BRPI1010297B1 - TRIVALENT BISECIFIC ANTIBODY AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

TRIVALENT BIESPECIFIC ANTIBODIES. The present invention relates to trivalent bispecific antibodies, methods for their production, pharmaceutical compositions containing said antibodies and uses thereof. The trivalent bispecific antibodies of the invention comprise a full-length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody chains and a VH domain fused to the C-terminus of one of said heavy chains and a VL domain fused to the C-terminus of the other said heavy chains, wherein said VH domain and said VL domain together form an antigen-binding site specifically binding to a second antigen.

Description

[0001] The present invention relates to bispecific, trivalent antibodies, methods for their production, pharmaceutical compositions containing said antibodies and uses thereof.

Background of the Invention

[0002] A wide variety of multispecific recombinant antibody formats have been developed in the recent past, e.g., tetravalent bispecific antibodies by fusion of e.g., an IgG antibody format and single chain domains (see e.g., Coloma, M. J. et al., Nature Biotech. 15 (1997) 159-163; WO 2001/077342; and Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234).

[0003] Also several other novel formats in which the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as dia-, tia- or tetrabodies, minibodies, various single chain formats (scFv, Bis-scFv), which are capable of binding two or more antigens, have been developed (Holliger, P. et al., Nature Biotech., 23 (2005) 1126-1136; Fischer, N. and Léger, O. Pathobiology 74 (2007) 3-14; Shen J. et al., Journal of Immunological Methods 318 (2007) 65-74; Wu C. et al., Nature Biotech. 25 (2007) 1290-1297).

[0004] All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to an additional binding protein (e.g. scFv) or to fuse, for example, two Fab fragments or scFvs (Fischer, N. and Léger, O., Pathobiology 74 (2007) 3-14). It should be kept in mind that one may want to retain effector functions, such as, for example, complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC), which are mediated by Fc receptor binding, by maintaining a high degree of similarity to naturally occurring antibodies.

[0005] In WO 2007/024715 dual variable domain immunoglobulins are reported as engineered multivalent and multispecific binding proteins. A process for the preparation of biologically active antibody dimers is reported in U.S. 6,897,044. Multivalent Fv antibody constructs having at least four variable domains that are linked to each other via peptide linkers are reported in U.S. 7,129,330. Dimeric and multimeric antigen-binding structures are reported in U.S. 2005/0079170. Tri- or tetravalent monospecific antigen-binding proteins comprising three or four Fab fragments linked to each other covalently through a connecting structure, a protein that is not a natural immunoglobulin, are reported in U.S. Pat. No. 6,511,663. In WO 2006/020258 tetravalent bispecific antibodies are reported, which can be efficiently expressed in prokaryotic and eukaryotic cells and are useful in therapeutic and diagnostic methods. A method of separating or preferably synthesizing dimers that are linked through at least one interchain disulfide bond from dimers that are not linked through at least one interchain disulfide bond from a mixture comprising the two types of polypeptide dimers is reported in U.S. Pat. No. 2005/0163782. Bispecific tetravalent receptors are reported in U.S. 5,959,083. Engineered antibodies with three or more functional antigen-binding sites are reported in WO 2001/077342.

[0006] Multispecific and multivalent antigen-binding polypeptides are reported in WO 1997/001580. WO 1992/004053 reports homoconjugates, typically prepared from monoclonal antibodies of the IgG class that bind to the same antigenic determinant are covalently linked by synthetic cross-linking. Oligomeric monoclonal antibodies with high avidity for antigen are reported in WO 1991/06305 such that the oligomers, typically of the IgG class, are secreted by having two or more immunoglobulin monomers associated together to form tetravalent or hexavalent IgG molecules. Sheep-derived antibodies and engineered antibody constructs are reported in U.S. 6,350,860, which can be used to treat diseases in which interferon gamma activity is pathogenic. In U.S. 2005/0100543 targetable constructs are reported that are multivalent vehicles of bispecific antibodies, i.e., each molecule of a targetable construct can serve as a vehicle for two or more bispecific antibodies. Genetically engineered tetravalent bispecific antibodies are reported in WO 1995/009917. In WO 2007/109254 stabilized binding molecules consisting of or comprising a stabilized scFv are reported.

Summary of the Invention

[0007] A first aspect of the present invention is a trivalent, bispecific antibody comprising: a) a full-length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains; b) a polypeptide consisting of (a) an antibody heavy chain variable domain (VH); or (b) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), wherein said polypeptide is fused to the N-terminus of the VH domain via a peptide linker to the C-terminus of the two heavy chains of said full-length antibody; (c) a polypeptide consisting of (a) an antibody light chain variable domain (VL), or (cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL); wherein said polypeptide is fused to the N-terminus of the VL domain via a peptide linker to the C-terminus of the other of the two heavy chains of said full-length antibody; and wherein the antibody heavy chain variable domain (VH) of the polypeptide under (b) and the antibody light chain variable domain (VL) of the polypeptide under (c) together form an antigen-binding site specifically binding to a second antigen.

[0008] A further aspect of the invention is a nucleic acid molecule encoding a trivalent, bispecific antibody according to the invention.

[0009] Additional aspects of the invention are a pharmaceutical composition comprising said trivalent, bispecific antibody.

[00010] The bispecific, trivalent antibodies according to the invention on the one hand show novel properties due to their binding to different antigens and on the other hand are suitable for pharmaceutical production and formulation due to their stability, low aggregation and pharmacokinetic and biological properties. Due to their Ig core they still retain the properties of natural antibodies such as ADCC and CDC.

Detailed Description of the Invention

[00011] One aspect of the invention is a bispecific, trivalent antibody comprising: a) a full-length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains; b) a polypeptide consisting of ba) an antibody heavy chain variable domain (VH); or (b) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), wherein said polypeptide is fused to the N-terminus of the VH domain via a peptide linker to the C-terminus of one of the two heavy chains of said full-length antibody; (c) a polypeptide consisting of (c) an antibody light chain variable domain (VL) or (cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL); wherein said polypeptide is fused to the N-terminus of the VL domain via a peptide linker to the C-terminus of the other of the two heavy chains of said full-length antibody; and wherein the antibody heavy chain variable domain (VH) of the polypeptide under (b) and the antibody light chain variable domain (VL) of the polypeptide under (c) together form an antigen-binding site specifically binding to a second antigen.

[00012] Optionally, the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via an interchain disulfide bridge by introducing a disulfide bond between the following positions: 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 (numbering always according to the Kabat EU index).

[00013] Techniques for introducing unnatural disulfide bridges for stabilization are described, for example, in WO 94/029350, Rajagopal, V. et al., Prot. Engin. (1997) 1453-59; Kobayashi, H. et al., Nuclear Medicine & Biology, Vol. 25 (1998) 387-393; or Schmidt, M. et al., Oncogene (1999) 18 1711-1721. In one embodiment the optional disulfide bond between the variable domains of the polypeptides under b) and c) is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment the optional disulfide bond between the variable domains of the polypeptides under b) and c) is between the position of the heavy chain variable domain 105 and the position of the light chain variable domain 43 (numbering always according to the Kabat EU index). In one embodiment a trivalent, bispecific antibody without said optional disulfide stabilization between the VH and VL variable domains of the single-chain Fab fragments is preferred.

[00014] The term "full-length antibody" denotes an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains" (see Figure 1). A "full-length antibody heavy chain" is a polypeptide consisting in the N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chain constant domain 4 (CH4) in the case of an antibody of the IgE subclass. Preferably the "full-length antibody heavy chain" is a polypeptide consisting in the N-terminal to C-terminal direction of VH, CH1, HR, CH2, and CH3. A "full-length antibody light chain" is a polypeptide consisting in the N-terminal to C-terminal direction of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated as VL-CL. The antibody light chain constant domain (CL) may be K (kappa) or Δ (lambda). The two full-length antibody chains are linked via interpolypeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge regions of the full-length antibody heavy chains. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE. The full-length antibodies according to the invention may be of a single species, e.g. human, or they may be chimerized or humanized antibodies. The full-length antibodies according to the invention comprise two antigen-binding sites, each formed by a pair of VH and VL, which both bind specifically to the same antigen. The C-terminus of the heavy or light chain of said full-length antibody denotes the last amino acid at the C-terminus of said heavy or light chain.

[00015] The N-terminus of the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) denote the last amino acid at the N-terminus of either the VH or VL domain.

[00016] The CH3 domains of said full-length antibody according to the invention may be altered by the "knobs-into-holes" technology which is described in detail with several examples in, for example, WO 96/027011, Ridgway, J. B. et al., Protein Eng. 9 (1996) 617-621; and Merchant, A. M. et al., Nat. Biotechnol. 16 (1998) 677-681. In this method the interaction surfaces of the two CH3 domains are altered to enhance the heterodimerization of both chains containing these CH3 domains. Each of the two CH3 domains (of the two heavy chains) may be the "knob", while the other is the "hole". The introduction of a disulfide bridge further stabilizes the heterodimers (Merchant, A. M. et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[00017] Then in one aspect of the invention said trivalent, bispecific antibody is further characterized by the fact that the CH3 domain of one heavy chain of the full-length antibody and the CH3 domain of the other heavy chain of the full-length antibody each encounter an interface comprising a native interface between the CH3 domains of the antibody; wherein said interface is altered to promote the formation of the bivalent, bispecific antibody, wherein the alteration is characterized by the fact that: a) the CH3 domain of one heavy chain is altered such that within the native interface the CH3 domain of one heavy chain encounters the native interface of the CH3 domain of the other heavy chain within the bivalent, bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain that is positionable in a cavity within the interface of the CH3 domain of the other heavy chain heavy(eb) the CH3 domain of the other heavy chain is altered so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the trivalent, bispecific antibody, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thus generating a cavity within the interface of the second CH3 domain within which a protrusion within the interface of the first CH3 domain is positionable.

[00018] Preferably, said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).

[00019] Preferably, said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).

[00020] In one aspect of the invention both CH3 domains are further altered by introducing cysteine (C) as amino acid at the corresponding positions of each CH3 domain, so that a disulfide bridge between both CH3 domains can be formed.

[00021] In a preferred embodiment, said trivalent, bispecific antibody comprises a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain". An additional interchain disulfide bridge between the CH3 domains may also be used (Merchant, A. M. et al., Nature Biotech. 16 (1998) 677-681), for example, by introducing a Y349C mutation in the CH3 domain of the "knob chain" and an E356C mutation or an S354C mutation in the CH3 domain of the "hole chain". Then, in another preferred embodiment said trivalent bispecific antibody comprises mutations Y349C, T366W in one of the two CH3 domains and mutations E356C, T366S, L368A, Y407V in the other of the two CH3 domains or said trivalent bispecific antibody comprises mutations Y349C, T366W in one of the two CH3 domains and mutations S354C, T366S, L368A, Y407V in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain forming an interchain disulfide bridge) (numbering always according to the EU Kabat index). But also other "knobs-into-hole" technologies as described by EP 1 870 459A1 can be used alternatively or additionally. A preferred example for said bispecific, trivalent antibody are mutations R409D; K370E in the CH3 domain of the "knob chain" and mutations D399K; E357K in the CH3 domain of the "hole chain" (numbering always according to the Kabat EU index).

[00022] In another preferred embodiment said trivalent bispecific antibody comprises a T366W mutation in the CH3 domain of the "knob chain" and mutations T366S, L368A, Y407V in the CH3 domain of the "hole chain" and additionally mutations R409D; K370E in the CH3 domain of the "knob chain" and mutations D399K; E357K in the CH3 domain of the "hole chain".

[00023] In another preferred embodiment said trivalent bispecific antibody comprises mutations Y349C, T366W in one of the two CH3 domains and mutations S354C, T366S, L368A, Y407V in the other of the two CH3 domains or said trivalent bispecific antibody comprises mutations Y349C, T366W in one of the two CH3 domains and mutations S354C, T366S, L368A, Y407V in the other of the two CH3 domains and further mutations R409D; K370E in the CH3 domain of the "knob chain" and mutations D399K; E357K in the CH3 domain of the "hole chain".

[00024] The bispecific antibody according to the invention comprises three antigen-binding sites (A) the full-length antibody according to comprises two identical antigen-binding sites specifically binding to the first antigen and B) the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) together form an antigen-binding site specifically binding to a second antigen). The term "binding site" or "antigen-binding site" as used herein denotes the region(s) of said bispecific antibody according to the invention to which the respective antigen actually specifically binds. The antigen binding sites on either the full-length antibody or the antibody heavy chain variable domain (VH) of the polypeptide under (b) and the antibody light chain variable domain (VL) of the polypeptide under (c) are each formed by a pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[00025] Antigen binding sites that specifically bind to the desired antigen may be derived a) from known antibodies to the antigen or b) from novel antibodies or antibody fragments obtained by immunization methods again using, inter alia, either the antigen protein or nucleic acid or fragments thereof or by phage display.

[00026] An antigen-binding site of an antibody of the invention contains six complementarity determining regions (CDRs) that contribute in varying degrees to the affinity of the binding site for antigen. There are three heavy chain variable domains (CDRH1, CDRH2, and CDRH3) and three light chain variable domains (CDRL1, CDRL2, and CDRL3). The extent of the CDR and backbone regions (FRs) is determined by comparing a compiled database of amino acid sequences in which these regions have been defined according to the variability among the sequences.

[00027] Antibody specificity refers to the selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. "Bispecific antibodies" according to the invention are antibodies that have two different antigen binding specificities. Where an antibody has more than one specificity, the recognized epitopes may be associated with a single antigen or with more than one antigen. The term "monospecific" antibody as used herein denotes an antibody that has one or more binding sites each of which binds to the same epitope of the same antigen. The term "valent" as used in the present application denotes the presence of a specified number of binding sites on an antibody molecule. A natural antibody, for example, or a full-length antibody according to the invention has two binding sites and is bivalent. Thus, the term "trivalent" denotes the presence of three binding sites on an antibody molecule. The bispecific antibodies according to the invention are "trivalent". The term "bispecific, trivalent" antibody as used herein denotes an antibody that has three antigen-binding sites of which two bind to the same antigen (or the same epitope of the antigen) and the third binds to a different antigen or a different epitope of the same antigen. The antibodies of the present invention have three binding sites and are bispecific.

[00028] Another embodiment of the present invention is a bispecific, trivalent antibody, comprising: c) a full-length antibody specifically binding to a first antigen and consisting of: aa) two antibody heavy chains consisting in the N-terminal to C-terminal direction an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody constant heavy chain domain 2 (CH2) and an antibody constant heavy chain domain 3 (CH3); and ab) two antibody light chains consisting in the N-terminal to C-terminal direction an antibody light chain variable domain (VL) and an antibody constant light chain domain (CL); and d) a polypeptide consisting of a) an antibody heavy chain variable domain (VH); or (bb) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), wherein said polypeptide is fused to the N-terminus of the VH domain via a peptide connector to the C-terminus of one of the two heavy chains of said full-length antibody, wherein said peptide connector is a peptide of at least 5 amino acids, preferably between 25 and 50 amino acids; (e) a polypeptide consisting of (ca) an antibody light chain variable domain (VL), or (cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL); wherein said polypeptide is fused to the N-terminus of the VL domain via a peptide connector to the C-terminus of the other of the two heavy chains of said full-length antibody; wherein said peptide connector is identical to the peptide connector under (b); and wherein said antibody heavy chain variable domain (VH) of the polypeptide under (b) and the antibody light chain variable domain (VL) of the polypeptide under c) together form an antigen binding site specifically binding to a second antigen.

[00029] In this embodiment, preferably the trivalent bispecific antibody comprises a T366W mutation in one of the two CH3 domains and T366S, L368A, Y407V mutations in the other of the two CH3 domains, and more preferably the trivalent bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains and D356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional D356C mutation in the other CH3 domain forming an interchain disulfide bridge).

[00030] In one embodiment of the invention the trivalent, bispecific antibody according to the invention is characterized in that a) said full-length antibody specifically binding to ErbB-3 comprises as heavy chain variable domain the sequence SEQ ID NO: 1 and as light chain variable domain the sequence SEQ ID NO: 2 b) said polypeptide under b) comprises as heavy chain variable domain the sequence SEQ ID NO: 3; and c) said polypeptide under c) comprises as the light chain variable domain the sequence SEQ ID NO: 4. In another aspect of the present invention the trivalent, bispecific antibody according to the invention comprises: a) a full-length antibody bound to a first antigen consisting of two antibody heavy chains VH-CH1-HR-CH2-CH3 and two antibody light chains VL-CL; (wherein preferably one of the two CH3 domains comprises mutations Y349C, T366W and the other of the two CH3 domains comprises mutations S354C, T366S, L368A, Y407V); b) a polypeptide consisting of (a) an antibody heavy chain variable domain (VH); or (bb) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), wherein said polypeptide is fused to the N-terminus of the VH domain via a peptide linker to the C-terminus of one of the two heavy chains of said full-length antibody; (c) a polypeptide consisting of (a) an antibody light chain variable domain (VL), or (cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL); wherein said polypeptide is fused to the N-terminus of the VL domain via a peptide linker to the C-terminus of the other of the two heavy chains of said full-length antibody; and wherein said antibody heavy chain variable domain (VH) of the polypeptide under (b) and the antibody light chain variable domain (VL) of the polypeptide under (c) together form an antigen-binding site specifically binding to a second antigen.

[00031] Another embodiment of the present invention is a bispecific, trivalent antibody comprising: a) a full-length antibody specifically binding to human ErbB-3 and consisting of: aa) two antibody heavy chains consisting in the N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody constant heavy chain domain 2 (CH2), and an antibody constant heavy chain domain 3 (CH3); and ab) two antibody light chains consisting in the N-terminal to C-terminal direction of an antibody light chain variable domain (VL) and an antibody constant light chain domain (CL) (VL-CL); and b) a single chain Fv fragment specifically binding to human c-Met), wherein said single chain Fv fragment under b) is fused to said full-length antibody under a) via a peptide connector at the C- or N-terminus of the heavy or light chain (preferably at the C-terminus of the heavy chain) of said full-length antibody; wherein said peptide connector is a peptide of at least 5 amino acids, preferably between 25 and 50 amino acids.

[00032] Preferably, such a trivalent, bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains of the full-length antibody and S354C (or E356C), T366S, L368A, Y407V mutations in the other of the two CH3 domains of the full-length antibody.

[00033] Another embodiment of the present invention is a bispecific, trivalent antibody, comprising: a) a full-length antibody specifically binding to human ErbB-3 and consisting of: aa) two antibody heavy chains in the N-terminal to C-terminal direction 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); and ab) two antibody light chains consisting in the N-terminal to C-terminal direction of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL); and b) a polypeptide consisting of: ba) an antibody heavy chain variable domain (VH); or (b) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), wherein said polypeptide is fused to the N-terminus of the VH domain via a peptide connector at the C-terminus of one of the two heavy chains of said full-length antibody wherein said peptide connector is a peptide of at least 5 amino acids, preferably between 25 and 50 amino acids; (c) a polypeptide consisting of: (ca) an antibody light chain variable domain (VL), or (cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL); wherein said polypeptide is fused to the V-terminus of the VL domain via a peptide connector to the C-terminus of the other of the two heavy chains of said full-length antibody; wherein said peptide connector is identical to the peptide connector under (b);

[00034] and wherein the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) together form an antigen binding site specifically binding to human c-Met.

[00035] Full-length antibodies of the invention comprise immunoglobulin constant regions of one or more immunoglobulin classes. Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE isotypes, and in the case of IgG and IgA, their subtypes. In a preferred embodiment, a full-length antibody of the invention has a constant domain structure of an IgG-type antibody.

[00036] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of a single amino acid composition.

[00037] The term "chimeric antibody" refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, generally prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially with respect to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-modified antibodies". Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques and are well known in the art. See, for example, Morrison, S. L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.

[00038] The term "humanized antibody" refers to antibodies in which the backbone or "complementarity determining regions" (CDRs) have been modified to comprise the CDR of an immunoglobulin of different specificity compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted onto the backbone region of a human antibody to prepare the "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 representing sequences recognizing the antigens mentioned above for chimeric antibodies. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been further modified or changed from that of the original antibody to generate the properties according to the invention, especially with respect to C1q binding and/or Fc receptor (FcR) binding.

[00039] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, M.A. and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germline immunoglobulin gene array into such a germline mutant mouse will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A. et al., Nature 362 (1993) 255-258; Brüggemann, M. et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R. and Winter, G. J. Mol. Biol. 227 (1992) 381-388; Marks, J. D. et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole, S. P. C. et al. and Boerner, P. et al. are also available for the preparation of human monoclonal antibodies (Cole, S. P. C. et al., Monoclonal Antibodies and Cancer Therapy, Alan, R. Liss, A. L., (1985) 77-96; Boerner, P. et al., J. Immunol. 147 (1991) 86-95). As already mentioned for chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially with regard to C1q binding and/or FcR binding, for example through "class switching", i.e. changing or mutating Fc parts (e.g. mutating IgG1 to IgG4 and/or IgG1/IgG4).

[00040] The term "recombinant human antibody" as used herein is intended to include all human antibodies that are prepared, expressed, raised or isolated by recombinant means, such as antibodies isolated from a host cell such as an NS0 or CHO cell or from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[00041] The "variable domain" (light chain variable domain (VL), heavy chain variable region (VH)) as used herein denotes each of the pair of light and heavy chains that is directly involved in antibody binding to antigen. The human light and heavy chain variable domains have the same general structure, and each domain comprises four framework (FR) regions whose sequences are largely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β-sheet conformation, and the CDRs can form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and together with the CDRs of the other chain form the antigen-binding site. The CDR3 regions of antibody heavy and light chains play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide an additional object of the invention.

[00042] The terms "hypervariable region" and "antigen-binding portion of an antibody" when used herein refer to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues of the "complementarity determining regions" or "CDRs". "Backbone" or "FR" regions are those regions of the variable domain other than the hypervariable region residues as defined herein. Thus, the light and heavy chains of an antibody comprise from the N-terminus to the C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs in each chain are separated by such backbone amino acids. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding. CDR and FR regions are determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[00043] As used herein, the term "binding" or "binding specifically" refers to the binding of the antibody to an epitope of the antigen in an in vitro assay, preferably in a plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) with purified wild-type antigen. Binding affinity is defined by the terms ka (rate constant for association of the antibody to the antibody/antigen complex), KD (dissociation constant) and KD (KD/Ka). Binding or binding specifically means a binding affinity (KD) of 10-8 moles/l or less, preferably 10-9 M to 10-3 moles/l. In this way, a trivalent, bispecific antibody according to the invention is specifically binding to each antigen for which it is specific with a binding affinity (KD) of 10-8 mols/l or less, preferably 10-9 M to 10-13 mols/l.

[00044] Antibody binding to FCYRIII can be investigated by a BIAcore assay (GE-Healthcare Uppsala, Sweden). Binding affinity is defined by the terms ka (rate constant for antibody association with the antibody/antigen complex), KD (dissociation constant) and KD (KD/Ka).

[00045] The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemically active surface molecule clusters such as amino acids, sugar, phosphoryl, or sulfonyl side chains, and 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.

[00046] 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.

[00047] The term "peptide connector" as used in the invention denotes a peptide with amino acid sequences, which is preferably of synthetic origin. Such peptide connectors according to the invention are used to fuse the polypeptide under b) and c) of the heavy chain C-termini of the full-length antibody to form the trivalent, bispecific antibody according to the invention. Preferably, said peptide connectors are peptides with an amino acid sequence with a length of at least 5 amino acids, preferably with a length of 10 to 100 amino acids, more preferably with a length of 25 to 50 amino acids. Preferably, said peptide connector under b) and c) are identical peptides with a length of at least 25 amino acids, preferably with a length between 25 and 50 amino acids and more preferably said peptide connector is (GxS)n or (GxS)nGm with G = glycine, S = serine and (x = 3, n = 6, 7 or 8 and m = 0, 1, 2 or 3) or (x = 4, n = 4, 5, 6 or 7 and m = 0, 1, 2 or 3), preferably x = 4 and n = 5, 6 or 7.

[00048] In a further embodiment the trivalent, bispecific antibody according to the invention is characterised in that said full-length antibody is of the human IgG1 subclass or of the human IgG1 subclass with the mutations L234A and L235A.

[00049] In a further embodiment the trivalent, bispecific antibody according to the invention is characterised in that said full-length antibody is of the human IgG2 subclass.

[00050] In a further embodiment the trivalent, bispecific antibody according to the invention is characterised in that said full-length antibody is of the human IgG3 subclass.

[00051] In a further embodiment the trivalent, bispecific antibody according to the invention is characterised in that said full-length antibody is of the human IgG4 subclass or of the human IgG4 subclass with the additional mutation S228P.

[00052] Preferably the trivalent, bispecific antibody according to the invention is characterized in that said full-length antibody is of the human IgG1 subclass, of the human IgG4 subclass with the additional S228P mutation.

[00053] It has now been found that the trivalent, bispecific antibodies according to the invention have improved characteristics such as biological or pharmacological activity, pharmacokinetic properties or toxicity. They can be used, for example, for the treatment of diseases such as cancer.

[00054] In a further embodiment the trivalent, bispecific antibody according to the invention is characterised in that it specifically binds to ErbB3 and c-Met. The term "constant region" as used in the present application denotes the sum of the domains of an antibody other than the variable region. The constant region is not directly involved in binding an antigen, but exhibits several effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are divided into the classes IgA, IgD, IgE, IgG and IgM and several of these can be further divided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to the different antibody classes are called α, δ, ε, Y and μ, respectively. The light chain constant (LC) regions that can be found in five classes of antibodies are called K (kappa) and À (lambda).

[00055] The term "human-derived constant region" as used herein denotes a human antibody constant heavy chain region of the IgG1, IgG2, IgG3, or IgG4 subclass and/or constant light chain kappa or lambda region. Such constant regions are well known in the art and, for example, described by Kabat, E.A. (see, for example, Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A. et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).

[00056] Although antibodies of the IgG4 subclass show reduced Fc receptor (FcyRIIIa) binding, antibodies of other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues that, if altered, also provide reduced Fc receptor binding (Shields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J. et al., FASEB J. 9 (1995) 115-119; Morgan, A. et al., Immunology 86 (1995) 319-324; EP 0 307 434).

[00057] In one embodiment an antibody according to the invention has reduced FcR binding compared to an IgG1 antibody and the full-length parent antibody is with respect to FcR binding of IgG4 subclass or IgG1 or IgG2 subclass with a mutation at S228, L234, L235 and/or D265, and/or contains the PVA236 mutation. In one embodiment the mutations in the full-length parent antibody are S228P, L234A, L235A, L235E and/or PVA236. In another embodiment the mutations in the full-length parent antibody are in IgG4 S228P and in IgG1 L234A and L235A.

[00058] The constant region of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by the binding of complement factor C1q to the constant region of most IgG antibody subclasses. Binding of C1q to an antibody is caused by defined protein-protein interactions at the so-called binding site. Such constant region binding sites are known in the art and described, for example, by Lukas, T. J. et al., J. Immunol. 127 (1981) 2555- 2560; Brunhouse, R. and Cebra, J. J., Mol. Immunol. 16 (1979) 907-917; Burton, D. R. et al., Nature 288 (1980) 338-344; Thommesen, J. E. et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, 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 0 307 434. Such constant region binding sites are, for example, characterized by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to Kabat's EU index).

[00059] 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 antigen-expressing cells with an antibody according to the invention in the presence of effector cells such as freshly isolated PBMC or effector cells purified from buffy coats, such as monocytes or natural killer (NK) cells or a permanently growing NK cell line.

[00060] The term "complement-dependent cytotoxicity (CDC)" denotes a process initiated by the binding of complement factor C1q to the Fc part of most IgG antibody subclasses. Binding of C1q to an antibody is caused by defined protein-protein interactions at the so-called binding site. Such Fc part binding sites are known in the prior art (see above). Such Fc part binding sites are, for example, characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU Kabat index). Antibodies of subclasses IgG1, IgG2 and IgG3 generally show complement activation including binding of C1q and C3, while IgG4 does not activate the complement system and does not bind C1q and/or C3.

[00061] Cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide component as described in Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180 and US 6,602,684. IgG1-type antibodies, the most frequently used therapeutic antibodies, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain. The two complex biantennary oligosaccharides linked to Asn297 are buried between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for the antibody to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, M. R. et al., Glycobiology 5 (1995) 813-822; Jefferis, R. et al., Immunol. Rev. 163 (1998) 59-76; Wright, A. and Morrison, S. L., TrendsBiotechnol. 15 (1997) 26-32). Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180 and WO 99/54342 have shown that overexpression in Chinese hamster ovary (CHO) cells of β(1,4)-N-acetylglucosaminyltransferase III ("GnTIII"), a glycosyltransferase that catalyzes the formation of bisected oligosaccharides, significantly increases the in vitro ADCC activity of antibodies. Changes in the composition of the carbohydrate Asn297 or its deletion also affect binding to FCYR and 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. Biol. Chem. 276 (2001) 45539-45547; Radaev, S. et al., J. Biol. Chem. 276 (2001) 16478-16483; Shields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604; Shields, R. L. et al., J. Biol. Chem. 277 (2002) 26733-26740; Simmons, L. C. et al., J. Immunol. Methods 263 (2002) 133-147).

[00062] Methods for enhancing the cell-mediated effector functions of monoclonal antibodies by reducing the amount of fucose are described in, for example, WO 2006/116260, WO 2005/011735, US 2006/0134709, WO 2003/035835, WO 2000/061739.

[00063] Surprisingly, the <ErbB3-c-Met> bispecific antibodies that are an embodiment of the invention show reduced downregulation and internalization of target antigen compared to their <ErbB3> and/or <c-Met> parent antibodies. Thus, in a preferred embodiment of the invention, the bispecific antibody is glycosylated (if it comprises an Fc part of subclass IgG1, IgG2, IgG3 or IgG4, preferably of subclass IgG1 or IgG3) with a sugar chain at Asn297 such that the amount of fucose within the sugar chain is 65% or less (Numbering according to Kabat). In another embodiment the amount of fucose within said sugar chain is between 5% and 65%, preferably between 20% and 40%. "Asn297" according to the invention means an asparagine amino acid located at about position 297 in the Fc region. Based on minor sequence variations of antibodies, Asn297 may be located a few amino acids (generally not more than ± 3 amino acids) upstream or downstream of position 297, i.e. between position 294 and position 300. In one embodiment the glycosylated antibody according to the invention of IgG subclass is of human IgG1 subclass, of human IgG1 subclass with mutations L234A and L235A or IgG3 subclass. In a further embodiment the amount of N-glycolylneuraminic acid (NGNA) is 1% or less and/or the amount of N-terminal alpha-1,3-galactose is 1% or less within the sugar chain. The sugar chain preferably shows the characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell.

[00064] The expression "the sugar chains show characteristics of N-linked glycan attached to Asn297 of an antibody recombinantly expressed in a CHO cell" denotes that the sugar chain at Asn297 of the full-length parent antibody according to the invention has the same structure and sugar residue sequence except for the fucose residue as those of the same antibody expressed in unmodified CHO cells, for example as those reported in WO 2006/103100.

[00065] The term "NGNA" as used in the present application denotes the sugar residue N-glycolylneuraminic acid.

[00066] Glycosylation of human IgG1 or IgG3 occurs at Asn297 as a biantennary core-fucosylated complex oligosaccharide glycosylation terminated with up to two Gal residues. Human constant heavy chain regions of the IgG1 and IgG3 subclasses are reported in detail by Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) and by Brüggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T.W. et al., Methods Enzymol. 178 (1989) 515-527. These structures are designated G0, G1 (α-1,6 or α-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 antibody Fc moieties is, for example, described by Routier, F.H., Glycoconjugate J. 14 (1997) 201-207. Antibodies that are recombinantly expressed in non-glycomodified CHO host cells are usually fucosylated at Asn297 in an amount of at least 85%. The modified oligosaccharides of the full-length parent antibody may be hybrid or complex. Preferably, bisected, reduced/non-fucosylated oligosaccharides are hybrids. In another embodiment, the bisected, reduced/non-fucosylated oligosaccharides are complex.

[00067] According to the invention "amount of fucose" means the amount of said sugar within the sugar chain at Asn297, related to the sum of all glycostructures linked to Asn297 (e.g. complex, hybrid and high-mannose structures) measured by MALDI-TOF mass spectrometry and calculated as the mean value. The relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures identified in a sample treated with FN-Glycosidase (e.g. complex, hybrid and oligo- and high-mannose structures, respectively) by MALDI-TOF.

[00068] The antibody according to the invention is produced by recombinant means. Accordingly, one aspect of the present invention is a nucleic acid encoding the antibody according to the invention and a further aspect is a cell comprising said nucleic acid encoding an antibody according to the invention. Methods for recombinant production are widely known in the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and generally purification to a pharmaceutically acceptable purity. For expression of the antibodies as mentioned above in a host cell, the nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods. Expression is carried out in prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.6 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 described, for example, in the reviewed articles by Makrides, S. C. Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-160; Wener, R. G., Drug Res. 48 (1998) 870-880.

[00069] The trivalent, bispecific antibodies according to the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA can be inserted into expression vectors, which are then transfected into host cells such as HEK 293 cells, CHO cells or myeloma cells that do not produce immunoglobulin protein, to achieve synthesis of recombinant monoclonal antibodies in the host cells.

[00070] Amino acid sequence variants (or mutants) of the trivalent, bispecific antibody are prepared by introducing appropriate nucleotide changes into the antibody DNA or by nucleotide synthesis. Such modifications can be carried out, however, only to a very limited extent, e.g., as described above. For example, the modifications do not alter the above-mentioned antibody characteristics such as IgG isotype and antigen binding, but may improve recombinant production yield, protein stability, or facilitate purification.

[00071] The term "host cell" as used herein denotes any type of cellular system that can be engineered to generate the antibodies according to the present invention. In one embodiment, HEK293 cells and CHO cells are used as host cells. As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably and all designations include progeny. Accordingly, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom with respect to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate and inadvertent mutations. Variant progeny that have the same function or biological activity as assessed in the originally transformed cell are included.

[00072] Expression in NS0 cells is described by, e.g., Barnes, L. M. et al., Cytotechnology 32 (2000) 109-123; Barnes, L. M. et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g., Durocher, Y. et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlangi, R. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P. et al., Proc. Natl. Acad. Sci., USA 89 (1992) 4285-4289; and Norderhaug, L. et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described by Schlaeger, E. J. and Christensen, K. in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E. J. in J. Immunol. Methods 194 (1996) 191-199.

[00073] Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.

[00074] A nucleic acid is "operably linked" when it is placed in a polyfunctional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it effects transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. In general, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[00075] Antibody purification is performed to eliminate cellular components or other contaminants, e.g., other nucleic acids or cellular proteins, by standard techniques, including alkaline/SDS treatment, CsCl strip separation, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F. et al., ed. 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 microbial protein affinity chromatography (e.g., protein A or protein G affinity chromatography), ion exchange chromatography (e.g., cation exchange (carboxymethyl resins), anion exchange (aminoethyl resins), and mixed-mode exchange), thiophilic adsorption (e.g., with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., with phenylsepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., with Ni(II) and Cu(II) affinity material), size exclusion chromatography, and electrophoretic methods (such as gel electrophoresis, capillary electrophoresis). (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).

[00076] One aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention. Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a pharmaceutical composition. A further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising an antibody according to the invention. In another aspect, the present invention provides a composition, e.g. a pharmaceutical composition, containing an antibody according to the present invention, formulated together with a pharmaceutical carrier.

[00077] One embodiment of the invention is the trivalent, bispecific antibody according to the invention for the treatment of cancer.

[00078] Another aspect of the invention is said pharmaceutical composition for the treatment of cancer.

[00079] Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a medicament for the treatment of cancer.

[00080] 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.

[00081] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[00082] A composition of the present invention may be administered by a variety of methods known in the art. As will be appreciated by one of skill in the art, the route and/or mode of administration will vary depending on the desired results. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in a suitable carrier, e.g., liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and buffered aqueous solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

[00083] The terms "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteral and topical administration, generally by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachinoid, intraspinal, epidural, and intrasternal injection and infusion.

[00084] The term cancer as used herein refers to proliferative diseases such as lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung cancer (NSCL), bronchioalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid gland cancer, parathyroid gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis carcinoma; mesothelioma, hepatocellular cancer, biliary cancer, central nervous system (CNS) neoplasms, spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymonomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, and Ewings sarcoma, including refractory versions of any of the above cancers or a combination of one or more of the above cancers.

[00085] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by the above sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride and the like, in the compositions. Further, prolonged absorption of the injectable pharmaceutical form may be achieved by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin.

[00086] Regardless of the route of administration selected, the compounds of the present invention, which can be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[00087] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to obtain the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[00088] The composition should be sterile and fluid to the point that the composition can be applied by syringe. In addition to water, the vehicle is preferably an isotonic buffered saline solution.

[00089] Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by maintaining the required particle size in the case of dispersion and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol and sodium chloride in the composition.

[00090] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably and all designations include progeny. Accordingly, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as assessed in the originally transformed cell are included. It will be clear from the context where distinct designations are intended.

[00091] The term "transformation" as used herein refers to the process of transferring vector/nucleic acid into a host cell. If cells without formidable cell wall barriers 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 for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells containing substantial cell wall constructs are used, for example, one method of transfection is calcium treatment using calcium chloride as described by Cohen, S. N. et al., PNAS 69 (1972) 2110-2114.

[00092] As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and/or the process by which the transcribed mRNA (also referred to as transcript) is subsequently translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectively referred to as the gene product. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the RNA.

[00093] A "vector" is a nucleic acid molecule, particularly a self-replicating one, that transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromosomal integration), replication vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one of the functions as described.

[00094] An "expression vector" is a polynucleotide that, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell comprised of an expression vector that can function to provide a desired expression product. Description of Amino Acid SequencesSEQ ID NO: 1 <ErbB3> heavy chain variable domain HER3 clone 29SEQ ID NO: 2 <ErbB3> light chain variable domain HER3 clone 29SEQ ID NO: 3 <c-Met> heavy chain variable domain Mab 5D5SEQ ID NO: 4 <c-Met> light chain variable domain Mab 5D5SEQ ID NO: 5 <ErbB3> heavy chain HER3 clone 29SEQ ID NO: 6 <ErbB3> light chain HER3 clone 29SEQ ID NO: 7 <c-Met> heavy chain Mab 5D5SEQ ID NO: 8 <c-Met> light chain Mab 5D5SEQ ID NO: 9 <c-Met> heavy chain Fab 5D5SEQ ID NO: 10 <c-Met> light chain Fab 5D5SEQ ID NO: 11 heavy chain 1 <ErbB3-c-Met> Her3/Met_KHSSSEQ ID NO: 12 heavy chain 2 <ErbB3-c-Met> Her3/Met_KHSSSEQ ID NO: 13 light chain <ErbB3-c-Met> Her3/Met_KHSSSEQ ID NO: 14 heavy chain 1 <ErbB3-c-Met> Her3/Met_SSKHSEQ ID NO: 15 heavy chain 2 <ErbB3-c-Met> Her3/Met_SSKHSEQ ID NO: 16 light chain <ErbB3-c-Met> Her3/Met_SSKHSEQ ID NO: 17 heavy chain 1 <ErbB3-c-Met> Her3/Met_SSKHSSSEQ ID NO: 18 heavy chain 2 <ErbB3-c-Met> Her3/Met_SSKHSSSEQ ID NO: 19 light chain <ErbB3-c-Met> Her3/Met_SSKHSSSEQ ID NO: 20 heavy chain 1 <ErbB3-c-Met> Her3/Met_1CSEQ ID NO: 21 heavy chain 2 <ErbB3-c-Met> Her3/Met_1CSEQ ID NO: 22 light chain <ErbB3-c-Met> Her3/Met_1CSEQ ID NO: 23 heavy chain 1 <ErbB3-c-Met> Her3/Met_6CSEQ ID NO: 24 heavy chain 2 <ErbB3-c-Met> Her3/Met_6CSEQ ID NO: 25 light chain <ErbB3-c-Met> Her3/Met_6CSEQ ID NO: 26 human IgG1 heavy chain constant regionSEQ ID NO: 27 human IgG1 heavy chain constant region SEQ ID NO: 28 human light chain kappa constant region SEQ ID NO: 29 human light chain lambda constant region

[00095] The following examples, sequence listings and figures are provided to aid in understanding the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications may be made to the procedures shown without departing from the spirit of the invention.

Description of figures

[00096] Figure 1 - Schematic structure of a full-length antibody lacking CH4 domain specifically binding to a first antigen 1 with two pairs of heavy and light chains comprising variable and constant domains in a typical order.

[00097] Figure 2 - Schematic representation of a bispecific, trivalent antibody according to the invention comprising a full-length antibody specifically binding to a first antigen 1 to which a) figure 2a two polypeptides VH and VL are fused (the VH and VL domains of both forming together an antigen binding site specifically binding to a second antigen 2; b) figure 2b two polypeptides VH-CH1 and VL-CL are fused (the VH and VL domains of both forming together an antigen binding site specifically binding to a second antigen 2)

[00098] Figure 3 - Schematic representation of a bispecific, trivalent antibody according to the invention comprising a full-length antibody specifically binding to a first antigen 1 to which two polypeptides VH and VL are fused (the VH and VL domains of both together forming an antigen binding site specifically binding to a second antigen 2) with "knobs and holes".

[00099] Figure 4 - Schematic representation of a bispecific, trivalent antibody according to the invention comprising a full-length antibody specifically binding to a first antigen 1 to which two VH and VL polypeptides are fused (the VH and VL domains of both together forming an antigen binding site specifically binding to a second antigen 2, wherein these VH and VL domains comprise an interchain disulfide bridge between positions VH44 and VL100) with "knobs and holes".

[000100] Figure 5 - Binding of specific antibodies to the cell surface of cancer cells

[000101] Figure 6 - Inhibition of HGF-induced c-Met receptor phosphorylation by Her3/c-Met bispecific antibody formats

[000102] Figure 7 - Inhibition of HGF-induced Her3 receptor phosphorylation by Her3/c-Met bispecific antibody formats

[000103] Figure 8 - Inhibition of HGF-induced HUVEC proliferation by Her3/c-Met bispecific antibody formats

[000104] Figure 9 - Inhibition of proliferation in the A431 cancer cell line by Her3/c-Met bispecific antibody formats

[000105] Figure 10 - Analysis of inhibition of HGF-induced cell-cell spread (spreading) in the A431 cancer cell line by Her3/c-Met bispecific antibody formats

Experimental Procedure Examples Materials & Methods Recombinant DNA techniques

[000106] Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

DNA and protein sequence analysis and sequence data processing

[000107] General information concerning nucleotide sequences of human immunoglobulin heavy and light chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242. Antibody chain amino acids are numbered according to EU numbering (Edelman, G.M. et al., PNAS 63 (1969) 78-85; Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242). The GCG (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and the Vector NTI Advance suite from Infomax 8.0 were used for sequence creation, mapping, analysis, annotation, and illustration.

DNA sequencing

[000108] DNA sequences were determined by double-stranded sequencing performed at SequiServe (Vaterstetten, Germany) and Geneart AG (Regensburg, Germany).

Gene synthesis

[000109] Desired gene segments were prepared by Geneert AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. Gene segments that are flanked by unique restriction endonuclease cleavage sites were cloned into pGA18(ampR) plasmids. Plasmid DNA was purified from transformed bacteria and the concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments encoding Her3 antibody heavy chain "knobs-into-hole" carrying a T366W mutation in the CH3 domain with a C-terminal 5D5 VH region linked by a (G4S)n peptide connector as well as Her3 antibody heavy chain (clone 29) "knobs-into-hole" carrying T366S, L368A, and Y407V mutations with a C-terminal 5D5 VL region linked by a (G4S)n peptide connector were synthesized with 5'-BamHI and 3'-XbaI restriction sites. In a similar manner, DNA sequences encoding Her3 (clone 29) antibody heavy chain "knobs-into-hole" carrying mutations S354C and T366W in the CH3 domain with a C-terminal 5D5 VH region linked by a (G4S)n peptide connector as well as Her3 (clone 29) antibody heavy chain "knobs-into-hole" carrying mutations Y349C, T366S, L368A, and Y407V with a C-terminal 5D5 VL region linked by a (G4S)n peptide connector were prepared by gene synthesis with flanking BamHI and XbaI restriction sites. Finally, DNA sequences encoding unmodified Her3 (clone 29) and 5D5 antibody heavy and light chains were synthesized with flanking BamHI and XbaI restriction sites. All constructs were designed with a 5' end DNA sequence encoding a leader peptide (MGWSCIILFLVATATGVHS), which targets proteins for secretion in eukaryotic cells.

Construction of expression plasmids

[000110] A Roche expression vector was used for the construction of all expression plasmids encoding the VH/VL heavy and light chain fusion protein encoding expression plasmids. The vector is composed of the following elements: - a hygromycin resistance gene as a selection marker, - an origin of replication, oriP, from Epstein-Barr virus (EBV), - an origin of replication from the pUC18 vector, which allows replication of this plasmid in E. coli, - a beta-lactamase gene that confers ampicillin resistance in E. coli, - the human cytomegalovirus (HCMV) immediate early enhancer and promoter, - the human immunoglobulin 1 ("poly A") polyadenylation signal sequence, and - unique BamHI and XbaI restriction sites.

[000111] Immunoglobulin fusion genes comprising the heavy or light chain constructs as well as "knobs-into-hole" constructs with C-terminal VH and VL domains were prepared by gene synthesis and cloned into pGA18(ampR) plasmids as described. The pG18(ampR) plasmids carrying the synthesized DNA segments and the Roche expression vector were digested with BamHI and XbaI restriction enzymes (Roche Molecular Biochemicals) and subjected to agarose gel electrophoresis. Purified heavy and light chain encoding DNA segments were then ligated to the BamHI/XbaI fragment of the isolated Roche expression vector resulting in the final expression vectors. The final expression vectors were transformed into E. coli cells, expression plasmid DNA was isolated (Miniprep) and subjected to restriction enzyme analysis and DNA sequencing. Correct clones were grown in 150 mL of LB-Amp medium, again plasmid DNA was isolated (Maxiprep) and sequence integrity confirmed by DNA sequencing.

Transient expression of immunoglobulin variants in HEK293 cells

[000112] Recombinant immunoglobulin variants were expressed by transient transfection of human embryonic kidney 293-F cells using the FreeStyle® 293 Expression System according to the manufacturer's instructions (Invitrogen, USA). Briefly, FreeStyle® 293-F cells in suspension were cultured in FreeStyle® 293 Expression Medium at 37°C/8% CO2 and cells were seeded in fresh medium at a density of 1-2x106 viable cells/ml on the day of transfection. DNA-293fectin® complexes were prepared in Opti-MEM® I medium (Invitrogen, USA) using 325 μl of 293fectin® (Invitrogen, Germany) and 250 μg of heavy and light chain plasmid DNA in a 1:1 molar ratio for a final transfection volume of 250 ml. DNA-293 fectin® "Knobs-into-hole" complexes were prepared in Opti-MEM® I medium (Invitrogen, USA) using 325 μl of 293fectin® (Invitrogen, Germany) and 250 μg of heavy chain 1 and 2 and light chain "Knobs-into-hole" plasmid DNA in a 1:1:2 molar ratio for a final transfection volume of 250 ml. Antibody-containing cell culture supernatants were collected 7 days after transfection by centrifugation at 14000 g for 30 min and filtered through a sterile filter (0.22 μm). Supernatants were stored at -20°C until purification.

Purification of trivalent and control bispecific antibodies

[000113] Trivalent and control bispecific antibodies were purified from cell culture supernatants by affinity chromatography using Protein A-Sepharose® (GE Healthcare, Sweden) and Superdex200 size exclusion chromatography. Briefly, sterile filtered cell culture supernatants were applied to a HiTrap ProteinA HP column (5 ml) equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins were washed away with equilibration buffer. Antibody and antibody variants were eluted with 0.1 M citrate buffer, pH 2.8, and protein-containing fractions were neutralized with 0.1 ml of 1 M Tris, pH 8.5. Then, the eluted protein fractions were pooled, concentrated with an Amicon Ultra centrifugal filter device (MWCO: 30 K, Millipore) to a volume of 3 ml, and loaded onto a 120 ml Superdex200 HiLoad 16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20 mM Histidine, 140 mM NaCl, pH 6.0. Fractions containing purified bispecific and control antibodies with less than 5% high molecular weight aggregates were pooled and stored as 1.0 mg/ml aliquots at −80°C. Fab fragments were generated by papain digestion of purified monoclonal antibody 5D5 and subsequent removal of contaminating Fc domains by Protein A chromatography. Unbound Fab fragments were further purified on a 120 ml 16/60 Superdex200 HiLoad gel filtration column (GE Healthcare, Sweden) equilibrated with 20 mM Histidine, 140 mM NaCl, pH 6.0, pooled and stored as 1.0 mg/ml aliquots at -80°C.

Analysis of purified proteins

[000114] The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence. Purity and molecular weight of bispecific and control antibodies were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM, 1,4-dithiothreitol) and staining with Coomassie brilliant blue. The NuPAGE® Pre-cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-20% Tris-Glycine gels). The aggregate content of bispecific and control antibody samples was analyzed by high-performance SEC using a Superdex 200 analytical size exclusion column (GE Healthcare, Sweden) in 200 mM KH2PO4, 250 mM KCl, pH 7.0 activity buffer at 25°C. 25 μg of protein was injected onto the column at a flow rate of 0.5 ml/min and eluted isocratically over 50 min. For stability analysis, 1 mg/ml concentrations of purified proteins were incubated at 4°C and 40°C for 7 days and then evaluated by high-performance SEC. The integrity of the amino acid backbone of reduced bispecific antibody light and heavy chains was verified by NanoElectrospray Q-TOF mass spectrometry after removal of N-glycans by enzymatic treatment with Peptide-N-Glycosidase F (Roche Molecular Biochemicals).

c-Met phosphorylation assay

[000115] 5x10e5 A549 cells were seeded per well of a 6-well plate the day before HGF stimulation in RPMI with 0.5% FCS (fetal bovine serum). The following day, the growth medium was replaced for one hour with RPMI containing 0.2% BSA (bovine serum albumin). 5 μg/mL of the bispecific antibody was then added to the medium and the cells were incubated for 10 minutes when HGF was added for an additional 10 minutes at a final concentration of 50 ng/mL. Cells were washed once with ice-cold PBS containing 1 mM sodium vanadate when they were placed on ice and lysed in a cell culture dish with 100 μL of lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% NP40, 0.5% DOC, aprotinin, 0.5 mM PMSF, 1 mM sodium vanadate). Cell lysates were transferred to eppendorf tubes and lysis was allowed to proceed for 30 min on ice. Protein concentration was determined using the BCA method (Pierce). 30–50 μg of lysate was separated on a 4–12% Bis-Tris NuPage gel (Invitrogen) and the proteins in the gel were transferred to a nitrocellulose membrane. Membranes were blocked for 1 h with TBS-T containing 5% BSA and developed with a phospho-specific c-Met antibody against Y1230-1234,1235 (44-888, Biosource) according to the manufacturer's instructions. Immunoblots were reprobed with an antibody binding to unphosphorylated c-Met (AF276, R&D).

Her3 (ErbB3) phosphorylation assay

[000116] 2x10e5 MCF7 cells were seeded per well of a 12-well plate in complete growth medium (RPMI 1640, 10% FCS). Cells were allowed to grow to 90% confluence within two days. Medium was then replaced with starvation medium containing 0.5% FCS. The following day the respective antibodies were supplemented at the indicated concentrations 1 hour before the addition of 500 ng/mL Heregulin (R&D). Upon addition of Heregulin the cells were cultured for an additional 10 minutes before the cells were harvested and lysed. Protein concentration was determined using the BCA method (Pierce). 30-50 μL of the lysate was separated on a 4-12% NuPage Bis-Tris gel and the proteins in the gel were transferred to a nitrocellulose membrane. Membranes were blocked for one hour with TBS-T containing 5% BSA and developed with a phosphospecific Her3/ErbB3 antibody specifically recognizing Tyr1289 (4791, Cell Signaling).

Spreading test

[000117] A549 (4000 cells per well) or A431 (8000 cells per well) were seeded the day before compound treatment in a total volume of 200 μL in 96-well E-Plates (Roche, 05232368001) in RPMI with 0.5% FCS. Cell adhesion and growth were monitored overnight with the Real Time Cell Analyzer machine with scans every 15 min monitoring the impedance. The following day, cells were pre-incubated with 5 μL of the respective antibody dilutions in PBS with scans every five minutes. After 30 minutes, 2.5 μL of an HGF solution giving a final concentration of 20 ng/mL was added and the experiment was allowed to proceed for another 72 hours. Immediate changes were monitored with scans every minute for 180 minutes followed by scans every 15 minutes for the remainder of the time.

FACS a) Bonding Test

[000118] A431 were sorted and counted. 1.5x10e5 cells were seeded per well of a 96-well conical plate. Cells were spun (1500 rpm, 4°C, 5 min) and incubated for 30 min on ice in 50 μL of a dilution series of the respective bispecific antibody in PBS with 2% FCS (fetal bovine serum). Cells were spun again and washed once with 200 μL of PBS containing 2% FCS followed by a second 30 min incubation with a phycoerythrin-coupled antibody directed against human Fc that was diluted in PBS containing 2% FCS (Jackson Immunoresearch, 109226098). Cells were spun down, washed twice with 200 μL of PBS containing 2% FCS, resuspended in BD CellFix solution (BD Bioscience), and incubated for at least 10 min on ice. Mean fluorescence intensity (mfi) of cells was determined by flow cytometry (FACS Canto, BD). Mfi was determined in at least duplicates of two independent stainings. Flow cytometry spectra were further processed using FlowJo software (TreeStar). Half-maximum binding was determined using XLFit 4.0 (IDBS) and the one-site dose-response model 205.

b) Internalization Test

[000119] Cells were sorted and counted. 5x10e5 cells were placed in 50 μL of complete medium in an eppendorf tube and incubated with 5 μg/mL of the respective bispecific antibody at 37°C. After the indicated time points the cells were stored on ice until the time course was complete. Then, the cells were transferred to FACS tubes, spun (1500 rpm, 4°C, 5 min), washed with PBS + 2% FCS and incubated for 30 minutes in 50 μL of phycoerythrin-coupled secondary antibody directed against human Fc that was diluted in PBS containing 2% FCS (Jackson Immunoresearch, 109116098). Cells were spun down again, washed with PBS + 2% FCS and fluorescence intensity was determined by flow cytometry (FACS Canto, BD).c) Crosslinking Experiment HT29 cells were sorted, counted and separated into two populations that were individually stained with PKH26 and PKH67 (Sigma) according to the manufacturer's instructions. From each of the stained populations 5x10e5 cells were picked, combined and incubated for 30 and 60 min with 10 μg/mL of the respective bispecific antibody in complete medium. After the indicated time points cells were stored on ice until the time course was completed. Cells were spun (1500 rpm, 4°C, 5 min), washed with PBS + 2% FCS and fluorescence intensity was determined by flow cytometry (FACS Canto, BD).

Cell Titer Brightness Assay

[000120] Cell viability and proliferation were quantified using the cell titer glow assay (Promega). The assay was performed according to the manufacturer's instructions. Briefly, cells were cultured in 96-well plates in a total volume of 100 μL for the desired period of time. For the proliferation assay, cells were removed from the incubator and placed at room temperature for 30 min. 100 μL of cell titer glow reagent was added and multivalent plates were placed on an orbital shaker for 2 min. Luminescence was quantified after 15 min on a microplate reader (Tecan).

Wst-1 Test

[000121] A Wst-1 cell viability and proliferation assay was performed as an endpoint analysis, detecting the number of metabolically active cells. Briefly, 20 μL of Wst-1 reagent (Roche, 116448070001) was added to 200 μL of culture medium. 96-well plates were further incubated for 30 minutes to 1 hour until robust dye development. Staining intensity was quantified on a microplate reader (Tecan) at a wavelength of 450 nm. Design of expressed and purified bispecific, trivalent <ErbB3-c-Met> antibodies In Table 1: Bispecific, trivalent <ErbB3-c-Met> antibodies based on a full-length ErbB-2 antibody (Her3 clone 29) and the VH and VL domains of an antibody to c-Met (c-Met 5D5) with the respective characteristics shown in Table 1 were expressed and purified according to the general methods described above. The corresponding VH and VL domains of Her3 clone 29 and c-Met 5D5 are given in the sequence listing. Table 1: Bispecific, trivalent <ErbB3-c-Met> antibodies with the VHVL-Ab nomenclature in Table 1 were expressed and purified (see also the Examples below and Figure 3c

Example 1 (figure 5): Binding of bispecific antibodies to the cell surface of cancer cells

[000122] The binding properties of bispecific antibodies to their respective cell surface receptor were analyzed on A431 cancer cells in a flow cytometry-based assay. Cells were incubated with the mono- or bispecific primary antibodies and binding of these antibodies to their cognate receptors was detected with a secondary antibody coupled to a fluorophore specifically binding to the Fc of the primary antibody. The mean fluorescence intensity of a dilution series of the primary antibodies was plotted against antibody concentration to obtain a sigmoidal binding curve. Cell surface expression of c-Met and Her3 was validated by incubation with 5D5 antibody and bivalent Her3 clone23 only. The Her3/c-Met_KHSS antibody readily binds to the cell surface of A431. Under these experimental settings, the antibody can bind only through its Her3 part and consequently the mean fluorescence intensity does not exceed that staining for Her3 clone29 alone.

Example 2 (figure 6): Inhibition of HGF-induced c-Met receptor phosphorylation by Her3/c-Met bispecific antibody formats

[000123] To confirm the functionality of the c-Met moiety in the bispecific antibodies a c-Met phosphorylation assay was performed. In this experiment, A549 lung cancer cells or HT29 colorectal cancer cells were treated with the bispecific antibodies or control antibodies prior to exposure to HGF. The cells were then lysed and phosphorylation of the c-Met receptor was examined. Both cell lines can be stimulated with HGF as can be observed by the occurrence of a specific band for phospho-c-Met in the immunoblot.

Example 3 (figure 6): Inhibition of HGR-induced Her3 receptor phosphorylation by Her3/c-Met bispecific antibody formats

[000124] To confirm the functionality of the Her3 moiety in the bispecific antibodies a Her3 phosphorylation assay was performed. In this experiment, MCF7 cells were treated with the bispecific antibodies or control antibodies prior to exposure to HRG (Heregulin). The cells were then lysed and phosphorylation of the Her3 receptor was examined. Her3/c-Met_KHSS inhibits Her3 receptor phosphorylation to the same degree as the parent Her3 clone29 indicating that binding and functionality to Her3 of the antibody are not compromised by the trivalent antibody format.

Example 4 (figure 8): Inhibition of HGF-induced HUVEC proliferation by Her3/c-Met bispecific antibody formats

[000125] HUVEC proliferation assays were performed to demonstrate the mitogenic effect of HGF. Addition of HGF to HUVEC leads to a two-fold increase in proliferation. Addition of control antibody to human IgG in the same concentration range as the bispecific antibodies has no impact on cell proliferation while the 5D5 Fab fragment inhibits HGF-induced proliferation. Titration of Her3/c-Met_KHSS demonstrates a weak inhibitory effect of the antibody (Figure 8). The effect is more pronounced for the Her3/Met-6C antibody indicating that a longer connector improves the efficacy of the antibody. This demonstrates the functionality of the c-Met component in the trivalent antibody format.

Example 5 (figure 9): Inhibition of proliferation in the A431 cancer cell line by Her3/c-Met bispecific antibody formats

[000126] If A431 were seeded in serum-reduced medium, addition of HGF induced a weak mitogenic effect on the spreading part. This was exploited to analyze the impact of Her3/c-Met_KHSS on proliferation of A431 treated with HGF. Indeed, bispecific antibodies could inhibit largely the HGF-induced increase in proliferation (15%). A control antibody to human IgG had no influence on HGF-promoted A431 cell growth.

Example 6 (figure 10): Analysis of inhibition of HGF-induced cell-cell spread (spreading) in A431 cancer cell line by Her3/c-Met bispecific antibody formats

[000127] HGF-induced spreading includes cell morphological changes, resulting in cell rounding, filopodia-like protrusions, spindle-like structures, and some cell motility. The Real Time Cell Analyzer (Roche) measures the impedance of a given cell culture well and can then indirectly monitor changes in cell morphology and proliferation. Addition of HGF to A431 and A549 cells resulted in impedance changes that were monitored as a function of time. Her3/c-Met_KHSS and Her3/Met-6C inhibited HGF-induced spreading with Her3/Met-6C being more effective (20.7% and 43.7% spreading inhibition) (Figure 10).

Claims (4)

1. A trivalent bispecific antibody comprising: (a) a full-length antibody comprising two ErbB-3 binding sites and consisting of two antibody heavy chains defined by SEQ ID NO: 1 and two antibody light chains defined by SEQ ID NO: 2; (b) a polypeptide consisting of (ba) an antibody heavy chain variable domain (VH) defined by SEQ ID NO: 3; or (bb) an antibody heavy chain variable domain (VH) defined by SEQ ID NO: 3 and an antibody constant domain 1 (CH1), wherein said polypeptide is fused to the N-terminus of the VH domain via a peptide linker to the C-terminus of the two heavy chains of said full-length antibody, wherein the linker is a peptide of at least 5 amino acids, preferably between 25 and 50 amino acids more preferably it is (GXS)n or (GXS)nGm, where (x = 3, n = 6, 7 or 8 and m = 0, 1, 2 or 3) or (x = 4, n = 4, 5, 6 or 7 and m = 0, 1, 2 or 3), preferably x = 4 and n = 5, 6 or 7; or (cb) an antibody light chain variable domain (VL) defined by SEQ ID NO: 4 and an antibody light chain constant domain (CL); wherein said polypeptide is fused to the N-terminus of the VL domain via a peptide linker to the C-terminus of the other of the two heavy chains of said full-length antibody, where the linker is a peptide of at least 5 amino acids, preferably between 25 and 50 amino acids more preferably it is (GXS)n or (GXS)nGm, where (x = 3, n = 6, 7 or 8 and m = 0, 1, 2 or 3) or (x = 4, n = 4, 5, 6 or 7 and m = 0, 1, 2 or 3), preferably x = 4 and n = 5, 6 or 7; and wherein the antibody heavy chain variable domain (VH) of the polypeptide under (b) and the antibody light chain variable domain (VL) of the polypeptide under (c) together form a second antigen binding site specifically binding to c-Met; wherein said full-length antibody that specifically binds to ErbB-3 comprises (i) a CH3 domain comprising a T366W mutation; and the other CH3 domain comprising mutations T366S, L368A, Y407V; or (ii) a CH3 domain comprising mutations Y349C, T366; and the other CH3 domain comprising mutations S354C, T366S, L368A, Y407V. 2. The trivalent bispecific antibody of claim 1, wherein: the antibody heavy chain variable domain (VH) of the polypeptide under (b) and the antibody light chain variable domain (VL) of the polypeptide under (c) are linked and stabilized via an interchain disulfide bridge by introducing a disulfide bond between the following positions: (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. 3. The trivalent bispecific antibody of claim 2, wherein: the antibody heavy chain variable domain (VH) of the polypeptide under (b) and the antibody light chain variable domain (VL) of the polypeptide under (c) are linked and stabilized via an interchain disulfide bridge by introducing a disulfide bridge between the following positions: (1) heavy chain variable domain position 44 to light chain variable domain position 100. 4. Pharmaceutical composition, characterized by the fact that it comprises a trivalent bispecific antibody, as defined in any one of claims 1 to 3.