HK1172354B

HK1172354B - Bispecific anti-vegf/anti-ang-2 antibodies

Info

Publication number: HK1172354B
Application number: HK12113232.7A
Authority: HK
Inventors: 莫妮卡．贝纳; 乌尔里希．布林克曼; 盖伊．乔治; 雷姆科．阿尔伯特．格里普; 萨拜因．伊姆霍夫-容; 阿妮塔．卡夫利; 休伯特．藤贝尔格; 克里斯蒂安．克莱因; 约尔格．托马斯．雷古拉; 沃尔夫冈.谢弗; 于尔根．迈克尔．尚策; 维尔纳．朔伊尔; 斯特凡．泽贝尔; 马库斯．托马斯
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2008-10-08
Filing date: 2009-10-07
Publication date: 2014-10-24

Abstract

The present invention relates to bispecific antibodies against human VEGF and against human ANG-2, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

Description

Bispecific anti-VEGF/anti-ANG-2 antibodies

The present invention relates to bispecific antibodies against human vascular endothelial growth factor (VEGF/VEGF-A) and against human angiopoietin-2 (ANG-2), 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; Klagsbrunu, 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, Lancet 340(1992) 1120-.

VEGF and anti-VEGF antibodies

Human vascular endothelial growth factor (VEGF/VEGF-A) (SEQ ID No: 105) is described, for example, in Leung, D.W., et al, Science 246(1989) 1306-9; keck, PJ., et al, Science 246(1989)1309-12 and Connolly, d.t., et al, j.biol.chem. (journal of biochemistry) 264(1989) 20017-24. VEGF is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with neoplastic and intraocular disorders (Ferrara, N., et al, endo cr. Rev. (endocrine review) 18(1997) 4-25; Berkman, R.A., et al, J.Clin.Invest. (J.Clin.Clin.) (J.Clin.) (153) 159; Brown, L.F., et al, Human Pathol. (Human pathology) 26(1995) 86-91; Brown, L.F., et al, Cancer Res. (Cancer research) 53(1993) 4727. 4735; Mattern, J.et al, Britt.J.cancer (British. Oncol.) (1996) 73 (102931; and Dvorak, H., et al, am.J.Pathol. (J.Pathol.) (1995) 1039). VEGF is a homodimeric glycoprotein that has been isolated from several sources. VEGF shows highly specific mitogenic activity on endothelial cells. VEGF has an important regulatory function in the neovascularization of angiogenesis during embryonic and adult life (Carmeliet, P., et al, Nature (Nature), 380(1996) 435-439; Ferrara, N., et al, Nature (Nature), 380(1996) 439-442; Ferrara and Davis-Smyth in Endocrine Rev., 18(1997)4-25 the importance of VEGF role was demonstrated in studies showing that inactivation of a single VEGF allele leads to embryonic lethality due to failure of the vasculature to develop (Carmeliet, P., et al, Nature (Nature), 380(1996) 435-439; Ferrara, N., et al, Nature (Nature), 380(1996) 439-442.) furthermore, VEGF has a strong chemoattractant activity for monocytes, is capable of inducing plasminogen and plasminogen activator inhibitors in endothelial cells and is also capable of inducing microvascular permeability, it is sometimes referred to as Vascular Permeability Factor (VPF). Isolation and properties of VEGF have been reviewed; see Ferrara, N., et al, J.Cellular Biochem (J.Cell. Biochem.), 47(1991) 211-. Alternative mRNA cleavage of a single VEGF gene produces five isoforms of VEGF.

anti-VEGF neutralizing antibodies inhibit the growth of various human tumor cell lines in mice (Kim, I., et al, Nature 362(1993) 841-844; Warren, S.R., et al, J.Clin.Invest. (J.Clin. Clin. J.Clin. 95(1995) 1789-1797; Borgstrom, P., et al, Cancer Res. 56(1996) 4032-4039; and Melnyk, O., et al, Cancer Res. (Cancer Res. 56 1996)921-924). WO 94/10202, WO 98/45332, WO 2005/00900 and WO 00/35956 refer to antibodies against VEGF. Humanized monoclonal antibody bevacizumab (bevacizumab under the trade name Avastin)Sold) is an anti-VEGF antibody WO 98/45331 for tumor therapy).

Ranibizumab (Ranibizumab, trade name Lucentis)) Are monoclonal antibody fragments derived from the same parent murine antibody as bevacizumab (avastin). It is much smaller than the parent molecule and affinity matured to provide stronger binding to VEGF-A (WO 98/45331). It is anti-angiogenic and has been approved for useIn the treatment of age-related macular degeneration of the "wet" type (ARMD), a common form of age-related vision loss. Another anti-VEGF antibody is, for example, humab 6-31, which is described, for example, in US 2007/0141065.

ANG-2 and anti-ANG-2 antibodies

Human angiopoietin-2 (ANG-2) (alternatively abbreviated ANGPT2 or ANG2) (SEQ ID NO: 106) is described in Maison pierre, P.C., et al, Science 277(1997)55-60 and Cheung, A.H., et al, Genomics 48(1998) 389-91. Angiopoietins-1 and-2 (ANG-1(SEQ ID No: 107) and ANG-2(SEQ ID No: 106)) 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 genetic locus in mice and humans. Kim, I., et al, FEBS Let, 443(1999) 353-56; kim, I., et al, J Biol Chem 274(1999) 26523-28. ANG-1 and ANG-2 were originally identified as agonists and antagonists, respectively, in tissue culture experiments (see: Davis, S., et al, Cell 87(1996)1161-69 for ANG-1; and Maison Pierre, P.C., et al, Science 277(1997)55-60 for ANG-2). 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) 1161-69; kwak, h.j., et al, FEBS Lett 448(1999) 249-53; suri, C., et al, Science 282(1998) 468-71; thurston, G., et al, Science 286(1999) 2511-14; thurston, g., et al, nat. med.6(2000)460-63, whereas ANG-2 has the opposite effect, promoting vessel destabilization and regression in the absence of the survival factors VEGF or basic fibroblast growth factor. 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, et al, Science 284 (Science) (1999) 1994-98; holash, J., et al, Oncogene 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.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.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.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.H. et al demonstrated that ANG-2 strongly upregulated the expression of MMPs-1, -9 and u-PA 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, to promote basal layer remodeling, proliferation and migration of endothelial cells, and to 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, p., et al, J clin. invest. (journal of clinical research) 09(2002) 777-85.

Studies of Tie-2 and angiopoietin-1 knockout mice have shown 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, J., et al, Genes & Development, 8(1994), 1897. 1909; Sato, T.N., Nature, 376(1995) 70-74; (Thurston, G., et al, Nature Medicine (Nature Medicine): 6(2000) 460) in adults where the effects of angiopoietin-1 are believed to be conserved among adults and are widely and constitutively expressed (Hanahan, D., Science, 1997) 48-50; Zzagg, D., Exp Neurology, 159, 1999: 391. compared to angiopoietin-400 (400), 463, 2-2 site of vascular remodeling, 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 sprouting signals (Hanahan, D.1997; Holash, J.et al, Orzcogerze 18(199) 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 research), 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) 124-29).

Angiopoietin-1, angiopoietin-2 and/or Tie-2 have been proposed in recent years as potential anti-cancer therapeutic targets. For example, each of US 6,166,185, US 5,650,490 and US 5,814,464 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, 1997; Lin 1998). Siemester, g., et al Siemeister, g., et al, Cancer Res, (Cancer research) 59(1999)3185-91 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 considered beneficial in the treatment of diseases (such as cancer) where progression is dependent on abnormal angiogenesis, where blockade of this process prevents disease progression (Follunan, 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, US 6166,185 and US 2003/10124129, WO 03/030833, WO 2006/068953, WO 03/057134 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, D., Science, 277(1997) 48-50). 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, thyroid tumor, Bunone, g., et al, Am JPathol 155(1999)1967-76, non-small cell Lung Cancer, Wong, m.p., et al, Lung Cancer 29(2000)11-22 and colon Cancer, Ahmad, s.a., et al, Cancer 92(2001)1138-43, and prostate Cancer Wurmbach, j.h., et al, anti Cancer res.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-2 mRNA 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) 228-35. ANG-2 mRNA was reported to be significantly associated with adjuvant lymph node invasion, short disease-free time and poor overall survival by detecting ANG-2 mRNA levels, sfigigio, c, et al, int.j. cancer (journal of international 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.cell 3(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.cell 3(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.cell 3(200) 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.cell 3(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 (proceedings of the american national academy of sciences) 95(1998) 8829-34; lin, p, et al, j.clin.invest. (journal of clinical research) 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 not being 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: 3(1999)3185-91. This is evidenced by the more recent disclosure of White, R., et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci.) 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.

Bispecific antibodies

A wide variety of recombinant antibody formats have recently been developed, such as tetravalent bispecific antibodies by fusion of, for example, an IgG antibody format and a single chain domain (see, e.g., Coloma, M.J., et al, Nature Biotech., 15(1997) 159-1234; WO 2001/077342 and Morrison, S.L., et al, Nature Biotech., 25(2007) 1233-1234).

Furthermore, several other novel forms have been developed which are capable of binding more than two antigens, wherein the antibody core structure (IgA, IgD, IgE, IgG or IgM) no longer retains, for example, diabodies, triabodies or tetrabodies, minibodies, single chain forms (scFv, bis-scFv) (Holliger P, et al, Nature Biotech (Nature Biotechnology) 23(2005) 1126. 11362005; Fischer N., and Leger, O., Pathiology (Pathiology) 74) 2007) 3-14; Shen J, et al, Journal of Immunological Methods (Journal of Immunological Methods)318(2007) 65-74; Wu, C. et al, Nature Biotech (25) (1290) 1297).

All such formats use linkers to fuse or fuse the antibody core (IgA, IgD, IgE, IgG or IgM) with other binding proteins (e.g. scFv), e.g. two Fab fragments or scFv (Fischer n., leger o., pathology (Pathobiology)74(2007) 3-14). One may always wish to maintain effector functions such as, for example, Complement Dependent Cytotoxicity (CDC) or Antibody Dependent Cellular Cytotoxicity (ADCC), which are mediated through Fc receptor binding by maintaining a high degree of similarity to naturally occurring antibodies.

In WO 2007/024715, dual variable domain immunoglobulins are reported as engineered multivalent and multispecific binding proteins. A method for the preparation of biologically active antibody dimers is reported in US 6,897,044. In US 7,129,330 multivalent F is reported having at least four variable domains connected to each other by a peptide linker_VAn antibody construct. Dimeric and multimeric antigen binding structures are reported in US 2005/0079170. Trivalent or tetravalent monospecific antigen binding proteins comprising three or four Fab fragments covalently bound to each other by a linking structure, which proteins are not native immunoglobulins, are reported in US 6,511,663. Tetravalent bispecific antibodies which can be efficiently expressed in prokaryotic and eukaryotic cells and which are used in therapeutic and diagnostic methods are reported in WO 2006/020258. In US 2005/0163782, a method is reported for separating or preferentially synthesizing dimers linked by at least one interchain disulfide bond from dimers not linked by at least one interchain disulfide bond in a mixture comprising two types of polypeptide dimers. Bispecific tetravalent receptors are reported in US 5,959,083. Engineered antibodies with three or more functional antigen binding sites are reported in WO 2001/077342.

Multispecific and multivalent antigen-binding polypeptides are reported in WO 1997/001580. WO 1992/004053 reports conjugate pairs (homoconjugates) typically prepared from monoclonal antibodies of the IgG class that bind the same antigenic determinant, covalently linked by synthetic cross-linking. Oligomeric monoclonal antibodies with high affinity for antigens are reported in WO 1991/06305, wherein oligomers, typically of the IgG class, are secreted with two or more immunoglobulin monomers that associate together to form tetravalent or hexavalent IgG molecules. Ovine derived antibodies and engineered antibody constructs are reported in US 6,350,860, which may be used for the treatment of diseases where interferon gamma activity is pathogenic. In US 2005/0100543, targetable constructs are reported which are multivalent vectors for bispecific antibodies, i.e. each molecule of a targetable construct can act as a vector for two or more bispecific antibodies. Genetically engineered bispecific tetravalent antibodies are reported in WO 1995/009917. In WO2007/109254, stable binding molecules consisting of or comprising stable scfvs are reported.

Combination of VEGF and ANG-2 inhibitors

WO 2007/068895 relates to a combination of an ANG-2 antagonist and a VEGF, KDR and/or FLTL antagonist. WO 2007/089445 relates to a combination of ANG-2 and a VEGF inhibitor.

WO 2003/106501 relates to fusion proteins that bind angiogenin and contain a multimerization domain. WO 2008/132568 relates to fusion proteins that bind angiogenin and VEGF.

Summary of The Invention

The first aspect of the invention is a bispecific antibody that specifically binds human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2), comprising a first antigen-binding site that specifically binds human VEGF and a second antigen-binding site that specifically binds human ANG-2.

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

i) the antigen binding sites are each a pair of an antibody heavy chain variable domain and an antibody light chain variable domain;

ii) said first antigen binding site that specifically binds VEGF comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO: 9, SEQ ID NO: 17, or SEQ ID NO: 94, CDR3 region of SEQ ID NO:2, SEQ ID NO: 10, SEQ ID NO: 18, or SEQ ID NO: 95 and the CDR2 region of SEQ ID NO:3, SEQ ID NO: 11, SEQ ID NO: 19, or SEQ id no: 96 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO:4, SEQ ID NO: 12, SEQ ID NO: 20, or SEQ ID NO: 97, the CDR3 region of SEQ ID NO:5, SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 98, and the CDR2 region of SEQ ID NO:6, SEQ ID NO: 14, SEQ ID NO: 22, or SEQ ID NO: 99, the CDR1 region;

iii) the second antigen-binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 38, SEQ ID NO:46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, or SEQ ID NO: 86, the CDR3 region of SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO:47, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, or SEQ ID NO: 87, and the CDR2 region of SEQ ID NO: 27, SEQ ID NO: 40, SEQ ID NO:48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, or SEQ ID NO: 88 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 28, SEQ ID NO: 28, SEQ ID NO: 41, SEQ ID NO:49, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81, or SEQ ID NO: 89, the CDR3 region of SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO:50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82 or SEQ ID NO: 90, and the CDR2 region of SEQ ID NO: 30, SEQ ID NO: 43, SEQ ID NO:51, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 91 CDR1 region.

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

ii) said first antigen binding site that specifically binds VEGF comprises the amino acid sequence of SEQ ID NO:1, CDR3 region of SEQ ID NO:2, and the CDR2 region of SEQ ID NO:3 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO:4, seq id NO:5, and the CDR2 region of SEQ ID NO:6, the CDR1 region;

or the first antigen binding site that specifically binds VEGF comprises in the heavy chain variable domain the amino acid sequence of SEQ ID NO: 9, CDR3 region of SEQ ID NO: 10, and the CDR2 region of SEQ id no: 11 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 12, CDR3 region of SEQ ID NO: 13, and the CDR2 region of SEQ ID NO: 14, CDR1 region;

or the first antigen binding site that specifically binds VEGF comprises in the heavy chain variable domain the amino acid sequence of SEQ ID NO: 17, CDR3 region of SEQ ID NO: 18, and the CDR2 region of SEQ id no: 19 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 20, CDR3 region of SEQ ID NO: 21, and the CDR2 region of SEQ ID NO: 22, CDR1 region; and

iii) the second antigen-binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 25, CDR3 region of SEQ ID NO: 26, and the CDR2 region of SEQ id no: 27, and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 28 or SEQ ID NO: 28, CDR3 region of SEQ ID NO: 29, and the CDR2 region of SEQ ID NO: 30, CDR1 region.

ii) the first antigen binding site that specifically binds VEGF comprises SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, or SEQ ID NO: 100 as a heavy chain variable domain, and SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, or SEQ ID NO: 101 as a light chain variable domain, and

iii) the second antigen binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84 or SEQ ID NO: 92 as a heavy chain variable domain, and SEQ id no: 32, SEQ ID NO: 32, SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 85 or SEQ ID NO: 93 as a light chain variable domain.

ii) said first antigen binding site that specifically binds VEGF comprises the amino acid sequence of SEQ ID NO:1, or SEQ ID NO: 94, CDR3 region of SEQ ID NO:2, or SEQ id no: 95 and the CDR2 region of SEQ ID NO:3, or SEQ ID NO: 96 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO:4, or SEQ ID NO: 97, seq id NO:5, or SEQ ID NO: 98, and the CDR2 region of SEQ ID NO:6, or SEQ ID NO: 99, the CDR1 region;

iii) the second antigen-binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 38, SEQ ID NO:46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, or SEQ ID NO: 86, the CDR3 region of SEQ ID NO: 39, SEQ ID NO:47, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, or SEQ ID NO: 87, and the CDR2 region of SEQ ID NO: 40, SEQ ID NO:48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, or SEQ ID NO: 88 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 41, SEQ ID NO:49, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81, or SEQ ID NO: 89, the CDR3 region of SEQ ID NO: 42, SEQ ID NO:50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82 or SEQ ID NO: 90, and the CDR2 region of SEQ ID NO: 43, SEQ ID NO:51, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 91 CDR1 region.

ii) the first antigen binding site that specifically binds VEGF comprises SEQ ID NO: 7, or SEQ ID NO: 100 as a heavy chain variable domain, and SEQ ID NO: 8 or SEQ ID NO: 101 as a light chain variable domain, and

iii) the second antigen binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84 or SEQ ID NO: 92 as a heavy chain variable domain, and SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 85 or SEQ ID NO: 93 as a light chain variable domain.

ii) said first antigen binding site that specifically binds VEGF comprises the amino acid sequence of SEQ ID NO:1, CDR3 region of SEQ ID NO:2, and the CDR2 region of SEQ ID NO:3 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO:4, seq id NO:5, and the CDR2 region of SEQ ID NO:6, the CDR1 region; and

iii) the second antigen-binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO:46, the CDR3 region of SEQ ID NO:47, and the CDR2 region of SEQ id no:48 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO:49, the CDR3 region of SEQ ID NO:50, and the CDR2 region of SEQ ID NO:51, CDR1 region.

ii) the first antigen binding site that specifically binds VEGF comprises SEQ ID NO: 7 as a heavy chain variable domain, and SEQ ID NO: 8 as a light chain variable domain, and

iii) the second antigen binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 52 as a heavy chain variable domain, and SEQ ID NO: 53 as light chain variable domains.

iii) the second antigen-binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 62, or SEQ ID NO: 86, the CDR3 region of SEQ ID NO: 63, or SEQ ID NO: 87, and the CDR2 region of SEQ ID NO: 64, or SEQ ID NO: 88 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 65, or SEQ ID NO: 89, the CDR3 region of SEQ ID NO: 66, or SEQ ID NO: 90, and the CDR2 region of SEQ ID NO: 67, or SEQ ID NO: 91 CDR1 region.

iii) the second antigen binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 68, or SEQ ID NO: 92 as a heavy chain variable domain, and SEQ ID NO: 69, or SEQ ID NO: 93 as a light chain variable domain.

iii) the second antigen-binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 62, the CDR3 region of SEQ ID NO: 63, and the CDR2 region of SEQ id no: 64, and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 65, the CDR3 region of SEQ ID NO: 66, and the CDR2 region of SEQ ID NO: 67, CDR1 region.

ii) the first antigen binding site that specifically binds VEGF comprises SEQ ID NO: 7 as a heavy chain variable domain, and SEQ ID NO: 8 as a light chain variable domain; and

iii) the second antigen binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 68 as a heavy chain variable domain, and SEQ ID NO: 69 as a light chain variable domain.

The bispecific antibody is at least bivalent and may be trivalent, tetravalent, or multivalent.

Preferably, the bispecific antibody according to the invention is bivalent, trivalent or tetravalent.

Another aspect of the invention is a nucleic acid molecule encoding a chain of said bispecific antibody.

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 the antibodies according to the invention.

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 bispecific 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 bispecific antibody is recovered from the cell or the cell culture supernatant. The invention also includes antibodies obtained by such recombinant methods.

Another aspect of the invention is a pharmaceutical composition comprising said bispecific antibody, said composition for use in the treatment of cancer, the use of said bispecific antibody for the manufacture of a medicament for the treatment of cancer, a method of treating a patient suffering from cancer by administering said bispecific antibody to a patient in need of such treatment.

The bispecific antibodies according to the invention show benefits for human patients in need of VEGF and ANG-2 targeted therapy. The antibodies according to the invention have new and inventive properties leading to benefits for patients suffering from said diseases, in particular patients suffering from cancer. It was surprisingly found that the combination of a bispecific antibody according to the invention with the respective monospecific parent antibody is more effective in tumor growth and/or in inhibiting tumor angiogenesis.

Detailed Description

One embodiment of the invention is a bispecific antibody specifically binding to human VEGF and human ANG-2, comprising a first antigen-binding site specifically binding to human VEGF and a second antigen-binding site specifically binding to human ANG-2, characterized in that:

iii) the second antigen-binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 38, SEQ ID NO:46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, or SEQ ID NO: 86, the CDR3 region of SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO:47, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, or SEQ ID NO: 87, and the CDR2 region of SEQ ID NO: 27, SEQ ID NO: 40, SEQ ID NO:48, SEQ ID NO: 56, SFQ IDNO: 64, SEQ ID NO: 72, SEQ ID NO: 80, or SEQ ID NO: 88 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 28, SEQ ID NO: 28, SEQ ID NO: 41, SEQ ID NO:49, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81, or SEQ ID NO: 89, the CDR3 region of SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO:50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82 or SEQ ID NO: 90, and the CDR2 region of SEQ ID NO: 30, SEQ ID NO: 43, SEQ ID NO:51, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 91 CDR1 region.

ii) the second antigen-binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 38, SEQ ID NO:46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, or SEQ ID NO: 86, the CDR3 region of SEQ ID NO: 39, SEQ ID NO:47, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, or SEQ ID NO: 87, and the CDR2 region of SEQ ID NO: 40, SEQ ID NO:48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, or SEQ ID NO: 88 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 41, SEQ ID NO:49, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81, or SEQ ID NO: 89, the CDR3 region of SEQ ID NO: 42, SEQ ID NO:50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82 or SEQ ID NO: 90, and the CDR2 region of SEQ ID NO: 43, SEQ ID NO:51, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 91 CDR1 region.

ii) the second antigen-binding site that specifically binds to ANG-2 comprises SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84 or SEQ ID NO: 92 as a heavy chain variable domain, and SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 85 or SEQ ID NO: 93 as a light chain variable domain.

iii) the second antigen-binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 78, the CDR3 region of SEQ ID NO: 79, and SEQ id no: 80, and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 81, the CDR3 region of SEQ ID NO: 82, and the CDR2 region of SEQ ID NO: 83 CDR1 region.

iii) the second antigen binding site that specifically binds ANG-2 comprises the amino acid sequence of SEQ ID NO: 84 as a heavy chain variable domain, and SEQ ID NO: 85 as the light chain variable domain.

Another embodiment of the invention is a bispecific antibody that specifically binds human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2), characterized in that the parent anti-ANG-2 antibody does not specifically bind human angiopoietin 1 (ANG-1). Typical maternal antibodies that specifically bind human ANG-2 but do not bind human ANG-1 are e.g. ANG2s _ R3_ LC03, ANG2s _ LC09, ANG2i _ LC06, ANG2i _ LC07, and preferably ANG2i _ LC10 or antibodies that bind the same epitope as ANG2s _ R3_ LC03, ANG2s _ LC09, ANG2i _ LC06, ANG2i _ LC07, ANG2i _ LC10, preferably antibodies that bind the same epitope as ANG2i _ LC06, or ANG2i _ LC 10. Thus in one embodiment of the invention, a bispecific antibody that specifically binds human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2) but does not bind human ANG-1 (or wherein the parent anti-ANG-2 antibody does not specifically bind human angiopoietin 1(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 _ LC06 or ANG2i _ LC 10. Such bispecific antibodies that specifically bind to human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2) but do not bind to human ANG-1 (or wherein the parent anti-ANG-2 antibody does not specifically bind to human angiopoietin-1 (ANG-1)) may have improved properties, such as e.g. biological or pharmacological activity, a lower toxicity or pharmacokinetic profile, compared to bispecific antibodies that specifically bind to human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2) and to human ANG-1.

A preferred embodiment is therefore a bispecific antibody specifically binding to human VEGF and human ANG-2 comprising a first antigen-binding site specifically binding to human VEGF and a second antigen-binding site specifically binding to human ANG-2, characterized in that: the second antigen binding site does not specifically bind to human angiopoietin 1 (ANG-1).

One embodiment of the invention is a bispecific antibody that specifically binds human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2), comprising a first antigen-binding site that specifically binds human VEGF and a second antigen-binding site that specifically binds human ANG-2, characterized in that: the ratio of binding affinity KD (antigen binding site specific for VEGF)/KD (antigen binding site specific for ANG-2) is 1.0-10.0, preferably 1.5-8.0 (in one embodiment 5.0-8.0) and preferably the absolute KD value is at 10^-8-10^-13In the range of mol/l. KD values were determined by binding BIACORE to ANG-2/VEGF (see example 2 and FIG. 15A). Because both human VEGF and human ANG-2 proteins are present in human serum at about the same concentration as soluble receptor ligands, blocking both receptor ligands by a bispecific antibody characterized by binding affinities KD (antigen binding site specific for VEGF)/KD (specific for ANG-2) may result in improved properties with respect to anti-angiogenic effects, tumor growth inhibition or resistance mechanisms in the treatment of cancer or vascular diseases with such bispecific antibodiesHeterologous antigen binding site) is 1.0 to 10.0, preferably 1.5 to 8.0, and in one embodiment 5.0 to 8.0. Preferably the bispecific antibody is characterized in that the ratio of binding affinity KD (antigen binding site specific for VEGF)/KD (antigen binding site specific for ANG-2) is 1.0-10.0, preferably 1.5-8.0 (in one embodiment 5.0-8.0), and the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 7 and the heavy chain variable domain of SEQ ID NO: 8 as a first antigen binding site that specifically binds VEGF, and a) the light chain variable domain of SEQ ID NO: 52 and the heavy chain variable domain of SEQ ID NO: 53 as the second antigen binding site that specifically binds ANG-2, or b) the light chain variable domain of SEQ ID NO: 84 and the heavy chain variable domain of SEQ ID NO: 85 as the second antigen binding site for the specific binding of ANG-2.

As used herein, "antibody" refers to a binding protein that comprises an antigen binding site. The term "binding site" or "antigen binding site" as used herein refers to the region of the antibody molecule to which an antibody actually binds. The term "antigen binding site" encompasses an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL) or a pair of VH/VL and may be derived from a whole antibody or antibody fragment such as a single chain Fv, VH domain and/or VL domain, Fab or (Fab)₂. In one embodiment of the invention, each antigen binding site comprises an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), and preferably is formed of a pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

Antigen binding sites that specifically bind human Vascular Endothelial Growth Factor (VEGF), and in particular the heavy chain variable domain (VH) and/or the antibody light chain variable domain (VL), can be obtained a) from known anti-VEGF antibodies, such as Kim et al, Nature (Nature) 362(1993) 841-844; warren, R.S., et al, J.Clin.Invest (J.Clin. Clin. J.Cl.) (1995) 1789-; borgstrom, P., et al, Cancer Res. (Cancer research) 56(1996) 4032-4039; melnyk, O., et al, cancer Res. (cancer research) 56(1996)921-924), WO 94/10202, WO 98/45332, WO 2005/00900, WO 00/35956 and US 2007/0141065 or b) from novel anti-VEGF antibodies obtained by de novo (de novo) immunization, wherein human VEGF proteins or nucleic acids or fragments thereof are used, or by phage display.

Antigen binding sites, in particular the heavy chain variable domain (VH) and/or the antibody light chain variable domain (VL), that specifically bind human angiopoietin-2 (ANG-2) can be obtained by a) from known anti-ANG-2 antibodies, such as WO 03/030833, WO 2006/068953, WO 2006/045049 or US 6,166,185; or b) from novel anti-ANG-2 antibodies obtained by a de novo immunization method in which human ANG-2 protein or nucleic acid or a fragment thereof is used or by phage display.

Antibody specificity refers to the selective recognition of a particular epitope of an antibody against an antigen. Natural antibodies, for example, are monospecific.

A "bispecific antibody" according to the invention is an antibody having two different antigen binding specificities. If the antibody has more than one specificity, the recognized epitope may be associated with a single antigen or more than one antigen. The antibodies of the invention are specific for two different antigens, VEGF as the first antigen and ANG-2 as the second antigen.

The term "monospecific" antibody, as used herein, refers to an antibody having one or more binding sites that each bind to the same epitope of the same antigen.

The term "valency" as used herein refers to the specific number of binding sites present on an antibody molecule. As such, the terms "divalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, on an antibody molecule. Bispecific antibodies according to the invention are at least "bivalent" and may be "trivalent" or "multivalent" (e.g., "tetravalent" or hexavalent). Preferably, the bispecific antibody according to the invention is bivalent, trivalent or tetravalent. In one embodiment, the bispecific antibody is bivalent. In one embodiment, the bispecific antibody is trivalent. In one embodiment, the bispecific antibody is tetravalent.

The antibodies of the invention have more than two binding sites and are bispecific. That is, the antibody may be bispecific even in the presence of more than two binding sites (i.e., the antibody is trivalent or multivalent). Bispecific antibodies of the invention include, for example, multivalent single chain, diabodies and triabodies, as well as antibodies having the constant domain structure of a full-length antibody to which an additional antigen-binding site (e.g., single chain Fv, VH and/or VL domains, Fab, or (Fab)2)) is linked by one or more peptide linkers. The antibody may be a full length antibody from a single species, or may be chimeric or humanized. For antibodies with more than two antigen binding sites, some binding sites may be the same, as long as the protein has binding sites for two different antigens. That is, when the first binding site is specific for VEGF, the second binding site is specific for ANG-2, and vice versa.

Human vascular endothelial growth factor (VEGF/VEGF-A) (SEQ ID No: 105) is described, for example, in Leung, D.W., et al, Science 246(1989) 1306-9; keck, P.J., et al, Science 246(1989)1309-12 and Connolly, D.T., et al, J.biol.chem. (J.Chem.Biol.264 (1989)) 20017-24. VEGF is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with neoplastic and intraocular disorders (Ferrara, N., et al, endo cr. Rev. (endocrine review) 18(1997) 4-25; Berkman, R.A., et al, J.Clin.Invest. (J.Clin.Clin.) (J.Clin.) (153) 159; Brown, L.F., et al, HumanPathol. (human pathology) 26(1995) 86-91; Brown, L.F., et al, Cancer Res. (Cancer research) 53(1993) 4727. 4735; Mattern, J.et al, Britt.J.cancer (British. Oncology) 73(1996) 934; 931; and Dvorak, H., et al, am.J.Pathol. (J.Pathol. (1995) 1039). VEGF is a homodimeric glycoprotein that has been isolated from several sources. VEGF shows highly specific mitogenic activity on endothelial cells.

Human angiopoietin-2 (ANG-2) (alternatively abbreviated ANGPT2 or ANG2) (SEQ ID NO: 106) is described in Maison pierre, P.C., et al, Science 277(1997)55-60 and Cheung, A.H., et al, Genomics 48(1998) 389-91. Angiopoietins-1 and-2 (ANG-1(SEQ ID No: 107) and ANG-2(SEQ ID No: 106)) 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 genetic locus in mice and humans. Kim, I., et al, FEBS Let, 443(1999) 353-56; kim, I., et al, J Biol Chem 274(1999) 26523-28. ANG-1 and ANG-2 were originally identified as agonists and antagonists, respectively, in tissue culture experiments (see: Davis, S., et al, Cell 87(1996)1161-69 for ANG-1; and maison Pierre, P.C., et al, Science 277(1997)55-60 for ANG-2). 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.

The antigen binding site of the antibodies of the invention may comprise six Complementarity Determining Regions (CDRs) that contribute to varying degrees to the affinity of the binding site for the antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 and CDRL 3). The extent of CDRs and Framework Regions (FRs) is determined by comparison with a compiled database of amino acid sequences in which those regions are determined by sequence-to-sequence variability. Also included within the scope of the invention are functional antigen binding sites comprising fewer CDRs (i.e., in cases where the binding specificity is determined by three, four, or five CDRs). For example, a complete set of fewer than 6 CDRs may be sufficient for binding. In some cases, a VH or VL domain is sufficient.

In certain embodiments, the antibodies of the invention further comprise an immunoglobulin constant region of one or more immunoglobulin classes. Immunoglobulin classes include the IgG, IgM, IgA, IgD, and IgE isotypes and, in the case of IgG and IgA, their subtypes. In a preferred embodiment, the antibody of the invention has the constant domain structure of an antibody of the IgG class, but has four antigen binding sites. This is accomplished, for example, by linking two intact antigen binding sites that specifically bind VEGF (e.g., single chain Fv) to the N-or C-terminal heavy or light chain of an intact antibody that specifically binds ANG-2. Alternatively this is achieved by fusing two intact binding peptides that specifically bind ANG-2 to the C-terminal heavy chain of a full length antibody that specifically binds VEGF. The four antigen binding sites preferably comprise two antigen binding sites for each of two different binding specificities.

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 that are currently well known in the art. See, for example, Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855; US 5,202,238 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; Bruggemann, 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, A.et al and Boemer, P.et al can also be used to prepare human Monoclonal Antibodies (Cole, A.et al, Monoclonal Antibodies and Cancer Therapy, Liss A.L., p.77 (1985); 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" (variable domain of a light chain (VL), variable region of a heavy chain (VH)) as used herein, denotes each pair of light and heavy chains that is directly involved in binding of an antibody to an antigen. The domains of variable human light and heavy chains have the same general structure and each domain comprises 4 Framework (FR) regions, the sequences of which are generally conserved, connected by 3 "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β -sheet conformation and the CDRs may form loops connecting the β -sheet structures. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain an antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and thus provide another object of the invention.

As used herein, the term "hypervariable region" or "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody which are responsible for antigen-binding. Hypervariable regions comprise amino acid residues from the "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 chains of an antibody comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The CDRs on each chain are separated by the framework amino acids. In particular, CDR3 of the heavy chain is the region most contributing to antigen binding. CDR and FR regions were 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 institute of Health, Bethesda, Md. (1991)) by Kabat et al.

Bispecific antibodies according to the present invention also include such antibodies with "conservative sequence modifications" (which refers to "variants" of bispecific antibodies). This means nucleotide and amino acid sequence modifications that do not affect or alter the above-mentioned characteristics of the antibodies according to the invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a bispecific < VEGF-ANG-2> antibody may preferably be replaced by another amino acid residue from the same side chain family. Thus, a "variant" bispecific < VEGF-ANG-2> antibody refers herein to a molecule whose amino acid sequence differs from the amino acid sequence of a "parent" bispecific < VEGF-ANG-2> antibody by up to 10, preferably about 2 to about 5, additions, deletions and/or substitutions in one or more variable or constant regions of the parent antibody. Amino acid substitutions may be made by mutagenesis based on molecular modeling, as described in Riechmann, L., et al, Nature (Nature)332(1988) 323-100327 and Queen, C., et al, Proc. Natl.Acad.Sci.USA 86(1989) 10029-10033. The "variant" bispecific < VEGF-ANG-2> antibodies according to the invention also include bispecific antibody formats in which the linker (if present) is modified or replaced by another linker.

As used herein, the term "binding" or "specific binding" refers to the binding of an antibody to an epitope of an antigen (human VEGF or human ANG-2) in an in vitro assay, preferably in a plasma assay with purified wild-type antigen (BIAcore, GE-Healthcare Uppsala, Sweden) (example 2). 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. 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 9).

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

In certain embodiments, an antibody is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

In one embodiment of the invention, the bispecific antibody comprises a full length parent antibody as a scaffold.

The term "full-length antibody" refers to an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains". A "full-length antibody heavy chain" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1(CH1), an antibody Hinge Region (HR), an antibody heavy chain constant domain 2(CH2), and an antibody heavy chain constant domain 3(CH3) in the N-terminal to C-terminal direction, abbreviated VH-CH1-HR-CH2-CH 3; and, in the case of antibodies of the IgE subclass, optionally also antibody heavy chain constant domain 4(CH 4). Preferably, a "full length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. A "full-length antibody light chain" is a polypeptide consisting of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated VL-CL, in the N-terminal to C-terminal direction. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). The two full-length antibody chains are linked together by interpeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge region of the full-length antibody heavy chain. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE. Full length antibodies according to the invention may be from a single species, e.g. human, or they may be chimeric or humanized antibodies. The full-length antibody according to the present invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which both specifically bind to the same antigen. Thus, a monospecific bivalent (═ full length) antibody comprising a first antigen binding site and consisting of two antibody light chains and two antibody heavy chains is a full length antibody. The C-terminus of the heavy or light chain of the full-length antibody refers to the last amino acid at the C-terminus of the heavy or light chain. The N-terminus of the heavy or light chain of the full-length antibody refers to the last amino acid at the N-terminus of the heavy or light chain.

For bispecific antibodies according to the invention which specifically bind human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2), preferred embodiments of the bispecific antibody format are bivalent antibodies with two different specificities, as e.g. a) described in WO 2009/080251, WO 2009/080252 or WO 2009/080253 (domain exchanged antibodies-see example 13), or b) scFab-based fusion antibodies, wherein one single-chain Fab fragment (eventually disulfide-stabilized) is specific for VEGF and the other single-chain Fab fragment (eventually disulfide-stabilized) is specific for ANG-2 (see example 14), or c) in Ridgway, j.b., Protein eng.9(1996) 617-621; WO 96/027011; merchant, A.M., et al, Nature Biotech 16(1998) 677-; atwell, S., et al, J.mol.biol. (1997) 270-35 and EP 1870459A 1.

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 121, SEQ ID NO: 122, SEQ ID NO: 123 and SEQ ID NO: 124 or a variant thereof.

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 125, SEQ ID NO: 126, SEQ ID NO: 127 and SEQ ID NO: 128 or a variant thereof.

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132 or a variant thereof.

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 133 and SEQ ID NO: 134 or a variant thereof.

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 135 and SEQ ID NO: 136, or a variant thereof.

These amino acid sequences are based on the amino acid sequence of SEQ id no: 7, and the heavy chain variable domain of SEQ ID NO: 8 (from bevacizumab (avastin)), and based on the amino acid sequence of seq id NO: 52, and the heavy chain variable domain of SEQ ID NO: 53 (from Ang2i _ LC 06).

In one embodiment, the bispecific antibody is trivalent, using for example a format based on a full length antibody specifically binding one of the two antigens VEGF or ANG-2, to which a scFab fragment is fused at only one C-terminus of one heavy chain, which specifically binds to the other of the two antigens VEGF or ANG-2, including the bump-in-hole technology (knobs-into-holes technology), as described in EP application No. 09004909.9 (see example 11), or a format based for example on a full length antibody specifically binding one of the two antigens VEGF or ANG-2, which at one C-terminus of one heavy chain, is fused to a VH or VH-CH1 fragment, and at the other C-terminus of the second heavy chain, which VL or VL-CL fragment specifically binds the other of the two antigens VEGF or ANG-2, including the bump-in-hole technique as described in EP application No. 09005108.7 (see example 12).

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 115, SEQ ID NO: 116 and SEQ ID NO: 117 or a variant thereof.

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 118, SEQ ID NO: 119 and SEQ ID NO: 120 or a variant thereof.

A preferred bispecific antibody format for a bispecific antibody according to the invention specifically binding to human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2) is a tetravalent antibody (TvAb) with two different specificities, as described for example in WO 2007/024715, or WO2007/109254 or EP application No. 09004909.9. Thus in one embodiment the bispecific antibody is tetravalent, using a format as described for example in WO 2007/024715, or WO2007/109254 or EP application No. 09004909.9 (see example 1 or 10).

In one embodiment of the invention, the bispecific tetravalent antibody TvAb-2441-bevacizumab-LC 06 is characterized by comprising SEQ ID NO: 102 and SEQ ID NO: 62 or a variant thereof.

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 109 and SEQ ID NO: 110 or a variant thereof.

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 111 and SEQ ID NO: 112 or a variant thereof.

In one embodiment, the bispecific antibody according to the invention is characterized by comprising the amino acid sequence of SEQ id no: 113 and SEQ ID NO: 114 or a variant thereof.

In one embodiment of the invention, the bispecific tetravalent antibody TvAb-2441-bevacizumab-LC 08 is characterized by comprising SEQ ID NO: 103 and SEQ ID NO: 62 or a variant thereof.

The binding sites in the antibodies according to the invention may each be formed by pairs of two variable domains, namely one heavy chain variable domain and one light chain variable domain. The smallest binding site determinant in an antibody is the heavy chain CDR3 region.

In one embodiment, the bispecific antibody according to the invention is tetravalent. In another embodiment, the tetravalent bispecific antibody has the following characteristics:

-it consists of:

a) a monospecific bivalent parent antibody consisting of two full-length antibody heavy chains and two full-length antibody light chains, wherein each chain comprises only one variable domain,

b) two peptide linkers are used to prepare the peptide,

c) two monospecific monovalent single chain antibodies, each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain and a single linker between said antibody heavy chain variable domain and said antibody light chain variable domain;

and preferably, the single chain antibody is linked to the same end (C-terminus and N-terminus) of the monospecific bivalent antibody heavy chain, or alternatively to the same end (preferably C-terminus) of the monospecific bivalent antibody light chain, and more preferably to the same end (C-terminus and N-terminus) of the monospecific bivalent antibody heavy chain.

In another embodiment, the bispecific antibody is tetravalent and consists of:

a) a full-length antibody comprising the antigen binding site and consisting of two antibody heavy chains and two antibody light chains; and

b) two identical single chain Fab fragments comprising the second antigen binding site,

wherein the single chain Fab fragment under b) is fused to the full length antibody under a) via a peptide linker at the C-or N-terminus of the heavy or light chain of the full length antibody.

In another embodiment, the bispecific antibody is tetravalent and consists of:

a) a full-length antibody comprising the second antigen-binding site and consisting of two antibody heavy chains and two antibody light chains; and

b) two identical single chain Fab fragments comprising the first antigen binding site,

Preferably, said single chain Fab fragment under b) is fused to said full length antibody by a peptide linker at the C-terminus of the heavy or light chain of said full length antibody.

In one embodiment, two identical single chain Fab fragments that bind a second antigen are fused to the full length antibody by a peptide linker at the C-terminus of each heavy or light chain of the full length antibody.

In one embodiment, two identical single chain Fab fragments that bind a second antigen are fused to the full length antibody by a peptide linker at the C-terminus of each heavy chain of the full length antibody.

In one embodiment, two identical single chain Fab fragments that bind a second antigen are fused to the full length antibody by a peptide linker at the C-terminus of each light chain of the full length antibody.

Such embodiments, including single chain Fab fragments, are described in more detail in, for example, EP application No. 09004909.9, which is incorporated herein by reference.

The term "peptide linker" as used in the present invention refers to a peptide having an amino acid sequence, which is preferably of synthetic origin. These peptide linkers according to the invention are used to link different antigen binding sites and/or antibody fragments (e.g. single chain Fv, full length antibody, VH domain and/or VL domain, Fab, (Fab)2, Fc part) eventually comprising different antigen binding sites to form together a bispecific antibody according to the invention. The peptide linker may comprise one or more of the following amino acid sequences listed in table 1 and additionally any selected amino acid. The peptide linker is a peptide having an amino acid sequence of at least 5 amino acids in length, preferably at least 10 amino acids in length, more preferably 10-50 amino acids in length. Preferably, said peptide linker under b) is a peptide having an amino acid sequence of at least 10 amino acids in length. In one embodiment, the peptide linker is (GxS) n, wherein G ═ glycine, S ═ serine, (x ═ 3 and n ═ 3, 4, 5 or 6) or (x ═ 4 and n ═ 2, 3, 4 or 5), preferably x ═ 4 and n ═ 2 or 3, more preferably x ═ 4, n ═ 2((G ═ 2 or 3), more preferably x ═ 4, n ═ 2 ═ n ═ 2₄S)₂). Additional G ═ glycine, e.g. GG, or GGG, may also be added to the (GxS) n peptide linker.

The term "single chain linker" as used in the present invention refers to a peptide having an amino acid sequence, which is preferably of synthetic origin. These single linkers according to the invention are used to link the VH and VL domains to form a single chain Fv. Preferably, said single linker under c) is a peptide having an amino acid sequence of at least 15 amino acids in length, more preferably at least 20 amino acids in length. In one embodiment, the single linker is (GxS) n, wherein G ═ glycine, S ═ serine, (x ═ 3 and n ═ 4, 5, or 6) or (x ═ 4 and n ═ 3, 4, or 5), preferably x ═ 4, n ═ 4 or 5, more preferably x ═ 4, n ═ 4.

Furthermore, the single chain (single chain Fv) antibodies are preferably disulfide-stabilized. Further disulfide stabilization of such single chain antibodies is achieved by introducing disulfide bonds between the variable domains of the single chain antibodies, and is described, for example, in WO 94/029350, Rajagopal, v., et al, prot.engin.10(12) (1997) 1453-59; kobayashi, H., et al, nucleic acid pharmaceuticals and Biology (nucleic acids Medicine & Biology)25(1998) 387-393; or Schmidt, M., et al, Oncogene (Oncogene)18(1999) 1711-1721.

In one embodiment of the disulfide-stabilized single chain antibody, the disulfide bonds between the variable domains of the single chain antibodies comprised in the antibody according to the invention are selected independently of each single chain antibody from:

i) heavy chain variable domain position 44 to light chain variable domain position 100,

ii) heavy chain variable domain position 105 to light chain variable domain position 43, or

iii) heavy chain variable domain position 101 to light chain variable domain position 100.

In one embodiment, the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is between heavy chain variable domain position 44 to light chain variable domain position 100.

In one embodiment, the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is between heavy chain variable domain position 105 to light chain variable domain position 43.

The structure of this tetravalent embodiment of the bispecific antibody according to the invention specifically binds VEGF and ANG-2, wherein one of the antigens a or B is VEGF and the other is ANG-2. The structure is based on a full-length antibody specifically binding to antigen a to which two (optionally disulfide-stabilized) single-chain Fv's specifically binding to antigen B are linked by a peptide linker, the structure being exemplified in the schematic diagrams of fig. 1 and 2.

In one embodiment, it is preferred that the optional disulfide stabilized single chain (single chain Fv) antibody is not present between the variable domains VH and VL of the single chain antibody (single chain Fv).

In another embodiment, the tetravalent bispecific antibody is characterized in that: the monospecific bivalent antibody moiety under a) specifically binds VEGF and the two monovalent monospecific single chain antibodies under c) bind ANG-2.

In another embodiment, the tetravalent bispecific antibody is characterized in that: the monospecific bivalent antibody moiety under a) specifically binds ANG-2 and the two monovalent monospecific single chain antibodies under c) bind VEGF.

A "single chain Fab fragment" (see FIG. 11) is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1(CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH 1-linker-VL-CL, b) VL-CL-linker-VH-CH 1, c) VH-CL-linker-VL-CH 1 or d) VL-CH 1-linker-VH-CL; and wherein the linker is a polypeptide of at least 30 amino acids, preferably a polypeptide of 32-50 amino acids. The single chain Fab fragment a) VH-CH 1-linker-VL-CL, b) VL-CL-linker-VH-CH 1, c) VH-CL-linker-VL-CH 1 and d) VL-CH 1-linker-VH-CL, stabilized by a natural disulfide bond between the CL domain and the CH1 domain. The term "N-terminal" refers to the last amino acid at the N-terminus. The term "C-terminal" refers to the last amino acid at the C-terminus.

In a preferred embodiment, said antibody domain and said linker in said single chain Fab fragment have one of the following sequences in the direction from N-terminus to C-terminus:

a) VH-CH 1-linker-VL-CL, or b) VL-CL-linker-VH-CH 1, more preferably VL-CL-linker-VH-CH 1.

In another preferred embodiment, said antibody domain and said linker in said single chain Fab fragment have one of the following sequences in the direction from N-terminus to C-terminus:

a) VH-CL-linker-VL-CH 1 or b) VL-CH 1-linker-VH-CL.

The term "peptide linker" as used in the present invention refers to a peptide having an amino acid sequence, which is preferably of synthetic origin. These peptide linkers according to the invention are used to fuse a single chain Fab fragment to the C-terminus or N-terminus of a full-length antibody to form a multispecific antibody according to the invention. Preferably, said peptide linker under b) is a peptide having an amino acid sequence of at least 5 amino acids in length, preferably a peptide having an amino acid sequence of 5-100 amino acids in length, more preferably a peptide having an amino acid sequence of 10-50 amino acids in length. In one embodiment, the peptide linker is (GxS) n or (GxS) nGm, wherein G ═ glycine, S ═ serine, and (x ═ 3, n ═ 3, 4, 5, or 6, and m ═ 0, 1, 2, or 3) or (x ═ 4, n ═ 2, 3, 4, or 5 and m ═ 0, 1, 2, or 3), preferably x ═ 4 and n ═ 2 or 3, more preferably x ═ 4, n ═ 2. In one embodiment, the peptide linker is (G)₄S)₂。

The term "linker" as used in the present invention refers to a peptide having an amino acid sequence, which is preferably of synthetic origin. These peptides according to the invention are used to link a) VH-CH1 with VL-CL, b) VL-CL with VH-CH1, c) VH-CL with VL-CH1 or d) VL-CH1 with VH-CL to form the following single chain Fab fragments according to the invention a) VH-CH 1-linker-VL-CL, b) VL-CL-linker-VH-CH 1, c) VH-CL-linker-VL-CH 1 or d) VL-CH 1-linker-VH-CL. The linker in the single chain Fab fragment is an amino acid sequence having a length of at least 30 amino acids, preferably an amino acid sequence having a length of 32-50 amino acids. In one embodiment, the linker is (GxS) n, wherein G ═ glycine, S ═ serine, (x ═ 3, n ═ 8, 9 or 10 and m ═ 0, 1, 2 or 3) or (x ═ 4 and n ═ 6, 7 or 8 and m ═ 0, 1, 2 or 3), preferably wherein x ═ 4, n ═ 6 or 7 and m ═ 0, 1, 2 or 3,2 or 3, more preferably x-4, n-7 and m-2. In one embodiment, the linker is (G)₄S)₆G₂。

Optionally in said single chain Fab fragment, disulfide-stabilizing the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL) by introducing a disulfide bond between:

iii) heavy chain variable domain position 101 to light chain variable domain position 100 (always numbered according to the EU index of Kabat).

These additional disulfide stabilization of the single chain Fab fragments is achieved by introducing a disulfide bond between the variable domains VH and VL of the single chain Fab fragment. Techniques for the introduction of non-natural disulfide bridges to stabilize single chain Fv are described, for example, in WO 94/029350, Rajagopal, V., et al, prot.Engin, (1997) 1453-59; kobayashi, h., etc.; nuclear medicine and Biology (nuclear medicine & Biology), Vol 25, (1998) 387-393; or Schmidt, M., et al, Oncogene (Oncogene) (1999)18, 1711-1721. In one embodiment, the optional disulfide bond between the variable domains of the single chain Fab fragments comprised in the antibody according to the present invention is between heavy chain variable domain position 44 and light chain variable domain position 100. In one embodiment, the optional disulfide bond between the variable domains of the single chain Fab fragments comprised in the antibody according to the invention is between heavy chain variable domain position 105 and light chain variable domain position 43 (always numbered according to EU index of Kabat).

In one embodiment it is preferred not to have said optional disulphide stabilized single chain Fab fragment between the variable domains VH and VL of the single chain Fab fragment.

Preferably, said second embodiment of the tetravalent bispecific antibody according to the invention comprises two identical single chain Fab fragments (preferably VL-CL-linker-VH-CH 1) fused both to the C-termini of the two heavy chains of said full length antibody under a) or fused both to the C-termini of the two light chains of said full length antibody under a). Such fusion results in the formation of two identical fusion peptides ((i) heavy chain and single chain Fab fragment or ii) light chain and single chain Fab fragment) which are co-expressed with i) the light or heavy chain of the full length antibody to provide a bispecific antibody according to the invention.

In another embodiment, the bispecific antibody is characterized in that the constant region is of human origin.

In another embodiment, the bispecific antibody is characterized in that the constant region of the bispecific antibody according to the invention is of the subclass human IgG1, or of the subclass human IgG1 with the mutations L234A and L235A.

In another embodiment, the bispecific antibody is characterized in that the constant region of the bispecific antibody according to the invention is of the human IgG2 subclass.

In another embodiment, the bispecific antibody is characterized in that the constant region of the bispecific antibody according to the invention is of the human IgG3 subclass.

In another embodiment, the bispecific antibody is characterized in that the constant region of the bispecific antibody according to the invention is of the subclass human IgG4, or of the subclass human IgG4 with the additional mutation S228P.

It has now been found that bispecific antibodies against human VEGF and human ANG-2 according to the invention have improved characteristics, such as biological or pharmacological activity, pharmacokinetic properties or toxicity. They show increased tumor growth inhibition and/or tumor angiogenesis inhibition in vivo when compared to monospecific parent antibodies directed against VEGF and ANG-2 (see examples 16, 17 and 18: comparison of the different bispecific < VEGF-ANG-2> antibodies bevacizumab-ANG 2i-LC06 with the monospecific antibodies avastin (bevacizumab) alone, ANG2i-LC06 alone or a combination of both).

In addition, the smaller toxic side effects (which are in response to increased body weight of the test animals and less death of the test animals during in vivo application) compared to the combined use of two corresponding single monospecific antibodies directed against VEGF and ANG-2 also represent an advantage of the bispecific antibody according to the invention.

Furthermore, bispecific antibodies according to the invention may provide benefits such as reduced dosage and/or frequency of administration and concomitant cost savings.

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, δ, ε, γ, and μ, respectively. The light chain constant regions that can be found in all 5 antibody species are called 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 or lambda 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 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 (full length) parent antibody is involved in FcR binding of the IgG4 subclass or of the IgG1 or IgG2 subclass with mutations S228, L234, L235 and/or D265, and/or comprises a PVA236 mutation. In one embodiment, the mutation in the monospecific bivalent (full-length) parent antibody is S228P, L234A, L235A, L235E and/or PVA 236. In another embodiment, the mutation in the monospecific bivalent (full length) parent antibody is S228P in IgG4 and L234A and L235A in IgG 1. Constant heavy chain region is set forth in SEQ ID NO: 35 and 36. In one embodiment, the constant heavy chain region of the monospecific bivalent (full-length) parent antibody is the heavy chain region of SEQ ID NO: 35, or a heavy chain constant region. In another embodiment, the constant heavy chain region of the monospecific bivalent (full-length) parent antibody is SEQ ID NO: 36, constant heavy chain region. In another embodiment, the constant light chain region of the monospecific bivalent (full-length) parent antibody is SEQ ID NO: 37 or the kappa light chain region of SEQ id no: 34 lambda light chain region. The constant heavy chain region of a preferred monospecific bivalent (full-length) parent antibody is SEQ ID NO: 35 or SEQ ID NO with mutation S228P: 36, constant heavy chain region.

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 127(1981) 2555-2560; brunhouse, r., and Cebra, j.j., molecular immunology 16(1979) 907-; burton, D.R., et al, Nature 288(1980) 338-344; thommesen, J.E., et al, molecular immunology (mol. Immunol.)37(2000) 995-1004; idusogene, E.E., et al, J.Immunol. 164(2000) 4178-4184; hezareh, M., et al, J.Virol., 75(2001) 12161-12168; morgan, A., et al, Immunology 86(1995) 319-324; and EP 0307434. 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 the antibody results from a defined protein-protein interaction at the 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 a 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 recombinant production of antibodies are well known in the art and are described, for example, in Makrides, S.C., Protein Expr. Purif.17(1999) 183-202; geisse, S., et al, Protein Expr. Purif.8(1996) 271-; kaufman, R.J., molecular biology techniques (mol.Biotechnol.)16(2000) 151-161; werner, R.G., Drug Res.48(1998) 870-.

The bispecific antibody is 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 bispecific antibodies 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, 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, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.usa)89(1992) 4285-; and Norderhaug, l., et al, journal of immunological methods (j.immunological. method)204(1997) 77-87. Preferred transient expression systems (HEK 293) 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., applied biochemistry technology (applied biochem. biotech.) 75(1998) 93-102).

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.

One embodiment of the invention is a bispecific antibody according to the invention for use in the treatment of cancer.

Another aspect of the invention is said pharmaceutical composition 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 clear to the skilled person, 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 (NSCL) cancer (non-small cell lung (NSCL) cancer), bronchoalveolar cell lung cancer (bronchololar cell lung cancer), bone cancer (bone cancer), pancreatic cancer (pancreatic cancer), skin cancer (skin cancer), head or neck cancer (cancer of the head, skin or intraocular melanoma (cutaneous or intraocular tumor cell), uterine cancer (uterine cancer), ovarian cancer (ovarian cancer), rectal cancer (rectal cancer), anal region cancer (cancer of the anal region), gastric cancer (gastric cancer), ovarian cancer (ovarian cancer), uterine cancer (ovarian cancer of the uterine cancer), ovarian cancer (ovarian cancer of the uterine cancer (colon cancer), uterine cancer (colon cancer, colon cancer (colon cancer), colon cancer (colon cancer, colon cancer (colon cancer, colon, cervical cancer (cancer of the cervical cancer), vaginal cancer (cancer of the vaginal), vulvar cancer (cancer of the vulva), Hodgkin's Disease (Hodgkin's Disease), esophageal cancer (cancer of the esophageal), small intestine cancer (cancer of the intestinal), 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 ovarian gland), soft tissue sarcoma (cancer of the soft tissue), urethral cancer (cancer of the urethral cancer), penile cancer (cancer of the lung), prostate cancer (cancer of the urinary tract), bladder cancer (cancer of the bladder, urethral cancer (renal cancer), renal cancer (cancer of the renal cell), hepatocellular carcinoma (hepatocellular carcinoma), cholangiocarcinoma (biliary carcinoma), Central Nervous System (CNS) tumors (neuroplasts of the Central Nervous System (CNS)), vertebral axis tumors (spinal axis tumors), brain stem glioma (braun stem glioma), glioblastoma multiforme (glioblastomas), astrocytoma (astrocytoma), schwannoma (schwannomas), ependymoma (epiymonas), meduloblastoma (meduloblastoma), meningioma (menias), squamous cell carcinoma (squamomus cell carcinosa), pituitary adenoma (pituitary adenosma), and Ewens sarcoma (Ewing's sarcoma), 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 a bispecific antibody according to the invention or 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.

One embodiment of the invention is a bispecific antibody according to the invention for use in the treatment of a vascular disease.

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

The term "Vascular diseases" includes Cancer (Cancer), Inflammatory diseases (Inflammatory diseases), Atherosclerosis (Atherosclerosis), Ischemia (Ischemia), Trauma (Trauma), Sepsis (Sepsis), COPD, Asthma (Asthma), Diabetes (diabets), AMD, Retinopathy (retinophathy), Stroke (Stroke), obesity (adipositis), Acute lung injury (Acute lung injury), Hemorrhage (Hemorrhage), Vascular leakage (Vascular leak) such as cytokine-induced Vascular leakage, Lupus Erythematosus (Allergy), Graves ' Disease, Hashimoto's Autoimmune Thyroiditis (Hashimoto's Autoimmune Thyroiditis), Idiopathic Thrombocytopenic Purpura (ischemic thrombosis, Systemic Lupus Erythematosus), Systemic Lupus Erythematosus (Systemic Lupus Erythematosus), multiple Sclerosis (Multiple Sclerosis), Ulcerative Colitis (Ulcerative Colitis), particularly solid tumors, intraocular neovascular syndromes such as proliferative retinopathies (proliferative retinopathies) or age-related macular degeneration (AMD), rheumatoid arthritis (psoriasis) and psoriasis (psoriasis). (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.Vasculardis (vascular diseases) in: Pathobiology of ocular disease, A dynamic approach (pathology of the ophthalmic disease, i.e., 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 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.

Suitable fluidity can be maintained, for example, by the use of a coating such as phosphatidylcholine, 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 calcium phosphate precipitation as described by Graham, F.L., van der Eb.A.J., Virology 52(1978)546 ff. 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 parenchymal cell wall structure are used, for example, one method of transfection is calcium treatment with calcium chloride, as described by Cohen, S.N, et al, PNAS (proceedings of the national academy of sciences USA) 69(1972) 2110-2114.

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.

Description of the amino acid sequence

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 understood that modifications may be made in the proposed method without departing from the spirit of the invention.

Description of the drawings

Figure 1A schematic structure of a tetravalent embodiment of a bispecific antibody according to the invention binding VEGF and ANG-2, wherein one of the antigens a or B is VEGF and the other is ANG-2. The structure is based on a full-length antibody that binds antigen a to which two (optionally disulfide-stabilized) single-chain Fv's that bind antigen B are linked by a peptide-linker.

Figure 1B schematic representation of bispecific tetravalent antibodies generated using TvAb nomenclature (see examples) -with or without disulfide stabilized scFv.

Figure 2A schematic representation of disulfide-stabilized < VEGF-ANG-2> bispecific tetravalent antibodies (═ VEGF-ANG-2> TvAb 6; No.2331, see table 3).

Figure 2B plasmid diagram of modified heavy and light chain vectors for expression of disulfide stabilized < VEGF-ANG-2> TvAb 6.

Figure 3 purified disulfide stabilized < VEGF-ANG-2> TvAb6 SDS-PAGE under reducing and non-reducing conditions compared to the "standard" human IgG1 antibody G6-31(< VEGF > HuMab G6-31).

Figure 4 comparison of size exclusion chromatography of purified disulfide-stabilized < VEGF-ANG-2> TvAb6 with "standard" human IgG1 antibody G6-31 shows that disulfide-stabilized TvAb6 no longer forms aggregates upon purification.

FIG. 5 results and schematic representation of VEGF binding ELISA. Disulfide-stabilized < VEGF-ANG-2> TvAb6 binds VEGF comparable to < VEGF > G6-31. < ANG-2> Mab536 did not bind VEGF.

FIG. 6A results and schematic of ANG-2 binding ELISA. Disulfide-stabilized < VEGF-ANG-2> TvAb6 binds ANG-2 in a manner comparable to < ANG-2> Mab 536. < VEGF > G6-31 did not bind ANG-2.

FIG. 6B schematic and results of ANG-2 binding analysis by surface plasmon resonance (Biacore.) disulfide stabilized < VEGF-ANG-2> TvAb6 binds ANG-2 with comparable affinity to < ANG-2> Mab 536.

FIG. 7 schematic and results of VEGF-ANG-2 bridging ELISA. Disulfide stabilized < VEGF-ANG-2> TvAb6 binds both VEGF and ANG-2, whereas < VEGF > G6-31 and < ANG-2> Mab536 are not able to bind both VEGF and ANG-2.

FIG. 8a Effect of disulfide stabilized < VEGF-ANG-2> TvAb6 in the Scid beige mouse staged subcutaneous Colo205 xenograft model compared to < ANG-2> Mab536, < VEGF > G6-31 and the combination of Mab536 and G6-31 (study ANG2_ Pz _ Colo205_ 003).

FIG. 8b Effect of disulfide stabilized < VEGF-ANG-2> TvAb6 in the Scid beige mouse staged subcutaneous Colo205 xenograft model compared to < ANG-2> Mab536, < VEGF > G6-31 and the combination of Mab536 and G6-31 (study ANG2_ Pz _ Colo205_ 005).

Figure 9 blocks VEGF-induced tube formation by the bispecific tetravalent antibody < VEGF-ANG-2> TvAb 6-results.

Figure 10A + B block VEGF-induced tube formation by B disulfide stabilized < VEGF-ANG-2> TvAb 6-quantitative analysis.

FIG. 11 schematic representation of VEGF binding analysis by surface plasmon resonance (Biacore).

Figure 12 kinetics of two < VEGF > antibodies < VEGF-Ang-2> TvAb6 and < VEGF > G6-31 in Ka-Kd graphs.

Figure 13 schematic representation of surface plasmon resonance (Biacore) assay to detect simultaneous binding of ANGPT2 and VEGF to bispecific antibodies.

Figure 14 results of surface plasmon resonance (Biacore) experiments show that TvAb6 binds to ANGPT2 and VEGF simultaneously.

Figure 15A + B A) < bispecific assay for VEGF-ANG-2> bispecific antibody and schematic of simultaneous Biacore binding assay. B) Biacore data demonstrated that ANG-2 and VEGF simultaneously bound to TvAb-2441-bevacizumab _ LC 06.

Figure 16A + B Tie2 phosphorylation of bispecific antibodies < VEGF-Ang-2> TvAb-2441-bevacizumab-LC 06 and < VEGF-Ang-2> TvAb-2441 compared to anti-Ang 2 antibodies < Ang-2> Ang2i _ LC06 and < Ang-2> Ang2_ 2k _ LC 08.

FIG. 17 schematic representation of human angiogenin interaction ELISA.

FIG. 18 VEGF-induced HUVEC proliferation of < VEGF-ANG-2> TvAb-2441-bevacizumab-LC 06 and < VEGF-ANG-2> TvAb-2441-bevacizumab-LC 08 and bevacizumab.

Figure 19 in vivo anti-angiogenic effect of bispecific antibody < VEGF-ANG-2> bevacizumab-LC 06 antibody monitored by relative change in labeled anti-CD 31 antibody and CD31 signal during treatment in Calu3 xenograft model (compared to < ANG-2> ANG2i-LC06, and the combination of < ANG-2> ANG2i-LC06 and avastin (bevacizumab)).

Experimental methods

Examples

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 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). The gene segments used encoding the light and heavy chains of the Ang-2/VEGF bispecific antibody were synthesized with the 5 ' terminal DNA sequence encoding the leader peptide (MGWSCIILFLVATATGVHS) targeted to a protein for secretion in eukaryotic cells and 5 ' -BamHI and 3 ' -XbaI restriction sites. DNA sequences with disulfide-stabilized "bulge-entry-hole" modified heavy chains were designed, with the S354C and T366W mutations in the "bulge" heavy chain and the Y349C, T366S, L368A and Y407V mutations in the "hole" heavy chain.

DNA sequencing

DNA sequence the DNA sequence was determined by double-strand sequencing performed 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 example 1)

For the expression of the antibodies, variants of expression plasmids for transient expression in cells (for example in HEK293 EBNA or HEK 293-F) or stable expression (for example in CHO cells) based on cDNA constructs with the CMV-intron a promoter (organization) or on genomic constructs with the CMV promoter (for example fig. 2B) are used.

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 and C-terminal scFv fusions described below were generated by PCR and/or gene synthesis and assembled by ligating the corresponding nucleic acid segments in a genomic heavy chain vector, for example, using unique NsiI and EcoRI sites, using known recombinant methods and techniques. 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.

Expression vector (for examples 10-14)

An expression vector consisting of the following elements was used:

-a hygromycin resistance gene as a selectable marker,

-origin of replication of Epstein-Barr virus (EBV), oriP,

the origin of replication of the vector pUC18, which allows the plasmid to replicate in E.coli (E.coli)

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

immediate early enhancer and promoter from Human Cytomegalovirus (HCMV),

-a human 1-immunoglobulin polyadenylation ("poly a") signal sequence, and

unique BamHI and XbaI restriction sites.

As described, an immunoglobulin fusion gene comprising a heavy or light chain construct and a "bulge-entry-hole" construct with C-terminal VH and VL domains was prepared by gene synthesis and cloned into the pGA18(ampR) plasmid. pG18(ampR) plasmid with the synthesized DNA segment and the Roche expression vector was digested with BamHI and XbaI restriction enzymes (Roche molecular Biochemicals) and subjected to agarose gel electrophoresis. The purified heavy and light chain encoding DNA segments were then ligated into the isolated Roche expression vector BamHI/XbaI fragment to generate the final expression vector. The final expression vector was transformed into E.coli cells, expression plasmid DNA (miniprep) was isolated and subjected to restriction enzyme analysis and DNA sequencing. The correct clones were grown in 150ml LB-Amp medium, and plasmid DNA (Maxiprep) was re-isolated and sequence integrity confirmed by DNA sequencing.

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 (for example 1)

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 (Invitrogen) grown in suspension in serum-free FreeStyle 293 expression medium (Invitrogen) in shake flasks or in stirred fermentors was 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 a mixture of ca.42ml, a) 20mL of Opti-MEM (invitrogen) with 600 μ g total plasmid DNA (1 μ g/mL) encoding equimolar ratios of heavy chain or modified heavy chain, respectively, and corresponding light chain, at a cell density of ca.1.5e 6 cells/mL, and B) a mixture of 20mL of Opti-MEM +1.2mL 293fectin or fectin (2 μ l/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.

Transient transfection in the HEK293-F System (for examples 10-14)

FreeStyle was used according to the manufacturer's instructions^TM293 expression System (Invitrogen, USA) by transient transformation of human embryonic Kidney 293-F cellsStaining to express recombinant immunoglobulin variants. Briefly, the suspension FreeStyle^TM293-F cells in FreeStyle^TM293 expression Medium at 37 ℃/8% CO₂Cultures were performed and cells were plated at 1-2X10 on the day of transfection⁶Viable cells/ml were seeded in fresh medium. 325. mu.l of 293fectin was used^TM(Invitrogen, Germany) and 250. mu.g of plasmid DNA for heavy and light chains in a 1: 1 molar ratio in Opti-MEMPreparation of DNA-293fectin I Medium (Invitrogen, USA)^TMThe final transfection volume was 250 ml. "bulge-entry-well" DNA-293fectin complexes with two heavy chains and one light chain were prepared in Opti-MEM as follows325. mu.l of 293fectin (Invitrogen, USA) was used in medium I^TM(Invitrogen, Germany) and 250. mu.g of "bulge-entry-hole" heavy chain 1 and 2 and light chain plasmid DNA at a 1: 2 molar ratio (for a final transfection volume of 250 ml). "bulge-entry-hole" DNA-293fectin complexes with two heavy chains were prepared in Opti-MEM as follows325. mu.l of 293fectin (Invitrogen, USA) was used in medium I^TM(Invitrogen, Germany) and 250. mu.g of "bulge-entry-hole" heavy chain 1 and 2DNA at a 1: 1 molar ratio (for a final transfection volume of 250 ml) were prepared. CrossMab DNA-293fectin complex in Opti-MEM325. mu.l of 293fectin (Invitrogen, USA) was used in medium I^TM(Invitrogen, Germany) and 250. mu.g of "bulge-entry-hole" heavy chain 1 and 2 and light chain plasmid DNA at a 1: 1 molar ratio (for a final transfection volume of 250 ml). 7 days after transfection, cell cultures containing the antibody were harvested by centrifugation at 14000g for 30 minutes and filtration through sterile filters (0.22 μm)And (4) supernatant fluid. The supernatant was stored at-20 ℃ until purification.

Protein determination

The Protein concentration of purified antibodies and derivatives was determined by determining the Optical Density (OD) at 280nm using a molar extinction coefficient calculated based on the amino acid sequence according to Pace et al, Protein Science, 1995, 4, 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, pH 7.5, 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 0.5mL 2XPBS (2xPBS, Roche (Roche)) and 4 simple washes with 0.5mL 100mM Na-citrate pH 5.0 on an Ultrafree-MC-filtration column (Amicon). By adding 35. mu.l NuPAGELDS sample buffer (Invitrogen) eluted bound antibody. Half of the samples were separately compared with NuPAGEThe sample reducing agents were mixed or left unreduced and heated at 70 ℃ for 10 min. Therefore, 20. mu.l was applied to 4-12% NuPAGEBis-Tris SDS-PAGE (Invitrogen) (with MOPS buffer for non-reducing SDS-PAGE, and with NuPAGEMES buffer (Invitrogen) against Oxidation running buffer additive, for reduced SDS-PAGE and CoomassieAnd (5) dyeing with 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 (GE Healthcare) in 50mM KH2PO4, 300mM NaCl, pH 7.3 and eluted from the matrix on a Dionex HPLC system with 550mM acetic acid, pH 2.5. Eluted protein was quantified by UV absorbance and integration of 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 (Superdex 200, 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. 3 and 4).

SDS-PAGE

NuPAGEThe preformed gel system (Invitrogen) was used according to the manufacturer's instructions. Specifically, 4-20% NuPAGE is usedNovexTRIS-Glycine Prefabricator (Pre-Cast) gel and NovexTRIS-Glycine SDS running buffer. (see, e.g., FIG. 3). Reduction of samples by adding NuPAGE 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 300mM NaCl on an Agilent (Agilent) HPLC 1100 system, 50mM KH2PO4/K2HPO4, TosohTSGGel G3000SW column in pH 7.5 or Superdex 200 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. (see, e.g., FIG. 4).

Mass spectrometry

The total deglycosylation mass of the exchange (crossover) 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 a protein concentration of at most 2mg/ml at 37 ℃ for 12-24h with 100mM KH2PO4/K2HPO4, pH 7, 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. mu.g of antibody in 115. mu.l was incubated with 60. mu.l of 1M TCEP and 50. mu.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 EliteMS system.

VEGF binding ELISA

The binding properties of the tetravalent antibody (TvAb) were evaluated in an ELISA assay using full-length VEGF165-His proteins (R & DSystems) (fig. 5). For this purpose, Falcon polystyrene anti-reflective (clear enhanced) microtiter plates were coated with 100 μ l of 2 μ g/mL recombinant human VEGF165(R & DSystems) in PBS for 2h at room temperature or overnight at 4 ℃. The wells were washed three times with 300. mu.l PBST (0.2% Tween 20) and blocked with 200. mu.l 2% BSA 0.1% Tween20 for 30min at room temperature and then washed three times with 300. mu.l PBST. Purified < VEGF-ANG-2> TvAb in PBS (Sigma) at 100. mu.L/well dilution series (40pM-0.01pM) and as reference human anti-ANG-2 antibody < ANG-2> antibody Mab536(Oliner et al, Cancer Cell. (Cancer cells) 2004 Nov; 6 (5): 507-16, US 2006/0122370) and anti-VEGF antibody < VEGF > antibody G6-31(Liang et al, J Biol Chem. (J. biochem.) 2006 Jan 13; 281 (2): 951-61; US 2007/0141065) were added to the wells and incubated on a microtiter plate shaker at room temperature for 1 h. The wells were washed three times with 300 μ L PBST (0.2% Tween 20) and bound antibody was detected with 100 μ L/well of 0.1 μ g/ml F (ab') < hFc γ > pod (immunoresearch) in 2% BSA 0.1% 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 of 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).

VEGF binding: characterization of VEGF binding by surface plasmon resonance (Biacore) at 37 ℃ kinetics

To further confirm the ELISA findings, < VEGF > antibody G6-31 or avastin and < VEGF-Ang-2> TvAb6 or TvAb-2441-bevacizumab-LC 06 or TvAb-2441-bevacizumab-LC 08 were quantitatively analyzed for binding to VEGF using the surface plasmon resonance technique on a Biacore T100 instrument according to the following protocol and analyzed using the T100 software package: briefly, < VEGF > antibody was captured on a CM 5-chip by binding to goat anti-human IgG (JIR 109-005-098). The capture antibody was immobilized by amine coupling using the following standard amine coupling: HBS-N buffer as running buffer, activation was performed by EDC/NHS mixture, targeting a ligand density of 700 RU. The capture-antibody was diluted in coupling buffer NaAc, pH 5.0, c ═ 2 μ g/mL, and finally the still activated carboxyl groups were blocked by injection of 1M ethanolamine. Capture of Mabs < VEGF > antibody was performed at a flow rate of 5 μ L/min and c (Mabs < VEGF >) -10 nM (diluted with running buffer +1mg/mL BSA); a capture level of about 30RU should be achieved. rhVEGF (rhVEGF, R & D-SystemsCat. -No, 293-VE) was used as the analyte. Kinetic characterization of VEGF binding to < VEGF > antibodies was performed at 37 ℃ in PBS + 0.005% (v/v) Tween20 as running buffer. Samples were injected at a flow rate of 50 μ L/min and associated with rhVEGF in a concentration series of 300-0.29nM for a period of 80 seconds and dissociated for a period of 1200 seconds. Regeneration of the free capture antibody surface was performed after each analyte cycle with 10mM p-hydroxyphenylglycine (glycine) pH 1.5 and a contact time of 60 seconds. The kinetic constants were calculated using the usual double reference method (control reference: rhVEGF binds to the capture molecule goat anti-human IgG, measurement of flow cell blank, rhVEGF concentration "0", model: Langmuir binding 1: 1, (due to capture molecule binding Rmax set to local) FIG. 11 shows a schematic of the Biacore assay.

ANG-2 binding ELISA

The binding properties of the tetravalent antibody (TvAb) were assessed in an ELISA assay using the full length angiopoietin-2-His protein (R & DSystems) (fig. 6 a). For this purpose, Falcon polystyrene anti-reflective microtiter plates were coated with 100. mu.l of 1. mu.g/mL recombinant human angiopoietin-2 (R & D Systems, without carrier) in PBS for 2h at room temperature or overnight at 4 ℃. The wells were washed three times with 300. mu.l PBST (0.2% Tween 20) and blocked with 200. mu.l 2% BSA 0.1% Tween20 for 30min at room temperature and then washed three times with 300. mu.l PBST. Purified < VEGF-ANG-2> TvAb in PBS (Sigma) in 100 μ L/well dilution series (40pM-0.01pM) and < ANG-2> antibody Mab536 and < VEGF > antibody G6-31 as references 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% Tween 20) 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% BSA 0.1% 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).

Comparison with ANG-1 and ANG-2 binding (ANG-1 and ANG-2 binding ELISA)

The binding properties of the antibodies were assessed in AN ELISA assay using either full-length angiopoietin-2-His protein (R & D Systems #623-AN/CF or internally produced material) 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 of 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. mu.l PBST (0.2% Tween 20) and blocked with 200. mu.l 2% BSA 0.1% Tween20 for 30min at room temperature and then washed three times with 300. mu.l PBST. Purified test antibody in PBS in a dilution series of 100. mu.L/well (40pM to 0.01pM) was 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% Tween 20) 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% BSA 0.1% 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 TecanFluor 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 (genegreat Biosciences AB, Uppsala, sweden). Briefly, for affinity measurements, goat < hIgG-Fcgamma > polyclonal antibodies were immobilized on CM5 chips via amine coupling for presentation of antibodies against human ANG-2 (fig. 6). Binding in HBS buffer (HBS-P (10mM HEPES, 150mM NaCl, 0.005% Tween20, ph 7.4), measured at 25 ℃ purified ANG-2-His (R & D system or internally purified) was added to the solution at different concentrations between 6.25nM and 200nM association was measured by ANG-2-injection for 3 minutes, dissociation was measured by washing the chip surface with HBS buffer for 3 minutes and the KD values were evaluated using the 1: 1 Langmuir binding model (Langmuir binding model). due to the heterogeneity of the ANG-2 formulation, no 1: 1 binding could be observed; therefore the KD values were only relative evaluation.negative control data (e.g.buffer curves) were subtracted from the sample curves for correcting the system intrinsic 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 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-2 (huAngiopoietin-2, R & D Systems (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, 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.

ANG-2-VEGF bridged ELISA

Binding properties of tetravalent antibodies (tvabs) were performed in ELISA assays using immobilized full-length VEGF165-His protein (R & D Systems) and human ANG-2-His protein (R & D Systems) to detect bound bispecific antibodies (fig. 7). Only bispecific < VEGF-ANG-2> TvAb should be able to bind VEGF and ANG-2 simultaneously and thereby bridge these two antigens, whereas monospecific "standard" IgG1 antibodies should not be able to bind VEGF and ANG-2 simultaneously (fig. 7).

For this purpose, Falcon polystyrene transparent reinforced microtiter plates were coated with 100. mu.l of 2. mu.g/mL recombinant human VEGF165(R & D Systems) in PBS for 2 hours at room temperature or overnight at 4 ℃. The wells were washed 3 times with 300 μ l PBST (0.2% tween 20) and blocked with 200 μ l 2% BSA 0, 1% tween20 for 30min at room temperature, and then washed 3 times with 300 μ l PBST. Purified < VEGF-ANG-2> TvAb in PBS (Sigma) and as reference < ANG-2> antibody Mab536 and VEGF > antibody G6-31 of a 100 μ L/well dilution series ((40pM-0.01pM)) were added to the wells and incubated for 1 hour at room temperature on a microtiter plate shaker. The wells were washed 3 times with 300 μ l PBST (0, 2% Tween 20) and bound antibody was detected by adding 100 μ l 0.5 μ g/ml human ANG-2-His in PBS (R & D Systems). The wells were washed 3 times with 300 μ l PBST (0, 2% Tween 20) and bound ANG-2 was detected with 100 μ l 0.5 μ g/mL < ANG-2> mIgG 1-biotin antibody (BAM0981, R & D Systems (R & D Systems)) for 1 hour at room temperature. Unbound detection antibody was washed off three times with 300. mu.l PBST (0, 2% Tween 20) and bound antibody was detected by adding 100. mu.l 1: 2000 streptavidin-POD conjugate (Roche Diagnostics GmbH, Cat. No.11089153) diluted 1: 4 in blocking buffer for 1 hour at room temperature. Unbound streptavidin-POD conjugate was washed off 3-6 times with 300. mu.l PBST (0, 2% Tween 20) and bound streptavidin-POD conjugate was detected by addition of 100. mu.L 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).

Demonstration of simultaneous binding of bispecific tetravalent antibody < VEGF-Ang-2> TvAb6 to VEGF-A and Ang-2 by Biacore

To further confirm the data from the bridging ELISA, additional assays were established using surface plasmon resonance techniques on a Biacore T100 instrument to confirm simultaneous binding to VEGF and Ang-2 according to the following protocol and analyzed using the T100 software package (T100 control, version 2.01, T100 evaluation, version 2.01, T100 kinetic Summary (Kinetics Summary), version 1.01): ang-2 was captured at a capture level of 2000-1700RU in PBS, 0.005% (v/v) Tween20 running buffer by a pentahistidine antibody (PentaHisAntibody, 5-His-Ab without BSA, Qiagen No.34660) immobilized on a CM5 chip via amine coupling (without BSA). During the coupling procedure, HBS-N buffer was used as running buffer and activation was performed by EDC/NHS mixture. 5-His-Ab BSA-free capture-antibody was diluted in coupling buffer NaAc, pH 4.5, c ═ 30 μ g/mL, and the final still activated carboxyl groups were blocked by injection of 1M ethanolamine; ligand densities of 5000 and 17000RU were tested. Ang-2 at a concentration of 500nM was captured by 5-His-Ab at a flow rate of 5. mu.L/min diluted with running buffer +1mg/mL BSA. Thereafter, the binding of < Ang-2, VEGF > bispecific antibody to Ang-2 and VEGF was demonstrated by incubation with rhVEGF and formation of a sandwich complex (sandwich complex). To this end, bispecific < VEGF-Ang-2> TvAb6 bound to Ang-2 at a flow rate of 50 μ L/min and a concentration of 100nM (diluted with running buffer +1mg/mL BSA), and simultaneous binding was detected by incubation with VEGF (rhVEGF, R & D-Systems Cat. -, 293-VE) at a flow rate of 50 μ L/min and a VEGF concentration of 150nM in PBS + 0.005% (v/v) Tween20 running buffer. Association time 120 seconds and dissociation time 1200 seconds. Regeneration was performed after each cycle with a flow rate of 50 μ L/min at 2X10mM p-hydroxyphenylglycine (Glycin) pH 2.0 and a contact time of 60 seconds. Sensorgrams were corrected using conventional double references (control references: binding of bispecific antibody and rhVEGF to the capture molecule 5-His-Ab). Blanks for each Ab were measured using rhVEGF concentration "0". The Biacore assay is shown in figure 13. An alternative Biacore assay format is shown in figure 15.

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-pcDNA3 Topo hTie2) encoding full-length human Tie2(SEQ ID 108) 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 DMEM 10% FCS, 500. mu.g/ml G418. Individual clones were isolated by cloning cylinders (cylinders) 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)&D systems#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 with 1mM NaV3O4, Sigma # S6508 for 5 minutes) and incubate at 37 ℃ for 5 minutes. 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, pH 8.0, 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 for 30 minutes at 4 ℃ and 100. mu.l of lysate were transferred directly to p-Tie2 ELISA 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 assay insert (plug-in) for Excel (dose-response single point, mode 205). IC50 values may be compared within an experiment but may vary from experiment to experiment.

VEGF-induced HUVEC proliferation assay

VEGF-induced proliferation of HUVECs (human umbilical vein endothelial cells, Promocell # C-12200) was selected to measure the cellular function of VEGF antibodies. Briefly, 5000HUVEC cells per 96 wells (low passage number, ≦ 5 passages) were incubated overnight in 100. mu.l starvation medium (EBM-2 endothelial basal medium 2, Promocell # C-22211, 0.5% FCS, penicillin/streptomycin) in collagen I-coated BDBiocoat collagen I96-well microtiter plates (BD # 354407/35640). Different concentrations of antibody were mixed with rhVEGF (30ng1/ml final concentration, BD #354107) and pre-incubated for 15 minutes at room temperature. Subsequently, the mixture was added to HUVEC cells and they were incubated at 37 ℃ for 72 hours in 5% CO 2. On the day of analysis the plates were equilibrated to room temperature for 30 minutes and cell viability/proliferation was measured according to the manual using the CellTiter-GloTM luminescent cell viability assay kit (Promega, # G7571/2/3). Luminescence was measured in a spectrophotometer.

Design of tetravalent bispecific and tetravalent monospecific antibodies

Bispecific antibodies that bind to VEGF (VEGF-a) and ANG-2 (angiopoietin-2) according to the invention comprise a first antigen-binding site that binds to VEGF and a second antigen-binding site that binds to ANG-2. For example, SEQ ID NO: 23, and the heavy chain variable domain of SEQ ID NO: 24 as a first antigen binding site for binding VEGF, both from the human phage display-derived anti-VEGF antibody G6-31, described in detail in Liang, w.c., et al, J Biol chem.281(2) (2006)951-61 and in US 2007/0141065. Alternatively, for example, the second antigen-binding site that specifically binds VEGF comprises SEQ ID NO: 7, or SEQ ID NO: 100 and the heavy chain variable domain of SEQ ID NO: 8 or SEQ ID NO: 101 from the anti-VEGF antibodies < VEGF > bevacizumab and < VEGF > B20-4.1, preferably from < VEGF > bevacizumab.

SEQ ID NO: 31, and the heavy chain variable domain of SEQ ID NO: 32 or SEQ ID NO with mutations T92L, H93Q and W94T (Kabat numbering): 32 as a second antigen-binding site, both from the human anti-ANG-2 antibody < ANG-2> Mab536, which is described in detail in olin, j., et al, Cancer cell.6(5) (2004)507-16 and in US 2006/0122370. Alternatively, for example, the second antigen-binding site that specifically binds ANG-2 comprises SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84 or SEQ ID NO: 92, and SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 85, SEQ ID NO: 93 from an anti-ANG-2 antibody < ANG-2> ANG2s _ R3_ LC03, < ANG-2> ANG2i _ LC06, < ANG-2> ANG2i _ LC07, < ANG-2> ANG2k _ LC08, < ANG-2> ANG2s _ LC09, < ANG-2> ANG2i _ LC10, or < ANG-2> ANG2k _ LC11, preferably from < ANG-2> ANG2i _ LC06, or < ANG-2> ANG2k _ LC 08.

To generate reagents that combine the characteristics of both antibodies, a new tetravalent bispecific antibody derived protein entity was constructed. Among these molecules, the recombinant single-chain binding molecule of one antibody was linked to another antibody by recombinant protein fusion techniques, which retained the form of full-length IgG 1. The second antibody has a desired second binding specificity.

By gene synthesis and recombinant molecular biology techniques, the heavy chain variable domain (VH) and light chain variable domain (VL) of the respective antibodies are linked by a glycine serine (G4S)3 or (G4S)4 single linker to provide a single chain fv (scfv) which is linked to the C-terminus of the heavy chain of another antibody using a (G) 6-or (G4S) 3-linker.

Furthermore, cysteine residues are introduced into the VH (including Kabat position 44) and VL (including Kabat position 100) domains of an scFv that binds ANG-2 or VEGF, as described previously (e.g., WO 94/029350; Reiter, Y., et al, Nature biotechnology (1996) 1239-1245; Young, N.M., et al, FEBS Letters (1995) 135-139; or Rajagopal, V.et al, Protein Engineering (Protein Engineering) (1997) 1453-59).

All these molecules were recombinantly produced, purified and characterized and protein expression, stability and biological activity were evaluated.

A summary of bispecific antibody designs for generating tetravalent, bispecific < VEGF-ANG-2>, < ANG-2-VEGF > antibodies and tetravalent monospecific < ANG-2> antibodies is provided in table 3. For this study, we used the term 'TvAb' to describe various tetravalent protein entities.

To obtain the bispecific tetravalent antibodies < VEGF-ANG-2> TvAb5 and TvAb6, single chain fv (scfv) binding to angiopoietin-2 (heavy chain variable domain (VH) derived from SEQ ID NO: 31, and light chain variable domain (VL) of SEQ ID NO: 32 with mutations T92L, H93Q and W94T, derived from the human anti-ANG-2 antibody < ANG-2> Mab536) were fused to a sequence corresponding to SEQ ID NO: 23, human anti-VEGF antibody < VEGF > G6-31, and a heavy chain vector C-terminal to the heavy chain vector based on SEQ ID NO: 24 are co-expressed together. A representation of the design form is shown in fig. 1B and listed in table 3.

To obtain the bispecific tetravalent antibodies TvAb9 and TvAb15, VEGF-binding single chain fv (scfv) (derived from the heavy chain variable domain (VH) of SEQ ID NO: 23, and the light chain variable domain (VL) of SEQ ID NO: 24, derived from the human anti-VEGF antibody < VEGF > G6-31) was fused to a sequence corresponding to SEQ ID NO: 31 of the heavy chain vector of human anti-ANG-2 antibody < ANG-2> Mab536 and is identical to the heavy chain vector based on SEQ ID NO: 32 are co-expressed together. A representation of the design form is shown in fig. 1B and listed in table 3.

Table 3-different bispecific tetravalent antibody formats with C-terminal scFv linker and corresponding TvAb-nomenclature. In the table "-" means "absent".

TvAb forms based on, for example

a) aa) human anti-VEGF antibodies < VEGF > G6-31 and ab) two single chain fv (scfv) that bind angiopoietin-2 (derived from SEQ ID NO: 31, and the heavy chain variable domain (VH) of SEQ ID NO: 32) linked to the heavy chain of an anti-VEGF antibody < VEGF > G6-31 (SEQ ID NO: 23) c-terminal of (1); or

b) ba) human anti-ANG-2 antibodies < ANG-2> Mab536 and bb) two single chain fvs (scfv) that bind VEGF (derived from SEQ ID NO: 23 (VH), and seq id NO: 24, light chain variable domain (VL)) linked to the heavy chain of an anti-ANG-2 antibody < ANG-2> Mab536 (SEQ ID NO: 31) c-terminal of (1); or

c) ca) human anti-VEGF antibody < VEGF > bevacizumab (avastin) and cb) two single chain fv (scfv) that bind angiopoietin-2 (derived from SEQ ID NO: 52 or SEQ ID NO: 68 (VH), and SEQ ID NO: 53 or SEQ ID NO: 69) linked to the C-terminus of the anti-VEGF antibody < VEGF > bevacizumab (avastin) heavy chain (the resulting fusion peptide sequence is SEQ ID NO: 102 or SEQ ID NO: 103, which is identical to bevacizumab light chain SEQ ID NO: 104 are co-expressed together. (alternatively two single chain fv (scFv) that bind angiopoietin-2 may also be attached to the C-terminus of the light chain or to the N-terminus of the heavy chain).

Instead of two single chain fv (scfv) alone, it is also possible to use single chain Fab fragments, as described above (using a peptide linker for fusion to the C-terminus or N-terminus), described in EP application No. 09004909.9 and in example 10.

Example 1

Double specificityExpression and purification of sexual tetravalent antibodies

< VEGF-ANG-2> TvAb5, TvAb6, TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08

The light and heavy chains of the corresponding tetravalent bispecific antibodies TvAb5 and TvAb6 were constructed in genomic expression vectors as described above. These plasmids were amplified in E.coli, purified, and then transfected for transient expression of recombinant proteins in HEK293-F cells (using the Invitrogen's FreeStyle 293 system). After 7 days, the HEK293-F cell supernatant was harvested, filtered and the bispecific antibody was purified by protein A and size exclusion chromatography. The homogeneity of all bispecific antibody constructs was confirmed by SDS-PAGE under non-reducing and reducing conditions and analytical size exclusion chromatography. Under reducing conditions (fig. 3), the polypeptide heavy chain of < VEGF-ANG-2> TvAb6 carrying the C-terminal scFv fusion showed an apparent molecular size of ca.75kda on SDS-PAGE (similar to the calculated molecular weight). The mass spectrum confirmed the identity of the purified antibody construct. All constructs were analyzed for expression levels by protein a HPLC, which is similar to the expression yield of 'standard' IgGs. Protein yields were up to 150mg of TvAb6< VEGF-ANG-2> (as determined by protein A HPLC) per liter of cell culture supernatant.

Size exclusion chromatography analysis of purified non-disulfide stabilized scFv fused at the C-terminus of the heavy chain construct TvAb5 showed an increased tendency to re-aggregate after purification of monomeric antibodies by size exclusion chromatography compared to 'standard' IgGs (the so-called "daisy chain" phenomenon). This finding is supported by other examples (Rajagopal, V., et al, prot. Engin. (1997)1453- & 1459; Kobayashi, H., et al, Nucl Med Biol. (1998)387-393 or Schmidt, M., et al, Oncogene (1999)18, 1711-1721) which show that molecules comprising scFvs which are not stabilized by interchain disulfides between VH and VL exhibit increased propensity to aggregation and decreased yield. To address the problem of aggregation of these bispecific antibodies, disulfide stabilization of the scFv moieties was used. In this regard, we introduced single cysteine substitutions within the VH and VL of the scFv at the indicated positions (positions VH44/VL100, according to the Kabat numbering scheme). These mutations enable the formation of stable interchain disulfides between VH and VL, which in turn stabilize the resulting disulfide-stabilized scFv module. Introduction of VH44/VL100 disulfide into scFvs C-terminal of Fv in TvAb6< VEGF-ANG-2> resulted in stable tetravalent antibodies that no longer displayed a propensity to aggregate and remained monomeric after purification (fig. 4). Furthermore, a concentration of 3mg/kg applied, for example, in vitro and in vivo, over repeated freeze-thaw cycles TvAb6< VEGF-ANG-2> showed no increase in the propensity for aggregation.

All other TvAb molecules described in table 3 (e.g., TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08) were prepared and similarly analytically characterized according to the described procedure.

Example 2

Bispecific tetravalent antibodies<VEGF-ANG-2>TvAb6, TvAb-2441-bevacizumab-LC 06 And simultaneous binding of TvAb-2441-bevacizumab-LC 08 to VEGF-A and ANG-2

The binding of the scFv module retained in the IgG-module of the different bispecific antibody formats and the binding of Fvs is compared to the binding of the "wild-type" IgGs from which the binding module and bispecific antibody are derived. These analyses were performed by performing biochemical binding ELISA and by using surface plasmon resonance (Biacore) at equimolar concentrations.

For < VEGF-ANG-2> TvAb6, it was shown to bind VEGF at an equimolar concentration of 0.625pM comparable to its parent antibody G6-31 by VEGF binding ELISA as described above (fig. 5). This finding was expected because the Fv region of the TvAb is identical to that of G6-31. The weak difference between < VEGF-ANG-2> TvAb6 and < VEGF > G6-31 is due to small differences in protein concentration and slight steric interference of C-terminal scFv binding to the < hFc > -POD detection antibody, which can be overcome by using the < hk > POD (Biozol cat. No.206005) detection antibody as used in ANG-2 binding ELISA.

These findings were confirmed using Biacore at 37 ℃ using a classical concentration series (figure 11). These data show fast K binding rates K (a) of 4.7-4.8E + 61/(Ms), saturating with the highest concentration of VEGF. The dissociation rate reached the limit of the specification (i.e. 5xE-6(s/s), probably because under this condition still due to the bivalent binding of the dimeric analyte rhVEGF (affinity effect), the final 10-15RU VEGF-response was produced despite the use of a very low ligand density. however, the kinetic constants of the different < VEGF > antibodies could be compared by this method and within the error of the method there was no significant difference in the detectable kinetic constants of the tetravalent bispecific antibody < VEGF-Ang-2> TvAb6 and the original antibody < VEGF > G6-31. the kinetic constants of < VEGF-Ang-2> TvAb6 and < VEGF > G6-31 under these conditions were practically identical by this method it could be concluded that TvAb6 completely retained its VEGF binding properties. table 4 shows the respective kinetic constants, figure 12 shows the kinetic characteristics of two < VEGF > antibodies < VEGF-Ang-2> TvAb6 and < VEGF > G6-31 in a Ka-Kd chart.

Table 14: kinetic Properties of < VEGF-Ang-2> TvAb6 and < VEGF > G6-31

Measurement at 37 ℃	Ka	kd	t1/2	KD
					Antibodies	[1/(Ms)]	[1/s]	[min]	[M]
<VEGF>G6-31	4.83E+06	9.33E-06	1237.8	1.93E-12
					<VEGF-Ang-2>TvAb6	4.72E+06	7.24E-06	1596.7	1.53E-12

In another experiment, ANG-2 binding ELISA using < hk > -POD detection antibody (Biozol commercial number 206005) as described above showed that < VEGF-ANG-2> TvAb6 bound ANG-2 at an equimolar concentration of 0.039pM in a manner comparable to Mab536 (fig. 6A). This shows that the scFv assembly of TvAb6 retained its binding properties in the TvAb construct.

To further confirm this finding, < ANG-2> Mab536 and < VEGF-ANG-2> TvAb6 were immobilized on biacore cm5 chips by secondary antibodies and the binding kinetics to human ANG-2 was measured. No 1: 1 binding could be observed due to heterogeneity of ANG-2 formulations; the KD values are therefore only relative estimates. Biacore analysis showed that < VEGF-ANG-2> TvAb6 had an estimated KD value of 4.4nM for ANG-2. In contrast, Mab536 has an estimated KD value of 1.6 nM. Within the error of this approach, no difference in binding pattern and affinity between < ANG-2> Mab536 and < VEGF-ANG-2> TvAb6 was observed (fig. 6B). Thus, it can be concluded that the scFv assembly of TvAb6 fully retained its binding properties in the TvAb construct.

To demonstrate that < VEGF-ANG-2> TvAb6 is capable of binding to both VEGF and ANG-2, the bridging ELISA assay and Biacore assay as described above were used.

It was shown by using the VEGF-ANG-2-bridging ELISA described above that only < VEGF-ANG-2> TvAb6 was able to bind VEGF and ANG-2 simultaneously at an equimolar concentration of 0.625pM, whereas the monospecific "standard" IgG1 antibodies < ANG-2> Mab536 and < VEGF > G6-31 were unable to bind VEGF and ANG-2 simultaneously (fig. 7).

Figure 14 shows the respective data from Biacore assays. For the tetravalent bispecific antibody < VEGF-Ang-2> TvAb6 simultaneous binding to both antigens Ang-2 and VEGF can be shown. Negative controls as expected: the monospecific antibody < Ang-2> Mab536 was shown to bind Ang-2 only, but not VEGF. The monospecific antibody < VEGF > G6-31 was shown to bind VEGF but not Ang-2 at all (data not shown). From the relative response units (relative response units) of binding of the tetravalent bispecific antibody < VEGF-Ang-2> TvAb6 to Ang-2 coated surfaces and subsequent binding to dimeric VEGF binding, the stoichiometry can be calculated to range from 1: 1 to 1: 1.4. In summary, it was shown by applying the ELISA and Biacore assays described that only < VEGF-Ang-2> TvAb6 was able to bind VEGF and Ang-2 simultaneously, whereas the monospecific "standard" IgG1 antibodies < Ang-2> Mab536 and < VEGFYG6-31 were not able to bind VEGF and Ang-2 simultaneously (fig. 15).

Similar results were obtained using constructs TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08 in a similar Biacore assay, shown in fig. 15A. Binding of antibodies to antigens (e.g., human ANG-2 and VEGF) was studied by surface plasmon resonance using biacore 100 instruments (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements, a goat < hIgG-Fc □ > polyclonal antibody was immobilized on a CM4 chip via amine coupling for displaying bispecific antibodies against human ANG-2 and VEGF. Binding was measured in HBS buffer (HBS-P (10mM HEPES, 150mM NaCl, 0.005% Tween20, ph 7.4), 25 ℃ purified ANG-2-His (R & D systems or internally purified) was added at various concentrations between 6.25nM and 200nM in solution association was measured by ANG-2-injection for 3 minutes, dissociation was measured by washing the chip surface with HBS buffer for 3 minutes, and KD values were estimated using a 1: 1 Langmuir (Langmuir) binding model.1: 1 binding could not be observed due to heterogeneity of the ANG-2 formulation, therefore KD values were only relatively estimated.

VEGF (R & D systems) was added at various concentrations between 6.25nM and 200nM in solution. Association was measured by VEGF-injection for 3 minutes; dissociation was measured by washing the chip surface with HBS buffer for 3 minutes, and KD values were estimated using a 1: 1 Langmuir (Langmuir) binding model.

The order of injection of binding partners may be changed, first VEGF then Ang2 or vice versa.

Negative control data (e.g., buffer curve) is subtracted from the sample curve to correct for baseline drift inherent to the system and for reduction of noise signal. Biacore T100 evaluation software version 1.1.1 was used to analyze sensorgrams (sensorgrams) and to calculate affinity data.

Antibodies	Affinity hAng-2	Affinity hVEGF

TvAb-2441-bevacizumab-LC 06	2.3nM	0.35nM
			TvAb-2441-bevacizumab-LC 08	0.7nM	0.34nM
G6-31	--	＜0.1nM
			MAb536	3nM	--
Bevacizumab	--	0.59nM

Finally, simultaneous binding of TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08 can be demonstrated by incubation with ANGPT2 and VEGF in a serial fashion. As shown in figure 15B, ANGPT2 and VEGF can bind to the bispecific antibody simultaneously.

Example 3

In the staged subcutaneous Colo205 xenograft model of Scid beige mice, with<ANG-2>Mab536， <VEGF>G6-31 and the combination of Mab536 and G6-31 are disulfide-stabilized Bispecific tetravalent antibodies<VEGF-ANG-2>In vivo Effect of TvAb6

In two phase subcutaneous Colo205 xenograft model studies (Ang2_ PZ _ Colo205_003 and Ang2_ PZ _ Colo205_005) in female Scid beige mice, different doses of purified disulfide stabilized < VEGF-Ang-2> TvAb6(n00.2331 see table 3) were compared to the combination of antibodies < Ang-2> Mab536, < VEGF > G6-31 and < Ang-2> Mab536 and < VEGF > G6-31.

Antibody: < ANG-2> Mab536 as a frozen stock (c ═ 4.5mg/mL), < VEGF > G6-31 as a frozen stock (c ═ 0.6mg/mL) and < VEGF-ANG-2> TvAb6 as a frozen stock (c ═ 0.5mg/mL) were provided in 20mM histidine, 140mM NaCl, pH 6.0. The antibody solution was appropriately diluted from stock in PBS before injection as needed, with PBS being used as an excipient.

Cell lines and culture conditions: colo205 human colorectal cancer cells were originally obtained from ATCC and were deposited in the Roche Penzberg internal cell bank after expansion. Tumor cell lines were cultured in RPMI 1640 medium (PAA, Laboratories, Austria) supplemented with 10% fetal bovine serum (PAA Laboratories, Austria) and 2mM L-glutamine at 37 ℃ in a water saturated atmosphere at 5% CO₂Conventionally. Passages 2-5 were used for transplantation.

Animals: female SCID beige mice; 4-5 weeks of age (purchased from Charles River Germany) were maintained under specific pathogen-free, 12h light/12 h dark cycle conditions per day according to the established guidelines (GV-Solas; Felassa; 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 Kliba 3337) and water (acidified pH 2.5-3) were provided ad libitum. The age of the mice at the beginning of the study was approximately 10 weeks.

Monitoring: animals were managed daily (control) for clinical symptoms and tested for side effects. For monitoring the body weight of the animals throughout the experiment was recorded 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 concentrates and CELL sizes 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 5.0x10E7 cells/ml, viability ca.90%. Followed by 2.5x10 per animal⁶100 μ l of this suspension of cells was injected s.c. into the right flank of the mice.

Animal treatment was started on days of randomization, 16 days post cell transplantation (study Ang2_ PZ _ Colo205_003), and 14 days post cell transplantation (study Ang2_ PZ _ Colo205_005) at mean tumor volumes of 100mm3 or 150mm3, respectively.

Study dosage regimen of Ang2_ PZ _ Colo205_003 up to 74 days (see fig. 8A):

in study Ang2_ PZ _ Colo205_003, < VEGF-Ang-2> TvAb6 was erroneously given an insufficient amount (underfeded) in terms of equimolar ratio. The dose of < VEGF-Ang-2> TvAb6 was adjusted in study Ang2_ PZ _ Colo205_005 so that animals received an equimolar ratio of < VEGF-Ang-2> TvAb6 and a combination of < VEGF > G6-31 and < Ang-2> Mab536 of Ang-2 and VEGF binding sites.

Tumor growth was inhibited until day 74 (see fig. 8 a). Ang2_ PZ _ Colo205_003 study:

the combination of < VEFG-ANG-2> TvAb6 at a dose of 7mg/kg showed comparable effects to < VEGF > G6-31 at 5mg/kg and < ANG-2> Mab536 at 6mg/kg, and < VEGF > G6-31 at a dose of 6mg/kg as a single dose (FIG. 8A) and better than < ANG-2> Mab536 at a dose of 6mg/kg as a single dose. Since the subcutaneous Colo205 model is very reactive to < VEGF > G6-31 antibodies blocking human as well as murine VEGF, leading to almost complete tumor growth inhibition, < VEGF-ANG-2> TvAb6 cannot be distinguished from G6-31(6mg/kg) as a single dose under the selected experimental conditions, whereas < VEGF-ANG-2> TvAb6 shows comparable inhibition to the combination of < ANG-2> Mab536 and < VEGF > G6-31 at a clearly lower cumulative dose (compare 40+48 to 88mg/kg antibody versus the combination of < ANG-2> Mab536 and < VEGF > G6-31 to 40+48 to 88mg/kg antibody).

Dosage regimen of Ang2_ PZ _ Colo205_005 was studied until day 63:

tumor growth inhibition was studied by Ang2_ PZ _ Colo205_005 until day 63:

the dose of 4mg/kg of < VEGF-ANG-2> TvAb6 showed comparable effects to the combination of < VEGF > G6-31 and < ANG-2> Mab536 at 3mg/kg each, and was better than the single dose of < VEGF > G6-31 and single dose of < ANG-2> Mab536 at 3mg/kg (fig. 8B). This is the first such example, which shows that lower doses (for total antibody concentration-the cumulative dose of this combination is 42 mg/kg-42 mg/kg versus 28mg/kg of bispecific antibody TvAb6) of bispecific antibodies targeting VEGF and ANG-2 can produce strong anti-tumor effects that are comparable to and superior to the combination of the respective single doses of blocking VEGF and ANG-2.

Example 4

Blocking VEGF-induced tube formation (tube formation)

To confirm that the anti-VEGF related activity was retained in the bispecific tetravalent < VEGF-ANG-2> TvAb6, it was shown that the dose dependent inhibition of vascular formation mediated by < VEGF-ANG-2> TvAb6 was comparable to the monospecific antibody < VEGF > G6-31 in the VEGF induced vascular formation assay AngioKit TCS CellWorks (CellSystems). The AngioKit TCS CellWorks assay was performed according to the following procedure: cells were stimulated with 2ng/ml VEGF before each antibody treatment on days 1, 4, 7 and 9. Blood vessels (Vascular tubes) were visualized by staining endothelial cells with CD31-PE antibody (BD Pharmingen #555446) on day 11. Photographs were taken at 4x magnification and the values of Tube length and the number of branch points were quantitatively analyzed using an Angiogenesis Tube formation application Module in metamorph (molecular devices). The numerical values and standard deviations were repeatedly calculated and 4 photographs were analyzed for each sample. Fig. 9 shows the respective results, and fig. 10A and B show the results of the quantitative analysis. Angiopoietin-2 had no effect on tube formation and therefore inhibition of ANG-2 was not investigated in this assay. The data show that bispecific < VEGF-ANG-2> TvAb6 and monospecific < VEGF > G6-31 antibodies were equally effective in inhibiting VEGF-stimulated tube formation.

Example 5

Tie2 phosphorylation

To confirm that anti-ANGPT 2-related activity was retained in the bispecific tetravalent < VEGF-ANGPT2> antibodies TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08, it was shown in the ANGPT2 stimulated Tie2 phosphorylation assay described above that TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08 interfered with ANGPT2 stimulated Tie2 phosphorylation in a comparable manner to their parent clones LC06 and LC 08.

In the first experiment, the bispecific antibodies TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08 both showed dose-dependent interference with ANGPT 2-stimulated Tie2 phosphorylation with IC50 values comparable to those of the parental clones LC06 and LC08 (as shown in fig. 16A). TvAb-2441-bevacizumab-LC 06 interfered with an IC50 value of about 721ng/ml for ANGPT2 stimulated Tie2 phosphorylation, while LC06 interfered with an IC50 value of about 508ng/ml for ANGPT2 stimulated Tie2 phosphorylation. TvAb-2441-bevacizumab-LC 08 interfered with an IC50 value of approximately 364ng/ml for ANGPT2 stimulated Tie2 phosphorylation, while LC08 interfered with an IC50 value of approximately 499ng/ml for ANGPT2 stimulated Tie2 phosphorylation.

In a second experiment, the bispecific antibodies TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08 both showed dose-dependent interference with ANGPT 2-stimulated Tie2 phosphorylation with IC50 values comparable to those of the parental clones LC06 and LC08 (as shown in fig. 16B). TvAb-2441-bevacizumab-LC 06 interfered with an IC50 value of about 488ng/ml for ANGPT2 stimulated Tie2 phosphorylation, while LC06 interfered with an IC50 value of about 424ng/ml for ANGPT2 stimulated Tie2 phosphorylation. TvAb-2441-bevacizumab-LC 08 interfered with an IC50 value of about 490ng/ml for ANGPT2 stimulated Tie2 phosphorylation, while LC08 interfered with an IC50 value of about 399ng/ml for ANGPT2 stimulated Tie2 phosphorylation.

Taken together, these data show that within the error of the present cellular assay, the bispecific tetravalent < VEGF-ANGPT2> antibodies TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08 interfere with ANGPT2 stimulated Tie2 phosphorylation in a comparable manner to their parental clones LC06 and LC 08.

Example 6

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

Ang2 interaction ELISA summary data:

example 7

Inhibition of binding of hVEGF to hVEGF receptor (ELISA)

The tests were carried out in 384 well microtiter plates (Microcoat, DE, Cat. No.464718) at RT. After each incubation step, plates were washed 3 times with PBST. Initially, plates were coated with 0.5. mu.g/ml of hVEGF-R protein (R & D Systems, Cat. No.321-FL) for at least 2 hours (h). Thereafter, the wells were blocked with PBS (Roche diagnostics GmbH, DE) supplemented with 0.2% tween-20 and 2% BSA for 1 hour. Dilutions of purified antibody in PBS were incubated with 0.15. mu.g/ml huVEGF 121(R & D Systems, UK, Cat. No.298-VS) for 1 hour at RT. After washing, a mixture of 0.5. mu.g/ml anti-VEGF clone Mab923(R & D Systems, UK) and 1: 2000 horseradish peroxidase (HRP) -conjugated F (ab') 2 anti-mouse IgG (GE Healthcare, UK, Cat. No. NA9310V) 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.

VEGF interaction ELISA summary data:

example 8

HUVEC proliferation

To confirm that the anti-VEGF related activity was retained in the bispecific tetravalent < VEGF-ANG2> antibodies TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08, TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08 were shown to interfere with VEGF-induced HUVEC proliferation in a comparable manner to their parent clones LC06 and LC08 in the VEGF-induced HUVEC proliferation assay described above.

Figure 18 shows that TvAb-2441-bevacizumab-LC 06 and TvAb-2441-bevacizumab-LC 08 did interfere with VEGF-induced HUVEC proliferation in a concentration-dependent manner comparable to the parent antibody bevacizumab.

Example 9

ELISA binding assay for human ANG-1 and human ANG-2

Mother body<ANG-2>Binding of antibodies Ang2i-LC06, Ang2i-LC07, and Ang2k-LC08 to human Ang-1 and human Ang-2 was measured in Ang-1 or Ang-2 binding ELISA, as described above (see comparison of binding to Ang-1 and Ang-2 (Ang-1 and Ang-2 binding ELISA)). Briefly, the ELISA-type assay is based on immobilization of human wild-type angiopoietin-1 or-2 in microtiter plates. Binding of antibodies against immobilized ANG-1 or ANG-2 by means of conjugates with POD<Human Fc>(anti-IgG) antibody measurement.<ANG-2>Serial dilutions of the antibody allowed for the measurement of EC₅₀And (4) concentration. Use of human anti-ANG-2 antibodies<ANG-2>Antibody Mab536(Oliner et al, cancer cell.2004 Nov; 6 (5): 507-16, US 2006/0122370) was used as a reference. The measured EC50 concentrations are summarized in the table below.

All antibodies specifically bind to ANG-2. MAb536 and Ang2k-LC08 also showed specific binding to Ang-1, whereas Ang2i-LC06 and Ang2i-LC07 did not specifically bind to Ang-1, as they had EC 50-values above 8000ng/ml (limit of detection).

Example 10

Bispecific, tetravalent single chain Fab<VEGF-ANG-2>Antibody molecule scFAb-avastin -LC06-2620, scFab-avastin-Ang 2i-LC06-2640 and scFab-avastin Expression and purification of Ang2i-LC06-2641

In analogy to the procedure described in example 1 and the materials and methods described above, the bispecific, tetravalent single chain Fab < VEGF-ANG-2> antibody molecules scFAb-avastin-LC 06-2620, scFAb-avastin-LC 06-2640 and scFAb-avastin-LC 06-2641 were expressed and purified, all three based on < VEGF > bevacizumab and < ANG-2> ANG2i-LC 06. Binding affinity and other properties were measured as described in the examples above. The relevant (possibly modified) light and heavy chain amino acid sequences of these bispecific antibodies are set forth in SEQ ID NO: 109-110 (scFAb-avastin-LC 06-2620) in SEQ ID NO: 111-112 (scFAb-avastin-LC 06-2640) and the sequences set forth in SEQ ID NO: 113-114 (scFAb-avastin-LC 06-2641).

Example 11

Bispecific, trivalent single chain Fab<VEGF-ANG-2>Antibody molecule avastin Expression and purification of-LC 06-KiH-C-scFab

In analogy to the procedure described in example 1 and the materials and methods described above, the bispecific, trivalent single chain Fab < VEGF-ANG-2> antibody molecule avastin-LC 06-KiH-C-scFab (based on < VEGF > bevacizumab and < ANG-2> ANG2i-LC06) was expressed and purified. Binding affinity and other properties were measured as described in the examples above. The relevant (possibly modified) light and heavy chain amino acid sequences of such bispecific antibodies are set forth in SEQ ID NO: 115-117 (Avastin-LC 06-KiH-C-scFab).

Example 12

Bispecific, trivalent<VEGF-ANG-2>Of the antibody molecule avastin-LC 06-C-Fab-6CSS Expression and purification

Similar to the procedure described in example 1 and the materials and methods described above (see also expression and purification of the bispecific, trivalent < VEGF-ANG-2> antibody molecule avastin-LC 06-C-Fab-6CSS (based on < VEGF > bevacizumab and < ANG-2> Ang2i-LC 06.) the binding affinity and other properties were measured as described in the examples above. the bispecific, trivalent antibody molecule in this form is generally described in EP application No. 09005108.7. the relevant (possibly modified) light and heavy chain amino acid sequences of this bispecific < VEGF-ANG-2> antibody are given in SEQ ID NO: 118-120 (avastin-LC 06-C-Fab-6 CSS).

Example 13

Bispecific, bivalent domain exchanged<VEGF-ANG-2>Antibody molecule avastin of-LC 06-CH1-CL, avastin-LC 06-VH-VL and avastin-LC 06-VH-VL-SS Expression and purification

In analogy to the procedure described in example 1 and the materials and methods described above, the bispecific, bivalent domain-exchanged < VEGF-ANG-2> antibody molecules avastin-LC 06-CH1-CL (CH-CL exchange performed as described in WO 2009/080253), avastin-LC 06-VH-VL (VH-VL exchange performed as described in WO 2009/080252) and avastin-LC 06-VH-VL-SS (VH-VL exchange performed as described in WO 2009/080252 and additionally introducing a VH44 VL100 disulfide bridge) based on < VEGF > bevacizumab and < ANG-2> ANG2i-LC06 were expressed and purified. Binding affinity and other properties were measured as described in the examples above. The relevant (possibly modified) light and heavy chain amino acid sequences of these bispecific antibodies are set forth in SEQ ID NO: 121-124 (Avastin-LC 06-CH1-CL) and the sequence given in SEQ ID NO: 125-128 (Avastin-LC 06-VH-VL) and the sequences shown in SEQ ID NO: 129-132 (Avastin-LC 06-VH-VL-SS).

Example 14

Bispecific, bivalent ScFab-Fc fusions<VEGF-ANG-2>Antibody molecule avastin Expression and purification of-LC 06-N-scFab and avastin-LC 06-N-scFabSS

In analogy to the procedure described in example 1 and the materials and methods described above, the bispecific, bivalent ScFab-Fc fusion < VEGF-ANG-2> antibody molecules avastin-LC 06-N-ScFab and avastin-LC 06-N-ScFab ss (based on < VEGF > bevacizumab and < ANG-2> ANG2i-LC06) were expressed and purified. Binding affinity and other properties were measured as described in the examples above. The relevant modified heavy chain amino acid sequences of these bispecific antibodies are set forth in SEQ ID NO: 133-: 135-.

Example 15

Bispecific of examples 10 to 14<VEGF-ANG-2>Antibody molecules inhibit hVEGF binding hVEGF receptor (ELISA), blocks VEGF-induced tube formation, inhibits huANG-2 binding to Tie-2 (ELISA), Tie2 phosphorylation and HUVEC proliferation

The bispecific < VEGF-ANG-2> antibody molecules of examples 10 to 14 inhibit hVEGF binding to hVEGF receptor (ELISA), block VEGF-induced tube formation, inhibit huANG-2 binding to Tie-2(ELISA), Tie2 phosphorylation and HUVEC proliferation can be similarly measured according to materials and methods and procedures described in examples 4 to 9 above.

Example 16

In the Scid beige mouse refractory Colo205 xenograft model (in the para-bevacizumab (avastin) After treatment resistance), with<ANG-2>ANG2i-LC06, and<ANG-2>ANG2i-LC06 bispecific antibodies compared to combinations of avastin<VEGF-ANG-2>In vivo effect of

Cell lines and culture conditions:

colo205 human colorectal cancer cells were originally obtained from ATCC and, after expansion, were deposited in the Roche Penzberg internal cell bank. Tumor cell lines were in RPMI 1640 medium (PAA, laboratory) supplemented with 10% fetal bovine serum (PAALabortors, Austria) and 2mM L-glutamines, Austria) at 37 ℃ in a water-saturated atmosphere at 5% CO₂Conventionally. Passages 2-5 were used for transplantation.

Animals:

female SCID beige mice; 4-5 weeks of age were reached (purchased from Charles River Germanyd) and 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 Kliba 3337) and water (acidified pH 2.5-3) 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, tumor cells (trypsin-EDTA) were harvested from culture flasks (Greiner) and transferred to 50ml of medium, washed once and resuspended in PBS. Washing step again with PBS and filtration (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 (BraunMelsungen) 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 prior to injection, animals were shaved and for cell injection, the skin of the anesthetized animals was carefully lifted with dissecting forceps and 100 μ l of cell suspension (═ 2.5x 10) was injected subcutaneously into the right abdomen of the animals⁶A cell).

Treatment of animals

Pretreatment:

animal treatment started 14 days after cell transplantation (study Ang2_ PZ _ Colo205_008) and the mean tumor volumes were 100mm each³To 150mm³. Mice were treated with avastin (10mg/kg) once a week over a period of 5 weeks.

Secondary treatment:

mice were then randomized for secondary treatment and divided into four groups (10 mice per group). Tumor volumes ranged from 336 to 341mm at the start of the secondary treatment (day 51)³. Mice were treated i.p. once a week with different compounds as shown in the table below.

Monitoring:

animals were managed 2x weekly for their health status. Body weights were recorded 2x weekly after cell injection. Tumor size was measured by caliper on the day of staging (on the staging day) and then 2 times per week throughout the treatment. Tumor volume according to the NCI method (tumor weight 1/2 ab)²Where "a" and "b" are the major and minor diameters of the tumor, respectively). The termination criteria were the critical tumor mass (up to 1.7g or) Weight loss greater than 20% of baseline, tumor ulceration or poor general condition in the animal.

As a result: tumor growth inhibition based on median (in percent) at day 91

The results show that in the Scid beige mouse bevacizumab (avastin) -resistant xenograft tumor model Colo205, the bispecific < VEGF-ANG-2> antibody TvAb-2441-bevacizumab-LC 06 showed higher tumor growth inhibition (at lower doses) compared to the monospecific antibody ANG2i-LC06 alone or ANG2i-LC06 and avastin combined treatment.

Example 17

In vivo inhibition of tumor angiogenesis in s.c.calu-3 NSCLC xenografts

Non-invasive in vivo imaging detection of angiogenesis using labeled anti-CD 31

Cell lines and culture conditions:

this human lung adenocarcinoma cancer cell line was established from a white man with lung cancer. Cells were obtained from Roche, Kamakura and passaged internally (in house) for working cell banks. Tumor cell lines were cultured in RPMI 1640 medium (PAN Biotech, Germany) supplemented with 10% fetal bovine serum (PAN Biotech, Germany) and 2mM L-glutamine (PANBIOTech, Germany) at 37 ℃ in a water-saturated atmosphere at 5% CO₂Conventionally. Cultures were passaged with trypsin/EDTA 1x (PAN) splits once a week.

Animals:

female BALB/c nude mice; 4-5 weeks of age were reached (purchased from Charles River Germanyd) and 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 Kliba 3337) and water (acidified pH 2.5-3) 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, tumor cells (trypsin-EDTA) were harvested from culture flasks (Greiner) and transferred to 50ml of medium, washed once and resuspended in PBS. Washing step again with PBS and filtration (cell Filter; Falcon)100 μm) was followed by adjusting the final cell titer to 5.0x10⁷And/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 (BraunMelsungen) 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 prior to injection, animals were shaved and for cell injection, the skin of the anesthetized animals was carefully lifted with dissecting forceps and 100 μ l of cell suspension (═ 5.0x 10) was injected subcutaneously into the right abdomen of the animals⁶A cell).

Treatment of animals

On study day 35, mice were randomized into statistically well distributed groups according to their body weight and tumor size. For the treatment with therapeutic antibody, each group consisted of 10 mice and the therapeutic antibody treatment was applied i.p. once a week over a period of 6 weeks. (see FIG. 19).

Group 1: excipient (omalizumab (Xolair))10mg/kg

Group 2: 10mg/kg of avastin

Group 3: combination of monospecific < VEGF > Avastin 10mg/kg + monospecific < ANG-2> Ang2i-LC0610mg/kg (═ Avastin/Ang 2i-LC06)

Group 4: bispecific < VEGF-ANG-2> antibody 2441-avastin-scFv-LC0613.3mg/kg

Monitoring:

Monitoring of blood vessels and angiogenesis with labeled anti-CD 31 antibodies

Preliminary studies revealed that anti-CD 31 antibodies are the best reagents for tumor vascular imaging. This agent targets the mouse endothelial CD31 receptor and shows a single vessel with a low signal-to-background ratio. Thus, anti-CD 31 antibody imaging represents a viable approach to tumor vascular imaging. Three mice from each treatment group were selected and injected i.v. at days 35, 49 and 79 with 50 μ g/mouse of anti-CD 3 antibody covalently labeled with organic fluorophore Alexa 610. Near infrared imaging was performed 24 hours after each application of labeled antibody under inhalation anesthesia. The increase or decrease of tumor vessels was shown by using the image comparison tool MAESTRO system. An increase in tumor vessels was observed at day 35 to day 79 under treatment with the control monoclonal antibody omalizumab (Xolair) and the therapeutic antibody avastin. In contrast, combined treatment with avastin + Ang2i-LC06 and 2441-avastin-scFv-LC 06 showed a decrease in tumor vasculature (FIG. 19).

Tumor areas were quantified by manually mapping the area of measurement and signal intensity was evaluated as intensity value (total signal/exposure time). The average changes in CD31 signal from days 35 to 49 and from days 49 to 79 are plotted in fig. 19. All treatment groups from day 35 to 49 revealed an increase in tumor vasculature. While the CD31 tumor signal steadily accelerated in group 1 (omalizumab (Xolair)) and group 2 (avastin), the tumor vasculature was significantly reduced in group 3 (avastin + < ANG-2> ANG2i-LC06 combination) and group 4 (bispecific < VEGF-ANG-2> antibody 2441-avastin-scFv-LC 06), group 4 clearly showed the most pronounced anti-angiogenic effect (fig. 19).

After the last in vivo imaging study, tumors were immediately removed (day 79), fixed in formalin and implanted in paraffin for ex vivo (ex vivo) studies. Fluorescence microscopy showed numerous well-defined capillaries in tumors treated with the control monoclonal antibody omalizumab (Xolair). Several tumor vessels were observed in the avastin-treated mice. In contrast, treatment groups 3 and 4 had significantly fewer and less distinct vessels in the tumor than treatment groups 1 and 2, while group 4 showed the clearest effect. Group 4 revealed lower microvascular density, capillary stem was generally smaller and disorganized (unstructured) and they showed weaker anti-CD 31 fluorescence signal compared to groups 1, 2 and 3. Histochemical HE-staining showed that intratumoral necrotic regions in the treatment group of group 4 bispecific antibody reached 90% of all regions, which was significantly higher than other treatment groups (data not shown).

Example 18

Bispecific antibodies in the Scid beige mouse staged subcutaneous Colo205 xenograft model <VEGF-ANG-2>And comparison with parent monospecific antibodies (alone or in combination)

Cell lines and culture conditions:

colo205 human colorectal cancer cells were originally obtained from ATCC and, after expansion, were deposited in the Roche Penzberg internal cell bank. Tumor cell lines were cultured in RPMI 1640 medium (PAA, Laboratories, Austria) supplemented with 10% fetal bovine serum (PAALabortors, Austria) and 2mM L-glutamine at 37 ℃ in waterSaturated atmosphere at 5% CO₂Conventionally. Passages 2-5 were used for transplantation.

Animals:

female SCID beige mice; 4-5 weeks of age (purchased from Charles River Germany) were maintained under specific pathogen-free, 12h light/12 h dark cycle conditions per day according to the established guidelines (GV-Solas; Felassa; 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 Kliba 3337) and water (acidified pH 2.5-3) 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 cells were centrifuged, washed once and resuspended in PBS. CELL concentrates and CELL sizes 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 5.0x10E7 cells/ml, viability ca.90%. Followed by 2.5x10 per animal⁶100 μ l of this suspension of cells was injected s.c. into the right flank of the mice.

Animal treatment was started on days of randomization, 16 days post cell transplantation (study Ang2_ PZ _ Colo205_009) at an average tumor volume of 100mm3, respectively.

Study dosage regimen of Ang2_ PZ _ Colo205_ 009:

monitoring:

As a result:

tumor Growth Inhibition (TGI) based on median (in percent) on day 61

The results show that in the Scid beige mouse xenograft tumor model Colo205, all three bispecific < VEGF-ANG-2> avastin (bevacizumab) -ANG2i-LC06 antibodies (all based on the bevacizumab sequences SEQ ID Nos 7 and 8 and on the ANG2i-LC06 sequence SEQ ID Nos 52 and 53) show higher tumor growth inhibition compared to treatment with the monospecific antibodies ANG2i-LC06 and avastin alone or in combination with ANG2i-LC06 and avastin.

Example 19

Dual specificity<VEGF-ANG-2>Antibody molecule scFAb-avastin-LC 10-2620, scFab-A Vastin-LC 10-2640 and scFab-avastin-LC 10-2641, avastin LC10-KiH-C-scFab, avastin-LC 10-C-Fab-6CSS, avastin -LC10-CH1-CL, avastin-LC 10-VH-VL and avastin-LC 10-VH-VL-SS, avastin-LC 10-N-scFab and avastin-LC 10-NExpression and purification of scFabSS and properties of

Bispecific < VEGF-ANG-2> antibody molecules scFAb-avastin-LC 10-2620, scFab-avastin-LC 10-2640 and scFab-avastin-LC 10-2641, avastin-LC 10-KiH-C-scFab, avastin-LC 10-C-Fab-6CSS, avastin-LC 10-CH1-CL, avastin-LC 10-VH-VL and avastin-LC 10-VH-VL-SS, were expressed and purified by replacing the VH and VL domains of Ang2i-LC06 (SEQ ID Nos 52 and 53) with the corresponding VH and VL domains of Ang2i-LC10 (SEQ ID Nos 84 and 85) using similar procedures and sequences as described in examples 10 to 14 (except for this replacement), avastin-LC 10-N-scFab and avastin-LC 10-N-scFabSS, both based on < VEGF > bevacizumab and < ANG-2> Ang2i-LC 10.

Binding affinity and other in vitro properties were determined as described in the above examples.

Example 20

Bispecific antibodies<VEGF-ANG-2>Molecular scFAb-avastin-LC 10-2620, scFab-avastin Vastin-LC 10-2640 and scFab-avastin-LC 10-2641, avastin LC10-KiH-C-scFab, avastin-LC 10-C-Fab-6CSS, avastin -LC10-CH1-CL, avastin-LC 10-VH-VL and avastin-LC 10-VH-VL-SS, in vivo Effect of avastin-LC 10-N-scFab and avastin-LC 10-N-scFabSS.

The in vivo effect of the bispecific antibody < VEGF-ANG-2> molecules scFAb-avastin-LC 10-2620, scFab-avastin-LC 10-2640 and scFab-avastin-LC 10-2641, avastin-LC 10-KiH-C-scFab, avastin-LC 10-C-Fab-6CSS, avastin-LC 10-CH1-CL, avastin-LC 10-VH-VL and avastin-LC 10-VH-VL-SS, avastin-LC 10-N-scFab and avastin-LC 10-N-scFabSS was similarly determined according to the corresponding examples above.

Claims

1. A bispecific antibody that specifically binds human vascular endothelial growth factor and human angiopoietin-2 comprising a first antigen-binding site that specifically binds human vascular endothelial growth factor and a second antigen-binding site that specifically binds human angiopoietin-2, characterized in that:

ii) the first antigen binding site that specifically binds human vascular endothelial growth factor 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 in the heavy chain variable domain and 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 the light chain variable domain; and

iii) the second antigen binding site that specifically binds human angiopoietin-2 comprises the CDR3 region of SEQ ID NO.46, the CDR2 region of SEQ ID NO. 47, and the CDR1 region of SEQ ID NO. 48 in the heavy chain variable domain, and the CDR3 region of SEQ ID NO. 49, the CDR2 region of SEQ ID NO. 50, and the CDR1 region of SEQ ID NO. 51 in the light chain variable domain.

2. The bispecific antibody according to claim 1, characterized in that the second antigen-binding site that specifically binds human angiopoietin-2 does not specifically bind human angiopoietin 1.

3. The bispecific antibody according to claim 1 or 2, characterized in that the ratio of the binding affinity KD for the antigen-binding site specific for vascular endothelial growth factor/the KD for the antigen-binding site specific for angiopoietin-2 is between 1.0 and 10.0.

4. The bispecific antibody according to claim 1 or 2, characterized in that said antibody is bivalent.

5. A pharmaceutical composition comprising the antibody of any one of claims 1-4.

6. The pharmaceutical composition of claim 5, for use in the treatment of cancer.

7. The pharmaceutical composition according to claim 5, for use in the treatment of vascular diseases.

8. The bispecific antibody of claim 1 or 2 for use in the treatment of cancer.

9. The bispecific antibody of claim 1 or 2 for use in the treatment of a vascular disease.

10. A nucleic acid encoding a bispecific antibody as defined in claim 1.

11. An expression vector containing the nucleic acid according to claim 10, which is capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell.

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

13. Method for the production of a bispecific antibody according to any one of claims 1 to 4, characterized in that a nucleic acid according to claim 10 is expressed in a prokaryotic or eukaryotic host cell and the bispecific antibody is recovered from the cell or the cell culture supernatant.