Angiopoietin-2-specific coupling agents

FIELD: medicine.

SUBSTANCE: there are offered versions of an angiopoietin-2 (Ang-2) specific antibody and a pharmaceutical antibody composition for treatment of various diseases associated with angiopoietin-2 overexpression. Also there are described methods of inhibition, modulation and treatment of various diseases mediated by angiopoietin-2 activity. There are offered: coding nucleic acid, an expression vector and a vector-transformed cell, as well as a method for producing antibodies.

EFFECT: use of the invention ensures new high-cytotoxicity antibodies (according to ELISA analysis IC50=0,35 nM) comparable with a common antibody Ab536 that further can find application in medicine.

22 cl, 2 dwg, 11 tbl, 6 ex

 

This application is a partial continuation of Application for U.S. patent number 10/269805, filed October 10, 2002, and PCT Application number PCT/US02/32613, filed October 11, 2002, which claims the priority of Provisional patent application U.S. serial number 60/328604, filed October 11, 2001, all of which are incorporated herein by reference. This application also claims the priority of Provisional patent application U.S. serial number 60/620161, filed October 19, 2004, and Claims the U.S. with the number 10/982440, filed November 4, 2004, which is incorporated herein by reference.

The SCOPE of the INVENTION

This invention relates to specific binding agents that recognize the angiopoietin-2 (Ang-2) and contact him. More specifically, this invention relates to the production, diagnostic use and therapeutic use of monoclonal and polyclonal antibodies and their fragments, which specifically bind Ang-2.

PRIOR art

Angiogenesis, the formation of new blood vessels from existing vessels, is important for many physiological and pathological processes. Normally, angiogenesis is tightly regulated by Pro - and antiangiogenic factors, but in the case of diseases such as cancer, ocular neovascular C the disease, arthritis and psoriasis, this process can be carried out in a distorted form. Folkman, J. Nat. Med., 1:27-31 (1995).

There are a number of diseases known that they are associated with improperly regulated or undesirable angiogenesis. Such diseases include, but are not limited to, ophthalmic neovascularization, such as retinopathy (including diabetic retinopathy), associated with age macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, such as rheumatoid or rheumatic inflammatory disease, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disorders such as chronic asthma, arterial or post-transplant atherosclerosis, endometriosis, and neoplastic diseases, for example so-called solid tumors and liquid (or hematopoietic) tumor (such as leukaemia and lymphoma). Specialists in this field will be known to other diseases associated with undesirable angiogenesis.

Although it was expected that many systems transduction of signals involved in the regulation of angiogenesis, one of the most well-characterized and most selective systems endothelial cells involves receptor tyrosinekinase Tie-2 (referred to as Tie-2 or Tie-2R” (also called“ORK”); murine Tie-2 also called “tek”) and its ligands, angiopoietin (Gale, N.W. and Yancopoulos, G.D., Genes Dev. 13:1055-1066 (1999)). There are 4 known angiopoietin: angiopoietin-1 (“Ang-1”) - angiopoietin-4 (“Ang-4”). These angiopoetin mean also "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)). While the binding of Ang-1 to Tie-2 stimulates the phosphorylation of the receptor in cultured endothelial cells, observed that Ang-2 is both agonist and antagonist phosphorylation of the receptor Tie-2 (Davis, S., et al., (1996), above; Maisonpierre, P.C. et al., (1997), above; 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 mice with a knockout Tie-2 and Ang-1 are similar and suggest that Ang-1-stimulated phosphorylation of Tie-2 uporabe the remodeling and stabilization of blood vessels in utero by maintaining adhesion of endothelial cells supporting cells (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), above). It is believed that the role of Ang-1 in the stabilization of vessels stored in adults, where it is expressed widely and constitutive (Hanahan, D., Science, 277:48-50 (1997); Zagzag, D., et al., Experimental Neurology, 159:391-400 (1999)). In contrast, expression of Ang-2 is primarily limited seats remodeling of blood vessels, where it is thought to block the function of Ang-1 inducyruya through this state of vascular plasticity favorable for angiogenesis (Hanahan, D., above; Holash, J., et al., Science, 284:1994-1998 (1999); Maisonpierre, P.C., et al., (1997), above).

Numerous published studies purposefully showed saludoslegion the expression of Ang-2 in pathological conditions 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 sharing the office (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,15:122-1225 (1999); Yuan, K.,et al.,Journal of Periodontal Research,35:165-171 (2000); Zagzag, D.,et al., (1999), above). Most of these studies focused on cancer, and many types of tumors, apparently, detect vascular expression of Ang-2. In contrast to its expression in pathological angiogenesis, expression of Ang-2 in normal tissues is extremely limited (Maisonpierre, P.C., et al., (1997), above, Mezquita, J., et al., Biochemical and Biophysical Research Communications, 260:492-498 (1999)). In a healthy adult, the three main areas of angiogenesis are the ovary, placenta, and uterus; they are the primary tissues in normal (i.e. non-cancerous) tissues, which was detected mRNA Ang-2.

Some functional studies suggest that Ang-2 may participate in tumor angiogenesis. Ahmad et al. (Cancer Res., 61:1255-1259 (2001)) describe the overexpression of Ang-2 and show that it is, presumably, is associated with increased tumor growth in mouse xenograft models. Cm. also Etoh et al., above, and Tanaka et al., above, where the result, presumably linking overexpression of Ang-2 with hypervascularity tumors. However, in contrast, Yu et al., (Am. J. Path., 158:563-570 (2001)) reported data showing that overexpression of Ang-2 in the cells of the carcinoma, Lewis lung and breast carcinoma TA3, apparently, prolongs the survival of mice, inj is qualified by relevant transfectants.

In the last few years, various publications have suggested that Ang-1, Ang-2 and/or Tie-2 as a possible target for anticancer therapy. For example, each of the U.S. Patents numbers 6166185, 5650490 and 5814464 describes the concept of antibodies against Tie-2 ligands and antibodies against Tie-2 receptors. Lin et al., (Proc. Natl. Acad. Sci. USA, 95:8829-8834 (1998)) mice were injected with adenovirus expressing soluble Tie-2; this soluble Tie-2 presumably reduced the number and size of tumors developed in mice. In a related study, Lin et al. (J. Clin. Invest., 100:2072-2078 (1997)) rats were injected with soluble form of Tie-2; this compound is believed to have reduced the size of tumors in rats. Siemeister et al. (Cancer Res., 59:3185-3189 (1999)) generated cell lines human melanoma expressing the extracellular domain of Tie-2, were injected with these cell lines naked mice and concluded that soluble Tie-2 presumably resulted in significant inhibition of growth of tumors and angiogenesis of tumors. Due to this information and due to the fact that both Ang-1 and Ang-2 bind to Tie-2, from these studies it is unclear whether Ang-1, Ang-2 or Tie-2 to be an attractive target for anticancer therapy.

The merging of some peptides with stable plasma protein, such as a constant region of the Ig, to improve the half-period of existence in the body of these molecules has been described, for example, in Publ is the ratification of the PCT WO 00/24782, published may 4, 2000.

This fusion protein or its fragment with a stable plasma protein, such as a constant region of the Ig, to improve the half-period of existence in the body of these molecules has been many times described (see, for example, U.S. Patent 5480981; 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. Patent 5808029, issued September 15, 1998; Capon et al., Nature, 337:525-531, (1989); Harvill et al., Immunotech., 1:95-105, (1995); WO 97/23614, published July 3, 1997; PCT/US 97/23183, filed December 11, 1997; Linsley, J. Exp. Med., 174:561-569, (1991); WO 95/21258, published on August 10, 1995).

Effective anti-Ang-2 therapy may be useful for enormous numbers of cancer patients, as most solid tumors require neovascularization to growth beyond 1-2 mm in diameter. Such therapy may have wide applicability in other related angiogenesis diseases, such as retinopathy, arthritis and psoriasis.

Has not received development need to identify new agents that specifically recognize and bind Ang-2. Such agents could be used for diagnostic screening and therapeutic intervention in pathological conditions that are associated with the activity of Ang-2.

Thus, the purpose of this invention is the provision of specific svyazyvayus the Ang-2 agents, which modulate the activity of Ang-2.

The INVENTION

This invention provides an antibody containing a heavy chain and light chain, where the specified heavy chain contains a variable region of the heavy chain 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 antigennegative fragments; and the light chain contains a variable region light chain selected from the group consisting of: 526 Kappa (SEQ ID NO: 2); 536 (THW) Kappa (SEQ ID NO: 12); 536 (LQT) Kappa (SEQ ID NO: 210); 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 lamb is a (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 antigennegative fragments.

This invention also provides a specific binding agent containing 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 its fragments.

It should be clear that the specific binding agent can be, for example, an antibody such as a polyclonal, monoclonal, chimeric, humanitariannet or fully human antibody. The antibody can be a single-chain antibody. In addition, this invention relates to hybridoma, which produces a monoclonal antibody according to this invention.

It should be clear that the invention relates to conjugates described herein. The conjugate can be, for example, specificeski binding agent (such as an antibody) according to the invention.

In addition, this invention also relates to nucleic acid molecules coding for specific binding agents (such as antibody) according to the invention, and vectors containing such nucleic acid molecule, as well as to the cell host containing the vector.

In addition, this invention provides a method of obtaining a specific binding agent, providing for: (a) transforming the host cell at least one nucleic acid molecule that encodes a specific binding agent under item 1; (b) the expression of the nucleic acid molecules in this cell the owner and (C) the allocation of the specified specific binding agent. In addition, this invention provides a method of obtaining antibodies that provide for: (a) transforming the host cell at least one nucleic acid molecule that encodes the antibody in accordance with this invention; (b) the expression of the nucleic acid molecules in this cell the owner and (C) the allocation of the specified bonding agent.

In addition, this invention relates to a method of inhibiting undesired angiogenesis in a mammal by introducing a therapeutically effective amount of a specific binding agent in accordance with this invention. This invention provides t is the train method of treating cancer in a mammal by introducing a therapeutically effective amount of a specific binding agent in accordance with this invention.

This invention relates also to a method of inhibiting undesired angiogenesis in a mammal, introducing a therapeutically effective amount of the antibodies in accordance with this invention. In addition, also provides a method of treating cancer in a mammal, introducing a therapeutically effective amount of the antibodies in accordance with this invention.

It should be clear that the invention relates also to pharmaceutical compositions containing a specific binding agent in accordance with this invention and pharmaceutically acceptable agent for the preparation of compositions. The pharmaceutical composition may contain the antibody in accordance with this invention and pharmaceutically acceptable agent for the preparation of compositions.

This invention provides a method of modulating or inhibiting the activity of angiopoietin-2 by introducing one or more specific binding agents of the present invention. The invention also provides methods of modulating or inhibiting the activity of angiopoietin-2 introduction antibodies according to the invention.

In addition, this invention relates to a method of modulating at least one property of vascular permeability or leakage of plasma in mammals is operauser, introducing a therapeutically effective amount of a specific binding agent in accordance with this invention. This invention relates also to a method of treating at least one disease of ocular neovascular disease, obesity, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory diseases, inflammatory disorders, atherosclerosis, endometriosis, and neoplastic diseases associated with bone disease or psoriasis in a mammal, introducing a therapeutically effective amount of a specific binding agent in accordance with this invention.

In addition, this invention provides a method of modulating at least one property of vascular permeability or leakage of plasma from the mammal, introducing a therapeutically effective amount of the antibodies in accordance with this invention. This invention relates also to a method of treating at least one disease of ocular neovascular disease, obesity, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory diseases, inflammatory disorders, atherosclerosis, endometriosis, and neoplastic diseases associated with bone disease or psoriasis in mlekovita is it introducing a therapeutically effective amount of the antibodies in accordance with this invention.

In addition, this invention relates to a method of treating cancer in a mammal, introducing a therapeutically effective amount of a specific binding agent in accordance with this invention and a chemotherapeutic agent. Professionals in this field should be clear that the specific binding agent and a chemotherapeutic agent should be administered concomitantly.

This invention relates also to a method of treating cancer in a mammal, introducing a therapeutically effective amount of the antibodies in accordance with this invention and a chemotherapeutic agent. Antibody and chemotherapeutic agent should be administered concomitantly.

This invention provides a specific binding agent containing the complementarity determining region 1 (CDR 1) any of the following sequences: 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 IDNO: 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 (THW) Kappa (SEQ ID NO: 12); 536 (LQT) Kappa (SEQ ID NO: 210); 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).

This invention provides a specific binding agent that contains a complementarity determining region 2 (CDR 2) any of the following sequences: 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 N: 57); IK-2E2 HC (SEQ ID NO: 59); IP-2C11 HC (SEQ ID NO: 61); 526 Kappa (SEQ ID NO: 2); 536 (THW) Kappa (SEQ ID NO: 12); 536 (LQT) Kappa (SEQ ID NO: 210); 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).

This invention provides a specific binding agent containing the complementarity determining region 3 (CDR 3) any of the following sequences: 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 (THW) Kappa (SEQ ID NO: 12); 536 (LQT) Kappa (SEQ ID NO: 210) 543 Kappa (SEQ ID NO: 18); 544 cap is and (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).

In addition, this invention provides a nucleic acid molecule encoding a specific binding agent in accordance with this invention.

In addition, this invention relates to a method of detecting the level of angiopoietin-2 in a biological sample (a) contacting a specific binding agent according to the invention with the sample and (b) determining the extent of binding of the specific binding agent with the sample. This invention relates also to a method of detecting the level of angiopoietin-2 in a biological sample (a) contacting the antibody according to the invention with the sample and (b) determining the extent of binding of the antibody with the sample.

This invention relates also to a method of inhibiting undesired angiogenesis in a mammal, involving the introduction of a therapeutically effective amount of the polypeptide or composition, as described here. This invention relates also to a method of modulating angiogenesis in a mammal, introducing a therapeutically effective amount of the polypeptide or composition described herein. This invention relates also to a method of inhibiting tumor growth, characterized by unwanted angiogenesis in a mammal, introducing a therapeutically effective amount of the polypeptide or composition described herein. In addition, this invention relates to a method of treating cancer in a mammal, introducing a therapeutically effective amount of the polypeptide or composition described herein, and a chemotherapeutic agent. In the preferred embodiment, this chemotherapeutic agent is at least one of 5-FU, CPT-11 and Taxotere. However, it should be clear that can be used and other suitable chemotherapeutic agents or other anti-cancer therapy.

It will be understood that the specific binding agents of the present invention can be used to treat a number of diseases associated with unregulated or undesirable angiogenesis. Such diseases include, but are not limited to, ophthalmic neovascularization, such as retinopathy (including diabetic retino atiu and associated with age macular degeneration), psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, such as rheumatoid or rheumatic inflammatory disease, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disorders such as chronic asthma, arterial or post-transplant atherosclerosis, endometriosis, and neoplastic diseases, for example so-called solid tumors and liquid tumors (such as leukemia). Specialists in this field will be obvious additional diseases that can be treated by the introduction of specific binding agents. Such additional diseases include, but are not limited to, obesity, vascular permeability, leakage of plasma and related bone disorders, including osteoporosis. Thus, in addition, this invention relates to methods for treating these diseases associated with unregulated or undesirable angiogenesis.

Other embodiments of this invention will be apparent from the present description.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 depicts a graph of tumor size (y-axis) depending on time (x-axis) in tumor bearing mice, which were treated with either anti-Ang-2 antibody (clone 533, 537 or 544) according to the invention, or control and what antibodies, either phosphate buffered saline (PBS). The details are described in the Examples.

Figures 2A, 2B and 2C depict the results of epitope mapping (OD 370) for full-length Ang-2 (hAng-2), relative to the N-end hAng-2 and C-end hAng-2, respectively, for peptide antibodies TN8-Con4-C, L1-7-N and 12-9-3 With in accordance with this invention and for a control peptide antibodies, Tie2-Fc, SV or V. The details are described in the Examples.

DETAILED description of the INVENTION

The section headings used here only for organizational purposes and should not be construed as limiting in any way the described subject matter.

To obtain recombinant DNA molecules, proteins and antibodies, as well as for tissue culture and transformation of cells can be used in a standardized way. Enzymatic reactions and purification techniques are typically performed in accordance with the descriptions of the manufacturer or, as usually performed in the field using conventional procedures, such as procedures described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), or as described herein. If there are no specific definitions used nomenclature and laboratory procedures and methods of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described C the ect, are well known and commonly used in this field. Standard methods can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, getting ready forms and delivery, and treatment of patients.

Definitions

In the context of this invention, the following terms, unless otherwise indicated, shall be understood as having the following values.

The term "Ang-2" refers to the polypeptide, are presented in figure 6 of U.S. Patent No. 6166185 ("Tie-2 ligand-2"), or its fragments, as well as related polypeptides that include allelic variants, splicing variants, derivatives, variants having substitutions, deletions and/or insertions, fused peptides and polypeptides, and interspecies homologues. Polypeptide Ang-2 may include or may not include additional terminal residues, for example, leader sequences, targeting sequences, aminobenzoyl methionine, aminobenzene residues of methionine and lysine and/or label or sequence fused proteins, depending on the method of their preparation.

The term "biologically active" in the application of relatively Ang-2 or Ang-2-specific binding agent refers to a peptide or polypeptide, having at least one characteristic activity of Ang-2 or Ang-2-specific binding is found agent. Specific binding Ang-2, the agent can have agonistic, antagonistic or neutralizing or blocking activity against at least one biological activity of Ang-2.

The term "specific binding agent" refers to a molecule, preferably a protein molecule that binds to Ang-2 (and its variants and derivatives defined here) with a higher affinity than other angiopoetin. The specific binding agent can be a protein, peptide, nucleic acid, carbohydrate, lipid or a compound with a low molecular weight, which is associated mainly with Ang-2. In a preferred embodiment, the specific binding agent in accordance with this invention is an antibody such as a polyclonal antibody, a monoclonal antibody (mAb), a chimeric antibody, a CDR-transplantirovannam antibody multispecificity antibody bespecifically antibody, a catalytic antibody, humanitariannet antibody, a human antibody, idiotypical (anti-Id) antibody and antibodies that can be labeled in soluble or bound form, as well as fragments, variants or derivatives either alone or in combination with other amino acid sequences, provided by known methods. Such methods include, but are not limited to yaytsa them enzymatic cleavage, chemical cleavage, peptide synthesis or recombinant methods. Anti-Ang-2-specific binding agents of the present invention are able to bind part of Ang-2 that modulate, e.g. inhibit or stimulate biological activity of Ang-2 and/or other Ang-2-associated activity.

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

The term "monoclonal antibody" refers to the concentration of the antibody encoded by the same nucleic acid molecule, which are not necessarily produced only hybridomas or another cell line or transgenic animal, so that each monoclonal antibody will normally recognize the same epitope on the antigen. The term "monoclonal" is not limited to any particular method for obtaining antibodies, and is not a term limited to antibodies produced in a particular form, such as a mouse, rat, etc.

The term "chimeric antibody" refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies, derived from a particular species or belonging to a particular class or subclass of antibody, while the remainder of the chain (these chains) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies. Also included are fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind Ang-2). Cm. U.S. patent No. 4816567 and Morrison et al., Proc Natl Acad Sci (USA), 81:6851-6855 (1985).

The term "CDR-transplantirovannam antibody" refers to an antibody in which the CDRs from one specific antibodies or isotype recombinante built into the frame of another antibody of the same or other species or isotype.

The term "multispecific antibody" refers to antibodies having variable regions that recognize more than one epitope on one or more antigens. A subclass of this type of antibody is bespecifically antibody, which recognize two different epitopes on the same or different antigens.

"Catalytic antibodies" refers to antibodies in which one or more cytotoxic or more often, one or more biologically active parts of molecules attached to a target binding agent.

The term "humanitariannet antibodies which refers to a specific type CDR-transplanted antibody in which a frame region of antibodies derived from human, but each CDR replaced by a CDR derived from another species, for example, murine CDR. The term "CDR" is defined below.

The term "fully human" antibody refers to an antibody in which the CDR and framework region originate from one or more DNA molecules of the person.

The term "antiidiotypic" antibody refers to any antibody that specifically binds with another antibody that recognizes the antigen. Getting antiidiotypic antibodies may be implemented by any of these methods of obtaining the Ang-2-specific antibodies, except that these antibodies may receive, for example, by immunization of an animal Ang-2-specific antibody or Ang-2 binding fragment, and not by Ang-2 polypeptide or fragment.

The term "variants" includes in this context, those polypeptides in which amino acid residues are embedded in the natural (or at least known) amino acid sequence of a binding agent, deleterows from this amino acid sequence or replaced in this amino acid sequence. Variants of the present invention include fused proteins, described below.

"Derivatives" include binding agents that have been chemically modified in some way, otlichayas is from variants with insertions, deletion or replacement.

"Specifically binds Ang-2" refers to the ability of a specific binding agent (such as an antibody or its fragment) according to the invention recognize and bind the Mature, full-length or having a partial-length polypeptide Ang-2, or its ortholog, so that its affinity (defined, for example, using assays ELISA or BIAcore as described here) or the ability to neutralize (defined, for example, analyses of neutralization ELISA described herein, or similar assays) is at least 10 times higher, but not necessarily 50 times higher, 100, 250 or 500 times higher, or even at least 1000 times higher than the affinity or ability to neutralize a specific binding agent in respect of any other of the angiopoietin or another peptide or polypeptide.

The term "antigennegative domain" or "antigennegative region" refers to the portion of the specific binding agent (such as an antibody molecule), which contains amino acid residues (or other parts of the molecule) specific binding agent that interact with the antigen and attach binding agent of its specificity and affinity for this antigen. The antibody antigennegative domain is usually referred to as the "defining the comp is mentornet area or CDR.

The term "epitope" means that portion of any molecule capable of being recognized and to be bound specific binding agent, e.g. antibody, in one or more antigenspecific areas of the agent. Epitopes usually consist of chemically active surface groupings of molecules such as, for example, side chains of amino acids or carbohydrates, and have specific three dimensional structural characteristics, as well as the specific characteristics of the charges. The epitopes in this context can be adjacent or nonadjacent. In addition, the epitopes can be mimetic in the sense that they contain the three-dimensional structure that is identical to the epitope used to generate antibodies, but does not contain any one of amino acid residues or contains only some of the amino acid residues found in Ang-2, used to stimulate the immune response in the form of antibodies.

The term "inhibiting and/or neutralizing epitope" refers to an epitope, which when binding a specific binding agent such as an antibody, leads to the loss (or at least reduce) the biological activity of the molecule, cell or organism containing such epitope, in vivo, in vitro or in situ. In the context of this invention, the neutralizing epitope localized on biologically asset is th district of Ang-2 or associated with a biologically active region of Ang-2. Alternatively, the term "activating epitope" refers to an epitope, which when binding a specific binding agent such as an antibody, leads to activation or at least maintaining the biologically active conformation of Ang-2.

The term "antibody fragment" refers to a peptide or polypeptide that contains less than the full, intact antibody. Full antibodies contain two functionally independent parts or two pieces: antigennegative fragment, known as "Fab", and carboxy-terminal kristallizuetsya fragment, known as "Fc"fragment. Fab-fragment includes the first constant domain, consisting of both heavy and light chain (SN and CL1) together with the variable regions from both the heavy and light chains that bind a specific antigen. Each of the variable regions of heavy and light chain comprises three complementarity determining region (CDR) and amino acid residues frame areas that share a separate CDR. Fc-region contains the second and third constant domains of the heavy chain (CH2 and CH3) and is involved in effector functions such as activation of complement and attack by phagocytic cells. In some antibodies, the areas of the Fc and Fab divided the hinge region of the antibody and, depending on how proteoliticeski cleaved full-size antibody, fixed the Naya region may be associated with either Fab-, or Fc fragment. For example, the decomposition of antibodies with a protease papain in the hinge region is associated with the receipt of the Fc-fragment, whereas cleavage by the protease pepsin provides the fragment in which the hinge region is associated with both Fab-fragments simultaneously. Since the two Fab-fragment actually covalently associated after cleavage by pepsin, the resulting fragment is called F(ab')2the fragment.

Fc-domain can have relatively long half-life existence in the serum, while Fab is short-lived (Capon et al., Nature, 337: 525-31 (1989)). When the expression as part of a fused protein Fc-domain can provide longer half-life existence in serum or enable functions such as binding to Fc receptor binding, Protein a, complement fixation, and perhaps even placental transfer protein, with which it merged. Fc-domain can be natural Fc-domain or can be modified to improve certain qualities, such as therapeutic or circulation time in the bloodstream.

The term "variable region" or "variable domain" refers to the part of the light and/or heavy chain antibodies, typically comprising about aminobenzene 120-130 amino acids in the heavy chain and approximately 100-110 aminobenzene amino acids in the light chain. Variable regions are usually the ilen differ in amino acid sequence, even among the antibodies of the same species. Variable region antibodies usually determines the binding and specificity of each particular antibody against the specific antigen. The variability in the sequence is concentrated in areas called complementarity determining regions (CDR), whereas the more highly conserved regions in the variable domain is called the frame regions (FR). CDR light and heavy chains contain amino acids, which are largely responsible for the direct interaction of the antibody with the antigen, however, amino acids in the FR can significantly affect the binding/recognition of antigen, as discussed here below.

The term "light chain" when referring to an antibody collectively refers to two distinct types, called Kappa (K) or lambda (L) based on the amino acid sequence of the constant domains.

The term "heavy chain" when referring to an antibody collectively refers to the five different types, called alpha, Delta, Epsilon, gamma and mu, based on the amino acid sequence of the constant domain of the heavy chain. The combination of the heavy and light chains can give five known classes of antibodies: IgA, IgD, IgE, IgG and IgM, respectively, including four well-known subclass of IgG, referred to as IgG1, IgG2, IgG3and IgG4.

The term "prirodnye" when used in connection with biological materials, such as molecules of nucleic acids, polypeptides, cells-owners and the like, refers to materials that are found in nature and is not modified by man.

The term "isolated" when used in relation to Ang-2, or in relation to specific binding Ang-2 agent refers to a compound that does not contain at least one contaminating polypeptide or compound that is found in its natural environment, and preferably essentially does not contain any other contaminating polypeptides mammals, which could interfere with its therapeutic or diagnostic use.

The term "Mature" when used in relation to Ang-2 and anti-Ang-2 antibodies or in respect of any other protein specific binding Ang-2 agent refers to a peptide or polypeptide, bereft of a leader or signal sequence. When expression of a binding agent according to the invention, for example, in prokaryotic cell-master, Mature peptide or polypeptide can include additional amino acid residues (but still not have a leader sequence), such as aminobenzoyl methionine, or one or more residues of methionine and lysine. The peptide or polypeptide, thus obtained, can be used with these additional amino acid is different residues or without these additional amino acid residues, they were removed.

The term "effective amount" and "therapeutically effective amount" when used in relation to specific binding Ang-2 agent refers to the amount of specific binding agent that is useful or necessary to maintain the observed changes in the level of one or more of the biological activities of Ang-2. This change can be either an increase or decrease in the activity level of Ang-2. Preferably, this change is a decrease in the activity of Ang-2.

Specific binding agents and antibodies

In this context, the term "specific binding agent" refers to a molecule that specifically recognizes and binds Ang-2, as described here. Suitable specific binding agents include, but are not limited to, antibodies and derivatives thereof, polypeptides and small molecules. Suitable specific binding agents can be obtained using known in the field of methods. An example of a specific binding agent polypeptide Ang-2 of the present invention are capable of binding a certain portion of the polypeptide Ang-2 and it is preferable to modulate the activity or function of the polypeptide of Ang-2.

Specific binding agents such as antibodies and antibody fragments that are specific the key bind polypeptides Ang-2, are within the scope of this invention. These antibodies can be polyclonal, including monospecific polyclonal, monoclonal (mAb), recombinant, chimeric, humanitarianism, for example, CDR-transplanted, human, single-stranded, catalytic, multispecificity and/or bespecifically, as well as fragments, variants and/or derivatives.

Polyclonal antibodies directed to a polypeptide Ang-2, are usually obtained in animals (such as rabbits, hamsters, goats, sheep, horses, pigs, rats, gerbils, Guinea pigs, mice or any other suitable mammal, as well as other non-mammals) by multiple subcutaneous or intraperitoneal injections of the polypeptide of Ang-2 or its fragment with adjuvant or without adjuvant. Such adjuvants include, but are not limited to, the complete and incomplete adjuvant's adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, the polyols of the type pluronics, polyanion, peptides, oil emulsions, hemocyanin fissurella and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corinebacterium parvum are potentially applicable adjuvants person. It may be useful conjugation of antigenic polypeptide with protein carrier, which is immunogenic in p is improper immunization form such as hemocyanin fissurella, serum albumin, bovine thyroglobulin, or trypsin inhibitor soybean. To enhance the immune response also use an aggregating agents, such as alum. After immunization of the animals were extracted blood and serum were analyzed to determine the titer of antibodies against the polypeptide of Ang-2, which could be determined using the assays described herein in the Examples section. Polyclonal antibodies can be used in the sera, in which they were detected, or can be purified from these sera using, for example, antigen-affinity chromatography or Protein a or G affinity chromatography.

Monoclonal antibodies directed against the polypeptides of Ang-2 may be obtained using, for example, but without limitation, the traditional "hybridoma" method or more of the new way "phage display". For example, the monoclonal antibodies according to the invention can be obtained hybridoma method described in Kohler et al., Nature 256:495 (1975); hybridoma method In human cells (Kosbor et al., Immunol Today 4:72 (1983); Cote et al., Proc Natl Acad Sci USA 80: 2026-2030 (1983); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, (1987)) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp. 77-96, (1985)). The invention also provides hybridoma of cleoc the s line, which produce monoclonal antibodies reactive with the polypeptides of Ang-2.

When using the hybridoma method can be used myeloma cell lines. Such cell lines suitable for use in producing hybridoma procedures merge, preferably are not producing antibodies lines, have high efficiency mergers and deficiencies of enzymes that make them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). For example, cell lines used in rat fusions are Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul; cell lines used in rat fusions are R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210. Other cell lines used for cell m, are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6. Hybridoma and other cell lines that produce monoclonal antibodies, are considered as being the new compositions of this invention.

Method of phage display can also be used to generate monoclonal antibodies of any kind. Preferably, this method is used to produce fully human monoclonal antibodies, in which polynucleotide encoding only Fab or Fv fragment of the antibody, xpresroute on the surface ragovoy particles (Hoogenboom et al., J Mol Biol 227: 381 (1991); Marks et al., J Mol Biol 222: 581 (1991); see also U.S. Patent No. 5885793)). Each phage can be subjected to screening using analyses of binding described here to identify fragments of antibodies having affinity against Ang-2. Thus, these processes mimic immune selection via the display (view) repertoires (spectra) fragments of antibodies on the surface of filamentous bacteriophage and the subsequent selection of the phage by its binding to Ang-2. One such procedure is described in PCT Application number PCT/US98/17364, filed on behalf of Adams et al., which describes the selection of high affinity and functional fragments agonistic antibodies for MPL - msk-receptors using this approach. In this approach, the full repertoire (range) genes of human antibodies can be generated by cloning the natural rearanging V-human genes from peripheral blood lymphocytes as described previously (Mullinax et al., Proc Natl Acad Sci (USA) 87: 8095-8099 (1990)).

After identification of polynucleotide sequences that encode each circuit full-size monoclonal antibodies or Fab or Fv fragments of this invention, the cells of the host, eukaryotic or prokaryotic, can be used for the expression of polynucleotides monoclonal antibodies using recombinant methods, well known for the x in the usual manner practiced in this area. Alternatively, obtain transgenic animals in which polynucleotide encoding the desired specific binding agent is introduced into the genome of the recipient animal, such as, for example, mouse, rabbit, goat or cow, in such a way that it creates the possibility of expression of polynucleotide molecules that encode a monoclonal antibody or other specific binding agent. In one aspect, polynucleotide encoding a monoclonal antibody or other specific binding agent can be legirovanyh with regulatory sequences that are specific to breast cancer, and these chimeric polynucleotide can be introduced into the germ line of the animal target. Then, the resulting transgenic animal produces the desired antibody in its milk (Pollock et al., J Immunol Meth 231:147-157 (1999); Little et al., Immunol Today 8:364-370 (2000)). In addition, can be used in plants for the expression and obtain the Ang-2-specific binding agents, such as monoclonal antibodies, transfection of suitable plants polynucleotide encoding these monoclonal antibodies or other specific binding agents.

In another embodiment of the present invention, monoclonal or polyclonal antibody or its fragment, which is obtained from non-human species can be "humanized" or "hee is risovannym". Methods of humanizing antibodies man well known in the art (see U.S. patent numbers 5859205, 5585089 and 5693762). Humanization is performed, for example, using the methods described in this region (Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by replacing at least part of, for example, complementarity determining regions (CDR) of the rodent corresponding regions of human antibodies. The invention also provides variants and derivatives of these antibodies person, as discussed here and is well known in this field.

This invention also includes a fully human antibodies that bind polypeptides Ang-2, as well as fragments, variants and/or derivatives. Such antibodies can be obtained using the above described method of phage display. Alternatively, to generate such antibodies can be used transgenic animals (e.g. mice)that are capable of producing a repertoire (range) human antibodies in the absence of producing endogenous immunoglobulin. This can be accomplished by immunization of an animal Ang-2 antigen or fragments, where the Ang-2-fragments have an amino acid sequence that is unique to Ang-2. Such immunogen can be optionally conjugated to a carrier. Cm. for example, 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 Measurement, 7: 33 (1993). In the same way such transgenic animals produced by loss of endogenous loci encoding the heavy and light chain immunoglobulin, and embedding in their gene loci encoding proteins of the heavy and light chains of human rights. Then partially modified animals, which are animals that have less than the full complement of these modifications are cross-breeding with obtaining an animal having all the desired modification of the immune system. With the introduction of the immunogen these transgenic animals capable of producing antibodies with variable regions of a human, comprising the amino acid sequence of human (but not mouse), which are immunospecificity against desired antigens. Cm. PCT application numbers PCT/US96/05928 and PCT/US93/06926. Additional methods are described in U.S. Patent No. 5545807, PCT Application numbers PCT/US91/245, PCT/GB89/01207 and in EP V and EP A. Human antibodies can also be obtained by expression of recombinant DNA in the cells of the host or expression in hybridoma cells, as described here.

Transgenes (gene transfer) is achieved by a number of different ways. See, for example, Bruggeman et al., Immunol Today 17:391-7 (1996). In one approach design minilogue, so gene segments which form the germ line artificially closer to each other. Due to size restrictions (i.e. usually when the amount is smaller than 30 TPN) received minilogue will contain a limited number of different gene segments, but is still able to produce a large repertoire (range) antibodies. Minilogue containing only the DNA sequence of human rights, including promoters and enhancers, are fully functional in transgenic mice.

When the desired is a larger number of gene segments in a transgenic animal, using yeast artificial chromosome (YAC). YAC can be in the range of a few hundred TPN to 1 MPN and they are introduced into the genome of a mouse (or other suitable animal) by microinjection directly into the egg, or by moving YAC in the line of embryonic stem (ES) cells. Usually YAC transferred into ES cells by lipofectin purified DNA or merge yeast spheroplasts, and the purified DNA is transferred into the micelles, and the merging is performed according to the protocols, similar to the Protocol of the fusion hybrid. The selection of the desired ES cells after DNA transfer is performed by using a power-on YAC any of breeding markers known in this field.

Alternatively, use vectors bacteriophage P1, which is propagated in a bacterial host E. coli. Although these vectors are usually less insertRow the Neu DNA than YAC, these clones can easily grow with high enough output for direct microinjection into the egg of a mouse. It was shown that the use of a mixture of different vectors P1 leads to high levels of homologous recombination.

After identifying a suitable transgenic mouse (or other suitable animal) using methods known in the field for detection of serum levels of circulating antibodies (e.g., ELISA), the transgenic animal is crossed with a mouse, which was destroyed endogenous Ig locus. The result is offspring, in which essentially all b cells Express human antibodies.

As another alternative, the full Ig locus of the animal is replaced by the Ig locus and received the animal expresses only human antibodies. In another approach, part of the locus of the animal substitute, specific and relevant areas in the locus of a person. In some cases, animals resulting from this procedure can Express the chimeric antibodies, in contrast to the fully human antibodies, depending on the nature of the substitutions in the Ig locus of the mouse.

Human antibodies can also be obtained by exposure of splenocytes person (or T cells) the action of antigen in vitro and then recreating exposed ant the gene cells in mice with weakened immune systems, for example, SCID or nod/SCID. Cm. Brams et al., J Immunol, 160: 2051-2058 (1998); Carballido et al., Nat Med, 6: 103-106 (2000). In one approach grafting fetal human tissues mouse SCID (SCID-hu) leads to long-term hematopoiesis and the development of T-cells (McCune et al., Science 241:1532-1639) (1988); Ifversen et al., Sem Immunol 8:243-248 (1996)). Any humoral immune response in these chimeric mice completely depends on the simultaneous development of T cells in these animals (Martensson et al., Immunol 83:1271-179 (1994)). In an alternative approach, the peripheral blood lymphocytes of human transplanted intraperitoneally (or otherwise) in SCID mice (Mosier et al., Nature 335:256-259 (1988)). In transplanted cells Primerose agent such as staphylococcal enterotoxin A (SEA) (Martensson et al., Immunol 84: 224-230 (1995)), or monoclonal antibodies against human CD40 (Murphy et al., Blood 86:1946-1953 (1995)), detects higher levels of production In the cells.

Alternative synthetic repertoire (range) heavy chains of the person creating from paranirvana segments V-gene Assembly each VHsegment D-segment random nucleotides together with the J-segment human (Hoogenboom et al., J. Mol. Biol., 227:381-388 (1992)). Similarly, the repertoire (range) of light chains design Association of each V-segment of a person with a J-segment (Griffiths et al., EMBO J 13:3245-3260 (1994)). The nucleotides encoding the full antibody (i.e. both heavy and Le the forge chains), link in the form of single-chain Fv fragment, and this polynucleotide are ligated with polynucleotides coding for minor envelope protein of filamentous phage. When the expression of this fused protein on the surface of phage polynucleotide encoding a specific antibody, identify the selection using immobilized antigen.

In another approach, antibody fragments collected in the form of two Fab fragments merge single chain protein of the phage and the secretion of another bacterial periplasm (Hoogenboom et al., Nucl Acids Res 19:4133-4137 (1991); Barbas et al., Proc Natl Acad Sci (USA) 88:7978-7982 (1991)).

Large-scale production of chimeric, humanized, CDR-transplanted and fully human antibodies or their fragments are usually performed by recombinant methods. Polynucleotide molecule or polynucleotide molecules encoding heavy and light chains of each antibody or fragments thereof, can be introduced into cells of the host and expressed with the use of these materials and procedures. In the preferred embodiment, these antibodies get into the cells of the host mammal, such as cells SNO. The details of such receipt described below.

Partners merge specific binding agents

In an additional embodiment of the invention, polypeptides containing variable romanianization sequence antibodies Ang-2, such as the variable region of the heavy chain with the amino acid sequence described herein, or the variable region of the light chain with the amino acid sequence described herein may be fused to the N-end or at the C-end with one or more domains of an Fc-region of IgG person. When designing together with a therapeutic protein, such as Fab Ang-2-specific antibody, an Fc domain can provide longer half-period of the existence or enable functions such as binding to Fc receptor binding, Protein a, complement fixation, and perhaps even placental transfer (Capon et al., Nature, 337: 525-531 (1989)).

In one example, the hinge region, CH2 and CH3-regions of the antibodies can be fused either to the N-end or at the C-end of the polypeptide specific binding agent, such as anti-Ang-2-Fab or Fv fragment (obtained, for example, from a library of phage display) using methods known to the person skilled in the art. The obtained protein can be purified using Protein a - or Protein G-affinity column. It was found that peptides and proteins, fused with the Fc region, are significantly higher half-period of the existence of in vivo than Nikita copy. Merge with Fc-region makes possible dimerization/multimerization fused polypeptide. Fc-region can be natural Fc-what Hayon or can be modified to improve certain qualities, such as therapeutic quality, circulation, reducing problems of aggregation, etc. are Other examples known in this area include examples in which the Fc-region, which may occur from human or other species, or may be synthetic, merge N-end CD30L, for the treatment of Hodgkin's disease, anaplastic lymphoma, T-cell leukemia (U.S. Patent No. 5480981), Fc-district merge with TNF-receptor for the treatment of septic shock (Fisher et al., N Engl J Med 334: 1697-1702 (1996) and Fc-region is drained from the Cd4 receptor for the treatment of AIDS (Capon et al., Nature, 337: 525-31 (1989)).

Catalytic antibodies are another type fused molecules include antibodies in which one or more cytotoxic or more often, one or more biologically active parts of molecules attached to a specific binding agent. See, for example (Rader et al., Chem Eur J 12:2091-2095 (2000)). Cytotoxic agents of this type improves mediated antibody cytotoxicity and include parts of molecules, such as cytokines, which directly or indirectly stimulate cell death, radioisotopes, chemotherapeutic drugs (including prodrugs), bacterial toxins (e.g., pseudomonas exotoxin, diphtheria toxin, and so on), the toxins of plants (e.g., ricin, gelonin etc), chemical conjugates (for example, maytansinoids toxins, Kalehe amicin etc), radioconjugates, conjugates with enzymes (conjugate with RNase, therapy using conjugate directed to antibody enzyme/prodrug (ADEPT))), etc. In one aspect, the cytotoxic agent can be "attached" to the same component especifismo or multispecific antibodies binding agent to one of the sites raspoznavaniya alternative antigens for the antibody. Alternatively, the protein cytotoxins can be expressed in the form of a fused protein with a specific binding agent after ligation of polynucleotides encoding this toxin with polynucleotides, crypto-binding agent. In another alternative method, the specific binding agent may be covalently modified to include the desired cytotoxin.

Examples of such fused proteins are immunogenic polypeptides, proteins with long half-periods of existence in the bloodstream, such as a constant region of immunoglobulins, marker proteins, proteins or polypeptides that facilitate purification of the desired polypeptide specific binding agent and the polypeptide sequence, which stimulate the formation of multimeric proteins (such as motives latinboy lightning that are applicable in education/improving the stability of the dimers).

This type of insertion the th option usually has full native molecule or a substantial portion of the native molecule, associated to the N-end-or-end, with a full second polypeptide or the second polypeptide. For example, fused proteins typically use a leader sequence from another species to create opportunities recombinant expression of a protein in a heterologous host. Other useful protein includes adding immunologically active domain, such as the epitope antibodies, for easier cleaning fused protein. The inclusion of a cleavage site at the border merger or near the border merger will facilitate the removal of the foreign polypeptide after purification. Other useful mergers include linking functional domains, such as active sites from enzymes, glycosylation domains, signals cell targeting or transmembrane regions.

There are various commercially available expression system, fused proteins, which may be used in this invention. Especially applicable systems include, but are not limited to, a system of glutathione-S-transferase (GST) (Pharmacia), the system maltsevazamkovaja protein (NEB, Beverly, MA), system FLAG (IBI, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA). These systems are capable of producing recombinant polypeptides, bearing only a small number of additional amino acids, which have a small probability of influence on the antigenic capacity of recombinant the polypeptide. For example, as a system FLAG, and the 6xHis system adds only a short sequence, and it is known that both of them are sebenteen and do not exert undesirable influence on the packing of the polypeptide in its natural conformation. Other N-terminal fusion, which is perceived as applicable, is the merger of the dipeptide Met-Lys at the N-terminal site of the protein or peptides. Such a merger may make a favorable increase of expression or activity of the protein.

Especially applicable slit design can be design in which the peptide specific binding agent attached to the hapten to enhance the immunogenicity of the slit design specific binding agent, which is applicable, for example, in obtaining antiidiotypic antibodies according to the invention. Such slit design to increase the immunogenicity are well known to specialists in this field, for example, merger-specific binding agent with helper antigen, such as hsp70 or peptide sequences, for example, from a chain of diphtheria toxin or cytokine, such as IL-2, will be applicable in the induction of immune responses. In other embodiments, the implementation can be created merged design, which will enhance the targeting compositions antigennegative agent to a specific site of the Il is on a specific cell.

Also considered other merged structure comprising heterologous polypeptides with desired properties, for example, the constant region of Ig for extension of the period of existence in serum or antibody or its fragment to target. Other fused systems produce polypeptide hybrids where it is desirable cut of a merge partner of the desired polypeptide. In one embodiment, the merge partner is linked to the recombinant polypeptide specific binding agent is a peptide sequence containing a specific sequence recognition for protease. Examples of suitable sequences are the sequence recognized by the protease of the virus engraving tobacco (Life Technologies, Gaithersburg, MD)or factor XA (New England Biolabs, Beverly, MA).

This invention also provides fused polypeptides containing the entire variable domain or part of a variable domain of Ang-2 antibodies, for example, the variable domain of the heavy chain with the amino acid sequence described herein, or variable domain of the light chain with the amino acid sequence described herein, in combination with the truncated tissue factor (tTF), vascular targeting agent, consisting of a shortened form inducing coagulation of protein a person who acts as the Ghent coagulation of the tumor blood vessels. Merge tTF with anti-Ang-2 antibody or its fragments can facilitate the delivery of anti-Ang-2 antibody to the target cells.

Options specific binding agents

Options specific binding agents of the present invention include insertion, deletion variants, and/or variants with substitutions. In one aspect of this invention, there is provided insertional variants in which one or more amino acid residues Supplement amino acid sequence specific binding agent. Insertions may be located at either or both ends of this protein or can be placed in the inner regions of amino acid sequence specific binding agent. Insertional variants with additional residues on either or both ends may include, for example, fused proteins or proteins comprising amino acid "tags" or labels. Insertional variants include polypeptides specific binding agent, in which one or more amino acid residues added to the amino acid sequence of the specific binding agent or fragment.

Variant products of the invention also include Mature products specific binding agent. Such products specific binding agent are deleted l the gender or signal sequence, however, the resulting protein has an additional aminobenzene residues compared to the polypeptide of Ang-2 wild type. Additional aminobenzene residues may be derived from another protein, or may include one or more residues that are not identified as derived from specific protein. Discusses products specific binding agent with an additional methionine residue at position 1 (Met-1-specific binding agent), as products of a specific binding agent with additional residues of methionine and lysine at positions -2 and -1 (Met-2-Lys-1-specific binding agent). Options specific binding agents with additional residues Met, Met-Lys, Lys (or one or more basic residues in General), especially applicable for enhanced production of recombinant protein in bacterial cells-hosts.

The invention also includes variants of the specific binding agent having additional amino acid residues, which receive, using specific expression systems. For example, using commercially available vectors that Express a desired polypeptide as part of the product fusion with glutathione-S-transferase (GST), provides the desired polypeptide having an additional balance of gli is in the position of amino acids -1 after removal of GST component from the desired polypeptide. Options that come from the expression in other vector systems are also considered, including ways in which polyhistidine labels included in the amino acid sequence, usually at the carboxy - and/or aminocore sequence.

Insertional variants also include fused proteins described above, in which the amino - and/or carboxylic polypeptide specific binding agent is fused with another polypeptide, fragment or amino acid sequences, which are usually not recognized as part of any specific sequence.

In another aspect, the invention provides deletion variants in which one or several amino acid residues in the polypeptide specific binding agent is removed. Deletions can be performed on one or both ends of the polypeptide specific binding agent or deleting one or more residues within the amino acid sequence specific binding agent. Deletion variants necessarily include all fragments of the polypeptide specific binding agent.

Fragments of antibodies include parts antibodies that bind to the epitope on the antigenic polypeptide. Examples of such fragments include Fab and F(ab')2fragments generated by, for example, enzymatic or chemical cleavage of the full-size antibodies. Other binding fragments include fragments generated by the methods of recombinant DNA, such as the expression of recombinant plasmids containing nucleic acid sequences encoding the variable regions of the antibodies. The invention also includes polypeptide fragments of Ang-2 binding agent, and fragments retain the ability to specifically bind the polypeptide Ang-2. Fragments containing at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 or more consecutive amino acids of the peptide or polypeptide of the present invention, included in this invention. Preferred polypeptide fragments exhibit immunological properties that are unique or specific in relation antigennegative agent according to the invention. Fragments according to the invention, having the desired immunological properties can be obtained by any means, well known and commonly practiced in the field.

In another aspect, the invention provides options specific binding agents with substitutions according to the invention. Variants with substitutions are usually considered as "similar" to the original polypeptide or as having a certain "percentage identity" with the original polypeptide, and include polypeptides in which one or several amino acid residues polypep the IDA removed and replaced with alternative residues. In one aspect of these substitutions are conservative by nature, however, the invention also includes substitutions are non-conservative.

Identity and similarity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, the methods described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991) and Carilloet al.,SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine the relationship or interest of the identity of the two polypeptides are designed to get the best match between the test sequences. Methods for determining the identity described in publicly available computer programs. The preferred methods of computer programs to determine 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, University of Wisconsin, Madison, WI, BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol., 215:403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources is s (BLAST Manual, Altschul et al., NCB/NLM/NIH Bethesda, MD 20894; Altschul et al., above (1990)). Identity can also be used a well-known algorithm of Smith-Waterman.

Some schemes alignment to align two amino acid sequences can lead to the match only in a short region of the two sequences, and this small leveled area may have very high sequence identity, even if there is no significant relationship between these two full-sized sequences. Thus, in some embodiments, the implementation, the selected alignment method (GAP) will lead to an alignment that extends at least ten percent of the full length polypeptide compared to the target, i.e. at least 40 contiguous amino acids, where comparing the sequence of at least 400 amino acids, 30 contiguous amino acids, where comparing the sequence of at least 300 to about 400 amino acids, at least 20 contiguous amino acids, where the compare sequence 200 to about 300 amino acids, at least 10 contiguous amino acids, where the compared sequences of approximately 100 to 200 amino acids.

For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, WI) two polypeptide, which is GNA to be defined percent identity, aligned for optimal matching of their respective amino acids (the"matching distance"determined by this algorithm). In some embodiments, the implementation, the penalty for opening a gap (which is usually calculated as 3X the average diagonal; the "average diagonal" is the average diagonal matrix used in the comparison; the "diagonal" is the score or number assigned to each exact amino acid specific matrix comparison) and the penalty for extending the gap (which is usually 1/10 of the penalty for opening a gap), and matrix comparisons, such as RAM 250 or BLOSUM 62, used in conjunction with this algorithm. In some embodiments, implementation, this algorithm is also used standard matrix comparison (see Dayhoff et al., Atlas of Protein Sequence and Structure, 5(3) (1978) instead of the matrix comparison FRAMES 250; Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919 (1992) instead of the matrix comparison BLOSUM 62).

In some embodiments, implementation, parameters for comparison of polypeptide sequences include the following:

Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);

Matrix comparison: BLOSUM 62 from Henikoff et al., higher (1992);

The penalty for gap: 12

The penalty for a gap length: 4

The threshold of similarity: 0

The GAP may be applicable with the current settings listed above. In some embodiments, implementation of the above-mentioned parameters are parameter and the default for comparisons of polypeptides (along with no penalty for end gaps) using the GAP algorithm.

In some embodiments, implementation, parameters for comparisons of sequences of polynucleotide molecules include the following:

Algorithm: Needleman et al., above (1970);

Matrix comparison: matches = +10, no match = 0

The penalty for gap: 50

The penalty for a gap length: 3

The GAP may be applicable with the current settings listed above. The aforementioned parameters are the default parameters for comparisons of polynucleotide molecules.

Can be used in other examples of algorithms that are penalties for the opening of the gap penalties for the lengthening of the gap matrix comparison, the thresholds of similarity, etc., including those provided in the Program Manual, Wisconsin Package, Version 9, September, 1997. Specific choices that must be made will be obvious to experts in this field and will depend on the specific comparison that should be made, for example, DNA to DNA, protein to protein, protein to DNA; and, furthermore, whether this comparison a comparison between specific pairs of sequences (in this case usually are preferred GAP or BestFit) or a comparison between one sequence and sequences from a large database (in this case, preferred are FASTA or BLASTA).

In this context, the twenty conventional amino acids and their abbreviations correspond to the standard application. the m manual Immunology-A Synthesis (2ndEdition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), incorporated herein by reference for any purpose.

The amino acid can be either L-or D-stereochemistry (except Gly, which is neither L nor D), and the polypeptides and compositions of this invention can contain a combination of stereohype. However, preferred is L-stereochemistry. This invention also provides the converted molecule in which the sequence of aminocore to carboxylic these amino acids is converted. For example, contacting the molecule with a normal sequence X1-X2-X3it would be X3-X2-X1. This invention also provides retrobyte molecule, which, as indicated above, the sequence from amino end to the carboxy-end is converted and residues, which are usually enantiomers “L”changed to stereoisomeric form “D”.

Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α, α-disubstituted amino acids, N-alkylaminocarbonyl, lactic acid and other unusual amino acids may also be suitable components for polypeptides according to the invention. Examples of unusual amino acids include, without limitation: aminoadipyl is islote, beta-alanine, beta-aminopropionic acid, aminobutyric acid, piperidine acid, aminocaproic acid, aminoheptanoic acid, aminoethanol acid, aminopimelic acid, diaminobutane acid, desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, ALLO-hydroxylysine, hydroxyproline, isodesmosine, ALLO-isoleucine, N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, Norvaline, norleucine, ornithine, 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetylized, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, δ-N-methylarginine and other similar amino acids and amino acids (for example, 4-hydroxyproline).

Similarly, if there are no other indications, the left end of the single-stranded polynucleotide sequence is 5'-end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5'-direction. Direction 5'-3'-attach the obtained RNA transcripts called the direction of transcription; the areas of the sequence on the DNA strand having the same sequence as the RNA and which are 5' (left) relative to the 5'-end of the RNA transcript, referred to as "sequences against the course of transcription" ("upstream"); the areas of the sequence n is the DNA chain, having the same sequence as the RNA and which are 3' (right) relative to the 3'-end of the RNA transcript, referred to as "sequences during transcription ("downstream").

Conservative amino acid substitutions may include non-natural amino acid residues, which are typically incorporated by chemical peptide synthesis, not by synthesis in biological systems. They include peptidomimetics and other inverted or inverted form amino acid portions of the molecule.

Natural residues can be divided into classes based on common properties of the side chains:

1) hydrophobic: Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may replace a member of one of these classes a member of another class. Such substituted residues may be introduced into regions of the human antibodies, which are homologous with antibodies not human, or non-homologous regions of this molecule.

When such changes, according to some variants of implementation, can be considered hydropathicity index (index of hydrophobicity) of the amino acids. Each amino acid was assigned hydropathicity index OS is ove its characteristics, hydrophobicity and charge. These indexes are: isoleucine (+4,5); valine (+4,2); leucine (is+3.8); phenylalanine (a+2.8); cysteine/cystine (+2,5); methionine (+1,9); alanine (+1,8); glycine (-0,4); threonine (a-0.7); serine (of-0.8); tryptophan (of-0.9); tyrosine (-1,3); Proline (of-1.6); histidine (-3,2); glutamate (for 3,5); glutamine (for 3,5); aspartate (for 3,5); asparagine (for 3,5); lysine (-3,9) and arginine (-4,5).

The importance hydropathical index of amino acids in conferring interactive biological function of the protein is recognized in this field. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids may be replaced with other amino acids having similar hydropathicity index or score and still retain a similar biological activity. In making changes based hydropathical index, in some embodiments, implementation, include substitution of amino acids, gidroopticheskie indices are within ±2. In some embodiments, implementation include the replacement, which are within ±1, and in some embodiments, implementation include replacement of ±0.5.

In this area it is also believed that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly when generated by this biologically functional protein or peptide is intended for use in immunological embodiments, as in this case. Some of the options that the exercise of the greatest local average hydrophilicity of a protein, created by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein.

The following hydrophilicity values have been assigned to the specified amino acid residues: arginine (+3,0); lysine (+3,0); aspartate (+3,0 ± 1); glutamate (+3,0 ± 1); serine (+0,3); asparagine (+0,2); glutamine (+0,2); glycine (0); threonine (-0,4); Proline at (- 0.5 ± 1); alanine (-0,5); histidine (-0,05); 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 making changes based on similar hydrophilicity values, in some embodiments, implementation, include substitution of amino acids, hydrophilicity values are within ±2, in some embodiments, implementation include the replacement, which are within ±1, and in some embodiments, implementation include replacement of ±0.5. You can also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These areas are also referred to as "regions of the inner part of the epitope".

Examples of amino acid substitutions shown in table 1.

Table 1
Amino acid substitutions
The original remainsApproximate replacement Preferred replacement
AlaVal, Leu, IleVal
ArgLys, Gln, AsnLys
AsnGln, Glu, AspGln
AspGLU, Gln, AsnGlu
CysSer, AlaSer
GlnAsn, Glu, AspAsn
GluAsp, Asn, GlnAsp
GlyPro AlaAla
HisAsn, Gln, Lys, ArgArg
IleLeu, Val, Met, Ala,
Phe, norleucine
Leu
LeuNorleucine, Ile,
Val, Met, Ala, Phe
Ile
LysArg, 1,4-diaminobutane acid, Gln, AsnArg
Met Leu, Phe, IleLeu
PheLeu, Val, Ile, Ala,
Tyr
Leu
ProAlaGly
SerThr, Ala, CYSThr
ThrSerSer
TrpTyr, PheTyr
TyrTrP, Phe, Thr, SerPhe
ValIle, Met, Leu, Phe,
Ala, norleucine
Leu

The person skilled in the art will be able to determine suitable variants of the polypeptide presented here, using well known methods. In some embodiments, implementation specialist in this area can identify suitable areas of the molecule that can be modified without destroying the activity by targeting areas known that they are not important for activity. In some embodiments, the implementation can identify the remains and parts of molecules, which are conservative environments is similar polypeptides. In some embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without undesirable effects on the structure of the polypeptide.

In addition, the person skilled in the art may view research on the structure-function identifying residues in similar polypeptides that are important for activity or structure. Because of this comparison, one can predict the importance of amino acid residues in the protein, which correspond to amino acid residues that are important for activity or structure in similar proteins. The person skilled in the art can choose a chemically similar amino acid substitutions for such predicted important amino acid residues.

The person skilled in the art may also analyze the three-dimensional structure and amino acid sequence relative to this structure in similar polypeptides. In view of such information, the person skilled in the art may predict the alignment of amino acid residues of the antibody with respect to its three dimensional structure. In some embodiments, implementation specialist in this area may choose not to make radical changes to the AMI is kislotnyh residues, which, as predicted, are on the surface of this protein, as these residues may be involved in important interactions with other molecules. In addition, the person skilled in the art can generate test variants containing a single amino acid substitution at each of the desired amino acid residue. Then these options may be subjected to screening using analyses of activity, known in this area. Such options could be used to gather information about suitable options. For example, if you find that change in relation to a specific amino acid residue led to impaired, reduced undesirable or inappropriate activity, it is possible to avoid such changes. In other words, based on the information collected from such routine experimentation, the skilled in the art can easily determine the amino acids that you should avoid additional substitutions, either alone or in combination with other mutations.

The number of scientific publications have been devoted to the prediction of secondary structure. Cm. Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):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., 47:251-276 and Chou et al., Biophys. J., 26:367-384 (1979). In addition, currently available computer programs to aid the prediction of the secondary structure. One method of secondary structure prediction based on homology modeling. For example, two of the polypeptide or protein, which have a sequence identity greater than 30%, or similarity, greater than 40%, often have similar structural topology. The recent increase in protein structural database (PDB) increased predictability of secondary structure, including the potential number of folds in the structure of the polypeptide or protein. Cm. Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). An assumption was made (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997))that there are a limited number of folds in a particular polypeptide or protein and that after the resolution of a critical number of structures structural prediction will become dramatically more accurate.

Additional methods of secondary structure prediction include “threading” (the way of replacing the side chains of the same protein side chains of another protein while maintaining constant the conformation of the main chain) (Jones, D.,Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sipplet al.,Structure, 4(1):15-19 (1996)), "profile analysis" (Bowieet al., Science, 253:164-170 (1991); Gribskovet al.,Meth. Enzym., 183:146-159 (1990); Gribskovet al.,Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage" (see Holm, above (1999) and Bennet, above (1997)).

In some embodiments, the implementation variants of antibodies include glycosylated variants in which the number is the Creator and/or type of glycosylation sites have been altered compared to the amino acid sequence of the original polypeptide. In some embodiments, the implementation of variants of the protein contain more or fewer N-linked glycosylation sites than the native protein. N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, where amino acid residue indicated by X may be any amino acid residue except Proline. Replacement of amino acid residues to create this sequence provides a new site for the addition of N-linked carbohydrate chain. Alternatively, the replacement, which helps eliminate this sequence will remove the existing N-linked carbohydrate chain. Provided also rurangirwa N-linked carbohydrate chains, in which one or more N-linked glycosylation sites (usually sites of glycosylation, which are natural) eliminated and created one or more new N-linked sites. Additional preferred options antibodies include cysteine variants in which one or more cysteine residues deleterows from the original amino acid sequence or replaced by another amino acid (e.g., serine) as compared to the original amino acid sequence. Cysteine variants can be applied when antibodies should be subjected to refolding in a biologically active conformation,for example, after removal of insoluble inclusion bodies. Cysteine variants usually have fewer cysteine residues than the natural protein, and usually have an even number of cysteine residues to minimize interactions originating from not paired cysteines.

In accordance with some of the options for the implementation of the amino acid substitutions are substitutions that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity of for the education of the protein complexes, (4) alter the affinity of binding and/or (5) add or modify other functional properties such polypeptides. In accordance with some of the options for the implementation of single or multiple substitutions of amino acids (in some embodiments, implementation, conservative substitutions of amino acids) can be obtained in the natural sequence (in some embodiments, implementation, parts of the polypeptide outside the forming domain (domains) intermolecular contacts). In some embodiments, the implementation of a conservative amino acid substitution is usually essentially cannot change the structural characteristics of the original sequence (for example, amino acid substitution should not try to break a helix that occurs in the source sequence, or once usati other types of secondary sequence, which characterizes the original sequence). Examples recognized in the field of secondary and tertiary structures of the polypeptide described in Proteins, Structures and Molecular Pinciples (Creighton, Ed., W. H. Freeman and Company, New York (1984)); 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).

Molecules of a specific binding agent of this invention, which are polypeptide or peptide variants with substitutions, can have up to approximately 10-12 percent replaced the original amino acid sequence. For variants of the antibody heavy chain may have 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 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 substituted amino acid, whereas the light chain may have 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 substituted amino acid.

Derivative specific binding agents

The invention also provides derivatives of polypeptides specific binding agent. Derivatives include polypeptides specific binding agent bearing modifications other than insertion, deletion or substitution of amino acid residues. Preferably, these modifications are covalent in nature and include, for example, the formation of chemical bonds with polymers, lipids, other organic and inorganic parts of the molecules. Derivatives of this invention can be prepared to increase the half-life existence in the blood of polypeptide specific binding agent or can be designed to improve the targeting ability of the polypeptide to desired cells, tissues or organs.

The invention further includes derivatives of binding agents, covalently modified to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethyleneglycol or polypropylenglycol, as described in U.S. Patent numbers 4640835, 4496689, 4301144, 4670417, 4791192 and 4179337. Other applicable polymers known in this area include monometoksipolietilenglikolya, dextran, cellulose or other polymers based on carbohydrates, poly(N-vinyl pyrrolidone)polyethylene glycol, homopolymers of propylene, a copolymer of polypropyleneoxide/ethylene oxide, polyoxyethylene polyols (e.g. glycerol) / polyvinyl alcohol, and mixtures of these polymers. Particularly preferred are the products of the specific binding agent covalently modified subunits of polyethylene glycol (PEG). Water-soluble polymers can be linked to specific provisions, for example, on aminocore products specific binding agent, or when uedineny randomly to one or more side chains of the polypeptide. The use of PEG to improve therapeutic capacity in relation to specific binding agent, and humanized antibodies, in particular, are described in U.S. Patent 6133426 issued by Gonzales et al. 17 October 2000.

Sites targeted for mutagenesis of antibodies

Certain strategies can be used to manipulate the intrinsic properties of Ang-2-specific antibody, such as the affinity of this antibody for its target. These strategies include the use of site-directed or random mutagenesis polynucleotide molecule that encodes the antibody, to generate variants of the antibodies with the subsequent stage of screening, designed to retrieve variants of antibodies that exhibit the desired change, for example, increased or decreased affinity.

Amino acid residues that are most commonly targeted in mutagenic strategies are amino acid residues in the CDR. As described above, these regions contain residues that actually interact with Ang-2 and other amino acids that affect the spatial distribution of these residues. However, it was also shown that amino acids in the framework regions of the variable domains outside the CDR regions contribute significantly antigennegative properties of this antibody and can serve as targets for mA is polerowanie such properties. Cm. Hudson, Curr Opin Biotech, 9:395-402 (1999) and references in this work.

Less and more efficiently scrinium library of variants of the antibodies can be obtained by restriction of random or site-directed mutagenesis sites in the CDR, which correspond to areas prone to "Hyper-mutations during somatic maturation process affinity. Cm. Chowdhury and Pastan, Nature Biotech, 17: 568-572 (1990) and references in this work. Types of DNA elements known that they define the sites of hypermutable thus include direct and inverted repeats, certain consensus sequence, secondary structure and palindromes. The consensus DNA sequences include the sequence of the four bases purine-G-pyrimidine-A/T (i.e. a or G - G - C or T a or t), and the serine codon AGY (where Y can be C or T).

Thus, one variant of the present invention includes mutagenic strategies with the goal of increasing the affinity of the antibody for its target. These strategies include mutagenesis of the entire variable region of the heavy chain and light chain, mutagenesis, only the CDR regions, mutagenesis of the consensus sites Hyper-mutations in CDR mutagenesis frame areas or a combination of these approaches ("mutagenesis" in this context could be random or site-directed). The final description of the CDR regions and identification of residues, provided the binding site of the antibody, can be done by disclosing the structure of the considered antibody and the complex of antibody-ligand, by methods known to experts in this field, such as x-ray crystallography, and can be used, although not completely, to approximate the CDR regions. Examples of such commonly known methods include definitions of Kabat, Chothia, AbM and contact definitions.

The Kabat definition is based on sequence variability and is the most frequently used definition for predicting the CDR regions. (Johnson and Wu, Nucleic Acids Res, 28: 214-8 (2000)). The Chothia definition is based on determining the location of the structural areas of loops (Chothia et al., Mol Biol, 196:901-17(1986); Chothia et al., Nature, 342: 877-83 (1989)). The AbM definition is a compromise between the definition of Kabat and Chothia. AbM is an integrated software Suite to simulate the structure of the antibody produced by Oxford Molecular Group (Martin et al., Proc Natl Acad Sci (USA) 86:9268-9272 (1989); Rees et al., ABM™, a computer program for modeling variable regions of antibodies, Oxford, UK; Oxford Molecular, Ltd.). Kit AbM simulates the tertiary structure of the antibody from the primary sequence using a combination of known databases and ab initio methods. Recently introduced additional definition, known as the contact definition (MacCallum et al., J Mol Biol, 5:732-45 (1996)). This definition is based on the analysis doctorrecommended crystal structures.

Under the agreement, the CDR regions in the heavy chain are typically referred to as N1, N2 and N3, and they are numbered sequentially from aminobenzo to carboxilic. CDR regions in the light chain are typically referred to as L1, L2 and L3, and they are numbered sequentially from aminobenzo to carboxilic.

CDR-H1 has a length of approximately 10-12 residues and usually starts after 4 residue Cys according to Chothia definitions and AbM or usually 5 residues later in accordance with the definition of Kabat. For N1 is usually followed by Trp, typically TRP-Val, but also Trp-Ile or Trp-Ala. The length H1 of approximately 10-12 residues in accordance with the definition of AbM, while the Chothia definition includes the last 4 residue.

CDR-H2 starts in 15 residues after the end of H1 in accordance with the definitions of Kabat and AbM. Residues preceding H2, are typically Leu-Glu-Trp-Ile-Gly, but there are a number of variations. For H2 is typically followed by the amino acid sequence Lys/Arg/-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. According to the Kabat definition, the length H2 of approximately 16 to 19 residues, while the definition of AbM predicts that this length is usually equal to 9-12 residues.

CDR-H3 is usually initiated via a 33 residue after the end of n, and it is usually preceded by the amino acid sequence (typically Cys-Ala-Arg). For N3 usually follows this amino acid sequence is Gly. The length of H3 can be either in the range 3-25 residues.

CDR-L1 usually where the at residue 24 and is usually followed Cys. The residue CDR-L1 is always a Trp, and he usually will be the beginning of the sequence Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln or Trp-Tyr-Leu. The length of the CDR-L1 of approximately 10-17 residues. CDR-L1 is the target for antibodies of the present invention closely follows this picture, with the Cys residue followed by 15 amino acids and then Trp-Tyr-Gln.

CDR-L2 begins approximately 16 residues after the end of L1. He usually follows the residues Ile-Tyr, Val-Tyr, Ile-Lys, or Ile-Phe. The length of the CDR-L2 of approximately 7 residues.

CDR-L3 usually begins after 33 residue after the end of L2, and it usually follows after Cys. For L3 is usually followed by the amino acid sequence Phe-Gly-XXX-Gly. The length L3 of approximately 7 to 11 residues.

In this area describes various methods for modifying antibodies. For example, U.S. Patent 5530101 (Queen et al., June 25, 1996) describes methods for obtaining humanized antibodies, in which the frame sequence field variable domain of the heavy chain gumanitarnogo immunoglobulin at 65-95% identical to the sequence of frame field variable domain of the heavy chain of the donor immunoglobulin. Each circuit gumanitarnogo immunoglobulin will typically contain, along with the CDR amino acids from the donor immunoglobulin framework, which, for example, capable of interacting with the CDR to influence the activity of binding, for example, one or more which of amino acids, which are directly adjacent to a CDR in the donor immunoglobulin, or amino acids located within about 3 angstroms, as predicted by molecular modeling. Heavy and light chain, each can easily be constructed using any one or all of the various criteria provisions. When combined into an intact antibody humanized immunoglobulin according to this invention are essentially non-immunogenic in humans and will retain essentially the same affinity to that of the donor immunoglobulin, against this antigen, such as a protein or other compound containing the epitope. Cm. related methods in U.S. patent 5693761 issued by Quinn et al. December 2, 1997 (“Polynucleotide, improved encoding humanized immunoglobulins”); U.S. Patent 5693762 issued by Quinn, et al. December 2, 1997 (“Superior immunoglobulins”); U.S. Patent 5585089 issued by Quinn, et al. December 17, 1996 (the“Superior immunoglobulins”).

In one example, the U.S. Patent 5565332 issued Hoogenboom October 15, 1996 (the"Receiving chimeric antibody - combined approach"), describes methods for producing antibodies and fragments of antibodies that have the same binding specificity as the original antibody, but which have increased features characteristic of human antibodies. GU is Anisimovna antibodies produced by shuffling circuit, using, for example, phage display technology resulting, and the polypeptide containing the variable domain of a heavy or light chain of the antibody is not person specific in respect of interest antibodies, together with a repertoire of complementary variable domains (light or heavy chain). Hybrid mating, which are specific in respect of interest antigen, identify and chains of human antibodies from selected matings combined with a range of complementary variable domains (heavy or light chain) of a person. In another embodiment, the component CDRs of the antibodies combine with the repertoire of the component parts CDRs of the antibodies of the person. From the library of dimer polypeptide antibodies are selected hybrids and use them in the second stage human nature and humanizing dicing. Alternatively, this second phase is passed, if the hybrid has quite a "human nature" for use in therapy. Methods of modification to increase human nature antibodies are also described. Cm. also Winter, FEBS Letts 430:92-92 (1998).

In another example, U.S. Patent 6054297, issued to Carter et al. April 25, 2000, describes a method of obtaining humanized antibodies replacement of the amino acid sequence of CDR of the corresponding amino acid follower of the awn CDR person and/or replacement of the amino acid sequence of FR corresponding amino acid sequences of FR of a human.

As another example, U.S. Patent 5766886 issued Studnicka et al., issued June 16, 1998 ("Modified variable domains of antibodies"), describes how the identification of amino acid residues of the variable domain of the antibody, which can be modified without reducing the native affinity of antigennegative domain while reducing its immunogenicity against heterologous species, and methods of obtaining these modified variable domains that are applicable for the introduction of heterologous species. Cm. also U.S. Patent 5869619 issued Studnicka February 9, 1999.

As discussed, the modification of antibodies by any of the methods known in this field, usually designed to obtain increased binding affinity towards the antigen and/or reduce immunogenicity of the antibody in the recipient. In one approach, the humanized antibodies can be modified to eliminate glycosylation sites to increase the affinity of this antibody with respect to its cognate antigen (Co et al., Mol Immunol 30:1361-1367 (1993)). Techniques such as "reverse engineering", "giperkeratose and lining/reconstruct the surface, gave humanized antibodies with a higher therapeutic potential. (Vaswami et al., Annals of Allergy, Asthma, & Immunol 81:105 (1998); Roguska et al., Prot Engineer 9:895-904 (1996)). Cm. also U.S. Patent 602035, issued by Hardman et al., June 6, 2000, which describes how reverse engineering (engineering) antibodies. Although these methods reduce the immunogenicity of antibodies due to the decrease of alien residues, they do not prevent antiidiotypic and antiaritmicheskie response after repeated administration of these antibodies. Alternative to these methods reduce immunogenicity described in Gilliland et al., J Immunol 62(6): 3663-71 (1999).

In many cases, the humanization of antibodies leads to the loss of antigennegative abilities. Therefore, preferably back to mutate" humanitariannet antibody to activate one or more amino acid residues found in the original antibody (most often the rodent antibody) in an attempt to restore the affinity of binding of this antibody. See, for example, Saldanha et al., Mol Immunol 36:709-19 (1999).

Ones analogues of specific binding agents/protein mimetics

In addition, are also considered ones analogues of peptides specific binding agent, which provide a stabilized structure or lessened biodegradation. Mimetic peptide analogs of the specific binding agent can be obtained on the basis of selected inhibitory peptide by replacement of one or more residues ones parts of the molecule. Preferably, these NEPA Tinnie part of the molecule allow the peptide to retain its natural conformation or stabilize preferred for example, the bioactive conformation that retains the ability to recognize and bind Ang-2. In one aspect, the received analogue, mimetic exhibits increased binding affinity of the ratio of Ang-2. One example of methods of obtaining ones mimetic analogs of peptides specific binding agent described in Nachman et al., Regul Pept 57:359-370 (1995). If desired, the peptides of the specific binding agent of the present invention can be modified, for example, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid additive salts, amides, esters, in particular C-terminal esters, and N-ACI-derivatives of the peptides of the present invention. Peptides specific binding agent can also be modified to generate peptide derivatives of the formation of covalent or non-covalent complexes with other parts of the molecule. Covalently linked complexes can be prepared by linking the chemical parts of the molecules with functional groups on the side chains of amino acids, peptides specific binding agent or the N - or C-end.

In particular, it is clear that the peptides of the specific binding agent may be conjugated with a reporter group, including, but not limited to, a radioactive label, fluorescent label, which can be settled (for example, an enzyme that catalyzes a colorimetric or fluorometric reaction), a substrate, a solid matrix, or carrier (e.g., Biotin or Avidya). Thus, this invention provides a molecule containing an antibody molecule, and this molecule is preferably further comprises a reporter group selected from the group consisting of a radioactive label, fluorescent label, an enzyme substrate, a solid matrix and media. Such labels are well known qualified in this field specialists, for example, are considered, in particular, bitenova label. The use of such labels is well known qualified in this field specialists and are described, for example, in U.S. Patent 3817837; U.S. Patent 3850752; U.S. Patent 3996345 and U.S. Patent 4277437. Other labels that will be applied include, but are not limited to, radioactive labels, fluorescent labels, and chemiluminescent labels. U.S. patents relating to the use of such labels include, for example, U.S. Patent No. 3817837; U.S. Patent No. 3850752; U.S. Patent No. 3939350 and U.S. Patent No. 3996345. Any of the peptides of this invention may contain one, two or more of any of these labels.

The methods of preparation of specific binding agents

The specific binding agents of the present invention, which are b what Lomi, can be obtained by chemical synthesis in solution or on a solid substrate in accordance with conventional ways. The existing limit for solid-phase synthesis is the synthesis of peptides with a length of 85-100 amino acids. However, the methods of chemical synthesis can often be used for chemical ligation of a number of peptides smaller to generate the full-length polypeptides. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Pase Peptide Synthesis, 2d. ed., Pierce Chemical Co., (1984); Tam et al., J Am Chem Soc, 105:6442, (1983); Merrifield, Science, 232:341-347, (1986); and 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); and U.S. Patent No. 5424398), incorporated herein by reference.

Methods of solid-phase peptide synthesis using a copolymer of styrene and divinylbenzene containing 0.1-1.0 mm amine per gram of polymer. These methods of peptide synthesis are used as protection of alpha-amino groups of butyloxycarbonyl (t-BOC) or 9-fluorenylmethoxycarbonyl (FMOC). Both methods involve stepwise syntheses whereby a single amino acid type at each stage, starting from the C-end of the peptide (see, Coligan et al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Upon completion of the chemical with which NASA synthetic peptide may be released from the blocking of the amino groups tBoc or FMOC and split-off from the polymer by treatment with an acid at low temperature (for example, liquid HF-10% anisole for about 0.25 to about 1 h at 0°C). After evaporation of the reagents, the peptides of the specific binding agent is extracted from the polymer with 1% acetic acid solution and then lyophilizer to obtain the crude material. He can usually be purified by means of gel filtration on Sephadex G-15 using 5% acetic acid as solvent. Lyophilization of appropriate fractions of the column will be to give a homogeneous peptide specific binding agent or derivative peptide, which can then be characterized by such standard methods as amino acid analysis, thin layer chromatography, high performance liquid chromatography, spectroscopy absorption in the ultraviolet, molecular rotation, solubility, and determining the amount of solid-phase degradation of Edman.

Chemical synthesis of anti-Ang-2 antibodies, derivatives thereof, variants and fragments, as well as other Ang-2-binding agents on the basis of protein allows to include in the agent unnatural amino acids.

Methods of recombinant DNA are a convenient means of obtaining a full-size antibodies and other large protein agents specific binding agent of the present invention or their fragments. cDNA molecule encoding this antibody or fragments which, can be insertion in expressing vector, which, in turn, can be integrated into the cell host to produce the antibody or fragment. It is clear that the cDNA encoding such antibodies can be modified to change from the "original" DNA (translated from mRNA) to ensure the degeneracy of codons, or to allow the use of preferences (preferences) codons in different cells of the host.

Typically, the DNA molecule encoding the antibody can be obtained using the procedures described in the Examples. When the desired is getting Fab molecules or CDR, which belong to the original molecule antibodies, can be subjected to screening a suitable library (library of phage display library lymphocytes etc) using standard methods for identification and cloning of related Fab/CDR. The probes used for this screening can be full-length or truncated Fab probes encoding the Fab-part of the original antibody, or other appropriate probes. When using DNA fragments as probes typical hybridization conditions are the conditions presented in Ausubel et al. (Current Protocols in Molecular Biology, Current Protocols Press (1994)). After hybridization probed blot can be washed with a suitable p the guests depending on such factors as the size of the probe, the expected homology of the probe relative to the clone library type, subjected to screening, and the number of clones subjected to screening. Examples of high stringency are 0.1 SSC and 0.1% LTOs at a temperature of 50-65°C.

Different systems of expression vector/host can be used for content and expression of polynucleotide molecules encoding polypeptides of the specific binding agent of the present invention. These systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosignee expressing the DNA vectors; yeast transformed with yeast expression vectors; insect cells infected with viral expression vectors (e.g. baculovirus); plant cell, transfetsirovannyh virus expression vectors (for example, the virus of the mosaic disease of cauliflower CaMV; the virus of the mosaic disease of tobacco, TMV) or transformed with bacterial expression vectors (e.g., Ti or plasmid pBR322); or animal cells.

Mammalian cells, which are applicable in recombinant get protein specific binding agent include, but are not limited to, VERO cells, HeLa cells, CL is the exact line of Chinese hamster (Cho), cells COS (for example COS-7)cells, W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293, as well as hybridoma cell lines described here. Mammalian cells are preferred to obtain those specific binding agents such as antibodies and antibody fragments, which are usually glycosylated and require proper installation for activity. Preferred mammalian cells include cells of Cho, hybridoma cells and myeloid cells.

Some exemplary protocols for the recombinant expression of proteins specific binding agent described herein below.

The term "expressing vector" refers to a plasmid, phage, virus or vector for expression of the polypeptide from a DNA (RNA)sequences. Expressing the vector may contain a transcriptional unit containing an ordered structure (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) the structure or sequence that encodes the binding agent, which is transcribed into mRNA and translated into protein, and (3) appropriate sequence of initiation and termination of transcription. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader placentas is required, enabling extracellular secretion of translated protein by the cell host. Alternatively, when a recombinant protein specific binding agent is expressed without a leader or transport sequence, it may include aminobenzoic the residue is methionine. This residue can be chipped off or may not be chipped off from the expressed recombinant protein to ensure the end product-specific binding agent.

For example, specific binding agents can recombinante to be expressed in yeast using a commercially available expression system, e.g., expression system Pichia (Invitrogen, San Diego, CA), in accordance with instrukciya manufacturer. This system can be based on pre-Pro-alpha sequence to control the secretion, but the transcription of the inserts is run by the promoter alcoholiday (OH) after induction with methanol.

Secreted peptide specific binding agent purified from the medium for growing yeast, for example, the methods used for purification of the peptide from supernatants bacterial cells and mammalian cells.

Alternatively, a cDNA encoding a peptide specific binding agent can be cloned into a baculovirus expressional vector pVL1393 (PharMingen, San Diego, CA). This is the vector may be used in accordance with the manufacturer's instructions (PharMingen) to infect cells of Spodoptera frugiperda in not containing sF9 protein environment and to obtain the recombinant protein. Protein specific binding agent can be purified and concentrated from the environment using heparin-sepharose column (Pharmacia).

Alternatively, the peptide can be expressed in the system of insects. System of insects for protein expression are well known qualified in this area specialists. In one such system, the nuclear polyhedrosis virus of Autographa californica NPV (AcNPV) can be used as a vector for expression of foreign genes in cells of Spodoptera frugiperda or Trichoplusia larvae. The coding sequence of the peptide specific binding agent can be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of this peptide specific binding agent will do polyhedrin gene inactive and to give the recombinant virus, devoid of envelope protein. These recombinant viruses can be used to infect cells Spodoptera frugiperda or Trichoplusia larvae in which the peptide is expressed (Smith et al., J Virol 46: 584 (1983); Engelhard et al., Proc Natl Acad Sci (USA) 91: 3224-7 (1994)).

In another example, a DNA sequence encoding a peptide specific binding agent, can be amplified using PCR and cloned into a suitable vector, for example, pGEX-3X (Pharmacia). Vector pGEX construe the IAOD for producing fused protein, glutathione-S-transferase (GST), encoded by this vector, and a protein specific binding agent encoded by a DNA fragment inserted into the cloning site of the vector. The primers for this PCR can be obtained in such a way that they include, for example, a suitable cleavage site. When the slit portion of the specific binding agent is used only to facilitate expression or it is otherwise undesirable in the form of joining of interest protein, recombinant protein specific binding agent may then be derived from GST-part of the fused protein. Construction of pGEX-3X/peptide specific binding agent to transform cells of E. coli XL-1 Blue (Stratagene, La Jolla CA), and individual transformants is isolated and grown. Plasmid DNA from individual transformants can be purified and partially sequenced using automated sequencing machine to confirm the presence of the desired encoding a specific binding agent inserts nucleic acids in the correct orientation.

Expression of polynucleotides encoding anti-Ang-2 antibodies and their fragments, using the above-described recombinant systems can lead to the production of antibodies or their fragments, which must be subjected to the "refolding" (for the correct creation of R is slichnih disulfide bridges), in order to be biologically active. Typical procedure for refolding of such antibodies is presented here as Examples and in the next section.

Specific binding agents obtained in bacterial cells, can be produced in the form of insoluble inclusion body in bacteria and can be cleaned in the following way. Cell owners can be killed by centrifugation; washed in 0.15 M NaCl, 10 mm Tris, pH 8, 1 M EDTA and treated with 0.1 mg/ml lysozyme (Sigma, St. Louis, MO) for 15 minutes at room temperature. This lysate can be clarified by sonication, and the remaining cells can be precipitated by centrifugation for 10 minutes at 12000×g. The precipitate containing the specific binding agent may be resuspendable in 50 mm Tris, pH 8, 10 mm EDTA, printed on 50% glycerol and centrifuged for 30 minutes at 6000×g. The precipitate can be resuspendable in standard phosphate buffered saline (PBS)containing no Mg++and Ca++. The specific binding agent may be further purified by fractionation resuspending sediment under denaturing LTO-polyacrylamide gel (Sambrook et al., above). This gel can be soaked in 0.4 M KCl for visualization of protein, which can be cut out and subjected electroelution in the buffer for separation in gel containing ordinator. If GST-f the th protein is produced in bacteria, in the form of a soluble protein, it may be cleaned using a Module GST-purification (Pharmacia).

System-mammalian hosts for expression of recombinant protein is a well-known qualified in this area specialists. Strains of host cells can be selected on a specific ability to processional expressed protein, or to produce certain post-translational modifications, which will be useful in ensuring the activity of the protein. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, limitirovanie and acylation. Different cells in a host, such as Cho, HeLa, MDCK, 293, WI38, and hybridoma cell lines, etc. have specific cellular apparatus and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and correct processing of introduced alien protein.

The number of sampling systems can be used to extract the cells, which were transformed to obtain the recombinant protein. Such a selection system include, but are not limited to, genes timedancing HSV genes hypoxanthineguanine and adrinfo.standortstr in cells tk -, hgprt-or aprt, respectively. Resistance to EN is metabolites can be used as the basis of selection for DHFR, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolate acid; neo, which confers resistance to the aminoglycoside G418 and confers resistance to chlorsulfuron; and hygro, which confers resistance to hygromycin. Additional breeding genes that may be applicable include trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to use gastinel instead of histidine. Markers that provide a visual indication to identify transformants include antizana, β-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.

Purification and refolding of specific binding agents

In some cases, specific binding agents produced using the procedures described above, may require the refolding and oxidation in the correct tertiary structure and the generation of disulfide bonds to be biologically active. Refolding can be performed using a number of procedures well known in the field. Such methods include, for example, the exposure action solubilizing polypeptide agent to a pH of usually more than 7 in the presence of chaotropes agent. The choice of chaotrope similar to the choice that is used to solubilize the inclusion bodies, however hotrod used usually at a lower concentration. Example chaotropes agent is guanidine. In most cases, the solution for refolding/oxidation will also contain a reducing agent and its oxidized form in a specific ratio to generate a particular redox potential, which will make possible the shuffling of disulfide for the formation of cysteine bridges. Some of the commonly used redox couples include cysteine/tsistamin, glutathione/dithiobis(GSH), chloride divalent copper, dithiothreitol DTT/gitian DDT and 2-mercaptoethanol (bME)/dithio-bME. In many cases, there may be used a co-solvent to increase the efficiency of refolding. The most commonly used co-solvents include glycerol, polyethylene glycol of various molecular masses and arginine.

Desirable would be cleaning protein specific binding agents or their variants according to the invention. Methods of protein purification well known to a skilled in this area specialists. These methods include, on one level, coarse fractionation polypeptide and polipeptidnyh faction. After separation of the polypeptide specific binding agent from other proteins of interest, the polypeptide may be further purified using chromatographic and electrophoretic SPO is mandatory to obtain partial or complete purification (or purification to homogeneity). Analytical methods are suitable, in particular, to obtain pure peptide specific binding agent, are ion-exchange chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly effective method of purification protein is liquid Express chromatography of proteins or even VGH.

Some aspects of this invention concern the purification, and in particular embodiments, implementation, substantial purification of the encoded protein or peptide specific binding agent. The term "purified protein or peptide specific binding agent" refers in this context to the composition, separated from the other components, in which the protein or peptide specific binding agent is purified to any degree relative to natural-received condition. Thus, the term protein or peptide specific binding agent refers to a specific binding protein or peptide that does not contain the environment in which it exists in nature.

Usually, the term "purified" will refer to the composition of the specific binding agent, which has been subjected to fractionation to remove various other components, and which essentially preserves the exercise of its biological activity. When using the term "su is estu purified" this term will refer to the composition of the specific binding agent, in which the protein or peptide of this specific binding agent forms a major component of this composition, for example, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95% or more of the proteins in this composition.

Various methods for quantifying the degree of purification of the specific binding agent will be known to the skilled in this field specialist in the light of this description. They include, for example, determining the specific activity of binding or assessing the number of polypeptides specific binding agent into fractions using electrophoresis with LTO-PAG. The preferred method of evaluation of the purity of the specific binding agent is the calculation of activity binding fraction compared to the binding activity of the original extract and, therefore, calculation of the degree of purification, measured here by the number of x-fold purification", where x is equal to this number. The actual units used to represent the magnitude of the activity of binding will, of course, depend on the specific method of analysis selected for follow-up treatment, and shows whether a protein or peptide specific binding agent detektiruya binding activity.

A variety of methods appropriate for a COI is whether in the purification of the protein specific binding agent, will be well-known qualified in this area specialists. They include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), etc. or denaturation by heating, followed by centrifugation; phase chromatography, such as affinity chromatography (for example, Protein a-Sepharose), ion-exchange chromatography; gel filtration, chromatography with reversed phase, hydroxyapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other methods. As is well known in this area, it is considered that the order of conducting the various purification stages can be changed, or that certain stages may be omitted, but this method is still suitable for the production of essentially purified specific binding agent.

There is no General requirement, namely, that this specific binding agent must always be provided in its most purified state. Indeed, it is assumed that less than essentially purified, products specific binding agent will be used in some embodiments of the invention. Partial purification can be performed using smaller stages of purification in combination stages or using other forms of the W is the most common way of cleaning. For example, it is clear that the cation-exchange column chromatography performed using VIH device will typically lead to more "x-fold" clearance than the same method using the system the low pressure chromatography. Methods that detect a lower degree of relative purification may have advantages in total output, the protein product of the specific binding agent or in maintaining the binding activity of the expressed protein specific binding agent.

It is known that the migration of a polypeptide can vary, sometimes significantly, depending on the different conditions of electrophoresis in LTOs/page (Capaldi et al., Biochem Biophys Res Comm, 76: 425 (1977)). Thus, it will be clear that under different conditions of electrophoresis, the average molecular weight of purified or partially purified product specific binding agent may vary.

Analyses bind

Immunological analyses linking typically use a gripping agent for specific binding and often immobilize the analyzed target antigen. Exciting agent is part of a molecule that specifically binds with the analyte. In one embodiment, the addictive agent is an antibody or fragment that specifically binds to Ang-2. the ti immunological analyses linking are well known in this field (see Asai, ed., Methods in Cell Biology, Vol. 37, Antibodies in Cell Biology, Academic Press, Inc., New York (1993)).

Immunological analyses of binding is often used aiming agent, which would signal the existence of the bound complex formed by exciting the agent and the antigen. This aiming agent can be one of the molecules that make up this linked complex: i.e. they may be labeled specific binding agent or a labeled antibody against the specific binding agent. Alternatively, this aiming agent may be a third molecule, typically another antibody that binds with an associated complex, aiming agent can be, for example, an antibody against a specific binding agent carrying a label. This second antibody specific against the associated complex may not have labels, but may be associated with the fourth molecule, specific in relation to the type of antibody, which member is this second antibody. For example, this second antibody may be modified detectable molecules, such as Biotin, which can then be related to the fourth molecule, such as a labeled enzyme streptavidin. Other proteins are able to specifically bind the constant region of immunoglobulin, such as protein a or protein G can also be used as aiming agent. E and binding proteins are common components of streptococcal cell walls of bacteria and exhibit a strong non-immunogenic reactivity with constant regions of immunoglobulins from different species (see, in General, Akerstrom, J Immunol, 135:2589-2542 (1985); and Chaubert, Mod Pathol, 10:585-591 (1997)).

In all of these analyses may be required under incubation and/or washing after each combination of reagents. The stage of incubation can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend on the format of the analysis of the analyte, volume of solution, concentrations, etc. Usually these analyses will be carried out at ambient temperature, although they can be conducted over a large temperature range.

A. non-Competitive analysis link:

Immunological analyses of binding can be analyses of non-competitive type. These analyses have a number of exciting analyte that can be measured. For example, in one preferred "sandwich"-the analysis of the exciting agent (antibody) may be connected directly to a solid substrate where it can be immobilized. Then these immobilized antibodies capture antigen (linked)present in the test sample. Then immobilized thus protein associated with aiming agent such as a second antibody having a label. In another preferred "sandwich"-the analysis of this second antibody does not have a label, but can the be associated with the labeled antibody, specific respect to antibodies of the species from which the second antibody. This second antibody may also be modified detectable molecules, such as Biotin, which can specifically bind a third labeled molecule, such as streptavidin. (See guide Harlow and Lane, Antibodies: A Laboratory Manual, Ch 14, Cold Spring Harbor Laboratory, NY (1988), incorporated herein by reference).

C. Competitive analysis link:

Immunological analyses of binding can be competitive analysis type. The amount of analyte present in the sample, is measured directly by measuring the amount of added analyte displaced or competitive remote from an agent by the analyte present in the sample. In one preferred competitive analysis of binding of a known amount of analyte, usually labeled add to the sample and then the sample is contacted with the antibody (breathtaking agent). The amount of labeled analyte bound to the antibody is inversely proportional to the concentration of analyte present in the sample. (See Harlow and Lane, Antibodies: A Laboratory Manual, Ch 14, pp. 579-583, above).

In another preferred competitive analysis of binding antibody immobilized on a solid substrate. The amount of protein that is associated with the antibody may be determined either by measurement to the number of protein present in a complex protein/antibody, or alternatively by measuring the amount of remaining not formed a complex protein. (See Harlow and Lane, Antibodies: A Laboratory Manual, Ch 14, above).

In another preferred competitive analysis of binding using inhibition of the hapten. Here, the known analyte immobilized on a solid substrate. A known amount of antibody is added to the sample and the sample in contact with the immobilized analyte. The number of antibodies bound to the immobilized analyte is inversely proportional to the amount of analyte present in the sample. The number of immobilized antibodies can be detected by detecting either immobilized fraction antibody, or fraction, which remains in solution. Detection may be direct, when the antibody target, or indirectly, by the subsequent addition of labeled parts of a molecule that specifically binds with the antibody, as described above.

C.Using competitive analysis link:

Competitive analysis of binding can be used to determine cross-reactivity, so that the specialist can determine whether a protein or enzyme complex, which is recognized by a specific binding agent of the present invention, the desired protein and does not cross-Raag is the dominant molecule, or to determine whether this antibody is specific for a given antigen and does not bind if it is undesirable antigens. In the analysis of this type of antigen can be immobilized on a solid carrier and an unknown mixture of proteins add to this analysis that could compete with the binding of the specific binding agents of this immobilized protein. Competing molecule binds to one or more antigens unrelated with respect to this antigen. The ability of these proteins to compete with the binding of the specific binding agents are antibodies to the immobilized antigen compared to binding of the same protein that was immobilized on a solid medium to determine the cross-reactivity of this protein mixture.

D.Other assays binding:

The invention also provides methods Western blotting for the detection or quantitative determination of the presence of Ang-2 in the sample. This method usually involves the separation of protein samples by gel-electrophoresis on the basis of molecular weight and transfer the proteins to a suitable solid substrate such as a nitrocellulose filter, a nylon filter, or derivationally nylon filter. This sample is incubated with the antibodies or fragments that specifically bind Ang-2, poluchenii complex detects. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies that specifically bind to the antibody.

Analyses of binding to detect those Ang-2 specific binding agents which disrupt binding of Ang-2 with its receptor, is presented here as Examples.

Diagnostic tests

Antibodies or fragments thereof of the present invention is applicable for the diagnosis of conditions or diseases characterized by expression of Ang-2 or subunits, or in assays to monitor patients undergoing treatment with inducers of Ang-2 and its fragments, agonists or inhibitors of the activity of Ang-2. Diagnostic tests for Ang-2 include methods using a specific binding agent and a label for detection of Ang-2 in the body fluids or extracts of cells or tissues. The specific binding agents of the present invention can be used with or without modification the modification. In the preferred diagnostic analysis of specific binding agents will be labeled by joining, for example, a label or reporter molecule. There are a large variety of labels and reporter molecules, some of which have already been described here. In particular, this invention is applicable for the diagnosis of diseases the project for a person.

In this area there are various protocols for measuring proteins Ang-2 using polyclonal or monoclonal antibodies specific for the corresponding protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and cell sorting with excitation fluorescence (FACS). Dvuhsvetny immunoassay based on monoclonal antibodies, using monoclonal antibodies reactive with two reiterferien the epitopes on Ang-2 are preferred, but can be used for competitive analysis link. These analyses are described, for example, in Maddox et al., J Exp Med, 158:1211 (1983).

To provide a framework for diagnosis, usually set to normal or standard values for the expression of Ang-2 people. This determination may be accomplished by combining body fluids or extracts of cells from normal subjects, preferably human, with a specific binding agent, for example, antibody to Ang-2, under conditions suitable for the formation of the complex, which is well known in this field. The standard value of the complex formation may be quantified by comparing the binding of the specific binding agents with known amounts of protein Ang-2, as with the control samples and samples with disease entities. Datastandards value, obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by the disease. The deviation between the standard values and the values of these entities assumes the role of Ang-2 in this pathological condition.

For diagnostic applications, in some embodiments, the implementation of specific binding agents usually mark detectable molecules. This detectable part of the molecule can be any molecule that is able to produce, directly or indirectly, the detected signal. For example, the detectable part of the molecule may be a radioisotope, such as3H,14C,32R35S or125I, a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase or horseradish peroxidase (Bayer et al., Meth Enz, 184: 138-163, (1990)).

Disease

This invention provides a specific binding agent that binds to Ang-2, which is applicable for the treatment of diseases and pathological States of the human. Agents that modulate Ang-2 binding activity, or other cellular activity, can be used in combination with other therapeutic agents to enhance their therapeutic action is s or decrease potential side effects of the disease.

In one aspect, the invention provides reagents and methods that are applicable for the treatment of diseases and conditions characterized by unwanted or abnormal levels of activity of Ang-2 in the cell. These diseases include cancer and other hyperproliferative conditions, such as hyperplasia, psoriasis, contact dermatitis, immune disorders and infertility.

The invention also provides methods of treating cancer in an animal, including humans, implying that the animal an effective amount of a specific binding agent that inhibits or reduces the activity of Ang-2. In addition, this invention relates to methods of inhibiting the growth of cancer cells, including the processes of cell proliferation, invasiveness and metastasis, in biological systems. Methods include the use of the compounds of this invention as a growth inhibitor of cancer cells. Preferably, these methods are used for inhibiting or reducing the growth of cancer cells, invasiveness, metastasis, or the occurrence of tumors in living animals, such as mammals. The methods of the present invention is easily adaptable for use in systems analyses, for example, in the analysis of the growth of cancer cells and their properties, as well as to identify which of soedinenii, which affect the growth of cancer cells.

Carcinoma, which magino be treated by the methods of the present invention, preferably are found in mammals. Mammals include, for example, humans and other primates, as well as loved ones or Pets, such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep and cattle.

Tumor or neoplasm include the growth of tissue in which the multiplication of cells is uncontrolled and progressive. In some cases this growth is benign, but in other cases it is malignant and can lead to death of the organism. Malignant neoplasm or carcinoma differ from benign growth that, along with the manifestation of aggressive cell proliferation, they may be embedded in the surrounding tissue and metastasize. In addition, malignant neoplasm are characterized by the fact that they reveal a great loss of differentiation (large dedifferentiate) and their arrangement relative to each other and their surrounding tissues. This property is also called "anaplasia".

Neoplasm, which can be treated according to the invention also include solid tumors, ie, carcinoma and sarcoma. Carcinoma include malignant the e neoplasma, originating from epithelial cells, which infiltrate (invaziruyut) the surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas originating from glandular (glandular) tissue, or carcinomas, which form glandularia patterns. Another broad category of cancers includes sarcomas are tumors whose cells immersed in a fibrillar or homogeneous substance that is similar to the embryonic connective tissue. This invention also allows you to treat cancer of the myeloid or lymphoid systems, including leukemia, lymphoma and other types of cancer that are not normally presented in the form tumor masses, and distributed in the vascular or lymphoreticular systems.

Types of cancer or tumor cells treated in accordance with this invention include, for example, producing ACTH (adrenocorticotropic hormone) tumor, acute lymphocytic leukemia, acute nelimfocitarnyi leukemia, cancer of the adrenal cortex, bladder cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic miliitary leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing sarcoma, gallbladder cancer, cancer of the hairy cells (reticuloendotheliosis), head and neck cancer, Hodgkin's lymphoma, sarcomatosis, kidney cancer, liver cancer, lung cancer (small cell and non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, nahodkinskuju lymphoma, osteosarcoma, ovarian cancer, cancer of the ovary (germ cells), sarcoma, pancreatic cancer, penile cancer, prostate cancer, retinoblastoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, trophoblastic neoplasm, uterine cancer, cancer of the vagina, vulvar cancer, and Wilms tumor.

The invention specifically illustrated here with reference to the treatment of some types of experimentally derived carcinomas. In these illustrative treatments used standard existing in this field models in vitro and in vivo. These methods can be used to identify agents that, according to the expectation, can be effective in treatment programmes in vivo. However, it will be clear that the method according to the invention is not limited to the treatment of these tumor types, and applies to any solid tumor formed from any organ system. Types of cancers, invasiveness and metastasis are associated with expression or activity of Ang-2, is particularly sensitive to inhibition or even inducyruetsa regression with this invention.

The invention may also be practiced switching in combination with the specific binding agent of the present invention, such as an antibody, other anti-cancer chemotherapeutic agent, such as a conventional chemotherapeutic agent. This combination of the specific binding agent with such other agents may potentiate the chemotherapeutic Protocol. Many chemotherapeutic protocols are known to the skilled in this field specialist as protocols that may be included in the method according to the invention. Can be used with any chemotherapeutic agent, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the connection according to the invention can be administered with antibiotics, such as doxorubicin and other analogues of anthracycline, nitrogen mustard analogues, such as cyclophosphamide, pyrimidine analogues such as 5-fluorouracil, cisplatin, hydroxyurea, Taxol and its natural and synthetic derivatives, etc. as another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where these tumors include gonadotropin-dependent and gonadotropin-independent cells, this compound may be injected together with lapro the home or goserelin (synthetic peptide analogues of LH-RH). Other antineoplastic protocols include the use of compounds of tetracycline with another modality, such as surgery, radiation, etc., also referred to here as the "auxiliary anticancer modalities". Thus, the method of the present invention may be used with such conventional schemes with the advantage of reducing side effects and enhancing efficiency.

Thus, this invention provides compositions and methods suitable for treating a variety of cancers, including solid tumors and leukemias. The types of cancers that can be treated include, but are not limited to: adenocarcinoma of the breast, prostate and colon; all forms of bronchogenic carcinoma of the lung; myeloid; melanoma; hepatoma; neuroblastoma; papilloma; apudoma; horitomo; branchiae; malignant carcinoid syndrome; carcinoid heart disease, carcinoma (e.g., Walker, basal cell carcinoma, carcinoma of the brown-Pier, running carcinoma, Ehrlich tumor, carcinoma, Krebs 2, carcinoma from Merkel cells, mulinuu carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, Scirroco (fibrous) carcinoma, bronchiolar, bronchiogenic, ploscockletocny the carcinoma and transitional cell carcinoma; Hodgkin's disease; immunoproliferative small cell lung carcinoma; nahodkinskuju lymphoma; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; a fibroma, fibrosarcoma; brain tumors; histiocytoma; lipoma, liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordomas; craniopharyngioma; dysgerminoma; hamartoma; mesenchymal; mesonephroma; myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; tenotomy; tofollow tumor. In addition, it is possible to treat the following types of cancer: adenoma; cholangioma; cholesteatoma; cylindroma; cystadenocarcinoma; cystadenoma; granulation tumor; androblastoma; hepatoma; hidradenoma; insuloma; swelling of Leydig cells; papilloma; their tumor Sertoli cells; TECOM; leiomyoma; leiomyosarcoma; myoblastoma; fibroids; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglionevroma; glioma; medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma; nephropathia paraganglioma; angiokeratoma; angiolymphoid hyperplasia with eosinophilia; sclerosing angioma; angiomatosis; glamamom; hemangioendothelioma; hemangioma; hemangiopericytoma; gemangiosarkoma; lymphangioma; lymphangioma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; l is stevena tumor; fibrosarcoma; gemangiosarkoma; leiomyosarcoma; leucosarcia; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; neoplasm; neurofibromatosis, and cervical dysplasia.

Another aspect of this invention consists in the application of materials and methods of the present invention to prevent and/or treat any hyperproliferative skin conditions, including psoriasis and contact dermatitis or other hyperproliferative diseases. It has been shown that patients with psoriasis and contact dermatitis had increased activity of Ang-2 in their damage (Ogoshi et al., J. Inv. Dermatol., 110:818-23 (1998)). Preferably, specific binding agents specific against Ang-2 will be used in combination with other pharmaceutical agents for the treatment of people who have these clinical symptoms. Specific binding agents can be delivered using any of a variety of media with techniques described herein, and other methods that are well known qualified in this area specialists.

Other aspects of this invention include the treatment of various retinopathy (including diabetic retinopathy and age-related macular degeneration), involving angiogenesis, and naru is events/diseases of the female genital tract, such as endometriosis, uterine fibroids and other conditions associated with impaired function of the proliferation of blood vessels (including endometrial microvascular growth) during the female reproductive cycle.

Another aspect of this invention relates to the treatment of abnormal growth of blood vessels, including cerebral arterio-venous malformation (AVM), the damage and the repair of gastrointestinal mucosa, ulceration of the gastroduodenal mucosa of patients with a medical history with peptic ulcers, including ischemia, originating from a stroke, a wide range of vascular disorders in liver disease and portal hypertension in patients with vnepechenochnoe portal hypertension.

Another aspect of this invention is the prevention of cancer, using the compositions and methods provided by the present invention. Such reagents will include specific binding agents against Ang-2.

The pharmaceutical composition

Pharmaceutical compositions Ang-2-specific binding agents are within the scope of this invention. Pharmaceutical compositions containing the antibodies, are described in detail, for example, in U.S. Patent 6171586 issued by Lam et al., January 9, 2001. Such compositions contain a therapeutically Il is prophylactically effective amount of a specific binding agent, such as an antibody or fragment, variant, derivative or fusion described here, in a mixture with a pharmaceutically acceptable agent. In a preferred embodiment, the pharmaceutical compositions contain antagonistic specific binding agents that modulate partially or completely at least one biological activity of Ang-2, in a mixture with a pharmaceutically acceptable agent. Usually, these specific binding agents will be sufficiently purified for administration to an animal.

These pharmaceutical compositions may contain needed to prepare materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or penetration of the composition. Suitable materials for the preparation of compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or hydrosulfite sodium); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate buffer) with other organic acids); bulk agents (such as mannitol or glycine), hepatoblastoma Agay is you (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring agents; flavoring agents and thinners; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; soleobrazutaya counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, finitely alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); polyalcohols, xylytol (such as mannitol or sorbitol); suspendresume agents; surfactants or wetting agents (such as pluronic, PEG, esters sorbitan, Polysorbate, such as Polysorbate 20, Polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapol); reinforcing the stability of the agents (sucrose or sorbitol); amplifying toychest agents (such as halides of alkali metals, preferably sodium chloride or potassium, mannitol sorbitol); delivering vehicles; diluents; excipients and/or pharmaceutical preparations is practical adjuvants. (Remington''s Pharmaceutical Sciences, 18thEdition, A.R. Gennaro, ed., Mack Publishing Company, 1990).

The optimal pharmaceutical composition will be determined by the skilled in this field specialist depending on, for example, from the intended method of administration, delivery format, and the desired dose. See, for example, Remington''s Pharmaceutical Sciences, supra. Such compositions may influence the physical state, stability, rate of release in vivo and speed of clearance in vivo specific binding agent.

Primary filler or carrier in the pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable filler or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Additional examples of fillers are neutral buffered saline or saline mixed with serum albumin. Other exemplary pharmaceutical compositions containing Tris buffer of about pH 7.0 to 8.5, or acetate buffer of about 4,0-5,5, which may further include sorbitol or a suitable substitute. In one embodiment of the present invention, the composition of the binding agent can be when is otopleni for storage by mixing the selected composition, having the desired degree of purity, with an optimal agents for preparation of compositions (Remington's Pharmaceutical Sciences, above) in the form of dried filter cake or an aqueous solution. In addition, the product of a binding agent can be prepared in the form of freeze-dried using appropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery. Alternatively, these compositions can be selected for inhalation or for enteric delivery, such as oral, aural, ocular, rectal or vaginal delivery. The preparation of such pharmaceutically acceptable compositions is within the skill in this field.

The components of the composition are present in concentrations that are acceptable for site-specific introduction. For example, buffers are used to maintain the composition at physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

When considering injecting therapeutic compositions for use in this invention can be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired specific binding agent in a pharmaceutically acceptable carrier. A particularly suitable filler for couples who naturalnego injection solution is sterile distilled water, in which the binding agent is prepared as a sterile, isotonic solution, properly preserved. In another preparation can be part of the finished form of the desired molecule with an agent, such as injectable microspheres, bio-degradable particles, polymeric compounds (polylactic acid, polyglycolic acid), beads or liposomes, that provides for the controlled or extended release of the product, which can then be delivered via a depot injection. Can also be used hyaluronic acid, and this can lead to the strengthening of the reception duration in the circulation. Other suitable means for introducing the desired molecules include implantable devices for drug delivery.

In another aspect, pharmaceutical compositions suitable for parenteral delivery, can be prepared in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In addition, suspensions of the active compounds can be prepared in the form of matching is alanah injectable suspensions. Suitable lipophilic solvents or fillers include fatty oils (oils with a high content of fat, such as sesame oil, or synthetic fatty acid esters, such as etiloleat, triglycerides, or liposomes. For delivery can also be used non-lipid poly-aminopolymers. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of these compounds and create opportunities to prepare highly concentrated solutions.

In another embodiment, the pharmaceutical composition may be prepared for inhalation. For example, the binding agent can be prepared in the form of a dry powder for inhalation. Solutions for inhalation polypeptide or nucleic acid can also be prepared with a propellant for aerosol delivery. In another embodiment, the solutions can be sprayed. In addition, pulmonary introduction described in PCT Application number PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.

It is also assumed that certain forms that can be administered orally. In one embodiment of the present invention, molecules of a binding agent, which is injected thus, can be prepared with fillers or b is C fillers, usually used in the compounding of solid dosage forms such as tablets and capsules. For example, can be made capsule for release of the active part of the composition in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. May include additional agents to facilitate absorption of the molecules of a binding agent. Can also be used diluents, flavouring agents, nikopensius waxes, vegetable oils, lubricating agents, suspendresume agents, dezintegriruetsja tablet agents and binders.

Pharmaceutical compositions for oral administration can be prepared using pharmaceutically acceptable carriers well known in this field, in dosages suitable for oral administration. Such carriers enable you to prepare pharmaceutical compositions in the form of tablets, pills, coated tablets, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients and processing the mixture of granules (optional after grinding) to obtain the core parts that the notches or pills. If desirable, may be added suitable excipients. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hypromellose or sodium carboxymethylcellulose; gums, including Arabian and tragacanth; and proteins such as gelatin and collagen. If desirable, can be added dezintegriruetsja or solubilizing agents, such as crosslinked polyvinylpyrrolidone, agar or alginic acid or its salt, such as sodium alginate.

The core area of the bean can be used together with suitable coatings, such as concentrated solutions of sugars, which may also contain the Arabian gum, talc, polyvinylpyrrolidone, carboloy gel, polyethylene glycol and/or titanium dioxide, solutions varnishes and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the coating tablets and dragee to identify the product or to characterize the quantity of active compound, i.e. the doses.

Pharmaceutical preparations which can be used orally, can also enable "push-fit"capsules (with thick posadka the halves of the capsules), made of gelatin, and also soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient mixed with fillers or binding agents such as lactose or starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid or liquid polyethylene glycol with or without stabilizers stabilizers.

Another pharmaceutical composition may involve an effective quantity of a binding agent in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. The dissolution of these tablets in sterile water or other suitable filler can be prepared solutions in a uniform dosage form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate; or binding agents such as starch, gelatin or the Arabian gum; or lubricating agents such as magnesium stearate, stearic acid or talc.

Additional pharmaceutical compositions will be evident, qualific is included in this area specialists including compositions comprising molecules of a binding agent in the composition with delayed or controlled delivery. Methods of making various other means of prolonged or controlled delivery, such as liposomal carriers, biologically degradiruete microparticles or porous granules and injection of substances delayed binding, also known qualified in this area specialists. See, for example, PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Additional examples of drugs slow release include semi-permeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. The slow release matrices may include polyesters, hydrogels, polylactide (US 3773919, EP 58481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Diopolymers, 22:547-556 (1983), poly(2-hydroxyethylmethacrylate) (Langer et al.,J Biomed Mater Res, 15:167-277, (1981)) and (Langer et al., Chem Tech, 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., above) or poly-D(-)-3-hydroxybutiric acid (EP 133988).

Composition of prolonged action also include liposomes, which can be manufactured in any of several ways known in this field. See, for example, Eppstein et al., Proc Natl Acad Sci (USA), 82:3688-392 (1985); EP 36676; EP 88046; EP 143949.

Pharmaceutical composition for use in the introduction in vivo typically must be sterile. This can be done by filtration through a sterile membrane filter. If the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and recreation. Compositions for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions generally are placed into a container having a sterile access hole, for example, a bag of IV solution or vial having a stopper, sharp needle for subcutaneous injection.

After preparation of pharmaceutical compositions, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or digidratirovannogo or lyophilized powder. Such preparations can be stored either in a ready to use form or in the form (e.g., lyophilized), should be recreated before the introduction.

In a specific embodiment, the present invention relates to kits for receiving units of a single dose injection. Each of these sets contains both a first container having a dried protein and a second container having an aqueous composition. In the volume of the m of this invention also includes kits, containing single - and multi-chamber pre-filled syringes (e.g., syringes for liquids and ListPrice).

An effective amount of the pharmaceutical composition to be used therapeutically will depend, for example, from therapeutic context and therapeutic purposes. Thus, qualified in this field specialist will be clear that suitable to ensure the dose levels will depend, in part, from the delivered molecules, indications for which the use of this molecule linking agent, the route of administration and the size (body weight, body surface or body size) and condition (the age and General health) of the patient. Thus, the Clinician may change the dosage and modify the route of administration to obtain the optimal therapeutic effect. The usual dose may be in the range from about 0.1 mg/kg to about 100 mg/kg or more, depending on the above factors. In other embodiments, implementation, this dose may be in the range from about 0.1 mg/kg to about 100 mg/kg or 1 mg/kg to about 100 mg/kg or 5 mg/kg to about 100 mg/kg

For any compound, therapeutically effective dose can be approximately estimated initially either in tests on cell cultures or in animal models, so the x as a mouse, rats, rabbits, dogs, or pigs. The animal model can also be used to determine the appropriate concentration range and route of administration. Then this information is used to determine the applicable doses and routes of administration in humans.

The exact dose is determined in light of factors related to the subject requiring treatment. The dose and the introduction adjusted to provide sufficient levels of the active compounds or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, General health of the subject, age, weight and sex of the subject, time and frequency of administration, combination (combination) of drugs, the sensitivity of the reaction and the response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week or every two weeks depending on half-life existence and rate of clearance of a particular composition.

Frequency of doses will depend upon the pharmacokinetic parameters of a molecule of a binding agent used in the composition. Typically, the composition is administered until dose is achieved, which gives the desired effect. Thus, this composition can be entered as a single dose or as multiple doses (at the same or at different concentrations) on length and time or as a continuous infusion. Additional improvement in appropriate doses perform routine way. Suitable doses can be obtained using the obtained data, the dose-response.

Route of administration of the pharmaceutical composition is consistent with the known methods, for example, orally, through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intra-arterial, intraportal ways, the introduction of damage, intramedullary, intrathecal (vnutriobolochechnoe), intraventricular, transdermal, intraperitoneal, intranasal, enteral, local, sublingual, urethral, vaginal or rectal way through systems of prolonged action or implantable devices. If desired, these compositions may be injected bolus injection or continuously by infusion or by an implanted device.

Alternative or additionally, the composition may be administered locally implantation of a membrane, sponge or other suitable material, which has been absorbed or encapsulated desired molecule. When using the implantable device this device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can done is take place by diffusion, bolus timing of the release or continuous introduction.

In some cases it may be desirable to use a pharmaceutical composition ex vivo. In such instances, cells, tissues or organs that have been removed from the patient, is subjected to the action of these pharmaceutical compositions after which the cells, tissues and/or organs are then implanted back into the patient.

In other cases, binding agent, which is a polypeptide can be delivered by implanting certain cells that have been genetically engineered, using methods such as the methods described herein for expression and secretion of the polypeptide. These cells may originate from animals or humans and can be autologous, heterologous or xenogeneic. Optionally, these cells can be immortality. To reduce the chance of an immunological reaction, these cells may be encapsulated to avoid infiltration of the surrounding tissues. The encapsulated materials are typically biocompatible, semipermeable polymeric enclosures or membranes that allow the release of the protein product (protein products), but prevent the destruction of these cells by the human immune system or by other detrimental factors from the surrounding tissue.

Combined Arabia

The specific binding agents of the present invention can be used in combination with other therapeutic tool in the treatment of Ang-2-pathologies. These other therapeutic agents include, for example, radiation treatment, chemotherapy and targeted therapy described here below. Additional combination therapy, not specifically listed here are also in the scope of this invention.

Thus, the invention includes the introduction of one or more specific binding agents of the present invention, administered to the same patient in combination with one or more additional suitable agents, each of which is administered in accordance with the scheme suitable for this medication. This introduction includes the joint introduction of a specific binding agent of the present invention and one or more suitable agents. In this context, the terms "added together" and "co-administration" includes essentially simultaneous introduction of one or more specific binding agents in accordance with this invention and one or more additional suitable agents.

In this context, the term "incompatible" introduction includes the introduction of one or more specific binding agents in accordance with the tvii with this invention and one or more additional suitable agents at different time points, in any order, regardless of whether they overlap or not. This includes, but is not limited to, sequential treatment (for example, pre-treatment before the introduction of the main component, the introduction after the main component or overlapping treatment) components of this combination and designs in which these medicines are interleaved, or introduction one long-term component and another component periodically. The components can be administered in the same composition or in separate compositions and the same or different routes of administration.

In some embodiments, implementation, combination therapy includes a specific binding agent capable of binding Ang-2, in combination with at least one additional antiangiogenic agent. These agents include, but are not limited to, in vitro synthetic chemical compositions, antibodies, antigennegative areas, radionuclides and their combinations and conjugates. In some embodiments, the implementation, the agent can act as an agonist, antagonist, allosteric modulator, or toxin. In some embodiments, the implementation, the agent can act as an inhibitor or stimulator of its target (e.g., activation or inhibition of the receptor or enzyme) and thereby facilitating the b cell death or growth arrest of cells.

Chemotherapy may be used anticancer agents, for example, alkylating agents including: nitrogen mustard analogues, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoanatabine, such as carmustine (BCNU), lomustin (CCNU) and semustine (methyl-CCNU); ethylenimines/methylmelamine, such as triethylenemelamine (THE), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (MMO altretamin); alkyl sulphonates such as busulfan; triazine, such as dacarbazine (DTIC); antimetabolites including analogs of folic acid, such as methotrexate and trimetrexate, pyrimidine analogues such as 5-fluorouracil, ftordezoksiuridin, gemcitabine, citizenoriented (AraC, cytarabine), 5-azacytidine, 2,2'-diverticulitis, purine analogues such as 6-mercaptopurine, 6-tioguanin, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydrobupropion (EHNA), fludarabine phosphate, and 2-chloromethoxypropyl (cladribine, 2-CdA); natural products including antimitoticescoy drugs, such as paclitaxel, vinylchloride, including vinblastine (VLB), vincristine and vinorelbine, Taxotere, estramustine and estramustine phosphate; epipodophyllotoxins, such as etoposide and teniposide; antibiotics such as actinomycin D, daunomycin (ripidolite), doxorubicin, mitoxantrone, idarubitsin, b is omicini, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; a variety of agents, including coordination complexes of platinum, such as cisplatin and carboplatin, anthracenediones, such as mitoxantrone, substituted urea such as hydroxyurea, derivatives methylhydrazine, including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressors, such as mitotane (o,p'-DDD) and aminoglutetimid; hormones and antagonists, including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutetimid; progestin, such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen, such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens, including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, analogues gonadotropinreleasing hormone and leuprolide; and non-steroidal antiandrogens such as flutamide.

Anti-cancer therapeutic agents that can be entered with Ang-2-specific binding agent include, but are not limited to, targeted therapeutic agent described herein. Examples aimed Ter is non-therapeutic means include, but not limited to, the use of therapeutic antibodies. Examples of therapeutic antibodies include, but are not limited to, mouse, mouse-human-chimeric, CDR-transplanted, humanized and fully human antibodies, synthetic antibodies, including, but not limited to, antibodies, selected by screening libraries of antibodies. Exemplary antibodies include, but are not limited to, antibodies that bind to proteins on the cell surface Her2, CDC20, CDC33, mucin-like glycoprotein and the receptor for epidermal growth factor (EGFR)present on tumor cells and not necessarily inducing cytostatic and/or cytotoxic effect on tumor cells that detect these proteins on their surface. Examples of antibodies include HERCEPTINE™ (trastuzumab), which can be used to treat breast cancer and other forms of cancer, and RITUXAN™ (rituximab), ZEVALIN™ (ibritumomab tiuxetan), GLEEVEC™ and LYMPHOCIDE™ (epratuzumab), which can be used to treat non-Hodgkin's lymphoma and other cancers. Some examples of antibodies include ERBITUX™ (IMC-C225); IRESSA™ (martinoli); BEXXAR™ (iodine-131-tositumomab); inhibitors of KDR (receptor kinase domain); anti-VEGF antibodies and antagonists (e.g., AVASTIN™ and VEGAF-TRAP); anti-VEGF receptor antibodies and antigennegative areas; anti-Ang-1-antic the La and antigennegative areas; antibodies against Tie-2 and other Ang-1 and Ang-2 receptors; Tie-2 ligands; antibodies against inhibitors of Tie-2 kinase and Campath® (alemtuzumab). In some embodiments, implementation, agents anticancer therapy are polypeptides that selectively induce apoptosis in tumor cells, including, but not only TNF-related polypeptides, such as TRAIL (apoptosis-inducing ligand TNF-receptor).

In some embodiments, implementation, use agents cancer therapy known that they are antiangiogenic. Some such agents include, but are not limited to, IL-8, Campath™, B-FGF; FGF antagonists; antagonists Tek (Ceretti et al., Publication of the U.S. No. 2003/0162712; Ceretti et al., U.S. patent No. 6413932 and Ceretti et al., U.S. patent No. 6521424, each of which is incorporated herein by reference for any purpose); anti-TWEAK-agents (which include, but are not limited to, antibodies and antigennegative areas); antagonists of soluble TWEAK receptor (Wiley, U.S. Patent No. 6727225); disintegrin ADAM (or their domains to counteract binding of integrin to its ligands (Fanslow et al., Publication of the U.S. No. 2002/0042368); anti-eph-receptor - and anti-ephrin-antibodies; antigennegative areas or antagonists (U.S. Patent№№ 5981245, 5728813, 5969110, 6596852, 6232447, 6057124 and family members of patent families); anti-VEGF agents, such as described herein (e.g., antibodies or antihistamie of t is s, which specifically bind VEGF, or soluble VEGF receptors or their landscapemode areas)such as AVASTIN™ or VEGF-TRAP™ and anti-VEGF receptor agents (e.g., antibodies or antihistamie areas that are specifically associated with them), inhibiting EGFR agents (e.g. antibodies or antihistamie areas that are specifically associated with them), IRESSA™ (gefitinib), TARCEVA™ (erlotinib), anti-Ang-1 and anti-Ang-2 agents (e.g. antibodies or antihistamie areas specifically communicate with them or their receptors, for example, Tie-2/TEK) and inhibiting anti-Tie-2-kinase agents (e.g. antibodies or antihistamie areas that specifically bind and inhibit the activity of growth factors, such as antagonists of growth factor hepatocyte (HGF, also known as Scatter Factor), and antibodies or antihistamie areas that specifically bind its receptor c-met”; anti-PDGF-BB-antagonists, antibody and antigennegative areas against PDGF-BB-ligands; and inhibitors DERIVED-kinase.

In some embodiments, implementing, cancer therapy agents are inhibitors of angiogenesis. Some such inhibitors include, but are not limited to, SD-7784 (Pfizer, USA); cilengitide (Merck KGaA, Germany, EPO 770622); pegaptanib of Octanate (Gilead Sciences, USA); AlphaStation (BioActa, UK); M-PGA (Celgene, USA, US 5712291); ilomastat (Arriva, USA, US 5892112); semakan the b (Pfizer, USA, US 5792783); vatalanib (Novartis, Switzerland); 2-methoxyestradiol (EntreMed, USA); TLC ELL-12 (Elan, Ireland); anecortave acetate (Alcon, USA); alpha-D148 Mab (Amgen, USA); CEP-7055 (Cephalon, USA); anti-Vn Mab (Crucell, Netherlands) DAC:antiangiogenic (ConjuChem, Canada); Angiocidin (InKine Pharmaceutical, USA); KM-2550 (Kyowa Hakko, Japan); SU-0879 (Pfizer(USA); CGP-79787 (Novartis, Switzerland, EP 970070); ARGENT technology (Ariad, USA); YIGSR-Stealth (Johnson & Johnson, USA); fragment of fibrinogen-E, (BioActa, UK); angiogenesis inhibitor, (Trigen, UK); TBC-1635, (Encysive Pharmaceuticals, USA); SC-236, (Pfizer, USA); ABT-567, (Abbott, USA); Metastatic, (EntreMed, USA); inhibitor angiogenesis, (Tripep, Sweden); maspin, (Sosei, Japan); 2-methoxyestradiol, (Oncology Sciences Corporation, USA); ER-68203-00, (IVAX, USA); Benefin, (Lane Labs, USA); Tz 93, (Tsumura, Japan); TAN-1120, (Takeda, Japan); FR-111142, (Fujisawa, Japan, JP 02233610); platelet factor 4, (RepliGen, USA, EP 407122); antagonist of vascular endothelial growth factor, (Borean, Denmark); anti-cancer therapeutic agent, (University of South Carolina, USA); bevacizumab (pINN), (Genentech, USA); angiogenesis inhibitors, (SUGEN, USA); XL 784, (Exelixis, USA); XL 647, (Exelixis, USA); MAb, alfaretta integrin, second generation, (Applied Molecular Evolution, USA and MedImmune, USA); gene therapy agent, retinopathy, (Oxford BioMedica, UK); enzastaurin hydrochloride (USAN), (Lilly, USA); CEP 7055, (Cephalon, USA and Sanofi-Synthelabo, France); BC 1, (Genoa Institute of Cancer Research, Italy); angiogenesis inhibitor, (Alchemia, Australia); VEGF antagonist, (Regeneron, USA); obtained from rBPI 21 and BPI-angiogenic agent, (XOMA, USA); PI 88, (Progen, Australia); cilengitide (pINN), (Merck KGaA; Munich Technical University, Germany, Scripps Clinic and Research Foundation, USA); cetuximab (INN), (Aventis, Frane); AVE 8062, (Ajinomoto, Japan); AS 1404, (Cancer Research Laboratory, New Zealand); SG 292 (Telios, USA); endostatin, (Boston Childrens Hospital, USA); ATN 161, (Attenuon, USA); ANGIOSTATIN, (Boston Childrens Hospital, USA); 2-methoxyestradiol, (Boston Childrens Hospital, USA); ZD 6474, (AstraZeneca, UK); ZD 6126, (Angiogene Pharmaceuticals, UK); PPI 2458, (Praecis, USA); AZD 9935, (AstraZeneca, UK); AZD 2171, (AstraZeneca, UK); vatalanib (pINN), (Novartis, Switzerland and Schering AG, Germany); inhibitors of the tissue factor pathway, (EntreMed, USA); pegaptanib (Pinn), (Gilead Sciences, USA); xanthorriza, (Yonsei University, South Korea); vaccine, on the basis of the gene, VEGF-2, (Scripps Clinic and Research Foundation, USA); SPV5.2, (Supratek, Canada); SDX 103, (University of California at San Diego, USA); PX 478, (ProlX, USA); METASTATIC, (EntreMed, USA); troponin I, (Harvard University, USA); SU 6668, (SUGEN, USA); OXI 4503, (OXiGENE, USA); o-guanidine, (Dimensional Pharmaceuticals, USA); motupore C, (British Columbia University, Canada); CDP 791, (Celltech Group, UK); atiprimod (pINN), (GlaxoSmithKline, UK); E 7820, (Eisai, Japan); CYC 381, (Harvard University, USA); AE 941, (Aeterna, Canada); vaccine, angiogenesis, (EntreMed, USA); inhibitor of plasminogen activator urokinase type, (Dendreon, USA); oglufanide (pINN), (Melmotte, USA); inhibitors of HIF-1alfa, (Xenova, UK); CEP 5214, (Cephalon, USA); BAY RES 2622, (Bayer, Germany); Angiocidin, (InKine, USA); A6, (Angstrom, USA); KR 31372, (Korea Research Institute of Chemical Technology, South Korea); GW-2286, (GlaxoSmithKline, UK); EHT 0101, (ExonHit, France); CP 868596, (Pfizer, USA); CP 564959, (OSI, USA); CP 547632, (Pfizer, USA); 786034, (GlaxoSmithKline, UK); KRN 633, (Kirin Brewery, Japan); the delivery system pharmaceuticals, intraocular, 2-methoxyestradiol, (EntreMed, USA); arinex, (Maastricht University, Netherlands, and Minnesota University, USA); ABT 510, (Abbott, USA); AAL-993, (Novartis, Switzerland); VEGI, (ProteomTech, USA); inhibitors of factor nikr is for tumors alpha, (National Institute on Aging, USA); SU 11248, (Pfizer, USA and SUGEN, USA); ABT 518, (Abbott, USA); YH16, (Yantai Rongchang, China); S-3APG, (Boston Childrens Hospital, USA and EntreMed, USA); MAb, KDR, (ImClone Systems, USA); MAb, Alfa beta, (Protein Design, USA); KDR kinase inhibitor, (Celltech Group, UK, and Johnson & Johnson, USA); GFB 116, (South Florida University, USA and Yale University, USA); CS 706, (Sankyo, Japan); a prodrug combretastatin A4, (Arizona State University, USA); chondroitinase AC, (IBEX, Canada); BAY RES 2690, (Bayer, Germany); AGM 1470, (Harvard University, USA, Takeda, Japan, and TAP, USA); AG 13925, (Agouron, USA); tetrathiomolybdate, (University of Michigan, USA); GCS 100, (Wayne State University, USA) CV 247, (Ivy Medical, UK); CKD 732, (Chong Kun Dang, South Korea); MAb, vascular endothelial 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); anti-cancer therapeutic agent, (Marinova, Australia); inhibitors of heparanase, (InSight, Israel); KL 3106, (Kolon, South Korea); Honokiol, (Emory University, 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); antagonists of VE-cadherin-2, (ImClone Systems, USA); Vasostatin, (National Institute of Health, USA); vaccine, Flk-1, (ImClone Systems, USA); TZ 93, (Tsumura, Japan); TumStatin, (Beth Israel Hospital, USA); truncated soluble FLT 1 (receptor 1 vascular endothelial growth factor), (Merck & Co, USA); Tie-2 ligands, (Regeneron, USA); inhibitor of thrombospondin 1, (Allegheny Health, Education and Research Foundation, USA); 4-(5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazole-1-yl)-2-benzosulfimide; Arriva; and C-Met. AVE 8062 (monohydrochloride (2S)-2-Amin is-3-hydroxy-N-[2-methoxy-5-[(1Z)-2-(3,4,5-trimethoxyphenyl)ethynyl]phenyl]propanamide); materialab (pINN) (immunoglobulin G4, anti-(transforming growth factor beta 1 person (gamma-chain monoclonal SAT 192 people)), disulfide with dimer Kappa-chain monoclonal SAT 192 people); Flt3 ligand; CD40 ligand; interleukin-2; interleukin-12; the ligand of 4-1BB; anti-4-1BB antibody; TNF-antagonists and antagonists of TNF-receptor including TNFR/Fc, TWEAK antagonist and TWEAK-R antagonists including TWEAK-R/Fc; TRAIL; VEGF-antagonists, including anti-VEGF antibodies, antagonists of VEGF-receptor (including VEGF-R1 and VEGF-R2, also known as Flt1 and Flk1 or KDR); antagonists of CD148 (also known as DEP-1, ECRTP, and PTPRJ, see Takahashi et al., J. Am. Soc. Nephrol. 10: 2135-45 (1999), hereby incorporated here by reference for any purpose); inhibitor of thrombospondin 1 and inhibitors of one or both of the Tie-2 or ligands of Tie-2 (such as Ang-2). A number of inhibitors of Ang-2 is known in this area, including some anti-Ang-2 antibodies described in published U.S. Patent No. 20030124129 (corresponding to the PCT Application No. WO03/030833), and U.S. Patent No. 6166185, the contents of which is incorporated herein by reference in full.

Additionally, Ang-2 peptide antibodies are also known in this field can be found, for example, in published patent Zaelke U.S. No. 20030229023 (corresponding to the Application number WO 03/057134) and opublikovannoi patent Zaelke U.S. No. 20030236193, the contents of which are included here as with ylki in full.

Some agents cancer therapies include, but are not limited to: thalidomide and thalidomide analogues (N-(2,6-dioxo-3-piperidyl)phthalamide); tecogen-sodium (sulfated polysaccharide peptidoglycan); TAN 1120 (8-acetyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-10-[[octahydro-5-hydroxy-2-(2-hydroxypropyl)-4,10-dimethylthieno[3,4-d]-1,3,6-doxazosin-8-yl]oxy]-5,12-naphthacenedione); coralista (Terentieva salt 7,7'-[carbonylbis[imino(1-methyl-1H-pyrrol-4,2-diyl)carbylamine(1-methyl-1H-pyrrol-4,2-diyl)carbylamine]]bis-1,3-naphthalenedisulfonic acid); SU 302; SU 301; SU 1498 ((E)-2-cyano-3-[4-hydroxy-3,5-bis(1-methylethyl)phenyl]-N-(3-phenylpropyl)-2-propenamide); SU 1433 (4-(6,7-dimethyl-2-honokalani)-1,2-benzodia); ST 1514; SR 25989; soluble Tie-2; derivatives SERM, Pharmos; semaxanib (pINN)(3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one); 836 S; RG 8803; RESTIN; R 440 (3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-1H-pyrrole-2,5-dione); R 123942 (1-[6-(1,2,4-thiadiazole-5-yl)-3-pyridazinyl]-N-[3-(trifluoromethyl)phenyl]-4-piperidine); inhibitor of prolylhydroxylase; progression elevated genes; prinomastat (INN) ((S)-2,2-dimethyl-4-[[n-(4-pyridyloxy)phenyl]sulfonyl]-3-tiokarbaminianowe acid); NV 1030; NM 3 (8-hydroxy-6-methoxy-alpha-methyl-1-oxo-1H-2-benzopyran-3-acetic acid); NF 681; NF 050; MIG; METH 2; METH 1; mansanti B (alpha-[1-[4-[5-[4-[2-(3,4-acid)-2-hydroxy-1-methylethoxy]-3-methoxyphenyl]tetrahydro-3,4-dimethy the-2-furanyl]-2-methoxyphenoxy]ethyl]-1,3-benzodioxol-5-methanol); monoclonal antibody against KDR; monoclonal antibody against integrin alfaretta; LY 290293 (2-amino-4-(3-pyridinyl)-4H-oil[1,2-b]Piran-3-carbonitrile); KP 0201448; KM 2550; integrin-specific peptides; INGN 401; GYKI 66475; GYKI 66462; Grinstein (101-354-plasminogen (person)); a gene therapy agent for rheumatoid arthritis, prostate cancer, ovarian cancer, gliomas, endostatin, colorectal cancer, ATF BTPI, genes antiangiogenesis, an inhibitor of angiogenesis or angiogenesis; inhibitor gelatinase, FR 111142 (5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2.5]Oct-6-silt ester of 4,5-dihydroxy-2-hexenoic acid); forenames (pINN) (S)-alpha-amino-3-hydroxy-4-(hydroxymethyl)venzolasca acid); antagonist fibronectin (1-acetyl-L-prolyl-L-histidyl-L-seryl-L-cysteinyl-L-aspartate); inhibitor of receptor fibroblast growth factor; an antagonist of fibroblast growth factor FCE 27164 (hexanetriol salt 7,7'-[carbonylbis[imino(1-methyl-1H-pyrrol-4,2-diyl)carbylamine(1-methyl-1H-pyrrol-4,2-diyl)carbylamine]]bis-1,3,5-naphthalenesulfonic acid); FCE 26752 (8,8'-[carbonylbis[imino(1-methyl-1H-pyrrol-4,2-diyl)carbylamine(1-methyl-1H-pyrrole-4,2-diyl)carbylamine]]bis-1,3,6-naphthalenesulfonic acid); endothelial activating monocytes polypeptide II; antisense oligonucleotide VEGFR; angiogenic and trophic factors; angiostatin agent NCHOR; endostatin; angiogenic protein Del-1; CT 3577; contortrostatin; CM 101; chondroitinase AC; CDP 845; Canstatin; BST 2002; BST 2001; BLS 0597; BIBF 1000; ARRESTIN; pomegran (collagen XV 1304-1388-type (predecessor of alpha 1-chain gene in human COL15A1)); angiogenin; aaATIII; 36; 9-alpha-formatexception acetate ((6-alpha)-17-(acetyloxy)-9-fluoro-6-methyl-pregn-4-ene-3,20-dione); 2-methyl-2-phthalimidopropyl acid (2-(1,3-dihydro-1-oxo-2H-isoindole-2-yl)-2-methylpentanediol acid); labeled with yttrium-90 monoclonal antibody BC-1; Semiaxis (3-(4,5-dimethylpyrrole-2-ylmethylene)indolin-2-one) (C15 H14 N2 O); PI 88 (sulfate postmenopause); Alvocidib (2-(2-chlorophenyl)-5,7-dihydroxy-8-(3-hydroxy-1-methyl-4-piperidinyl)-CIS-(-)-4H-1-benzopyran-4-one) (C21 H20 Cl N O5); E 7820; SU 11248 (2-diethylaminoethyl)amide) 5-[3-fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (C22 H27 F N4 O2); Squalamine (Cholestan-7,24-diol, 3-[[3-[(4-aminobutyl)aminopropyl]amino]-, 24-(hydrosulfate), (beta.,Alpha.,Alpha.)-) (C34 H65 N3 O5 S); Eriochrome Black T; AGM 1470 (Karamanova acid, (chloroacetyl)-, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]Oct-6-silt ester, [3R-[3-alpha, 4-alpha(2R, 3R), 5-beta, 6-beta]]) (C19 H28 Cl N O6); AZD 9935; BIBF 1000; AZD 2171; ABT 828; KS-interleukin-2; Uteroglobin; A 6; NSC 639366 (1-[3-(diethylamino)-2-hydroxypropylamino]-4-(oxiran-2-ylmethylamino)anthraquinone fumarate) (C24 H29 N3 O4. C4 H4 O4); ISV 616; anti-ED-B-fused proteins; HUI 77; Troponin I; monoclonal the second antibody BC-1; SPV 5.2; ER 68203; CKD 731 (3R,4S,5S,6R)-4-[2(R)-methyl-3(R)-3(R)-(3-methyl-2-butenyl)oxiran-2-yl]-5-methoxy-1-oxaspiro[2.5]Oct-6-silt ester 3-(3,4,5-trimethoxyphenyl)-2(E)-propanolol acid) (C28 H38 O8); IMC-1C11; aaATIII; SC 7; CM 101; Angical; Kringle 5; CKD 732 (3-[4-[2-(dimethylamino)ethoxy]phenyl]-2(E)-Papanova acid)(C29 H41 N O6); U 995; Canstatin; 885 SQ; CT 2584 (1-[11-(dodecylamino)-10-hydroxyphenyl]was 3.7-dimethylxanthine)(C30 H55 N5 O3); Salasin; EMAP II; TX 1920 (1-(4-methylpiperazine)-2-(2-nitro-1H-1-imidazolyl)-1-Etalon) (C10 H15 N5 O3); inhibitor of alpha-v beta-x; CHIR 11509 (N-(1-PROPYNYL)glycyl-[N-(2-naphthyl)]glycyl-[N-(carbamoylmethyl)]glycine bis(4-methoxyphenyl)methylamide)(C36 H37 N5 O6); BST 2002; BST 2001; B 0829; FR 111142; (3R,4S,5S,6R)-4-[1(R),2(R)-epoxy-1,5-dimethyl-4-hexenyl]-5-methoxy-1-oxaspiro[2.5]octane-6-silt ester of 4,5-dihydroxy-2(E)-hexenoic acid (C22 H34 O7); and inhibitors of kinases, including, but not limited to N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-1-phthalazinone; 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-ocso-3H-indol-3-ilidene)methyl]-2,4-dimethyl-1H-pyrrol-3-carboxamide; 3-[(4-bromo-2,6-differenl)methoxy]-5-[[[[4-(1-pyrrolidinyl)butyl]amino]carbonyl]amino]-4-isothiazolinone; N-(4-bromo-2-forfinal)-6-methoxy-7-[(1-methyl-4-piperidinyl)methoxy]-4-hinazolinam 3-[5,6,7,13-tetrahydro-9-[(1-methylethoxy)methyl]-5-oxo-12H-indeno[2,1-a]pyrrolo[3,4-c]carbazole-12-yl]propyl ester N,N-dimethylglycine, N-[5-[[[5-(1,1-dim is teletel)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxylic; N-[3-chloro-4-[(3-forfinal)methoxy]phenyl]-6-[5-[[[2-(methylsulphonyl)ethyl]amino]methyl]-2-furanyl]-4-hinazolinam; 4-[(4-methyl-1-piperazinil)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamide;N-(3-chloro-4-forfinal)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-hinahayaan;N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-hinazolinam; N-(3-((((2R)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-2-((3-(1,3-oxazol-5-yl)phenyl)amino)-3-pyridinecarboxamide; 2-(((4-forfinal)methyl)amino)-N-(3-((((2R)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-3-pyridinecarboxamide; N-[3-(azetidin-3-ylethoxy)-5-triptoreline]-2-(4-forbindelsen)nicotinamide; 6-fluoro-N-(4-(1-methylethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide; 2-((4-pyridinylmethyl)amino)-N-(3-(((2S)-2-pyrrolidinyl)oxy)-5-(trifluoromethyl)phenyl)-3-pyridinecarboxamide; N-(3-(1,1-dimethylethyl)-1H-pyrazole-5-yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide; N-(3,3-dimethyl-2,3-dihydro-1-benzofuran-6-yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide; N-(3-((((2S)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide; 2-((4-pyridinylmethyl)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-pyridinylmethyl)amino)-3-pyridinecarboxamide; N-(4-(pentafluoroethyl)-3-(((2S)-2-pyrrolidinyl)is XI)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide; N-(3-((3-azetidinol)oxy)-5-(trifluoromethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide; N-(3-(4-piperidinyloxy)-5-(trifluoromethyl)phenyl)-2-((2-(3-pyridinyl)ethyl)amino)-3-pyridinecarboxamide; N-(4,4-dimethyl-1,2,3,4-tetrahydroisoquinoline-7-yl)-2-(1H-indazol-6-ylamino)nicotinamide; 2-(1H-indazol-6-ylamino)-N-[3-(1-methylpyrrolidine-2-ylethoxy)-5-triptoreline]nicotinamide; N-[1-(2-dimethylaminoacetyl)-3,3-dimethyl-2,3-dihydro-1H-indol-6-yl]-2-(1H-indazol-6-ylamino)nicotinamide; 2-(1H-indazol-6-ylamino)-N-[3-(pyrrolidin-2-ylethoxy)-5-triptoreline]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-tetrahydroisoquinoline-7-yl)-2-(1H-indazol-6-ylamino)nicotinamide; N-[4-(tert-butyl)-3-(3-piperidinyl)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 described in U.S. Patent numbers 6258812; 6235764; 6630500; 6515004; 6713485; 5521184; 5770599; 5747498; 5990141; U.S. Publication No. US20030105091; and the Patent Cooperation Treaty publication nos. WO01/37820; WO01/32651; WO02/68406; WO02/66470; WO02/55501; WO04/05279; WO04/07481; WO04/07458; WO04/09784; WO02/59110; WO99/45009; WO98/35958; WO00/59509; WO99/61422; WO00/12089 and WO00/02871, and each of these publications is incorporated herein by reference for any purpose.

Combination therapy with growth factors may include CIT is Kini, lymphokines, growth factors, or other hematopoietic factors such as M-CSF, G-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 erythropoietin. Other compositions may include known angiopoetin, for example, Ang-1, -2, -3, -4, -Y, and/or Ang-like polypeptide, and/or vascular endothelially growth factor (VEGF). Growth factors include angiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein-IA bone morphogenetic protein IB, obtained from brain-derived neurotrophic factor, ciliary neurotrophic factor, the receptor for ciliary neurotrophic factor induced by cytokines chemotactic factor-1 neutrophils induced by cytokines chemotactic factor-2 of neutrophils, the growth factor is vascular endothelial cells, endothelin-1, epidermal growth factor, derived from epithelial neutrophil attractant, fibroblast growth factor-4, fibroblast growth factor-5, fibroblastic growth factor-6, fibroblast growth factor-7, fibroblast growth factor-8, fibroblast growth factor-8b, fibroblast growth factor-8s, fibroblastic growth factor-9, fibroblast growth factor-10, fibroblast growth factor acidic, fibroblast growth factor basic, receptor-1 obtained from the glial cell line derived neurotrophic factor receptor-2 obtained from the glial cell line-derived neurotrophic factor, associated with the growth of the protein associated with increasing protein-2, associated with the increase in protein-2, associated with the increase in protein-3, heparinase epidermal growth factor, hepaticotomy growth factor, insulin-like growth factor I receptor insulin-like growth factor, insulin-like growth factor II binding protein insulin-like growth factor, keratinocyte growth factor, leukemia inhibitory factor, receptor-1 leukemia inhibitory factor, nerve growth factor, growth factor receptor nerves, neurotrophin-3, neurotrophin-4, growth factor, placenta, growth factor placental-2, platelet-derived growth factor, endothelial cells, platelet-derived growth factor A-chain of platelet-derived growth factor derived from platelets factor R is a hundred AA, derived from platelet growth factor AB, In-circuit derived from platelet factor derived from platelet growth factor BB, receptor-1 obtained from platelet-growth factor receptor-2 obtained from platelet growth factor, the factor stimulating the growth of pre-B-cell factor stem cell factor receptor stem cells, 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 binding protein I, transforming factor growth 1, binding protein II, transforming growth factor 1 binding protein III transforming growth factor 1 receptor type I, tumor necrosis factor (TNF-R1), receptor type II tumor necrosis factor (TNF-R2), receptor activator of plasminogen urokinase type, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments.

It is clear that the specific binding agents of this invention can be administered with one or more anti-inflammatory agents. In this context, the term "anti-inflammatory agent" refers generally to any agent that reduces inflammation or swelling in the patient. Here perejil what is the approximate number of anti-inflammatory agents, but it should be clear that there may be additional suitable anti-inflammatory agents that are not listed here, but included in the scope of this invention.

Anti-inflammatory agent may be, for example, a compound that inhibits the interaction of inflammatory cytokines with their receptors. Examples of inhibitors of cytokines, applicable in combination with the specific binding agents of the present invention include, for example, antagonists (such as antibodies), TGF-β, as well as antagonists (such as antibodies)directed against interleukin involved in inflammation. These interleukins 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. Cm. Feghali, et al., Frontiers in Biosci., 2:12-26 (1997).

The specific binding agents of the present invention can also be administered in combination with inhibitors of protein kinase type 1 to enhance the proliferation of T-cells in HIV-infected patients receiving antiretroviral therapy.

Nerve growth factors (NGF) can also be combined with the specific binding agents of the present invention for the treatment of certain conditions. Such conditions include neurodegenerative diseases, spinal cord injury and multiple sclerosis. Other conditions that can be treated with the same combination, are glaucoma and diabetes.

A preferred combination therapy refers to the specific binding agent of the present invention, administered to the patient in combination with one or more suitable inhibitors of IL-1. Inhibitors of IL-1 include, but are not limited to, receptornegative peptide fragments of IL-1, antibodies directed against IL-1 or IL-1-beta or receptor type I IL-1, and recombinant proteins containing the full receptor or parts thereof to IL-1 or their modified versions, including genetically modified mutiny, multimeric forms and form extended release. Specific antagonists include IL-1ra-polypeptides, inhibitors of IL-1-beta converting enzyme (ICE), antibodies to the receptor of IL-1 antagonist of type I, IL-binding form of the receptor IL-1 type I receptor, IL-1 type II, antibodies to IL-1, including IL-1 alpha and IL-1 beta and other members of the family of IL-1, and the drug, known as IL-1 Trap (Regeneron). The polypeptides of IL-1ra include forms of IL-ra, described in U.S. Patent No. 5075222, and modified forms and variants, including forms and variants described in U.S. Patent 5922573, WO 91/17184, WO 92 16221 and WO 96 09323. The converting inhibitors of IL-1 beta enzyme (ICE) include peptidyl and low molecular weight inhibitors of ICE, including what is described in Patent Applications PCT WO 91/15577; WO 93/05071; W 93/09135; WO 93/14777 and WO 93/16710; and European published patent Application 0547699. Nematodirinae compounds include compounds described in the patent Application PCT WO 95/26958, U.S. Patent No. 5552400, U.S. Patent No. 6121266 and Dolle et al., J. Med. Chem., 39, pp. 2438-2440 (1996). Additional ICE inhibitors are described in U.S. Patents numbers 6162790, 6204261, 6136787, 6103711, 6025147, 6008217, 5973111, 5874424, 5847135, 5843904, 5756466, 5656627, 5716929. IL-1-binding forms of IL-1 receptor Type I and IL-1 receptor Type II is described in U.S. Patent numbers 4968607, 4968607, 5081228, Re 35450, 5319071 and 5350683. Other suitable antagonists of IL-1 include, but are not limited to, peptides derived from IL-1, which is able to communicate competitive with receptor signal IL-1, IL-1 R type I. Additional guidance in respect of certain antagonists of IL-1 (and other cytokines) can be found in U.S. Patent No. 6472179.

Also suitable are inhibitors of TNF, and they include, but are not limited to, receptornegative peptide fragments TNFα, antisense oligonucleotides and ribozymes that inhibit the production of TNFα antibodies directed against TNFα, and recombinant proteins containing the full receptor or portions of receptors for TNFα or their modified versions, including their modified variants, including genetically modified mutiny, multimeric forms and shapes prolonged released who I am. Also applicable TAS (inhibitors of the enzyme that turns necrosis factor-α), such as TAPI (Immunex Corp.) and GW-3333X (Glaxo Wellcome Inc.). Suitable are also molecules that inhibit the formation of complexes of IgA-α1At, for example, the peptides described in EP 0614464 IN, or antibodies against this complex. In addition, suitable molecules include, but are not limited to, TNFα-inhibiting disaccharides, sulfated derivatives of glucosamine or other similar carbohydrates, described in U.S. Patent No. 6020323. Additional suitable molecules include peptide inhibitors of TNFα, are described in U.S. Patents numbers 5641751 and 5519000, and containing D-amino acid peptides described in U.S. Patent No. 5753628. Also suitable are also inhibitors of TNFα converting enzyme. In addition, WO 01/03719 describes additional agents that can be used in combination in accordance with this invention.

Additional suitable compounds include, but are not limited to, small molecules, such as thalidomide or analogs of thalidomide, pentoxifylline, or inhibitors of matrix metalloproteinases (MMP) or other small molecule. Suitable MMP inhibitors for this purpose include, for example, the inhibitors described in U.S. Patents numbers 5883131, 5863949 and 5861510 and mercaptoacetyltriglycine described in Patent No. 587214. Other small molecules capable of reducing the production of TNFα, include, for example, the molecules described in U.S. Patent numbers 5508300, 5596013 and 5563143. Additional suitable small molecules include, but are not limited to, MMP inhibitors described in U.S. Patents numbers 5747514 and 5691382, as well as derivatives of hydroxamic acids, such as described in U.S. Patent No. 5821262. Additional suitable molecules include, for example, small molecules that inhibit the production of phosphodiesterase IV and TNFα, for example, substituted derivatives Asimov (WO 96/00215), chinaincorporated (U.S. Patent No. 5834485), allpurpose (WO 99/18095 and heterobicyclic derivatives (WO 96/01825); GB 2291422). Applicable also thiazole derivatives that inhibit TNFα and IFNγ (WO 99/15524), and xanthine derivatives that inhibit TNFα and other proinflammatory cytokines (see, for example, U.S. Patent numbers 5118500, 5096906 and 5196430). Additional small molecules, applicable to the treatment described here States include small molecules described in U.S. Patent No. 5547979.

Additional examples of drugs and types of drugs that can be entered through combination therapy include, but are not limited to, antiviral agents, antibiotics, analgesics (eg, acetaminophen, codeine, propoxyphene NAPs the lat, of oxycodone hydrochloride, hydrocodone bitartrate, is tramadol), corticosteroids, antagonists of inflammatory cytokines, disease modifying Antirheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAID) and Antirheumatic drugs slow action (SAARD).

Examples of disease modifying Antirheumatic means (DMARDs) include, but are not limited to: RheumatrexTM(methotrexate); Enbrel® (etanercept); Remicade® (infliximab); HumiraTM(adalimumab); Segard® (afelimomab); AravaTM(Leflunomide); KineretTM(anakinra); AravaTM (Leflunomide); D-penicillamine; Myocrisin; Plaquenil; RidauraTM(auranofin); Solganol; lenercept (Hoffman-La Roche); CDP870 (Celltech); CDP571 (Celltech), as well as antibodies, are described in EP 0 516 785 B1, U.S. Patent No. 5656272, EP 0 492 448 A1; onercept (Serono; CAS reg. no. 199685-57-9); MRA (Chugai); ImuranTM(azathioprine); inhibitors NEKB; CytoxanTM(cyclophosphamide; cyclosporine; hydroxychloroquine sulfate; minocycline; sulfasalazine; and gold compounds, such as oral gold, thiomalate gold-sodium and aurothioglucose.

In addition, suitable molecules include, for example, soluble TNFR derived from extracellular regions of the receptor molecules TNFα, other than TNFR P55 and P75, such as, for example, TNFR, described in WO 99/04001, including TNFR-Ig obtained from this TNFR. Additional suitable inhibitors of TNF is, suitable for use here, is described here. They include the use of not only antibodies against TNFα and TNFR, but also obtained from TNFα peptide that can act as a competitive inhibitor of TNFα (e.g., such as described in U.S. Patent No. 5795859 or U.S. Patent No. 6107273), fused proteins TNFR-IgG, such as a fusion protein containing the extracellular part of the receptor TNFα P55 soluble TNFR, other than IgG-protein or other molecules that reduce endogenous levels of TNFα, for example, inhibitors of TNFα converting enzyme (see, for example, US 5594106), or small molecules or inhibitors of TNFα, some of which are described here.

As regards antibodies to TNF, although the dose should ideally be determined by a qualified provider in accordance with the specific needs of the intended patient, one example of a preferred dose range for antibodies against TNFα is the range 0.1-20 mg/kg, more preferably 1-10 mg/kg. Another preferred dose range for an anti-TNFα antibody is in the range of 0.75 to 7.5 mg/kg of body weight.

This invention can also use a specific binding agent and any of one or more of the nonsteroidal anti-inflammatory drugs (NSAID). NSAIDs have anti-inflammatory effects, at least partially, in relation to inhibition of the Intesa prostaglandin. Goodman and Gilman, The Pharmacological Basis of Therapeutics, MacMillan 7thEdition (1985). NSAID can be divided into nine groups: (1) salicylic acid derivatives; (2) the propionic acid derivatives; (3) derivatives of acetic acid; (4) the derivative Funambol acid; (5) derivatives of carboxylic acids; (6) derivatives of butyric acid; (7) oxicam; (8) pyrazoles and (9) pyrazolones. Examples of NSAIDs include, but are not limited to: AnaproxTM, Anaprox DSTM(naproxen sodium); AnsaidTM(flurbiprofen); ArthrotecTM(diclofenac sodium + misoprostil); CataflamTM/VoltarenTM(diclofenac-potassium); ClinorilTM(sulindac); DayproTM(oxaprozin); DisalcidTM(salsalate); DolobidTM(diflunisal); EC NaprosynTM(naproxen sodium); FeldeneTM(piroxicam); IndocinTM, Indocin SRTM(indometacin); LodineTM, Lodine XLTM(etodolac); MotrinTM(ibuprofen); NaprelanTM(naproxen); NaprosynTM(naproxen); OrudisTM(Ketoprofen); OruvailTM(Ketoprofen); RelafenTM(nabumetone); TolectinTM(tolmetin-sodium); TrilisateTM(choline-magnesium trisalicylate); Cox-1 inhibitors; Cox-2 inhibitors such as VioxxTM(rofecoksib); Arcoxiatm(etoricoxib), CelebrexTM(celecoxib); Mobic™(meloxicam); BextraTM(valdecoxib), DynastatTM(parecoxib-sodium); Prexige™ (lumiracoxib), nabumeton. Additional suitable NSAIDs include, but are not limited what they are, the following: ε-acetamidophenol acid, S-adenosylmethionine, 3-amino-4-hydroxybutiric acid, amixetrine, nitrazepan, bendazac, bendazac-lisinac, benzydamine, barosin, properly, bukola, buttala, coprokazan, classimat, dasadmin, debacle, detomidine, divinename (difenpiramide), divinename (difenpyramide), cifically, diazol, emorfazone, fanatical mesilate, penfluridol, floctafenine, flunizol, flunixin, fluprofen, papertole, posposil, hvymetal, guaiazulene, solixir, lefetamine-HCl, Leflunomide, levamisol, latifat, lysine-clonixinate, Meselson, nabumeton, Nintendo, nimesulide, orgotein, herpanacine, oxaceprol, hexapedal, phrenilin, personsal, personsal citrate, pivoxil, piperoxan, pyrazole, pirfenidone, proquazone, proxitol, talvin In, diflunisal, timelady, tolectin, solpadol, tryptamide and other NSAID, denoted by the code numbers of the company, such as 480156S, AA861, AD1590, AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CN100, EB382, EL508, F1044, FK-506, GV3658, ITF182, KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, ONO3144, PR823, PV102, PV108, R830, RS2131, SCR152, SH440, SIR133, SPAS510, SQ27239, ST281, SY6001, TA60, TAI-901 (4-benzoyl-1-indocaribbean acid), TVX2706, U60257, UR2301 and WY41770. Structurally related NSAID having similar analgesic and anti-inflammatory properties in comparison with NSAID, also included in this group.

Suitable SAARD or DMARDs include, but are not limited to: allocateed on the Sabbath.' auranofin, aurothioglucose, aurothioglucose, azathioprine, brequinar sodium, bucillamine, 3 Eurotia-2-propanol-1-sulfonate calcium, chlorambucil, chloroquine, kabutari, kupresanin, cyclophosphamide, cyclosporine, Dapsone, 15-desoxypeganine, diacerein, glucosamine, gold salts (for example, salt cycloben-gold, thiomalate gold sodium thiosulfate gold-sodium), hydroxychloroquine, hydroxyurea, Cabazon, levamisole, lobenzarit, melittin, 6-mercaptopurine, methotrexate, mizoribine, mycophenolate-mofetil, moral, the nitrogen analog of the compound, D-penicillamine, pyridinedimethanol, such as SKNF86002 and SB203580, rapamycin, thiols, thymopoietin and vincristine. It is assumed that the structurally related SAARD or DMARDs, having similar analgesic and anti-inflammatory properties, are also included in this group.

Inhibitors of kinases in the cascade signal transmission are also suitable agents for combination with the specific binding agents of this invention. They include, but are not limited to, agents that are able to inhibit P-38 (also known as “RK” or “SARK-2”), Lee et al., Nature, 372:739 (1994). P-38 is described as serine/trionychinae (see Han et al., Biochimica Biophysica Acta, 1265:224-227 (1995). It has been shown that inhibitors of P-38 intervene between the extracellular stimulus and the secretion of IL-1 and TNFα from cells, and this intervention involves blocking transduction with whom drove through inhibition of the kinase, which is in this way signal transmission.

Also suitable are also the MK2 inhibitors and inhibitors, tpl-2. Also suitable are also inhibitors of T cells, including, for example, ctla-4, CsA, Fk-506, OX40, OX40R-Fc, OX40 antibody, ligand OH, the antibody against the ligand OH, lck and ZAP70. Suitable are also retinoids, including oral retinoids, as well as antagonists of TGF-β.

In addition, appropriate agents for combination with the specific binding agents of the present invention include, for example, any one or more derivatives of salicylic acid, esters as prodrugs or pharmaceutically acceptable salts. These salicylic acid derivatives, esters as prodrugs or pharmaceutically acceptable salts include acetaminophenol, aloxiprin, aspirin, benorilate, bromocriptin, atsetilsalitsilata calcium, choline-magnesium trisalicylate-diflunisal, Etisalat, fendosal, entityname acid, glycol salicylate, imidazole salicylate, lysine atsetilsalitsilata, mesalamine, research salicylate, 1-afterselect, olsalazine, parceled, phenylacetylcarbinol, fenilsalitsilat, saleclomid, salicylamide O-acetic acid, salsalate and sulfasalazin. It is assumed that the structurally related derivatives of salicylic acid with similar analgesics is tiraumea and anti-inflammatory properties, included in this group. In addition, suitable agents include, for example, derivatives of propionic acid, esters as prodrugs or pharmaceutically acceptable salts. Such derivatives of propionic acid, esters as prodrugs or pharmaceutically acceptable salts include alminoprofen, benoxaprofen, bulochnikov acid, carprofen, dexindoprofen, fenoprofen, flunoxaprofen, fluprofen, flurbiprofen, perclorate, ibuprofen, ibuprofen, aluminum, ibuproxam, indoprofen, ibuprofen, Ketoprofen, loxoprofen, miroprofen, naproxen, oxaprozin, Pittodrie, timeproven, pirprofen, pranoprofen, proteinovyy acid, pyridoxine, suprofen, tiaprofenic acid and tioxaprofen. It is assumed that the structurally related salicylic acid derivatives having similar analgesic and anti-inflammatory properties that are included in this group. In addition, suitable agents include, for example, derivatives of acetic acid, esters as prodrugs or pharmaceutically acceptable salts. Such derivatives of acetic acid, esters as prodrugs or pharmaceutically acceptable salts include: acemetacin, alclofenac, amfenac, bufexamac, cinmetacin, clairac, deleteln, diclofenac sodium, etodolac, felbinac, fenclofenac, fenk the ORAK, enclosure acid, fentiazac, furofenac, glucometry, ibufenac, indomethacin, isophtalic, isoxepac, lonazolac, medicinebuy acid, oxiplatin, Xiping, peltatin, proglumetacin, sulindac, tolmetin, tiaramide, tiopinac, tolmetin, zidometacin, zomepirac. It is assumed that the structurally related derivatives of acetic acid having similar analgesic and anti-inflammatory properties that are included in this group. In addition, suitable agents include, for example, derivatives of Funambol acid esters as prodrugs or pharmaceutically acceptable salts. These derivatives Funambol acid esters as prodrugs or pharmaceutically acceptable salts include antenova acid, etofenamate, flufenamic acid, sonksen, meclofenamic acid, meclofenamate sodium, mediasavvy acid, mefenamico acid, niflumova acid, talniflumate, meropenam, tolfenamic acid and openmath. It is assumed that the structurally related derivatives of Funambol acid having similar analgesic and anti-inflammatory properties that are included in this group.

Suitable are derivatives of carboxylic acids, esters as prodrugs or pharmaceutically acceptable salts, which can be used, and they include clida the AK, diflunisal, flufenisal, yorigin, Ketorolac, and tinoridine. It is assumed that the structurally related derivatives of carboxylic acids having similar analgesic and anti-inflammatory properties that are included in this group. Also suitable are derivatives of butyric acid, esters as prodrugs or pharmaceutically acceptable salts. These derivatives of butyric acid, esters as prodrugs or pharmaceutically acceptable salts include boydson, butibufen, fenbufen and xenmotion. It is assumed that the structurally related derivatives of butyric acid having similar analgesic and anti-inflammatory properties that are included in this group. Suitable are also oxicam, esters as prodrugs or pharmaceutically acceptable salts. Oxicam, esters as prodrugs or pharmaceutically acceptable salts include droxicam, analika, isoxicam, piroxicam, sudoxicam, tenoxicam and 4-hydroxy-1,2-benzothiazine-1,1-dioxide-4-(N-phenyl)carboxamide. It is assumed that the structurally related oxicam having similar analgesic and anti-inflammatory properties that are included in this group. Suitable are also pyrazoles, esters as prodrugs or pharmaceutically acceptable salts. These pyrazoles, with whom you esters as prodrugs or pharmaceutically acceptable salts, which can be used include: dipannita and epirizole. It is assumed that the structurally related pyrazoles having similar analgesic and anti-inflammatory properties, are also included in this group. Also suitable are also pyrazolones, esters as prodrugs or pharmaceutically acceptable salts. These pyrazolones, esters as prodrugs or pharmaceutically acceptable salts, which can be used include: Amazon, azapropazone, benzoperylene, feprazone, mofebutazone, Morison, oxyphenbutazone, phenylbutazone, pipeuse, propyphenazone, ramification, skibotn and titledbutton. It is assumed that the structurally related pyrazolones having similar analgesic and anti-inflammatory properties, are also included in this group.

Esters as prodrugs or pharmaceutically acceptable salts are also suitable for the treatment of TNF-mediated diseases. Corticosteroids, esters as prodrugs and their pharmaceutically acceptable salts include hydrocortisone and compounds that are derived from hydrocortisone, such as the 21-acetoxyphenyl, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, betamethasone valerate, budesonide, chloroprednisone, clobetasol, clobetasol propionate, CLOB tazon, clobetasone butyrate, clocortolone, cloprednol, corticosterone, cortisone, cortisol, deflation, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, flashcart, fluchloralin, flumetazon, flumetazon pivalate, flunisolide, fluocinolone acetonide, fluocinonide, ftorhinolona acetonide, fluocortin-butyl, perkantoran, forcertain hexanoate, deformation valerate, formation, flaperon acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, farmacita, halcinonide, halobetasol, halopedia acetate, hydrocortamate, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone 21-sodium succinate, hydrocortisone tebutate, mazipredon, Madison, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 21-dicriminated, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone sodium 21st-sulfobenzoate, prednisolone sodium 21-stearoptene, prednisolone tebutate, prednisolone 21-trimetilatsetat, prednisone, prednesol, prednisone, prednisolone 21-diethylaminoacetate, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetone and triamcinolone of hexatonic. It is assumed that the structurally related steroids with similar analgesia the s and anti-inflammatory properties, also included in this group.

Antimicrobial agents (and esters as prodrugs or pharmaceutically acceptable salts) are also suitable for the combined use described here. Suitable antimicrobial agents include, for example, ampicillin, amoxicillin, aureomycin, bacitracin, ceftazidime, Ceftriaxone, Cefotaxime, cefaclor, cephalexin, cefradine, ciprofloxacin, clavulanic acid, cloxacillin, dicloxacillin, erythromycin, Flucloxacillin, gentamicin, gramicidin, methicillin, neomycin, oxacillin, penicillin and vancomycin. It is assumed that the structurally related antimicrobial agents having similar analgesic and anti-inflammatory properties, are also included in this group.

Additional suitable compounds include, but are not limited to: BN 50730; tenidap; F 5531; thepatent PCA 4248; nimesulide; Panavir; rolipram; RP 73401; peptide T; MDL 201 449A; hydrochloride (1R,3S)-CIS-1-[9-(2,6-diaminopurine)]-3-hydroxy-4-cyclopentene; (1R,3S)-TRANS-1-[9-(2,6-diamino)purine]-3-acetoxysilane; hydrochloride (1R,3S)-TRANS-1-[9-adenyl]-3-azidocillin and (1R,3S)-TRANS-1-[6-hydroxypurine-9-yl]-3-azidocillin.

It was found that IL-4 can induce inflammatory effect in some cases, such as asthma, in which overexpression of IL-4 in the lungs causes hypertrophy epic liannah cells and accumulation of lymphocytes, eosinophils and neutrophils. This reaction is characteristic of the main symptoms of proinflammatory response induced by other Th2-cytokines. Thus, as noted above, inhibitors of IL-4 are also applicable in accordance with this invention. In addition, it should be clear that some immunosuppressive drugs can also be used in the treatment of arthritis, including, but not limited to, inhibitors of iNOS and inhibitors of 5-lipoxygenase.

It was shown that ginger has some anti-inflammatory properties and, therefore, suitable for use as an anti-inflammatory agent in accordance with this invention, and chondroitin.

In some embodiments, implementation, Ang-2-specific binding agent can be administered before, together or after treatment with the agent of cancer treatment. Exemplary cancers include, but are not limited to, breast cancer, colorectal cancer, gastric cancer, glioma, squamous cell head and neck cancer, hereditary or accidental papillary kidney cancer, leukemia, lymphoma syndrome Li-o Fraumeni, malignant pleural mesothelioma, melanoma, multiple melanoma, non-small cell carcinoma of the lung, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, small cell carcinoma of the lung, si is vialou sarcoma, thyroid cancer and transitional cell bladder cancer.

In some embodiments, implementation, Ang-2-specific binding agent may be used alone or with at least one additional therapeutic agent for the treatment of cancer. In some embodiments, implementation, Ang-2-specific binding agent is used together with a therapeutically effective amount of an additional therapeutic agent. Exemplary therapeutic agents that may be administered with Ang-2-specific binding agent include, but are not limited to, a member of the family geldanamycin anisometropic antibiotics; Pro-HGF; NK2; a peptide inhibitor of c-Met; antagonist domain Src homology 2 (SH2) Grb2; modulator Gab1; dominant-negative Src; inhibitor of von-Hippel-Landau, including, but not limited to, wortmannin; inhibitors of P13 kinase, other agents antireceptor therapy, anti-EGFR, COX-2 inhibitor, Celebrex™, Vioxx™; vascular endothelial growth factor (VEGF), a modulator of VEGF, fibroblast growth factor (FGF), epidermal growth factor (EGF); modulator EGF; keratinocyte growth factor (KGF), KGF-related molecule modulator KGF; modulators of matrix metalloproteinases (MMP).

In some embodiments, implementation, this invention relates to therapeutic agents containing Ang-2-specific binding agent and less than the least one inhibitor of serine proteases, and methods of treatment using such therapeutic agents. In some embodiments, implementation of therapeutic agent contains the Ang-2-specific binding agent and the inhibitor of serine protease and at least one additional molecule described herein.

In some embodiments, the implementation, the imbalance of protease/protease inhibitor can lead to protease-mediated degradation of tissue, including, but not limited to, invasion of tumor-normal tissue, leading to metastasis.

In some embodiments, implementation, Ang-2-specific binding agent can be used with at least one anti-inflammatory therapeutic agent. In some embodiments, implementation, Ang-2-specific binding agent can be used with at least one therapeutic agent in respect of immune disorders. Examples of therapeutic agents for inflammation and immune disorders include, but are not limited to, inhibitors of cyclooxygenase type 1 (COX-1) and cyclooxygenase type 2 (COX-2), small molecule modulators of mitogen-activated protein kinase 38 kDa (R-MARK); small molecule modulators of intracellular molecules involved in inflammatory pathways, where such intracellular molecules include, but are not limited to, jnk, IKK, NF-κB, ZAP70 and lck. Some of the s examples of anti-inflammatory therapeutic agents described, for example, in C.A. Dinarello, and L.L. Moldawer of Proinflammatory and Anti-Inflammatory Cytokines in Rheumatoid Arthritis. A Primer for Clinicians Third Edition (2001) Amgen Inc. Thousand Oaks, CA.

In some embodiments, the implementation, the pharmaceutical compositions will include more than one other Ang-2-specific binding agent. In some embodiments, the implementation, the pharmaceutical compositions will include more than one Ang-2-specific binding agent, and these Ang-2-specific binding agents bind to Ang-2 in more than one epitope.

Immunotherapy

Immunotherapy is usually based on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors may be, for example, the antibody of the present invention, which recognize any marker on the surface of target cells. This antibody, one may serve as an effector of therapy or it may recruit other cells to actually perform the killing cells. This antibody may also be conjugated with a drug or toxin (chemotherapeutic agent, a radionuclide, A-chain of ricin, by cholera toxin, pertussis toxin, and so on) and, therefore, can only serve as a targeting agent.

According to this invention, or antibodies or conjugates of the antibodies the present invention can be targeted to the mutant form of Ang-2 by immunotherapy. In particular, it has been discussed that the composition of antibodies of this invention can be used in combination therapy approach together with Ang-2-targeted therapy.

Passive immunotherapy has proven to be particularly effective against a number of cancers. See, for example, WO 98/39027.

The following examples are only for illustration purposes and should not be construed as limiting in any way the invention.

EXAMPLE 1

The expression of Ang-2 in pathological and normal tissue

The expression of Ang-2 was investigated in normal and patologicheskoi tissue using in situ hybridization. Fragments of Ang-2 sequences of human (Genbank Accession Number: AF004327, nucleotides 1274-1726) and mouse (Genbank Accession Number: AF004326, nucleotides 1135-1588) amplified PCR with reverse transcriptase from cDNA fetal lung human or mouse, cloned in plasmid pGEM-T and confirmed by sequencing.33P-labeled antisense RNA probes were transcribable from linearized plasmid matrices using33P-UTP and RNA polymerase. Blocks fixed with formaldehyde, embedded in paraffin tissue used for making sections at 5 μm, and the sections were collected on charged glass slides. Before in situ hybridization tissues were processed to increase the permeability of 0.2 M HCl, followed by splitting what proteinases To and acetylation by triethanolamine and acetic anhydride. Sections hybridized with radioactively labeled probe overnight at 55°C, then subjected to cleavage by RNase and flushing high severity in approximately 0,1X SSC at 55°C. slides were dipped in emulsion (Kodak NTB2, exhibited at 4°C for 2-3 weeks, and showed clearly stained. Slices were examined with a dark field and standard lighting for the simultaneous measurement of tissue morphology and hybridization signal.

The results showed that the normal postnatal human expression of Ang-2 is limited to a small number of tissues containing angiogenic vasculature, such as the ovary, placenta and uterus. The expression of Ang-2 was not detected in heart, brain, kidney, liver, lung, pancreas, spleen, muscle, tonsil, thymus, Appendix, lymph node, gall bladder, prostate or testicular normal adult. In the five-week mouse (but not in adult monkey or a human kidney was found notable expression of Ang-2 in the vasa recta. To determine whether this expression remnant of embryonic development, the experiment was repeated on the kidneys obtained from mice with an age range of up to one year using mouse Ang-2 probe and the above-described conditions. Observed that the expression of Ang-2 was decreased in time for catalogo development but was still evident in the kidney annual mice.

The expression of Ang-2 was also detected in virtually all tested tumors, including primary human tumors, such as cancer of the colon (5 cases), breast cancer (10 cases), lung cancer (8 cases), glioblastoma (1 case), metastatic human tumors, such as breast cancer (2 cases), lung cancer (2 cases) and ovarian cancer (2 cases), which metastasized to the brain, and models of rodent tumors, such as C6 (rat glioma), NT (cancer the colon of a person), Colo-205 (colon cancer cancer man), NST (cancer of the colon of a person), A431 (epidermoid cancer man), A (human rhabdomyosarcoma), NT (human fibrosarcoma) PC-3 (prostate cancer man), B16F10 (murine melanoma), MethA (mouse sarcoma) and Lewis lung cancer mets. In addition, the expression of Ang-2 were detected in the new vessels growing in the tube of Matrigel in response to VEGF, and in a murine model of hypoxia retrolateral fibroplasia (syndrome Terry).

EXAMPLE 2

The preparation of recombinant protein mAng-2 and polyclonal anti-Ang-2 antisera rabbit

Full-length, His-tagged cDNA of mouse Ang-2 was obtained using PCR (Clontech Advantage PCR Kit, Cat. # K1905-01) from the cDNA library of mouse 15-day embryo (Marathon-Ready cDNA, Cat. # 7459-1, Clontech, Inc.) using PCR-PRA is the size for full-length Ang-2 people. The PCR product is ligated into the expression vector with a CMV promoter and the resulting plasmid was transfusional cells fibrosarcoma person NT (obtained from ATSS) using the reagent for transfection FuGENE6 (Roche, Cat. # 1814443). Stabilire clones were isolated by selection using G418. Anti-His-tag-ELISA and Western blotting were used for screening mAng-2-His-expressing clones.

Recombinant polypeptide mAng-2 was purified from the conditioned media (S.M.) of these cells. S.M. containing mAng-2-His was purified using a two-step chromatographic Protocol. In short, this air-conditioned environment was titrated to a pH of 8.9 by adding Tris buffer pH 9.5 to a final concentration of approximately 20 mm. In addition, added the detergent CHAPS to a final concentration of approximately 5 mm. Then that S.M. was applied directly onto the anion exchange column, Q-sepharose ff (Pharmacia). Then the column was washed approximately 10 mm Tricom pH 8.0, containing approximately 350 mm NaCl and approximately 5 mm CHAPS.

The eluate from the column Q-sepharose brought up to approximately 4 mm imidazole and was applied to the affinity column with immobilized metal (Ni-NTA superflow (Qiagen)). Bound protein was suirable PBS containing approximately 5 mm CHAPS and approximately 100 mm imidazole. Then the eluate was concentrated to approximately 1.0 mg/ml) followed by dialysis against PBS. mAng-2-His was greater than 90%, when measuring the staining of Kumasi after electrophoresis in LTO-PAG.

Rabbits were immunized approximately 0.2 mg mAng-2 for injection to obtain antibodies. Rabbits were injected with approximately 1 ml of TiterMax® hunter (Sigma) and mAng-2 at a ratio of 1:1. After four weeks each rabbit received a second injection or booster; two weeks after they received their next booster injection, and 7 week took the serum and evaluated on the titer against mAng-2. If serum titer was high, production krovoisliania 50 ml was performed once a week for six consecutive weeks. However, if the titer of the serum was low, the rabbits were injected additional booster injection and production krovoisliania 50 ml was performed once a week for six consecutive weeks, starting from week 9. After six consecutive production chromosphere rabbits were allowed to rest for six weeks. If required a large number of sera, the rabbits were again introduced a booster injection after one month after the last production of blood collection.

Using analysis of neutralization ELISA (described below) observed that the rabbit polyclonal anti-mAng-2 antisera from two rabbits, 5254 and 5255, neutralized interaction mAng-2:Tie-2.

EXAMPLE 3

Molecular anal the PS for the assessment of Ang-2 antibodies

Molecular analyses (analysis of affinity ELISA analysis of neutralization ELISA and BIAcore) were designed to assess direct binding of antibodies to Ang-2 and related members of this family and the impact on the interaction of Ang-2:Tie-2. These in vitro and cell-based is described as follows.

A. Analysis of the affinity ELISA

For initial screening of candidate anti-Ang-2 antibodies, used purified Ang-2 (R&D Systems, Inc.; catalog number 623-AN; Ang-2 is provided in the form of a mixture of 2 shorter versions) or murine polypeptide Ang-2 (obtained as described above). For confirming the binding assays Ang-2 was obtained from conditioned media of cells T person, transfected with full-length DNA Ang-2, and cultured in serum-free DMEM containing approximately 50 μg per ml of bovine serum albumin (BSA).

Using microtiter tablets, approximately 100 μl per well of Ang-2 was added to each well and the plates were incubated for approximately 2 hours, after which these tablets were washed in phosphate buffered saline (PBS)containing 0.1% Tween-20 four times. Then these plates were blocked using approximately 250 microliters per well of approximately 5% BSA in PBS and the plates were incubated at room temperature for CA is approximately 2 hours. After incubation, excess blocking solution was discarded and each well was added approximately 100 microliters candidate anti-Ang-2 antibodies in a series of dilutions, starting at a concentration of approximately 40 nm, followed by serial dilution in 4 times in PBS containing about 1% BSA. Then these plates were incubated overnight at room temperature. After incubation tablets were washed in PBS containing 0.1% Tween-20. The washing was repeated four more times, after which was added approximately 100 microliters per well of goat antibodies against IgG(Fc) man (Pierce Chemical Co., catalog number 31416), pre-diluted 1:5000 in PBS containing 1% BSA (bovine serum albumin). The plates were incubated for approximately 1 hour at room temperature. Then the tablets were washed five times in PBS containing 0.1% tween-20, after which was added approximately 100 microliters per well of TMB substrate (system containing 3,3',5,5'-tetramethylbenzidine liquid substrate; Sigma Chemicals Company, St. Louis, MO, catalog number T) and the plates were incubated for approximately 5-15 minutes, until it developed a blue color. Then the optical density was read in a spectrophotometer at approximately 370 nm.

C. Analysis of neutralization ELISA

Microtiter plates, with which is was associated polypeptide Ang-2 person received as described for affinity ELISA. Candidate anti-Ang-2 antibodies were prepared in serial dilutions, as described for the analysis of affinity ELISA above, in a solution of PBS containing about 1% BSA and about 1 nm Tie-2 (provided in the form of molecules of Tie-2-Fc, where Tie-2-part contains only the soluble extracellular portion of the molecule; R&D Systems, Inc.; catalog number 313-TI). After adding to each well approximately 100 microliters solution antibody/Tie-2 these plates were incubated overnight at room temperature and then washed five times in PBS containing 0.1% Tween-20. After washing was added approximately 100 microliters per well of anti-Tie-2 antibody (Pharmingen Inc., catalog number 557039) to a final concentration of about 1 microgram per ml and the plates were incubated for approximately 1 hour at room temperature. Then add approximately 100 microliters goat antibodies against IgG-HRP mouse (Pierce Chemical Co., catalog number 31432) at a dilution of 1:10000 in PBS containing 1% BSA. The plates were incubated for approximately 1 hour at room temperature, after which they were washed five times with PBS containing 0.1% Tween-20. Then add approximately 100 microliters per well of TMB substrate (described above) and allowed to manifest coloring. The optical density was read in range of the photometer at 370 nm.

C. analysis of the BIAcore affinity

Analysis of the affinity of each candidate anti-Ang-2 antibodies was performed on a BIAcore® 2000 (Biacore, Inc., Piscataway, NJ) with PBS and 0.005% surfactant substance P20 (BIAcore, Inc.) as a working buffer. Recombinant protein G (Repligen, Needham, MA) was immobilized on the sensor (the perceiver) "chip (Biacore, Inc.) through the primary amino group with a set of binding amino groups (Biacore, Inc.) in accordance with the proposed manufacturer Protocol.

Analyses linking was performed by attaching first approximately 100 Ru of each candidate anti-Ang-2 antibody to immobilized Protein G, and then various concentrations (0-100 nm) huAng-2 or mAng-2 were injected with the surface bound antibodies at a speed of current of approximately 50 μl/min for about 3 minutes. The binding kinetics of antibodies, including ka(the rate constant of the Association), kd(the rate constant of dissociation) and KD(the equilibrium constant of dissociation) was determined using a computer program BIA evaluation 3.1 (BIAcore, Inc.). Lower the equilibrium constant for the dissociation indicates high affinity antibodies against Ang-2.

EXAMPLE 4

Getting a full-sized anti-Ang-2 antibodies using phage display

Full anti-Ang-2-human antibodies generated by penninga Fab-bi is liteky phage display Target Quest (Quest Target, Inc.) against Ang-2 polypeptide person (R & D Systems Inc., catalog 623-AN) in accordance with the following Protocol.

Ang-2 was immobilized on the surface of magnetic polystyrene granules in two ways: (1) direct coating Ang-2 at 50 µg/ml at 4°C during the night and (2) indirect grasping Ang-2 goat anti-Ang-2 antibody at 50 µg/ml at 4°C over night. The surface of the granules blocked with 2% milk in PBS (MPBS). Phage library Fab person was previously selected for deletion phage clones reactive with uncoated magnetic granules or goat anti-Ang-2 antibody. Then Ang-2-coated magnetic granules were incubated with the phage library at room temperature for 1.5 hours. After the stage of binding phage, the surface was washed 6 times MPBS containing approximately 0.1% tween-20 followed by washing 6 times with PBS containing 0.1% tween-20, and then 2 times with PBS. Bound phage were suirable first approximately 100 μg/ml of Tie-2-Fc man (R and D Systems, Minneapolis, MN) and then approximately 100 mm triethanolamine. Suirvey phage were infected into cells of E. coli TG1, amplified and saved for the next round of screening. The selection pressure was increased in subsequent rounds the inclusion of more rigorous leaching and reduction of phage. After 3 rounds of selection identified 18 unique, Ang-2 binding Fab clones, factual and all of which recognize the Ang-2 person Ang-2 and mouse Ang-2 rats when measured using analysis of affinity ELISA described above. Approximately ten percent of this phage was also associated Ang-1 person. These clones were transformed into IgG1 antibodies, as described below.

For more unique phage conducted a second round of screening using the same library, but slightly different Protocol. In this Protocol Ang-2 were sown in the buffer NaHCO3when the pH of 9.6 in immunoresearch Nunc maxisorp at approximately 4°C over night. Ang-2 were sown at approximately 1.5, and 0.3 to 0.74 mg/ml for rounds of panning 1, 2, and 3, respectively. The surface of immunoprobes blocked using approximately 2% milk in PBS (MPBS), before incubation with approximately 3 trillion of phage particles (approximately 50 copies of each unique phage in the library) from the same library phage display technique mentioned above (Quest Target) in approximately 4 ml of 2% MPBS. After the stage of incubation of the phage surface was washed 20 times with PBS 0.1% Tween-20 and then 20 times with PBS. Bound phage were suirable using 1 μm hAng-2 or 1 µm Tie-2 (R and D Systems, described above). Suirvey phage were infected into cells of E. coli TG1 (secured ragovoy library), amplified and saved for the next round of screening. Sixteen unique, Ang-2 to tie the living Fab clones were identified by PCR amplification of all phages with whom was associated hAng-2 or Tie-2, and these clones were analyzed by restriction cleavage. DNA of each clone sequenced.

The sequence encoding the variable region of each heavy chain of each phage amplified with complementary primers. These primers were designed in such a way as to include the site HindIII, XbaI site, a Kozak sequence and a signal sequence (translated peptide is a MDMRVPAQLLGLLLLWLRGARC; SEQ ID NO:202) at the 5'-end of the variable regions, whereas the BSMBI site added at the 3'-end of the PCR product. As an example, how could clone the heavy chain, matrix DNA phage to clone 544 (SEQ ID NO:19) amplified using primers 2248-21 (GTG GTT GAG AGG TGC CAG ATG TCA GGT CCA GCT GGT GCA G; SEQ ID NO:203), which was added last 7 amino acids of the signal sequence, and 2502-31 (ATT ACG TCT CAC AGT TCG TTT GAT CTC CAC; SEQ ID NO:204), which added a BsmBI site at the end of the variable region. The resulting product amplified using primers 2148-98 (CCG CTC AGC TCC TGG GGC TCC TGC TAT TGT GGT TGA GAG GTG CCA GAT; SEQ ID NO:205), which added nine amino acids of the signal peptide (AQLLGLLLL; SEQ ID NO:206), and then 2489-36 (CAG CAG AAG CTT CTA GAC CAC CAT GGA CAT GAG GGT CCC CGC TCA GCT CCT GGG; SEQ ID NO:207) and 2502-31. Added primer 2489-36, from 5' to 3', the site HindIII, XbaI site, a Kozak sequence and the first 6 amino acids of the signal sequence. PCR prod the points were digested XbaI and BsmBI and then cloned in expressing vector mammal, containing a constant region of human IgG1. This vector contains the SV40 promoter and allows DHFR selection.

The light chain of each phage were light chain class Kappa or lambda. For each light chain designed complementary primers to add, from 5' to 3', HindIII site and XbaI site, a Kozak sequence and a signal sequence (above). The chains that had error-free coding region, was cloned as full-length products. As an example, the light chain of the phage clone 536 (SEQ ID NO:11 and SEQ ID NO:210) amplified the full-length coding region using primers 2627-69 (GTG GTT GAG AGG TGC CAG ATG TGA CAT TGT GAT GAC TCA GTC TCC; SEQ ID NO:208), which was added last seven amino acids of the signal sequence, and primer 2458-54 (CTT GTC GAC TTA TTA ACA CTC TCC CCT GTT G; SEQ ID NO:209), which added a SalI site after the stop codon. Then, the PCR product amplified as described above, with an additional 5'-primers, 2148-98 and 2489-36, respectively, paired with a primer 2458-54, to complete the addition of the signal sequence and the cloning sites. These full-size light chains were cloned as XbaI-SalI fragments in expressing vector mammal described above.

Some lambda clones had errors in their constant regions when compared with the natural sequence of the constant is Noah area. To correct for these differences, carried out overlapping PCR using DNA that encodes a precise constant region of the lambda, and variable regions derived from phage. These clones also cloned as XbaI-SalI fragments, as described above.

If variable region Kappa cloned separately from their constant regions, have added a BsmBI site to the 3'-end PCR product. After cleavage of the PCR product with XbaI and BsmBI variable region chain Kappa cloned in expressing the vector containing the constant region of Kappa man.

The paired design of light and heavy chains of each converted phage was cotranslationally cells SNO using the kit for transfection using calcium phosphate (Invitrogen Corp.) typically, in accordance with the proposed manufacturer's Protocol. The medium was replaced after 14-16 hours after transfection and cells were passively in cups for tissue culture for selection after approximately 48 hours in accordance with the manufacturer's recommendations. Transfetsirovannyh cells were isolated HT-selection for about 3 weeks, then colonies of transfected cells SNO was trypsinization and combined into a pool of transfected cells.

Air-conditioned environment of small scale were collected 48 hours later and analyzed for the production of antibodies by the analysis of the Western blot is in using antibodies against Fc man antibodies against chain Kappa or antibody against the chain lambda man. Then the selected cell population was passively when selection pressure using standard sterile fashion tissue culture, has not yet received sufficient cells for seeding four roller bottles at 850 cm22×107viable cells and to obtain the original frozen cell lines using DMSO. After seeding these cells are supported in roller bottles with DMEM containing approximately 10% serum (Gibco/BRL, Inc.), supplemented with glutamine and nonessential amino acids. The cells were maintained for two to three days until they reached confluently cells by approximately 80%. At this point, the environment in roller bottles were switched to serum-free medium (50% DMEM, 50% F12, Gibco), supplemented with glutamine and nonessential amino acids. Conditioned medium was collected after seven days, and added to fresh serum-free medium for one or two additional charges environment.

Antibody was purified Protein G-affinity chromatography directly from the air-conditioned environment using standard protocols. The elution of the Protein G column was performed using a buffer with low pH (about pH 3), then suirvey protein antibodies neutralized using the receiving of 1 M Tris, pH 8.5, and then concentrated using a centrifuge concentrates with cut-off molecular weight of 10 KD. Then, the original material of the concentrated antibody was subjected to replacement of buffer PBS buffer.

Were created thirty-one antibody, and each consists of two heavy chains and two light (Kappa or lambda) chains, as presented in table 2.

Table 2
Heavy chain antibodiesLight chain antibodies
526 HC*526 Kappa
528 HC*528 lambda C1
531 HC*531 lambda C3
533 HC*533-Kappa
535 HC*535 lambda C3
536 HC*536 Kappa
537 HC*537 lambda C3 (G 107A R)
540 HC*540 lambda C3
543 HC*543 Kappa
544 HC*544-lambda C3
545 HC* 545 lambda C2
546 HC*546 lambda C1 (G 107A S, 112 N A, T114 S)
551 HC*551 Kappa
553 HC*553 Kappa
555 HC*555 Kappa
558 HC558 Kappa
559 HC559 lambda C1 (N 112 A, T 114 S)
565 HC*565 Kappa
F1-C6 HCF1-C6 lambda C2
FB1-A7 HCFB1-A7 lambda C2 (G 107A S)
FD-B2 HCFD-B2 lambda C3 (G 107A S)
FE-B7 HCFE-B7 Kappa
FJ-G11 HCFJ-G11 Kappa
FK-E3 HCFK-E3 Kappa
G1D4 HC*G1D4 lambda C2
GC1E8 HCGC1E8 lambda C3 (K 149 R)
H1C12 HCH1C12 lambda C2
IA1-1E7 HCIA1-1E7 Kappa
IF-1C10 HCIF-1C10 lambda C3 (T 212 (A)
IK-2E2 HCIK-2E2 lambda C2 (T 212 (A)
IP-2C11 HCIP-2C11 Kappa
* Experienced in linking to hAng-2, mAng-2 and hAng-1.
# Some constant region of the light chain lambda are, apparently, chimeras from more than one gene constant region lambda germline. Nearest gene constant region of the lambda genes germline listed along with amino acids that differs from the amino acids encoded by the genome of the cells of the germ line, numbered using the Kabat system.

The following four tables present the sequence and SEQ ID NO: heavy and light (Kappa and lambda) chains 31 anti-Ang-2 antibodies, converted from a phage in full-IgG1-antibodies. Define complementarity regions (CDR) of these monoclonal antibodies were predicted using the VBASE database, which uses the method described by Kabat et al., in: Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242; U.S. Dept. Health and Human Services, 5thed.). Fab-areas were compared with sequences in the database with the closest sequence genes in the germ line (gametic cells) using the tools available from the MRC Centre for ProteinEngineering, Cambridge, UK, and then visually compared with such sequences. CDR for each variable region (heavy or light chain) are presented in Table 7.

Table 3
Variable regions of the heavy chain
HC (heavy chain) antibodySequence
526 HC
(SEQ ID NO: 1)
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGPYAYWGQGTLVTVSS
528 HC
(SEQ ID NO: 3)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGVVGDFDWLSFFDYWGQGTLVTVSS
531 HC
(SEQ ID NO: 5)
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPILGIANYAQKFQGRVTITADKSTNTAYMELTSLTSDDTAVYYCARDREDTAMVFNYWGQGTLVTVSS
533 HC
(SEQ ID NO: 7)
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSS
535 HC
(SEQ ID NO: 9)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAAFSPFTETDAFDIWGQGTMVTVSS
536 HC
(SEQ ID NO: 11)
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSS
537 HC
(SEQ ID NO: 13)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPILGIANYAQKFQGRVTITADKSTSTAYMELSGLGSEDTAVYYCARGSSDAAVAGMWGQGTLVTVSS
540 HC
(SEQ ID NO: 15)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPILGIANYAQKFQGRVTITADKFTSTAYMELSSLGSEDTAVYYCARAVPGTEDAFDIWGQGTMVTVSS
543 HC
(SEQ ID NO: 17)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARPYYDFWSGPGGMDVWGQGTTVTVSS

544 HC
(SEQ ID NO: 19)
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARFESGYWGDAFDIWGQGTMVTVSS
545 HC
(SEQ ID NO: 21)
QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGPVDFDYGDYAIDYWGQGTLVTVSS
546 HC
(SEQ ID NO: 23)
EVQLVDSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKETISFSTFSGYFDYWAQGTLVTVSS
551 HC
(SEQ ID NO: 25)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGYDFWSGYSLDAFDIWGQGTMVTVSS
553 HC
(SEQ ID NO: 27)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSGLRSEDTAVYYCARGVDDYGGNSWAFDIWGQGTMVTVSS
555 HC
(SEQ ID NO: 29)
QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSASDHYYDSSGYYSDAFDIWGQGTMVTVSS
558 HC
(SEQ ID O: 31)
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPGKGLEWIGEINHSGSTNFNPSLKSRITISVDTSNNQFSLKLSSVTAADTAAYYCARGHDWGMGIGGAAYDIWGQGTMVTVSS
559 HC
(SEQ ID NO: 33)
QVQLVQSGAEVKKPGASVKVSCKVSGYTLTESSMHWVRQAPGKGLEWMGGFDPEHGETIYAQKFQGRLTMTEDTSTDTAYMELSSLRSEDTAVYFCARGVQVTSGYHYFDHWGQGTLVTVSS
565 HC
(SEQ ID NO: 35)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSPIYYDILTGIDAFDIWGQGTMVTVSS
F1-C6 HC
(SEQ ID NO: 37)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDPIPSGWYFDLWGRGTLVTVSS
FB1-A7 HC
(SEQ ID NO: 39)
QVQLVESGGGLVKPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREVGNYYDSSGYGYWGQGTLVTVSS
FD-B2 HC
(SEQ ID NO: 41)
QVQLQQSGPGLVKPSQTLSLTCAISGDTVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAVSLRGRITINLDTDTSKNQFSLQLNSVTPEDTAVYYCARDRGGYIDSWGQGTLVTVSS
FE-B7 HC
(SEQ ID NO: 43)
EVQLVESGGGLGQPGGSLRLSCAATGFSLDDYEMNWVRQAPGRGLEWVSYIIGSGKTIFYADSVKGRFTISRDNGKNSVYLQMNSLRAEDTAIYYCARGGGSAYYLNTSDIWGQGTMVTVSS
FJ-G11 HC
(SEQ ID NO: 45)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDRGIAARSAYYYGMDVWGQGTTVTVSS
FK-E3 HC
(SEQ ID NO: 47)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDLNWVRQASGQGLEWMGWMNPTSGNTGYAQKFQGRITMTRNTSISTAYMELRSLRSDDTAVYYCARDPPSGGWEFDYWGQGTLVTVSS
G1D4 HC
(SEQ ID NO: 49)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATSRLEWLLYLDYWGQGTLVTVSS
GC1E8 HC
(SEQ ID NO: 51)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMEVRSLRSDDTAVYYCARGGSPYGGYAYPFDYWGQGTLVTVSS
H1C12 HC
(SEQ ID NO: 53)
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSS
IA1-1E7 HC
(SEQ ID NO: 55)
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPGKGLEWIGEINHSGSTNFNPSLKSRITISVDTSNNQFSLKLSSVTAADTAVYYCARGHDWGMGIGGAAYDIWGQGTMVTVSS
IF-1C10 HC
(SEQ ID NO: 57)
QVQLVESGGGLVQPGGSLRLSCAASGFTFFSTYAMTWVRQAPGKGLEWVSVIRSNGGTDYADFVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCMTDYYWGQGTLVTVSS
IK-2E2 HC
(SEQ ID NO: 59)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKETISFSTFSGYFDYWGQGTLVTVSS
IP-2C11 HC
(SEQ ID NO: 61)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCAKEIAVAGTRYGMDVWGQGTTVTVSS

Table 4
Variable region chain Kappa
LC (light chain) antibodySequence
526 Kappa
(SEQ ID NO: 2)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPTFGGGTKVIK
533 Kappa
(SEQ ID NO: 8)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLNWYLQKPGQSPQILIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTPPTFGQGTKLEIK
536 Kappa (THW)
(SEQ ID NO: 12)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK
536 Kappa (LQT)
(SEQ ID NO: 210)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDF
TLKISRVEAEDVGVYYCMQGLQTPPTFGQGTKLEIK
543 Kappa
(SEQ ID NO: 18)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK
551 Kappa
(SEQ ID NO: 26)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK
553 Kappa
(SEQ ID NO: 28)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFTGSGSATDFTLRISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK
555 Kappa
(SEQ ID NO: 30)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLASNRASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQTLQIPITFGPGTKVDIK
558 Kappa
(SEQ ID NO: 32)
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSSLAWYQQKPGQAPRLLVYAASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPRTFGQGTKVEIK
565 Kappa
(SEQ ID NO: 36)
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSSLAWYQQKPGQAPRLLVYAASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPRTFGQGTKVEIK
565 Kappa (2)
(SEQ ID NO: 211)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIFLGSSRASGVPDRFSGSGSGTDFTLKISKVEADDVGIYYCMQALDTPPTFGPGTKVEIK
FE-B7 Kappa
(SEQ ID NO: 44)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSKGDNYLDWYLQKPGQSPQLLIYLGSHRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK
FJ-G11 Kappa
(SEQ ID NO: 46)
DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGKTYLDWYLQRPGQSPQLLMYTTSSRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQATQFPYTFGQGTKLEIK
FK-E3 Kappa
(SEQ ID NO: 48)
DIVMTQTPLSSTVTLGQPASISCRSSQSLVHEDGNTYLNWLHQRPGQPPRLLIYKISKRFSGVPDRFSGSGAGTDFTLKISRVEPEDVGVYYCMQSTRFPRTFGQGTKLEIK
IA1-1E7 Kappa
(SEQ ID NO: 56)
EIVLTQSPATLSLSPGERATLSCRASQSVSSSFLAWYQQKAGQAPRLLIYDTSTRATGIADRFSGSGSGTDFTLTISRLEAEDSAVYYCQQYDFSPLTFGGGTKVEIK
IP-2C11 Kappa
(SEQ ID NO: 62)
EIVLTQSPGTLSLSPGERATLSCRASQSISTFLAWYQQKPGQAPRLLIYDASNRATGIPGRFSGSGSGTDFTLTISNLEPEDFAVYYCQHRINWPLTFGGGTKVEIK

Table 5
Variable region chain lambda
LC (light chain) antibodySequence
528 lambda
(SEQ ID NO: 4)
SYELTQPPSVSVSPGQTASITCSGDKLGYTYTSWFQQKPGQSPVLVIFQDFKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSTTAVVFGTGTKVTVL
531 lambda
(SEQ ID NO: 6)
QSVLTQPPSVSAAPGQKVTVSCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAFWVFGGGTKLTVL
535 lambda
(SEQ ID NO: 10)
QSVLTQPPSVSAAPGQKVTISCSGSNSNIGNNFVSWYQQLPGTAPKLLVYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAAEVVFGGGTKLTVL
537 lambda
(SEQ ID NO: 14)
QSVLTQPPSVSAAPGQDVTISCSGNNSNIGNNYVSWYQQVPGTAPKLLVYDNHKRPSGISDRFSGSKSDTSATLDITGLQPGDEADYYCGTWDTSLSANWVFGGGTKLTVL
540 lambda
(SEQ ID NO: 16)
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGANYVSWYQQLPGTAPKLLIYNNNKRPSGIPDRFSGSKSDTSATLGITGLQTGDEADYYCGAWDSSLSASWVFGGGTKLTVL
544 lambda
(SEQ ID NO: 20)
SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSHVVFGGGTKLTVL
545 lambda
(SEQ ID NO: 22)
QSVLTQPSSVSGAPGQRVTISCTGQSSNIGAGYDVHWYQQFPGRAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQPEDEADYYCQSYDSRLSGSVFGGGTKLTVL
546 lambda
(SEQ ID NO: 24)
QSVLTQPSSVSEAPRQRVTISCSGSASNIGANGVSWYHQVPGKAPRLLLSHDGLVTSGVPDRLSVSKSGTSASLAISGLHSDDEGDYYCAVWDDSLNAVVFGGGTKLTVL
559 lambda
(SEQ ID NO: 34)
QSALTQPPSASGSPGQSITISCTGTNSDIGSYPFVSWYQRHPGKAPKLLIYDVSNRPSGVSDRFSGSKSGNTASLTISGLQAEDEGDYYCSSFTMNSFVIFGGGTKLTVL
F1-C6 lambda
(SEQ ID NO: 38)
QSVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLSGWVFGGGTKLTVL
FB1-A7 lambda
(SEQ ID NO: 40)
NFMLTQPHSVSESPGKTVTISCTRSGGGIGSSFVHWFQQRPGSSPTTVIFDDNQRPTGVPDRFSAAIDTSSSSASLTISGLTAEDEADYYCQSSHSTAVVFGGGTKLTVL
FD-B2 lambda
(SEQ ID NO: 42)
NFMLTQPHSVSESPGKTVTISCTRSSGSIATNYVQWYQQRPGSSPATVIYEDNQRPSGVPDRFSGSIDTSSNSASLTISGLTTEDEADYFCQSYGDNNWVFGGGTKLTVL
G1D4 lambda
(SEQ ID NO: 50)
NFMLTQPHSVSESPGKTVIIPCTRSSGSIASNYVQWYQKRPGSAPSIVIYEDKQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYNSRGVMFGGGTKLTVL
GC1E8 lambda
(SEQ ID NO: 52)
NFMLTQPHSVLESAGKTVTISCTRSSGSIASNYVQWYQQRPGTSPTNVIFEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYFCQSYDSNIWVFGGGTKLTVL
H1C12 lambda
(SEQ ID NO: 54)
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQHLPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSASLAIAGLQAEDEADYYCQSYDSSLSGSLVFGGGTKLTVL
IF-1C10 lambda
(SEQ ID NO: 58)
NFMLTQPHSVSESPGKTVTISCTGSGGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSTWVFGGGTKLTVL
IK-2E2 lambda
(SEQ ID NO: 60)
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWFQQHPGKAPKLMIYKVNNRPSGLSNRFSGSQSGNTASLTISGLQAEDEADYYCSSYTSSSTLGFGGGTKLTVL

Table 6
Constant region (CR) of a person
CR (constant region) antibodiesSequence
Constant region 1 lambda person (C1)
(SEQ ID NO: 63)
GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
Constant region 2 lambda man (C2)
(SEQ ID NO: 64)
GQPKAAPSVTLFPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
Constant region 3 lambda man (C3)
(SEQ ID NO: 65)
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS
Constant region 7 lambda man (C7)
(SEQ ID NO: 66)
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS
The constant region of human Kappa
(SEQ ID NO: 67)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
The constant region of human IgG1
(SEQ ID NO: 68)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Table 7
Define complementarity regions (CDR) of the heavy chains (HC) and light chains (LC) of the anti-Ang-2 antibodies
CDR 1CDR 2CDR 3
AntibodyResiduesResiduesResidues
Ab 526 HC SYGMH
(SEQ ID NO: 69)
AISGSGGSTYYADSVKG
(SEQ ID NO: 105)
DLLDYDILTGPYAY
(SEQ ID NO: 144)
Ab 526 KCRSSQSLLHSNGYNYLD
(SEQ ID NO: 70)
LGSNRAS -
(SEQ ID NO: 106)
MQALQTPPT
(SEQ ID NO: 145)
Ab 528 HCSYAIS
(SEQ ID NO: 71)
GIIPIFGTANYAQKFQG
(SEQ ID NO: 107)
GVVGDFDWLSFFDY
(SEQ ID NO: 146)
Ab 528 LCSGDKLGYTYTS
(SEQ ID NO: 72)
QDFKRPS
(SEQ ID NO: 108)
QAWDSTTAVV
(SEQ ID NO: 147)
Ab 531 HCSYAIS
(SEQ ID NO: 71)
GIIPILGIANYAQKFQG
(SEQ ID NO: 109)
DREDTAMVFNY
(SEQ ID NO: 148)
Ab 531 LCSGSSSNIGNNYVS
(SEQ ID NO: 73)
DNNKRPS
(SEQ ID NO: 110)
GTWDSSLSAFWV
(SEQ ID NO: 149)
Ab 533 HCSYGMH
(SEQ ID NO: 69)
YISSSGSTIYYADSVKG
(SEQ ID NO: 111)
DLLDYDILTGYGY
(SEQ ID NO: 150)
Ab 533 KCRSSQSLLHSNGYNYLN
(SEQ ID NO: 74)
LGSNRAS
(SEQ ID NO: 106)
MQGLQPPT
(SEQ ID NO: 151)
Ab 535 HCSYAIS
(SEQ ID NO: 71)
GIIPIFGTANYAQKFQG
(SEQ ID NO: 107)
FSPFTETDAFDI
(SEQ ID NO: 152)
Ab 535 LCSGSNSNIGNNFVS
(SEQ ID NO: 75)
DNNKRPS
(SEQ ID NO: 110)
GTWDSSLSAAEVV
(SEQ ID NO: 153)
Ab 536 HCSYGMH
(SEQ ID NO: 69)
YISSSGSTIYYADSVKG
(SEQ ID NO: 111)
DLLDYDILTGYGY
(SEQ ID NO: 150)
Ab 536 (THW) KCRSSQSLLHSNGYNYLD
(SEQ ID NO: 70)
LGSNRAS
(SEQ ID NO: 106)
MQGTHWPPT
(SEQ ID NO: 154)
Ab 536 (LQT) KCRSSQSLLHSNGYNYLD
(SEQ ID NO: 70)
LGSNRAS
(SEQ ID NO: 106)
MQGLQTPPT
(SEQ ID NO: 212)
Ab 537 HCSYAIS
(SEQ ID NO: 71)
GIIPILGIANYAQKFQG
(SEQ ID NO: 109)
GSSDAAVAGM
(SEQ ID NO: 155)
Ab 537 LCSGNNSNIGNNYVS
(SEQ ID NO: 76)
DNHKRPS
(SEQ ID NO: 112)
GTWDTSLSANWV
(SEQ ID NO: 156)
Ab 540 HCSYAIS
(SEQ ID NO: 71)/td>
GIIPILGIANYAQKFQG
(SEQ ID NO: 109)
AVPGTEDAFDI
(SEQ ID NO: 157)
Ab 540 LCSGSSSNIGANYVS
(SEQ ID NO: 77)
NNNKRPS
(SEQ ID NO: 113)
GAWDSSLSASWV
(SEQ ID NO: 158)
Ab 543 HCSYAIS
(SEQ ID NO: 71)
RIIPILGIANYAQKFQG
(SEQ ID NO: 114)
PYYDFWSGPGGMDV
(SEQ ID NO: 159)
Ab 543 KCRSSQSLLHSNGYNYLD
(SEQ ID NO: 70)
LGSNRAS
(SEQ ID NO: 106)
MQALQTPLT
(SEQ ID NO: 160)
Ab 544 HCSYAIS
(SEQ ID NO: 71)
GIIPIFGTANYAQKFQG
(SEQ ID NO: 107)
FESGYWGDAFDI
(SEQ ID NO: 161)
Ab 544 LCSGDALPKQYAY
(SEQ ID NO: 78)
KDSERPS
(SEQ ID NO: 115)
QSADSSHVV
(SEQ ID NO: 162)
Ab 545 HCSYGMH
(SEQ ID NO: 69)
VISYDGSNKYYADSVKG
(SEQ ID NO: 116)
GPVDFDYGDYAIDY
(SEQ ID NO: 163)
Ab 545 LCTGQSSNIGAGYDVH
(SEQ ID NO: 79)
GNSNRPS
(SEQ ID NO: 117)
QSYDSRLSGSV
(SEQ ID NO: 164)
Ab 546 HCSYAMS
(SEQ ID NO: 80)
AISGSGGSTYYADSVKG
(SEQ ID NO: 105)
ETISFSTFSGYFDY
(SEQ ID NO: 165)
Ab 546 LCSGSASNIGANGVS
(SEQ ID NO: 81)
HDGLVTS
(SEQ ID NO: 118)
AVWDDSLNAVV
(SEQ ID NO: 166)
Ab 551 HCSYAIS
(SEQ ID NO: 71)
GIIPIFGTANYAQKFQG
(SEQ ID NO: 107)
GYDFWSGYSLDAFDI
(SEQ ID NO: 167)
Ab 551 KCRSSQSLLHSNGYNYLD
(SEQ ID NO: 70)
LGSNRAS
(SEQ ID NO: 106)
MQALQTPLT
(SEQ ID NO: 160)
Ab 553 HCSYAMH
(SEQ ID NO: 82)
WINAGNGNTKYSQKFQG
(SEQ ID NO: 119)
GVDDYGGNSWAFDI
(SEQ ID NO: 168)
Ab 553 KCRSSQSLLHSNGYNYLD
(SEQ ID NO: 70)
LGSNRAS
(SEQ ID NO: 106)
MQALQTPLT
(SEQ ID NO: 160)
Ab 555 HCSYAMH
(SEQ ID NO: 82)
VISYDGSNKYYADSVKG
(SEQ ID NO: 116)
SASDHYYDSSGYYSDAFDI
(SEQ ID NO: 169)
Ab 555 KCRSSQSLLHSNGYNYLD
(SEQ ID NO: 70)
LASNRAS
(SEQ ID NO: 120)
MQTLQIPIT
(SEQ ID NO: 170)
Ab 558 HCGYYWS
(SEQ ID NO: 83)
EINHSGSTNFNPSLKS
(SEQ ID NO: 121)
GHDWGMGIGGAAYDI
(SEQ ID NO: 171)
Ab 558 KCRASQSVSSSSLA
(SEQ ID NO: 84)
AASSRAT
(SEQ ID NO: 122)
QHYGSSPRT
(SEQ ID NO: 172)
Ab 559 HCESSMH
(SEQ ID NO: 85)
GFDPEHGETIYAQKFQG
(SEQ ID NO: 123)
GVQVTSGYHYFDH
(SEQ ID NO: 173)
Ab 559 LCTGTNSDIGSYPFVS
(SEQ ID NO: 86)
DVSNRPS
(SEQ ID NO: 124)
SSFTMNSFVI
(SEQ ID NO: 174)
Ab 565 HCSYAIS
(SEQ ID NO: 71)
GIIPIFGTANYAQKFQG
(SEQ ID NO: 107)
SPIYYDILTGIDAFDI
(SEQ ID NO: 175)
Ab 565 KCRASQSVSSSSLA
(SEQ ID NO: 213)
AASSRAT
(SEQ ID NO: 214)
QHYGSSPRT
(SEQ ID NO: 215)
Ab 565 (2) KCRSSQSLLHSNGYNYLD
(SEQ ID NO: 70)
LGSSRAS
(SEQ ID NO: 125)
MQALDTPPT
(SEQ ID NO: 176)
A F1-C6 HC SYAIS
(SEQ ID NO: 71)
RIIPILGIANYAQKFQG
(SEQ ID NO: 114)
DPIPSGWYFDL
(SEQ ID NO: 177)
Ab F1-C6 LCSGSSSNIGNNAVN
(SEQ ID NO: 87)
YDDLLPS
(SEQ ID NO: 126)
ATWDDSLSGWV
(SEQ ID NO: 178)
Ab FB1-A7 HCSYGMH
(SEQ ID NO: 69)
VIWYDGSNKYYADSVKG
(SEQ ID NO: 127)
EVGNYYDSSGYGY
(SEQ ID NO: 179)
Ab FB1-A7 LCTRSGGGIGSSFVH
(SEQ ID NO: 88)
DDNQRPT
(SEQ ID NO: 128)
QSSHSTAVV
(SEQ ID NO: 180)
Ab FD-B2 HCSNSAAWN
(SEQ ID NO: 89)
RTYYRSKWYSDYAVSLRG
(SEQ ID NO: 129)
DRGGYIDS
(SEQ ID NO: 181)
Ab FD-B2 LCTRSSGSIATNYVQ
(SEQ ID NO: 90)
EDNQRPS
(SEQ ID NO: 130)
QSYGDNNWV
(SEQ ID NO: 182)
Ab FE-B7 HCDYEMN
(SEQ ID NO: 91)
YIIGSGKTIFYADSVKG
(SEQ ID NO: 131)
GGGSAYYLNTSDI
(SEQ ID NO: 183)
Ab FE-B7 KCRSSQSLLHSKGDNYLD
(SEQ ID NO: 92)
LGSHRAS
(SEQ ID NO: 132)
MQALQTPLT
(SEQ ID NO: 160)
Ab FJ-G11 HCSYGIS
(SEQ ID NO: 93)
WISAYNGNTNYAQKLQG
(SEQ ID NO: 133)
DRGIAARSAYYYGMDV
(SEQ ID NO: 184)
Ab FJ-G11 KCRSSQSLLDSDDGKTYLD
(SEQ ID NO: 94)
TTSSRAS
(SEQ ID NO: 134)
MQATQFPYT
(SEQ ID NO: 185)
Ab FK-E3 HCSYDLN
(SEQ ID NO: 95)
WMNPTSGNTGYAQKFQG
(SEQ ID NO: 135)
DPPSGGWEFDY
(SEQ ID NO: 186)
Ab FK-E3 KCRSSQSLVHEDGNTYLN
(SEQ ID NO: 96)
KISKRFS
(SEQ ID NO: 136)
MQSTRFPRT
(SEQ ID NO: 187)
Ab G1D4 HCSHAIS
(SEQ ID NO: 97)
RIIPILGIANYAQKFQG
(SEQ ID NO: 114)
SRLEWLLYLDY
(SEQ ID NO: 188)
Ab G1D4 LCTRSSGSIASNYVQ
(SEQ ID NO: 98)
EDKQRPS
(SEQ ID NO: 137)
QSYNSRGVM
(SEQ ID NO: 189)
Ab GC1E8 HCSYGIS
(SEQ ID NO: 93)
WISAYNGNTNYAQKLQG
(SEQ ID NO: 133)
GGSPYGGYAYPFDY
(SEQ ID NO: 190)
Ab GC1E8 LC TRSSGSIASNYVQ
(SEQ ID NO: 98)
EDNQRPS
(SEQ ID NO: 130)
QSYDSNIWV
(SEQ ID NO: 191)
Ab H1C12 HCSYGMH
(SEQ ID NO: 69)
YISSSGSTIYYADSVKG
(SEQ ID NO: 111)
DLLDYDILTGYGY
(SEQ ID NO: 150)
Ab H1C12 LCSGSSSNIGNNYVS
(SEQ ID NO: 73)
GNTNRPS
(SEQ ID NO: 138)
QSYDSSLSGSLV
(SEQ ID NO: 192)
Ab IA1-1E7 HCGYYWS
(SEQ ID NO: 83)
EINHSGSTNFNPSLKS
(SEQ ID NO: 121)
GHDWGMGIGGAAYDI
(SEQ ID NO: 171)
Ab IA1-1E7 KCRASQSVSSSFLA
(SEQ ID NO: 99)
DTSTRAT
(SEQ ID NO: 139)
QQYDFSPLT
(SEQ ID NO: 193)
Ab IF-1C10 HCSTYAMT
(SEQ ID NO: 100)
VIRSNGGTDYADFVKG
(SEQ ID NO: 140)
DYY
(SEQ ID NO: 194)
Ab IF-1C10 LCTGSGGSIASNYVQ
(SEQ ID NO: 101)
EDNQRPS
(SEQ ID NO: 130)
QSYDSSTWV
(SEQ ID NO: 195)
Ab IK-2E2 HCSYAMS
(SEQ ID NO: 80)
AISGSGGSTYYADSVKG
(SEQ ID NO: 105)
EISFSTFSGYFDY
(SEQ ID NO: 165)
Ab IK-2E2 LCTGTSSDVGGYNYVS
(SEQ ID NO: 102)
KVNNRPS
(SEQ ID NO: 141)
SSYTSSSTLG
(SEQ ID NO: 196)
Ab IP-2C11 HCSYDIN
(SEQ ID NO: 103)
WMNPNSGNTGYAQKFQG
(SEQ ID NO: 142)
EIAVAGTRYGMDV
(SEQ ID NO: 197)
Ab IP-2C11 KCRASQSISTFLA
(SEQ ID NO: 104)
DASNRAT
(SEQ ID NO: 143)
QHRINWPLT
(SEQ ID NO: 198)

Seventeen of these antibodies and negative control IgG1 (called RDB1) were tested using analysis of affinity and neutralization ELISA (as described in Example 3 above), as well as analysis of neutralization BIAcore to determine their affinity, the ability to neutralize and specificity. These results are presented in table 8, and they were calculated using standard protocols.

td align="center"> IC50 (nm)td align="center"> 1,41
Table 8
ES and IC50 of the anti-Ang-2 antibodies
hAng-2mAng-2hAng-1
AntibodyEC50 (nm)IC50 (nm)EC50 (nm)IC50 (nm)EC50 (nm)
Ab 536 (THW/LQT mix)0,080,0050,050,01114,6530
Ab 5650,260,26No inhibition
Ab 5460,371,09No inhibition
Ab 5430,510,24No inhibition
Ab 5330,30,08No inhibition
Ab 5370,560,62No inhibition
Ab 5400,701,53No inhibition
Ab 5440,971,8223,32
Ab 5451,040,021,300,058,312
Ab 5281,370,73No inhibition
Ab G1D41,390,6069,48
Ab 5512,88No inhibition

Ab 5531,471,41No inhibition
Ab 5261,830,27243,15
Ab 5312,151,67No inhibition
Ab 555of 2.211,76No inhibition
Ab 5352,812,45No inhibition
RDB1No inhibitionNo bindingNo inhibitionNo bindingNo inhibitionNo binding

Two antibodies, clone 536 and clone 545, were evaluated using BIAcore analysis as described above. Antibody binding was determined as described above for BIAcore analysis, and lower KDindicate a higher affinity, and the results are presented in table 9.

Table 9
The affinity of antibodies for hAng-2 and mAng-2
hAng-2mAng-2
AbKD(nm)ka(1/MS)kd(1/s)KD(nm)ka(1/MS)kd(1/s)
Ab 536 (THW/LQT mix)0,12of 3.2×105 of 3.8×10-50,156,2×1059,5×10-5
Ab 5451,2of 3.3×1053,9×10-40,9of 5.9×1055,3×10-4

Clone 536 analyzed above, contained a mixture of two variants of antibodies, which are shown in table 4 as SEQ ID NO:12 (536 Kappa THW) and SEQ ID NO:210 (536 Kappa LQT). These two options 536 were divided and analyzed separately on the activity using ELISA assays and HTRF.

For ELISA defenestration tablets were coated with recombinant angiopoietin in air-conditioned environment of the cells T (DMEM/50 μg/ml BSA) at 37°C for 1 hour. The air-conditioned environment used at concentrations of angiopoietin, which gave 80% of the maximum attainable binding of 1 nm hTie2-Fc (R&D Systems, catalog # 313-TI). The tablets were washed in PBS/0.1% tween-20 and then blocked for 2 hours at room temperature in PBS/5% BSA. The angiopoietin-neutralizing agents, titrated from 100 nm to 0.4 PM in a solution of PBS/1% BSA/1 nm Tie-2, was added to the coated angiopoietin tablets, which were incubated overnight at room temperature and then washed with PBS/0.1% tween-20. Received from the ISA anti-Tie-2 antibody (BD Pharmingen Inc., catalog # 557039) was added to each plate at a final concentration of 1 µg/ml (1 hour incubation at room temperature), after which the tablets were washed in PBS/0.1% tween-20. Goat antibody against mouse IgG-HRP (Pierce, catalog # 31432) was added at a dilution of 1:10000 in PBS/1% BSA (1 h were incubated at room temperature), after which the tablets were washed several times in PBS/1% tween-20. Added TMB-substrate (Sigma, catalog # T8665), was measured by optical density O.D. 370 nm and a degree of neutralization of complex angiopoietin:Tie-2 was determined by comparison against a standard curve of Tie-2.

For analysis HTRF defenestration microtiter "combined tablet was prepared by adding 50 μl of HTRF buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.05% tween-20, 0.1% BSA)containing 0.8 nm Europium-conjugated streptavidin (PERKIN ELMER LIFE SCIENCES INC. Catalog # AD0062) and 4.0 nm biotinylated angiopoietin 1 person (R&D Systems) or angiopoietin 2 (Amgen Inc.) in each well. In a separate microtiter tablet, the angiopoietin-neutralizing agents were titrated from 400 nm to 20 theorm in the HTRF buffer and then 50 µl of each serially diluted angiopoietin-neutralizing agent is transferred and mixed with a mixed tablet containing streptavidin-Europium/angiopoietin. Then this tablet incubated on a shaker at room temperature for one hour. Then, 20 μl from each well of "mixed planches is the" transferred in "tablet for analysis", containing 20 μl conjugated with 10 nm allophycocyanin human Tie-2-Fc man in HTRF buffer in each of the ninety-six wells. This is the ultimate tablet for analysis were incubated at room temperature with shaking for two hours. The final concentration tablet for analysis were: 1,0 nm angiopoietin, 5,0 nm Tie-2-Fc man and 100 nm to 5.0 PM serially diluted angiopoietin-neutralizing agents. Tablets for analysis were analyzed using a tablet reader Rubystar (BMG Labtechnologies, Offenberg, Germany). The degree of neutralization of complex angiopoietin:Tie-2 was determined by calculating the percentage inhibition of each breeding angiopoietin-neutralizing agent using the control without the angiopoietin-neutralizing agent (representing zero inhibition) and control without angiopoietin" (complete inhibition). Then the expected value IC50through analysis of the percentage inhibition using the program GRAFIT 5.0 (Erithacus Software Ltd.).

All results were expressed as curves IC50calculated from samples that were tested in duplicate, using the following formulas. The results of the IC50provide evidence of inhibition by 2-parametric equation, where the lower threshold data was equal to 0 (i.e., these data were corrected relative to the background), and Vyssi the threshold data was equal to 100 (i.e. these data were corrected for range).

In this equation, s denotes the coefficient of the slope. This equation implies that y decreases with increasing H. This equation is used in the program GRAFIT 5.0 (Erithacus Software Limited).

These results are shown in tables 10 and 11.

Table 10
IC50 results ELISA option Ab536
SampleIC50 human Ang1 (nm)IC50 human Ang2 (nm)IC50 Ang2 mouse (nm)
Ab536 LQT>1000,350,10
Ab536 THW>1000,310,088

Table 11
IC50 results HTRF option Ab536
SampleIC50 human Ang1 (nm)IC50 human Ang2 (nm)
Ab536 LQT>1000,072
Ab536 THW>100 0,071

EXAMPLE 5

Study of therapeutic effectiveness of using an anti-Ang-2 antibodies

The pharmacokinetics of Protein G-purified rabbit polyclonal anti-Ang-2 antibodies was investigated in mice. Twenty-four mice were treated with polyclonal rabbit anti-Ang-2 antibody (1 mg per mouse). Four treated animals were killed at each of the following time points after injection of antibodies: 1 hour, 6 hours, 1 day, 3 days, 7 days and 14 days.

The results showed that the total rabbit IgG had a half-life in serum is about 19 days, whereas anti-Ang-2-IgG-component of the total IgG had a half-life in serum is approximately eight days.

To evaluate therapeutic efficacy of mice (10 animals per group)bearing transplants of tumor A431, were introduced 10 doses (approximately 10 mg IgG per mouse per dose) administered intraperitoneally Protein G-purified polyclonal anti-Ang-2 antibodies in the days 1, 5, 6, 7, 8, 12, 13, 14, 15 and 18 after xenotransplantation. Tumor size was measured on days 7, 12, 15, 19 and 21. Body weight was measured on days 0, 7, 15 and 21, and body weight was not changed by treatment. The results showed that polyclonal anti-Ang-2 antibody inhibited the growth of transplant tumor A431 by approximately 50% with p=0.008 relative to control non-purified polyclonal antisera (10 mg IgG mouse on the RAM) and media (PBS) according to repeated measurements ANOVA.

To test the effectiveness of full monoclonal anti-Ang-2 antibodies of human in vivo to mice (10 animals per group)bearing transplants of tumor A431, were injected intraperitoneally clone anti-Ang-2 antibodies 533, 537 or 544 or negative control PBS or IgG1-Kappa man. Introduced approximately 420 µg of protein per mouse for the first dose, about 140 μg of protein per mouse for each of the next three doses and approximately 55 μg of protein per mouse for each of the subsequent four doses, a total of 8 doses per mouse. The volume of tumor and body weight was recorded twice a week. At the end of this study the animals were killed and their serum was collected to measure the level of antibodies using ELISA. The tumor and a panel of normal tissues were collected from all groups.

Were found clear differences in tumor growth between the anti-Ang-2 antibody-treated and control groups, as shown in figure 1. All three treatment anti-Ang-2 antibody inhibited tumor growth compared to controls (p < 0,005 against hIgG1-control in all treatments using repeated measure ANOVA for all 3 antibodies). In contrast, tumors in the control groups continued to grow at a much greater speed.

EXAMPLE 6

Mapping of epitopes

Full-size (amino acids 1-495), N-terminal (amino acids 1-254) and C-terminal (aminoxy is lots 255-495) proteins Ang-2 (hAng-2) human cloned into the CMV-started expressing vector mammals with C-terminal 6×His-tags. Three received designs and vector control transtorno was transfusional cells T. Then the conditioned medium was collected from the transfected cells and the expression level of Ang-2 in these environments was determined by anti-6×His-ELISA and Western blot testing.

The binding epitope of the anti-Ang-2 antibodies and peptide antibodies was determined by their ability to bind three versions of hAng-2 using ELISA in accordance with the following Protocol: 96-well plate for analysis of high binding were covered with 100 μl of conditioned medium per well and incubated at 37°C for 1 hour. Air-conditioned environment aspirated and the plate was blocked with 200 μl per well of 5% BSA in PBS at room temperature for 1 hour. Then the blocking solution was aspirated. 100 μl per well of antibody, peptide antibodies or Tie-2-Fc was added at 1 μg/ml in 1% BSA in PBS and incubated at room temperature for 1 hour. The wells were washed 4 times with 200 μl of 0.1% Tween in PBS. Added 100 μl per well of HRP-conjugated goat antibodies against human IgG or goat antibodies against mouse IgG and incubated at room temperature for 45 minutes Then the wells were washed with 200 μl of 0.1% Tween in PBS 4 times. Then add 100 μl per well of TMB substrate. O.D. was recorded at 370 nm.

These results are shown in figure 2A, figure 2B and figure 2C.

1. The antibody that is specificeski binds angiopoietin-2, containing a heavy chain and light chain, where the specified heavy chain contains a variable region heavy chain SEQ ID NO:11 or its antigennegative fragments of the light chain contains a variable region light chain containing the amino acid sequence of SEQ ID NO:210 or her antigennegative fragment, where antigennegative fragments contain at least CDR1, CDR2 and CDR3 SEQ ID NO:11 or SEQ ID NO:210.

2. The antibody that specifically binds angiopoietin-2, containing a heavy chain and light chain, where the light chain contains a variable region light chain containing the amino acid sequence of SEQ ID NO:210 or her antigennegative fragment.

3. The antibody or antigennegative fragment that specifically binds angiopoietin-2, containing a heavy chain and light chain, where
the heavy chain contains the frame region of the heavy chain and the variable region of the heavy chain, where the variable region of the heavy chain contains CDR1 (SEQ ID NO:69), CDR2 (SEQ ID NO:111), CDR3 (SEQ ID NO:150); and
light chain contains the frame region of the light chain and the variable region of light chain, where the variable region of the light chain contains CDR1 (SEQ ID NO:70), CDR2 (SEQ ID NO:106), CDR3 (SEQ ID NO:212).

4. Antibody containing a heavy chain and light chain, where
a) a heavy chain contains a variable region containing SEQ ID NO:11, and a constant region, sotiriadou is SEQ ID NO:68; and
b) the light chain contains a variable region containing SEQ ID NO:210, and a constant region containing SEQ ID NO:67;
where the antibody binds angiopoietin-2 (Ang-2).

5. The antibody or antigennegative fragment according to any one of claims 1 to 4, which is a polyclonal, monoclonal, chimeric, humanized or fully human antibody.

6. Antigennegative fragment of the antibody according to claim 5, containing single-chain Fv-antibody.

7. Antigennegative fragment of the antibody according to claim 5, containing the Fab fragment of the antibody.

8. Antigennegative fragment of the antibody according to claim 5, containing Fab'antibody fragment.

9. Antigennegative fragment of the antibody according to claim 5, containing F(AB')2-a fragment of an antibody.

10. The antibody according to any one of claims 1 to 4, representing an IgG1 antibody.

11. Pharmaceutical composition for treatment of conditions associated with increased expression of angiopoietin-2, containing the antibody according to claims 1 to 10 and a pharmaceutically acceptable required for the preparation of the agent.

12. Method of inhibiting undesired angiogenesis in a mammal, involving the administration to a mammal a therapeutically effective amount of the antibody according to any one of claims 1 to 10, where subject to inhibition of angiogenesis is associated with enhanced expression of angiopoietin-2.

13. A method of treating cancer in a mammal, involving the introduction therapeutically effective amount of the antibody according to any one of claims 1 to 10, where the recoverable cancer is associated with increased expression of angiopoietin-2.

14. A method of modulating at least one of the symptoms of vascular permeability or leakage of plasma from a mammal, involving the administration to a mammal a therapeutically effective amount of the antibody according to any one of claims 1 to 10, where vascular permeability or leakage of plasma associated with increased expression of angiopoietin-2.

15. A method of treating at least one condition of ocular neovascular diseases, hemangioblastoma, hemangioma, inflammatory diseases, chronic inflammatory disorders, endometriosis, and neoplastic diseases associated with bone disease or psoriasis in a mammal, involving the administration to a mammal a therapeutically effective amount of the antibody according to any one of claims 1 to 10, where the condition is associated with increased expression of angiopoietin-2.

16. The method according to item 13, additionally introducing a therapeutically effective amount of a chemotherapeutic agent in combination with the antibody.

17. The method according to clause 16, where the antibody and the chemotherapeutic agent are administered simultaneously.

18. The method according to clause 16, where the antibody and the chemotherapeutic agent are not administered simultaneously.

19. The nucleic acid molecule encoding the antibody according to any one of claims 1 to 10.

20. Expresser the store vector containing the nucleic acid molecule according to claim 19.

21. A host cell containing the vector of claim 20.

22. A method of obtaining antibodies, including:
(a) transforming the host cell, the selected nucleic acid molecule according to claim 19;
(b) the expression of the nucleic acid molecules in this cell the owner and
(c) the selection of the indicated antibodies.



 

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36 cl, 14 dwg, 6 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention relates to anti-M-CSF-specific antibodies based on RX1 or originating from RX1, and which more than 785% compete with monoclonal antibodies RX1, MC1 and/or MC3 for bonding with M-CSF (macrophagal colony-stimulating factor). The non-mouse antibody is two-stranded, contains a certain amino acid sequence (given in the formula of invention and list of sequences) and retains high affinity towards M-CSF. The invention discloses an isolated nucleic acid which codes the said antibody, an expression vector, a host cell and a method of producing the anti-M-CSF-antibody using a host cell or hybridome, particularly ATCC PTA-6263 or ATCC PTA-6264 hybridome. The invention describes a pharmaceutical composition containing said antibodies, sets containing pharmaceutical compositions and methods of preventing and treating osteoporosis in a person suffering from an osteolytic disease.

EFFECT: disclosed antibodies can inhibit osteoclast differentiation, which facilitates their use as highly effective preparations for treating osteolysis, cancer with metastases and osteoporosis associated with cancer metastases.

131 cl, 44 dwg, 12 tbl, 16 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention discloses a monomer single-strand V93 nanoantibody which can bind and inhibit the human vascular endothelial growth factor. The invention describes a nucleotide sequence which codes the V93 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V93 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V93 nanoantibody, as well as use of the V93 nanoantibody for qualitative and quantitative determination of VEGF in a sample.

EFFECT: use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

7 cl, 7 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention describes a nucleotide sequence which codes the V9 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V9 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V9 nanoantibody, as well as use of the V9 nanoantibody for qualitative and quantitative determination of VEGF in a sample. Use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

EFFECT: invention discloses a monomer single-strand V9 nanoantibody which can bind and inhibit the human vascular endothelial growth factor.

7 cl, 7 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology and immunology. An antibody against angiopoietin-2 is proposed. Versions of the antibody are disclosed, which are produced by hybridome ATCC PTA-7258, ATCC PTA-7259, ATCC PTA-7260. The corresponding coding nucleic acid and expression vector are disclosed. A host cell which produces the antibody based on the said vector is described. The disclosed antibodies have Kd of the order of 10-10-10-12 M, for the antibody 3.19.3 (from ATCC PTA-7260) IC50=99 nM. The said antibody properties can be used in treating human tumours.

EFFECT: design of a method of treating pathological angiogenesis based on an antibody and use of the antibody to prepare a medicinal agent for treating pathological angiogenesis.

33 cl, 18 dwg, 18 tbl, 24 ex

FIELD: medicine.

SUBSTANCE: method facilitates linkage of sequences, coding immunoglobulin variable regions, T-cells receptors or B-cells receptors. Method is instrument of higher effectivity for making sequence data libraries. Capability of multiple RT-PCR with chain extension by interruption with employment of matrix, derived from single cell, provides highly effective creation of sister pairs libraries.

EFFECT: method is effective for linkage of two or few nucleotide sequences, coding domens or subunits of heteromeric protein as a result of single reaction performance.

51 cl, 25 dwg, 27 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.

EFFECT: applicable in medicine for preparing a vaccine.

52 cl, 32 dwg, 7 tbl, 29 ex

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. Described are versions of the humanised antibody CD45RO/RB which carry a light and a heavy strand. Versions of the following are disclosed: isolated polynucleotide, coding antibody, expression vector containing a polynucleotide and host cells containing the expression vector. Described also is use of the antibody to treat and/or prevent various diseases, including as a component of a pharmaceutical composition.

EFFECT: invention provides antibodies identified as CD45RO and CD45RB, which can find use in medicine.

9 cl, 14 dwg, 2 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention discloses a monomer single-strand V93 nanoantibody which can bind and inhibit the human vascular endothelial growth factor. The invention describes a nucleotide sequence which codes the V93 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V93 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V93 nanoantibody, as well as use of the V93 nanoantibody for qualitative and quantitative determination of VEGF in a sample.

EFFECT: use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

7 cl, 7 dwg, 6 ex

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