Antibodies against angiopoietin-2 and use thereof

This application claims priority to US Provisional Application reg. Number 60/638354, filed December 21, 2004, and US Provisional Application reg. Number 60/711289, filed August 25, 2005 which are incorporated herein by reference.

<u>Field of the Invention</u>

The present invention relates to monoclonal antibodies against Angiopoietin-2 (Ang-2) and the use of such antibodies. More particularly, the present invention relates to fully human monoclonal antibodies against Ang-2 and uses of these antibodies. In other aspects, the present invention also relates to hybridomas or other cell lines expressing such antibodies. The described antibodies are useful as diagnostics and for the treatment of diseases associated with the activity and / or overproduction Ang-2.

<u>BACKGROUND ART</u>

Angiogenesis is the process of forming new capillaries from existing blood vessels and is an essential component of embryogenesis processes, normal physiological growth, tissue repair and tumor growth. While the responses of endothelial cells (EC)<i>in vitro</i> and blood vessel growth <i>in vivo</i> can be modulated by various factors, however, obvious that vascular EC only affect almost exclusively members of the family of vascular endothelial growth factor (VEGF) and the angiopoietins. Yancopoulos et al, Nature 407:. 242-48 (2000).

Angiopoietins were discovered as ligands for the Tie, that is, for members of the family of tyrosine kinases that is selectively expressed in the vascular endothelium. Yancopoulos et al, Nature 407:. 242-48 (2000). At present there are four different members of the angiopoietin family. Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely varying counterparts of the same gene locus in mouse and man. Kim et al.,<i>FEBS Let</i>, 443: 353-56 (1999); Kim et al, J Biol Chem 274:. 26523-28 (1999). Ang-1 and Ang-2 were first identified in tissue culture experiments for both agonist and antagonist, respectively. Davis et al.,<i>Cell</i> 87:1161-69 (1996); Maisonpierre et al., <i>Science</i> 277: 55- 60 (1997). All of the known angiopoietins bind primarily to Tie-2, and both Ang-1 and Ang-2 bind to Tie-2 with an affinity of 3 nM (Kd). Maisonpierre et al.,<i>Science</i> 277: 55-60 (1997). It was demonstrated that Ang-1 promotes an increase in life span of endothelial cells and preservation of endothelial integrity, Davis et al.,<i>Cell</i> 87:1161-69 (1996); Kwak et al., <i>FEBS</i> Lett 448: 249-53 (1999); Suri et al.<i>Science</i> 282:468-71 (1998); Thurston et al., <i>Science</i> 286: 2511-14 (1999); Thurston et al., <i>Night. With.</i> 6: 460-63 (2000), whereas Ang-2 has the opposite effect and promotes destabilization and regression of blood vessels in the absence of VEGF factors responsible for cell survival, or basic fibroblast growth factors. Maisonpierre et al, Science 277:. 55-60 (1997). However, in many studies, Ang-2 function has been suggested a more complex role of Ang-2. Ang-2 might be a complex regulator of vascular remodeling that plays a role in vascular development, and in their degradation. To confirm a role of Ang-2 were analyzed for its expression, which showed that a rapid induction of Ang-2 with VEGF leads to the development of angiogenesis in adults, and induction of Ang-2 in the absence of VEGF leads to vascular destruction. Holash et al.,<i>Science</i> 284: 1994-98 (1999); Hols et al.,<i>Oncogene</i> 18: 5356-62 (1999). In accordance with its environment dependent on a role, Ang-2 binds to the same specific for endothelium receptor Tie-2, which is activated by Ang-1, but this activation is dependent on its environment. Maisonpierre et al.,<i>Science</i> 277:55-60 (1997).

Angiogenesis in the cornea assays showed that Ang-1 and Ang-2 have similar effects, i.e. their effect with VEGF, which stimulates the growth of new blood vessels, it is synergistic. Asahara et al., Circ. Res. 83: 233-40 (1998). The possibility of increasing the dose-dependent endothelial response indicates observation of the fact that Ang-2 can also be pro-angiogenic in a high concentration<i>in vitro</i>. Kim et al, Oncogene 19:. 4549-52 (2000). At high concentration of Ang-2 acts as an endothelial cell survival factor in serum-deprivation induced apoptosis induced activation of Tie-2 kinase in the cascade of reactions PI-3 and Akt. Kim et al.,<i>Oncogene</i> 19:4549-52 (2000).

In other <i>in vitro</i> experiments have suggested that the long-term effects Ang-2 effects may gradually change from antagonicheskogo agonistic effect on the action with respect to the Tie-2, and in most later stages, they may directly lead to the formation and stabilization of microtubules vascular neovascularization. Teichert-Kuliszewska et al.,<i>Cardiovasc. Res.</i> 49: 659-70 (2001). In addition, when cultured in a fibrin gel-EC was also observed Tie-2 activation under the action of Ang-2 that is likely to indicate that the action of Ang-2 could depend on EC differentiation level. Teichert-Kuliszewska et al.,<i>Cardiovasc. Res</i>. 49: 659-70 (2001). The microvascular endothelial cells cultured in three-dimensional collagen gel, Ang-2 can also induce activation of Tie-2 and stimulate the formation of capillary-like structures. Mochizuki et al., J. Cell. Sci. 115: 175-83 (2002). Using three-dimensional spheroid co-culture as<i>in vitro</i> Model vessels maturation demonstrated that direct contacting endothelial cells with mesenchymal cells suppresses susceptibility to VEGF, whereas the presence of VEGF and Ang-2 induced proliferation of blood vessels. Korff et al.,<i>Faseb J.</i> 15: 447-57 (2001). Etoh et al. Demonstrated that the EC, which constitutively express the Tie-2, the expression of MMP-1, -9 and u-PA is highly activated by Ang-2 in the presence of Angiopoietin VEGF. Etoh, et al.,<i>Cancer Res.</i> 61: 2145-53 (2001). On<i>in vivo</i> model membrane pupil Lobov et al. showed that Ang-2 in the presence of endogenous VEGF induces a rapid increase in capillary diameter, remodeling of the basal plate, proliferation and migration of endothelial cells, and promotes the formation of new blood vessels. Lobov et al.,<i>Proc. Natl. Acad. Sci.</i> USA 99: 11205-10 (2002). In contrast, in the absence of endogenous VEGF, Ang-2 promotes endothelial cell death and destruction of the vessels. Lobov et al., Proc. Natl. Acad. Sci. USA 99: 11205-10 (2002). , In a similar manner<i>in vivo</i> tumor model, Vajkoczy et al. demonstrated that multicellular aggregates initiate vascular growth with the development of angiogenesis by VEGFR-2 and Ang-2 expression in a host simultaneous endothelium and endothelium in tumors. Vajkoczy et al., J. Clin. Invest. 109: 777-85 (2002). This model illustrated that the developing network of capillary vessels growing tumors is characterized by a continuous remodeling, probably mediated expression of VEGF and Ang-2. Vajkoczy et al., J. Clin. Invest. 109: 777-85 (2002).

The Tie-2 research and Angiopoietin-1 deficient for these proteins mice showed similar phenotypes, and it was suggested that stimulated by angiopoietin-1 phosphorylation of Tie-2 mediates remodeling and stabilization of developing vessels, it stimulates the development of blood vessels during angiogenesis and promotes adhesion endothelial cells to "anchor" cells (Dumont et al, Genes &amp; Development, 8:. 1897-1909 (1994); Sato, Nature, 376: 70-74 (1995); (Thurston, G. et al, 2000 Nature. Medicine: 6, 460-463)). Clearly, the role of Angiopoietin-1 is preserved in adult individuals, where it is constitutively expressed in a wide variety of different tissues (Hanahan, Science, 277: 48-50 (1997); Zagzag, et al, Exp Neurology, 159: 391-400. (1999)). In contrast, Angiopoietin-2 expression is restricted,<i>Oncogene</i> 18: 5356-62 (1999); Maisonpierre, 1997). Studies of Angiopoietin-2 expression in pathological angiogenesis have shown that many cancer types show expression of vascular Angiopoietin-2 (Maisonpierre et al.,<i>Science</i> 277: 55-60 (1997)). Functional studies performed in a murine xenograft model, suggest that Angiopoietin-2 is involved in tumor angiogenesis and associate Angiopoietin-2 overexpression with an increase in tumor growth (Ahmad, et al.,<i>Cancer Res</i>., 61: 1255-1259 (2001)). Other studies have shown that overexpression of Angiopoietin-2 is associated with tumor hypervascularity (Etoh, et al, Cancer Res 61: 2145-53 (2001); Tanaka et al, Cancer Res 62:.. 7124-29 (2002)..).

In recent years, there is provided the use of Angiopoietin-1, Angiopoietin-2 and / or Tie-2 as a possible anti-cancer therapeutic targets. Thus, for example, in U.S. Pat №№ 6166185, 5650490 and 5814464 describe ligand against Tie-2 and anti-receptor.

In studies conducted using soluble Tie-2 were reported to decrease the number and size of tumors in rodents (Lin, 1997; Lin 1998). Siemester et al. (1999) received human cell melanoma lines expressing the extracellular domain of Tie-2, and then these cells were injected "nude" mice, after which it was concluded that soluble Tie-2 strongly inhibits the growth of tumor and its angiogenesis . If we consider that Angiopoietin-1 and Angiopoietin-2 bind to Tie-2, then, based on these studies, it is unclear whether Angiopoietin-1 may, Angiopoietin-2 or Tie-2 serve as an attractive target for anticancer therapy. However, it is obvious that therapy directed against Angiopoietin-2 may be effective for the treatment of diseases such as cancer, progression of which depends on the degree of angiogenesis disorders, where blocking the process can lead to prevention of disease progression (Folkman, J., Nature Medicine 1:. 27-31 (1995)). In addition, some research groups have reported the use of antibodies that bind to Angiopoietin-2, see. E.g., U.S. Patent 6,166,185 and № Application Publication U.S. 2003/0124129 Al №. Investigation of the local expression of Angiopoietin-2 effect showed that antagonize Angiopoietin-1 / Tie-2 signal leads to a weakening of dense vascular structure and, thereby, to the transmission of activating signals to the endothelial cells (EC) inducers of angiogenesis, for example, VEGF. (Hanahan, 1997). Such pro-angiogenic effect induced inhibition of Angiopoietin-1 indicates that the therapy is directed against Angiopoietin-1, apparently

Ang-2 is expressed during vascular development at sites where blood vessel remodeling occurs. Maisonpierre et al, Science 277:. 55-60 (1997). In adult individuals Ang-2 expression is restricted to sites of vascular remodeling as well as in tumors with a high degree of vascularization, including glioma (Osada et al.,<i>Int. J. Oncol.</i> 18:305-09 (2001); Koga et al., Cancer Res. 61:6248-54 (2001)), гепатоцеллюлярную карциному (Tanaka et al., <i>J. Clin. Invest.</i> 103: 341-45 (1999)), карциному желудка (Etoh, et al. <i>Cancer Res.</i> 61:2145-53 (2001); Lee et al., <i>Int. J. Oncol.</i> 18: 355-61 (2001)), thyroid tumor (Bunone et al,. <i>Am. J. Pathol.</i> 155: 1967-76 (1999)), non-small cell lung cancer (Wong et al,. <i>Lung Cancer </i>29: 11-22 (2000)), colon cancers (Ahmad et al,. <i>Cancer</i> 92: 1138-43 (2001)), and prostate (Wurmbach et al,. <i>Anticancer Res</i>. 20: 5217-20 (2000)). It has been found that some tumor cells express Ang-2. For example, Tanaka et al. (<i>J. Clin. Invest</i>. 103: 341-45 (1999)) was detected Ang-2 mRNA in 10 out of 12 specimens of human hepatocellular carcinoma (HCC). Ellis group reported that Ang-2 is expressed ubiquitously in tumor epithelium. Ahmad et al, Cancer 92:. 1138-43 (2001). Other researchers have reported similar findings. Chen et al., J. Tongji Med. Univ. 21: 228-30, 235 (2001). Based on the determination of mRNA levels of Ang-2 samples in archived human breast cancer tissues, Sfilogoi et al (Int J. Cancer 103:. 466-74 (2003)). Reported that Ang-2 mRNA is associated mainly with additional lymph node invasion, short latency periods of the disease and, in general, the short duration of life. Tanaka et al. (<i>Cancer Res.</i> 62: 7124-29 (2002)) was observed in all 236 patients with non-small cell lung cancer (NSCLC) on pathological stage I-IIIA of the disease, respectively. Conducted their immunohistochemical analysis showed that 16.9% of patients with NSCLC were positive for Ang-2. Microvessel density in the Ang-2 positive tumor is significantly higher than in the Ang-2-negative tumors. Such an angiogenic effect of Ang-2 was only observed in the case of a high level of expression of VEGF. In addition, positive specimens expression of Ang-2 is an important factor, which is an indicator of poor prognosis concerning the duration of life after operation. Tanaka et al., Cancer Res. 62: 7124-29 (2002). However, any significant correlation between Ang-1 expression and the microvessel density was found. Tanaka et al.,<i>Cancer Res.</i> 62: 7124-29 (2002). These results suggest that Ang-2 is an indicator of poor prognosis for patients with cancer of various types.

Recently, a group of Yancopoulos, conducted the study on the Ang-2-deficient mouse models, reported that Ang-2 is required for postnatal angiogenesis. Gale et al.,<i>Dev. Cell</i> 3: 411-23 (2002). These researchers have shown that genetically programmed regression of vasculature eye vitreous body is absent in Ang-2^-/-mice and their retinal blood vessels were not developed from the central retinal artery. Gale et al., Dev. Cell 3: 411-23 (2002). These researchers also found that deletion of Ang-2 leads to serious disruption of the development and function of the lymphatic vessels. Gale et al.,<i>Dev. Cell</i> 3: 411-23 (2002). Saving gene Ang-1 corrects the defects of lymph vessels, but does not eliminate the angiogenesis defects. Gale et al.,<i>Dev. Cell</i> 3:411-23 (2002).

Peters (Peters) and colleagues reported that soluble Tie-2, when delivered either as recombinant protein or in a viral expression vector, inhibited <i>in vivo</i> the growth of breast carcinoma and a murine melanoma in mouse models. Lin et al., Proc. Natl Acad. Sci USA 95: 8829-34 (1998); Lin et al., J. Clin. Invest. 100: 2072-78 (1997). vascular density in the thus treated tumor tissues is significantly reduced. Moreover, the soluble Tie-2 blocked angiogenesis in the rat corneal stimulated by conditioned medium from tumor cells. Lin et al., J. Clin. Invest. 100: 2072-78 (1997). Furthermore, Isner and his team demonstrated that attachment of Ang-2 to VEGF induces significantly longer and more extensive neovascularization than one VEGF. Asahara et al.,<i>Circ. Res</i>. 83: 233-40 (1998). Excess soluble Tie-2 receptor prevents modulation of VEGF-induced neovascularization by Ang-2 action. Asahara et al.,<i>Circ. Res</i>. 83: 233-40 (1998). Siemeister et al. (<i>Cancer Res.</i> 59: 3185-91 (1999)) in the studies conducted on the "naked" mice with xenografts showed that overexpression of the extracellular ligand-binding domains of FIt-I or Tie-2 in these xenografts results in significant inhibition of the pathways of reactions that do not can compensate for each other, which suggests that the VEGF receptor pathway and Tie-2 pathway should be considered as two independent neurotransmitter playing an important role in angiogenesis <i>in vivo</i>. Siemeister et al., <i>Cancer Res</i>. 59: 3185-91 (1999). This is confirmed later publications White et al.,<i>Proc. Natl. Acad. Sci. USA</i> 100: 5028-33 (2003). In this study it was demonstrated that a nuclease-resistant RNA aptamer that specifically binds to Ang-2 and inhibits Ang-2 significantly inhibited neovascularization induced by bFGF in the angiogenesis model in the rat corneal micropocket.

<u>SUMMARY OF THE INVENTION</u>

In one embodiment, the present invention relates to the aimed at target binding agents that specifically bind to Angiopoietin-2 and thereby inhibit tumor angiogenesis and reduce tumor growth. The mechanisms by which may be achieved, this effect can be, but are not limited to, inhibition of Ang-2 binding to its receptor, Tie-2, inhibition of Ang-2-induced transmission Tie-2 signal or a gain in clearance Ang-2, and thereby, reducing the effective concentration of Ang-2.

In one embodiment, aiming binding agent is a fully human antibody that binds to Ang-2 and prevents Ang-2 binding to Tie-2. In yet another embodiment, the present invention relates to fully human monoclonal antibody that binds to Ang-2 and Ang-1, and also inhibits Ang-2 induced Tie-2 phosphorylation. The antibody may bind Ang-2 with a K_d less than 100 pM, 30 pM, 20 pM, 10 pM or 5 pM.

The antibody may comprise a heavy chain amino acid sequence having a complementarity determining region (complementarity-determining region (CDR)), which is one of the sequences presented in Table 11. It should be noted that one skilled in the art can readily determine such CDR. See. Eg, Kabat et al manual.,<i>Sequences of Proteins of Immunological Interest</i>, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols.1-3.

One embodiment of the present invention comprises fully human monoclonal antibodies 3.3.2 (accession number ATCC PTA-7258), 3.19.3 (accession number ATCC PTA-7260) and 5.88.3 (accession number ATCC PTA-7259), which specifically bind to Ang-2, as discussed in more detail below.

In yet another embodiment, the present invention relates to an antibody that binds to Ang-2 and comprises a light chain amino acid sequence having a CDR, comprising one of the sequences shown in Table 12. In certain embodiments, the antibody is a fully human monoclonal antibody .

In another embodiment, the present invention relates to an antibody that binds to Ang-2 and comprises the amino acid sequence of a heavy chain having one of the sequences of CDR set forth in Table 11, and the amino acid sequence of the light chain, having one of the CDR sequences shown in Table 12. In some embodiments, the antibody is a fully human monoclonal antibody. Another embodiment of the present invention is an antibody that competes with the fully human antibodies of the invention for cross-linking with Ang-2, and preferably, an antibody comprising the amino acid sequence of a heavy chain having one of the sequences of CDR, shown in Table 11, and the amino acid sequence of the light chain .

Other embodiments of the invention include human monoclonal antibodies that specifically bind to Angiopoietin-2, wherein said antibodies comprise complementarity determining region 1 (CDRl) of the heavy chain corresponding to canonical class 1. The antibodies described herein can also include a complementarity determining region 2 (CDR2) of the heavy chain, corresponding to canonical class 3, the hypervariable region 1 (CDR1) of the light chain corresponding to canonical class 2, complementarity determining region 2 (CDR2) of the light chain corresponding to the canon nical class 1 and complementarity determining region 3 (CDR3) of the light chain corresponding to canonical class 1.

The present invention also relates to methods for analyzing the level of Angiopoietin-2 (Ang-2) in a sample from a patient, wherein said method comprises contacting an anti-Ang-2 antibody with a biological sample taken from a patient and detecting the level of binding of said antibody to Ang -2 in said sample. In more specific embodiments of the invention, said biological sample is blood.

In further embodiments, the present invention relates to compositions comprising an antibody or functional fragment thereof and a pharmaceutically acceptable carrier.

In further embodiments, the present invention relates to methods for effectively treating an animal suffering from a disease associated with angiogenesis, wherein the methods include selecting an animal in need of treatment of neoplastic or non-neoplastic disease, and administering to said animal a therapeutically effective dose of a fully human monoclonal antibodies that specifically binds to Angiopoietin-2 (Ang-2).

Associated with angiogenesis disorders treatable may be neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), thyroid tumor, cancer of the gastrointestinal tract (stomach) cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, esophageal carcinoma, head and neck cancer, mesothelioma, sarcomas, biliary olangiokartsinoma, adenocarcinoma of the small intestine, cancer in children and squamous cell carcinoma.

In further embodiments, the present invention relates to methods of inhibiting Angiopoietin-2 induced (Ang-2) angiogenesis in animals. Such methods include selecting an animal in need of treatment of Ang-2 induced angiogenesis, and administering to said animal a therapeutically effective dose of a fully human monoclonal antibody wherein said antibody specifically binds to Ang-2.

In further embodiments, the present invention relates to the use of antibodies for preparation of a medicament for the treatment of diseases associated with angiogenesis in an animal, wherein said monoclonal antibody specifically binds to Angiopoietin-2 (Ang-2). Associated with angiogenesis disorders treatable may be neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), thyroid tumor, cancer of the gastrointestinal tract (stomach) cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, esophageal carcinoma, head and neck cancer, mesothelioma, sarcomas, biliary olangiokartsinoma,

In other embodiments, the antibodies described herein may be used for the preparation of a medicament for the effective treatment of Angiopoietin-2 induced angiogenesis in an animal, wherein said monoclonal antibody specifically binds to Angiopoietin-2 (Ang-2).

In other embodiments described herein, the present invention relates to monoclonal antibodies that bind Ang-2 and affect Ang-2 function. In further embodiments, the present invention relates to fully human antibodies against Ang-2, and to preparations of antibodies against Ang-2 possessing useful and promising therapeutic point of view of properties, including high binding affinity for Ang-2, the ability to neutralize Ang-2<i>in vitro</i> and <i>in vivo</i> and the ability to inhibit Ang-2 induced angiogenesis.

In a preferred embodiment, antibodies described herein bind to Ang-2 with very high affinities (Kd). Thus, for example, human, rabbit, mouse, chimeric or humanized antibody capable of binding to Ang-2 with a Kd, having a value of, but not limited to, at least 10^-5, 10^-6, 10^-7, 10^-8, 10^-9, 10^-10, 10^-11, 10^-12, 10^-13 or 10^-14 M, or any range of values ​​indicated, or any value in this range. Affinity and / or avidity can be measured by analysis using KinExA® and / or BIACORE®, as described below.

Accordingly, in one embodiment described herein, the present invention relates to isolated antibodies, or fragments of those antibodies, that bind to Ang-2. As known in the art, such antibodies can advantageously be, for example, polyclonal, oligoclonal, monoclonal, chimeric, humanized and / or fully human antibodies. In other embodiments described herein, the present invention also relates to cells producing such antibodies.

In another embodiment, the present invention relates to fully human antibody that binds to other members of the family to which it belongs angiopoietin-2, including but not limited to, Angiopoietin-1, Angiopoietin-3 and Angiopoietin-4. In another embodiment, the present invention relates to an antibody that competes with the fully human antibody of the invention for cross-linking with Tie-2 / Ang-2. In one embodiment, the antibody binds Angiopoietin-2 and neutralizes Angiopoietin-2, and also binds to Angiopoietin-1 and Angiopoietin-1 and cures.

It should be noted that embodiments of the present invention is not limited to any particular form of an antibody or method of generation or production of it. For example, anti-Ang-2 antibody may be a full length antibody (e.g., antibody, having an intact human Fc-region) or an antibody fragment (e.g., Fab, Fab 'or F (ab')₂). Furthermore, the antibody can be obtained from a hybridoma that secretes the antibody, or from a recombinantly-derived cell that has been transformed or transfected with a gene or genes encoding the antibody.

Other embodiments of the invention include isolated nucleic acid molecules encoding any of the antibodies described herein; vectors comprising the isolated nucleic acid molecule; encoding anti-Ang-2 antibody; or a host cell transformed with any of such nucleic acid molecules. Furthermore, in one embodiment, the present invention relates to a method of producing an anti-Ang-2 antibody by culturing host cells under conditions favoring expression of the nucleic acid molecule and production of antibodies, and subsequent isolation of such antibodies. It should be noted that embodiments of the present invention also include any nucleic acid molecule that encodes the antibody of the invention or fragment thereof, including a nucleic acid sequence,

Another embodiment of the present invention includes a method of producing high affinity antibodies to Ang-2 by immunizing a mammal with human Ang-2 or a fragment thereof and one or more orthologous sequences or fragments thereof.

Other embodiments of the invention are based on the generation and identification of isolated antibodies that bind specifically to Ang-2. In diseases associated with angiogenesis such as tumor diseases, Ang-2 is expressed at elevated levels. Inhibition of the biological activity of Ang-2 can prevent Ang-2 induced angiogenesis and other desired effects.

Another embodiment of the present invention includes a method of diagnosing diseases or conditions in which an antibody prepared as described herein, is used to detect the level of Ang-2 in a sample from the patient. In one embodiment, the samples taken from the patient is blood or blood serum. In other embodiments, the invention describes methods for the identification of risk factors, diagnosis of disease, and staging of disease, wherein said method comprises identification of the overexpression of Ang-2 using anti-Ang-2 antibodies.

Another embodiment of the present invention includes a method of diagnosing a condition associated with the expression of Ang-2 in the cells, by contacting the serum or a cell with an anti-Ang-2 antibody, followed by detection of the presence of Ang-2. Preferred conditions include diseases associated with angiogenesis, including but not limited to, neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), glioblastoma, and carcinoma of the thyroid, stomach, prostate prostate, breast, ovary, bladder, lung, uterus, kidney, colon, pancreas, salivary gland and colon.

In another embodiment, the present invention includes an assay kit for detecting Angiopoietin-2 and members of the angiopoietin family in tissues, cells or body fluids of mammals in order to screen for diseases associated with angiogenesis. The kit includes an antibody that binds to Angiopoietin-2, and means, which indicator antibody reaction with Angiopoietin-2, if it occurs. A preferred antibody is a monoclonal antibody. In one embodiment, the antibody that binds to Ang-2 is labeled. In another embodiment, the antibody is unlabeled "first" antibody, and wherein said kit further comprises means for detection of said "first" antibody. In one embodiment of the invention, said means includes a labeled the "second" antibody which is an antibody against immunoglobulin. A preferred antibody is an antibody labeled with a marker selected from the group consisting of a fluorochrome, an enzyme, a radionuclide and a material which is impermeable to radiation.

Another embodiment of the present invention includes methods for treating diseases or conditions associated with the expression of Ang-2 in a patient by administering to said patient an effective amount of an anti-Ang-2 antibody. Anti-Ang-2 antibody can be administered alone or it can be administered in combination with other antibodies or chemotherapeutic drug or in combination with radiotherapy. For example, a mixture of monoclonal, oligoclonal or polyclonal antibodies against Ang-2 that block angiogenesis can be administered in combination with a drug which has been shown to directly inhibit the proliferation of tumor cells. The method may be carried out<i>in vivo</i>And said patient, preferably a human. In a preferred embodiment, the method comprises treatment associated with angiogenesis disorders, including but not limited to, neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), glioblastoma, and carcinoma of the thyroid prostate, stomach, prostate, breast, ovary, bladder, lung, uterus, kidney, colon, pancreas, salivary gland and colon.

In another embodiment, the present invention relates to an article of manufacture comprising a container. Such a container includes a composition containing an anti-Ang-2 antibody, and a package insert or label on this packaging, which indicates that the composition can be used to treat angiogenesis associated with diseases characterized by the overexpression of Ang-2.

In some embodiments, the patient is administered an anti-Ang-2 antibody, and then the cleaning agent to remove excess antibodies from the bloodstream.

In another embodiment, the present invention relates to the use of anti-Ang-2 antibody for the preparation of a medicament for the treatment of diseases such as diseases associated with angiogenesis. In one embodiment, associated with angiogenesis diseases include carcinoma, such as breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, colon, esophageal, thyroid, pancreatic, prostate and bladder bubble. In another embodiment of the invention associated with angiogenesis diseases include but are not limited to, neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), sarcoma, head and neck cancer, mesothelioma, biliary cholangiocarcinoma,

Ang-2 is an important "switch" of angiogenesis. In accordance with this, it is assumed that the suppression of the activity of this molecule leads to an inhibition of pathophysiological processes and therefore can serve as an effective treatment for various angiogenesis-dependent diseases. In addition to solid tumors and their metastases, angiogenesis-dependent cancers are also the blood such as leukemia, lymphoma and multiple myeloma. Excessive vascular growth leads to a different non-tumor dist . Roystv These non-neoplastic angiogenesis-dependent diseases include: atherosclerosis, hemangioma, hemangioendothelioma, angiofibroma, impaired vascular development (e.g., hereditary hemorrhagic telangiectasia (HHT), or Osler-Weber syndrome), warts, pyogenic granulomas, excessive hair growth, Kaposi's sarcoma, keloids, allergic edema, psoriasis, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation, endometriosis, respiratory distress, ascites, peritoneal sclerosis in a patient undergoing dialysis, the formation of adhesions following surgery, abdominal obesity, rheumatoid arthritis, synovitis, osteomyelitis, pannus overgrowth, osteophyte, hemophilic arthropathy, inflammatory and infektsirnnye processes (e.g. hepatitis, pneumonia, glomerulonephritis), asthma, poly nN nasal, liver regeneration, pulmonary hypertension, retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, leukomalacia, neovascular glaucoma, corneal graft neovascularization, trachoma, thyroiditis,

<u>BRIEF DESCRIPTION OF THE DRAWINGS</u>

Figure 1 shows a Western blot analysis which showed that mAb against Ang-2 inhibits Ang-2 induced phosphorylation of Tie-2 ectopically expressed in HEK293F cells.

Figure 2 shows the linear dose-response curve of inhibitory effect of monoclonal anti-Ang-2 antibodies on the Ang-2-induced phosphorylation of Tie-2.

Figure 3 is a line graph showing dose-dependent inhibition of binding of Ang-1 (top graph) and Ang-2 (bottom graph) from Tie-2 in the presence of mAb 3.19.3, or Tie-2 / Fc.

Figure 4 shows a Western blot analysis showing inhibition of Angiopoietin-1 stimulated phosphorylation of Tie-2 in endothelial cells Eahy 926 under the action of mAb 3.19.3. In this system, there is inhibition of Angiopoietin-1 induced phosphorylation of Tie-2. Antibody concentrations are shown in nM.

Figure 5 is a line graph showing inhibition of Angiopoietin-1 stimulated phosphorylation of Tie-2 in endothelial cells Eahy 926 under the action of mAb 3.19.3. IC50 = 99 nM. The axis x is the concentration of mAb 3.19.3, and y axis represents response.

Figure 6 schematically shows a diagram of human protein structure Ang-2 and Ang-2₄₄₃. The upper numbers denote amino acid sequences (this diagram is taken from the publication Injune et al, (2000) JBC 275:. 18550).

Figure 7 shows the amino acid sequence of the chimeric molecules of mouse / human (SEQ ID NO: 1). Human residues (cloned as<i>Stu</i>I<i>-Tfi</i>I-fragment) 310-400 are underlined.

Figure 8 shows a comparison of the amino acid sequences of proteins of human Ang-1 (SEQ ID NO: 2), human Ang-2 (SEQ ID NO: 3) and mouse Ang-2 (SEQ ID NO: 4). Sites attachment chimeric Ang-2 molecules and point mutations are indicated in bold.

Figure 9 shows a comparison of the amino acid sequences of mouse Ang-1 (SEQ ID NO: 5), human Ang-1 (SEQ ID NO: 2), mouse Ang-2 (SEQ ID NO: 4) and human Ang-2 (SEQ ID NO: 3). Arrows indicate the cleavage site for hydrophobic leader sequences. These arrows define the limits supercpiralizovannyh and fibrinogen-like domains. Shaded circles indicate conserved cysteine ​​residues (this graph was taken from a publication Maisonpierre et al., 1997,<i>Science</i> 277:55).

Figure 10 is a line graph showing dose-dependent cross-reactivity in mice. Monoclonal antibody clones 5.2.1 Also shown, 5.28.1, 3.19.3 and 3.31.2.

Figure 11 is a line graph showing dose-dependent inhibition of human (black triangles) and mouse (black squares) Ang-2 with the human Tie-2 in the presence of mAb 3.19.3.

Figure 12 is a bar graph illustrating the influence of antibodies on analysis angiogenesis induced MCF-7 cells. In Figure 12A show the influence of anti-Ang-2 antibodies on the number of vascular branching, wherein the axle<i><u>x</u></i> given the experimental group, and the axis <i><u>Y</u></i> the average number of vascular branches (± SEM). In Figure 12B show the influence of anti-Ang-2 antibodies on blood vessel length where the axle<i><u>x</u></i> given the experimental group, and the axis <i><u>Y</u></i> It contains the average length of the blood vessels (± SEM).

Figure 13 is a line graph showing the antitumor effect of clone 3.19.3, monoclonal anti-Ang-2 antibody, analyzed in a mouse xenograft model of human squamous cell carcinoma of the skin using the cell line A431. on the axis<i><u>x</u></i> indicates the number of days after tumor cell implantation, and on the axis <i><u>in</u></i> indicates the mean tumor volume (± SEM in cm³). Black triangles indicate the tumor volume measured after tumor cell implantation the mice injected with clone 3.19.3 monoclonal anti-Ang-2 antibody; and the black circles indicate the tumor volume measured after the implantation of tumor cells into mice, which were injected isotype control antibody PKl 6.3.1.

Figure 14A is a line graph showing prevention of tumor growth in a xenograft model of human adenocarcinoma LoVo with said tumor size in mice treated with 0.5, 2 and 10 mg / kg of antibody or isotype control antibody. on the axis<i><u>x</u></i> indicates the number of days after tumor cell implantation, and on the axis <i><u>in</u></i> indicates the mean tumor volume (± SEM in cm³). Figure 14B is a line graph showing the effect of inhibiting tumor growth by the action of mAb in a xenograft model of human adenocarcinoma SW480 colon.

In Figure 15A is a line graph showing prevention of tumor growth in the HT29 xenograft model. on the axis<i><u>x</u></i> indicates the number of days after tumor cell implantation, and on the axis <i><u>in</u></i> indicates the mean tumor volume (± SEM in cm³). Figure 15B is a line graph showing prevention of tumor growth in a xenograft model Calu-6, which for mice treated 10 mg / kg clone mAb 3.3.2 or 3.19.3, or isotype control antibody, specified tumor size. Figure 15C is a line graph showing the density of CD31⁺-okrashivaniya tumors in tumor-bearing mice MDA-MB-231 and treated with control IgG or 10mg / kg 3.19.3 mAb. Also present the results obtained by calculating a threshold method and manual counting using a grid.

<u>DETAILED DESCRIPTION OF THE INVENTION</u>

Embodiments of the invention described herein relate to monoclonal antibodies that bind Ang-2. In certain embodiments, the antibodies bind to Ang-2 and inhibit the binding of Ang-2 to its receptor, Tie-2. In further embodiments, the present invention relates to fully human anti-Ang-2 antibodies and antibody preparations which can be used for therapeutic purposes. These preparations of anti-Ang-2 antibodies preferably possess desirable therapeutic properties, including high binding affinity for Ang-2, the ability to neutralize Ang-2<i>in vitro</i> and the ability to inhibit Ang-2-induced angiogenesis <i>in vivo</i>.

In one embodiment, the present invention relates to an antibody that binds to Ang-2, and neutralizes Ang-2, but does not bind to Ang-1. In another embodiment, the antibody binds to Ang-2 and Ang-1, but only neutralizes Ang-2. In another embodiment, the antibody binds to both Ang-2 and Ang-1, and neutralizes binding of both Ang-1 and Ang-2 with Tie-2.

Embodiments of the invention also include isolated binding fragments of anti-Ang-2 antibodies. Such binding moieties are preferably derived from full-length human anti-Ang-2 antibodies. Illustrated fragments include Fv, Fab 'or other well-known antibody fragments, which will be described below in more detail. Embodiments of the invention also include cells expressing full-length human antibodies against Ang-2. Examples of such cells include hybridoma cells, or by recombinant methods, such as Chinese hamster ovary cells (CHO), variants of CHO cells (e.g., DG44) and NSO cells that produce antibodies against Ang-2. Additional information about variants of CHO cells can be found in the publication Andersen and Reilly (2004) Current Opinion in Biotechnology 15, 456-462,

Moreover, in further embodiments, the present invention relates to methods of using these antibodies for treating diseases. Anti-Ang-2 antibodies may be used for preventing Ang-2 mediated Tie-2 transmission signal, and thereby for inhibiting angiogenesis. The mechanism of action of this inhibition may include inhibition of binding of Ang-2 to its receptor, Tie-2; inhibition of Ang-2 induced Tie-2 transmission signal; or enhanced clearance of Ang-2, leading to a decrease in the effective concentration of Ang-2 binding to Tie-2. Diseases that are treatable through this inhibition mechanism include, but are not limited to, neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), glioblastoma,

Other embodiments of the invention include diagnostic assays, particularly to determine the amount of Ang-2 in a biological sample. The assay kit can include anti-Ang-2 antibodies along with the necessary labels for detecting such antibodies. Such diagnostic assays can be used to screen for angiogenesis-related diseases including, but not limited to, neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), glioblastoma, and carcinoma of the thyroid prostate, stomach, prostate, breast, ovary, bladder, lung, uterus, kidney, colon, pancreas, salivary gland and colon.

According to one of its aspects, the present invention relates to an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2 wherein the antagonist does not bind to ATP-binding site of Tie-2.

In accordance with another aspect, the present invention relates to an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2 wherein the antagonist binds to Angiopoietin-1 and Angiopoietin-2.

In accordance with another aspect, the present invention relates to an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2 wherein the antagonist is not a compound.

In one embodiment, the present invention relates to an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2 wherein the antagonist activity directed against Angiopoietin-1 and said antagonistic activity directed against Angiopoietin-2 are enclosed in a single molecule. In its alternate embodiment, the present invention relates to the antagonist, wherein the antagonist activity against Angiopoietin-1 and said antagonistic activity against Angiopoietin-2 are enclosed in more than one molecule.

In one embodiment, the present invention relates to an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2 wherein the antagonist may bind to:

i) to the receptor Tie-2;

ii) from Angiopoietin-1 and / or Angiopoietin-2;

iii) a "Tie-2 receptor - Angiopoietin-1" complex; or

iv) a "Tie-2 receptor - angiopoietin-2" complex; or

with any combination thereof.

In one embodiment, an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2 may bind to Angiopoietin-1 and / or Angiopoietin-2 and / or Tie-2 and thereby prevent mediated by angiopoietin-1 and angiopoietin-2 transmitting Tie-2 signal and to inhibit angiogenesis. The mechanism of action of this inhibition may include:

i) binding of the antagonist to Angiopoietin-1 and inhibiting the binding of Angiopoietin-1 to its receptor, Tie-2, and / or

ii) binding of the antagonist to Angiopoietin-2 and the inhibition of binding of Angiopoietin-2 to its receptor, Tie-2, and / or

iii) enhancement of the clearance of Angiopoietin-1 and / or Angiopoietin-2 and, thereby, decrease the effective concentration of Angiopoietin-1 and / or Angiopoietin-2 available for binding to Tie-2, or

any combination thereof, sufficient to inhibit the biological activity of Angiopoietin-1 and Angiopoietin-2.

Without claiming any theoretical conclusions should be noted that the mechanisms through which the inhibition of the biological activity of Angiopoietin-1 and Angiopoietin-2 may be accomplished include, but are not limited to, inhibition of binding of Angiopoietin-1 and Angiopoietin-2 to the receptor Tie -2; induced inhibition of Angiopoietin-1 and Angiopoietin-2 transmission Tie-2 signal or enhancing the clearance of Angiopoietin-1 and Angiopoietin-2 and, thereby, decrease the effective concentration of Angiopoietin-1 and Angiopoietin-2.

In one embodiment, the present invention relates to an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2 wherein the antagonist is an antibody. Preferably, the antibody is capable of inhibiting the biological activity of Angiopoietin-1 and / or Angiopoietin-2<i>in vitro</i> and <i>in vivo</i>. A preferred antibody is a polyclonal antibody or a monoclonal antibody. More preferably the antibody is a monoclonal antibody and more preferably the antibody is a fully human monoclonal antibody. Most preferably the antibody is a fully human monoclonal antibody 3.19.3.

In one embodiment, the present invention relates to an antibody that binds to the same epitope or the same epitope as the fully human monoclonal antibody 3.19.3.

In one embodiment, the present invention relates to fully human antibody that binds to Angiopoietin-1 and prevents Angiopoietin-1 binding to Tie-2. In another embodiment, the present invention relates to fully human monoclonal antibody that binds to Angiopoietin-1 and inhibits Angiopoietin-1 induced phosphorylation of Tie-2. In one embodiment, the antibody binds Angiopoietin-1 with a K_dOf less than 1 nanomolar (nM). More preferably, the antibody binds with a K_dOf less than 500 picomolar (pM). More preferably, the antibody binds with a K_dOf less than 100 picomolar (pM). Even more preferably, the antibody binds with a K_dOf less than 30 picomolar (pM). Even more preferably, the antibody binds with a K_dOf less than 20 pM. Most preferably, the antibody binds with a K_dOf less than 10 or 5 pM.

In one embodiment, the present invention relates to fully human antibody that binds to Angiopoietin-2 and prevents Angiopoietin-2 binding to Tie-2. In another embodiment, the present invention relates to fully human monoclonal antibody that binds to Angiopoietin-2 and inhibits Angiopoietin-2 induced Tie-2 phosphorylation. In one embodiment, the antibody binds Angiopoietin-1 with a K_dOf less than 1 nanomolar (nM). More preferably, the antibody binds with a K_dOf less than 500 picomolar (pM). More preferably, the antibody binds with a K_dOf less than 100 picomolar (pM). Even more preferably, the antibody binds with a K_dOf less than 30 picomolar (pM). Even more preferably, the antibody binds with a K_dOf less than 20 pM. Most preferably, the antibody binds with a K_dOf less than 10 or 5 pM.

In one embodiment, the present invention relates to a hybridoma producing the light chain and / or heavy chain of the antibody described above. Preferably, the hybridoma produces the light chain and / or heavy chain of a fully human monoclonal antibody. More preferably, the hybridoma produces the light chain and / or heavy chain of a fully human monoclonal antibody 3.19.3, 3.3.2 or 5.88.3. Alternatively, the hybridoma produces an antibody that binds to the same epitope or the same epitope as the fully human monoclonal antibody 3.19.3, 3.3.2 or 5.88.3.

In one embodiment, the present invention relates to a nucleic acid molecule encoding the light chain or the heavy chain of the antibody described above. Preferably, the present invention relates to a nucleic acid molecule encoding the light chain or the heavy chain of a fully human monoclonal antibody. More preferably, the present invention relates to a nucleic acid molecule encoding the light chain or the heavy chain of a fully human monoclonal antibody 3.19.3.

In one embodiment, the present invention relates to a vector comprising a nucleic acid molecule or molecules as described above, wherein said vector encodes a light chain and / or heavy chain of an antibody, as defined above.

In one embodiment, the present invention relates to a host cell comprising a vector described above. Alternatively, the host cell may comprise more than one vector.

Furthermore, in one embodiment, the present invention relates to a method for producing an antibody by culturing host cells under conditions which favor the expression of nucleic acid molecules, the production of the antibody and its subsequent release.

In one embodiment, the present invention relates to a method of producing an antibody comprising transfecting at least one host cell with at least one nucleic acid molecule encoding the antibody as described above; expression of said nucleic acid molecule in said host cell; and isolating said antibody.

In accordance with another aspect, the present invention relates to a method of inhibiting the biological activity of Angiopoietin-1 and Angiopoietin-2 comprising administering an antagonist as described above. The method may include selecting an animal in need of preventing it disease associated with angiogenesis, and administering to said animal a therapeutically effective dose of an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2.

In accordance with another aspect, the present invention relates to a method of inhibiting the biological activity of Angiopoietin-1 and Angiopoietin-2 comprising administering an antibody as described above. The method may include selecting an animal in need of preventing it angiogenesis associated with disease, and administering to said animal a therapeutically effective dose of an antibody inhibiting a biological activity of Angiopoietin-1 and Angiopoietin-2.

In accordance with another aspect, the present invention relates to a method for preventing a disease associated with angiogenesis in a mammal comprising administering a therapeutically effective amount of an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2. The method may include selecting an animal in need of preventing it disease associated with angiogenesis, and administering to said animal a therapeutically effective dose of an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2.

In accordance with another aspect, the present invention relates to a method for preventing a disease associated with angiogenesis in a mammal comprising administering a therapeutically effective amount of an antibody that inhibits the biological activity of Angiopoietin-1 and Angiopoietin-2. The method may include selecting an animal in need of prevention of a disease associated with angiogenesis, and administering to said animal a therapeutically effective dose of an antibody inhibiting a biological activity of Angiopoietin-1 and Angiopoietin-2. The antibody can be administered alone or it can be administered in combination with other antibodies or chemotherapeutic drug or radiation therapy.

In accordance with another aspect, the present invention relates to a method of treating cancer in a mammal comprising administering a therapeutically effective amount of an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2. The method may include selecting an animal in need of treatment of cancer, and administering to said animal a therapeutically effective dose of an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2. Said antagonist may be administered separately or can be administered in combination with other antibodies or chemotherapeutic drug or radiation therapy.

In accordance with another aspect, the present invention relates to a method of treating cancer in a mammal comprising administering a therapeutically effective amount of an antibody that inhibits the biological activity of Angiopoietin-1 and Angiopoietin-2. The method may include selecting an animal in need of treatment of cancer, and administering to said animal a therapeutically effective dose of an antibody inhibiting a biological activity of Angiopoietin-1 and Angiopoietin-2. The antibody can be administered alone or in combination with other antibodies, or in combination with a chemotherapeutic agent or with radiation therapy.

In accordance with another aspect, the present invention relates to the use of an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2 for the preparation of a medicament for the treatment of a disease associated with angiogenesis.

In accordance with another aspect, the present invention relates to the use of an antibody that inhibits the biological activity of Angiopoietin-1 and Angiopoietin-2 for the preparation of a medicament for the treatment of a disease associated with angiogenesis.

In a preferred embodiment, the present invention may be in particular applied for the inhibition of Angiopoietin-1 or Angiopoietin-2 in patients with a tumor which is dependent alone, or in part from the receptor Tie-2.

In another embodiment, the present invention includes an assay kit for detecting Angiopoietin-1 and / or Angiopoietin-2 in tissues, cells or body fluids of mammals in order to screen for diseases associated with angiogenesis. The kit includes an antibody that binds to Angiopoietin-1 and / or Angiopoietin-2, and means, which indicator antibody reaction with Angiopoietin-1 and / or Angiopoietin-2, if it occurs. The antibody may be a monoclonal antibody. In one embodiment, an antibody that binds to Ang-2 is labeled. In another embodiment, the antibody is unlabeled "first" antibody, and wherein said kit further comprises means for detection of said "first" antibody. In one embodiment of the invention, said means includes a labeled the "second" antibody which is an antibody against immunoglobulin. A preferred antibody is an antibody labeled with a marker selected from the group consisting of a fluorochrome, an enzyme, a radionuclide and a material which is impermeable to radiation.

Other embodiments, features and the like, related to the anti-Ang-2 antibodies described in more detail below.

<u>sequence Listing</u>

Embodiments of the invention include the specific anti-Ang-2 antibodies listed below in Table 1. In this table, the identification number of each anti-Ang-2 antibodies along with the identification number (SEQ ID) genes corresponding heavy chain and light chain.

Each antibody has been given an identification number that includes two or three numbers separated by a dot, typically used in decimal numbers. For the majority of antibodies is only two identification numbers, separated by periods.

However, in some cases, several clones of one antibody were prepared. Although the clones have the nucleotide sequence and amino acid sequence identical to the corresponding original sequences, however, they may also be provided separately, with the clone number indicated by reference numeral to the right of the second dividing point. For example, the sequence of the nucleic acid and amino acid sequences of antibody 5.35 are identical to the sequences of antibody 5.35.1, 5.35.2 and 5.35.3.

<u>define</u>

If not otherwise defined, scientific and technical terms used herein have the conventional values ​​known to one of ordinary skill in the art. In addition, if it is not obvious from the context of the description, the nouns that are used in the singular can mean a noun in the plural, and vice versa. In general, the nomenclature and methods relating to tissue culture, molecular biology, and chemical properties of the protein and hybridizing oligo- or polynucleotides are well known and widely used in the art.

Standard methods are techniques of recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's instructions or according to standard procedures or procedures described herein. The above procedures are usually performed by standard techniques well known in the art and described in various general and special guides, cited and discussed herein. See., Eg, Sambrook et al manual.<i>Molecular Cloning: A Laboratory Manual</i> (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)), which is incorporated herein by reference. As used herein, the nomenclature and the laboratory procedures and methods relating to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry and described herein, are well known and widely used in the art. For chemical syntheses, chemical analyzes, pharmaceutical preparations, of formulation, delivery, and treatment of patients standard methods are used.

In the present specification the following terms are used which, if not stated otherwise, are as defined below.

Antagonist can be a polypeptide, nucleic acid, carbohydrate, lipid, small molecule, an oligonucleotide, an oligopeptide, interfering RNA (RNAi), an antisense molecule, a recombinant protein, an antibody, or conjugates or fusion proteins. Description of the mRNA may be found in Milhavet O, Gary DS, Mattson MP. (Pharmacol Rev. 2003 Dec; 55 (4): 629-48 Review..), While the description of antisense molecules can be found in the publication Opalinska JB, Gewirtz AM (Sci STKE 2003 Oct 28; 2003 (206):. P.47. )

Angiogenesis associated with said disease, can be any abnormal, undesirable or pathological angiogenesis, e.g., tumor-associated angiogenesis. Angiogenesis-related diseases include, but are not limited to, non-solid tumors such as leukemia, multiple myeloma or lymphoma, and also solid tumors such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver ) carcinoma, glioblastoma, carcinoma of the thyroid, bile duct, bone, gastrointestinal tract, brain / CNS, head and neck, hepatic, stomach, prostate, breast, renal, testicular, ovarian, skin, cervical, le soft, muscles, nerve cells, esophagus, bladder, lung, uterus, vulva, endometrial, kidney, colon, pancreas,

The term "compound" refers to any low molecular compound having a molecular weight of less than about 2000 Daltons.

The term "Ang-2" refers to a molecule Angiopoietin-2.

The term "neutralizing" referring to an antibody refers to its ability to eliminate or significantly reduce the activity of a target antigen. Accordingly, "neutralizing" anti-Ang-2 antibody is capable of eliminating or significantly reducing the activity of Ang-2. The neutralizing anti-Ang-2 antibody may, for example, act by blocking the binding of Ang-2 to its receptor Tie-2. By blocking this binding, Tie-2 mediated signal transduction is significantly, or completely eliminated. Ideally, a neutralizing antibody against Ang-2 inhibits angiogenesis.

As used herein, "isolated polynucleotide" denotes a polynucleotide that has been isolated from its environment. Such polynucleotides may be genomic, cDNA or synthetic. The isolated polynucleotides preferably are not associated with all of the polynucleotides or a portion of a polynucleotide with which they are associated in nature. The isolated polynucleotides may be operably linked to another polynucleotide with which they are associated in nature. In addition, isolated polynucleotides preferably do not occur in nature as part of a larger sequence.

As used herein, the term "isolated protein" temperature is a general term meaning a protein which has been isolated from its natural environment. Such proteins may be produced from genomic DNA, cDNA, recombinant DNA, recombinant RNA, or they may be synthesized or obtained by a combination of these and other ways, wherein the "isolated protein" in its origin or source of origin (1) is not associated with natural proteins, (2) isolated from other proteins from the same source, to example of murine proteins, (3) is expressed by various types of cells, or (4) does not occur in nature.

The term "polypeptide" as used herein is a generic term meaning the native protein or fragments or analogs of a polypeptide sequence. Hence, native protein, as well as fragments and analogs are molecules such as polypeptides. Preferred polypeptides of the invention include molecules of the heavy chain of human immunoglobulin molecule light of human immunoglobulin kappa, as well as antibody molecules formed by combinations comprising the molecule an immunoglobulin heavy chain molecule immunoglobulin light chain, such as molecules kappa light chain or lambda immunoglobulin, or vice versa , as well as fragments and analogues. Preferred polypeptides of the invention may also comprise only human immunoglobulin molecule heavy chain or fragments thereof.

The term "naturally occurring" as used herein referring to a particular object, it means that this object can exist in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) can be considered as natural, if it is isolated from its natural source, and if it has not been specifically modified by man in the laboratory or in any other conditions.

The term "operably linked" as used herein refers to positions of components described herein, attached to each other means ensuring their "correct" operation. For example, a control sequence "operably linked" to a coding sequence is linked to this sequence so that expression of the coding could be carried out in sequence under conditions suitable for the functioning regulatory sequences.

As used herein, the term "regulatory sequence" Oznachaet polynucleotide sequences necessary for expression and processing or to affect the expression and processing of coding sequences to which they are attached. The nature of such control sequences will vary depending upon the host organism; wherein in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; and eukaryotes, such regulatory sequences generally include promoters, enhancers, introns, transcription termination sequences, polyadenylation signal sequences, and 5 'and 3' untranslated regions. The term "regulatory sequences" includes, at a minimum, all components whose presence is essential for expression and processing,

The term "polynucleotide" as used herein means a polymeric form of nucleotide sequence length, at least 10 nucleotides or ribonucleotide or deoxynucleotide sequence, or a modified form of nucleotides of any type, or of the RNA-DNA hetero-duplexes. The term includes single and double stranded forms of DNA.

The term "oligonucleotide" as used herein means natural and modified nucleotides linked together by natural and non-natural linkages. Oligonucleotides are a polynucleotide subsequence, typically having a length of 200 nucleotides or less. Preferably, the oligonucleotides are 10-60 nucleotides, and most preferably a length of 12, 13, 14, 15, 16, 17, 18, 19 or 20-40 nucleotides. Typically, the oligonucleotides are single stranded, e.g., the oligonucleotides used as probes, although sometimes they Oguta be double stranded, e.g., the oligonucleotides used to construct the mutant gene. The oligonucleotides may be sense or antisense.

The term "naturally occurring nucleotides" as used herein refers to deoxyribonucleotides and ribonucleotides. As used herein, the term "modified nucleotides" means nucleotides with modified or substituted sugar groups and the like The term "oligonucleotide linkages" as used herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, fosforselenoat, fosfordiselenoat, fosforanilotioat, fosforanilidat, phosphoramidate, and the like See., E.g., LaPlanche et al.,<i>Nucl. Acids. Res.</i> 14:9081 (1986); Stec et al., <i>J. Am. Chem. Soc. </i>106:6077 (1984); Stein et al., <i>Nucl. Acids. Res.</i> 16: 3209 (1988); Zon et al.,<i>Anti-Cancer Drug Design</i> 6: 539 (1991); Zon et al.,<i>Oligonucleotides and Analogues: A Practical Approach</i>, pp.87-108 (F.Eckstein Ed., Oxford University Press, Oxford England (1991)); Stec et al., патент США № 5151510; Uhlmann &amp; Peyman <i>Chemical Reviews</i>90: 543 (1990). If necessary, the oligonucleotide can include a label for detection.

the term "selective hybridization" as used herein refers to detectable and specific binding. Polynucleotides, oligonucleotides and fragments thereof of the present invention selectively hybridize to nucleic acid strands under conditions of hybridization and washing, which greatly minimizes the amount of detectable binding to nonspecific nucleic acids. To provide selective hybridization, known in the art and discussed herein may be used conditions of high stringency. In general, the homology between the sequences of the polynucleotides, oligonucleotides, or fragments of the invention and the sequence of the nucleic acid of interest is at least 80%, and more preferably at least 85%, 90%, 95%, 99% and 100 %.

Two amino acid sequences are homologous if their sequences are partially or completely identical. For example, 85% sequence homology means that the alignment of the two sequences to compare their maximum correspondence, 85% of the amino acids are identical. "Gaps" (in either of the two sequences being compared) allow to maximize matching; wherein the gap length is a preferred 5 amino acids or less, and more preferably 2 amino acids or less. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from these sequences, and having a length of at least 30 amino acids) are considered homologous in the conventional sense of the word, if they have a price alignment more than 5 (in standard deviation units) in the comparison performed using the program ALIGN with the mutation data matrix and of a penalty for the "gap" of 6 or more. (See. Publication MO Dayhoff,<i>Atlas of Protein Sequence and Structure</i>, Pp. 101-110 (Volume 5, National Biomedical Research Foundation, 1972)) and the appendix 2 to this volume (Supplement 2 to this volume, pp. 1-10). The two sequences or parts thereof are more preferred homologous if their amino acids are identical at 50% or more when comparing their optimal alignment by using the ALIGN program. It should be noted that in the two orthologous sequences can be present differing regions of homology. For example, the functional sites of mouse and human orthologues may have a higher degree of homology than non-functional regions.

The term "corresponds" as used herein means that a polynucleotide sequence is homologous (i.e., identical but evolutionarily unrelated) to all or a portion of a reference polynucleotide sequence, or that term means that the polypeptide sequence is identical to a reference polypeptide sequence.

In contrast, the term "complementary" as used herein mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For example, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to sequence "GTATA".

For a description of the similarities between two or more polynucleotide or amino acid sequences, the following terms: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". The term "reference sequence" means a sequence that is used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, the segment of the full-length cDNA or gene sequences present in the Sequence Listing, or the sequence may comprise the full-length cDNA or gene sequence. Generally speaking, the reference sequence has a length of at least 18 nucleotides or 6 amino acids, typically at least 24 nucleotides or 8 amino acids, more often at least 48 nucleotides or 16 amino acids. Since each of the two polynucleotides or amino acid sequences (1) may comprise a sequence (i.e., part of the full-length polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that differs at these two polynucleotide or amino acid sequences, the sequence comparisons between two (or more) molecules typically performed by comparing sequences of the two molecules over "of NUC comparison "to identify and compare local regions of sequence homology. As used herein, "comparison window" means a conceptual segment consisting of at least of 18 contiguous nucleotides or 6 amino acids wherein a polynucleotide sequence or amino acid sequence can be compared to a reference sequence consisting of at least 18 contiguous nucleotides or 6 amino acids, and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, replacement etc. (I.e., spaces) that account for 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) and which are used for optimal alignment of the two sequences. Optimal alignment of sequences for their comparison carried out by comparison window may be performed using the local homology algorithm (Smith and Waterman wherein the polynucleotide sequence or amino acid sequence can be compared to a reference sequence consisting of at least 18 contiguous nucleotides or 6 amino acids, and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, etc. (I.e., spaces) that account for 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) and which are used for optimal alignment of the two sequences. Optimal alignment of sequences for their comparison carried out by comparison window may be performed using the local homology algorithm (Smith and Waterman wherein the polynucleotide sequence or amino acid sequence can be compared to a reference sequence consisting of at least 18 contiguous nucleotides or 6 amino acids, and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, etc. (I.e., spaces) that account for 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) and which are used for optimal alignment of the two sequences. Optimal alignment of sequences for their comparison carried out by comparison window may be performed using the local homology algorithm (Smith and Waterman at least 18 contiguous nucleotides or 6 amino acids, and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, etc. (I.e., spaces) that account for 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) and which are used for optimal alignment of the two sequences. Optimal alignment of sequences for their comparison carried out by comparison window may be performed using the local homology algorithm (Smith and Waterman at least 18 contiguous nucleotides or 6 amino acids, and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, etc. (I.e., spaces) that account for 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) and which are used for optimal alignment of the two sequences. Optimal alignment of sequences for their comparison carried out by comparison window may be performed using the local homology algorithm (Smith and Waterman which constitute 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) and which are used for optimal alignment of the two sequences. Optimal alignment of sequences for their comparison carried out by comparison window may be performed using the local homology algorithm (Smith and Waterman which constitute 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) and which are used for optimal alignment of the two sequences. Optimal alignment of sequences for their comparison carried out by comparison window may be performed using the local homology algorithm (Smith and Waterman<i>Adv. Appl. Math.</i> 2: 482 (1981)), the regions of homology alignment algorithm (Needleman and Wunsch, <i>J. Mol. Biol. </i>48: 443 (1970)), the similarity search method (Pearson and Lipman, <i>Proc. Natl. Acad. Sci. </i>(USA) 85:2444 (1988)) путем компьютерной реализации этих алгоритмов (GAP, BESTFIT, FASTA и TFASTA в Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), GENEWORKS^TM or packages of programs MACVECTOR^®) Or using control and build the program best alignment (i.e., obtain the highest percentage homology "comparison window") achieved by various methods.

The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide or amino acid level) in the window of comparison. The term "percentage of sequence identity" means the percentage which is calculated by comparing two optimally aligned sequences by comparison window; determining the number of positions identical nucleic acid bases (e.g., A, T, C, G, U or I) or residue occurs in both sequences to yield the number of matched positions; dividing this number of matched positions by the total number of positions in the comparison window (ie the window size); and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein said polynucleotide or said amino acid constitute a sequence that is at least 85%, preferably at least 90-95%, and more preferably at least 99% identical to the reference sequence over the comparison window is at least 18 nucleotide (6 amino acids), and often in the comparison window is at least 24-48 nucleotide (8-16 amine acids), wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which in general constitute 20 percent or less as compared to the reference sequence over the comparison window.

As used herein, the twenty main amino acids have the common abbreviation. See. Publication<i>Immunology - A Synthesis</i> (2nd Edition, ES Golub and DR Gren. Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α, α-disubstituted amino acid; N-alkyl-substituted amino acids, lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides according to the invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N, N, N-trimetillizin, ε-N-atsetillizin, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). As used herein, refer to a system of the polypeptides,

Similarly, unless otherwise specified, the left end of single-stranded polynucleotide sequences is the 5 'end, and from left to right direction is considered to 5'-direction (5' →) for a double-stranded polynucleotide sequences. The direction of attachment of growing RNA transcript 5 '→ 3' direction of transcription is called, the sequence regions on the DNA strand having the same sequence as the RNA and which are from the 5'-end with respect to the 5'-end RNA transcript are referred to as "preceding sequences (i.e., situated upstream of the transcription)", and sequence regions on the DNA strand having the same sequence as the RNA and which are located on the 3'-end with respect to the 3 'tra' end of the RNA script,

The term "substantially identical" used with respect to polypeptides means that two peptide sequences, when optimally aligned, for example using GAP or BESTFIT programs using "weights" default gap, share at least 80 % sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity to the serial nostey. In this case, it is preferred that residue positions which are not identical differ by conservative amino acid substitutions. The term "conservative amino acid substitution" is interchangeable residues comprising similar side chains. For example, the amino group, having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having sulfur-containing side chains comprise cysteine ​​and methionine. Preferred conservative amino acid substitutions are substitutions within groups such as valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid, aspartic acid, and asparagine-glutamine. alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having sulfur-containing side chains comprise cysteine ​​and methionine. Preferred conservative amino acid substitutions are substitutions within groups such as valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid, aspartic acid, and asparagine-glutamine. alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having sulfur-containing side chains comprise cysteine ​​and methionine. Preferred conservative amino acid substitutions are substitutions within groups such as valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid, aspartic acid, and asparagine-glutamine. comprise serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having sulfur-containing side chains comprise cysteine ​​and methionine. Preferred conservative amino acid substitutions are substitutions within groups such as valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid, aspartic acid, and asparagine-glutamine. comprise serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having sulfur-containing side chains comprise cysteine ​​and methionine. Preferred conservative amino acid substitutions are substitutions within groups such as valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid, aspartic acid, and asparagine-glutamine. a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having sulfur-containing side chains comprise cysteine ​​and methionine. Preferred conservative amino acid substitutions are substitutions within groups such as valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid, aspartic acid, and asparagine-glutamine. a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having sulfur-containing side chains comprise cysteine ​​and methionine. Preferred conservative amino acid substitutions are substitutions within groups such as valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid, aspartic acid, and asparagine-glutamine.

As discussed herein, minor changes in amino acid sequences of antibody molecules or immunoglobulins are considered as modifications within the scope of the present invention, provided that such changes will be at least 75% amino acid sequence described herein, antibody molecules or immunoglobulins more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, it is also considered conservative amino acid substitutions. Conservative substitutions are those which are part of a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids = aspartate, glutamate; (2) basic amino acids = lysine, arginine, histidine; (3) nepolyarnыe aminokislotы = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) nezaryazhennыe polyarnыe aminokislotы = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Disease predpochtitelynыmi semeystvami yavlyayutsya: serine and threonine, prinadlezhashtie k semeystvu alifaticheskih oksikislot; asparagine and glutamine prinadlezhashtie k semeystvu amide soderzhashtih Chisloth; alanine, valine, leucine and isoleucine, prinadlezhashtie k semeystvu alifaticheskih aminokislot and phenylalanine, tryptophan and tyrosine, prinadlezhashtie k semeystvu aromaticheskih aminokislot. Tak, for example, sleduet ozhidaty, Chto otdelynaya replace leucine izoleytsinom or valinom, substitute aspartate glutamatom, replacing threonine with a serine, or a similar replacement of any amino acid with a structurally related amino acid will not have a significant effect on the binding function or properties of the resulting molecule, especially if this replacement is not the amino acid substitution in the framework region. Production of a functional peptide resulting from such amino acid substitutions may be readily ascertained by analysis on specific activity of the polypeptide derivative. Such assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of ordinary skill. Preferably, the amino- and carboxy-termini of fragments or analogs were near boundaries of functional domains. Structural and functional domains can be identified by comparing the data of the nucleotide and / or amino acid sequences with the sequence data available in certain public databases or databases that are privately owned. Preferably, to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function applicable methods of computer comparisons. Methods to identify protein sequences that form a known three-dimensional structure are known in the art and are described in Bowie et al., to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function applicable methods of computer comparisons. Methods to identify protein sequences that form a known three-dimensional structure are known in the art and are described in Bowie et al., to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function applicable methods of computer comparisons. Methods to identify protein sequences that form a known three-dimensional structure are known in the art and are described in Bowie et al.,<i>Science</i> 253: 164 (1991). For example, examples described above demonstrated that one skilled in the art can readily identify sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the antibodies described herein.

Preferred amino acid substitutions are those that result in: (1) reduced sensitivity to proteolysis, (2) reduced sensitivity to oxidation, (3) a change in binding affinity for forming protein complexes, (4) a change in binding affinity, and (5) to the message or modify other physicochemical or functional properties of such analogs. Such analogs may be various muteins of a sequence differing from the natural peptide sequence. For example, in native sequence or a plurality of amino acid substitutions (preferably conservative amino acid substitutions) may be made (preferably in the portion of the polypeptide that is located outside the domain-forming intermolecular contacts). A conservative amino acid substitution should not substantially affect the structural properties of the parent sequence (e.g., a replacement amino acid should not lead to break a helix present in the parent sequence, or to the disturbance of other types of secondary structure that characterizes the parent sequence). Examples of known secondary and tertiary structures of the polypeptide are described in<i>Protein, Structures and Molecular Principles </i>(Creighton ed., W.H. Freeman and Company, New York 1984); Introduction to Protein Structure (C. Branden &amp; J. Tooze eds., Garland Publishing, New York, N,Y. (1991)) и в работе Thornton et al., Nature 354:105 (1991), каждая из которых вводится в настоящее описание посредством ссылки.

The term "polypeptide fragment" as used herein means a polypeptide that has an amino-terminal and / or carboxy-terminal deletion, but where the remaining amino acid sequence corresponding to amino acid positions is identical to the native amino acid sequence derived, e.g., from full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids, preferably at least 14 amino acids, more preferably at least 20 amino acids, usually at least 50 amino acids, and even more preferably at least 70 amino acids. The term "analog" as used herein refers to polypeptides which are comprised of segments having at least 25 amino acids, mainly, parts identical deduced amino acid sequences and which possess at least one of the following properties, namely, they are (1) specifically bind to Ang-2 under suitable binding conditions, (2) capable of blocking the corresponding binding to Ang-2, or (3) ability to inhibit Ang -2. Typically, polypeptide analogs, as compared with the native sequence, comprise a conservative amino acid change (addition or deletion). Analogs typically have a length of at least 20 amino acids, preferably at least 50 amino acids or more, and most often, they have the same length as the full-length natural polypeptide. (2) capable of blocking the corresponding binding Ang-2, or (3) ability to inhibit Ang-2. Typically, polypeptide analogs, as compared with the native sequence, comprise a conservative amino acid change (addition or deletion). Analogs typically have a length of at least 20 amino acids, preferably at least 50 amino acids or more, and most often, they have the same length as the full-length natural polypeptide. (2) capable of blocking the corresponding binding Ang-2, or (3) ability to inhibit Ang-2. Typically, polypeptide analogs, as compared with the native sequence, comprise a conservative amino acid change (addition or deletion). Analogs typically have a length of at least 20 amino acids, preferably at least 50 amino acids or more, and most often, they have the same length as the full-length natural polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs having properties analogous to those of the template peptide. Non-peptide compounds of this type are called "peptide mimetics" or "peptidomimetics". Cm. Publication Fauchere,<i>J. Adv. Drug. Res.</i> 3:29 p.m. (1986); Veber &amp; Freidinger,<i>TINS</i> p.392 (1985) и Evans et al. <i>J. Med. Chem.</i> 30: 1229 (1987) which are incorporated herein by reference. Such compounds are often developed with the aid of a computer program molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to achieve an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a representative polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as a human antibody, and they usually have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of - CH₂OG, -SHSN₂S, -SN₂-SN₂-, -SN = MV (cis and trans), -SOSN₂- -SN (ON) SN₂- and -SN₂SO-, according to well known techniques. Systematic substitution of one or more amino acids of a consensus sequence with D-amino acid of the same type (e.g., replacement of L-lysine D-lysine) may be used to generate more stable peptides. Additionally, peptides with conformational constraints comprising a consensus sequence or variant sequences substantially identical consensus sequence variation may be generated by known methods (Rizo &amp; Gierassh publication,<i>Ann.Rev. Biochem.</i> 61: 387 (1992) which is incorporated herein by reference), e.g., by attaching internal cysteine ​​residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

The term "antibody" as used herein refers to a polypeptide or polypeptides group consisting of at least one binding domain that is formed by the stacking of polypeptide chains having three-dimensional binding regions with internal surface structures and charge distribution complementary to the structural features of an antigenic determinant of the antigen. Typically, an antibody has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each pair of light / heavy chains form the binding site of the antibody.

As used herein, a "targeted binding agent" is an antibody or a binding fragment thereof that preferentially bind to the target site. In one embodiment, said targeted binding agent is specific for only one target site. In other embodiments of the invention, said targeted binding agent is specific for more than one target site. In one embodiment, said aiming binding agent may be a monoclonal antibody and the said target site may be an epitope.

"Binding fragments" of an antibody prepared by recombinant DNA techniques or by enzymatic or chemical hydrolysis of intact antibodies. Binding fragments are Fab, Fab ', F (ab')₂, Fv and single chain antibodies. Antibodies, if they are not a "bispecific" or "bifunctional", considered as antibodies, each of which has identical binding sites. An antibody substantially inhibits adhesion receptor to a counterreceptor when an antibody excess reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60% or 80% and often approximately more than 85 % (as measured in a competitive binding assay<i>in vitro</i>).

Antitelo may be oligoclonal, a polyclonal or a monoclonal antibody, a chimeric antibody, CDR-hybrid antibody, a multispecific antibody, a bispecific antibody, a catalytic antibody, a chimeric antibody, humanized antibody, fully human antibody, an anti-idiotypic antibody and antibodies that can be labeled in soluble or in bound form, as well as fragments, variants or derivatives, alone or in combination with other amino acid sequences derived bubbled known methods. The antibody may be derived from any kind of organism. The term "antibody" also includes binding fragments of antibodies according to the invention, and such representative fragments include Fv, Fab, Fab ', single chain antibody (svFC),

The term "epitope" means any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and may, but not always, have specific three-dimensional structure, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is ≤1 uM, preferably, ≤100 nM and most preferably, ≤10 nM.

The term "agent" as used herein refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract obtained from biological materials.

As used herein, "active" or "activity" if it belongs to Ang-2 polypeptide, means a portion of Ang-2 polypeptide that has a biological or immunological activity of a native Ang-2 polypeptide. The term "biological" as used herein refers to a biological function caused by the activity of the native Ang-2 polypeptide. A preferred Ang-2 biological activity includes, for example, Ang-2 induced angiogenesis.

The term "mammal" as used herein means any animal belonging to the class of mammals. Preferably, the mammal is a human.

Hydrolysis of the antibodies with papain enzyme results in the production of two identical antigen-binding fragments, also known as "Fab" fragments, and a "Fc" fragment, having no antigen-binding activity but having the ability to crystallize. Hydrolysis of antibodies with pepsin enzyme leads to the formation of F (ab ')₂fragment in which the two domains of the antibody molecule are linked and contain two antigen-binding sites. Such F (ab ')₂fragment has the ability to cross-bind to the antigen.

The term "Fv" as used herein refers to the minimum antibody fragment comprising an antigen recognition sites and antigen-binding sites.

As used herein, "Fab" means an antibody fragment comprising a light chain constant domain and the CH1 domain of the heavy chain.

The term "mAb" is a monoclonal antibody.

The term "liposome" as used herein refers to a small vesicle that may be used to deliver drugs to a mammal, which may include Ang-2 polypeptide of the invention or an antibody against said polypeptide Ang-2.

The term "label" or "labeled" as used herein refers to incorporation into a polypeptide detectable group, e.g., a radiolabel, fluorescent label, enzymatic label, a chemiluminescent label or a biotinyl group. Radioisotopes or radionuclides may be³n, ¹⁴FROM, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁴Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, fluorescent labels may be rhodamine complex "lanthanide phosphor" or FITC and enzymatic labels may be horseradish peroxidase, β-galactosidase, luciferase and alkaline phosphatase.

As used herein, the term "pharmaceutical agent or drug" refers to a chemical compound or composition capable of inducing a desired therapeutic effect when administered to a patient appropriate. Other conventional chemical terms used herein are described in the user manual<i> The McGraw-Hill Dictionary of Chemical Terms</i> (Parker S. Ed, McGraw-Hill, San Francisco (1985), which is incorporated herein by reference).

The term "substantially pure" as used herein refers to the considered molecule which is the predominant molecule (i.e., is present in a higher molar concentration than any other individual molecules existing in the composition), and preferably, the term means "in substantially purified fraction ", i.e. a composition in which the considered molecule is at least about 50 percent (on a molar level) of all present this type of macromolecules. Typically, substantially pure composition is greater than about 80 percent of all macromolecules present in the composition, and more preferably greater than about 85%, 90%, 95% and 99%. Most preferably, the molecule of interest was purified almost to homogeneity (where impurities

The term "patient" includes human and animal.

<u>Human Antibodies and "humanization" of antibodies</u>

The use of human antibodies avoids some of the problems encountered in the use of antibodies having murine or rat variable and / or constant regions. The presence of such murine or rat protein may lead to rapid removal of the antibodies or can lead to generating an immune response in a patient against the antibody. In order to avoid having to use mouse or rat antibodies, they can be generated fully human antibodies through the introduction of functional human antibody portion of a rodent, other mammal or animal so that they develop a fully human antibody.

One method for generating fully human antibodies is to use a XenoMouse® strains of mice which were derived so that they comprise a suitably configured fragments of the human heavy chain locus and kappa light chain locus germline size to less than 1000 kb See. Mendez et al.<i>Nature Genetics</i> 15:146-156 (1997) и Green and Jakobovits <i>J. Exp. Med.</i> 188: 483-495 (1998). XenoMouse strains^® supplied by Abgenix, Inc. (Fremont, CA).

Preparation of XenoMouse strains of mice^® as discussed and described in the following US Patent Applications: Application in Identification No 07/466008, filed January 12, 1990, 07/610515, filed November 8, 1990, 07/919297, filed July 24, 1992, 07/922649, filed July 30 1992 08/031801, filed March 15, 1993, 08/112848, filed August 27, 1993, 08/234145, filed April 28, 1994, 08/376279, filed January 20, 1995, 08/430938, filed April 27, 1995, 08/464584 filed 5 June 1995, 08/464582, filed 5 June 1995, 08/463191, filed 5 June 1995, 08/462837, filed 5 June 1995, 08/486853, filed 5 June 1995, 08/486857, filed 5 June 1995 , 08/486859, filed June 5, 1995, 08/462513, filed June 5, 1995, 08/724752, filed October 2, 1996 and 08/759620, filed on December 3, 1996; Application Publication U.S. 2003/0093820, filed November 30, 2001; in US patents №№ 6162963, 6150584, 6114598, 6075181 and 5939598, and in Japanese Patent Applications №№ 3068180 B2, 3,068,506 B2 and 3,068,507 B2. Cm. And European patent number EP 0463151 Bl, issued and published on June 12, 1996, International Patent Application № WO 94/02602, published February 3, 1994, International Patent Application № WO 96/34096, published October 31, 1996, WO 98/24893, published June 11, 1998 and WO 00/76310, published December 21, 2000. A description of all the above-mentioned patents, applications and publications in their entireties are hereby incorporated by reference.

In an alternative method, other researchers, including researchers GenPharm International, Inc., used "miniloci". This approach is using minilocus that was exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the locus Ig. Thus, one or more V gene_nOne or more genes D_nOne or more genes J_n, A mu constant region, and usually a second constant region (preferably a gamma constant region) design was established for administration to the animal. This method is described in U.S. Patent № 5545807, Surani et al., And U.S. Pat №№ 5545806, 5625825, 5625126, 5633425, 5661016, 5770429, 5789650, 5814318, 5877397, 5874299 and 6255458, Lonberg and Kay, U.S. Patent № № 5591669 and 6023010, Krimpenfort &amp; Berns, U.S. patent №№ 5612205, 5721367 and 5789215, Berns et al, and U.S. patent № 5643763, Choi &amp; Dunn, and in International applications US patent, GenPharm.; in the application registration. Number 07/574748, filed August 29, 1990, patent application 07/575962, filed August 31, 1990, patent application 07/810279, filed December 17, 1991, patent application 07/853408, filed March 18, 1992, patent application 07/904068, filed June 23, 1992, application 07/990860, filed December 16, 1992, patent application 08/053131, filed April 26, 1993, patent application 08/096762, filed July 22, 1993, application 08/155301, filed November 18, 1993, patent application 08/161739, filed on December 3, 1993, patent application 08/165699, filed December 10, 1993, and application 08/209741 filed March 9, 1994, the disclosure of which are incorporated herein by reference. See. Also Europatent № 0546073 B1, International Patent Applications WO №№ 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96 / 14436, WO 97/13852 and WO 98/24884, and U.S. patent № 5,981,175, the disclosures of which in their entireties are hereby incorporated by reference. See. Publications Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Londberg et al., 1994, Taylor et al. 1994 and Tuaillon et al., 1995, Fishwild et al., 1996,

Kirin has also demonstrated the generation of human antibodies in mice through microcell fusion, large pieces of chromosomes administered or full chromosome. See. European patent applications №№ 773 288 and 843 961, the disclosure of which in its entirety is hereby incorporated by reference. Furthermore, as a result of cross-breeding Tc Kirin mice and mice with Medarex minilocus (Humab) mice were obtained KM^TM. These mice had human IgH-transchromosome Kirin mice and the kappa chain transgene Genpharm mice (Ishida et al, Cloning Stem Cells, (2002) 4:. 91-102).

Human antibodies can also be prepared by <i>in vitro</i>. Suitable methods include, but are not limited to phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphoge n, Alexion (formerly Proliferon), Affimed), ribosome representation (CAT), yeast, etc. Introduction

<u>Preparation of antibodies</u>

Antibodies described herein were obtained using XenoMouse® mice removal technology described below. Such mice are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. The technology used for these purposes is described in the patents, patent applications and papers cited in the "Background Art" herein. However, in a particularly preferred embodiment of the production of transgenic mice and the antibodies disclosed in U.S. Patent Identification No 08/759620, filed on 3 December 1996, and in International Patent Applications: Application in № WO 98/24893, published 11 June 1998, and in patent application WO 00/76310, published 21 December 2000, the disclosure of which are incorporated herein by reference. See. Also Mendez et al publication.,<i>Nature Genetics</i> 15: 146-156 (1997), the disclosure of which is incorporated herein by reference.

Through the use of such technologies have been produced fully human monoclonal antibodies against different antigens. Substantially XenoMouse® strains of mice immunized with the antigen of interest (e.g., Ang-2) and then lymphatic cells (such as B-cells) were isolated from the hyperimmunized mice and lymphocytes obtained was fused with a myeloid-type cell line to give immortal hybridoma cell lines. Such hybridoma cell lines were screened and selected to identify hybridoma cell lines that produce antibodies specific to a desired antigen. The present application describes methods for producing a plurality of hybridoma cell lines that produce antibodies specific to Ang-1 and Ang-2. Furthermore, characterization of the antibodies provided herein

Alternatively, instead of the fusion with myeloma cells to produce hybridomas, direct analysis of B cells can be conducted. For example, CD19⁺-B-cells can be isolated from hyperimmune XenoMouse® mice and subjected to the proliferation and differentiation to give antibody-secreting plasma cells. antibodies are then isolated from the cell supernatants were screened using ELISA for reactivity with Ang-2 immunogen. These supernatants might also be screened for immunoreactivity with the Ang-2 fragments in order to further map the different antibodies for binding assays with the functional domains of interest on Ang-2. Such antibodies may also be screened for binding to Ang-1, Ang-3 or Ang-4 and other related human chemokines, as well as the rat and mouse Ang-2 with Ang-2 primates, non-human, such as cynomolgus monkey, orthologues and Ang-2, wherein the orthologs are used to determine the species crossreactivity. The cells in wells containing an antibody of interest may be immortalized by various methods including fusion of cells to obtain hybridomas, or in separate wells, or wells with a pool of cells or by infection with EBV or transfection by known immortalizing genes and then plating cells in a suitable medium. Alternatively, single plasma cells secreting antibodies with the desired specificities are then isolated using the hemolytic plaque assay method for Ang-1 or Ang-2-specific antibodies (see. For example, Babcook et al., including cell fusion to obtain hybridoma, or in separate wells, or wells with a pool of cells or by infection with EBV or transfection by known immortalizing genes of the virus, followed by plating the cells in a suitable medium. Alternatively, single plasma cells secreting antibodies with the desired specificities are then isolated using the hemolytic plaque assay method for Ang-1 or Ang-2-specific antibodies (see. For example, Babcook et al., including cell fusion to obtain hybridoma, or in separate wells, or wells with a pool of cells or by infection with EBV or transfection by known immortalizing genes of the virus, followed by plating the cells in a suitable medium. Alternatively, single plasma cells secreting antibodies with the desired specificities are then isolated using the hemolytic plaque assay method for Ang-1 or Ang-2-specific antibodies (see. For example, Babcook et al.,<i>Proc. Natl. Acad. Sci.</i> USA, 93: 7843-7848 (1996)). The cells were lysed are preferably sheep red blood cells (SRBC), coated with Ang-2 antigen. For screening antibodies capable of inhibiting Angiopoietin-1, with the same success the above methods, where the angiopoietin-1 is used instead of Angiopoietin-2 may be applied.

In the presence of B-cell culture containing plasma cells secreting the desired immunoglobulin and complement, the formation of plaque indicates specific Ang-1 / Ang-2-mediated lysis of sheep erythrocytes surrounding the plasma cell of interest. It can be isolated single antigen-specific plasma cell present in the center of the plaque, and then from this single plasma cell genetic information on the specificity of the antibody can be obtained. By performing reverse transcriptase PCR (RT-PCR) can be cloned DNA encoding the variable regions of the heavy and light chains of the antibody. Such cloned DNA can then be inserted into an appropriate expression vector, preferably a vector in the cluster, such as rsDNA and more preferably rsDNA, containing the constant domains of immunoglobulin heavy and light chains. The generated vector can then be transfected into host cells such as HEK 293 cells or CHO cells, and cultured in a suitable nutrient media modified, if necessary, for inducing transcription, selecting transformants, or amplifying the genes encoding the desired sequences.

In general, antibodies produced by the hybridomas were human IgG2 heavy chains with full-length light chain kappa or lambda human antibody. Described herein are antibody heavy chain of human IgG4, as well as the human heavy chain IgG2. Such antibodies may also be human antibodies of other isotypes, including IgG1. Antibodies possessing high affinity typically have K_D from about 10^-6 10^-12 M or less, as shown by measurements in the solid phase and in solution. Antibodies having K_DAt least 10^-11 M are preferred to inhibit the activity of Ang-1 and / or Ang-2.

It should be noted that the antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies can be used to transform a suitable mammalian host cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotides in a virus (or into a viral vector) and transfecting a host cell with the virus (or vector) or it may be carried out in accordance with the transfection procedures It is known in the art and described in U.S. Pat №№ 4399216, 4912040, 4740461 and 4959455 (which is incorporated herein by reference). This procedure depends on the transformation of a transformed host. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection; calcium phosphate precipitation; polybrene-mediated transfection; protoplast fusion; electroporation; encapsulation of the polynucleotide (s) in liposomes, and direct microinjection of DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known in the art, and such cell lines include many immortalized cell lines that can be drawn from the American Type Culture Collection (ATCC), including but not limited to, ovarian cells of Chinese hamster ( CHO), HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells and various other cell whether NII. Particularly preferred cell lines may be selected by identifying cell lines that express high levels and produce antibodies capable of constitutively bind to Ang-2.

Based on mAb ability largely neutralize the activity of Angiopoietin-1 and angiopoietin-2 (as demonstrated in the following examples), it can be concluded that these antibodies will have therapeutic effects, aimed at elimination of symptoms and treatment of conditions associated with the expression of the angiopoietin-1 and / or Angiopoietin-2. In specific embodiments, antibodies described herein and methods may be used to treat symptoms associated with angiogenesis induced by angiopoietin-1 and / or Angiopoietin-2.

In accordance with another aspect, the present invention relates to a pharmaceutical composition comprising an antagonist of the biological activity of Angiopoietin-1 and Angiopoietin-2 and a pharmaceutically acceptable carrier. In one embodiment, said antagonist comprises an antibody. In accordance with another aspect, the present invention relates to a pharmaceutical composition comprising an antagonist of the biological activity of Angiopoietin-2 and a pharmaceutically acceptable carrier. In one embodiment, said antagonist comprises an antibody.

Anti-Ang-2 antibodies may be used to detect Ang-2 in the samples taken from the patient, and, accordingly, they can be used as diagnostic agents for the treatment of pathological conditions described herein. Furthermore, based on the ability of these antibodies is significantly neutralize Ang-2 activity (as demonstrated in the following examples), it can be concluded that the anti-Ang-2 antibodies will have therapeutic effects, aimed at elimination of symptoms and treatment of conditions associated with the expression of Ang-2. In specific embodiments, the antibodies and methods described herein may be applied to eliminate symptoms associated with Ang-2 induced angiogenesis.

<u>Therapeutic compositions and administration</u>

Embodiments of the invention include sterile pharmaceutical compositions which comprise anti-Ang-2 antibodies or antibodies which bind to both Ang-1 and Ang-2 with and which may be used to treat diseases. Such formulations would inhibit the binding of Ang-2 or Ang-1 and Ang-2 to its receptor Tie-2, and so they should be effective for treating pathological conditions where, for example, an anomalous increase in levels of Ang-1 and / or Ang -2 in serum or tissue. Anti-Ang-2 antibodies preferably possess adequate affinity to neutralize Ang-2, and preferably have an adequate duration of action, which allows a person to inject these antibodies are much less likely. Anti-Ang-1 / Ang-2 antibodies preferably possess adequate affinity, allowing to neutralize Ang-1 and Ang-2, and preferably have an adequate duration of action, which allows a person to inject these antibodies are much less likely. Prolonged effect allows the use of said antibodies regimen less frequent, or more comfortable way of introduction doses Alternatively, parenteral administration such as subcutaneous or intramuscular injection.

Sterile formulations may be prepared, for example, by filtration through sterile filtration membranes, prior to or following lyophilization followed by dilution of the antibody. The antibody is typically stored in lyophilized form or in solution. Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, a package or container for intravenous administration, having a device for supplying the composition, such as a stopper pierceable by a hypodermic injection needle.

Administration of the antibody can be carried out by known methods, e.g., by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarticular, intrathecal injection or infusion; by inhalation or by injection into the affected parts, or by using sustained release systems described below. The antibody is preferably administered continuously by infusion or by bolus injection.

An effective amount of therapeutic antibody used depends, for example, by treatment objectives, the route of administration and the condition of the patient. In a preferred embodiment, the treating physician can set the desired dose by titration and modify the route of administration, if needed to obtain the optimal therapeutic effect. Typically, the clinician administering the dose assigns antibodies suitable for achieving the desired effect. Monitoring the effectiveness of this therapy can be readily implemented using standard tests or assays described herein.

Antibodies described herein can be formulated in admixture with a pharmaceutically acceptable carrier. This therapeutic composition can be administered intravenously, intranasally or intrapulmonalno, preferably as a liquid or powder aerosol (lyophilized form). The composition, if desired, can also be administered parenterally or subcutaneously. When administered systemically, such a therapeutic composition should be sterile and pyrogen-free, and it can be administered in a parenterally acceptable solution having the desired pH, isotonicity and stability desired. These conditions are well known in the art. Briefly, in the embodiments of the invention described herein, the dosage formulations of compounds intended for storage or administration is prepared by mixing the compound having the desired degree of purity, with physiologically acceptable carriers, excipients or stabilizers. These substances are non-toxic to recipients at the dosages and concentrations employed, and they may be buffers, such as Tris-HCl, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight peptides (having less than about ten residues) such as polyarginine; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counter,

Sterile compositions for injection can be formulated in accordance with standard pharmaceutical practice as described in the user manual <i>Remington: The Science and Practice of Pharmacy </i>(20^th ed., Lippincott Williams &amp; Wilkens Publishers (2003)). For example, it may be desirable dissolving or suspending the active compound in a vehicle such as water or a natural vegetable oil such as sesame oil, peanut oil or cottonseed oil or a synthetic fatty vehicle such as ethyl oleate or the like Buffers, preservatives, antioxidants, etc. It may be administered in accordance with standard pharmaceutical practice.

Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices obtained in the form of shaped articles, films or microcapsules. Examples of matrices for sustained release are polyesters, hydrogels (e.g., poly (2-hydroxyethylmethacrylate) as described Langer et al.,<i>J. Biomed. Mater. Res</i>., 15: 167-277 (1981) и Langer, <i>Chem. Tech.</i>(1982) 12: 98-105, or polyvinyl alcohol), polylactides (U.S. Patent № 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al,. <i>Biopolymers</i>, 22: 547-556 (1983)), nondegradable ethylene vinyl acetate (Langer et al, supra), degradable copolymers of lactic and glycolic acid, such as the LUPRON Depot.^TM (Injectable microspheres consisting of a copolymer of lactic and glycolic acids and leuprolide acetate), and poly-D - (-) - 3-hydroxybutyric acid (EP 133988).

Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for a time period of over 100 days, certain hydrogels release proteins provide for shorter time intervals. When encapsulated proteins present in the body for a long period of time, then at 37 ° C they may denature or aggregate under the action of moisture, which leads to loss of biological activity and possible changes in immunogenicity. Rational strategies depending on the mechanism can be devised for protein stabilization. For example, if the aggregation mechanism is SS intermolecular bond by disulfide exchange, the protein stabilization may be achieved by modifying sulfhydryl residues,

Sustained release compositions also include preparations of crystals of the antibody suspended in suitable formulations capable of maintaining crystals in suspension. These drugs, with their subcutaneous or intraperitoneal injection, can produce a sustained release effect. Other compositions also include liposomes captured antibody. Liposomes containing such antibodies are prepared by methods known<i>for himself</i> and described in U.S. Patent № DE 3218121; in papers Epstein et al.,<i>Proc. Natl. Acad. Sci.</i>, USA, (1985) 82:3688-3692; Hwang et al., <i>Proc. Natl. Acad. Sci</i>., USA, (1980) 77: 4030-4034; in EP 52322; in EP 36676; in EP 88046, EP 143 949 and 142 641; in Japanese patent application 83-118008; in U.S. Patents 4485045 and №№ 4544545 and in EP 102324.

Doses of the antibody formulation for a given patient must be determined by the attending physician, considering various factors which are known to affect the action of drugs, e.g., such factors, the severity and type of disease, body weight, sex, diet of the patient, time and method of administration drug and other medical conditions, and other relevant clinical factors. Therapeutically effective dosages may be determined by methods<i>in vitro</i> or <i>in vivo</i>.

An effective amount of antibody according to the invention used in therapy will depend, e.g., upon the therapeutic treatment objectives, the route of administration, and health of the patient. Accordingly, in a preferred embodiment, the practitioner can independently select the desired dosage and modify the route of administration, if necessary to achieve optimum therapeutic effect. A typical daily dosage may range from about 0.001 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. Typically, the clinician assigns the administration of a therapeutic antibody as long until the desired effect is achieved. Monitoring the effectiveness of this therapy can be readily implemented using standard tests or assays described herein.

It should be noted that therapeutic agents used in the herein described compositions and methods, may be administered together with suitable carriers, excipients, and other agents included in the formulations to improve the data transfer and delivery, and enhance tolerance etc. Such agents are, for example, powders, pastes, ointments, jelly, waxes, oils, lipids, vesicles, lipid-containing (or catiogenic aniogennye) (such as Lipofectin^TM), DNA conjugates, anhydrous absorbent paste, emulsion type "oil in water" and "water in oil" emulsion carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. any of the above mixture, with the proviso that the active ingredient in the composition will not be inactivated, and this composition is physiologically compatible and is well tolerated in this method of administration for treatment and therapy of the invention may be used. See also the publication Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance".,<i>Regul. Toxicol. Pharmacol</i>. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals", <i>Int. J. Pharm.</i> 203(1-2): 1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts", <i>J. Pharm. Sci.</i> 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA <i>J. Pharm. Sci. Technol.</i> 52: 238-311 (1998) and is listed in their additional information related to drugs, excipients and carriers well known to pharmaceutical chemists.

<u>combinations</u>

treatment directed at preventing angiogenesis described herein may be applied as a monotherapy, or may, in addition to administration of the compounds of the invention include conducting conventional surgery, radiation therapy or chemotherapy. Such chemotherapy may comprise administration of one or more of the following categories of antitumor agents:

(I) cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoksifen) inhibitors, estrogen receptor (e.g., fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), antagonists of LHRH or agonist LHRH (for example goserelin, leuprorelin and buserelin), progesterones (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5 * -reductase such as finasteride;

(Ii) agents which inhibit cancer cell invasion (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function);

(Iii) inhibitors of growth factor function, for example, inhibitors that are antibodies against growth factors, antibodies against receptors for growth factors (e.g., anti-erbb2 antibody trastuzumab [Herceptin^TM] And anti-<i>erbbl</i> antibody cetuximab [C225]), farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine / threonine kinase inhibitors, for example inhibitors of the family of epidermal growth factors (e.g., family tyrosine kinase inhibitors, EGFR, such as N- (3-chloro-4-fluorophenyl) -7-methoxy 6- (3-morpholinopropoxy) quinazolin-4-amine (gefitinib, AZDl839), N- (3-ethynylphenyl) -6,7-bis (2-methoxyethoxy) quinazolin-4-amine (erlotinib, OSI 774) and 6 acrylamide-N- (3-chloro-4-fluorophenyl) -7- (3-morpholinopropoxy) quinazolin-4-amine (CI 1033)), for example inhibitors of platelet-derived growth factor family and for Reamer, inhibitors of the hepatocyte growth factor family;

(Iv) antiangiogenic agents such as agents which inhibit the action of vascular endothelial growth factor (e.g., anti-growth factor vascular endothelial cell bevacizumab [Avastin^TM], Antibodies against the receptor vascular endothelial growth factor such as anti-KDR antibodies and anti-flt1 antibodies, compounds described in International Patent Applications WO 97/22596, WO 97/30035, WO 97/3285, WO 98/13354, WO 00/47212 and WO 01/32651) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin avb3 function and angiostatin);

(V) means that deplete vessels such as combretastatin A4, and compounds disclosed in International Patent Applications WO 99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213 ;

(Vi) therapeutic agents containing antisense sequences, e.g., therapeutic agents directed to the targets listed above, such as ISIS 2503, and anti-therapeutic<i>ras</i> means containing antisense sequences;

(Vii) means used in methods of gene therapy, including, for example, methods replace aberrant genes such as aberratny p53 or aberrant BRCAl or BRCA2, GDEPT (gene therapy using enzyme prodrug), for example, methods using cytosine deaminase, thymidine kinase or a bacterial enzyme nitroreductase and the methods used to improve the tolerance to chemotherapy or radiation therapy, such as therapy with resistance genes for multidrug; and

(Viii) means used in immunotherapy methods, including, for example, <i>ex vivo</i> and <i>in vivo</i> methods to increase the immunogenicity of patient tumor cells, such as transfection with cytokines such as interleukin-2, interleukin 4 or granulocyte-macrophage colony stimulating factor, methods of reducing the energy of T cells, approaches using transfected immune cells such as transfected cytokine dendritic cells, methods using cytokine-transfected tumor cell lines and approaches using anti-idiotypic antibodies.

In one embodiment, the antiangiogenic treatment means according to the invention is carried out in combination with other agents which inhibit the effects of vascular endothelial growth factor (VEGF) (e.g., anti-vascular endothelial cell growth factor, bevacizumab [Avastin^TM], Antibodies against the receptor vascular endothelial growth factor, such as an anti-KDR antibody, and anti-flt1 antibody; the compounds disclosed in International Patent Applications WO 97/22596, WO 97/30035, WO 97/3285, WO 98/13354, WO 00/47212 and WO 01/32651), and compounds that work by other mechanisms (for example linomide , inhibitors of integrin avb3 function and angiostatin). In other embodiments, the antiangiogenic treatment means according to the invention is carried out in combination with other drugs that inhibit the tyrosine kinase activity of the receptor vascular endothelial growth factor, KDR (for example, AZD2171 or AZD6474). AZD2171 detailed description can be found in the publications Wedge et al. (2005) Cancer Research. 65 (10): 4389-400. AZD6474 A detailed description can be found in the publications of Ryan &amp; Wedge (2005) British Journal of Cancer. 92 Suppl 1: S6-13. Both of these publications in their entireties are hereby incorporated by reference. In another embodiment, the fully human antibodies 3.19.3, 3.3.2 or 5.88.3 are administered alone or in combination with Avastin^TM, AZD2171 or AZD6474.

Such conjoint treatment may be carried out by simultaneous, sequential or separate dosing of the individual components. Such combination products include the compounds of the invention or a pharmaceutically acceptable salt thereof in the above dosage ranges and the other pharmaceutically active agent within the allowable range of doses.

<b>EXAMPLES</b>

The following Examples, including the description of the experiments and the results obtained are given for illustrative purposes only and should not be construed as limiting the present invention.

EXAMPLE 1

IMMUNIZATION AND TITRATION

<u>Immunization</u>

Recombinant human Ang-2, obtained from R &amp; D Systems, Inc. (Minneapolis, MN Cat. № 623-AM / CF), was used as antigen. Monoclonal antibodies against Ang-2 produced by sequentially immunizing mice XenoMouse® (strains of XenoMouse XMG2 and XMG4 (3C-1 strain), Abgenix, Inc. Fremont, CA). XenoMouse animals were immunized by administration of all injections in the footpad. The total volume of each injection was 50 l per mouse, 25 l per footpad. The first injection was 2.35 ug of recombinant human Ang-2 (rhAng-2, cat № 623-AM / CF; lot № BN023202A.) In pyrogen-free Dulbecco's PBS (DPBS), a mixture (1: 1 v / v) with 10 g CpG (15 l of mouse adjuvant ImmunEasy, cat № 303101;. lot № 11553042; Qiagen) per mouse. 6 was then introduced booster injections containing 2.35 g rhAng-2 in pyrogen-free DPBS, in admixture with 25 micrograms Adju-Phos (aluminophosphate gel;. Cat № 1452-250, batch № 8937, HCI Biosector) and 10 pg CpG per mouse, after which was added last booster injection containing 2.35 g rhAng-2 in pyrogen-free DPBS, without adjuvant. In accordance with this protocol, XenoMouse mice were immunized on days 0, 3, 6, 10, 13, 17, 20 and 24 and the fusion was performed on day 29.

<u>Selection of animals for antibody titration by</u>

Titers of anti-Ang-2 antibodies in the serum obtained from mice immunized XenoMouse, was determined by ELISA. Briefly, polystyrene 96-well plates Costar Labcoat Universal Binding (Corning, Acton, MA) were coated with recombinant Ang-2 (1 ug / ml) overnight at 4 ° C in antigen coating buffer (0,1 M carbonate buffer, pH 9.6, NaHCO₃ 8.4 g / l). The next day the plates were washed 3 times with wash buffer (0.05% Tween 20 in 1 × PBS) using a Biotek apparatus for washing tablets. Plates were then blocked with 200 ul / well of blocking buffer (0,05% BSA, 0,1% Tween 20, 0.01% thimerosal in 1 × PBS) and incubated at room temperature for 1 hour. After blocking for one hour, plates were washed 3 times with washing buffer using a Biotek apparatus for washing tablets. Serum from the Ang-2 immunized XenoMouse mice or in neimmunizovannyh XenoMouse animals, were titrated in 0,5% BSA / PBS-buffer at dilutions of 1: 3 in duplicates, since dilution of 1: 100. The last well was left for controls. These plates were incubated at room temperature for 2 hours and then washed 3 times with washing buffer using a Biotek apparatus for washing tablets. Thereafter was added horseradish peroxidase-conjugated Fc-specific goat anti-human IgG (HRP, Pierce, Rockford, IL) at a final concentration of 1 ug / ml and incubated for 1 hour at room temperature. Plates were then washed 3 times with washing buffer using a Biotek apparatus for washing tablets.

After washing, plates were developed by adding a chromogenic substrate TMB (BioFx BSTP-0100-01) for 10-20 minutes or as long as the negative control wells started to appear no coloration. ELISA-analysis was then stopped by adding solution to terminate the reaction (650 nM blocking reagent for TMB (BioFx BSTP-0100-01), diluted with 100 ml H₂O per vial). The specific titer of each animal XenoMouse® determined by absorbance at 650 nm and the results are summarized in the following Tables 2 and 3. The magnitude titer represents the reciprocal value of the highest serum dilution with a value OD, which is twice the background value. Therefore, the higher the value, the higher the level of humoral immune response to Ang-2.

<tables num="1"><table frame="all"><tgroup cols="4" rowsep="1" colsep="1"><colspec colname="c0" colwidth="27mm" /><colspec colname="c1" colwidth="69mm" /><colspec colname="c2" colwidth="5mm" /><colspec colname="c3" colwidth="62mm" /><tbody><row><entry align="right" namest="c0" nameend="c3" rowsep="1" colsep="0">Table 2Gruppa 1: 10 mice (XMG2 strain)</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="justify" namest="c1" nameend="c2" rowsep="1" colsep="1">After 4 injections</entry><entry align="justify" rowsep="1" colsep="0">After 6 injections</entry></row><row><entry align="justify" rowsep="1" colsep="1">mouse ID</entry><entry align="justify" namest="c1" nameend="c3" rowsep="1" colsep="0">Reactivity to rhAng-2</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="justify" namest="c1" nameend="c3" rowsep="1" colsep="0">Antibody titers against hIgG</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-1</entry><entry align="justify" rowsep="1" colsep="1">92000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">231000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-2</entry><entry align="justify" rowsep="1" colsep="1">56000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">212000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-3</entry><entry align="justify" rowsep="1" colsep="1">73000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">331000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-4</entry><entry align="justify" rowsep="1" colsep="1">16000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">175000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-5</entry><entry align="justify" rowsep="1" colsep="1">95000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">236000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-6</entry><entry align="justify" rowsep="1" colsep="1">27000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">119000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-7</entry><entry align="justify" rowsep="1" colsep="1">100000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">239000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-8</entry><entry align="justify" rowsep="1" colsep="1">25000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">165000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-9</entry><entry align="justify" rowsep="1" colsep="1">68000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">136000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O825-10</entry><entry align="justify" rowsep="1" colsep="1">120000</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">264000</entry></row><row><entry align="justify" rowsep="1" colsep="1">NC</entry><entry align="justify" rowsep="1" colsep="1">35</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">65</entry></row><row><entry align="justify" rowsep="1" colsep="1">RS</entry><entry align="justify" rowsep="1" colsep="1">Sensitivity: 10 ng / ml</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">Sensitivity: 9 ng / ml</entry></row><row><entry align="justify" namest="c0" nameend="c3" rowsep="0" colsep="0">* NC = штамм xmg2, ova gp2, fp* PC = козье антитело против huAng-2 (R&amp;D Systems, Catalog No. AF623), 1 мг/мл</entry></row></tbody></tgroup></table></tables>

<tables num="2"><table frame="all"><tgroup cols="4" rowsep="1" colsep="1"><colspec colname="c0" colwidth="27mm" /><colspec colname="c1" colwidth="69mm" /><colspec colname="c2" colwidth="5mm" /><colspec colname="c3" colwidth="62mm" /><tbody><row><entry align="right" namest="c0" nameend="c3" rowsep="1" colsep="0">3Gruppa Table 2: 10 mice (XMG4 strain)</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="justify" namest="c1" nameend="c2" rowsep="1" colsep="1">After 4 injections</entry><entry align="justify" rowsep="1" colsep="0">After 6 injections</entry></row><row><entry align="justify" rowsep="1" colsep="1">mouse ID</entry><entry align="justify" namest="c1" nameend="c3" rowsep="1" colsep="0">Reactivity to rhAng-2</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="justify" namest="c1" nameend="c3" rowsep="1" colsep="0">Antibody titers against hIgG</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-1</entry><entry align="justify" rowsep="1" colsep="1">750</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">4600</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-2</entry><entry align="justify" rowsep="1" colsep="1">200</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">5800</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-3</entry><entry align="justify" rowsep="1" colsep="1">500</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">7400</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-4</entry><entry align="justify" rowsep="1" colsep="1">225</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">4700</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-5</entry><entry align="justify" rowsep="1" colsep="1">300</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">5800</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-6</entry><entry align="justify" rowsep="1" colsep="1">550</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">7400</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-7</entry><entry align="justify" rowsep="1" colsep="1">1600</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">11000</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-8</entry><entry align="justify" rowsep="1" colsep="1">45</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">2400</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-9</entry><entry align="justify" rowsep="1" colsep="1">600</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">6900</entry></row><row><entry align="justify" rowsep="1" colsep="1">O824-10</entry><entry align="justify" rowsep="1" colsep="1">225</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">2300</entry></row><row><entry align="justify" rowsep="1" colsep="1">NC</entry><entry align="justify" rowsep="1" colsep="1">&lt;100</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">35</entry></row><row><entry align="justify" rowsep="1" colsep="1">RS</entry><entry align="justify" rowsep="1" colsep="1">Sensitivity: 12 ng / ml</entry><entry align="justify" namest="c2" nameend="c3" rowsep="1" colsep="0">Sensitivity: 8 ng / ml</entry></row><row><entry align="justify" namest="c0" nameend="c3" rowsep="0" colsep="0">* NC = 3c-l N128-3* PC = козье антитело против huAng-2 (R&amp;D Systems, Catalog No. AF623), 1 мг/мл</entry></row></tbody></tgroup></table></tables>

EXAMPLE 2

ISOLATION lymphocytes isolated B cells, cell fusion and generation of hybridomas.

Immunized mice were sacrificed by cervical dislocation, and the mice of each group were taken draining lymph nodes, and then they were pooled. Lymphoid cells were disrupted by grinding in DMEM to release the cells from the tissues and the cells were suspended in DMEM. Cells were counted, and the cell precipitate was added 0.9 ml of DMEM to 100 million lymphocytes for soft but full resuspend cells. Using 100 l of CD90 magnetic⁺-spheres to 100 million cells, the cells were labeled by incubating them with the magnetic beads at 4 ° C for 15 minutes. The cell suspension was labeled with using magnetic fields and containing up to 10⁸ positive cells (or whole to 2 × 10⁹ cells) was loaded onto the column LS +, and the column washed with DMEM. The total effluent was collected as the CD90-negative fraction (most of these cells, presumably formed B cells).

Cell fusion was performed by mixing washed enriched B cells described above with non-secretory myeloma cells P3X63Ag8.653, purchased from ATCC, cat. # CRL 1580 (Kearney et al., J. Immunol. 123, 1979, 1548-1550), in the ratio of 1: 1. The cell mixture was gently pelleted by centrifugation at 800 × g. After removal of the supernatant the cells were treated with 2-4 mL of Pronase solution (CalBiochem, cat # 53702;. 0.5 mg / ml in PBS) for no more than 2 minutes. Then, to block the enzymatic activity of 3-5 ml of FBS was added and the suspension was adjusted to a total volume of 40 ml by adding cells electrofusion solution (ECFS, 0,3M sucrose, Sigma, Cat # S7903, 0.1 mM Magnesium Acetate, Sigma, Cat # M2545, 0,1 mM calcium acetate, Sigma, Cat # C4705). After centrifugation, the supernatant was removed and the cells were resuspended in 40 ml ECFS.⁶ cells / ml.

cells Electrofusion was performed using a fusion generator (Model ESM2001, Genetronic, Inc., San Diego, Ca). generator fusion chamber size was 2.0 ml, and said device has the following parameters:

Measurement conditions: voltage: 50 V, time: 50 seconds

Diaphragm: voltage: 3000 V, time: 30 microseconds

Holding time after the merger: 3 seconds

After electrofusion, cell suspensions were carefully removed from the fusion chamber under sterile conditions and transferred into a sterile tube containing the same volume of the medium for culturing hybridomas (DMEM, JRH Biosciences) with 15% FBS (Hyclone), to which was added L-glutamine, penicillin / streptomycin, OPI (oxaloacetate, pyruvate, bovine insulin) (all reagents were obtained from Sigma) and IL-6 (Boehringer Mannheim). Cells were incubated for 15-30 minutes at 37^aboutC, and then centrifuged at 400 × g (1000 rpm / min) for five minutes. These cells were gently resuspended in a small volume of Hybridoma Selection medium (medium for culturing hybridomas that were added 0,5x HA (Sigma, cat. # A9666)), and this amount was adjusted appropriately by adding additional amounts of the medium for the selection of hybridomas from calculating final culturing 5 × 10⁶ B-cells in a 96-well plate and 200 l per well. The cells were mixed gently and pipetted into 96-well plates, and then allowed to grow. On the 7th or 10th day half the medium was removed, and the cells were fed media for the selection of hybridomas.

EXAMPLE 3

SELECTION OF CANDIDATE ANTIBODIES BY-ELISA.

After 14 days of culture, hybridoma supernatants were screened for Ang-2-specific monoclonal antibody. ELISA-plates (Fisher, Cat. № 12-565-136) were coated with 50 .mu.l / well of human Ang-2 (2 mg / ml) in coating buffer (0.1 M carbonate buffer, pH 9.6, NaHCO₃, 8.4 g / l) and then incubated at 4 ° C overnight. After incubation, the plates 3 times induwali wash buffer (0.05% Tween-20 in PBS). Then, 200 ul / well of blocking buffer (0,5% BSA, 0,1% Tween-20, 0.01% Thimerosal in 1x PBS) and the plates were incubated at room temperature for 1 hour. After incubation, the plates were washed three times with wash buffer. Then 50 .mu.l / well of hybridoma supernatants, and positive and negative controls, the plates were then incubated at room temperature for 2 hours. During the whole of the experiment as a positive control, a serum taken from Ang-2 mice immunized XenoMouse, Ang-2 Group 1 XMG2, footpad (fp) which is designated N160-7, and the serum was used as a negative control, taken from the KLH-immunized XenoMouse KLH-mice of group 1 XMG2, footpad (fp) which is designated L627-6.

After incubation, the plates were washed three times with wash buffer. Thereafter, 100 .mu.l / well of detection of HRP-conjugated goat anti huIgGFc (Caltag, Cat. № H10507), and plates were incubated at room temperature for 1 hour. The second positive samples screening procedure in the first screening were screened in two stages, one - for detecting hIgG, and in the other - for the detection of kappa light chain of human Ig (HRP-conjugated goat anti-kappa hIg) (Southern Biotechnology, Cat №. 2060-05), in order to demonstrate that the composition comprises a fully human antibody IgG and Ig-kappa. After incubation, the plates were washed three times with wash buffer. Then, 100 ul / well of TMB (BioFX Lab. Cat. № TMSK-0100-01) and the plates were left for color development approximately 10 minutes (as long as the negative control wells barely noticeable not detectable dye). Then 50 l / well to stop the reaction solution (a blocking solution of TMB (BioFX Lab. Cat. # STPR-0100-01), and the plates were read on a ELISA-plate reader at a wavelength of 450 nm. There was obtained 185 fully human IgG antibodies kappa against Ang-2.

All antibodies that bind to ELISA-analysis, were kontrskrinirovaniyu binding to Ang-1 by ELISA in order to eliminate antibodies cross-reactive with Ang-1. ELISA-plates (Fisher, Cat. № 12-565-136) were coated with 50 .mu.l / well of recombinant Ang-1 (2 ug / ml, obtained from R &amp; D Systems, Cat. № 293-AN-025 / CF) in coating buffer (0,1 M carbonate buffer, pH 9,6, NaHCO₃, 8.4 g / l) and then incubated at 4 ° C overnight. In the experimental conditions described here, when the recombinant Ang-1 molecule was immobilized on the ELISA-plate, the binding of antibodies to Ang-1 was not observed. However, the method described here kontrskrinirovaniya has some technical limitations. First, the antibodies were obtained from cell lines, but not from cloned hybridoma. Binding signals from a particular clone, which is only a small percentage of the line, may be below the detection sensitivity threshold. Secondly, in this experiment, certain epitopes in the antigen may be concealed from the antibodies due to slight conformational changes induced by immobilization of the antigen. For these reasons, the cross-reactivity of each antibody to Ang-1 was further evaluated using cloned antibodies and a Biacore system (see. Example 8). As described in Example 8, it was found that only one clone (mAb 3.19.3) actually had a strong cross-reactivity to human recombinant Ang-1 (Examples 8, 9 and 12).

EXAMPLE 4

INHIBITION OF ANG-2 BINDING Since TIE-2

As discussed above, the biological effect of Ang-2 is its binding to the receptor Tie-2. Monoclonal antibodies that inhibit the binding of Ang-2 / Tie-2 have been identified by a competitive binding assay using a modified ELISA. MAb used are the products of 50 ml mikroochistki depleting the pool of supernatants Ang-2-specific hybridomas (see. Example 3). 96-well plates Nunc Immplates^TM coated with 100 .mu.l of human recombinant fusion protein Tie-2 / Fc (R &amp; D Systems, Inc., Cat. № 313-TI-100) at a density of 4 g / ml by incubating overnight at 4 ° C. Plates were washed four times with phosphate buffered saline (PBS) for the washer Skan^TM Washer 300 (SKATRON). Wells were blocked by adding 100 .mu.l of ABX-blocking buffer (0,5% BSA, 0,1% Tween, 0.01% Thimerosal in PBS) for 1 hour.

To each well, containing or not containing an anti-Ang-2 mAb at 100 ug / ml biotinylated recombinant human Ang-2 (R &amp; D Systems, Inc. Cat. No. BT623) at 100 ng / ml. Plates were incubated at room temperature for two hours and then the unbound molecules were removed by washing. Thereafter, a related biotinylated Ang-2 was detected with 100 ul / well streptavidin-HRP conjugate at 1: 200 by incubating at room temperature for an hour. After two rinsing linked streptavidin was detected using HRP substrate (R &amp; D Systems, Cat. DY998 №). Plates were incubated for 30 minutes and then added to stop the reaction 450 blocking solution (100 l / well, BioFX, Cat # BSTP-0100-01). The absorbance at 450 nm was determined on a plate reader Spectramax Plus.

As a positive control, a recombinant soluble fusion protein Tie-2 / Fc with 10-fold molar excess with respect to Ang-2. At this concentration, the fusion protein Tie-2 / Fc inhibited by 80% the binding of Ang-2 to immobilized Tie-2. If this fact is used as an arbitrary criterion, we can say that 74 out of 175 Ang-2 binding mAb exhibited inhibitory activity. For the convenience of the 27 best neutralizing antibodies were selected procedures for subsequent hybridoma cloning.

Each hybridoma was cloned by limiting dilution in accordance with the following standard procedures. Of each hybridoma was taken three sister clones. For each clone, the supernatant was tested by ELISA-analysis of binding to human Ang-2 and cross-linking with Ang-1, as described above, in order to ensure that each antibody is specific only to Ang-2. IgG concentrations were then determined in the supernatants debilitating, and one clone of the three sister clones from each hybridoma, which has the highest yield, was selected for purification of IgG. For further characterization of each supernatant was allocated 0.5-1 mg IgG.

To quantify the activity of the mAb, directed to inhibition of binding Ang-2 with Tie-2, the titer of purified mAb was determined from the best of all 27 candidates using a competitive binding assay. Each concentration of mAb was tested in duplicate. Dependence "concentration - response" set on the curve plotted using a graphical computer program Graphpad Prism^TM (Nonlinear sigmoid curve). Maximum inhibition (efficacy) and IC₅₀ (Activity) was calculated using a computer program. Ten monoclonal antibodies having relative high efficacy and potency were selected for further study. The efficacy and potency of these 10 mAb illustrated in Table 4.

<tables num="3"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="30mm" /><colspec colname="c1" colwidth="53mm" /><colspec colname="c2" colwidth="48mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 4Effektivnost best activity and mAb 10</entry></row><row><entry align="center" rowsep="1" colsep="1">branch</entry><entry align="center" rowsep="1" colsep="1">Efficiency^*</entry><entry align="center" rowsep="1" colsep="0">EC₅₀ (Ug / ml)</entry></row><row><entry align="center" rowsep="1" colsep="1">3.31.2</entry><entry align="center" rowsep="1" colsep="1">0,3751</entry><entry align="center" rowsep="1" colsep="0">0,04169</entry></row><row><entry align="center" rowsep="1" colsep="1">5.16.3</entry><entry align="center" rowsep="1" colsep="1">0,3279</entry><entry align="center" rowsep="1" colsep="0">0,08532</entry></row><row><entry align="center" rowsep="1" colsep="1">5.86.1</entry><entry align="center" rowsep="1" colsep="1">0,3844</entry><entry align="center" rowsep="1" colsep="0">0,1331</entry></row><row><entry align="center" rowsep="1" colsep="1">5.88.3</entry><entry align="center" rowsep="1" colsep="1">0,4032</entry><entry align="center" rowsep="1" colsep="0">0,1557</entry></row><row><entry align="center" rowsep="1" colsep="1">3.3.2</entry><entry align="center" rowsep="1" colsep="1">0,3881</entry><entry align="center" rowsep="1" colsep="0">0,1684</entry></row><row><entry align="center" rowsep="1" colsep="1">5.103.1</entry><entry align="center" rowsep="1" colsep="1">0,2317</entry><entry align="center" rowsep="1" colsep="0">0,3643</entry></row><row><entry align="center" rowsep="1" colsep="1">5.101.1</entry><entry align="center" rowsep="1" colsep="1">0,3639</entry><entry align="center" rowsep="1" colsep="0">0,3762</entry></row><row><entry align="center" rowsep="1" colsep="1">3.19.3</entry><entry align="center" rowsep="1" colsep="1">0,3945</entry><entry align="center" rowsep="1" colsep="0">0,7976</entry></row><row><entry align="center" rowsep="1" colsep="1">5.28.1</entry><entry align="center" rowsep="1" colsep="1">0,3892</entry><entry align="center" rowsep="1" colsep="0">2,698</entry></row><row><entry align="center" rowsep="1" colsep="1">5.78.3</entry><entry align="center" rowsep="1" colsep="1">0,2621</entry><entry align="center" rowsep="1" colsep="0">5,969</entry></row><row><entry align="left" namest="c0" nameend="c2" rowsep="0" colsep="0">* Efficacy is expressed as the ratio of Ang-2 bound to mAb (30 ug / ml) to Ang-2, not associated with the mAb.</entry></row></tbody></tgroup></table></tables>

EXAMPLE 5

MAPPING OF ANTIBODIES

Epitope mapping was performed to determine which of the anti-Ang-2 antibodies are competing with each other for crosslinking, and therefore likely to bind to the same epitope on Ang-2. mapping process is described in US Patent Application 20030175760, and in the publication Jia et al., J. Immunol. Methods, (2004)<u>288</u>: 91-98, which in its entirety are herein incorporated by reference. Briefly, Luminex scope associated with mouse anti-hulgG antibody (Pharmingen # 555784) according to the protocol binding proteins, which can be found on the web-site Luminex. Pre associated spheres obtained for binding to the "first" unknown antibody in accordance with the following procedure, said spheres were stored in a place protected from light. For each unknown supernatant individual tubes. The required volume of the supernatant was calculated using the following formula: (n × 2 + 10) × 50 l (where n = total number of samples). In this assay concentration of 0.1 ug / ml. The mother liquor was lightly stirred for each area, and diluted in supernatant to a concentration of 2500 or 0,5 × 10⁵ spheres / ml in 50 l / well.

Samples were incubated on a shaker in the dark at room temperature overnight.

The filter plate was pre-wetted by adding 200 l wash buffer per well, and then the buffer was aspirated. To each well of the filter plate was added 50 ul of each sphere. Samples were washed once by adding 100 ul / well wash buffer and suction. In said filter plate was added to antigen and control at 50 l / well. Then, the plate sealed, incubated in the dark for 1 hour on a shaker, and then samples were washed 3 times. Thereafter was added the "second" unknown antibody at 50 l / well. The concentration of the "first" antibody was 0.1 .mu.g / ml. The plates were then incubated in the dark for 2 hours at room temperature on a shaker, and then samples were washed 3 times. Then 50 .mu.l / well of biotinylated mouse anti-human IgG (Pharmingen # 555785),

The samples were washed 3 times. Then 50 l / well Streptavidin-PE diluted 1: 1000, and the samples were incubated in the dark for 15 minutes at room temperature with shaking. After two wash cycles on the Luminex apparatus 100, samples were washed 3 times. The contents of each well were resuspended in 80 ul blocking buffer. Samples were gently mixed by pipetting several times to resuspend the spheres. Samples were then analyzed on the device Luminex 100. Results are shown in Table 5 below.

<tables num="4"><table frame="all"><tgroup cols="8" rowsep="1" colsep="1"><colspec colname="c0" colwidth="19mm" /><colspec colname="c1" colwidth="21mm" /><colspec colname="c2" colwidth="21mm" /><colspec colname="c3" colwidth="21mm" /><colspec colname="c4" colwidth="21mm" /><colspec colname="c5" colwidth="21mm" /><colspec colname="c6" colwidth="21mm" /><colspec colname="c7" colwidth="19mm" /><tbody><row><entry align="right" namest="c0" nameend="c7" rowsep="1" colsep="0">Table 5Rezultaty mapping (Bin) for the best 24 positive anti-Ang-2 antibodies in a functional assay</entry></row><row><entry align="justify" rowsep="1" colsep="1">Bin1</entry><entry align="justify" rowsep="1" colsep="1">sins.2</entry><entry align="justify" rowsep="1" colsep="1">Bin3</entry><entry align="justify" rowsep="1" colsep="1">Bin4</entry><entry align="justify" rowsep="1" colsep="1">BIN5</entry><entry align="justify" rowsep="1" colsep="1">Bin6</entry><entry align="justify" rowsep="1" colsep="1">Bin7</entry><entry align="justify" rowsep="1" colsep="0">Bin8</entry></row><row><entry align="justify" rowsep="0" colsep="1">3.3 </entry><entry align="justify" rowsep="0" colsep="1">3.38 </entry><entry align="justify" rowsep="0" colsep="1">5.56* </entry><entry align="justify" rowsep="0" colsep="1">5.28 </entry><entry align="justify" rowsep="0" colsep="1">5.78 </entry><entry align="justify" rowsep="0" colsep="1">3.19 </entry><entry align="justify" rowsep="0" colsep="1">6.3 </entry><entry align="justify" rowsep="0" colsep="0">5.35</entry></row><row><entry align="justify" rowsep="0" colsep="1">3.28</entry><entry align="justify" rowsep="1" colsep="1">5.103 </entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="justify" rowsep="1" colsep="0">5.40</entry></row><row><entry align="justify" rowsep="0" colsep="1">3.31</entry><entry align="left" rowsep="1" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.14</entry><entry align="justify" rowsep="1" colsep="1">5.2</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.16</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.39</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.41</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.49</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.54</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.62</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.83</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.86</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.88</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.101</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" rowsep="0" colsep="1">5.108</entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" namest="c0" nameend="c7" rowsep="0" colsep="0">* Note: mAb 5.56 show the same binding pattern, with only minor differences, as well as mAb 3.38 and 5.103, and gave a much lower signal.</entry></row></tbody></tgroup></table></tables>

EXAMPLE 6

Determination of the Affinity ANTI-ANG-2 ANTIBODIES USING BIACORE ANALYSIS

<u>Screening low resolution for 27 purified monoclonal antibodies</u>

To measure the antibody affinity to the antigen used surface plasmon resonance without labeling (SPR), or Biacore. For these purposes, the surface of CM5 Biacore chip coated goat anti-human antibody at a high density using routine amine coupling method. All purified mAb were diluted to approximately 8 .mu.g / ml in running buffer HBS-P, containing 100 ug / ml BSA and 10 mg / mL carboxymethyldextran. Each mAb was immobilized on a separate surface for contacting time 42 seconds and washed for 5 minutes to stabilize the baseline mAb.

On all surfaces for one minute Ang-2 was injected at 90.9 nM and then dissociating performed for 10 minutes. These dual reference binding was obtained by subtracting the signal magnitude from the magnitude of the signal obtained to a control flow cell and subtracting the baseline level "drift" buffer injected just prior to injection of Ang-2. Data for the binding of Ang-2 with each mAb were normalized for the amount of mAb, immobilized on each surface, and the normalized and adjusted for drift answers for 27 mAb. According to reports built common curve for the model reaction of 1: 1 in order to determine the binding kinetics. The results of kinetic analysis of binding Ang-2, carried out at 25 ° C, are given in the table below, mAb listed in order of decreasing affinity.

<tables num="5"><table frame="all"><tgroup cols="5" rowsep="1" colsep="1"><colspec colname="c0" colwidth="27mm" /><colspec colname="c1" colwidth="52mm" /><colspec colname="c2" colwidth="27mm" /><colspec colname="c3" colwidth="31mm" /><colspec colname="c4" colwidth="25mm" /><tbody><row><entry align="right" namest="c0" nameend="c4" rowsep="1" colsep="0">Table 6Skrining low resolution Ang-2 using Biacore to 27 purified monoclonal antibodies</entry></row><row><entry align="justify" rowsep="1" colsep="1">Sample</entry><entry align="justify" rowsep="1" colsep="1">The amount of immobilized antibody (RU)</entry><entry align="justify" rowsep="1" colsep="1">k_a (M^-1from^-1)</entry><entry align="justify" rowsep="1" colsep="1">k_d (from^-1)</entry><entry align="justify" rowsep="1" colsep="0">TO_d (PM)</entry></row><row><entry align="left" rowsep="1" colsep="1">5.16</entry><entry align="left" rowsep="1" colsep="1">157</entry><entry align="left" rowsep="1" colsep="1">3,6×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">27</entry></row><row><entry align="left" rowsep="1" colsep="1">5.41</entry><entry align="left" rowsep="1" colsep="1">152</entry><entry align="left" rowsep="1" colsep="1">3,6×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">28</entry></row><row><entry align="left" rowsep="1" colsep="1">5.35</entry><entry align="left" rowsep="1" colsep="1">138</entry><entry align="left" rowsep="1" colsep="1">3,4×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">29</entry></row><row><entry align="left" rowsep="1" colsep="1">3.38</entry><entry align="left" rowsep="1" colsep="1">143</entry><entry align="left" rowsep="1" colsep="1">3,4×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">30</entry></row><row><entry align="left" rowsep="1" colsep="1">5.108</entry><entry align="left" rowsep="1" colsep="1">66</entry><entry align="left" rowsep="1" colsep="1">3,2×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">31</entry></row><row><entry align="left" rowsep="1" colsep="1">3.3</entry><entry align="left" rowsep="1" colsep="1">125</entry><entry align="left" rowsep="1" colsep="1">3,0×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">33</entry></row><row><entry align="left" rowsep="1" colsep="1">5.49</entry><entry align="left" rowsep="1" colsep="1">260</entry><entry align="left" rowsep="1" colsep="1">3,0×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">33</entry></row><row><entry align="left" rowsep="1" colsep="1">3.28</entry><entry align="left" rowsep="1" colsep="1">280</entry><entry align="left" rowsep="1" colsep="1">2,7×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">37</entry></row><row><entry align="left" rowsep="1" colsep="1">5.88</entry><entry align="left" rowsep="1" colsep="1">65</entry><entry align="left" rowsep="1" colsep="1">2,7×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">37</entry></row><row><entry align="left" rowsep="1" colsep="1">5.28</entry><entry align="left" rowsep="1" colsep="1">136</entry><entry align="left" rowsep="1" colsep="1">2,5×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">40</entry></row><row><entry align="left" rowsep="1" colsep="1">5.78</entry><entry align="left" rowsep="1" colsep="1">222</entry><entry align="left" rowsep="1" colsep="1">2,4×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">42</entry></row><row><entry align="left" rowsep="1" colsep="1">5.39</entry><entry align="left" rowsep="1" colsep="1">166</entry><entry align="left" rowsep="1" colsep="1">2,3×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">43</entry></row><row><entry align="left" rowsep="1" colsep="1">5.103</entry><entry align="left" rowsep="1" colsep="1">127</entry><entry align="left" rowsep="1" colsep="1">2,2×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">45</entry></row><row><entry align="left" rowsep="1" colsep="1">5.13</entry><entry align="left" rowsep="1" colsep="1">78</entry><entry align="left" rowsep="1" colsep="1">2,1×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">47</entry></row><row><entry align="left" rowsep="1" colsep="1">5.14</entry><entry align="left" rowsep="1" colsep="1">471</entry><entry align="left" rowsep="1" colsep="1">2,0×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">49</entry></row><row><entry align="left" rowsep="1" colsep="1">3.31</entry><entry align="left" rowsep="1" colsep="1">196</entry><entry align="left" rowsep="1" colsep="1">1,9×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">51</entry></row><row><entry align="left" rowsep="1" colsep="1">5.56</entry><entry align="left" rowsep="1" colsep="1">144</entry><entry align="left" rowsep="1" colsep="1">1,9×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">52</entry></row><row><entry align="left" rowsep="1" colsep="1">5.2</entry><entry align="left" rowsep="1" colsep="1">111</entry><entry align="left" rowsep="1" colsep="1">1,6×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">62</entry></row><row><entry align="left" rowsep="1" colsep="1">5.62</entry><entry align="left" rowsep="1" colsep="1">126</entry><entry align="left" rowsep="1" colsep="1">1,5×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">65</entry></row><row><entry align="left" rowsep="1" colsep="1">5.54</entry><entry align="left" rowsep="1" colsep="1">131</entry><entry align="left" rowsep="1" colsep="1">1,5×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">66</entry></row><row><entry align="left" rowsep="1" colsep="1">6.3</entry><entry align="left" rowsep="1" colsep="1">221</entry><entry align="left" rowsep="1" colsep="1">1,4×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">73</entry></row><row><entry align="left" rowsep="1" colsep="1">3.19</entry><entry align="left" rowsep="1" colsep="1">252</entry><entry align="left" rowsep="1" colsep="1">9,0×10⁴</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">111</entry></row><row><entry align="left" rowsep="1" colsep="1">5.40</entry><entry align="left" rowsep="1" colsep="1">130</entry><entry align="left" rowsep="1" colsep="1">7,8×10⁴</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">129</entry></row><row><entry align="left" rowsep="1" colsep="1">5.83</entry><entry align="left" rowsep="1" colsep="1">157</entry><entry align="left" rowsep="1" colsep="1">6,8×10⁴</entry><entry align="left" rowsep="1" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="1" colsep="0">147</entry></row><row><entry align="left" rowsep="1" colsep="1">5.101</entry><entry align="left" rowsep="1" colsep="1">217</entry><entry align="left" rowsep="1" colsep="1">1,5×10⁵</entry><entry align="left" rowsep="1" colsep="1">8,7×10^-5</entry><entry align="left" rowsep="1" colsep="0">581</entry></row><row><entry align="left" rowsep="1" colsep="1">5.86</entry><entry align="left" rowsep="1" colsep="1">126</entry><entry align="left" rowsep="1" colsep="1">1,5×10⁵</entry><entry align="left" rowsep="1" colsep="1">1,1×10^-4</entry><entry align="left" rowsep="1" colsep="0">744</entry></row><row><entry align="left" rowsep="0" colsep="1">5.52</entry><entry align="left" rowsep="0" colsep="1">114</entry><entry align="left" rowsep="0" colsep="1">1,3×10⁵</entry><entry align="left" rowsep="0" colsep="1">1,0×10^-5*</entry><entry align="left" rowsep="0" colsep="0">750</entry></row></tbody></tgroup></table></tables>

Asterisk appearing after the values ​​of k_d for all of the mAb, with the exception of mAb 5.101 and 5.86 indicate that these k value_d They are constants and represent the best estimate for the order of magnitude characterizing parameters for low dissociation rate. Selection of the model for these samples did not show any significant changes in the rate of dissociation of such a relatively short time of dissociation, and therefore the selection of the data rate of association requires that the value of k_d It was constant. Data for these antibodies indicated that these antibodies with such values ​​k_d They operate in the same manner as an antibody to k_d about 10^-6 from^-1And therefore the interactions may be 10 times or more stronger than the interaction mentioned above.

These dissociation normally measured 4-6 hours for experiments on the kinetics of high resolution using mAb, with affinity below 100 pM. The maximum dissociation time that can be measured without artifacts "drift" from the immobilized on mAb surface is 20 minutes. Attenuation almost undetectable signals for high affinity mAb was measured over a relatively short period of time, and therefore, the values ​​of k_d may vary considerably within the values ​​constituting two orders of magnitude.

EXAMPLE 7

Determination of the Affinity ANTI-ANG-2 ANTIBODIES USING BIACORE ANALYSIS

<u>Screening Medium / High Resolution Ang-2 using the three purified monoclonal antibodies</u>

Purified mAb 5.16, 5.35, and 5.41 were diluted to approximately 8 .mu.g / ml in 10 mM sodium acetate, pH 5,0. Then, each diluted mAB was immobilized on different flow cells surfaces (chip CM5 Biacore) by routine amine coupling.

To obtain association rate data randomly injected eight concentrations (2-fold dilutions) Ang-2 in the range 90,9-0,71 nM for 90 seconds in triplicate, with several buffer injections done alternately with dual control injections, followed by dissociation within four minutes. antibody surface was regenerated by two 9-second injection of 10 mM glycine-HCl, pH 1,5, after each injection cycle.

For data rate diassotsiatsii three samples were injected with 90.9 nM Ang-2 in the running buffer HBS-P, containing 100 ug / ml BSA, as described above and dissociation values ​​recorded over eight hours. Injection of the sample made alternately with three cycles of blind control injections. Regeneration was performed as described above.

According to reports built common curve for the model reaction of 1: 1 with mass transport using CLAMP (David G. Myszka and Thomas Morton (1998) "CLAMP ©: a biosensor kinetic data analysis program," TIBS 23, 149-150). The resulting binding constants are shown in Table 7.

<tables num="6"><table frame="all"><tgroup cols="5" rowsep="1" colsep="1"><colspec colname="c0" colwidth="27mm" /><colspec colname="c1" colwidth="35mm" /><colspec colname="c2" colwidth="33mm" /><colspec colname="c3" colwidth="33mm" /><colspec colname="c4" colwidth="33mm" /><tbody><row><entry align="right" namest="c0" nameend="c4" rowsep="1" colsep="0">Table 7Skrining medium resolution Ang-2 using Biacore 3 purified monoclonal antibodies</entry></row><row><entry align="justify" rowsep="1" colsep="1">Sample</entry><entry align="justify" rowsep="1" colsep="1">R_max</entry><entry align="justify" rowsep="1" colsep="1">k_a (M^-1from^-1)</entry><entry align="justify" rowsep="1" colsep="1">k_d (from^-1)</entry><entry align="justify" rowsep="1" colsep="0">TO_d (PM)</entry></row><row><entry align="justify" rowsep="1" colsep="1">5.16</entry><entry align="justify" rowsep="1" colsep="1">36</entry><entry align="justify" rowsep="1" colsep="1">3,41×10⁵</entry><entry align="justify" rowsep="1" colsep="1">2,77×10^-6</entry><entry align="justify" rowsep="1" colsep="0">8,13</entry></row><row><entry align="justify" rowsep="1" colsep="1">5.35</entry><entry align="justify" rowsep="1" colsep="1">54</entry><entry align="justify" rowsep="1" colsep="1">5,64×10⁵</entry><entry align="justify" rowsep="1" colsep="1">1,87×10^-6</entry><entry align="justify" rowsep="1" colsep="0">3,31</entry></row><row><entry align="justify" rowsep="0" colsep="1">5.41</entry><entry align="justify" rowsep="0" colsep="1">44</entry><entry align="justify" rowsep="0" colsep="1">4,69×10⁵</entry><entry align="justify" rowsep="0" colsep="1">8,31×10^-6*</entry><entry align="justify" rowsep="0" colsep="0">17,7*</entry></row></tbody></tgroup></table></tables>

Significant attenuation was measured over the 8-hour dissociation. Using the data obtained for the 8-hour dissociation, CLAMP software can more accurately determine the value of k_d for each mAb. In this case, k_d is about 10 for antibodies 5.16 and 5.35^-6 from^-1.

Then the cross-reactivity of antibodies to Ang-1 was investigated by measuring the binding affinity of mAb with Ang-1, as described in Example 8.

EXAMPLE 8

Determination of the Affinity ANTI-ANG-1 ANTIBODY USING BIACORE ANALYSIS

Cross-reactivity of antibodies to Ang-1 further investigated by measuring the binding affinity of mAb with Ang-1. In this case, instead of immobilizing Ang-1, as performed in the described ELISA-assay on crosslinking (Example 3) was carried out immobilization of anti-Ang-2 mAb on chip CM5 Biacore, and then injected with Ang-1 in solution to determine assotsiiatsii speed and dissociation. Six mAb, including 3.3.2, 3.31.2, 5.16.3, 5.86.1, 5.88.3 and 3.19.3, were tested in this experiment as described below to determine the level of cross-linking with Ang-1.

<u>Screening medium resolution for the six purified monoclonal antibodies</u>

To measure antibody affinity to Ang-1 using surface plasmon resonance without labeling (SPR), or Biacore 2000. device for this purpose on CM5 Biacore chip surface coated goat anti-human antibody at a high density using routine amine coupling method. For carrying out scientific experiments, purified mAb (clones 3.19.3, 3.3.2, 5.88.3, ​​5.86.1, 3.31.2, 5.16.3) were diluted to a concentration of about 2.5-3.5 mg / ml in running buffer HBS-P, containing 100 ug / ml BSA. capture level for each mAb was approximately 150 RU. After each cycle, the capture was performed 5-minute wash for stabilization of the core layer mAb.

On all the surfaces to capture antibody for 1 minute injected one Ang-1 sample diluted to 87.4 nM in the running buffer. For five mAb any detectable binding was observed, although it was found that Ang-1 binds to the mAb 3.19.3. This experiment was repeated with increased levels of mAb binding to the wells within 500-600 RU and injecting 380 nM Ang-1 for one minute. And in this case it was found that mAb 3.19.3 was associated with Ang-1.

Because Ang-1 only showed binding activity with mAb 3.19.3, the measured binding affinity of the mAb with Ang-1 and Ang-2. Since during the above scientific experiment Ang-1 showed low dissociation rate, the experiment on screening medium resolution capture antibody did not give a sufficient amount of the dissociation rate data based on which to accurately calculate the value k_d. And therefore, binding of Ang-1 and Ang-2 to mAb 3.19.3 was measured by high resolution Biacore analysis conditions.

EXAMPLE 9

Determining the binding affinity MAB 3.19.3 C ANG-1 AND ANG-2 USING HIGH-RESOLUTION BIACORE ANALYSIS

Purified mAb 3.19.3 was diluted to about 12.5 .mu.g / ml in 10 mM sodium acetate, pH 4,0. Each mAb was then immobilized on flow cells 1-3 (chip CM5 Biacore) routine amine coupling method, and flow cell 4 was used as a control.

To obtain association rate data randomly injected eight concentrations (2-fold dilutions) Ang-1 within 39,8-0,31 nM (in running buffer HBS-P, containing 100 ug / ml BSA) for 90 seconds (flow rate 100 l / min) in triplicate, with several buffer injections done alternately with injections for dual control, followed by dissociation for four minutes. antibody surfaces were regenerated by 6-second injection of 10 mM NaOH after each injection cycle.

For data rate diassotsiatsii three samples were injected with 19.9 nM Ang-1, as described above, and the value of the dissociation were recorded over six hours. Injection of the sample made alternately with three cycles of blind control injections. Regeneration was performed as described above.

According to reports built common curve for the model reaction of 1: 1 with mass transport using CLAMP (David G. Myszka and Thomas Morton (1998) "CLAMP ©: a biosensor kinetic data analysis program," TIBS 23, 149-150).

<u>Ang-2 study conducted by high resolution Biacore analysis using the purified MAb 3.19.3</u>

Purified mAb 3.19.3 was diluted to about 12.5 .mu.g / ml in 10 mM sodium acetate, pH 4,0. MAB was then immobilized on flow cells 1-3 (chip CM5 Biacore) routine amine coupling method, and flow cell 4 was used as a control.

To obtain association rate data randomly injected eight concentrations (2-fold dilutions) Ang-2 in the range 30,0-0,23 nM (in running buffer HBS-P, containing 100 ug / ml BSA) for 90 seconds (flow rate 100 l / min) in triplicate, with several buffer injections done alternately with injections for dual control, followed by dissociation for four minutes. antibody surfaces were regenerated by 6-second injection of 15 mM NaOH after each injection cycle.

For data rate diassotsiatsii three samples were injected with 15.0 nM Ang-2 as described above, and the values ​​of dissociation was recorded for six hours. Injection of the sample made alternately with three cycles of blind control injections. After each injection cycle, each surface was regenerated by a 6-second injection of 15 mM NaOH for estimation of the dissociation rate.

According to reports built common curve for the model reaction of 1: 1 with mass transport using CLAMP (David G. Myszka and Thomas Morton (1998) "CLAMP ©: a biosensor kinetic data analysis program", TIBS 23, 149-150).

<u>Results and Discussion: The study of binding Ang-1 and Ang-2 with MAb 3.19.3, conducted by high resolution Biacore analysis</u>

For each antigen two independent experiments were performed. The results of these experiments are given in Table 8 below.

<tables num="7"><table frame="all"><tgroup cols="4" rowsep="1" colsep="1"><colspec colname="c0" colwidth="41mm" /><colspec colname="c1" colwidth="41mm" /><colspec colname="c2" colwidth="41mm" /><colspec colname="c3" colwidth="41mm" /><tbody><row><entry align="right" namest="c0" nameend="c3" rowsep="1" colsep="0">Table 8Rezultaty study binding Ang-1 and Ang-2 with purified mAb 3.19.3, conducted by high resolution Biacore analysis</entry></row><row><entry align="center" rowsep="1" colsep="1">Antigen</entry><entry align="center" rowsep="1" colsep="1">k_a (M^-1from^-1)</entry><entry align="center" rowsep="1" colsep="1">k_d (from^-1)</entry><entry align="center" rowsep="1" colsep="0">TO_d (PM)</entry></row><row><entry align="justify" rowsep="1" colsep="1">A-1 (1^st)</entry><entry align="center" rowsep="1" colsep="1">1,33×10⁵</entry><entry align="center" rowsep="1" colsep="1">4,05×10^-6</entry><entry align="center" rowsep="1" colsep="0">30,4</entry></row><row><entry align="justify" rowsep="1" colsep="1">A-1 (2^nd)</entry><entry align="center" rowsep="1" colsep="1">1,82×10⁵</entry><entry align="center" rowsep="1" colsep="1">5,51×10^-6</entry><entry align="center" rowsep="1" colsep="0">30,2</entry></row><row><entry align="justify" rowsep="1" colsep="1">A-2 (1^st)</entry><entry align="center" rowsep="1" colsep="1">1,89×10⁵</entry><entry align="center" rowsep="1" colsep="1">1,00×10^-6*</entry><entry align="center" rowsep="1" colsep="0">5,3</entry></row><row><entry align="justify" rowsep="0" colsep="1">A-2 (2^nd)</entry><entry align="center" rowsep="0" colsep="1">1,78×10⁵</entry><entry align="center" rowsep="0" colsep="1">1,00×10^-6*</entry><entry align="center" rowsep="0" colsep="0">5,6</entry></row></tbody></tgroup></table></tables>

In the above table the values ​​of k_d to Ang-2 are marked by asterisks because these values ​​were held constant during the simulation of interaction of 1: 1 using CLAMP computer program. In experiments to Ang-2 any appreciable dissociation of the signal was not registered, and therefore the best value of the dissociation rate k_d remained constant at about 1 × 10^-6 from^-1. Dissociation period of time for Ang-2 actually tended to increase in its constant data registration process carried out for six hours. This trend was repeated for the analysis on two different sensor chips with two different devices, then both of these devices were subjected to "superochistke" in accordance with the protocol. For a more accurate measurement of binding affinity of mAb 3.19.3 to Ang-1 and Ang-2 was conducted additional experiment (see. Example 10) to determine the Kd mAb 3.19.3 towards these antigens.

Interestingly, when Ang-1 was immobilized on the ELISA-plate, mAb 3.19.3 did not bind to Ang-1 in the ELISA-binding assay (Example 3). Nesootvestvie This can be explained by the fact that when Ang-1 was immobilized on a plastic surface, to the minor epitope plays an important role in the binding of mAb 3.19.3, was not provided with appropriate access. However, when Ang-1 was in the liquid phase, for example, provided by experimental conditions Biacore, then this epitope became accessible to mAb 3.19.3, whereby binding occurred.

EXAMPLE 10

Determine the affinity of MAB 3.19.3 to the human Ang-2 by high performance ANALYSIS KINEXA (KINETIC ANALYSIS exclusion)

When measuring the affinity of mAb 3.19.3 against human Ang-2 with the high-resolution analysis Biacore (Example 9) any significant dissociation signal was detected. dissociation time period for Ang-2 showed a tendency to increase during continuous data recording carried out for six hours. Based on this, k_d mAb 3.19.3, bind to human Ang-2 was determined using KinExA technology, in order to get a more reliable value k_d. For this purpose the KinExA device 3000. First, in 1 ml of formulation (<b>~</b> 271 .mu.g) of starting Ang-2 (R &amp; D Systems, Inc., Lot # BNO32510A), the buffer was replaced with 1 × PBS, pH 7,0, using the Pierce D-Salt a 10 ml desalting column (column polyacrylamide^TM cutoff molecular weight of 6000, Lot # GF97965). Concentration of the combined fractions was 1.7 pM as determined by calculating the protein concentration described C. Nick Pace (Pace, et al.,<i>Protein Science</i>, Vol. 4: 2411-2423, 1995). Thereafter, 450 .mu.l (<b>~</b> 122 .mu.g) of starting Ang-2 was associated with 200 mg of spheres of polymethylmethacrylate (PMMA, Lot # 206-01), overnight at 24ºS. Then the spheres were centrifuged and washed once with blocking buffer (1 × PBS, 10 mg / ml BSA), after which they were centrifuged again, and then incubated in blocking buffer for one hour at 24ºS. After blocking sphere diluted in approximately 30 ml of buffer HBS (0,01 M Hepes, 0,15 M NaCl, pH 7,4) in a standard vessel-container with spheres KinExA and placed in said device.

k_d<u>-controlled titration</u>

Twelve solutions containing a mAb 3.19.3 binding site concentration of 25.3 pM were titrated with increasing concentrations of Ang-2. For the preparation of Ang-2 samples were buffer exchanged. Each solution had a total volume of 25 ml, and this solution is equilibrated for 5 days at<b>~</b>24ºS. titration solutions were prepared in volumetric glassware, the Ang-2 concentrations varied from 5.09 nM to 99.3 the FM. A method conducted on KinExA apparatus for the analysis of these solutions consisted of a step of packing the areas in which the PMMA-spheres packed in glass capillary tubes and equilibrated solutions were passed through a column with spheres at a rate of 0.25 ml / min for 6 min (1 , 5 mL) in duplicate. Then, fluorescently labeled goat anti-human (Fc-spetsifichechskoe) polyclonal antibody Cy-5 at 3.4 nM was passed through a packing of spheres for 1 minute at a rate of 0.5 ml / min to label the free mAb binding sites immobilized on the spheres. Fluorescent emission packing spheres was measured at 670 nm with excitation at 620 nm._d-controlled titration curve was constructed using KinExA software, and the curve was an equilibrium isotherm 1: 1 with a correction coefficient "drift". The optimal value of k_dWhich correspond to these data, was 86.4 pM with low and high 95% confidence limits, was 64.3 pM and 98.7 pM, respectively. Curve mAb-controlled titration omitted.

EXAMPLE 11

BLOCKING ANG-2-INDUCED PHOSPHORYLATION TIE-2 ectopically expressed in HEK293 CELLS

As discussed above, Tie-2 is a receptor tyrosine kinase that is specific for endothelial cells. <i>In vitro</i> experiments performed using vascular endothelial cells show that Ang-1 induces phosphorylation of Tie-2 and Ang-2 inhibits the receptor phosphorylation induced by Ang-1. However, if the Tie-2 is expressed ectopically, such as it occurs in fibroblasts, the Ang-2 is also able to induce the phosphorylation of Tie-2 under certain conditions, including but not limited to, prolonged action of angiopoietin-2 or high angiopoietin concentrations -2.

Ang-2-induced phosphorylation of Tie-2 also occurs when the receptor is expressed in HEK293F cells. The ability of anti-Ang-2 mAb to block Ang-2 induced Tie-2 phosphorylation was assessed using HEK293F cells transfected with human Tie-2 receptor. The plasmid vector pORK / pBS-SK, having Tie-2 cDNA was obtained from ATCC (sequence BC033514:. Cat № 69003, Genbank). The cDNA sequence was verified by nucleotide sequencing. 3.9 kb fragment comprising 3375 bp cDNA kodiruschuyu human Tie-2, was removed from the vector by EcoRI enzyme hydrolysis. This fragment was subcloned in a functional orientation into the vector pCR3.1, was digested with EcoRI according to standard procedures.

Tie-2 comprising a structure obtained according to the above procedures was transfected into HEK293F cells by transfection using calcium phosphate. 1 × 10⁶ HEK293F cells were cultured in 100-mm tissue culture plates for coated 1% gelatin at 37 ° C with 5% CO₂. Prior to transfection, cells were fed with fresh media for 2-3 hours. 10 ug of plasmid DNA dissolved in 248 mM calcium phosphate solution. Transfection was performed by standard methods. Stable transfectants were selected by incubation in 0.5 mg / ml G418. Stable transfectants expressing Tie-2 were identified by FACS-analysis carried out using a mouse anti-Tie-2 mAb (R &amp; D Cat. № MAB313) and PE-conjugated goat anti-mouse IgG (Caltag, Cat. № M30004-4 ) for detection.

To perform analysis on the phosphorylation of Tie-2, HEK293F / Tie-2-transfectants were cultured in 60 mm Petri cell culture at a density of 2 × 10⁶ cells / plate with complete medium at 37 ° C with 5% CO₂ until subconfluent. The complete medium in each plate was replaced with 2 ml serum free medium. Cells were incubated for an additional 16 hours. The medium was then again replaced with 2 ml serum free medium. After incubation for 2 more hours, the cells were treated with 0.1 mM sodium orthovanadate (Sigma, Cat. № S 6508) for 20 minutes. Cells were treated with Ang-2 (2 mg / ml) in the presence or absence of mAb at 100 ug / ml. The treatment was performed in duplicate. When this was used a negative control without Ang-2 treatment. Cells were washed with ice-cold TBS containing vanadate and lysed by addition of 300 .mu.l / dish of cooled lysis buffer NP-40 (50 mM Hepes, pH 7,2, which in 0,15 M NaCl were added, 10% glycerol, 10 mM pyrophosphate, 50 mM NaF, 1% NP40, 100 U / ml aprotinin, 1 mM PMSF, 0,1 mM orthovanadate, 10 .mu.M 10 .mu.M leupeptin and pepstatin A), and the plates were placed for 10 minutes on ice. The treated cells were scraped from these plates into a microtube pre-chilled on ice.

Cell lysates were subjected to brief sonication and centrifuged at 12,000 × g for 10 minutes at 4 ° C in a tabletop microfuge. Supernatants were collected into fresh microtubes, and then the supernatant was added 1.5 ug of anti-Tie-2 mAb (R &amp; D Systems, Inc.), and the mixture was subjected to light rocking for 2 hours at 4 ° C. The resulting mixture was added 50 .mu.l of protein A immobilized, ImmunoPure (PIERCE Cat. № 20333), and the mixture was incubated for at least 3 hours at 4 ° C on a rocking platform. Complexes were collected by centrifugation at 12,000 × g for 10 minutes. After carefully removing the supernatant, the complexes were washed twice with lysis buffer by centrifuging (12,000 × g, 4 ° C) for 4 minutes. The pellet was resuspended in 50 ul of 2 × electrophoresis sample buffer (Invitrogen, Cat. № LC-2676) containing 1 mM β-mercaptoethanol or DTT, and boiled for 5 minutes and then centrifuged (12,000 × g, 4 ° C) for 5 minutes. The supernatants were transferred to fresh tubes.

The samples were loaded into wells of SDS-PAGE gel (e.g., 4-20% Tris-Glycine gel, Invitrogen, Cat. № EC 6025). Electrophoresis was performed in Tris-glycine buffer system. After electrophoresis, the gel is blotted onto PVDF-membrane (Invitrogen, Cat. № LC 2005) following the standard protocol. Tyrosine phosphorylation was confirmed using as a probe 4G10 anti-phosphotyrosine antibody at a concentration of 1 .mu.g / ml (Upstate, Cat. № 05-321) by incubation for 1 hour at room temperature with shaking, and followed by washing three times with 1 × TBST (TBS with 0.1% Tween-20). Bound antibodies were detected by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz, Cat № sc-2302.) At a dilution of 1: 10,000, for 1 hour at room temperature, and subsequent amplification reaction using the chemiluminescent substrate system SuperSignal West Dura Extended Duration Substrate system (PIERCE Cat. № 34075). The blot was then purified by washing with a reducing buffer western blots (Restore Western Blot Stripping Buffer) (PIERCE, Cat. № 21059) and re-probed with specific antibodies against RTK to verify sample loading level.

It has been found that if the human Tie-2 ectopically expressed in HEK293F cells, autophosphorylation of Tie-2 have not been observed. In response to Ang-2 treatment (2 mg / ml), a significant level of tyrosine phosphorylation was detected using mAb against phosphorylated tyrosine (4G10) Tie-2, subjected to immunoprecipitation with the specific mAb. At a concentration of 100 .mu.g / ml of all tested anti-Ang-2 mAb significantly inhibited phosphorylation of Tie-2, whereas the isotype control mAb showed no inhibitory effect (Figure 1). Monoclonal antibody 5.103.1, which is not shown in Figure 1, has a similar inhibitory effect.

To assess the ability of anti-Ang-2 mAb to inhibit Ang-2-induced phosphorylation of Tie-2 <i>in vitro</i>, A method was developed ELISA-based analysis to quantify the phosphorylation of Tie-2. Briefly, cell lysates were obtained from HEK293F / Tie-2-transfectants were treated with Ang-2 with mAb at various concentrations. The whole Tie-2, obtained from said lysate contacted with the wells of 96-well ELISA-plate that had been sensitized mouse anti-hTie-2 mAb. The phosphorylated Tie-2 were detected using HRP-conjugated "first" antibody 4G10 (purchased from Upstate) and HRP substrate solution. OD at 650 nm was determined on a plate reader SpectraMax. Dependence "concentration - response" has been established using curve constructed using the graphics computer program Graphpad Prism^TM (Nonlinear sigmoid curve). Maximum inhibition (efficacy) and IC₅₀ (Activity) was calculated, as shown in Figure 2. EC₅₀ calculated as shown in Table 9.

<tables num="8"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="55mm" /><colspec colname="c1" colwidth="55mm" /><colspec colname="c2" colwidth="55mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 9</entry></row><row><entry align="justify" rowsep="1" colsep="1">mAb</entry><entry align="justify" rowsep="1" colsep="1">EC50 (ug / ml)</entry><entry align="justify" rowsep="1" colsep="0">95% DI</entry></row><row><entry align="justify" rowsep="1" colsep="1">3.19.3</entry><entry align="justify" rowsep="1" colsep="1">0,006</entry><entry align="justify" rowsep="1" colsep="0">0,004-0,009</entry></row><row><entry align="justify" rowsep="1" colsep="1">5.86.1</entry><entry align="justify" rowsep="1" colsep="1">0,008</entry><entry align="justify" rowsep="1" colsep="0">0,007-0,011</entry></row><row><entry align="justify" rowsep="1" colsep="1">5.88.3</entry><entry align="justify" rowsep="1" colsep="1">0,016</entry><entry align="justify" rowsep="1" colsep="0">0,011-0,024</entry></row><row><entry align="justify" rowsep="1" colsep="1">3.31.2</entry><entry align="justify" rowsep="1" colsep="1">0,043</entry><entry align="justify" rowsep="1" colsep="0">0,029-0,064</entry></row><row><entry align="justify" rowsep="1" colsep="1">3.3.2</entry><entry align="justify" rowsep="1" colsep="1">0,046</entry><entry align="justify" rowsep="1" colsep="0">0,020-0,105</entry></row><row><entry align="justify" rowsep="1" colsep="1">5.16.3</entry><entry align="justify" rowsep="1" colsep="1">0,089</entry><entry align="justify" rowsep="1" colsep="0">0,046-0,174</entry></row><row><entry align="justify" rowsep="1" colsep="1">5.103.3</entry><entry align="justify" rowsep="1" colsep="1">0,095</entry><entry align="justify" rowsep="1" colsep="0">0,046-0,199</entry></row><row><entry align="justify" rowsep="0" colsep="1">5.101.1</entry><entry align="justify" rowsep="0" colsep="1">0,733</entry><entry align="justify" rowsep="0" colsep="0">0,487-1,105</entry></row></tbody></tgroup></table></tables>

As reported above, it was found that mAb 3.19.3 cross-reacts with Ang-1. However, the results of initial experiments did not reveal any inhibition of Tie-2 phosphorylation induced by angiopoietin-1 by the action of mAb 3.19.3. It should be noted that the ectopic expression of Tie-2 may affect its susceptibility to activation by different ligands, as indicated by the fact that Ang-2 did not induce phosphorylation of Tie-2 in HUVEC cells, however, and it was found that it induces persistent phosphorylation of Tie-2 in the event that said receptor ectopically expressed in HEK293 cells.

Based on these results, additional experiments were carried out to determine precisely whether mAb 3.19.3 is able to inhibit the binding of Ang-1 and Ang-2 to cell-associated Tie-2. In addition, inhibition of Angiopoietin-1 induced Tie-2 phosphorylation by the action of mAb 3.19.3 was investigated in more detail by the method described in Example 12.

EXAMPLE 12

ANTIBODY INHIBITS THE BINDING OF MAB 3.19.3 angiopoietin-1 P TIE-2 AND ANG-1 INDUCED PHOSPHORYLATION OF TIE-2

mAb 3.19.3 cross-reacts with human Ang-1 (Examples 8 and 9). However, preliminary experiments indicated that mAb 3.19.3 did not inhibit Ang-1 induced phosphorylation of Tie-2. This contradiction may be explained by the fact that: (1) for generating a stable phosphorylation of Tie-2 signal may require a high concentration of Ang-1, which may be far above physiological concentration; or (2) ectopic expression of Tie-2 in HEK293 may alter the conformation of Tie-2, and thus change its susceptibility to a variety of ligands. To test these hypotheses mAb 3.19.3 was tested in a binding assay, wherein Ang-1 or Ang-2 (3 nM) associated with Tie-2 cell surface, had a low concentration. In this experiment, it was found that mAb 3.19.3 inhibited binding of both Ang-1 and Ang-2. Besides, for research Ang-1 induced phosphorylation of Tie-2 have used immortalized endothelial cells (EA.hy926 / B3). The results of this experiment, described in more detail below, demonstrate that mAb 3.19.3 inhibits Ang-1 induced phosphorylation of Tie-2 in a dose dependent manner.

HEK293F / Tie-2 transfectants were allowed to grow to 95% confluence in culture flasks, and then collected. It was obtained cell suspension of 4 million cells / ml FAC buffer and then the suspension was divided into aliquots and distributed in 96-well polypropylene plate at 50 ul per well. The cell suspension was added to mAb 3.19.3 at the indicated concentrations. Then, this cell suspension was added to solutions of recombinant human Ang-1 and Ang-2, followed by incubation at room temperature for 2 hours. Cells were washed by centrifuging the plate at 1200 rev / min for 5 minutes, after which the supernatant was removed by aspiration, and the cells were resuspended in 200 ul per well of FACS Buffer. Washing procedures were repeated twice. The cells were then resuspended in 100 ul of mouse anti-6X-histidine, diluted to 2 ug / ml in FACS buffer and incubated at room temperature for 30 minutes. After washing, cells were suspended in 100 ul of PE-conjugated goat anti-mouse IgG, diluted 1: 100 in FACS buffer, and incubated at room temperature for 30 minutes. The sample volumes were adjusted to 300 .mu.l by the addition of FACS Buffer, and measured on the device FACS Calibur.

The results are shown in Figure 3 and summarized in Table 10. As shown above, the soluble Tie-2 / Fc showed dose-dependent inhibition of binding of Ang-1 and Ang-2 by blocking the ligands, whereas the isotype control mAb PK16.3.1 not involved in binding with any of these ligands. mAb 3.19.3 has detected a concentration-dependent inhibition of Ang-1 and Ang-2. Interestingly, as Tie-2 / Fc activity, used as standard, mAb 3.19.3 activity in binding to Ang-2 binding activity to greater than Ang-1.

<tables num="9"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="40mm" /><colspec colname="c1" colwidth="52mm" /><colspec colname="c2" colwidth="52mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 10Ingibirovanie binding Ang-1 and Ang-2 with Tie-2</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" namest="c1" nameend="c2" rowsep="1" colsep="0"><b>EC50 (nM)</b></entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1"><b>A-1</b></entry><entry align="center" rowsep="1" colsep="0"><b>A-2</b></entry></row><row><entry align="center" rowsep="1" colsep="1"><b>Tie-2/Fc</b></entry><entry align="center" rowsep="1" colsep="1">18,73</entry><entry align="center" rowsep="1" colsep="0">25,70</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>3.19.3</b></entry><entry align="center" rowsep="0" colsep="1">218,5</entry><entry align="center" rowsep="0" colsep="0">0,7310</entry></row></tbody></tgroup></table></tables>

The results showed that mAb 3.19.3 not only bound to human Ang-1, but also block its binding to the receptor, Tie-2. This again confirmed the inhibition of Ang-1 induced Tie-2 phosphorylation in immortalized endothelial cells as described below.

The activity of mAb 3.19.3, aimed at the inhibition of Ang-1 induced phosphorylation of Tie-2 was quantified as described below. mAb show a noticeable increase in the level of inhibition of Tie-2 phosphorylation with increasing antibody concentration, as shown in Figures 4 and 5. IC₅₀Calculated "dose - response" depending on the curve was 99 nM.

<u>Analysis of receptor phosphorylation stimulated by angiopoietin-1 ligand Tie-2</u>

Cells EA.hy926 / B3 were seeded in 6-well plates at a density of 2,5 × 10⁵ EA.hy926 cells per well in 2 ml volume DMEM medium containing HAT and 10% FCS, and incubated for 3 days under standard mammalian cell culture conditions.

The culture medium was replaced with 2 ml DMEM medium (without FCS) and cells were maintained in serum-free medium during the 2 hours. Test compounds doubly diluted in DMEM medium containing 1% FCS, to achieve the desired final concentration. After incubation of cells in serum-free medium for 1 hour and 40 minutes, the medium was removed and replaced with 1 ml of test compound dilutions. Similarly, for the samples that represent 100% standards ligand stimulation, was used as control, not treated with compound.

Incubation was performed for another 10 minutes and then added to each well 100 l of 10 mM orthovanadate in DMEM solution to each well to achieve a final concentration of 1 mM orthovanadate. The cells were then incubated for the last 10 minutes of the 2 hour period of culture in serum-free medium.

After completion of the 2-hour period of cultivation in serum-free medium to each well, 1 ml of Angiopoietin-1 (diluted to the appropriate concentration in DMEM medium containing 1 mM orthovanadate) and incubated at 37 ° C for another 10 minutes.

Then 6-well (s), the plate (s) cooled by placing them in an ice-cooled metal plate (which itself was on ice). The cell medium was removed, and the cell layer washed with 5 ml cold PBS, containing 1 mM orthovanadate. Then to each well was added 1 ml of ice-cold lysis buffer (20 mM Tris, pH 7,6, 150 mM NaCl, 50 mM NaF, 0,1% SDS, 1% NP40, 0,5% DOC, 1 mM orthovanadate , 1 mM EDTA, 1 mM PMSF, 30 .mu.l / ml aprotinin, 10 ug / ml pepstatin, 10 ug / ml leupeptin), and the plate was allowed to stand for 10-20 minutes on ice. Cells were scraped from the plate using means for scraping the cells, after which the whole lysate solution was transferred to a 1.5-mL Eppendorf tube and kept on ice. Then the samples were centrifuged for 3 minutes at 13000 rev / min at 4 ° C and all subsequent steps were performed at 4 ° C.

50 ul of each lysate was allowed for subsequent analysis on protein BCA (Pierce, Cat. № 23225) (in Greiner polypropylene microtiter plates for the binding assay with a low molecular weight protein). Protein concentration was determined in a standard assay conditions provided the data set. Another 800 l of each sample lysate was transferred to fresh 2 ml Eppendorf tube for immunoprecipitation (IP). Then the lysates were added 15 l (3 mg) of anti-PY antibody (Santa Cruz Cat. № E2203) and left for 2 hours at 4 ° C for an incubation, after which was added 600 .mu.l of spheres Magnabind (goat anti-mouse IgG, Pierce Cat. № 21354). Spheres Magnabind prepared as follows. The required volume was transferred into 15-mL conical tubes. Then these tubes create a magnetic field, and fluid was removed. After this fresh PBS was added to the original volume, and the sphere is taken. This procedure was repeated twice. Then the lysate-containing solution was mixed with the spheres, and the tubes were left overnight at 4 ° C with rotation on a rotary mixer.

Samples for about 1 minute, exposed to a magnet and the liquid carefully removed. Then, 1 ml of lysis buffer, and the tubes rotated for 5 min for washing. The washing steps were repeated twice. The liquid was completely removed, and the spheres were resuspended in 12 ul of hot (94ºS) 2 × Laemmli sample loading buffer + bME, then they were left for 15 minutes at room temperature. The tubes for 1 minute, exposed to magnet, and the liquid which separated from the spheres was analyzed by SDS-PAGE.

Samples were analyzed by SDS-PAGE on 15 wells with 4-12% Bis-Tris gels NuPAGE / MOPS (Novex). Per well they were charged with only 12 microliters of each immunoprecipitate. Gels were electrophoresed at 200 V / 120 mA / 25 Watts for 55 minutes and then the samples were Western blotted onto nitrocellulose membrane for 1 h 30 min at 50 V / 250 mA. All blots were then treated with 5% Marvel in PBS-Tween for 1 hour at room temperature, then washed with PBS-Tween.

Rabbit anti-Tie-2 antibody (. Santa Cruz Cat № C1303) was diluted 1: 500 in 0,5% Marvel / PBS-Tween and each blot was added 12.5 mL of the antibody, and allowed to stand overnight at 4 ° C. Then the blots were washed with PBS-Tween and each blot was added goat anti-rabbit-POD (Dako Cat № P 0448.) (1: 5000 dilution in 0.5% Marvel / PBS-Tween) and then allowed to stand for 1 hour at room temperature. The blots were washed with PBS-Tween and each blot showed over 10 minutes with 12.5 ml (equal volumes of solution A and B) reagent Supersignal (PIERCE Cat. № 34080). The blots were then transferred to an X-ray cassette and exposed to film (5 sec / 15 sec / 30 sec / 60 sec / 150 seconds). Figure 4 shows a western blot showing the results of this analysis.

Then each sample image on the film was evaluated using the system for analysis FluorS BioRad image. pixel density was measured as OD / mm² and expressed as a percentage of volume. The results, expressed as a volume percentage, normalized to 1 mg protein / immunoprecipitation using the protein concentration determined in the BCA assay and the lysate volume of each sample used in the immunoprecipitation. The percentage phosphorylation of each sample was calculated on the assumption that the 100% phosphorylation value received untreated control sample on each gel and the percentage inhibition of each sample was calculated on the assumption that the 100% phosphorylation value taken, which is itself represented 0% bonus inhibition). These values ​​are shown graphically in Figure 5 and indicate that IC₅₀ for inhibition of Angiopoietin-1 stimulated Tie-2 phosphorylation is 99 nM.

Overall, these data indicate that in this system mAb inhibits Angiopoietin-1 induced phosphorylation of Tie-2.

EXAMPLE 13

STRUCTURAL ANALYSIS OF ANTI-ANG-2 ANTIBODIES

The variable regions of heavy chains and light chains of the antibodies were sequenced to determine their DNA sequences. Full details of the anti-Ang-2 antibody sequences contained in the list of nucleotide sequences and amino acid sequences for each combination of gamma and kappa chains. The sequences of the variable regions of the heavy chain sequences were analyzed to determine the VH-family, D-region and the J-region. The sequences were then broadcast to determine the primary amino acid sequence and compared to the sequences of VH-family, D-region and the J-region germline to assess somatic hypermutations.

Table 11 shows a comparison of the heavy chain antibodies to their cognate region of the heavy chain germline. Table 12 shows a comparison of the kappa light chain regions to their cognate antibody light chain germline.

The variable (V) region of immunoglobulin chains are encoded by a plurality of DNA germline segments, which are connected to the functional variable regions (V_HDJ_H or V_KJ_K) During B-cell ontogeny. Molecular and genetic differences in the antibody response to Ang-2 have been investigated in more detail. These assays revealed several regions that are specific to anti-Ang-2 antibodies.

Analysis of 152 individual antibodies specific to Ang-2, revealed that the antibodies are derived from 21 different germline VH genes, and 112 of these antibodies belong to the VH3 family, with 46 antibodies derived from VH3-33 gene segment. Tables 11 and 12 present the results of this analysis.

It should be noted that the amino acid sequence of the sister clones collected from each hybridoma are identical. For example, a heavy chain sequence and light chain mAb 3.19.3 are identical to the sequences of mAb 3.19 and 3.19.1., Presented in Tables 11 and 12.

EXAMPLE 14

Definition of the canonical classes of antibodies

Chothia et al. Have described antibody structure, and in this specification, for the hypervariable regions of each immunoglobulin chain, they have used the term "canonical classes" (<i>J. Mol. Biol.</i> 1987 Aug 20; 196 (4): 901-17). Fab- atomic structures were analyzed and VL-fragments of different immunoglobulins to determine the relationship between their amino acid sequences and three dimensional structures of their antigen binding sites. Chothia et al. Found that there is a relatively small number of residues, which due to their packing, hydrogen bonding or the ability to buy unusual fi-, psi or omega conformations, primarily responsible for the main chain conformation of the hypervariable regions. It has been found that these residues are present in the sites of these hypervariable regions and in the conserved beta-sheet framework structure. By assessing immunoglobulin sequences with unknown structure, Chothia et al. Found that many immunoglobulins have hypervariable regions that

It is their discovery led to the idea that these hypervariable regions have conformations similar to the conformations of known structures. For five of the hypervariable regions of the repertoire of conformations obviously limited to a relatively small number of discrete structural classes. These common conformation of the main chain hypervariable regions are called "canonical structures." Subsequent work by Chothia et al. (<i>Nature</i> 1989 Dec 21-28; 342 (6252): 877-83) and others (Martin, et al.<i>J. Mol. Biol.</i> 1996 Nov 15; 263 (5): 800-15) confirmed that at least five of the six hypervariable regions of antibodies a small set of main chain conformations.

CDR each antibody described above were analyzed to determine their canonical class. As is known, canonical classes have been assigned a CDRL and CDR2 of the antibody heavy chain and CDRL, CDR2 and CDR3 of the light chains of the antibody. The results of this analysis are summarized in the following table (Table 13). These canonical classes are presented in the form of * HCDR1-HCDR2-LCDR1-LCDR2-LCDR3, wherein "HCDR" means the heavy chain CDR and "LCDR" means the light chain CDR. For example, a canonical class of 1-3-2-1-5 assigns antibody that has HCDRl, corresponding to canonical class 1, HCDR2, corresponding to canonical class 3, LCDRl, corresponding to canonical class 2, LCDR2, corresponding to canonical class 1, and a LCDR3 corresponding to canonical class 5.

A specific canonical class assigned in the event that the amino acids in said antibody at 70% or more identical to the amino acids defined for each canonical class. If the identity is less than 70%, the assigned canonical class is marked with an asterisk ( "*") which indicates that for determining the true canonical class was made the best estimate, based on the length of each CDR, and the data obtained were pooled. If the length of a CDR does not correspond to any canonical class, then assigned to the canonical class designated "Y". The amino acids defined for each antibody can be found, for example, in articles Chothia et al., Mentioned above. Table 13 presents data for each canonical class of anti-Ang-2 antibodies.

<tables num="10"><table frame="all"><tgroup cols="2" rowsep="1" colsep="1"><colspec colname="c0" colwidth="42mm" /><colspec colname="c1" colwidth="57mm" /><tbody><row><entry align="right" namest="c0" nameend="c1" rowsep="1" colsep="0">Table 13Kanonicheskie classes of antibodies against Ang-2</entry></row><row><entry align="center" rowsep="1" colsep="1">Antibody</entry><entry align="center" rowsep="1" colsep="0">The canonical class</entry></row><row><entry align="center" rowsep="1" colsep="1">5.18</entry><entry align="center" rowsep="1" colsep="0">1-1-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.81</entry><entry align="center" rowsep="1" colsep="0">1-1-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.66</entry><entry align="center" rowsep="1" colsep="0">1-1-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.48</entry><entry align="center" rowsep="1" colsep="0">1-1-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.23</entry><entry align="center" rowsep="1" colsep="0">1-1-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.33</entry><entry align="center" rowsep="1" colsep="0">1-1-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.15</entry><entry align="center" rowsep="1" colsep="0">1-1-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">4.3</entry><entry align="center" rowsep="1" colsep="0">1-1-4-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.12</entry><entry align="center" rowsep="1" colsep="0">1-1-4-1-5*</entry></row><row><entry align="center" rowsep="1" colsep="1">4.16</entry><entry align="center" rowsep="1" colsep="0">1-1-4-1-5*</entry></row><row><entry align="center" rowsep="1" colsep="1">3.13</entry><entry align="center" rowsep="1" colsep="0">1-1-4-1-5*</entry></row><row><entry align="center" rowsep="1" colsep="1">3.7</entry><entry align="center" rowsep="1" colsep="0">1-1-4-1-5*</entry></row><row><entry align="center" rowsep="1" colsep="1">3.32</entry><entry align="center" rowsep="1" colsep="0">1-1-4-1-5*</entry></row><row><entry align="center" rowsep="1" colsep="1">3.37</entry><entry align="center" rowsep="1" colsep="0">1-1-4-1-Y</entry></row><row><entry align="center" rowsep="1" colsep="1">5.30</entry><entry align="center" rowsep="1" colsep="0">1-2-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.6</entry><entry align="center" rowsep="1" colsep="0">1-2-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">6.6</entry><entry align="center" rowsep="1" colsep="0">1-2-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.34</entry><entry align="center" rowsep="1" colsep="0">l-2 * -2-ll</entry></row><row><entry align="center" rowsep="1" colsep="1">5.74</entry><entry align="center" rowsep="1" colsep="0">l-2 * -2-ll</entry></row><row><entry align="center" rowsep="1" colsep="1">5.38</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.28.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.6</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.44</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.28</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.86.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.35.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.35</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.8</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.22</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">4.15</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.67</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.87</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.10</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.71</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.21</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.80</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.18</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.11</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.26</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.31</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.76</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">4.9</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.17</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.39</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.37</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.22</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.29</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.73</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.12</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.11</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">4.8</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.24</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">6.3.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.56.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-1*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.111</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1*-!</entry></row><row><entry align="center" rowsep="1" colsep="1">4.13</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1*-!</entry></row><row><entry align="center" rowsep="1" colsep="1">5.52</entry><entry align="center" rowsep="1" colsep="0">1-3*-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.16.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.39.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.103.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.101.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.54.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.83.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.62</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.88.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.40.2</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.109</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.64</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.13</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">3.3</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.41</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">3.3.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">3.31.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.41.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.62.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.108.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.13.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.97</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-5*</entry></row><row><entry align="center" rowsep="1" colsep="1">5.52.1</entry><entry align="center" rowsep="1" colsep="0">1-3-2-1-Y</entry></row><row><entry align="center" rowsep="1" colsep="1">5.78.1</entry><entry align="center" rowsep="1" colsep="0">1-3-3-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.60</entry><entry align="center" rowsep="1" colsep="0">1-3-3-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.43</entry><entry align="center" rowsep="1" colsep="0">1-3-3-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.90</entry><entry align="center" rowsep="1" colsep="0">1-3-3-1*-!</entry></row><row><entry align="center" rowsep="1" colsep="1">3.42</entry><entry align="center" rowsep="1" colsep="0">1-3-3-1*-!</entry></row><row><entry align="center" rowsep="1" colsep="1">4.11</entry><entry align="center" rowsep="1" colsep="0">1-3-4-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.40</entry><entry align="center" rowsep="1" colsep="0">1-3-4-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.82</entry><entry align="center" rowsep="1" colsep="0">1-3-4-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">4.18</entry><entry align="center" rowsep="1" colsep="0">1-3-4-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.41</entry><entry align="center" rowsep="1" colsep="0">1-3-4-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.92</entry><entry align="center" rowsep="1" colsep="0">1-3-4-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">3.10</entry><entry align="center" rowsep="1" colsep="0">3-4-l * -ll</entry></row><row><entry align="center" rowsep="1" colsep="1">4.14</entry><entry align="center" rowsep="1" colsep="0">1-3-4-1-5*</entry></row><row><entry align="center" rowsep="1" colsep="1">3.19.1</entry><entry align="center" rowsep="1" colsep="0">3-8-l * -ll</entry></row><row><entry align="center" rowsep="1" colsep="1">6.2</entry><entry align="center" rowsep="1" colsep="0">3-8-l * -ll</entry></row><row><entry align="center" rowsep="1" colsep="1">3.8</entry><entry align="center" rowsep="1" colsep="0">3-8-l * -ll</entry></row><row><entry align="center" rowsep="1" colsep="1">5.58</entry><entry align="center" rowsep="1" colsep="0">1-3-8-1-1*</entry></row><row><entry align="center" rowsep="1" colsep="1">4.5</entry><entry align="center" rowsep="1" colsep="0">l-4 * 2-ll</entry></row><row><entry align="center" rowsep="1" colsep="1">5.1</entry><entry align="center" rowsep="1" colsep="0">l-4 * 2-ll</entry></row><row><entry align="center" rowsep="1" colsep="1">4.2</entry><entry align="center" rowsep="1" colsep="0">l-4 * 2-ll</entry></row><row><entry align="center" rowsep="1" colsep="1">3.9</entry><entry align="center" rowsep="1" colsep="0">l-4 * 2-ll</entry></row><row><entry align="center" rowsep="1" colsep="1">5.45</entry><entry align="center" rowsep="1" colsep="0">l-4 * 2-ll</entry></row><row><entry align="center" rowsep="1" colsep="1">3.17</entry><entry align="center" rowsep="1" colsep="0">l-4 * 2-ll</entry></row><row><entry align="center" rowsep="1" colsep="1">5.72</entry><entry align="center" rowsep="1" colsep="0">l-ll-4 * -2 *</entry></row><row><entry align="center" rowsep="1" colsep="1">3.14</entry><entry align="center" rowsep="1" colsep="0">l-4 * -2-lY</entry></row><row><entry align="center" rowsep="1" colsep="1">5.115</entry><entry align="center" rowsep="1" colsep="0">3-1-2-1-1</entry></row><row><entry align="center" rowsep="1" colsep="1">5.36</entry><entry align="center" rowsep="1" colsep="0">3-l-ll * -2</entry></row><row><entry align="center" rowsep="1" colsep="1">3.2</entry><entry align="center" rowsep="1" colsep="0">3-1-2-1-3*</entry></row><row><entry align="center" rowsep="1" colsep="1">3.21</entry><entry align="center" rowsep="1" colsep="0">3-1-8-1-1*</entry></row><row><entry align="center" rowsep="0" colsep="1">5.61</entry><entry align="center" rowsep="0" colsep="0">3-Y-2-1-3 *</entry></row></tbody></tgroup></table></tables>

Table 14 illustrates the analysis performed to determine the number of antibodies per class. Canonical classes of antibodies are shown in the left column, and the number of antibodies belonging to a particular canonical class is given in the right column.

<tables num="11"><table frame="all"><tgroup cols="2" rowsep="1" colsep="1"><colspec colname="c0" colwidth="67mm" /><colspec colname="c1" colwidth="57mm" /><tbody><row><entry align="right" namest="c0" nameend="c1" rowsep="1" colsep="0">Table 14Chislo anti-Ang-2 antibodies in each canonical class</entry></row><row><entry align="center" rowsep="1" colsep="1">H1-H2-L1-L2-L3</entry><entry align="center" rowsep="1" colsep="0">Номер mAbs</entry></row><row><entry align="center" rowsep="1" colsep="1">1-1-2-1-1</entry><entry align="center" rowsep="1" colsep="0">6</entry></row><row><entry align="center" rowsep="1" colsep="1">1-1-2-1-3*</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="center" rowsep="1" colsep="1">1-1-4-1-1</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="center" rowsep="1" colsep="1">1-1-4-1-5*</entry><entry align="center" rowsep="1" colsep="0">5</entry></row><row><entry align="center" rowsep="1" colsep="1">1-1-4-1-Y</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="center" rowsep="1" colsep="1">1-2-2-1-1</entry><entry align="center" rowsep="1" colsep="0">5</entry></row><row><entry align="center" rowsep="1" colsep="1">1-3-2-1-1</entry><entry align="center" rowsep="1" colsep="0">38</entry></row><row><entry align="center" rowsep="1" colsep="1">1-3-2-1-3*</entry><entry align="center" rowsep="1" colsep="0">21</entry></row><row><entry align="center" rowsep="1" colsep="1">1-3-2-1-5*</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="center" rowsep="1" colsep="1">1-3-2-1-Y</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="center" rowsep="1" colsep="1">1-3-3-1-1</entry><entry align="center" rowsep="1" colsep="0">5</entry></row><row><entry align="center" rowsep="1" colsep="1">1-3-4-1-1</entry><entry align="center" rowsep="1" colsep="0">7</entry></row><row><entry align="center" rowsep="1" colsep="1">1-3-4-1-5*</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="center" rowsep="1" colsep="1">3-8-l * -ll</entry><entry align="center" rowsep="1" colsep="0">4</entry></row><row><entry align="center" rowsep="1" colsep="1">l-4 * 2-ll</entry><entry align="center" rowsep="1" colsep="0">7</entry></row><row><entry align="center" rowsep="1" colsep="1">l-4 * -2-lY</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="center" rowsep="1" colsep="1">3-1-2-1-1</entry><entry align="center" rowsep="1" colsep="0">2</entry></row><row><entry align="center" rowsep="1" colsep="1">3-1-2-1-3*</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="center" rowsep="1" colsep="1">3-1-8-1-1*</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="center" rowsep="1" colsep="1">3-Y-2-1-3 *</entry><entry align="center" rowsep="1" colsep="0">1</entry></row><row><entry align="justify" namest="c0" nameend="c1" rowsep="0" colsep="0">Notes: 1. The figures marked with * indicate that the antibody has the best compliance with this class, although certain provisions of this antibody has some otkloneniya.2. Y means that the length of a CDR does not match the canonical class.</entry></row></tbody></tgroup></table></tables>

EXAMPLE 15

Epitope mapping of antibodies against Ang-2

binding domain of 27 antibodies was analyzed to neutralize Ang-2 activity.

Recombinant human Ang-2 was purchased from the company R &amp; D systems (623-AN). Goat polyclonal antibodies against human Ang-2 (R &amp; D systems AF623) were selected on their ability to recognize rhAng-2 in direct ELISA and Western blot assays. The polyclonal antibodies were biotinylated for detection with HRP-conjugated streptavidin.

All restriction enzymes were purchased from New England Biolabs and were used by the firm in accordance with the manufacturer's instructions. All plasmid DNA was purified by centrifugal minicolumn (Invitrogen, Carlsbad, CA). Oligonucleotide primers used for cloning and site directed mutagenesis were synthesized by Qiagen Operon.

Antibodies: 27 human anti-Ang-2 antibodies derived from the hybridomas were selected based on their ability to inhibit binding of rhAng-2 to its receptor. These antibodies are listed below in Table 15.

<tables num="12"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="19mm" /><colspec colname="c1" colwidth="52mm" /><colspec colname="c2" colwidth="52mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 15</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1">code hybridoma</entry><entry align="center" rowsep="1" colsep="0">OD650 in the inhibition assay</entry></row><row><entry align="justify" rowsep="0" colsep="1">1</entry><entry align="justify" rowsep="0" colsep="1">x5.56</entry><entry align="justify" rowsep="1" colsep="0">0,0863</entry></row><row><entry align="justify" rowsep="0" colsep="1">2</entry><entry align="justify" rowsep="0" colsep="1">kh3.38</entry><entry align="justify" rowsep="1" colsep="0">0,0792</entry></row><row><entry align="justify" rowsep="0" colsep="1">3</entry><entry align="justify" rowsep="0" colsep="1">kh3.19</entry><entry align="justify" rowsep="1" colsep="0">0,0633</entry></row><row><entry align="justify" rowsep="0" colsep="1">4</entry><entry align="justify" rowsep="0" colsep="1">kh3.28 *</entry><entry align="justify" rowsep="1" colsep="0">0,0588</entry></row><row><entry align="justify" rowsep="0" colsep="1">5</entry><entry align="justify" rowsep="0" colsep="1">X3.3</entry><entry align="justify" rowsep="1" colsep="0">0,0558</entry></row><row><entry align="justify" rowsep="0" colsep="1">6</entry><entry align="justify" rowsep="1" colsep="1">kh3.31 *</entry><entry align="justify" rowsep="1" colsep="0">0,0516</entry></row><row><entry align="justify" rowsep="0" colsep="1">7</entry><entry align="justify" rowsep="0" colsep="1">kh5.88 *</entry><entry align="justify" rowsep="0" colsep="0">0,0874</entry></row><row><entry align="justify" rowsep="0" colsep="1">8</entry><entry align="justify" rowsep="0" colsep="1">kh5.49 *</entry><entry align="justify" rowsep="0" colsep="0">0,0856</entry></row><row><entry align="justify" rowsep="0" colsep="1">9</entry><entry align="justify" rowsep="0" colsep="1">kh5.101</entry><entry align="justify" rowsep="0" colsep="0">0,0824</entry></row><row><entry align="justify" rowsep="0" colsep="1">10</entry><entry align="justify" rowsep="0" colsep="1">kh5.41 *</entry><entry align="justify" rowsep="0" colsep="0">0,0776</entry></row><row><entry align="justify" rowsep="0" colsep="1">11</entry><entry align="justify" rowsep="0" colsep="1">kh5.108 *</entry><entry align="justify" rowsep="0" colsep="0">0,0688</entry></row><row><entry align="justify" rowsep="0" colsep="1">12</entry><entry align="justify" rowsep="0" colsep="1">kh5.62</entry><entry align="justify" rowsep="0" colsep="0">0,0650</entry></row><row><entry align="justify" rowsep="0" colsep="1">13</entry><entry align="justify" rowsep="0" colsep="1">kh5.39</entry><entry align="justify" rowsep="0" colsep="0">0,0519</entry></row><row><entry align="justify" rowsep="0" colsep="1">14</entry><entry align="justify" rowsep="0" colsep="1">kh5.16 *</entry><entry align="justify" rowsep="0" colsep="0">0,0500</entry></row><row><entry align="justify" rowsep="0" colsep="1">15</entry><entry align="justify" rowsep="0" colsep="1">kh5.83</entry><entry align="justify" rowsep="0" colsep="0">0,0484</entry></row><row><entry align="justify" rowsep="0" colsep="1">16</entry><entry align="justify" rowsep="0" colsep="1">kh5.54</entry><entry align="justify" rowsep="0" colsep="0">0,0440</entry></row><row><entry align="justify" rowsep="0" colsep="1">17</entry><entry align="justify" rowsep="0" colsep="1">kh5.14</entry><entry align="justify" rowsep="0" colsep="0">0,0430</entry></row><row><entry align="justify" rowsep="0" colsep="1">18</entry><entry align="justify" rowsep="1" colsep="1">kh5.86</entry><entry align="justify" rowsep="1" colsep="0">0,0419</entry></row><row><entry align="justify" rowsep="0" colsep="1">19</entry><entry align="justify" rowsep="1" colsep="1">kh5.78</entry><entry align="justify" rowsep="1" colsep="0">0,0984</entry></row><row><entry align="justify" rowsep="0" colsep="1">20</entry><entry align="justify" rowsep="1" colsep="1">kh5.103 *</entry><entry align="justify" rowsep="1" colsep="0">0,1013</entry></row><row><entry align="justify" rowsep="0" colsep="1">21</entry><entry align="justify" rowsep="0" colsep="1">kh5.28</entry><entry align="justify" rowsep="0" colsep="0">0,0821</entry></row><row><entry align="justify" rowsep="0" colsep="1">22</entry><entry align="justify" rowsep="0" colsep="1">kh5.40</entry><entry align="justify" rowsep="0" colsep="0">0,0691</entry></row><row><entry align="justify" rowsep="0" colsep="1">23</entry><entry align="justify" rowsep="0" colsep="1">kh5.35 *</entry><entry align="justify" rowsep="0" colsep="0">0,0663</entry></row><row><entry align="justify" rowsep="0" colsep="1">24</entry><entry align="justify" rowsep="1" colsep="1">kh6.3</entry><entry align="justify" rowsep="1" colsep="0">0,0617</entry></row><row><entry align="justify" rowsep="0" colsep="1">25kh</entry><entry align="justify" rowsep="1" colsep="1">kh5.13</entry><entry align="justify" rowsep="1" colsep="0">0,0744</entry></row><row><entry align="justify" rowsep="0" colsep="1">26</entry><entry align="justify" rowsep="1" colsep="1">kh5.2</entry><entry align="justify" rowsep="1" colsep="0">0,0690</entry></row><row><entry align="justify" rowsep="0" colsep="1">27</entry><entry align="justify" rowsep="0" colsep="1">kh5.52</entry><entry align="justify" rowsep="0" colsep="0">0,0627</entry></row></tbody></tgroup></table></tables>

<u>Characterization of Epitope for 27 neutralizing anti-Ang-2 antibodies</u>

<u>Dot-bloty</u>

RhAng-2 (R &amp; D systems) were spotted on nitrocellulose membrane in its native or reduced form, using an apparatus for microfiltration Bio-Dot. All human monoclonal antibodies (MAb), produced against a human Ang-2 bind to unreduced Ang-2, but did not bind to its reduced form, indicating that all mAb recognize conformational epitopes, which are apparently destroyed upon protein recovery .

<u>Cloning and expression of proteins Angl and Ang-2 </u>

To better understand the structural basis of the interaction of mAb with Ang-2 series of chimeric molecules Angl / Ang-2 was used. This method has the advantage that members of the angiogenic proteins are structurally related. Although the protein sequence Ang-2 and Angl have only 60% homology, however, they both have the same modular structure composed of an amino-terminal coiled-coil domain and a carboxy-terminal fibrinogen-like domain.

<u>Cloning of human Ang-1 and Ang-2</u>

Two alternatively spliced ​​forms of the cDNA of human Ang-2 was amplified from human umbilical vein endothelial cell line (HUVEC). PCR amplification of HUVEC cDNA was carried out using the Ang-2-specific primers indicated the presence of full length Ang-2 (1491 bp) and variant Ang-2₄₄₃Consisting of 1330 bp (Injune et al, (2000) JBC 275:. 18550). Ang-2₄₄₃ It is a variant generated by alternative splicing of exon B and removing a portion of supercoiled domain (amino acids 96-148). Both Ang-2 cDNAs were cloned into pCR3.1 expression vector and expressed in 293F cells as shown in Figure 6. The cDNA of human Ang-1 was obtained by RT-PCR was carried out using total RNA extracted from a cell line of human breast MDA -MB-231. 1.5 kb cDNA was cloned into the expression vector pCR3.1, and its expression was detected in the supernatant of transiently transfected 293F cells.

<u>ELISA</u>

Coupling of 27 with mAb supernatants obtained as a result of Ang-2 and Ang-l cDNA transient transfection were tested by ELISA with antibody capture. Ang-2, Ang-2₄₄₃ Ang-1 and contacted with goat polyclonal antibodies against Ang-2 or Ang-1 (respectively) present on the ELISA-plate. Binding best 27 human monoclonal antibodies was detected with HRP-conjugated goat anti-human antibody, followed by colorimetric horseradish peroxidase substrate (active substrate Enhanced K-Blue TMB, Neogen Corporation). The absorbance in each well ELISA-plates was measured at 450 nm on a plate reader for microtitre plates.

<u>Transfection kletok 293F</u>

Human embryonic kidney 293F cells were maintained in 10% fetal bovine serum in Dulbecco's modified Eagle's medium, to which were added penicillin and streptomycin. 293F cells were transiently transfected using Calcium phosphate. After 72 hours, the medium was harvested and filtered for ELISA and Western blot analyzes.

It was shown that all 27 antibodies specifically bind to an antigen Ang-2 / Ang-2₄₄₃. Any crossreactivity with human Ang-1 was found. Amino acids 96-148 in the coiled-coil domain of Ang-2 that were absent in the protein sequence of Ang-2₄₄₃ and which constituted a binding domain in each of the 27 antibodies were excluded.

<u>The Конструирование химерных молекул-l / A-2</u>

To construct hybrid Angiopoietin chimeric proteins were used restriction sites normally present in the genes of human Ang-1 and Ang-2 in the same reading frame.

Four constructs were made: Human Ang-1/2<i>Bsm</i>l, Ang-2 / l<i>Bsm</i>I, A-1/2<i>Ssp</i>The I-и 2/1<i>Ssp</i>l. All proteins were expressed and secreted in detectable levels as measured by ELISA-assay using polyclonal antibodies against human Ang-1 and Ang-2.

Sites are joining amino acids in the following positions:

Bsml- 117 (A-2) / l19 (A-1)

Sspl- 353 (A-2) / 354 (A-l)

The difference in one amino acid is due to the fact that human Ang-1 is present 497 residues, and human Ang-2 is present 496 residues. All constructs were expressed in 293F cells, and detected using goat anti-human polyclonal antibodies against Ang-1 and Ang-2. The best 27 antibodies were tested for their ability to bind chimeric Ang-molecules half. All 27 antibodies showed a similar pattern of binding only to the structure Ang-1/2<i>Bsm</i>I. The results of these experiments showed that the binding domain for all antibodies is residues at positions 117-496, and most likely in the fibrinogen binding domain, wherein in the hybrid proteins <i>SspI</i>-Ang epitope is located in close proximity to the amino acid position 353, was destroyed.

<u>Construction of chimeric molecules "Mouse / human Ang-2"</u>

Since the amino acid sequence of Ang-2 by about 55% identical to the amino acid sequence of Ang-1, it is rather difficult to find a common restriction site that can be used for cloning of chimeric molecules. Sequences of mouse and human Ang-2 are more similar, i.e. They are homologous to about 85%. cDNA of mouse Ang-2 cloned into the expression vector pCMCsport, was purchased from the company Invitrogen. 27 selected antibodies were tested for their immunoreactivity with recombinant mouse Ang-2. 6 out of 27 antibodies cross-reacting with mouse Ang-2, possessed 100% immunoreactivity towards human Ang-2, indicating that the murine antigen retains most level immmunoreaktivnosti human Ang-2 (Data are summarized in Table 16).

The chimeric "human-mouse" was chosen for epitope mapping based on the fact that most antibodies bind specifically to the human Ang-2 antigen and do not cross react with mouse Ang-2. Various cDNA constructs of Ang-2 were generated and cloned into a mammalian expression vector.

Construction of mouse / human Ang-2 was obtained using the general restriction <i>Stu</i>I-site located in the fibrinogen-binding domain, wherein the junction point of murine and human amino acid residue located at position 311. All mAb, specific to human Ang-2 had the ability to bind to <i>Stu</i>1 site of the murine / human Ang-2, indicating that the binding domain is in the fibrinogen-binding domain residues at positions 311-496. To narrow the binding domain was prepared a new design in which the murine cDNA sequence of mouse Ang-2 was replaced with a fragment of human<i>Stu</i>I-<i>Tfi</i>I (Figure 9).

All antibodies specific to human Ang-2 showed a positive ELISA-signal, and their immunoreactivity towards human Ang-2 was 15-100%. The binding domain of two antibodies with a unique gene, occurring in the VH, designated 5.35.1 (VH3-20) and 5.28.1 (VH3-43) and shown in Table 17, was mapped to the amino acid region 310-400.

It has been shown that antibodies that cross react with mouse Ang-2, as expected, have 100% reactivity and could not be mapped using chimeric constructs mouse / human.

<u>Site-directed mutagenesis napravlennыy</u>

To determine the residues which play an important role in binding and present in the binding site of different antibodies, a few human Ang-2 were mutated residues and screened across the panel of antibodies for binding to ELISA-analysis.

Because direct binding detected by using ELISA, it is insensitive to small and moderate changes in affinity, significant changes in binding observed after substitution of one amino acid probably identify key sites allow that interact with the antibody. In addition, polyclonal antibodies against human Ang-2 maintain 100% reactivity with each construct, indicating that such a procedure does not introduce mutagenesis any significant structural replacement Ang-2 molecule. All 27 antibodies have any effect on the two independent substitution VaI to Met at position 345 (V345M) and His to GIn at position 375 (H375Q) was not observed, indicating that these residues are not reactive, or the fact it takes more than one amino acid substitution for the implementation of changes in the conformational epitopes. Replacement of two residues at positions 365 and 367 resulted in the drastic change in binding of one antibody Mab 5.35.1. Analysis revealed the sequence of the antibody 5.35.1 VH3-20 one region and one region CDR3 in the heavy and light chains. All points of attachment chimeric Ang-2 molecules and point mutations are highlighted in Figure 8. Figure 9 shows a comparison of the amino acid sequences of mouse Ang-1 (SEQ ID NO: 5), human Ang-1 (SEQ ID NO: 2), murine Ang- 2 (SEQ ID NO: 4), and human Ang-2 (SEQ ID NO: 3). Arrows indicate the cleavage site for hydrophobic leader sequences. These arrows define the limits supercpiralizovannyh and fibrinogen-like domains.<i>Science</i> 277:55).

Binding data to all Ang-2 molecules systematized in Table 16 below:

<tables num="13"><table frame="all"><tgroup cols="11" rowsep="1" colsep="1"><colspec colname="c0" colwidth="17mm" /><colspec colname="c1" colwidth="12mm" /><colspec colname="c2" colwidth="22mm" /><colspec colname="c3" colwidth="24mm" /><colspec colname="c4" colwidth="16mm" /><colspec colname="c5" colwidth="14mm" /><colspec colname="c6" colwidth="14mm" /><colspec colname="c7" colwidth="14mm" /><colspec colname="c8" colwidth="13mm" /><colspec colname="c9" colwidth="17mm" /><colspec colname="c10" colwidth="12mm" /><tbody><row><entry align="right" namest="c0" nameend="c10" rowsep="1" colsep="0">Table 16</entry></row><row><entry align="center" rowsep="1" colsep="1">branch</entry><entry align="center" rowsep="1" colsep="1">Bin</entry><entry align="center" rowsep="1" colsep="1">binding domain</entry><entry align="center" rowsep="1" colsep="1">Man ang-2-443</entry><entry align="center" rowsep="1" colsep="1">Myšinyj SH</entry><entry align="center" rowsep="1" colsep="1"><i>M/H Bsml</i></entry><entry align="center" rowsep="1" colsep="1"><i>M/H Stul 310-496</i></entry><entry align="center" rowsep="1" colsep="1"><i>Stu-Tfil 310-400</i></entry><entry align="center" rowsep="1" colsep="1">V345M</entry><entry align="center" rowsep="1" colsep="1">N365Q367</entry><entry align="center" rowsep="1" colsep="0">H375Q</entry></row><row><entry align="center" rowsep="1" colsep="1">5.39.1</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">25%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.16.1*</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">30%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.86.1</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">23%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.54.1</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">77%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.14.1</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">31%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.83.1</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">86%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.101.1</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">28%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">6.3.1</entry><entry align="center" rowsep="1" colsep="1">7</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">27%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.103.1*</entry><entry align="center" rowsep="1" colsep="1">2</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">40%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.78.1</entry><entry align="center" rowsep="1" colsep="1">5</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">Yes</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.35.1*</entry><entry align="center" rowsep="1" colsep="1">8</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">15%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.40.2*</entry><entry align="center" rowsep="1" colsep="1">8</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">65%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">3.19.1</entry><entry align="center" rowsep="1" colsep="1">6</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">Yes</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.108.1*</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">36%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.52.1</entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">Yes</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.56.1</entry><entry align="center" rowsep="1" colsep="1">3</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">Yes</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.2</entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">Yes</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.28.1</entry><entry align="center" rowsep="1" colsep="1">4</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.41.1*</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">30%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.13.1</entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">27%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">3.3.1</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">15%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">3.31.1*</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">15%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.62.1</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">30%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">3.28.1*</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">31%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">5.88.1*</entry><entry align="center" rowsep="1" colsep="1">1</entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">No</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">20%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="1" colsep="1">3.38</entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">Yes</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="justify" rowsep="1" colsep="1">not determined</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="1">100%</entry><entry align="center" rowsep="1" colsep="0">100%</entry></row><row><entry align="center" rowsep="0" colsep="1">5.49*</entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="1">The fibrinogen-like domain</entry><entry align="center" rowsep="0" colsep="1">100%</entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">100%</entry><entry align="center" rowsep="0" colsep="1">100%</entry><entry align="center" rowsep="0" colsep="1">35%</entry><entry align="center" rowsep="0" colsep="1">100%</entry><entry align="center" rowsep="0" colsep="1">100%</entry><entry align="center" rowsep="0" colsep="0">100%</entry></row></tbody></tgroup></table></tables>

Data are presented as percent binding compared to human Ang-2.

<tables num="14"><table frame="all"><tgroup cols="9" rowsep="1" colsep="1"><colspec colname="c0" colwidth="18mm" /><colspec colname="c1" colwidth="25mm" /><colspec colname="c2" colwidth="20mm" /><colspec colname="c3" colwidth="19mm" /><colspec colname="c4" colwidth="19mm" /><colspec colname="c5" colwidth="19mm" /><colspec colname="c6" colwidth="19mm" /><colspec colname="c7" colwidth="19mm" /><colspec colname="c8" colwidth="15mm" /><tbody><row><entry align="right" namest="c0" nameend="c8" rowsep="1" colsep="0">Table 17Analiz sequence and cross-reactivity with mouse Ang-2</entry></row><row><entry align="center" rowsep="1" colsep="1"><b>code hybridoma</b></entry><entry align="center" rowsep="1" colsep="1">FROM₆₅₀ in the inhibition assay</entry><entry align="center" rowsep="1" colsep="1">Mouse Ang-2</entry><entry align="center" rowsep="1" colsep="1">Bin</entry><entry align="center" rowsep="1" colsep="1">VH</entry><entry align="center" rowsep="1" colsep="1">DH</entry><entry align="center" rowsep="1" colsep="1">JH</entry><entry align="center" rowsep="1" colsep="1">VK</entry><entry align="center" rowsep="1" colsep="0">JK</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>x5.56</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0863</b></entry><entry align="center" rowsep="0" colsep="1">Yes</entry><entry align="center" rowsep="0" colsep="1">3</entry><entry align="center" rowsep="0" colsep="1">VH3-33</entry><entry align="center" rowsep="0" colsep="1">D1-7</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">A20</entry><entry align="center" rowsep="0" colsep="0">JK3</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>kh3.38</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0792</b></entry><entry align="center" rowsep="0" colsep="1">Yes</entry><entry align="center" rowsep="0" colsep="1">2</entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="center" rowsep="0" colsep="1"><b>kh3.19</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0633</b></entry><entry align="center" rowsep="0" colsep="1">Yes</entry><entry align="center" rowsep="0" colsep="1">6</entry><entry align="center" rowsep="0" colsep="1">VH3-30</entry><entry align="center" rowsep="0" colsep="1">D3-3</entry><entry align="center" rowsep="0" colsep="1">J5b</entry><entry align="center" rowsep="0" colsep="1">A27</entry><entry align="center" rowsep="0" colsep="0">JK5</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>kh3.28 *</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0588</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH3-7</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>X3.3</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0558</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH3-7</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="1" colsep="1"><b>kh3.31 *</b></entry><entry align="center" rowsep="1" colsep="1"><b>0,0516</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH3-7</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.88 *</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0874</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH3-7</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.49 *</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0856</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.101</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0824</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.41 *</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0776</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH3-7</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.108 *</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0688</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH3-33</entry><entry align="center" rowsep="0" colsep="1">D1-7</entry><entry align="center" rowsep="0" colsep="1">J5b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>kh5.62</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0650</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH3-7</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>kh5.39</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0519</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D6-13</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.16 *</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0500</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D6-13</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">12</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.83</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0484</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">12</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.54</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0440</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">12</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.14</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0430</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="center" rowsep="1" colsep="1"><b>kh5.86</b></entry><entry align="center" rowsep="1" colsep="1"><b>0,0419</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">1</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D6-13</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">O12</entry><entry align="center" rowsep="0" colsep="0">JK3</entry></row><row><entry align="center" rowsep="1" colsep="1">kh5.78</entry><entry align="center" rowsep="1" colsep="1">0,0984</entry><entry align="center" rowsep="0" colsep="1">Yes</entry><entry align="center" rowsep="0" colsep="1">5</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D2-2</entry><entry align="center" rowsep="0" colsep="1">J6b</entry><entry align="center" rowsep="0" colsep="1">B3</entry><entry align="center" rowsep="0" colsep="0">JK5</entry></row><row><entry align="center" rowsep="1" colsep="1">kh5.103 *</entry><entry align="center" rowsep="1" colsep="1">0,1013</entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">2</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4a</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.28</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0821</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">4</entry><entry align="center" rowsep="0" colsep="1">VH3-43</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK3</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>kh5.40</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0691</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">8</entry><entry align="center" rowsep="0" colsep="1">VH3-23</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4B</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1"><b>Kh5.35 *</b></entry><entry align="center" rowsep="0" colsep="1"><b>0,0663</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">8</entry><entry align="center" rowsep="0" colsep="1">VH3-20</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J2</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK4</entry></row><row><entry align="center" rowsep="1" colsep="1"><b>kh6.3</b></entry><entry align="center" rowsep="1" colsep="1"><b>0,0617</b></entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="center" rowsep="0" colsep="1">7</entry><entry align="center" rowsep="0" colsep="1">VH1-2</entry><entry align="center" rowsep="0" colsep="1">D1-7</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">O12</entry><entry align="center" rowsep="0" colsep="0">JK2</entry></row><row><entry align="center" rowsep="0" colsep="1">Kh5.13</entry><entry align="center" rowsep="0" colsep="1">0,0744</entry><entry align="center" rowsep="0" colsep="1">No</entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="center" rowsep="0" colsep="1">VH3-7</entry><entry align="center" rowsep="0" colsep="1">D6-19</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L2</entry><entry align="center" rowsep="0" colsep="0">JK1</entry></row><row><entry align="center" rowsep="0" colsep="1">kh5.2</entry><entry align="center" rowsep="0" colsep="1">0,0690</entry><entry align="center" rowsep="0" colsep="1">Yes</entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="center" rowsep="0" colsep="1">VH3-33</entry><entry align="center" rowsep="0" colsep="1">D5-12</entry><entry align="center" rowsep="0" colsep="1">J6b</entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="center" rowsep="0" colsep="1">kh5.52</entry><entry align="center" rowsep="0" colsep="1">0,0627</entry><entry align="center" rowsep="0" colsep="1">Yes</entry><entry align="left" rowsep="0" colsep="1"> </entry><entry align="center" rowsep="0" colsep="1">VH3-33</entry><entry align="center" rowsep="0" colsep="1">D1-1</entry><entry align="center" rowsep="0" colsep="1">J4b</entry><entry align="center" rowsep="0" colsep="1">L5</entry><entry align="center" rowsep="0" colsep="0">JK4</entry></row></tbody></tgroup></table></tables>

The analysis of IgH and IgL sequences was performed using the computer programs for sequence analysis, and by sospostavleniya VH gene sequences to the germline sequences, available in the database. This computer program also allows to analyze the elements D, reading frame region of the insert N, P accession nucleotides, nucleotide loss and CDR3 length. Analysis of 27 individual antibodies specific to CR64, revealed that only 7 VH genes of these antibodies belong to the germline, and 10 genes of these antibodies belong to the same family VHl. Selection of neutralizing antibodies showed that these antibodies were expressed the same V_n Ig, and in some cases same V rearrangement_HDJ_HAnd that a pair of H- and L-chains were conservative. These results suggest that, for any given epitope, to form the corresponding paratope involves only a few members of the germ line repertoire, and for each antigenic epitope a limited number of pairs of genes L- and H-chains may participate in the formation of a specific paratope.

The periodic occurrence of similar structures V_H, V_K and hypervariable region (complementarity-determining region, CDR) in different monoclonal antibodies is due to the fact that all Ang-2 neutralizing activity is restricted fibrinogen-like domain, and that output coincides with the view expressed in a paper published Procopio et al. (1999,<i>JBC</i> 274: 30196), suggesting that the effect of Ang-2 in the Tie-2 depends on the fibrinogen-like domain of presence. These epitope mapping showed that the monoclonal antibodies bind Ang-2 through a broad boundary region that includes most fibrinogen-like domains.

EXAMPLE 16

DETERMINATION OF CROSS REACTIVITY WITH ANG-2 murine

MAb cross-reactivity against human Ang-2 with mouse Ang-2 was analyzed by ELISA. For this purpose, we constructed an expression vector containing the murine Ang-2, and eukaryotic cells were subjected to transient transfection to produce mouse Ang-2.

Expression construct of murine Angiopoietin-2 (mAng-2) was obtained from Research Genetics, a distributor consortium IMAGE consortium (see. Www / image.llnl.gov site). cDNA of mouse Ang-2 (GenBank Accession № BC027216, IMAGE: 3494566) was obtained from NCI_CGAP_Lu29 library which is a library of lung tumor cells. cDNA was cloned into the expression vector pCMV-SPORT6 (Invitrogen Carlsbad, CA) through SalI (5 ') - and Notl (3') - sites, and this vector contained an open reading frame of 496 amino acids of the full length mouse Ang-2 (mAng-2) as well as 5'- and 3'-untranslated flanking regions for all 2471 bp.

10 g of the above mAng-2 plasmid was transfected into HEK293F cells by the calcium phosphate. Approximately 1 × 10⁶ HEK293F cells were seeded on 10 cm tissue culture plate one day before the initiation of culture. After 5 hours after transfection or after an overnight transfection, the medium was replaced and the cells were cultured for a further 2-3 days, and then collected supernatants containing the secreted mAng-2 protein. The expression of mAng-2 was confirmed by ELISA-analysis carried out using a polyclonal antibody obtained from the company R &amp; D Systems (catalog № AF623).

96-well plates were coated Nunc Immplates conditioned medium collected from transfectants HEK293F / mouse Ang-2, 100 .mu.l in each well. Plates were incubated at 4 ° C overnight and then washed four times with phosphate buffered saline for washing apparatus Skan Washer 300 (SKATRON). The wells were blocked with 100 l of ABX-blocking buffer (0,5% BSA, 0,1% Tween, 0.01% Thimerosal in PBS) for 1 hour. Is then added to the wells of anti-Ang-2 mAb at appropriate concentrations diluted in blocking buffer, in a volume of 100 .mu.l / well and incubated at room temperature for at least 1 hour. mAb and each of their dilutions were tested in duplicate. After two rinsing associated mAb was detected using HPPO-conjugated goat anti-human Fc (Caltag, Code Hl 0507) at 1/1000 dilution at room temperature for 1 hour. For chromogenic detection reaction was added 100 l of substrate TMB (TMB-microwell, BioFX, Cat. № TMSK-1000-01), and then the wells were washed three times with PBS. Plates were incubated for 30 minutes and then the reaction was stopped by adding 650 of blocking solution (100 l / well, BioFX, Cat. № BSTP-0100-01). The absorbance at 650 nm was determined on a plate reader Spectramax Plus.

This assay has been tested 27 best neutralizing mAb. The optical density showed that monoclonal antibodies 3.19.3, 3.38, 5.2.1, 5.52.1, 5.56.1 and 5.78.1 are capable of binding to mouse Ang-2 under the experimental conditions. To confirm this, each binding antibody was titrated by ELISA. Figure 10 is a plot of OD650 nm depending on the average values ​​(± Wed kv.ot.) from logarithmic mAb concentrations (ug / ml). In this figure are also represented the clones 5.2.1, 5.28.1, 3.19.3 and 3.31.2. Dose-dependent binding of monoclonal antibodies 5.2.1 and 3.19.3 to the murine Ang-2 reaches saturation at about 10 mcg / ml (Figure 10). The binding curves for these two mAb were typical sigmoidal curves dependence "dose-response". Dose-dependency and saturation was not observed in the tested antibodies in a certain concentration range, except for clones 5.2.1 and 3.19.3. Based on these data it can be concluded that cross-reactivity to mouse Ang-2 had only mAb 5.2.1 and 3.19.3.

EXAMPLE 17

INHIBITION OF BINDING OF MOUSE ANG-2 TO HUMAN TIE-2

The monoclonal antibody 3.19.3 was selected for further analysis of its ability to inhibit the binding of mouse Ang-2 with the human Tie-2. For this purpose, ELISA-plates were coated with 4 ug / ml hTie-2 / Fc (R &amp; D Systems, Inc.) at 100 l / well, and the wells were blocked with a routine method at 4 ° C overnight. Recombinant murine Ang-2 (mAng-2) was used in the culture supernatant of 293T / mAng-2 transfectants described above. The pre-coated wells were added 100 .mu.l of mAng-2 containing supernatant with mAb 3.19.3 at various concentrations and incubated at room temperature for 1 hour.

As a control, we used recombinant human Ang-2 (R &amp; D Systems, Inc.), mixed with the antibody. Each concentration of mAb was tested in triplicate. The bound mouse and human Ang-2 was detected with goat polyclonal antibodies against human Ang-2 (Santa Cruz Biotechnology, Santa Cruz, CA), which cross-reacts with mouse Ang-2, associated with a "second" HRP konyugirovannym rabbit anti-goat IgG. After 30 minutes after addition of HRP-substrate was determined by OD650. It was discovered that mAb 3.19.3 inhibited binding of human and murine Ang-2 with the human Tie-2 in a dose dependent manner (Figure 11).

EXAMPLE 18

The cross-reactivity with CAPILLARY NETWORK OF MONKEYS

Because Ang-2 is specifically expressed in angiogenic endothelial cells, monkey cells were subjected to immunohistochemical staining with anti-Ang-2 antibodies in order to indirectly determine whether such an antibody may cross react with monkey Ang-2.

In this experiment, the best assessed 10 neutralizing mAb, selected as described in Example 4 (Table 4) using isolated from monkey ovary tissue enriched endothelial cells. Completely drained 6-micron sections were frozen ovarian tissue monkeys (cynomolgus) were fixed with acetone at 4 ° C for 5 minutes. After three times washing with PBS-slides, tissue endogenous peroxidase blocked with 0,3% H₂O₂ for 10 minutes. The tissues were then washed with PBS and blocked with 10 ug / ml goat anti-human IgG Fab for 15 minutes. Tissue sections were washed again with PBS, then treated with 10% normal goat serum for 10 minutes. After draining the serum was applied to each of the sections 10 anti-Ang-2 mAb (10 .mu.g / ml) and incubated for 2 hours. Bound anti-Ang-2 mAb was detected with 10 pg / ml mouse anti-human IgG for 15 minutes and then incubated with peroxidase conjugated goat anti-mouse IgG for 30 minutes. For optimum results, staining was performed using AEC-substrate system (DAKO, Cat. № 3464) under a microscope.

It was found that all 10 mAb stained angiogenic vascular endothelial cells of the ovary tissue, whereas the isotype control mAb did not give such staining. This indicates that the 10 mAb, presented in Table 4, the cross-react with monkey Ang-2.

EXAMPLE 19

MAB 3.19.3 INHIBITS <i>IN VIVO</i> Angiogenesis in the analyzes available in the MATRIX BLOCK MATRIGEL

For <i>in vivo</i> assessing the potential antiangiogenic ability of monoclonal anti-Ang-2 antibodies were analyzed for angiogenesis in Matrigel matrix block. It was found that MCF-7 cells, when cultured<i>in vitro</i> or when implanted into immunodeficient mice as xenograft produce Ang-2. When administered in MCF-7 Matrigel matrix and subcutaneous implantation "nude" mice a steady increase in gel vessels. For models 6-8 week old females were used block matrix Matrigel Mouse BALB / c / nu / nu weighing 18 to 20 g (Charles River Laboratories, Wilmington, MA). "Nude" mice were injected subcutaneously in the right flank only 0.5 ml Matrigel, containing 2 × 10⁶ MCF-7 cells with the anti-Ang-2 antibodies, or without these antibodies, or control agents (including one Matrigel, isotype control Tie-2 / Fc, IgG2 and IgG4, and anti-VEGF mAb). Each test group comprised five mice. The concentration of the test mAb was adjusted to 100 ug / ml.

After seven days, Matrigel blocks are collected and evaluated for blood vessel density. For this purpose, mice were sacrificed by cervical dislocation under deep anesthesia. Blocks Matrigel opened by removal of the covering skin flap these blocks. Then Matrigel blocks were removed and digital images were obtained. Blocks Matrigel was excised carefully and cut into two parts. One part was snap frozen in TissueTek, and the other part was fixed in buffered formalin. Thereafter, the two parts embedded in paraffin for sectioning. Three cut 5-7 microns thickness derived from each mouse were cut and stained with hematoxylin and eosin. Sections were then evaluated in a phase contrast microscope. representative photomicrographs were obtained [in two formats (100 X and 400 X)], which was evaluated on the infiltration of endothelial cells and blood vessels.

Frozen blocks Matrigel was used to produce slices (10-micron slice) using a microtome Cryocut. We produced two separate cut-off, which was used for the staining for each mouse. Sections were blocked with BSA (0,1%), and then treated with monoclonal antibody reactive to the murine CD31, conjugated with phycoerythrin (at dilutions recommended by the manufacturer). After thorough washes, sections were placed under the reagent that prevents discoloration (Vecta Shield), and observed under a UV microscope using a red filter. As a result representative digital images were obtained [in two formats (two images with magnification of 100 X and 200 X)]. Nuclei were counterstained with DAPI. Immunofluorescent staining image CD31 were analyzed using on skeleting program. The obtained data were processed to calculate the average density value, the number of units and length of vessels for each group. Results are shown in Figures 12A and 12B, which illustrates the effect of anti-Ang-2 antibodies on the number of branches of blood vessels (12A figure) and the length of the blood vessels (12B figure).

This experiment showed that compared with one matrix Matrigel, MCF-7 cells that were incorporated in the Matrigel, possessed the ability to induce a significant level of angiogenesis. The induced angiogenesis could be inhibited by anti-VEGF antibody used as positive control. This also significantly inhibited angiogenesis soluble recombinant protein Tie-2 / Fc, suggesting that Ang-2 produced by MCF-7 cells plays a role in the angiogenesis in this model. By binding to any Ang-2, Tie-2 / Fc must effectively reduce the level of Ang-2 that was available for the MCF-7 cells.

However, it remains unclear how the antibody isotype IgG2, PK16.1.3, used as a negative control, affect the angiogenesis, although it was also found that the antibody is sometimes prevents tumor growth in some xenograft models (data not shown). IgG4 isotype control antibody did not have any effect on angiogenesis in ketoy model. As shown in Figures 12A and 12B, clones 5.88.3, ​​3.3.2, 3.19.3 and 5.28.1 significantly inhibited angiogenesis (P &lt;0,05, t-test was conducted by the specialists VasculoGen), whereas other clones gave a smaller Effect.

It is well established that Ang-2 is expressed by endothelial cells in the tumor, and therefore it rassmtarivaetsya as an autocrine angiogenic factor. However, it was also found that Ang-2 is expressed in many types of tumor cells<i>in vitro</i> and <i>in vivo</i>. Tested here mAb, except 3.19.3 showed no cross-react with mouse Ang-2. In this model,<i>in vivo</i>, The mAb only neutralized human Ang-2 produced by MCF-7 cells, but not the mouse Ang-2. The inhibitory effect of mAb suggesting that the expressed Ang-2 in the tumor can be a paracrine angiogenesis factor. The overall anti-angiogenic activity of the mAb, besides neutralizing Ang-2 is expressed by vascular endothelium, also makes some contribution to the neutralization of the tumor Ang-2.

EXAMPLE 20

Determining the therapeutically effective MAB 3.19.3 IN MODEL with pre-injected cells A431 xenografts AS

Clone of anti-Ang-2 mAb 3.19.3 not only binds the murine Ang-2, but also inhibited binding of mouse Ang-2 to human Tie-2. The antitumor activity of this monoclonal antibody was tested in mouse model ksenotransplanta human cutaneous squamous carcinoma using the A431 cell line.

A431 cells were routinely cultured in flasks method as long as these cells reached subconfluent. immunodeficiency 6-8 week old female mice were employed for model development (Balb / c / nu / nu). A431 cells were harvested and suspended in the matrix Matrigel. The cell suspension containing 5 × 10⁶ cells, mice were injected intradermally in the flank. Mice were randomly distributed in different groups, each containing 11 mice. On the same day, then twice a week, the mice were injected intraperitoneally with 0.5 mg of mAb 3.19.3, or isotype control antibody. The size of each tumor was measured twice a week. Tumor volume was calculated according to the formula: volume = length × (width)² × 0,5 (cm³).

As illustrated in Figure 13, mAb 3.19.3 significantly slowed the growth of tumor xenograft A431. The average tumor volume of the group administered with the antibody isotype control, reached approximately 1.5 cm³ at the end of the experiment, whereas 10 days after the start eksprimenta and at the end of the experiment the growth rate of tumors in the treated group significantly slowed down, and the size of the tumor was about 0.5 cm³. On the 23rd day of the volume ratio T / C (treatment / control) was 1/3, which corresponds to 66% inhibition of tumor growth -th.

These results suggest that the dose used in this experiment, by binding to mouse Ang-2 and blocking the binding of this ligand to its receptor Tie-2, mAb 3.16.3 can significantly slow the growth of A431 xenograft in "nude" mice. It is likely that the antitumor effect of the monoclonal antibody is due to inhibiting angiogenesis in the host, as demonstrated in the assays using Matrigel matrix block. The mechanism of action directed to the inhibition of angiogenesis is further illustrated in Example 22, where as the pharmacodynamic marker microvessel density (MVD) tumors has been used.

The mechanism of action of mAb 3.19.3 may not be limited to its blockage of association and subsequent transfer of Ang-2 / Tie-2 signal. As shown in Example 7, it was also found that this mAb binds to Ang-1 and block binding of Ang-1 to Tie-2. It is interesting to note that this mAb also blocks Ang-1-induced phosphorylation of Tie-2. It is known that Ang-1 is involved in vascular development. Comparison of mAb 3.19.3 activity on the inhibition of Ang-1 binding to Tie-2, with activity directed at inhibiting the binding of Ang-2 with Tie-2 (Example 12) indicated that mAb 3.19.3 is predominantly an antagonist Ang- 2. Without being limited to any particular theory, it is merely noted that dual blockade of signal transmission of Ang-2 and Ang-1 inhibits angiogenesis and consequently tumor growth.

EXAMPLE 21

MAB 3.19.3 inhibits tumor growth in xenograft models with engraftment

Ang-2 is stimulated by angiogenic endothelial cells, which leads to the development of many types of tumors. There is every reason to believe that a monoclonal antibody that blocks binding of Ang-2 Association / Tie-2 has the ability to inhibit angiogenesis and consequently tumor growth. In this experiment, the therapeutic efficacy of anti-Ang-2 mAb was demonstrated. Since mAb 3.19.3 cross-reacts with mouse Ang-2 / Tie-2 signal transduction and neutralizes mouse Ang-2 / Tie-2, such a mAb was chosen to illustrate<i>in vivo</i> efficacy of inhibiting tumor growth.

To determine whether anti-Ang-2 mAb 3.19.3 also inhibits the growing tumor and other tumors, i.e. no A-431 xenograft model of human adenocarcinoma LoVo colon was used. Doses of Mab 3.19.3, constituting 0.5, 2 and 10 mg / kg, administered intraperitoneally twice a week. Such administration started only when the tumors reached the average volume of 0.2 cm³. In developing these tumors it was also demonstrated that mAb 3.19.3 has an inhibitory effect as compared to the isotype control. In Figure 14A shows that 79% inhibition was achieved at 0.5 and 2 mg / kg (p values ​​were 0.022 and 0.027, respectively), and a 75% tumor growth inhibition was achieved at 10 mg / kg (p = 0.006 ).

The effect of inhibition of tumor growth was reproduced on additional xenograft model of human adenocarcinoma SW480 colon cancer, the size of which is adjusted to the average volume of 0.2 cm³. Although mAb 3.19.3 did not give a significant effect at 0.5 mg / kg, however, it was found that at the 53 th day after tumor implantation indicated mAb at a concentration of 2 and 10 mg / kg has detected 60% inhibition of tumor growth ( p = 0,003 and 0.006, respectively) (14B figure).

In general, the above results revealed that anti-Ang-2 mAb 3.19.3 significantly inhibited tumor growth in the three models tested. Interestingly, LoVo and SW480 express human Ang-2. However, two other mAb, which did not have cross-reactivity to mouse Ang-2 does not appear to have any significant inhibitory effect on tumor growth (data not shown), despite the fact that human Ang-2 was expressed by tumor cells. The results suggest that Ang-2 antagonist host needs to block angiogenesis and tumor growth.

As discussed above, Mab 3.19.3 cross-reacts with Ang-1. However, mAb 3.19.3 activity to bind to Ang-1 / Tie-2 was much lower than the activity to bind Ang-2 / Tie-2 (Example 12). From this, we can conclude that the therapeutic efficacy seen in these models is due primarily an antagonistic effect on Ang-2. However, we can not completely exclude the blocking of Ang-1 in these models<i>in vivo</i>. During the entire experiment any significant toxic effect, as well as weight loss or bleeding in the animals were observed.

EXAMPLE 22

<i>IN VIVO</i> EFFICACY OF MAB 3.19.3 IN OTHER models with tumor xenografts

The antitumor activity of monoclonal antibody 3.19.3 was tested in mouse xenograft models of human cancer by using 9 different tumor cell lines.

Cells colon adenocarcinoma (Lovo, SW480, Colo205, HT29, HCTl 16), squamous carcinoma (A431), lung carcinoma (Calu-6) and breast adenocarcinoma (MCF7, MDA-MB-231) were cultured in flasks routine method to those long as the cells reached subconfluent. immunodeficiency 7-10 week old female mice were used to develop the model. Cells were harvested, suspended in Matrigel matrix and each mouse injected subcutaneously. The mice were then randomly assigned to groups so that each group contained 10-12 mice. On the same day, then twice a week, the mice were injected intraperitoneally with 0.5 mg of mAb 3.19.3, or isotype control antibody. In all experiments performed isotype control antibody treatment. The size of each tumor was measured twice a week. Tumor volume was calculated by the formula:² × 0,5 (cm³). Graphical comparisons of tumor growth inhibition levels illustrated xenografts HT29 (15A figure) and Calu6 (15B figure).

As shown in Table 18, mAb 3.19.3 has detected significant activity in all 7 xenograft subcutaneous models tested and both ortotopnyh models with non-optimized dose and administration scheme.

<tables num="15"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="52mm" /><colspec colname="c1" colwidth="50mm" /><colspec colname="c2" colwidth="61mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 18Sistematizirovannaya <i>in vivo</i> the effectiveness of mAb 3.19.3</entry></row><row><entry align="center" rowsep="1" colsep="1">tumor models</entry><entry align="center" rowsep="1" colsep="1">% inhibition</entry><entry align="center" rowsep="1" colsep="0">% inhibition</entry></row><row><entry align="center" rowsep="1" colsep="1">Ксенотрансплантаты SubQ</entry><entry align="center" rowsep="1" colsep="1">2 mg / kg, 2 times per week</entry><entry align="center" rowsep="1" colsep="0">10 mg / kg, 2 times per week</entry></row><row><entry align="justify" rowsep="1" colsep="1">Colo205</entry><entry align="center" rowsep="1" colsep="1">35</entry><entry align="center" rowsep="1" colsep="0">46</entry></row><row><entry align="justify" rowsep="1" colsep="1">A431</entry><entry align="center" rowsep="1" colsep="1">43</entry><entry align="center" rowsep="1" colsep="0">66</entry></row><row><entry align="justify" rowsep="1" colsep="1">HT29</entry><entry align="center" rowsep="1" colsep="1">ND</entry><entry align="center" rowsep="1" colsep="0">54</entry></row><row><entry align="justify" rowsep="1" colsep="1">Calu6</entry><entry align="center" rowsep="1" colsep="1">ND</entry><entry align="center" rowsep="1" colsep="0">38</entry></row><row><entry align="justify" rowsep="1" colsep="1">HCT116</entry><entry align="center" rowsep="1" colsep="1">ND</entry><entry align="center" rowsep="1" colsep="0">33</entry></row><row><entry align="justify" rowsep="1" colsep="1">Ortotopnıe model</entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="0"> </entry></row><row><entry align="justify" rowsep="1" colsep="1">MCF7</entry><entry align="center" rowsep="1" colsep="1">35*</entry><entry align="center" rowsep="1" colsep="0">74</entry></row><row><entry align="left" rowsep="1" colsep="1">MDA-MB-231</entry><entry align="center" rowsep="1" colsep="1">50</entry><entry align="center" rowsep="1" colsep="0">58</entry></row><row><entry align="left" namest="c0" nameend="c2" rowsep="0" colsep="0">In all cases, P &lt;0,05 * Growth inhibition was not statistically znachimym.ND - not determined</entry></row></tbody></tgroup></table></tables>

Tumor tissue MDA-MB-231 was analyzed by staining density of CD31⁺-sosudov. CD31-staining density was determined by computing a threshold method and manual counting using a grid. Eleven tumors per group were analyzed and at least 20 tumor imaging. As shown in Figure 15C, treatment of mice resulted in antibody 3.19.3 40% reduction density CD31-staining as compared to staining with control antibody IgG. This result was statistically significant for both counting methods, namely the threshold method (p &lt;0,015) and manual counting method using a grid (p &lt;0,00004) in accordance with one-t-criterion. Similar calculations CD31⁺-sosudov were made on the fabric <i>ex vivo</i> xenografts Colo205 and HCTl16. These samples also exhibited a similar significant reduction in CD31 density⁺-sosudov.

EXAMPLE 23

Application anti-Ang-2 antibodies to help prevent angiogenesis-related diseases

For rate <i>in vivo</i> effectiveness of treatment of a human with various solid tumors using anti-Ang-1 and anti-Ang-2 antibodies are periodically administered to patients an effective amount of anti-Ang-1 and anti-Ang-2 antibody. At certain periods of the course of treatment of patients seen in the suppression of tumor growth. After the course of treatment, it was found that patients that were treated with anti-Ang-1 and anti-Ang-2 antibody in comparison to patients who did not pass this treatment, there was a relative improvement in one or more indicators, including, but not limited to, a reduction in tumor size, slowing of tumor progression or an increase in life expectancy.

EXAMPLE 24

USE OF ANTI-Ang-2 ANTIBODIES AS A DIAGNOSTIC TOOLS

<u>Detection of Ang-2 antigen in a sample</u>

For detection of Ang-1 or Ang-2 antigen in the sample enzyme linked immunosorbent assay (ELISA) can be developed. In this assay microtiter wells, such as 96-well microtiter plate or 384-well microtiter plate, for several hours, adsorbed "first" fully human monoclonal anti-Ang-1 and Ang-2. The immobilized antibody served as a capture antibody for any of the antigen that may be present in the test sample. The wells were then rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.

The wells were then treated with a test sample suspected of containing the antigen or a solution containing a standard amount of the antigen. Thus the sample may be, e.g., serum sample taken from an individual suspected to have high levels of antigen in the blood stream that are indicative of disease at diagnosis.

After washing the test sample or standard, the wells are treated with "second" fully human monoclonal anti-Ang-1 and anti-Ang-2 antibodies which have been labeled by conjugation with biotin. It may also be used murine monoclonal antibodies or antibodies derived from other species. The labeled anti-Ang-1 and anti-Ang-2 antibodies served as the detection antibody. After washing the excess of the "second" antibody wells are treated with avidin-conjugated horseradish peroxidase (HRP) and a suitable chromogenic substrate. The concentration of antigen in the test samples was determined by comparison with a standard curve constructed from the data obtained for standard samples.

This ELISA-assay is highly specific and highly sensitive assay for the detection of antigens Ang-1 and Ang-2 in a test sample.

<u>Determination of Ang-2 antigen concentration in patients</u>

To quantify the levels of Ang-1 and Ang-2 in human serum was developed by "sandwich" ELISA. In this "sandwich" -ELISA two fully human monoclonal anti-Ang-2 antibodies recognize different epitopes on the Ang-2 molecule. Alternatively, there may also be used murine monoclonal antibodies or antibodies derived from other species. In this case, but not necessarily, ELISA-analysis can be used, conducted as described below. 50 ul of anti-Ang-2 antibodies for capture in coating buffer (0,1 M NaHCO₃, PH 9,6) at a concentration of 2 mg / ml was applied to ELISA-plates (Fisher). After incubation at 4 ° C overnight, plates were treated with 200 .mu.l of blocking buffer (0,5% BSA, 0,1% Tween 20, 0.01% thimerosal in PBS) for 1 hour at 25 ° C. Plates were washed (3 ×) 0,05% Tween 20 in PBS (washing buffer, WB). Normal serum or patient sera (Clinomics, Bioreclaimation) diluted in blocking buffer containing a 50% human serum. Plates were incubated with serum samples overnight at 4 ° C, washed with WB, and then incubated with 100 ul / well of biotinylated detection anti-Ang-2 antibody for 1 hour at 25 ° C. After washing, plates were incubated with HRP-Streptavidin for 15 minutes, washed as described above and then treated with 100 .mu.l / well of o-phenylenediamine in H₂O₂ (Sigma developing solution) for color development. Then the reaction was stopped by adding 50 l / well H₂SO₄ (2M) and analyzed for ELISA-plate reader at 492 nm. The concentration of Ang-2 antigen in serum samples is calculated by comparison to dilutions of purified Ang-2 antigen using a program for constructing a 4-parameter curve.

Incorporated herein by reference INVENTION

All sources cited herein, including patents, patent applications, documents, manuals, etc., as well as the publications cited in these sources, and not yet published work in its entirety is incorporated herein by reference.

EQUIVALENTS

The present inventors believe that the above description of the invention it is sufficient to implement one of ordinary skill in the art. In the above description and the examples illustrate some preferred embodiments of the invention and described in detail the best mode considered by the applicants. However, it should be noted that, irrespective of the detailed description provided above, the present invention can be practiced in many ways and should be interpreted in accordance with the appended claims and any equivalents thereof.

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