Angiopoietin-2 specific binding agents Number:7,521,053 from the United States Patent and Trademark Office (PTO) owispatent Disclosed are specific binding agents, such as fully human antibodies, that bind to angiopoietin-2. Also disclosed are heavy chain fragments, light chain fragments, and CDRs of the antibodies, as well as methods of making and using the antibodies.<H Angiopoietin, specific, Angiopoietin, 2, , 7521053.html, Patent, pto, 7521053

Title: Angiopoietin-2 specific binding agents

Abstract: Disclosed are specific binding agents, such as fully human antibodies, that bind to angiopoietin-2. Also disclosed are heavy chain fragments, light chain fragments, and CDRs of the antibodies, as well as methods of making and using the antibodies.

Patent Number: 7,521,053 Issued on 04/21/2009 to Oliner

Inventors: Oliner; Jonathan Daniel (Newbury Park, CA)
Assignee: Amgen Inc. (Thousand Oaks, CA)
Medarex, Inc. (Princeton, NJ)

Appl. No.: 10/269,805

Filed: October 10, 2002

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60328604	Oct., 2001

Current U.S. Class: 424/155.1 ; 530/387.7; 530/388.85

Current International Class: C07K 16/00 (20060101); C07K 16/30 (20060101); A61K 39/395 (20060101)

Field of Search: 530/300,350 424/155.1

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Primary Examiner: Fetterolf; Brandon J
Attorney, Agent or Firm: McDonnell Boehnen Hulbert & Berghoff LLP

Parent Case Text

This application claims benefit to U.S. Provisional Application Ser. No. 60/328,604, filed Oct. 11, 2001, which is incorporated herein by reference.

Claims

What is claimed is:

1. An isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 of SEQ ID NO. 11, and wherein the light chain comprises a light chain variable region comprising CDR1, CDR2, and CDR3 of SEQ ID NO. 12, wherein the antibody binds angiopoietin-2 (Ang-2).

2. The antibody of claim 1, wherein the heavy chain variable region comprises SEQ ID NO. 11; and the light chain variable region comprises CDR1, CDR2, and CDR3 of SEQ ID NO. 12.

3. The antibody of claim 1, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of SEQ ID NO. 11; and the light chain variable region comprises of SEQ ID NO. 12.

4. The antibody of claim 1, wherein the heavy chain variable region comprises SEQ ID NO. 11, and the light chain variable region comprises SEQ ID NO. 12.

5. An isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, wherein the antibody binds angiopoietin-2 (Ang-2).

6. An isolated antibody 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, wherein the antibody binds angiopoietin-2 (Ang-2).

7. An isolated antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, or an antigen-binding fragment thereof, wherein the antigen-binding fragment comprises at least CDR1, CDR2, and CDR3 of SEQ ID NO. 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12, or an antigen-binding fragment thereof, wherein the antigen-binding fragment comprises at least CDR1, CDR2, and CDR3 of SEQ ID NO. 12, wherein the antibody binds angiopoietin-2 (Ang-2).

8. An isolated antibody comprising: a. heavy chain framework regions, a heavy chain CDR1 region comprising CDR1 of SEQ ID NO. 11, a heavy chain CDR2 region comprising CDR2 of SEQ ID NO. 11, and a heavy chain CDR3 region comprising CDR3 of SEQ ID NO. 11; and b. light chain framework regions, a light chain CDR1 region comprising CDR1 of SEQ ID NO. 12, a light chain CDR2 region comprising CDR2 of SEQ ID NO. 12, and a light chain CDR3 region comprising CDR3 of SEQ ID NO. 12; wherein the antibody binds angiopoietin-2 (Ang-2).

9. The antibody of claim 1, 5, 6, or 7 that is a polyclonal, monoclonal, chimeric, humanized, or fully human antibody.

10. The antibody of claim 9 that is a single chain antibody.

11. The antibody of claim 9, or 10, wherein the antibody is covalently attached to a molecule selected form the group consisting of a reporter group, a water soluble polymer, an Fc region, and a cytotoxic agent.

12. The antibody of claim 10, which is a single-chain Fv antibody.

13. The antibody of claim 10, which is a Fab antibody fragment.

14. The antibody of claim 10, which is a Fab' antibody fragment.

15. The antibody of claim 10, which is a (Fab').sub.2 antibody fragment.

16. A pharmaceutical composition comprising the isolated antibody of claim 9 and a pharmaceutically acceptable formulation agent.

17. A pharmaceutical composition comprising the antibody of claim 9 and a pharmaceutically acceptable carrier.

Description

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

Angiogenesis, the formation of new blood vessels from existing ones, is essential to many physiological and pathological processes. Normally, angiogenesis is tightly regulated by pro- and anti-angiogenic factors, but in the case of diseases such as cancer, ocular neovascular diseases, arthritis, and psoriasis, the process can go awry. Folkman, J., Nat. Med., 1:27-31 (1995).

There are a number of diseases known to be associated with deregulated or undesired angiogenesis. Such diseases include, but are not limited to, ocular neovascularisation, such as retinopathies (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, such as a rheumatoid or rheumatic inflammatory disease, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disorders, such as chronic asthma, arterial or post-transplantational atherosclerosis, endometriosis, and neoplastic diseases, for example so-called solid tumors and liquid (or hematopoietic) tumors (such as leukemias and lymphomas). Other diseases associated with undesired angiogenesis will be apparent to those skilled in the art.

Although many signal transduction systems have been implicated in the regulation of angiogenesis, one of the best-characterized and most endothelial cell-selective systems involves the Tie-2 receptor tyrosine kinase (referred to as "Tie-2" or "Tie-2R" (also referred to as "ORK"); murine Tie-2 is also referred to as "tek") and its ligands, the angiopoietins (Gale, N. W. and Yancopoulos, G. D., Genes Dev. 13:1055-1066 [1999]). There are 4 known angiopoietins; angiopoietin-1 ("Ang-1") through angiopoietin-4 ("Ang-4"). These angiopoietins are also referred to as "Tie-2 ligands". (Davis, S., et al., Cell, 87:1161-1169 [1996]; Grosios, K., et al., Cytogenet Cell Genet, 84:118-120 [1999]; Holash, J., et al., Investigative Ophthalmology & Visual Science, 42:1617-1625 [1999]; Koblizek, T. I., et al., Current Biology, 8:529-532 [1998]; Lin, P., et al., Proc Natl Acad Sci USA, 95:8829-8834 [1998]; Maisonpierre, P. C., et al., Science, 277:55-60 [1997]; Papapetropoulos, 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]; Valenzuela, D. M., et al., Proceedings of the National Academy of Sciences of the USA, 96:1904-1909 [1999]; Witzenbichler, B., et al., J Biol Chem, 273:18514-18521 [1998]). Whereas Ang-1 binding to Tie-2 stimulates receptor phosphorylation in cultured endothelial cells, Ang-2 has been observed to both agonize and antagonize Tie-2 receptor phosphorylation (Davis, S., et al., [1996], supra; Maisonpierre, P. C., et al., [1997], supra; Kim, I., J. H. Kim, et al., Oncogene 19(39): 4549-4552 (2000); Teichert-Kuliszewska, K., P. C. Maisonpierre, et al., Cardiovascular Research 49(3): 659-70 (2001)).

The phenotypes of mouse Tie-2 and Ang-1 knockouts are similar and suggest that Ang-1-stimulated Tie-2 phosphorylation mediates remodeling and stabilization of developing vessels in utero through maintenance of endothelial cell-support cell adhesion (Dumont, D. J., et al., Genes & Development, 8:1897-1909 [1994]; Sato, T. N., et al., Nature, 376:70-74 [1995]; Suri, C., et al., [1996], supra). The role of Ang-1 in vessel stabilization is thought to be conserved in the adult, where it is expressed widely and constitutively (Hanahan, D., Science, 277:48-50 [1997]; Zagzag, D., et al., Experimental Neurology, 159:391-400 [1999]). In contrast, Ang-2 expression is primarily limited to sites of vascular remodeling, where it is thought to block Ang-1 function, thereby inducing a state of vascular plasticity conducive to angiogenesis (Hanahan, D., [1997], supra; Holash, J., et al., Science, 284:1994-1998 [1999]; Maisonpierre, P. C., et al., [1997], supra).

Numerous published studies have purportedly demonstrated vessel-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, 155:1967-1976 [1999]; Etoh, T., et al., Cancer Research, 61:2145-2153 [2001]; Hangai, M., et al., Investigative Ophthalmology & Visual Science, 42:1617-1625 [2001]; Holash, J., et al., [1999] supra; Kuroda, K., et al., Journal of Investigative Dermatology, 116:713-720 [2001]; Otani, A., et al., Investigative Ophthalmology & Visual Science, 40:1912-1920 [1999]; Stratmann, A., et al., American Journal of Pathology, 153:1459-1466 [1998]; Tanaka, S., et al., J Clin Invest, 103:34-345 [1999]; Yoshida, Y., et al., International Journal of Oncology, 15:1221-1225 [1999]; Yuan, K., et al., Journal of Periodontal Research, 35:165-171 [2000]; Zagzag, D., et al., [1999] supra). Most of these studies have focused on cancer, in which many tumor types appear to display vascular Ang-2 expression. In contrast with its expression in pathological angiogenesis, Ang-2 expression in normal tissues is extremely limited (Maisonpierre, P. C., et al., [1997], supra; Mezquita, J., et al., Biochemical and Biophysical Research Communications, 260:492-498 [1999]). In the normal adult, the three main sites of angiogenesis are the ovary, placenta, and uterus; these are the primary tissues in normal (i.e., non-cancerous) tissues in which Ang-2 mRNA has been detected.

Certain functional studies suggest that Ang-2 may be involved in tumor angiogenesis. Ahmad et al. (Cancer Res., 61:1255-1259 [2001]) describe Ang-2 over-expression and show that it is purportedly associated with an increase in tumor growth in a mouse xenograft model. See also Etoh et al., supra, and Tanaka et al., supra, wherein data is presented purportedly associating Ang-2 over expression with tumor hypervascularity. However, in contrast, Yu et al. (Am. J. Path., 158:563-570 [2001]) report data to show that overexpression of Ang-2 in Lewis lung carcinoma and TA3 mammary carcinoma cells purportedly prolonged the survival of mice injected with the corresponding transfectants.

In the past few years, various publications have suggested Ang-1, Ang-2 and/or Tie-2 as a possible target for anti-cancer therapy. For example, U.S. Pat. Nos. 6,166,185, 5,650,490, and 5,814,464 each disclose the concept of anti-Tie-2 ligand antibodies and receptor bodies. Lin et al. (Proc. Natl. Acad. Sci USA, 95:8829-8834 [1998]) injected an adenovirus expressing soluble Tie-2 into mice; the soluble Tie-2 purportedly decreased the number and size of the tumors developed by the mice. In a related study, Lin et al (J. Clin. Invest., 100:2072-2078 [1997]) injected a soluble form of Tie-2 into rats; this compound purportedly reduced tumor size in the rats. Siemeister et al. (Cancer Res., 59:3185-3189 [1999]) generated human melanoma cell lines expressing the extracellular domain of Tie-2, injected these cell lines into nude mice, and concluded that soluble Tie-2 purportedly resulted in a "significant inhibition" of tumor growth and tumor angiogenesis. In view of this information, and given that 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 would be an attractive target for anti-cancer therapy.

The fusion of certain peptides to a stable plasma protein such as an Ig constant region to improve the half-life of these molecules has been described in, for example, PCT publication WO 00/24782, published May 4, 2000.

The fusion of a protein or fragment thereof to a stable plasma protein such as an Ig constant region to improve the half-life of these molecules has been variously described (see, for example, U.S. Pat. No. 5,480,981; Zheng et al., J. Immunol., 154:5590-5600, (1995); Fisher et al., N. Engl. J. Med., 334:1697-1702, (1996); Van Zee, K. et al., J. Immunol., 156:2221-2230, (1996); U.S. Pat. No. 5,808,029, issued Sep. 15, 1998; Capon et al., Nature, 337:525-531, (1989); Harvill et al., Immunotech., 1:95-105, (1995); WO 97/23614, published Jul. 3, 1997; PCT/US 97/23183, filed Dec. 11, 1997; Linsley, J. Exp. Med., 174:561-569, (1991); WO 95/21258, published Aug. 10, 1995).

An effective anti-Ang-2 therapy might benefit a vast population of cancer patients because most solid tumors require neovascularization to grow beyond 1-2 millimeters in diameter. Such therapy might have wider application in other angiogenesis-associated diseases as well, such as retinopathies, arthritis, and psoriasis.

There is an undeveloped need to identify new agents that specifically recognize and bind Ang-2. Such agents would be useful for diagnostic screening and therapeutic intervention in disease states that are associated with Ang-2 activity.

Accordingly, it is an object of the present invention to provide specific binding agents of Ang-2 that modulate Ang-2 activity.

SUMMARY OF THE INVENTION

The present invention provides an antibody comprising a heavy chain and a light chain, wherein said heavy chain comprises a heavy chain variable region selected from the group consisting of 526 HC (SEQ ID NO. 1); 528 HC (SEQ ID NO. 3); 531 HC (SEQ ID NO. 5); 533 HC (SEQ ID NO. 7); 535 HC (SEQ ID NO. 9); 536 HC (SEQ ID NO. 11); 537 HC (SEQ ID NO. 13); 540 HC (SEQ ID NO. 15); 543 HC (SEQ ID NO. 17); 544 HC (SEQ ID NO. 19); 545 HC (SEQ ID NO. 21); 546 HC (SEQ ID NO. 23); 551 HC (SEQ ID NO. 25); 553 HC (SEQ ID NO. 27); 555 HC (SEQ ID NO. 29); 558 HC (SEQ ID NO. 31); 559 HC (SEQ ID NO. 33); 565 HC (SEQ ID NO. 35); F1-C6 HC (SEQ ID NO. 37); FB1-A7 HC (SEQ ID NO. 39); FD-B2 HC (SEQ ID NO. 41); FE-B7 HC (SEQ ID NO. 43); FJ-G11 HC (SEQ ID NO. 45); FK-E3 HC (SEQ ID NO. 47); G1D4 HC (SEQ ID NO. 49); GC1E8 HC (SEQ ID NO. 51); H1C12 HC (SEQ ID NO. 53); IA1-1E7 HC (SEQ ID NO. 55); IF-1C10 HC (SEQ ID NO. 57); IK-2E2 HC (SEQ ID NO. 59); IP-2C11 HC (SEQ ID NO. 61); and antigen binding fragments thereof; and said light chain comprises a light chain variable region selected from the group consisting of: 526 kappa (SEQ ID NO. 2); 536 kappa (SEQ ID NO. 12); 543 kappa (SEQ ID NO. 18); 544 kappa (SEQ ID NO. 20); 551 kappa (SEQ ID NO. 26); 553 kappa (SEQ ID NO. 28); 555 kappa (SEQ ID NO. 30); 558 kappa (SEQ ID NO. 32); 565 kappa (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 kappa (SEQ ID NO. 56); IP-2C11 kappa (SEQ ID NO. 62); 528 lambda (SEQ ID NO. 4); 531 lambda (SEQ ID NO. 6); 533 lambda (SEQ ID NO. 8); 535 lambda (SEQ ID NO. 10); 537 lambda (SEQ ID NO. 14); 540 lambda (SEQ ID NO. 16); 545 lambda (SEQ ID NO. 22); 546 lambda (SEQ ID NO. 24); 559 lambda (SEQ ID NO. 34); F1-C6 lambda (SEQ ID NO. 38); FB1-A7 lambda (SEQ ID NO. 40); FD-B2 lambda (SEQ ID NO. 42); G1D4 lambda (SEQ ID NO. 50); GC1E8 lambda (SEQ ID NO. 52); H1C12 lambda (SEQ ID NO. 54); IF-1C10 lambda (SEQ ID NO. 58); IK-2E2 lambda (SEQ ID NO. 60); and antigen binding fragments thereof.

The invention also provides a specific binding agent comprising at least one peptide 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; 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; SEQ ID NO. 32; SEQ ID NO. 36; SEQ ID NO. 44; SEQ ID NO. 46; SEQ ID NO. 48; SEQ ID NO. 56; SEQ ID NO. 62; 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; SEQ ID NO. 58; and SEQ ID NO. 60, and fragments thereof.

It will be appreciated that the specific binding agent can be, for example, an antibody, such as a polyclonal, monoclonal, chimeric, humanized, or a fully human antibody. The antibody may also be a single chain antibody. The invention further relates to a hybridoma that produces a monoclonal antibody according to the invention.

It will also be appreciated that the invention relates to conjugates as described herein. The conjugate can be, for example, a specific binding agent (such as an antibody) of the invention.

The invention further relates to nucleic acid molecules encoding the specific binding agents (such as an antibody) of the invention, as well as a vector comprising such nucleic acid molecule, as well as a host cell containing the vector.

Additionally, the invention provides a method of making a specific binding agent comprising, (a) transforming a host cell with at least one nucleic acid molecule encoding the specific binding agent of claim 1; (b) expressing the nucleic acid molecule in said host cell; and (c) isolating said specific binding agent. The invention further provides a method of making an antibody comprising: (a) transforming a host cell with at least one nucleic acid molecule encoding the antibody according to the invention; (b) expressing the nucleic acid molecule in said host cell; and (c) isolating said specific binding agent.

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

The invention also relates to a method of inhibiting undesired angiogenesis in a mammal comprising by administering a therapeutically effective amount of an antibody according to the invention. The invention additionally provides a method of treating cancer in a mammal comprising administering a therapeutically effective amount of antibody according to the invention.

It will be appreciated that the invention further relates to pharmaceutical compositions comprising the specific binding agent according to the invention and a pharmaceutically acceptable formulation agent. The pharmaceutical composition may comprise an antibody according to the invention and a pharmaceutically acceptable formulation agent.

The invention provides a method of modulating or inhibiting angiopoietin-2 activity by administering one or more specific binding agents of the invention. The invention also provides a method of modulating or inhibiting angiopoietin-2 activity by administering an antibody of the invention.

The invention further relates to a method of modulating at least one of vascular permeability or plasma leakage in a mammal comprising administering a therapeutically effective amount of the specific binding agent according to the invention. The invention also relates to a method of treating at least one of ocular neovascular disease, obesity, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, inflammatory disorders, atherosclerosis, endometriosis, neoplastic disease, bone-related disease, or psoriasis in a mammal comprising administering a therapeutically effective amount of a specific binding agent according to the invention.

The invention further provides a method of modulating at least one of vascular permeability or plasma leakage in a mammal comprising administering a therapeutically effective amount of an antibody according to the invention. The invention also relates to a method of treating at least one of ocular neovascular disease, obesity, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, inflammatory disorders, atherosclerosis, endometriosis, neoplastic disease, bone-related disease, or psoriasis in a mammal comprising administering a therapeutically effective amount of an antibody according to the invention.

Furthermore, the invention relates to a method of treating cancer in a mammal comprising administering a therapeutically effective amount of a specific binding agent according to the invention and a chemotherapeutic agent. It will be appreciated by those in the art that the specific binding agent and chemotherapeutic agent need not be administered simultaneously.

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

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

The invention further relates to a specific binding agent comprising complementarity determining region 2 (CDR 2) of any of: 526 HC (SEQ ID NO. 1); 528 HC (SEQ ID NO. 3); 531 HC (SEQ ID NO. 5); 533 HC (SEQ ID NO. 7); 535 HC (SEQ ID NO. 9); 536 HC (SEQ ID NO. 11); 537 HC (SEQ ID NO. 13); 540 HC (SEQ ID NO. 15); 543 HC (SEQ ID NO. 17); 544 HC (SEQ ID NO. 19); 545 HC (SEQ ID NO. 21); 546 HC (SEQ ID NO. 23); 551 HC (SEQ ID NO. 25); 553 HC (SEQ ID NO. 27); 555 HC (SEQ ID NO. 29); 558 HC (SEQ ID NO. 31); 559 HC (SEQ ID NO. 33); 565 HC (SEQ ID NO. 35); F1-C6 HC (SEQ ID NO. 37); FB1-A7 HC (SEQ ID NO. 39); FD-B2 HC (SEQ ID NO. 41); FE-B7 HC (SEQ ID NO. 43); FJ-G11 HC (SEQ ID NO. 45); FK-E3 HC (SEQ ID NO. 47); G1D4 HC (SEQ ID NO. 49); GC1E8 HC (SEQ ID NO. 51); H1C12 HC (SEQ ID NO. 53); IA1-1E7 HC (SEQ ID NO. 55); IF-1C10 HC (SEQ ID NO. 57); IK-2E2 HC (SEQ ID NO. 59); IP-2C11 HC (SEQ ID NO. 61); 526 kappa (SEQ ID NO. 2); 536 kappa (SEQ ID NO. 12); 543 kappa (SEQ ID NO. 18); 544 kappa (SEQ ID NO. 20); 551 kappa (SEQ ID NO. 26); 553 kappa (SEQ ID NO. 28); 555 kappa (SEQ ID NO. 30); 558 kappa (SEQ ID NO. 32); 565 kappa (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 kappa (SEQ ID NO. 56); IP-2C11 kappa (SEQ ID NO. 62); 528 lambda (SEQ ID NO. 4); 531 lambda (SEQ ID NO. 6); 533 lambda (SEQ ID NO. 8); 535 lambda (SEQ ID NO. 10); 537 lambda (SEQ ID NO. 14); 540 lambda (SEQ ID NO. 16); 545 lambda (SEQ ID NO. 22); 546 lambda (SEQ ID NO. 24); 559 lambda (SEQ ID NO. 34); F1-C6 lambda (SEQ ID NO. 38); FB1-A7 lambda (SEQ ID NO. 40); FD-B2 lambda (SEQ ID NO. 42); G1D4 lambda (SEQ ID NO. 50); GC1E8 lambda (SEQ ID NO. 52); H1C12 lambda (SEQ ID NO. 54); IF-1C10 lambda (SEQ ID NO. 58); and IK-2E2 lambda (SEQ ID NO. 60).

The invention also relates to a specific binding agent comprising complementarity determining region 3 (CDR 3) of any of: 526 HC (SEQ ID NO. 1); 528 HC (SEQ ID NO. 3); 531 HC (SEQ ID NO. 5); 533 HC (SEQ ID NO. 7); 535 HC (SEQ ID NO. 9); 536 HC (SEQ ID NO. 11); 537 HC (SEQ ID NO. 13); 540 HC (SEQ ID NO. 15); 543 HC (SEQ ID NO. 17); 544 HC (SEQ ID NO. 19); 545 HC (SEQ ID NO. 21); 546 HC (SEQ ID NO. 23); 551 HC (SEQ ID NO. 25); 553 HC (SEQ ID NO. 27); 555 HC (SEQ ID NO. 29); 558 HC (SEQ ID NO. 31); 559 HC (SEQ ID NO. 33); 565 HC (SEQ ID NO. 35); F1-C6 HC (SEQ ID NO. 37); FB1-A7 HC (SEQ ID NO. 39); FD-B2 HC (SEQ ID NO. 41); FE-B7 HC (SEQ ID NO. 43); FJ-G11 HC (SEQ ID NO. 45); FK-E3 HC (SEQ ID NO. 47); G1D4 HC (SEQ ID NO. 49); GC1E8 HC (SEQ ID NO. 51); H1C12 HC (SEQ ID NO. 53); IA1-1E7 HC (SEQ ID NO. 55); IF-1C10 HC (SEQ ID NO. 57); IK-2E2 HC (SEQ ID NO. 59); IP-2C11 HC (SEQ ID NO. 61); 526 kappa (SEQ ID NO. 2); 536 kappa (SEQ ID NO. 12); 543 kappa (SEQ ID NO. 18); 544 kappa (SEQ ID NO. 20); 551 kappa (SEQ ID NO. 26); 553 kappa (SEQ ID NO. 28); 555 kappa (SEQ ID NO. 30); 558 kappa (SEQ ID NO. 32); 565 kappa (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 kappa (SEQ ID NO. 56); IP-2C11 kappa (SEQ ID NO. 62); 528 lambda (SEQ ID NO. 4); 531 lambda (SEQ ID NO. 6); 533 lambda (SEQ ID NO. 8); 535 lambda (SEQ ID NO. 10); 537 lambda (SEQ ID NO. 14); 540 lambda (SEQ ID NO. 16); 545 lambda (SEQ ID NO. 22); 546 lambda (SEQ ID NO. 24); 559 lambda (SEQ ID NO. 34); F1-C6 lambda (SEQ ID NO. 38); FB1-A7 lambda (SEQ ID NO. 40); FD-B2 lambda (SEQ ID NO. 42); G1D4 lambda (SEQ ID NO. 50); GC1E8 lambda (SEQ ID NO. 52); H1C12 lambda (SEQ ID NO. 54); IF-1C10 lambda (SEQ ID NO. 58); and IK-2E2 lambda (SEQ ID NO. 60).

The invention further provides a nucleic acid molecule encoding a specific binding agent according to the invention.

Moreover, the invention relates to a method of detecting the level of angiopoietin-2 in a biological sample by (a) contacting a specific binding agent of the invention with the sample; and (b) determining the extent of binding of the specific binding agent to the sample. The invention also relates to a method of detecting the level of angiopoietin-2 in a biological sample by (a) contacting an antibody of the invention with the sample; 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 administering a therapeutically effective amount of a polypeptide or composition as described herein. The invention also relates to a method of modulating angiogenesis in a mammal comprising administering a therapeutically effective amount of a polypeptide or composition as described herein. The invention further relates to a method of inhibiting tumor growth characterized by undesired angiogenesis in a mammal comprising administering a therapeutically effective amount of a polypeptide or composition as described herein. Additionally, the invention relates to a method of treating cancer in a mammal comprising administering a therapeutically effective amount of a polypeptide or composition as described herein, and a chemotherapeutic agent. In a preferred embodiment, the chemotherapeutic agent is at least one of 5-FU, CPT-11, and Taxotere. It will be appreciated, however, that other suitable chemotherapeutic agents and other cancer therapies can be used.

It will be appreciated that the specific binding agents of the invention can be used to treat a number of diseases associated with deregulated or undesired angiogenesis. Such diseases include, but are not limited to, ocular neovascularisation, such as retinopathies (including diabetic retinopathy and age-related macular degeneration) psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, such as a rheumatoid or rheumatic inflammatory disease, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disorders, such as chronic asthma, arterial or post-transplantational atherosclerosis, endometriosis, and neoplastic diseases, for example so-called solid tumors and liquid tumors (such as leukemias). Additional diseases which can be treated by administration of the specific binding agents will be apparent to those skilled in the art. Such additional diseases include, but are not limited to, obesity, vascular permeability, plasma leakage, and bone-related disorders, including osteoporosis. Thus, the invention further relates to methods of treating these diseases associated with deregulated or undesired angiogenesis.

Other embodiments of this invention will be readily apparent from the disclosure provided herewith.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a graph of tumor size (y-axis) versus time x-axis) in tumor bearing mice treated with either an anti-Ang-2 antibody (clone 533, 537 or 544) of the invention, with a control antibody, or with phosphate buffered saline (PBS). Details are described in the Examples.

FIGS. 2A, 2B, and 2C depict epitope mapping data (O.D. 370) for full-length human Ang-2 (hAng-2), to the N-terminus of hAng-2, and to the C-terminus of hAng-2, respectively, for peptibodies TN8-Con4-C, L1-7-N, and 12-9-3-C according to the invention, as well as for control peptibody, Tie2-Fc, C2B8, or 5B 12. Details are described in the Examples.

DETAILED DESCRIPTION OF INVENTION

The section headings are used herein for organizational purposes only, and are not to be construed as in any way limiting the subject matter described.

Standard techniques may be used for recombinant DNA molecule, protein, and antibody production, as well as for tissue culture and cell transformation. Enzymatic reactions and purification techniques are typically performed according to the manufacturer's specifications or as commonly accomplished in the art using conventional procedures such as those set forth in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1989]), or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

DEFINITIONS

As utilized in accordance with the present disclosure, the following terms unless otherwise indicated, shall be understood to have the following meanings:

The term "Ang-2" refers to the polypeptide set forth in FIG. 6 of U.S. Pat. No. 6,166,185 ("Tie-2 ligand-2") or fragments thereof as well as related polypeptides which include allelic variants, splice variants, derivatives, substitution, deletions, and/or insertion variants, fusion peptides and polypeptides, and interspecies homologs. The Ang-2 polypeptide may or may not include additional terminal residues, e.g., leader sequences, targeting sequences, amino terminal methionine, amino terminal methionine and lysine residues, and/or tag or fusion proteins sequences, depending on the manner in which it is prepared.

The term "biologically active" when used in relation to Ang-2 or an Ang-2 specific binding agent refers to a peptide or polypeptide having at least one activity characteristic of Ang-2 or of an Ang-2 specific binding agent. A specific binding agent of Ang-2 may have agonist, antagonist, or neutralizing or blocking activity with respect to at least one biological activity of Ang-2.

The term "specific binding agent" refers to a molecule, preferably a proteinaceous molecule, that binds Ang-2 (and variants and derivatives thereof as defined herein) with a greater affinity than other angiopoietins. A specific binding agent may be a protein, peptide, nucleic acid, carbohydrate, lipid, or small molecular weight compound which binds preferentially to Ang-2. In a preferred embodiment, the specific binding agent according to the present invention is an antibody, such as a polyclonal antibody, a monoclonal antibody (mAb), a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a humanized antibody, a human antibody, an anti-idiotypic (anti-Id) antibody, and antibodies that can be labeled in soluble or bound form, as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences, provided by known techniques. Such 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 present invention are capable of binding portions of Ang-2 that modulate, e.g., inhibit or promote, the biological activity of Ang-2 and/or other Ang-2-associated activities.

The term "polyclonal antibody" refers to a heterogeneous mixture of antibodies that recognize and bind to different epitopes on the same antigen. Polyclonal antibodies may be obtained from crude serum preparations or may be purified using, for example, antigen affinity chromatography, or Protein A/Protein G affinity chromatography.

The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule which are optionally produced by a single hybridoma or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same epitope on the antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

The term "chimeric antibodies" refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies, that exhibit 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].

The term "CDR grafted antibody" refers to an antibody in which the CDR from one antibody of a particular species or isotype is recombinantly inserted into the framework of another antibody of the same or different species or isotype.

The term "multi-specific antibody" refers to an antibody having variable regions that recognize more than one epitope on one or more antigens. A subclass of this type of antibody is a "bi-specific antibody" which recognizes two distinct epitopes on the same or different antigens.

"Catalytic" antibodies refers to antibodies wherein one or more cytotoxic, or more generally one or more biologically active, moieties are attached to the targeting binding agent.

The term "humanized antibody" refers to a specific type of CDR-grafted antibody in which the antibody framework region is derived from a human but each CDR is replaced with that derived from another species, such as a murine CDR. The term "CDR" is defined infra.

The term "fully human" antibody refers to an antibody in which both the CDR and the framework are derived from one or more human DNA molecules.

The term "anti-idiotype" antibody refers to any antibody that specifically binds to another antibody that recognizes an antigen. Production of anti-idiotype antibodies can be performed by any of the methods described herein for production of an Ang-2-specific antibodies except that these antibodies arise from e.g., immunization of an animal with an Ang-2-specific antibody or Ang-2-binding fragment thereof, rather than Ang-2 polypeptide itself or a fragment thereof.

The term "variants," as used herein, include those polypeptides wherein amino acid residues are inserted into, deleted from and/or substituted into the naturally occurring (or at least a known) amino acid sequence for the binding agent. Variants of the invention include fusion proteins as described below.

"Derivatives" include those binding agents that have been chemically modified in some manner distinct from insertion, deletion, or substitution variants.

"Specifically binds Ang-2" refers to the ability of a specific binding agent (such as an antibody or fragment thereof) of the present invention to recognize and bind mature, full-length or partial-length human Ang-2 polypeptide, or an ortholog thereof, such that its affinity (as determined by, e.g., Affinity ELISA or BIAcore assays as described herein) or its neutralization capability (as determined by e.g., Neutralization ELISA assays described herein, or similar assays) is at least 10 times as great, but optionally 50 times as great, 100, 250 or 500 times as great, or even at least 1000 times as great as the affinity or neutralization capability of the same for any other angiopoietin or other peptide or polypeptide.

The term "antigen binding domain" or "antigen binding region" refers to that portion of the specific binding agent (such as an antibody molecule) which contains the specific binding agent amino acid residues (or other moieties) that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen. In an antibody, the antigen-binding domain is commonly referred to as the "complementarity-determining region, or CDR."

The term "epitope" refers to that portion of any molecule capable of being recognized by and bound by a specific binding agent, e.g. an antibody, at one or more of the binding agent's antigen binding regions. Epitopes usually consist of chemically active surface groupings of molecules, such as for example, amino acids or carbohydrate side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes as used herein may be contiguous or non-contiguous. Moreover, epitopes may be mimetic in that they comprise a three dimensional structure that is identical to the epitope used to generate the antibody, yet comprise none or only some of the amino acid residues found in the Ang-2 used to stimulate the antibody immune response.

The term "inhibiting and/or neutralizing epitope" is an epitope, which when bound by a specific binding agent such as an antibody, results in the loss of (or at least the decrease in) biological activity of the molecule, cell, or organism containing such epitope, in vivo, in vitro, or in situ. In the context of the present invention, the neutralizing epitope is located on or is associated with a biologically active region of Ang-2. Alternatively, the term "activating epitope" is an epitope, which when bound by a specific binding agent of the invention, such as an antibody, results in activation, or at least maintenance of a biologically active conformation, of Ang-2.

The term "antibody fragment" refers to a peptide or polypeptide which comprises less than