HK1195775B - Antibodies against human angiopoietin 2 - Google Patents

Description

Antibodies against human angiopoietin 2

The present application is a divisional application of chinese patent application No.201210237207.1 entitled "antibody against human angiopoietin 2" filed on 12/14/2009.

The present invention relates to antibodies against human angiopoietin 2 (anti-ANG-2 antibodies), methods for their preparation, pharmaceutical compositions comprising said antibodies, and uses thereof.

Background

Angiogenesis is involved in the pathogenesis of a variety of conditions including solid tumors, intraocular neovascular syndromes such as proliferative retinopathy or age-related macular degeneration (AMD), rheumatoid arthritis and psoriasis (Folkman, J., et al, J. biol. chem. (J. chem. biol.) (J. chem.) (1992) 10931-) -10934; Klagsbrun, M., et al, Annu. Rev. Physiol. (review of Physics) 53(1991) 217-. In the case of solid tumors, neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to normal cells. Thus, in breast cancer, as well as in some other tumors, a correlation between microvascular density in tumor sections and patient survival has been observed (Weidner, N., et al, N.Engl. J. Med. (New England journal of medicine) 324(1991)1-8; Horak, E.R., et al, Lancet340(1992) 1120-.

ANG-2 and anti-ANG-2 antibodies

Human angiopoietin-2 (ANG-2) (alternatively abbreviated ANGPT2 or ANG2) (SEQ ID NO:107) is described in Maison pierre, P.C., et al, Science 277(1997)55-60 and Cheung, A.H., et al, Genomics48(1998) 389-91. Angiopoietins-1 and-2 (ANG-1(SEQ ID NO:108) and ANG-2(SEQ ID NO:107)) were found to be ligands for Ties, a family of tyrosine kinases that are selectively expressed in vascular endothelium. Yancopoulos, G.D., et al, Nature (Nature) 407(2000) 242-48. There are now four well-defined members of the angiogenin family. Angiopoietins-3 and-4 (Ang-3 and Ang-4) may represent very different counterparts of the same locus in mice and humans. Kim, I.D., et al, FEBS Let,443(1999)353-56, Kim, I.D., et al, JBiol Chem (J. Biochem) 274(1999) 26523-28. ANG-1 and ANG-2 were originally identified as agonists and antagonists, respectively, in tissue culture experiments (for ANG-1, see: Davies, S., et al, Cell (Cell), 87(1996)1161-1169; for ANG-2, see: Maison Pierre, P.C., et al, Science 277(1997) 55-60). All angiogenin is known to bind essentially to Tie2, and Ang-1 and-2 bind to Tie2 with an affinity of 3nM (Kd). Maison pierre, p.c., et al, Science 277 (Science) (1997) 55-60. Ang-1 was shown to support EC survival and promote endothelial integrity, Davis, S., et al, Cell 87(1996) 1161-. Maison pierre, p.c., et al, Science 277 (Science) (1997) 55-60. However, many studies of ANG-2 function have shown more complex situations. ANG-2 may be a complex regulator of vascular remodeling, playing a role in both vascular sprouting and vascular regression. In support of this effect of ANG-2, expression analysis revealed that ANG-2 was rapidly induced together with VEGF in the angiogenic sprouting environment of adults, whereas ANG-2 was induced in the absence of VEGF in the angiogenic environment. Holash, J.On.et al, Science 284(1999)1994-98, Holash, J.On. 18(1999) 5356-62. Consistent with the background-dependent effect, ANG-2 specifically binds to the same endothelial-specific receptor Tie-2, Tie-2 being activated by ANG-1 but with a background-dependent effect upon activation. Maison pierre, p.c., et al, Science 277 (Science) (1997) 55-60.

Corneal angiogenesis assays have shown that ANG-1 and ANG-2 both have similar effects, acting synergistically with VEGF to promote the growth of new blood vessels. Asahara, T., et al, circ.Res.,83(1998) 233-40. The observation that high concentrations of ANG-2 also promote angiogenesis in vitro suggests the possibility that a dose-dependent endothelial response exists. Kim, I.et al, Oncogene 19(2000) 4549-52. High concentrations of ANG-2 act as an apoptotic survival factor for endothelial cells in serum starvation apoptosis, which activates Tie2 via PI-3 kinase and the Akt pathway. Kim, I.et al, Oncogene 19(2000) 4549-52.

Other in vitro experiments indicate that the effect of ANG-2 can progressively switch from an antagonist of Tie2 to its activator during sustained exposure, and that at a later point in time ANG-2 can directly promote vascular tube formation and new vessel stabilization. Teichert-Kuliszewska, k., et al, cardiovasc. res. (cardiovascular studies) 49(2001) 659-70. In addition, ANG-2 activation of Tie2 was also observed if EC was cultured on fibrin gel, possibly suggesting that ANG-2 effects are dependent on EC differentiation status. Teichert-Kuliszewska, k., et al, cardiovasc. res. (cardiovascular studies) 49(2001) 659-70. ANG-2 also induces Tie2 activation and promotes the formation of capillary-like structures in microvascular ECs cultured in three-dimensional collagen gel. Mochizuki, y, et al, j.cell.sci. (journal of cell science) 115(2002) 175-83. The use of 3-D globular co-culture as an in vitro model of vascular maturation demonstrated that direct contact of EC and mesenchymal cells abolished reactivity to VEGF, whereas the presence of VEGF and ANG-2 induced sprouting. Korff, T., et al, Faseeb J.15(2001) 447-57. Etoh, T, et al demonstrated that ANG-2 strongly upregulated MMP-1, -9 and u-PA expression in the presence of VEGF in ECs constitutively expressing Tie 2. Etoh, T., et al, Cancer Res. (Cancer research) 61(2001) 2145-53. ANG-2 in the presence of endogenous VEGF was shown by the in vivo pupillary membrane model, Lobov, i.b., et al, to promote rapid increase in capillary diameter, promote basal layer remodeling, proliferation and migration of endothelial cells, and stimulate the sprouting of new blood vessels. Lobov, I.B., et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci. USA) 99(2002) 11205-10. In contrast, ANG-2 promotes endothelial cell death and vascular degeneration in the absence of endogenous VEGF. Lobov, I.B., et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci. USA) 99(2002) 11205-10. Similarly, it was demonstrated by in vivo tumor models, Vajkoczy, P, et al, that multicellular aggregates initiate vascular growth through angiogenic sprouting that occurs via both host and tumor endothelium expression of VEGFR-2 and ANG-2. Vajkoczy, p., et al, j.clin.invest. (journal of clinical research) 109(2002) 777-85. This model suggests that established microvessels of growing tumors are characterized by continuous remodeling, presumably mediated by the expression of VEGF and ANG-2. Vajkoczy, m.a., et al, J clin. invest. (journal of clinical research) 09(2002) 777-85.

Studies of Tie-2 and angiopoietin-1 knockout mice show similar phenotypes and suggest that angiopoietin-1 stimulated Tie-2 phosphorylation mediates developmental vascular remodeling and stabilization, promotes maturation of blood vessels during angiogenesis and maintains endothelial cell-supporting cell adhesion (Dumont, D.J., et al, Genes & Development, 8(1994)1897 1909; Sato, T.N., Nature, 376(1995)70-74; (Thurston, G., et al, Nature (Nature medicine) 6(2000) 460. 463.) the role of angiopoietin-1 is believed to be conserved in adults and is widely and constitutively expressed in adults (Hanahan, D.D., Science, 1997) (277) 48-50; Zaggag, D.D., et al, ExpNeology, 159) compared to angiopoietin-2, 400-2, 2. remodeling site expression, where angiopoietin-2 is thought to block the constitutive, stable or mature function of angiopoietin-1, allowing the vessels to recover and remain in a plastic state that is more responsive to the sprouting signal (Hanahan, D.1997; Holash, J.et al, Orzcogerze18(1999)5356-62; Maison pierre, P.C., 1997). Studies of angiopoietin-2 expression in pathological angiogenesis have found that many tumor types display angiopoietin-2 expression in blood vessels (Maison pierre, P.C., et al, Science 277(1997) 55-60). Functional studies indicate that angiopoietin-2 is involved in tumor angiogenesis and that angiopoietin-2 overexpression is associated with increased tumor growth in a mouse xenograft model (Ahmad, s.a., et al, Cancer Res. (Cancer studies), 61(2001) 1255-1259). Other studies have linked angiopoietin-2 overexpression to tumor vascularization (Etoh, T., et al, Cancer Res. (Cancer research) 61(2001)2145-53; Tanaka, F., et al, Cancer Res. (Cancer research) 62(2002) 7124-29).

Angiopoietin-1, angiopoietin-2 and/or Tie-2 have been proposed in recent years as potential anti-cancer therapeutic targets. For example, each of US6,166,185, US5,650,490 and US5,814,464 discloses anti-Tie-2 ligands and receptor antibodies. Studies using soluble Tie-2 have been reported to reduce the number and size of tumors in rodents (Lin, P,1997; Lin, P., 1998). Siemester, g., et al (1999) generated a human melanoma cell line expressing the extracellular domain of Tie-2, which was injected into nude mice and reported that soluble Tie-2 caused significant inhibition of tumor growth and tumor angiogenesis. Given that both angiopoietin-1 and angiopoietin-2 bind Tie-2, it is not clear from these studies whether angiopoietin-1, angiopoietin-2 or Tie-2 would be attractive targets for anti-cancer therapy. However, effective anti-angiopoietin-2 therapy is thought to be beneficial in the treatment of diseases (such as cancer) where progression is dependent on abnormal angiogenesis, where blockade of this process prevents disease progression (Folkman, j., Nature Medicine 1, (1995) 27-31).

In addition, several groups have reported the use of antibodies and peptides that bind to angiopoietin-2. See, for example, US6,166,185 and US2003/10124129, WO03/030833, WO2006/068953, WO03/057134 or US 2006/0122370.

Studies of the focal (focal) expression effect of angiopoietin-2 have shown that antagonizing angiopoietin-1/Tie-2 signaling relaxes the tight vasculature thereby exposing EC to activation signals from angiogenesis inducers such as VEGF (Hanahan, 1997). The pro-angiogenic effect resulting from this inhibition of angiopoietin-1 indicates that anti-angiopoietin-1 therapy will not be an effective anti-cancer therapy.

ANG-2 is expressed during development at sites where vascular remodeling occurs. Maison pierre, p.c., et al, Science 277 (Science) (1997) 55-60. In adult individuals, ANG-2 expression is restricted to sites of vascular remodeling and to highly vascularized tumors, including gliomas, Osada, h, et al, int.j.oncol.18(2001)305-09; koga, k, et al, Cancer Res, (Cancer research) 61(2001)6248-54, hepatocellular carcinoma, Tanaka, s, et al, j.clin. invest, (journal of clinical research) 103(1999)341-45, gastric Cancer, Etoh, t, et al, Cancer Res, (Cancer research) 61(2001)2145-53; lee, J.H., et al, int.J.Oncol.18(2001)355-61, thyroma, Bunone, G.et al, AmJPathol155(1999)1967-76, non-small cell Lung Cancer, Wong, M.P., et al, Lung Cancer (Lung Cancer) 29(2000)11-22 and colon Cancer, Ahmad, S.A., et al, Cancer (Cancer) 92(2001)1138-43, and prostate Cancer Wurmbach, J.H., et al, Anti Cancer Res (Anti-Cancer research) 20(2000) 5217-20. Some tumor cells were found to express ANG-2. For example, Tanaka, s., et al, j.clin.invest. (journal of clinical research) 103(1999)341-45 detected ANG-2mRNA in 10 of 12 samples of human hepatocellular carcinoma (HCC). The Ellis group reported that ANG-2 is widely expressed in tumor epithelium. Ahmad, s.a., et al, Cancer 92(2001) 1138-43. Other researchers reported similar findings. Chen, L., et al, J.Tongji Med.Univ.21(2001) 228-. ANG-2mRNA was reported to be significantly associated with adjuvant lymph node invasion, short disease-free time and poor overall survival by detecting ANG-2mRNA levels, sfilogi, c, et al, int.j. cancer (international journal of cancer) 103(2003)466-74 in archived human breast cancer samples. Tanaka, f., et al, Cancer Res, (Cancer research) 62(2002)7124-29 reviewed a total of 236 patients with pathological stage I-to stage IIIA non-small cell lung Cancer (NSCLC), respectively. Using immunohistochemistry, they found 16.9% of NSCLC patients to be ANG-2 positive. Microvascular density of ANG-2 positive tumors was significantly higher than that of ANG-2 negative tumors. This angiogenic effect of ANG-2 is only visible when VEGF expression is high. Furthermore, positive expression of ANG-2 is a significant factor in predicting poor post-operative survival. Tanaka, f., et al, Cancer Res, (Cancer research) 62(2002) 7124-29. However, they did not find a significant correlation between Ang-1 expression and microvascular density. Tanaka, f., et al, Cancer Res, (Cancer research) 62(2002) 7124-29. These results indicate that ANG-2 is an indicator of patients with poor prognosis for several types of cancer.

Recently, using the ANG-2 knockout mouse model, the Yancopoulos research group reported that ANG-2 was required in postnatal angiogenesis. Gale, n.w., et al, dev.cell3(2002) 411-23. They showed that developmental programmed degeneration of the vitreous vasculature in the eye did not occur in ANG-2 knockout mice and that their retinal blood vessels did not sprout out of the central retinal artery. Gale, n.w., et al, dev.cell3(2002) 411-23. They also found that loss of ANG-2 results in a severe defect in the patterning and function of the lymphatic vasculature. Gale, n.w., et al, dev.cell3(2002) 411-23. The lymphoid defect was corrected by genetic rescue of Ang-1, but the angiogenic defect was not corrected. Gale, n.w., et al, dev.cell3(2002) 411-23.

Peters and colleagues reported that soluble Tie2, when delivered as a recombinant protein or in a viral expression vector, inhibited the in vivo growth of murine breast cancer (mammary carcinoma) and melanoma in mouse models. Lin, P.et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci.) 95(1998)8829-34; Lin, P.et al, J. Clin. invest. (J. Clin. Clest. (J. Clin. Res.) 100(1997) 2072-78. The vascular density in the tumor tissue thus treated is greatly reduced. In addition, soluble Tie2 blocked angiogenesis in rat corneas stimulated by tumor cell conditioned media. Lin, p, et al, j.clin.invest. (journal of clinical research) 100(1997) 2072-78. In addition, Isner and its panel demonstrated that addition of ANG-2 to VEGF significantly promoted longer and more peripheral neovascularization than VEGF alone. Asahara, T., et al, circ.Res.,83(1998) 233-40. Excess soluble Tie2 receptor prevented VEGF-induced neovascularization from being modulated by ANG-2. Asahara, T., et al, circ.Res.,83(1998) 233-40. Siemeister, g., et al, Cancer Res, (Cancer research) 59(1999)3185-91 the overexpression of the extracellular ligand-binding domain of Flt-1 or Tie2 in xenografts by nude mouse xenograft has been shown to result in significant inhibition of the pathway that cannot be compensated by the other, suggesting that the VEGF receptor pathway and Tie2 pathway should be considered essential mediators in two separate in vivo angiogenic processes. Siemeister, g., et al, cancer res, (cancer research) 59(1999) 3185-91. This is evidenced by the more recent disclosure of White, r.r., et al, proc.natl.acad.sci.usa (proceedings of the american national academy of sciences) 100(2003) 5028-33. In their studies, nuclease-resistant RNA aptamers (aptamers) that specifically bind to and inhibit ANG-2 were demonstrated to significantly inhibit bFGF-induced neovascularization in the rat corneal micro-pocket angiogenesis model.

Summary of The Invention

The present invention includes antibodies that specifically bind human angiopoietin-2 (ANG-2) characterized by comprising the CDR3 region of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41 or SEQ ID NO:49 as the heavy chain variable domain CDR3 region.

Preferably, the antibody is characterized by:

a) the heavy chain variable domain comprises the CDR3 region of SEQ ID NO 1, 9, 17, 25, 33, 41 or 49, the CDR2 region of SEQ ID NO 2, 10, 18, 26, 34, 42 or 50 and the CDR2 region of SEQ ID NO 3, 11, 19, 27, 35, 43 or 51 and the CDR1 region of SEQ ID NO 51 and

b) the light chain variable domain comprises the CDR3 region of SEQ ID NO 4, SEQ ID NO 12, SEQ ID NO 20, SEQ ID NO 28, SEQ ID NO 36, SEQ ID NO 44 or SEQ ID NO 52, CDR2 region of SEQ ID NO 5, SEQ ID NO 13, SEQ ID NO 21, SEQ ID NO 29, SEQ ID NO 37, SEQ ID NO 45 or SEQ ID NO 53, and the CDR1 region of SEQ ID NO 6, SEQ ID NO 14, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 38, SEQ ID NO 46 or SEQ ID NO 54.

Preferably, the antibody is characterized by comprising:

a)7, 15, 23, 31, 39, 47 or 55 and

b) 8, 16, 24, 32, 40, 48 or 56.

Preferably, the antibody is characterized in that the antibody does not specifically bind to angiopoietin 1 (ANG-1).

Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention.

Another embodiment of the invention is the use of an antibody according to the invention for the preparation of a pharmaceutical composition.

Another embodiment of the invention is the use of an antibody according to the invention for the prevention of metastasis.

Another embodiment of the invention is the use of an antibody according to the invention for the treatment of cancer.

Another embodiment of the invention is the use of an antibody according to the invention for the treatment of a vascular disease.

Another embodiment of the invention is the use of an antibody according to the invention for the treatment of retinopathy.

Another embodiment of the invention is a nucleic acid encoding a heavy chain variable domain and/or a light chain variable domain of an antibody according to the invention.

The invention also provides expression vectors containing the nucleic acids according to the invention, which are capable of expressing said nucleic acids in prokaryotic or eukaryotic host cells, and host cells containing such vectors for the recombinant production of said antibodies.

The invention also encompasses prokaryotic or eukaryotic host cells comprising a vector according to the invention.

The invention also comprises a method for producing a recombinant human or humanized antibody according to the invention, characterized in that a nucleic acid according to the invention is expressed in a prokaryotic or eukaryotic host cell and the antibody is recovered from the cell or the cell culture supernatant. The invention also includes antibodies obtainable by such recombinant methods.

The antibodies according to the invention are particularly useful for the prevention of secondary tumors/metastases or for the treatment of vascular diseases such as retinopathy.

Detailed Description

The present invention includes antibodies that specifically bind to human angiopoietin-2 (ANG-2) characterized by comprising the CDR3 region of SEQ ID NO 1, SEQ ID NO 9, SEQ ID NO 17, SEQ ID NO 25, SEQ ID NO 33, SEQ ID NO 41 or SEQ ID NO 49 as the heavy chain variable domain CDR3 region.

In one embodiment of the invention, the antibody is characterized in that

a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO 1, 9, 17, 25, 33, 41 or 49, the CDR2 region of SEQ ID NO 2, 10, 18, 26, 34, 42 or 50 and the CDR2 region of SEQ ID NO 3, 11, 19, 27, 35, 43 or 51 and the CDR1 region of SEQ ID NO 51 and

b) the light chain variable domain comprises the CDR3 region of SEQ ID NO 4, SEQ ID NO 12, SEQ ID NO 20, SEQ ID NO 28, SEQ ID NO 36, SEQ ID NO 44 or SEQ ID NO 52, CDR2 region of SEQ ID NO 5, SEQ ID NO 13, SEQ ID NO 21, SEQ ID NO 29, SEQ ID NO 37, SEQ ID NO 45 or SEQ ID NO 53, and the CDR1 region of SEQ ID NO 6, SEQ ID NO 14, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 38, SEQ ID NO 46 or SEQ ID NO 54.

Preferably, the antibody is characterized by comprising

a)7, 15, 23, 31, 39, 47 or 55 and

b) 8, 16, 24, 32, 40, 48 or 56.

Another embodiment of the invention is an antibody that specifically binds to human ANG-2, characterized in that said antibody does not specifically bind to human angiopoietin 1 (ANG-1). Typical antibodies that specifically bind to human ANG-2, but not to human ANG-1 are for example ANG2s _ R3_ LC03, ANG2s _ LC09, ANG2i _ LC06, ANG2i _ LC07, or antibodies that bind to the same epitope as ANG2s _ R3_ LC03, ANG2s _ LC09, ANG2i _ LC06, ANG2i _ LC07, ANG2i _ LC10, preferably to the same epitope as ANG2i _ LC 06. Thus, in one embodiment of the invention, an antibody that specifically binds to human angiopoietin-2 (ANG-2), but not specifically binds to human ANG-1 binds to the same epitope as ANG2s _ R3_ LC03, ANG2s _ LC09, ANG2i _ LC06, ANG2i _ LC07, ANG2i _ LC10, preferably to the same epitope as ANG2i _ LC 06. These antibodies that specifically bind to ANG-2, but do not specifically bind to ANG-1, may have improved properties such as efficacy, less toxicity, pharmacokinetic properties compared to ANG-2 and ANG-1 specific antibodies.

Thus, in one embodiment of the invention, an antibody that specifically binds to human angiopoietin-2 (ANG-2) but does not specifically bind to human ANG-1 is characterized

a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO 1, SEQ ID NO 9, SEQ ID NO 25, SEQ ID NO 33 or SEQ ID NO 49, the CDR2 region of SEQ ID NO 2, SEQ ID NO 10, SEQ ID NO 26, SEQ ID NO 34 or SEQ ID NO 50 and the CDR1 region of SEQ ID NO 3, SEQ ID NO 11, SEQ ID NO 27, SEQ ID NO 35 or SEQ ID NO 51 and

b) the light chain variable domain comprises the CDR3 region of SEQ ID NO 4, 12, 28, 36 or 52, the CDR2 region of SEQ ID NO 5, 13, 29, 37 or 53 and the CDR1 region of SEQ ID NO 6, 14, 30, 38 or 54.

Preferably, said antibody that specifically binds to human angiopoietin-2 (ANG-2) but does not specifically bind to human ANG-1 is characterized by comprising:

a)7, 15, 31, 39 or 55, and

b) 8, 16, 32, 40 or 56.

In one embodiment, the antibody according to the invention is characterized in that:

a) the heavy chain variable domain comprises the CDR3 region of SEQ ID NO 1 or SEQ ID NO 9, the CDR2 region of SEQ ID NO 2 or SEQ ID NO 10, and the CDR1 region of SEQ ID NO 3 or SEQ ID NO 11, and

b) the light chain variable domain comprises the CDR3 region of SEQ ID NO.4 or SEQ ID NO. 12, the CDR2 region of SEQ ID NO. 5 or SEQ ID NO. 13, and the CDR1 region of SEQ ID NO.6 or SEQ ID NO. 14.

In one embodiment, the antibody according to the invention is characterized by comprising

a) The heavy chain variable domain of SEQ ID NO 7 or SEQ ID NO 15, and

b) the light chain variable domain of SEQ ID NO 8 or SEQ ID NO 16.

In one embodiment, the antibody according to the invention is characterized in that:

a) the heavy chain variable domain comprises the CDR3 region of SEQ ID NO.1, the CDR2 region of SEQ ID NO.2, and the CDR1 region of SEQ ID NO.3, and

b) the light chain variable domain comprises the CDR3 region of SEQ ID NO.4, the CDR2 region of SEQ ID NO. 5, and the CDR1 region of SEQ ID NO. 6.

In one embodiment, the antibody according to the present invention is characterized by comprising:

a) the heavy chain variable domain of SEQ ID NO 7, and

b) the light chain variable domain of SEQ ID NO 8.

In one embodiment, the antibody according to the invention is characterized in that:

a) the heavy chain variable domain comprises the CDR3 region of SEQ ID NO 17, the CDR2 region of SEQ ID NO 18, and the CDR1 region of SEQ ID NO 19, and

b) the light chain variable domain comprises the CDR3 region of SEQ ID NO:20, the CDR2 region of SEQ ID NO:21, and the CDR1 region of SEQ ID NO: 22.

In one embodiment, the antibody according to the present invention is characterized by comprising:

a) the heavy chain variable domain of SEQ ID NO 23, and

b) 24, and a light chain variable domain of SEQ ID NO.

Preferably, the antibody according to the invention is characterized in that the antibody is of the subclass human IgG1 or human IgG 4.

The term "antibody" encompasses various forms of antibody structures, including, but not limited to, intact antibodies and antibody fragments. The antibodies according to the invention are preferably humanized, chimeric or otherwise genetically engineered antibodies, as long as the characteristic properties according to the invention are still retained.

An "antibody fragment" comprises a portion of a full length antibody, preferably its variable domain, or at least its antigen binding site. Examples of antibody fragments include diabodies, single chain antibody molecules (scFv or scFab) and multispecific antibodies (e.g., bispecific) formed from antibody fragments. ScFv antibodies are described, for example, in Houston, J.S., Methods in Enzymol (Methods in enzymology) 203(1991) 46-88. Furthermore, antibody fragments comprise single chain polypeptides having V_HThe characteristics of the domains are such that,

i.e. can be connected with V_LThe domains are assembled together to form a functional antibodyOriginal binding site and thus providing properties, or having V binding to ANG-2_LFeatures of the domains, i.e. capable of interacting with V_HThe domains assemble together to form a functional antigen binding site and thereby provide properties. The ScFvs may be stabilized using the following: a) disulfide stabilization (see, e.g., WO94/029350, Rajagopal, V., et al, prot. Engin. (1997)1453-59; Kobayashi, H., et al, Nuclear Medicine&Biology (nuclear medicine and Biology), Vol 25, (1998)387-393; or Schmidt, M., et al, Oncogene (1999) 181711-1721) or b) a stable framework (e.g.by specific mutagenesis, see for example WO2007/109254, specific stable frameworks, see for example US7,258,985, Furrer, F., et al, invest. Ophthalmol. Vis. Sci.50(2009), p.771-778 or Ottiger, M., et al, invest. Ophthalmol. Vis. Sci.50(2009), p.779-786.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules consisting of a single amino acid.

The term "chimeric antibody" refers to an antibody that includes a variable, i.e., binding, region from one source or species, and at least a portion of a constant region from a different source or species, typically prepared by recombinant DNA techniques. Chimeric antibodies comprising murine variable regions and human constant regions are preferred. Other preferred forms of "chimeric antibodies" encompassed by the invention are those in which the constant regions have been modified or altered from the constant regions of the original antibody to produce properties according to the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding. Such "chimeric" antibodies are also referred to as "class switch antibodies". Chimeric antibodies are the product of an expressed immunoglobulin gene that includes DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for making chimeric antibodies include conventional recombinant DNA and gene transfection techniques well known in the art. See, for example, Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA 81(1984)6851-6855, US5,202,238 and 5,204,244.

The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDRs) have been modified to include CDRs from an immunoglobulin that are specifically different from the parent immunoglobulin. In a preferred embodiment, murine CDRs are grafted onto the framework regions of a human antibody to make a "humanized antibody". See, e.g., Riechmann, L., et al, Nature 332(1988) 323-327; and Neuberger, M.S., et al, Nature 314(1985) 268-270. Particularly preferred CDRs correspond to those representative sequences which recognize the antigens indicated above for the chimeric antibodies. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have additionally been modified or altered from the constant regions of the original antibody to produce properties in accordance with the present invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.

As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, m.a., and van de Winkel, j.g., current chemical biology views (curr. opin. chem.biol.). 5(2001) 368-. Human antibodies can also be produced in transgenic animals (e.g., mice) that, when immunized, are capable of producing all or selected portions (selections) of the human antibody in the absence of endogenous immunoglobulin production. Transfer of a human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci. USA) 90(1993)2551-2555; Jakobovits, A., et al, Nature (Nature) 362(1993)255-258; Brueggemann, M., et al, Yeast Immunol. (Annuology Year 7 (1993)) 33-40). Human antibodies can also be generated in phage display libraries (Hoogenboom, H.R., and Winter, G., J.mol.biol. (J.Mol.Biol.) (J.M.biol.) 227(1992) 381-. The techniques of Cole, S.P.C., et al and Boerner, et al can also be used to prepare human Monoclonal Antibodies (Cole, S.P.C., et al, Monoclonal Antibodies and Cancer Therapy, Liss A.R., (1985)77-96; and Boerner, P.et al, J.Immunol.147 (1991) 86-95). As already mentioned for the chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein also includes antibodies which are modified in the constant region to produce the properties according to the invention, in particular with regard to C1q binding and/or FcR binding, for example by "class switching", i.e.by altering or mutating the Fc part (for example by mutation from IgG1 to IgG4 and/or IgG1/IgG 4.)

As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies isolated from host cells, such as NS0 or CHO cells, or from transgenic animals (e.g., mice) of human immunoglobulin genes, or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions in rearranged form. Recombinant human antibodies according to the invention have undergone somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of a recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur in vivo in human antibody germline repertoires.

"variable Domain" (light chain (V)_L) Of (2), the heavy chain (V)_H) Variable domains of (a) variable light and heavy chains have the same general structure and each domain comprises 4 Framework (FR) regions, the sequences of which are generally conserved, which are linked by 3 "hypervariable regions" (or complementarity determining regions, CDRs).

As used herein, the term "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody includes amino acid residues from "complementarity determining regions" or "CDRs". The "framework" or "FR" regions are those variable domain regions other than the hypervariable region residues defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. In particular, the CDR3 of the heavy chain is the region that is most conducive to antigen binding and defines the properties of the antibody. The CDR and FR regions, and/or those residues from the "hypervariable loops", are determined according to the standard definition of the protein sequence of Immunological Interest (Sequences of Proteins of Immunological Interest), 5 th edition, Public Health services, National Institutes of Health, Bethesda, Md. (1991), and National Health research institute.

The term "nucleic acid" or "nucleic acid molecule", as used herein, is intended to include both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

The term "amino acid" as used herein refers to the group of naturally occurring carboxy alpha amino acids, which includes alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide, provided that it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence, provided that it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence, provided that it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are co-linear and, in the case of secretory leader sequences, contiguous and in open reading frame. However, enhancers need not be contiguous. Ligation is achieved by ligation at convenient restriction sites. If the site is not present, synthetic oligonucleotide aptamers or linkers are used according to conventional practice.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all of these designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and the culture derived therefrom, regardless of the number of transfers. It is also understood that the DNA content of all progeny may not be exactly the same due to deliberate or inadvertent mutations. Variant progeny selected for the same function or biological activity in the originally transformed cell are included.

As used herein, the term "binding" or "specific binding" refers to the binding of an antibody to an epitope of an antigen (ANG-2) in an in vitro assay, preferably in a plasma resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) with a purified wild-type ANG-2 antigen (example 3). The affinity of binding is defined by the term ka (rate constant for association of antibody from antibody/antigen complex), k_D(dissociation constant) and K_D(kD/ka). Binding or specific binding means 10^-8Less than mol/l, preferably 10^-9M to 10^-13Binding affinity (K) in mol/l_D)。

Binding of antibodies to Fc γ RIII can be studied by BIAcore assay (GE-Healthcare, Uppsala, Sweden). The affinity of binding is defined by the term ka (rate constant for association of antibody from antibody/antigen complex), k_D(dissociation constant) and K_D(k_D/ka) definition.

As used herein, the term "does not bind ANG-1" or "does not specifically bind ANG-1" means that the antibody has an EC50 value of greater than 8000ng/ml in an in vitro ANG-1 binding ELISA assay (according to example 2).

The term "epitope" includes any polypeptide determinant capable of specifically binding to an antibody. In certain embodiments, epitope determinants include chemically active surface groupings (groups) of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, which may, in certain embodiments, have particular three-dimensional structural characteristics, and/or particular charge characteristics. An epitope is a region of an antigen that is bound by an antibody.

The "Fc portion" of an antibody is not directly involved in binding of the antibody to an antigen, but rather exhibits a distinct effector function. The "Fc portion of an antibody" is a term well known to the skilled artisan and is defined based on the papain cleavage of the antibody. Antibodies or immunoglobulins are classified into the following classes according to the amino acid sequence of the constant region of their heavy chains: IgA, IgD, IgE, IgG, and IgM, and some of them can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA 2.

Depending on the heavy chain constant region, different classes of immunoglobulins are designated α, γ and μ, respectively. The Fc portion of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, C1q binding and Fc receptor binding. Complement activation (CDC) is initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. Although the effect of antibodies on the complement system depends on certain conditions, binding to C1q is caused by a defined binding site in the Fc portion. Such binding sites are known in the art and are described, for example, by Boakle, R.J., et al Nature 282 (1975) 743, Lukas, T.J., et al, J.Immunol 127(1981) 2555-. Such binding sites are for example L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat, see below). Antibodies of subclasses IgG1, IgG2 and IgG3 generally show complement activation, and bind to C1q and C3, whereas IgG4 does not activate the complement system and does not bind to C1q and C3.

The antibody according to the invention preferably comprises an Fc part from human origin, which is that of a human antibody of subclass IgG 1.

The antibodies according to the invention are characterized by constant chains of human origin. Such invariant chains are well known in the art and are described, for example, by Kabat, E.A. (see, e.g., Johnson, G. and Wu, T.T., Nucleic Acids Res. (Nucleic Acids research) 28(2000) 214-218). For example, a useful human heavy chain constant region comprises the amino acid sequence of SEQ ID NO. 57 or the amino acid sequence of SEQ ID NO. 58. For example, useful human light chain constant regions comprise the amino acid sequence of the kappa-light chain constant region of SEQ ID NO:59 or the amino acid sequence of the lambda-light chain constant region of SEQ ID NO: 60.

The term "constant region" as used herein refers to the sum of the domains of an antibody, except for the variable region. The constant regions are not directly involved in antigen binding, but exhibit different effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are classified into the following categories: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses such as IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA 2. The heavy chain constant regions corresponding to different classes of antibodies are referred to as α, γ and μ, respectively. The light chain constant regions that can be found in all 5 antibody species are termed kappa (kappa) and lambda (lambda).

The term "constant region from human origin" as used herein refers to the constant heavy chain region and/or constant light chain kappa region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG 4. Such constant regions are well known in the art and are described, for example, by Kabat, E.A. (see, e.g., Johnson, G. and Wu, T.T., Nucleic Acids research (Nucleic Acids Res.)28(2000) 214-.

While antibodies of the IgG4 subclass showed reduced Fc receptor (Fc γ RIIIa) binding, antibodies of the other IgG subclasses showed strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc sugar), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues which, if altered, also provide reduced Fc receptor binding (Shields, R.L., et al, J.Biol.Chem. (J.Chem.) (276 (2001)6591-6604; Lund, J., et al, FASEB J.9(1995)115-119; Morgan, A., et al, Immunology (Immunology)86 1995)319-324; EP 0307434).

In one embodiment, the antibody according to the invention has reduced FcR binding compared to the IgG1 antibody, and the monospecific bivalent parent antibody is involved in FcR binding of the IgG4 subclass or of the IgG1 or IgG2 subclass with mutations in S228, L234, L235 and/or D265, and/or comprises a PVA236 mutation. In one embodiment, the mutation in the monospecific bivalent parent antibody is S228P, L234A, L235A, L235E and/or PVA 236. In another embodiment, the mutation in the monospecific bivalent parent antibody is S228P in IgG4 and L234A and L235A in IgG 1. The constant heavy chain regions are shown in SEQ ID NO:57 and 58. In one embodiment, the constant heavy chain region of the monospecific bivalent parent antibody is the constant heavy chain region of SEQ ID No. 57 with mutations L234A and L235A. In another embodiment, the constant heavy chain region of the monospecific bivalent parent antibody is the constant heavy chain region of SEQ ID NO 58 with the mutation S228P. In another embodiment, the constant light chain region of the monospecific bivalent parent antibody is a kappa light chain region of SEQ ID NO 59 or a lambda light chain constant region of SEQ ID NO 60. In one embodiment of the invention, the constant heavy chain region of the monospecific bivalent parent antibody is the constant heavy chain region of SEQ ID NO. 57 or SEQ ID NO. 58 with mutation S228P.

The constant regions of antibodies are directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by the binding of complement factor C1q to the constant regions of most IgG antibody subclasses. Binding of C1q to antibodies results from defined protein-protein interactions at the so-called binding site. Such constant region binding sites are known in the art and are described, for example, by Lukas, T.J., et al, J.Immunol. (J.Immunol.). 127(1981)2555- & 2560; Brunhouse, R., and Cebra, J.J., molecular Immunology (mol.Immunol.). 16(1979) 907; Burton, D.R., et al, Nature (Nature)288(1980)338- & 344; Thommesen, J.E., et al, molecular Immunology (mol.Immunol. & 37 & 2000)995- & 1004; usogie, E.E., Id.J. (J.Mumunol. & 164) 4178- & 4184; Hezareh, M.et al, J.Virol. & 68; 2001.164) 164; Immunol. & 12186; EP 12186; Brunhusung, J.J.Immunol. & 324; III. & 83; EP 12134). The constant region binding site is for example characterized by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).

The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to the lysis of human target cells by an antibody according to the invention in the presence of effector cells. ADCC is preferably measured by treating a preparation of CCR5 expressing cells with an antibody according to the invention in the presence of effector cells, such as freshly isolated PBMCs or purified effector cells from a dark yellow overlay, such as monocytes or Natural Killer (NK) cells or permanently growing NK cell lines.

The term "Complement Dependent Cytotoxicity (CDC)" refers to the process initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. Binding of C1q to antibodies results from defined protein-protein interactions at the so-called binding site. These Fc part binding sites are known in the art (see above). These Fc moiety binding sites are for example characterized by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat). Antibodies of subclasses IgG1, IgG2, and IgG3 generally show complement activation including C1q and C3 binding, while IgG4 does not activate the complement system and does not bind C1q and/or C3.

The antibodies according to the invention are produced by recombinant means. Thus, one aspect of the invention is a nucleic acid encoding an antibody according to the invention, and another aspect is a cell comprising said nucleic acid encoding an antibody according to the invention. Methods for recombinant production are widely known in the art and involve protein expression in prokaryotic and eukaryotic cells, followed by antibody isolation and often purification to pharmaceutical purity. For expression of the foregoing antibodies in a host cell, the nucleic acids encoding the respective modified light and heavy chains are inserted into the expression vector by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER. C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or lysed cells). General methods for the recombinant production of antibodies are well known in the art and are described, for example, in the review articles by Makrides, S.C., Protein Expr.Purif.17(1999) 183-.

The antibodies according to the invention are suitably isolated from the culture medium by conventional immunoglobulin purification methods such as, for example, protein a-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional methods. Hybridoma cells can serve as a source of the DNA and RNA. Once isolated, the DNA may be inserted into an expression vector which is then transfected into host cells that do not otherwise produce immunoglobulins, such as HEK293 cells, CHO cells, or myeloma cells, to obtain synthesis of recombinant monoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of the antibodies according to the invention are prepared by introducing appropriate nucleotide changes into the antibody DNA, or by nucleotide synthesis. However, such modifications may be made only within a very limited range, for example as described above. For example, the modifications do not alter the antibody characteristics mentioned above, such as IgG isotype and antigen binding, but may improve the yield of recombinant production, protein stability or facilitate purification.

The term "host cell" as used herein refers to any kind of cellular system that can be engineered to produce antibodies according to the present invention. In one embodiment, HEK293 cells and CHO cells are used as host cells. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all of these designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and the culture from which it was derived, regardless of the number of transfers. It is also understood that the DNA content of all progeny may not be exactly consistent due to deliberate or inadvertent mutations. Variant progeny selected for the same function or biological activity in the originally transformed cell are included.

Expression in NS0 cells is described, for example, in Barnes, L.M., et al, cytotechnologics (Cytotechnology)32(2000)109-123; Barnes, L.M., et al, Biotechnology and bioengineering (Biotech. Bioeng.)73(2001) 261-270. Transient expression is described, for example, in Durocher, y., et al, nucleic acid research (nucleic acids. res.)30(2002) E9. Cloning of variable domains is described in Orlandi, R.et al, Proc.Natl.Acad.Sci.USA 86(1989)3833-3837, Carter, P.et al, Proc.Natl.Acad.Sci.USA 89(1992)4285-4289, and Norderhaug, L.et al, J.Immunol.methods 204(1997) 77-87. Preferred transient expression systems (HEK293) are described in Schlaeger, E.J., and Christensen, K., in Cytotechnology (Cytotechnology)30(1999)71-83 and Schlaeger, E.J., in journal of immunological methods (J.Immunol.methods)194(1996) 191-199.

Control sequences suitable for use in prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide, provided that it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence, provided that it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence, provided that it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Ligation is achieved by ligation at convenient restriction sites. If the site is not present, synthetic oligonucleotide aptamers or linkers are used according to conventional practice.

Purification of the antibody to eliminate cellular components or other contaminants, such as other cellular nucleic acids or proteins, is performed by standard techniques, including alkali/SDS treatment, CsCl fractionation (CsCl banding), column chromatography, agarose gel electrophoresis, and other techniques known in the art. See Ausubel, F., et al, eds., methods in modern Molecular Biology (Current protocols in Molecular Biology), Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widely used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein a or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin) and mixed mode exchange), thiophilic adsorption (e.g. with β -mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resin, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with ni (ii) -and cu (ii) -affinity materials), size exclusion chromatography and electrophoretic methods (e.g. gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, m., a. application biochemistry technology (appl. biochem. biotech).

The invention comprises a method for treating a patient in need of treatment, characterized in that a therapeutically effective amount of an antibody according to the invention is administered to said patient.

The invention includes the use of an antibody according to the invention for therapy.

The invention includes the use of an antibody according to the invention for the preparation of a medicament for the prevention of metastasis.

The invention includes the use of an antibody according to the invention for the manufacture of a medicament for the treatment of cancer.

One aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention. Another aspect of the invention is the use of an antibody according to the invention for the preparation of a pharmaceutical composition. Another aspect of the invention is a method for preparing a pharmaceutical composition comprising an antibody according to the invention. In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising an antibody according to the invention formulated with a pharmaceutically acceptable carrier.

Another aspect of the invention is said pharmaceutical composition for preventing metastasis.

Another aspect of the invention is an antibody according to the invention for use in the prevention of metastasis.

Another aspect of the invention is the use of an antibody according to the invention for the preparation of a medicament for the prevention of metastasis.

Another aspect of the invention is a method of preventing metastasis in a patient suffering from a primary cancer by administering an antibody according to the invention to a patient in need of such prophylactic treatment.

We can show highly effective prevention in vivo of spontaneous metastasis/secondary tumors in situ and subcutaneous cancer models (see example 9) (in contrast to experimental models in which tumor cells are injected intravenously). This is similar to the clinical situation where cells disseminate from a primary tumor and metastasize to a secondary organ such as the lung or liver where the secondary tumor is located.

The term "metastasis" according to the invention refers to the spread of cancer cells from a primary tumor to one or more other sites in a patient where secondary tumors then develop. Metastatic modalities (MetastasMeans) to determine whether a cancer metastasizes are known in the art and include bone scans, chest X-ray examinations, CAT scans, MRI scans, and tumor marker detection.

The term "preventing metastasis" or "preventing secondary tumor" as used herein has the same meaning and refers to a prophylactic agent against metastasis in a patient suffering from recurrent HER2 positive cancer in such a way as to inhibit or reduce further spread of cancer cells from the primary tumor to one or more other sites in the patient. This means that metastasis of the primary tumor or cancer is prevented, delayed or reduced and thus the development of secondary tumors is prevented, delayed or reduced. Preferably, metastasis of the lung, i.e. secondary tumors of the lung, is prevented or reduced, which means that metastatic spread of cancer cells from the primary tumor to the lung is prevented or reduced.

Another aspect of the invention is said pharmaceutical composition for use in the treatment of cancer.

Another aspect of the invention is an antibody according to the invention for use in the treatment of cancer.

Another aspect of the invention is the use of an antibody according to the invention for the preparation of a medicament for the treatment of cancer.

Another aspect of the invention is a method of treating a patient suffering from cancer by administering an antibody according to the invention to a patient in need of such treatment.

As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and adsorption delaying agents, and the like, that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).

The compositions of the present invention may be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or manner of administration will vary depending on the desired result. In order to administer a compound of the present invention by certain routes of administration, it may be desirable to coat the compound with, or co-administer the compound with, a material that prevents its inactivation. For example, the compound may be administered to a subject in a suitable carrier, such as a liposome or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Such media and agents are known in the art for pharmaceutically active substances.

The terms "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, sub-cuticular (subcuticular), intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

The term cancer as used herein refers to proliferative diseases such as lymphoma (lymphoma), lymphocytic leukemia (lymphocytic leukemia), lung cancer (lung cancer), non-small cell lung (NSL) cancer (non-small cell lung (NSCL) cancer), bronchoalveolar cell lung cancer (bronchololar lung cancer), bone cancer (bone cancer), pancreatic cancer (pancreatic cancer), skin cancer (skin cancer), head or neck cancer (cancerous the head cancer), skin or intraocular melanoma (cutaneous or intraocular tumor), uterine cancer (uterine cancer), ovarian cancer (ovarian cancer), rectal cancer (rectal cancer), anal region cancer (cancerous the ovarian cancer), gastric cancer (gastric cancer), ovarian cancer (ovarian cancer), uterine cancer (cervical cancer), uterine cancer (uterine cancer of uterine cancer), uterine cancer (cervical cancer), uterine cancer (uterine cancer of the colon cancer), uterine cancer (uterine cancer of the colon cancer (uterine cancer), uterine cancer (uterine cancer of the colon cancer), uterine cancer (uterine cancer of the uterine cancer (uterine cancer), uterine cancer of the uterine cancer (uterine cancer of the, vaginal cancer (cancer of the vagina), vulvar cancer (cancer of the vulva), Hodgkin's Disease, esophageal cancer (cancer of the esophagus), small intestine cancer (cancer of the small intestine), endocrine system cancer (cancer of the endocrine system), thyroid cancer (cancer of the thyroid gland), parathyroid cancer (cancer of the parathyroid gland), adrenal cancer (cancer of the adrenal gland), soft tissue sarcoma (cancer of the bladder), urethral cancer (cancer of the urethra), penile cancer (cancer of the penile cancer), prostate cancer (prostate), bladder cancer (cancer of the bladder, renal cancer or central mucosa), renal cancer (cancer of the renal mucosa of the renal system), renal cancer (cancer of the renal carcinoma of the renal mucosa), renal cancer of the renal cancer (cancer of the renal mucosa of the renal system, renal cancer (cancer of the renal carcinoma of the biliary system), renal cancer (cancer of the renal carcinoma of the biliary system, renal carcinoma (cancer of the renal carcinoma of the biliary system, renal carcinoma of the urinary tract, renal carcinoma of the urinary tract of the urinary system, renal carcinoma of the urinary tract, vertebral axial tumors (vertebral axistmoms), brain stem glioma (brain stem glioma), glioblastoma multiforme (glioblastomas), astrocytoma (astrocytoma), schwanomas (schwanomas), ependymomas (ependoymonas), medulloblastomas (medulloblastomas), meningiomas (meningiomas), squamous cell carcinoma (squamomus cell carcinomas), pituitary adenomas (pituitary adenomas), and Ewing's tumor (Ewing's), including refractory forms of any of the foregoing cancers, or combinations of one or more of the foregoing cancers.

Another aspect of the invention is said pharmaceutical composition as an anti-angiogenic agent. Such anti-angiogenic agents are useful in the treatment of cancer (particularly solid tumors) and other vascular diseases.

Another aspect of the invention is the use of an antibody according to the invention for the preparation of a medicament for the treatment of a vascular disease.

Another aspect of the invention is an antibody according to the invention for use in the treatment of a vascular disease.

A preferred embodiment is an antibody according to the invention for use in the treatment of retinopathy.

A preferred embodiment is the use of an antibody according to the invention for the preparation of a medicament for the treatment of retinopathy.

Another aspect of the invention is a method of treating a patient suffering from a vascular disease by administering an antibody according to the invention to a patient in need of such treatment.

The term "vascular diseases" includes Cancer, Inflammatory diseases, Atherosclerosis, Ischemia (Ischemia),

trauma (Trauma), Sepsis (Sepsis), COPD, Asthma (athma), Diabetes (Diabetes), AMD, Retinopathy (retinitis), Stroke (Stroke), obesity (adiposis), Acute lung injury (Acute lung), Hemorrhage (Hemorrhage), Vascular leakage (Vascular leak) such as cytokine-induced Vascular leakage, Allergy (Allergy), Graves ' Disease, Hashimoto ' Autoimmune Thyroiditis (Hashimoto ' Autoimmune Thyroiditis), Idiopathic thrombocytopenic purpura (idiophathic purpura), Giant Cell Arteritis (Giant Arteritis), rheumatoid arthritis (Rheumatoid arthritis), Systemic Lupus Erythematosus (Systemic Lupus Erythematosus), ileocervous Sclerosis (Systemic Sclerosis), neonatal macular degeneration (Systemic Sclerosis), Acute lung injury (Acute macular degeneration), Acute macular degeneration (Systemic Sclerosis), Systemic Sclerosis, or Multiple Sclerosis), particularly Acute macular degeneration (Systemic Sclerosis), Systemic Sclerosis (Systemic Sclerosis, or Multiple Sclerosis) (Systemic Sclerosis, such as Systemic Sclerosis, Acute macular degeneration of the lung Disease, Acute macular degeneration (Systemic Sclerosis, rheumatoid arthritis), rheumatoid arthritis (Systemic Lupus Erythematosus, rheumatoid arthritis, Systemic Lupus Erythematosus, Systemic Macromolecular Degeneration (AMD)), rheumatoid arthritis (rhematoid arthritis), and psoriasis (psioriasis). (Folkman, J., et al, J.biol. chem. (J. chem. Biol.) (267 (1992) 10931-), (Klagsbrun, M., et al, Annu.Rev. Physiol. (annual review of Physics) 53 (1991)) 217-) -239; and Garner, A.vascurdiseas (vascular disease) in: Pathobiology of ocular disease, A dynamic assessment (pathology of the ophthalmic disease, i.e. of the kinetic pathway), Garner, A.and Klintworth, G.K. (eds.) second edition Marcel Dekker, New York, (1994), page 1625-.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization methods, see above and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay adsorption, such as aluminum monostearate and gelatin.

Regardless of the route of administration chosen, the compounds of the invention may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the invention may be formulated into pharmaceutical dosage forms by conventional means known to those skilled in the art.

The actual dosage level of the active ingredient in the pharmaceutical composition of the invention may be varied so as to obtain an amount of the active ingredient which is effective to obtain the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, body weight, condition, general health and previous medical history of the patient to be treated, and like factors known in the medical arts.

The composition must be sterile and flowable to the extent that the composition can be delivered by syringe. In addition to water, the carrier is preferably an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all of these designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and the culture derived therefrom, regardless of the number of transfers. It is also understood that the DNA content of all progeny may not be exactly the same due to deliberate or inadvertent mutations. Variant progeny selected for the same function or biological activity in the originally transformed cell are included. Where different names are intended, they will be clear by context.

The term "transformation" as used herein refers to the process of transferring a vector/nucleic acid into a host cell. If cells without a difficult cell wall barrier are used as host cells, transfection is carried out, for example, by the calcium phosphate precipitation method as described by Graham, F.L., and van der Eb.virology 52(1973) 456-467. However, other methods of introducing DNA into cells may also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells comprising a substantial cell wall structure are used, one method of transfection is, for example, calcium treatment with calcium chloride, as described by Cohen, F.N, et al, PNAS (proceedings of the national academy of sciences USA) 69(1972)7110 ff.

As used herein, "expression" refers to the process of transcribing a nucleic acid into mRNA and/or the subsequent translation of the transcribed mRNA (also referred to as a transcript) into a peptide, polypeptide or protein. The transcripts and the encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA.

A "vector" is a nucleic acid molecule, particularly self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily to insert DNA or RNA into a cell (e.g., chromosomal integration), replicating vectors that function primarily to replicate DNA or RNA, and expression vectors that function to transcribe and/or translate DNA or RNA. Also included are vectors that provide more than one of the above functions.

An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, is capable of being transcribed and translated into a polypeptide. An "expression system" generally refers to an appropriate host cell that includes an expression vector that can be manipulated to produce a desired expression product.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is to be understood that modifications may be made to the proposed method without departing from the spirit of the invention.

Description of the amino acid sequence

1 heavy chain CDR3, < ANG-2> Ang2i _ LC06 of SEQ ID NO

2 heavy chain CDR2, < ANG-2> Ang2i _ LC06 of SEQ ID NO

3 heavy chain CDR1, < ANG-2> Ang2i _ LC06 of SEQ ID NO

4 light chain CDR3, < ANG-2> Ang2i _ LC06 of SEQ ID NO

5 light chain CDR2, < ANG-2> Ang2i _ LC06 of SEQ ID NO

6 light chain CDR1, < ANG-2> Ang2i _ LC06 of SEQ ID NO

7 heavy chain variable Domain, < ANG-2> Ang2i _ LC06

8 light chain variable Domain, < ANG-2> Ang2i _ LC06

9 heavy chain CDR3, < ANG-2> Ang2i _ LC07 of SEQ ID NO

10 heavy chain CDR2, < ANG-2> Ang2i _ LC07 of SEQ ID NO

11 heavy chain CDR1, < ANG-2> Ang2i _ LC07 of SEQ ID NO

12 light chain CDR3, < ANG-2> Ang2i _ LC07 of SEQ ID NO

13 light chain CDR2, < ANG-2> Ang2i _ LC07 of SEQ ID NO

14 light chain CDR1, < ANG-2> Ang2i _ LC07 of SEQ ID NO

15 heavy chain variable domain, < ANG-2> Ang2i _ LC07

16 light chain variable Domain, < ANG-2> Ang2i _ LC07

17 heavy chain CDR3, < ANG-2> Ang2k _ LC08 of SEQ ID NO

18 heavy chain CDR2, < ANG-2> Ang2k _ LC08 of SEQ ID NO

19 heavy chain CDR1, < ANG-2> Ang2k _ LC08 of SEQ ID NO

20 light chain CDR3, < ANG-2> Ang2k _ LC08 of SEQ ID NO

21 light chain CDR2, < ANG-2> Ang2k _ LC08 of SEQ ID NO

22 light chain CDR1, < ANG-2> Ang2k _ LC08 of SEQ ID NO

23 heavy chain variable domain, < ANG-2> Ang2k _ LC08

24 light chain variable domain, < ANG-2> Ang2k _ LC08

25 heavy chain CDR3, < ANG-2> Ang2s _ LC09 of SEQ ID NO

26 heavy chain CDR2, < ANG-2> Ang2s _ LC09 of SEQ ID NO

27 heavy chain CDR1, < ANG-2> Ang2s _ LC09 of SEQ ID NO

28 light chain CDR3, < ANG-2> Ang2s _ LC09 of SEQ ID NO

29 light chain CDR2, < ANG-2> Ang2s _ LC09 of SEQ ID NO

30 light chain CDR1, < ANG-2> Ang2s _ LC09 of SEQ ID NO

31 heavy chain variable domain, < ANG-2> Ang2s _ LC09

32 light chain variable domain, < ANG-2> Ang2s _ LC09

33 heavy chain CDR3, < ANG-2> Ang2i _ LC10 of SEQ ID NO

34 heavy chain CDR2, < ANG-2> Ang2i _ LC10 of SEQ ID NO

35 heavy chain CDR1, < ANG-2> Ang2i _ LC10 of SEQ ID NO

36 light chain CDR3, < ANG-2> Ang2i _ LC10 of SEQ ID NO

37 light chain CDR2, < ANG-2> Ang2i _ LC10 of SEQ ID NO

38 light chain CDR1, < ANG-2> Ang2i _ LC10 of SEQ ID NO

39 heavy chain variable domain, < ANG-2> Ang2i _ LC10

40 light chain variable domain, < ANG-2> Ang2i _ LC10

41 heavy chain CDR3, < ANG-2> Ang2k _ LC11 of SEQ ID NO

42 heavy chain CDR2, < ANG-2> Ang2k _ LC11 of SEQ ID NO

43 heavy chain CDR1, < ANG-2> Ang2k _ LC11 of SEQ ID NO

44 light chain CDR3, < ANG-2> Ang2k _ LC11 of SEQ ID NO

45 light chain CDR2, < ANG-2> Ang2k _ LC11 of SEQ ID NO

46 light chain CDR1, < ANG-2> Ang2k _ LC11 of SEQ ID NO

47 heavy chain variable domain, < ANG-2> Ang2k _ LC11

48 light chain variable domain, < ANG-2> Ang2k _ LC11

49 heavy chain CDR3, < ANG-2> Ang2s _ R3_ LC03 of SEQ ID NO

SEQ ID NO 50 heavy chain CDR2, < ANG-2> Ang2s _ R3_ LC03

51 heavy chain CDR1, < ANG-2> Ang2s _ R3_ LC03 of SEQ ID NO

52 light chain CDR3, < ANG-2> Ang2s _ R3_ LC03 of SEQ ID NO

53 light chain CDR2, < ANG-2> Ang2s _ R3_ LC03 of SEQ ID NO

54 light chain CDR1, < ANG-2> Ang2s _ R3_ LC03 of SEQ ID NO

55 heavy chain variable domain, < ANG-2> Ang2s _ R3_ LC03

56 light chain variable domain, < ANG-2> Ang2s _ R3_ LC03

SEQ ID NO 57 human heavy chain constant region from IgG1

SEQ ID NO 58 human heavy chain constant region from IgG4

59 kappa light chain constant region of SEQ ID NO

60 Lambda light chain constant region of SEQ ID NO

61 human Tie-2 receptor of SEQ ID NO

62 human angiopoietin-2 (ANG-2) with leader sequence and His tag

Human angiopoietin-1 (ANG-1) with leader sequence and His tag of SEQ ID NO:63

Description of the drawings

FIG. 1 cloning of IgGs for transient expression in an expression vector, transient expression A) Ang2i-LC06 (FIG. 1A) B.) Ang2i-LC06 (FIG. 1B)

FIG. 2 SDS-PAGE gels of purified anti-ANG-2 antibodies Ang2i-LC06, Ang2i-LC07 and Ang2k-LC08

FIG. 3 angiogenin-Tie 2 interaction ELISA

FIG. 4 inhibits ANG-2 binding to Tie2 by Ang2i-LC06 and Ang2k-LC08

FIG. 5 inhibits ANG-1 binding to Tie2 by Ang2i-LC06 and Ang2k-LC08

FIG. 6 Colo205 xenograft model for testing in vivo efficacy of anti-ANG-2 antibodies

FIG. 7 KPL-4 xenograft model for testing in vivo efficacy of anti-ANG-2 antibodies

FIG. 8 ANG-1 binding by Biacore sensorgram

FIG. 9 prevention of lung metastasis/secondary tumor by antibodies according to the invention in Primary Colon tumor xenografts (9A) and Primary Breast cancer xenografts (9B)

FIG. 10 inhibition of retinopathy by antibodies according to the invention.

Experimental method 1

Materials and general methods

General information on the nucleotide Sequences of human immunoglobulin light and heavy chains is provided in Kabat, e.a., et al, Sequences of Proteins of immunological Interest (published of immunological Interest), 5 th edition, Public Health Service (Public Health Service), National Health institute (National Institutes of Health), Bethesda, MD (1991). Amino acids of an antibody chain are numbered and referenced according to EU numbering (Edelman, G.M., et al, Proc. Natl. Acad. Sci. USA 63(1969)78-85; Kabat, E.A., et al, protein Sequences of Immunological Interest (Sequences of Proteins of Immunological Interest), 5 th edition, public Health Service (public Health Service), National Institutes of Health, Bethesda, MD (1991).

Recombinant DNA technology

Standard methods are used for manipulating DNA, such as in Sambrook, j., et al, molecular cloning: a laboratory Manual (Molecular cloning: A laboratory Manual), Cold Spring harbor laboratory Press (Cold Spring harbor laboratory Press), Cold Spring harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Gene synthesis

The desired gene fragment is prepared from oligonucleotides prepared by chemical synthesis. The gene fragments flanking the single restriction endonuclease cleavage site were assembled by annealing and oligonucleotide ligation including PCR amplification and subsequently cloned into pPCRScript (Stratagene) based on the pGA4 cloning vector via a designated restriction enzyme cleavage site, e.g., KpnI/SacI or AscI/PacI. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. The gene synthesis fragments were ordered according to the instructions specified in Geneart (Regensburg, Germany).

DNA sequencing

The DNA sequence was determined by double-strand sequencing in MediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH (Vaterstetten, Germany).

DNA and protein sequence analysis and sequence data processing

Version 10.2 of the GCG's (Genetics Computer group, Madison, Wisconsin) software package and version 8.0 of Infmax's Vector NT1 Advance suite were used for sequence generation, mapping, analysis, annotation and illustration.

Expression vector

For the expression of the antibodies, variants of expression plasmids for transient expression (e.g. in HEK293 EBNA or HEK 293-F) or stable expression (e.g. in CHO cells) based on cDNA constructs with the CMV-intron a promoter (organization) or on genomic constructs with the CMV promoter were used (e.g. fig. 1).

In addition to the antibody expression cassette, the vector comprises:

an origin of replication which allows the plasmid to replicate in E.coli, and

-a beta-lactamase gene conferring ampicillin resistance in E.coli.

The transcription unit of the antibody gene consists of the following elements:

unique restriction sites at the 5' end

Immediate early enhancer and promoter from human cytomegalovirus,

in the case of cDNA organization, followed by an intron A sequence,

-the 5' -untranslated region of a human antibody gene,

an immunoglobulin heavy chain signal sequence,

human antibody chains (heavy, modified or light) as cDNA or as genomic organization with an immunoglobulin exon-intron organization

-a 3' untranslated region having a polyadenylation signal sequence, and

-a unique restriction site at the 3' end.

The fusion genes comprising the selected antibody heavy chain sequences described below were generated by PCR and/or gene synthesis and assembled using known recombinant methods and techniques by ligating the corresponding nucleic acid segments in the genomic heavy chain vector, for example, using unique NsiI and EcoRI sites. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient and stable transfection, larger quantities of plasmid (Nucleobond AX, Macherey-Nagel) were prepared by plasmid preparation from transformed e.

Cell culture technique

Standard Cell culture techniques are used as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J.and Yamada, K.M, (ed.), John Wiley & Sons, Inc.

Transient transfection in the HEK293-F System

Antibodies were generated by transient transfection of two plasmids (encoding the heavy chain or modified heavy chain and the corresponding light chain, respectively) using the HEK293-F system (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293-F cells (Invitrogen) grown in suspension in serum-free FreeStyle293 expression medium (Invitrogen) in shake flasks or in stirred fermentors were transfected with a mixture of the two respective expression plasmids and 293fectin or fectin (Invitrogen). For example, 2L shake flasks (Corning), 600mL HEK293-F cells were seeded at a density of 1.0E 6 cells/mL and incubated at 120rpm,8% CO 2. The next day, cells were transfected with about 42mL of a mixture of a) 20mL of Opti-MEM (invitrogen) with 600 μ g total plasmid DNA (1 μ g/mL) encoding the heavy or modified heavy chain, respectively, and the corresponding light chain, respectively, in equimolar ratios, and B)20mL of Opti-MEM +1.2mL293fectin or fectin (2 μ l/mL), at a cell density of about 1.5E 6 cells/mL. Glucose solution was added during the fermentation process according to the glucose consumption. The supernatant containing the secreted antibody is harvested after 5-10 days, and the antibody is purified directly from the supernatant or the supernatant is frozen and stored.

Protein determination

The Protein concentration of purified antibodies and derivatives was determined by determining the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence according to Pace, C.N., et al, Protein Science (Protein Science),4(1995), 2411-1423.

Determination of antibody concentration in supernatant

The concentration of antibodies and derivatives in cell culture supernatants was assessed by immunoprecipitation using protein a sepharose-beads (Roche). 60 μ L protein A agarose beads were washed three times in TBS-NP40(50mM Tris, pH7.5,150mM NaCl,1% Nonidet-P40). Subsequently, 1-15mL of cell culture supernatant was loaded onto protein a agarose beads pre-equilibrated in TBS-NP 40. After 1h incubation at room temperature, the beads were washed 1 time with 0.5mL TBS-NP40, 2 times with 0.5mL2x phosphate buffer (2xPBS, Roche (Roche)) and 4 times with 0.5mL100mM sodium citrate pH5.0 on an Ultrafree-MC-filtration column (Amicon). By adding 35. mu.lLDS sample buffer (Invitrogen) eluted bound antibody. Half of the sample was separately prepared withThe sample reducing agents were mixed or left unreduced and heated at 70 ℃ for 10 min. Therefore, will20 μ l applied to 4-12%Bis-Tris SDS-PAGE (Invitrogen) (with MOPS buffer for non-reducing SDS-PAGE, and withMES buffer (Invitrogen) against running buffer additives for reduced SDS-PAGE and stained with Coomassie blue.

The concentration of antibodies and derivatives in the cell culture supernatant was measured by protein a-HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind protein a were applied to HiTrap protein a columns (GEHealthcare) eluted from the matrix with 50mM acetic acid, ph2.5 in 50mM k2hpo4,300mm NaCl, ph7.3 and on a Dionex HPLC system. Eluted protein was quantified by integration of UV absorbance and peak area. Purified standard IgG1 antibody was used as a standard.

Alternatively, the concentration of antibodies and derivatives in the cell culture supernatant is measured by sandwich-IgG-ELISA. Briefly, StreptaWell High binding streptavidin (StreptaWell High Bind streptavidin) a-96 well microtiter plates (Roche) were coated with 100 μ L/well biotinylated anti-human IgG capture molecule F (ab')2< h-Fc γ > bi (dianova) at 0.1 μ g/mL for 1h at room temperature or alternatively overnight at 4 ℃, and then washed 3 times with 200 μ L/well PBS,0.05% tween (PBST, Sigma (Sigma)). A dilution series of 100 μ L/well of cell culture supernatant containing the various antibodies in PBS (Sigma) was added to the wells and incubated on a microtiter plate shaker for 1-2h at room temperature. The wells were washed three times with 200. mu.L/well of PBST and bound antibody was detected on a microtiter plate shaker at room temperature for 1-2h with 100. mu.l of F (ab')2< hFc γ > POD (Dianova) as detection antibody at a concentration of 0.1. mu.g/mL. Unbound detection antibody was washed off in three washes with 200 μ L/well PBST and bound detection antibody was detected by addition of 100 μ LABTS/well. The determination of the absorbance was carried out on a Tecan Fluor spectrometer at a measurement wavelength of 405nm (reference wavelength 492 nm).

Protein purification

The protein was purified from the filtered cell culture supernatant with reference to standard procedures. Briefly, the antibody was applied to a protein a sepharose column (GE Healthcare) and washed with PBS. Antibody elution was performed at acidic pH and the samples were immediately neutralized subsequently. Aggregated proteins were separated from monomeric antibodies by size exclusion chromatography (Superdex200, GE healthcare) in 20mM histidine, 140mM NaCl ph 6.0. The monomeric antibody fractions are pooled, concentrated if necessary using, for example, a MILLIPORE Amicon Ultra (30MWCO) centrifugal concentrator, and stored at-80 ℃. Portions of the sample are provided for subsequent protein analysis and analytical characterization, for example by SDS-PAGE, size exclusion chromatography, mass spectrometry, and endotoxin measurement (see fig. 2).

SDS-PAGE

The preformed gel system (Invitrogen) was used according to the manufacturer's instructions. Specifically, 4-20% of the total weight of the composition is usedTRIS-Glycine Prep (Pre-Cast) gel andTRIS-Glycine SDS running buffer. (see, e.g., FIG. 1). Reduction of samples by addition before running the gelSample reducing agent was completed.

Analytical size exclusion chromatography

Size exclusion chromatography to determine the aggregation and oligomerization status of the antibody was performed by HPLC chromatography. Briefly, protein A purified antibody was loaded on a 300mM NaCl,50mM KH2PO4/K2HPO4, Tosoh TSKgel G3000SW column in pH7.5 or Superdex200 column in 2 × PBS on a Dionex HPLC-system (GE Healthcare). Eluted protein was quantified by integration of UV absorbance and peak area. The BioRad gel filtration Standard 151-1901 served as a standard.

Mass spectrometry

The total deglycosylation mass of the antibody was determined and verified by electrospray ionization mass spectrometry (ESI-MS). Briefly, 100 μ g of purified antibody was deglycosylated with 50mU of N-glycosidase F (PNGaseF, ProZyme) at 37 ℃ at protein concentrations of up to 2mg/ml with 100mM KH2PO4/K2HPO4, pH7 for 12-24h and subsequently desalted by HPLC on a Sephadex G25 column (GE healthcare). The mass of the various heavy and light chains was determined by ESI-MS after deglycosylation and reduction. Briefly, 50 μ g of antibody in 115 μ l was incubated with 60 μ l of 1M TCEP and 50 μ l of 8M guanidine hydrochloride and subsequently desalted. The total mass and the mass of the reduced heavy and light chains were determined by ESI-MS on a NanoMate Source equipped Q-Star Elite MS system.

ANG-1 and ANG-2 binding ELISA

The binding properties of antibodies to ANGPTs (angiopoietin 1 or angiopoietin 2) were assessed in AN ELISA assay using full-length angiopoietin-2-His protein (R & D Systems #623-AN/CF or materials produced internally) or angiopoietin-1-His (R & D Systems # 923-AN). Thus, 96-well plates (Falcon polystyrene anti-reflective microtiter plates or Nunc Maxisorb) were coated with 100. mu.l 1. mu.g/mL recombinant human angiopoietin-1 or angiopoietin-2 (without carrier) in PBS (Sigma) for 2h at room temperature or overnight at 4 ℃. The wells were washed three times with 300 μ l PBST (0.2% Tween20) and blocked with 200 μ l2% BSA0.1% Tween20 for 30min at room temperature and then washed three times with 300 μ l PBST. A100. mu.L/well dilution series (40pM-0.01pM) of purified test antibody against < ANG-2> in PBS and, as a reference, Mab536(Oliner, J., et al, cancer cell, 11.6.2004 507-16, US2006/0122370) were added to the wells and incubated for 1h at room temperature on a microtiter plate shaker. The wells were washed three times with 300 μ L PBST (0.2% Tween20) and bound antibody was detected with 100 μ L/well of 0.1 μ g/ml F (ab') < hk > POD (Biozol Cat. No.206005) in 2% BSA0.1% Tween20 as detection antibody on a microtiter plate shaker at room temperature for 1 h. Unbound detection antibody was washed off in three washes using 300 μ L/well PBST, and bound detection antibody was detected by adding 100 μ L ABTS/well. The determination of the absorbance was carried out on a Tecan Fluor spectrometer at a measurement wavelength of 405nm (reference wavelength 492 nm).

ANG-2 binding BIACORE

Binding of antibodies to antigens (e.g., human ANG-2) was studied by surface plasmon resonance using BIACORE T100 instrument (ge healthcare biosciences AB, Uppsala, sweden). Briefly, for affinity measurements, goat < hIgG-Fc γ > polyclonal antibodies were immobilized on CM4 chips via amine coupling for presentation of antibodies directed to human ANG-2. Binding in HBS buffer (HBS-P (10mM HEPES,150mM NaCl,0.05% Tween20, ph7.4), measurement at 25 ℃ purified ANG-2-His (R & D system or internally purified) was added to the solution at different concentrations between 0.41nM and 200nM association was measured by ANG-2-injection for 3 minutes, dissociation was measured by washing the chip surface with HBS buffer for 5 minutes and KD values were evaluated using the 1:1 Langmuir binding model (Langmuir binding model). due to the heterogeneity of ANG-2 formulation, no 1:1 binding could be observed; therefore KD values were only relative evaluation.negative control data (e.g. buffer curves) were subtracted from the sample curves for correcting the system-inherent baseline drift and for the reduction of noise signals. Ang-2 was captured at a capture level of 2000-1700RU by a pentahistidine antibody (5-His-Ab without BSA, Qiagen No.34660) immobilized on a CM5 chip via amine coupling (without BSA) (see below).

Inhibition of binding of huANG-2 to Tie-2 (ELISA)

The interaction ELISA was performed in 384 well microtiter plates (Microcoat, DE, Cat. No.464718) at RT. After each incubation step, plates were washed 3 times with PBST. ELISA plates were coated with 0.5. mu.g/ml Tie-2 protein (R & D Systems, UK, Cat. No.313-TI) for at least 2 hours (h). Thereafter, the wells were blocked for 1 hour with PBS supplemented with 0.2% tween-20 and 2% BSA (roche diagnostics GmbH, DE). Dilutions of the purified antibody in PBS were incubated with 0.2. mu.g/ml human angiopoietin-2 (huAngiopoietin-2) (R & D Systems, UK, Cat. No.623-AN) for 1 hour at RT. After washing, a mixture of 0.5. mu.g/ml biotinylated anti-angiopoietin-2 clone BAM0981(R & D Systems, UK) and 1:3000 diluted streptavidin HRP (Roche diagnostics GmbH, Roche diagnostics, DE, Cat. No.11089153001) was added for 1 hour. Thereafter, the plates were washed 6 times with PBST. The plates were developed with freshly prepared ABTS reagents (Roche Diagnostics GmbH, DE, buffer #204530001, tablet #11112422001) for 30 minutes at RT. The absorbance was measured at 405 nm.

Inhibition of binding of huANG-1 to Tie-2 (ELISA)

The interaction ELISA was performed in 384-well microtiter plates (MaxiSorb Nunc #442768) at RT. After each incubation step, plates were washed 3 times with PBST. ELISA plates were coated with 0.5. mu.g/ml Tie-2 protein (R & D Systems, UK, Cat. No.313-TI or materials produced internally) for at least 2 hours (h). Thereafter, the wells were blocked with PBS supplemented with 0.2% tween-20 and 2% BSA (Roche diagnostics GmbH, DE) for 1 hour. Dilutions of the purified antibody in PBS were incubated with 0.2. mu.g/ml human angiopoietin-1 (huAngiopoietin-1) (R & DSsystems #923-AN/CF or materials produced internally)) for 1 hour at RT. After washing, a mixture of 0.5. mu.g/ml biotinylated anti-angiopoietin-1 clone (R & D Systems # BAF923) and 1:3000 dilution of streptavidin HRP (Roche diagnostics GmbH, DE, Cat. No.11089153001) was added for 1 hour. Thereafter, the plates were washed 6 times with PBST. The plates were developed with freshly prepared ABTS reagents (Roche Diagnostics GmbH, DE, buffer #204530001, tablet #11112422001) for 30 minutes at RT. The absorbance was measured at 405 nm.

Generation of HEK293-Tie2 cell line

To determine that angiopoietin-2 antibodies interfered with ANGPT 2-stimulated Tie2 phosphorylation and ANGPT2 binding to Tie2 on cells, a recombinant HEK293-Tie cell line was generated. Briefly, a pcDNA 3-based plasmid (RB22-pcDNA3TopohTie2) encoding full-length human Tie2(SEQ ID61) and a neomycin resistance marker under the control of the CMV promoter was transfected into HEK293 cells (ATCC) using Fugene (Roche Applied science) as a transfection reagent and resistant cells were selected in DMEM10% FCS, 500. mu.g/ml G418. Individual clones were isolated by cloning columns (cloning cylinder) and subsequently analyzed for Tie2 expression by FACS. Clone 22 was identified as a clone with high and stable Tie2 expression (even in the absence of G418) (HEK293-Tie2 clone 22). HEK293-Tie2 clone 22 was subsequently used in cellular assays: ANGPT 2-induced Tie2 phosphorylation and ANGPT2 cell ligand binding assays.

ANGPT2 Induction of Tie2 phosphorylation assay

Inhibition of ANGPT2 antibody on ANGPT 2-induced Tie2 phosphorylation was measured according to the following assay principle. HEK293-Tie2 clone 22 was stimulated with ANGPT2 for 5 minutes in the absence or presence of ANGPT2 antibody and P-Tie2 was quantitated by sandwich ELISA. Briefly, 2 × 10 per well⁵HEK293-Tie2 clone 22 cells were cultured overnight in 100. mu.l DMEM,10% FCS, 500. mu.g/ml Geneticin (Geneticin) in poly-D-lysine coated 96-well microtiter plates. The next day a row of titrated ANGPT2 antibody (4-fold concentration, 75 μ l final volume/well, in duplicate) was prepared in a microtiter plate and mixed with 75 μ l ANGPT2 (R)&Dsystems#623-AN]Dilutions (3.2. mu.g/ml as 4-fold concentrate) were mixed. The antibody and ANGPT2 were preincubated for 15 minutes at room temperature. Add 100. mu.l of the mixture to HEK293-Tie2 clone 22 cells (preincubated for 5 minutes with 1mM NaV3O4, Sigma # S6508) and incubate for 5 minutes at 37 ℃. Subsequently, the cells were washed with 200. mu.l of ice-cold PBS +1mM NaV3O4 per well and lysed by adding 120. mu.l of lysis buffer (20mM Tris, pH8.0,137mM NaCl,1% NP-40,10% glycerol, 2mM EDTA,1mM NaV3O4,1mM PMSF and 10. mu.g/ml aprotinin) per well on ice. Cells were lysed on a microtiter plate shaker at 4 ℃ for 30min, and 100. mu.l of lysate was transferred directly to p-Tie2ELISA microdropletsFixed plate (R)&D Systems,R&D # DY990), no prior centrifugation and no total protein measurement were performed. The amount of P-Tie2 was quantified according to manufacturer's instructions, and the IC50 value of inhibition was measured using XLfit4 assay insert for Excel (plug-in) (dose-response single point, mode 205). IC50 values may be compared within an experiment but may vary from experiment to experiment.

ANGPT1 Induction of Tie2 phosphorylation assay

Inhibition of ANGPT1 antibody on ANGPT 1-induced Tie2 phosphorylation was measured according to the following assay principle. HEK293-Tie2 clone 22 was stimulated with ANGPT1 for 5 minutes in the absence or presence of ANGPT1 antibody and P-Tie2 was quantitated by sandwich ELISA. Briefly, 2 × 10 per well⁵HEK293-Tie2 clone 22 cells were cultured overnight in 100. mu.l DMEM,10% FCS, 500. mu.g/ml Geneticin (Geneticin) in poly-D-lysine coated 96-well microtiter plates. The next day a row of titrated ANGPT1 antibody (4-fold concentration, 75 μ l final volume/well, in duplicate) was prepared in a microtiter plate and mixed with 75 μ l ANGPT1 (R)&Dsystems #923-AN) dilutions (0.8. mu.g/ml as 4-fold concentrate) were mixed. The antibody and ANGPT1 were preincubated for 15 minutes at room temperature. Add 100. mu.l of the mixture to HEK293-Tie2 clone 22 cells (preincubated for 5 minutes with 1mM NaV3O4, Sigma # S6508) and incubate for 5 minutes at 37 ℃. Subsequently, the cells were washed with 200. mu.l of ice-cold PBS +1mM NaV3O4 per well and lysed by adding 120. mu.l of lysis buffer (20mM Tris, pH8.0,137mM NaCl,1% NP-40,10% glycerol, 2mM EDTA,1mM NaV3O4,1mM PMSF and 10. mu.g/ml aprotinin) per well on ice. Cells were lysed on a microtiter plate shaker at 4 ℃ for 30min, and 100. mu.l of lysate were transferred directly to p-Tie2ELISA microtiter plates (R)&D Systems,R&D # DY990), no prior centrifugation and no total protein measurement were performed. The amount of P-Tie2 was quantified according to manufacturer's instructions and the IC50 value of inhibition was measured using XLfit4 analytical insert for Excel (dose-response single point, mode 205). IC50 values may be compared within an experiment but may vary from experiment to experiment.

Example 1

Expression and purification of monoclonal < ANG-2> antibodies Ang2i-LC06, Ang2i-LC07 and Ang2k-LC08

The light and heavy chains of the corresponding antibodies Ang2i-LC06, Ang2i-LC07 and Ang2k-LC08 were constructed in expression vectors as described above. The heavy and kappa light chains were cloned in genomic expression cassettes, while the lambda light chain was cloned as cDNA containing intron a (fig. 1B). The plasmids were amplified in E.coli, purified and subsequently transfected for transient expression of recombinant proteins in HEK293-F cells (using the Invitrogen's FreeStyle293 system). After 7 days, HEK293-F cell supernatants were collected, filtered and the antibodies were purified by protein A and size exclusion chromatography. Homogeneity of all antibodies was confirmed by SDS-PAGE under non-reducing and reducing conditions and analytical size exclusion chromatography. Under reducing conditions (fig. 1), the polypeptide heavy chain of the < ANG-2> antibody showed an apparent molecular size of about 50kDa on SDS-PAGE, similar to the calculated molecular weight, and the polypeptide light chain showed an apparent molecular weight of 25kDa according to their expected size. Mass spectrometry confirmed the identity of the purified antibody. Expression levels of all constructs were analyzed by protein a HPLC.

Size exclusion chromatography analysis of purified antibodies. All antibodies were prepared and characterized analytically similar to the method described above. The SEC data for the corresponding antibodies are summarized in the table below.

Example 2

ELISA binding assays with human ANG-1 and with human ANG-2

< ANG-2> binding of antibodies Ang2i-LC06, Ang2i-LC07 and Ang2k-LC08 to human ANG-1 and human ANG-2 was determined as described above in ANG-1 or ANG-2 binding ELISA. Briefly, ELISA-type assays are based on the immobilization of human wild-type angiopoietin-1 or-2 in microtiter plates. The binding of the antibody to immobilized ANG-1 or ANG-2 was measured by a < human Fc > (anti-IgG) antibody with POD conjugate. Serial dilutions of < ANG-2> antibodies allowed determination of EC50 concentration. Human anti-ANG-2 antibody < ANG-2> antibody Mab536 was used as a reference (olin et al, cancer cell. (cancer cells) 11 months 6 days (2004)507-16, US 2006/0122370). The measured EC50 concentrations are summarized in the table below.

All antibodies specifically bound to ANG-2. MAb536 and Ang2k-LC08 also showed specific binding to Ang-1, whereas Ang2i-LC06 and Ang2i-LC07 bound non-specifically to Ang-1, since their EC 50-values exceeded 8000ng/ml (detection limit).

Example 3:

binding to ANG-2 by Biacore

The affinity of binding to human ANGPT2 was examined using the Biacore assay described above. Briefly, in this assay, a capture antibody (anti-Fc) is immobilized on the surface of a Biacore chip, which captures and presents the corresponding antibody (e.g., Ang2i-LC 06). The ligand (here ANGPT2) was captured from solution. The putative 1:1 interaction was used to determine affinity for this interaction. Details on this experiment can be found in the general methods section. The affinities determined for ANGPT 2-binding (KD) are summarized in the table below.

The antibodies Ang2i-LC06 and Ang2k bind to ANGPT2 with high affinity.

Example 4:

neutralization of the ANGPT1/2-Tie2 interaction (human)

Blocking of the human ANGPT 1/2/human Tie2 interaction was shown by receptor interaction ELISA. 384-well Maxisorp plates (Nunc) were coated with 0.5. mu.g/ml human Tie2(R & D Systems, UK, Cat. No.313-TI or materials produced internally) for 2 hours at room temperature and blocked with PBS supplemented with 0.2% Tween-20 and 2% BSA (Roche Diagnostics GmbH, Roche Diagnostics, DE) for 1 hour at room temperature with shaking. At the same time, a dilution of the purified antibody in PBS was incubated with 0.2. mu.g/ml human angiopoietin-1/2 (R & D Systems #923-AN/CF, R & DSsystems, UK, Cat. No.623-AN or internally produced material) at room temperature for 1 hour. After washing, a mixture of 0.5. mu.g/ml biotinylated anti-angiopoietin-1/2 clone (R & DSsystems # BAF923, BAM0981R & D Systems, UK) and 1:3000 dilution of streptavidin HRP (Roche Diagnostics GmbH, DE, Cat. No.11089153001) was added for 1 hour. Thereafter, the plates were washed 6 times with PBST. The plates were developed with freshly prepared ABTS reagents (Roche Diagnostics GmbH, DE, buffer #204530001, tablet #11112422001) for 30 minutes at RT. The absorbance was measured at 405 nm.

The inhibitory concentrations obtained are summarized in the table below.

The table above shows different selectivity patterns for the two antibodies Ang2i-LC06 and Ang2k-LC 08. Ang2i-LC06 is selective for ANGPT2, while Ang2k-LC08 is cross-reactive for ANGPT1/2 in inhibiting the ANGPT1/2Tie2 interaction.

Example 5:

tie2 phosphorylation

The ability of the identified ANGPT2 antibodies to interfere with ANGPT2 and ANGPT1 mediated Tie2 phosphorylation was examined in ANGPT2 and ANGPT1 induced Tie2 phosphorylation assays as described above. A schematic of the assay setup is shown in figure 3.

Both antibodies Ang2i-LC06 and Ang2k-LC08 showed dose-dependent interference on ANGPT 2-stimulated Tie2 phosphorylation with comparable IC50 values as shown in figure 4. Ang2i-LC06 interfered with ANGPT 2-stimulated Tie2 phosphorylation with an IC50 value of approximately 508ng/ml, while Ang2k-LC08 interfered with ANGPT 2-stimulated Tie2 phosphorylation with an IC50 value of approximately 499 ng/ml. In contrast, Ang2k-LC08 alone interfered with ANGPT 1-stimulated Tie2 phosphorylation with an IC50 value of about 391ng/ml, whereas Ang2i-LC06 did not interfere with ANGPT 2-stimulated Tie2 phosphorylation within the same range of tested concentrations (fig. 5).

Example 6 in vivo efficacy

Effect of anti-ANGPT antibodies on Colo205 xenograft growth

In a staged (staged) subcutaneous Colo205 xenograft model, < ANGPT2> antibodies Ang2i-LC06 and Ang2k-LC08 compared to < ANGPT2> Mab536 in vivo efficacy.

In female Scid beige mice, purified Ang2i-LC06 and Ang2k-LC08 antibodies were compared to antibody Mab536 in a staged subcutaneous Colo205 xenograft model (Ang2_ PZ _ Colo205_ 006).

Antibody Mab536 was provided as a frozen stock solution (c =4.5mg/mL), Ang2i-LC06 and Ang2k-LC08 were provided as frozen stock solutions (c =1mg/mL) in 20mM histidine, 140mM NaCl, ph 6.0. If necessary, the antibody solution is suitably diluted in PBS from a stock solution before injection, and PBS is used as an excipient. Humanized IgG1 anti-IgE antibody Xolair (omalizumab) was used as a positive control and taken from the pharmacy.

Cell lines and culture conditions Colo205 human colorectal cancer cells were originally obtained from ATCC and, after expansion, deposited in the Roche Penzberg internal cell bank. At 37 ℃ in a water-saturated atmosphere at 5% CO₂In (1), the tumor is thinnedCell lines were routinely cultured in RPMI1640 medium (PAA, laboratories, Austria) supplemented with 10% fetal bovine serum (PAA laboratories, Austria) and 2mM L-glutamine. Passage 3 was used for transplantation.

Animals female SCID beige mice (purchased from Charles River Germany) were maintained under specific pathogen-free, daily 12h light/12 h dark cycle conditions according to the defined guidelines (committed guidiness) (GV-Solas; Felasa; TierschG). Experimental study protocol was reviewed and approved by local government. Animals were maintained in the animal laboratory for one week after arrival at the quarantine section to accommodate new environments and for observation. Continuous health monitoring was performed periodically. Diet (provimiKliba3337) and water (acidified pH2.5-3) were provided ad libitum. The age of the mice at the beginning of the study was approximately 12-14 weeks.

Monitoring: animals were managed daily (control) for clinical symptoms and tested for side effects. The body weight of the animals was recorded for the entire experimental monitoring and tumor volume was measured with calipers after staging.

Tumor cell injection: on the day of injection, Colo205 cells were centrifuged, washed once and resuspended in PBS. CELL concentration and CELL size after washing again with PBS were measured using a CELL counter and analysis system (Vi-CELL, Beckman Coulter). For injection of Colo205 cells, the final titer was adjusted to 5.0x10⁷Cells/ml, about 90% viability. Followed by 2.5x10 per animal⁶100 μ l of this suspension of cells was injected s.c. into the right flank of the mouse.

Animal treatment: animal treatment on randomized days, 16 days post cell transplantation (study Ang2_ PZ _ Colo205_006) mean tumor volume 178mm³It is started.

Study of dosage regimen of Ang2PZColo 205006:

tumor growth inhibition up to day 50 is shown in figure 6. The data show that ANGPT2 selective antibody Ang2i-LC06 is the most active antibody (tumor control quantification (TCR) value 0.39). Ang2i-LC06 was more effective in inhibiting tumor growth than the ANGPT1 cross-reactive antibody Ang2k-LC08(TCR value 0.46) selective to antibody MAb536(TCR value 0.47) and ANGPT 2.

Effect of anti-ANGPT antibodies on KPL-4 xenograft growth

In stage in situ KPL-4 xenograft model, < ANGPT2> antibodies Ang2i-LC06 and Ang2k-LC08 compared in vivo efficacy with < ANGPT2> Mab536

Purified Ang2i-LC06 and Ang2k-LC08 antibodies were compared to antibody Mab536 in a staged orthotopic KPL-4 xenograft model (Ang2_ PZ _ KPL-4_002) in female Scid beige mice.

Antibody: mab536 was provided as a frozen stock (c =4.5mg/mL) and Ang2i-LC06 and Ang2k-LC08 were provided as frozen stocks (c =1mg/mL) in 20mM histidine, 140mM NaCl, ph 6.0. If necessary, prior to injection, the antibody solution is suitably diluted from stock in PBS and PBS is used as an excipient.

Cell lines and culture conditions KPL-4 human breast cancer cells were initially established from malignant pleural effusion in breast cancer patients with inflammatory skin metastases. KPL-4 cells were awarded by professor J.Kurebayashi (Kawasaki Medical School, Kurashiki, Japan). Tumor cells were cultured in DMEM medium (PAN Biotech, Germany) supplemented with 10% fetal bovine serum (PAN Biotech, Germany) and 2 mML-glutamine (PAN Biotech, Germany) at 37 ℃ in a water-saturated atmosphere at 5% CO₂Conventionally. Cultures were passaged with trypsin/EDTA 1x (PAN) and split 3 times per week.

Animals: female SCID beige mice (purchased from Charles River Germany) were maintained under specific pathogen-free, 12h light/12 h dark cycle conditions per day according to the defined guidelines (committed guidelines) (GV-Solas; Felasa; TierschG). Experimental study protocol was reviewed and approved by local government. Animals were maintained in the animal laboratory for one week after arrival at the quarantine section to accommodate new environments and for observation. Continuous health monitoring was performed periodically. Diet (provimiKliba3337) and water (acidified pH2.5-3) were provided ad libitum. The age of the mice at the beginning of the study was approximately 12 weeks.

Monitoring: animals were managed daily (control) for clinical symptoms and tested for side effects. The body weight of the animals was recorded for the entire experimental monitoring and tumor volume was measured with calipers after staging.

Tumor cell injection: on the day of injection, tumor cells (trypsin-EDTA) were harvested from culture flasks (Greiner TriFlask) and transferred to 50ml of medium, washed once and resuspended in PBS. The washing step and filtration were again carried out with PBS (cell Filter; Falcon;)^TM100 μm) was followed by adjusting the final cell titer to 1.5 × 10⁸And/ml. The tumor cell suspension was carefully mixed with a pipette to avoid cell aggregation. Anesthesia was performed on small animals in a closed circulation system using a Stephens inhalation device with a pre-incubation chamber (polymethylmethacrylate), a nose-mask (silicon) for individual mice and using isoflurane (Pharmacia-Upjohn, Germany), an anesthetic compound that does not burn or explode. Two days prior to injection, animals were shaved. For i.m.f.p. injection, cells were injected at 20 μ l (3 x 10)⁶Animals) were injected in situ into the right penultimate inguinal mammary fat pad of each anesthetized mouse. For in situ implantation, the cell suspension was injected through the skin under the nipple using a Hamilton microliter syringe and a 30Gx1/2 "needle.

Animal treatment 35 days after cell transplantation (study Ang2_ PZ _ KPL-4_002), tumor range was 60-180mm³Starting animal treatment on randomized days, mean tumor volume about 90mm³. Study dosage regimen of Ang2_ PZ _ KPL-4_ 002:

tumor growth inhibition up to day 64 is shown in figure 7. The data show that the ANGPT2 selective antibody Ang2i-LC06 is the most active antibody in the KPL-4 model (TCR value 0.55). Ang2i-LC06 was more effective in inhibiting tumor growth than the ANGPT1 cross-reactive antibody Ang2k-LC08(TCR value 0.57) selective to antibody MAb536(TCR value 0.57) and ANGPT 2.

Example 7:

binding of ANG-1 by Biacore

The affinity of binding to human ANG-1 was examined with Biacore assay: huAng-1 was immobilized on a CM5 biosensor chip using amine coupling chemistry. The protein was injected at 10. mu.g/ml in pH4.5 sodium acetate over 20 minutes at a flow rate of 5. mu.l/min. This gives a surface density of about 20000 RU. BSA was immobilized on reference flow cells under the same conditions. Antibodies were diluted to 100nM in HBS-P and injected for 3 min (association phase). After washing with running buffer for 3 minutes (dissociation phase), the surface was regenerated by injection of 10mM sodium hydroxide at 5. mu.l/min for 1 minute. The results are shown in fig. 8: ang2k _ LC08 had a complex dissociation half-life of about 50s, Ang2i _ LC06 had a complex dissociation half-life of about 5s, and Ang2i _ LC10 did not show binding to Ang-1.

Example 8 prevention of metastasis/Secondary tumors in vivo when carrying Primary tumors

a) Prevention of metastasis/Secondary tumors in mice xenografted with Primary Colo205 tumors

Cell lines and culture conditions:

colo205 human colorectal cancer cells were initially obtained from ATCC and, after expansion, deposited in the Roche Penzberg internal cell bank. At 37 ℃ in a water-saturated atmosphere at 5% CO₂In (1), tumor cell lines were routinely cultured in RPMI1640 medium (PAA, laboratories, Austria) supplemented with 10% fetal bovine serum (PAA laboratories, Austria) and 2mM L-glutamine. Passage 3 was used for transplantation.

Animals:

female SCID beige mice, reached at 4-5 weeks of age (purchased from Charles River Germanyd), were maintained under specific pathogen-free, 12h light/12 h dark cycle conditions per day according to the guidelines for determination (GV-Solas; Felasa; TierschG). Experimental study protocol was reviewed and approved by local government. Animals were maintained in the animal laboratory for one week after arrival at the quarantine section to accommodate new environments and for observation. Continuous health monitoring was performed periodically. Diet (Provimi Kliba3337) and water (acidified ph2.5-3) were provided ad libitum. The age of the mice at the beginning of the study was approximately 10 weeks.

Tumor cell injection:

on the day of injection, Colo205 tumor cells (trypsin-EDTA) were harvested from culture flasks (Greiner) and transferred to 50ml of medium, washed once and resuspended in PBS. The washing step and filtration were again carried out with PBS (cell Filter; Falcon;)100 μm) was followed by adjusting the final cell titer to 2.5 × 10⁷And/ml. The tumor cell suspension was carefully mixed with a pipette to avoid cell aggregation. Thereafter, the cell suspension was loaded into a 1.0ml tuberculin syringe (Braun Melsungen) using a wide needle (wide needle,1.10x40 mm); for injections, the needle size was changed (0.45x25mm) and a new needle was used for each injection. Anesthesia was performed on small animals in a closed circulatory system using a Stephens inhalation device with a pre-incubation chamber (polymethylmethacrylate), a nose-mask (silicon) for individual mice, and isoflurane (cp-pharma) as an anesthetic compound that does not burn or explode. Two days before injection, animals were shaved, and for cell injection, the skin of anesthetized animals was carefully lifted with dissecting forceps and 100 μ l of cell suspension (=2.5x 10) was injected subcutaneously in the right flank of the animals⁶A cell). Tumor growth of the primary tumor was monitored (data not shown).

Monitoring of secondary tumors, e.g. in the lung, by quantification of human Alu sequences

At the end of the study (day 103), lungs were collected from all groups of animals. Briefly, the samples were immediately transferred to liquid nitrogen. In a further step, the total DNA was isolated from the sample using a MagNAPure LC instrument according to the manufacturer's instructions. Human Alu-specific primers were selected for selective amplification of Alu sequences by quantitative PCR (LightCycler instrument). (T.Schneider et al, Clin.Exp.Metas.2002;19: 571-.

Treatment of animals

14 days after cell transplantation (study Ang2_ PZ _ Colo205_008), the mean tumor volume was 340mm³Animals were initially treated with Avastin (Avastin) (10mg/kg i.p. once a week). After 7 weeks, on day 51, mice were randomized to begin subsequent secondary treatment with the compounds listed in the table below. Secondary treatment was started on day 51 of study Ang2_ PZ _ Colo205_ 008.

The results of preventing metastasis/secondary tumors (in the lung) are listed in the table below and shown in fig. 9A

TABLE 1 quantification of human ALU DNA in the lungs of mice starting to carry the primary Colo205 tumor after treatment with different antibodies

The results show that ANG2i-LC06 significantly improved prevention of secondary tumors/metastases compared to avastin.

b) Prevention of metastasis/Secondary tumors in mice xenografted with Primary KPL-4 tumors

Tumor cell lines

A human breast cancer cell line KPL-4 (a gift from professor j. kurebayashi) was established from malignant pleural effusion in breast cancer patients with inflammatory skin metastases. Tumor cells were supplemented with 10% fetal bovine serum (PAN Biotech, G)ermann) and 2mM L-glutamine (PAN Biotech, Germany) in DMEM medium (PAN Biotech, Germany) at 37 ℃ in a water-saturated atmosphere at 5% CO₂Conventionally. Cultures were passaged with trypsin/EDTA 1x (PAN) and split 3 times per week.

Mouse

After this was achieved, female SCID beige mice (10-12 weeks old; 18-20g weight) Charles River, Sulzfeld, Germany) were maintained for one week at the quarantine section of the animal laboratory to accommodate new circumstances and for observation. Continuous health monitoring was performed. Mice were maintained under SPF, 12h light/12 h dark cycle conditions daily according to International guidelines (GV-Solas; Felasa; TierschG). Diet (KlibaProvimi3347) and water (filtered) were provided ad libitum. Experimental study protocol was reviewed and approved by local government. (Regierung von Oberbayorn; registration No.211.2531.2-22/2003).

Tumor cell injection

On the day of injection, tumor cells (trypsin-EDTA) were harvested from culture flasks (Greiner TriFlask) and transferred to 50ml of medium, washed once and resuspended in PBS. The washing step and filtration were again carried out with PBS (cell Filter; Falcon;)100 μm) was followed by adjusting the final cell titer to 1.5 × 10⁸And/ml. The tumor cell suspension was carefully mixed with a pipette to avoid cell aggregation. Anesthesia was performed on small animals in a closed circulation system using a Stephens inhalation device with a pre-incubation chamber (polymethylmethacrylate perspex), individual mouse nasal mask (silicon) and isoflurane (Pharmacia-Upjohn, Germany) as an anesthetic compound that did not burn or explode. Two days prior to injection, animals were shaved. For i.m.f.p. injection, cells were injected in situ in a volume of 20 μ Ι into the penultimate right inguinal mammary fat pad of each anesthetized mouse. For in situ implantation, the cell suspension was injected through the skin under the nipple using a Hamilton microliter syringe and a 30Gx1/2 "needle. Tumor growth of the primary tumor was monitored (data not shown).

Monitoring of secondary tumors, e.g. in the lung, by quantification of human Alu sequences

At the end of the study (day 103), lungs were collected from all groups of animals. Briefly, the samples were immediately transferred to liquid nitrogen. In a further step, the total DNA was isolated from the sample using a MagNAPure LC instrument according to the manufacturer's instructions. Human Alu-specific primers were selected for selective amplification of Alu sequences by quantitative PCR (LightCycler instrument). (T.Schneider et al, Clin.Exp.Metas.2002;19: 571-.

Treatment of animals

The mean tumor volume was 60-160mm 35 days after cell transplantation³The treatment of the animals was started. The compounds and dosage regimes are listed in the table below.

The results of preventing metastasis/secondary tumor (in lung) are listed in the table below and shown in fig. 9B

TABLE 2 quantification of human ALUDNA in the lungs of mice initially bearing the primary KPL4 tumor after treatment with different antibodies

The results show that ANG2i-LC06, ANG2i-LC07, ANG2k-LC08 are very effective in preventing secondary tumors/metastases.

Example 9 Effect in the treatment of retinopathy

Method of producing a composite material

C57/Bl6 puppies were exchanged with CD1 care dams and exposed to 75% oxygen from P7 to P12 (PRO-OX110 chamber oxygen manipulator, Biospherix Ltd, Redfield, NY), which induces vascular occlusion and capillary disruption in the center of the retina (procession). Puppies and nursing dams were placed in normal air, resulting in relative hypoxia and induction of neovascularization. At P13, puppies were anesthetized with isoflurane (5% induction, 3% maintenance in combination with 1.5% oxygen), the eyes were exposed and 1 μ l of intraocular injection was injected into the left eye using a Nanofil syringe equipped with a 35 gauge needle (WPI, Sarasota, FL). At P17, both eyes were dissected, fixed in 4% paraformaldehyde at 4 ℃ for 4h, and the retina was dissected. The retinas were permeabilized in PBS containing 0.5% Triton X-100 and 1% Bovine Serum Albumin (BSA), with 0.1mM CaCl in PBS pH6.8,1% Triton X-100, and₂,0.1mM MgCl₂20. mu.g/ml biotinylated isolectin B4(Sigma Aldrich, Gillingham, UK), followed by staining with 20. mu.g/ml ALEXA 488-streptavidin (Molecular Probes, Eugene, OR) and flat-mounting in Vectashield (Vector laboratories, Burlingame, Calif.). The retina was imaged at 4x magnification using a Nikon epifluorescence microscope (epifluorescence). Quantification of new vessels and ischemic areas was performed in a blind manner (bound washion) using PhotoshopCS3 and Image J (NIH) and expressed as a percentage of the total retinal area (= normal + ischemia + new vessels).

Results

Figure 10A shows a representative flat-packed retina with retinal vasculature, visualized by isolectin staining. The central ischemic region induces neovascularization and regenerates retinal vessels through upregulation of angiogenic inducers. The neovascular front is hyperproliferative, resulting in tortuous vessels in an irregular vascular pattern. Most of the outer region contains normal unaffected vessels. Quantification of retinal plain patches showed that inhibition of VEGF with avastin reduced retinal neovascularization (see FIG. 10B, 36.7 + -1.8% for non-injections versus 22.4 + -3.0% for injections), as expected. Inhibition of Ang2 using antibodies LC06 or LC08 also resulted in a reduction in neovascularization (31.5 ± 1.1% versus 18.8 ± 1.3% and 34.0 ± 3.1% versus 25.4 ± 3.4%). Control injections of human Ig G had no effect on neovascularization (see FIG. 10B, 38.3. + -. 1.1% vs 38.3. + -. 0.8%).

Claims (12)

1. An antibody specifically binding to human angiopoietin-2, wherein

a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO 33, the CDR2 region of SEQ ID NO 34 and the CDR1 region of SEQ ID NO 35, and

b) the light chain variable domain comprises the CDR3 region of SEQ ID NO:36, the CDR2 region of SEQ ID NO:37 and the CDR1 region of SEQ ID NO: 38.

2. The antibody of claim 1, characterized by comprising

a) 39; and

b) 40, light chain variable domain of SEQ ID NO.

3. The antibody according to any one of claims 1-2, wherein the antibody does not bind to human angiopoietin 1.

4. The antibody according to any one of claims 1-2, wherein the antibody is an antibody of the human IgG4 subclass or of the human IgG1 subclass.

5. A diabody, a single-chain antibody molecule or a multispecific antibody that specifically binds to human angiopoietin-2 formed from an antibody fragment of an antibody according to claim 1 or claim 2.

6. Pharmaceutical composition, characterized in that it comprises an antibody according to any one of claims 1 to 4 or a diabody, a single-chain antibody molecule or a multispecific antibody according to claim 5.

7. Use of an antibody according to any one of claims 1 to 4 or a diabody, a single-chain antibody molecule or a multispecific antibody according to claim 5 for the manufacture of a medicament for the prevention of metastasis of a solid tumor.

8. Use of an antibody according to any one of claims 1 to 4 or a diabody, a single-chain antibody molecule or a multispecific antibody according to claim 5 for the manufacture of a medicament for the treatment of a solid tumor.

9. Use of an antibody according to any one of claims 1 to 4 or a diabody, a single-chain antibody molecule or a multispecific antibody according to claim 5 for the manufacture of a medicament for the treatment of a vascular disease whose progression is dependent on abnormal angiogenesis.

10. Nucleic acid encoding the heavy chain of an antibody specifically binding to human angiopoietin-2, characterized in that said antibody comprises a heavy chain variable domain and a light chain variable domain according to claim 1.

11. Expression vector, characterized in that it comprises a nucleic acid according to claim 10, for expressing an antibody specifically binding to human angiopoietin-2 in a prokaryotic or eukaryotic host cell.

12. A prokaryotic or eukaryotic host cell comprising the vector according to claim 11.