Antibodies against angiopoietin-2 and use thereof

FIELD: chemistry.

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

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

33 cl, 18 dwg, 18 tbl, 24 ex

 

This application claims the priority of provisional application U.S. reg. No. 60/638354, filed December 21, 2004, and provisional application of U.S. reg. No. 60/711289, filed August 25, 2005, entered into the present description by reference.

The scope to which the invention relates

The present invention relates to monoclonal antibodies against angiopoietin-2 (Ang-2) and to the use of such antibodies. More specifically, the present invention relates to fully human monoclonal antibodies against Ang-2 and to the use of these antibodies. In other aspects, the present invention also relates to hybridomas or other cell lines expressing the indicated antibodies. Described antibodies may be used as diagnostic tools and for treatment of diseases associated with the activity and/or with overproduction Ang-2.

Prior art

Angiogenesis is the process of formation of new capillaries from existing blood vessels and is an essential component of the processes of embryogenesis, normal physiological growth, repair of tissues and tumor growth. Although the responses of endothelial cells (EC)in vitroand the growth of blood vesselsin vivocan be modulated by various factors, however, it is evident that with Aditya EK almost exclusively affect only members of the family of vascular endothelial growth factors (VEGF) and angiopoietin. Yancopoulos et al., Nature 407:242-48 (2000).

Angiopoetin were identified as ligands for the Tie, that is for family members tyrosinekinase, which is selectively expressed in vascular endothelium. Yancopoulos et al., Nature 407:242-48 (2000). Currently, there are four different family member angiopoetin. Angiopoietin-3 and -4 (Ang-3 and Ang-4) can represent widely differing analogues of the same gene locus in mice and in humans. Kim et al.,FEBS Let, 443:353-56 (1999); Kim et al., J Biol Chem 274:26523-28 (1999). Ang-1 and Ang-2 were first identified in experiments on tissue culture as agonist and antagonist, respectively. Davis et al.,Cell87:1161-69 (1996); Maisonpierre et al.,Science277:55 - 60 (1997). All known angiopoetin associated mainly with Tie-2, and both Ang-1 and Ang-2 bind to Tie-2 with an affinity of 3 nm (Kd). Maisonpierre et al.,Science277:55-60 (1997). It has been shown that Ang-1 promotes the increase of the life expectancy of endothelial cells and maintaining the integrity of the endothelium, Davis et al.,Cell87:1161-69 (1996); Kwak et al.,FEBSLett 448:249-53 (1999); Suri et al.,Science282:468-71 (1998); Thurston et al.,Science286: 2511-14 (1999); Thurston et al.,Nat. Med.6:460-63 (2000), whereas Ang-2 has the opposite effect and stimulates the destabilization and regression of blood vessels in the absence of factors VEGF, responsible for the survival of cells, or basic fibroblast growth factors. Maisonpierre et al., Science 277:55-60 (1997). One is about the many studies of the function of Ang-2 suggested a more complex role of Ang-2. Ang-2 may represent a complex regulator remodeling of blood vessels, which plays a role in the development of blood vessels, and in their destruction. To confirm such a role of Ang-2 were conducted analyses on his expression, have shown that the rapid induction of Ang-2 with VEGF leads to the development of angiogenesis in adults, and the induction of Ang-2 in the absence of VEGF leads to destruction of vessels. Holash et al.,Science284:1994-98 (1999); Holash et al.,Oncogene18:5356-62 (1999). In accordance with its dependent on the environment the role of Ang-2 is associated with the same specific endothelial receptor Tie-2, which is activated under the action of Ang-1, but this activation depends on its environment. Maisonpierre et al.,Science277:55-60 (1997).

Analysis of angiogenesis in the cornea showed that Ang-1 and Ang-2 have similar effects, i.e. their action, together with VEGF, which stimulates the growth of new blood vessels, is synergistic. Asahara et al., Circ. Res. 83:233-40 (1998). On the possibility of dose-dependent increase in endothelial response indicates the observation of the fact that Ang-2 may also be Pro-angiogenic when high concentrations ofin vitro. Kim et al., Oncogene 19:4549-52 (2000). At high concentrations of Ang-2 acts as a survival factor for endothelial cells during induced by serum deprivation apoptosis induced by activation of Tie-2 in the cascade of reactions kinase PI-3 and Akt. Kim et al.,Oncogene19:454-52 (2000).

In otherin vitroexperiments it has been suggested that the long-term effects effects of Ang-2 may change gradually from antagonismo steps on agonistic action against Tie-2, and in the later stages they can directly lead to the formation of vascular microtubules and stabilization of new blood vessels. Teichert-Kuliszewska et al.,Cardiovasc. Res.49:659-70 (2001). In addition, under cultivation EC on fibrin gel was also observed activation of Tie-2 under the action of Ang-2, which probably indicates that the effect of Ang-2 may depend on the level of differentiation of EC. Teichert-Kuliszewska et al.,Cardiovasc. Res. 49:659-70 (2001). In microvascular endothelial cells cultured in three-dimensional collagen gel, Ang-2 can also induce the activation of Tie-2, and to encourage the formation of capillaries such structures. Mochizuki et al., J. Cell. Sci. 115:175-83 (2002). The use of three-dimensional spheroid coculture asin vitromodel maturation vessels demonstrated that the direct contacting endothelial cells with mesenchymal cells suppresses sensitivity to VEGF, whereas the presence of VEGF and Ang-2 induces the proliferation of blood vessels. Korff et al.,Faseb J.15:447-57 (2001). Etoh and others have demonstrated that in EC, which constitutively Express Tie-2, expression of MMP-1, -9 and u-PA in a high degree is activated by angiopoietin Ang-2 is the presence of VEGF. Etoh, et al.,Cancer Res.61:2145-53 (2001). Onin vivomodel membrane pupil, Lobov and others have shown that Ang-2 in the presence of endogenous VEGF induces a rapid increase of the diameter of the capillaries, remodeling of the basal plate, proliferation and migration of endothelial cells, and stimulates the formation of new blood vessels. Lobov et al.,Proc. Natl. Acad. Sci.USA 99:11205-10 (2002). In contrast, in the absence of endogenous VEGF, Ang-2 stimulates the death of endothelial cells and the destruction of the vessels. Lobov et al., Proc. Natl. Acad. Sci. USA 99:11205-10 (2002). Similarly,in vivomodel tumor, Vajkoczy et al. demonstrated that the multicellular aggregates initiate the growth of blood vessels in the development of angiogenesis by simultaneous expression of VEGFR-2 and Ang-2 in the endothelium of the owner and in the endothelium of the tumor. Vajkoczy et al., J. Clin. Invest. 109:777-85 (2002). This model illustrated that developing network of capillary vessels in the growing tumor is characterized by constant remodeling, likely mediated expression of VEGF and Ang-2. Vajkoczy et al., J. Clin. Invest. 109:777-85 (2002).

In studies of Tie-2 and angiopoietin-1, deficient for these proteins mouse found similar phenotypes, and it has been suggested that induced by angiopoietin-1 phosphorylation of Tie-2 mediates remodeling and stabilization of developing blood vessels, stimulates the development of blood the blood vessels during angiogenesis and promotes adhesion of endothelial cells to “anchor” cells (Dumont et al., Genes & Development, 8:1897-1909 (1994); Sato, Nature, 376:70-74 (1995); (Thurston, G. et al., 2000 Nature Medicine: 6, 460-463)). It is obvious that the role of angiopoietin-1 is stored in the body of adult individuals, where it is constitutively expressed in various tissues of a wide range (Hanahan, Science, 277:48-50 (1997); Zagzag et al., Exp Neurology, 159:391-400 (1999)). In contrast, expression of angiopoietin-2 is limited, mainly, lots of remodeling of blood vessels, where it probably blocks the constitutive function of angiopoietin-1, responsible for the stabilization and development of blood vessels, which allows these vessels to return in a plastic condition and to remain in the condition in which they may be more susceptible to the transmission of signals (Hanahan, 1997; Holash et al.,Oncogene18:5356-62 (1999); Maisonpierre, 1997). Study the expression of angiopoietin-2 in pathological angiogenesis showed that the tumors of many types detect the expression of vascular angiopoietin-2 (Maisonpierre et al.,Science277:55-60 (1997)). Functional studies performed in murine xenograft models suggest that angiopoietin-2 is involved in tumor angiogenesis, and to associate the overexpression of angiopoietin-2 with the increase of tumor growth (Ahmad, et al.,Cancer Res., 61:1255-1259 (2001)). Other studies have shown that overexpression of angiopoietin-2 is associated with hypervascularization tumors (Etoh, et al., Cancer Res. 61:2145-53 (2001); Tnaka et al., Cancer Res. 62:7124-29 (2002)).

In recent years the application of angiopoietin-1, angiopoietin-2 and/or Tie-2 as a possible anti-cancer therapeutic targets. For example, in U.S. patent No. 6166185, 5650490 and 5814464 described ligand against Tie-2 and antibodies against receptors.

In studies conducted using soluble Tie-2, have been reported to reduce the number and size of tumors in rodents (Lin, 1997; Lin, 1998). Siemester and others (1999) received a human cell lines melanoma expressing the extracellular domain of Tie-2, and then these cells were injected with “Nude” mice, after which it was concluded that soluble Tie-2 significantly inhibits tumor growth and angiogenesis. Considering that and the angiopoietin-1 and angiopoietin-2 are associated with Tie-2, then, based on these studies, it remains unclear whether the angiopoietin-1, angiopoietin-2 or Tie-2 to serve as an attractive target for anticancer therapy. However, it is clear that therapy directed against angiopoietin-2, can be effective for the treatment of diseases such as cancer, the progression of which depends on the degree of violation of angiogenesis, where the blocking of this process can lead to the prevention of disease progression (Folkman, J., Nature Medicine. 1: 27-31 (1995)). In addition, some groups of researchers have reported on the use of antibodies, which is nazyvautsa with angiopoietin-2, see for example, U.S. patent No. 6166185 and publication of the patent application U.S. No. 2003/0124129 Al. The study of the effect of local expression of angiopoietin-2 showed that the antagonistic action of signal angiopoietin-1/Tie-2 leads to a weakening of dense vascular structures and, thus, to transmit an activating signal applied to endothelial cells (EC) inducers of angiogenesis, for example, VEGF. (Hanahan, 1997). Such Pro-angiogenic effect induced by inhibition of angiopoietin-1, indicates that therapy directed against angiopoietin-1, apparently, may not be effective anti-cancer therapy.

Ang-2 is expressed during the development of blood vessels in areas where there is remodeling of blood vessels. Maisonpierre et al., Science 277:55-60 (1997). In adult individuals, the expression of Ang-2 is limited to areas of remodeling of blood vessels, as well as in tumors with a high degree of vascularization, including glioma (sada et al.,Int. J. Oncol.18:305-09 (2001); Koga et al., Cancer Res. 61:6248-54 (2001)), hepatocellular carcinoma (Tanaka et al.,J. Clin. Invest.103:341-45 (1999)), gastric carcinoma (Etoh, et al.,Cancer Res.61:2145-53 (2001); Lee et al.,Int. J. Oncol.18:355-61 (2001)), swelling of the thyroid gland (Bunone et al.,Am. J. Pathol.155:1967-76 (1999)), non-small cell lung cancer (Wong et al.,Lung Cancer29:11-22 (2000)), colon cancer (Ahmad et al.,Cancer92:1138-43 (2001)) and prostate cancer (Wurmbach et al.,Anticancer Res. 20:5217-20 (2000)). B is lo detected, some tumor cells Express Ang-2. For example, Tanaka and others (J. Clin. Invest. 103:341-45 (1999)) was detected mRNA Ang-2 in 10 of 12 samples of human hepatocellular carcinoma (HCC). Group Ellis reported that Ang-2 is expressed throughout the tumor epithelium. Ahmad et al., Cancer 92:1138-43 (2001). Other researchers reported similar data. Chen et al., J. Tongji Med. Univ. 21:228-30, 235 (2001). Based on the determination of mRNA levels of Ang-2 in archived samples of cancer tissues in the breast of man, Sfilogoi and other (Int. J. Cancer 103:466-74 (2003)) reported that mRNA Ang-2 is associated mainly with the additional invasion of lymph nodes with a short latent period of the disease and, in General, small lifetime. Tanaka and others (Cancer Res.62:7124-29 (2002)) was observed only 236 patients with non-small cell lung cancer (NSCLC) in pathological stage I-IIIA disease, respectively. They carried out the immunohistochemical analysis showed that 16.9% of patients with NSCLC were positive for Ang-2. The density of microvessels in the Ang-2-positive tumors was significantly higher than in Ang-2-negative tumors. Such angiogenic effect of Ang-2 was observed only in case of high level of expression of VEGF. In addition, placitella expression of Ang-2 is an important factor, which is an indicator of poor prognosis relative to the duration of the spine of life after surgery. Tanaka et al., Cancer Res. 62:7124-29 (2002). However, any noticeable correlation between the expression of Ang-1 and density of microvessels were detected. Tanaka et al.,Cancer Res.62:7124-29 (2002). These results suggest that Ang-2 is an indicator of poor prognosis for patients with cancer of various types.

Recently, the group Yancopoulos, conducted studies on Ang-2-deficient mouse models, reported that Ang-2 is required for postnatal angiogenesis. Gale et al.,Dev. Cell3:411-23 (2002). These researchers have shown that genetically programmed regression of the vascular network of the vitreous body of the eye is absent in Ang-2-/--mice, and their blood vessels of the retina did not develop from the Central artery of the retina. Gale et al., Dev. Cell 3:411-23 (2002). These researchers also found that deletion of Ang-2 leads to a serious breach of the development and function of the lymphatic vessels. Gale et al.,Dev. Cell3:411-23 (2002). Conservation of gene Ang-1 corrects the defects of the lymphatic vessels, but does not eliminate the defects in angiogenesis. Gale et al.,Dev. Cell3:411-23 (2002).

Peters (Peters) and colleagues reported that soluble Tie-2, when it is delivered either as recombinant protein or a viral expression vector inhibitsin vivothe growth of murine breast carcinoma and melanoma in murine models. Lin et al., Proc. Natl Acad. Sci USA 95:8829-34 (1998); Lin et al., J. Clin. Invest. 100:2072-78 (1997) the Density of vessels in the thus treated tumor tissues was significantly decreased. In addition, soluble Tie-2 blocked angiogenesis in the cornea of rats, stimulated by the environment, air-conditioned tumor cells. Lin et al., J. Clin. Invest. 100:2072-78 (1997). In addition, Isner and his group demonstrated that the accession of Ang-2 to VEGF induces a much longer and more extensive neovascularization than one VEGF. Asahara et al.,Circ. Res. 83:233-40 (1998). Excess soluble receptor Tie-2 prevents modulation of VEGF-induced neovascularization under the action of Ang-2. Asahara et al.,Circ. Res. 83:233-40 (1998). Siemeister, and other (Cancer Res.59:3185-91 (1999)research on “Nude” mice with xenografts showed that overexpression of the extracellular ligand-binding domain FIt-I or Tie-2 in these xenografts leads to a significant inhibition of the ways their reactions, which cannot compensate for each other, suggesting that the path of the VEGF receptor and the path of the Tie-2 should be considered as two independent mediator, which play an important role in the process of angiogenesisin vivo. Siemeister et al.,Cancer Res. 59:3185-91 (1999). This is confirmed by more recent publications White et al.,Proc. Natl. Acad. Sci. USA100:5028-33 (2003). In this study, it was demonstrated that resistant to nuclease RNA aptamers that specifically binds to Ang-2 and inhibits Ang-2 significantly inhibits the neovascularization induced bFG in a model of angiogenesis in microturbine cornea of rats.

A brief description of the invention

In one of its variants, the present invention relates to aim at the target-binding agents that specifically bind to angiopoietin-2 and, thereby, inhibit tumor angiogenesis and reduce tumor growth. The mechanisms through which can be achieved this effect can be, but are not limited to, inhibition of binding Ang-2 with its receptor Tie-2, inhibition of Ang-2-induced signal transmission Tie-2 or enhanced clearance of Ang-2, thereby reducing the effective concentration of Ang-2.

In one embodiment of the invention, the targeted binding agent is a fully human antibody that binds to Ang-2 and prevents the binding of Ang-2, Tie-2. In yet another embodiment, the present invention relates to fully human monoclonal antibody that binds to Ang-2 and Ang-1 and inhibits Ang-2-induced phosphorylation of Tie-2. The specified antibody can bind to Ang-2 with Kdless than 100 PM, 30 PM, 20 PM, 10 PM or 5 PM.

The antibody may contain the amino acid sequence of the heavy chain having hypervariable region (complementarity-determining region (CDR), one of the sequences of which are presented in table 11. It should be noted that a specialist in this OBLASTNOE easily identify such CDR. Cm. for example, the guide Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols.1-3.

One of the variants of the present invention includes a fully human monoclonal antibodies 3.3.2 (access number in ATCC PTA-7258), 3.19.3 (access number in ATCC PTA-7260) and 5.88.3 (access number in ATCC PTA-7259), which are specifically associated with Ang-2, as further discussed below.

In yet another embodiment, the present invention relates to an antibody that binds to Ang-2 and contains the amino acid sequence of light chain having CDRs containing one of the sequences shown in table 12. In some embodiments of the invention, the specified antibody is a fully human monoclonal antibody.

In another embodiment, the present invention relates to an antibody that binds to Ang-2 and contains the amino acid sequence of the heavy chain, having one of the CDR sequences shown in table 11, and the amino acid sequence of light chain, having one of the CDR sequences shown in table 12. In some embodiments of the invention, the specified antibody is a fully human monoclonal antibody. Another option of the present invention is an antibody that competes with a fully human antibodies is according to the invention for cross-linking with Ang-2, and preferably, the antibody containing the amino acid sequence of the heavy chain, having one of the CDR sequences shown in table 11, and the amino acid sequence of light chain, having one of the CDR sequences shown in table 12. Another option of the present invention is an antibody that binds to the same epitope on Ang-2, and fully human antibodies according to the invention, and preferably, the antibody containing the amino acid sequence of the heavy chain, having one of the CDR sequences shown in table 11, and the amino acid sequence of light chain, having one of the CDR sequences shown in table 12.

Other variants of the present invention include human monoclonal antibodies that specifically bind to angiopoietin-2, where these antibodies contain hypervariable region 1 (CDRl) the heavy chain corresponding to canonical class 1. These antibodies can also include hypervariable region 2 (CDR2) of the heavy chain corresponding to canonical class 3, hypervariable region 1 (CDR1) of the light chain corresponding to canonical class 2, hypervariable region 2 (CDR2) of the light chain corresponding to canonical class 1 and hypervariable region is here 3 (CDR3) of the light chain, corresponding to canonical class 1.

The present invention also relates to methods of analysis of the level of angiopoietin-2 (Ang-2) in the sample taken from the patient, where the method includes contacting an anti-Ang-2 antibody with a biological sample obtained from the patient, and detecting the level of binding of the specified antibodies to Ang-2 in the specified pattern. In more specific embodiments of the invention, the specified biological sample is blood.

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

In other embodiments, the present invention relates to methods of effectively treating an animal suffering from a disease associated with angiogenesis, where these methods include selecting an animal in need of treatment of neoplastic or non-neoplastic disease, and the introduction of specified animal a therapeutically effective dose of a fully human monoclonal antibody that specifically binds to angiopoietin-2 (Ang-2).

Associated with angiogenesis diseases, treatable, can be neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), SDA is the role of the thyroid gland, cancer of the gastrointestinal tract (stomach), prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, carcinoma of the esophagus, head and neck cancer, mesothelioma, sarcomas, biliary cholangiocellular carcinoma, adenocarcinoma of the small intestine, malignant tumors in children and squamous cell carcinoma.

In other embodiments, the present invention relates to methods of inhibiting induced by angiopoietin-2 (Ang-2) angiogenesis in animals. Such methods include selecting an animal in need of treatment of Ang-2-induced angiogenesis, and the introduction of specified animal a therapeutically effective dose of a fully human monoclonal antibodies, where the specified antibody specifically binds to Ang-2.

In other embodiments, the present invention relates to the use of antibodies for the preparation of drugs for treatment-associated angiogenesis diseases in an animal, where the specified monoclonal antibody specifically binds to angiopoietin-2 (Ang-2). Associated with angiogenesis diseases, treatable, can be neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatically the popular carcinoma (liver), swelling of the thyroid gland, cancer of the gastrointestinal tract (stomach), prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, carcinoma of the esophagus, head and neck cancer, mesothelioma, sarcomas, biliary cholangiocellular carcinoma, adenocarcinoma of the small intestine, malignant tumors in children and squamous cell carcinoma.

In other embodiments of the invention described herein, the antibodies can be used to prepare medicines for effective treatment induced angiopoietin-2 angiogenesis in an animal, where the specified monoclonal antibody specifically binds to angiopoietin-2 (Ang-2).

In other embodiments described herein, the present invention relates to monoclonal antibodies that bind to Ang-2 and affect the function of Ang-2. In other embodiments, the present invention relates to fully human antibodies against Ang-2 and to preparations of antibodies against Ang-2, with the right and perspective from a therapeutic standpoint properties, including high affinity of binding to Ang-2, the ability to neutralize Ang-2in vitroandin vivoand the ability to inhibit Ang-2-induced angiogenesis.

In a preferred embodiment of the invention, opican the e here antibody binds to Ang-2 with a very high appendectomy (Kd). So, for example, human, rabbit, mouse, chimeric or gumanitarnoe antibody have the ability to bind to Ang-2 with a Kd that have values, but not limited to, less than 10-5, 10-6, 10-7, 10-8, 10-9, 10-10, 10-11, 10-12, 10-13or 10-14M or any interval of these values, or any value in this interval. The affinity and/or avidity can be measured by analysis using KinExA® and/or BIACORE®, as described below.

Accordingly, in one of the options described here, the present invention relates to selected antibodies or fragments of these antibodies that bind to Ang-2. As is known from the prior art, such antibodies preferably can 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 that produce such antibodies.

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

It should be noted that variants of the present invention is not limited to any specific form of antibodies or the method of its generation or production. For example, the anti-Ang-2 antibody may be a full-sized antibody (e.g., antibody, having an intact human Fc region) or an antibody fragment (e.g., Fab, Fab' or F(ab')2). In addition, the antibody can be obtained from hybridoma, which secretes such an antibody, or recombinante-derived cells that had been transformed or transliterowany gene or genes encoding such an antibody.

Other variants of the present invention include the selected nucleic acid molecule encoding any of the antibodies described here; the vectors containing the selected nucleic acid molecule; encoding anti-Ang-2 antibodies; or the cell host transformed by any such nucleic acid molecules. In addition, in one of its variants, the present invention relates to a method for producing antiad-2 antibodies by culturing host cells under conditions conducive to the expression of the nucleic acid molecules and the production of antibodies, and subsequent allocation of such antibodies. It should be noted that variants of the present invention include any nucleic acid molecule that encodes the antibody according to the invention or its fragment, including nucleic acid sequence that is optimized to increase yield of antibodies or their fragments by transfection of such sequences in cell-hosts for the production of antibodies.

Another variant of the present invention includes a method of obtaining high-affinity antibodies against Ang-2 by immunization of a mammal human Ang-2 or its fragment and one or more ontologyname sequences or their fragments.

Other variants of the invention are based on the generation and identification of selected antibodies that specifically bind to Ang-2. Diseases associated with angiogenesis, such as tumor diseases, Ang-2 is expressed at higher levels. Inhibition of the biological activity of Ang-2 can prevent Ang-2-induced angiogenesis and other desirable effects.

Another variant of the present invention includes a method for diagnosing diseases or conditions in which the antibody obtained as described in the present application, used to detect the level of Ang-2 in a sample obtained from the patient. In one embodiment of the invention, a sample taken from the patient is blood or serum. In other embodiments, the invention describes methods of identifying risk factors, diagnosis and determine the stage of the disease, where these methods include the identification of shorecrest Ang-2 using anti-Ang-2 antibodies.

Another variant of the present invention includes a method for diagnosing the condition, associated with expression of Ang-2 in the cells, by contacting the serum or cells with anti-Ang-2 antibody, followed by detection of the presence of Ang-2. The preferred conditions are 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 cancer), glioblastoma, and carcinoma of the thyroid, stomach, prostate, breast, ovary, bladder, lung, uterus, kidney, colon, pancreas, salivary glands and colon.

In another embodiment, the present invention includes an analytical kit for detection of angiopoietin-2 and family members angiopoetin in tissues, cells or the physical and the ideological liquids mammals in screening for disease, associated with angiogenesis. Specified the kit includes an antibody that binds to angiopoietin-2, and the tool, which is an indicator of antibody response to angiopoietin-2, if it happens. The preferred antibody is a monoclonal antibody. In one embodiment of the invention, the specified antibody, binding to Ang-2 is labeled. In another embodiment of the invention, the specified antibody is its “first” antibody, and a specified set further includes means for detecting the specified “first” antibody. In one embodiment of the invention, the tool includes a labeled second antibody which is an antibody against immunoglobulin. The preferred antibody is an antibody labeled with a marker selected from the group consisting of fluorochrome, an enzyme, a radionuclide, and a material which is impermeable to radiation.

Another variant of the present invention includes methods of treating diseases or conditions associated with expression of Ang-2 in a patient, by introducing a specified patient an effective amount of anti-Ang-2 antibodies. Anti-Ang-2 antibody may be introduced separately, or it can be introduced in combination with other antibodies or chemotherapeutic agent, or in combination with radiation therapy is Oia. So, for example, a mixture of monoclonal, oligoclonal or polyclonal antibodies against Ang-2 that block angiogenesis, can be introduced in combination with a medicine that has been shown to be directly inhibits proliferation of tumor cells. The method may be carried outin vivoand the specified patient, preferably a human being is. In a preferred embodiment of the invention, the method includes treatment-associated 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 cancer), glioblastoma, and carcinoma of the thyroid, stomach, prostate, breast, ovary, bladder, lung, uterus, kidney, colon, pancreas, salivary glands and colon.

In another embodiment, the present invention relates to industrial products, including the container. Such a container includes a composition containing an anti-Ang-2 antibody, and an insert in the packaging or label on the packaging stating that the composition can be used to treat associated with angiogenesis diseases characterized by overexpression of Ang-2.

In some embodiments, from whom retene, the patient is given anti-Ang-2 antibody, and then a cleansing agent to remove excess antibodies from the bloodstream.

In another embodiment, the present invention relates to the use of anti-Ang-2 antibodies for the preparation of drugs for the treatment of diseases, such as diseases associated with angiogenesis. In one embodiment of the invention, associated with angiogenesis diseases are cancer, such as breast cancer, ovarian, stomach, endometrial, salivary glands, lungs, kidneys, colon, colon, esophagus, thyroid gland, pancreas, prostate and bladder. 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 cholangiocellular carcinoma, adenocarcinoma of the small intestine, malignant tumors in children and glioblastoma.

Ang-2 is an important “switch" angiogenesis. In line with this, it is assumed that the suppression of activity of this molecule leads to inhibition of pathophysiological processes, and therefore can serve as an effective means d is I the treatment of various angiogenesis-dependent diseases. In addition to solid tumors and their metastases, angiogenesis-dependent are also malignant tumors of the blood, such as leukemia, lymphoma and multiple myeloma. Excessive growth of blood vessels leads to the emergence of various non-neoplastic disorders. Such non-neoplastic angiogenesis-dependent diseases are atherosclerosis, hemangioma, hemangioendothelioma, angiofibroma, developmental disorders of blood vessels (e.g., hereditary hemorrhagic telangiectasia (NGT) or syndrome Osler-Weber), warts, pyogenic granulomas, excessive hair growth, Kaposi's sarcoma, keloid scars, allergic edema, psoriasis, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation, endometriosis, respiratory distress syndrome, ascites, peritoneal sclerosis in patients subjected to dialysis, the formation of adhesions after surgery on the abdomen, obesity, rheumatoid arthritis, synovitis, osteomyelitis, excessive growth of pannus, osteofit, hemophilic arthropathy, inflammatory and infekcionnye processes (e.g., hepatitis, pneumonia, glomerulonephritis, asthma, polyps in the nasal cavity, liver regeneration, pulmonary hypertension, retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, leucomalacia, neovascular glaucoma, neovasc laritate transplant of a cornea, trachoma, thyroiditis, thyroid enlargement, and lymphoproliferative disorders.

Brief description of the graphical material

The figure 1 shows the Western blot analysis, which showed that mAb against Ang-2 inhibit Ang-2-induced phosphorylation of Tie-2, ectopiceski expressed in HEK293F cells.

The figure 2 presents the linear curve dose-dependent inhibitory effect of monoclonal anti-Ang-2 antibodies to Ang-2-induced phosphorylation of Tie-2.

The figure 3 presents the linear curve illustrating the dose-dependent inhibition of binding of Ang-1 (upper graph) and Ang-2 (lower graph) with Tie-2 in the presence of mAb 3.19.3 or Tie-2/Fc.

The figure 4 shows a Western blot analysis illustrating the inhibition induced by angiopoietin-1 phosphorylation of Tie-2 in endothelial cells Eahy 926 under the action of mAb 3.19.3. In this system, the observed inhibition induced by angiopoietin-1 phosphorylation of Tie-2. Antibody concentrations are presented in nm.

The figure 5 presents the linear curve illustrating the inhibition induced by angiopoietin-1 phosphorylation of Tie-2 in endothelial cells Eahy 926 under the action of mAb 3.19.3. IC50=99 nm. The x axis represents the concentration of mAb 3.19.3 and the y-axis represents the response.

Figure 6 schematically shows the structure diagram of the human proteins is in Ang-2 and Ang-2 443. Top numbers indicate amino acid sequence (this chart was taken from the publication Injune et al., (2000) JBC 275: 18550).

The figure 7 shows the amino acid sequence of the chimeric molecules of the mouse/human (SEQ ID NO: 1). Human remains (cloned asStuI-TfiI-slice) 310-400 underlined.

The figure 8 shows a comparison of amino acid sequences of the 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 joining chimeric molecules Ang-2 and point mutations are indicated in bold.

The figure 9 shows the comparison of 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 site of cleavage for hydrophobic leader sequences. These arrows determine the limits superspiritual and fibrinogen-like domains. Shaded circles indicate conservative cysteine residues (this graph was taken from the publication Maisonpierre et al., 1997,Science277:55).

The figure 10 shows the linear curve illustrating the dose-dependent cross-reactivity in mice. Also shown clones of monoclonal antibodies 5.2.1, 5.28.1, 3.19.3 and 3.31.2.

The figure 11 presents the linear curve illustrating the dose-dependent inhibition of binding of h is human (black triangles) and mice (black squares) Ang-2 with human Tie-2 in the presence of mAb 3.19.3.

The figure 12 presents a histogram showing the analysis of the impact of antibodies on angiogenesis induced MCF-7 cells. Figure 12A shows the effects of anti-Ang-2 antibodies on the number of vascular branches, where on the axisxspecify the experimental group, and on the axisyindicates the average number of vascular branches (± srcpath.). Figure 12B shows the effects of anti-Ang-2 antibodies on the length of the blood vessels, where on the axisxspecify the experimental group, and on the axisyspecify the average length of blood vessels (± srcpath.).

The figure 13 shows the linear curve illustrating the antitumor effect of clone 3.19.3, monoclonal anti-Ang-2 antibodies, and analyzed on a murine xenograft models of human squamous cell carcinoma of the skin using a cell line A431. On the axisxspecified number of days after implantation of tumor cells, and on the axistheindicated the average tumor volume (± srcpath. in cm3). Black triangles indicate the tumor volume measured after implantation of tumor cells to mice that were injected clone 3.19.3 monoclonal anti-Ang-2 antibodies; and black circles denote tumor volume measured after implantation of tumor cells to mice who were injected control antibody isotype PKl6.3.1.

Figure 14A presents a linear curve illustrating the prevention of tumor growth in xenograft models of human adenocarcinoma LoVo with the specified tumor size in mice treated with 0.5, 2 and 10 mg/kg of the antibodies or antibody isotype control. On the axisxspecified number of days after implantation of tumor cells, and on the axistheindicated the average tumor volume (± srcpath. in cm3). Figure 14B presents a linear curve illustrating the effect of inhibition of tumor growth under the action of mAb in xenograft models of human colon adenocarcinoma SW480.

Figure 15A presents the linear curve illustrating the prevention of tumor growth in xenograft models NT. On the axisxspecified number of days after implantation of tumor cells, and on the axistheindicated the average tumor volume (± srcpath. in cm3). Figure 15 presents a linear curve illustrating the prevention of tumor growth in xenograft models Calu-6, where for mice treated with 10 mg/kg of mAb clone 3.3.2 or 3.19.3 or control isotype antibody, specified the size of the tumor. In the figure 15C presents a linear curve illustrating the density of CD31+-staining of tumors in mice with tumor MDA-MB-231 and processed by control IgG or 10 mg/kg of mAb 3.19.3. Presents also the results of the s, obtained by calculating the threshold method and the method of manual counting using a coordinate grid.

Detailed description of the invention

Described herein embodiments of the present invention relate to monoclonal antibodies that bind to Ang-2. In some embodiments of the invention, the specified antibodies bind to Ang-2 and inhibit the binding of Ang-2 with its receptor, Tie-2. In other embodiments, the present invention relates to fully human anti-Ang-2 antibodies and antibody preparations, which can be used for therapeutic purposes. These drugs are anti-Ang-2 antibodies preferably possess desirable therapeutic properties, including high affinity of binding to Ang-2, the ability to neutralize Ang-2in vitroand the ability to inhibit Ang-2-induced angiogenesisin vivo.

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

Variants of the present invention also include a dedicated binding fragments of the anti-Ang-2 and the antibodies. Such binding fragments, preferably, derived from a full-sized human anti-Ang-2 antibodies. Illustrated fragments are Fv, Fab' or other known fragments of antibodies, which will be described in detail below. Variants of the present invention also include cells expressing full-sized human antibodies against Ang-2. Examples of such cells are hybridoma or cells created recombinant methods, such as cells of the Chinese hamster ovary (CHO), variants of CHO cells (e.g., DG44) and NSO cells producing antibodies against Ang-2. For more information on variants of CHO cells can be found in the publication Andersen and Reilly (2004) Current Opinion in Biotechnology 15, 456-462, which in its entirety is introduced into the present description by reference.

In addition, in other embodiments, the present invention relates to methods of using these antibodies for the treatment of diseases. Anti-Ang-2 antibodies can be used to prevent Ang-2-mediated signal transmission Tie-2 and thereby inhibiting angiogenesis. The mechanism of action of this inhibition may involve the inhibition of binding Ang-2 with its receptor, Tie-2; inhibition of Ang-2-induced signal transmission Tie-2; or enhanced clearance of Ang-2, leading to the reduction of the effective concentration of Ang-2, svyazivayuschego is with Tie-2. Diseases that are treatable through such a mechanism of inhibition are, but are not limited to, neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver cancer), glioblastoma, as well as cancer and tumors of the thyroid gland, stomach, prostate, breast, ovary, bladder, lung, uterus, kidney, colon, pancreas, salivary glands and colon.

Other variants of the present invention include diagnostic tests, in particular, to determine the amount of Ang-2 in the biological sample. The test kit may include anti-Ang-2 antibodies together with the necessary labels for the detection of such antibodies. Such diagnostic tests can be applied for screening associated with angiogenesis diseases, including, but not limited to, neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver cancer), glioblastoma, and carcinoma of the thyroid, stomach, prostate, breast, ovary, bladder, lung, uterus, kidney, colon, pancreas, salivary glands and colon.

In accordance with one of its aspects, the crust is ASEE the invention relates to an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2, where specified, the antagonist is not associated with the ATP-binding site Tie-2.

In accordance with another of its aspects, the present invention relates to an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2, where specified antagonist binds angiopoietin-1 and angiopoietin-2.

In accordance with another of its aspects, the present invention relates to an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2, where specified, the antagonist is not a connection.

In one of its variants, the present invention relates to an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2, where this antagonistic activity directed against angiopoietin-1, and the specified antagonistic activity directed against angiopoietin-2, are enclosed in a single molecule. In its alternative embodiment, the present invention relates to an antagonist, where this antagonistic activity against angiopoietin-1 and the specified antagonistic activity against angiopoietin-2 made in more than one molecule.

In one of its variants, the present invention relates to an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2, where specified antagonist can be contacted at:

i) receptor Tie-2;

ii) angiopoietin-1 and/or with angioma is Tina-2;

iii) complex receptor Tie-2 - angiopoietin-1”; or

iv) complex of the receptor Tie-2 - angiopoietin-2”; or

with any combinations thereof.

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

i) binding of the antagonist with angiopoietin-1 and inhibition of binding of angiopoietin-1 with its receptor, Tie-2, and/or

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

iii) increased clearance of angiopoietin-1 and/or angiopoietin-2, thereby reducing the effective concentration of angiopoietin-1 and/or angiopoietin-2, is available for binding to Tie-2, or

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

Without pretending to any theoretical conclusions, it should be noted that the mechanisms through which this may be done inhibition of the biological activity of angiopoietin-1 and angiopoietin-2 are, but are not limited to, inhibition of binding of angiopoietin-1 and angio Atina-2 receptor Tie-2; the inhibition induced by angiopoietin-1 and angiopoietin-2 signal transmission Tie-2, or enhance clearance of angiopoietin-1 and angiopoietin-2, thereby reducing the effective concentration of angiopoietin-1 and angiopoietin-2.

In one of its variants, the present invention relates to an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2, where the specified antagonist is an antibody. Preferably, this antibody has the ability to inhibit the biological activity of angiopoietin-1 and/or angiopoietin-2in vitroandin vivo. The preferred antibody is a polyclonal antibody or a monoclonal antibody. The preferred antibody is a monoclonal antibody, and still more preferred antibody is a fully human monoclonal antibody. The preferred antibody is a fully human monoclonal antibody 3.19.3.

In one of its variants, the present invention relates to an antibody that binds to the same epitope or with the same epitopes as fully human monoclonal antibody 3.19.3.

In one of its variants, the present invention relates to fully human antibody that binds to angiopoietin-1 and prevents the binding of angiopoietin-1 and Tie-2. Each is m its option, the present invention relates to fully human monoclonal antibody that binds to angiopoietin-1 and inhibits induced by angiopoietin-1 phosphorylation of Tie-2. In one embodiment of the invention, the specified antibody binds to angiopoietin-1 with Kdconstituting less than 1 nanomoles (nm). More preferably, this antibody is associated with Kdconstituting less than 500 picomole (PM). More preferably, this antibody is associated with Kdconstituting less than 100 picomol (PM). Even more preferably, this antibody is associated with Kdconstituting less than 30 picomol (PM). Even more preferably, this antibody is associated with Kdconstituting less than 20 PM. Most preferably, this antibody is associated with Kdconstituting less than 10 or 5 PM.

In one of its variants, the present invention relates to fully human antibody that binds to angiopoietin-2 and prevents the binding of angiopoietin-2, Tie-2. In another embodiment, the present invention relates to fully human monoclonal antibody that binds to angiopoietin-2 and inhibits induced by angiopoietin-2 phosphorylation of Tie-2. In one embodiment of the invention, the specified antibody is vyzyvaetsya with angiopoietin-1 with K dconstituting less than 1 nanomoles (nm). More preferably, this antibody is associated with Kdconstituting less than 500 picomole (PM). More preferably, this antibody is associated with Kdconstituting less than 100 picomol (PM). Even more preferably, this antibody is associated with Kdconstituting less than 30 picomol (PM). Even more preferably, this antibody is associated with Kdconstituting less than 20 PM. Most preferably, this antibody is associated with Kdconstituting less than 10 or 5 PM.

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

In one of its variants, the present invention relates to a nucleic acid molecule that encodes became the th chain or heavy chain antibodies, described above. Preferably, the present invention relates to a nucleic acid molecule that encodes a light chain or heavy chain of a fully human monoclonal antibodies. More preferably, the present invention relates to a nucleic acid molecule that encodes a light chain or heavy chain of a fully human monoclonal antibody 3.19.3.

In one of its variants, the present invention relates to a vector containing the molecule or nucleic acid molecules described above, where the specified vector encodes the light chain and/or heavy chain antibodies, as defined above.

In one of its variants, the present invention relates to the cell host containing the vector described above. Alternative specified a host cell may contain more than one vector.

In addition, in one of its variants, the present invention relates to a method for producing antibodies by culturing host cells under conditions that favor expression of the nucleic acid molecules, producing the indicated antibodies and subsequent selection.

In one of its variants, the present invention relates to a method for producing antibodies, including transfection, at least one host cell, at least one molecule of nucleic acid is you, encoding the above antibody; the expression of the indicated molecules of nucleic acid in said cell host; and the selection of the indicated antibodies.

In accordance with another of its aspects, the present invention relates to a method of inhibiting the biological activity of angiopoietin-1 and angiopoietin-2, including the introduction described above antagonist. The method may include selecting an animal in need of prevention it is associated with disease angiogenesis, and the introduction of specified animal a therapeutically effective dose of an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2.

In accordance with another of its aspects, the present invention relates to a method of inhibiting the biological activity of angiopoietin-1 and angiopoietin-2, including the introduction of the above-described antibodies. The method may include selecting an animal in need of prevention it is associated with disease angiogenesis, and the introduction of specified animal a therapeutically effective dose of an antibody that inhibits the biological activity of angiopoietin-1 and angiopoietin-2.

In accordance with another of its aspects, the present invention relates to a method of preventing associated with the disease angiogenesis in a mammal, comprising introducing terapevticheskii 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 prevention it is associated with disease angiogenesis, and the introduction of specified animal a therapeutically effective dose of an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2.

In accordance with another of its aspects, the present invention relates to a method of preventing associated with the disease angiogenesis in a mammal, comprising introducing 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 associated with disease angiogenesis, and the introduction of specified animal a therapeutically effective dose of an antibody that inhibits the biological activity of angiopoietin-1 and angiopoietin-2. The specified antibody may be introduced separately, or it can be introduced in combination with other antibodies or chemotherapeutic agent or radiation therapy.

In accordance with another of its aspects, the present invention relates to a method of treating cancer in a mammal, comprising introducing a therapeutically effective amount of an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2. This method can on the part the choice of the animal, in need of cancer treatment, and the introduction of specified animal a therapeutically effective dose of an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2. Specified antagonist may be introduced separately, or it can be introduced in combination with other antibodies or chemotherapeutic agent or radiation therapy.

In accordance with another of its aspects, the present invention relates to a method of treating cancer in a mammal, comprising introducing 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 the introduction of specified animal a therapeutically effective dose of an antibody that inhibits the biological activity of angiopoietin-1 and angiopoietin-2. The specified antibody may be introduced separately or in combination with other antibodies or in combination with a chemotherapeutic agent or radiation therapy.

In accordance with another of its aspects, the present invention relates to the use of an antagonist of the biological activity of angiopoietin-1 and angiopoietin-2 for the preparation of drugs for the treatment associated with the disease angiogenesis.

In accordance with another of its aspects, the present of Britanie refers to the use of antibodies, inhibiting the biological activity of angiopoietin-1 and angiopoietin-2, for preparing a medicinal product for the treatment associated with the disease angiogenesis.

In a preferred embodiment, the present invention can be particularly applied for the inhibition of angiopoietin-1 and angiopoietin-2 in patients with tumor, which depends solely or in part from the receptor Tie-2.

In another embodiment, the present invention includes an analytical kit for detection of angiopoietin-1 and/or angiopoietin-2 in tissues, cells or body fluids of mammals in screening for diseases associated with angiogenesis. Specified the kit includes an antibody that binds to angiopoietin-1 and/or angiopoietin-2, and the tool, which is an indicator of antibody response to angiopoietin-1 and/or angiopoietin-2, if it happens. The specified antibody may be a monoclonal antibody. In one embodiment of the invention, the antibody that bind to Ang-2 is labeled. In another embodiment of the invention, the specified antibody is its “first” antibody, and the specified set also includes a means for detecting the specified “first” antibody. In one embodiment of the invention, the tool includes a labeled second antibody, which is the raised antibody against immunoglobulin. The preferred antibody is an antibody labeled with a marker selected from the group consisting of fluorochrome, an enzyme, a radionuclide, and a material which is impermeable to radiation.

Other options, features, etc. related to anti-Ang-2 antibody, described in more detail below.

The list of sequences

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

Each antibody was assigned a unique identification number, which consists of two or three numbers, separated by a period, usually used in decimal numbers. For most antibodies is only two ID numbers, separated by dots.

However, in some cases there were several clones of the same antibodies. Although these clones have the nucleotide sequence and amino acid sequence identical to the corresponding original sequences, however, they can also be presented separately, with the number of the clone is indicated by the number on the right side of the second dividing point. For example, the sequence of nucleic key is lots and amino acid sequences of antibodies 5.35 identical sequences of antibodies 5.35.1, 5.35.2 and 5.35.3.

Definitions

If it is not specifically mentioned, it is used here of scientific and technical terms have common values, well known to the average person skilled in the art. In addition, if it is not clear from context, the nouns used in the singular can mean the nouns in the plural, and Vice versa. In General terms, nomenclature and methods related to tissue culture, molecular biology, chemical properties and hybridization of protein and oligo - or polynucleotides, well known in the art and are widely used in this field.

Standard methods are the methods of recombinant DNA, oligonucleotide synthesis, and the cultivation and transformation of tissues (e.g., electroporation, lipofection). Enzymatic reactions and purification methods is carried out in accordance with the manufacturer's instructions or in accordance with standard procedures or procedures described in this application. The above procedure is usually carried out by standard methods, well known in the art and described in various General and specific guidelines, cited and discussed in the present description. See, for example, the manual Sambrook et al.Molecular Cloning: A Laboratory Manual(3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001)), which is th is introduced into the present description by reference. The nomenclature used here and laboratory procedures, and methods related to analytical chemistry, chemistry of organic synthesis and medicinal and pharmaceutical chemistry described in this application are well known in the art and are widely used in this field. For chemical synthesis, chemical analysis, preparation of pharmaceuticals, compositing, delivery and treatment of patients used standard methods.

In the description of the present invention uses the following terms, which, if it is not specifically mentioned, shall have the meanings defined below.

The antagonist can be a polypeptide, nucleic acid, carbohydrate, lipid, small molecule compound, an oligonucleotide, Oligopeptide, interfering RNA (mRNA), antisense molecule, a recombinant protein, an antibody, or conjugates or hybrid proteins. Description mRNA can be found in the publication Milhavet O, Gary DS, Mattson MP. (Pharmacol Rev. 2003 Dec; 55(4):629-48. Review.), and the description of antisense molecules can be found in the publication Opalinska J.B., A.M. Gewirtz (Sci STKE. 2003 Oct 28; 2003(206):p.47.)

The angiogenesis associated with the specified disease, can be any abnormal, undesirable or pathological angiogenesis, for example, tumor-associated angiogenesis. Associated with angiogenesis diseases include, but are not limited, and is, and non-solid tumors such as leukemia, multiple myeloma or lymphoma, and solid tumors, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver)carcinoma, glioblastoma, carcinoma of the thyroid gland, bile duct, bone, gastrointestinal tract, brain/CNS, head and neck, liver, stomach, prostate, breast, kidney, testicular, ovarian, skin, cervical, lung, muscles, nerves, esophagus, bladder, lung, uterus, vulva, endometrium, kidney, colon, pancreatic, pleural/peritoneal membranes and salivary glands and squamous cell tumors.

The term “connection” means any small molecule compound, the molecular weight of which is less than about 2000 daltons.

The term “Ang-2" means a molecule of angiopoietin-2.

The term “neutralizing"related to the antibody indicates its ability to eliminate or significantly reduce the activity of a target antigen. Accordingly, a “neutralizing" anti-Ang-2 antibody has the ability to eliminate or significantly reduce the activity of Ang-2. Neutralizing anti-ADF-2 antibody may, for example, act by blocking the binding of Ang-2 with its receptor Tie-2. At block such swazilan what I Tie-2-mediated signal transmission substantially or completely eliminated. In the ideal case, a neutralizing antibody against Ang-2 inhibits angiogenesis.

Used herein, the term “isolated polynucleotide” means polynucleotide, which was isolated from its environment. Such polynucleotide may be of genomic, cDNA or synthetic. Selected polynucleotide, preferably not associated with all polynucleotide or part of polynucleotides with which they are associated in nature. Selected polynucleotide can be functionally linked to another polynucleotide with which they are associated in nature. In addition, the selected polynucleotide, preferably, does not occur in nature as part of a larger sequence.

Used herein, the term “isolated protein" when the temperature is a General term meaning a protein which has been isolated from its natural environment. Such proteins can be produced from genomic DNA, cDNA, recombinant DNA, recombinant RNA, or they can be synthesized or obtained by a combination of those and other ways in which the “isolated protein”, according to their origin or source of origin (1) is not associated with natural proteins, (2) is isolated from other proteins, derived from the same history is nick, for example, murine proteins, (3) is expressed by cells of different types or (4) does not occur in nature.

Used herein, the term "polypeptide" is a General term meaning the native protein, or fragments or analogs of the polypeptide sequence. Therefore, natural protein, and fragments and analogs are molecules such as polypeptides. Preferred polypeptides according to the invention include molecules of the heavy chain of human immunoglobulin and molecules Kappa light-chain of human immunoglobulin, as well as antibody molecules formed by combinations containing molecules of the heavy chain of an antibody and molecules of the light chain of the immunoglobulin, such as molecule light chain Kappa or lambda immunoglobulin, and Vice versa, as well as their fragments and analogues. Preferred polypeptides according to the invention can also include only the molecules of the heavy chain of human immunoglobulin or fragments thereof.

Used herein, the term “natural”that apply to a specific object means that the object can exist in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses)that can be considered natural if it is isolated from its natural source, and if it was specially modified by man in the laboratory or in any other conditions.

Used herein, the term “functionally linked” refers to the provisions described here, components that are attached to each other by way of providing them the “correct” functioning. For example, the regulatory sequence functionally linked” to the coding sequence, is associated with this sequence so that could be the expression of the coding sequence under conditions suitable for the operation of data regulatory sequences.

Used herein, the term “regulatory sequence" means a polynucleotide sequence that is required for the implementation of the expression and processing or for effects on the expression and processing of coding sequences to which they are linked. The nature of such regulatory sequences varies depending on the host body; in prokaryotes, such regulatory sequences generally include promoter, the binding site with the ribosome and the sequence termination of transcription; in eukaryotes, such regulatory sequences are usually promoters, enhancers, introns, sequences termination of transcription, sequences, polyadenylation signal and 5'- and 3'-noncoding region. The term “regulatory consequences of the successive" includes, as a minimum, all components whose presence is essential for expression and processing, and it may also include additional components whose presence is preferred, for example, leader sequences, and sequences that are partners for hybrid sequences.

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

Used herein, the term “oligonucleotide" means natural and modified nucleotides linked together natural and non-natural links. Oligonucleotides represent a polynucleotide subsequence that has a length of typically 200 nucleotides or less. Preferably, the oligonucleotides have a length of 10 to 60 nucleotides, and most preferably, the length in 12, 13, 14, 15, 16, 17, 18, 19 or 20-40 nucleotides. Typically, the oligonucleotides are single-stranded, for example, the oligonucleotides used as probes, although sometimes they can be double-stranded, for example, the oligonucleotide is, used to construct gene mutants. Oligonucleotides can be semantic or antimuslim.

Used herein, the term “natural nucleotide” means the deoxyribonucleotides and ribonucleotides. Used herein, the term “modified nucleotide” refers to the nucleotides with modified or substituted sugar groups, etc. Used herein, the term “oligonucleotide communication” includes oligonucleotide communication, as phosphorotioate, phosphorodithioate, phosphoresent, phosphorescent, phosphoramidothioate, phosphoramidate, phosphoramidate etc. See for example, LaPlanche et al.,Nucl. Acids. Res.14:9081 (1986); Stec et al.,J. Am. Chem. Soc.106:6077 (1984); Stein et al.,Nucl. Acids. Res.16:3209 (1988); Zon et al.,Anti-Cancer Drug Design6:539 (1991); Zon et al.,Oligonucleotides and Analogues: A Practical Approach, pp.87-108 (F.Eckstein Ed., Oxford University Press, Oxford England (1991)); Stec et al., U.S. patent No. 5151510; Uhlmann &PeymanChemical Reviews, 90:543 (1990). If necessary, the oligonucleotide can include a label for detection.

Used herein, the term “selective hybridization” refers to detectivemisa and specific binding. Polynucleotides, oligonucleotides and fragments thereof of the present invention selectively hybridize with nucleic acid chains in such conditions of hybridization and washing, which greatly minimizes the number of detected binding to nonspecific NUS is Inouye acids. To ensure selective hybridization, known in the art and discussed in this application can be used in conditions of high stringency. In General, the homology between the sequences polynucleotides, oligonucleotides or fragments according to the invention and the sequence of interest, the nucleic acid 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. So, for example, 85%homology sequences means that the alignment of these two sequences to map their maximum satisfaction, 85% of the amino acids are identical. Gaps (in either of the two compared sequences) to maximize compliance; however, the preferred length of the gap is 5 amino acids or less, and more preferably 2 amino acids or less. Alternative and preferably, two sequences of the protein (or polypeptide sequences derived from these sequences and having a length of at least 30 amino acids) are considered to be homologous in the conventional sense of the word, if they have a price alignment over h is m 5 (in standard deviation units) when mapping, carried out using the program ALIGN with the data matrix for the mutation and fine for the space of 6 or more. (See publication M.O. Dayhoff,Atlas of Protein Sequence and Structurepp. 101-110 (Volume 5, National Biomedical Research Foundation, 1972)) and Annex 2 to this volume (Supplement 2 to this volume, pp. 1-10). Two sequences or parts thereof are more preferred homologues, if their amino acids are identical at 50% or more at their optimal comparison by alignment using the ALIGN program. It should be noted that in two ontologica sequences, can be present in different areas of homology. For example, the functional sites of mouse and human orthologues may have a higher degree of homology than the non-functional area.

Used herein, the term “corresponds” means that a polynucleotide sequence is homologous (i.e. identical, but the evolutionary unrelated) all or a portion of a reference polynucleotide sequence, or this term means that the polypeptide sequence identical to a reference polypeptide sequence.

In contrast, as used herein, the term “complementary” means that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. So, for example, the R, nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC and complementary sequences “G”.

To describe the similarities between two or more polynucleotide or amino acid sequences, the following terms are used: “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”. The term “reference sequence” means a sequence that is used as the basis for comparison of sequences; a reference sequence may be a subsequence of a larger sequence, for example, the segment of full-size cDNA or gene sequences present in the list of sequences, or the sequence can contain a full-sized cDNA or gene sequence. Generally speaking, the reference sequence has a length of at least 18 nucleotides or 6 amino acids, usually at least 24 nucleotides, or 8 amino acids, and more usually at least 48 nucleotides or 16 amino acids. Because each of the two polynucleotide or amino acid sequence (1) may contain a sequence (i.e. part of the full size p is dinucleotides or amino acid sequence), which is similar in these two molecules, and (2) may, in addition, contain a sequence that differs from these two polynucleotide or amino acid sequences, the comparison of the sequences of the two (or more) molecules usually carried out by comparing the sequences of these two molecules on a “comparison window” to identify and compare local regions of homology sequences. Used herein, the term “comparison window” refers to a conceptual segment, consisting of at least 18 contiguous nucleotides or 6 amino acids, where the polynucleotide sequence or amino acid sequence can be compared with a reference sequence comprising at least 18 contiguous nucleotides or 6 amino acids, and where the portion of the polynucleotide sequence in the comparison window may include additions, deletions, substitutions and the like (i.e. gaps), which constitute 20 percent or less compared to the reference sequence (which does not include additions or deletions) and used for optimal alignment of these two sequences. Optimal alignment of sequences necessary for their mapping on the comparison window may be conducted using the algorithm level is th homology (Smith and Waterman Adv. Appl. Math.2:482 (1981)), the algorithm alignment regions of homology (Needleman and Wunsch,J. Mol. Biol.48:443 (1970))by finding similarities (Pearson and Lipman,Proc. Natl. Acad. Sci.(USA) 85:2444 (1988)), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), GENEWORKSTMor software packages MACVECTOR®) or by using the control program and build the best alignment (that is, receiving the highest percentage of homology over a “comparison window”)achieved by various methods.

The term “sequence identity” means that two polynucleotide or amino acid sequences are identical (i.e. nucleotide or amino acid level) in the comparison window. The term “percentage of sequence identity” means the percentage, which is calculated by comparing two optimally aligned sequences over the window of comparison; determine the number of provisions of the identical nucleic acid base (e.g., a, T, C, G, U, or I) or residues in both sequences with the receipt number of the relevant provisions; dividing this number of the relevant provisions on the total number of positions in the comparison window (i.e. window size), and multiplying the result by 100 to obtain the percentage identity of posledovatel the values. Used herein, the term “substantial identity” means a characteristic of a polynucleotide or amino acid sequence, where the specified polynucleotide or the specified amino acids comprise a sequence that is at least 85%, preferably at least 90-95%, and more preferably at least 99% identical to a reference sequence in the comparison window, comprising at least 18 nucleotides (6 amino acids), and often in the comparison window, comprising at least 24-48 nucleotides (8-16 amino acids), where the percentage of sequence identity is calculated by comparing the reference sequence with the sequence which may include deletions or additions which are generally constitute 20 percent or less compared to the reference sequence in the comparison window. The reference sequence may be a subsequence of a larger sequence.

Used here twenty major amino acids are generally accepted abbreviations. Cm. the publication ofImmunology - A Synthesis(2nd Edition, E.S. Golub and D.R Gren. Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is introduced in the present description by reference. Stereoisomers (e.g., D-amino acids) of the twenty major amino acids, unnatural amino acids such as α,α-diseasemanagement; N-alkyl-substituted amino acids, lactic acid and other rare amino acids may also be suitable components for polypeptides according to the invention. Examples of rare amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetylized, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and other similar amino acids and aminokisloty (for example, 4-hydroxyproline). In the system used here refer to polypeptides, in accordance with standard practice and with the adopted agreement on terminology, the left end of the amino acids is called the amino end and the right end of the amino acids is called the carboxy end.

Similarly, if it is not specifically mentioned, the left end of the single-stranded polynucleotide sequence is 5'-end, and the direction from left to right for a double-stranded polynucleotide sequence is 5'-direction (5' →). The direction of joining the growing RNA transcript 5' → 3' direction is called transcription in this region sequences on a DNA chain having the same sequence as the RNA chain, which are from 5'-end towards the 5'-end of the RNA transcript are referred to as “upstream sequences (i.e. above what about during transcription)”, and region sequences on a DNA chain having the same sequence as the RNA chain, which are from the 3'end toward the 3'-end of the RNA transcript are referred to as “the subsequent sequence that is located below during transcription)”.

The term “identical”as used with respect to a polypeptide, means that two peptide sequences, when optimally aligned, for example, using the programs GAP or BESTFIT using “weights” whitespace by default, have 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. In this case, it is preferable that the position of the residues that are not identical, differed in the result of conservative amino acid substitutions. The term “conservative amino acid substitution” means interchangeable residues comprising similar side chains. So, for example, a group of amino acids having aliphatic side chains, are glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic hydroxyl side chains, are the Erin and threonine; a group of amino acids having amide-containing side chains, are asparagine and glutamine; a group of amino acids having aromatic side chains, are phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains, are lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chains, are 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 in this application, small changes in the amino acid sequences of the molecules are antibodies or immunoglobulins are treated as changes included in the scope of the present invention, provided that such changes in the amino acid sequence described herein molecules, antibodies or immunoglobulins will be at least 75%, more preferably at least, 80%, 90%, 95%, and most preferably 99%. In particular, are also considered conservative amino acid substitutions. Conservative substitutions are the replacements, which are part of a family of amino acids that are related in their side chains. Genetically encoded amanakis what the notes are usually divided into the following families: (1) acidic amino acids = aspartate, glutamate; (2) basic amino acids = lysine, arginine, histidine; (3) non-polar amino acids = alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The preferred collections are: serine and threonine, belonging to the family of aliphatic hydroxy acid; asparagine and glutamine, belonging to the family of amide-containing acids; alanine, valine, leucine and isoleucine, belonging to the family of aliphatic amino acids, phenylalanine, tryptophan and tyrosine, belonging to the family of aromatic amino acids. For example, it should be expected that a single replacement of leucine with isoleucine or valine, replacement of aspartate glutamate, replacement of threonine with serine, or a similar replacement of any amino acids structurally related amino acid will not have a significant impact on the function of binding or properties of these molecules, especially if this replacement is not an amino acid substitution in the frame area. Producing a functional peptide as a result of such amino acid substitutions may readily be ascertained by analysis of the specific activity of the polypeptide is derived. Such analyses are described in detail in this application. Fragments or analogs of molecules of antibody is whether immunoglobulins can be easily obtained by the average specialist. Preferably, the amino - and carboxy ends of these fragments or analogues was near boundaries of functional domains. Structural and functional domains can be identified by comparing data on nucleotide and/or amino acid sequences with the data on the sequences available in the well-known public databases or databases that are privately owned. Preferably, to identify sequence motifs or conformational domains predicted proteins, which are found in other proteins of known structure and/or function, using methods of computer comparison. Methods identification of protein sequences, which form a known three-dimensional structure, known in the art and described in Bowie et al.,Science253:164 (1991). For example, the previously described examples demonstrated that the person skilled in the art can easily identify sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the above description of antibodies.

Preferred amino acid substitutions are substitutions that result in: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) to change the of the affinity of binding for the formation of protein complexes, (4) to change the affinity of binding and (5) to the message or modify other physicochemical or functional properties of such analogs. These analogues may be different mutiny sequence, different from the natural peptide sequence. For example, in the natural sequence (preferably in the portion of the polypeptide, which is located outside the domain-forming intermolecular contacts) can be made of one or multiple amino acid substitutions (preferably conservative amino acid substitutions). Conservative amino acid substitution should not significantly affect the structural properties of the parent sequence (e.g., amino acid substitution should not lead to the destruction of a helix that occurs in the parent sequence, or to a breach of secondary structures of other types, which characterizes the parent sequence). Examples of known secondary and tertiary structures of the polypeptide described inProtein, Structures and Molecular Principles(Creighton, ed., W.H. Freeman and Company, New York 1984); Introduction to Protein Structure (C. Branden &J. Tooze, eds., Garland Publishing, New York, N,Y. (1991)) and in the work of Thornton et al., Nature 354:105 (1991), each of which is introduced into the present description by reference.

Used herein, the term “polypeptide fragment” means a polypeptide which kelamin-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence in the relevant provisions of amino acids identical to the natural amino acid sequence, occurring, for example, from full-size cDNA sequences. Fragments typically have a length of at least 5, 6, 8 or 10 amino acids, and 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. Used herein, the term “analog” means polypeptides that consist of a segment having at least 25 amino acids, basically, identical parts of the derived amino acid sequence, and which have at least one of the following properties, namely, they are (1) specifically associated with Ang-2 in suitable binding conditions, (2) is able to block the corresponding binding Ang-2, or (3) is able to inhibit the activity of Ang-2. Typically, the polypeptide analogues, compared to the natural sequence, contain conservative amino acid substitution (addition or deletion). Counterparts usually have a length of at least 20 amino acids, preferably at least 50 amino acids or more, and more often than not, they have the same length as a full-sized natural polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry as ones drugs with properties analogous to properties of the matrix peptide. Ones connection of this type are termed “peptide mimetics” or “peptidomimetics”. Cm. publication Fauchere,J. Adv. Drug. Res.15:29 (1986); Veber & Freidinger,TINSp.392 (1985) and Evans et al.,J. Med. Chem.30:1229 (1987), which are introduced in the present description by reference. Such compounds are often developed using computer programs for molecular modeling. Peptide mimetics that are structurally similar to therapeutically valuable peptides, can be used to achieve equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to the representative polypeptide (a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, and they usually have one or more peptide bonds, optionally replaced by a linkage selected from the group consisting of-CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (CIS and TRANS), -the PINES2-, -CH(OH)CH2- and-CH2SO, in accordance with well known methods. System replacement of one or several amino acids of the consensus pic is egovernance D-amino acid of the same type (for example, replacement of L-lysine D-lysine) can be used to generate more stable peptides. In addition, peptides with conformational constraints containing the consensus sequence or the variant sequence, basically, identical to the consensus sequences, can be generated by known methods (publication Rizo & Gierash,Ann.Rev. Biochem.61:387 (1992), which is introduced in the present description by reference), for example, by attaching an internal cysteine residues capable of forming intramolecular disulfide bridges which cyclist this peptide.

Used herein, the term “antibody” means a polypeptide or group of polypeptides consisting of at least one binding domain that is formed by the stacking of polypeptide chains having a three-dimensional connecting areas with the internal surface structure and charge distribution complementary structural features of the antigenic determinants of the antigen. Typically, the antibody tetramer has a form that contains two identical pairs of polypeptide chains, where each pair has one light and one heavy chain. Variable regions of each pair of light and heavy chains form the binding site from an antibody.

Used herein, the term “targeted binding agent is the mean antibody or its binding fragment, mainly associated with site-targeted. In one embodiment of the invention, the specified targeted binding agent is specific to only one target site. In other embodiments of the invention, the specified targeted binding agent is specific to more than one target site. In one embodiment of the invention, the specified targeted binding agent may be a monoclonal antibody, as specified site-the target may be an epitope.

“Binding fragments of the antibodies to be made by methods of recombinant DNA, 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 “bespecifically” or “bifunctional”, are considered as antibodies, each of which has an identical binding sites. Antibody mainly inhibits the adhesion receptor contraceptor, if an excess of antibody reduces the quantity of receptor associated with contraception, at least about 20%, 40%, 60% or 80%, and often about more than 85% (as measured in the analysis of competitive bindingin vitro).

The antibody can be an oligoclonal, a polyclonal or monoclonal antibody, a chimeric antibody, a CDR-hybrid antibody, mult the specific antibody, bespecifically antibody, a catalytic antibody, chimeric antibody, gumanitarnoe antibody, a fully human antibody, antiidiotypic antibody and antibodies that can be labeled in soluble or bound form, as well as fragments, variants or derivatives, alone or in combination with other amino acid sequences obtained by the known methods. The antibody may occur from the body of any kind. The term “antibody” also includes binding fragments of the antibodies according to the invention, and such representative fragments are Fv, Fab, Fab', single-chain antibody (svFC), dimeric variable region (dianthicola) and stabilized by disulfide variable region (dsFv).

The term “epitope” means any of the determinants of protein that can specifically bind to an immunoglobulin or T-cell receptor. Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and can, but not always, have a specific three-dimensional structure, as well as specific charge characteristics. It is believed that the antibody specifically binds to the antigen when the dissociation constant is ≤1 μm, preferably ≤100 nm, and most preferably ≤10 nm.

Use what has been created here, the term “agent” means a chemical compound, the mixture of chemical compounds, a biological macromolecule, or an extract derived from biological materials.

Used herein, the term “active” or “activity”if it refers to the polypeptide Ang-2, means a portion of the polypeptide Ang-2, which has the biological or immunological activity of a native polypeptide Ang-2. Used herein, the term “biological” refers to a biological function caused by the activity of the native polypeptide Ang-2. Preferred biological activity of Ang-2 includes, for example, Ang-2-induced angiogenesis.

Used herein, the term “mammal” means any animal belonging to the class of mammals. The preferred mammal is a human.

Hydrolysis of the antibody with the enzyme papain leads to the production of two identical antigen-binding fragments, also known as “Fab”fragments, “Fc”fragment, not having antigennegative activity, but with the ability to crystallization. Hydrolysis of the antibody with the enzyme pepsin leads to the formation of F(ab')2fragment in which the two domains of the molecule antibodies are related and contain two antigenspecific site. This F(ab')2the fragment has the ability to cross-contact with the antigen.

Used herein, the term “Fv” OSN which denotes the minimum antibody fragment, containing the recognition sites of antigen and antigennegative sites.

Used herein, the term “Fab” refers to the fragment of the antibody containing the constant domain of the light chain domain and SN heavy chain.

The term “mAb” means a monoclonal antibody.

Used herein, the term “liposome” means a small vesicles, which can be used for delivery to the mammal medicines, which may include the polypeptide of Ang-2 according to the invention or antibodies against the indicated polypeptide Ang-2.

Used herein, the terms “label” or “labeled” refers to incorporation into the polypeptide detectable group, for example, a radioactive label, fluorescent label, enzymatic label, a chemiluminescent label, or biotinylate group. Radioisotopes or radionuclides can be3H,14C,15N35S90Y94Tc111In125I131I, fluorescent labels may be rhodamine, complex lanthanide-phosphorus or FITZ, and enzymatic labels may include horseradish peroxidase, β-galactosidase, luciferase, and alkaline phosphatase.

Used herein, the term “pharmaceutical agent or drug” means a chemical compound or composition capable of inducing a desired therapeutic effect when their corresponding introduction is the patient. Other common chemical terms used in the present application is described in theThe McGraw-Hill Dictionary of Chemical Terms(S. Parker Ed, McGraw-Hill, San Francisco (1985), which is incorporated into the present description by reference).

Used herein, the term “net” refers to the molecule, which is the predominant molecule (i.e. is present at a higher molar concentration than any other individual molecules present in the composition), and preferably, the term means “basically, the purified fraction”, i.e. a composition in which the considered molecule is at least about 50 percent (on a molar level) from all present macromolecules of this type. Usually, mostly pure composition is approximately more than 80 percent of all macromolecules present in the composition, and more preferably more than about 85%, 90%, 95% and 99%. Most preferably, this molecule was purified almost to full homogeneity (where impurities which are present in this composition cannot be detected by standard methods of detection), so that this composition consisted mainly of macromolecules of the same species.

The term “patient” includes humans and animals.

The human antibody is a “humanized” antibodies

The use of human antibodies avoids some of the problems arising from the use of antibodies having murine or rat variable and/or constant region. The presence of mouse or rat proteins can lead to rapid removal of antibodies, or it can lead to the production of the patient's immune response against the antibody. In order to avoid having to use a mouse or rat antibodies, can be generated fully human antibodies by introducing functional areas of human antibodies to rodent, other mammal or animal so that they develop fully human antibodies.

One method for generating fully human antibodies is to use mice strains XenoMouse®, have been bred so that they contain correspondingly configured fragments locus, the human heavy chain and light chain locus Kappa germline size to less than 1000 TPN Cm. Mendez et al.Nature Genetics15:146-156 (1997) and Green and JakobovitsJ. Exp. Med.188:483-495 (1998). The XenoMouse strains®are delivered by the company Abgenix, Inc. (Fremont, CA).

Getting XenoMouse strains of mice®also discussed and described in the following patents, U.S.: in the application reg. # 07/466008, filed January 12, 1990, 07/610515, poda is Noah 8 November 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 June 5, 1995, 08/464582 filed June 5, 1995, 08/463191 filed June 5, 1995, 08/462837 filed June 5, 1995, 08/486853 filed June 5, 1995, 08/486857 filed June 5, 1995, 08/486859 filed June 5, 1995, 08/462513, filed June 5, 1995, 08/724752, filed October 2, 1996 and 08/759620, filed December 3, 1996; publication of patent application U.S. 2003/0093820, filed November 30, 2001; U.S. patent No. 6162963, 6150584, 6114598, 6075181 and 5939598, and in Japanese patent applications No. 3068180 B2, 3068506 B2 and 3068507 B2. Cm. also European patent number EP 0463151 Bl, issued and published June 12, 1996, International patent application no WO 94/02602, published February 3, 1994, International patent application no WO 96/34096, published October 31, 1996, WO 98/24893, published June 11, 1998 and WO 00/76310, published on 21 December 2000. Description all of the above patents, applications and publications in its entirety is introduced into the present description by reference.

In an alternative method, other researchers, including researchers GenPharm International, Inc. used “minilogue”. This approach minitokyo is that was simulated locus exogenous Ig by incorporating fragments (individual genes) of the Ig locus. So about what atom, of one or more genes VNone or more genes DNone or more genes JN, constant mu-region and a second constant region (preferably, a constant gamma region) was created the design for the introduction of the animal. This method is described in U.S. patent No. 5545807, Surani et al., and in the U.S. patents№№ 5545806, 5625825, 5625126, 5633425, 5661016, 5770429, 5789650, 5814318, 5877397, 5874299 and 6255458, Lonberg and Kay, in U.S. patent No. 5591669 and 6023010, Krimpenfort & Berns, U.S. patent No. 5612205, 5721367 and 5789215, Berns et al., and in U.S. patent No. 5643763, Choi & Dunn, and in International patent applications U.S., GenPharm; in the application reg. No. 07/574748, filed August 29, 1990, the application 07/575962, filed August 31, 1990, the application 07/810279, filed December 17, 1991, the application 07/853408 filed March 18, 1992, the application 07/904068, filed June 23, 1992, the application 07/990860, filed December 16, 1992, the application 08/053131 filed April 26, 1993, the application 08/096762 filed July 22, 1993, the application 08/155301, filed November 18, 1993, the application 08/161739, filed December 3, 1993, the application 08/165699, filed December 10, 1993, and the application 08/209741 filed March 9, 1994, the description of which is introduced in the present application by reference. Cm. also Europatent No. 0546073 B1, International patent application no 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 No. 5981175, the description of which in its entirety is introduced into the present application by reference. Cm. also the ublications 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, the description of which in its entirety is introduced into the present application by reference.

Kirin also demonstrated the generation of human antibodies in mice, which by the merger of microclear, introduced a large fragments of chromosomes or complete chromosomes. Cm. European patent application No. 773288 and 843961, the description of which in its entirety is introduced into the present application by reference. In addition, as a result of cross-breeding mice Kirin TC and Medarex mice with minitokyo (Humab) were obtained mouse KMTM. These mice had human IgH-transfromation Kirin mice and transgene Kappa-chain Genpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).

Human antibodies can also be obtained byin vitro. Suitable methods include, but are not limited to, phage representation (CAT, Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosomal representation (CAT), yeast representation, etc.

Antibodies

These antibodies were obtained by applying the following techniques breeding mice XenoMouse®. Such mice are able to produce molecules of human immunoglobulin and antibodies and are deficient in the production of molecules of mouse immunoglobulin and antibodies. The technology used for these purposes, described in the patents, patent applications and in the works cited in the section “Prior art” of this application. However, in particular, the preferred option for obtaining transgenic mice and antibodies described in application for U.S. patent, reg.№ 08/759620, filed December 3, 1996, and in International patent applications: application number WO 98/24893, published June 11, 1998, and in the application WO 00/76310, published December 21, 2000, the description of which is introduced into the present application by reference. Cm. publication Mendez et al.,Nature Genetics15:146-156 (1997), the description of which is introduced into the present application by reference.

Thanks to the use of such technology were produced fully human monoclonal antibodies against different antigens. Essentially, XenoMouse mice® were immunized interest antigen (e.g., Ang-2), and then the lymphatic cells (such as b-cells) were isolated from hyperimmunized mice, and the resulting cells were subjected to fusion with the cell line myeloid type, the result has been immortalized hybridoma cell lines. Such hybridoma cell lines were skanirovaniya and selected to identify hybridoma cell lines that produce antibodies specific to the desired antigen. In the present application describes methods for getting multiple hybridoma glue the face-to-face lines, producing antibodies that are specific for Ang-1 and Ang-2. In addition, the present application provides a characterization of the antibodies produced by such cell lines, including analyses on nucleotide and amino acid sequences of the heavy and light chains of these antibodies.

Alternatively, instead of fusion with myeloma cells to obtain hybridomas can be carried out by direct analysis of b-cells. So, for example, CD19+-B cells can be isolated from hyperimmunizing mice of the XenoMouse® and undergo proliferation and differentiation to obtain antibody-secreting plasma cells. Then antibodies isolated from cell supernatants, sceneroot using ELISA for reactivity with the immunogen Ang-2. These supernatant can also be skanirovaniya on immunoreactivity with fragments of Ang-2 for additional mapping of different antibodies for analysis of binding interest functional domains on Ang-2. Such antibodies can also be skanirovaniya on the binding of Ang-1, Ang-3 or Ang-4 and other related human chemokines, as well as with rat and mouse Ang-2, Ang-2 primates, non-human, such as abacadabra monkeys, and orthologues of Ang-2, where these orthologues are used to determine species cross-reactivity. B-cells in the wells containing the x of interest antibodies can be immortality various methods, including cell fusion with getting a hybrid or in a separate hole or in the hole with the pool of cells, or by virus infection EBV or transfection are known immortalizing genes, with subsequent seeding of the cells in a suitable environment. Alternatively, single plasma cells secreting antibodies with the desired specificity, then allocate using hemolytic assays, plaque Ang-1 or Ang-2-specific antibodies (see, for example, Babcook et al.,Proc. Natl. Acad. Sci.USA, 93:7843-7848 (1996)). Cells subjected to lysis, preferably, are sheep erythrocytes (SRBC)antigen coated Ang-2. For the screening of antibodies capable of inhibiting angiopoietin-1, with the same success can be applied to the above methods, where instead of angiopoietin-2 is used angiopoietin-1.

In the presence of b-cell culture containing plasma cells secreting the desired immunoglobulin and complement, the formation of a plaque indicates specific Ang-1/Ang-2-mediated lysis of sheep erythrocytes surrounding of interest plasma cells. Can be allocated to a single antigen specific plasma cell is present in the center of the plaque, and then from this single plasma cells can be obtained genetic is the information on the specificity of this antibody. By carrying out PCR with reverse transcriptase (RT-PCR) can be cloned DNA encoding variable regions of the heavy and light chains of this antibody. Such cloned DNA can then be incorporated into a suitable expression vector, preferably a vector cluster, such as DNA, and more preferably DNA containing the constant domains of the heavy and light chains of immunoglobulin. The generated vector can then be transfected into cells-owners, for example, in cells NECK or in cells SNO, and cultivated in a suitable nutrient medium, modified, if necessary, to induce transcription, selection of transformants, or amplifying the genes encoding the desired sequences.

In General, antibodies produced by hybridomas, are the heavy chain of human IgG2 with a full-sized light chain Kappa or lambda human antibodies. These antibodies have a heavy chain of human IgG4, as well as the heavy chain of human IgG2. Such antibodies may be human antibodies of other isotypes, including IgG1. Antibodies with high affinity, usually have ToDabout 10-6up to 10-12M or less, as shown by the measurements in the solid phase and in solution. Antibodies having ToDat least 10-11M, are preferred for inhibiting the activity of Ang-1 and/or Ang-2.

It should be noted that antibodies can be expressed in cell lines that are not hybridoma cell lines. Sequence encoding a specific antibody, can be used to transform a suitable host cell of the mammal. The transformation can be carried out by any known method of introducing polynucleotides cells of the host, including, for example, packaging polynucleotide in a virus (or into a viral vector) and the transfection of the host cell with the virus (or vector), or it can be carried out in accordance with the procedures transfection, known in the art and are described in U.S. patent No. 4399216, 4912040, 4740461 and 4959455 (which are introduced in the present description by reference). This procedure transformation depends on convertible owner. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran mediated by transfection; the precipitation of calcium phosphate; mediated polybrene transfection; the fusion of protoplasts, electroporation; encapsulating polynucleotide(s) in liposomes, and direct microinjection DNA in the nucleus.

Mammalian cell lines that are suitable as hosts for expression are well known JV is the experts, such cell lines include many immortalized cell lines, which can be obtained from the American type culture collection (ATSC), including, but not limited to, cells of the Chinese hamster ovary (Cho), HeLa cells, kidney cells baby hamster (KSS)cells, monkey kidney (COS)cells, human hepatocellular carcinoma (e.g., Hep G2), epithelial cells, 293 human kidney and various other cell lines. Particularly preferred cell line can be selected by identifying cell lines, which Express and secrete high levels of antibodies capable of constitutive to connect with Ang-2.

Based on the ability of mAb significantly neutralize the activity of angiopoietin-1 and angiopoietin-2 (as shown in the following examples), we can conclude that these antibodies will have a therapeutic effect, aimed at eliminating the symptoms and treatment of conditions associated with expression of angiopoietin-1 and/or angiopoietin-2. In specific embodiments of the invention described herein, the antibodies and methods can be applied for the treatment of symptoms associated with angiogenesis, induced by angiopoietin-1 and/or angiopoietin-2.

In accordance with another of its aspects, the present invention relates to pharmaceutical the composition, containing antagonist of the biological activity of angiopoietin-1 and angiopoietin-2 and a pharmaceutically acceptable carrier. In one embodiment of the invention, the antagonist contains the antibody. In accordance with another of its aspects, the present invention relates to a pharmaceutical composition comprising the antagonist of the biological activity of angiopoietin-2 and a pharmaceutically acceptable carrier. In one embodiment of the invention, the antagonist contains the antibody.

Anti-Ang-2 antibodies can be used for detection of Ang-2 in samples taken from the patient, and, accordingly, they can be used as diagnostic tools for the treatment described herein pathological conditions. In addition, based on the ability of these antibodies to substantially neutralize the activity of Ang-2 (as shown in the following examples), we can conclude that these anti-Ang-2 antibodies will have a therapeutic effect, aimed at eliminating the symptoms and treatment of conditions associated with expression of Ang-2. In specific embodiments of the invention described herein, the antibodies and methods can be applied for the relief of symptoms associated with Ang-2-induced angiogenesis. Other variants of the invention include the application described here, the antibodies and methods for treatment of oregamax angiogenesis diseases, including neoplastic diseases, such as melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver)tumor of the thyroid gland, cancer of the gastrointestinal tract (stomach), prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer and pancreatic cancer.

Therapeutic introduction and composition

Variants of the present invention include sterile pharmaceutical compositions, which contain anti-Ang-2 antibodies or antibodies that bind with both Ang-1 and Ang-2, and which can be used for the treatment of diseases. Such compositions should inhibit the binding of Ang-2 or Ang-1 and Ang-2 and their receptor Tie-2, and therefore, they should be effective for treating pathological conditions where, for example, there is an abnormal 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 that allows you to enter these antibodies person is much less likely. Anti-Ang-1/Ang-2 antibodies preferably possess adequate affinity to neutralize Ang-1 and Ang-2, and preferably, the region is up adequate duration of action, that allows you to enter these antibodies person is much less likely. Prolonged action of these antibodies allows the use of regimens with less frequent or more convenient dose method, alternative parenteral introduction, such as subcutaneous or intramuscular injection.

Sterile compositions can be obtained, for example, by filtration through sterile filtration membranes, prior to or after lyophilization and subsequent dilution of antibodies. The antibody is usually stored in liofilizovannyh form or in solution. Therapeutic compositions of the antibodies is usually placed in a container having a sterile inlet, for example, a package or container for intravenous administration, with the device for feeding the composition, such as cork, protykaya needle for subcutaneous injection.

Introduction antibodies can be carried out with known methods, for example, by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarticular, intrathecal injection or infusion; by inhalation, or by injection into the affected area, either through the use of systems, sustained release, described below. The antibody is preferably administered continuously by infusion or injection loading dose.

The effective amount used terapevticheskoj the antibodies depends for example, goals of therapy, route of administration and the condition of the patient. In a preferred embodiment, the medical doctor can adjust the dose by titration and modify the route of administration, if it is necessary to obtain the optimal therapeutic effect. Typically, the Clinician assigns a dose of antibodies suitable for achieving the desired effect. Monitoring the effectiveness of this therapy can be easily implemented using standard tests or analyses described in this application.

These antibodies can be prepared in a mixture with a pharmaceutically acceptable carrier. Such a therapeutic composition can be administered intravenously, intranasally or intrapulmonary, and preferably in the form of liquid or powder aerosol (in liofilizovannyh form). This composition, if necessary, can also be introduced parenterally or subcutaneously. Upon systemic administration, such therapeutic composition must be sterile and pyrogen-free, and it can be put in the form of a parenterally acceptable solution having the desired pH, the desired isotonicity and stability. These conditions are well known in the art. Briefly, described here in embodiments of the invention, pharmaceutical compositions of the compounds for the storage or introduction, the floor is given by mixing compounds having the desired degree of purity with physiologically acceptable carriers, excipients or stabilizers. These substances are non-toxic to recipients at the used doses and concentrations, and they can be buffers, such as Tris-HCl, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight peptides (approximately less than ten residues), such as polyalanine; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; 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; hepatoblastoma agents such as EDTA; the spirits of a number of sugars, such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as tween, pluronic or polyethylene glycol.

Sterile compositions for injection can be prepared in accordance with standard pharmaceutical practice as described in the manualRemington: The Science and Practice of Pharmacy(20thed., Lippincott Williams &Wilkens Publishers (2003)). For example, it may be desirable dissolution or suspension of the active connection is placed in the carrier, such as water or natural vegetable oil such as sesame oil, peanut oil or oil from the seeds of cotton or synthetic fatty media, such as etiloleat or the like, Buffers, preservatives, antioxidants, etc. can be entered in accordance with generally accepted pharmaceutical practice.

Suitable examples of sustained release formulations release are semi-permeable matrices of solid hydrophobic polymers containing the polypeptide, where the above matrix receive in the form of molded articles, films, or microcapsules. Examples of matrices for sustained release include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate), described by Langer et al.,J. Biomed. Mater. Res., 15:167-277 (1981) and Langer,Chem. Tech., (1982) 12:98-105 or polyvinyl alcohol), polylactide (U.S. patent No. 3773919, EP 58481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers, 22:547-556 (1983)), the non-biodegradable ethylene vinyl acetate (Langer et al., supra), degradable copolymers of lactic and glycolic acid, such as LUPRON DepotTM(microspheres for injection, consisting of a copolymer of lactic and glycolic acid and acetate leuprolide), and poly-D-(-)-3-hydroxipropionic acid (EP 133988).

Such polymers as vinyl acetate and lactic acid-glycolic acid, provide for the release of molecules within a time period of over 100 days, some hydrogels provide release proteins for shorter time periods. When encapsulated proteins are present in the body for a long period of time, when 37º they can denaturirate or aggregated under the influence of moisture, which leads to loss of biological activity and to a reasonably possible change in immunogenicity. For stabilization of the protein can be a rational strategy depending on the mechanism used. For example, if the aggregation mechanism is the formation of intermolecular bonds S-S through disulfide interchange, stabilization of the protein can be achieved by modifying sulfhydryl residues, lyophilization from acid solutions, regulating the moisture content, using appropriate additives, and developing specific composition of the polymer matrix.

Compositions prolonged release also include preparations crystals antibodies suspended in suitable compositions capable of supporting the crystals in suspension. These drugs, when they are subcutaneous or intraperitoneal injections, can produce the effect of a prolonged release. Other compositions also include captured liposomes antibodies. Liposomes containing such antibodies, to be made by methods knownper seand description of nami in U.S. patent No. DE 3218121; in the work of Epstein et al.,Proc. Natl. Acad. Sci., USA (1985) 82:3688-3692; Hwang et al.,Proc. Natl. Acad. Sci., USA (1980) 77:4030-4034; EP 52322; EP 36676; EP 88046, in EP 143949 and 142641; in Japanese patent application 83-118008; U.S. patent No. 4485045 and 4544545, and in EP 102324.

The dosage of the antibodies for a given patient must be determined by the attending physician considering various factors which are known to affect the action of drugs, for example, such factors, the severity and type of disease, body weight, sex, diet of the patient, time and route of administration of drugs, other medical conditions, and other relevant clinical factors. A therapeutically effective dose can be determined by methodsin vitroorin vivo.

An effective amount of an antibody according to the invention, used in therapy depends on the goals of therapy, route of administration and the health of the patient. Accordingly, in a preferred embodiment, the practitioner can find the right dosage and modify the route of administration, if necessary to achieve optimal therapeutic effect. A typical daily dose may vary from about 0.001 mg/kg to 100 mg/kg or more, depending on the above factors. Typically, the Clinician assigns introduction therapeutic antibodies to tejpar, until you achieve the desired effect. Monitoring the effectiveness of this therapy can be easily implemented using standard tests or analyses described in this application.

It should be noted that a therapeutic agent used in the compositions described herein and the methods that can be put together with suitable carriers, fillers and other means, included in these drugs to improve transfer and delivery, and increase tolerance and other Such preparations are, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (ketiganya or biogenie) (such as lipofectinTM), DNA conjugates, anhydrous absorbent pastes, emulsions of the type oil-in-water” and “water in oil”emulsion carbowax (glycols with different molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. For treatment and therapy according to the invention can be used with any of the above mixture, provided that the active ingredient in this composition will not inaktivirovanie, and the song is physiologically compatible and will be well tolerated with this method of introduction. Cm. publications Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance",Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid prtein pharmaceuticals", Int. J. Pharm.203(1-2): 1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts",J. Pharm. Sci.89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDAJ. Pharm. Sci. Technol.52:238-311 (1998) and found them additional information relating to drugs, the fillers and carriers, well known to chemists pharmacists.

Combination

This treatment is aimed at preventing angiogenesis, can be used as monotherapy, or it may, in addition to the compounds according to the invention, to include carrying out standard surgery, radiation therapy or chemotherapy. Such chemotherapy may include one or more anticancer agents the following categories:

(i) cytotoxic tools such as antiestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and idoxifene), inhibitors of estrogen receptors (for example fulvestrant), antiandrogens (for example, bikalutamid, flutamide, nilutamide and acetate ciproteron), LHRH antagonists or LHRH agonists (for example goserelin, leiprorelina, buserelin), progesterones (for example, acetate megestrol), aromatase inhibitors (e.g. anastrozole, letrozole, varsol and exemestane) and inhibitors of 5*-reductase such as finasteride;

(ii) means of inhibiting the invasion of cancer cells (for example, ingebi the Directors metalloproteinases, such as marimastat, and inhibitors function urokinase receptor plasminogen activator);

(iii) inhibitors of the function of growth factors, for example, inhibitors that are antibodies against growth factors, antibodies against receptors of growth factors (for example the anti-erbb2 antibody trastuzumab [HerceptinTM] and anti-erbblantibody cetuximab [C225]), inhibitors farnesyltransferase, tyrosine kinase inhibitors and inhibitors of serine/trionychinae, for example, inhibitors of the family of epidermal growth factors (e.g., inhibitors of protein tyrosine kinase EGFR, such as N-(3-chloro-4-forfinal)-7-methoxy-6-(3-morpholinopropan)hinzelin-4-amine (gefitinib, AZDl839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)hinzelin-4-amine (erlotinib, OSI-774) and 6-acrylamide-N-(3-chloro-4-forfinal)-7-(3-morpholinopropan)hinzelin-4-amine (CI 1033)), for example, inhibitors of the family of growth factors, platelets, and, for example, inhibitors of the family of growth factors hepatocyte;

(iv) antiangiogenic tools such as inhibiting the action of vascular endothelial growth factor (for example, antibodies against growth factor vascular endothelial cells, bevacizumab [AvastinTM], antibodies against the receptor for vascular endothelial growth factor, such as anti-KDR antibodies and anti-flt1 antibodies, compounds described in ' Between the national patent applications WO 97/22596, WO 97/30035, WO 97/3285, WO 98/13354 and WO 00/47212 and WO 01/32651), and compounds that act by a different mechanism (e.g., linomide, inhibitors of the function of integrin avb3 and angiostatin);

(v) means destroying vessels, such as combretastatin A4 and compounds described 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 products containing the antisense sequence, for example, therapeutic agents aimed at the targets listed above, such as ISIS 2503, and therapeutic anti-rasproducts containing the antisense sequence;

(vii) means used in methods of gene therapy, including, for example, methods replace aberrant genes such as Abernathy p53 or aberrant BRCAl or BRCA2, GDEPT (gene therapy using enzyme prodrug), for example, methods using citizendiumat, timedancing or bacterial enzyme nitroreductase, and methods used to increase tolerance to chemotherapy or radiation therapy, such as therapy with the use of resistance genes to many medicines; and

(viii) the funds used in the methods of immunotherapy, including, for example,ex vivoandin vivomethods for enhancing the immunogenicity of tumor cells, such as transfection with cytokines such as interleukin-, interleukin 4 or granulocyte-macrophage colony-stimulating factor, methods of reducing energy T cells, methods of using transfected immune cells such as transfected cytokines dendritic cells, methods of using transfected cytokines tumor cell lines and methods of using antiidiotypic antibodies.

In one embodiment of the invention, the antiangiogenic treatment means according to the invention is carried out in combination with other agents that inhibit the action of vascular endothelial growth factor (VEGF) (e.g., antibodies against vascular endothelial growth factor cells, bevacizumab [AvastinTM], antibodies against the receptor for vascular endothelial growth factor, such as an anti-KDR antibody and anti-flt1 antibody; compounds described in International patent applications WO 97/22596, WO 97/30035, WO 97/3285, WO 98/13354 and WO 00/47212 and WO 01/32651), and compounds that act by a different mechanism (e.g., linomide, inhibitors of the function of integrin avb3 and angiostatin). In other embodiments of the invention, the antiangiogenic treatment means according to the invention is carried out in combination with other agents that inhibit tyrosinekinase activity of the receptor vascular endothelial growth factor, KDR (for example, AZD2171 or AD6474). A detailed description of AZD2171 can be found in publications Wedge et al. (2005) Cancer Research. 65(10):4389-400. Detailed description AZD6474 can be found in publications Ryan & Wedge (2005) British Journal of Cancer. 92 Suppl 1:S6-13. Both of these publications in their entirety are introduced in the present description by reference. In another embodiment of the invention, a fully human antibody 3.19.3, 3.3.2 or 5.88.3 administered alone or in combination with AvastinTM, AZD2171 or AZD6474.

This combined treatment may be carried out by simultaneous, sequential or separate doses of the individual components. These combined products include compounds according to the invention or pharmaceutically acceptable salts of the above intervals of doses and the other pharmaceutically active agent within the acceptable range of doses.

EXAMPLES

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

EXAMPLE 1

IMMUNIZATION AND TITRATION

Immunization

Recombinant human Ang-2, obtained from firm R&D Systems, Inc. (Minneapolis, MN cat. No. 623-AM/CF)was used as antigen. Monoclonal antibodies against Ang-2 was produced by sequential immunization of mice XenoMouse® (XenoMouse strains XMG2 and MG4 (strain 3C-1), Abgenix, Inc. Fremont, CA). XenoMouse animals were immunitary by putting all injection into the foot pad. Total volume of each injection was 50 µl per mouse, 25 µl of the foot pad. The first injection was 2,35 µg recombinant human Ang-2 (rhAng-2, cat. No. 623-AM/CF; lot # BN023202A) in pyrogen-free PBS, Dulbecco (DPBS), in a mixture (1:1 V/V) with 10 μg of CpG (15 ál of rat adjuvant ImmunEasy, cat. No. 303101; lot # 11553042; Qiagen) on the mouse. Then typed 6 booster injection containing 2,35 µg rhAng-2 in pyrogen-free DPBS, mixed with 25 μg Adju-Phos (aluminophosphate gel; cat. No. 1452-250, batch No. 8937, HCI Biosector) and 10 μg CpG on the mouse, after which he entered the last booster injection containing 2,35 µg rhAng-2 in pyrogen-free DPBS, in the absence of adjuvant. In accordance with the specified Protocol, XenoMouse mouse were immunitary in the days 0, 3, 6, 10, 13, 17, 20 and 24, and the fusion was performed on day 29.

The selection of animals to generate antibodies by titration

The titers of anti-Ang-2 antibodies in serum obtained from immunogenic XenoMouse mice, were determined using ELISA. Briefly, polystyrene 96-well tablets Costar Labcoat Universal Binding (Corning, Acton, MA) were senzibilizirani recombinant Ang-2 (1 μg/ml) overnight at 4°C in buffer for sensitization antigen (0.1 M carbonate buffer, pH 9,6, Panso38,4 g/l). The next day tablets 3 times washed with wash buffer (0.05% tween-20 in 1 × PBS) using a device for p is mevki tablets Biotek. Then the plates were blocked with 200 µl/well blocking buffer (0.05% of BSA, 0.1% tween-20, 0.01% thimerosal in 1 × PBS) and incubated at room temperature for 1 hour. After blocking for one hour tablets 3 times washed with wash buffer using a device for washing tablets Biotek. Serum taken from Ang-2-immunogenic XenoMouse mice or reimmunization XenoMouse animals, were titrated in 0.5% BSA/PBS-buffer at dilutions of 1:3 in duplicate, starting with a dilution of 1:100. The last hole was left for control. These plates were incubated at room temperature for 2 hours and then 3 times washed with wash buffer using a device for washing tablets Biotek. After this was added, conjugated to horseradish peroxidase Fc-specific goat antibody against human IgG (HRP, Pierce, Rockford, IL) at a final concentration of 1 μg/ml and incubated for 1 hour at room temperature. Then the tablets 3 times washed with wash buffer using a device for washing tablets Biotek.

After washing tablets showed the addition of a chromogenic substrate TMB (BioFx BSTP-0100-01) for 10-20 minutes or until while in the holes with the negative control did not begin to appear in color. Then ELISA analysis was stopped by the addition of the solution to terminate the reaction (650 nm blocking re the Ghent for TMB (BioFx BSTP-0100-01), diluted with 100 ml of H2O on the vessel). Specific titers for each animal XenoMouse® was determined by optical density at 650 nm, and the results are systematized in the following tables 2 and 3. The magnitude of the titer represents the reciprocal value of the highest dilution of sera with OD value, which is twice the background value. Therefore, the higher the value, the higher the level of humoral immune response to Ang-2.

Table 2
Group 1: 10 mice (strain XG2)
After 4 injectionsAfter 6 injections
Mouse IDReactivity towards rhAng-2
Antibody titers against hIgG
A-192000231000
A-256000212000
A-373000331000
A-4 16000175000
A-595000236000
A-627000119000
A-7100000239000
O-825000165000
A-968000to 136,000
A-10120000264000
NC3565
RSSensitivity: 10 ng/mlSensitivity: 9 ng/ml
* NC = the strain xmg2, ova gp2, fp
* PC = goat antibody against huAng-2 (R&D Systems, Catalog No. AF623), 1 mg/ml

225
Table 3
Group 2: 10 mice (strain XG4)
After 4 injectionsAfter 6 injections
Mouse IDReactivity towards rhAng-2
Antibody titers against hIgG
A-17504600
A-22005800
A-35007400
A-42254700
A-53005800
A-65507400
A-7160011000
O-8452400
A-96006900
A-102300
NC<10035
RSSensitivity: 12 ng/mlSensitivity: 8 ng/ml
* NC = 3c-l N128-3
* PC = goat antibody against huAng-2 (R&D Systems, Catalog No. AF623), 1 mg/ml

EXAMPLE 2

THE SELECTION OF LYMPHOCYTES, THE SELECTION OF B-CELLS, MERGING CELLS AND THE GENERATION OF HYBRIDOMAS.

Immunogenic mice were killed by displacement of the cervical vertebrae, and mice of each group were taken draining lymph nodes, and then they were United. Lymphoid cells are destroyed by shredding in DMEM to release cells from the tissues, and cells suspended in DMEM. Cells were counted, and the cell sediment was added to 0.9 ml of DMEM 100 million lymphocytes to implement soft, but full of resuspendable cells. Using 100 ál of magnetic CD90+-areas of 100 million cells, the cells were labeled by incubation with magnetic fields at 4°C for 15 minutes. Cell suspension labeled with magnetic fields and containing up to 108positive cells (or up to 2×109cells)were loaded on the column LS+, and this column were washed in DMEM. General you shall odasi stream was collected as CD90-negative fraction (most of these cells, presumably, was In the cells).

The cell fusion was performed by mixing washed enriched b-cells, described above, with non-secretory myeloma P3X63Ag8.653 cells 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 besieged by centrifugation at 800×g. After removal of the supernatant the cells were treated with 2-4 ml of a solution pronase (CalBiochem, cat. # 53702; 0.5 mg/ml in PBS) for a period of not more than 2 minutes. Then to block the enzymatic activity was added 3-5 ml of FBS and the suspension is brought to a total volume of 40 ml by adding the solution to electroline cells (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 cells resuspendable in 40 ml of ECFS. This stage of washing was repeated and the cells again resuspendable in ECFS to a concentration of 2×106cells/ml

Electroline cells was performed using a generator merge (Model ECM, Genetronic, Inc., San Diego, CA). The size of the camera generator merger was 2.0 ml, and the specified device had the following parameters:

Measuring conditions: voltage: 50 V, time: 50 sec

Fracture membrane: voltage: 3000 V, time: 30 μs

Aging time after the merger: 3 seconds

After electroline cell suspension with caution in the occupy of the camera to merge under sterile conditions and transferred into a sterile test tube, containing the same volumes of media for culturing hybridomas (DMEM, JRH Biosciences) with 15% FBS (Hyclone), which were 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 37aboutC, and then centrifuged at 400×g (1000 rpm) for five minutes. These cells gently resuspendable in a small volume of medium for selection of hybridomas (media for culturing hybridomas, which was added 0,5x HA (Sigma, cat.# A9666)), and this volume is appropriately corrected by adding additional quantities environment for hybrid selection, based end-culturing 5×106B-cells to 96-well plate and 200 μl per well. These cells are gently mixed and pietravalle in 96-well plates, and then left for growth. On the 7th or 10th day half of the medium was removed and cells were fed environment for hybrid selection.

EXAMPLE 3

SELECTION OF ANTIBODY CANDIDATES USING ELISA.

After 14 days of cultivation, supernatant hybrid was skanirovali on Ang-2-specific monoclonal antibodies. ELISA-plates (Fisher, Cat. No. 12-565-136) was senzibilizirani 50 µl/well of human Ang-2 (2 μg/ml) in buffer for sensitization (0.1 M carbonate buffer, pH 9,6, Panso3, 8,4 g/l), and then incubated at 4ºC for the night. After incubation tablets 3 times washed with wash buffer (0.05% tween-20 in PBS). Then add 200 µl/well blocking buffer (0.5% of BSA, 0.1% tween-20, 0.01% thimerosal in 1x PBS) and the plates were incubated at room temperature for 1 hour. After incubation tablets three times washed with wash buffer. Then add 50 μl/well of supernatant hybridoma and positive and negative control, after which the plates were incubated at room temperature for 2 hours. During the whole experiment as a positive control was used serum taken from Ang-2-immunogenic XenoMouse mice, Ang-2 group 1 XMG2, foot pad (fp) which meant N160-7, and as a negative control was used serum taken from KLH-immunogenic XenoMouse mice KLH group 1 XMG2, foot pad (fp) which meant L627-6.

After incubation tablets three times washed with wash buffer. After this was added 100 μl/well detection of HRP-conjugated goat antibodies against huIgGFc (Caltag, Cat. No. H10507), and the plates were incubated at room temperature for 1 hour. In the second procedure of screening positive samples in the first screening were skanirovali in two stages, one for the detection of hIgG and the other for detecting light chain Kappa human Ig (HRP-conjugated goat antibodies to p is otiv chain Kappa hIg)(Southern Biotechnology, Cat. No. 2060-05), in order to demonstrate that the composition of the fully human antibody contains IgG and Ig-Kappa. After incubation tablets three times washed with wash buffer. Then add 100 μl/well TMB (BioFX Lab. Cat no. TMSK-0100-01) and tablets left for the manifestation of color for about 10 minutes (until, while in the wells of negative control were not found barely noticeable coloration). Then add 50 μl/well of a solution to terminate the reaction (blocking solution of TMB (BioFX Lab. Cat.# STPR-0100-01), and the tablets were read on an ELISA-tablet-reader at a wavelength of 450 nm. Received 185 fully human antibodies IgG Kappa against Ang-2.

All antibodies, which were associated in the ELISA analysis were subjected to kontrstimulirovaniya on the binding of Ang-1 using ELISA to exclude antibodies cross-reacting with Ang-1. ELISA-plates (Fisher, Cat. No. 12-565-136) was senzibilizirani 50 µl/well of recombinant Ang-1 (2 µg/ml, obtained from R&D Systems, Cat. No. 293-AN-025/CF) in the buffer for sensitization (0.1 M carbonate buffer, pH 9,6, Panso3, 8,4 g/l), and then incubated at 4ºC overnight. In this experimental conditions, if the recombinant molecule Ang-1 was immobilized on the ELISA-plate, that of antibody binding to Ang-1 was not observed. However, the method described here kontrstimulirovaniya has some technical limitations. In the first place, these antibodies were obtained from cell lines, but not from cloned hybridoma. The binding signals coming from a particular clone, which is only a small percentage of this line may be below the threshold of detection. Second, in this experiment, some epitopes in the antigen can be hidden from antibodies, due to small conformational changes induced by immobilization of the antigen. For these reasons, the cross-reactivity of each antibody with Ang-1 was further evaluated using the cloned antibodies and Biacore system (see example 8). As described in example 8, it was found that only one clone (mAb 3.19.3) really had a strong cross-reactivity with recombinant human Ang-1 (examples 8, 9 and 12).

EXAMPLE 4

INHIBITION of BINDING ANG-2, TIE-2

As discussed above, the biological effects of Ang-2 is its binding to the receptor Tie-2. Monoclonal antibodies that inhibit the binding of Ang-2/Tie-2, were identified through analysis of competitive binding using a modified ELISA. Used mAb are products microcode of 50 ml of debilitating supernatants Pula Ang-2-specific hybridomas (see example 3). 96-well tablets Nunc ImmplatesTMwas senzibilizirani 100 ál human is someone recombinant hybrid protein Tie-2/Fc (R& D Systems, Inc., Cat. No. 313-TI-100) at a density of 4 mcg/ml by incubation over night at 4ºC. Tablets four times washed with phosphate buffered saline (PBS) to wash the device scanTMWasher 300 (SKATRON). The wells were blocked by adding 100 μl of ABX-blocking buffer (0,5% BSA, 0.1% tween, 0.01% thimerosal in PBS) for 1 hour.

In each well, whether or not containing anti-Ang-2 mAb at 100 µg/ml, was added biotinylated recombinant human Ang-2 (R&D Systems, Inc. Cat. No. BT623) at 100 ng/ml. the plates were incubated at room temperature for two hours, and then unbound molecules were removed by washing. After that, associated biotinylated Ang-2 were detected using 100 μl/well of conjugate streptavidin-HRP in 1:200 by incubation at room temperature for half an hour. After twice washing the bound streptavidin was detected using HRP-substrate (R&D Systems, Cat. No. DY998). The plates were incubated for 30 minutes, and then to terminate the reaction, was added to the blocking solution 450 (100 µl/well, BioFX, Cat# BSTP-0100-01). The optical density at 450 nm was determined on a plate reader Spectramax Plus.

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

Each hybridoma cloned by the method of limiting dilution, in accordance with the following standard procedures. From each hybridoma was taken three nursing clone. For each clone supernatant were tested using 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 for Ang-2. Then determined the concentration of IgG in exhausting supernatant, and one clone from three nursing clones from each hybridoma, which has the largest output was selected for purification of IgG. For additional characterization of each supernatant was allocated 0.5-1 mg of IgG.

To quantify the activity of mAb directed to the inhibition of binding Ang-2, Tie-2, determined the titer of purified mAb best of all 27 candidates through analysis of competitive binding. Each mAb concentration was tested in duplicate. The dependence of the “concentration - response” was established along the curve, constructed using the graphics to mutarnee program Graphpad Prism TM(nonlinear sigmoid curve). Maximum inhibition (efficiency) and IC50(activity) was calculated using a computer program. Ten monoclonal antibodies with relatively high efficiency and activity, was selected for subsequent studies. The efficiency and activity of these 10 mAb illustrated in table 4.

Table 4
The efficiency and activity of the top 10 mAb
CloneEfficiency*EC50(ág/ml)
3.31.20,37510,04169
5.16.30,32790,08532
5.86.10,38440,1331
5.88.30,40320,1557
3.3.20,38810,1684
5.103.10,23170,3643
5.101.10,3639 0,3762
3.19.30,39450,7976
5.28.10,38922,698
5.78.30,26215,969
* Efficiency is expressed as the ratio of Ang-2, associated with mAb (30 µg/ml), Ang-2, is not associated with the mAb.

EXAMPLE 5

MAPPING of ANTIBODIES

Mapping of epitopes was performed in order to determine which of the anti-Ang-2 antibodies compete with each other for cross-linking, and therefore likely to bind to the same epitope on Ang-2. The mapping process described in application for U.S. patent 20030175760, as well as in the publication Jia et al., J. Immunol. Methods, (2004)288:91-98, which in its entirety are introduced in the present description by reference. Briefly, areas Luminex associated with mouse anti-hulgG antibody (Pharmingen# 555784) in accordance with the Protocol binding proteins, which can be found on the web site Luminex. Pre-connected areas received to associate with the “first” unknown antibody in accordance with the following procedure, these spheres were stored in a place protected from exposure to light. For each unknown supernatant was used to separate the tubes. The required volume of the supernatant was calculated by the following formula: (n×2+10)×50 μl (where n = total number of samples). In this assay was 0.1 µg/ml of the mother solution for each area was gently mixed and dissolved in the supernatant to a concentration of 2500 or 0.5×105spheres/ml 50 ál/well.

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

Filtering the tablet was pre-wetted by adding 200 ál wash buffer per well, and then the buffer was aspirated. To each well of the filter tablet was added 50 μl of each sphere. The samples once washed by adding 100 µl/well of wash buffer and extraction. In the specified filtering tablet was added to the antigen and control with 50 µl/well. Then the tablet was covered, incubated in the dark for 1 hour on a shaker, after which the samples 3 times washed. After that, he added, “second” unknown antibody in 50 μl/well. The concentration of the first antibody was 0.1 µg/ml Then the plates were incubated in the dark for 2 hours at room temperature on a shaker, after which the samples 3 times washed. Then add 50 μl/well biotinylated mouse antibodies against human IgG (Pharmingen #555785)diluted 1:500, and the samples were incubated with shaking in the dark for 1 hour at whom atoi temperature.

Samples 3 times washed. Then add 50 μl/well of streptavidin-Feh, 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 device 100, the samples 3 times washed. The contents of each well resuspendable in 80 ál of blocking buffer. The samples gently mixed by pipetting several times to resuspendable areas. Then the samples were analyzed on the Luminex device 100. The results are shown below in table 5.

Table 5
The results of mapping (Bin) for best 24 positive anti-Ang-2 antibodies in functional analysis
Bin1Bin2Bin3Bin4Bin5Bin6Bin7Bin8
3.33.385.56*5.285.783.196.35.35
3.285.103 5.40
3.31
5.145.2
5.16
5.39
5.41
5.49
5.54
5.62
5.83
5.86
5.88
5.101
5.108
*Note: mAb 5.56 found the same pattern of binding, with only minor differences, like mAb 3.38 and 5.103, and gave a much smaller signal.

EXAMPLE 6

DETERMINATION of the affinity of ANTI-ANG-2 ANTIBODIES USING BIACORE ANALYSIS

Screening low-resolution 27 of purified monoclonal antibodies

To measure the affinity of the antibody to the antigen used surface plasmon resonance without ringing (SPR) or Biacore. For these purposes, on the surface of the chip CM5 Biacore inflicted goat anti-human antibody at high density using a routine method of bonding with the amine. All purified mAb was diluted to about 8 μg/ml working buffer HBS-P containing 100 µg/ml BSA and 10 mg/ml carboxymethylcysteine. Each mAb was immobilized on a separate surface for time who I contact 42 seconds and washed for 5 minutes to stabilize the baseline mAb.

On all surfaces within one minute were injected with Ang-2 in 90,9 nm, and then carried dissociation within 10 minutes. Data double reference the binding was obtained by subtracting the signal magnitude from the magnitude of the signal received to control the flow cell, and subtracting the level of the base of the “drift” of the buffer injected immediately before injection of Ang-2. Data for the binding of Ang-2 with each mAb were normalized to the amount of mAb immobilized on each surface, and determine the normalized and adjusted for drift responses for 27 mAb. Reportedly built a General curve for the model of interaction of 1:1 in order to determine the kinetics of binding. The results of kinetic analysis of binding Ang-2, conducted at 25 º C, are given in the table below, mAb listed in descending order of affinity.

Table 6
Screening low-resolution Ang-2 using Biacore for 27 of purified monoclonal antibodies
SampleThe amount of immobilized
antibodies (EN)
kand(M-1with-1)kd(with-1)Tod(PM)
5.16 157of 3.6×1051,0×10-5*27
5.41152of 3.6×1051,0×10-5*28
5.35138of 3.4×1051,0×10-5*29
3.38143of 3.4×1051,0×10-5*30
5.10866of 3.2×1051,0×10-5*31
3.31253,0×1051,0×10-5*33
5.492603,0×1051,0×10-5*33
3.28280of 2.7×1051,0×10-5*37
5.88 65of 2.7×1051,0×10-5*37
5.28136a 2.5×1051,0×10-5*40
5.782222.4 x 1051,0×10-5*42
5.39166of 2.3×1051,0×10-5*43
5.103127of 2.2×1051,0×10-5*45
5.13782,1×1051,0×10-5*47
5.14471of 2.0×1051,0×10-5*49
3.31196of 1.9×1051,0×10-5*51
5.56 144of 1.9×1051,0×10-5*52
5.2111of 1.6×1051,0×10-5*62
5.62126of 1.5×1051,0×10-5*65
5.54131of 1.5×1051,0×10-5*66
6.3221of 1.4×1051,0×10-5*73
3.192529,0×1041,0×10-5*111
5.40130of 7.8×1041,0×10-5*129
5.83157of 6.8×1041,0×10-5*147
5.101 217of 1.5×105of 8.7×10-5581
5.86126of 1.5×105of 1.1×10-4744
5.52114of 1.3×1051,0×10-5*750

Asterisk appearing after the values of kdfor all mAb, with the exception of mAb 5.101 and 5.86, indicate that these values of kdare permanent and represent the best estimate for the magnitude characterizing the performance for low speed dissociation. Selection models for these samples did not show any noticeable changes in the speed of such a dissociation in a relatively short time of dissociation, and therefore for the selection of the data rate of the Association it is necessary that the value of kdwas constant. Data for these antibodies showed that these antibodies with such values of kdact as antibodies with kdorder 10-6with-1and so these interactions can be 10 times or more stronger than the interaction mentioned above.

Data dissociation is usually measured for 4-6 hours in the Expo is iontach on kinetics of high resolution using a mAb, having an affinity of less than 100 PM. The maximum time of dissociation, which can be measured without artifacts “drift” from immobilized on the surface of the mAb, is 20 minutes. The attenuation is almost redetection signals for mAb with high affinity was measured over a relatively short period of time, and therefore, the values of kdvary considerably in the range of values, comprising two orders of magnitude.

EXAMPLE 7

DETERMINATION of the affinity of ANTI-ANG-2 ANTIBODIES USING BIACORE ANALYSIS

The screening medium/high resolution Ang-2 using three purified monoclonal antibodies

Purified mAb 5.16, 5.35 and 5.41 diluted to approximately 8 mg/ml in 10 mm sodium acetate, pH 5.0. Then each diluted mAB was immobilized on different surfaces of the flow-through cuvettes (chip CM5 Biacore) routine method of bonding with the amine.

To retrieve the data rate of the Association were randomly injected with eight concentrations (2-fold dilution) Ang-2 within 90,9 is 0.71 nm for 90 seconds with three replications, with did a few of injection buffer alternately with injections dual control, with subsequent dissociation within four minutes. Surface antibodies was regenerated by two 9-second injection of 10 mm glycine-HCl, pH 1,5, after each cycle of injection.

To get Dan what's the speed of diassociative were injected with three samples with 90,9 nm Ang-2 in the working buffer HBS-P, containing 100 μg/ml BSA, as described above, and the values of dissociation were recorded for eight hours. Injection of the sample was done alternately with three injection cycles blind control. Regeneration was carried out as described above.

Reportedly built a General curve for the model of interaction of 1:1 by mass transfer using CLAMP (David G. Myszka and Thomas Morton (1998) "CLAMP©: a biosensor kinetic data analysis program," TIBS 23, 149-150). The obtained binding constants are given in table 7.

Table 7
The screening medium resolution Ang-2 using Biacore 3 purified monoclonal antibodies
SampleRmaxkand(M-1with-1)kd(with-1)Tod(PM)
5.1636to 3.41×1052,77×10-68,13
5.35545,64×105of 1.87×10-63,31
5.4144of 4.9×10 58,31×10-6*17,7*

Significant attenuation of the signal measured during an 8-hour dissociation. Using the data obtained for the 8-hour dissociation, the program CLAMP allows you to more accurately determine the value of kdfor each mAb. In this case, kdfor antibodies 5.35 5.16 and is of the order of 10-6with-1.

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

EXAMPLE 8

DETERMINATION of the affinity of ANTI-ANG-1 ANTIBODIES USING BIACORE ANALYSIS

The cross-reactivity of antibodies against Ang-1 additionally investigated by measuring the affinity of mAb binding to Ang-1. In this case, instead of immobilization of Ang-1, as was performed in the described ELISA analysis on cross-linking (example 3), was carried out by immobilization of anti-Ang-2 mAb on chips CM5 Biacore, and then were injected with Ang-1 in solution to determine the speed and obviously reminds dissociation. Six mAb, including 3.3.2, 3.31.2, 5.16.3, 5.86.1, 5.88.3 and 3.19.3, tested in this experiment, as described below, to determine their level of cross-linking with Ang-1.

The screening medium resolution for the six purified monoclonal antibodies

To measure the affinity of antibodies to Ang-1 used surface PLA is Manny response without ringing (SPR) or device Biacore 2000. For these purposes, on the surface of the chip CM5 Biacore inflicted goat anti-human antibody at high density using a routine method of bonding with the amine. To conduct scientific experiments, purified mAb (clone 3.19.3, 3.3.2, 5.88.3, 5.86.1, 3.31.2, 5.16.3) was diluted approximately to a concentration of 2.5-3.5 μg/ml working buffer HBS-P containing 100 μg/ml BSA. The level of capture for each mAb was approximately 150 RU. After each loop seizure conducted 5-minute rinses to stabilize the baseline mAb.

On all surfaces to capture antibodies for 1 minute were injected with one sample of Ang-1, diluted to 87,4 nm in the working buffer. For five mAb any detectable binding was not observed, although it was found that Ang-1 binds to mAb 3.19.3. The experiment was repeated with increasing levels of binding of mAb with holes within 500-600 EN 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.

Since only Ang-1 was detected activity of binding to mAb 3.19.3, we determined the affinity of binding of this mAb with Ang-1 and Ang-2. Because during the above scientific experiment Ang-1 was found low dissociation rate, the experiment on screening medium resolution on the capture antibody did not give enough data speed dissociate is, on the basis of which it is possible to accurately calculate the value of kd. Therefore, the level of binding of Ang-1 and Ang-2 with mAb 3.19.3 was measured using high-resolution analysis in Biacore conditions.

EXAMPLE 9

DETERMINATION of AFFINITY of binding of MAB 3.19.3 WITH ANG-1 AND ANG-2 USING high-resolution BIACORE ANALYSIS

Purified mAb 3.19.3 was diluted to approximately 12.5 µg/ml in 10 mm sodium acetate, pH of 4.0. Then each mAb was immobilized on flow cells 1-3 (chip CM5 Biacore) routine method of binding an amine, and flow cell 4 was used as control.

To retrieve the data rate of the Association were randomly injected with eight concentrations (2-fold dilution) Ang-1 within 39,8-0,31 nm (in the working buffer HBS-P containing 100 μg/ml BSA) for 90 seconds (flow rate of 100 µl/min) with three replications, with did a few of injection buffer alternately with injections for dual control, with subsequent dissociation within four minutes. Surface antibodies were regenerating through a 6-second injection of 10 mm Paon after each cycle of injection.

To obtain data speed diassociative were injected with three samples from 19.9 nm Ang-1, as described above, and the values of the dissociation registered for six hours. Injection of the sample was done alternately with three injection cycles blind control. Regeneration was carried out as op is Sano above.

Reportedly built a General curve for the model of interaction of 1:1 by mass transfer using CLAMP (David G. Myszka and Thomas Morton (1998) "CLAMP©: a biosensor kinetic data analysis program," TIBS 23, 149-150).

The study of Ang-2 by using high-resolution Biacore analysis using purified MAb 3.19.3

Purified mAb 3.19.3 was diluted to approximately 12.5 µg/ml in 10 mm sodium acetate, pH of 4.0. Then mAB was immobilized on flow cells 1-3 (chip CM5 Biacore) routine method of binding an amine, and flow cell 4 was used as control.

To retrieve the data rate of the Association were randomly injected with eight concentrations (2-fold dilution) Ang-2 in the range of 30.0-0,23 nm (in the working buffer HBS-P containing 100 μg/ml BSA) for 90 seconds (flow rate of 100 µl/min) with three replications, with did a few of injection buffer alternately with injections for dual control, with subsequent dissociation within four minutes. Surface antibodies were regenerating through a 6-second injection of 15 mm Paon after each cycle of injection.

To obtain data speed diassociative were injected with three samples from 15.0 nm Ang-2, as described above, and the values of the dissociation registered for six hours. Injection of the sample was done alternately with three injection cycles blind control. After each injection cycle, each p is the surface was regenerated by a 6-second injection of 15 mm Paon to assess the rate of dissociation.

Reportedly built a General curve for the model of interaction of 1:1 by mass transfer using CLAMP (David G. Myszka and Thomas Morton (1998) "CLAMP©: a biosensor kinetic data analysis program", TIBS 23, 149-150).

Results and discussion: the study of the binding of Ang-1 and Ang-2 with MAb 3.19.3 conducted using high-resolution Biacore analysis

For each antigen were conducted by two independent experiment. The results of these experiments are given below in table 8.

Table 8
The results of the study of the binding of Ang-1 and Ang-2 with purified mAb 3.19.3 conducted using high-resolution Biacore analysis
Antigenkand(M-1with-1)kd(with-1)Tod(PM)
Ang-1 (1st)of 1.33×105of 4.05×10-630,4
Ang-1 (2nd)is 1.82×105the 5.51×10-630,2
Ang-2 (1st)1,89×1051,00×10-6*5,3
Ang-2 (2nd)of 1.78×1051,00×10-6*5,6

In the above table, the values of kdfor Ang-2 are marked with an asterisk, because these values have remained constant in the process of modeling the interaction of 1:1 using a computer program CLAMP. In experiments for Ang-2 any noticeable signal dissociation was not registered, and therefore the best value rate of dissociation (kdremained constant at about 1×10-6with-1. The period of time of dissociation for Ang-2 in fact tended to his constant increase in the registration process the data held within six hours. This trend was repeated in the analysis on two different sensor chips using two different devices, and then both of these devices were subjected to superoxide” in accordance with the Protocol. For a more accurate measurement of the affinity of binding of mAb 3.19.3 with Ang-1 and Ang-2 conducted an additional experiment (see example 10) in order to determine the Kd of mAb 3.19.3 in relation to these antigens.

Interestingly, when Ang-1 was immobilized on ELISA tablet, mAb 3.19.3 was not associated with Ang-1 in ELISA analysis on binding (example 3). This inconsistency can be explained by the fact that if Ang-1 was immobilizer is sowen on a plastic surface, to minor epitope, which plays an important role in the binding of mAb 3.19.3, he was not provided with appropriate access. If, however, Ang-1 was in the liquid phase, for example, provide experimental conditions Biacore, this epitope was accessible to mAb 3.19.3, resulting happened binding.

EXAMPLE 10

DETERMINATION of the affinity of the MAB 3.19.3 IN RELATION TO HUMAN Ang-2 USING high-resolution ANALYSIS KINEXA (KINETIC EXCLUSION ANALYSIS)

When measuring the affinity of mAb 3.19.3 against human Ang-2 using high-resolution Biacore analysis (example 9) any significant signal dissociation was found. The period of time of dissociation for Ang-2 was found to steadily increase during the registration process the data held within six hours. Hence, kdmAb 3.19.3, bind to human Ang-2 was determined using KinExA technology, in order to obtain a more accurate value of kd. For this purpose used the device KinExA 3000. First, in the preparation of 1 ml (~271 µg) background Ang-2 (R&D Systems, Inc., Lot# BNO32510A), the buffer was replaced with 1 × PBS, pH 7.0, using a 10-ml desalting column (polyacrylamide columns Pierce D-SaltTMwith a cutoff molecular weight of 6000, Lot# GF97965). Concentration of the combined fractions was 1.7 μm, as op is Adelino by calculating the concentration of the protein, described C. Nick Pace (Pace, et al.,Protein ScienceVol. 4: 2411-2423, 1995). Then, 450 µl (~122 µg) source of Ang-2 was associated with 200 mg polymetylmetacrylate areas (PMMA, Lot# 206-01), during the night when 24º. Then these areas were centrifuged and once washed with the blocking buffer (1 × PBS, 10 mg/ml BSA), then centrifuged, and then incubated in blocking buffer for one hour at 24º. After blocking spheres were diluted in approximately 30 ml of HBS buffer (0.01 M Hepes, 0.15 M NaCl, pH 7,4) standard vessel-the vessel with the spheres KinExA and placed in the specified device.

kd-controlled titration

Twelve solutions containing the binding site mAb 3.19.3 in concentration of 25.3 PM, was titrated with increasing concentrations of Ang-2. For sample preparation of Ang-2 was conducted buffer exchange. Each solution had a total volume of 25 ml, and this solution was balanced within 5 days when~24º. Solutions for titration were prepared in volumetric glassware, the concentrations of Ang-2 ranged from 5,09 nm to FM 99,3. The method is carried out on the device KinExA analysis of these solutions consisted of the packing stage of the spheres, in which PMMA sphere Packed in glass capillary tubes and equilibrated solutions were passed through the column with the spheres at a speed of 0.25 ml/min for 6 minutes (1.5 ml) in duplicates. Then Fluor is Santo labeled goat anti-human (Fc-spetsificheskoe) polyclonal antibody su-5 3.4 nm was passed through a packing of spheres in for 1 minute at a speed of 0.5 ml/min for marking free mAb binding sites, immobilized on the fields. Fluorescent radiation packing of spheres was measured at 670 nm with excitation at 620 nm. The results of measurement of fluorescence was converted to “% of free binding site mAb" in relation to the total concentration of an antigen using the supplied software package KinExA (version 1.0.3, Sapidyne, Inc.). Total kd-controlled titration curve was constructed using KinExA, and this curve represented the isotherm equilibrium 1:1 with a correction factor of “drift.” The optimal value of kdthat is consistent with these data, amounted to 86.4 PM with lower and upper 95% confidence limits components 64,3 PM 98,7 and PM, respectively. Curve mAb-controlled titration is not given.

EXAMPLE 11

BLOCKING ANG-2-INDUCED PHOSPHORYLATION of TIE-2, ECTOPICESKI EXPRESSED IN HEK293 CELLS

As discussed above, the Tie-2 is tyrosinekinase receptor specific for endothelial cells.In vitroexperiments conducted using vascular endothelial cells, showed that Ang-1 induces the phosphorylation of Tie-2 and Ang-2 inhibits the phosphorylation of the receptor induced by Ang-1. However, if the Tie-2 is expressed ectopiceski, for example, as it occurs in fibroblasts, Ang-2 is also able to induce FOS is arilirovaniya Tie-2 in some conditions, including, but not limited to, the prolonged action of angiopoietin-2 or high concentrations of angiopoietin-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 phosphorylation of Tie-2 was evaluated using HEK293F cells, transfected with the human receptor Tie-2. Plasmid vector pORK/pBS-SK with cDNA Tie-2, was obtained from ATCC (sequence BC033514: Cat. No. 69003, Genbank). The cDNA sequence was confirmed by nucleotide sequencing. 3,9 TPN fragment containing 3375 digested cDNA, coderush human Tie-2 were removed from the vector by hydrolysis with the enzyme EcoRI. This fragment was subcloned into the functional orientation in the vector pCR3.1, hydrolyzed by the enzyme EcoRI in accordance with standard procedures. Selected plasmids amplified and purified according to standard protocols.

Tie-2-containing structure obtained in accordance with the above procedures, transferrable in HEK293F cells by the method of transfection using calcium phosphate. 1×106HEK293F cells were cultured in 100 mm plates for culturing fabric coated with 1% gelatin, when 37º with 5% CO2. Before transfection the cells were fed fresh medium for 2-3 hours. 10 μg of PL is Sidney DNA was dissolved in 248 mm solution of calcium phosphate. The transfection was carried out by standard methods. Stable transfectants were selected by incubation in 0.5 mg/ml G418. Stable transfectants expressing Tie-2, was performed using FACS analysis using mouse anti-Tie-2 mAb (R&D Cat. No. MAB313) and PV-conjugated goat antibodies against mouse IgG (Caltag, Cat. No. M30004-4) for detection.

For analysis on the phosphorylation of Tie-2, HEK293F/Tie-2-transfectants were cultured in 60 mm plates for culturing cells at a density of 2×106cells/plate with complete medium at 37º with 5% CO2to achieve subconfluent. Complete medium in each dish was replaced with 2 ml serum-free medium. Cells were incubated for another 16 hours. Then the medium was again replaced with 2 ml serum-free medium. After incubation for a further 2 hours the cells were treated with 0.1 mm of orthovanadate sodium (Sigma, Cat. No. 6508 S) for 20 minutes. Cells were treated with Ang-2 (2 μg/ml) in the presence or in the absence of mAb at 100 µg/ml Treatment was performed in duplicate. Was used a negative control without Ang-2 treatment. Cells were washed in chilled on ice TBS containing Vanadate, and subjected to lysis by adding 300 μl/Cup chilled buffer for lysis NP-40 (50 mm Hepes, pH 7.2, were added 0.15 M NaCl, 10% glycerol, 10 mm pyrophosphate, 50 mm NaF, 1% NP40, 100 u/ml Aprotinin, 1 mm PMSF, and 1 mm of orthovanadate, 10 μm leupeptin and 10 μm of pepstatin A), and the cups were placed for 10 minutes on ice. The treated cells were scraped off with these cups in microprobing, pre-cooled on ice.

Cell lysates were subjected to brief sonication and centrifuged at 12000×g for 10 minutes at 4ºC in the table microcentrifuge. Supernatant was collected in a fresh microtube, and then the supernatant was added to 1-5 μg of anti-Tie-2 mAb (R&D Systems, Inc.), then the mixture was subjected to gentle shaking for 2 hours at 4ºC. To the mixture was added 50 μl of immobilized protein a, ImmunoPure (PIERCE Cat. No. 20333), and the mixture is incubated at least for 3 hours at 4ºC on a platform shaker. The complexes were collected by centrifugation at 12000×g for 10 minutes. After careful removal of the supernatant, these complexes twice washed with buffer for lysis by centrifugation (12000×g, 4ºC) for 4 minutes. Sediment resuspendable in 50 μl 2 × buffer for electrophoresis samples (Invitrogen, Cat. No. LC-2676)containing 1 mm β-mercaptoethanol or DTT and boiled for 5 minutes and then centrifuged (12000×g, 4ºC) for 5 minutes. Supernatant was transferred into a fresh tube.

The samples were loaded into the wells with LTO-SDS page gel (e.g., 4-20% Tris-glycine gel, Invitrogen, Cat. No. EC 6025). Electrophoresis was performed in Tris-glycine buffer system is IU. After electrophoresis the gel was blokirovala on a PVDF-membrane (Invitrogen, Cat. No. LC 2005) in accordance with the standard Protocol. Phosphorylation of tyrosine was confirmed using as a probe antibody against phosphotyrosine 4G10 at a concentration of 1 µg/ml (Upstate, Cat. No. 05-321) by incubation for 1 hour at room temperature with shaking, and followed by three rinses in 1 × TBST (TBS with 0.1% tween-20). Bound antibodies were detected by incubation with conjugated with horseradish peroxidase goat antibody against mouse IgG (Santa Cruz, Cat. No. sc-2302) at a dilution of 1:10000 for 1 hour at room temperature, and subsequent amplification of the chemiluminescent reaction using the substrate system SuperSignal West Dura Extended Duration Substrate system (PIERCE Cat. No. 34075). Then the blot was purified regenerating buffer for washing the Western blots (Restore Western Blot Stripping Buffer (PIERCE, Cat. No. 21059) and re-probed with specific antibodies against RTK to check the level of loading of the sample.

It was found that if the human Tie-2 ectopiceski expressively in HEK293F cells, autophosphorylation Tie-2 was not observed. In response to the processing of Ang-2 (2 μg/ml), a significant level of tyrosine phosphorylation was detected using mAb against phosphorylated tyrosine (4G10) Tie-2, thus subjected peculiar is m mAb. At a concentration of 100 μg/ml of the tested anti-Ang-2 mAb significantly inhibited the phosphorylation of Tie-2, whereas mAb isotype control did not show inhibitory effect (figure 1). Monoclonal antibody 5.103.1, which is not shown in figure 1, had a similar inhibitory effect.

To assess the ability of anti-Ang-2 mAb to inhibit Ang-2-induced phosphorylation of Tie-2in vitroa method was developed based on the ELISA analysis to quantify the phosphorylation of Tie-2. Briefly, cell lysates obtained from HEK293F/Tie-2-transfectants were treated with Ang-2 using mAb at various concentrations. The whole Tie-2, derived from the specified lysate was bound to the wells 96-well ELISA tablet, which was sensitized murine anti-hTie-2 mAb. Phosphorylated Tie-2 were detected using HRP-conjugated “first” antibody 4G10 (purchased from Upstate) and a solution of HRP-substrate. OD at 650 nm was determined on a plate reader SpectraMax. The dependence of the “concentration - response” was established through a curve, constructed using a graphical computer program Graphpad PrismTM(nonlinear sigmoid curve). Maximum inhibition (efficiency) and IC50(activity) was calculated, as shown in figure 2. EC50was calculated, as shown in table 9.

Table 9
mAbEC50 (ág/ml)95% CI
3.19.30,0060,004-0,009
5.86.10,0080,007-0,011
5.88.30,0160,011-0,024
3.31.20,0430,029-0,064
3.3.20,0460,020-0,105
5.16.30,0890,046-0,174
5.103.30,0950,046-0,199
5.101.10,7330,487-1,105

As reported above, it was found that mAb 3.19.3 cross-reacts with Ang-1. However, the results of the initial experiments did not reveal any inhibition of the phosphorylation of Tie-2, induced by angiopoietin-1 under the action of mAb 3.19.3. It should be noted that ectopic expression of Tie-2 may influence its susceptibility to the asset is of various ligands, as indicated by the fact that Ang-2 induces phosphorylation of Tie-2 in HUVEC cells, however, it was found that it induces persistent phosphorylation of Tie-2 in that case, if the specified receptor ectopiceski is expressed in HEK293 cells.

From these results, were conducted additional experiments in order to accurately determine whether mAb 3.19.3 to inhibit the binding of Ang-1 and Ang-2 with cell-associated Tie-2. In addition, the inhibition induced by angiopoietin-1 phosphorylation of Tie-2 under the action of mAb 3.19.3 was more thoroughly investigated by the method described in example 12.

EXAMPLE 12

The ANTIBODY INHIBITS MAB 3.19.3 BINDING of ANGIOPOIETIN-1 TO 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 showed that mAb 3.19.3 did not inhibit Ang-1-induced phosphorylation of Tie-2. This contradiction can be explained by the fact that: (1) to generate a stable signal of the phosphorylation of Tie-2 may require high concentrations of Ang-1, which can be much higher than the physiological concentration; or (2) ectopic expression of Tie-2 in HEK293 may change the conformation of Tie-2 and thereby change its susceptibility to the action of various ligands. To test these hypotheses mAb 3.19.3 tested in anal the se on linking, where 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. In addition, for studies of Ang-1-induced phosphorylation of Tie-2 was used an immortalized endothelial cells (EA.hy926/B3). The results of this experiment, described in more detail below, demonstrated that mAb 3.19.3 inhibits Ang-1-induced phosphorylation of Tie-2, depending on the dose.

HEK293F/Tie-2-transfectants were left for growth up to 95% of confluently in the culture flasks, and then collected. Was obtained cell suspension of 4 million cells/ml in FACS buffer, and then this suspension was divided into aliquots and placed in the holes 96-well polypropylene plate at a concentration of 50 μl per well. The cell suspension was added mAb 3.19.3 in the indicated concentrations. Then this cell suspension was added a solution of recombinant human Ang-1 and Ang-2, followed by incubation at room temperature for 2 hours. Cells were washed by centrifugation of the plate at 1200 rpm for 5 minutes, after which the supernatant was removed by aspiration, and cells resuspendable 200 µl per well of FACS buffer. Of the washing procedure was repeated twice. The cells are then suspended in 100 μl of mouse antibodies p is otiv 6X-histidine, diluted to 2 μg/ml in FACS buffer, and incubated at room temperature for 30 minutes. After washing, cells suspended in 100 μl of PV-conjugated goat antibodies against mouse IgG, diluted 1:100 in FACS buffer, and incubated at room temperature for 30 minutes. Sample size was made up to 300 μl by adding FACS buffer and were measured on the device FACS Calibur.

The obtained results are shown in figure 3 and systematized in table 10. As was shown above, soluble Tie-2/Fc was detected dose-dependent inhibition of binding of Ang-1 and Ang-2 by blocking ligands, whereas mAb isotype control PK16.3.1 was not involved in binding with any of these ligands. mAb 3.19.3 was found to be dependent on the concentration inhibition of Ang-1 and Ang-2. It is interesting to note that as the activity of Tie-2/Fc, used as standard, the activity of mAb 3.19.3 in binding to Ang-2 was greater than activity of binding to Ang-1.

Table 10
Inhibition of binding of Ang-1 and Ang-2, Tie-2
ES (nm)
Ang-1Ang-2
Tie-2/Fc 18,7325,70
3.19.3218,50,7310

The results showed that mAb 3.19.3 not only binds to human Ang-1, but also blocks its binding to the receptor Tie-2. This is again confirmed by the inhibition of Ang-1-induced phosphorylation of Tie-2 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 detected a noticeable increase in the level of inhibition of the phosphorylation of Tie-2 by increasing the concentration of the antibody, as shown in figures 4 and 5. IC50calculated from the curve of dependence “dose - response”, was 99 nm.

Analysis of the phosphorylation of receptor-stimulated ligand angiopoietin-1 Tie-2

EA.hy926 cells/B3 were sown in 6-hole tablets at a density of 2.5×105EA.hy926 cells per well in 2 ml volume of DMEM medium containing HAT and 10 % FCS, and incubated for 3 days under standard conditions of cultivation of mammalian cells.

The culture medium was replaced with 2 ml of DMEM medium (without FCS) and cells were kept in serum-free medium during the whole 2 hours. Test connection twice diluted in DMEM, steriade the 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 the dilutions of the test compounds. Similarly, to obtain samples that represent the standards 100% stimulation of ligand, also used a control not treated with the compound.

Incubation was carried out for another 10 minutes, and then each well was added 100 μl of 10 mm orthovanadate in a solution of DMEM to achieve in each hole a final concentration of 1 mm orthovanadate. Then cells were incubated for the last 10 minutes of the 2 hour period of cultivation in serum-free medium.

After 2-hour period of cultivation in serum-free medium in each well was added 1 ml of angiopoietin-1 (diluted to the appropriate concentration in DMEM containing 1 mm orthovanadate) and incubated at 37º for another 10 minutes.

Then 6-hole(s) tablet(s) were cooled by placing them on ice metal plate (which itself was on the ice). The cell medium was removed and the cell layer washed with 5 ml of cold PBS containing 1 mm orthovanadate. Then to each well was added 1 ml of chilled on ice buffer for lysis (20 mm Tris, pH of 7.6, 150 mm NaCl, 50 mm NaF, 0.1 % of LTOs, 1% NP40, AND 0.5% DOC, 1 mm orthovanadate, 1 mm EDTA, 1 mm PMSF, 30 μl/ml and retinene, 10 μg/ml of pepstatin, 10 μg/ml leupeptin), and the plate was left on ice for 10-20 minutes. Cells scraped from the tablet using tool for scraping cells, and then the entire solution of the lysate was transferred into a 1.5-ml Eppendorf tube and kept on ice. Then the samples were centrifuged for 3 minutes at 13,000 rpm at 4ºC, and all subsequent phases was carried out at 4ºC.

50 µl of each lysate was left for subsequent analysis on protein BCA (Pierce, Cat. No. 23225) (in polypropylene microtiter tablets Greiner for analysis on the binding of low molecular weight protein). The protein concentration was determined by standard analytical conditions provided by the set. 800 ál of each sample lysate was transferred into a fresh 2-ml Eppendorf tube for holding thus (PI). Then the lysates were added to 15 μl (3 mg), anti-P-Y antibody (Santa Cruz Cat. No. E2203) and left for 2 hours at 4°C for incubation, after which was added 600 μl of spheres Magnabind (with goat antibody against mouse IgG, Pierce Cat. No. 21354). Sphere Magnabind was prepared as follows. The required amount was transferred into a 15-ml conical tubes. Then these tubes were created magnetic field, and the liquid was removed. Then added fresh PBS to the original volume, and areas resuspendable. This procedure was repeated twice and Then the lysate-containing solution was mixed with the spheres, and the tubes were left overnight at 4ºC with rotation on a rotary mixer.

The sample for approximately 1 minute and subjected to the action of the magnet, and the liquid was carefully removed. Then added 1 ml of buffer for lysis, and the tube was rotated for 5 minutes to flush. Stage washing was repeated twice. Then the liquid was completely removed, and areas resuspendable in 12 µl of hot (94º) 2 × buffer laemmli's method for loading samples + bME, after which they were left for 15 minutes at room temperature. The tubes for 1 minute and subjected to the action of the magnet, and the liquid which was separated from the spheres were analyzed by electrophoresis in LTO-PAG.

The samples were analyzed by electrophoresis in LTO-page 15 holes with 4-12% bis-Tris NuPAGE gels/MOPS (Novex). On one hole was downloaded a total of 12 µl of each immunoprecipitate. The gels were subjected to electrophoresis at 200 V/120 mA/25 Watts for 55 minutes and then the samples were subjected to Western-blotting on a nitrocellulose membrane for 1 hour 30 minutes at 50 V/250 mA. Then, the blots were 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. No. C1303) diluted 1:500 in 0.5% Marvel/PBS-tween and each blot was added 12.5 ml of this antibody, and left overnight at 4°C. afterwards, the blots were washed in PBS-tween, and each blot was added cozy the antibody against rabbit POD (Dako Cat. No. P 0448) (from 1:5000 dilution in 0.5% Marvel/PBS-tween), and then left for 1 hour at room temperature. The blots were washed in PBS-tween, and each blot was shown for 10 minutes using 12.5 ml (equal volumes of solution A and B) Supersignal reagent (PIERCE Cat. No. 34080). Then the blots moved in the x-ray cassette and exposed to film (5 sec/15 sec/30 sec/60 sec/150 seconds). The figure 4 shows a Western blot illustrating the results of this analysis. In this system was observed inhibition induced by angiopoietin-1 phosphorylation of Tie-2 under the action of mAb 3.19.3.

Then the image of each sample film was evaluated using the system for image analysis BioRad FluorS. Pixel density was measured as OD/mm2and expressed in percent volume. The results, expressed in per cent volume, normalized to 1 mg protein/immunoprecipitate using protein concentration determined in the analysis of the EGM and the volume of the lysate of each sample used in thus. The percentage of phosphorylation of each sample was calculated, based on the fact that 100% accepted value phosphorylation untreated control sample on each gel, and the percentage inhibition of each sample was calculated, based on the fact that 100% accepted value phosphorylation, which itself was 0 %inhibition). These values are graficheskiy presented in figure 5 and indicate what IC50for inhibition induced by angiopoietin-1 phosphorylation of Tie-2 is 99 nm.

Overall, these data showed that in this system mAb inhibits induced by angiopoietin-1 phosphorylation of Tie-2.

EXAMPLE 13

STRUCTURAL ANALYSIS of ANTI-ANG-2 ANTIBODIES

Variable regions of the heavy chains and light chains of antibodies sequenced to determine their DNA sequences. Full information about the sequences of the anti-Ang-2 antibody is contained in the list of nucleotide sequences and amino acid sequences for each combination of circuits gamma and Kappa. Sequences of the variable regions of the heavy chain were analyzed to determine the sequences of the VH family, the D-region and J-region. Then these sequences are broadcasted to determine the primary amino acid sequence was compared with sequences of VH families, the D-region and J-region of the germ line for the assessment of somatic seperately.

Table 11 illustrates the comparison of the regions of the heavy chain of the antibodies with their cognates region heavy chain germline. Table 12 illustrates the comparison of the regions of the light chain of the Kappa antibodies with their cognates region of the light chain germline.

Variable (V) region of the chain immunoglobulin encoded by a lot of the NC-segments of the germ line, which are connected to the functional variable region (VHDJHor VKJKduring B-cell ontogeny. Molecular and genetic differences in humoral responses to Ang-2 were investigated in more detail. These analyses identified several areas that are specific to the anti-Ang-2 antibodies.

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

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

EXAMPLE 14

The DEFINITION of the CANONICAL CLASS of ANTIBODIES

CATIA and others have described the structure of antibodies, and in this description, for the hypervariable regions in each chain immunoglobulin they used the term “canonical classes” (J. Mol. Biol.1987 Aug 20; 196(4):901-17). Analyzed the atomic structure of Fab - and VL fragments of various immunoglobulins to determine the relationship between their amino acid sequences and three-dimensional the mi structures and their antigenspecific sites. Chothia et al. found that there is a relatively small number of residues, which due to their packaging, hydrogen bonds or the ability to acquire unusual Phi, psi - or omega-conformation, responsible, mainly, for the conformation of the main chain hypervariable regions. It was found that these residues are present in the websites mentioned hypervariable regions and conservative beta-folded frame structure. By evaluating sequences of immunoglobulins of unknown structure, Chothia et al. found that many of the immunoglobulins are hypervariable region, which, in size similar to the one of the famous structures and, in addition, contain identical residues in the sites responsible for the observed conformation.

It is their discovery has suggested that these hypervariable regions have a conformation similar to the conformations of known structures. For five hypervariable regions repertoire of conformations, obviously, is limited to a relatively small number of discrete structural classes. These are the most common conformation of the main chain hypervariable regions are called “canonical structures”. Subsequent work of Chothia and other (Nature1989 Dec 21-28; 342(6252):877-83) and other authors (Martin, et al.J. Mol. Biol.1996 Nov 15; 263(5):800-15) confirmed that,at least, in five of the six hypervariable regions of the antibodies have a small set of conformations of the main circuit.

The CDR of each of the above antibodies were analyzed to determine their canonical classes. As is known, canonical classes were assigned only CDRl and CDR2 of the heavy chain antibodies, as well as CDRl, CDR2 and CDR3 light chain antibodies. The results of this analysis are systematized in the following table (table 13). Data canonical classes represented in the form *HCDR1-HCDR2-LCDR1-LCDR2-LCDR3, where “HCDR" means the CDR of the heavy chain, and “LCDR" means the CDR of the light chain. For example, the canonical class 1-3-2-1-5 prisvaivaetsya the 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 LCDR3, corresponding to canonical class 5.

Specific canonical class is assigned if the amino acids in the indicated antibody at 70% or more identical to amino acids, specific for each of the canonical class. If identity is less than 70%, it is assigned the canonical class marked with an asterisk (“*”), which indicates that to determine the true canonical class was made a best estimate, based on the length of each CDR, and the resulting data were combined. In the case of the EU and the length of any CDR does not match any of the canonical class then assigned the canonical class was designated “Y”. Amino acids, specific for each antibody, can be found, for example, in the articles of CATIA and others mentioned above. Table 13 provides data canonical classes for each of the anti-Ang-2 antibodies.

Table 13
Canonical classes of antibodies against Ang-2
AntibodyThe canonical class
5.181-1-2-1-1
5.811-1-2-1-1
5.661-1-2-1-1
5.481-1-2-1-1
5.231-1-2-1-1
3.331-1-2-1-1
5.151-1-2-1-3*
4.31-1-4-1-1
3.121-1-4-1-5*
4.161-1-4-1-5*
3.131-1-4-1-5*
3.71-1-4-1-5*
3.321-1-4-1-5*
3.371-1-4-1-Y
5.301-2-2-1-1
3.61-2-2-1-1
6.61-2-2-1-1
3.34l-2*-2-l-l
5.74l-2*-2-l-l
5.381-3-2-1-1
5.28.11-3-2-1-1
5.61-3-2-1-1
5.441-3-2-1-1
5.281-3-2-1-1
5.86.11-3-2-1-1
5.35.11-3-2-1-1
5.351-3-2-1-1
5.81-3-2-1-1
5.221-3-2-1-1
4.151-3-2-1-1
5.671-3-2-1-1
5.875.101-3-2-1-1
5.711-3-2-1-1
5.211-3-2-1-1
5.801-3-2-1-1
3.181-3-2-1-1
3.111-3-2-1-1
3.261-3-2-1-1
5.311-3-2-1-1
5.761-3-2-1-1
4.91-3-2-1-1
5.171-3-2-1-1
3.391-3-2-1-1
5.371-3-2-1-1
3.221-3-2-1-1
5.291-3-2-1-1
5.731-3-2-1-1
5.121-3-2-1-1
3.11-3-2-1-1
5.111-3-2-1-1
4.81-3-2-1-1
5.241-3-2-1-1
6.3.11-3-2-1-1*
5.56.11-3-2-1-1*
5.1111-3-2-1*-!
4.131-3-2-1*-!
5.521-3*-2-1-3*
5.16.11-3-2-1-3*
5.39.11-3-2-1-3*
5.103.11-3-2-1-3*
5.101.11-3-2-1-3*
5.54.11-3-2-1-3*
5.83.11-3-2-1-3*
5.621-3-2-1-3*
5.88.11-3-2-1-3*
5.40.21-3-2-1-3*
5.1091-3-2-1-3*
5.641-3-2-1-3*
5.131-3-2-1-3*
3.3 1-3-2-1-3*
5.411-3-2-1-3*
3.3.11-3-2-1-3*
3.31.11-3-2-1-3*
5.41.11-3-2-1-3*
5.62.11-3-2-1-3*
5.108.11-3-2-1-3*
5.13.11-3-2-1-3*
5.971-3-2-1-5*
5.52.11-3-2-1-Y
5.78.11-3-3-1-1
5.601-3-3-1-1
5.431-3-3-1-1
5.901-3-3-1*-!
3.421-3-3-1*-!
4.111-3-4-1-1
3.401-3-4-1-1
5.821-3-4-1-1
4.181-3-4-1-1
3.415.921-3-4-1-1
3.10l-3-4*-l-l
4.141-3-4-1-5*
3.19.1l-3-8*-l-l
6.2l-3-8*-l-l
3.8l-3-8*-l-l
5.581-3-8-1-1*
4.5l-4*-2-l-l
5.1l-4*-2-l-l
4.2l-4*-2-l-l
3.9l-4*-2-l-l
5.45l-4*-2-l-l
3.17l-4*-2-l-l
5.72l-4*-2-l-l*
3.14l-4*-2-l-Y
5.1153-1-2-1-1
5.363-l*-2-l-l
3.23-1-2-1-3*
3.213-1-8-1-1*
5.613-Y-2-1-3*

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

Table 14
The number of anti-Ang-2 antibodies in each canonical class
H1-H2-L1-L2-L3Room mAbs
1-1-2-1-16
1-1-2-1-3*1
1-1-4-1-11
1-1-4-1-5*5
1-1-4-1-Y1
1-2-2-1-15
1-3-2-1-138
1-3-2-1-3*21
1-3-2-1-5*1
1-3-2-1-Y1
1-3-3-1-15
1-3-4-1-1 7
1-3-4-1-5*1
l-3-8*-l-l4
l-4*-2-l-l7
l-4*-2-l-Y1
3-1-2-1-12
3-1-2-1-3*1
3-1-8-1-1*1
3-Y-2-1-3*1
Notes:
1. Figures marked with *indicate that this antibody has the best fit to this class, although in certain provisions of this antibody are some deviations.
2. Y means that the length of any CDR does not match the given canonical class.

EXAMPLE 15

MAPPING the EPITOPES of ANTIBODIES AGAINST Ang-2

Was analyzed binding domain for 27 antibodies that neutralize the activity of Ang-2.

Recombinant human Ang-2 were purchased from firm R&D systems (623-AN). Goat polyclonal antibodies against human Ang-2 (R&D systems AF623) were selected on their ability to recognize rhAng-2 in direct ELISA and Western blot analyses. Polyclonal antibodies were biotinilated for detection used with the eating of HRP-conjugated streptavidin.

All restrictive enzymes were purchased from a company New England Biolabs and were used according to manufacturer's instructions. All plasmid DNA was purified on centrifuge minicolumnar (Invitrogen, Carlsbad, CA). Oligonucleotide primers used for cloning and site-directed mutagenesis were synthesized by the company Qiagen Operon.

Antibodies: 27 human anti-Ang-2 antibodies, derived from hybridoma were selected based on their ability to inhibit the binding rhAng-2 with its receptor. These antibodies listed below in table 15.

Table 15
Code hybridomaOD650 in the analysis of inhibition
1x5.560,0863
2h0,0792
3h0,0633
4h*0,0588
5h0,0558
6h*/td> 0,0516
7h*0,0874
8h*0,0856
9h0,0824
10h*0,0776
11h*0,0688
12h0,0650
13h0,0519
14h*0,0500
15h0,0484
16h0,0440
17h0,0430
18h0,0419
19h0,0984
20h*0,1013
21h0,0821
22h0,0691
23h*0,0663
24h0,0617
25xh0,0744
26h0,0690
27h0,0627

Characterization of epitopes for neutralizing 27 anti-Ang-2 antibodies

Dot-blots

RhAng-2 (R&D systems) were marked spots on the nitrocellulose membrane in native or restored using the device for microfiltration Bio-Dot. All human monoclonal antibodies (MAb)generated against human Ang-2 was associated with unrecovered Ang-2, but did not correlate with its restored form, which indicates that all mAb recognize conformational epitopes, which, obviously, are destroyed after recovery of the protein.

Cloning and expression of proteins Angl and Ang-2

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

Cloning of human Ang-1 and Ang-2

Two alternative splanirowannya form cDNA of human Ang-2 amplified from endothelial cell lines of human umbilical vein (HUVEC). PCR amplification of cDNA HUVEC conducted using Ang-2-specific primers indicated the presence of full-Ang-2 (1491 BP) and a variant of Ang-2443consisting of 1330 base pairs (Injune et al., (2000) JBC 275: 18550). Ang-2443is a variant, generated through alternative splicing of exon B and remove part superspiritual domain (amino acids 96-148). Both cDNA Ang-2 was cloned in the expression vector pCR3.1 and expressed in 293F cells, as shown in figure 6. cDNA-human Ang-1 were obtained using RT-PCR conducted using a full-sized RNA extracted the Oh from cell lines of human breast cancer MDA-MB-231. 1,5 TPN cDNA cloned in the expression vector pCR3.1, and her expression was detected in the supernatant temporarily transfected 293F cells.

ELISA

Linking 27 mAb with supernatants resulting from temporary transfection of cDNA Ang-2 and Ang-l, were tested using ELISA with capture antibodies. Ang-2, Ang-2443and Ang-1 was associated with goat polyclonal antibodies against Ang-2 or Ang-1 (respectively), present at ELISA tablet. Linking 27 best human monoclonal antibodies were detected using HRP-conjugated goat anti-human antibody, and then using the colorimetric substrate of horseradish peroxidase (activated substrate Enhanced K-Blue TMB, Neogen Corporation). The optical density in each well of ELISA-plates were measured at 450 nm on a microtiter plate reader for tablets.

Transfection of 293F cells

Embryonic kidney cells human 293F maintained in 10% fetal bovine serum in a modified method of Dulbecco environment the Needle, to which were added penicillin and streptomycin. 293F cells were subjected to temporary transfection using calcium phosphate. After 72 hours, the medium was collected and filtered for carrying out ELISA and Western blot assays.

It was shown that all 27 of the antibodies specifically bind to the antigens Ang-2/Ang-2443. A is for cross-reactivity with human Ang-1 was not detected. Amino acids 96-148 in superspiritual domain of Ang-2, which was absent in the protein sequence of Ang-2443and that was a binding domain in each of the 27 antibodies were excluded.

Construction of chimeric molecules Ang-l/Ang-2

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

Were made four designs: human Ang-1/2Bsml, Ang-2/lBsmI, Ang-1/2SspI and Ang-2/1Sspl. All proteins expressibility and secretarials at detectable levels as determined by ELISA analysis using polyclonal antibodies against human Ang-1 and Ang-2.

The sites of attachment of amino acids are in the following positions:

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

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

The difference in one amino acid 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. Best 27 antibodies were tested for their ability to bind to the chimeric molecules Ang-1/2. All 27 of the antibodies detected a similar pattern of binding only with the design of Ang-1/2Bsm/i> I. the Results of these experiments showed that the binding domain for all antibodies is in the positions of residues 117-496, and most likely, in the fibrinogen-binding domain, and hybrid proteinsSspIAng epitope located in close proximity to the position of 353 amino acids, was destroyed.

Construction of chimeric molecules “mouse/human Ang-2”

Since the amino acid sequence of Ang-2 by about 55% identical to the amino acid sequence of Ang-1, it is quite difficult to find a common restriction site, which can be used for cloning of chimeric molecules. The sequence of mouse and human Ang-2 are more similar, i.e. they homology of about 85%. cDNA of mouse Ang-2, cloned in the expression vector pCMCsport, was purchased from the company Invitrogen. 27 selected antibodies were tested for their immunoreactivity with recombinant murine Ang-2. 6 of 27 antibodies cross-reacting with mouse Ang-2 had a 100% immunoreactivity against human Ang-2, which indicates that this murine antigen retains the highest level of immunoreactivity human Ang-2 (the data are summarized in table 16).

Chimera system “man-mouse” was selected for epitope mapping, based on the fact that the majority of antibodies specific the Eski contact with a human antigen Ang-2 and not cross-react with murine Ang-2. Various cDNA constructs Ang-2 was generated and cloned in the expression vector mammals.

Design murine/human Ang-2 were obtained using a General restrictionStuI-site localized in the fibrinogen-binding domain, where the point of junction of the mouse and human amino acid is in position of rest 311. All mAb specific to human Ang-2, had the ability to contact theStu1 site mouse/human Ang-2, which indicates that the binding domain is located in the fibrinogen-binding domain in the positions of residues 311-496. To narrow binding domain has received a new design, in which the murine sequence in the cDNA of mouse Ang-2 was replaced by the human fragmentStuI-TfiI (figure 9).

All antibodies specific to human Ang-2 were found positive ELISA signal, and their immunoreactivity against human Ang-2 was 15-100%. Binding domain of the two antibodies with unique genome, occurring in the field of VH, denoted 5.35.1 (VH3-20) and 5.28.1 (VH3-43) and shown in table 17, was mapped to the region of amino acids 310-400.

It was shown that antibodies cross-reacting with mouse Ang-2, as expected, have a 100% reactive and cannot be mapped using chimeric constructs of the mouse/human.

Site-directed the military mutagenesis

To identify residues that play an important role in linking and present in the binding site of different antibodies, several residues of human Ang-2 were motivovany and skanirovaniya across the panel of antibodies for binding in ELISA analysis.

Because direct linking, detective using ELISA, is insensitive to small and moderate changes in the affinity, a significant change in binding observed after replacement of one amino acid will likely allow you to identify key sites that interact with the antibody. In addition, polyclonal antibodies against human Ang-2 save 100% reactivity in relation to each design, which indicates that this mutagenesis procedure does not introduce any significant structural substitutions in the molecule Ang-2. All 27 of the antibody of any effect from two independent substitutions VaI at the Met at position 345 (V345M) and on His GIn at position 375 (H375Q) was not observed, indicating that these residues are not reactive, or that to effect change in conformational epitopes requires more than one amino acid replacement. The substitution of two residues in positions 365, and 367 were led to a sharp change in the level of binding of one antibody Mab 5.35.1. Sequence analysis of antibody 5.35.1 revealed one area VH3-20, and one CDR region heavy and light chains. All the connecting points of the chimeric molecules of Ang-2 and point mutations are highlighted in figure 8. The figure 9 illustrates the comparison of 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 site of cleavage for hydrophobic leader sequences. These arrows determine the limits superspiritual and fibrinogen-like domains. Shaded circles indicate conservative cysteine residues (these data were taken from publications Maisonpierre et al., 1997,Science277:55).

Data binding with all molecules Ang-2 is systematized in the following table 16:

Table 16
CloneBinBinding domainHuman ang-2-443Murine CXM/H BsmlM/H Stul 310-496Stu-Tfil 310-400V345MN365Q367H375Q
5.39.11The fibrinogen-like domain100% No100%100%25%100%100%100%
5.16.1*1The fibrinogen-like domain100%No100%100%30%100%100%100%
5.86.11The fibrinogen-like domain100%No100%100%23%100%100%100%
5.54.11The fibrinogen-like domain100%No100%100%77%100%100%100%
5.14.11 The fibrinogen-like domain100%No100%100%31%100%100%100%
5.83.11The fibrinogen-like domain100%No100%100%86%100%100%100%
5.101.11The fibrinogen-like domain100%No100%100%28%100%100%100%
6.3.17The fibrinogen-like domain100%No100%100%27%100%100% 100%
5.103.1*2The fibrinogen-like domain100%No100%100%40%100%100%100%
5.78.15The fibrinogen-like domain100%Yes100%100%100%100%100%100%
5.35.1*8The fibrinogen-like domain100%No100%100%100%100%15%100%
5.40.2*8The fibrinogen-like domain100%No100%100% 65%100%100%100%
3.19.16The fibrinogen-like domain100%Yes100%100%100%100%100%100%
5.108.1*1The fibrinogen-like domain100%No100%100%36%100%100%100%
5.52.1The fibrinogen-like domain100%Yes100%100%100%100%100%100%
5.56.13The fibrinogen-like domain100% Yes100%100%100%100%100%100%
5.2The fibrinogen-like domain100%Yes100%100%100%100%100%100%
5.28.14The fibrinogen-like domain100%No100%100%100%100%100%100%
5.41.1*1The fibrinogen-like domain100%No100%100%30%100%100%100%
5.13.1 The fibrinogen-like domain100%No100%100%27%100%100%100%
3.3.11The fibrinogen-like domain100%No100%100%15%100%100%100%
3.31.1*1The fibrinogen-like domain100%No100%100%15%100%100%100%
5.62.11The fibrinogen-like domain100%No100%100%30%100%100% 100%
3.28.1*1The fibrinogen-like domain100%No100%100%31%100%100%100%
5.88.1*1The fibrinogen-like domain100%No100%100%20%100%100%100%
3.38The fibrinogen-like domain100%Yes100%100%Not defined
delali
100%100%100%
5.49*The fibrinogen-like domain100%No100%10% 35%100%100%100%

Data are presented as the percentage of binding compared to human Ang-2.

JH6b
Table 17
The sequence analysis and cross-reactivity with murine Ang-2
Code hybridomaOD650in the analysis of inhibitionMouse Ang-2BinVHDHJHVKJK
x5.560,0863Yes3VH3-33D1-7JH4bA20JK3
h0,0792Yes2
h 0,0633Yes6VH3-30D3-3JH5bA27JK5
h*0,0588No1VH3-7D6-19JH4bL21
h0,0558No1VH3-7D6-19JH4bL21
h*0,0516No1VH3-7D6-19JH4bL21
H*0,0874No1VH3-7D6-19JH4b L21
H*0,0856No1
H0,0824No1VH1-2D6-19JH4bL21
H*0,0776No1VH3-7D6-19JH4bL21
H*0,0688No1VH3-33D1-7JH5bL21
h0,0650No1 VH3-7D6-19JH4bL21
h0,0519No1VH1-2D6-13JH4bL21
H*0,0500No1VH1-2D6-13JH4b121
H0,0484No1VH1-2D6-19JH4b121
H0,0440No1VH1-2D6-19JH4b121
H 0,0430No1VH1-2D6-19JH4b
h0,0419No1VH1-2D6-13JH4bØ12JK3
h0,0984Yes5VH1-2D2-2JH6bB3JK5
h*0,1013No2VH1-2D6-19JH4aL21
H0,0821No4VH3-43D6-19JH4bL2 JK3
h0,0691No8VH3-23D6-19JH4BL21
H*0,0663No8VH3-20D6-19JH2L2JK4
h0,0617No7VH1-2D1-7JH4bØ12JK2
H0,0744NoVH3-7D6-19JH4bL21
h0,0690YesVH3-33D5-12
h0,0627YesVH3-33D1-1JH4bL5JK4

Sequence analysis of IgH and IgL was performed using computer programs for sequence analysis and by sopostavlenija sequences of VH genes with germline sequences in the database. This computer program also allows you to analyze the elements of D, the frame is read, the paste area N, the joining of nucleotides R., loss of nucleotides and the length of the CDR3. Analysis of 27 individual antibodies specific to CR64, revealed that only 7 VH genes of these antibodies belong to the germ line, and 10 genes of these antibodies belong to the same family of VHl. The selection of neutralizing antibodies showed that these antibodies expressed the same VNIg, and in some cases, the same rearrangement of the VHDJHand that pair of H - and L-chains were conservative. These results suggest that for any given epitope, in the formation of the corresponding paratope are only a few members of reperto the RA germ line, and for each antigenic epitope, in the formation of specific paratope can participate, only a limited number of pairs of genes for L - and H-chain.

Periodic occurrence of similar structures VHVKand hypervariable regions (complementarity-determining region CDR) in different monoclonal antibodies due to the fact that all of Ang-2-neutralizing activity is limited to the fibrinogen-like domain, and this conclusion is consistent with the view expressed in the work, published Procopio et al. (1999,JBC274: 30196), which suggests that the effect of Ang-2, Tie-2 depends on the presence of fibrinogen-like domain. Data epitope mapping showed that the monoclonal antibody binds to Ang-2 through an extensive border areas, which includes most of the fibrinogen-like domain.

EXAMPLE 16

DETERMINATION of CROSS-REACTIVITY WITH MURINE ANG-2

Cross reactivity mAb against human Ang-2 from murine Ang-2 was analyzed using ELISA. For this purpose designed expression vector containing the mouse Ang-2, and eukaryotic cells were subjected to temporary transfection for producing murine Ang-2.

Expression design mouse angiopoietin-2 (mAng-2) was obtained from Research Genetics, distributor consortium I.M.A.G.E consortium (see website www/image.llnl.gov). cDNA mouse An-2 (GenBank Accession No. BC027216, IMAGE:3494566) received from the library NCI_CGAP_Lu29, which is a library of tumor cells in the lungs. cDNA cloned in the expression vector pCMV-SPORT6 (Invitrogen Carlsbad, CA) by SalI(5')and Notl(3')sites, and this vector contained an open reading frame of 496 amino acids of a full-sized mouse Ang-2 (mAng-2)and 5'- and 3'-noncoding flanking region for all 2471 pairs.

10 μg of the above mAng-2-plasmids were transferrable in HEK293F cells calcium phosphate method. Approximately, 1×106HEK293F cells were sown in 10 cm-tablet for the cultivation of tissues in one day before the start of the cultivation. 5 hours after transfection or night after transfection, the medium was replaced and cells were cultured for 2-3 days and then collected supernatant containing secreted protein mAng-2. The expression of mAng-2 was confirmed using ELISA analysis, conducted using polyclonal antibodies obtained from firm R&D Systems (catalog No. AF623).

96-well tablets Nunc Immplates was senzibilizirani conditioned medium collected from transfectants HEK293F/mouse Ang-2, 100 ál in each well. The plates were incubated at 4ºC overnight, and then four times washed with phosphate buffered saline to the flushing device scan Washer 300 (SKATRON). The wells were blocked with 100 μl of ABX-blocking buffer (0.5% of BA, 0.1% tween, 0.01% thimerosal in PBS) for 1 hour. Then the wells were added to the anti-Ang-2 mAb in appropriate concentrations, diluted in blocking buffer at a volume of 100 μl/well, and incubated at room temperature for at least 1 hour. mAb and each of their dilutions were tested in duplicate. After two washing associated mAb was detected using the HPPO-conjugated goat antibodies against human Fc (Caltag, Code Hl 0507) at 1/1000 dilution at room temperature for 1 hour. For detection chromogenic reaction was added 100 μl of TMB substrate (TMB-microwell, BioFX, Cat. No. TMSK-1000-01), and then the wells three times washed with PBS. The plates were incubated for 30 minutes and then the reaction was stopped by adding blocking solution 650 (100 µl/well, BioFX, Cat. No. BSTP-0100-01). The optical density at 650 nm was determined on a plate reader Spectramax Plus.

This analysis was tested 27 best neutralizing mAb. The optical density showed that monoclonal antibody 3.19.3, 3.38, 5.2.1, 5.52.1, 5.56.1 and 5.78.1 have the ability to bind to murine Ang-2 in experimental conditions. To confirm this, each linking antibody was titrated by ELISA. The figure 10 presents a plot of the average values OD650 nm (± cf. quot.) from the logarithmic concentrations of mAb (μg/ml). This figure also presents CLO the s 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 mouse Ang-2 was reached saturation at about 10 μg/ml (figure 10). Curves binding for these two mAb were typical sigmoidal curves of dependence “dose-response”. The dependence on the dosage and saturation was not observed in the tested antibodies in a certain range of concentrations, with the exception of clones 5.2.1 and 3.19.3. Based on these data we can conclude that cross-reactivity with murine Ang-2 had only mAb 5.2.1 and 3.19.3.

EXAMPLE 17

INHIBITION of BINDING of MOUSE ANG-2 WITH HUMAN TIE-2

Monoclonal antibody 3.19.3 was selected for further analysis of its ability to inhibit the binding of mouse Ang-2 with human Tie-2. For this purpose ELISA tablet was senzibilizirani 4 µg/ml hTie-2/Fc (R&D Systems, Inc.) at 100 μl/well, and the wells were blocked by a routine method at 4ºC overnight. Recombinant murine Ang-2 (mAng-2) was used in the supernatant of the culture 293T/mAng-2-transfectants described above. In pre-sensitised well) was added 100 μl of mAng-2-containing supernatant with mAb 3.19.3 in various concentrations and incubated at room temperature for 1 hour.

As control is also used recombinant human Ang-2 (R&D Systems, Inc.), mixed with the antibody. Each mAb concentration fathers-in-law is ovali with three replications. Related mouse and human Ang-2 were detected using goat polyclonal antibodies against human Ang-2 (Santa Cruz Biotechnology, Santa Cruz, CA), which cross-reacts with murine Ang-2, associated with the “second” HRP-anygiven rabbit antibody against goat IgG. After 30 minutes add HRP-substrate OD650 was determined. It was found that mAb 3.19.3 inhibits the binding of human and mouse Ang-2 with human Tie-2, depending on the dose (figure 11).

EXAMPLE 18

DETERMINATION of CROSS-REACTIVITY WITH the CAPILLARY NETWORK of the APES

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 this antibody cross-react with monkey Ang-2.

In this experiment evaluated the top 10 neutralizing mAb selected as described in example 4 (table 4), using isolated from monkey ovarian tissue enriched in endothelial cells. Completely drained 6 μm-slices of frozen ovarian tissue monkeys (abacadabra monkeys) were fixed with acetone at 4ºC for 5 minutes. After three PBS-wash slides, endogenous tissue peroxidase was blocked with 0.3% H2O2within 10 minutes. C is the tissue was washed in PBS and blocked 10 μg/ml goat Fab against human IgG for 15 minutes. The tissue sections were again washed in PBS, and then treated with 10% normal goat serum for 10 minutes. After draining the whey on the cuts inflicted each of the 10 anti-Ang-2 mAb (10 μg/ml) and incubated for 2 hours. Related anti-Ang-2 mAb were detected using 10 µg/ml mouse antibodies against human IgG for 15 minutes, and then incubated with conjugated with peroxidase goat antibody against mouse IgG for 30 minutes. For optimum results, staining was performed using AEC-substrate system (DAKO, Cat. No. 3464) under a microscope.

It was found that all 10 were stained with mAb angiogenic vascular endothelial cells of ovarian tissue, whereas mAb isotype control did not give such a coloring. This indicates that 10 mAb presented in table 4 cross-react with monkey Ang-2.

EXAMPLE 19

MAB 3.19.3 INHIBITSIN VIVOANGIOGENESIS IN the ANALYSIS carried out IN the MATRIX BLOCK MATRIGEL

Forin vivoassessment of possible antiangiogenic ability of a monoclonal anti-Ang-2 antibodies was performed the analysis of angiogenesis in the matrix block Matrigel. It was found that cells MCF-7, when their cultivationin vitroor when implantation in immunodeficient mice as xenograft produce Ang-2. When vvedeniye MCF-7 in the Matrigel matrix and subcutaneous implantation of the naked mice there was a steady growth of blood vessels in the gel. To obtain the model matrix block Matrigel were used 6-8-week-old female mice BALB/c/nu/nu weighing 18 to 20 g (Charles River Laboratories, Wilmington, MA). “Nude” mice were subcutaneously injected with in the right side only 0.5 ml of Matrigel containing 2×106the MCF-7 cells with anti-Ang-2 antibodies or without these antibodies, or control agents (including one Matrigel, control isotypes Tie-2/Fc, IgG2 and IgG4, and anti-VEGF mAb). Each test group consisted of five mice. The concentration of all tested mAb brought up to 100 mcg/ml

After seven days the blocks Matrigel were collected and evaluated by the density of blood vessels. For this purpose, mice were killed by displacement of the cervical vertebrae during deep anesthesia. Blocks Matrigel was exposed by removing the covering those blocks of the skin flap. Then the blocks Matrigel was removed and receive digital pictures. Blocks Matrigel carefully cut out and cut in two parts. One part was quickly frozen in TissueTek, and the other part was fixed in buffered formalin. After this, both parts were placed in paraffin to obtain slices. Three slice thickness of 5-7 microns, obtained from each mouse, cut and stained with hematoxylin and eosin. Then the sections were evaluated in a phase-contrast microscope. Were obtained representative micrograph [in two formats (100 X and 400 X)], which was evaluated for infiltration of endothelial cells and blood vessels.

p> Frozen blocks of Matrigel were used for making slices (10 mm slices) using a Cryocut microtome. For each mouse were made by two independent cutoff that was used for staining. Slices were blocked with BSA (0.1%)and then was treated with a monoclonal antibody reactive with murine CD31, conjugated with phycoerythrin (at dilutions recommended by the manufacturer). After thorough washes, the sections were placed under a reagent that prevents discoloration (Vecta Shield), and observed under UV microscope using a red filter. This has resulted in a representative digital image [in two formats (two images with magnification of 100 X and 200 X)]. Cores were subjected to contrast DAPI staining. Staining immunofluorescent images of CD31 was analyzed using the program skeletron. The obtained data were processed to calculate the values of average density, number of nodes and length of vessels for each group. The results are shown in figures 12A and 12B, where is illustrated the effect of anti-Ang-2 antibodies on the number of branches of the blood vessels (figure 12A) and on the length of the blood vessels (figure 12B).

This experiment demonstrated that, compared with the same matrix Matrigel, cells MCF-7, which were introduced in Matrigel, were able to induce a significant level of angiogen the see. Induced angiogenesis can be Engibarov anti-VEGF antibody used as a positive control. Such angiogenesis also significantly inhibited soluble recombinant protein Tie-2/Fc, suggesting that Ang-2, produced by MCF-7 cells, plays a role in angiogenesis in this model. When linking with any Ang-2, Tie-2/Fc should effectively reduce the level of Ang-2, which was available for MCF-7 cells.

However, it remains unclear how the antibody isotype IgG2, PK16.1.3 used as a negative control, affects angiogenesis, although it was also found that this antibody is sometimes prevents tumor growth in some models xenografts (data not shown). Control antibody isotype IgG4 did not exert any effect on angiogenesis in katoi model. As shown in figures 12A and 12B, the clones 5.88.3, 3.3.2, 3.19.3 and 5.28.1 significantly inhibited angiogenesis (P<0,05, t-test was conducted by the specialists of the company VasculoGen), whereas other clones gave a lesser effect.

It was well established that Ang-2 is expressed by endothelial cells in the tumor, and therefore he rassmatrivaetsya as autocrine angiogenic factor. However, it was also discovered that Ang-2 is expressed in the tumor cells of many types ofin vitroandin vivo. Tested here mAb, except the receiving 3.19.3, did not cross-react with murine Ang-2. In this model,in vivo, mAb neutralized only human Ang-2, produced by MCF-7 cells but not mouse Ang-2. The inhibitory activity of these mAb suggests that downregulation in tumor Ang-2 may be a paracrine factor in angiogenesis. General antiangiogenic activity of mAb, in addition to the neutralization of Ang-2, expressed by vascular endothelium, also makes some contribution to the neutralization of tumor Ang-2.

EXAMPLE 20

Determination of theRAPEUTIC efficacy of MAB 3.19.3 IN the MODEL WITH the PREVIOUSLY ENTERED A431 CELLS as a XENOGRAFT

Clone anti-Ang-2 mAb 3.19.3 not only been associated with murine Ang-2, but also inhibited the binding of mouse Ang-2 with human Tie-2. The antitumor activity of this monoclonal antibody has been tested in mice with model xenotransplant human cutaneous squamous cell carcinoma stem cell line A431.

The A431 cells were cultured in flasks routine method until such time as these cells did not reach subconfluent. For model development were used immunodeficiency 6-8-week-old female mice (Balb/c/nu/nu). The A431 cells were collected and suspended in Matrigel matrix. The cell suspension containing 5×106cells intradermally mice were injected with the side of the Mice were randomly distributed in different groups, each of which contained 11 mice. On the same day, and then twice a week, the mice was intraperitoneally injected with 0.5 mg of mAb 3.19.3 or antibody isotype control. 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 A431 xenograft. The average tumor volume in the group that was administered the antibody isotype control, reached approximately 1.5 cm3at the end of the experiment, whereas after 10 days after the start of experiment and at the end of the experiment the growth rate of tumors in the treated group was significantly slowed down, and the tumor size was approximately 0.5 cm3. On the 23rd day, the ratio of the volume T/C (treatment/control) was 1/3, which corresponds to 66%inhibition of tumor growth.

The results suggest that the dose used in this experiment, binding of mouse Ang-2 and blocking the binding of this ligand with its receptor Tie-2, mAb 3.16.3 able to significantly slow the growth of A431 xenograft in “Nude” mice. It is likely that the antitumor effects of monoclonal antibodies due to the inhibition of angiogenesis in the host, as demonstrated in the analyses using mA is exnovo block Matrigel. The mechanism of action, aimed at the inhibition of angiogenesis, further illustrated in example 22, where as a pharmacodynamic marker was used, the density of microvessels (MVD) of the tumor.

The mechanism of action of mAb 3.19.3 may not be limited to its blockade of the Association and subsequent signal transmission Ang-2/Tie-2. As shown in example 7, it was also discovered that this mAb binds to Ang-1 and blocks the binding of Ang-1 to Tie-2. It is interesting to note that this mAb also inhibits Ang-1-induced phosphorylation of Tie-2. It is known that Ang-1 is involved in the development of blood vessels. Comparison of activity of mAb 3.19.3 aimed at the inhibition of binding of Ang-1 to Tie-2 activity, aimed at the inhibition of binding Ang-2, Tie-2 (example 12), showed that mAb 3.19.3 is an antagonist mostly Ang-2. Not limited to any specific theory, it can be noted that dual blockade of signal transmission from Ang-2 and Ang-1 inhibits angiogenesis and thus tumor growth.

EXAMPLE 21

MAB 3.19.3 INHIBITS TUMOR GROWTH IN MODELS WITH ENGRAFTMENT by XENOGRAFT

Ang-2 is stimulated by angiogenic endothelial cells, which leads to the development of tumors of many types. There is every reason to assume that a monoclonal antibody that blocks the binding of the Association of Ang-2/Tie-2, has the ability ing biavati angiogenesis, consequently, the growth of the tumor. In this experiment it was demonstrated therapeutic efficacy of anti-Ang-2 mAb. Because mAb 3.19.3 cross-reacts with murine Ang-2/Tie-2 and neutralizes the signal transmission of mouse Ang-2/Tie-2, this mAb was selected for illustrationin vivothe efficiency of inhibition of tumor growth.

In order to determine inhibits whether anti-Ang-2 mAb 3.19.3 well as developing tumor and other tumors, that is not A-431, was used a xenograft model of human colon adenocarcinoma LoVo. Doses of Mab 3.19.3, constituting 0.5, 2 and 10 mg/kg, was injected intraperitoneally twice a week. This introduction began only when the average tumor volume reached 0.2 cm3. In developing these tumors was also demonstrated that mAb 3.19.3 has inhibitory effect compared with the control isotype. Figure 14A shows that 79%inhibition was achieved at 0.5 and 2 mg/kg (p value amounted to 0.022 and 0.027, respectively), and 75%inhibition of tumor growth was achieved at 10 mg/kg (p=0.006).

The effect of inhibition of tumor growth was reproduced on additional xenograft model of human colon adenocarcinoma SW480, which brought up to the average volume of 0.2 cm3. Although mAb 3.19.3 did not give significant effect at 0.5 mg/kg, however, was about arozena, on the 53rd day after implantation of the tumor indicated mAb at a concentration of 2 and 10 mg/kg was found in 60%inhibition of tumor growth (p=0.003 and 0.006, respectively) (figure 14B).

In General, the above results showed that the anti-Ang-2 mAb 3.19.3 significantly inhibited tumor growth in the three tested models. Interestingly, LoVo and SW480 Express human Ang-2. However, two other mAb, which had no cross-reactivity with murine Ang-2, did not provide any significant inhibitory effect on tumor growth (data not shown), despite the fact that the human Ang-2 expressively tumor cells. The obtained results allow to conclude that the antagonist of Ang-2 owner's necessary to block angiogenesis and tumor growth.

As discussed above, Mab 3.19.3 cross-reacts with Ang-1. However, the activity of mAb 3.19.3 in relation to the binding of Ang-1/Tie-2 was much lower than the activity against the binding of Ang-2/Tie-2 (Example 12). Based on this, we can conclude that therapeutic efficacy observed in these models, due mainly antagonistic effect on Ang-2. However, we cannot completely exclude the blocking of Ang-1 in these models,in vivo. During the whole experiment any noticeable toxic effects, and weight loss or what remotekey animals was not observed.

EXAMPLE 22

IN VIVOThe EFFECTIVENESS of MAB 3.19.3 IN OTHER MODELS WITH TUMOR XENOGRAFT

Antitumor activity of a monoclonal antibody 3.19.3 tested in mice with xenograft models of human cancer using 9 different tumor cell lines.

Cell adenocarcinoma of the colon (Lovo, SW480, Colo205, HT29, HCTl 16), epidermoid carcinoma (A431), carcinoma of the lung (Calu-6) and adenocarcinoma of the breast (MCF7, MDA-MB-231) were cultured in flasks routine method up until the cells reached subconfluent. For model development were used immunodeficiency 7-10-week-old female mice. Cells were collected, suspended in the matrix of the Matrigel and subcutaneously were injected with each mouse. Then mice were randomly divided into groups so that each group contained 10-12 mice. On the same day, and then twice a week, the mice was intraperitoneally injected with 0.5 mg of mAb 3.19.3 or antibodies isotype control. All experiments were conducted in the processing of the antibody isotype control. The size of each tumor was determined twice a week. Tumor volume was calculated according to the formula: volume = length × (width)2× 0,5 (cm3). A graphical comparison of the levels of inhibition of tumor growth are illustrated for HT29 xenografts (figure 15A) and Calu6 (figure 15B).

As the show is about in table 18, mAb 3.19.3 was found significant activity in all 7 tested models of subcutaneous xenografts and in both orthotopic models with sub-optimal dose and scheme administration.

Table 18
Systematicin vivothe effectiveness of mAb 3.19.3
Tumor model% inhibition% inhibition
The xenografts SubQ2 mg/kg, 2 times per week10 mg/kg, 2 times per week
Colo2053546
A4314366
HT29N.D.54
Calu6N.D.38
HCT116N.D.33
Orthopnea model
MCF735*74
MDA-MB-2315058
In all cases, P<0,05
* Inhibition of growth was not statistically significant.
N.D. - not determined

Tumor tissue MDA-MB-231 were analysed by the density of CD31 staining+-vessels. The density of CD31-staining was determined by calculating the threshold method and the method of manual counting using a coordinate grid. Analyzed eleven tumors per group and at least 20 images of the tumor. As shown in figure 15C, treatment of mice with antibody 3.19.3 led to 40%reduction in density of CD31-staining compared with staining with control antibody IgG. This result was statistically significant for both methods of calculation, namely the threshold method (p<0.015 g) and manual counting method using a coordinate grid (p<0,00004) in accordance with the one-sided t-test. Similar calculations CD31+-vessels were made on fabricex vivofor Colo205 xenografts and HCTl16. These samples were also found similar significant decrease in the density of CD31+-vessels.

EXAMPLE 23

The USE of ANTI-Ang-2 ANTIBODIES TO PREVENT ASSOCIATED WITH ANGIOGENESIS DISEASES

To assessin vivothe effectiveness of treatment of a person with different solidn the mi tumors using anti-Ang-1 and anti-Ang-2 antibody, patients were periodically introduced effective amount of anti-Ang-1 and anti-Ang-2 antibodies. In certain periods of treatment the patients seen at suppressing tumor growth. After treatment it was found that patients who were treated with anti-Ang-1 and anti-Ang-2 antibodies, compared with patients who never underwent such treatment, was observed relative improvement in one or more indicators, including, but not limited to, reduction in tumor size, slow the progression of the tumor or increased life expectancy.

EXAMPLE 24

The USE of ANTI-Ang-2 ANTIBODIES AS DIAGNOSTIC TOOLS

Detection of antigen ADF-2 sample

For detection of antigen Ang-1 or Ang-2 in a sample may be developed enzyme-linked immunosorbent assay (ELISA). In this assay on microtiter wells tablet, such as 96-well microtiter tablet or 384-well microtiter tablet, within a few hours, adsorbing first fully human monoclonal antibody against Ang-1 and Ang-2. Immobilized antibody served as antibodies to capture any of the antigen that may be present in the test sample. Then the wells were rinsed and treated with a blocking agent such as milk protein or albumi is, to prevent nonspecific adsorption of the analyte.

Then the wells were treated with a test sample, presumably containing the antigen, or a solution containing a standard quantity of a specified antigen. This sample can be, for example, a serum sample taken from the individual with suspected high levels of antigen in the bloodstream, which are indicators of pathology in the diagnosis.

After laundering the test sample or standard, the wells were treated “second” fully human monoclonal anti-Ang-1 and anti-Ang-2 antibodies, which were marked by conjugation with Biotin. Can also be used monoclonal mouse antibodies or antibodies derived from other species. Labeled anti-Ang-1 and anti-Ang-2 antibodies were used as detecting antibodies. After washing off the excess second antibody, the wells were treated with conjugated with Avidya horseradish peroxidase (HRP) and a suitable chromogenic substrate. The concentration of antigen in the test samples was determined by comparison to a standard curve constructed according to the data obtained for standard samples.

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

Determining the concentration of antigen Ang-2 in patients

For the quantitative assessment of the levels of Ang-1 and Ang-2 in human serum was developed sandwich ELISA. This “sandwich”ELISA two fully human monoclonal anti-Ang-2 antibodies recognize different epitopes on the molecule Ang-2. Alternatively, can also be used monoclonal mouse antibodies or antibodies derived from other species. At the same time, but not necessarily, may be used ELISA-analysis as described below. 50 μl of anti-Ang-2 antibodies for capture in sensitizing buffer (0.1 M NaHCO3pH of 9.6) at a concentration of 2 μg/ml were applied to the ELISA-plates (Fisher). After incubation at 4ºC overnight, tablets was treated with 200 μl of blocking buffer (0,5% BSA, 0.1% tween-20, 0.01% thimerosal in PBS) for 1 hour at 25°C. the Tablets were washed (3×) 0.05% tween-20 in PBS (washing buffer, WB). Normal serum or serum of the patient (Clinomics, Bioreclaimation) diluted in blocking buffer, containing 50%human serum. The plates were incubated with serum samples overnight at 4ºC, washed with WB, and then incubated with 100 μl/well of biotinylated detection anti-Ang-2 antibodies for 1 h at 25ºC. After washing, the plates were incubated with HRP-streptavidin for 15 minutes, washed as described above and then treated with 100 μl/well of o-phenylenediamine in H2O2(developing solution Sigma) for development of color. Then the reaction was stopped by adding 50 µl/well N2SO (2M) and analyzed on ELISA-tablet-reader at 492 nm. The concentration of antigen Ang-2 serum samples was calculated by comparison with dilutions of purified antigen Ang-2 using build 4-parametric curve.

Inclusion IN the DESCRIPTION of the INVENTION REFERENCE MATERIAL

All sources cited here, including patents, patent applications, documents, manuals, etc. and publications cited in these sources, and not yet published work, in its entirety introduced into the present description by reference.

EQUIVALENTS

The authors of the present invention believe that the above description is sufficient for realization of an average person skilled in the art. In the above description and the examples illustrated some preferred embodiments of the invention and described in detail the best way considered by the applicants. However, it should be noted that regardless of the detailed description above, the present invention can be implemented in many ways and should be interpreted in accordance with the attached claims and any equivalents.

1. Monoclonal antibody that binds to angiopoietin-2 (Ang-2), where the specified antibody contains
variable region light chain selected from the group consisting of:
A. the sequence of the light chain containing SEQ ID NO:81,
b. the sequence of the light chain containing SEQ ID NO:25,
and
C. the sequence of the light chain containing SEQ ID NO471,
and
variable region of the heavy chain selected from the group consisting of:
A. the sequence of the heavy chain, containing SEQ ID NO:79,
b. the sequence of the heavy chain, containing SEQ ID NO:23, and
C. sequence of the heavy chain, containing SEQ ID NO:469.

2. Monoclonal antibody according to claim 1, where the specified antibody contains a sequence of variable region of the light chain containing SEQ ID NO:81.

3. Monoclonal antibody according to claim 1, where the specified antibody contains a sequence of variable region of the light chain containing SEQ ID NO:25.

4. Monoclonal antibody according to claim 1, where the specified antibody contains a sequence of variable region of the light chain containing SEQ ID NO:471.

5. Monoclonal antibody according to claim 1, where the specified antibody contains a sequence of the heavy chain, containing SEQ ID NO:79.

6. Monoclonal antibody according to claim 1, where the specified antibody contains a sequence of the heavy chain, containing SEQ ID NO:23.

7. Monoclonal antibody according to claim 1, where the specified antibody contains a sequence of the heavy chain, containing SEQ ID NO:469.

8. Monoclonal antibody according to claim 1, where the specified antibody contains a component selected from the group consisting of:
A. the sequence of the variable region of the light chain containing SEQ ID NO:81, and a sequence of variable regions of the heavy chain, containing the th SEQ ID NO:79;
b. the sequence of the variable region of the light chain containing SEQ ID NO:25, and sequence of the variable region of the heavy chain, containing SEQ ID NO:23; and
C. sequence of the variable region of the light chain containing SEQ ID NO:471, and sequence of the variable region of the heavy chain, containing SEQ ID NO:469.

9. Monoclonal antibody of claim 8, where the specified antibody contains a sequence of variable region of the light chain containing SEQ ID NO:81, and a sequence of variable regions of the heavy chain, containing SEQ ID NO:79.

10. Monoclonal antibody of claim 8, where the specified antibody contains a sequence of variable region of the light chain containing SEQ ID NO:25, and the sequence of the variable region of the heavy chain, containing SEQ ID NO:23.

11. Monoclonal antibody of claim 8, where the specified antibody contains a sequence of variable region of the light chain containing SEQ ID NO:471, and the sequence of the variable region of the heavy chain, containing SEQ ID NO:469.

12. The antibody of claim 8, where the specified antibody selected from the group consisting of:
a. mAb 3.19.3, where the aforementioned antibody is produced by hybridoma deposited under the access number in ATSC MOUTH-7260;
b. mAb 3.3.2, where the aforementioned antibody is produced by hybridoma deposited under the access number in ATSC MOUTH-7258;
and
C. mAb 5.88.3, where the specified antibodies is about is produced by hybridomas, deposited under the access number in ATSC MOUTH-7259.

13. The antibody according to item 12, where the aforementioned antibody is an antibody produced by hybridomas deposited under the access number in ATSC MOUTH-7260.

14. The antibody according to item 12, where the aforementioned antibody is an antibody produced by hybridomas deposited under the access number in ATSC MOUTH-7258.

15. The antibody according to item 12, where the aforementioned antibody is an antibody produced by hybridomas deposited under the access number in ATSC MOUTH-7259.

16. Monoclonal antibody that binds to angiopoietin-2 (Ang-2), where the specified antibody contains:
A. three sites CDR heavy chain antibodies 3.19.1 presented in tabl;
b. three sites CDR light chain antibodies 3.19.1 presented in tabl.

17. The antibody according to any one of claims 1 to 16, is present in combination with a pharmaceutically acceptable carrier.

18. The nucleic acid molecule encoding the antibody according to any one of claims 1 to 16.

19. The expression vector containing the nucleic acid molecule according p.

20. A host cell to obtain antibodies containing the vector according to claim 19.

21. A method of treating a malignant tumor in an animal, comprising the introduction of a specified animal in need, a therapeutically effective dose of the antibody according to any one of claims 1 to 17.

22. The method according to item 21, where a specified animal who is the man.

23. The method according to item 21, where the specified antibody is a monoclonal antibody 3.19.3 (access number in ATSC MOUTH-7260)or 3.3.2 (access number in ATSC MOUTH-7258), or 5.88.3(access number in ATSC MOUTH-7259).

24. The method according to item 21, where the specified malignant tumor selected from the group consisting of melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), tumors of the thyroid gland, cancer of the gastrointestinal tract (stomach), prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer and squamous cell carcinoma.

25. Treatment-induced angiopoietin-2 pathological angiogenesis, including the introduction of an animal in need, a therapeutically effective dose of the antibody according to any one of claims 1 to 17.

26. The method according A.25, where a specified animal is man.

27. The method according A.25, where the specified antibody is humanitariannet monoclonal antibody.

28. The method according A.25, where the specified antibody binds to angiopoietin-1.

29. The use of antibodies according to any one of claims 1 to 17 for the preparation of drugs for the treatment of malignant tumors.

30. The application of clause 29, where the specified malignant tumor is selected is from the group consisting of: melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (liver), tumors of the thyroid gland, cancer of the gastrointestinal tract (stomach), prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer and squamous cell carcinoma.

31. The application of clause 29, where the specified antibody is a monoclonal antibody.

32. Use p, where the specified antibody is humanitariannet monoclonal antibody.

33. Use p, where the specified fully human monoclonal antibody is a fully human antibody 3.19.3 (access number in ATSC MOUTH-7260)or 3.3.2 (access number in ATSC MOUTH-7258), or 5.88.3 (access number in ATSC MOUTH-7259).
Priority items:

19.12.2005 on PP-15, 23, 33;

21.12.2004 according to claims 1-11, 16-22, 24-32;

25.08.2005 according to claims 1-11, 16-22, 24-32.



 

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