HK1178177B

HK1178177B - Bispecific, bivalent anti-vegf/anti-ang-2 antibodies

Info

Publication number: HK1178177B
Application number: HK13105057.4A
Authority: HK
Inventors: Monika Baehner; Sabine Imhof-Jung; Anita Kavlie; Hubert Kettenberger; Christian Klein; Joerg Thomas Regula; Wolfgang Schaefer; Juergen Michael Schanzer; Werner Scheuer; Kay-Gunnar Stubenrauch; Markus Thomas
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2010-03-26
Filing date: 2011-03-24
Publication date: 2018-04-20

Description

Bispecific bivalent anti-VEGF/anti-ANG-2 antibodies

The present invention relates to bispecific bivalent antibodies against human vascular endothelial growth factor (VEGF/VEGF-A) and against human angiopoietin-2 (ANG-2), methods for their production, pharmaceutical compositions containing said antibodies and uses thereof.

Background

Angiogenesis is implicated in the pathogenesis of a variety of disorders including solid tumors, intraocular neovascular syndromes such as proliferative retinopathy or age-related macular degeneration, rheumatoid arthritis, and psoriasis (Folkman, J. et al, J. biol. chem.267(1992) 10931-. In the case of solid tumors, neovascularization allows tumor cells to acquire a growth advantage and proliferative autonomy compared to normal cells. Thus, a correlation between the density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors has been observed (Weidner, N. et al, N Engl J Med.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, Science246(1989) 1306-9; Keck, P.J. et al, Science246(1989)1309-12 and Connolly, D.T. et al, J.biol.chem.264(1989) 20017-24. VEGF has been implicated in regulating normal and abnormal angiogenesis and neovascularization associated with tumor and intraocular disorders (Ferrara, N. et al, endo cr. Rev.18(1997)4-25; Berkman, R.A. et al, J.Clin. Invest.91(1993) 153-. VEGF is a homodimeric glycoprotein that has been isolated from several sources. VEGF exhibits highly specific mitogenic activity against endothelial cells. VEGF has important regulatory functions in neovascularization during embryonic angiogenesis and angiogenesis during adulthood (Carmeliet, P. et al, Nature,380(1996) 435-. The significance of the role played by VEGF has been demonstrated in studies showing that inactivation of a single VEGF allele leads to embryonic lethality due to the inability to form vasculature (vasculure) (Carmeliet, P. et al, Nature,380(1996)435-439; Ferrara, N. et al, Nature,380(1996) 439-442). In addition, VEGF has strong chemoattractant activity for monocytes, induces plasminogen activators and plasminogen activator inhibitors in endothelial cells, and also induces microvascular permeability. Due to the latter activity, it is sometimes referred to as Vascular Permeability Factor (VPF). The isolation and properties of VEGF have been reviewed; see Ferrara, N.et al, J.Cellular biochem.,47(1991) 211-. Alternative splicing of mRNA from a single VEGF gene produces 5 VEGF isoforms.

anti-VEGF neutralizing antibody inhibitionGrowth of various human tumor cell lines in mice (Kim, K.J. et al, Nature362(1993)841-844; Warren, S.R. et al, J.Clin.Invest.95(1995)1789-1797; Borgstrom, P. et al, Cancer Res.56(1996)4032-4039; and Melnyk, O. et al, Cancer Res.56(1996)921 924). WO94/10202, WO 98/45332, WO 2005/00900 and WO 00/35956 relate to antibodies against VEGF. Humanized monoclonal antibody bevacizumab (bevacizumab) (tradename avastin)Marketed) is an anti-VEGF antibody used in the treatment of tumors (WO 98/45331).

Ranibizumab (trade name)) Is a monoclonal antibody fragment derived from the same parent murine antibody as bevacizumab (avastin). It is much smaller than the parent molecule and has undergone affinity maturation, providing stronger binding to VEGF-A (WO 98/45331). It is an antiangiogenic agent that has been approved for use in the treatment of age-related macular degeneration (ARMD), the "wet" form (i.e., a common form of age-related vision loss). Another anti-VEGF antibody is HuMab G6-31, 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, Science277(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 Tie, a family of tyrosine kinases that are selectively expressed in vascular endothelium. Yancopoulos, G.D., et al, Nature 407(2000) 242-48. There are currently four defined members of the angiogenin family. Angiopoietins-3 and-4 (Ang-3 and Ang-4) may represent widely divergent counterparts of the same locus in mice and humans. Kim, I.et al, FEBSLet,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, for ANG-1: Davis, S. et al, Cell87(1996)1161-69; and for ANG-2: Maison Pierre, P.C. et al, Science277(1997) 55-60). All known angiopoietins bind predominantly to Tie2, while Ang-1 and-2 both bind to Tie2 with an affinity of 3nM (Kd). Maison pierre, p.c. et al, Science277(1997) 55-60. Ang-1 was shown to support EC survival and promote endothelial integrity, Davis, S. et al, Cell87(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) -2514, Thurston, G. et al, nat. Med.6(2000)460-63, while ANG-2 has the opposite effect and promotes vessel destabilization and regression in the absence of survival factor VEGF or basic fibroblast growth factor. Maison pierre, p.c. et al, Science277(1997) 55-60. However, many studies of ANG-2 function have suggested more complex situations. ANG-2 may be a complex regulator of vascular remodeling, which plays a role in both vessel sprouting and vessel regression. In support of such effects of ANG-2, expression analysis revealed that ANG-2 was rapidly induced with VEGF in the adult background of angiogenic sprouting, whereas ANG-2 was induced in the absence of VEGF in the background of vessel regression. Holash, J.et al, Science284(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 (which is activated by ANG-1), but has a background-dependent effect on its activation. Maison pierre, p.c. et al, Science277(1997) 55-60.

Corneal angiogenesis assays have shown that both ANG-1 and ANG-2 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. ANG-2 may also be pro-angiogenic due to the observation of high concentrations in vitro, suggesting the possibility of a dose-dependent endothelial response. Kim, I.et al, Oncogene19(2000) 4549-52. At high concentrations, ANG-2 acts as an apoptotic survival factor for endothelial cells during serum deprivation of apoptosis via the PI-3 kinase and Akt pathways via activation of Tie 2. Kim, I.et al, Oncogene19(2000) 4549-52.

Other in vitro experiments suggest that the effect of ANG-2 may gradually switch from an antagonist to an agonist of Tie2 during sustained exposure, and that it may directly contribute to the formation of vessels and the stabilization of new vessels at later time points. Teichert-Kuliszewska, k. et al, cardiovasc. res.49(2001) 659-70. Furthermore, if EC were cultured on fibrin gel, activation of Tie2 with ANG-2 was also observed, possibly suggesting that the effect of ANG-2 may depend on the state of EC differentiation. 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 gels. Mochizuki, y, et al, j.cell.sci.115(2002) 175-83. The use of 3-D spherical co-cultures as an in vitro model of vascular maturation indicates that direct contact between EC and mesenchymal cells abolished responsiveness to VEGF, while 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 the expression of EC, MMP-1, -9, and u-PA, which constitutively express Tie2, is strongly upregulated by ANG-2 in the presence of VEGF. Etoh, T, et al, Cancer Res.61(2001) 2145-53. By means of the in vivo pupillary membrane model, Lobov, i.b. et al showed that ANG-2 promotes rapid increase in capillary diameter, remodeling of the basal layer, proliferation and migration of endothelial cells, and stimulation of sprouting of new blood vessels in the presence of endogenous VEGF. Lobov, I.B. et al, Proc. Natl. Acad. Sci. USA 99(2002) 11205-10. In contrast, ANG-2 promoted endothelial cell death and vessel regression in the absence of endogenous VEGF. Lobov, I.B. et al, Proc. Natl. Acad. Sci. USA 99(2002) 11205-10. Similarly, by virtue of an in vivo tumor model, Vajkoczy, p. et al demonstrated that multicellular aggregates initiate vascular growth through angiogenic sprouting via simultaneous expression of VEGFR-2 and ANG-2 by the host and tumor endothelium. Vajkoczy, P., et al, J.Clin.invest.109(2002) 777-85. This model exemplifies that the microvasculature established by growing tumors is characterized by continuous remodeling, which is inferred to be mediated by the expression of VEGF and ANG-2. Vajkoczy, P., et al, J.Clin.invest.109(2002) 777-85.

Studies in mice knockout of Tie-2 and angiopoietin-1 show similar phenotypes and suggest that angiopoietin-1 stimulated Tie-2 phosphorylation mediates remodeling and stabilization of developing blood vessels, which promotes vascular maturation and maintenance of endothelial cell-supporting cell adhesion during angiogenesis (Dumont, D.J. et al, Genes & Development,8(1994) 1897-. The role of angiopoietin-1 is believed to be conserved in adulthood, where it is widely and constitutively expressed (Hanahan, D.D., Science277(1997) 48-50; Zagzag, D.et al, Exp Neurology 159(1999) 391-400). In contrast, angiopoietin-2 expression is primarily restricted to sites of vascular remodeling where it is thought to block the constitutive stabilizing or maturation function of angiopoietin-1, allowing the vessels to revert to and remain in a plastic state that can be more responsive to sprouting signals (Hanahan, D.,1997; Holash, J. et al, Oncogene 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 (Maisonpierre, P.C. et al, Science277(1997) 55-60). Functional studies suggest that angiopoietin-2 is involved in tumor angiogenesis and correlate angiopoietin-2 overexpression with increased tumor growth in mouse xenograft models (Ahmad, S.A., et al, Cancer Res.,61(2001) 1255-. Other studies have linked angiopoietin-2 overexpression to tumor hypervascularity (hypervascularity) (Etoh, T. et al, Cancer Res.61(2001)2145-53; Tanaka, F. et al, Cancer Res.62(2002) 7124-.

In recent years, angiopoietin-1, angiopoietin-2 and/or Tie-2 have been proposed as potential anti-cancer therapeutic targets. For example, US 6,166,185, US5,650,490 and US5,814,464 each disclose 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). Siemeister, g. et al, Cancer res.59:3(1999)3185-91 generated a human melanoma cell line expressing the extracellular domain of Tie-2, these were injected into nude mice, and it was reported that soluble Tie-2 resulted in significant inhibition of tumor growth and tumor angiogenesis. Given that angiopoietin-1 and angiopoietin-2 both bind Tie-2, it is unclear from these studies whether angiopoietin-1, angiopoietin-2 or Tie-2 would be attractive targets for anti-cancer therapy. However, effective anti-angiopoietin-2 therapy is thought to be beneficial for the treatment of diseases such as cancer, where progression is dependent on aberrant angiogenesis, where blocking this process can lead to arrest disease progression (Folkman, j., Nature medicine.1(1995) 27-31).

In addition, several groups have reported the use of antibodies and peptides that bind to angiopoietin-2. See, for example, US 6,166,185 and US 2003/10124129, WO 03/030833, WO 2006/068953, WO 03/057134 or US 2006/0122370.

Studies of the effects of focal expression (focal expression) of angiopoietin-2 have shown that antagonizing angiopoietin-1/Tie-2 signaling relaxes tight vascular structures, thereby exposing EC to activation signals from angiogenesis inducers such as VEGF (Hanahan, D.A., Science,277(1997) 48-50). This pro-angiogenic effect resulting from inhibition of angiopoietin-1 indicates that anti-angiopoietin-1 therapy would not be an effective anti-cancer treatment.

ANG-2 is expressed during development at sites where vascular remodeling occurs. Maison pierre, p.c. et al, Science277(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.61(2001)6248-54), hepatocellular carcinoma (Tanaka, S.et al, J.Clin.invest.103(1999)341-45), gastric Cancer (Etoh, T.et al, Cancer Res.61(2001)2145-53; Lee, J.H.et al, int.J.Oncol.18(2001)355-61), thyroid tumors (Bunone, G.et al, Am J Pathol 155(1999)1967-76), non-small cell lung Cancer (WoM.P. et al, Lungcer 29(2000)11-22), colon Cancer and Cancer (Ahmad, S.92. A.92, S.92. et al, Cancer 2001 7-76), prostate Cancer (Wancer) 5220, W.H.H.H.et al, J.20). Some tumor cells were found to express ANG-2. For example, Tanaka, S. et al, J.Clin.invest.103(1999)341-45 detected ANG-2mRNA in 10 samples of 12 specimens of human hepatocellular carcinoma (HCC). The group of Ellis reported ubiquitous expression of ANG-2 in tumor epithelium. Ahmad, s.a., et al, Cancer 92(2001) 1138-43. Other researchers reported similar findings. Chen, l., et al, j.tongjimed.univ.21(2001) 228-35. ANG-2mRNA was reported to be significantly associated with adjuvant lymph node invasion, short disease-free time and poor overall survival by examining ANG-2mRNA levels, sfigigio, c, et al, int.j. cancer 103(2003)466-74 in archived human breast cancer specimens. Tanaka, f, et al, Cancer res.62(2002)7124-29 examined a total of 236 non-small cell lung Cancer (NSCLC) patients in stages I to IIIA of pathology, respectively. Using immunohistochemistry, they found 16.9% of NSCLC patients positive for ANG-2. The microvascular density of ANG-2 positive tumors was significantly higher than that of ANG-2 negative tumors. Such angiogenic effects of ANG-2 are only seen when VEGF expression is high. Furthermore, positive expression of ANG-2 is an important factor in predicting poor post-operative survival. Tanaka, F. et al, cancer Res.62(2002)7124 and 7129. However, they did not find a significant correlation between Ang-1 expression and microvascular density. Tanaka, F. et al, Cancer Res.62(2002)7124 and 7129. These results suggest that ANG-2 is an indicator of poor prognosis in several cancer patients.

Recently, using the ANG-2 knockout mouse model, the Yancopoulos group reported that ANG-2 was required for postnatal angiogenesis. Gale, N.W., et al, Dev.cell3(2002) 411-23. They showed that ANG-2 knockout mice did not develop a developmentally programmed regression of the vitreous (hyaloid) vasculature in the eye, and that their retinal vessels failed to sprout from the central retinal artery. Gale, N.W., et al, Dev.Cell3(2002) 411-23. They also found that loss of ANG-2 results in profound defects in patterning and function of the lymphatic vasculature. Gale, N.W., et al, Dev.cell3(2002) 411-23. Genetic rescue with Ang-1 corrected lymphoid, but not angiogenic defects. Gale, N.W., et al, Dev.cell3(2002) 411-23.

Peters and his colleagues reported that soluble Tie2 inhibited the growth of murine breast cancer and melanoma in vivo in a mouse model when delivered as a recombinant protein or in a viral expression vector. Lin, P.et al, Proc.Natl.Acad.Sci.USA 95(1998)8829-34; Lin, P.et al, J.Clin.Invest.100(1997) 2072-78. The vascular density in the tumor tissue thus treated is greatly reduced. In addition, soluble Tie2 blocks angiogenesis in rat corneas stimulated by tumor cell conditioned media (Lin, p. et al, j.clin.invest.100(1997) 2072-78). Furthermore, Isner and his group demonstrated that the addition of ANG-2 to VEGF promoted significantly longer and more circumferential new blood vessel distribution than VEGF alone. Asahara, T, et al, circ. Res.83(1998) 233-40. Excess soluble Tie2 receptor abrogated VEGF-induced neovascularization by ANG-2 regulation. Asahara, T, et al, circ. Res.83(1998) 233-40. Siemeister, g. et al, Cancer res.59:3(1999)3185-91 with nude mouse xenografts showed that overexpression of the extracellular ligand binding domain of Flt-1 or Tie2 in xenografts resulted in significant inhibition of the pathway that could not be compensated by another pathway, suggesting that the VEGF receptor pathway and Tie2 pathway should be considered as two independent mediators essential for the angiogenic process in vivo. Siemeister, G. et al, Cancer Res.59:3(1999) 3185-91. This is demonstrated by recent publications by White, r. et al, proc.natl.acad.sci.usa100(2003) 5028-33. In their studies, nuclease resistant RNA aptamers that specifically bind to and inhibit ANG-2 were demonstrated to significantly inhibit bFGF-induced neovascularization in the rat corneal micro pocket (micropocket) angiogenesis model.

Bispecific antibodies

A wide variety of recombinant antibody formats have recently been developed, such as tetravalent bispecific antibodies obtained 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)159-163; WO 2001/077342; and Morrison, S.L., Nature Biotech25(2007) 1233-1234).

Several other new forms have also been developed which are capable of binding two or more antigens and no longer retain the core structure of the antibody (IgA, IgD, IgE, IgG or IgM), such as diabodies, triabodies or tetrabodies, minibodies, several single chain forms (scFv, bis-scFv) (Holliger, P. et al, Nature Biotech23(2005)1126-1136; Fischer, N., Leger, O., Pathiology 74(2007)3-14; Shen, J. et al, Journal of Immunological Methods318(2007)65-74; Wu, C. et al, Nature Biotech.25(2007) 1290-1297).

All such formats use linkers to fuse or fuse an antibody core (IgA, IgD, IgE, IgG or IgM) with another binding protein (e.g. scFv), e.g. two Fab fragments or scFv (Fischer, n., leger, o., Pathobiology 74(2007) 3-14). It must be kept in mind that it may be desirable to retain effector functions mediated via Fc receptor binding, such as, for example, Complement Dependent Cytotoxicity (CDC) or Antibody Dependent Cellular Cytotoxicity (ADCC), by maintaining a high degree of similarity to naturally occurring antibodies.

In WO 2007/024715 it is reported that dual variable domain immunoglobulins are engineered as multivalent and multispecific binding proteins. A method for preparing biologically active antibody dimers is reported in US 6,897,044. In US 7,129,330 a multivalent FV antibody construct with at least four variable domains linked to each other via a peptide linker is reported. Dimeric and multimeric antigen binding structures are reported in US 2005/0079170. In US 6,511,663 a tri-or tetravalent monospecific antigen binding protein comprising three or four Fab fragments covalently bound to each other by a linking structure is reported, which protein is not a native immunoglobulin. Tetravalent bispecific antibodies which can be efficiently expressed in prokaryotic and eukaryotic cells and which are useful in therapeutic and diagnostic methods are reported in WO 2006/020258. In US 2005/0163782 a method is reported for the isolation or preferential synthesis of dimers linked via at least one interchain disulfide bond from a mixture comprising two types of polypeptide dimers relative to dimers which are not linked via at least one interchain disulfide bond. Bispecific tetravalent receptors are reported in US5,959,083. 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 homoconjugates, which are usually prepared by synthetic cross-linking of monoclonal antibodies of the IgG class covalently bound to the same antigenic determinant. Oligomeric monoclonal antibodies with high affinity for antigens are reported in WO 1991/06305, wherein oligomers, usually of the IgG class, are secreted with two or more immunoglobulin monomers linked together to form tetravalent or hexavalent IgG molecules. Sheep derived antibodies and engineered antibody constructs that may be used to treat pathogenic diseases with interferon gamma activity are reported in US 6,350,860. Targetable constructs that are multivalent vectors for bispecific antibodies, i.e. each molecule of the targetable construct can act as a vector for two or more bispecific antibodies, are reported in US 2005/0100543. Genetically engineered bispecific tetravalent antibodies are reported in WO 1995/009917. Stabilized binding molecules consisting of or comprising stabilized scfvs are reported in WO 2007/109254.

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 ANG-2 and VEGF inhibitor combinations.

WO 2003/106501 relates to fusion proteins that bind angiogenin and contain a multimerization domain. WO2008/132568 relates to fusion proteins that bind angiogenin and VEGF. WO 2003/020906 relates to multivalent protein conjugates having multiple ligand binding domains of a receptor.

WO 2009/136352 relates to anti-angiogenic compounds.

Summary of The Invention

The present invention relates to a bispecific bivalent antibody 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:

i) the first antigen binding site comprises SEQ ID NO 1 as the heavy chain variable domain (VH),

and SEQ ID NO 2 as the light chain variable domain (VL); and is

ii) the second antigen binding site comprises SEQ ID NO 3 as the heavy chain variable domain (VH) and SEQ ID NO 4 as the light chain variable domain (VL).

In one aspect of the invention, a bispecific antibody according to the invention is characterized by comprising:

a) heavy and light chains of a first full length antibody that specifically binds VEGF; and

b) a modified heavy chain and a modified light chain of a full-length antibody that specifically binds ANG-2, wherein the constant domains CL and CH1 are replaced with each other.

In one embodiment, such bispecific bivalent antibodies are characterized by comprising:

a) amino acid sequence SEQ ID NO 7 as the heavy chain and amino acid sequence SEQ ID NO 5 as the light chain of said first full-length antibody, and

b) amino acid sequence SEQ ID NO 8 as the modified heavy chain and amino acid sequence SEQ ID NO 6 as the modified light chain of said second full length antibody.

a) amino acid sequence SEQ ID NO 11 as the heavy chain and amino acid sequence SEQ ID NO 9 as the light chain of said first full-length antibody, and

b) amino acid sequence SEQ ID NO 12 as the modified heavy chain and amino acid sequence SEQ ID NO 10 as the modified light chain of the second full-length antibody.

a) amino acid sequence SEQ ID NO 15 as the heavy chain and amino acid sequence SEQ ID NO 13 as the light chain of said first full-length antibody, and

b) amino acid sequence SEQ ID NO 16 as the modified heavy chain of the second full-length antibody and amino acid sequence SEQ ID NO 14 as the modified light chain of the second full-length antibody.

Further aspects of the invention are a pharmaceutical composition comprising said bispecific antibody, said composition for 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.

Further aspects of the invention are a pharmaceutical composition comprising said bispecific antibody, said composition for the treatment of a vascular disease, the use of said bispecific antibody for the manufacture of a medicament for the treatment of a vascular disease, a method of treating a patient suffering from a vascular disease by administering said bispecific antibody to a patient in need of such treatment.

Yet another aspect of the invention is a nucleic acid molecule encoding a chain of a bispecific antibody according to the invention.

The invention further provides expression vectors comprising said nucleic acid according to the invention, capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell, and host cells comprising said vectors for the recombinant production of bispecific antibodies according to the invention.

The invention further comprises a prokaryotic or eukaryotic host cell comprising a vector according to the invention.

The invention further comprises a method for the production of 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 said cell or cell culture supernatant. The invention further includes antibodies obtained by such methods for generating bispecific antibodies.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 9, 10, 11 and 12.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.

The bispecific bivalent antibody according to the invention shows benefits for human patients in need of VEGF and ANG-2 targeted therapy. The antibodies according to the invention have highly valuable properties which confer benefits to patients suffering from such diseases, in particular from cancer. The bispecific antibodies according to the invention are highly effective in tumor growth inhibition and/or inhibition of tumor angiogenesis or vascular disease. The bispecific bivalent antibody according to the invention, the bispecific bivalent < VEGF-ANG-2> antibody according to the invention shows valuable pharmacokinetic/pharmacodynamic properties such as e.g. stability, good (i.e. slow) clearance (e.g. at low doses).

The bispecific antibodies according to the invention are highly effective in:

a) tumor growth inhibition (e.g., with a bispecific antibody according to the invention, tumor arrest (statis) can already be achieved at lower concentrations compared to the combination of two monospecific antibodies (e.g., in the Colo205 and KPL-4 tumor models of examples 9 and 10, tumor arrest has already been achieved with 10mg/kg of XMAb1 compared to the combination of 10mg/kg of ANG2i-LC06+10mg/kg of avastin), and/or

b) Inhibiting tumor angiogenesis or vascular disease (e.g., the maximal anti-angiogenic effect has been achieved at a lower concentration with a bispecific antibody according to the invention compared to the combination of two monospecific antibodies (e.g., the maximal anti-angiogenic effect has been achieved with 10mg/kg of XMAb1 compared to the combination of 10mg/kg of ANG2i-LC06+10mg/kg of avastin in the mouse corneal angiogenesis assay of example 8).

Brief Description of Drawings

FIG. 1 is an exemplary bivalent, bispecific antibody format of the Xmab example, comprising a CH3 domain modified by protuberance-entry-cavities (Knobs-into-Holes).

Figure 2a exemplary bivalent bispecific antibody format of the OAscFab example comprising a protuberance-entry-hole modified CH3 domain.

Figure 2b is an exemplary bivalent bispecific antibody format of oascfab1 comprising a CH3 domain modified by a protuberance-into-hole.

Figure 2c illustrates exemplary bivalent bispecific antibody formats of oascfab2 and oascfab3, comprising a protrusion-entry-hole modified CH3 domain.

FIG. 3 simultaneous binding of < VEGF-Ang-2> XMab1 to VEGF (step 1) followed by binding to hAng-2 (step two).

Figure 4 ELISA principle for quantification of binding-active mab < Ang2/VEGF > antibodies.

Figure 5 ELISA calibration curves for quantification of binding activity < Ang2/VEGF > XMab1 antibodies.

Figure 6 mouse corneal angiogenesis assay: the growth of the vessel from the limbus (limbus) towards the VEGF gradient (outgrowth) is inhibited by administration of a bispecific antibody according to the invention.

Figure 7 mouse corneal angiogenesis assay: angiogenesis/vascular-comparison of the limbic (limbic) to VEGF gradient by administration of bispecific antibodies according to the invention bispecific < Ang2/VEGF > antibodies XMab1, < Ang2> mabs Ang2i-LC06(LC06), < VEGF > mabbevacizumab (avastin) and a combination of Ang2i-LC06 and < VEGF > mabbevacizumab (avastin).

Figure 8 in vivo tumor growth inhibition by bispecific antibodies according to the invention in mouse xenografts (small tumors) of human colorectal cancer Colo 205: the combination of the bispecific < Ang2/VEGF > antibodies XMab1, < Ang2> mabs Ang2i-LC06(LC06), < VEGF > mabbevacizumab (avastin), and Ang2i-LC06 and < VEGF > mabbevacizumab (avastin) were compared.

Figure 9 in vivo tumor growth inhibition by bispecific antibodies according to the invention in mouse xenografts (large tumors) of human colorectal cancer Colo 205: the bispecific < Ang2/VEGF > antibodies XMab1, < Ang2> Mab Ang2i-LC06(LC06), < VEGF > Mab bevacizumab (avastin), and the combination of Ang2i-LC06 and < VEGF > Mab bevacizumab (avastin) were compared.

Figure 10 in vivo tumor growth inhibition by bispecific antibodies according to the invention in mouse xenografts (small tumors) of human breast cancer KPL-4: the combination of the bispecific < Ang2/VEGF > antibody XMab1, < Ang2> mab Ang2i-LC06(LC06), < VEGF > mab bevacizumab (avastin), and Ang2i-LC06 and < VEGF > mab bevacizumab (avastin) were compared.

Figure 11 in vivo tumor growth inhibition by bispecific antibodies according to the invention in mouse xenografts (large tumors) of human breast cancer KPL-4: the combination of the bispecific < Ang2/VEGF > antibody XMab1, < Ang2> mab Ang2i-LC06(LC06), < VEGF > mab bevacizumab (avastin), and Ang2i-LC06 and < VEGF > mab bevacizumab (avastin) were compared.

Figure 12 in vivo tumor growth inhibition by bispecific antibodies according to the invention in mouse xenografts of gastric cancer N87: the bispecific < Ang2/VEGF > antibodies XMab1, < Ang2> mabs Ang2i-LC06(LC06), < VEGF > mabbevacizumab (avastin), and the combination of Ang2i-LC06 and < VEGF > mabbevacizumab (avastin) were compared.

Detailed Description

i) the first antigen binding site comprises SEQ ID NO 1 as the heavy chain variable domain (VH) and SEQ ID NO 2 as the light chain variable domain (VL); and is

a) heavy and light chains of a first full length antibody that specifically binds VEGF;

b) a modified heavy chain and a modified light chain of a full-length antibody that specifically binds ANG-2, wherein the constant regions CL and CH1 are replaced with each other.

This bispecific bivalent antibody format of a bispecific antibody specifically binding to human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2) is described in WO 2009/080253 (see fig. 1 for an exemplary scheme including a protrusion-entry-hole modified CH3 domain). Antibodies based on this bispecific bivalent antibody format were designated Xmab in the examples of the invention.

a) amino acid sequence SEQ ID NO 19 as the heavy chain and amino acid sequence SEQ ID NO 17 as the light chain of said first full-length antibody, and

b) amino acid sequence SEQ ID NO 20 as the modified heavy chain of the second full-length antibody and amino acid sequence SEQ ID NO 18 as the modified light chain of the second full-length antibody.

a) amino acid sequence SEQ ID NO 23 as the heavy chain and amino acid sequence SEQ ID NO 21 as the light chain of said first full-length antibody, and

b) amino acid sequence SEQ ID NO. 24 as the modified heavy chain and amino acid sequence SEQ ID NO. 22 as the modified light chain of the second full-length antibody.

a) amino acid sequence SEQ ID NO 27 as the heavy chain and amino acid sequence SEQ ID NO 25 as the light chain of said first full-length antibody, and

b) amino acid sequence SEQ ID NO 28 as the modified heavy chain of the second full-length antibody and amino acid sequence SEQ ID NO 26 as the modified light chain of the second full-length antibody.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 17, 18, 19 and 20.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 25, 26, 27 and 28.

In another aspect of the invention, a bispecific antibody according to the invention is characterized by comprising:

b) a heavy chain and a light chain of a second full-length antibody that specifically binds ANG-2, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker.

An exemplary scheme of this bispecific bivalent antibody format of this bispecific antibody specifically binding to human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2) comprising a protuberance-into-hole modified CH3 domain is shown in fig. 2 a. An antibody based on this bispecific bivalent antibody format was named OascFab in the examples of the present invention.

a) amino acid sequence SEQ ID NO 30 as the heavy chain and amino acid sequence SEQ ID NO 31 as the light chain of said first full-length antibody, and

b) amino acid sequence SEQ ID NO 29 as the heavy chain of the second full length antibody and the light chain of the second full length antibody connected via a peptide linker.

a) amino acid sequence SEQ ID NO 33 as the heavy chain and amino acid sequence SEQ ID NO 34 as the light chain of said first full-length antibody, and

b) the amino acid sequence of SEQ ID NO 32 as the heavy chain of the second full length antibody and the light chain of the second full length antibody connected via a peptide linker.

In one embodiment, the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL) of the heavy and light chains of the second full length antibody are disulfide stabilized by introducing a disulfide bond between: the heavy chain variable domain from position 44 to position 100 of the light chain variable domain (always numbered according to the EU index of Kabat; Kabat, E.A. et al, Sequences of proteins of Immunological Interest, fifth edition, public Health Service, national institutes of Health, Bethesda, MD (1991)). Such further disulfide stabilization is achieved by introducing disulfide bonds between the variable regions VH and VL of the heavy and light chains of the second full length antibody. Techniques for stabilization by the introduction of non-natural disulfide bridges are described, for example, in WO 94/029350, Rajagopal, V.et al, prot.Engin.10(1997)1453-59, Kobayashi et al, nucleic Medicine & Biology, Vol.25 (1998)387-393, or Schmidt, M.et al, Oncogene 18(1999) 1711-1721.

Thus, in one embodiment, such bispecific bivalent antibodies are characterized as comprising a disulfide bond between the variable domains of the heavy and light chains of the second full length antibody, i.e. between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain, and comprising

a) Amino acid sequence SEQ ID NO 36 as the heavy chain and amino acid sequence SEQ ID NO 37 as the light chain of said first full-length antibody, and

b) 35 as the heavy chain of the second full length antibody and the light chain of the second full length antibody connected via a peptide linker.

In another aspect of the invention, a bispecific antibody according to the invention is characterized in comprising

b) a heavy chain and a light chain of a second full-length antibody that specifically binds ANG-2,

wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker; and wherein the variable domains VL and VH are substituted for one another.

An exemplary scheme of this bispecific bivalent antibody format of this bispecific antibody specifically binding to human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2) is shown in fig. 2b, which includes a protuberance-into-hole modified CH3 domain. An antibody based on this bispecific bivalent antibody format was designated as oascfab1 in the examples of the invention.

In one embodiment, such bispecific antibodies are characterized by comprising

a) 39 as the heavy chain of said first full-length antibody and SEQ ID NO 40 as the light chain of said first full-length antibody, and

b) 38 as the heavy chain of the second full length antibody and the light chain of the second full length antibody connected via a peptide linker.

wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker; and is

Where the constant domains CL and CH1 are replaced by each other.

An exemplary scheme of this bispecific bivalent antibody format of this bispecific antibody specifically binding to human Vascular Endothelial Growth Factor (VEGF) and human angiopoietin-2 (ANG-2) is shown in fig. 2c, which includes a protuberance-into-hole modified CH3 domain. Antibodies based on this bispecific bivalent antibody format were designated in the examples as oascxab 2 and oascxab 3.

In one embodiment, such bispecific antibodies are characterized by comprising

a) 42 as the heavy chain of said first full-length antibody and SEQ ID NO 43 as the light chain of said first full-length antibody, and

b) 41 as the heavy chain of the second full length antibody and the light chain of the second full length antibody connected via a peptide linker.

In one embodiment, such bispecific antibodies are characterized by comprising

a) 45 as the heavy chain of said first full-length antibody and SEQ ID NO 46 as the light chain of said first full-length antibody, and

b) 44 as the heavy chain of the second full length antibody and the light chain of the second full length antibody connected via a peptide linker.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 29, SEQ ID NO 30 and SEQ ID NO 31.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 35, SEQ ID NO 36 and SEQ ID NO 37.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 38, SEQ ID NO 39 and SEQ ID NO 40.

Thus, one embodiment of the invention is a bispecific bivalent antibody 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 comprising the amino acid sequences of SEQ ID NO 41, 42 and 43.

Thus, one embodiment of the invention is a bispecific bivalent antibody comprising a first antigen-binding site specifically binding to human VEGF and a second antigen-binding site specifically binding to ANG-2, characterized in comprising the amino acid sequences of SEQ ID NO 44, SEQ ID NO 45 and SEQ ID NO 46.

Preferably, the CH3 domain of the bispecific bivalent antibody according to the invention is altered by the "protrusion-entry-cavity" technique which is described in more detail in, for example, WO 96/027011, RidgwayJ.B. et al, Protein Eng 9(1996) 617-681 and Merchant, A.M. et al, Nat Biotechnol 16(1998)677-681, to name a few. In this approach, the interaction surface of the two CH3 domains is altered to enhance heterodimerization of the two heavy chains containing the two CH3 domains. The two CH3 domains (of both heavy chains) may each be a "protuberance" and the other a "hole". Introduction of disulfide bridges stabilizes the heterodimer (Merchant, A.M et al, Nature Biotech 16(1998)677-681; Atwell, S., et al J.mol.biol.270(1997)26-35) and increases yield.

In a preferred aspect of the invention, all bispecific antibodies according to the invention are characterized in that

The CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each meet at an interface comprising the initial interface between the antibody CH3 domains;

wherein the interface is altered to facilitate formation of a bispecific antibody, wherein the alteration is characterized by:

a) the CH3 domain of one heavy chain is altered,

so that within the initial interface where the CH3 domain of one heavy chain meets the initial interface of the CH3 domain of the other heavy chain within the bispecific antibody,

the amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating an overhang in the interface of the CH3 domain of one heavy chain that can be located in a pocket in the interface of the CH3 domain of the other heavy chain, and

b) the CH3 domain of the other heavy chain is altered,

so that within the initial interface where the second CH3 domain meets the initial interface of the first CH3 domain within the bispecific antibody,

the amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a pocket within the interface of the second CH3 domain within which the protrusion within the interface of the first CH3 domain may be located.

Thus, preferably, the antibodies according to the invention are characterized in that

a) The heavy chain CH3 domain of the full-length antibody of b) and the heavy chain CH3 domain of the full-length antibody of b) each meet at an interface comprising a change in the initial interface between the antibody CH3 domains;

wherein i) in the CH3 domain of one heavy chain,

the amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion within the interface of the CH3 domain of one heavy chain, which protrusion may be located in a pocket within the interface of the CH3 domain of the other heavy chain,

and wherein

ii) in the CH3 domain of the other heavy chain,

Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of: arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).

Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of: alanine (a), serine (S), threonine (T), valine (V).

In one aspect of the invention, the two CH3 domains are further altered by introducing cysteine (C) as an amino acid in the corresponding position of each CH3 domain, such that a disulfide bridge between the two CH3 domains can be formed.

In one embodiment, the bispecific antibody comprises a T366W mutation in the CH3 domain of the "protruding chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "hole chain". Other interchain disulfide bridges between the CH3 domains may also be used (Merchant, A.M et al, Nature Biotech 16(1998) 677-.

In another embodiment, the bispecific antibody according to the invention comprises a Y349C, T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In another preferred embodiment, the bispecific antibody comprises a Y349C, T366W mutation in one of the two CH3 domains and a S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains (an additional Y349C mutation in one CH3 domain and an additional E356C or S354C mutation in the other CH3, which form inter-chain disulfide bridges) (always numbered according to the EU index of Kabat; Kabat, e.a. et al, Sequences of Proteins of Immunological Interest, fifth edition, public Health Service, n.ionon. of Health, Bethesda, MD (1991)). Also alternatively/additionally, other protrusion-entry-cavity techniques may be used, as described in EP 1870459a 1. Thus, another example of a bispecific antibody is the R409D in the CH3 domain of the "protruding chain", the K370E mutation and the D399K in the CH3 domain of the "hole chain", the E357K mutation (numbering always according to the EU index of Kabat; Kabat, E.A. et al, Sequences of Proteins of immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, Md (1991)).

In another embodiment, the bispecific antibody comprises a T366W mutation in the CH3 domain of the "protruding chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "hole chain" and additionally a R409D, K370E mutation in the CH3 domain of the "protruding chain" and a D399K, E357K mutation in the CH3 domain of the "hole chain".

In another embodiment, the bispecific antibody comprises a Y349C, T366W mutation in one of the two CH3 domains and a S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains, or the trivalent, bispecific antibody comprises a Y349C, T366W mutation in one of the two CH3 domains and a S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains and additionally a R409D in the CH3 domain of the "bulge chain", a K370E mutation and a D399K in the CH3 domain of the "hole chain", an E357K mutation.

In one embodiment of the invention, the bispecific antibody according to the invention is characterized by having one or more of the following properties (determined in the assays as described in examples 3to 7):

-the bispecific bivalent antibody binds VEGF with a KD value of binding affinity of 5nM or less;

-the bispecific bivalent antibody binds ANG-2 with a KD value of binding affinity of 5nM or less;

-the bispecific bivalent antibody inhibits ANG-2 induced Tie2 phosphorylation in Tie2 transfected HEK293 cells with an IC50 of 15nM or less (in one embodiment, with an IC50 of 10nM or less);

-the bispecific bivalent antibody inhibits the binding of ANG-2 to Tie2 with an IC50 of 20nM or less (in one embodiment, with an IC50 of 15nM or less);

-the bispecific bivalent antibody inhibits VEGF binding to a VEGF receptor with an IC50 of 20nM or less (in one embodiment, with an IC50 of 15nM or less);

bispecific bivalent antibodies inhibit VEGF-induced HUVEC cell proliferation with an IC50 of 10nM or less (in one embodiment, with an IC50 of 5nM or less).

In one embodiment, the bispecific bivalent antibody is characterized by 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

ii) the second antigen binding site comprises SEQ ID NO 3 as the heavy chain variable domain (VH) and SEQ ID NO 4 as the light chain variable domain (VL);

and having one or more of the following characteristics (determined in assays as described in examples 3to 7):

In one aspect of the invention, such bispecific antibodies according to the invention are characterized in comprising

b) a modified heavy chain and a modified light chain of a full-length antibody that specifically binds ANG-2, wherein constant regions CL and CH1 are replaced with each other;

In one embodiment, the bispecific bivalent antibody is characterized by 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:

i) the first antigen binding site comprises SEQ ID NO 1 with NO more than 1 amino acid residue substitutions in the CDRs as a heavy chain variable domain (VH), and SEQ ID NO 2 with NO more than 1 amino acid residue substitutions in the CDRs as a light chain variable domain (VL); and is

ii) the second antigen binding site comprises SEQ ID NO 3 with NO more than 1 amino acid residue substitutions in the CDRs as a heavy chain variable domain (VH), and SEQ ID NO 4 with NO more than 1 amino acid residue substitutions in the CDRs as a light chain variable domain (VL).

ii) the second antigen binding site comprises SEQ ID NO 3 with NO more than 1 amino acid residue substitutions in the CDRs as a heavy chain variable domain (VH) and SEQ ID NO 4 with NO more than 1 amino acid residue substitutions in the CDRs as a light chain variable domain (VL);

and having one or more of the following characteristics (determined in assays such as those described in examples 3to 7):

In one aspect of the invention, a bispecific antibody according to the invention is characterized in comprising

and wherein the heavy chain of the first full-length antibody comprises the amino acid sequence of SEQ ID NO 7 and has NO more than 1 amino acid residue substitution in a CDR and the light chain of the first full-length antibody comprises the amino acid sequence of SEQ ID NO 5 and has NO more than 1 amino acid residue substitution in a CDR, and

b) a modified heavy chain and a modified light chain of a full-length antibody that specifically binds ANG-2, wherein constant domains CL and CH1 are replaced with each other,

and wherein the modified heavy chain of the second full length antibody comprises the amino acid sequence of SEQ ID NO 8 and has NO more than 1 amino acid residue substitution in a CDR and the modified light chain of the second full length antibody comprises the amino acid sequence of SEQ ID NO 6 and has NO more than 1 amino acid residue substitution in a CDR.

a) Heavy and light chains of a first full-length antibody that specifically binds VEGF,

and wherein the modified heavy chain of the second full length antibody comprises the amino acid sequence of SEQ ID NO 8 and has NO more than 1 amino acid residue substitution in a CDR and the modified light chain of the second full length antibody comprises the amino acid sequence of SEQ ID NO 6 and has NO more than 1 amino acid residue substitution in a CDR;

As used herein, "antibody" refers to a binding protein that comprises an antigen binding site. As used herein, the term "binding site" or "antigen binding site" refers to the region of an antibody molecule to which a ligand actually binds. The term "antigen-binding site" encompasses an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) (VH/VL pair).

Antibody specificity refers to the selective recognition of a particular epitope by an antibody. For example, natural antibodies are monospecific.

A "bispecific antibody" according to the invention is an antibody having two different antigen binding specificities. The antibodies of the invention are specific for two different antigens, namely VEGF as the first antigen and ANG-2 as the second antigen.

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

As used within this application, the term "valency" refers to the presence of a defined number of binding sites in an antibody molecule. Thus, the terms "bivalent", "tetravalent", and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antibody molecule. Bispecific antibodies according to the invention are "bivalent".

As used herein, the term "VEGF" refers to human vascular endothelial growth factor (VEGF/VEGF-A) (SEQ ID NO:47), which is described, for example, in Leung, D.W. et al, Science246(1989) 1306-9, Keck, P.J. et al, Science246(1989)1309-12 and Connolly, D.T. et al, J.biol.chem.264(1989) 20017-24. VEGF has been implicated in regulating normal and abnormal angiogenesis and neovascularization associated with tumor and intraocular disorders (Ferrara, N. et al, endo cr. Rev.18(1997)4-25; Berkman, R.A. et al, J.Clin. Invest.91(1993) 153-. VEGF is a homodimeric glycoprotein that has been isolated from several sources. VEGF exhibits highly specific mitogenic activity against endothelial cells.

As used herein, the term "ANG-2" refers to angiopoietin-2 (ANG-2) (or abbreviated ANGPT2 or ANG2) (SEQ ID NO:48) as described, for example, in Maison pierre, P.C. et al, Science277(1997)55-60 and Cheung, A.H. et al, Genomics 48(1998) 389-91. Angiopoietins-1 and-2 were found to be ligands for Tie, a family of tyrosine kinases that are selectively expressed in the vascular endothelium. Yancopoulos, G.D., et al, Nature 407(2000) 242-48. There are currently four defined members of the angiogenin family. Angiopoietins-3 and-4 (Ang-3 and Ang-4) can represent widely divergent counterparts of the same gene 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, for ANG-1: Davis, S. et al, Cell87(1996)1161-69; and for ANG-2: Maison Pierre, P.C. et al, Science277(1997) 55-60). All known angiopoietins bind predominantly to Tie2, while Ang-1 and-2 both bind to Tie2 with an affinity of 3nM (Kd). Maison pierre, p.c. et al, Science277(1997) 55-60.

The antigen binding site of the bispecific antibodies of the present invention contains 6 Complementarity Determining Regions (CDRs) that contribute to varying degrees to the affinity of the binding site for the antigen. There are 3 heavy chain variable domain CDRs (CDRH 1, CDRH2 and CDRH 3) and 3 light chain variable domain CDRs (CDRL 1, CDRL2 and CDRL 3). The extent of the CDRs and Framework Regions (FRs) is determined by comparison with a compiled database of amino acid sequences in which those regions have been defined according to the variability between sequences. Also included within the scope of the invention are functional antigen binding sites that are composed of fewer CDRs (i.e., wherein binding specificity is determined by 3, 4, or 5 CDRs). For example, less than a complete set of 6 CDRs may be sufficient for binding. In some cases, a VH or VL domain may be sufficient.

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, the subtypes thereof.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single amino acid composition.

The term "chimeric antibody" refers to an antibody comprising at least a portion of a variable, i.e., binding, region from one source or species and a constant region derived from a different source or species, which is 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 generate properties according to the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also known as "class switch antibodies". Chimeric antibodies are the expression product of an immunoglobulin gene comprising a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for generating chimeric antibodies involve conventional recombinant DNA and gene transfection techniques, which are well known in the art. See, e.g., Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA 81(1984)6851-6855, US5,202,238 and US5,204,244.

The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDRs) have been modified to comprise immunoglobulin CDRs of different specificity compared to the specificity of the parent immunoglobulin. In a preferred embodiment, murine CDRs are grafted into the framework regions of a human antibody to make a "humanized antibody". See, for example, 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 representing sequences that recognize the antigens noted above for the chimeric antibodies. Other forms of "humanized antibodies" encompassed by the invention are those in which the constant regions have been additionally modified or altered from the constant regions of the original antibody to generate properties in accordance with the 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., curr. opin. chem. biol.5(2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing a complete repertoire or selection of human antibodies after immunization without endogenous immunoglobulin production. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice results in the production of human antibodies after antigen challenge (see, e.g., Jakobovits, A. et al, Proc. Natl. Acad. Sci. USA 90(1993)2551-2555; Jakobovits, A. et al, Nature362(1993) 255-258; Brueggemann, M. et al, Yeast Immunol.7(1993) 33-40). Human antibodies can also be generated in phage display libraries (Hoogenboom, H.R., and Winter, G.J., mol.biol.227(1992) 381-59388; Marks, J.D., et al, J.mol.biol.222(1991) 581-597). The techniques of Cole, A. et al and Boerner, P et al can also be used to prepare human monoclonal antibodies (Cole, A. et al, monoclonal antibodies and Cancer Therapy, Liss, A.L., page 77 (1985); and Boerner, P. et al, J.Immunol.147(1991) 86-95). As already mentioned in relation to the chimeric and humanized antibodies according to the invention, the term "human antibody", as used herein, also comprises antibodies which are modified in the constant region to generate the properties according to the invention (in particular with respect to C1q binding and/or FcR binding), for example by "class switching", i.e. a change or mutation of the Fc part (for example from IgG1 to IgG4 and/or IgG1/IgG4 mutation).

As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, created or isolated by recombinant means, 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 rearranged forms of variable and constant regions. Recombinant human antibodies according to the invention have been subject to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur within the human antibody germline repertoire in vivo.

As used herein, "variable domain" (light chain variable domain (VL), heavy chain variable domain (VH)) means each pair of light and heavy chains directly involved in antibody-to-antigen binding. The human light and heavy chain variable domains have the same general structure, and each domain comprises 4 sequence-wide conserved Framework (FR) regions connected by 3 "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β -sheet conformation, and the CDRs can form loops connecting the β -sheet structure. The CDRs in each chain retain their three-dimensional structure through 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 a further object of the invention.

The term "antigen-binding portion of an antibody" or "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. Hypervariable regions comprise amino acid residues from "complementarity determining regions" or "CDRs". The "framework" or "FR" regions are those variable domain regions which differ from the hypervariable region residues as defined herein. Thus, the light and heavy chains of the antibody comprise the N-to C-terminal domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR 4. The CDRs on each chain are separated by such framework amino acids. In particular, CDR3 of the heavy chain is the region most contributing to antigen binding. The CDR and FR regions (including numbering according to the EU index of Kabat, abbreviated hereinafter as numbering according to Kabat) were determined according to the standard definition of Sequences of Proteins of immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

As used herein, the term "binding" or "specific binding" refers to an in vitro assay that is superiorBinding of the antibody to an epitope of an antigen (human VEGF or human ANG-2) is optionally performed in a plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) (example 3) using purified wild-type antigen. By the term ka (binding rate constant of antibody from antibody/antigen complex), k_D(dissociation constant), and K_D(k_D/ka) to define binding affinity. In one embodiment, binding or specific binding means 10^-8mol/l or less, preferably 10^-9M to 10^-13Binding affinity (K) in mol/l_D)。

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

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

The term "full-length antibody" refers to an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains" (see fig. 1). "full-length antibody heavy chain" is composed of an antibody heavy chain variable domain (VH), an antibody heavy chain constant 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) (abbreviated VH-CH1-HR-CH2-CH3) in the N-terminal to C-terminal direction; and optionally, antibody heavy chain constant domain 4(CH4) (in the case of antibodies of subclass IgE). 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. "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) in the N-terminal to C-terminal direction, abbreviated VL-CL. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). The two full-length antibody chains are linked together via interpoly 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 (chimeric) or humanized antibodies. A full-length antibody according to the invention comprises two antigen-binding sites, each formed by a pair of VH and VL, which both specifically bind to the same antigen. The C-terminus of the heavy or light chain of the full-length antibody refers to the last amino acid of 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 of the N-terminus of the heavy or light chain.

As used within the present invention, the term "peptide linker" 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 the C-terminus of the light chain to the N-terminus of the heavy chain of a second full-length antibody (which specifically binds to a second antigen) via a peptide linker. The peptide linkers within the second full length antibody heavy and light chains are peptides having an amino acid sequence of at least 30 amino acids in length, preferably 32 to 50 amino acids in length. In one embodiment, the linker is a peptide having an amino acid sequence of 32 to 40 amino acids in length. 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 x =4, n =6 or 7 and m =0, 1, 2 or 3, more preferably x =4, n =7 and m =2. In one embodiment, the linker is (G)₄S)₆G₂。

As used within this application, the term "constant region" means the sum of the domains in an antibody that differ from the variable region. The constant region is not directly involved in antigen binding, but exhibits multiple effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant region into categories: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses, such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA 2. The heavy chain constant regions corresponding to different classes of antibodies are referred to as α, γ, and μ, respectively. The light chain constant regions that can be found in all 5 antibody classes are called kappa (kappa) and lambda (lambda).

As used herein, the term "constant region derived from human origin" means the heavy chain constant region and/or the light chain kappa or lambda constant 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-.

Preferably, the bispecific bivalent antibody according to the present invention has a constant region of the subclass human IgG 1.

While antibodies of the IgG4 subclass showed reduced Fc receptor (FcgRIIIa) binding, antibodies of other IgG subclasses showed binding. Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues which when altered also provide reduced Fc receptor binding (Shields, R.L. et al, J.biol.chem.276(2001)6591-6604; Lund, J.et al, FASJ.9 (1995)115-119; Morgan, A. et al, 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, whereas the bispecific diabody is of the subclass IgG4 or of the subclass IgG1 with mutations in S228, L234, L235 and/or D265 with respect to FcR binding and/or contains a PVA236 mutation. In one embodiment, the mutation in the full-length parent antibody is S228P, L234A, L235A, L235E, and/or PVA 236. In another embodiment, the mutations in the bispecific bivalent antibody are in IgG 4S 228P and L235E and in IgG1L234A and L235A.

In another aspect of the invention, a bispecific bivalent antibody is characterized by comprising

a) A heavy chain and a light chain of a first full-length antibody that specifically binds a first antigen;

b) a heavy chain and a light chain of a second full-length antibody that specifically binds a second antigen,

In which the variable domains VL and VH or the constant domains CL and CH1 are substituted for each other.

Preferably, the CH3 domain of this bispecific bivalent antibody format is altered by the "protrusion-entry-cavity" technique described in more detail in, for example, WO 96/027011, Ridgway J.B. et al, Protein Eng 9(1996)617-621, and Merchant, A.M. et al, Nat Biotechnol 16(1998)677-681, to name a few. In this approach, the interaction surface of the two CH3 domains is altered to enhance heterodimerization of the two heavy chains containing the two CH3 domains. The two CH3 domains (of the two heavy chains) may each be a "protuberance" and the other a "hole". Introduction of disulfide bridges stabilizes the heterodimer (Merchant, A.M et al, Nature Biotech 16(1998)677-681; Atwell, S., et al J.mol.biol.270(1997)26-35) and increases yield. For further details and embodiments, see above.

Wherein the variable domains VL and VH are substituted for each other.

An exemplary scheme of this bispecific bivalent antibody, which includes a CH3 domain modified by a protuberance-into-cavity, is shown in figure 2 b. An antibody based on this bispecific bivalent antibody format is named in the examples as oascfab 1.

In one embodiment, such bispecific antibodies are characterized by comprising

wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker; and wherein the constant domains CL and CH1 replace each other.

An exemplary scheme of this bispecific diabody format antibody is shown in figure 2c, which includes a protuberance-into-cavity modified CH3 domain. Antibodies based on this bispecific bivalent antibody format are designated in the examples as oascxmab 2 and oascxmab 3.

In one embodiment, such bispecific antibodies are characterized by comprising

The antibodies according to the invention are generated by recombinant means. Thus, one aspect of the invention is a nucleic acid encoding an antibody according to the invention, and yet another aspect is a cell comprising said nucleic acid encoding an antibody according to the invention. Methods for recombinant production are generally known in the art and include protein expression in prokaryotic and eukaryotic cells, followed by isolation of the antibody and usually purification to a pharmaceutically acceptable purity. For expression of the antibody in a host cell as described above, the nucleic acids encoding each of the modified light and heavy chains are inserted into an 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 review articles Makrides, S.C., Protein Expr. Purif.17(1999)183- "202; Geisse, S.et al, Protein Expr. Purif.8(1996) 271-" 282; Kaufman, R.J., mol. Biotechnol.16(2000)151- "160; Werner, R.G., Drug Res.48(1998) 870-" 880).

Thus, one embodiment of the invention is a method for preparing a bispecific antibody according to the invention, comprising the following steps:

a) transforming a host cell with a vector comprising a nucleic acid molecule encoding the antibody;

b) culturing said host cell under conditions that allow synthesis of said antibody molecule; and are

c) Recovering the antibody molecule from the culture.

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

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 can only be carried out in a very limited range. 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.

As used herein, the term "host cell" means any kind of cellular system that can be engineered to produce an antibody according to the 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 such designations include progeny. As such, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of deliveries. It is also understood that all progeny may not be exactly identical in DNA content due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

For example, Barnes, L.M. et al, Cytotechnology 32(2000)109-123; Barnes, L.M. et al, Biotech.Bioeng.73(2001)261-270 describe expression in NS0 cells. Transient expression is described, for example, by Durocher, Y., et al, Nucl. acids. Res.30(2002) E9. Orlandi, R.et al, Proc.Natl.Acad.Sci.USA86(1989) 3833-. A preferred transient expression system (HEK 293) is described by Schlaeger, E.J. and Christensen, K., in Cytotechnology 30(1999)71-83 and Schlaeger, E.J., in J.Immunol. methods 194(1996) 191-199.

Suitable control sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

A nucleic acid is "operably linked" when it is 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 if 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 if it affects the transcription of the sequence; or operably linked to a coding sequence if the ribosome binding site is positioned so as to facilitate translation. In general, "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 such sites are not present, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

Antibody purification 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 banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See, Ausubel, F. et al, 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, azaarene resin (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 (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, m.a., appl.biochem.biotech.75(1998) 93-102).

It has now been found that bispecific antibodies against human VEGF and human ANG-2 according to the invention have valuable features, such as high stability and valuable pharmacokinetic/pharmacodynamic properties, e.g. good (i.e. slow) clearance (e.g. at low doses).

The bispecific bivalent antibody according to the invention shows benefits for human patients in need of VEGF and ANG-2 targeted therapy.

Furthermore, they have biological or pharmacological activity and show in vivo inhibition of tumor growth and/or inhibition of tumor angiogenesis.

The bispecific antibody according to the present invention is highly effective for

a) Tumor growth inhibition (e.g., tumor arrest has been achieved at lower concentrations with a bispecific antibody according to the invention compared to a combination of two monospecific antibodies (e.g., tumor arrest has been achieved with 10mg/kg of XMAb1 compared to a combination of 10mg/kg of ANG2i-LC06+10mg/kg of avastin in COLO205 and KPL-4 tumor models of examples 9 and 10), and/or

b) Inhibition of tumor angiogenesis or vascular disease (e.g., the maximal anti-angiogenic effect has been achieved with a bispecific antibody according to the invention at a lower concentration compared to the combination of two monospecific antibodies (e.g., the maximal anti-angiogenic effect has been achieved with 10mg/kg of XMAb1 compared to the combination of 10mg/kg of ANG2i-LC06+10mg/kg of avastin in the mouse corneal angiogenesis assay of example 8).

Finally, the bivalent bispecific against human VEGF and human ANG-2 according to the invention may have a valuable efficacy/toxicity profile and may provide benefits to patients in need of anti-VEGF and anti-ANG-2 therapy.

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 manufacture of a pharmaceutical composition. Yet another aspect of the invention is a method of manufacturing 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 the 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 manufacture 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.

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

The present invention includes bispecific antibodies according to the invention for use in the prevention of metastasis.

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

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

We can show highly effective prevention of spontaneous metastasis/secondary tumors in vivo in orthotopic (orthotic) and subcutaneous cancer models (see example 9) (in contrast to experimental models of i.v. injection of tumor cells, this is similar to the clinical situation where cells spread from a primary tumor and metastasize to a secondary organ, such as the lung or liver (secondary tumor).

The term "metastasis" in accordance with the present invention refers to the transmission of cancerous cells from a primary tumor elsewhere in a patient where secondary tumors form at one or more sites. Means of determining whether a cancer has metastasized are known in the art and include bone scans, chest X-rays (chest X-ray), CAT scans, MRI scans, and tumor marker tests.

As used herein, the terms "prevent metastasis" or "prevent secondary tumor" have the same meaning and refer to a prophylactic agent against metastasis in a patient with cancer in such a way as to inhibit or reduce further transport of cancerous cells from the primary tumor to one or more sites elsewhere in the patient. This means that metastasis of the primary tumor or cancer is prevented, delayed or reduced, and thus the formation of secondary tumors is prevented, delayed or reduced. Preferably, lung metastasis, i.e. secondary tumors, is prevented or reduced, which means that metastatic transmission of cancerous cells from the primary tumor to the lung is prevented or reduced.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption 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 the skilled artisan will appreciate, the route and/or pattern of administration will vary with the desired result. In order to administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with a material or to co-administer the compound with a material to prevent its inactivation. For example, the compounds can 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 in situ preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

As used herein, the term cancer refers to a proliferative Disease such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer (bronchoolliololalecular cancer), bone cancer, pancreatic cancer, skin cancer, head and neck cancer (cancer of the head or the kidney), skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region (cancer of the anal region), stomach cancer (stomach cancer), stomach cancer (gastric cancer), colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes (carcinoma of the villopacities), endometrial cancer (carcinoma of the endometrium), cervical cancer (carcinoma of the esophagus), vaginal cancer (carcinoma of the esophagus), carcinoma of the vulva (carcinoma of the parathyroid), carcinoma of the thyroid, carcinoma of the adrenal gland, carcinoma of the thyroid, adrenal gland carcinoma, thyroid carcinoma, bladder carcinoma of the head carcinoma, bladder carcinoma of the head, Soft tissue sarcoma, urethral carcinoma, penile carcinoma, prostate cancer, bladder cancer, renal or ureteral cancer, renal cell carcinoma, carcinoma of the renal pelvis (carcinoma of the renal pellis), mesothelioma, hepatocellular carcinoma, cholangiocarcinoma (biliary cancer), Central Nervous System (CNS) neoplasms, spinal axis tumors (spinal axis tumors), brain stem gliomas, glioblastoma multiforme (glioblastomas), astrocytomas (astrocytoma), schwanomas (schwanomas), ependymomas (ependomoma), medulloblastomas (medulloblastomas), meningiomas (meniginoma), squamous cell carcinoma (squamous carcinoma), pituitary adenomas (pituitary adenomas), and ewingsis sarcoma, including refractory comas of any of the above cancers, or a combination of one or more of the above 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 can be used to treat 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 manufacture 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 an antibody according to the invention to a patient in need of such treatment.

The term "Vascular disease" includes cancer, inflammatory diseases, Atherosclerosis (Atherosclerosis), Ischemia (Ischemia), trauma, sepsis, COPD, asthma, diabetes, AMD, retinopathy, stroke, obesity, acute lung injury, hemorrhage, Vascular leakage (Vascaleak), e.g., cytokine-induced, allergy, Graves ' disease, Hashimoto's autoimmune thyroiditis, idiopathic thrombocytopenic purpura, giant cell arteritis, rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), lupus nephritis, Crohn's disease, multiple sclerosis, ulcerative colitis, particularly of 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.267(1992)10931-, Annu.Rev.Physiol.53(1991) 217-: pathiology of annular disease, A dynamic approach, Garner, A. and Klintworth, G.K. (eds.), second edition, Marcel Dekker, New York (1994), pp.1625-1710).

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

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

The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response, composition, and mode of administration for a particular patient, yet is non-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, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

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

Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferred to include isotonic agents (e.g., sugars, polyols 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 such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be exactly identical in DNA content due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a unique name is intended, it may be evident from the context.

As used herein, the term "transformation" refers to the process of transferring a vector/nucleic acid into a host cell. If cells without an intractable 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(1973) 546-467. However, other methods for introducing DNA into cells may also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells containing rigid cell wall structures are used, one method of transfection is, for example, calcium treatment with calcium chloride, as described by Cohen, S.N. et al, PNAS.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 called 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 the eukaryotic cell may include splicing of mRNA.

"vector" refers to a nucleic acid molecule, particularly self-replicating, that transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily in the insertion of DNA or RNA into a cell (e.g., chromosomal integration), replicating vectors that function primarily in the replication of DNA or RNA, and expression vectors that function in the transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one function, as described.

An "expression vector" refers to a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" generally refers to a suitable host cell containing an expression vector that can function to produce a desired expression product.

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

Description of the sequence listing (amino acid sequence)

1< VEGF > heavy chain variable domain VH of bevacizumab of SEQ ID NO

2< VEGF > light chain variable domain VL of bevacizumab of SEQ ID NO

3< ANG-2> E6Q heavy chain variable domain VH

4< ANG-2> E6Q light chain variable domain VL

SEQ ID NO 5 XMab1- < VEGF > light chain

6 XMab1- < ANG2> light chain

7 XMab1- < VEGF > heavy chain of SEQ ID NO

8 XMab1- < ANG2> heavy chain of SEQ ID NO

9 XMab2- < VEGF > light chain

10 XMab2- < ANG2> light chain

11 XMab2- < VEGF > heavy chain of SEQ ID NO

12 XMab2- < ANG2> heavy chain of SEQ ID NO

13 XMab3- < VEGF > light chain

14 XMab3- < ANG2> light chain

15 XMab3- < VEGF > heavy chain of SEQ ID NO

16 XMab3- < ANG2> heavy chain of SEQ ID NO

17 XMab4- < VEGF > light chain

18 XMab4- < ANG2> light chain of SEQ ID NO

19 XMab4- < VEGF > heavy chain of SEQ ID NO

20 XMab4- < ANG2> heavy chain of SEQ ID NO

21 XMab5- < VEGF > light chain

22 XMab5- < ANG2> light chain

23 XMab5- < VEGF > heavy chain of SEQ ID NO

24 XMab5- < ANG2> heavy chain of SEQ ID NO

25 XMab6- < VEGF > light chain of SEQ ID NO

26 XMab6- < ANG2> light chain

27 XMab6- < VEGF > heavy chain of SEQ ID NO

28 XMab6- < ANG2> heavy chain of SEQ ID NO

29 OAscFab 1-peptide Linked < ANG2> heavy and light chains

30 OAscFab1- < VEGF > heavy chain

31 OAscFab1- < VEGF > light chain

32 OAscFab 2-peptide Linked < ANG2> heavy and light chains

33 OAscFab2- < VEGF > heavy chain

SEQ ID NO 34 OAscFab2- < VEGF > light chain

35 OAscFab 3-peptide Linked < ANG2> heavy and light chains

36 OAscFab3- < VEGF > heavy chain of SEQ ID NO

37 OAscFab3- < VEGF > light chain

38 OAscXFab 1-peptide Linked < ANG2> heavy and light chains

39 OAscXtab 1- < VEGF > heavy chain of SEQ ID NO

SEQ ID NO 40 OAscXtab 1- < VEGF > light chain

41 OAscXFab 2-peptide Linked < ANG2> heavy and light chains

42 OAscXtab 2- < VEGF > heavy chain of SEQ ID NO

43 OAscXtab 2- < VEGF > light chain of SEQ ID NO

44 OAscXFab 3-peptide Linked < ANG2> heavy and light chains

SEQ ID NO 45 OAscXtab 3- < VEGF > heavy chain

46 OAscXFab3- < VEGF > light chain of SEQ ID NO

47 human Vascular Endothelial Growth Factor (VEGF) SEQ ID NO

Human angiopoietin-2 (ANG-2) of SEQ ID NO 48

Examples

Materials and general methods

For general information on the nucleotide sequences of human immunoglobulin light and heavy chains see: kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health service, National Institutes of Health, Bethesda, Md. (1991). The amino acids of the antibody chain are numbered and referred to according to EU numbering (Edelman, G.M. et al, Proc. Natl.Acad. Sci. USA 63(1969)78-85; Kabat, E.A. et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national institutes of Health, Bethesda, Md. (1991)).

Recombinant DNA technology

The DNA is manipulated using standard procedures, such as those described in Sambrook, J.et al, Molecular cloning, Alaberration manual, 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 segment can be prepared from oligonucleotides generated by chemical synthesis. Gene segments flanked by single restriction endonuclease cleavage sites are assembled by annealing and ligation of oligonucleotides, including PCR amplification, followed by cloning into pcrscript (stratagene) -based pGA4 cloning vectors via designated restriction sites, such as 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 given specifications in Geneart (Regensburg, Germany). All gene segments encoding the light and heavy chains of the Ang-2/VEGF bispecific antibody are synthesized with a 5 ' DNA sequence encoding a leader peptide (MGWSCIILFLVATATGVHS) that targets secretion of the protein in eukaryotic cells, and unique restriction sites at the 5 ' and 3 ' ends of the synthesized gene. The DNA sequence of the heavy chain modified with "protuberance-entry-cavity" carrying disulfide stabilization was designed with S354C and T366W in the "protuberance" heavy chain and Y349C, T366S, L368A and Y407V mutations in the "cavity" heavy chain.

DNA sequencing

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

DNA and protein sequence analysis and sequence data management

Sequence creation, localization, analysis, annotation, and instantiation were performed using the GCG (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Vector NT1 advanced suite version 8.0 of Infmax.

Expression vector

For the expression of the described antibodies, variants of expression plasmids for transient expression (e.g. in HEK293EBNA or HEK293-F cells) or for stable expression (e.g. in CHO cells) are applied, which are based on cDNA constructs with a CMV-intron a promoter or on genomic constructs with a CMV promoter (see fig. 2B).

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

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

-a beta-lactamase gene conferring ampicillin resistance in E.coli. The transcription unit of the antibody gene is composed of the following elements:

unique restriction sites at the 5' end

Immediate early enhancer and promoter from human cytomegalovirus,

in the case of cDNA construction, 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 chains) as cDNA or as genomic construct with an immunoglobulin exon-intron construct

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

a unique restriction site at the 3' end.

For transient and stable transfection, larger quantities of plasmid (Nucleobond AX, Macherey-Nagel) were prepared by plasmid preparations from transformed e.

Cell culture technique

Standard Cell culture techniques are used, such as those described in Current Protocols in Cell Biology (2000), Bonifacino, j.s., Dasso, m., Harford, j.b., Lippincott-Schwartz, j.and Yamada, K.M (eds.), John Wiley & Sons, Inc.

Transient transfection in the HEK293-F System

FreeStyle was used according to the manufacturer's instructions (Invitrogen, USA)^TM293 expression system expression of recombinant immunoglobulin variants by transient transfection of human embryonic kidney 293-F cells. Briefly, at 37 ℃ C/8% CO₂In FreeStyle^TMCultivation of suspended FreeStyle in 293 expression Medium^TM293-F cells, and the cells were transfected at 1-2X10⁶The density of individual viable cells/ml was inoculated in fresh medium. For a final transfection volume of 250ml for monospecific parent antibody 325. mu.l 293fectin was used in a 1:1 molar ratio^TM(Invitrogen, Germany) and 250. mu.g of heavy and light chain plasmid DNAPreparation of DNA-293fectin I Medium (Invitrogen, USA)^TMAnd (c) a complex. For a final transfection volume of 250ml (OAscFab and OAscXFab), 325. mu.l of 293fectin are generally used in a 1:1 molar ratio^TM(Invitrogen, Germany) and 250. mu.g of "protrusion-entry-hole" heavy chain 1 and 2 and light chain plasmid DNA"protrusion-entry-hole" DNA-293fectin with two heavy chains and one light chain was prepared in I Medium (Invitrogen, USA)^TMAnd (c) a complex. The ratio can be varied for expression yield optimization. For a final volume of 250ml transfection, 325. mu.l of 293fectin was used at a 1:1:1:1 molar ratio^TM(Invitrogen, Germany) and 250. mu.g of "protrusion-entry-hole" heavy chain 1 and 2 and light chain plasmid DNAXMabDNA-293fectin complex was prepared in I medium (Invitrogen, USA). The ratio can be varied for expression yield optimization. Cell culture supernatants containing the antibodies were harvested 7 days post-transfection by centrifugation at 14000g for 30 minutes and filtered through sterile filters (0.22 μm). The supernatant was stored at-20 ℃ until purification.

Protein assay

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

Determination of antibody concentration in supernatant

The concentration of antibodies and derivatives in cell culture supernatants was assessed by immunoprecipitation with protein a agarose beads (Roche). mu.L of protein A agarose beads were washed three times in TBS-NP40(50mM Tris, pH7.5,150mM NaCl,1% Nonidet-P40). Subsequently, 1-15mL of cell culture supernatant was applied to protein a agarose beads pre-equilibrated in TBS-NP 40. After 1 hour incubation at room temperature, the beads were applied to an Ultrafree-MC-Filter column (Amicon)]The column was washed once with 0.5mL of PBS-NP 40, twice with 0.5mL of 2 Xphosphate buffered saline (2xPBS, Roche), and 4 brief 0 washes with 0.5mL of 100mM sodium citrate pH 5. By adding 35. mu.lLDS sample buffer (Invitrogen) to elute bound antibody. Half of the sample was separately mixed withThe sample reducing agents were combined or left unreduced and heated at 70 ℃ for 10 minutes. Therefore, 20. mu.l was applied to 4-12%Bis-Tris SDS-PAGE (Invitrogen) (MOPS buffer for non-reducing SDS-PAGE and MOPS buffer for reducing SDS-PAGE)Antioxidant running buffer additive (Invitrogen) MES buffer) and stained with coomassie blue.

The concentration of antibodies and derivatives in the cell culture supernatant was measured by protein a-HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind protein a were applied to HiTrap protein a columns (GE Healthcare) in 50mM k2hpo4,300mm NaCl, ph7.3 on a Dionex HPLC system and eluted from the matrix with 550mM citric acid, ph 2.5. Eluted protein was quantified by UV absorbance and peak area integration. Purified standard IgG1 antibody served as standard.

Alternatively, the concentration of antibodies and derivatives in the cell culture supernatant was measured by sandwich-IgG-ELISA. Briefly, StreptaWell high binding streptavidin a-96 well microtiter plates (Roche) were coated with 100 μ L/well of 0.1 μ g/mL biotinylated anti-human IgG capture molecule F (ab') 2< h-fcy > bi (dianova), either at room temperature for 1 hour or alternatively overnight at 4 ℃, followed by three washes with 200 μ L/well PBS, 0.05% Tween (PBST, Sigma). Dilution series of 100 μ L/well each antibody-containing cell culture supernatant in pbs (sigma) were added to the wells and incubated on a microtiter plate shaker at room temperature for 1-2 hours. The wells were washed three times with 200 μ L/well PBST and bound antibody was detected on a microtiter plate shaker at room temperature for 1-2 hours with 100 μ L of 0.1 μ g/mL F (ab') 2< hFc γ > POD (Dianova) as the detection antibody. Unbound detection antibody was washed off three times with 200 μ Ι/well PBST and bound detection antibody was detected by adding 100 μ Ι ABTS/well. The absorbance measurements were carried out on a Tecan Fluor spectrometer at a measurement wavelength of 405nm (reference wavelength 492 nm).

Purification of bispecific antibodies

By using protein A-Sepharose^TMAffinity chromatography and Superdex200 size exclusion chromatography of (GE Healthcare, Sweden) bispecific antibodies were purified from cell culture supernatants. Briefly, sterile filtered cell culture supernatant was applied to a buffer solution with PBS (10mM Na)₂HPO₄,1mMKH₂PO₄137mM NaCl and 2.7mM KCl, pH7.4) on a HiTrap protein A HP (5ml) column. Unbound protein was washed away with equilibration buffer. The antibodies and antibody variants were eluted with 0.1M citrate buffer, pH2.8 and the protein containing fractions were neutralized with 0.1ml 1M Tris, pH 8.5. The eluted protein fractions were then combined, concentrated to a 3ml volume using an Amicon Ultra centrifugal filter device (MWCO:30K, Millipore) and loaded on a Superdex200HiLoad 120ml 16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20mM histidine, 140mM NaCl, pH 6.0. Fractions containing purified bispecific antibody with less than 5% high molecular weight aggregates were combined and stored at-80 ℃ in 1.0mg/ml aliquots.

SDS-PAGE

Used according to the instructions of the manufacturerPre-gel systems (Invitrogen). Specifically, 4-20% of the total weight of the composition is usedTRIS-Glycine precast gels andTRIS-glycine SDS running buffer (see, e.g., fig. 3). By addingThe sample reducing agent effects reduction of the sample, after which the gel is run.

Size exclusion chromatography for analysis

Size exclusion chromatography for determination of the aggregation and oligomerization status of the antibodies was performed by HPLC chromatography. Briefly, protein A purified antibody was applied to an Agilent HPLC 1100 system at 300mM NaCl, 50mM KH₂PO₄/K₂HPO₄Tosoh TSKgel G3000SW column at pH7.5, or Superdex200 column in 2 × PBS on a Dionex HPLC system (GE Healthcare). Eluted protein was quantified by UV absorbance and peak area fractions. The BioRad gel filtration standard 151-.

Mass spectrometry

The total deglycosylation mass of the exchanged antibody was determined and confirmed via electrospray ionization mass spectrometry (ESI-MS). Briefly, 100. mu.g of purified antibody was applied at 50mU at 100mM KH₂PO₄/K₂HPO₄N-glycosidase F (PNGaseF, ProZyme) in pH7 was deglycosylated at 37 ℃ for 12-24 hours at protein concentrations up to 2mg/ml, followed by desalting via HPLC on a Sephadex G25 column (GE Healthcare). After deglycosylation and reduction, the respective masses of the heavy and light chains were determined by ESI-MS. 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, followed by desalting. The total mass and the mass of the reduced heavy and light chains were determined via ESI-MS on a Q-Star Elite MS system equipped with a NanoMate source.

Generation of HEK293-Tie2 cell line

To determine the interference of angiopoietin-2 antibodies on ANGPT 2-stimulated Tie2 phosphorylation and the binding of ANGPT2 to Tie2 on cells, a recombinant HEK293-Tie cell line was generated. Briefly, a pcDNA 3-based plasmid (RB22-pcDNA3Topo hTie2) encoding full-length human Tie2(seq id 108) under the control of the CMV promoter and neomycin resistance marker (seq id 108) was transfected into HEK293 cells (ATCC) using fugene (roche applied science) as transfection reagent and resistant cells were selected in DMEM 10% FCS,500 μ G/ml G418. Individual clones were isolated via cloning 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). Subsequently, cell assays were performed using HEK293-Tie2 clone 22: ANGPT 2-induced Tie2 phosphorylation and ANGPT2 cell ligand binding assays.

ANGPT 2-induced Tie2 phosphorylation assay

The 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 quantified by sandwich ELISA. Briefly, 2 × 10 per well⁵The individual HEK293-Tie2 clone 22 cells were cultured overnight in 100. mu.l DMEM, 10% FCS, 500. mu.g/ml geneticin poly-D-lysine coated 96-well microtiter plates. The following day, titration lines of ANGPT2 antibody (4-fold concentrated, 75 μ Ι final volume/well in duplicate) were prepared in microtiter plates and were combined with 75 μ Ι ANGPT2 (R)&D systems#623-AN]Dilutions (3.2. mu.g/ml as 4-fold concentrated solution) were mixed. The antibody and ANGPT2 were preincubated for 15 minutes at room temperature. Mu.l of the mixture was added to HEK293-Tie2 clone 22 cells (with 1mM NaV)₃O₄Sigma # S6508 pre-incubated for 5 minutes) and incubated at 37 ℃ for 5 minutes. Subsequently, cells were plated with 200. mu.l of ice-cold PBS +1mM NaV per well₃O₄Washed and lysed by adding 120. mu.l lysis buffer (20mM Tris, pH8.0, 137mM NaCl,1% NP-40, 10% glycerol, 2mM EDTA, 1mM NaV) per well on ice₃O₄1mM PMSF and 10. mu.g/ml Aprotinin (Aprotinin)). Cells were lysed at 4 ℃ for 30 min on a microtiter plate shaker, and 100. mu.l of the lysate were transferred directly into p-Tie2ELISA microtiter plates (R) without prior centrifugation and without total protein determination&D Systems,R&D # DY 990). The amount of P-Tie2 was quantified according to manufacturer's instructions and used ExcXLfit4 of el analyzes the plug-in program (dose-response one-site, 205 model) to determine IC50 values for inhibition. IC50 values may be compared within one experiment, but there may be variations between experiments.

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, 5000 HUVEC cells per 96 wells (low passage number, 5 passages or less) were incubated in collagen I coated BD Biocoat collagen I96 well microtiter plates (BD #354407/35640) overnight in 100. mu.l starvation medium (EBM-2 endothelial basal medium 2, Promocell # C-22211,0.5% FCS, penicillin/streptomycin). Varying 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 ℃,5% CO₂Incubate for 72 hours. On the day of analysis, the plates were equilibrated to room temperature for 30 minutes and cell viability/proliferation was determined using the CellTiter-GloTM luminescent cell viability assay kit according to the manual (Promega, # G7571/2/3). Luminescence was measured in a spectrometer.

Example 1a

Expression and purification of bispecific, bivalent domain-exchanged < VEGF-ANG-2> antibody molecule Xmab

Bispecific, bivalent domain-exchanged < VEGF-ANG-2> antibody molecules XMab1, XMab2, and XMab3 were expressed and purified according to the procedures described in the materials and methods above. < VEGF > the VH and VL of the moieties (SEQ ID NO:1 and SEQ ID NO:2) are based on bevacizumab. The VH (SEQ ID NO:3) of the < ANG2> portion was derived by the E6Q mutation (substitution of the initial amino acid glutamic acid (E) at position 6 with glutamine (Q)) of the VH sequence of ANG2i-LC06, which is described in PCT application No. PCT/EP2009/007182(WO2010/040508) and which is a further mature sequence fragment obtained via phage display. The VL (SEQ ID NO:4) of the < ANG2> portion is derived from the VL sequence of ANG2i-LC06 (see PCT application No. PCT/EP2009/007182(WO 2010/040508)). Expression and purification of bispecific, bivalent domain-exchanged < VEGF-ANG-2> antibody molecules XMab1, XMab2 and XMab 3. The relevant light and heavy chain amino acid sequences of these bispecific diabodies are given in SEQ ID NO:5-8(XMab1), SEQ ID NO:9-12(XMab2) and SEQ ID NO:13-16(XMab 3). For an exemplary structure, see fig. 1.

Similarly, the bispecific, bivalent < VEGF-ANG-2> antibodies XMab4, XMab5, and XMab6 (relevant light and heavy chain amino acid sequences are given in SEQ ID NOS: 17-20(XMab4), SEQ ID NOS: 21-24(XMab5), and SEQ ID NOS: 25-28(XMab6) were expressed and purified).

Binding affinity and other properties were determined as described.

Example 1b

Expression and purification of bispecific, bivalent < VEGF-ANG-2> antibody molecule OAscFab

The bispecific, bivalent < VEGF-ANG-2> antibody molecules OAscFab1, OAscFab2, OAscFab3 were expressed and purified according to the procedures described in the materials and methods above. < VEGF > the VH and VL of the portions (SEQ ID NO:1 and SEQ ID NO:2) are based on bevacizumab. < ANG2> the VH of part E6Q (SEQ ID NO:3) was derived by the E6Q mutation (substitution of the initial amino acid glutamic acid (E) at position 6 with glutamine (Q)) of the VH sequence of ANG2i-LC06, which is described in PCT application No. PCT/EP2009/007182(WO2010/040508), and which is a further mature sequence fragment obtained via phage display. < ANG2> VL (SEQ ID NO:4) of the E6Q portion was derived from the VL sequence of ANG2i-LC06 (see PCT application No. PCT/EP2009/007182(WO 2010/040508)). The relevant light and heavy chain amino acid sequences of these bispecific diabodies are given in SEQ ID NO:29-31(OAscFab1), SEQ ID NO:32-34(OAscFab2) and SEQ ID NO:35-37(OAscFab 3). For an exemplary structure, see fig. 2 a. Expression of OAscFab1, OAscFab2 and OAscFab3 was confirmed by Western blotting. Purification of OAscFab2 and OAscFab3 yielded the following yields.

Binding affinity and other properties were determined as described.

Example 1c

Expression and purification of bispecific, bivalent domain-exchanged < VEGF-ANG-2> antibody molecule OAscXFab

Bispecific, bivalent domain exchanged < VEGF-ANG-2> antibody molecules oascfab1, oascfab2, oascfab3 were expressed and purified according to the procedures described in the materials and methods above. < VEGF > the VH and VL of the portions (SEQ ID NO:1 and SEQ ID NO:2) are based on bevacizumab. < ANG2> the VH of part E6Q (SEQ ID NO:3) was derived by the E6Q mutation (substitution of the initial amino acid glutamic acid (E) at position 6 with glutamine (Q)) of the VH sequence of ANG2i-LC06, which is described in PCT application No. PCT/EP2009/007182(WO2010/040508), and which is a further mature sequence fragment obtained via phage display. < ANG2> VL (SEQ ID NO:4) of the E6Q portion was derived from the VL sequence of ANG2i-LC06 (see PCT application No. PCT/EP2009/007182(WO 2010/040508)). The relevant light and heavy chain amino acid sequences of these bispecific diabodies are given in SEQ ID NO:38-40(OAscXFab1), SEQ ID NO:41-43(OAscXFab2) and SEQ ID NO:44-46(OAscXFab 3). For exemplary structures, see fig. 2b (oascftab 1) and fig. 2c (oascftab 2, oascftab 3). Expression was confirmed by Western blot.

Key data	OAscXFab1	OAscXFab2	OAscXFab3
				Expression (yield)	23μg/mL	23μg/mL	26μg/mL

Binding affinity and other properties were determined as described.

Example 2

Stability of bispecific antibodies

Denaturation temperature (SYPRO orange method)

To determine the temperature at which protein denaturation (i.e. temperature-induced loss of protein structure) occurs, a method is used that relies on a hydrophobic fluorescent dye (SYPRO orange, Invitrogen) that exhibits strong fluorescence in a hydrophobic environment. After denaturation of the protein, the hydrophobic plaque becomes exposed to the solvent, resulting in increased fluorescence. At temperatures above the denaturation temperature, the fluorescence intensity decreases again, so the temperature at which the maximum intensity is reached is defined as the denaturation temperature. The method is described by Ericsson, U.B. et al, AnalBiochem 357(2006)289-298 and He, F. et al, Journal of Pharmaceutical Sciences 99(2010) 1707-1720.

Protein samples at a concentration of about 1mg/mL in 20mM His/HisCl, 140mM NaCl, pH6.0 were mixed with SYPRO orange (5000X stock solution) to achieve a final dilution of 1: 5000. Transfer 20 μ L volume to 384 well plates andtemperature dependent fluorescence was recorded in a 480 real-time PCR system (Roche applied sciences) at a heating rate of 0.36 ℃ per min.

Aggregation temperature by Dynamic Light Scattering (DLS)

The temperature at which heat-induced protein aggregation occurs was determined by Dynamic Light Scattering (DLS). DLS yields information about the size distribution of macromolecules in solution, which is derived from fluctuations in scattered light intensity on the order of microseconds. As the sample is gradually heated, aggregation begins at a certain temperature, resulting in a growing particle size. The temperature at which the particle size begins to increase is defined as the aggregation temperature. The aggregation and denaturation temperatures need not necessarily be the same, as denaturation may not necessarily be a prerequisite for aggregation.

For the aggregation temperature measurement, a DynaPro DLS plate reader (Wyatt technologies) was used. Before measurement, the samples were filtered through 384-well filter plates (Millipore MultiScreen 384-well filtration system, 0.45 μm) into optical 384-well plates (Corning # 3540). A sample volume of 35 μ Ι _ at a protein concentration of about 1mg/mL was used in formulation buffer (20mM citrate, 180mM sucrose, 20mM arginine, 0.02% polysorbate 20). Each well was covered with 20 μ L of paraffin oil (Sigma) to avoid evaporation. The samples were heated from 25 ℃ to 80 ℃ at a rate of 0.05 ℃ per minute and DLS data were obtained continuously for a maximum number of 15 samples per run.

Aggregation Rate according to DLS

DLS is a sensitive method for detecting aggregates of macromolecules in solution because the aggregates produce a strong light scattering signal. Thus, the tendency of the molecules to aggregate can be followed over time by repeated collection of DLS data. To accelerate the potential aggregation to the actual rate, measurements were made at 50 ℃.

Sample preparation was performed as described above. DLS data were recorded for up to 100 hours. The aggregation rate (nm/day) was calculated as the slope of a linear fit of the mean diameter over time.

Stability in formulation buffer

To assess the stability of the bispecific molecule with respect to aggregation/fragmentation, the samples were incubated in formulation buffer (20mM citrate, 180mM sucrose, 20mM arginine, 0.02% polysorbate 20) at a protein concentration of about 1mg/mL at 40 ℃ for 3 weeks. The control samples were stored at-80 ℃ for 3 weeks.

Size exclusion chromatography for quantification of aggregates and Low Molecular Weight (LMW) species was performed by HPLC. An amount of 25-100. mu.g of protein was applied to a Tosoh TSKgel G3000SWXL column in 300mM NaCl, 50mM potassium phosphate, pH7.5 on an Ultimate3000 HPLC system (Dionex). Eluted proteins were quantified by UV absorbance at 280 nm.

As a result:

example 3

Binding characteristics of bispecific antibody < VEGF-Ang-2>

A) Binding characteristics characterized by Surface Plasmon Resonance (SPR) analysis

Simultaneous binding to both antigens was confirmed by application of Surface Plasmon Resonance (SPR) using a BIAcore T100 instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). VEGF was immobilized to the CM5 sensor chip using standard amine coupling chemistry. In a first step, < VEGF-Ang-2> XMAb was injected at 25 ℃ at a concentration of 10. mu.g/ml in HBS buffer (10mM HEPES,150mM NaCl,0.05% Tween 20, pH 7.4). After the antibody binds to the immobilized VEGF, hAng-2 was injected at 10. mu.g/ml in the second step (FIG. 3).

In further experiments, the affinity and binding kinetics of < VEGF-Ang-2> Xmab were determined. Briefly, goat < hIgG-Fc γ > polyclonal antibodies were immobilized via amine coupling on CM4 chips to present bispecific antibodies against Ang-2 and VEGF. Binding was measured in HBS buffer at 25 ℃ or 37 ℃. Purified Ang-2-His (R & D Systems or purified from scratch) or VEGF (R & DSystems or purified from scratch) was added to the solution at various concentrations between 0.37nM and 30nM or between 3.7nM and 200 nM. Binding was measured by 3 min injection; dissociation was measured by washing the chip surface with HBS buffer for 10 min, and KD values were estimated using a 1:1 Langmuir (Langmuir) binding model. Due to the heterogeneity of the Ang-2 preparation, no 1:1 binding could be observed. Thus, the KD values are apparent values. The measured affinity of < VEGF-Ang-2> Xmab for VEGF was extremely high, and the calculated off-rate was outside the Biacore specification even at 37 ℃. In table 1, the binding constants for the two antigens are summarized.

Table 1: kinetic parameters for VEGF-Ang-2> XMab1 binding to Ang-2 and VEGF

Analyte	Apparent ka (1/Ms)	Apparent kd (1/s)	Apparent KD (M)
				Ang-2	2.7E+06	6.3E-04	2.4E-10
VEGF	1.2E+05	<1E-06	<1E-10

B) Assay for quantifying the binding activity bispecific < Ang2/VEGF > XMab1

As an alternative to SPR analysis, an ELISA was set up to quantify the binding activity of bispecific mAbs<Ang2/VEGF>The amount of antibody. In this assay, hAng2 was coated directly to the wells of a Maxisorp microtiter plate (MTP) in a first step. At the same time, the sample/reference standard (monoclonal antibody)<Ang2/VEGF>) In wells of another MTP were preincubated with digoxigenylated (digoxigenylated) VEGF. After pre-incubation and coating, excess unbound Ang2 was removed by washing the Ang 2-coated MTP. Then, will<Ang2/VEGF>The preincubation mixture with VEGF-Dig was transferred to hAng 2-coated MTP and incubated. After incubation, excess pre-incubation solution was removed by washing, followed by incubation with horseradish peroxidase-labeled anti-digoxigenin antibody. Antibody-enzyme conjugate catalysisColor reaction of the substrate. By ELISA reader at 405nm wavelength (reference wavelength: 490nm ([405/490 ]]nm)) measurement signals. The absorbance values for each sample were determined in duplicate. (a scheme illustrating this test system is shown in FIG. 4, while an ELISA calibration curve for quantification is shown in FIG. 5.)

Example 4

Tie2 phosphorylation

To confirm that anti-ANGPT 2 related activity was retained in the bispecific bivalent < VEGF-ANGPT2> antibody XMAb1, a Tie2 phosphorylation assay was performed. The potency of XMAb1 was determined in an ANGPT2 stimulated Tie2 phosphorylation assay as described above.

XMAb1 is shown to interfere with ANGPT2 stimulated Tie2 phosphorylation in the ANGPT2 stimulated Tie2 phosphorylation assay as described above. IC50 of XMAb1 is 7.4nM +/-2.3.

Example 5

Inhibition of huANG-2 binding 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, the plates were washed 3 times with PBST. ELISA plates were coated with 5. mu.g/ml Tie-2 protein for 1 hour (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 purified bispecific Xmab antibody in PBS were incubated with 0.2. mu.g/ml human angiopoietin-2 (R & DSsystems, UK, catalog No. 623-AN) at RT for 1 hour. After washing, a mixture of 0.5. mu.g/ml biotinylated anti-angiopoietin-2 clone BAM0981(R & D Systems, UK) and 1:3000 dilution of streptavidin HRP (Roche diagnostics GmbH, DE, Cat. catalog No. 11089153001) was added for 1 hour. Thereafter, the plates were washed 3 times with PBST. Plates were visualized with freshly prepared ABTS reagent (RocheDiagnostics GmbH, DE, buffer #204530001, tablet #11112422001) for 30 min at RT. Absorbance was measured at 405nm and IC50 was determined. XMab1 showed 12nM IC50 to inhibit ANG-2 binding to Tie-2.

Example 6

Inhibition of hVEGF binding 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, the plates were washed 3 times with PBST. At the beginning, plates were coated with 1. mu.g/ml hVEGF-R protein (R & D Systems, UK, Cat. No. 321-FL) for 1 hour (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 purified bispecific Xmab antibody in PBS were incubated for 1 hour at RT with 0.15. mu.g/ml huVEGF121(R & D Systems, UK, Cat. No. 298-VS). After washing, a mixture of 0.5. mu.g/ml anti-VEGF cloning mAb 923(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. Plates were visualized with freshly prepared ABTS reagent (Roche Diagnostics GmbH, D E, buffer #204530001, tablet #11112422001) for 30 min at RT. Absorbance was measured at 405nm and IC50 was determined. XMab1 showed inhibition of VEGF binding to VEGF receptors at 10nM IC 50.

Example 7

HUVEC proliferation

To confirm that anti-VEGF related activity was retained in the bispecific bivalent < VEGF-ANG2> antibody XMAb1, VEGF-induced HUVEC proliferation assay was performed. XMAb1 is shown to interfere with VEGF-induced HUVEC proliferation in a comparable manner to bevacizumab in the VEGF-induced HUVEC proliferation assay described above. XMAb1 interfered with VEGF-induced HUVEC proliferation in a concentration-dependent manner, comparable to the parent antibody bevacizumab (avastin). IC50 was 1.1nM (for bevacizumab) and 2.3nM (for XMAb).

Example 8

Mouse cornea micro-bag angiogenesis determination method

Female Balb/c mice 8-10 weeks old were purchased from Charles River, Sulzfeld, Germany. According to a modification of the method described by Rogers, M.S. et al, Nat Protoc 2(2007) 2545-. Briefly, a micro-bag of about 500 μm width was prepared under a microscope at about 1mm from limbus to the top using a scalpel blade and sharp forceps in anesthetized mice. Implant a disc with a diameter of 0.6 mm: (Pall corporation, Michigan) and smoothens the surface of the implanted region. Make the disc correspond toIs incubated for at least 30 minutes in the growth factor or vehicle. After 3, 5 and 7 days (or only after 3 days), the eyes were photographed and the vascular response was measured. The assay was quantified by calculating the percentage of new blood vessel area to total corneal area.

The discs were loaded with 300ng VEGF or PBS as control and implanted for 7 days. Outgrowth of vessels from the rim to the disc was monitored over time on days 3, 5 and 7. Antibodies (< Ang-2/VEGF > XMAb1, < hVEGF > avastin (bevacizumab)) were administered intravenously at a dose of 10mg/kg for avastin and XMAb 11 day prior to disc implantation. Animals in the control group received vehicle. The volume applied was 10 ml/kg.

To test the effect of XMAb1 on VEGF-induced angiogenesis in vivo, we performed a mouse corneal angiogenesis assay. In this assay, VEGF-soaked Nylaflo discs are implanted into a pocket of avascular cornea at a fixed distance from the limbal vessels. The blood vessels immediately grow into the cornea towards the developing VEGF gradient. Our results demonstrate that systemic administration of XMAb1(10mg/kg) almost completely inhibited vessel outgrowth from study day 3to day 5 towards a VEGF gradient (fig. 6). In further experiments, direct comparative studies were performed. The discs were loaded with 300ng VEGF or PBS as control and implanted for 3 days. Vessel outgrowth from the rim to the disc was monitored over time on day 3. Antibodies (bispecific < ANG-2/VEGF > antibody XMAb1, parent < VEGF > antibody bevacizumab (avastin), parent < ANG-2> antibody ANG2i-LC06, and a combination of < VEGF > antibody bevacizumab (avastin) and < ANG-2> antibody ANG2i-LC 06) were administered intravenously at a dose of 10mg/g for bevacizumab (avastin), 10mg/g for XMAb1, 10mg/kg for bevacizumab (avastin), and 10mg/kg for ANG2i-LC06 1 days before disc implantation. The combination of bevacizumab (avastin) and ANG2i-LC06 was administered at 10mg/kg for bevacizumab (avastin) and 10mg/kg for ANG2i-LC 06. Animals in the control group received vehicle. The volume applied was 10 ml/kg.

Our results (see figure 7 and table below) demonstrate that systemic administration of XMAb1(10mg/kg) almost completely inhibits vessel outgrowth towards VEGF gradient at study day 3, comparable to bevacizumab and ANG2i-LC06 combination. In contrast, anti-Ang-2 monotherapy only slightly inhibited VEGF-induced angiogenesis (fig. 7). The maximum effect can already be achieved with a lower concentration of 10mg/kg XMAb1 compared to the combination of 10mg/kg ANG2i-LC06+10mg/kg bevacizumab (avastin).

Table: percent inhibition of VEGF-induced angiogenesis on day 3 in mouse corneal micro-pocket angiogenesis assay

Example 9

In vivo efficacy of bispecific antibody < VEGF-ANG-2> antibody in Colo205 xenograft model of Scid beige mice

Cell lines and culture conditions:

colo205 human colorectal cancer cells (ATCC No. CCL-222). In RPMI 1640 medium (PAA, Laboratories, Austria) supplemented with 10% fetal bovine serum (PAALabortors, Austria) and 2mM L-glutamine at 37 ℃ in a water saturated atmosphere at 5% CO₂Tumor cells were routinely cultured. Transplantation was performed using generations 2-5.

Animals:

female SCID beige mice (4-5 weeks old at arrival) (purchased from Charles River Germany) were maintained under pathogen-free conditions at a daily period of 12 hours light/12 hours dark according to the promised principles (GV-Solas; Felasa; TierschG). Experimental study protocol was reviewed and approved by the local government. After arrival, animals were maintained in the quarantine part of the animal house for 1 week to acclimatize to the new environment and observed. Continuous health monitoring is performed periodically. Dietary food (Provimi Kliba 3337) and water (acidified ph2.5-3) were provided ad libitum. Mice at the start of the study were about 10 weeks old.

Tumor cell injection:

on the day of injection, tumor cells were harvested (trypsin-EDTA) from culture flasks (Greiner) and transferred into 50ml of medium, washed 1 time, and resuspended in PBS. After a further washing step with PBS and filtration (cell strainer;) Thereafter, the final cell titer was adjusted to 2.5x10⁷And/ml. The tumor cell suspension was carefully mixed with a transfer pipette to avoid cell aggregation. Thereafter, the cell suspension was filled into 1.0ml tuberculin (tuboculin) syringe (Braun Melsungen) using a wide needle (1.10 × 40 mm); for the injections, the needle size (needle size) was changed (0.45x25mm) and for each injection a new needle was used. Anesthesia was performed using a Stephens inhalation unit for small animals with a pre-incubation chamber (plexiglass), a separate mouse nasal mask (silicon), and isoflurane (cp-pharma), a non-flammable or explosive anesthetic compound in a closed circulation system. Two days before injection, the animal's hairs were scraped off and for cell injection, the skin of the anesthetized animal was carefully lifted with dissecting forceps and 100 μ l of the cell suspension (=2.5x 10)⁶Individual cells) were injected subcutaneously in the right flank of the animal.

Treatment of animals

Animal treatment was started on the day of randomization at a mean tumor volume of about 100mm3, respectively. Mice were treated once weekly with different compounds i.p, as indicated in the table below.

Monitoring:

animals were controlled for health 2 times per week. After cell injection, body weight was recorded 2x weekly. Starting on staging days and then throughout the treatmentTumor size was measured by caliper 2 times per week during the session. Tumor volume was calculated according to NCI protocol (tumor weight =1/2 ab)²Where "a" and "b" are the major and minor diameters, respectively, of the tumor). Termination criteria are the critical tumor mass of the animal (up to 1.7g or) More than 20% body weight loss from baseline, tumor ulceration, or a poor general condition.

The results (see figure 8) show that the bispecific bivalent < VEGF-ANG-2> antibody XMAb1 shows higher tumor growth inhibition in the xenograft tumor model Colo205 of Scid beige mice compared to treatment with monospecific antibody. The potency of ANG2i-LC06 and bevacizumab combination showed comparable results to XMAb 1. The maximum potency of XMAb1 has been reached with 10 mg/kg.

In a second experiment, the effect of XMAb1 on larger tumors was analyzed.

Treatment of animals

Animal treatment was started on the day of randomization at a mean tumor volume of about 400mm3, respectively. Mice were treated once weekly with different compounds i.p, as indicated in the table below.

Monitoring:

animals were controlled for health 2 times per week. After cell injection, body weight was recorded 2x weekly. Tumor size was measured by caliper starting on the staging day and then 2 times per week throughout the treatment period. Tumor volume was calculated according to NCI protocol (tumor weight =1/2 ab)²Where "a" and "b" are the major and minor diameters, respectively, of the tumor). Termination criteria were the critical tumor mass of the animals (up to 1.7 g)Or) More than 20% body weight loss from baseline, tumor ulceration, or a poor general condition.

The results (see figure 9) show that the bispecific bivalent < VEGF-ANG-2> antibody XMAb1 shows higher tumor growth inhibition in the xenograft tumor model Colo205 of Scid beige mice compared to treatment with monospecific antibody, which showed no efficacy in large tumors compared to control. The potency of ANG2i-LC06 and bevacizumab combination showed comparable results to XMAb 1. The maximum potency of XMAb1 has been reached with 10 mg/kg.

In summary, the results demonstrate that XMAb1 shows superior efficacy compared to treatment with monospecific antibody, independent of tumor size.

Compared to the combination of 10mg/kg ANG2i-LC06+10mg/kg avastin, tumor arrest can already be achieved in these models at lower concentrations of 10mg/kg XMAb 1.

Example 10

In vivo efficacy of bispecific antibody < VEGF-ANG-2> antibody in orthotopic KPL-4 xenograft model in Scid beige mice

Tumor cell lines

The human breast cancer cell line KPL-4(Kurebayashi, J. et al, Br.J. cancer 79(1999)707-17) has been established from malignant pleural effusions in breast cancer patients with inflammatory skin metastases. In DMEM medium (PANBIOTech, Germany) supplemented with 10% fetal bovine serum (PANBIOTech, Germany) and 2mM L-glutamine (PAN Biotech, Germany) at 37 ℃ in a water-saturated atmosphere at 5% CO₂Tumor cells were routinely cultured. Cultures were passaged three times/week apart with trypsin/EDTA 1x (pan).

Mouse

Upon arrival, female SCID beige mice (10-12 weeks of age; 18-20g in weight, Charles river, Sulzfeld, Germany) were maintained in the quarantine section of the AALAAC-approved animal house for 1 week to acclimatize and observed. Continuous health monitoring was performed. Mice were kept under SPF conditions according to international guidelines (GV-Solas; Felasa; TierschG) with a daily cycle of 12 hours of light/12 hours of darkness. Dietary food (Kliba Provimi3347) and water (filtered) were provided ad libitum. The experimental study protocol was reviewed and approved by the local government (Regierung von Oberbayorn; accession number 211.2531.2-22/2003).

Tumor cell injection

On the day of injection, tumor cells were harvested (trypsin-EDTA) from culture flasks (Greiner TriFlask) and transferred into 50ml of medium, washed 1 time, and resuspended in PBS. After a further washing step with PBS and filtration (cell strainer;) Thereafter, the final cell titer was adjusted to 1.5x10⁸And/ml. The tumor cell suspension was carefully mixed with a transfer pipette to avoid cell aggregation. Anesthesia was performed using a Stephens inhalation unit for small animals with a pre-incubation chamber (plexiglass), a separate mouse nasal mask (silicon), and isoflurane (Pharmacia-Upjohn, Germany), a non-flammable or explosive anesthetic compound in a closed circulation system. Two days prior to injection, the animal's hair was scraped. For i.m.f.p. injection, cells were injected in a volume of 20 μ Ι into the right penultimate inguinal breast fat pad of each anesthetized mouse. For orthotopic implantation, the cell suspension was injected through the skin under the nipple using a Hamilton microliter syringe and a 30Gx1/2 "needle.

Treatment of animals

Animal treatment was started on the day of randomization at a mean tumor volume of about 80mm3, respectively. Mice were treated once weekly with different compounds i.p, as indicated in the table below.

Monitoring of tumor growth

Animals were controlled for health 2x weekly. After cell injection, body weight was recorded 2x weekly. On staging days, tumor size was measured by caliper 2 times per week at the beginning of treatment session. Tumor volumes (tumor weight =1/2ab2, where "a" and "b" are the major and minor diameters of the tumor, respectively) were calculated according to the NCI protocol (b.teicher; anticancer degradation guide, human Press,1997, chapter 5, page 92).

Termination criteria are the critical tumor mass of the animal (up to 1.7g or) More than 20% body weight loss from baseline, tumor ulceration, or a poor general condition.

The results (see figure 10) show that the bispecific bivalent < VEGF-ANG-2> antibody XMAb1 shows higher tumor growth inhibition in the xenograft tumor model Colo205 of Scid beige mice compared to treatment with monospecific antibody. The potency of ANG2i-LC06 and bevacizumab combination showed comparable results to XMAb 1. The maximum potency of XMAb1 has been reached with 10 mg/kg.

In a second experiment, the effect of XMAb1 on larger tumors was analyzed.

Treatment of animals

Animal treatment was started on the day of randomization at a mean tumor volume of about 160mm3, respectively. Mice were treated once weekly with different compounds i.p, as indicated in the table below.

Monitoring:

animals were controlled for health 2x weekly. After cell injection, body weight was recorded 2x weekly. On staging days, tumor size was measured by caliper 2 times per week at the beginning of treatment session. Tumor volume (tumor weight =1/2 ab) was calculated according to NCI protocol (B.Teicher; anticancer definition guide, Humana Press,1997, Chapter 5, page 92)²Where "a" and "b" are the long and short diameters of the tumor, respectively).

The results (see figure 11) show that the bispecific bivalent < VEGF-ANG-2> antibody XMAb1 shows higher tumor growth inhibition in the xenograft tumor model Colo205 of Scid beige mice compared to treatment with monospecific antibody. The potency of ANG2i-LC06 and bevacizumab combination showed comparable results to XMAb 1. The maximum potency of XMAb1 has been reached with 10 mg/kg.

In summary, the results demonstrate that XMAb1 shows superior efficacy compared to treatment with monospecific antibodies, independent of tumor size.

Compared to the combination of 10mg/kg Ang2i-LC06+10mg/kg avastin, a lower concentration of 10mg/kg XMAb1 can already achieve tumor arrest in these models.

Example 11

Effect of treatment with XMAb1 on microvascular density in s.c. colo205 xenografts

All angiometers by tumor slideThe blood vessel density was evaluated. Vessels were labeled with fluorescent anti-mouse CD34 antibody (clone MEC14.7) on paraffin-embedded sections. Vessels were quantified and microvessel density was measured per mm²The vessel of (4) is calculated. All results are expressed as mean ± SEM. To define significant differences for the experimental groups, the Dunnetts method was used. Consider p<0.05 was statistically significant. The results show that total intratumoral MVD is reduced in the treated tumors. Treatment with ANG2i-LC06 reduced MVD by 29% and bevacizumab reduced MVD</=0%, 15% reduction with bevacizumab + ANG2i-LC06 and 28% reduction with XMAb 1.

Example 12

In vivo efficacy of bispecific antibody < VEGF-ANG-2> antibody in s.c.N87 xenograft model of Scid beige mice

Tumor cell lines

Human gastric cancer cell line N87 cancer cell (NCI-N87(ATCC No. CRL 5822)). In RPMI 1640 supplemented with 10% fetal bovine serum (PAN Biotech, Germany) and 2 mML-glutamine (PAN Biotech, Germany) at 37 ℃ in a water saturated atmosphere at 5% CO₂Tumor cells were routinely cultured. Cultures were passaged three times/week apart with trypsin/EDTA 1x (pan).

Mouse

Upon arrival, female SCID beige mice (10-12 weeks of age; 18-20g weight, charles river, Sulzfeld, Germany) were maintained in the aalac approved animal house quarantine department for 1 week to acclimatize to the new environment and observed. Continuous health monitoring is performed. Mice were kept under SPF conditions according to international guidelines (GV-Solas; Felasa; TierschG) with a daily cycle of 12 hours of light/12 hours of darkness. Dietary food (Kliba Provimi3347) and water (filtered) were provided ad libitum. The experimental study protocol was reviewed and approved by the local government (Regierung von Oberbayorn; accession number 211.2531.2-22/2003).

Tumor cell injection

On the day of cell injection, cells were harvested from culture flasks (Greiner T75), transferred to 50ml of medium, washed 1 time, and resuspended in PBS. After rewashing with PBS, Vi-Cell was used^TM(cell viability Analyzer, BeckmanCoulter, Madison, Wisconsin, U.S.A.). Tumor cell suspensions (PBS) were carefully mixed (to reduce cell aggregation) and kept on ice. The cell suspension was filled into a 1.0ml syringe. For injection, a needle gauge of 0.45x25mm was used. To generate primary tumors, a volume of 100 μ l of 5 × 10 in PBS was used⁶A number of N87 tumor cells were injected subcutaneously into the right flank of each mouse.

Treatment of animals

Animal treatment was started separately at mean tumor volume of about 130mm3 on the day of randomization. Mice were treated once weekly with different compounds i.p, as indicated in the table below.

Number of animals	Compound (I)	Dosage (mg/kg)	Route/mode of administration
				10	Xolair	10	I.p once a week.
10	<VEGF>Avastine	10	I.p once a week.
				10	<ANG-2>Ang2i-LC06	10	I.p once a week.
10	XMAb1	10	I.p once a week.

Monitoring of tumor growth

Animals were controlled for health 1 time per week. After cell injection, body weight was recorded at 1x weekly. On staging days, tumor size was measured by caliper 1 time per week at the beginning of treatment session. Tumor volume (tumor weight =1/2 ab) was calculated according to NCI protocol (B.Teicher; anticancer definition guide, Humana Press,1997, Chapter 5, page 92)²Where "a" and "b" are the long and short diameters of the tumor, respectively).

The results show that the bispecific bivalent < VEGF-ANG-2> antibody XMAb1 shows higher tumor growth inhibition in the xenograft tumor model Colo205 of Scid beige mice compared to treatment with monospecific antibody (fig. 12).

Claims

1. A bispecific bivalent antibody 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 antibody comprises:

a) heavy and light chains of a first full-length antibody that specifically binds VEGF, the first antigen-binding site comprising SEQ ID NO:1 as the heavy chain variable domain (VH) and SEQ ID NO:2 as the light chain variable domain (VL); and is

b) A modified heavy chain and a modified light chain of a second full-length antibody that specifically binds ANG-2, wherein constant domains CL and CH1 are replaced with each other, the second antigen-binding site comprising SEQ ID NO:3 as the heavy chain variable domain (VH) and SEQ ID NO:4 as the light chain variable domain (VL),

and the antibody is characterized in that:

wherein

i) In the CH3 of one heavy chain,

one amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance in the interface of the CH3 domain of one heavy chain, which protuberance can be located in a cavity in the interface of the CH3 domain of the other heavy chain,

wherein the CH3 domain comprises a T366W mutation,

and wherein

ii) in the CH3 domain of the other heavy chain,

one amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity in the interface of the second CH3 domain, into which cavity a protuberance in the interface of the first CH3 domain may be located,

wherein the CH3 domain comprises T366S, L368A, and Y407V mutations.

2. The bispecific antibody according to claim 1, characterized by having a constant region of the subclass human IgG 1.

3. The bispecific antibody according to claim 1 or 2, characterized in comprising:

a) SEQ ID NO 7 as the heavy chain of said first full-length antibody and SEQ ID NO 5 as the light chain of said first full-length antibody, and

b) SEQ ID NO 8 as the modified heavy chain and SEQ ID NO 6 as the modified light chain of said second full length antibody.

4. The bispecific antibody according to claim 1 or 2, characterized in comprising:

a) 11 as the heavy chain of said first full-length antibody and SEQ ID NO 9 as the light chain of said first full-length antibody, and

b) 12 as the modified heavy chain of the second full-length antibody and 10 as the modified light chain of the second full-length antibody.

5. The bispecific antibody according to claim 1 or 2, characterized in comprising:

a) 15 as the heavy chain of said first full-length antibody and SEQ ID NO 13 as the light chain of said first full-length antibody, and

b) 16 as the modified heavy chain of the second full-length antibody and 14 as the modified light chain of the second full-length antibody.

6. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 5.

7. Use of a bispecific antibody according to any one of claims 1 to 5 for the manufacture of a medicament for the treatment of cancer.

8. Use of a bispecific antibody according to any one of claims 1 to 5 for the manufacture of a medicament for the treatment of a disorder selected from intraocular neovascular syndrome, rheumatoid arthritis or psoriasis.

9. Use according to claim 8, wherein the medicament is for the treatment of proliferative retinopathy or age-related macular degeneration.

10. A nucleic acid encoding the bispecific antibody according to any one of claims 1 to 5.

11. An expression vector comprising 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. A process for the preparation of a bispecific antibody according to any one of claims 1 to 5, comprising the steps of:

c) Recovering the antibody from the culture.