HK1171765A

HK1171765A - Bispecific anti-egfr/anti-igf-1r antibodies

Info

Publication number: HK1171765A
Application number: HK12112190.9A
Authority: HK
Inventors: 乌尔里希．布林克曼; 丽贝卡．克罗斯代尔; 维尔马．劳; 克里斯蒂安．吉尔德斯; 埃克．霍夫曼; 克里斯蒂安．克莱因; 克劳斯-彼得．金克勒; 沃尔夫冈.谢弗; 扬．奥拉夫．斯特拉克; 巴勃罗．乌马纳
Original assignee: 罗氏格黎卡特股份公司
Priority date: 2008-09-26
Filing date: 2012-11-28
Publication date: 2013-04-05

Abstract

Bispecific antibodies against EGFR and against IGF-1R, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

Description

Bispecific anti-EGFR/anti-IGF-1R antibodies

The application is a divisional application with the international application number of PCT/EP2009/006782, the international application date is 2009, 9, 21, the date of entering China is 2011, 3, 24, the Chinese national application number is 200980137723.3, and the invention name is 'bispecific anti-EGFR/anti-IGF-1R antibody'.

The present invention relates to bispecific antibodies against EGFR and against IGF-1R, methods for their preparation, pharmaceutical compositions comprising said antibodies, and uses thereof.

Background

EGFR and anti-EGFR antibodies

The human epidermal growth factor receptor (also known as HER-1 or Erb-B1, and referred to herein as "EGFR") is a 170kDa transmembrane receptor that is encoded by the c-erbB proto-oncogene and that exhibits intrinsic tyrosine kinase activity (Modjtahedi, H., et al, British journal of Cancer (Br. J. Cancer)73 (1996): 228-. The sequence of EGFR is provided by SwissProt database accession number P00533. Isoforms and variants of EGFR also exist (e.g., variable RNA transcripts, truncated forms, polymorphisms, etc.), including but not limited to those identified by Swissprot database accession numbers P00533-1, P00533-2, P00533-3, and P00533-4. EGFR is known to bind ligands including: epidermal Growth Factor (EGF), transforming growth factor-alpha (TGf-alpha), amphiregulin, heparin-binding EGF (hb-EGF), betacellulin, and epiregulin (Herbst, R.S. and Shin, D.M., Cancer (Cancer)94(2002) 1593-. EGFR regulates many cellular processes via tyrosine kinase-mediated Signal transduction pathways, including but not limited to activation of Signal transduction pathways that control cell proliferation, differentiation, cell survival, programmed cell death, angiogenesis, mitogenesis and metastasis (Atalay et al, Ann. Oncology 14 (2003)) 1346-.

Overexpression of EGFR has been reported in a number of human malignant conditions, including bladder, brain, head and neck, pancreatic, lung, breast, ovarian, colon, prostate and renal cancers. (Atalay, G., et al, Ann. Oncology 14(2003) 1346-. In many of these disorders, overexpression of EGFR correlates or correlates with poor prognosis in patients. (Herbst, R.S., and Shin, D.M., Cancer (Cancer)94(2002) 1593-235; Modjtahedi, H., et al, British journal of Cancer (Br. J.cancer)73(1996) 228-235). EGFR is also expressed in cells of normal tissues, particularly epithelial tissues of the skin, liver and gastrointestinal tract, although usually at lower levels than in malignant cells (Herbst, r.s., and Shin, d.m., Cancer)94(2002) 1593-.

Unconjugated monoclonal antibodies (mAbs) may be useful drugs for treating cancer, as evidenced by the following drugs approved by the U.S. food and Drug Administration: trastuzumab (Herceptin) for the treatment of advanced breast cancer^TM(ii) a Genentech Inc.) (Grillo-Lopez, a. -j., et al, semin. oncol.26(1999) 66-73; golden nberg, m.m., clinical treatment (clin.ther.)21 (1999): 309-18) for treating CD20 positive B cells, low grade or follicular non-hodgkin's lymphoma^TM(ii) a IDECpharmaceuticals, San Diego, CA, and Genentech Inc., San Francisco, CA), Gituzumab (Mylotarg) for the treatment of relapsed acute myeloid leukemia^TMCelltech/Wyeth-Ayerst), and alemtuzumab (CAMPATH) for the treatment of B-cell chronic lymphocytic leukemia^TMMillenium Pharmaceuticals/Schering AG). The success of these products depends not only on their efficacy, but also on their outstanding safety profile (Grillo-Lopez, A. -J., et al., Semin. Oncol.26 (1999): 66-73; Goldenberg, M.M., clinical treatment (Clin. Ther).21(1999) 309-18). Despite the achievements achieved with these drugs, there is currently great interest in obtaining higher specific antibody activities than typically provided by unconjugated mAb therapy.

The results of many studies show that the Fc-receptor dependent mechanism is rather favorable for the action of cytotoxic antibodies against tumors and indicate that optimal antibodies against tumors will preferentially bind to activate the Fc receptor and bind to the inhibitory partner fcyriib to a minimal extent. (Clynes, R.A., et al, Nature Medicine (Nature Medicine)6 (4): 443-.

Various strategies have been reported to target EGFR and block the EGFR signaling pathway. Small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and CI-1033 block autophosphorylation of EGFR within the intracellular tyrosine kinase domain, thereby inhibiting downstream signaling events (Tsao, a.s., and Herbst, r.s., Signal 4(2003) 4-9). Monoclonal antibodies, on the other hand, target the extracellular portion of EGFR, resulting in blocking ligand binding and thereby inhibiting downstream events such as cell proliferation (Tsao, a.s., and Herbst, r.s., Signal 4(2003) 4-9).

Several murine monoclonal antibodies have been generated which obtain this in vitro blockade and have been evaluated for their ability to affect tumor growth in a mouse xenograft model (Masui et al, Cancer research (Cancer Res.)46(1986) 5592-. For example, EMD 55900(EMD pharmaceuticals) is a murine anti-EGFR monoclonal antibody raised against the human epidermoid carcinoma cell line a431 and tested in clinical studies in patients with advanced squamous cell carcinoma of the larynx or hypopharynx (Bier, h., et al, eur. arch. otohilolaryngol.252 (1995) 433-9). In addition, rat monoclonal antibodies ICR16, ICR62, and ICR80, which bind to the extracellular domain of EGFR, have been shown to effectively inhibit the binding of EGF and TGF- α receptor (Modjtahedi, H., et al, int. J. cancer 75(1998) 310-316). Murine monoclonal antibody 425 is another Mab raised against the human a431 cancer cell line and was found to bind to a polypeptide epitope on the external domain of the human epidermal growth factor receptor. (Murthy, U.S., et al, Biophys.) 252(2), (1987)549- > 560 the potential problem of using murine antibodies in therapeutic treatments is that non-human monoclonal antibodies can be recognized by the human host as foreign proteins, and thus repeated injections of these foreign antibodies can result in the induction of an immune response, resulting in a deleterious hypersensitivity reaction for murine-based monoclonal antibodies, often referred to as human anti-mouse antibody responses, or "HAMA" responses, or human anti-rat antibodies, or "HARA" responses.

Chimeric antibodies comprising portions of antibodies from two or more different species (e.g., mouse and human) have been developed as replacements for "conjugated" antibodies. For example, US5,891,996 (matero de Acosta del Rio, c.m., et al) discusses a mouse/human chimeric antibody, R3, against EGFR, and US5,558,864 discusses the generation of chimeric and humanized forms of murine anti-EGFR MAb 425. Further, IMC-C225ImClone) is a chimeric mouse/human anti-EGFR monoclonal antibody (based on the mouse M225 monoclonal antibody, which results in HAMA response in human clinical trials), which has been reported to show anti-tumor efficacy in various human xenograft models. (Herbst, R.S., and Shin, D.M., Cancer 94(2002) 1593-1611). The efficacy of IMC-C225 has been attributed to several mechanisms, including inhibition of cellular events mediated through the EGFR signaling pathway, and possibly through increased antibody-dependent cellular cytotoxicity (ADCC) activity (Herbst, r.s., and Shin, d.m., Cancer (Cancer)94(2002) 1593-. IMC-C225 is also used in clinical trials, including in combination with radiation therapy and chemotherapy (Herbst, r.s., and Shin, d.m., Cancer 94(2002) 1593-1611). Recently, Abgenix, Inc (Frem)ont, CA) developed ABX-EGF for cancer treatment. ABX-EGF is a fully human anti-EGFR monoclonal antibody. (Yang, X.D., et al, crit. Rev. Oncol./Hematol.38(2001) 17-23).

WO2006/082515 relates to humanized anti-EGFR monoclonal antibodies derived from the rat monoclonal antibody ICR62 and to glycoengineered forms thereof for use in cancer therapy.

IGF-1R and anti-IGF-1R antibodies

Insulin-like growth factor I receptor (IGF-1R, IGF-IR, CD 221 antigen) belongs to the family of transmembrane protein tyrosine kinases) (LeRoith, D., et al, reviews in Endocrin. Rev.). 16(1995) 143-163; and Adams, T.E., et al, cell. mol. Life Sci 57(2000) 1050-. IGF-IR binds IGF-I with high affinity and initiates physiological responses to this ligand in vivo. IGF-IR also binds to IGF-II, but with slightly lower affinity. Overexpression of IGF-IR promotes neoplastic transformation of cells, and there is evidence that IGF-IR is involved in malignant transformation of cells and is therefore a useful target for the development of therapeutics for the treatment of cancer (Adams, t.e., et al, cell. mol. life sciences) 57(2000) 1050-.

Antibodies to IGF-IR are well known in the art and are studied for their anti-tumor efficacy in vitro and in vivo (Benini, S., et al, clinical Cancer research (Clin. Cancer Res.)7(2001) 1790-1797; Scotlandi, K., et al, Cancer Gene therapy (Cancer GeneTherr.) 9(2002) 296; Scotlandi, K., et al, International journal of Cancer (int. J. Cancer)101(2002) 11-16; Brunetti, A., et al, Biochemical Physics research communication (biochem. Biophys. Res. Commun.)165(1989) 165; Prigent, S243A., et al, Biochemicophysis. chem. (1990)9970, 9977; Li, S.L., 12431, S.243A., 1989; Biophys. chem.) 265; Biophys. J. Immun. chem. J., 1989; Biophys. chem. Press, S.81, S.J. Immunol. 75, J. 1989; Biophys. chem. Press, S.75, J. Immunol. Press, J. 1989, Biophys. J. Press, 1989, J. 1989, Biophys. J. Press, 1989, J. 1989, J. Immunol. Press, 1989, J. Press, J. chem. Press, 1989, M.A., et al, J.Biol.chem., 267(1992) 12955-12963; soos, M.A., et al, Proc. Natl.Acad.Sci.USA 86(1989) 5217-; o' Brien, R., M., et al, EMBO J.6(1987) 4003-; taylor, R., et al, J.Biochem.J.) 242(1987) 123-129; soos, m.a., et al, journal of biochemistry (biochem.j.)235(1986) 199-208; li, s.l., et al, biochemical biophysical communication (biochem. biophysis. res.commun.)196(1993) 92-98; delafontaine, P., et al, J.mol.cell.Cardiol.26(1994) 1659-; kull, F.C., Jr, et al J.Biol.chem.) -258 (1983) 6561-6566; morgan, d.o., and Roth, r.a., Biochemistry 25(1986) 1364-; forsayeth, J.R., et al, Proc. Natl.Acad.Sci.USA 84(1987) 3448-; schaefer, E.M., et al, J.Biol.chem. 265(1990) 13248-13253; gustafson, t.a., and Rutter, w.j., journal of biochemistry (j.biol.chem.)265(1990) 18663-18667; hoyne, p.a., et al, FEBS communication (FEBS Lett.)469(2000) 57-60; tulloch, P.A., et al, J.Structure biol. (J.struct.biol.)125(1999) 11-18; rohlik, Q.T., et al, Biochemical Biophysical research communications (biochem. Biophys. Res. Comm.)149(1987) 276-281; and Kalebic, T., et al, Cancer research (Cancer Res.)54(1994) 5531-; adams, T.E., et al, cell. mol. Life Sci 57(2000) 1050-; dricu, A., et al, Glycobiology (Glycobiology)9(1999) 571-; Kanter-Lewensohn, L., et al, Melanoma research (Melanoma Res.)8(1998) 389-397); li, s.l., et al, Cancer immunological immunotherapy (Cancer immunol. immunotherapy) 49(2000)243- "252). Antibodies to IGF-IR are described in many other documents, such as artemia, c.l., et al, Breast Cancer research treatment (Breast Cancer res. treatment)22(1992) 101-106; and Hailey, J., et al, molecular cancer therapy (mol. cancer Ther.)1(2002) 1349-1353.

In particular, monoclonal antibodies directed against IGF-IR, known as α IR3, are widely used in the study of IGF-IR mediated processes and IGF-I mediated diseases such as cancer. alpha-IR-3 is described by Kull, F.C., J.Biol.chem.)258(1983) 6561-6566. At the same time, about a hundred published publications relate to the study and therapeutic application of the antitumor effect of α IR3, in which α IR3 is treated alone and together with cytostatics such as doxorubicin (doxorubicin) and vincristine (vincristine). Alpha IR3 is a murine monoclonal antibody known to inhibit the binding of IGF-I to IGF receptors, but not IGF-II to IGF-IR. Alpha IR3 stimulates tumor cell proliferation and IGF-IR phosphorylation at high concentrations (Bergmann, U.S., et al, Cancer research (Cancer Res.)55(1995) 2007) -2007-262011; Kato, H.et al, J.Biol.chem.)268(1993) 2655-2661). There are other antibodies (e.g., 1H7, Li, s., l., etc., Cancer immunological immunotherapy 49(2000)243-252) that inhibit IGF-II binding more effectively than IGF-I binding. Brief description of the prior art for antibodies and their properties and characteristics is described by Adams, t.e., et al, cell molecular life sciences (cell. mol. life Sci.)57(2000) 1050-.

Most antibodies described in the prior art are of mouse origin. Such antibodies, as is well known in the art, are ineffective for treatment of human patients without further modification such as chimerization or humanization. Based on these deficiencies, it is clearly preferred that human antibodies be used as therapeutic agents for the treatment of human patients. Human antibodies are well known in the art (van Dijk, m.a., and van de Winkel, j.g., curr. opin. pharmacol.5(2001) 368-374). Based on these techniques, human antibodies can be generated against a variety of targets. Examples of human antibodies to IGF-IR are described in WO 02/053596.

WO 2005/005635 relates to the human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC2587) or < IGF-1R > HUMAB clone 22(DSM ACC 2594) and their use in cancer therapy.

Bispecific antibodies

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

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

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

In WO 2007/024715, dual variable domain immunoglobulins are reported as engineered multivalent and multispecific binding proteins. A method for the preparation of biologically active antibody dimers is reported in US 6,897,044. In US 7,129,330 multivalent F is reported having at least four variable domains connected to each other by a peptide linker_VAn antibody construct. Dimeric and multimeric antigen binding structures are reported in US 2005/0079170. Trivalent or tetravalent monospecific antigen binding proteins comprising three or four Fab fragments covalently bound to each other by a linking structure, which proteins are not native immunoglobulins, are reported in US 6,511,663. Such tetravalent is reported in WO 2006/020258Bispecific antibodies which can be efficiently expressed in prokaryotic and eukaryotic cells and are useful in therapeutic and diagnostic methods. In US 2005/0163782, a method is reported for separating or preferentially synthesizing dimers linked by at least one interchain disulfide bond from dimers not linked by at least one interchain disulfide bond in a mixture comprising two types of polypeptide dimers. Bispecific tetravalent receptors are reported in 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 a conjugated pair complex (homoconjugate) typically prepared from monoclonal antibodies of the IgG class that bind the same antigenic determinant, covalently linked by synthetic cross-linking. Oligomeric monoclonal antibodies with high affinity for antigens are reported in WO 1991/06305, wherein oligomers, typically of the IgG class, are secreted with two or more immunoglobulin monomers that associate together to form tetravalent or hexavalent IgG molecules. Ovine derived antibodies and engineered antibody constructs are reported in US 6,350,860, which may be used for the treatment of diseases where interferon gamma activity is pathogenic. In US 2005/0100543, targetable constructs are reported which are multivalent vectors for bispecific antibodies, i.e. each molecule of a targetable construct can act as a vector for two or more bispecific antibodies. Genetically engineered bispecific tetravalent antibodies are reported in WO 1995/009917. In WO 2007/109254, stable binding molecules consisting of or comprising stable scfvs are reported.

From Lu, D., et al, Biochemical and biophysical Research Communications 318(2004) 507-; journal of biochemistry (j.biol.chem.), 279(2004) 2856-2865; and journal of biochemistry (J.biol Chem.)280(2005)19665-72 bispecific antibodies against EGFR and IGF-1R are known. However, these bispecific anti-EGFR/anti-IGF-1R antibodies clearly show reduced tumor growth inhibition when compared to the combination of the parent monospecific antibodies (especially in tumor cells where both EGFR and IGF-1R have equal (high) expression levels).

Brief description of the invention

We have now surprisingly found that novel bispecific anti-EGFR/anti-IGF-1R antibodies show at least similar tumor growth inhibition (using only a reduced amount of bispecific antibody) compared to the combination of the parent monospecific antibodies (especially in tumor cells where both, i.e. EGFR and IGF-1R, have equal (high) expression levels).

A first aspect of the invention is a bispecific antibody that binds EGFR and IGF-1R, comprising a first antigen-binding site that binds EGFR and a second antigen-binding site that binds IGF-1R, characterized in that the bispecific antibody is

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

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

iii) the second antigen binding site comprises the amino acid sequence of SEQ ID NO: 11, CDR3 region of SEQ ID NO: 12, and the CDR2 region of SEQ ID NO: 13, and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 14, CDR3 region of SEQ ID NO: 15, and the CDR2 region of SEQ ID NO: 16, CDR1 region;

or the second antigen binding site comprises the amino acid sequence of SEQ ID NO: 17, CDR3 region of SEQ ID NO: 18, and the CDR2 region of SEQ ID NO: 19 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 20, CDR3 region of SEQ ID NO: 21, and the CDR2 region of SEQ ID NO: 22, CDR1 region.

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

i) the first antigen binding site comprises SEQ ID NO: 7 or SEQ ID NO: 8 as a heavy chain variable domain, and comprises SEQ ID NO: 9 or SEQ ID NO: 10 as a light chain variable domain, and,

ii) the second antigen binding site comprises SEQ ID NO: 23 or SEQ ID NO: 24 as a heavy chain variable domain, and comprises SEQ ID NO: 25 or SEQ ID NO: 26 as a light chain variable domain.

i) the first antigen binding site comprises SEQ ID NO: 8 as a heavy chain variable domain, and comprises SEQ ID NO: 10 as a light chain variable domain, and,

ii) the second antigen binding site comprises SEQ ID NO: 23 as a heavy chain variable domain, and comprises SEQ ID NO: 25 as light chain variable domain.

The bispecific antibody is at least bivalent and may be trivalent, tetravalent, or multivalent. Preferably, the bispecific antibody according to the invention is bivalent, trivalent or tetravalent.

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

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

The bispecific antibodies according to the invention show benefits for patients in need of EGFR and IGF-1R targeted therapy. The antibodies according to the invention have new and inventive properties leading to benefits for patients suffering from said diseases, in particular patients suffering from cancer.

Detailed Description

One embodiment of the invention is a bispecific antibody that binds EGFR and IGF-1R, comprising a first antigen-binding site that binds EGFR and a second antigen-binding site that binds IGF-1R, said bispecific antibody being characterized in that:

iii) the second antigen binding site comprises the amino acid sequence of SEQ ID NO: 11, CDR3 region of SEQ ID NO: 12, and the CDR2 region of SEQ ID NO: 13, and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 14, CDR3 region of SEQ ID NO: 15, and the CDR2 region of SEQ ID NO: 16, CDR1 region.

Another embodiment of the invention is a bispecific antibody that binds EGFR and IGF-1R, comprising a first antigen-binding site that binds EGFR and a second antigen-binding site that binds IGF-1R, said bispecific antibody being characterized in that:

iii) the second antigen binding site comprises the amino acid sequence of SEQ ID NO: 17, CDR3 region of SEQ ID NO: 18, and the CDR2 region of SEQ ID NO: 19 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 20, CDR3 region of SEQ ID NO: 21, and the CDR2 region of SEQ ID NO: 22, CDR1 region.

ii) the first antigen binding site comprises SEQ ID NO: 7 or SEQ ID NO: 8 as a heavy chain variable domain, and comprises SEQ ID NO: 9 or SEQ ID NO: 10 as light chain variable domain

iii) the second antigen binding site comprises SEQ ID NO: 23 or SEQ ID NO: 24 as a heavy chain variable domain, and comprises SEQ ID NO: 25 or SEQ ID NO: 26 as a light chain variable domain.

ii) the first antigen binding site comprises SEQ ID NO: 7 as a heavy chain variable domain, and comprising SEQ ID NO: 10 as light chain variable domain

iii) the second antigen binding site comprises SEQ ID NO: 23 as a heavy chain variable domain, and comprises SEQ ID NO: 25 as light chain variable domain.

ii) the first antigen binding site comprises SEQ ID NO: 8 as a heavy chain variable domain, and comprises SEQ ID NO: 10 as light chain variable domain

iii) the second antigen binding site comprises SEQ ID NO: 24 as a heavy chain variable domain, and comprises SEQ ID NO: 26 as a light chain variable domain.

As used herein, "antibody" refers to a binding protein that comprises an antigen binding site. The term "binding site" or "antigen binding site" as used herein refers to the region of the antibody molecule to which an antibody actually binds. The binding sites in the antibodies according to the invention may each be formed by pairs of two variable domains, namely one heavy chain variable domain and one light chain variable domain. The smallest binding site determinant in an antibody is the heavy chain CDR3 region. In one embodiment of the invention, each binding site comprises an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), and preferably is formed of a pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

Antibody specificity refers to the selective recognition of a particular epitope of an antibody against an antigen. Natural antibodies, for example, are monospecific. A "bispecific antibody" according to the invention is an antibody having two different antigen binding specificities. If the antibody has more than one specificity, the recognized epitope may be associated with a single antigen or more than one antigen. The antibodies of the invention are specific for two different antigens, namely EGFR as the first antigen and IGF-1R as the second antigen.

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

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

The antibodies of the invention have more than two binding sites and are bispecific. That is, the antibody may be bispecific even in the presence of more than two binding sites (i.e., the antibody is trivalent or multivalent). Bispecific antibodies of the invention include, for example, multivalent single chain, diabodies and triabodies, as well as antibodies having the constant domain structure of a full-length antibody to which an additional antigen-binding site (e.g., single chain Fv, VH and/or VL domains, Fab, or (Fab)2)) is linked by one or more peptide linkers. The antibody may be a full length antibody from a single species, or may be chimeric or humanized. For antibodies with more than two antigen binding sites, some binding sites may be the same, as long as the protein has binding sites for two different antigens. That is, when the first binding site is specific for EGFR, the second binding site is specific for IGF-1R.

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

In certain embodiments, the antibodies of the invention further comprise an immunoglobulin constant region of one or more immunoglobulin classes. Immunoglobulin classes include the IgG, IgM, IgA, IgD, and IgE isotypes and, in the case of IgG and IgA, their subtypes. In a preferred embodiment, the antibody of the invention has the constant domain structure of an antibody of the IgG class, but has four antigen binding sites. This is accomplished by linking two intact antigen binding sites that specifically bind EGFR (e.g., single chain Fv) with the N-or C-terminal heavy or light chain of an intact antibody that specifically binds IGF-1R. The four antigen binding sites preferably comprise two binding sites for each of two different binding specificities.

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

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

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

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

As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies isolated from host cells, such as NS0 or CHO cells, or from transgenic animals (e.g., mice) of human immunoglobulin genes, or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions in rearranged form. Recombinant human antibodies according to the invention have undergone somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of a recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur in vivo in human antibody germline repertoires. "variable domain" (variable domain of a light chain (VL), variable region of a heavy chain (VH)) as used herein, denotes each pair of light and heavy chains that is directly involved in binding of an antibody to an antigen. The domains of variable human light and heavy chains have the same general structure and each domain comprises 4 Framework (FR) regions, the sequences of which are generally conserved, connected by 3 "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β -sheet conformation and the CDRs may form loops connecting the β -sheet structures. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain an antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and thus provide another object of the invention.

As used herein, the term "hypervariable region" or "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody which are responsible for antigen-binding. Hypervariable regions comprise amino acid residues from the "complementarity determining regions" or "CDRs". The "framework" or "FR" regions are those variable domain regions other than the hypervariable region residues defined herein. Thus, the light and heavy chains of an antibody comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The CDRs on each chain are separated by the framework amino acids. In particular, CDR3 of the heavy chain is the region most contributing to antigen binding. CDR and FR regions were determined according to the standard definition of the protein sequence of immunological Interest (Sequences of Proteins of immunological Interest), 5 th edition, public Health services, National Institutes of Health, Bethesda, Md. (1991), by Kabat et al.

Bispecific antibodies according to the present invention also include such antibodies with "conservative sequence modifications" (which refers to "variants" of bispecific antibodies). This means nucleotide and amino acid sequence modifications that do not affect or alter the above-mentioned characteristics of the antibodies according to the invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the predicted non-essential amino acid residue in the bispecific < EGFR-IGF1R > antibody may preferably be replaced by another amino acid residue from the same side chain family. Thus, a "variant" bispecific < EGFR-IGF1R > antibody is herein a molecule whose amino acid sequence differs from the amino acid sequence of a "parent" bispecific < EGFR-IGF1R > antibody by up to 10, preferably from about 2 to about 5, additions, deletions and/or substitutions in one or more variable or constant regions of the parent antibody. Amino acid substitutions may be made by mutagenesis based on molecular modeling, as described in Riechmann, L., et al, Nature (Nature)332(1988) 323-100327 and Queen, C., et al, Proc. Natl.Acad.Sci.USA 86(1989) 10029-10033.

Identity or homology with respect to the sequences is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to the parent sequence, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity. No N-terminal, C-terminal or internal extension, deletion or insertion of antibody sequences should be considered to affect sequence identity or homology. The variants retain the ability to bind to human EGFR and human IGF-1R.

As used herein, the term "binding" or "specific binding" refers to the binding of an antibody to an epitope of an antigen in an in vitro assay, preferably in a cell-based ELISA performed with CHO cells expressing the wild-type antigen. Bound means 10^-8M is less, preferably 10^-13M to 10^-9Binding affinity of M (K)_D). Binding of antibodies to antigen or Fc γ RIII can be studied by BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden). The affinity of binding is defined by the term ka (rate constant for association of antibody from antibody/antigen complex), k_D(dissociation constant)Number) and K_D(k_D/ka) definition.

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

The human epidermal growth factor receptor (also known as HER-1 or Erb-B1, and referred to herein as "EGFR") is a 170kDa transmembrane receptor that is encoded by the c-erbB proto-oncogene and that exhibits intrinsic tyrosine kinase activity (Modjtahedi, H., et al, British journal of Cancer (Br. J. Cancer)73 (1996): 228-. The sequence of EGFR is provided by SwissProt database accession number P00533. Isoforms and variants of EGFR also exist (e.g., variable RNA transcripts, truncated forms, polymorphisms, etc.), including but not limited to those identified by Swissprot database accession numbers P00533-1, P00533-2, P00533-3, and P00533-4. EGFR is known to bind ligands including: α), Epidermal Growth Factor (EGF), transforming growth factor- α (TGf- α), amphiregulin, heparin-binding EGF (hb-EGF), β -zoocellulose, and epiregulin (Herbst, R.S. and Shin, D.M., Cancer (Cancer)94(2002) 1593-1611; mendelsohn, J., and Baselga, J., Oncogene (Oncogene)19(2000) 6550-6565). EGFR regulates many cellular processes via tyrosine kinase-mediated Signal transduction pathways, including but not limited to activation of Signal transduction pathways that control cell proliferation, differentiation, cell survival, programmed cell death, angiogenesis, mitogenesis and metastasis (Atalay, g., et al, ann. oncology 14(2003) 1346-.

Insulin-like growth factor I receptor (IGF-IR, CD 221 antigen) belongs to the family of transmembrane protein tyrosine kinases) (LeRoith, D., et al, reviews in Endocrin. Rev.) -16 (1995) 143-163; and Adams, T.E., et al, cell. mol. Life Sci 57(2000) 1050-. SwissProt database accession number P08069 provides the sequence of IGF-IR. IGF-IR binds IGF-I with high affinity and initiates physiological responses to this ligand in vivo. IGF-IR also binds to IGF-II, but with slightly lower affinity. Overexpression of IGF-IR promotes neoplastic transformation of cells, and there is evidence that IGF-IR is involved in malignant transformation of cells and is therefore a useful target for the development of therapeutics for the treatment of cancer (Adams, t.e., et al, cell. mol. life Sci.) -57 (2000) 1050-.

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

The term "full-length antibody" refers to an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains" (see fig. 10, schematic structure of a "full-length antibody" without the CH4 domain, see also fig. 1 and 12, full-length portions with a single-chain Fv-linker (XGFR) and a tetravalent bispecific version with a single-chain Fab-linker (scFab-XGFR)). A "full-length antibody heavy chain" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1(CH1), an antibody Hinge Region (HR), an antibody heavy chain constant domain 2(CH2), and an antibody heavy chain constant domain 3(CH3) in the N-terminal to C-terminal direction, abbreviated VH-CH1-HR-CH2-CH 3; and, in the case of antibodies of the IgE subclass, optionally also antibody heavy chain constant domain 4(CH 4). Preferably, a "full length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. A "full-length antibody light chain" is a polypeptide consisting of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated VL-CL, in the N-terminal to C-terminal direction. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). The two full-length antibody chains are linked together by interpeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge region of the full-length antibody heavy chain. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE. Full length antibodies according to the invention may be from a single species, e.g. human, or they may be chimeric or humanized antibodies. The full-length antibody according to the present invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which both specifically bind to the same antigen. Thus, a monospecific bivalent (═ full length) antibody comprising a first antigen binding site and consisting of two antibody light chains and two antibody heavy chains is a full length antibody. The C-terminus of the heavy or light chain of the full-length antibody refers to the last amino acid at the C-terminus of the heavy or light chain. The N-terminus of the heavy or light chain of the full-length antibody refers to the last amino acid at the N-terminus of the heavy or light chain.

In one embodiment, the bispecific antibody is bivalent-using a format as described, for example, a) in WO 2009/080251, WO 2009/080252 or WO 2009/080253 (domain exchanged antibody-see example 14) or a format based on scFab-Fc fusion antibodies, wherein one single chain Fab fragment is specific for EGFR and the other single chain Fab fragment is specific for IGF-1R (see example 17) or c) in EP application No. 07024867.9(WO 2009/080251), Ridgway, j.b., Protein eng.9(1996) 617-621; WO 96/027011; merchant A.M, et al, Nature Biotech 16(1998) 677-681; atwell, S., et al, J.Mol.biol. (1997) 270-35 and EP1870459A 1. In one embodiment, the bispecific antibody according to the invention is characterized in comprising SEQ ID NO: 30, seq id NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33 or a variant thereof. In one embodiment, the bispecific antibody according to the invention is characterized in comprising SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37 or a variant thereof. In one embodiment, the bispecific antibody according to the invention is characterized in comprising seq id NO: 38, and SEQ ID NO: 39 or a variant thereof. In one embodiment, the bispecific antibody according to the invention is characterized in comprising SEQ ID NO: 38, and SEQ id no: 39 or a variant thereof. In one embodiment, the bispecific antibody according to the invention is characterized in comprising SEQ ID NO: 40, and SEQ ID NO: 41 or a variant thereof. In one embodiment, the bispecific antibody according to the invention is characterized in comprising SEQ ID NO: 42, and SEQ ID NO: 43 or a variant thereof. These amino acid sequences are based on the amino acid sequences of SEQ ID NOs: 8, and the heavy chain variable domain of SEQ ID NO: 10 (from humanized < EGFR > ICR62) and is based on the amino acid sequence of seq id NO: 23, and the heavy chain variable domain of SEQ ID NO: 25 (from the human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSMACC 2587)).

In one embodiment, the bispecific antibody is trivalent, using for example a form based on a full length antibody specifically binding to one of the two receptors EGFR or IGF-1R to which only one C-terminal scFab fragment of one heavy chain is fused, which scFab fragment specifically binds to the other of the two receptors EGFR or IGF-1R, including the knob-into-hole technology, as described in EP application No. 09004909.9, or a form based for example on a full length antibody specifically binding to one of the two receptors EGFR or IGF-1R to which at one C-terminus of one heavy chain the full length antibody is fused a VH or VH-CH1 fragment and at the other C-terminus of the second heavy chain a VL or VL-CL fragment specifically binding to the other of the two receptors EGFR or IGF-1R, including the projection-entry-hole technique as described in EP application No. 09005108.7. See also Ridgway, J.B., Protein Eng.9(1996) 617-; WO 96/027011, MerchantA.M., et al, Nature Biotech 16(1998) 677-681; atwell, S., et al, J.mol.biol. (1997) 270 (26-35); and EP1870459a 1. In one embodiment, the bispecific trivalent antibody according to the invention is characterized in comprising SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 or a variant thereof. In one embodiment, the bispecific trivalent antibody according to the invention is characterized in comprising SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49 or a variant thereof. These amino acid sequences are based on the amino acid sequence of SEQ id no: 8, and the heavy chain variable domain of SEQ ID NO: 10 (from humanized < EGFR > ICR62), and is based on the amino acid sequence of SEQ ID NO: 23, and the heavy chain variable domain of SEQ ID NO: 25 (from human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC 2587)).

In one embodiment, the bispecific antibody is tetravalent, using a format as described, for example, in WO 2007/024715, or WO 2007/109254 or EP application No. 09004909.9 (full length antibody binding to a first antigen, to which two scFab fragments binding to other antigens are fused) (see, e.g., example 1 or 9)

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

a) a monospecific bivalent antibody comprising the first antigen binding site and consisting of two antibody light chains and two antibody heavy chains comprising only one variable domain per chain,

b) two peptide linkers, and

c) two monovalent monospecific single chain antibodies (monospecific monovalent single chain Fv) comprising the second antigen-binding site, consisting of a light chain variable domain, a heavy chain variable domain and a single linker between the light chain variable domain and the heavy chain variable domain, respectively;

wherein the single chain antibody (the single chain Fv) is linked to the same end of a monospecific bivalent antibody light chain or antibody heavy chain.

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

a) a monospecific bivalent antibody comprising the second antigen binding site and consisting of two antibody light chains and two antibody heavy chains comprising only one variable domain per chain,

b) two peptide linkers, and

c) two monovalent monospecific single chain antibodies (monospecific monovalent single chain Fv) comprising the first antigen binding site, consisting of a light chain variable domain, a heavy chain variable domain and a single linker between the light chain variable domain and the heavy chain variable domain, respectively;

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

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

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

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

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

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

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

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

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

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

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

In another embodiment, the tetravalent bispecific antibody has the following characteristics: -it consists of:

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

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

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

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

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

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

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

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

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

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

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

In one embodiment, the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is between heavy chain variable domain position 44 to light chain variable domain position 100. In one embodiment, the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is between heavy chain variable domain position 105 to light chain variable domain position 43.

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

In another embodiment, the bispecific antibody is characterized by:

two antigen binding sites are formed by two pairs of heavy and light chain variable domains of a monospecific bivalent parent antibody, respectively, and both bind to the same epitope,

two further antigen binding sites are formed by the heavy and light chain variable domains of a single chain antibody,

-the single chain antibodies are linked to one heavy chain or one light chain, respectively, by peptide linkers, wherein each antibody chain end is linked to a single chain antibody only.

In another embodiment, the tetravalent bispecific antibody is characterized in that: the monospecific bivalent (full-length) antibody part under a) binds EGFR and the two monovalent monospecific single-chain antibodies under c) bind IGF-1R.

In another embodiment, the tetravalent bispecific antibody is characterized in that: the monospecific bivalent (full-length) antibody under a) binds in part to IGF-1R and the two monovalent monospecific single-chain antibodies under c) bind to EGFR.

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

In a second tetravalent embodiment, said tetravalent, bispecific antibody comprises

a) A full length antibody that specifically binds to the first antigen (one of the two antigens EGFR or IGF-1R) and consists of two antibody heavy chains and two antibody light chains; and

b) two identical single chain Fab fragments which specifically bind to said second antigen (the other of the two antigens EGFR or IGF-1R),

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

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

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

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

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

The term "peptide linker" as used in the present invention refers to a peptide having an amino acid sequence, which is preferably of synthetic origin. These peptide linkers according to the invention are used to fuse a single chain Fab fragment to the C-terminus or N-terminus of a full-length antibody to form a multispecific antibody according to the invention.

Preferably, said peptide linker under b) is a peptide having an amino acid sequence of at least 5 amino acids in length, preferably a peptide having an amino acid sequence of 5-100 amino acids in length, more preferably a peptide having an amino acid sequence of 10-50 amino acids in length. In one embodiment, the peptide linker is (GxS) n or (GxS) nGm, wherein G ═ glycine, S ═ serine, and (x ═ 3, n ═ 3, 4, 5, or 6, and m ═ 0, 1, 2, or 3) or (x ═ 4, n ═ 2, 3, 4, or 5 and m ═ 0, 1, 2, or 3), preferably x ═ 4 and n ═ 2 or 3, more preferably x ═ 4, n ═ 2. In one embodiment, the peptide linker is (G)₄S)₂。

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

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

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

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

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

Preferably, said second embodiment of the tetravalent bispecific antibody according to the invention comprises two identical single chain Fab fragments (preferably VL-CL-linker-VH-CH 1) fused both to the C-termini of the two heavy chains of said full length antibody under a) or fused both to the C-termini of the two light chains of said full length antibody under a). Such fusion results in the formation of two identical fusion peptides ((i) heavy chain and single chain Fab fragments or ii) light chain and single chain Fab fragments) which are co-expressed with i) the light or heavy chain of the full length antibody to provide a bispecific antibody according to the invention (see fig. 12, 13 and 14).

In another embodiment, the tetravalent bispecific antibody is characterized in that the full length antibody moiety under a) binds to EGFR and the two single chain Fab fragments under b) bind to IGF-1R.

In another embodiment, the tetravalent bispecific antibody is characterized in that the full length antibody moiety under a) binds to IGF-1R and the two single chain Fab fragments under b) bind to EGFR.

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

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

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

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

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

It has now been found that bispecific antibodies according to the invention have improved characteristics. Comparison with the use of only one individual antibody or a combination of two individual antibodies, or with Lu, D, et al, Biochemical and Biophysical research communications (Biochemical and Biophysical research) 318(2004) 507-; journal of biochemistry (j.biol.chem.), 279(2004) 2856-2865; they show at least the same or increased antitumor activity/efficacy in vitro and in vivo compared to bispecific antibodies of the journal of biochemistry (j.biol Chem.)280(2005) 19665-72. 507-513 in communication with Lu, D, et al, Biochemical and biophysical Research Communications 318 (2004); journal of biochemistry (j.biol.chem.), 279(2004) 2856-2865; they show improved in vivo pharmacokinetic stability compared to bispecific antibodies of the journal of biochemistry (j.biol.chem.)280(2005) 19665-72. In addition, bispecific antibodies according to the invention show modulated receptor downregulation/internalization compared to the use of only one individual antibody or a combination of two individual antibodies. Furthermore, bispecific antibodies according to the invention may provide benefits such as reduced dosage and/or frequency of administration and concomitant cost savings.

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

The term "constant region from human origin" as used herein refers to the constant heavy chain region and/or constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG 4. Such constant regions are well known in the art and are described, for example, by Kabat, E.A. (see, e.g., Johnson, G. and Wu, T.T., Nucleic Acids research (Nucleic Acids Res.)28(2000) 214-. While antibodies of the IgG4 subclass showed reduced Fc receptor (Fc γ RIIIa) binding, antibodies of the other IgG subclasses showed strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc sugar), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues which, if altered, also provide reduced Fc receptor binding (Shields, R., L., et al, J. Biol.Chem. (J.biol.chem.) -276 (2001) 6591-6604; Lund, J., et al, FASEB J.9(1995) 115-119; Morgan, A., et al, Immunology (Immunology)86(1995) 319-324; EP 0307434). In one embodiment, the antibody according to the invention has reduced FcR binding compared to the IgG1 antibody, and the monospecific bivalent (full length) parent antibody is involved in FcR binding of the IgG4 subclass or of the IgG1 or IgG2 subclass with mutations S228, L234, L235 and/or D265, and/or comprises a PVA236 mutation. In one embodiment, the mutation in the monospecific bivalent (full-length) parent antibody is S228P, L234A, L235A, L235E and/or PVA 236. In another embodiment, the mutation in the monospecific bivalent (full length) parent antibody is S228P in IgG4 and L234A and L235A in IgG 1. Constant heavy chain region is set forth in SEQ ID NO: 27 and 28. In one embodiment, the constant heavy chain region of the monospecific bivalent (full-length) parent antibody is the heavy chain region of SEQ ID NO: 27, constant heavy chain region. In another embodiment, the constant heavy chain region of the monospecific bivalent (full-length) parent antibody is SEQ ID NO: 28, and a constant heavy chain region. In another embodiment, the constant light chain region of the monospecific bivalent (full-length) parent antibody is SEQ ID NO: 29 constant light chain region.

The constant regions of antibodies are directly involved in ADCC (antibody-dependent cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by the binding of complement factor C1q to the constant regions of most IgG antibody subclasses. Binding of C1q to antibodies results from defined protein-protein interactions at the so-called binding site. Such constant region binding sites are known in the art and are described, for example, by Lukas, T.J., et al, J.Immunol 127(1981) 2555-2560; brunhouse, r., and Cebra, j.j., molecular immunology 16(1979) 907-; burton, D.R., et al, Nature 288(1980) 338-344; thommesen, J.E., et al, molecular immunology (mol. Immunol.)37(2000) 995-1004; idusogene, E.E., et al, J.Immunol. 164(2000) 4178-4184; hezareh, M., et al, J.Virol., 75(2001) 12161-12168; morgan, A., et al, Immunology 86(1995) 319-324; and EP 0307434. The constant region binding site is for example characterized by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).

The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to the lysis of human target cells by an antibody according to the invention in the presence of effector cells. ADCC is preferably measured by treating a preparation of IGF-1R and EGFR expressing cells with an antibody according to the invention in the presence of effector cells, such as freshly isolated PBMCs or purified effector cells from a buffy coat, such as monocytes or Natural Killer (NK) cells or permanently growing NK cell lines.

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

The cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide components as described in Umana, P., et al, Nature Biotechnol. 17(1999)176-180, and U.S. Pat. No. 6,602,684. IgG 1-type antibodies are the most commonly used therapeutic antibodies, which are glycoproteins with conserved N-linked glycosylation sites at Asn297 of each CH2 domain. Two complex biantennary oligosaccharides attached to Asn297 are hidden between the CH2 domains, making extensive contact with the polypeptide backbone, and their presence is essential for antibody-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, m.r., et al, Glycobiology (Glycobiology)5 (1995): 813-822; Jefferis, r., et al, immunological reviews (immunorev.) (1998): 59-76; Wright, a., and Morrison, s.l., biotechnological trends (trends biotechnol.)15 (1997): 26-32). Umana, p., et al, nature biotechnology (nature biotechnol.)17(1999)176-180 and WO 99/54342 show that β (1, 4) -N-acetylglucosaminyltransferase III ("GnTIII"), a glycosyltransferase that catalyzes the formation of bifurcated oligosaccharides, is overexpressed in Chinese Hamster Ovary (CHO) cells significantly increasing the in vitro ADCC activity of the antibody. Alterations in the composition of Asn297 sugars or their elimination also affect the binding of Fc γ R to C1q (Umana, P., et al, Nature Biotechnol. 17(1999) 176-180; Davies, J., et al, Biotechnol. Bioeng. 74(2001) 288-294; Mimura, Y., et al, J. Biochem. J. biol. chem. 276) 276(2001) 45539-45452001; Rad v, S., et al, J. Biochem. chem. 276 (276) 2001-16478-16483; Shields, R.L., et al, Biochem. 2001) 276- (6591-6604; Shields, R.L., Chel. 733, et al, Biochel. 2002) 147-277-75-12, Met. J. 2635, J. immunol. 35, J. Immunol. 547. 35, J. Immunol. 133, J. 35, S., G.J. 35, D. 35, D.J. 12, D.E.J. 547.).

Methods for enhancing cell-mediated effector function of monoclonal antibodies are described, for example, in WO 2005/044859, WO 2004/065540, WO2007/031875, Umana, p., et al, Nature biotechnology (Nature Biotechnol.) 17: 176-180(1999), WO 99/154342, WO 2005/018572, WO 2006/116260, WO 2006/114700, WO 2004/065540, WO 2005/011735, WO 2005/027966, WO 1997/028267, US 2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835 and WO 2000/061739 or, for example, in Niwa, R.et al, J.Immunol. methods)306(2005) 151-160; shinkawa, T., et al, J Biol Chem, 278(2003) 3466-; reported in WO 03/055993 and US 2005/0249722.

Thus, in one embodiment of the invention, the bispecific antibody is glycosylated (if it comprises an Fc part of the subclass IgG1, IgG2, IgG3 or IgG4, preferably of the subclass IgG1 or IgG 3) having a sugar chain at Asn297, wherein the amount of fucose in said sugar chain is 65% or less (numbering according to Kabat). In another embodiment, the amount of fucose in said sugar chain is between 5% and 65%, preferably between 20% and 40%. "Asn 297" according to the invention means the amino acid asparagine located at about position 297 of the Fc region. Based on minor sequence variations of the antibody, Asn297 may also be at some amino acid (typically no more than ± 3 amino acids) located upstream or downstream of position 297, i.e. between positions 294-300. In one embodiment, the glycosylated antibody IgG subclass according to the invention is of human IgG1 subclass, of human IgG1 subclass with mutations L234A and L235A, or of IgG3 subclass. In another embodiment, the amount of N-glycolylneuraminic acid (NGNA) in the sugar chain is 1% or less and/or the amount of N-terminal alpha-1, 3-galactose is 1% or less. The sugar chain preferably exhibits the characteristics of an N-linked glycan attached to Asn297 of an antibody recombinantly expressed in CHO cells.

The term "the sugar chain shows the characteristics of an N-linked glycan attached to Asn297 of an antibody recombinantly expressed in CHO cells" means that the sugar chain at Asn297 of the constant region of the bispecific antibody according to the invention has the same structure and sugar residue sequence as the same antibody expressed in unmodified CHO cells, e.g. as those antibodies reported in WO 2006/103100, except for the fucose residue.

The term "NGNA" as used herein refers to the sugar residue N-glycolylneuraminic acid.

Human IgG1 or IgG3 was glycosylated at Asn297, such as a core fucosylated biantennary complex oligosaccharide, terminating with up to two Gal residues. The human constant heavy chain region of the IgG1 or IgG3 subclasses is represented by Kabat, E.A., et al, Sequences of Proteins of immunological Interest (Sequences of Proteins of immunological Interest), 5 th edition Public Health Service (Public Health Service), National Institutes of Health (National Institutes of Health), Bethesda, Md. (1991), and by Brugreemann, M.et al, J.exp.Med.166(1987) 1351-; love, T.W., et al, Methods in enzymology (Methods Enzymol.)178(1989) 515-527. These structures are referred to as G0, G1 (. alpha. -1, 6-or. alpha. -1, 3-), or G2 glycan residues depending on the amount of terminal Gal residues (Raju, T.S., Bioprocess int.1(2003) 44-53). CHO-type glycosylation of the Fc part of antibodies is described, for example, by Router, F.H., J.glycoconjugate (Glycoconjugate J) 14(1997) 201-207. Antibodies recombinantly expressed in CHO host cells without sugar modification are typically fucosylated at Asn297 in an amount of at least 85%. The modified oligosaccharides of the constant region of the bispecific antibody according to the invention may be hybrid or complexed. Preferably, the branched, reduced/nonfucosylated oligosaccharides are hybrid. In another embodiment, the bisected, reduced/nonfucosylated oligosaccharides are complex.

According to the present invention, "amount of fucose" means an amount of the sugar in a sugar chain of Asn297, which is measured by MALDI-TOF mass spectrometry and calculated as an average value, as compared to the sum of all sugar structures attached at Asn297 (e.g., complex, hybrid and high mannose structures). The relative amount of fucose is the percentage of structures comprising fucose relative to all sugar structures (e.g., complex, hybrid and oligomeric and high mannose structures, respectively) identified by MALDI-TOF in the N-glycosidase F treated sample.

For all bispecific antibodies according to the invention, "GE" means glycoengineered.

In one other aspect of the invention, the bispecific antibody according to the invention is an antibody with ADCC and/or CDC and having a constant region from the IgG1 or IgG3 (preferably IgG1) subclass of human origin, which binds to Fc γ receptor and/or complement factor C1 q. Such antibodies that bind Fc receptors and/or complement factor C1q do elicit antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).

The antibodies according to the invention are produced by recombinant means. Thus, one aspect of the invention is a nucleic acid encoding an antibody according to the invention, and another aspect is a cell comprising a nucleic acid encoding an antibody according to the invention. Methods for recombinant production are widely known in the art and involve protein expression in prokaryotic and eukaryotic cells, followed by antibody isolation and often purification to pharmaceutical purity. For expression of the foregoing antibodies in a host cell, the nucleic acids encoding the respective modified light and heavy chains are inserted into the expression vector by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER. C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). General methods for recombinant production of antibodies are well known in the art and are described, for example, in Makrides, S.C., Protein Expr. Purif.17(1999) 183-202; geisse, S., et al, Protein Expr. Purif 8(1996) 271-282; kaufman, R.J., molecular biology techniques (mol.Biotechnol.)16(2000) 151-160; werner, R.G., Drug Res.48(1998) 870-.

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

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

Bispecific antibodies that bind to EGFR and IGF-1R according to the invention down-regulate EGFR. In a549 cells, the downregulation of EGFR is at least about 30% in one embodiment, at least about 35% in another embodiment, and at least about 40% in another embodiment.

The bispecific antibodies that bind EGFR and IGF-1R according to the invention down-regulate IGF-1R. In H322M cells, bispecific Cross-Mab (VH/VL) or Cross-Mab (CH/CL) down-regulated IGF-1R up to about 15% in one embodiment, up to about 20% in another embodiment, and up to 40% in another embodiment (75. mu.g protein/mL).

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

Expression in NS0 cells is described, for example, in Barnes, L.M., et al, Cytotechnology 32(2000) 109-123; barnes, L.M., et al, Biotechnology and bioengineering (Biotech.Bioeng.)73(2001) 261-270. Transient expression is described, for example, in Durocher, y., et al, nucleic acid research (nucl. acids. res.)30E9 (2002). Cloning of variable domains is described in Orlandi, R.et al, Proc.Natl.Acad.Sci.USA 86(1989) 3833-3837; carter, p., et al, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.usa)89(1992) 4285-; and Norderhaug, l., et al, journal of immunological methods (j.immunological. method)204(1997) 77-87. Preferred transient expression systems (HEK 293) are described in Schlaeger, E.J., and Christensen, K., in Cytotechnology (Cytotechnology)30(1999)71-83 and Schlaeger, E.J., in journal of immunological methods (J.Immunol.methods)194(1996) 191-199.

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

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

Antibodies according to the present invention having a reduced amount of fucose may be expressed in a carbohydrate-modified host cell engineered to express at least one nucleic acid encoding a polypeptide having GnTIII activity and a polypeptide having ManII activity in an amount that fucosylates oligosaccharides in the Fc region according to the present invention. In one embodiment, the polypeptide having GnTIII activity is a fusion polypeptide. Alternatively, the α 1, 6-fucosyltransferase activity of the host cell may be reduced or eliminated according to US 6,946,292 to produce a sugar-modified host cell. The amount of antibody fucosylation may be predetermined, for example, by fermentation conditions or by the combination of at least two antibodies with different amounts of rock glycosylation.

The antibodies according to the invention with reduced amounts of fucose can be produced in a host cell by a method comprising the steps of: (a) culturing a host cell engineered to express at least one polynucleotide encoding a fusion polypeptide having GnTIII activity and/or man ii activity under conditions permitting production of said antibody and permitting fucosylation of oligosaccharides present on the Fc region of said antibody in an amount according to the invention; and (b) isolating the antibody. In one embodiment, the polypeptide having GnTIII activity is a fusion polypeptide, preferably comprising the catalytic domain of GnTIII and the golgi localization domain of a heterologous golgi colonization (residual) polypeptide, said localization domain being selected from the group consisting of: the localization domain of mannosidase II, the localization domain of beta (1, 2) -N-acetylglucosaminyltransferase I ("GnTI"), the localization domain of mannosidase I, the localization domain of beta (1, 2) -N-acetylglucosaminyltransferase II ("GnTII"), and the localization domain of alpha 1-6 core fucosyltransferase. Preferably, the golgi localization domain is from mannosidase II or GnTI.

As used herein, a "polypeptide having GnTIII activity" refers to a polypeptide that is capable of catalyzing the addition of N-acetylglucosamine (GlcNAc) residues to the β -linked mannosides of the trimannosyl core of an N-linked oligosaccharide in a β -1-4 linkage. This includes fusion polypeptides that exhibit an enzymatic activity similar to, but not necessarily identical to, that of β (1, 4) -N-acetylglucosaminyltransferase III, with or without dose-dependence as measured in a particular bioassay, also known as β -1, 4-mannosyl-glycoprotein 4- β -N-acetylglucosaminyltransferase (EC 2.4.1.144), according to the international committee for nomenclature of biochemistry and molecular biology (NC-IUBMB). In cases where dose-dependence does exist, it need not be the same as dose-dependence of GnTIII, but is substantially similar to dose-dependence in a given activity as compared to GnTIII activity (i.e., the candidate polypeptide will exhibit greater activity or no more than about 25-fold less activity, and preferably, no more than about 10-fold less activity, and most preferably, no more than about three-fold less activity relative to GnTIII). As used herein, the term "golgi localization domain" refers to the amino acid sequence of a golgi-colonizing polypeptide that is responsible for anchoring the polypeptide in place of the golgi complex. Typically, the localization domain comprises the amino-terminal "tail" of the enzyme.

For the production of antibodies according to the invention with reduced amounts of fucose, host cells capable of and adapted to allow the production of antibodies with modified glycoforms can likewise be used. The host cell may be further manipulated to express increased levels of one or more polypeptides having GnTIII activity. CHO cells are preferred as such host cells. Likewise, cells producing antibody compositions with increased ADCC are reported in US 6,946,292.

Purification of the antibody to eliminate cellular components or other contaminants, such as other cellular nucleic acids or proteins, is performed by standard techniques, including alkali/SDS treatment, CsCl fractionation (CsCl banding), column chromatography, agarose gel electrophoresis, and other techniques known in the art. See Ausubel, F., et al, eds, methods in modern Molecular Biology (Current Protocols in Molecular Biology), Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widely used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein a or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin) and mixed mode exchange), thiophilic adsorption (e.g. with β -mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resin, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with ni (ii) -and cu (ii) -affinity materials), size exclusion chromatography and electrophoretic methods (e.g. gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, m., a., applied biochemical biotechnology (biochemical applied. biotech.) 75(1998) 93-102).

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

It has surprisingly been found that when compared to monospecific parent anti-EGFR antibodies and anti-IGF-1R antibodies, or to Biochemical and biophysical Research Communications from Lu, D., et al (Biochemical and biological Research Communications)318(2004) 507-; journal of biochemistry (j.biol.chem.), 279(2004) 2856-2865; and the journal of biochemistry (j.biol.chem.)280(2005)19665-72 known bispecific antibodies against EGFR and against IGF-1R (as a combined comparison of these bispecific antibodies with the respective parent antibody shows only a reduced efficacy in EGFR/IGF-1R expressing tumor cells), the bispecific antibodies against EGFR and against IGF-1R according to the invention have an improved anti-proliferative property against cancer cells.

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

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

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

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

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

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

The term cancer as used herein refers to proliferative diseases such as lymphoma (lymphoma), lymphocytic leukemia (lymphocytic leukemia), lung cancer (lung cancer), non-small cell lung (NSCL) cancer (non-small cell lung (NSCL) cancer), bronchoalveolar cell lung cancer (bronchololar cell lung cancer), bone cancer (bone cancer), pancreatic cancer (pancreatic cancer), skin cancer (skin cancer), head or neck cancer (cancer of the head, skin or intraocular melanoma (cutaneous or intraocular tumor cell), uterine cancer (uterine cancer), ovarian cancer (ovarian cancer), rectal cancer (rectal cancer), anal region cancer (cancer of the anal region), gastric cancer (gastric cancer), ovarian cancer (ovarian cancer), uterine cancer (ovarian cancer of the uterine cancer), ovarian cancer (ovarian cancer of the uterine cancer (colon cancer), uterine cancer (colon cancer, colon cancer (colon cancer), colon cancer (colon cancer, colon cancer (colon cancer, colon, cervical cancer (cancer of the cervical cancer), vaginal cancer (cancer of the vagina), vulvar cancer (cancer of the vulcana), Hodgkin's Disease, esophageal cancer (cancer of the esophageal), small intestine cancer (cancer of the small intestine), endocrine system cancer (cancer of the endocrine system), thyroid cancer (cancer of the thyroid gland), parathyroid cancer (cancer of the parathyroid gland), adrenal cancer (cancer of the cancere gland), soft tissue sarcoma (cancer of the soft tissue), urethral cancer (cancer of the urethra), penile cancer (cancer of the lung), prostate cancer (cancer of the prostate), bladder cancer (cancer of the bladder, renal cancer (cancer of the ureter), renal cancer (cancer of the renal cell), bile duct carcinoma (biliary cancer), Central Nervous System (CNS) tumors (neoplasms of the Central Nervous System (CNS)), vertebral axis tumors (spinal axis tumors), brain stem glioma (brain stem glioma), glioblastoma multiforme (gliobastoma), astrocytoma (astrocytoma), schwanomas (schwanomas), ependymomas (ependomas), medulloblastoma (medulloblastoma), meningioma (menigiomas), squamous cell carcinoma (squamomus cell carcinomas), pituitary adenoma (pituitary adenoma), and evans's sarcoma, including refractory forms of any of the foregoing cancers, or combinations of one or more of the foregoing cancers.

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

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

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

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

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

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

The term "transformation" as used herein refers to the process of transferring a vector/nucleic acid into a host cell. If cells without a difficult cell wall barrier are used as host cells, transfection is carried out, for example, by the calcium phosphate precipitation method as described by Graham, F.L. and van der Eb.A.J., Virology 52(1973) 456-467. However, other methods of introducing DNA into cells may also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells comprising a parenchymal cell wall structure are used, for example, one method of transfection is calcium treatment with calcium chloride, as described by Cohen, S.N, et al, PNAS (proceedings of the national academy of sciences USA) 69(1972) 2110-2114.

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

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

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

Description of the amino acid sequence

SEQ ID NO: 1 heavy chain CDR3, humanized < EGFR > ICR62

SEQ ID NO: 2 heavy chain CDR2, humanized < EGFR > ICR62

SEQ ID NO: 3 heavy chain CDR1, humanized < EGFR > ICR62

SEQ ID NO: 4 light chain CDR3, humanized < EGFR > ICR62

SEQ ID NO: 5 light chain CDR2, humanized < EGFR > ICR62

SEQ ID NO: 6 light chain CDR1, humanized < EGFR > ICR62

SEQ ID NO: 7 heavy chain variable domain, humanized < EGFR > ICR62-I-HHB

SEQ ID NO: 8 heavy chain variable domain, humanized < EGFR > ICR62-I-HHD

SEQ ID NO: 9 light chain variable domain, humanized < EGFR > ICR62-I-KA

SEQ ID NO: 10 light chain variable domain, humanized < EGFR > ICR62-I-KC

SEQ ID NO: 11 heavy chain CDR3, < IGF-1R > HUMAB-clone 18

SEQ ID NO: 12 heavy chain CDR2, < IGF-1R > HUMAB-clone 18

SEQ ID NO: 13 heavy chain CDR1, < IGF-1R > HUMAB-clone 18

SEQ ID NO: 14 light chain CDR3, < IGF-1R > HUMAB-clone 18

SEQ ID NO: 15 light chain CDR2, < IGF-1R > HUMAB-clone 18

SEQ ID NO: 16 light chain CDR1, < IGF-1R > HUMAB-clone 18

SEQ ID NO: 17 heavy chain CDR3, < IGF-1R > HUMAB-clone 22

SEQ ID NO: 18 heavy chain CDR2, < IGF-1R > HUMAB-clone 22

SEQ ID NO: 19 heavy chain CDR1, < IGF-1R > HUMAB-clone 22

SEQ ID NO: 20 light chain CDR3, < IGF-1R > HUMAB-clone 22

SEQ ID NO: 21 light chain CDR2, < IGF-1R > HUMAB-clone 22

SEQ ID NO: 22 light chain CDR1, < IGF-1R > HUMAB-clone 22

SEQ ID NO: 23 heavy chain variable domain, < IGF-1R > HUMAB-clone 18

SEQ ID NO: 24 heavy chain variable domain, < IGF-1R > HUMAB-clone 22

SEQ ID NO: 25 light chain variable domain, < IGF-1R > HUMAB-clone 18

SEQ ID NO: 26 light chain variable domain, < IGF-1R > HUMAB-clone 22

SEQ ID NO: 27 human heavy chain constant region from IgG1

SEQ ID NO: 28 human heavy chain constant region from IgG4

SEQ ID NO: 29kappa light chain constant region

SEQ ID NO: 30 bispecific, bivalent domain exchanged < EGFR-IGF1R > antibody molecules: heavy chain 1 of Cross-Mab (VH/VL)

SEQ ID NO: 31 bispecific, bivalent domain exchanged < EGFR-IGF1R > antibody molecule: heavy chain 2 of Cross-Mab (VH/VL)

SEQ ID NO: 32 bispecific, bivalent domain exchanged < EGFR-IGF1R > antibody molecules: Cross-Mab (VH/VL) light chain 1

SEQ ID NO: 33 bispecific, bivalent domain exchanged < EGFR-IGF1R > antibody molecule: light chain 2 of Cross-Mab (VH/VL)

SEQ ID NO: 34 bispecific, bivalent domain exchanged < EGFR-IGF1R > antibody molecule: heavy chain 1 of Cross-Mab (CH/CL)

SEQ ID NO: 35 bispecific, bivalent domain exchanged < EGFR-IGF1R > antibody molecule: heavy chain 2 of Cross-Mab (CH/CL)

SEQ ID NO: 36 bispecific, bivalent domain exchanged < EGFR-IGF1R > antibody molecule: Cross-Mab (CH/CL) light chain 1

SEQ ID NO: 37 bispecific, bivalent domain exchanged < EGFR-IGF1R > antibody molecule: Cross-Mab (CH/CL) light chain 2

SEQ ID NO: 38 bispecific, bivalent scFab-Fc fusion < EGFR-IGF1R > antibody molecule: heavy chain 1 of scFab-Fc

SEQ ID NO: 39 bispecific, bivalent scFab-Fc fusion < EGFR-IGF1R > antibody molecules: heavy chain 2 of scFab-Fc

SEQ ID NO: 40 bispecific, bivalent scFab-Fc fusion < EGFR-IGF1R > antibody molecules: heavy chain 1 of N-scFabSS-salt bridge-s 3

SEQ ID NO: 41 bispecific, bivalent scFab-Fc fusion < EGFR-IGF1R > antibody molecules: heavy chain 2 of N-scFabSS-salt bridge-s 3

SEQ ID NO: 42 bispecific, bivalent scFab-Fc fusion < EGFR-IGF1R > antibody molecules: heavy chain 1 of N-scFabSS-salt bridge-w 3C

SEQ ID NO: 43 bispecific, bivalent scFab-Fc fusion < EGFR-IGF1R > antibody molecules: heavy chain 2 of N-scFabSS-salt bridge-w 3C

SEQ ID NO: 44 bispecific, trivalent scFab-IgG fusion < EGFR-IGF1R > antibody molecules: heavy chain 1 of KiH-C-scFab-1

SEQ ID NO: 45 bispecific, trivalent scFab-IgG fusion < EGFR-IGF1R > antibody molecules: heavy chain 2 of KiH-C-scFab-1

SEQ ID NO: 46 bispecific, trivalent scFab-IgG fusion < EGFR-IGF1R > antibody molecules: light chain of KiH-C-scFab-1

SEQ ID NO: 47 bispecific, trivalent scFab-IgG fusion < EGFR-IGF1R > antibody molecules: heavy chain 1 of KiH-C-scFab-2

SEQ ID NO: 48 bispecific, trivalent scFab-IgG fusion < EGFR-IGF1R > antibody molecules: heavy chain 2 of KiH-C-scFab-2

SEQ ID NO: 49 bispecific, trivalent scFab-IgG fusion < EGFR-IGF1R > antibody molecules: light chain of KiH-C-scFab-2

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

Brief Description of Drawings

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

FIG. 2 schematic structures of four possible tetravalent embodiments A-D of the bispecific antibody according to the invention binding EGFR and IGF-1R, wherein one of the antigens A or B is EGFR and the other is IGF-1R. The structure is based on a full-length antibody that binds antigen a to which two (optionally disulfide-stabilized) single-chain Fv's that bind antigen B are linked by peptide-linkers at the following positions:

a: c-terminus of full-length antibody heavy chain

B: n-terminus of full-length antibody heavy chain

C: c-terminus of full-length antibody light chain

D: c-terminus of full-length antibody light chain

Fig. 3.3 a: SDS-PAGE of purified bispecific antibody XGFR1-2421

3 b: HP-Size Exclusion Chromatography (SEC) analysis of purified bispecific antibody XGFR1-2421(3 mg/ml)

3 c: HP-Size Exclusion Chromatography (SEC) analysis of purified bispecific antibody XGFR1-2421(1 mg/ml)

Fig. 4.4 a: HP-Size Exclusion Chromatography (SEC) purification of bispecific antibody XGFR1-2320 (disulfide free stabilized) (8.7% aggregates)

4 b: HP-Size Exclusion Chromatography (SEC) purification (0% aggregates) of bispecific antibody XGFR1-2321 (disulfide stabilized)

FIG. 5 Simultaneous binding of bispecific anti-EGFR/anti-IGF-1R antibody (XGFR1-2320) to EGFR and IGF1R in a Biacore assay with immobilized XGFR1-2320

FIG. 6 Down-Regulation of IGFR (6a) and EGFR (6b) in A549NSCLC tumor cell line by bispecific antibodies

FIG. 7 bispecific anti-EGFR/anti-IGF-1R antibody molecule (XGFR) inhibits IGF-1R phosphorylation (7a) and EGFR phosphorylation (7b) in H322M NSCLC tumor cell line

7 a: Phospho-EGF-R-ELISA after inhibition in H322MNSCLC tumor cells with various bispecific antibody XGFR molecules and their parent antibodies, the antibody concentration being dependent on the incubation, in the case of stimulation with IGF1/EGF antibody, the concentration being diluted to half the initial concentration

7 b: Phospho-EGF-R-ELISA after inhibition in H322MNSCLC tumor cells with various bispecific antibody XGFR molecules and their parent antibodies, the antibody concentration being dependent on the incubation, in the case of stimulation with IGF1/EGF antibody, the antibody concentration being diluted to half the initial concentration

FIG. 8 anti-tumor growth inhibition of EGFR-and IGF-1R expressing H322M NSCLC tumor cells by bispecific anti-EGFR/anti-IGF-1R antibody molecules (XGFR) and their parent antibodies

FIG. 9 in vitro ADCC Activity of bispecific anti-EGFR/anti-IGF-1R antibody molecule (XGFR)

FIG. 10 schematic structure of a full length antibody without the CH4 domain that specifically binds EGFR or IGF1-R, having two pairs of heavy and light chains comprising variable and constant domains in typical order.

FIG. 11 schematic structures of four possible single chain Fab fragments specifically binding to, for example, EGFR or IGF1-R

FIG. 12 schematic structure of a tetravalent, bispecific antibody according to the invention, comprising a full length antibody specifically binding to one of the two antigens EGFR or IGF1-R and two single chain Fabs (scFab-XGFR molecules) specifically binding to the other of the two antigens EGFR or IGF1-R

FIG. 13, bispecific antibody-ScFab-XGFR 1 molecules A, B, C, and D according to the invention comprising a full length antibody specifically binding IGF-1R and two identical single chain Fabs specifically binding EGFR and expression levels after purification

A: scFab fused to the C-terminus of the heavy chain (VH-CH 1-linker-VL-CL)

B: scFab fused to the C-terminus of the heavy chain (VH-CH 1-linker-VL-CL, fused to an additional VH44-VL100 disulfide bridge)

C: scFab fused to the C-terminus of the light chain (VH-CH 1-linker-VL-CL)

D: scFab fused to the C-terminus of the light chain (VH-CH 1-linker-VL-CL, fused to an additional VH44-VL100 disulfide bridge)

FIG. 14 bispecific antibody-ScFab-XGFR 2 molecules A, B, C, and D according to the invention comprising a full length antibody specifically binding EGFR and two identical single chain Fabs specifically binding IGF-1R

A: scFab fused to the C-terminus of the heavy chain (VH-CH 1-linker-VL-CL)

C: scFab fused to the C-terminus of the light chain (VH-CH 1-linker-VL-CL)

FIG. 15 SDS-PAGE analysis of the bispecific antibody derivative scFab-XGFR1 comprising single chain Fab

1: scFab-XGFR1_4720 (unreduced)

2: scFab-XGFR1_4721 (unreduced)

3: scFab-XGFR1_4720 (reduction)

4: scFab-XGFR1_4721 (reduction)

FIG. 16 HP-SEC analysis of the bispecific antibody derivative scFab-XGFR1 comprising scFab

FIG. 16 a: scFab-XGFR 1-4720; 7.7% of aggregate (marked with box)

FIG. 16 b: scFab-XGFR 1-4721; 3.5% of aggregate (marked with box)

FIG. 17 binding of scFab-XGFR1 and scFab-XGFR2 to EGFR and IGF1R

FIG. 17 a: binding of Biacore map-scFab-XGFR 1_2720 to EGFR with KD of 2nM

FIG. 17 b: binding of Biacore map-scFab-XGFR 1_2720 to IGF-1R with KD of 2nM

FIG. 17 c: binding of Biacore map-scFab-XGFR 2_2720 to EGFR with KD ═ 0.5nM

FIG. 17 d: binding of Biacore map-scFab-XGFR 2_2720 to IGF-1R with KD of 11nM

Figure 18 schematic-binding of scFab-XGFR to cells analyzed with FACS competition assay using the following general method:

parallel addition of < IGF1R > Mab + unlabelled scFab-XGFR (100. mu.g/mL-0,001. mu.g/mL) labeled with Alexa647 (1. mu.g/mL)

Incubation on ice for 45 minutes, washing and removal of unbound antibody

Immobilization with 1% HCHO, followed by FACS

FIG. 19 binding of scFab-XGFR _2721 and parent < IGF1R > clone 18 to cells analyzed by FACS competition assay

FIG. 19 a: comparison of IC50 values for < IGF-1R > clone 18(0, 18. mu.g/ml) and scFab-XGFR _2721(0, 15. mu.g/ml)

FIG. 19 b: < binding curve of IGF-1R > clone 18 (turning point 0, 11 μ g/ml) -y-axis ═ RLU; x-axis antibody concentration μ g/ml)

FIG. 19 c: binding curve of scFab-XGFR _2721 (turning point 0, 10 μ g/ml) -y-axis ═ RLU; x-axis antibody concentration (μ g/ml)

FIG. 20 Down-Regulation of IGF1-R on H322M cells after 24H incubation with different bispecific anti-EGFR/anti-IGF-1R antibody molecules (scFab-XGFR; 100nM)

FIG. 21 upregulation of EGFR in H322M cells after 24H incubation with different bispecific anti-EGFR/anti-IGF-1R antibody molecules (scFab-XGFR; 100nM)

FIG. 22 inhibition of H322M-cell proliferation with different bispecific anti-EGFR/anti-IGF-1R antibody molecules (scFab-XGFR; 100nM)

Experimental procedures

Examples

Design of bispecific < EGFR-IGF-1R > antibodies

The bispecific antibody that binds to EGFR and IGF-1R according to the present invention comprises a first antigen-binding site that binds to EGFR and a second antigen-binding site that binds to IGF-1R. SEQ ID NO: 7 or SEQ ID NO: 8, and the heavy chain variable domain of SEQ ID NO: 9 or SEQ ID NO: 10 as a first antigen binding site for binding to EGFR, both from the humanized rat anti-EGFR antibody ICR62, which is described in detail in WO 2006/082515.

SEQ ID NO: 23 or SEQ ID NO: 24, and the heavy chain variable domain of SEQ ID NO: 25 or SEQ ID NO: 26 as a second antigen binding site for binding IGF-1R, both from the human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC2587) or < IGF-1R > HUMAB clone 22(DSM ACC 2594), which is described in detail in WO 2005/005635.

In all of the following examples 1-20, the bispecific < EGFR-IGF-1R > antibody is based on the amino acid sequence of SEQ ID NO: 8, and the heavy chain variable domain of SEQ ID NO: 10 (from humanized < EGFR > ICR62), and is based on the amino acid sequence of SEQ ID NO: 23, and the heavy chain variable domain of SEQ ID NO: 25 (from human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC 2587)).

A) Designing bispecific < EGFR-IGF-1R > antibodies with scFv linker (attachment) (XGFR1 and XGFR2 nomenclature, refer to scFv-XGFR molecules)

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

Human anti-IGF-1R antibody based < IGF-1R > HUMAB clone 18(DSM ACC2587) and a nucleic acid sequence derived from SEQ ID NO: 8 and the heavy chain variable domain (VH) of SEQ ID NO: a summary of the designed versions of EGFR-binding single chain fv (scfv) of the light chain variable domain (VL) of 10 is shown in figure 1 and listed in tables 1 and 2.

By gene synthesis and recombinant molecular biology techniques, the nucleotide sequence of SEQ ID NO: 8 and the heavy chain variable domain (VH) of SEQ ID NO: 10 are linked by a glycine serine (G4S) n single linker to provide a single chain fv (scfv) that binds EGFR, linked to an anti-IGF-1R antibody<IGF-1R>HUMAB clone 18(DSM ACC2587) variable position at the N-or C-terminus of the light or heavy chain. Furthermore, cysteine residues are introduced at different positions in the VH domain (including Kabat position 44) and the VL domain (including Kabat position 100) of an scFv that binds EGFR, as described previously (e.g., WO 94/029350; Reiter, Y., et al, Nature biotechnology 14(1996)1239-<IGF1R>The length of the (glycine 4-serine) n-containing peptide linker between the C-terminus of the heavy or light chain of the antibody and the scFv that binds EGFR varies. In addition, glycine 4-serine (G), an integral part of a single chain Fv module that binds EGFR₄S) the length of the single link is varied. All these molecules were recombinantly produced, purified and characterized. Bispecific for generating tetravalent are provided in tables 1 and 2<EGFR-IGF1R>Summary of all bispecific antibody designs for antibodies. For this study we used the term'XGFR' describes various protein entities that recognize both EGFR and IGF1R and comprise a full-length antibody that specifically binds to one of EGFR or IGF1R and two scFv fragments that specifically bind to the other of EGFR or IGF 1R.

Table 1-different bispecific < EGFR-IGF1R > antibody formats with N-and C-terminal scFv linkers, and corresponding XGFR 1-nomenclature and XGFR 2-nomenclature.

The XGFR1 format is based on a) the human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC2587) and b) a sequence derived from SEQ ID NO: 8 and seq id NO: 10, linked to the same end (C-or N-terminus) of the Heavy Chain (HC) or Light Chain (LC) of an anti-IGF-1R antibody < IGF-1R > HUMAB clone 18.

The XGFR2 format is based on a) the variable region VH of the humanized rat anti-EGFR antibody ICR62 (SEQ ID NO: 8) and VL (SEQ ID NO: 10) and b) human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC2587) binding to anti-IGF-1R antibody < IGF-1R > HUMAB clone 18 (SEQ ID NO: 23) and VL (SEQ ID NO: 25) two single chain fv (scFv) of (1).

In the table, "-" means "absence"

Table 2-XGFR 1 bispecific antibodies with variable single-chain linker and peptide linker lengths. In the table, "-" indicates "absence".

Examples 1-8 relating to tetravalent XGFR1 and XGFR2 molecules B with scFv linker) design of tetravalent, bispecific < EGFR-IGF-1R > antibodies with Single chain Fab (scFab) linker (scFab-XGFR1 and scFab-XGFR2 nomenclature)

The term scFab-XGFR is used to describe a full length antibody that recognizes both EGFR and IGF1R and comprises one that specifically binds to EGFR or IGF 1R; and various protein entities that specifically bind to two scFab fragments of the other of EGFR or IGF 1R.

In one embodiment of the invention below, tetravalent bispecific antibodies are listed, comprising a full-length antibody binding to a first antigen (IGF-1R or EGFR), with two single-chain Fab fragments binding to a second, different antigen (the other of IGF-1R or EGFR) linked to the full-length antibody (two single-chain Fab fragments at both C-termini of the heavy chain or two single-chain Fab fragments at both C-termini of the light chain) by a peptide linker. The antibody domains and linkers in the single chain Fab fragments have the following order in the N-terminal to C-terminal direction: VL-CL-linker-VH-CH 1.

Using SEQ ID NO: 23 as < IGF-1R > antigen binding site. Using SEQ ID NO: 25 light chain variable domain VL as < IGF-1R > antigen binding site.

Using SEQ ID NO: 8 the heavy chain variable domain VH which serves as the < EGFR > antigen binding site. Using SEQ ID NO: 10 light chain variable domain VL as < EGFR > antigen binding site.

VL-CL and VH-CH1, which contain VH and VL of the respective antigen binding sites, are linked by a glycine serine (G4S) nGm linker to provide a single chain Fab fragment VL-CL-linker-VH-CH 1, which is linked to the C-terminus of the antibody heavy or light chain using a (G4S) n peptide linker, by gene synthesis and recombinant molecular biology techniques.

Optionally, cysteine residues are introduced into the VH (including Kabat position 44) and VL (including Kabat position 100) domains of the single-chain Fab fragment according to the previously described techniques (e.g., WO 94/029350; Reiter, Y., et al, Nature biotechnology (1996) 1239-.

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

A summary of the antibody design for the production of tetravalent, bispecific scFab < EGFR-IGF-1R >, < IGF-1R-EGFR > antibodies is provided in table 3. For this study, we used the term 'scFab' to describe various tetravalent protein entities. A representation of the form of the design is shown in fig. 13 and 14 and listed in table 3.

TABLE 3-different bispecific anti-IGF 1R and anti-EGFR scFab-tetravalent antibody formats with C-terminal single chain Fab fragment linkers and corresponding scFab-Ab-nomenclature

Examples 9-13 relate to tetravalent scFab-XGFR1 and scFab-XGFR2 molecules with single chain Fab linker

Materials and general methods

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

Recombinant DNA technology

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

Gene synthesis

The desired gene fragment is prepared from oligonucleotides prepared by chemical synthesis. The 600-1800bp long gene fragment flanked by single restriction endonuclease cleavage sites was assembled by annealing and oligonucleotide ligation including PCR amplification and subsequently cloned into the pUC based cloning vector based pcDNA 3.1/Zeo (+) (Invitrogen) through the indicated restriction enzyme cleavage sites, e.g., BamHI/BstEII, BamHI/BsiWI, BstEII/NotI or BsiWI/NotI. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. The gene synthesis fragments were ordered according to the instructions specified in Geneart (Regensburg, Germany).

DNA sequencing

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

DNA and protein sequence analysis and sequence data processing

GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Invitrogen vector Advance suite version 9.1 were used for sequence generation, mapping, analysis, annotation and illustration.

Cell culture technique

Standard Cell culture techniques are used as described in Current methods in Cell Biology (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 expression of immunoglobulin variants in HEK293F cells

FreeStyle was used according to the manufacturer's instructions^TMThe 293 expression system (Invitrogen, USA) expresses bispecific antibodies by transient transfection of human embryonic kidney 293-F cells. Briefly, the suspension FreeStyle^TM293-F cells in FreeStyle^TM293 expression Medium at 37 ℃/8% CO₂Cultures were performed and cells were plated at 1-2X10 on the day of transfection⁶Viable cells/ml were seeded in fresh medium. 333 μ l of 293fectin was used^TM(Invitrogen, Germany) and 250. mu.g of 1: 1 molar ratio of heavy and light chain plasmid DNAPreparation of DNA-293fectin I Medium (Invitrogen, USA)^TMThe final transfection volume was 250 ml. 7 days after transfection, cell culture supernatants containing bispecific antibody were clarified by centrifugation at 14000g for 30 minutes and filtration through sterile filters (0.22 μm). The supernatant was stored at-20 ℃ until purification.

Protein determination

The Protein concentration of purified antibodies and derivatives was determined by determining the Optical Density (OD) at 280nm (using OD at 320nm as a background correction) using the molar extinction coefficient calculated based on the amino acid sequence according to Pace, C.N., et al, Protein Science (Protein Science), 4(1995) 2411-1423.

Determination of antibody concentration in supernatant

The concentration of antibodies and derivatives in the cell culture supernatant was measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind protein a were Applied to Applied Biosystems (Applied Biosystems) Poros a/20 columns in 200mM KH2PO4, 100mM sodium citrate, pH7.4 and eluted from the matrix with 200mM NaCl, 100mM citric acid, pH2,5 on an UltiMate 3000HPLC system (Dionex). Eluted protein was quantified by UV absorbance and integration of peak area. Purified standard IgG1 antibody was used as a standard.

Protein purification

By using protein A-agarose^TM(Protein A-Sepharose^TM) (GE healthcare, Sweden) affinity chromatography and Superdex200 size exclusion chromatography, the secreted antibody was purified from the supernatant in two steps. Briefly, clarified culture supernatants containing bispecific and trispecific antibodies were applied to a HiTrap protein a HP (5ml) column with PBS buffer (10mM Na) buffer₂HPO₄，1mM KH₂PO₄137mM NaCl and 2.7mM KCl, pH 7.4). Unbound protein was washed out with equilibration buffer. Bispecific antibody with 0.1M citrate buffer, pH2.8 elution, and protein containing fractions with 0.1ml 1M Tris, pH8.5 neutralization. Subsequently, the eluted protein fractions were pooled, concentrated to a volume of 3ml using an Amicon ultracentrifuge filter unit (MWCO: 30K, Millipore) and loaded onto a Superdex200HiLoad 120ml16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20mM histidine, 140mM NaCl, pH 6.0. Monomeric antibody fractions were pooled, snap frozen and stored at-80 ℃. Portions of the sample are provided for subsequent protein analysis and characterization.

Analysis of purified proteins

The protein concentration of the purified protein sample was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. By reducing the presence and absence of a reducing agent (5mM 1, 4-bisStachyose) were subjected to SDS-PAGE and stained with coomassie brilliant blue to analyze the purity of the bispecific antibody. Used according to the manufacturer's instructionsPre-Cast gel system (Invitrogen, USA) (4-20% Tris-glycine gel). At 25 deg.C, 200mM KH₂PO₄Superdex200 in 250mM KCl, pH7.0 running buffer was analyzed on size exclusion columns (GE Healthcare, Sweden) and aggregate content of bispecific antibody samples was analyzed by high performance SEC on an Ultimate 3000HPLC system (Dionex). Mu.g of protein was injected onto the column at a flow rate of 0.5ml/min and eluted isocratically over 50 minutes. For stability analysis, purified protein was prepared at concentrations of 0.1mg/ml, 1mg/ml and 3mg/ml and incubated at 4 ℃ for 7 days at 37 ℃ followed by evaluation by high performance SEC. The integrity of the amino acid backbone of the reduced bispecific antibody light and heavy chains was confirmed by NanoElectrospray Q-TOF mass spectrometry after removal of the N-glycans by enzymatic treatment with peptide-N-glycosidase F (Roche Molecular Biochemicals).

Example 1

Expression and purification of bispecific < EGFR-IGF1R > antibody XGFR1 molecules

The light and heavy chains of the corresponding bispecific antibodies were constructed in expression vectors carrying prokaryotic and eukaryotic selection markers. These plasmids were amplified in E.coli, purified, and then transfected to perform transient expression of recombinant proteins in HEK293F cells using the Invitrogen's free System (Invitrogen's freesystem). After 7 days, HEK293 cell supernatants were harvested and purified by protein a and size exclusion chromatography. The homogeneity of all bispecific antibody constructs was confirmed by SDS-PAGE under non-reducing and reducing conditions. Under reducing conditions (FIG. 2a), the polypeptide chains carrying the C-terminal and N-terminal scFv fusions showed an apparent molecular size on SDS-PAGE similar to the calculated molecular weight. All constructs were analyzed by protein AHPLC for expression levels that were similar to the expression yield of 'standard' IgGs, or in some cases slightly lower. In such non-optimized transient expression experiments (FIG. 3), the average protein yield was 1-36mg of purified protein per liter of cell culture supernatant. Non-disulfide stable constructs of scFvs with C-terminal fusions in either the light chain (XGFR-4320) or the heavy chain (XGFR-2320) showed higher amounts of recovered protein of the desired size after protein A purification compared to the N-terminally attached scFvs (XGFR1-3320 and XGFR 1-5320).

HP-size exclusion chromatography analysis of the purified proteins showed some tendency to aggregate (compared to 'normal' IgGs) molecules containing scFvs that were not stabilized by interchain disulfides between VH and VL. To address the problem of aggregation of these bispecific antibodies, disulfide stabilization of the scFv moieties was used. In this regard, we introduced single cysteine substitutions within the VH and VL of the scFv at the indicated positions (positions VH44/VL100, according to the Kabat numbering scheme). These mutations enable the formation of stable interchain disulfides between VH and VL, which in turn stabilize the resulting disulfide-stabilized scFv module. Introduction of the VH44/VL100 disulfide into scFvs at the N-and C-termini of the Fv resulted in an increase in protein expression levels for all constructs (see figure 4).

FreeStyle was used according to the manufacturer's instructions^TMThe 293 expression system (Invitrogen, USA) expresses bispecific antibodies by transient transfection of human embryonic kidney 293-F cells. Briefly, the suspension FreeStyle^TM293-F cells in FreeStyle^TM293 expression Medium at 37 ℃/8% CO₂Cultures were performed and cells were plated at 1-2X10 on the day of transfection⁶Viable cells/ml were seeded in fresh medium. 333 μ l of 293fectin was used^TM(Invitrogen, Germany) and 250. mu.g of 1: 1 molar ratio of heavy and light chain plasmid DNAPreparation of DNA-293fectin I Medium (Invitrogen, USA)^TMThe final transfection volume was 250 ml. 7 days post transfection, inclusion of BsAb was performed by centrifugation at 14000g for 30 min and filtration through sterile filter (0.22 μm)Cell culture supernatant of sex antibodies was clarified. The supernatant was stored at-20 ℃ until purification.

After purification, XGFR1-2320 had a final yield of 0.27mg, while XGFR1-2321 had a final yield of 13.8 mg.

Exemplary SDS-PAGE and HP-Size Exclusion Chromatography (SEC) purification and analysis of bispecific antibodies are shown in FIGS. 3 and 4.

Example 2

In vitro stability of bispecific < EGFR IGF1R > antibody XGFR1 molecules HP-size exclusion chromatography

At 25 ℃ using 200mM KH₂PO₄Superdex200 analytical size exclusion column (GE Healthcare, Sweden) in 250mM KCl, pH7.0 running buffer, by high performance SEC fractionation on an Ultimate 3000HPLC system (Dionex)Samples of bispecific antibodies were analyzed for aggregate content. Mu.g of protein was injected onto the column at a flow rate of 0.5ml/min and eluted isocratically over 50 minutes. For stability analysis, purified protein was prepared at concentrations of 0.1mg/ml, 1mg/ml and 5mg/ml and incubated at 4 ℃, 37 ℃ and 40 ℃ for 7 days or 28 days, followed by evaluation by high performance SEC. The integrity of the amino acid backbone of the reduced bispecific antibody light and heavy chains was confirmed by nanoelectrospray q-TOF mass spectrometry after removal of the N-glycans by enzymatic treatment with peptide-N-glycosidase F (roche molecular Biochemicals).

HP-size exclusion chromatography analysis of the purified proteins under different conditions (different concentrations and times) showed-a greatly increased tendency of the molecules comprising scFvs to aggregate compared to normal IgGs-0.

For this work, we define that the "monomeric molecule" required consists of 2 heterodimers of heavy and light chains, with scFvs attached to either the heavy or light chain.

HP size exclusion analysis of the VH44/VL100 disulfide stabilized construct showed a much smaller tendency to aggregate compared to the strong tendency to aggregate of entities comprising unmodified scFvs.

Exemplary HP-Size Exclusion Chromatography (SEC) purification and analysis of bispecific antibodies are shown in figure 4.

Example 3

Bispecific < EGFR-IGF1R > antibody XGFR1 analysis for simultaneous binding to RTKs EGFR and IGF1R

The binding of the scFv module and the binding of Fvs retained in the IgG-module of the different bispecific antibody formats was compared to the binding of the binding module and the 'wild-type' IgGs from which the bispecific antibody was derived. These analyses were performed by applying Surface Plasmon Resonance (Surface Plasmon Resonance) (Biacore), and cell-ELISA.

The binding properties of the bispecific anti-IGF-1R/anti-EGFR antibodies were analyzed by Surface Plasmon Resonance (SPR) techniques using a Biacore T100 instrument (Biacore AB, Uppsla). This system is well established for studying molecular interactions. This allows for continuous real-time monitoring of ligand/analyte binding in various assay settings and thus determination of association rate constant (ka), dissociation rate constant (KD), and equilibrium constant (KD). SPR-technique is based on the measurement of the refractive index close to the gold coated biosensor chip surface. The change in refractive index indicates a mass change on the surface caused by the interaction of the immobilized ligand and the injected analyte in solution. If the molecule binds to an immobilized ligand on the surface, the mass increases, and if dissociated, the mass decreases.

Using the chemical principle of amine-coupling, capture anti-human IgG antibodies were immobilized on the surface of a CM5 biosensor chip. Flow cells were activated with a 1: 1 mixture of 0.1M N-hydroxysuccinimide and 0.1M 3- (N, N-dimethylamino) propyl-N-ethylcarbodiimide at a flow rate of 5. mu.l/min. Anti-human IgG antibodies were injected at 10. mu.g/ml in sodium acetate, pH 5.0, which resulted in a surface density of about 12000 RU. The reference control flow cells were treated in the same manner, but only the vehicle buffer was substituted for the capture antibody. The surface was blocked with 1M ethanolamine/HCl injection pH 8.5. Bispecific antibodies were diluted in HBS-P and injected at a flow rate of 5. mu.l/min. For EGFR-ECD binding, the contact time (association phase) was 1 minute for antibodies at concentrations of 1-5nM, and for antibodies with 1-5nM concentrations

For IGF-1R interaction, the contact time (association phase) was 1 min for the antibody at 20nM concentration. EGFR-ECD was injected at increasing concentrations of 3.125, 6.25, 12.5, 25, 50 and 100nM, IGF-1R was injected at concentrations of 0.21, 0.62, 1.85, 5.6, 16.7 and 50 nM. For both molecules at a flow rate of 30. mu.l/min, the contact time (association phase) was 3 minutes and the dissociation time (washing with running buffer) was 5 minutes. All interactions were performed at 25 ℃ (standard temperature). After each binding period, a 3M regeneration solution of magnesium chloride was injected at a flow rate of 5. mu.l/min for 60 seconds to remove any non-covalently bound proteins. The signals are detected at a rate of 1 signal per second. The sample is injected at increasing concentration.

Exemplary simultaneous binding of bispecific antibodies to EGFR and IGF1R is shown in figure 5.

TABLE 4 affinity (KD) of bispecific antibody (XGFR-nomenclature) for EGFR and IGF-1R

Example 4

Down-regulation of EGFR-and IGF1R by bispecific < EGFR-IGF1R > antibody XGFR1 molecules

The human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC2587) inhibits IGFR 1-signaling and the humanized rat anti-EGFR antibody < EGFR > ICR62 inhibits EGFR signaling. To assess the potential inhibitory activity of the different XGFR1 variants, the extent to which both down-regulate the receptor was analyzed.

To examine the effect of the antibodies of the invention on the amount of IGF-I receptor (IGF-IR) in tumor cells, time course experiments and subsequent ELISA analyses were performed with IGF-IR and EGFR-specific antibodies.

Human tumor cells (A549, 2x 10)⁵Cells/ml) were cultured in RPMI-VM medium (PAA, batch No. E15-039) supplemented with 1% PenStrep (in a 6-well plate), and 4ml of cells were seeded in each medium for each experiment at 37 ℃ and 5% CO₂The culture was carried out for 24 hours.

The medium was carefully removed and replaced with 2ml of 0.01mg/ml XGFR antibody diluted in RPMI-VM medium. In 3 control wells, medium without antibody, medium with control antibody: (<IGF-1R>HUMAB clones 18 and<EGFR>ICR62, final concentration 0.01mg/ml) was substituted for the medium, and one well contained only buffer. Cells were incubated at 37 ℃ and 5% CO₂Incubate and remove each plate after 24 hours for further processing.

Will cultivateThe nutrient was carefully removed by aspiration and the cells were washed with 1ml PBS. Add 300. mu.l/well of cold MES-lysis buffer (MES, 10mM Na)₃VO₄And are andprotease inhibitors). The cells were detached using a cell scraper (Corning, cat. No.3010) and the contents of the wells were transferred to an Eppendorf reaction tube. Cell debris was removed by centrifugation at 13000rpm and 4 ℃ for 10 minutes.

For EGFR detection

A 96-well streptavidin microtiter plate (MTP) was prepared according to the protocol (DuoSet ELISA for human EGFR, RnD system batch No. DY 231). 144 μ g/ml of human EGFR goat antibody in PBS was diluted 1: 180 in PBS and added to MTP at 100 μ l/well. The MTP was incubated at 4 ℃ overnight with agitation. The plates were supplemented with 0.1%20 PBS wash 3 times, and 300 u l/hole with 3% BSA and 0.1%The 20 solution of PBS was blocked at Room Temperature (RT) for 1 hour with agitation. The plates were supplemented with 0.1%20 PBS washes 3 times.

The amount of protein in the cell lysate was determined using the BCA protein assay kit (Pierce), and the cell lysate was then supplemented with 100mM Na₃VO₄1: 100 andprotease inhibitor 1: 20 MES-lysis buffer was adjusted to a protein concentration of 0.1mg/ml and 100. mu.l/well of lysate was added to the previously prepared MTP.

The second cell lysate concentration used was 0.05mg/ml and the lysate was diluted 1: 2 and added to the pre-prepared MTP at 100. mu.l/well. The MTP was incubated at room temperature for a further 2 hours with agitation, then with a solution of 0.1%20 solutions of PBS were washed 3 times.

The detection antibody for EGFR was a human EGFR goat biotinylated antibody at a concentration of 36. mu.g/ml diluted 1: 180 with 3% BSA and 0.2%20 in PBS. Add 100. mu.l/well and incubate for 2 hours at room temperature with agitation. The MTP was then diluted with 200. mu.l/well with 0.1%20 solutions in PBS were washed 3 times. Followed by addition of a secondary antibody at 3% BSA and 0.2%20 in PBS 1: 200 streptavidin-HRP, added to 100 u l/hole and at room temperature 20 minutes, with stirring. Next, the plates were dipped with a solution of 0.1%20 solutions in PBS were washed 6 times. 100. mu.l/well of 3, 3 '-5, 5' -tetramethylbenzidine (Roche, BM-Blue ID No.: 11484581) was added and incubated at room temperature for 20 minutes with agitation. By adding 25. mu.l/well of 1M H₂SO₄The color reaction was stopped and incubated for an additional 5 minutes at room temperature. Absorbance was measured at 450 nm.

For IGF-1R detection

streptavidin-MTP (Roche ID. No.: 11965891001) was prepared by adding 100. mu.l/well of AK1 a-biotinylated antibody (Genmab, Denmark), which was incubated with 3% BSA and 0.2%20 in PBS at a 1: 200 dilution. streptavidin-MTP was incubated for 1 hour at room temperature with agitation, followed by 200. mu.l/well with 0.1%20 solutions in PBS were washed 3 times.

The amount of protein in the cell lysate was determined using the BCA protein assay kit (Pierce), followed by subjecting the cell lysate to 50mM Tris pH7.4, 100mM Na₃VO₄1: 100 andprotease inhibitor was adjusted 1: 20 to a protein concentration of 0.04mg/ml and 100. mu.l/well of lysate was added to the pre-prepared streptavidin-MTP.

The second cell lysate concentration used was 0.02mg/ml, the lysate was diluted and added to the pre-prepared streptavidin-MTP at 100. mu.l/well. Positive controls containing unstimulated cells were supplemented with 50mM Tris pH7.4, 100mM Na₃VO₄1: 100 andprotease inhibitor 1: 20 in lysis buffer was diluted to 1: 4000 and 100. mu.l/well of lysate was added to the pre-prepared streptavidin-MTP. For negative control, 100. mu.l lysis buffer was added to the wells of streptavidin-MTP.

The MTP was incubated at room temperature for a further 1h with agitation, then with a solution of 0.1%20 solutions in PBS were washed 3 times.

The detection antibody for IGF-1R is a human IGF-1R beta rabbit antibody (St. Cruis Biotechnology)(Santa Cruz Biotechnology, batch sc-713) in the presence of 3% BSA and 0.2%20 in PBS at a dilution of 1: 750. Added at 100. mu.l/well and incubated for 1 hour at room temperature with agitation. Next, the MTP was measured at 0.1% in 200. mu.l/well20 solutions in PBS were washed 3 times. Next, a secondary antibody was added in the presence of 3% BSA and 0.2%20 of rabbit IgG-POD (Cell signaling) batch 7074 at 1: 4000 in PBS was added at 100. mu.l/well and incubated for 1 hour at room temperature with agitation. Next, the plates were dipped with a solution of 0.1%20 solutions in PBS were washed 6 times. 100. mu.l/well of 3, 3 '-5, 5' -tetramethylbenzidine (Roche, BM-Blue ID No.: 11484581) was added and incubated at room temperature for 20 minutes with agitation. By adding 25. mu.l/well of 1M H₂SO₄The color reaction was stopped and incubated for an additional 5 minutes at room temperature. Absorbance was measured at 450 nm.

FIG. 12 shows the results of receptor downregulation assays comparing the bispecific antibody XGFR to the parent monospecific antibodies < EGFR > ICR62 and < IGF-1R > HUMAB-clone 18 in A549 cells. The bispecific antibody XGFR down regulates EGFR as well as IGF 1R. Surprisingly, the bispecific antibody XGFR shows improved down-regulation of EGFR compared to the parent < EGFR > ICR62 antibody.

Example 5

Bispecific < EGFR IGF1R > antibody XGFR1 molecule inhibits EGFR and IGF1R signaling pathways

Human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSMAC 2587) inhibits IGFR 1-signaling and humanized rat anti-EGFR antibody ICR62 inhibits EGFR signaling. To assess the potential inhibitory activity of the different XGFR1 variants, the degree of inhibition of signaling for both pathways was analyzed.

Human tumor cells (H322M, 2X 10)⁵Cells/ml) were cultured in RPMI medium (PAA, lot No. E15-039) supplemented with 1% PenStrep (in a 6-well plate), and 4ml of cells were seeded in each medium for each experiment at 37 ℃ and 5% CO₂The culture was carried out for 24 hours.

The medium was carefully removed and replaced with 2ml of 0.01mg/ml XGFR antibody diluted in RPMI-VM medium. In 3 control wells, with control antibody in antibody-free medium: (<IGF-1R>HUMAB clones 18 and<EGFR>ICR62, final concentration 0.01mg/ml) was substituted for the medium, and one well contained only buffer. Cells were incubated at 37 ℃ and 5% CO₂Incubate and remove each plate after 24 hours for further processing.

Assays for EGFR phosphorylation

Use ofIC human Phospho-EGF R, RnD System batch No. DYC 1095-5. The plate was prepared by diluting the phosphoEGF R capture antibody (batch No. 841402) to a concentration of 0.8. mu.g/ml. 100 μ l/well of MTP was added, plates were sealed and incubated overnight at room temperature.

Next, the capture antibody was aspirated, and each well was washed with 400. mu.l of washing buffer (0.05% in PBS)20, pH 7.2-7.4 lot No. WA126) 5 times, after the last wash the plate WAs blotted dry on clean paper towels.

The plates were prepared by adding 300. mu.l of blocking buffer (1% BSA, 0.05% NaN in PBS)₃pH 7.2-7.4) sealClosed and incubated at room temperature for 2 hours. Next, the solution was aspirated and each well was washed with 400. mu.l of washing buffer (0.05% in PBS)20pH 7.2-7.4 lot No. WA126) 5 times, after the last wash the plates were blotted dry on clean paper towels.

Cells were rinsed with PBS and lysed with lysis buffer 9 (1% NP-40, 20mM Tris pH8.0, 137mM NaCl, 10% glycerol, 2mM EDTA, 1mM activated sodium orthovanadate, 10. mu.g/ml bovine trypsin inhibitor and 10. mu.g/ml zymoaldehyde-inhibiting peptide) at 1X 10⁷Cells were lysed at a cell density of cells/ml and incubated at 4 ℃ for 30 minutes with gentle agitation. The sample was then centrifuged at 14,000g for 5 minutes. Next, the sample was transferred to a clean test tube.

The amount of protein in the cell lysate was determined using the BCA protein assay kit (Pierce), followed by adjustment of the cell lysate to protein concentrations of 0.1mg/ml and 0.05mg/ml with IC diluent 12 (1% NP-40, 20mM Tris pH8.0, 137mM NaCl, 10% glycerol, 2mM EDTA, 1mM activated sodium orthovanadate). 100 μ l/well of lysate was added to the pre-prepared MTP and the plate was sealed and incubated for 2 hours at room temperature.

Immediately prior to use, the detection antibody was applied to IC diluent 14(20mM Tris, 137mM NaCl, 0.05%20, 0.1% BSA, pH 7.2-7.4) to the working concentration established on the bottle. 100 μ l of detection antibody was added to each well, the plate was sealed, and incubated at room temperature for 2 hours in the dark. Next, the detection antibody was aspirated and each well was washed with 400. mu.1 wash buffer (0.05% in PBS)20pH 7.2-7.4 lot No. WA126) 5 times, after the last wash the plates were blotted dry on clean paper towels.

100 μ l of substrate solution (batch No. DY999) was added to each well and the plates were incubated in the dark for an additional 20 minutes. Stop solution 2N H by adding 50. mu.l₂SO₄(batch No. DY994) and thoroughly mixed to terminate the reaction.

The absorbance at 450nm was measured.

Detection of IGF-1R phosphorylation

streptavidin-MTP (Roche ID. No.: 11965891001) was prepared by adding 100. mu.l/well of AK1 a-biotinylated antibody (Genmab, Denmark) in a solution with 3% BSA and 0.2%20 in PBS at a 1: 200 dilution. streptavidin-MTP was incubated for 1 hour at room temperature with agitation, followed by 200. mu.l/well with 0.1%20 solutions in PBS were washed 3 times.

The amount of protein in the cell lysate was determined using the BCA protein assay kit (Pierce), followed by subjecting the cell lysate to 50mM Tris pH7.4, 100mM Na₃VO₄1: 100 andprotease inhibitors were adjusted 1: 20 to a protein concentration of 1. mu.M, and 100. mu.l/well of lysate was added to the pre-prepared streptavidin-MTP.

Positive controls containing unstimulated cells were supplemented with 50mM Tris pH7.4, 100mM Na₃VO₄1: 100 andprotease inhibitor 1: 20 in lysis buffer diluted to 1: 4000 and 100. mu.l/well lysate added to the pre-prepared streptavidinIn the vegetarian protein-MTP. For negative control, 100. mu.l lysis buffer was added to the wells of streptavidin-MTP.

The detection antibody for IGF-1R was a human IGF-1R (Tyr 1135/1136)/insulin receptor beta (Tyr1150/1151) (19H7) antibody (Cell signaling, batch No. 3024L) with 3% BSA and 0.2%20 in PBS at a 1: 500 dilution. Added at 100. mu.l/well and incubated for 1 hour at room temperature with agitation. Next, the MTP was measured at 0.1% in 200. mu.l/well20 solutions in PBS were washed 3 times. Next, a secondary antibody was added in the presence of 3% BSA and 0.2%20 of rabbit IgG-POD (Cell signaling) batch 7074 at 1: 4000 in PBS was added at 100. mu.l/well and incubated for 1 hour at room temperature with agitation. Next, the plates were dipped with a solution of 0.1%20 solutions in PBS were washed 6 times. 100. mu.l/well of 3, 3 '-5, 5' -tetramethylbenzidine (Roche, BM-BlueID No.: 11484581) was added and incubated at room temperature for 20 minutes with agitation. By adding 25. mu.l/well of 1M H₂SO₄The color reaction was stopped and incubated for an additional 5 minutes at room temperature. Absorbance was measured at 450 nm.

FIGS. 7a and 7b show that administration of < IGF-1R > HUMAB-clone 18 strongly reduced specific phosphorylation signals in the IGFR 1-signaling assay, but was not effective in the corresponding assay measuring EGFR-signaling. Vice versa, administration of < EGFR > ICR62 reduced specific phosphorylation signals in EGFR-signaling assays, but showed no effect in corresponding assays measuring IGF 1R-signaling. The XGFR1 variants #2421, 3421, and 4421 showed the same or better activity in both assays as the wild type antibody when applied to the same assay at the same molarity. Therefore, the molecule XGFR1 is able to interfere with both signaling pathways.

Example 6

XGFR 1-mediated inhibition of growth of tumor cell lines in vitro

Human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSMACC 2587) inhibits growth of tumor cell lines expressing IGF1R (WO 2005/005635). In a similar manner, the humanized rat anti-EGFR antibody < EGFR > ICR62 was shown to inhibit the growth of EGFR-expressing tumor cell lines (WO 2006/082515). To evaluate the potential inhibitory activity of the different XGFR1 variants in growth assays of tumor cell lines, the extent of inhibition in H322M cells expressing EGFR as well as IGF1R was analyzed.

H322M cells were cultured on poly-HEMA (poly (2-hydroxyethyl methacrylate)) coated dishes in RPMI1640 medium supplemented with 0.5% FCS to prevent adhesion to plastic surfaces. Under these conditions, H322M cells formed dense spheres that grew in three dimensions (a property known as anchorage independence). These spheres closely resemble the three-dimensional tissue structure and organization of solid tumors in situ. The sphere cultures were incubated for 5 days in the presence of increasing amounts of antibody from 50 or 100 nM. The WST conversion assay was used to measure growth inhibition. Growth inhibition was observed when H322M sphere cultures were treated with < IGF-1R > HUMAB-clone 18.

FIG. 8 shows that administration of 50nM < IGF-1R > HUMAB-clone 18 reduced cell growth by 53%, whereas in the same assay, administration of 50nM < EGFR > ICR62 reduced cell growth by 53%.

Simultaneous administration of both antibodies (at the same concentration) resulted in a further reduction in cell viability to 26% (74% inhibition). This suggests that interfering with both RTK pathways simultaneously has a more pronounced effect on tumor cell lines than interfering with only one pathway.

Administration of the various XGFR 1-variants at a molarity of 50nM resulted in higher growth inhibition, which was more pronounced than the inhibition observed with a single molecule at a concentration of 50 nM.

Indeed, at an antibody concentration of 50nM, various XGFR 1-variants showed improved anti-proliferative activity compared to the combination of the original < EGFR > and < IGF1R > antibodies at double antibody concentration of 100nM (50nM < IGF-1R > HUMAB-clone 18 and 50nM < EGFR > ICR 62).

We conclude that the XGFR1 molecule has a significantly increased growth inhibitory activity compared to IgGs that interfere with EGFR signaling or IGF1R signaling. Furthermore, if the activity of the XGFR1 molecule is compared to the activity of a mixture of < IGF-1R > HUMAB-clone 18 and < EGFR > ICR62 antibody, the same or better activity can be obtained at significantly lower concentrations (molar and mass) than the mixture.

TABLE 5 anti-proliferative Activity of bispecific antibodies (XGFR-nomenclature) against H322M tumor cells (survival and inhibition)

Example 7

Preparation of derivatives of glycoform XGFR1-2421, XGFR1-3421, XGFR1-4421 and XGFR1-5421 (XGFR1-2421-GE, XGFR1-3421-GE, XGFR1-4421-GE and XGFR1-5421-GE)

The resulting complete antibody heavy and light chain DNA sequences were subcloned into mammalian expression vectors (one for the light chain and one for the heavy chain) under the control of the MPSV promoter and upstream of the synthetic poly a site, each vector carrying the EBV OriP sequence.

Antibodies were produced by co-transfecting HEK293-EBNA cells with mammalian antibody heavy and light chain expression vectors using a calcium phosphate-transfection method. Exponentially growing HEK293-EBNA cells were transfected by the calcium phosphate method. To produce unmodified antibody, cells were transfected with only antibody heavy and light chain expression vectors in a 1: 1 ratio. To produce glycoengineered antibodies, cells were co-transfected with four plasmids, two for antibody expression, one for fusion GnTIII polypeptide expression, and one for mannosidase II expression, each at a ratio of 4: 1. Cells were cultured in T-flasks as adherent monolayer cultures, using DMEM medium supplemented with 10% FCS, and transfected when they were between 50% and 80% confluency. For transfection in T75 flasks, 8 million cells were seeded 24 hours prior to transfection in 14ml DMEM medium supplemented with FCS (at 10% V/V final concentration), 250. mu.g/ml neomycin, and cells were plated at 37 ℃ in 5% CO₂The atmosphere was maintained in an incubator overnight. For each T75 flask to be transfected, 235. mu.l of 1M CaCl was prepared by bisecting 47. mu.g of total plasmid vector DNA between light and heavy chain expression vectors₂The solutions were mixed and water was added to a final volume of 469. mu.l to prepare DNA, CaCl₂And a solution of water. To this solution was added 469. mu.l of 50mM HEPES, 280mM NaCl, 1.5mM Na₂HPO₄The solution was pH 7.05, mixed immediately for 10 seconds and left at room temperature for 20 seconds. The suspension was diluted with 12ml of DMEM supplemented with 2% FCS and added to T75 in place of the existing medium. Cells were incubated at 37 ℃ with 5% CO₂Incubation was carried out for about 17 to 20 hours, followed by replacement of the medium with 12ml DMEM, 10% FCS. Conditioned media were harvested 5-7 days post transfection, centrifuged at 1200rpm for 5 minutes followed by a second centrifugation at 4000rpm for 10 minutes and held at 4 ℃.

Secreted antibodies were purified by protein a affinity chromatography, followed by cation exchange chromatography and a final size exclusion chromatography step exchanging buffer to phosphate buffer on a Superdex200 column (Amersham Pharmacia) and collecting pure monomeric IgG1 antibody. The antibody concentration was estimated from the absorbance at 280nm using a spectrophotometer. The antibody was formulated in 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine solution, pH 6.7.

Glycoengineered variants of humanized antibodies were produced by co-transfection of: antibody expression plasmid and GnT-III glycosyltransferase expression vector, or GnT-III expression vector and Golgi mannosidase II expression vector. Purification and formulation of glycoengineered antibodies are as described above for the non-glycoengineered antibodies. Oligosaccharides attached to the Fc region of the antibody were analyzed by MALDI/TOF-MS as described below.

Oligosaccharides are enzymatically released from antibodies by PNGaseF digestion, wherein the antibodies are immobilized on PVDF membranes or in solution.

The resulting digestion solution containing the released oligosaccharides was prepared directly for MALDI/TOF-MS analysis or further digested with EndoH glycosidase prior to sample preparation for MALDI/TOF-MS analysis.

For all bispecific antibodies according to the invention, GE means glycoengineering.

Example 8

Binding and ADCC Capacity of XGFR1 molecule to FcgRIIIIa

The aglycosylated modified humanized rat anti-EGFR antibody ICR62 (from WO2006/082515) mediates its anti-tumor activity not only by interfering with RTK mediated growth stimulatory signals, but also to a significant extent by inducing ADCC on tumor cells. In a similar manner, other antibodies, such as the anti-IGF-1R antibody < IGF-1R > HUMAB clone 18 are also capable of inducing ADCC. The degree of ADCC mediated by a given antibody depends not only on the antigen bound, but also on the affinity of the constant region for FcgRIIIa, which is known to be the Fc receptor that elicits ADCC responses.

Since ADCC is a mechanism required for the XGFR1 molecules, it is important that these molecules can bind FcgRIIIa in the same way as 'normal' antibodies and that these molecules have good ADCC capability. To analyze the binding of various molecules of XGFR1 to bfcgrlia, we applied the previously established Biacore technique (ref). By this technique, the binding of the XGFR 1-molecule to the recombinantly produced FcgRIIIa domain was evaluated.

All surface plasmon resonance measurements were performed on a BIAcore 3000 instrument (GE healthcare biosciences ab), Sweden) at 25 ℃. The running and dilution buffer was PBS (1mM KH)₂PO₄，10mM Na₂HPO₄137mM NaCl, 2.7mM KCl), pH6.0, 0.005% (v/v) Tween 20. Soluble human FcgRIIIa was diluted in 10mM sodium citrate, pH 5.0 and immobilized on a CM5 biosensor chip using a standard amine coupling kit (GE Healthcare Biosciences ab, (GE Healthcare Biosciences ab), Sweden) to obtain an FcgRIIIa surface density of about 1000 RU. HBS-P (10mM HEPES, pH7.4, 150mM NaCl, 0.005% surfactant P20; GE Healthcare Biosciences AB (GE Healthcare Biosciences AB), Sweden) was used as a running buffer during the immobilization. XGFR bispecific antibody was diluted to a concentration of 450nM with PBS, 0.005% (v/v) Tween20, pH6.0 and injected over 3 minutes at a flow rate of 30. mu.l/min. Next, the sensor chip was regenerated with PBS, pH8.0, 0.005% (v/v) Tween20 for 1 minute. Data analysis was performed using BIA evaluation software (BIAcore, Sweden).

The results of these experiments are summarized in table 7.

TABLE 6 binding affinities of bispecific antibodies (XGFR-nomenclature) to Fc γ RIIIa and FcRn

These analyses revealed that binding to FcgRIIIa for the XGFR1 molecule without antigen binding was indistinguishable from that of the wild-type IgG1 molecule. Thus, these biochemical assays represent the complete ability of XGFR1-2421, and XGFR1-4421, to bind the ADCC-mediated receptor FcgRIIIa without antigen binding.

The reproducibility of these experiments using the XGFR1 molecule in the presence of antigen revealed no effect on the ability of soluble FcgRIII to bind.

Another set of such Biacore experiments was performed with XGFR 1-molecules (see example 7) that have been sugar modified by the aforementioned techniques (Umana, P., et al, Nature Biotechnol. 17(1999)176-180 and WO 99/54342). This sugar modification increases the affinity of the Fc-region to FcgRIIIa and thereby increases ADCC on the target cell. Comparing the fcgrima-binding ability of the sugar modified XGFR 1-molecule without antigen binding to the fcgrima-binding ability of the sugar modified wild-type IgG without antigen binding shows that the sugar modified XGFR1 molecule without antigen binding has increased binding affinity compared to the wild-type antibody.

TABLE 7 binding affinities of bispecific antibodies (XGFR-nomenclature) to Fc γ RIIIa and FcRn

Molecule	Affinity for Fc γ RIIIa	Binding affinity to FcRn
			XGFR1-2421-GE	Is that	Is that
XGFR1-3421-GE	Is that	Is that
			XGFR1-4421-GE	Is that	Is that
XGFR1-5421-GE	Is that	Is that

To analyze the extent to which the binding ability of the XGFR 1-molecule to FcgRIIIa also translates into ADCC activity in vitro against tumor cells, we determined ADCC ability in a cellular assay. For these assays, sugar modified derivatives of XGFR1-2421, XGFR1-3421, XGFR1-4421 and XGFR1-5421 (XGFR1-2421-GE, XGFR1-3421-GE, XGFR1-4421-GE and XGFR1-5421-GE) were prepared (see example 6) and tested in the BIAcore ADCC-capacity assay format described above and in the in vitro ADCC assay described below.

Human Peripheral Blood Mononuclear Cells (PBMCs) were used as effector cells and prepared using Histopaque-1077(Sigma Diagnostics inc., st. louis, MO63178USA) and essentially following the manufacturer's instructions. Briefly, venous blood was drawn from healthy volunteers using a heparinized syringe. With PBS (Ca-free)⁺⁺Or Mg⁺⁺) Blood was diluted 1: 0.75-1.3 and plated on Histopaque-1077. The gradient was centrifuged uninterruptedly at 400x g for 30 min at Room Temperature (RT). The intermediate phase containing PBMCs was collected and washed with PBS (50 ml of cells from each of the two gradients) and collected by centrifugation at 300x g for 10 minutes at RT. After resuspending the pellet with PBS, the PBMCs were counted and passedA second wash was performed by centrifugation at 200x g for 10min at RT. The cells are then resuspended in an appropriate medium for the next operation.

For PBMC, the ratio of effector cells to target for ADCC assay was 25: 1. Effector cells were prepared in AIM-V medium at appropriate concentrations to add to wells of a 50. mu.l/round bottom 96 well plate. The target cells were human EGFR/IGFR-expressing cells (e.g., H322M, A549, or MCF-7) grown in DMEM containing 10% FCS. Target cells were washed in PBS, counted and resuspended in AIM-V at 0.3 million/ml to add 30,000 cells at 100. mu.l/well. The antibody was diluted in AIM-V, 50. mu.l was added to the pre-plated target cells and allowed to bind to the target at RT for 10 min. Effector cells were then added and incubated at 37 ℃ with 5% CO₂The plates were incubated for 4 hours in a humidified atmosphere. Killing of target cells was assessed by measuring Lactate Dehydrogenase (LDH) released by damaged cells using a cytotoxicity detection kit (roche diagnostics, Rotkreuz, Switzerland). After 4 hours incubation the plates were centrifuged at 800x g. Transfer 100 μ l of supernatant from each well to a new clear flat bottom 96 well plate. Add 100. mu.l of color substrate buffer from the kit to each well. The Vmax value of the color reaction was determined at 490nm in an ELISA reader for at least 10min using SOFTMax PRO software (Molecular Devices), Sunnyvale, CA94089, USA). Spontaneous LDH release was determined from wells containing only target and effector cells but no antibody. Maximum release was determined from wells containing only target cells and 1% Triton X-100. The percent killing mediated by specific antibodies was calculated as follows: ((x-SR)/(MR-SR) × 100, where x is the average value of Vmax at a particular antibody concentration, SR is the average value of spontaneously released Vmax, and MR is the average value of maximally released Vmax.

In these assays, ADCC ability was also compared to that of the sugar-modified wild-type antibody. The results of these assays showed excellent ADCC capacity for the carbohydrate-modified XGFR1-3421-GE/XGFR1-4421-GE/XGFR1-5421-GE (see FIG. 9).

Example 9

Expression and purification of bispecific < EGFR-IGF1R > antibody scFab-XGFR1 molecule

The light and heavy chains of the corresponding bispecific antibodies were constructed in expression vectors carrying prokaryotic and eukaryotic selection markers. These plasmids were amplified in E.coli, purified, and then transfected to perform transient expression of recombinant proteins in HEK293F cells using the Invitrogen's free System (Invitrogen's freesystem). After 7 days, HEK293 cell supernatants were harvested and purified by protein a and size exclusion chromatography. The homogeneity of all bispecific antibody constructs was confirmed by SDS-PAGE under non-reducing and reducing conditions. Under reducing conditions (fig. 15), the polypeptide chains carrying the C-terminal and N-terminal scFab fusions displayed an apparent molecular size on SDS-PAGE similar to the calculated molecular weight. All constructs were analyzed by protein AHPLC for expression levels that were similar to the expression yield of 'standard' IgGs, or in some cases slightly lower. In such non-optimized transient expression experiments, the average protein yield was 1.5-10mg protein per liter cell culture supernatant (FIGS. 13 and 14).

HP-size exclusion chromatography analysis of the purified protein showed some tendency of the recombinant molecule to aggregate. To address the problem of aggregation of these bispecific antibodies, disulfide stabilization between VH and VL using additional binding moieties. In this regard, we introduced single cysteine substitutions within the VH and VL of scfabs at the indicated positions (positions VH44/VL100, according to the Kabat numbering scheme). These mutations enable the formation of stable interchain disulfides between VH and VL, which in turn stabilize the resulting disulfide-stabilized scFab modules. The introduction of VH44/VL100 disulfide into scFabs did not significantly interfere with protein expression levels, and in some cases even improved expression yields (see fig. 13 and 14).

FreeStyle was used according to the manufacturer's instructions^TMThe 293 expression system (Invitrogen, USA) expresses bispecific antibodies by transient transfection of human embryonic kidney 293-F cells. Briefly, the suspension FreeStyle^TM293-F cells in FreeStyle^TM293 expression Medium at 37 ℃/8% CO₂Cultures were performed and cells were plated at 1-2X10 on the day of transfection⁶Viable cells/ml were seeded in fresh medium. 333 μ l of 293fectin was used^TM(Invitrogen, Germany) and 250. mu.g of 1: 1 molar ratio of heavy and light chain plasmid DNAPreparation of DNA-293fectin I Medium (Invitrogen, USA)^TMThe final transfection volume was 250 ml. 7 days after transfection, cell culture supernatants containing recombinant antibody derivatives were clarified by centrifugation at 14000g for 30 minutes and filtration through sterile filters (0.22 μm). The supernatant was stored at-20 ℃ until purification.

By using protein A-agarose^TM(Protein A-Sepharose^TM) Affinity chromatography (GE healthcare, Sweden) and Superdex200 size exclusion chromatography, secreted antibody derivatives were purified from the supernatant in two steps. Briefly, clarified culture supernatants containing bispecific and trispecific antibodies were applied to a HiTrap protein a HP (5ml) column with PBS buffer (10mM Na) buffer₂HPO₄，1mM KH₂PO₄137mM NaCl and 2.7mM KCl, pH 7.4). Unbound protein was washed out with equilibration buffer. The antibody derivative was eluted with 0.1M citrate buffer, pH2.8 and the protein containing fraction was neutralized with 0.1ml 1M Tris, pH 8.5. Subsequently, the eluted protein fractions were pooled, concentrated to a volume of 3ml using an Amicon ultracentrifuge filter unit (MWCO: 30K, Millipore) and loaded onto a Superdex200HiLoad 120ml16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20mM histidine, 140mM NaCl, pH 6.0. Monomeric antibody fractions were pooled, snap frozen and stored at-80 ℃. Portions of the sample are provided for subsequent protein analysis and characterization. Exemplary SDS-PAGE analyses of the purified proteins and HP-Size Exclusion Chromatography (SEC) profiles of the bispecific antibody derivatives are shown in fig. 15 and fig. 16.

FIGS. 13 and 14 list the expression yields observed in the transient expression system: all specified antibody derivatives can be expressed and purified in sufficient quantities for further analysis. The expression yield per liter of supernatant ranged from less than 1mg to > 30 mg. For example, scFab-XGFR1-2720 has a final yield of less than 1mg after purification, while scFab-XGFR1-2721 has a final yield of 13.8 mg. This difference also shows the positive effect of VH44-VL100 disulfide stabilization on the expression yield we observed for certain proteins.

Example 10

In vitro stability of bispecific < EGFR IGF1R > antibody scFab XGFR1 molecule

Stability and aggregation propensity of bispecific < EGFR-IGF1R > antibody scFab molecules

HP size exclusion chromatography analysis was performed to determine the amount of aggregates present in the preparation of recombinant antibody derivatives. For this purpose, size exclusion columns (GE healthcare, Sweden) were analyzed using Superdex200, and bispecific antibody samples were analyzed by high performance SEC on an UltiMate 3000HPLC system (Dionex). Fig. 16 shows examples of these analyses. The aggregates appear as a separate peak or shoulder before the fraction comprising the monomeric antibody derivative. For this work, we define that the "monomer molecule" required consists of 2 heterodimers of heavy and light chains, with scFabs attached to either the heavy or light chain. The integrity of the reduced bispecific antibody light and heavy chains and the amino acid backbone of the fusion protein was confirmed by NanoElectrospray Q-TOF mass spectrometry after removal of the N-glycans by enzymatic treatment with peptide N-glycosidase F (Roche Molecular Biochemicals). HP-size exclusion chromatography analysis of the purified proteins under different conditions (different concentrations and times) showed a slightly increased tendency of the molecules comprising scFabs to aggregate compared to normal IgGs-. We observed that for certain molecules this aggregation propensity can be improved by introducing a VH44/VL100 interchain disulfide bond in the scFab module.

Example 11

Binding of bispecific < EGFR-IGF1R > antibody scFab-molecules to RTKs EGFR and IGF1R

The binding of the scFab module and the binding of the antigen binding site remaining in the full-length IgG-module of the different bispecific antibody format scFab-XGFR is compared to the binding of the binding module and the 'wild type' IgGs from which the bispecific antibody is derived. These analyses were performed by applying Surface Plasmon Resonance (Surface Plasmon Resonance) (Biacore), and cell-ELISA.

The binding properties of the bispecific < IGF-1R-EGFR > antibodies were analyzed by Surface Plasmon Resonance (SPR) technique using a Biacore T100 instrument (GE healthcare bios-Sciences AB, Uppsala). This system is well established for studying molecular interactions. This allows for continuous real-time monitoring of ligand/analyte binding in various assay settings and thus determination of association rate constant (ka), dissociation rate constant (KD), and equilibrium constant (KD). SPR-technique is based on the measurement of the refractive index close to the gold coated biosensor chip surface. The change in refractive index indicates a mass change on the surface caused by the interaction of the immobilized ligand and the injected analyte in solution. If the molecule binds to an immobilized ligand on the surface, the mass increases, and if dissociated, the mass decreases.

Using the chemical principle of amine-coupling, capture anti-human IgG antibodies were immobilized on the surface of the C1 biosensor chip. Flow cells were activated with a 1: 1 mixture of 0.1M N-hydroxysuccinimide and 0.1M 3- (N, N-dimethylamino) propyl-N-ethylcarbodiimide at a flow rate of 5. mu.l/min. Anti-human IgG antibodies were injected at 5. mu.g/ml into sodium acetate, pH 5.0, which resulted in a surface density of about 200 RU. The reference control flow cells were treated in the same manner, but only the vehicle buffer was substituted for the capture antibody. The surface was blocked with 1M ethanolamine/HCl injection pH 8.5. Bispecific antibodies were diluted in HBS-P and injected at a flow rate of 5. mu.l/min. For the antibody concentration of 1-5nM, the contact time (association phase) is 1 minute. EGFR-ECD was injected at increasing concentrations of 1.2, 3.7, 11.1, 33.3, 100 and 300nM, IGF-1R was injected at concentrations of 0.37, 1.11, 3.33, 10, 30 and 90 nM. For both molecules at a flow rate of 30. mu.l/min, the contact time (association phase) was 3 minutes and the dissociation time (washing with running buffer) was 5 minutes. All interactions were performed at 25 ℃ (standard temperature). After each binding cycle, a regeneration solution of 0.85% phosphoric acid and 5mM sodium hydroxide, respectively, was injected at a flow rate of 5. mu.l/min for 60 seconds to remove any non-covalently bound protein. The signals are detected at a rate of 1 signal per second. The sample is injected at increasing concentration.

Exemplary simultaneous binding of the bispecific antibody < IGF-1R-EGFR > antibody to EGFR and IGF1R is shown in 17 a-d.

Table 8: affinity (KD) of bispecific antibodies (scFab-XGFR1_2720 and scFab-XGFR2_2720) to EGFR and IGF-1R

FACS-based binding and competition assays on cultured cells can also be applied to assess the binding capacity of bispecific antibody derivatives to RTKs exposed on the cell surface. Figure 18 shows our experimental setup for testing the binding capacity of bispecific XGFR derivatives comprising scFab to a549 carcinoma cells. For these cell competition assays, a549 cells expressing the antigens EGFR as well as IGF1R were detached and counted. Mix 1.5x10⁵Cells were seeded into each well of a conical 96-well plate. Cells were centrifuged (1500rpm, 4 ℃, 5min) and incubated on ice for 45 min in 50 μ L of a dilution series of each bispecific antibody in PBS with 2% FCS (fetal calf serum) containing 1 μ g/mL of Alexa 647-labeled IGFIR-specific antibody. The cells were again centrifuged and washed twice with 200 μ L PBS containing 2% FCS. Finally, cells were resuspended in BD CellFix solution (BD Biosciences) and incubated on ice for at least 10 minutes. Determination of the mean fluorescence intensity of the cells by flow cytometry (FACS Canto)Degrees (mfi). At least two independent staining replicates were performed to determine Mfi. Flow cytometry spectra were further processed using FlowJo software (TreeStar). Half maximal binding was determined using XLFit 4.0(IDBS) and a dose reaction single site model (one site model) 205.

The results of these assays, shown in fig. 19a-c, show the binding functionality of the antibody derivatives comprising the bispecific scFab on the surface of tumor cells. For example, the IC50 in a competition experiment with the bispecific antibody derivative scFab-XGFR1_2721 was 0.11. mu.g/ml, whereas the IC50 of the monospecific antibody was > 50% higher (0.18. mu.g/ml). This increased activity of the bispecific scFab-XGFR _2721 derivative in a competition assay compared to the parent antibody suggests that the bispecific molecule binds better to the cell surface compared to the monospecific antibody.

Example 12

The bispecific < EGFR-IGF-1R > antibody scFab-XGFR molecule down-regulates EGFR-and IGF-1R-human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC2587) inhibits IGFR 1-signaling while the humanized rat anti-EGFR antibody < EGFR > ICR62 inhibits EGFR signaling. To assess the potential inhibitory activity of the different scFab-XGFR1 variants, the extent to which both down-regulate the receptor was analyzed.

Human tumor cells (H322M, 5X 10) in RPMI1640 supplemented with 10% FCS (PAA, batch No. E15-039) and 1% PenStrep⁵Cells/ml) 6 well plates were seeded at 1 ml/well. 3ml of medium was added to each well and the cells were incubated at 37 ℃ and 5% CO₂The culture was carried out for 24 hours.

The medium was carefully removed and replaced with 2ml of 100nMXGFR antibody diluted in RPMI-VM medium. In control wells, with medium and buffer without antibody and with control antibody<IGF-1R>HUMAB clones 18 and<EGFR>ICR62, endConcentration 100nM) was substituted for the medium. Cells were incubated at 37 ℃ and 5% CO₂Incubate and remove each plate after 24 hours for further processing.

The medium was carefully removed by aspiration and the cells were washed with 1ml PBS. Add 300. mu.l/well of cold MES-lysis buffer (MES, 10mM Na)₃VO₄And are andprotease inhibitors). After 1 hour, the cells were detached on ice using a cell scraper (Corning, batch 3010) and the contents of the wells were transferred to Eppendorf reaction tubes. Cell debris was removed by centrifugation at 13000rpm and 4 ℃ for 10 minutes.

For EGFR detection

A 96-well microtiter plate (MTP) was prepared according to the protocol (DuoSet ELISA for human EGFR, RnD system batch No. DY 231). 144 μ g/ml of human EGFR goat antibody in PBS was diluted 1: 180 in PBS and 100 μ l/well was added to the MTP. The MTP was incubated overnight at room temperature with agitation. The plates were supplemented with 0.1%20 PBS wash 3 times, and 300 u l/hole with 3% BSA and 0.1%The 20 solution of PBS was blocked at Room Temperature (RT) for 1 hour with agitation. The plates were supplemented with 0.1%20 PBS washes 3 times.

The amount of protein in the cell lysate was determined using the BCA protein assay kit (Pierce), and the cell lysate was then supplemented with 100mM Na₃VO₄1: 100 andprotease inhibitor 1: 20 MES-lysis buffer was adjusted to a protein concentration of 0.04mg/ml and 100. mu.l/well of lysate was added to the previously prepared MTP. For background measurements, 100. mu.l of lysis buffer was added to the wells of the MTP.

The second cell lysate concentration used was 0.025mg/ml and the lysate was diluted 1: 2 and added to the pre-prepared MTP at 100. mu.l/well. The MTP was incubated at room temperature for a further 2 hours with agitation, then with a solution of 0.1%20 solutions of PBS were washed 3 times.

The detection antibody for EGFR was a human EGFR goat biotinylated antibody at a concentration of 36. mu.g/ml diluted 1: 180 with 3% BSA and 0.2%20 in PBS. Added at 100. mu.l/well and incubated for 2 hours at room temperature with agitation. The MTP was then diluted with 200. mu.l/well with 0.1%20 solutions in PBS were washed 3 times. Followed by addition of a solution of 3% BSA and 0.2%20 in PBS streptavidin-HRP 1: 200, 100 u l/hole and at room temperature 20 minutes, with stirring. Next, the plates were dipped with a solution of 0.1%20 solutions in PBS were washed 6 times. 100. mu.l/well of 3, 3 '-5, 5' -tetramethylbenzidine (Roche, BM-Blue ID No.: 11484581) was added and incubated at room temperature for 20 minutes with stirring. By adding 25. mu.l/well of 1M H₂SO₄The color reaction was stopped and incubated for an additional 5 minutes at room temperature. Absorbance was measured at 450 nm.

For IGF-1R detection

The amount of protein in the cell lysate was determined using the BCA protein assay kit (Pierce), followed by subjecting the cell lysate to 50mM Tris pH7.4, 100mM Na₃VO₄1: 100 andprotease inhibitor was adjusted 1: 20 to a protein concentration of 0.3mg/ml and 100. mu.l/well of lysate was added to the pre-prepared streptavidin-MTP.

The second cell lysate concentration used was 0.15mg/ml, the lysate was diluted and added to the pre-prepared streptavidin-MTP at 100. mu.l/well. 100 μ l of lysis buffer was added to the wells of the streptavidin-MTP for background measurements.

The detection antibody for IGF-1R was a human IGF-1R beta rabbit antibody (Santa Cruz Biotechnology, batch No. sc-713) with 3% BSA and 0.2%20 in PBS at a dilution of 1: 750. Added at 100. mu.l/well and incubated for 1 hour at room temperature with agitation. Next, the MTP was measured at 0.1% in 200. mu.l/well20 solutions in PBS were washed 3 times. Next, a secondary antibody was added in the presence of 3% BSA and 0.2%20 of rabbit IgG-POD (Cell signaling) batch 7074 at 1: 4000 in PBS was added at 100. mu.l/well and incubated for 1 hour at room temperature with agitation. Next, the plates were dipped with a solution of 0.1%20 solutions in PBS were washed 6 times. 100. mu.l/well of 3, 3 '-5, 5' -tetramethylbenzidine (Roche, BM-Blue ID No.: 11484581) was added and incubated at room temperature for 20 minutes with stirring. By adding 25. mu.l/well of 1M H₂SO₄The color reaction was stopped and incubated for an additional 5 minutes at room temperature. Absorbance was measured at 450 nm.

The results of the receptor downregulation assay for the XGFR molecules comprising the bispecific scFab compared to the parent monospecific antibodies < EGFR > ICR62 and < IGF-1R > HUMAB-clone 18 in H322M cells are shown in figures 20 and 21. The bispecific antibody scFab-XGFR down-regulates both EGFR-and IGF 1R. This shows that the full functionality (biological functionality) and phenotypic modulation of the binding modules is retained. Figure 21 also shows that surprisingly the bispecific antibody scFab-XGFR _2720 shows improved down-regulation of EGFR compared to the parent < EGFR > ICR62 antibody alone.

The fact that the XGFR1 variant comprising scFab showed the same or better activity compared to the wild type antibody when applied in the same assay at the same molar concentration suggests that the scFab-XGFR1 molecule is able to interfere with both signalling pathways.

Example 13

scFab-XGFR1 and scFab-XGFR 2-mediated inhibition of growth in vitro on tumor cell lines

Human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSMACC 2587) inhibits growth of tumor cell lines expressing IGF1R (WO 2005/005635). In a similar manner, the humanized rat anti-EGFR antibody < EGFR > ICR62 was shown to inhibit the growth of EGFR expressing tumor cell lines (WO 2006/082515). To evaluate the potential inhibitory activity of the different scFab-XGFR1 variants in growth assays of tumor cell lines, the extent of inhibition in H322M cells expressing EGFR and IGF1R was analyzed.

H322M cells (5000 cells/well) were cultured on poly-HEMA (poly (2-hydroxyethyl methacrylate)) coated dishes in RPMI1640 medium supplemented with 10% FCS to prevent adhesion to plastic surfaces. Under these conditions, H322M cells formed dense spheres that grew in three dimensions (a property known as anchorage independence). These spheres closely resemble the three-dimensional tissue structure and organization of solid tumors in situ. The sphere cultures were incubated for 7 days in the presence of 100nM antibody. The Celltiter Glow luminescence assay was used to measure growth inhibition. Growth inhibition was observed when H322M sphere cultures were treated with < IGF-1R > HUMAB-clone 18.

FIG. 22 shows that administration of 100nM < IGF-1R > HUMAB-clone 18 reduced cell growth by 72%, and administration of 100nM < EGFR > ICR62 reduced cell growth by 77% in the same assay. Simultaneous administration of both antibodies (both at the same concentration, 100nM) resulted in a complete reduction in cell viability (100% inhibition). This shows that interfering with both RTK pathways simultaneously has a more pronounced effect on tumor cell lines than interfering with only one pathway. Administration of the different scFab-XGFR 1-variants at a molar concentration of 100nM resulted in higher growth inhibition, which was more pronounced than the inhibition observed with the individual molecules alone. Indeed, at an antibody concentration of 100nM, the various scFab-XGFR 1-variants showed complete (100%) inhibition of cell growth, whereas administration of single components resulted in partial inhibition.

We conclude that the scFab-XGFR1 molecule has significantly increased growth inhibitory activity compared to IgGs that only interfere with EGFR signalling or IGF1R signalling.

Example 14

Bispecific, bivalent domain exchanged<EGFR-IGF1R>Antibody moleculesCross-Mab (VH/VL)(VH/VL domain swapping) orCross-Mab(CH/CL)Expression and purification of (CH/CL Domain exchange)

In analogy to the methods described in examples 1 and 9, the bispecific, bivalent domain-exchanged < EGFR-IGF1R > antibody molecules Cross-Mab (VH/VL) (VH/VL exchange as described in WO 2009/080252) and Cross-Mab (CH/CL) (CH/CL exchange as described in WO 2009/080253) were expressed and purified. Two bispecific < EGFR-IGF-1R > antibodies are based on the amino acid sequence of SEQ ID NO: 8, and the heavy chain variable domain of SEQ ID NO: 10 (from humanized < EGFR > ICR62) and is based on the amino acid sequence of SEQ id no: 23 and the heavy chain variable domain of SEQ ID NO: 25 (from human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC 2587)).

By using protein A-agarose^TM(Protein A-Sepharose^TM) The expression yields after affinity chromatography and Superdex200 size exclusion chromatography (GE healthcare, Sweden) were 29.6mg/L for Cross-Mab (VH/VL) and 28.2mg/L for Cross-Mab (CH/CL).

For Cross-Mab (VH/VL) in SEQ ID NO: the relevant complete (partially modified) light and heavy chain amino acid sequences of the corresponding bispecific antibodies are provided in fig. 30-33, and the amino acid sequences in SEQ ID NOs: 34-37 provide the relevant complete (partially modified) light and heavy chain amino acid sequences of the corresponding bispecific antibody.

Example 15

Downregulation of EGFR-and IGF-1R-

Determination of the bispecific, bivalent Domain exchange of example 14 analogously to example 12<EGFR-IGF1R>Antibody molecules Cross-Mab (VH/VL) (VH/VL crossover) and Cross-Mab (CH/CL) (CH/CL crossover) vs H322MOn tumor cellsDown-regulation of EGFR-and IGF-1R.

The downregulation of EGFR by the bispecific, bivalent domain-exchanged < EGFR-IGF1R > antibodies Cross-Mab (VH/VL) and Cross-Mab (CH/CL) was similar (Cross-Mab (VH/VL) about 41%) or slightly higher (Cross-Mab (VH/VL) about 49%) compared to the downregulation of monospecific < EGFR > ICR62 (about 41%; at 9,38 μ g protein/ml).

The downregulation of IGF-1R by the bispecific, bivalent domain-exchanged < EGFR-IGF1R > antibodies Cross-Mab (VH/VL) and Cross-Mab (CH/CL) was surprisingly significantly lower (about 17% of Cross-Mab (VH/VL)) (about 20% of Cross-Mab (VH/VL)) compared to the downregulation of monospecific < IGF-1R > HUMAB-clone 18 ((about 85%; at 75. mu.g protein/ml)).

Example 16

Bispecific, bivalent domain exchanged<EGFR-IGF1R>Antibody moleculesCross-Mab(VH/VL)OrCross-Mab(CH/CL)In vitro tumor growth inhibition of the H322M tumor cell line

Determination of the bispecific, bivalent Domain exchange of example 14 analogously to example 13<EGFR-IGF1R>Antibody molecules Cross-Mab (VH/VL) (VH/VL crossover) and Cross-Mab (CH/CL) (CH/CL crossover) pairsH322M tumor cellThe tumor growth inhibition of (3).

At 100nM, the monospecific antibody < IGF-1R > HUMAB-clone 18 reduced cell growth by 75%, while administration of 100nM < EGFR > ICR62 reduced cell growth by 89%.

The simultaneous administration of both antibodies (both at the same concentration of 100nM, resulting in a total antibody concentration of 200 nM) resulted in a complete reduction in cell viability (> 100% inhibition).

The bispecific, bivalent domain-exchanged < EGFR-IGF1R > antibody molecules Cross-Mab (VH/VL) and Cross-Mab (CH/CL) (only at 100nM concentration) also individually showed complete (. gtoreq.100%) inhibition of cell growth.

This demonstrates that the bispecific antibodies according to the invention can completely inhibit tumor cell growth at lower antibody concentrations than the combination of the corresponding monospecific maternal antibodies, whereas the monospecific maternal antibodies alone only lead to partial inhibition.

Example 17

Bispecific, bivalent ScFab-Fc fusions<EGFR-IGF1R>Antibody moleculesscFab-FcExpression and purification of

In analogy to the methods described in examples 1 and 9, the bispecific, bivalent ScFab-Fc fusion < EGFR-IGF1R > antibody ScFab-Fc was expressed and purified. This bispecific < EGFR-IGF-1R > antibody is also based on the amino acid sequence of SEQ ID NO: 8, and the heavy chain variable domain of SEQ ID NO: 10 (from humanized < EGFR > ICR62) and is based on the amino acid sequence of seq id NO: 23 and the heavy chain variable domain of SEQ ID NO: 25 (from human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSM ACC 2587)).

By using protein A-agarose^TM(Protein A-Sepharose^TM) The expression yield after affinity chromatography and Superdex200 size exclusion chromatography of (GE healthcare, Sweden) was 29.7mg/L for scFab-Fc.

Table 10: yield of bispecific, bivalent ScFab-Fc fused < EGFR-IGF1R > antibody molecule scFab-Fc after expression and purification

In SEQ ID NO: the relevant complete (modified) heavy chain amino acid sequences of the bispecific antibody scFab-Fc are provided in 38-39.

Example 18

Down-regulation of EGFR-and IGF-1R by bispecific, bivalent ScFab-Fc fusion < EGFR-IGF1R > antibody molecules

Similar to example 12, the bispecific, bivalent ScFab-Fc fusion < EGFR-IGF1R > antibody of example 17 was determined to cause down-regulation of EGFR-as well as IGF-1R on H322M tumor cells.

Example 19

In vitro tumor growth inhibition of tumor cell lines by bispecific, bivalent ScFab-Fc fusion < EGFR-IGF1R > antibody molecules

Tumor growth inhibition of H322M tumor cells by the bispecific, bivalent ScFab-Fc fusion < EGFR-IGF1R > antibody of example 17 was determined, similarly to example 13.

Example 20

Survival analysis in an orthotopic A549 xenograft model

Cell culture

A549 adenocarcinoma cells (NSCLC) were initially obtained from ATCC and, after expansion, were deposited in an internal cell bank. Tumor cell lines were routinely cultured in DMEM medium (GIBCO, Switzerland) supplemented with 10% fetal bovine serum (Invitrogen, Switzerland) and 2mM L-glutamine (GIBCO, Switzerland) at 37 ℃ in a water-saturated atmosphere at 5% CO 2. Cultures were passaged every three days with trypsin/EDTA 1X (GIBCO, Switzerland) split. Passage 10 was used for injection.

Animal(s) production

SCID beige female mice (purchased from Charles River, Sulzfeld, Germany) aged 8-9 weeks at the start of the experiment were maintained without specific pathogens, following the indicated guidelines (committed guideline) (GV-Solas; Felasa; TierschG), with a period of 12 hours light/12 hours dark per day. Experimental study protocol was reviewed and approved by the local government (P2005086). After the animals arrived, they were maintained for 1 week to acclimate to the new environment and facilitate observation. Continuous health monitoring is routinely performed.

Tumor cell injection

On the day of injection, a549 tumor cells were harvested from culture flasks (Greiner Bio-One) using trypsin-EDTA (Gibco, Switzerland), transferred to 50ml of medium, washed 1 time, and resuspended in AIMV (Gibco, Switzerland). After washing again with AIM V, cell concentration was determined using a cytometer. For injection of a549 cells, final titer was adjusted to 5.0x 10⁶Cells/ml. Subsequently, 200. mu.l of this mixture was injected into the lateral tail vein of the mice using a 1.0ml conjugated rhzomorph syringe (BD Biosciences, Germany).

Treatment of

Animal treatment was started two weeks after tumor cell inoculation of 10 animals per group. Bispecific anti-EGFR/anti-IGF 1R antibodies XGFR1-4421GE, XGFR1-2421GE, XGFR1-3421GE, < EGFR > ICR62GE, < IGF-1R > HUMAB-clone 18 and corresponding vehicle were administered intravenously once a week at the indicated doses. Monthly doses were administered until termination of the experiment. Prior to use, antibody dilutions were freshly prepared from stock solutions.

Table 11: study design of survival analysis in orthotopic A549 xenograft model

GE & sugar remodeling

Monitoring

Animals were controlled daily for clinical symptoms and tested for adverse effects, i.e., dyspnea, impaired mobility, and dirty fur. The study exclusion criteria for animals are described and approved in the corresponding project permit.

Identification/grading (staging)

Mice were randomly distributed when ranked. Animals were placed in M3 size cages.

Autopsy

Mice were sacrificed according to terminal criteria (dirty fur, arched back, impaired exercise). Lung tumors were collected from all animals for subsequent histopathological analysis (PFA, frozen).

Survival assay

The survivability data includes the time that lasts until a specific event occurs and is sometimes referred to as time-to-event data. The event may be, for example, death of the patient. An observation is considered to be checked (censored) if, for an observation, the event did not occur before the end of the study or when the study object left the study before the event occurred. The exact survival time is not known but is known to be greater than a specific value.

Survival data need to be analyzed by specialized methods, but they have specialized abnormal distributions, such as exponential or Weibull (Weibull) distributions. Furthermore, the observed observation cannot be ignored without deviating from the analysis.

The Kaplan-Meier curve provides an estimate of the survival function for one or more sets of correct examination data.

Table 12: subtraction of quantiles-median survival

GE & sugar remodeling

The quantile chart shows median survival time. It can be seen from table Y that median survival times were higher for days treated with the bispecific < EGFR-IGF1R > antibodies XGFR1-4421GE, XGFR1-2421GE, XGFR1-3421GE when compared to treatment with monospecific < EGFR > ICR62GE and higher or at least the same when compared to combined treatment with < EGFR > ICR62GE and < IGF-1R > HUMAB-clone 18.

Example 21

Expression and purification of bispecific, bivalent ScFab-Fc fusion < EGFR-IGF-1R > antibody molecules N-scFabSS-salt bridge-s 3 and N-scFabSS-salt bridge-w 3C, in vitro and in vivo Properties

In analogy to the methods described in examples 17, 1 and 9, the bispecific, bivalent ScFab-Fc fusion < EGFR-IGF1R > antibody molecules N-ScFab-salt bridge-s 3 and N-ScFab-salt bridge-w 3C were expressed and purified. These bispecific < EGFR-IGF-1R > antibodies are also based on the amino acid sequence of SEQ ID NO: 8, and seq id NO: 10 (from humanized < EGFR > ICR62) and is based on the amino acid sequence of SEQ ID NO: 23 and the heavy chain variable domain of SEQ ID NO: 25 (from the human anti-IGF-1R antibody < IGF-1R > HUMAB clone 18(DSMACC 2587)).

The relevant complete (modified) heavy chain amino acid sequence for the bispecific antibody molecule of N-scFabSS-salt bridge-s 3 is SEQ ID NO: 40-41, whereas the relevant complete (modified) heavy chain amino acid sequence for the bispecific antibody molecule of N-scFabSS-salt bridge-w 3C is SEQ ID NO: 42-43.

Expression yield, purity, in vitro and in vivo properties of the bispecific, bivalent ScFab-Fc fusion < EGFR-IGF1R > antibody molecules N-ScFab-salt bridge-s 3 and N-ScFab-salt bridge-w 3C were determined according to the above examples.

Example 22

Expression and purification of bispecific, trivalent ScFab-IgG fusion < EGFR-IGF1R > antibody molecules KiH-C-scFab-1 and KiH-C-scFab-2, in vitro and in vivo Properties

Similar to the methods described in examples 1 and 9, 17, for bispecific,TrivalentScFab-IgG fusion<EGFR-IGF1R>Antibody molecules KiH-C-scFab-1 and KiH-C-scFab-2 (scFab specific for IGF1R fused to the C-terminus of only one heavy chain of a full-length EGFR-specific antibody (or vice versa) using bulge-entry-hole technology) were expressed and purified. These dual specificities<EGFR-IGF-1R>The antibodies are also based on the amino acid sequence of seq id NO: 8, and the heavy chain variable domain of SEQ ID NO: 10 (from humanization)<EGFR>ICR62) and based on the amino acid sequence of SEQ ID NO: 23 and the heavy chain variable domain of SEQ ID NO: 25 (from a human anti-IGF-1R antibody)<IGF-1R>HUMAB clone 18(DSMACC 2587)).

The relevant complete (modified) heavy and light chain amino acid sequences for the bispecific antibody molecule N-scFabSS are SEQ ID NO: 44-46, and the relevant complete (modified) heavy and light chain amino acid sequences for N-scFabSS-salt bridge-s 3c are SEQ ID NOs: 47-49.

Expression yield, purity, in vitro and in vivo properties of the bispecific, bivalent ScFab-Fc fusion < EGFR-IGF1R > antibody molecules N-ScFab, N-ScFab-salt bridge-s 3 and N-ScFab-salt bridge-w 3C were determined according to the above examples.

Claims

1. Bispecific antibody binding to EGFR and IGF-1R, comprising a first antigen-binding site binding to EGFR and a second antigen-binding site binding to IGF-1R, characterized in that said bispecific antibody is characterized in that

2. The bispecific antibody of claim 1, characterized in that:

i) the first antigen binding site comprises in the heavy chain variable domain the amino acid sequence of SEQ ID NO: 1, CDR3 region of SEQ ID NO: 2, and the CDR2 region of SEQ ID NO: 3 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 4, CDR3 region of SEQ ID NO: 5, and the CDR2 region of SEQ ID NO: 6, CDR1 region; and

ii) the second antigen binding site comprises the amino acid sequence of SEQ ID NO: 11, CDR3 region of SEQ ID NO: 12, and the CDR2 region of SEQ ID NO: 13, and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 14, CDR3 region of SEQ ID NO: 15, and the CDR2 region of SEQ ID NO: 16, CDR1 region.

3. The bispecific antibody of claim 1, characterized in that:

ii) the second antigen binding site comprises the amino acid sequence of SEQ ID NO: 17, CDR3 region of SEQ ID NO: 18, and the CDR2 region of SEQ ID NO: 19 and comprises in the light chain variable domain the CDR1 region of SEQ ID NO: 20, CDR3 region of SEQ ID NO: 21, and the CDR2 region of SEQ ID NO: 22, CDR1 region.

4. The bispecific antibody of claim 1, characterized in that:

5. The bispecific antibody of claim 1, characterized in that:

6. Bispecific antibody according to any of claims 1 to 5, characterized in that said antibody is bivalent, trivalent or tetravalent.

7. The bispecific antibody according to any one of claims 1-6, characterized in that said antibody is glycosylated with a sugar chain at Asn297, wherein the amount of fucose in said sugar chain is 65% or less.

8. A pharmaceutical composition comprising the bispecific antibody of claims 1-7.

9. The pharmaceutical composition according to claim 8, for use in the treatment of cancer.

10. The bispecific antibody of any one of claims 1-7 for use in the treatment of cancer.

11. Use of a bispecific antibody according to claims 1-7 for the preparation of a medicament for the treatment of cancer.

12. A method of treating a patient suffering from cancer by administering a bispecific antibody according to claims 1-7 to a patient in need of such treatment.