol-6-ylamino) (3-pyridyl) ] carboxamide; and N- [4- (tert-butyl) phenyl ] [2- (1H-indazol-6-ylamino) (3-pyridyl) ] carboxamide and kinase inhibitors, see U.S. patent No.6,258,812; 6,235,764, respectively; 6,630,500, respectively; 6,515,004, respectively; 6,713,485, respectively; 5,521,184, respectively; 5,770,599, respectively; 5,747,498; 5,990,141, respectively; U.S. publication No. us 20030105091; and PCT publication No. wo 01/37820; WO 01/32651; WO 02/68406; WO 02/66470; WO 02/55501; WO 04/05279; WO 04/07481; WO 04/07458; WO 04/09784; WO 02/59110; WO 99/45009; WO 98/35958; WO 00/59509; WO 99/61422; WO 00/12089; and WO00/02871, both incorporated herein by reference.

Therapeutic agents that can be used in combination with growth factors include cytokines, growth factors or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor and erythropoeitin. Other components may include known angiopoietins such as Ang-1, -2, -4, -Y and/or human angiopoietin polypeptides, and/or Vascular Endothelial Growth Factor (VEGF). (P50-58) growth factors include angiogenin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor-IA, bone morphogenetic protein receptor IB, brain neurotrophic factor, ciliary neurotrophic factor receptor, cytokine-induced neutrophilic chemokine-1, alpha-glucosidase, alpha-2, alpha-glucosidase, alpha-2, cytokine-induced neutrophile chemotactic factor, endothelial cell growth factor, endothelin-1, epidermal growth factor, neutrophil chemotactic substance derived from epithelial cells, fibroblast growth factor-4, fibroblast growth factor-5, fibroblast growth factor-6, f-fibroblast growth factor-7, fibroblast growth factor-8 b, fibroblast growth factor-8 c, fibroblast growth factor-9, fibroblast growth factor-10, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell-derived neurotrophic factor-1, glial cell-derived neurotrophic factor-2, growth-related protein-2, epidermal growth factor, fibroblast chemotactic substance derived from epithelial cells, fibroblast growth factor-8, fibroblast growth factor-9, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell-derived neurotrophic factor-1, glial cell-derived neurotrophic factor-2, growth-related protein-2, epidermal, Growth-related protein-3, heparin-binding epidermal growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor receptor-1, nerve growth factor, factor-in-quartz receptor, neurotrophic factor-3, neurotrophic factor-4, placenta growth factor-2, platelet-derived endothelial growth factor, platelet-derived growth factor A chain, platelet-derived growth factor AA, platelet-derived growth factor AB, platelet-derived growth factor B chain, platelet-derived growth factor BB, platelet-derived growth factor receptor-1, Platelet-derived growth factor receptor-2, stimulating factor for promoting B-cell proliferation, hepatocyte factor receptor, transforming growth factor-1, transforming growth factor-2, transforming growth factor-3, transforming growth factor-1.2, transforming growth factor-4, transforming growth factor-5, latent transforming growth factor-1, transforming growth factor-1 binding protein I, transforming growth factor-1 binding protein II, transforming growth factor-1 binding protein III, tumor necrosis factor type I receptor (TNF-R1), tumor necrosis factor type II receptor (TNF-R2), urokinase-type plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments of the above molecules.

Of course, specific binding agents of the present invention may be administered in combination with one or more anti-inflammatory agents. As used herein, "anti-inflammatory factor" refers generally to any drug that reduces the inflammatory response or swelling of a patient. Many examples of anti-inflammatory agents are mentioned herein, although there are certainly other anti-inflammatory agents not mentioned, but such agents are also contemplated within the scope of the present invention.

The anti-inflammatory factor may be, for example, a compound that inhibits the interaction of inflammatory cytokines with their receptors. Cytokine inhibitors that may be used in combination with specific binding agents of the invention include, for example, TGF- β antagonists (e.g., antibodies) and antagonists (e.g., antibodies) directed against interleukins involved in inflammation. These interleukins are described herein, and preferably include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, and IL-18. See Feghali, et al, Frontiers in biosci, 2: 12-26(1997).

Specific binding agents of the invention may be administered in combination with a first class of protein kinase a inhibitors to enhance T cell proliferation in HIV-infected patients receiving antiretroviral therapy.

Specific binding agents of the invention may also be used in combination with Nerve Growth Factor (NGF) to treat certain diseases. These include neurodegenerative diseases, spinal cord injury and multiple sclerosis, and also include glaucoma and diabetes.

Preferably, one or more IL-1 inhibitors are used in combination therapy with a specific binding agent of the invention. IL-1 inhibitors include, but are not limited to, peptide fragments of the IL-1 receptor, antibodies directed against IL-1 or IL-1 β or the first class of IL-1 receptor, and recombinant proteins (or modified variants thereof) comprising all or part of the IL-1 receptor, including genetically modified muteins, multimeric forms, and sustained release formulations. Specific antagonists include IL-1ra polypeptides, IL-1 beta converting enzyme (ICE) inhibitors, antagonistic antibodies to the first class of IL-1 receptors, IL-1-binding forms of the first class of IL-1 receptors and the second class of IL-1 receptors, antibodies to IL-1 (including IL-1 α, IL-1 β and other IL-1 family members), and the therapeutic IL-1trap (Regeneron). IL-1ra includes forms of IL-1ra and modified and variant forms described in U.S. Pat. No.5,075,222, including U.S. Pat. No.5,922,573, WO 91/17184, WO 9216221 and WO 9609323. IL-1 beta converting enzyme (ICE) inhibitors include peptidyl and small molecule ICE inhibitors, including those described in PCT patent applications WO 91/15577, WO 93/05071, WO93/09135, WO 93/14777, WO 93/16710, European patent application 0547699. Non-peptidic compounds include such compounds as described in PCT application WO 95/26958, U.S. Pat. No.5,552,400, U.S. Pat. No.6,121,266, and Dolle et al, J.Med.chem., 39, pp.2438-2440 (1996). Other ICE inhibitors are described in U.S. patent nos. 6,162,790, 6,204,261, 6,136,787, 6,103,711, 6,025,147, 6,008,217, 5,973,111, 5,874,424, 5,847,135, 5,843,904, 5,756,466, 5,656,627, 5,716,929. IL-1 binding forms of type I IL-1 receptors and type II IL-1 receptors are described in U.S. Pat. Nos. 4,968,607, 4,968,607, 5,081,228, Re 35,450, 5,319,071, and 5,350,683. Other suitable IL-1 antagonists include, but are not limited to, IL-1 derived peptides, type I IL-1 receptors, that competitively bind to IL-1 signaling receptors. Other antagonists of IL-1 (and other cytokines) are described in U.S. Pat. No.6,472,179.

In addition, TNF inhibitors are also suitable, including, but not limited to, receptor-binding peptide fragments of TNF α, antisense oligonucleotides or ribozymes that inhibit TNF α production, antibodies directed against TNF α, recombinant proteins containing all or part of the TNF α receptor, or modified variants thereof, including chimeric forms of genetically modified variant proteins, and sustained release formulations. TACE (tumor necrosis factor-alpha converting enzyme) inhibitors are also suitable, such as TAPI (Immunex Corp.) and GW-3333X (Glaxo Wellcome Inc.). Inhibition of IgA-alpha₁Molecules formed from AT complexes (as described in EP 0614464B) or antibodies directed against such complexes are also suitable. Other suitable molecules include, but are not limited to, TNF α -inhibiting disaccharides, sulfate derivatives of glucosamine, or other similar carbohydrates, see U.S. patent No.6,020,323, and TNF α peptide inhibitors (U.S. patent nos. 5,641,751 and 5,519,000), peptides containing D-amino acids (U.S. patent No.5,753,628). In addition, inhibitors of TNF α converting enzyme are also suitable. Further to WO 01/03719The medicaments which may be used in combination in the present invention are described.

Other suitable molecules include, but are not limited to, small molecules such as thalidomide or thalidomide analogs, pentoxifylline (pentoxifylline), Matrix Metalloproteinase (MMP) inhibitors, or other small molecules. MMP inhibitors suitable for the present invention include molecules as described in U.S. patent nos. 5,883,131, 5,863,949 and 5,861,510, and alkylpeptidyl compounds as described in U.S. patent No.5,872,146. Other small molecules capable of reducing TNF α production include molecules as described in U.S. patents 5,508,300, 5,596,013, and 5,563,143. Other suitable small molecules include, but are not limited to, MMP inhibitors described in U.S. patent 5,747,514, and 5,691,382, and hydroxamic acid derivatives described in U.S. patent No.5,821,262. Other suitable molecules include, for example, small molecules that inhibit phosphodiesterase IV and TNF α production, such as substituted oxime derivatives (WO 96/00215), quinoline sulfonamide (U.S. Pat. No.5,834,485), aryl furan derivatives (WO 99/18095), heterobicyclic (WO 96/01825; GB 2291422A). Thiazole derivatives which inhibit TNF α and IFN γ (WO 99/15524) and xanthine derivatives which inhibit TNF α and other inflammatory cytokines (see, e.g., U.S. patent nos. 5,118,500, 5,096,906 and 5,196,430) are also suitable. The molecules described in U.S. Pat. No.5,547,979 are also suitable for use in the present invention.

Examples of other Drugs and drug types that may be used in combination therapy include, but are not limited to, antiviral Drugs, antibiotics, analgesics (such as acetaminophen, codeine, propoxyphene naphthalenesulfonate, oxycodone hydrochloride, dihydrocodeinone bitartrate, tramadol), corticosteroids, inflammatory cytokine antagonists, Disease-Modifying Anti-inflammatory-rheumatoid Drugs (DMARDs), non-steroidal Anti-inflammatory Drugs (NSAIDs), slow-acting antirheumatic Drugs (SAARDs).

Disease modifying antirheumatic drugs (DMARDs) include, for example but not limited to Rheumatrex^TM(methotrexate);(etanercept);(infliximab)；Humira^TM(Adalimumab);(Afelmomab); arava (r)^TM(leflunomide); kineret^TM(anakinra); arava (r)^TM(leflunomide); d-penicillamine; sodium thiosulfate; hydroxychloroquine; ridaura^TM(Auranofin); a thiodextrose gold finish (Solganil); lencept (Hoffman-La Roche); CDP870 (Celltech); CDP571(Celltech), and the drugs described in EP 0516785B1, U.S. patent No.5,656,272, EP 0492448a 1; p55 tumor necrosis factor connexin (onercept) (Serono; CAS reg. No. 199685-57-9); mra (chugai); imuran ^TM(imidazothiopurine); an NFKB inhibitor; cytoxan^TM(cyclophosphamide); cyclosporine; hydroxychloroquine sulfate; minocycline; sulfasalazine (sulfasalazine); and gold compounds such as oral gold, gold sodium thiomalate, and gold thioglucose.

Other suitable molecules include, for example, TNFR derivatives derived from the extracellular region of a TNF α receptor molecule (other than p55 and p75TNFR), such as the TNFR described in WO 99/04001, including TNFR-Ig derived from such TNFR. Suitable TNF α inhibitors are also suitable for the uses described herein. This includes not only use as antibodies to TNF α or TNFR as described herein, but also use as TNF α -derived peptides that act as competitive inhibitors of TNF α (as described in U.S. patent No.5,795,859 or No.6,107,273), as TNFR-IgG fusion proteins (e.g., a protein containing the extracellular portion of the p55TNF α receptor), as soluble TNFRs (not IgG fusion proteins), as other molecules that reduce endogenous levels of TNF α, such as TNF α converter inhibitors (see e.g., U.S. patent 5,594,106), or small molecules or TNF α inhibitors. Many of the above have descriptions herein.

Although the optimal dose of TNF antibody will need to be determined by skilled medical personnel based on the specific needs of the patient, a preferred dose range for the antibody against TNF α is 0.1-20mg/kg, more preferably 1-10 mg/kg. Other suitable anti-TNF alpha antibody doses are 0.75-7.5mg/kg body weight.

Specific binding agents and any one or more non-steroidal anti-inflammatory drugs (NSAIDs) may also be used in the present invention. The anti-inflammatory function of NSAIDs depends at least in part on their inhibition of prostaglandin synthesis. (Goodman and Gilman, The Pharmacological Basis of therapeutics, MacMillan 7th Edition (1985)). NSAIDs have 9 characteristic groups: (1) a salicylic acid derivative; (2) a propionic acid derivative; (3) a crude acid derivative; (4) a fenamic acid derivative; (5) a carboxylic acid derivative; (6) a butyric acid derivative; (7) 2-benzothiazines; (8) pyrazole and (9) pyrazolone. NSAIDs include, for example (but not limited to) Anaprox^TM、AnaproxDS^TM(sodium naproxen); ansoid^TM(flurbiprofen sodium); arthrotec^TM(diclofenac sodium + misoprostol); cataflam^TM/Voltaren^TM(diclofenac potassium); clinoril^TM(misoprostol); daypro^TM(oxaprozin)); disalcid^TM(salicylic acid salicylate); dolobid^TM(Diflunisal (Diflunisal)); EC Napro syn^TM(sodium naproxen); feldene^TM(piroxicam)); indocin^TM、Indocin SR^TM(indomethacin); lodine^TM、Lodine XL^TM(etodolac); motrin^TM(ibuprofen); naprelan^TM(naproxen); naprosyn ^TM(naproxen); orudis^TMKetoprofen (ketoprofen)); oruvail^TM(ketoprofen); relafen^TM(nabumetone); tolectin^TM(tolmetin sodium); trilisate^TM(magnesium trisilanide); a Cox-1 inhibitor; cox-2 inhibitors such as Vioxx^TM(rofecoxib)；Arcoxia^tm(etoricoxib) and Celebrex^TM(celecoxib)；Mobic^TM(meloxicam)); bextra^TM(valdecoxib)、Dynastat^TM(sodium naproxen); prexige^TM(lumiracoxib) and nabumetone. Other suitable NSAIDs include (but are not limited to): epsilon-acetamido hexanoic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amitriptyline (amitriptrine), anitrazafen (anitrazafen), atrafenine (antrafenine), bendazac lysine (bendazac lysine), benzohydramine (beprozin), beprolin, bromopaminole (bropamole), bucolone (bucolome), butylbenzoic acid (bufazolac), cycloquinazone (ciprozine), cloxime, daclizamine (dazidazidamine), dibozamamide (deboxamine), detomidine, pyridylbiphenylacetamide, difenoxamine, difilamine, ditazone, dimetazone, emofazone (emofazone), fazidine, fenflurazole (fenflurazone), flufenamide (flufenacetone), flufenacetone (flufenacetone, flufenacetone, Nicotinazole (nicotindole), Nimesulide (Nimesulide), orgotein, orpanoxin (orpanoxin), oxaproperolm, oxapradol (oxapradol), renitoline (paranyline), perisoxazole (perisoxal), piperadone citrate, piperafosxime (pifoxime), naproxen piperazinamic acid (Piproxen), pirazoic acid (pirazolac), pirfenidone, proquinquazone (proquazone), propaxazole (proxazole), thielavin B, tiflozolizole (tiflamizole), timegadine (egtimadine), tolitin (tollectin), topadol (tolpadol), trtamid and drugs allocated as encoded by 480156S, AA AD 861, ONO, AFP 1590, 36802, 860, AP 42, SAI (CHIP) 2842, BW 1, SAL 2811, MRS 31, PSN-11, PSK-11, PSN-11, PSK-, TVX2706, U602 57. UR2301 and WY 41770. NSAID structural relatives having similar analgesic and anti-inflammatory functions may also be included.

Suitable SAARD or DMARDS include, but are not limited to, Sodium allose (allopurin Sodium), Auranofin (Auranofin), aurothioglucose (aurothioglucose), alpha-thiomethylanilide (aurothioglycide), imidazothioprine, Brequinar (Brequinar Sodium), bucinamine (bucillamine), calcium 3-thiogold (aurothio) -2-propanol-1-Sodium sulfonate, chlorambucil, chloroquine, chlorobutyryl (clozarit), cuproxyline (cuproxolin), cyclophosphamide, cyclosporine, dapsone, 15-deoxyspergualin (15-deoxyspergualin), Diacerein (Diacerein), glucosamine, gold salts (such as cycloaureolin, Sodium thiomorpholate, Sodium thiosulfate), hydroxylated urea, procarbazine (benzalkonium), benzalkonium chloride, thiohydramine (6-mebendazole), methoxazole (methoxazole), meclozine, methoprene (methothrene, methothrexate, mechlorethazine, mechlorethamine, thioprimidone, etc, Mycophenolate mofetil (mycophenolate mofetil), calcium thioglycolate, nitrogen mustard, D-penicillamine, hydroxypyridine pyrazoles such as SKNF86002 and SB203580, Rapamycin (Rapamycin), thiols, thymopoietin and vincristine. SAARD or DMARD structural relatives with similar analgesic and anti-inflammatory functions may also be included.

Kinase inhibitors in the signaling cascade are also suitable for use in combination with the specific binding agents of the invention. This includes, but is not limited to, drugs that inhibit P-38 (also known as "RK" or "SAPK-2", Lee et al, Nature, 372: 739 (1994)). P-38 is described as a serine/threonine kinase (see Han et al, Biochimica Biophysica Acta, 1265: 224-. It has been found that P-38 inhibitors interfere with the association between extracellular stimulation and IL-1 and TNF α and secretion in cells involved in blocking signaling by inhibiting signal pathway dependent kinases.

MK-2 inhibitors and tp1-2 inhibitors are also suitable. Also suitable are T cell inhibitors including e.g. ctla-4, CsA, Fk-506, OX40, OX40R-Fc, OX40 antibodies, OX40 ligand, antibodies to OX40 ligand, ck and ZAP 70. Retinoids, including oral retinoids and TGF-beta antagonists are also suitable.

Other suitable drugs for use in combination with the specific binding agents of the present invention include, for example, any one or more salicylic acid derivatives, and ester prodrugs or pharmaceutically acceptable salts thereof. This includes: acexamol (acetaminosalol), aloprine (aloxiprin), aspirin, acetaminophen (benorilate), bromosaligenin, calcium acetylsalicylate, magnesium trisilanide, diflusinal, etisalate (etaneralate), fensal (fendosal), 2, 5-dihydroxybenzoic acid, ethylene glycol salicylate, imidazole salicylate, lysine, mesalamine, morpholine salicylate, 1-naphthyl salicylic acid, salzine, pasamide (parsalmide), phenylacetic salicylic acid, phenyl salicylic acid, salicylamide (salacetamide), salicylamide O-acetic acid, salicylate, and salicylazine. Structural relatives of salicylic acid with similar analgesic and anti-inflammatory functions may also be included. Other suitable drugs include, for example, propionic acid derivatives, and ester prodrugs or pharmaceutically acceptable salts thereof, including: aminoprofen (Alminoprofen), benoxaprofen (benoxaprofen), Bucloxic acid (Bucloxic acid), Carprofen (Carprofen), Right Indoprofen (dexindoprofen), Fenprofen (fenoprofen), Fluoroprofen (fluunoprofen), flurprofen (flubiprofen), flurbiprofen (flurbiprofen), furprofen (furcloprofen), ibuprofen, aluminum ibuprofen, isopropyl ibuprofen, indoprofen (indoprofen), Isoprofen (isoprofen), ketoprofen (ketoprofen), loxoprofen (loxoprofen), misoprofen (miroprofen), naproprionic acid, oxaprozin, pyroprofen (piketoprofen), pimefen (pirprofen), piroprofen (propfen), Proprofen (Pronoprofen), propionic acid, oxyprofen (propiprofen), suprofen (piketoprofen), pimefen (pirprofen), proprofen (proprofen), propiprofen (proprofen), proprofen, suloprofen (suloprofen), suprofen (tioprofen), and tioprofen (tioprofen). Propionic acid structural relatives having similar analgesic and anti-inflammatory functions may also be included. Other suitable drugs include, for example, acetic acid derivatives, and ester prodrugs or pharmaceutically acceptable salts thereof, including: acemetacin, alclofenac (alclofenac), aminophenylacetic acid, bufexamac, indomethacin (cinmetacin), clopidogrel (cyclopirac), dimemethacin (delmetacin), diclofenac sodium (diclofenacacsodium), etodolac (etodolac), FELBINAC (FELBINAC), fenclofenac (fenclofenac), fenac (fenclorac), fenclorac (fenclozic acid), fentiazac (fentiazac), ethyldihydrobenzofuranacetic acid, meglumine (glucametacin), isobutylphenylacetic acid (ibufenac), indomethacin, triafenac (isofenac), Isoxepac, linac (linaclofenac), chlorfenac (zizoxazo), metrizac (isoxemacin), meturamethacin (oxamethacin), pimetacin (acetominometacin), propicin (acetofenac), doxetacin (sultaine), doxazone (sultaine (valcanin). Acetate structural relatives with similar analgesic and anti-inflammatory functions may also be included. Also suitable are fenamic acid derivatives, and ester prodrugs or pharmaceutically acceptable salts thereof, which include: phenylanthranilic Acid, etofenamate, flufenamic Acid, isonixin, meclofenamic Acid, meclofenamate sodium, meclofenamic Acid, mefenamic Acid, niflumic Acid, talniflumate, terfenamate, tolfenamic Acid, and ifenate. Also included are fenamic structural relatives having similar analgesic and anti-inflammatory functions.

Also suitable are carboxylic acid derivatives, and ester prodrugs or pharmaceutically acceptable salts thereof, which include: clavulanic acid (clindanac), Diflunisal (Diflunisal), flufenisal (flufenisal), inoridine, ketorolac (ketorolac) and tinolidine (tinoridine). Structural associations of carboxylic acids having similar analgesic and anti-inflammatory functions may also be included. Also suitable are butyric acid derivatives, and ester prodrugs or pharmaceutically acceptable salts thereof, which include: butapropiophenone (bumadizon), butibufen (butibufen), benbufen (fenbufen), and biphenyl butyric acid (xenbucin). Butyric acid structural relatives having similar analgesic and anti-inflammatory functions may also be included. Also suitable are oxicam derivatives, and ester prodrugs or pharmaceutically acceptable salts thereof, which include: deraxicam (droxicam), enoxicam (enoicam), isoxazolidamide (isoxicam), piroxicam (piroxicam), daminoxicam (Sudoxicam), tenoxicam (tenoxicam) and 4-hydroxy-1, 2-benzothiazine 1, 1-dioxo 4- (N-phenyl) -carboxamide. Oxicam structural associates having similar analgesic and anti-inflammatory functions may also be included. Also suitable are pyrazole derivatives, and ester prodrugs or pharmaceutically acceptable salts thereof, which include: diphenyl-imidazole (difenamizole) and epiprazole (epirizole). Pyrazole structural related compounds having similar analgesic and anti-inflammatory functions may also be included. Also suitable are pyrazolone derivatives, and ester prodrugs or pharmaceutically acceptable salts thereof, which include: azapropazone (apazone), azapropazone (azapropazone), benperidol (benzopiperidone), feprazone (feprazone), mofetizone (mofebutazone), morazone (morazone), oxyphenbutazone, phenylbutazone, pipobuzone (pipobuzone), propylphenazone, raminone (ramifenazone), succinbuperazone (suxzoxazone), and thialinobutazone. Pyrazolone structural relatives having similar analgesic and anti-inflammatory functions may also be included.

In addition, corticosteroid ester prodrugs and pharmaceutically acceptable salts are suitable for use in the treatment of TNF mediated disorders. Corticosteroid ester prodrugs and pharmaceutically acceptable salts include hydrocortisone and compounds derived from hydrocortisone such as 21-acetoxy-pregnenolone, alclomerase, algestrone (algestone), amcinonide (amcinonide), beclomethasone (beclomethasone), betamethasone (beta-methasone), betamethasone valerate (betamethasone valerate), budesonide (budesonide), prednisone (chlorprednisone), clobetasol (clobetasol), clobetasol propionate (clobetasol propionate), clobetasol (clobetasol), clobetasol butyrate (clobetasol), clobetasol (clobetasol), prednisolone (clobetasol), adrenal ketone, cortisone (codevazole), desoxacone (desoxydione), desoxyquinone (desoxychloride), and hydrocortisone (beclomethasone), and compounds derived from hydrocortisone, such as 21-acetoxy-pregnenolone, alclomerase (clobetasol), betasol (clobetasol, betasol, and pharmaceutically acceptable salts thereof? Dexamethasone, diflunisal (diflurasone), diflucortolone (diflucortolone), difluprednate (difluprednate), glycyrrhetinic acid (enoxolone), fluzacort (fluzacort), fluocinolone chloride (fluloronate), flumethasone pivalate (flumethasone pivalate), Flunisolide (Flunisolide), fluocinolone acetonide (flunisolone acetate), fluocinonide (Flunisolide), fluocinolone acetonide acetate (fluxolone acetate), fluocinolone acetonide (fluxolone acetonide), fluocortolide (fluxolone buthyl), fluocortolone (fluxolone caproate), fluocortolone hexanoate (fluxolone hexanoate), fluocortolone valerate (fluxolone valerate), flunisolone (fluxolone), fluocinolone acetate (fluxolone acetate), fluxolone (fluxolone acetate), fluxolone (fluxolone acetate (fluxolone, fluxolone (fluxolone acetate), fluxolone (fluxolone, fluxolone acetate (fluxolone, fluxolone (fluxolone acetate), fluxolone (fluxolone, fluxolone acetate), flunisole, fluxolone (fluxolone acetate), fluxolone (fluxolone, fluxolone (fluxolone acetate), fluxolone (fluxolone acetate, Hydrocortisone 21-sodium succinate, tert-butylacetate, hydrocortisone, maprepredenone (mazipreddone), medrysone (medrysone), methylprednisolone (meprednisone), methylprednisolone, mometasone furoate (mometasone furoate), paramethasone (paramethasone), prednisone ester, prednisolone, 21-diethylaminoacetic acid (diedryaminoacetate) prednisolone, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone 21-m-sodium benzoate, sodium 21-stearoyl glycerate, prednisolone tert-butyl ethyl ester, 21-trimethylprednisolone acetate, prednisone, prednisolone valerate (prednival), prednisolone (prednylidene), prednisolone 21-diethylaminoacetate, prednisolone mercaptohydrocortisone (tixocortol), triamcinolone acetonide (triamcinolone benetonide), and triamcinolone acetonide (triamcinolone hexacetonide). Corticosteroid structural relatives with similar analgesic and anti-inflammatory functions may also be included.

Antimicrobial agents (and ester prodrugs and pharmaceutically acceptable salts thereof) may also be employed in the combinations described herein. Suitable antibacterial agents include, for example, ampicillin, amoxicillin, aureomycin, bacitracin, ceftazidime, ceftriaxone, cefotaxime, cephaclor, cephalexin, cephradine, ciprofloxacin, clavulanic acid, cloxacillin, dicloxacillin, erythromycin, flucloxacillin, gentamicin, gramicin, methicillin, neomycin, oxacillin, penicillin and vancomycin. Structural associations of antimicrobial agents with similar analgesic and anti-inflammatory functions may also be included.

Other suitable compounds include, but are not limited to, BN 50730; tenidap (teidap); e5531; tiapafant PCA 4248; nimesulide (Nimesulide); panavir; rolipram (Rolipram); RP 73401; peptide T (peptide T); MDL 201, 449A; (1R, 3S) -cis-1- [9- (2, 6-diaminopurine) ] -3-hydroxy-4-cyclopentene hydrochloride; (1R, 3R) -trans-1- [9- (2, 6-diamino) purine ] -3-acetoxycyclopentane; (1R, 3R) -trans-1- [ 9-adenine) -3-azidocyclopentanecarboxylic acid and (1R, 3R) -trans-1- [ 6-hydroxy-purin-9-yl) -3-azidocyclopentane.

IL-4 has been found to cause inflammatory responses in certain circumstances, such as in asthma where over-expression of IL-4 in the lung causes epithelial hypertrophy, and accumulation of lymphocytes, eosinophils, neutrophils. This response represents the main feature of the pro-inflammatory response caused by the other Th2 cytokine. Thus, as described above, inhibitors of IL-4 may also function in the present invention. In addition, certain immunosuppressant drugs are of course also useful in the treatment of arthritis, including, but not limited to, iNOS inhibitors and 5-lipoxygenase inhibitors.

Ginger has been found to have a certain anti-inflammatory action and is therefore also suitable as an anti-inflammatory drug in the present invention, and similarly chondroitin.

In certain embodiments, administration of the Ang-2 specific binding agent may occur prior to, concurrently with, or subsequent to treatment with the anti-cancer drug. Cancers include, for example (but are not limited to, breast cancer, intestinal cancer, gastric cancer, glioma, head and neck squamous cell carcinoma, hereditary papillary renal cell carcinoma, leukemia, lymphoma, Li-Fraumeni syndrome, pleural malignant mesothelioma, melanoma, multiple myeloma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, synovial sarcoma, thyroid cancer, bladder transitional cancer.

In certain embodiments, Ang-2 specific binding agents may be used alone or in combination with at least one other cancer treatment drug. In certain embodiments, the Ang-2 specific binding agent is used in combination with an effective dose of a cancer treatment drug. Such cancer therapeutics that can be used in combination with Ang-2 specific binding agents include, for example, but are not limited to, members of the geldanamycin family of anidamycin antibiotics; Pro-HGF; NK 2; a c-methionine peptide inhibitors; antagonists of the growth factor receptor binding protein 2(Grb2) SH2(Src homology 2) domain; a regulator of Gab 1; dominant negative Src; von-Hippel-Landau inhibitors, including (but not limited to) wortmannin; p13 kinase inhibitors, other anti-receptor (anti-receptor) therapies, anti-EGFR, COX-2 inhibitors, Celebrex^TM、Vioxx^TM(ii) a Vascular Endothelial Growth Factor (VEGF), VEGF modulator, Fibroblast Growth Factor (FGF), FGF modulator, Endothelial Growth Factor (EGF); an EGF modulator; keratinocyte Growth Factor (KGF), KGF-related molecules, KGF modulators; a Matrix Metalloprotease (MMP) modulator.

In certain embodiments of the invention, the agents used include Ang-2 specific binding agents and at least one serine protease inhibitor, and are used for treatment. In certain embodiments, the agent used comprises an Ang-2 specific binding agent, a serpin, and at least one other molecule described herein.

In some cases, disruption of the protease/protease balance can lead to protease-mediated tissue destruction, including (but not limited to) tumor invasion of normal tissue, thereby causing tumor metastasis.

In certain embodiments, Ang-2 specific binding agents may be used in combination with at least one inflammatory therapeutic agent. In certain embodiments, Ang-2 specific binding agents may be used in combination with at least one immune disorder treatment agent. Therapeutic agents for inflammation and immune disorders include, for example, but are not limited to, cyclooxygenase 1(COX-1) and cyclooxygenase 2(COX-2) inhibitors; a small molecule modulator of a 38kDa mitogen-activated protein kinase (p 38-MAPK); small molecule modulators of intracellular molecules involved in inflammatory pathways, including but not limited to jnk, IKK, NF-. kappa. B, ZAP70, and lck. Examples of certain inflammatory treatment drugs can be found in pro-inflammatory and anti-inflammatory cytokines as in c.a. dinarello and l.l.moldawer "rheumatoid arthritis: the therapeutic criteria "Proinflmat and Anti-inflammation Cytokines in Rheumatoid arthritis: a Primer for Clinicians Third Edition (2001) Amgen Inc. Thousand Oaks, Calif.

In certain embodiments, the pharmaceutical composition comprises more than one different Ang-2 specific binding agent. In certain embodiments, the pharmaceutical composition comprises more than one Ang-2 specific binding agent that binds to more than one epitope.

Immunotherapy

Immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. The immune influencer may be, for example, an antibody of the invention that recognizes a number of markers on the surface of a target cell. Such antibodies may act as therapeutic influences alone, or may stimulate other cells to function to actually kill the cells. Such antibodies may also be used in combination with drugs or toxins (chemotherapeutic drugs, radiotherapeutic drugs, ricin A chain, cholera toxin, pertussis toxin, etc.), the antibodies merely playing the role of a targeting agent.

According to the invention, immunotherapeutic agents (antibodies or antibody conjugates of the invention) can target an Ang-2 in the form of a localized mutant. At a particularly desirable expense, the antibody compositions of the invention can be used in combination therapy with Ang-2 targeting agents.

Passive immunotherapy has proven to be particularly effective for many cancers. See, e.g., WO 98/39027.

The following examples are given solely for the purpose of illustration and are not intended to limit the invention in any way.

Example 1

Expression of Ang-2 in pathological and normal tissues

Expression of Ang-2 in normal and pathological tissues was detected using in situ hybridization. A human Ang-2 sequence fragment (Genbank accession number: AF004327, nucleotide 1274-minus 1726) and a mouse Ang-2 fragment (Genbank accession number: AF004326, nucleotide 1135-minus 1588) were amplified from human or mouse fetal lung cDNA by reverse transcription PCR and cloned into pGEM-T plasmid vector, and confirmed by sequencing. Use of³³The P-UTP and RNA polymerase are obtained by transcription by using linearized plasmid as a template³³P-labeled antisense RNA probes. Tissues blocked with formaldehyde and embedded in paraffin were sectioned (5 μm) and collected on charged (charged) slides. Prior to in situ hybridization, the tissue was soaked (permeabilized) with 0.2M HCl, then digested with proteinase K and acetylated with triethanolamine and acetic anhydride. The radiolabeled probe was hybridized to the sections overnight at 55 deg.C, then digested with RNase and subjected to a high stringency wash with about 0.1 XSSC at 55 deg.C. Slides were immersed in Kodak NTB2 solution and exposed for 2-3 weeks at 4 deg.C, developed and counterstained. Sections were examined using dark field and standard light to simultaneously assess tissue morphology and hybridization signals.

The results indicate that Ang-2 expression is restricted to a few neovascular containing tissues, such as ovary, placenta and uterus, in normal postnatal humans. Normal adult hearts, brains, kidneys, livers, lungs, pancreas, spleen, muscles, tonsils, thymus, appendix, lymph nodes, gall bladder, prostate and testes are devoid of Ang-2 expression. Significant Ang-2 expression was observed in the direct small vessels of the kidney of 5-week old mice, but not in adult monkeys or humans. To confirm whether this expression is a residual in embryonic development, the kidneys of mice of various sizes (up to 1 year old) were examined using the mouse Ang-2 probe and the above reaction conditions. It was found that Ang-2 expression decreased with postnatal growth, but was still evident in the kidney of 1 year old mice.

Ang-2 expression was detected in almost all tumor types tested, including human primary tumors such as colon cancer (5 cases), breast cancer (10 cases), lung cancer (8 cases), glioblastoma multiforme (1 case), human metastases such as breast cancer (2 cases), lung cancer (2 cases) and ovarian cancer (2 cases) (metastatic to the brain), and rodent tumor models such as C6 (rat glioma), HT29 (human colon cancer), Colo-205 (human colon cancer), HCT116 (human colon cancer), a431 (human epidermoid carcinoma), a673 (human rhabdomyosarcoma), HT1080 (human fibrosarcoma), PC-3 (human prostate cancer), B16F10 (murine melanoma), MethA (murine sarcoma), and Lewis lung carcinoma (Lewis lung cancer metastases). In addition, Ang-2 expression was also tested in the neonatal blood vessel grown into a matrix plug (Matrigel plug) in response to VEGF, and in the hypoxia model of mouse retinopathy of prematurity.

Example 2

Preparation of recombinant mAng-2 protein and rabbit polyclonal anti-Ang-2 antiserum

His-tagged full-length murine Ang-2cDNA was obtained by PCR (Clontech Advantage PCR Kit, Cat. # K1905-01) from a 15-day-old murine embryo cDNA library (Marathon-Ready-cDNA, Cat. #7459-1, Clonetech, Inc.) using PCR primers used to amplify full-length human Ang-2. The PCR products were ligated into CMV promoter expression vectors and the resulting plasmids were transfected into HT1080 human fibrosarcoma cells (obtained from ATCC) using FuGENE6 transfection reagent (Roche, Cat. # 1814443). Stable clones were isolated by G418 selection. Clones expressing the mAng-2-His were screened using ELASA against the His tag and Western blotting.

The recombinant mAng-2 polypeptide was purified from the conditioned medium (C.M.) of these cells. Two-step chromatography was used to purify conditioned media containing mAng-2-His. Briefly, Tris buffer (pH 9.5) was added to the conditioned medium to a final concentration of about 20mM, adjusting the pH to 8.9. The detergent CHAPS was also added to a final concentration of about 5 mM. The medium was then directly applied to an anion exchange column Q-sepharose ff (Pharmacia). The column was washed with Tris buffer (pH 8.0, containing about 50mM NaCl) at a concentration of about 10 mM. The recombinant mAng-2-His was eluted in one step using Tris buffer (pH 8.0) at a concentration of 10mM, containing about 350mM NaCl and 5mM CHAPS.

The eluate from the Q-sepharose column was adjusted with imidazole at a concentration of about 4mM and passed through a fixed metal affinity column (Ni-NTA superflow (Qiagen)). Bound protein was eluted with PBS containing CHAPS at a concentration of about 5mM and imidazole at about 100 mM. The eluate was then concentrated to about 1.0mg/ml and dialyzed against PBS. Coomassie blue staining on SDS-PAGE indicated that the purity of mAng-2-His was higher than 90%.

Rabbits were immunized by injection of about 0.2mg of mAng-2 to generate antibodies. Injection of 1mL Hunter's into rabbits(Sigma) and mAng-2 in a ratio of 1: 1. After 4 weeks, each rabbit received repeated injections or booster immunizations; two weeks later, receive boosters again; blood was drawn at week seven and the mAb-2 titer was evaluated. If the serum titer is high, 50mL of blood is drawn weekly for 6 weeks. However, if the serum titer is low, the rabbits are given an additional booster and 50mL of blood is drawn weekly for 6 weeks starting at week nine. After 6 weeks from blood draw, the rabbits were not treated for 6 weeks. Rabbits were boosted again one month after the last blood draw if more serum was required.

The rabbit anti-bang-2 polyclonal antisera obtained from rabbits 5245 and 5255 were observed against bang-2 using a neutralization ELISA (see below): neutralization of Tie2 interaction.

Example 3

Molecular experiments to evaluate Ang-2 antibodies

Molecular experiments (affinity ELISA, neutralization ELISA and BIAcore) were developed to directly evaluate antibodies bound to Ang-2 and related family members and to evaluate antibodies against the positive effects of hong-2: influence of Tie2 interaction. These cell-based in vitro experiments are described below.

A.Affinity ELISA

For initial screening of candidate anti-Ang-2 antibodies, purified human Ang-2(Rand D Systems, Inc; catalog No. 623-AN; where Ang-2 is provided as a mixture of two short fragments) or murine Ang-2 polypeptide (prepared as described above) was used. For confirmatory binding experiments, human 293T cells were transfected with full-length human Ang-2DNA and cultured in serum-free DMEM (containing approximately 50. mu.g/ml Bovine Serum Albumin (BSA)), from which human Ang-2 was obtained.

Using microtiter plates, add about 100 microliter Ang-2 to each well and incubate for about 2 h. The microtiter plates were then washed 4 times with phosphate buffered saline (PBS, containing about 0.1% Tween-20). Blocking was performed using about 250 microliters of about 5% BSA in PBS per well, followed by incubation at room temperature for about 2 h. After incubation, excess blocking solution was removed and about 100 microliters of candidate anti-Ang-2 antibody was added to each well with a dilution gradient of about 40nmol initially, followed by four-fold serial dilutions with PBS containing about 1% BSA. Then incubated at room temperature overnight. After incubation, the microtiter plates were washed 5 times with PBS containing about 0.1% Tween-20, then about 100 microliters of goat anti-human IgG (Fc) -HRP (Pierce Chemical Co., catalog #31416, pre-diluted 1: 5000 with 1% BSA in PBS) were added to each well. After incubation of the microtiter plates for about 1h at room temperature, the plates were washed 5 times with PBS containing about 0.1% Tween-20, then about 100 microliters of TMB (3, 3 ', 5, 5' -tetramethylbenzidine Liquid substrate System; Sigma chemical Company, St. Louis, Mo., Cat. No. T8665) substrate was added to each well and incubated for 5-15min until blue color appeared. The absorbance was then measured at 370 nm.

B.Neutralization ELISA

Human Ang-2 polypeptide was immobilized on microtiter plates as described in the affinity ELISA section. Candidate anti-Ang-2 antibodies were gradient diluted with PBS solution containing about 1% BSA and about 1nM Tie2 (provided as a Tie2-Fc molecule in which the Tie2 moiety contains only the soluble extracellular portion of the molecule, R and D systems, cat # 313-TI) as described in the affinity ELISA section above. After addition of approximately 100 microliters of antibody/Tie 2 solution to each well, incubated overnight at room temperature and washed 5 times with PBS containing 0.1% Tween-20. Approximately 100 microliters of anti-Tie 2 antibody (Pharmingen inc., catalog No. 557039) was then added to each well to a final concentration of approximately 1 microgram/ml and incubated at room temperature for approximately 1 h. Approximately 100 microliters of goat anti-mouse IgG-HRP (Pierce Chemical Co., catalog No. 31432, pre-diluted 1: 10000 in PBS with 1% BSA) was then added to each well. After incubation of the microtiter plates at room temperature for about 1h, the plates were washed 5 times with PBS containing 0.1% Tween-20, then about 100 microliters of TMB substrate (as described above) was added to each well and incubated until color appeared. The absorbance was then measured at 370 nm.

C.Affinity BIAcore

PBS and 0.005% P20 surfactant (BIAcore, Inc.) as running buffer inAffinity analysis was performed on each candidate Ang-2 antibody on a 2000(Biacore, inc., Piscataway, NJ). Recombinant G proteins (Repligen, needlem, MA) were immobilized on a research grade CM5 sensor chip (Biacore, Inc.) via a primary Amine group using the Amine Coupling Kit (Biacore, Inc.) according to the instructions for use.

The binding assay procedure is as follows. Approximately 100Ru of each candidate anti-Ang-2 antibody was first attached to immobilized G-protein, and then different concentrations (0-100nM) of huAng-2 or mAng-2 were injected over the immobilized antibody surface at a flow rate of approximately 50 μ l/min for approximately 3 min. Determination of antibody binding kinetics using the BIA evaluation version 3.1 computer program (BIAcore, Inc.), including k_a(binding Rate constant), k_d(dissociation Rate constant) and K_D(dissociation equilibrium constant). A low dissociation equilibrium constant indicates a higher affinity for the Ang-2 antibody.

Example 4

Preparation of full-length human Ang-2 antibody by phage display method

Full-length human Ang-2 antibodies were panned against human Ang-2 polypeptides (R and D Systems Inc., catalog No. 623-AN) from a targeted query Phage Fab library (Target Quest phase Display library, Inc.).

Human Ang-2 was immobilized on the surface of polystyrene magnetic beads by two methods: (1) directly coating, and reacting overnight at Ang-24 ℃; (2) indirect capture, capture Ang-2 with 50. mu.g/ml goat anti-Ang-2 antibody overnight at 4 ℃. The bead surface was blocked with 2% milk in pbs (mpbs). The human Fab phage library was pre-selected to remove phage that reacted with uncoated magnetic beads or reacted with goat anti-Ang-2 antibody. The Ang-2 coated beads were then incubated with the phage library for 1.5h at room temperature. After the phage binding step, the beads were washed 6 times with MPBS containing 0.1% Tween-20, 6 times with PBS containing 0.1% Tween-20, and 2 times with PBS. The immobilized phage was first eluted with about 10. mu.g/ml Tie2-Fc (R and D Systems, Minneapolis, MN) followed by about 100mM triethanolamine. The eluted phage was used to infect E.coli TG1 cells, which were recovered after amplification for further screening. In successive screening rounds, the selection pressure was increased by using more stringent wash conditions and reducing phage input. After 3 rounds of screening 18 unique Ang-2 binding Fab clones were identified, which recognized almost all human Ang-2, rat Ang-2 and mouse Ang-2 as detected by the ELISA affinity assay described above. About 10% of these phages also bound human Ang-1. These clones were converted to IgG1 antibody as described below.

To obtain additional unique phage, a second round of screening was performed, using the same library only slightly different. In this method, human Ang-2 is added to a solution containing NaHCO₃Buffer (pH 9.6) in Nunc maxisorp immune tubes, 4 degrees C overnight. The Ang-2 concentrations in the 1 st, 2 nd and 3 rd panning were 1.5, 0.74 and 0.3. mu.g/ml, respectively. Blocking the surface of the immune tubes with 2% milk in PBS (MPBS), and then mixing with the same library (T) as described abovearget Quest) were incubated in about 4ml 2% MPBS (about 50 copies of each unique clone in the library). After the phage incubation step, the surface was washed 20 times with PBS plus about 0.1% Tween-20, followed by 20 washes with PBS. The immobilized phage was eluted using 1. mu.M hAng-2 or 1. mu.M human Tie2(R and D Systems, supra). The phage obtained from the elution were used to infect E.coli TG1 cells (carrying the phage library) and recovered after amplification for the next round of screening. 16 unique Ang-2 binding Fab clones were identified by PCR amplification of all phage immobilized either hAng-2 or Tie2 and analyzed using restriction enzyme analysis. The DNA of all 16 clones was sequenced.

The coding sequence of each heavy chain variable region of each phage was amplified using complementary primers. The primers were designed to contain HindIII and XbaI sites, Kozak sequence and signal sequence (the translated peptide is MDMRVPAQLLGLLLLWLRGARC; SEQ ID NO: 202) at the 5 'end of the variable region, and a BsmBI site at the 3' end of the PCR product. For example, the heavy chain was cloned by amplifying the phage DNA template from clone No. 544 (SEQ ID NO: 19) using the 2248-21 primer (GTG GTT GAG AGG TGC CAG ATG TCAGGT CCA GCT GGT GCA G; SEQ ID NO: 203, last 7 amino acids with added signal sequence), the 2502-31 primer (ATT ACG TCT CAC AGT TCG TTTGAT CTC CAC; SEQ ID NO: 204, variable region ends with added BsmBI sites). The resulting product was re-amplified using 2148-98 primer (CCG CTC AGC TCC TGGGGC TCC TGC TAT TGT GGT TGA GAG GTG CCA GAT; SEQ ID NO: 205, adding 9 amino acids to the signal peptide (AQLLGLLLL; SEQ ID NO: 206)) and 2502-31 primer, and then using 2489-36 primer (CAG CAG AAG CTT CTAGAC CAC CAT GGA CAT GAG GGT CCC CGC TCA GCT CCTGGG; SEQ ID NO: 207) and 2502-31 primer. 2498-36 primer adds HindIII and XbaI sites, Kozak sequence and the first 6 amino acids of the signal peptide from 5 'to 3'. The PCR product was digested with XbaI and BsmBI and then cloned into a mammalian expression vector containing the human IgG1 constant region. This vector contains the SV40 promoter and DHFR selection region.

The light chains from each phage were either kappa-or lambda-type. For the light chain, the complementary primers were designed to contain, from 5 'to 3', HindIII and XbaI sites, Kozak sequence and signal peptide (see above). Those strands with error-free coding regions were cloned as full-length products. For example, the light chain from the 536 phage clone (SEQ ID NO: 11 and SEQ ID NO: 210) was amplified as the full-length coding region using 2627-69 primers (GTG GTT GAG AGGTGC CAG ATG TGA CAT TGT GAT GAC TCA GTC TCC; SEQ ID NO: 208 with the last 7 amino acids of the signal peptide added) and 2458-54 primers (CTTGTC GAC TTA TTA ACA CTC TCC CCT GTT G; SEQ ID NO: 209 with the SalI site added after the stop codon). The PCR product was then amplified using additional 5' primers 2148-98 and 2489-36 (paired with primers 2458-54), respectively, as described above, to complete addition of the signal peptide sequence and cloning site. The full length light chain was cloned into a mammalian expression vector as an XbaI-SalI fragment as described above.

Some lambda clones have errors in their constant regions compared to the native human constant region sequence. To correct these errors, overlapping PCR was performed using DNA encoding error-free lambda constant regions and variable regions from these phages. These clones were cloned into mammalian expression vectors as XbaI-SalI fragments as described above.

When the kappa variable regions were independently cloned from their constant regions, a BsmBI site was added to the 3' end of the PCR product. After digestion of the PCR product with XbaI and BsmBI, the kappa chain variable region was cloned into an expression vector containing the human kappa constant region.

CHO cells were co-transfected with the light/heavy chain pairing constructs from each transformed phage by calcium phosphate Transfection Kit (Invitrogen Corp.) essentially following the instructions for use. The medium was changed 14-16h after transfection and cells were passaged in cell culture dishes for selection after about 48h according to the instructions. Transfected cells were isolated using HT selection for about 3 weeks, at which time the transfected CHO cell clones were trypsinized (trypsinized) and pooled into a "pool" of transfected cells.

A small amount of conditioned medium was collected after 48h and antibody production was detected by Western blotting using anti-human Fc antibody, anti-human kappa antibody or anti-human lambda antibody. The selected cell population was then passaged using standard tissue culture aseptic techniques under selective pressure until sufficient cells were obtained to inoculate 4 850cm of cells²In roller bottles (2X 10 per bottle)⁷Live cells) and cell line lyophilized stocks were prepared using DMSO. Cells were maintained in roller bottles after inoculation using medium (DMEM with 10% serum (Gibco/BRL, Inc) with addition of glutamate and non-essential amino acids). Cells were maintained for 2-3 days until cell confluency reached approximately 80%. At this point the medium in the roller bottle was replaced and a serum-free medium mixture (50% DMEM, 50% F12, Gibco, with addition of glutamic acid and non-essential amino acids) was used. After 7d, the conditioned medium was collected and collected by adding fresh serum-free medium, and additional 1-2 times as same.

Antibodies were purified directly from conditioned media by protein G affinity chromatography using standard methods. The antibody was eluted from the G protein column using a low pH buffer (pH about 3), and then the eluted antibody was neutralized with 1M Tris (pH8.5), followed by concentration using a 10kD molecular weight cut-off centrifugal concentrator. The concentrated antibody was then transferred to PBS by buffer exchange.

31 antibodies were established, each consisting of two heavy chains and two light chains (κ or λ), as shown in Table 2 below.

TABLE 2

Antibody heavy chain	Antibody light chains#
		526HC*	526κ
528HC*	528λC1
		531HC*	531λC3
533HC*	533-κ
		535HC*	535λC3
536HC*	536κ
		537HC*	537λC3(G 107A R)
540HC*	540λC3
		543HC*	543κ
544HC*	544-λC3
		545HC*	545λC2
546HC*	546λC1(G 107A S，N 112A，T114S)
		551HC*	551κ
553HC*	553κ
		555HC*	555κ
558HC	558κ
		559HC	559λC1(N 112 A，T 114 S)
565HC*	565κ
		F1-C6HC	F1-C6λC2
FB1-A7HC	FB1-A7λC2(G 107 A S)
		FD-B2HC	FD-B2λC3(G 107 A S)
FE-B7HC	FE-B7κ
		FJ-G11HC	FJ-G11κ
FK-E3HC	FK-E3κ
		G1D4HC*	G1D4λC2
GC1E8HC	GC1E8λC3(K 149 R)
		H1C12HC	H1C12λC2
IA1-1E7HC	IA1-1E7κ
		IF-1C10HC	IF-1C10λC3(T 212 A)
IK-2E2HC	IK-2E2λC2(T 212 A)
		IP-2C11HC	IP-2C11κ

^*Binding to hAng-2, mAng-2 and hAng-1 was detected as described herein.

^#Some lambda light chain constant regions appear as chimeras of more than 1 germline lambda constant region gene. The closest lambda constant region germline gene and amino acids different from this germline gene are indicated, numbered with the Kabat system.

31 Ang-2 antibodies (from phage transfer toFull length IgG1 antibody) and SEQ ID No. see 4 tables below. The VBASE database (methods used for it are described in Kabat et al, Sequences of Proteins of immunological Interest (NIH publication No. 91-3242; U.S. Dept. health and human Services, 5)^thed.)) predicted the Complementarity Determining Regions (CDRs) of the polyclonal antibody. The Fab region was aligned with the most closely related germline sequences to sequences in the database using tools from MRC Centre for Protein Engineering (Cambridge, UK) and these sequences were then visually compared. The CDRs of each variable region (heavy or light chain) are listed in Table 7.

TABLE 3

Heavy chain variable region

TABLE 4

Kappa chain variable region

TABLE 5

Variable region of lambda

TABLE 6

Human Constant Region (CR)

Antibody Constant Region (CR)	Sequence of
		Human lambda constant region 1(C1) (SEQ ID NO: 63)	GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
Human lambda constant region 2(C2) (SEQ ID NO: 64)	GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
		Human lambda constant region 3(C3) (SEQ ID NO: 65)	GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS
Human lambda constant region 7(C7) (SEQ ID NO: 66)	GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS
		Human kappa constant region (SEQ ID NO: 67)	RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Human IgG1 constant region (SEQ ID NO: 68)	ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE 7

Complementarity Determining Regions (CDRs) of Heavy (HC) and Light (LC) chains of Ang-2 antibody

	CDR1	CDR2	CDR3
				Antibodies	Residue of	Residue of	Residue of
Ab 526HC	SYGMH(SEQ ID NO：69)	AISGSGGSTYYADSVKG(SEQ ID NO：105)	DLLDYDILTGPYAY(SEQ ID NO：144)
				Ab 526

KC	RSSQSLLHSNGYNYLD(SEQ ID NO：70)	LGSNRAS-(SEQ ID NO：106)	MQALQTPPT(SEQ ID NO：145)
				Ab 528HC	SYAIS(SEQ ID NO：71)	GIIPIFGTANYAQKFQG(SEQ ID NO：107)	GVVGDFDWLSFFDY(SEQ ID NO：146)
Ab 528LC	SGDKLGYTYTS(SEQ ID NO：72)	QDFKRPS(SEQ ID NO：108)	QAWDSTTAVV(SEQ ID NO：147)
				Ab 531HC	SYAIS(SEQ ID NO：71)	GIIPILGIANYAQKFQG(SEQ ID NO：109)	DREDTAMVFNY(SEQ ID NO：148)
Ab 531LC	SGSSSNIGNNYVS(SEQ ID NO：73)	DNNKRPS(SEQ ID NO：110)	GTWDSSLSAFWV(SEQ ID NO：149)
				Ab 533HC	SYGMH(SEQ ID NO：69)	YISSSGSTIYYADSVKG(SEQ ID NO：111)	DLLDYDILTGYGY(SEQ ID NO：150)
Ab 533KC	RSSQSLLHSNGYNYLN(SEQ ID NO：74)	LGSNRAS(SEQ ID NO：106)	MQGLQTPPT(SEQ ID NO：151)
				Ab 535HC	SYAIS(SEQ ID NO：71)	GIIPIFGTANYAQKFQG(SEQ ID NO：107)	FSPFTETDAFDI(SEQ ID NO：152)
Ab 535LC	SGSNSNIGNNFVS(SEQ ID NO：75)	DNNKRPS(SEQ ID NO：110)	GTWDSSLSAAEVV(SEQ ID NO：153)
				Ab 536HC	SYGMH(SEQ ID NO：69)	YISSSGSTIYYADSVKG(SEQ ID NO：111)	DLLDYDILTGYGY(SEQ ID NO：150)
Ab 536(THw)KC	RSSQSLLHSNGYNYLD(SEQ ID NO：70)	LGSNRAS(SEQ ID NO：106)	MQGTHWPPT(SEQ ID NO：154 )
				Ab 536(LQT)KC	RSSQSLLHSNGYNYLD(SEQ ID NO：70)	LGSNRAS(SEQ ID NO：106)	MQGLQTPPT(SEQ ID NO：212)
Ab 537HC	SYAIS(SEQ ID NO：71)	GIIPILGIANYAQKFQG(SEQ ID NO：109)	GSSDAAVAGM(SEQ ID NO：155)
				Ab 537LC	SGNNSNIGNNYVS(SEQID NO：76)	DNHKRPS(SEQ ID NO：112)	GTWDTSLSANWV(SEQ ID NO：156)
Ab 540HC	SYAIS(SEQ ID NO：71)	GIIPILGIANYAQKFQG(SEQ ID NO：109)	AVPGTEDAFDI(SEQ IDNO：157)
				Ab 540LC	SGSSSNIGANYVS(SEQID NO：77)	NNNKRPS(SEQ ID NO：113)	GAWDSSLSASWV(SEQ IDNO：158)
Ab 543HC	SYAIS(SEQ ID NO：71)	RIIPILGIANYAQKFQG(SEQ ID NO：114)	PYYDFWSGPGGMDV(SEQ ID NO：159)
				Ab 543KC	RSSQSLLHSNGYNYLD(SEQ ID NO：70)	LGSNRAS(SEQ ID NO：106)	MQALQTPLT(SEQ ID NO：160)
Ab 544HC	SYAIS(SEQ ID NO：71)	GIIPIFGTANYAQKFQG(SEQ ID NO：107)	FESGYWGDAFDI(SEQ ID NO：161)

Ab 544LC	SGDALPKQYAY(SEQID NO：78)	KDSERPS(SEQ ID NO：115)	QSADSSHVV(SEQ ID NO：162)
				Ab 545HC	SYGMH(SEQ ID NO：69)	VISYDGSNKYYADSVKG(SEQ ID NO：116)	GPVDFDYGDYAIDY(SEQ ID NO：163)
Ab 545LC	TGQSSNIGAGYDVH(SEQ ID NO：79)	GNSNRPS(SEQ ID NO：117)	QSYDSRLSGSV(SEQ ID NO：164)
				Ab 546HC	SYAMS(SEQ ID NO：80)	AISGSGGSTYYADSVKG(SEQ ID NO：105)	ETISFSTFSGYFDY(SEQ ID NO：165)
Ab 546LC	SGSASNIGANGVS(SEQID NO：81)	HDGLVTS(SEQ ID NO：118)	AVWDDSLNAVV(SEQ ID NO：166)
				Ab 551HC	SYAIS(SEQ ID NO：71)	GIIPIFGTANYAQKFQG(SEQ ID NO：107)	GYDFWSGYSLDAFDI(SEQ ID NO：167)
Ab 551KC	RSSQSLLHSNGYNYLD(SEQ ID NO：70)	LGSNRAS(SEQ ID NO：106)	MQALQTPLT(SEQ ID NO：160)
				Ab 553HC	SYAMH(SEQ ID NO：82)	WINAGNGNTKYSQKFQG(SEQ ID NO：119)	GVDDYGGNSWAFDI(SEQ ID NO：168)
Ab 553KC	RSSQSLLHSNGYNYLD(SEQ ID NO：70)	LGSNRAS(SEQ ID NO：106)	MQALQTPLT(SEQ ID NO：160)
				Ab 555HC	SYAMH(SEQ ID NO：82)	VISYDGSNKYYADSVKG(SEQ ID NO：116)	SASDHYYDSSGYYSDAFDI(SEQ ID NO：169)
Ab 555KC	RSSQSLLHSNGYNYLD(SEQ ID NO：70)	LASNRAS(SEQ ID NO：120)	MQTLQIPIT(SEQ ID NO：170)
				Ab 558HC	GYYWS(SEQ ID NO：83)	EINHSGSTNFNPSLKS(SEQID NO：121)	GHDWGMGIGGAAYDI(SEQ ID NO：171)
Ab 558KC	RASQSVSSSSLA(SEQID NO：84)	AASSRAT(SEQ ID NO：122)	QHYGSSPRT(SEQ ID NO：172)
				Ab 559HC	ESSMH(SEQ ID NO：85)	GFDPEHGETIYAQKFQG(SEQ ID NO：123)	GVQVTSGYHYFDH(SEQ ID NO：173)
Ab 559LC	TGTNSDIGSYPFVS(SEQ ID NO：86)	DVSNRPS(SEQ ID NO：124)	SSFTMNSFVI(SEQ ID NO：174)
				Ab 565HC	SYAIS(SEQ ID NO：71)	GIIPIFGTANYAQKFQG(SEQ ID NO：107)	SPIYYDILTGIDAFDI(SEQ ID NO：175)
Ab 565KC	RASQSVSSSSLA(SEQID NO：213)	AASSRAT(SEQ ID NO：214)	QHYGSSPRT(SEQ ID NO：215)
				Ab 565(2)KC	RSSQSLLHSNGYNYLD(SEQ ID NO：70)	LGSSRAS(SEQ ID NO：125)	MQALDTPPT(SEQ ID NO：176)
Ab F1-C6HC	SYAIS(SEQ ID NO：71)	RIIPILGIANYAQKFQG(SEQ ID NO：114)	DPIPSGWYFDL(SEQ ID NO：177)

Ab F1-C6LC	SGSSSNIGNNAVN(SEQID NO：87)	YDDLLPS(SEQ ID NO：126)	ATWDDSLSGWV(SEQ ID NO：178)
				AbFB1-A7HC	SYGMH(SEQ ID NO：69)	VIWYDGSNKYYADSVKG(SEQ ID NO：127)	EVGNYYDSSGYGY(SEQ ID NO：179)
AbFB1-A7LC	TRSGGGIGSSFVH(SEQID NO：88)	DDNQRPT(SEQ ID NO：128)	QSSHSTAVV(SEQ ID NO：180)
				Ab FD-B2HC	SNSAAWN(SEQ ID NO：89)	RTYYRSKWYSDYAVSLRG(SEQ ID NO：129)	DRGGYIDS(SEQ ID NO：181)
Ab FD-B2LC	TRSSGSIATNYVQ(SEQID NO：90)	EDNQRPS(SEQ ID NO：130)	QSYGDNNWV(SEQ ID NO：182)
				Ab FE-B7HC	DYEMN(SEQ ID NO：91)	YIIGSGKTIFYADSVKG(SEQ ID NO：131)	GGGSAYYLNTSDI(SEQ ID NO：183)
Ab FE-B7KC	RSSQSLLHSKGDNYLD(SEQ ID NO：92)	LGSHRAS(SEQ ID NO：132)	MQALQTPLT(SEQ ID NO：160)
				Ab FJ-G11HC	SYGIS(SEQ ID NO：93)	WISAYNGNTNYAQKLQG(SEQ ID NO：133)	DRGIAARSAYYYGMDV(SEQ ID NO：184)
Ab FJ-G11KC	RSSQSLLDSDDGKTYLD(SEQ ID NO：94)	TTSSRAS(SEQ ID NO：134)	MQATQFPYT(SEQ ID NO：185)
				Ab FK-E3HC	SYDLN(SEQ ID NO：95)	WMNPTSGNTGYAQKFQG(SEQ ID NO：135)	DPPSGGWEFDY(SEQ ID NO：186)
Ab FK-E3KC	RSSQSLVHEDGNTYLN(SEQ ID NO：96)	KISKRFS(SEQ ID NO：136)	MQSTRFPRT(SEQ ID NO：187)
				AbG1D4HC	SHAIS(SEQ ID NO：97)	RIIPILGIANYAQKFQG(SEQ ID NO：114)	SRLEWLLYLDY(SEQ ID NO：188)
AbG1D4LC	TRSSGSIASNYVQ(SEQID NO：98)	EDKQRPS(SEQ ID NO：137)	QSYNSRGVM(SEQ ID NO：189)
				AbGC1E8HC	SYGIS(SEQ ID NO：93)	WISAYNGNTNYAQKLQG(SEQ ID NO：133)	GGSPYGGYAYPFDY(SEQ ID NO：190)
AbGC1E8LC	TRSSGSIASNYVQ(SEQID NO：98)	EDNQRPS(SEQ ID NO：130)	QSYDSNIWV(SEQ ID NO：191)
				AbH1C12HC	SYGMH(SEQ ID NO：69)	YISSSGSTIYYADSVKG(SEQ ID NO：111)	DLLDYDILTGYGY(SEQ ID NO：150)
AbH1C12LC	SGSSSNIGNNYVS(SEQ ID NO：73)	GNTNRPS(SEQ ID NO：138)	QSYDSSLSGSLV(SEQ ID NO：192)
				Ab IA1-1E7HC	GYYWS(SEQ ID NO：83)	EINHSGSTNFNPSLKS(SEQID NO：121)	GHDWGMGIGGAAYDI(SEQ ID NO：171)
Ab IA1-1E7KC	RASQSVSSSFLA(SEQ	DTSTRAT(SEQ ID NO：139)	QQYDFSPLT(SEQ ID NO：

	ID NO：99)		193)
				Ab IF-1C10HC	STYAMT(SEQ ID NO：100)	VIRSNGGTDYADFVKG(SEQ ID NO：140)	DYY(SEQ ID NO：194)
Ab IF-1C10LC	TGSGGSIASNYVQ(SEQID NO：101)	EDNQRPS(SEQ ID NO：130)	QSYDSSTWV(SEQ ID NO：195)
				Ab IK-2E2HC	SYAMS(SEQ ID NO：80)	AISGSGGSTYYADSVKG(SEQ ID NO：105)	ETISFSTFSGYFDY(SEQ ID NO：165)
Ab IK-2E2LC	TGTSSDVGGYNYVS(SEQ ID NO：102)	KVNNRPS(SEQ ID NO：141)	SSYTSSSTLG(SEQ ID NO：196)
				Ab IP-2C11HC	SYDIN(SEQ ID NO：103)	WMNPNSGNTGYAQKFQG(SEQ ID NO：142)	EIAVAGTRYGMDV(SEQ ID NO：197)
Ab IP-2C11KC	RASQSISTFLA(SEQID NO：104)	DASNRAT(SEQ ID NO：143)	QHRINWPLT(SEQ ID NO：198)

The affinity, neutralization and specificity of 17 of these antibodies and the negative control IgG1 (referred to as RDB1) were determined using affinity ELISA and neutralization ELISA (as described in example 3 above) as well as BIAcore neutralization experiments. The results are shown in Table 8 below and calculated using standard methods.

TABLE 8

EC50 and IC50 of Ang-2 antibody

As hereinbefore described for useBIAcore analysis detected antibodies to clone 536 and clone 545. Binding of the antibody, K, was determined using BIAcore as described above_DsLower indicates higher affinity and the results are shown in Table 9.

TABLE 9

Affinity of antibody to hAng-2 and mAng-2

Clone 536 analyzed above contained a mixture of two variant antibodies, respectively SEQ ID NOs: 12(536 κ THW) and SEQ ID NO: 210(536 kLQT). The two 536 variants were independent and separately subjected to ELISA and HTRF experiments to determine their potency.

ELISA was performed using 96-well plates coated with 293T cell conditioned medium containing recombinant angiopoietin (DMEM/50. mu.g/ml BSA) at 37 ℃ for 1 h. The concentration of angiopoietin in conditioned media was adjusted to allow for 1nM hTie2-Fc (R)&DSystems, catalog number 313-TI) was 80% of the maximum binding. Plates were washed with PBS/0.1% Tween-20 and then blocked with PBS/5% BSA at room temperature for 2 h. Angiopoietin neutralizer was added dropwise to the PBS/1% BSA/1nM Tie2 solution at concentrations from 100nM to 0.4pM, then this solution was added to angiopoietin-coated multi-well plates, incubated overnight at room temperature and washed with PBS/0.1% Tween-20. Murine anti-Tie 2 antibody (BD Pharmingen Inc., Cat. No. 557039) was added to each well until a final concentration of 1. mu.g/ml was reached, and after incubation for 1h at room temperature, the plates were washed with PBS/0.1% Tween-20. To PBS/1% BSA was added goat anti-mouse IgG-HRP (Pierce, Cat. No. 31432) diluted 1: 10000, and after 1h incubation at room temperature, the plates were washed several times with PBS/0.1% Tween-20. Addition of TMB substrate (Sigma, cat # T8665) and measurement of OD_370nmAngiogenin was determined by comparison to the Tie2 standard curve: degree of neutralization of Tie 2.

For HTRF experiments, 50 μ l of HTRF buffer (50mM Tris-HCl, pH 7.5, 100mM NaCl, 0.05% Tween 20, 0.1% BSA) containing also 0.8nM europium-coupled streptavidin (PERKIN ELMERLIFE SCIENCES Inc., catalog number AD0062) and 4.0nM biotinylated human angiopoietin 1(R & D Systems) or angiopoietin 2(Amgen Inc.) was added to each well of a 96-well plate to prepare a mixed plate (mix plate). On the other plate, an angiopoietin neutralizer (from 400nM to 20pM) was added dropwise to the HTRF buffer, then 50. mu.l of each serially diluted angiopoietin neutralizer was transferred to a mixing plate (containing streptavidin-europium/angiopoietin) and mixed. The plate was then incubated on a shaker for 1h at room temperature. Next 20. mu.l of each well of the mixed plate was transferred to an "experimental plate" (96-well plate with 20. mu.l of HTFR buffer solution in each well containing 10nM of human allophycocyanin-conjugated human Tie 2-Fc). The resulting plates were incubated for 2h at room temperature on a shaker. The final concentration of the solution on the test plate was: 1.0nM of angiopoietin, 5.0nM of human Tie2-Fc and 100nM to 5.0pM gradient diluted angiopoietin neutralizing agent. The assay plates were analyzed using a Rubystar plate reader (BMG Labtechnologies, Offenberg, Germany). Using the "no angiopoietin neutralizer" control (representing zero inhibition) and the "no angiopoietin" control (representing complete inhibition), angiopoietin was determined by calculating the percent inhibition of each concentration of angiopoietin neutralizer: degree of neutralization of Tie 2. The percent inhibition was analyzed using the GRAFIT 5.0 program (Erithocus Software Ltd.) to calculate IC50 values.

All results are expressed as IC50 curves calculated from samples tested in duplicate using the following formula. The IC50 results in fitting the suppressed data to a two parameter equation with a lower data limit of 0 (i.e., the data is background corrected) and an upper limit of 100 (i.e., the data is range corrected).

In this equation, s is the slope (slope factor). The equation assumes that y decreases as x increases, using the Software GRAFIT 5.0 (Erithocus Software Limited).

The results are shown in tables 10 and 11.

Watch 10

Ab536 variant ELISA IC50 results

Sample (I)	Human Ang1 IC50(nM)	Human Ang2 IC50(nM)	Mouse Ang2 IC50(nM)
				Ab536LQT	＞100	0.35	0.10
Ab536THW	＞100	0.31	0.088

TABLE 11

Ab536 variant HTRF IC50 results

Sample (I)	Human Ang1 IC50(nM)	Human Ang2 IC50(nM)
			Ab53 6LQT	＞100	0.072
Ab536THW	＞100	0.071

Example 5

Therapeutic Studies Using anti-Ang-2 antibodies

The pharmacokinetics of rabbit anti-Ang-2 polyclonal antibodies (purified using G protein) were tested using mice. 24 mice (1 mg each) were treated with rabbit anti-Ang-2 polyclonal antibody. 4 treated mice were sacrificed at 1h, 6h, 1d, 3d, 7d and 14d, respectively, after antibody injection.

The results indicate that the circulating serum half-life of total rabbit IgG is about 19d, while the half-life of the anti-Ang-2 IgG component of total IgG is about 8 d.

To evaluate the efficacy, mice xenografted with a431 tumors (10 per group) were used. Mice were injected intraperitoneally with 10 doses of rabbit anti-Ang-2 polyclonal antibody (purified using G protein) (each dose was approximately 10mg IgG) at 1d, 5d, 6d, 7d, 8d, 12d, 13d, 14d, 15d, 18d after transplantation. Tumor sizes were measured at 7d, 12d, 15d, 19d, 21 d. Body weight was measured at 0d, 7d, 15d, 21d and found to be unaffected. Repeated measures ANOVA results indicated that anti-Ang-2 polyclonal antibodies inhibited the growth of a431 tumor xenografts to about 50% compared to controls treated with immunocompromised purified polyclonal antisera (approximately 10mg IgG per dose) and vehicle (PBS), with p ═ 0.008.

To evaluate the in vivo efficacy of full-length human anti-Ang-2 mab, mice xenografted with a431 tumors (10 per group) were used. Mice were injected intraperitoneally with anti-Ang-2 antibody clones 533, 537, 544, or negative controls (PBS or human IgG1- κ, respectively). The injection amounts were about 420 μ g protein per injection 1, about 140 μ g protein per injection three times, about 55 μ g protein per injection four times, and 8 times total. Tumor size and body weight were recorded twice weekly. Animals were sacrificed at the end of the study and serum was collected and serum antibody levels were measured by ELISA. Tumors and a set of normal tissues were collected from each group of animals.

There was a clear difference between tumor growth in the control group treated with the anti-Ang-2 antibody, see fig. 1. Tumor growth was inhibited in all three groups treated with anti-Ang-2 antibody compared to control (p <.005 for all 3 antibody treated groups versus hIgG1 control, ANOVA was measured repeatedly). In contrast, the tumors of the control group continued to grow at a higher rate.

Example 6

Epitope mapping

The full-length protein (amino acids 1-495), the N-terminus (amino acids 1-254), and the C-terminus (amino acids 255-495) of human Ang-2(hAng-2) were cloned into a CMV-driven mammalian expression vector containing a C-terminal 6XHis tag. The resulting 3 constructs were transiently expressed in 293T cells, along with a control vector. Conditioned media from transfected cells were then collected and anti-6 xHis ELISA and Western blot were used to estimate Ang-2 expression levels in the media.

The binding epitopes of anti-Ang-2 antibodies and peptibodies were determined by ELISA for three human hAng-2 binding capacities as follows: each well of the high efficiency binding assay 96 well plate was coated with 100. mu.l of conditioned medium and incubated at 37 ℃ for 1 h. Conditioned medium was blotted (asparated) and each well blocked with 200. mu.l PBS containing 5% BSA at room temperature for 1 h. Then the blocking liquid is sucked dry. To each well, 100. mu.l of PBS solution containing 1. mu.g/ml antibody, peptibody or Tie2-Fc and 1% BSA was added and incubated at room temperature for 1 h. The plate was washed 4 times with 200. mu.l of PBS solution containing 0.1% Tween. Mu.l of HRP-conjugated goat anti-human IgG or goat anti-mouse IgG was added to each well and incubated at room temperature for 45 min. The plate was then washed 4 times with 200. mu.l of PBS solution containing 0.1% Tween. OD was measured by adding 100. mu.l of TMB substrate to each well_370nm。

The results are shown in FIGS. 2A, 2B and 2C.

Sequence listing

<110>Oliner，John

Graham，Kevin

<120> angiopoietin-2 specific binding agents

<130>04-881-A

<140>10/982,440

<141>2004-11-04

<150>60/620,161

<151>2004-10-19

<160>215

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<213> human (Homo sapiens)

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50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

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85 90 95

Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>19

<211>121

<212>PRT

<213> human (Homo sapiens)

<400>19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

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20 25 30

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50 55 60

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65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Glu Ser Gly Tyr Trp Gly Asp Ala Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210>20

<211>106

<212>PRT

<213> human (Homo sapiens)

<400>20

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

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20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser His Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210>21

<211>123

<212>PRT

<213> human (Homo sapiens)

<400>21

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Pro Val Asp Phe Asp Tyr Gly Asp Tyr Ala Ile Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>22

<211>111

<212>PRT

<213> human (Homo sapiens)

<400>22

Gln Ser Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Gln Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Phe Pro Gly Arg Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Arg

85 90 95

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

100 105 110

<210>23

<211>123

<212>PRT

<213> human (Homo sapiens)

<400>23

Glu Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Thr Ile Ser Phe Ser Thr Phe Ser Gly Tyr Phe Asp Tyr

100 105 110

Trp Ala Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>24

<211>110

<212>PRT

<213> human (Homo sapiens)

<400>24

Gln Ser Val Leu Thr Gln Pro Ser Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

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

20 25 30

Gly Val Ser Trp Tyr His Gln Val Pro Gly Lys Ala Pro Arg Leu Leu

35 40 45

Leu Ser His Asp Gly Leu Val Thr Ser Gly Val Pro Asp Arg Leu Ser

50 55 60

Val Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu His

65 70 75 80

Ser Asp Asp Glu Gly Asp Tyr Tyr Cys Ala Val Trp Asp Asp Ser Leu

85 90 95

Asn Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>25

<211>124

<212>PRT

<213> human (Homo sapiens)

<400>25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Asp Phe Trp Ser Gly Tyr Ser Leu Asp Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210>26

<211>112

<212>PRT

<213> human (Homo sapiens)

<400>26

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>27

<211>123

<212>PRT

<213> human (Homo sapiens)

<400>27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Gly Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Asp Asp Tyr Gly Gly Asn Ser Trp Ala Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210>28

<211>112

<212>PRT

<213> human (Homo sapiens)

<400>28

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>29

<211>128

<212>PRT

<213> human (Homo sapiens)

<400>29

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ala Ser Asp His Tyr Tyr Asp Ser Ser Gly Tyr Tyr Ser

100 105 110

Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210>30

<211>112

<212>PRT

<213> human (Homo sapiens)

<400>30

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Ala Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Thr

85 90 95

Leu Gln Ile Pro Ile Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105 110

<210>31

<211>123

<212>PRT

<213> human (Homo sapiens)

<400>31

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Phe Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Ser Val Asp Thr Ser Asn Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys Ala

85 90 95

Arg Gly His Asp Trp Gly Met Gly Ile Gly Gly Ala Ala Tyr Asp Ile

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210>32

<211>108

<212>PRT

<213> human (Homo sapiens)

<400>32

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

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

20 25 30

Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Val Tyr Ala Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Ser Ser Pro

85 90 95

Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>33

<211>122

<212>PRT

<213> human (Homo sapiens)

<400>33

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

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

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Gly Phe Asp Pro Glu His Gly Glu Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Leu Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Val Gln Val Thr Ser Gly Tyr His Tyr Phe Asp His Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>34

<211>110

<212>PRT

<213> human (Homo sapiens)

<400>34

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Asn Ser Asp Ile Gly Ser Tyr

20 25 30

Pro Phe Val Ser Trp Tyr Gln Arg His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Gly Asp Tyr Tyr Cys Ser Ser Phe Thr Met Asn

85 90 95

Ser Phe Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>35

<211>125

<212>PRT

<213> human (Homo sapiens)

<400>35

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Ile Tyr Tyr Asp Ile Leu Thr Gly Ile Asp Ala Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210>36

<211>108

<212>PRT

<213> human (Homo sapiens)

<400>36

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

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

20 25 30

Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Val Tyr Ala Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Ser Ser Pro

85 90 95

Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>37

<211>120

<212>PRT

<213> human (Homo sapiens)

<400>37

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Pro Ile Pro Ser Gly Trp Tyr Phe Asp Leu Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>38

<211>110

<212>PRT

<213> human (Homo sapiens)

<400>38

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu

85 90 95

Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>39

<211>122

<212>PRT

<213> human (Homo sapiens)

<400>39

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Gly Asn Tyr Tyr Asp Ser Ser Gly Tyr Gly Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>40

<211>110

<212>PRT

<213> human (Homo sapiens)

<400>40

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Arg Ser Gly Gly Gly Ile Gly Ser Ser

20 25 30

Phe Val His Trp Phe Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val

35 40 45

Ile Phe Asp Asp Asn Gln Arg Pro Thr Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ala Ile Asp Thr Ser Ser Ser Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

Leu Thr Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ser His Ser

85 90 95

Thr Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>41

<211>122

<212>PRT

<213> human (Homo sapiens)

<400>41

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

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

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Ser Asp Tyr Ala

50 55 60

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

65 70 75 80

Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Gly Tyr Ile Asp Ser Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>42

<211>110

<212>PRT

<213> human (Homo sapiens)

<400>42

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1 5 10 15

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

20 25 30

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

35 40 45

Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Thr Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

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

85 90 95

Asn Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>43

<211>122

<212>PRT

<213> human (Homo sapiens)

<400>43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Gly Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Thr Gly Phe Ser Leu Asp Asp Tyr

20 25 30

Glu Met Asn Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ile Gly Ser Gly Lys Thr Ile Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Ser Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Gly Ser Ala Tyr Tyr Leu Asn Thr Ser Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210>44

<211>112

<212>PRT

<213> human (Homo sapiens)

<400>44

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Lys Gly Asp Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>45

<211>125

<212>PRT

<213> human (Homo sapiens)

<400>45

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Ile Ala Ala Arg Ser Ala Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210>46

<211>113

<212>PRT

<213> human (Homo sapiens)

<400>46

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Lys Thr Tyr Leu Asp Trp Tyr Leu Gln Arg Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Met Tyr Thr Thr Ser Ser Arg Ala Ser Gly Val

50 55 60

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

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln

85 90 95

Ala Thr Gln Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210>47

<211>120

<212>PRT

<213> human (Homo sapiens)

<400>47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asp Leu Asn Trp Val Arg Gln Ala Ser Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Thr Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ile Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Pro Pro Ser Gly Gly Trp Glu Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>48

<211>112

<212>PRT

<213> human (Homo sapiens)

<400>48

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Thr Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Glu

20 25 30

Asp Gly Asn Thr Tyr Leu Asn Trp Leu His Gln Arg Pro Gly Gln Pro

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Ile Ser Lys Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser

85 90 95

Thr Arg Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>49

<211>120

<212>PRT

<213> human (Homo sapiens)

<400>49

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser His

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Leu Glu Trp Leu Leu Tyr Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>50

<211>110

<212>PRT

<213> human (Homo sapiens)

<400>50

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1 5 10 15

Thr Val Ile Ile Pro Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn

20 25 30

Tyr Val Gln Trp Tyr Gln Lys Arg Pro Gly Ser Ala Pro Ser Ile Val

35 40 45

Ile Tyr Glu Asp Lys Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

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

85 90 95

Arg Gly Val Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>51

<211>123

<212>PRT

<213> human (Homo sapiens)

<400>51

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Pro Tyr Gly Gly Tyr Ala Tyr Pro Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>52

<211>110

<212>PRT

<213> human (Homo sapiens)

<400>52

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

1 5 10 15

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

20 25 30

Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Thr Ser Pro Thr Asn Val

35 40 45

Ile Phe Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

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

85 90 95

Asn Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>53

<211>122

<212>PRT

<213> human (Homo sapiens)

<400>53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Leu Asp Tyr Asp Ile Leu Thr Gly Tyr Gly Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>54

<211>111

<212>PRT

<213> human (Homo sapiens)

<400>54

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ala Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

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

100 105 110

<210>55

<211>123

<212>PRT

<213> human (Homo sapiens)

<400>55

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Phe Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Ser Val Asp Thr Ser Asn Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly His Asp Trp Gly Met Gly Ile Gly Gly Ala Ala Tyr Asp Ile

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210>56

<211>108

<212>PRT

<213> human (Homo sapiens)

<400>56

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

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

20 25 30

Phe Leu Ala Trp Tyr Gln Gln Lys Ala Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Thr Ser Thr Arg Ala Thr Gly Ile Ala Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Ala Glu Asp Ser Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Phe Ser Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>57

<211>112

<212>PRT

<213> human (Homo sapiens)

<400>57

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Phe Ser Thr

20 25 30

Tyr Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ser Val Ile Arg Ser Asn Gly Gly Thr Asp Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Met Thr Asp Tyr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210>58

<211>110

<212>PRT

<213> human (Homo sapiens)

<400>58

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1 5 10 15

Thr Val Thr Ile Ser Cys Thr Gly Ser Gly Gly Ser Ile Ala Ser Asn

20 25 30

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

35 40 45

Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser

85 90 95

Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>59

<211>123

<212>PRT

<213> human (Homo sapiens)

<400>59

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Thr Ile Ser Phe Ser Thr Phe Ser Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>60

<211>110

<212>PRT

<213> human (Homo sapiens)

<400>60

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val SerTrp Phe Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Lys Val Asn Asn Arg Pro Ser Gly Leu Ser Asn Arg Phe

50 55 60

Ser Gly Ser Gln Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

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

100 105 110

<210>61

<211>122

<212>PRT

<213> human (Homo sapiens)

<400>61

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Ile Ala Val Ala Gly Thr Arg Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>62

<211>107

<212>PRT

<213> human (Homo sapiens)

<400>62

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Thr Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Gly Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Arg Ile Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>63

<211>106

<212>PRT

<213> human (Homo sapiens)

<400>63

Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210>64

<211>106

<212>PRT

<213> human (Homo sapiens)

<400>64

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210>65

<211>106

<212>PRT

<213> human (Homo sapiens)

<400>65

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

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

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210>66

<211>106

<212>PRT

<213> human (Homo sapiens)

<400>66

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro

35 40 45

Val Lys Val Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Ala Glu Cys Ser

100 105

<210>67

<211>107

<212>PRT

<213> human (Homo sapiens)

<400>67

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>68

<211>330

<212>PRT

<213> human (Homo sapiens)

<400>68

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

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

225 2302 35 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>69

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>69

Ser Tyr Gly Met His

1 5

<210>70

<211>16

<212>PRT

<213> human (Homo sapiens)

<400>70

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210>71

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>71

Ser Tyr Ala Ile Ser

1 5

<210>72

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>72

Ser Gly Asp Lys Leu Gly Tyr Thr Tyr Thr Ser

1 5 10

<210>73

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>73

Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser

1 5 10

<210>74

<211>16

<212>PRT

<213> human (Homo sapiens)

<400>74

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asn

1 5 10 15

<210>75

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>75

Ser Gly Ser Asn Ser Asn Ile Gly Asn Asn Phe Val Ser

1 5 10

<210>76

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>76

Ser Gly Asn Asn Ser Asn Ile Gly Asn Asn Tyr Val Ser

1 5 10

<210>77

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>77

Ser Gly Ser Ser Ser Asn Ile Gly Ala Asn Tyr Val Ser

1 5 10

<210>78

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>78

Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala Tyr

1 5 10

<210>79

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>79

Thr Gly Gln Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10

<210>80

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>80

Ser Tyr Ala Met Ser

1 5

<210>81

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>81

Ser Gly Ser Ala Ser Asn Ile Gly Ala Asn Gly Val Ser

1 5 10

<210>82

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>82

Ser Tyr Ala Met His

1 5

<210>83

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>83

Gly Tyr Tyr Trp Ser

1 5

<210>84

<211>12

<212>PRT

<213> human (Homo sapiens)

<400>84

Arg Ala Ser Gln Ser Val Ser Ser Ser Ser Leu Ala

1 5 10

<210>85

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>85

Glu Ser Ser Met His

1 5

<210>86

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>86

Thr Gly Thr Asn Ser Asp Ile Gly Ser Tyr Pro Phe Val Ser

1 5 10

<210>87

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>87

Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn

1 5 10

<210>88

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>88

Thr Arg Ser Gly Gly Gly Ile Gly Ser Ser Phe Val His

1 5 10

<210>89

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>89

Ser Asn Ser Ala Ala Trp Asn

1 5

<210>90

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>90

Thr Arg Ser Ser Gly Ser Ile Ala Thr Asn Tyr Val Gln

1 5 10

<210>91

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>91

Asp Tyr Glu Met Asn

1 5

<210>92

<211>16

<212>PRT

<213> human (Homo sapiens)

<400>92

Arg Ser Ser Gln Ser Leu Leu His Ser Lys Gly Asp Asn Tyr Leu Asp

1 5 10 15

<210>93

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>93

Ser Tyr Gly Ile Ser

1 5

<210>94

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>94

Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Lys Thr Tyr Leu

1 5 10 15

Asp

<210>95

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>95

Ser Tyr Asp Leu Asn

1 5

<210>96

<211>16

<212>PRT

<213> human (Homo sapiens)

<400>96

Arg Ser Ser Gln Ser Leu Val His Glu Asp Gly Asn Thr Tyr Leu Asn

1 5 10 15

<210>97

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>97

Ser His Ala Ile Ser

1 5

<210>98

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>98

Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln

1 5 10

<210>99

<211>12

<212>PRT

<213> human (Homo sapiens)

<400>99

Arg Ala Ser Gln Ser Val Ser Ser Ser Phe Leu Ala

1 5 10

<210>100

<211>6

<212>PRT

<213> human (Homo sapiens)

<400>100

Ser Thr Tyr Ala Met Thr

1 5

<210>101

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>101

Thr Gly Ser Gly Gly Ser Ile Ala Ser Asn Tyr Val Gln

1 5 10

<210>102

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>102

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210>103

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>103

Ser Tyr Asp Ile Asn

1 5

<210>104

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>104

Arg Ala Ser Gln Ser Ile Ser Thr Phe Leu Ala

1 5 10

<210>105

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>105

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>106

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>106

Leu Gly Ser Asn Arg Ala Ser

1 5

<210>107

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>107

Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210>108

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>108

Gln Asp Phe Lys Arg Pro Ser

1 5

<210>109

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>109

Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210>110

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>110

Asp Asn Asn Lys Arg Pro Ser

1 5

<210>111

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>111

Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>112

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>112

Asp Asn His Lys Arg Pro Ser

1 5

<210>113

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>113

Asn Asn Asn Lys Arg Pro Ser

1 5

<210>114

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>114

Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210>115

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>115

Lys Asp Ser Glu Arg Pro Ser

1 5

<210>116

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>116

Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>117

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>117

Gly Asn Ser Asn Arg Pro Ser

1 5

<210>118

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>118

His Asp Gly Leu Val Thr Ser

1 5

<210>119

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>119

Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln

1 5 10 15

Gly

<210>120

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>120

Leu Ala Ser Asn Arg Ala Ser

1 5

<210>121

<211>16

<212>PRT

<213> human (Homo sapiens)

<400>121

Glu Ile Asn His Ser Gly Ser Thr Asn Phe Asn Pro Ser Leu Lys Ser

1 5 10 15

<210>122

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>122

Ala Ala Ser Ser Arg Ala Thr

1 5

<210>123

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>123

Gly Phe Asp Pro Glu His Gly Glu Thr Ile Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210>124

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>124

Asp Val Ser Asn Arg Pro Ser

1 5

<210>125

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>125

Leu Gly Ser Ser Arg Ala Ser

1 5

<210>126

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>126

Tyr Asp Asp Leu Leu Pro Ser

1 5

<210>127

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>127

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>128

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>128

Asp Asp Asn Gln Arg Pro Thr

1 5

<210>129

<211>18

<212>PRT

<213> human (Homo sapiens)

<400>129

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Ser Asp Tyr Ala Val Ser Leu

1 5 10 15

Arg Gly

<210>130

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>130

Glu Asp Asn Gln Arg Pro Ser

1 5

<210>131

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>131

Tyr Ile Ile Gly Ser Gly Lys Thr Ile Phe Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>132

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>132

Leu Gly Ser His Arg Ala Ser

1 5

<210>133

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>133

Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln

1 5 10 15

Gly

<210>134

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>134

Thr Thr Ser Ser Arg Ala Ser

1 5

<210>135

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>135

Trp Met Asn Pro Thr Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210>136

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>136

Lys Ile Ser Lys Arg Phe Ser

1 5

<210>137

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>137

Glu Asp Lys Gln Arg Pro Ser

1 5

<210>138

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>138

Gly Asn Thr Asn Arg Pro Ser

1 5

<210>139

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>139

Asp Thr Ser Thr Arg Ala Thr

1 5

<210>140

<211>16

<212>PRT

<213> human (Homo sapiens)

<400>140

Val Ile Arg Ser Asn Gly Gly Thr Asp Tyr Ala Asp Phe Val Lys Gly

1 5 10 15

<210>141

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>141

Lys Val Asn Asn Arg Pro Ser

1 5

<210>142

<211>17

<212>PRT

<213> human (Homo sapiens)

<400>142

Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210>143

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>143

Asp Ala Ser Asn Arg Ala Thr

1 5

<210>144

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>144

Asp Leu Leu Asp Tyr Asp Ile Leu Thr Gly Pro Tyr Ala Tyr

1 5 10

<210>145

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>145

Met Gln Ala Leu Gln Thr Pro Pro Thr

1 5

<210>146

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>146

Gly Val Val Gly Asp Phe Asp Trp Leu Ser Phe Phe Asp Tyr

1 5 10

<210>147

<211>10

<212>PRT

<213> human (Homo sapiens)

<400>147

Gln Ala Trp Asp Ser Thr Thr Ala Val Val

1 5 10

<210>148

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>148

Asp Arg Glu Asp Thr Ala Met Val Phe Asn Tyr

1 5 10

<210>149

<211>12

<212>PRT

<213> human (Homo sapiens)

<400>149

Gly Thr Trp Asp Ser Ser Leu Ser Ala Phe Trp Val

1 5 10

<210>150

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>150

Asp Leu Leu Asp Tyr Asp Ile Leu Thr Gly Tyr Gly Tyr

1 5 10

<210>151

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>151

Met Gln Gly Leu Gln Thr Pro Pro Thr

1 5

<210>152

<211>12

<212>PRT

<213> human (Homo sapiens)

<400>152

Phe Ser Pro Phe Thr Glu Thr Asp Ala Phe Asp Ile

1 5 10

<210>153

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>153

Gly Thr Trp Asp Ser Ser Leu Ser Ala Ala Glu Val Val

1 5 10

<210>154

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>154

Met Gln Gly Thr His Trp Pro Pro Thr

1 5

<210>155

<211>10

<212>PRT

<213> human (Homo sapiens)

<400>155

Gly Ser Ser Asp Ala Ala Val Ala Gly Met

1 5 10

<210>156

<211>12

<212>PRT

<213> human (Homo sapiens)

<400>156

Gly Thr Trp Asp Thr Ser Leu Ser Ala Asn Trp Val

1 5 10

<210>157

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>157

Ala Val Pro Gly Thr Glu Asp Ala Phe Asp Ile

1 5 10

<210>158

<211>12

<212>PRT

<213> human (Homo sapiens)

<400>158

Gly Ala Trp Asp Ser Ser Leu Ser Ala Ser Trp Val

1 5 10

<210>159

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>159

Pro Tyr Tyr Asp Phe Trp Ser Gly Pro Gly Gly Met Asp Val

1 5 10

<210>160

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>160

Met Gln Ala Leu Gln Thr Pro Leu Thr

1 5

<210>161

<211>12

<212>PRT

<213> human (Homo sapiens)

<400>161

Phe Glu Ser Gly Tyr Trp Gly Asp Ala Phe Asp Ile

1 5 10

<210>162

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>162

Gln Ser Ala Asp Ser Ser His Val Val

1 5

<210>163

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>163

Gly Pro Val Asp Phe Asp Tyr Gly Asp Tyr Ala Ile Asp Tyr

1 5 10

<210>164

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>164

Gln Ser Tyr Asp Ser Arg Leu Ser Gly Ser Val

1 5 10

<210>165

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>165

Glu Thr Ile Ser Phe Ser Thr Phe Ser Gly Tyr Phe Asp Tyr

1 5 10

<210>166

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>166

Ala Val Trp Asp Asp Ser Leu Asn Ala Val Val

1 5 10

<210>167

<211>15

<212>PRT

<213> human (Homo sapiens)

<400>167

Gly Tyr Asp Phe Trp Ser Gly Tyr Ser Leu Asp Ala Phe Asp Ile

1 5 10 15

<210>168

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>168

Gly Val Asp Asp Tyr Gly Gly Asn Ser Trp Ala Phe Asp Ile

1 5 10

<210>169

<211>19

<212>PRT

<213> human (Homo sapiens)

<400>169

Ser Ala Ser Asp His Tyr Tyr Asp Ser Ser Gly Tyr Tyr Ser Asp Ala

1 5 10 15

Phe Asp Ile

<210>170

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>170

Met Gln Thr Leu Gln Ile Pro Ile Thr

1 5

<210>171

<211>15

<212>PRT

<213> human (Homo sapiens)

<400>171

Gly His Asp Trp Gly Met Gly Ile Gly Gly Ala Ala Tyr Asp Ile

1 5 10 15

<210>172

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>172

Gln His Tyr Gly Ser Ser Pro Arg Thr

1 5

<210>173

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>173

Gly Val Gln Val Thr Ser Gly Tyr His Tyr Phe Asp His

1 5 10

<210>174

<211>10

<212>PRT

<213> human (Homo sapiens)

<400>174

Ser Ser Phe Thr Met Asn Ser Phe Val Ile

1 5 10

<210>175

<211>16

<212>PRT

<213> human (Homo sapiens)

<400>175

Ser Pro Ile Tyr Tyr Asp Ile Leu Thr Gly Ile Asp Ala Phe Asp Ile

1 5 10 15

<210>176

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>176

Met Gln Ala Leu Asp Thr Pro Pro Thr

1 5

<210>177

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>177

Asp Pro Ile Pro Ser Gly Trp Tyr Phe Asp Leu

1 5 10

<210>178

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>178

Ala Thr Trp Asp Asp Ser Leu Ser Gly Trp Val

1 5 10

<210>179

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>179

Glu Val Gly Asn Tyr Tyr Asp Ser Ser Gly Tyr Gly Tyr

1 5 10

<210>180

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>180

Gln Ser Ser His Ser Thr Ala Val Val

1 5

<210>181

<211>8

<212>PRT

<213> human (Homo sapiens)

<400>181

Asp Arg Gly Gly Tyr Ile Asp Ser

1 5

<210>182

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>182

Gln Ser Tyr Gly Asp Asn Asn Trp Val

1 5

<210>183

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>183

Gly Gly Gly Ser Ala Tyr Tyr Leu Asn Thr Ser Asp Ile

1 5 10

<210>184

<211>16

<212>PRT

<213> human (Homo sapiens)

<400>184

Asp Arg Gly Ile Ala Ala Arg Ser Ala Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210>185

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>185

Met Gln Ala Thr Gln Phe Pro Tyr Thr

1 5

<210>186

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>186

Asp Pro Pro Ser Gly Gly Trp Glu Phe Asp Tyr

1 5 10

<210>187

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>187

Met Gln Ser Thr Arg Phe Pro Arg Thr

1 5

<210>188

<211>11

<212>PRT

<213> human (Homo sapiens)

<400>188

Ser Arg Leu Glu Trp Leu Leu Tyr Leu Asp Tyr

1 5 10

<210>189

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>189

Gln Ser Tyr Asn Ser Arg Gly Val Met

1 5

<210>190

<211>14

<212>PRT

<213> human (Homo sapiens)

<400>190

Gly Gly Ser Pro Tyr Gly Gly Tyr Ala Tyr Pro Phe Asp Tyr

1 5 10

<210>191

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>191

Gln Ser Tyr Asp Ser Asn Ile Trp Val

1 5

<210>192

<211>12

<212>PRT

<213> human (Homo sapiens)

<400>192

Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Leu Val

1 5 10

<210>193

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>193

Gln Gln Tyr Asp Phe Ser Pro Leu Thr

1 5

<210>194

<211>3

<212>PRT

<213> human (Homo sapiens)

<400>194

Asp Tyr Tyr

1

<210>195

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>195

Gln Ser Tyr Asp Ser Ser Thr Trp Val

1 5

<210>196

<211>10

<212>PRT

<213> human (Homo sapiens)

<400>196

Ser Ser Tyr Thr Ser Ser Ser Thr Leu Gly

1 5 10

<210>197

<211>13

<212>PRT

<213> human (Homo sapiens)

<400>197

Glu Ile Ala Val Ala Gly Thr Arg Tyr Gly Met Asp Val

1 5 10

<210>198

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>198

Gln His Arg Ile Asn Trp Pro Leu Thr

1 5

<210>199

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> CDR1 region

<220>

<221>misc_feature

<222>(1)..(1)

<223> Xaa R, S, T, G, E or D

<220>

<221>misc_feature

<222>(2)..(2)

<223> Xaa is S, G, A, R, N, T, Y or H.

<220>

<221>misc_feature

<222>(3)..(3)

<223> Xaa S, D, N, Q, T, Y, A, G or E

<220>

<221>misc_feature

<222>(4)..(4)

<223> Xaa Q, K, S, N, A, G, I, M, W or L

<220>

<221>misc_feature

<222>(5)..(5)

<223> Xaa S, L, G, A, M, H or N

<220>

<221>misc_feature

<222>(6)..(6)

<223> Xaa is L, G, N, P, V, D, W, S, T, I, or absent

<220>

<221>misc_feature

<222>(7)..(7)

<223> Xaa is L, Y, I, K, S, N, V, or absent

<220>

<221>misc_feature

<222>(8)..(8)

<223> Xaa is H, T, G, Q, S, A, D, or absent

<220>

<221>misc_feature

<222>(9)..(9)

<223> Xaa is S, Y, N, A, T, E, G, F, or absent

<220>

<221>misc_feature

<222>(10)..(10)

<223> Xaa is N, T, A, G, S, Y, K, D, F, L, or absent

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa is G, S, Y, F, L, P, A, F, D, N, or absent

<220>

<221>misc_feature

<222>(12)..(12)

<223> Xaa is Y, V, D, A, F, G, N, or absent

<220>

<221>misc_feature

<222>(13)..(13)

<223> Xaa is N, S, V, H, Q, K, T, or absent

<220>

<221>misc_feature

<222>(14)..(14)

<223> Xaa is Y, H, S, T, or absent

<220>

<221>misc_feature

<222>(15)..(15)

<223> Xaa is L, Y, or absent

<220>

<221>misc_feature

<222>(16)..(16)

<223> Xaa is D, N, L, or absent

<220>

<221>misc_feature

<222>(17)..(17)

<223> Xaa is D, or is absent

<400>199

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa

<210>200

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> CDR2 region

<220>

<221>misc_feature

<222>(1)..(1)

<223> Xaa L, Q, D, N, K, G, H, A, Y, E, T, R, V or W

<220>

<221>misc_feature

<222>(2)..(2)

<223> Xaa G, D, N, A, V, T, I, F or M

<220>

<221>misc_feature

<222>(3)..(3)

<223> Xaa S, F, N, H, G, D, K, T, I, W, Y or R

<220>

<221>misc_feature

<222>(4)..(4)

<223> Xaa N, K, E, L, S, Q, H, T, G, P, Y or A

<220>

<221>misc_feature

<222>(5)..(5)

<223> Xaa R, V, L, S, I, D, G, E, Y, T or N

<220>

<221>misc_feature

<222>(6)..(6)

<223> Xaa A, P, T, F, G, L, N, H or S

<220>

<221>misc_feature

<222>(7)..(7)

<223> Xaa S, T, G or K

<220>

<221>misc_feature

<222>(8)..(8)

<223> Xaa is S, T, I, N, E, W, or absent

<220>

<221>misc_feature

<222>(9)..(9)

<223> Xaa is T, A, I, K, N, Y, D, or absent

<220>

<221>misc_feature

<222>(10)..(10)

<223> Xaa is Y, N, K, F, I, S, G, or absent

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa is Y, N, D, A, or absent

<220>

<221>misc_feature

<222>(12)..(12)

<223> Xaa is A, S, P, Y, D, or absent

<220>

<221>misc_feature

<222>(13)..(13)

<223> Xaa is D, Q, S, A, F, or absent

<220>

<221>misc_feature

<222>(14)..(14)

<223> Xaa is S, K, L, V, or absent

<220>

<221>misc_feature

<222>(15)..(15)

<223> Xaa is V, F, K, S, L, or absent

<220>

<221>misc_feature

<222>(16)..(16)

<223> Xaa is K, Q, S, L, F, G, or absent

<220>

<221>misc_feature

<222>(17)..(17)

<223> Xaa is G, R, or absent

<220>

<221>misc_feature

<222>(18)..(18)

<223> Xaa is G, or is absent

<400>200

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa

<210>201

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> CDR3 region

<220>

<221>misc_feature

<222>(1)..(1)

<223> Xaa D, M, G, Q, G, A, P, E or S

<220>

<221>misc_feature

<222>(2)..(2)

<223> Xaa L, Q, V, A, R, T, S, Y, E, P, H, G or I

<220>

<221>misc_feature

<222>(3)..(3)

<223> Xaa L, A, V, W, E, P, G, S, Y, I, D, T, Q, F or R

<220>

<221>misc_feature

<222>(4)..(4)

<223> Xaa D, L, G, F, T, S, W, V, Y, P, N, H, I or E

<220>

<221>misc_feature

<222>(5)..(5)

<223> Xaa Y, Q, D, S, T, H, A, F, W, G, M, R or N

<220>

<221>misc_feature

<222>(6)..(6)

<223> Xaa D, T, F, A, S, E, W, R, G, Y, I, M, N or L

<220>

<221>misc_feature

<222>(7)..(7)

<223> Xaa I, P, D, T, M, L, S, V, G, H, Y, W, A, N or R

<220>

<221>misc_feature

<222>(8)..(8)

<223> Xaa is any natural amino acid

<220>

<221>misc_feature

<222>(9)..(9)

<223> Xaa is any natural amino acid

<220>

<221>misc_feature

<222>(10)..(10)

<223> Xaa is any natural amino acid

<220>

<221>misc_feature

<222>(11)..(11)

<223> Xaa is any natural amino acid

<220>

<221>misc_feature

<222>(12)..(12)

<223> Xaa is any natural amino acid

<220>

<221>misc_feature

<222>(13)..(13)

<223> Xaa is any natural amino acid

<220>

<221>misc_feature

<222>(14)..(14)

<223> Xaa is any natural amino acid

<220>

<221>misc_feature

<222>(15)..(15)

<223> Xaa is I, D, or absent

<220>

<221>misc_feature

<222>(16)..(16)

<223> Xaa is A, I, V, or absent

<220>

<221>misc_feature

<222>(17)..(17)

<223> Xaa is F, or is absent

<220>

<221>misc_feature

<222>(18)..(18)

<223> Xaa is D, or is absent

<220>

<221>misc_feature

<222>(19)..(19)

<223> Xaa is I, or is absent

<400>201

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa

<210>202

<211>22

<212>PRT

<213> human (Homo sapiens)

<400>202

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys

20

<210>203

<211>40

<212>DNA

<213> human (Homo sapiens)

<400>203

gtggttgaga ggtgccagat gtcaggtcca gctggtgcag 40

<210>204

<211>30

<212>DNA

<213> human (Homo sapiens)

<400>204

attacgtctc acagttcgtt tgatctccac 30

<210>205

<211>48

<212>DNA

<213> human (Homo sapiens)

<400>205

ccgctcagct cctggggctc ctgctattgt ggttgagagg tgccagat 48

<210>206

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>206

Ala Gln Leu Leu Gly Leu Leu Leu Leu

1 5

<210>207

<211>54

<212>DNA

<213> human (Homo sapiens)

<400>207

cagcagaagc ttctagacca ccatggacat gagggtcccc gctcagctcc tggg 54

<210>208

<211>45

<212>DNA

<213> human (Homo sapiens)

<400>208

gtggttgaga ggtgccagat gtgacattgt gatgactcag tctcc 45

<210>209

<211>31

<212>DNA

<213> human (Homo sapiens)

<400>209

cttgtcgact tattaacact ctcccctgtt g 31

<210>210

<211>112

<212>PRT

<213> human (Homo sapiens)

<400>210

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Leu Gln Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>211

<211>112

<212>PRT

<213> human (Homo sapiens)

<400>211

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Phe Leu Gly Ser Ser Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Lys Val Glu Ala Asp Asp Val Gly Ile Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Asp Thr Pro Pro Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>212

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>212

Met Gln Gly Leu Gln Thr Pro Pro Thr

1 5

<210>213

<211>12

<212>PRT

<213> human (Homo sapiens)

<400>213

Arg Ala Ser Gln Ser Val Ser Ser Ser Ser Leu Ala

1 5 10

<210>214

<211>7

<212>PRT

<213> human (Homo sapiens)

<400>214

Ala Ala Ser Ser Arg Ala Thr

1 5

<210>215

<211>9

<212>PRT

<213> human (Homo sapiens)

<400>215

Gln His Tyr Gly Ser Ser Pro Arg Thr

1 5

Claims (47)

1. An isolated polypeptide that specifically binds to angiopoietin-2, wherein said polypeptide comprises at least one Complementarity Determining Region (CDR), wherein said CDR is:

a) a CDR1 region comprising an amino acid sequence of the formula:

X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹X¹⁰X¹¹X¹²X¹³X¹⁴X¹⁵X¹⁶X¹⁷(SEQ ID NO: 199) in which

X¹R, S, T, G, E, D;

X²s, G, A, R, N, T, Y, H;

X³s, D, N, Q, T, Y, A, G, E;

X⁴q, K, S, N, A, G, I, M, W, L;

X⁵s, L, G, A, M, H, N;

X⁶l, G, N, P, V, D, W, S, T, I, or absent;

X⁷l, Y, I, K, S, N, V, or absent;

X⁸h, T, G, Q, S, A, D, or absent;

X⁹s, Y, N, A, T, E, G, F, or absent;

X¹⁰n, T, A, G, S, Y, K, D, F, L, or absent;

X¹¹g, S, Y, F, L, P, A, F, D, N, or absent;

X¹²y, V, D, A, F, G, N, or absent;

X¹³n, S, V, H, Q, K, T, or absent;

X¹⁴y, H, S, T, or absent;

X¹⁵l, Y, or absent;

X¹⁶d, N, L, or absent;

X¹⁷is D, or is absent;

b) a CDR2 region comprising an amino acid sequence of the formula:

X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹X¹⁰X¹¹X¹²X¹³X¹⁴X¹⁵X¹⁶X¹⁷X¹⁸(SEQ ID NO: 200) wherein,

X¹l, Q, D, N, K, G, H, A, Y, E, T, R, V, W;

X²G, D, N, A, V, T, I, F, M;

X³s, F, N, H, G, D, K, T, I, W, Y, R;

X⁴n, K, E, L, S, Q, H, T, G, P, Y, A;

X⁵r, V, L, S, I, D, G, E, Y, T, N;

X⁶a, P, T, F, G, L, N, H, S;

X⁷s, T, G, K;

X⁸s, T, I, N, E, W, or absent;

X⁹t, A, I, K, N, Y, D, or absent;

X¹⁰y, N, K, F, I, S, G, or absent;

X¹¹y, N, D, A, or absent;

X¹²a, S, P, Y, D, or absent;

X¹³d, Q, S, A, F, or absent;

X¹⁴s, K, L, V, or absent;

X¹⁵v, F, K, S, L, or absent;

X¹⁶k, Q, S, L, F, G, or absent;

X¹⁷g, R, or absent;

X¹⁸is G, or is absent; or

c) A CDR3 region comprising an amino acid sequence of the formula:

X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹X¹⁰X¹¹X¹²X¹³X¹⁴X¹⁵X¹⁶X¹⁷X¹⁸X¹⁹(SEQ ID NO: 201) wherein,

X¹d, M, G, Q, F, A, P, E, S;

X²l, Q, V, A, R, T, S, Y, E, P, H, G, I;

X³l, A, V, W, E, P, G, S, Y, I, D, T, Q, F, R;

X⁴d, L, G, F, T, S, W, V, Y, P, N, H, I, E;

X⁵y, Q, D, S, T, H, A, F, W, G, M, R, N;

X⁶D, T, F, A, S, E, W, R, G, Y, I, M, N, L;

X⁷i, P, D, T, M, L, S, V, G, H, Y, W, A, N, R;

X⁸l, P, W, A, V, S, D, G, F, N, Y, I, R, E, T;

X⁹t, L, V, F, A, G, P, D, S, H, N, M, Y, or absent;

X¹⁰g, S, V, N, F, A, M, D, Y, L, W, I, T, or absent;

X¹¹p, F, Y, W, D, E, I, G, A, V, L, S, M, or absent;

X¹²y, F, V, G, I, V, M, A, D, or absent;

X¹³a, D, Y, V, F, H, I, G, or absent;

X¹⁴y, V, D, I, S, F, M, or absent;

X¹⁵i, D, or absent;

X¹⁶a, I, V, or absent;

X¹⁷is F, or is absent;

X¹⁸is D, or is absent; and

X¹⁹is I, or is absent.

2. The polypeptide of claim 1, wherein said polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ id nos: 69-104 and SEQ ID NO: 213.

3. The polypeptide of claim 1, wherein said polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ id nos: 105-143 and SEQ ID NO: 214, or a pharmaceutically acceptable salt thereof.

4. The polypeptide of claim 1, wherein said polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ id nos: 144-198, SEQ ID NO: 212 and SEQ ID NO: 215, or a pharmaceutically acceptable salt thereof.

5. The isolated polypeptide of claim 1, wherein the polypeptide is an antibody.

6. The antibody of claim 5, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

7. The antibody of claim 6, wherein the antibody is a single chain antibody.

8. A hybridoma that produces the monoclonal antibody of claim 6.

9. A nucleic acid molecule encoding the polypeptide of claim 1.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A host cell comprising the vector of claim 10.

12. A nucleic acid molecule encoding the antibody of claim 5, 6 or 7.

13. A vector comprising the nucleic acid molecule of claim 12.

14. A host cell comprising the vector of claim 13.

15. A method of making an antibody, wherein the method comprises:

(a) transforming a host cell with at least one nucleic acid molecule encoding the antibody of claim 5, 6 or 7;

(b) expressing a nucleic acid molecule in said host cell; and

(c) isolating the specific binding agent.

16. A method of inhibiting undesired angiogenesis in a mammal, said method comprising administering a therapeutically effective amount of the isolated polypeptide of claim 1.

17. A method of treating cancer in a mammal comprising administering a therapeutically effective amount of the isolated polypeptide of claim 1.

18. A method of inhibiting undesired angiogenesis in a mammal, said method comprising administering a therapeutically effective amount of the antibody of claim 5, 6, or 7.

19. A method of treating cancer in a mammal comprising administering a therapeutically effective amount of the antibody of claim 5, 6 or 7.

20. A pharmaceutical composition comprising the isolated polypeptide of claim 1 and a pharmaceutically acceptable excipient.

21. A pharmaceutical composition comprising the antibody of claim 5, 6 or 7 and a pharmaceutically acceptable excipient.

22. A method of modulating or inhibiting angiopoietin-2 activity comprising administering to a patient the isolated polypeptide of claim 1.

23. A method of modulating or inhibiting angiopoietin-2 activity comprising administering to a patient the antibody of claim 5, 6 or 7.

24. A method of modulating vascular permeability and/or plasma leakage in a mammal comprising administering to the mammal a therapeutically effective amount of the isolated polypeptide of claim 1.

25. A method of treating at least one of the following mammalian diseases: an ocular neovascular disease, obesity, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, inflammation, atherosclerosis, endometriosis, neoplastic disease, bone-related disease or psoriasis, comprising administering a therapeutically effective amount of the isolated polypeptide of claim 1.

26. A method of modulating vascular permeability and/or plasma leakage in a mammal comprising administering a therapeutically effective amount of the antibody of claim 5, 6 or 7.

27. A method of treating at least one of the following mammalian diseases: an ocular neovascular disease, obesity, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, inflammation, atherosclerosis, endometriosis, neoplastic disease, bone-related disease or psoriasis, comprising administering a therapeutically effective amount of an antibody of claim 5, 6 or 7.

28. A method of treating cancer in a mammal comprising administering a therapeutically effective amount of the isolated polypeptide of claim 1 and a chemotherapeutic agent.

29. The method of claim 28, wherein the isolated polypeptide and the chemotherapeutic agent are administered simultaneously.

30. The method of claim 28, wherein the isolated polypeptide and the chemotherapeutic agent are not administered simultaneously.

31. A method of treating cancer in a mammal comprising administering a therapeutically effective amount of the antibody of claim 5, 6 or 7 and a chemotherapeutic agent.

32. An isolated antibody that binds to angiopoietin-2, comprising CDR1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 69-104 or SEQ ID NO: 213.

33. An isolated antibody that binds to angiopoietin-2, comprising CDR2, wherein CDR2 comprises the amino acid sequence of SEQ ID NO: 105-143 or SEQ ID NO: 214, or a pharmaceutically acceptable salt thereof.

34. An isolated antibody that binds to angiopoietin-2, comprising CDR3, wherein CDR3 comprises the amino acid sequence of SEQ ID NO: 144-198, SEQ ID NO: 212 or SEQ ID NO: 215, or a pharmaceutically acceptable salt thereof.

35. A nucleic acid molecule encoding the antibody of claim 32, 33 or 34.

36. A vector comprising the nucleic acid molecule of claim 35.

37. A host cell comprising the vector of claim 36.

38. A method of detecting the level of angiopoietin-2 in a biological sample, comprising:

(a) contacting the isolated polypeptide of claim 1 with the sample; and

(b) determining the extent of binding of a specific binding agent to said sample.

39. A method of detecting the level of angiopoietin-2 in a biological sample, comprising:

(a) contacting the antibody of claim 32, 33 or 34 with the sample; and

(b) determining the extent of binding of the antibody to the sample.

40. An antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region selected from the group consisting of:

SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 33. SEQ ID NO: 35. SEQ ID NO: 37. SEQ ID NO: 39. SEQ ID NO: 41. SEQ ID NO: 43. SEQ ID NO: 45. SEQ ID NO: 47. SEQ ID NO: 49. SEQ ID NO: 51. SEQ ID NO: 53. SEQ ID NO: 55. SEQ ID NO: 57. SEQ ID NO: 59. SEQ ID NO: 61; and antigen binding fragments thereof;

and the light chain comprises a light chain variable region comprising SEQ ID NO: 12. SEQ ID NO: 210 or SEQ ID NO: 211, or an antigen-binding fragment thereof.

41. A nucleic acid molecule encoding the antibody of claim 40.

42. A vector comprising the nucleic acid molecule of claim 41.

43. A host cell comprising the vector of claim 42.

44. An antibody that specifically binds to angiopoietin-2, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. SEQ ID NO: 210 or SEQ ID NO: 211, or an antigen-binding fragment thereof.

45. A nucleic acid molecule encoding the antibody of claim 44.

46. A vector comprising the nucleic acid molecule of claim 45.

47. A host cell comprising the vector of claim 46.