Antibody that binds ERBB-2 and ERBB-3

Abstract

The invention relates among others to antibodies comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3. The antibodies can typically reduce a ligand-induced receptor function of ErbB-3 on a ErbB-2 and ErbB-3 positive cell. Also described are method for the treatment and use of the antibodies in imaging and in the treatment of subjects having an ErbB-2, ErbB-3 or ErbB-2 / 3 positive tumor.

Description

(19) *EP004644425A1* (11) EP 4 644 425 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 05.11.2025 Bulletin 2025 / 45 (21) Application number: 24216946.4 (22) Date of filing: 27.02.2015 (51) International Patent Classification (IPC): C07K 16 / 32 (2006.01) A61P 43 / 00 (2006.01) (52) Cooperative Patent Classification (CPC): C07K 16 / 32; A61P 9 / 04; A61P 35 / 00; A61P 35 / 04; A61P 43 / 00; C07K 2317 / 31; C07K 2317 / 55; C07K 2317 / 565; C07K 2317 / 732; C07K 2317 / 76; C07K 2317 / 92 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR Designated Extension States: BA ME (30) Priority: 28.02.2014 EP 14157360 05.05.2014 EP 14167066 (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 20191815.8 / 3 805 268 15708907.9 / 3 110 849 (71) Applicant: Merus N.V. 3584 CT Utrecht (NL) (72) Inventors: • GEUIJEN, Cecilia Anna Wilhelmina 3584 CT Utrecht (NL) • DE KRUIF, Cornelis Adriaan 3584 CT Utrecht (NL) • THROSBY, Mark 3584 CT Utrecht (NL) • LOGTENBERG, Ton 3584 CT Utrecht (NL) • BAKKER, Alexander Berthold Hendrik 3584 CT Utrecht (NL) (74) Representative: V.O. P.O. Box 87930 2508 DH Den Haag (NL) Remarks: •This application was filed on 02.12.2024 as a divisional application to the application mentioned under INID code 62. •Claims filed after the date of filing of the application (Rule 68(4) EPC). (54) Antibody that binds ErbB‑2 and ErbB‑3 (57) The invention relates among others to antibo- dies comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3. The antibodies can typically reduce a ligand- induced receptor function of ErbB‑3 on a ErbB‑2 and ErbB‑3 positive cell. Also described are method for the treatment and use of the antibodies in imaging and in the treatment of subjects having an ErbB‑2, ErbB‑3 or ErbB‑2 / 3 positive tumor. EP 4 64 4 42 5 A 1 Processed by Luminess, 75001 PARIS (FR) Description

[0001] The invention relates to the field of antibodies. In particular it relates to the field of therapeutic (human) antibodies for the treatment of diseases involvingaberrant cells.More in particular it relates to antibodies that bindErbB‑2andErbB‑3 and their use in the binding of ErbB‑2 and ErbB‑3 positive cells, particularly tumor cells.

[0002] The human epidermal growth factor receptor family (HER, also collectively referred to as the ErbB signaling network) is a family of transmembrane receptor tyrosine kinases (RTK). The family includes the epidermal growth factor receptor (EGFR), also known as ErbB‑1 (or HERl), and the homologous receptors ErbB‑2 (HER2), ErbB‑3 (HER3) and ErbB‑4 (HER4). The receptors (reviewed inYardenandPines2012) arewidely expressedonepithelial cells. Upregulation ofHER receptors or their ligands, such as heregulin (HRG) or epidermal growth factor (EGF), is a frequent event in human cancer (Wilson, Fridlyandet al. 2012).OverexpressionofErbB‑1andErbB‑2 in particular occurs in epithelial tumors and is associated with tumor invasion, metastasis, resistance to chemotherapy, and poor prognosis (Zhang, Berezov et al. 2007). In the normal breast, ErbB‑3has been shown to be important in the growth and differentiation of luminal epithelium. For instance, loss / inhibition of ErbB‑3 results in selective expansion of the basal over the luminal epithelium (Balko, Miller et al. 2012). Binding of ligand to the extracellular domain of the RTKs induces receptor dimerization, both between the same (homodimerization) anddifferent (heterodimerization) receptor subtypes.Dimerization canactivate the intracellular tyrosine kinase domains, which undergo autophosphorylation and, in turn, can activate a number of downstream pro- proliferative signaling pathways, including those mediated by mitogen-activated protein kinases (MAPK) and the prosurvival pathway Akt (reviewed in Yarden and Pines, 2012). No specific endogenous ligand has been identified for ErbB‑2, which is therefore assumed to normally signal through heterodimerization (Sergina, Rausch et al. 2007). ErbB‑3 canbeactivatedbyengagement of its ligands. These ligands includebut arenot limited toneuregulin (NRG)andheregulin (HRG).

[0003] Variousmodesof activationof signaling of theErbB receptor family havebeen identified.Among theseare ligand dependentand ligand independentactivationof signaling.Over-expressedErbB‑2 isable togenerateoncogenicsignaling through the ErbB‑2:ErbB‑3 heterodimer even in the absence of the ErbB‑3 ligand (Junttila, Akita et al. 2009). ErbB‑2 activity can be inhibited by ErbB‑2 specific antibodies. Such ErbB‑2 specific antibodies are for instance used in the treatment of ErbB‑2 positive (HER2+) tumors. A problem with such treatments is that often tumors escape the ErbB‑2 specific treatment and continue to grow even in the presence of the inhibiting antibody. It has been observed that ErbB‑2 positive tumors, such as breast, ovarian, cervical and gastric tumors can escape treatment by the selective outgrowth of a subpopulation of tumor cells that exhibit upregulated ErbB‑3 expression (Ocana, Vera-Badillo et al. 2013) and / or ErbB‑3 ligand expression (Wilson, Fridlyand et al. 2012). Also activating mutations in the ErbB‑3 receptor have been identified.

[0004] The anti-ErbB‑2 monoclonal antibody trastuzumab (Herceptin) and the ErbB‑1 specific cetuximab (Erbitux) are among several monoclonal antibodies approved for clinical application. Trastuzumab has a proven survival benefit in metastatic breast cancer (Arteaga, Sliwkowski et al. 2011). The precisemechanismof action of trastuzumab has not been unequivocally established. Suggestedmodes of action are the inhibition of RTK signaling and the recruitment of antibody dependent cellular cytotoxicity (ADCC).Othermechanismsofaction thathavebeendescribed includeblockingproteolytic cleavage of the ErbB‑2 extracellular domain, inhibition of angiogenic factors and enhancement of receptor endocytosis. Other agents that interfere with ErbB‑2 signaling have been approved or are under development for treatment of breast andotherErbB‑2overexpressioncancers.Forexample, thechemical compound lapatinib inhibitsbothErbB‑1andErbB‑2 tyrosine kinase activity and is used in first line treatment of ErbB‑2 amplified breast cancer.

[0005] In patients with HER2+ metastatic breast cancer, resistance to trastuzumab either as single-agent or in combination with chemotherapy, commonly occurs within months of starting therapy. Only a fraction of patients with HER2+metastatic breast cancer respond to single agent trastuzumab, suggesting de novomechanisms of resistance in advanced cancers. These mechanisms include, among others, signaling from other HER family of receptors and compensatory signaling from RTKs outside of the HER family (Thery et al., Resistance to human epidermal growth factor receptor type 2-targeted therapies, Eur J Cancer (2014), Vol. 50, Issue 5, pages 892‑901 (ttp: / / dx.doi.org / 10.1016 / j.ejca.2014.01.003)). For example, overexpression of HER3 or its ligands along with HER2 leads to the formation of HER‑2 / HER‑3 heterodimers and acquired resistance to trastuzumab. Thus, the antibody trastuzumab is thought to be ineffective in blocking signaling driven by ErbB‑3 ligands (Wehrman, Raab et al. 2006, Junttila, Akita et al. 2009, Thery et al. 2014).

[0006] Recently the monoclonal antibody pertuzumab was approved for use in combination with trastuzumab on the basis of an extra 5months progression-free survival benefit (Baselga, Cortes et al. 2012). Pertuzumab also binds ErbB‑2 but at a different position than trastuzumab.

[0007] Other strategies to treat ErbB‑2 positive tumors are directed towards ErbB‑3. ErbB‑3 binding monoclonal antibodies have demonstrated activity in preclinical studies (Schoeberl, Faber et al. 2010). Some ErbB‑3 binding monoclonal antibodies can inhibit proliferation and growth of a variety of cancers.

[0008] Another strategy involves binding of both the ErbB‑2 and ErbB‑3 receptor. The molecule MM‑111, is an artificial biological molecule containing two single chain Fv (scFv) fragments that bind ErbB‑2 and ErbB‑3. The two scFv are 2 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 associated with a mutated human serum albumin (HSA) protein to increase the half-life of the molecule. In preclinical testing the molecule was shown to inhibit ErbB‑3 signaling and proliferation. This effect was predominantly measured on ErbB‑3 positive cell lines that expressed relatively high amounts of ErbB‑2. SUMMARY OF THE INVENTION

[0009] The invention provides a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, andwherein the antibody can reduce a ligand-induced receptor function of ErbB‑3 on a ErbB‑2 and ErbB‑3 positive cell. Said first antigen-binding site is preferably present in a variable domain comprising aVHchainwith theaminoacid sequenceofVHchainMF2926;MF2930;MF1849;MF2973;MF3004;MF3958 (is humanized MF2971); MF2971; MF3025; MF2916; MF3991 (is humanized MF3004); MF3031; MF2889; MF2913; MF1847; MF3001; MF3003 or MF1898 as depicted in Figure 16A or Figure 16E. Said second antigen-binding site is preferably present in a variable domain comprising a VH chain with the amino acid sequence of VH chain MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074 as depicted in Figure 16B or Figure 16E or Figure 37. The immunoglobulin light chain in the variable domain preferably comprises the amino acid sequence of figure 16C.

[0010] An antibody of the invention is, unless otherwise specifically specified, preferably a bispecific antibody.

[0011] The invention further provides a pharmaceutical composition comprising an antibody according to the invention.

[0012] Further provided is an antibody according to the invention that further comprises a label, preferably a label for in vivo imaging.

[0013] The invention also provides a method for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor or at risk of having said tumor comprising administering to the subject a bispecific antibody according to the invention. Also provided is a bispecific antibody according to the invention for use in the treatment of a subject having or at risk of having an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor. DETAILED DESCRIPTION OF THE INVENTION

[0014] The invention provides a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein the bispecific antibody reduces or can reduce a ligand-induced receptor function of ErbB‑3 on a ErbB‑2 and ErbB‑3 positive cell.

[0015] As used herein, the term "antigen-binding site" refers to a site derived from and preferably as present on a bispecific antibody which is capable of binding to antigen. An unmodified antigen-binding site is typically formed by and present in the variable domain of the antibody. The variable domain contains said antigen-binding site. A variable domain that binds an antigen is a variable domain comprising an antigen-binding site that binds the antigen.

[0016] In one embodiment an antibody variable domain of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). The antigen-binding site can be present in the combined VH / VL variable domain, or in only the VH region or only the VL region. When the antigen-binding site is present in only one of the two regions of the variable domain, the counterpart variable region can contribute to the folding and / or stability of the binding variable region, but does not significantly contribute to the binding of the antigen itself.

[0017] As used herein, antigen-binding refers to the typical binding capacity of an antibody to its antigen. An antibody comprising an antigen-binding site that binds toErbB‑2, binds toErbB‑2and, under otherwise identical conditions, at least 100-fold lower to the homologous receptors ErbB‑1 and ErbB‑4 of the same species. An antibody comprising an antigen- binding site that binds to ErbB‑3, binds to ErbB‑3 and, under otherwise identical conditions, not to the homologous receptorsErbB‑1andErbB‑4of thesamespecies.Considering that theErbB-family isa familyof cell surface receptors, the binding is typically assessed on cells that express the receptor(s). Binding of an antibody to an antigen can be assessed in various ways. One way is to incubate the antibody with the antigen (preferably cells expressing the antigen), removing unbound antibody (preferably by a wash step) and detecting bound antibody bymeans of a labeled antibody that binds to the bound antibody.

[0018] Antigenbindingbyanantibody is typicallymediated through the complementarity regions of the antibodyand the specific three-dimensional structure of both the antigen and the variable domain allowing these two structures to bind togetherwith precision (an interaction similar to a lockand key), as opposed to random,non-specific sticking of antibodies. As an antibody typically recognizes an epitope of an antigen, and as such epitopemay be present in other compounds as well, antibodies according to the present invention that bind ErbB‑2 and / or ErbB‑3may recognize other proteins aswell, if such other compounds contain the same epitope. Hence, the term "binding" does not exclude binding of the antibodies to another protein or protein(s) that contain the same epitope. Such other protein(s) is preferably not a human protein. An ErbB‑2 antigen-binding site and an ErbB‑3 antigen-binding site as defined in the present invention typically do not bind to other proteins on the membrane of cells in a post-natal, preferably adult human. A bispecific antibody according to the 3 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 present invention is typically capable of bindingErbB‑2 andErbB‑3with a binding affinity of at least 1x10e‑6M, as outlined in more detail below.

[0019] The term "interfereswith binding" as usedhereinmeans that the antibody is directed to anepitope onErbB‑3and the antibody competes with ligand for binding to ErbB‑3. The antibody may diminish ligand binding, displace ligand when this is alreadybound toErbB‑3or itmay, for instance through steric hindrance, at least partially prevent that ligandcanbind to ErbB‑3.

[0020] The term "antibody" as used herein means a proteinaceous molecule, preferably belonging to the immunoglo- bulin class of proteins, containing one or more variable domains that bind an epitope on an antigen, where such domains are derived from or share sequence homology with the variable domain of an antibody. Antibodies for therapeutic use are preferably as close to natural antibodies of the subject to be treated as possible (for instance human antibodies for human subjects). Antibody binding can be expressed in terms of specificity and affinity. The specificity determines which antigen or epitope thereof is specifically bound by the binding domain. The affinity is a measure for the strength of binding to a particular antigen or epitope. Specific binding, is defined as binding with affinities (KD) of at least 1x10e‑6 M, more preferably 1x10e‑7 M, more preferably higher than 1x10e‑9 M. Typically, antibodies for therapeutic applications have affinities of up to 1x10e‑10 M or higher. Antibodies such the bispecific antibodies of the present invention comprise the constant domains (Fc part) of a natural antibody. An antibody of the invention is typically a bispecific full length antibody, preferably of the human IgG subclass. Preferably, an antibody of the present invention is of the human IgG1 subclass. Such antibodies of the invention have good ADCC properties, have favorable half life upon in vivo administration to humans and CH3 engineering technology exists that can provide for modified heavy chains that preferentially form heterodimers over homodimers upon co-expression in clonal cells.

[0021] Anantibodyof the invention ispreferably a "full length" antibody.The term ’full length’ according to the invention is definedascomprisinganessentially completeantibody,whichhowever doesnot necessarily haveall functionsof an intact antibody. For the avoidance of doubt, a full length antibody contains two heavy and two light chains. Each chain contains constant (C) and variable (V) regions, which can be broken down into domains designated CH1, CH2, CH3, VH, and CL, VL. An antibody binds to antigen via the variable domains contained in the Fab portion, and after binding can interact with molecules and cells of the immune system through the constant domains, mostly through the Fc portion. The terms ’variable domain’, ‘VH / VL pair’, ‘VH / VL’ are used herein interchangeably. Full length antibodies according to the invention encompass antibodieswhereinmutationsmaybepresent that provide desired characteristics. Suchmutations should not be deletions of substantial portions of any of the regions. However, antibodieswherein one or several amino acid residues are deleted, without essentially altering the binding characteristics of the resulting antibody are embraced within the term "full length antibody". For instance, an IgG antibody can have 1‑20 amino acid residue insertions, deletions or a combination thereof in the constant region. For instance, ADCC activity of an antibody can be improved when the antibody itself has a lowADCCactivity, by slightlymodifying the constant region of the antibody (Junttila, T. T., K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70(11): 4481‑4489)

[0022] Full length IgG antibodies are preferred because of their favourable half life and the need to stay as close to fully autologous (human) molecules for reasons of immunogenicity. An antibody of the invention is preferably a bispecific IgG antibody, preferably a bispecific full length IgG1 antibody. IgG1 is favoured based on its long circulatory half life in man. In order topreventany immunogenicity inhumans it is preferred that thebispecific IgGantibodyaccording to the invention isa human IgG1.

[0023] The term ‘bispecific’ (bs) means that one part of the antibody (as defined above) binds to one epitope on an antigenwhereas a second part binds to a different epitope. The different epitope is typically present on a different antigen. According to the present invention, said first and second antigens are in fact two different proteins. A preferred bispecific antibody isanantibody that comprisespartsof twodifferentmonoclonal antibodiesandconsequently binds to twodifferent types of antigen. One arm of the bispecific antibody typically contains the variable domain of one antibody and the other arm contains the variable domain of another antibody. The heavy chain variable regions of the bispecific antibody of the invention are typically different from each other, whereas the light chain variable regions are preferably the same in the bispecific antibodies of the invention. A bispecific antibody wherein the different heavy chain variable regions are associated with the same, or a common, light chain is also referred to as a bispecific antibody with a common light chain. Further provided is therefore a bispecific antibody according to the invention, wherein both arms comprise a common light chain.

[0024] Preferred bispecific antibodies can be obtained by co-expression of two different heavy chains and a common light chain in a single cell. When wildtype CH3 domains are used, co-expression of two different heavy chains and a common light chain will result in three different species, AA, AB and BB. To increase the percentage of the desired bispecific product (AB) CH3 engineering can be employed, or in other words, one can use heavy chains with compatible heterodimerization domains, as defined hereunder.

[0025] The term ’compatible heterodimerization domains’ as used herein refers to protein domains that are engineered such that engineered domain A’ will preferentially form heterodimers with engineered domain B’ and vice versa, whereas 4 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 homodimerization between A’-A’ and B’-B’ is diminished.

[0026] The term ’common light chain’ according to the invention refers to light chains which may be identical or have some amino acid sequence differences while the binding specificity of the full length antibody is not affected . It is for instance possible within the scope of the definition of common light chains as used herein, to prepare or find light chains that are not identical but still functionally equivalent, e.g., by introducing and testing conservative amino acid changes, changes of amino acids in regions that do not or only partly contribute to binding specificity when paired with the heavy chain, and the like. The terms ’common light chain’, ‘commonVL’, ‘single light chain’, ‘singleVL’,withorwithout theaddition of the term ‘rearranged’ are all used herein interchangeably. It is an aspect of the present invention to use as common light chain a human light chain that can combine with different heavy chains to form antibodies with functional antigen binding domains (WO2004 / 009618,WO2009 / 157771,Merchant et al. 1998 andNissim et al. 1994). Preferably, the common light chain hasagermline sequence.Apreferredgermline sequence is a light chain variable region that is frequently used in the human repertoire and has good thermodynamic stability, yield and solubility. A preferred germline light chain is O12, preferably the rearranged germline human kappa light chain IgVκ1‑39*01 / IGJκ1*01 or a fragment or a functional equivalent (i.e. same IgVκ1‑39 gene segment but different IGJκ gene segment) thereof (nomenclature according to the IMGT database worldwide web at imgt.org). Further provided is therefore a bispecific antibody according to the invention, wherein said common light chain is a germline light chain, preferably a rearranged germline human kappa light chain comprising the IgVKl‑39 gene segment, most preferably the rearranged germline human kappa light chain IgVKI‑39*01 / IGJKI*01. The terms rearranged germline human kappa light chain IgVκ1‑39*01 / IGJκ1*01, IGKV1‑39 / IGKJ1, huVκ1‑39 light chain or in short huVκ1‑39 are used interchangeably throughout the application. Obviously, those of skill in the art will recognize that "common" also refers to functional equivalents of the light chain of which the amino acid sequence is not identical. Many variants of said light chain exist wherein mutations (deletions, substitutions, additions) are present that do not materially influence the formation of functional binding regions. The light chain of the present invention can also be a light chain as specified herein above, having 1‑5 amino acid insertions, deletions, substitutions or a combination thereof.

[0027] Also contemplated are antibodies wherein a VH is capable of specifically recognizing a first antigen and the VL, paired with the VH in a immunoglobulin variable domain, is capable of specifically recognizing a second antigen. The resultingVH / VLpairwill bindeither antigen1or antigen2.Suchsocalled "two-in-oneantibodies", described in for instance WO 2008 / 027236, WO 2010 / 108127 and Schaefer et al (Cancer Cell 20, 472‑486, October 2011), are different from bispecific antibodies of the invention and are further referred to as "two-in-one" antibodies. Such "two-in-one" antibodies have identical arms and are not antibodies of the present invention.

[0028] The term ’ErbB‑2’ as usedherein refers to theprotein that in humans is encodedby theERBB‑2gene.Alternative names for the gene or protein include CD340; HER‑2; HER‑2 / neu; MLN 19; NEU; NGL; TKR1. The ERBB‑2 gene is frequently called HER2 (from human epidermal growth factor receptor 2). Where reference is made herein to ErbB‑2, the reference refers tohumanErbB‑2.Anantibodycomprisinganantigen-bindingsite thatbindsErbB‑2,bindshumanErbB‑2. The ErbB‑2 antigen-binding site may, due to sequence and tertiary structure similarity between human and other mammalian orthologs, also bind such an ortholog but not necessarily so. Database accession numbers for the human ErbB‑2 protein and the gene encoding it are (NP_001005862.1, NP_004439.2 NC_000017.10 NT_010783.15 NC_018928.2). The accession numbers are primarily given to provide a further method of identification of ErbB‑2 as a target, the actual sequence of the ErbB‑2 protein bound the antibodymay vary, for instance because of amutation in the encoding gene such as those occurring in some cancers or the like. The ErbB‑2 antigen binding site binds ErbB‑2 and a variety of variants thereof, such as those expressed by some ErbB‑2 positive tumor cells.

[0029] The term ’ErbB‑3’ as usedherein refers to theprotein that in humans is encodedby theERBB‑3gene.Alternative names for the gene or protein are HER3; LCCS2; MDA-BF‑1; c-ErbB‑3; c-erbb‑3; erbb‑3-S; p180-Erbb‑3; p45-sErbb‑3; and p85-sErbb‑3. Where reference is made herein to ErbB‑3, the reference refers to human ErbB‑3. An antibody comprising an antigen-binding site that binds ErbB‑3, binds human ErbB‑3. The ErbB‑3 antigen-binding site, may, due to sequence and tertiary structure similarity between human and othermammalian orthologs, also bind such an ortholog but not necessarily so. Database accession numbers for the human ErbB‑3 protein and the gene encoding it are (NP_001005915.1 NP_001973.2, NC_000012.11 NC_018923.2 NT_029419.12 ). The accession numbers are primarily given toprovide a furthermethodof identificationofErbB‑3asa target, theactual sequenceof theErbB‑3protein boundby anantibodymay vary, for instancebecauseof amutation in theencoding gene suchas those occurring in somecancers or the like. The ErbB‑3 antigen binding site binds ErbB‑3 and a variety of variants thereof, such as those expressed by some ErbB‑2 positive tumor cells.

[0030] A bispecific antibody of the invention that comprises a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, can reduce or reduces a ligand-induced receptor function of ErbB‑3 on an ErbB‑2 and ErbB‑3 positive cell. In the presence of excess ErbB‑2, ErbB‑2 / ErbB‑3 heterodimers may provide a growth signal to the expressing cell in the absence of detectable ligand for the ErbB‑3 chain in the heterodimer. This ErbB‑3 receptor function is herein referred as a ligand-independent receptor function of ErbB‑3. The ErbB‑2 / ErbB‑3 heterodimer also provide a growth signal to the expressing cell in the presence of an ErbB‑3 ligand. This ErbB‑3 receptor function is herein 5 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 referred to as a ligand-induced receptor function of ErbB‑3.

[0031] The term "ErbB‑3 ligand" as used herein refers to polypeptides which bind and activate ErbB‑3. Examples of ErbB‑3 ligands include, but arenot limited to neuregulin 1 (NRG)andneuregulin 2, betacellulin, heparin-bindingepidermal growth factor, and epiregulin. The term includes biologically active fragments and / or variants of a naturally occurring polypeptide.

[0032] In a preferred embodiment of the invention the ligand-induced receptor function of ErbB‑3 is ErbB‑3 ligand- induced growth of an ErbB‑2 and ErbB‑3 positive cell. In a preferred embodiment said cell is an MCF‑7 cell (ATCC® HTB‑22™); an SKBR3 (ATCC® HTB‑30™) cell; an NCI‑87 (ATCC® CRL‑5822™) cell; a BxPC‑3-luc2 cell (Perkin Elmer 125058), a BT‑474 cell (ATCC® HTB‑20™) or a JIMT‑1 cell (DSMZ no.: ACC 589).

[0033] In apreferredembodiment theErbB‑2andErbB‑3positive cell comprisesat least 50.000ErbB‑2 receptorson the cell surface. In a preferred embodiment at least 100.000 ErbB‑2 receptors. In one preferred embodiment, the ErbB‑2 and ErbB‑3 positive cell comprises at least 1.000.000 ErbB‑2 receptors on the cell surface. In another preferred embodiment the ErbB‑2 and ErbB‑3 positive cell comprises no more than 1.000.000 ErbB‑2 receptors on the cell surface. Currently used therapies such as trastuzumab (Herceptin) and pertuzumab are only prescribed for patients with malignant ErbB‑2 positive cells that have more than 1.000.000 ErbB‑2 receptors on their cell surface, in order to obtain a clinical response. Patients with ErbB‑2 positive tumor cells with more than 1.000.000 ErbB‑2 receptors on their cell surface are typically classified as ErbB‑2 [+++]. Patients are for instance classified using the HercepTest™ and / or HER2 FISH (pharm Dx™), marketed both by Dako Denmark A / S, and / or using a HERmark® assay, marketed by Monogram Biosciences. Trastu- zumab and pertuzumab are only prescribed to ErbB‑2 [+++] patients because patients with lower ErbB‑2 concentrations typically do not exhibit a sufficient clinical response when treated with trastuzumab and pertuzumab. The invention, however, provides bispecific antibodies that also have an improved binding affinity for cells with a lower ErbB‑2 receptor concentration, as compared to trastuzumab. As shown in the Examples, proliferation of such cells with lower ErbB2 expression is effectively counteracted with an antibody according to the invention. Such lower ErbB‑2 receptor con- centration is present on malignant cells of patients that are classified as ErbB‑2 [++] or ErbB‑2 [+]. Also, relapsed ErbB‑2 positive tumors often have anErbB‑2 receptor concentration of lower than 1.000.000 receptors per cell. Such ErbB‑2 [++] or ErbB‑2 [+] patients, as well as patients with a relapsed ErbB‑2 positive tumor, are therefore preferably treated with a bispecific antibody according to the present invention. Further provided is therefore a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein the antibody can reduce ligand-induced growth of an ErbB‑2 and ErbB‑3 positive cell that has less than 1.000.000 ErbB‑2 cell-surface receptors. Also provided is a method for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumoror at risk of having said tumor,wherein said tumorhas less than1.000.000ErbB‑2cell-surface receptors per cell, the method comprising administering to the subject a bispecific antibody or pharmaceutical composition according to the invention. A bispecific antibody according to the invention for use in the treatment of a subject having or at risk of having an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, wherein said tumor has less than 1.000.000 ErbB‑2 cell-surface receptors per cell, is also herewith provided. Said antibody according to the present invention is typically capable of reducing a ligand-induced receptor function, preferably ligand induced growth, of ErbB‑3 on aErbB‑2 andErbB‑3 positive cell. Said antibodyaccording to the inventionpreferably comprisesafirst antigen-binding site that bindsdomain I ofErbB‑2 and a second antigen-binding site that binds domain III of ErbB‑3. In one preferred embodiment, the affinity of said second antigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB‑2positive cell, as explainedherein below inmoredetail. Theaffinity of said secondantigen-binding site for anErbB‑3 positive cell is preferably lower than or equal to 2.0 nM, more preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. The affinity of said first antigen-binding site for an ErbB‑2 positive cell is preferably lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM preferably lower than or equal to 4.0 nM.

[0034] In one preferred embodiment, said antibody according to the invention comprises an antigen-binding site that binds at least one amino acid of domain I of ErbB‑2 selected from the group consisting of T144, T164, R166, P172, G179, S180andR181, and surface-exposedaminoacid residues that are locatedwithin about 5 aminoacid positions fromT144, T164, R166, P172, G179, S180 or R181. In one preferred embodiment, said antibody according to the invention preferably comprises an antigen-binding site that binds at least one amino acid of domain III of ErbB‑3 selected from the group consisting of R426 and surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB‑3 protein.

[0035] To establish whether a tumor is positive for ErbB‑3 the skilled person can for instance determine the ErbB‑3 amplificationand / or staining in immunohistochemistry. At least 10% tumor cells in a biopt should bepositive. Thebiopt can also contain 20%, 30% 40% 50% 60% 70% or more positive cells.

[0036] As used herein the ligand-induced receptor function is reduced by at least 20%, preferably at least 30, 40, 50 60, or at least 70% in a particularly preferred embodiment the ligand-induced receptor function is reduced by 80, more preferably by 90%. The reduction is preferably determined by determining a ligand-induced receptor function in the presence of a bispecific antibody of the invention, and comparing it with the same function in the absence of the antibody, under otherwise identical conditions. The conditions comprise at least the presence of an ErbB‑3 ligand. The amount of 6 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 ligand present is preferably an amount that induces half of themaximumgrowth of an ErbB‑2 andErbB‑3 positive cell line. The ErbB‑2 and ErbB‑3 positive cell line for this test is preferably the MCF‑7 cell line (ATCC® HTB‑22™), the SKBR3 cell line (ATCC®HTB‑30™) cells, the JIMT‑1 cell line (DSMZ ACC 589) or the NCI‑87 cell line (ATCC®CRL‑5822™). The test and / or the ligand for determining ErbB‑3 ligand-induced receptor function is preferably a test for ErbB‑3 ligand induced growth reduction as specified in the examples.

[0037] TheErbB‑2protein contains several domains (see for referencefigure1ofLandgraf,RBreastCancerRes. 2007; 9(1): 202‑). The extracellular domains are referred to as domains I-IV. The place of binding to the respective domains of antigen-binding sites of antibodies described herein has been mapped (see examples). A bispecific antibody of the invention with an antigen-binding site (first antigen-binding site) that binds domain I or domain IVof ErbB‑2 (first antigen- binding site) comprises a heavy chain variable region that maintains significant binding specificity and affinity for ErbB‑2 when combined with various light chains. Bispecific antibodies with an antigen-binding site (first antigen-binding site) that binds domain I or domain IVof ErbB‑2 (first antigen-binding site) and an antigen-binding site for ErbB‑3 (second antigen- binding site) were found to bemore effective in reducing a ligand-induced receptor function of ErbB‑3when compared to a bispecific antibody comprising an antigen-binding site (first antigen-binding site) that binds to another extra-cellular domain of ErbB‑2. A bispecific antibody comprising an antigen-binding site (first antigen-binding site) that binds ErbB‑2, wherein saidantigen-bindingsitebinds todomain I or domain IVofErbB‑2 ispreferred.Preferably saidantigen-bindingsite binds to domain IV of ErbB‑2. A bispecific antibody with an antigen-binding site (first antigen-binding site) that binds ErbB‑2, and that further comprisesADCCwas found to bemore effective than other ErbB‑2 binding antibodies that did not have significant ADCCactivity, particularly in vivo.Abispecific antibody according to the inventionwhich exhibits ADCC is therefore preferred. It was found that antibodies wherein said first antigen-binding site binds to domain IV of ErbB‑2 had intrinsic ADCCactivity. A domain I bindingErbB‑2 binding antibody that has low intrinsic ADCCactivity can be engineered to enhance the ADCC activity Fc regions mediate antibody function by binding to different receptors on immune effector cells such as macrophages, natural killer cells, B-cells and neutrophils. Some of these receptors, such as CD16A (FcyRIIIA) andCD32A (FcyRIIA), activate the cells to build a responseagainst antigens.Other receptors, suchasCD32B, inhibit the activation of immune cells. By engineering Fc regions (through introducing amino acid substitutions) that bind to activating receptors with greater selectivity, antibodies can be created that have greater capability to mediate cytotoxic activities desired by an anti-cancer Mab.

[0038] One technique forenhancingADCCofanantibody isafucosylation. (See for instanceJunttila,T.T.,K.Parsons,et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." CancerResearch70(11): 4481‑4489). Further provided is therefore abispecific antibodyaccording to the invention,which is afucosylated. Alternatively, or additionally, multiple other strategies can be used to achieve ADCC enhancement, for instance including glycoengineering (KyowaHakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) andmutagenesis (Xencor and Macrogenics), all of which seek to improve Fc binding to low-affinity activating FcyRIIIa, and / or to reduce binding to the low affinity inhibitory FcyRIIb.

[0039] Several in vitromethods exist for determining the efficacy of antibodies or effector cells in elicitingADCC.Among thesearechromium‑51 [Cr51] releaseassays, europium [Eu] releaseassays, andsulfur‑35 [S35] releaseassays.Usually, a labeled target cell line expressing a certain surface-exposed antigen is incubated with antibody specific for that antigen. Afterwashing, effector cells expressingFc receptorCD16are typically co-incubatedwith theantibody-labeled target cells. Target cell lysis is subsequently typicallymeasuredby releaseof intracellular label, for instancebyascintillation counter or spectrophotometry. A preferred test is detailed in the Examples.

[0040] Oneadvantageof thepresent invention is the fact that bindingof antibodiesaccording to the invention suchas for instance PB4188 to ErbB‑2 and ErbB‑3 positive cells results in internalization that is to the same extent as compared to trastuzumab. If a combination of trastuzumab and pertuzumab is used, internalization of these antibodies is enhanced. Thisenhanced internalization, however, results in reducedADCC.Anantibodyaccording to thepresent invention resulting in internalization that is essentially to the same extent as compared to trastuzumab is, therefore, preferred over a combination of trastuzumab and pertuzumab because with such antibody the ADCC activity is better maintained.

[0041] An antibody of the invention comprising an antigen-binding site that binds ErbB‑3, interferes with binding of an ErbB‑3 ligand toErbB‑3.Suchantibodiesaremoreeffective in reducinga ligand-induced receptor functionofErbB‑3onan ErbB‑2 and ErbB‑3 positive cell line, particularly in the context of an bi-specific antibody that also comprises an antigen- binding site that binds ErbB‑2.

[0042] Preferred embodiments of the current invention provide a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site binds domain I of ErbB‑2. As shown in the Examples, bispecific antibodies having these characteristics are well capable of binding ErbB‑2 and ErbB‑3 positive cells and counteracting their activity (such as the ligand-induced receptor function of ErbB‑3 and the ligand-induced growth of an ErbB‑2 and ErbB3 positive cell). Moreover, bispecific antibodies according to the invention comprising a first antigen-binding site that binds domain I of ErbB‑2 are particularly suitable for use in combinationwithexistinganti-ErbB‑2 therapies like trastuzumabandpertuzumab, because trastuzumabandpertuzumab bind different domains of ErbB‑2. Trastuzumab binds domain IV of ErbB‑2 and pertuzumab binds domain II of ErbB‑2. 7 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Hence, bispecific antibodies according to the invention that bind domain I of ErbB‑2 are preferred because they do not compete with trastuzumab and pertuzumab for the same epitope.

[0043] Another preferred embodiment provides a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said second antigen-binding site binds domain III of ErbB‑3. Such antibody according to the invention is particularly suitable for combination therapy with currently used anti‑ ErbB‑3 binding molecules that do not bind domain III of ErbB‑3, such as MM‑121 (Merrimack Pharmaceuticals; also referred to as #Ab6) and RG7116 (Roche) that bind domain I of ErbB‑3, because then the different binding molecules do not compete with each other for the same epitope.

[0044] Preferably, a bispecific antibody is provided that comprises a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site binds domain I of ErbB‑2 and said second antigen-binding site binds domain III of ErbB‑3. Such antibody is particularly suitable for combination therapywith anti‑ ErbB‑2 binding molecules that do not bind domain I of ErbB‑2, such as trastuzumab and pertuzumab, and with anti‑ ErbB‑3 binding molecules that do not bind domain III of ErbB‑3, such as MM‑121 (#Ab6) and RG7116.

[0045] One preferred embodiment provides a bispecific antibody that comprises a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site binds domain I of ErbB‑2 and said second antigen-binding site binds domain III of ErbB‑3 and wherein the antibody can reduce a ligand- induced receptor function of ErbB‑3 on a ErbB‑2 and ErbB‑3 positive cell. Said antibody can preferably reduce ligand- induced growth of an ErbB‑2 and ErbB‑3 positive cell.

[0046] Further embodiments of the invention provide a bispecific antibody comprising a first antigen-binding site that bindsErbB‑2andasecondantigen-binding site that bindsErbB‑3,wherein theaffinity (KD) of said secondantigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB‑2 positive cell.Contrary to prior art bispecific compoundssuchas for instanceMM‑111 fromMerrimackPharmaceuticals,whichhave a higher affinity for ErbB‑2 than for ErbB‑3, the present invention provides bispecific antibodies which have an ErbB‑3- specific armwith an affinity for ErbB‑3 on cells that is higher than the affinity of the ErbB‑2-specific arm for ErbB‑2 on cells. Such bispecific antibodies are better capable of binding ErbB‑3, despite the low cell surface concentration of ErbB‑3. This provides the advantage that the functional activity against ErbB‑3 is enhanced as compared to prior art compounds, meaning that these bispecific antibodies according to the invention are better capable of counteracting ErbB‑3 activity (such as ligand-induced growth).

[0047] As used herein, the term "affinity" refers to the KD value.

[0048] Theaffinity (KD) of said secondantigen-binding site for anErbB‑3positive cell is preferably lower thanor equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, more preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. In one preferred embodiment, the affinity of said second antigen-binding site for ErbB‑3 on SK-BR‑3cells is lower thanor equal to2.0nM,morepreferably lower thanor equal to 1.5nM,morepreferably lower thanor equal to1.39nM,preferably lower thanor equal to0.99nM. Inoneembodiment, saidaffinity iswithin the rangeof 1.39‑0.59 nM. In one preferred embodiment, the affinity of said second antigen-binding site for ErbB‑3 on BT‑474 cells is lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, more preferably lower than or equal to 1.0 nM, more preferably lower than 0.5 nM, more preferably lower than or equal to 0.31 nM, more preferably lower than or equal to 0.23 nM. In one embodiment, said affinity is within the range of 0.31‑0.15 nM. The above-mentioned affinities are preferably as measured using steady state cell affinity measurements, wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell-bound radioactivity is measured, as described in the Examples.

[0049] Theaffinity (KD) of said first antigen-binding site for anErbB‑2 positive cell is preferably lower than or equal to 5.0 nM, more preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 3.9 nM. In one preferred embodiment, the affinity of said first antigen-binding site for ErbB‑2 on SK-BR‑3 cells is lower than or equal to 5.0 nM, preferably lower thanor equal to4.5nM,morepreferably lower thanor equal to4.0nM,morepreferably lower thanor equal to 3.5 nM, more preferably lower than or equal to 3.0 nM, more preferably lower than or equal to 2.3 nM. In one embodiment, said affinity is within the range of 3.0‑1.6 nM. In one preferred embodiment, the affinity of said first antigen- binding site for ErbB‑2 on BT‑474 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 3.9 nM. In one embodiment, said affinity is within the range of 4.5‑3.3 nM. The above- mentionedaffinitiesarepreferably asmeasuredusingsteadystate cell affinitymeasurements,wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell-bound radioactivity is measured, as described in the Examples.

[0050] In one preferred embodiment, a bispecific antibody according to the invention is provided, wherein the affinity (KD) of said bispecific antibody for BT‑474 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM, morepreferably lower thanor equal to 4.0nM,morepreferably lower thanor equal to 3.5nM,morepreferably lower thanor equal to 3.7 nM, preferably lower than or equal to 3.2 nM. In oneembodiment, said affinity iswithin the rangeof 3.7‑2.7 nM. In one preferred embodiment, a bispecific antibody according to the invention is provided, wherein the affinity of said bispecific antibody for SK-BR‑3 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM, more preferably lower thanor equal to4.0nM,morepreferably lower thanor equal to3.5nM,morepreferably lower thanor equal 8 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 to 3.0 nM, preferably lower than or equal to 2.5 nM,morepreferably lower thanor equal to 2.0 nM. In oneembodiment, said affinity is within the range of 2.4‑1.6 nM. Again, the above-mentioned affinities are preferably as measured using steady state cell affinity measurements, wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell- bound radioactivity is measured, as described in the Examples.

[0051] Further preferred embodiments of the invention provide a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein the affinity (KD) of said second antigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB‑2 positive cell, and wherein the antibody can reduce a ligand-induced receptor function of ErbB‑3 on a ErbB‑2 and ErbB‑3 positive cell. Said antibody can preferably reduce ligand-induced growth of an ErbB‑2 and ErbB‑3 positive cell.

[0052] Theabove-mentionedantibodies according to the inventionwith a highaffinity for ErbB‑3preferably binddomain I of ErbB2 and / or domain III of ErbB‑3. Further provided is, therefore, a bispecific antibody according to the invention that comprises a first antigen-binding site that binds domain I of ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein theaffinity (KD)of said secondantigen-binding site for anErbB‑3positive cell is equal to, or higher than, theaffinity of said first antigen-binding site for anErbB‑2 positive cell. Also provided is a bispecific antibody according to the invention that comprises a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds domain III of ErbB‑3, wherein the affinity of said second antigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB‑2 positive cell. In a particularly preferred embodiment a bispecific antibody according to the invention is provided that comprises a first antigen-binding site that binds domain I of ErbB‑2and a secondantigen-binding site that binds domain III of ErbB‑3,wherein theaffinity of said secondantigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB‑2 positive cell.

[0053] Said second antigen-binding site preferably binds domain III of ErbB‑3 and has an affinity (KD) for an ErbB‑3 positive cell that is lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. In one preferred embodiment, said second antigen- binding site binds domain III of ErbB‑3 and has an affinity for ErbB‑3 onSK-BR‑3 cells that is lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. In one embodiment, said affinity is within the range of 1.39‑0.59 nM. In one preferred embodiment, said second antigen-binding site binds domain III of ErbB‑3 and has an affinity for ErbB‑3 on BT‑474 cells that is lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, more preferably lower than or equal to 1.0 nM, more preferably lower than or equal to 0.5 nM, more preferably lower than or equal to 0.31 nM, more preferably lower than or equal to 0.23 nM. In one embodiment, said affinity is within the range of 0.31‑0.15 nM.

[0054] Said first antigen-binding site preferably binds domain I of ErbB‑2 and has an affinity (KD) for an ErbB‑2 positive cell that is lower than or equal to 5.0 nM,morepreferably lower thanor equal to 4.5 nM,morepreferably lower thanor equal to 3.9 nM. In one preferred embodiment, said first antigen-binding site binds domain I of ErbB‑2 and has an affinity for ErbB‑2 on SK-BR‑3 cells that is lower than or equal to 5.0 nM, more preferably lower than or equal to 4.5 nM, more preferably lower thanor equal to4.0nM,morepreferably lower thanor equal to3.5nM,morepreferably lower thanor equal to 3.0 nM, more preferably lower than or equal to 2.5 nM, more preferably lower than or equal to 2.3 nM. In one embodiment, said affinity is within the range of 3.0‑1.6 nM. The affinity of said bispecific antibody for SK-BR‑3 cells is preferably lower thanor equal to5.0nM,morepreferably lower thanor equal to4.5nM,morepreferably lower thanor equal to 4.0 nM, more preferably lower than or equal to 3.5 nM, more preferably lower than or equal to 3.0 nM, more preferably lower thanor equal to 2.5 nM,morepreferably lower thanor equal to 2.4 nM,morepreferably lower thanor equal to 2.0 nM. In one embodiment, said affinity is within the range of 2.4‑1.6 nM.

[0055] In one preferred embodiment, said first antigen-binding site binds domain I of ErbB‑2 and has an affinity (KD) for ErbB‑2onBT‑474cells that is lower thanorequal to5.0nM,morepreferably lower thanor equal to4.5nM,preferably lower than or equal to 3.9 nM. In one embodiment, said affinity is within the range of 4.5‑3.3 nM. The affinity of said bispecific antibody for BT‑474 cells is preferably lower than or equal to 5.0 nM, more preferably lower than or equal to 4.5 nM, more preferably lower thanor equal to4.0nM,morepreferably lower thanor equal to3.7nM,morepreferably lower thanor equal to 3.2 nM. In one embodiment, said affinity is within the range of 3.7‑2.7 nM.

[0056] Again, the above-mentioned affinities are preferably asmeasured using steady state cell affinitymeasurements, wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell-bound radioactivity is measured, as described in the Examples.

[0057] Another preferred embodiment provides a bispecific antibody according to the invention comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein the antibody can reducea ligand-induced receptor function of ErbB‑3onaErbB‑2andErbB‑3positive cell, wherein said bispecific antibody does not significantly affect the survival of cardiomyocytes. Cardiotoxicity is a known risk factor in ErbB‑2 targeting therapies and the frequency of complications is increased when trastuzumab is used in conjunction with anthracyclines thereby inducing cardiac stress. For instance, the combination of doxycycline (DOX) with trastuzumab induces severe cardiac side effects. Clinical studies have estimated that 5% to 10% of patients who receive trastuzumab in the adjuvant settingofbreast cancerdevelopcardiacdysfunction (Guarneri et al., JClinOncol., 1985,3:818‑26;EwerMSetal.,NatRev 9 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Cardiol 2010;7:564‑75). However, in a retrospective study, it was demonstrated that the risk for developing asymptomatic cardiac dysfunction is actually as high as about 25%when trastuzumab is used in the adjuvant settingwithDOX (Wadhwa et al., Breast Cancer Res Treat 2009;117:357‑64). As shown in the Examples, the present invention provides antibodies that target ErbB‑2 and that do not, or to a significantly lesser extent as compared to trastuzumab and pertuzumab, affect the survival of cardiomyocytes. This provides an important advantage since cardiotoxicity is reduced. This is already advantageous for people who do not suffer from an impaired cardiac function, and evenmore so for people who do suffer froman impaired cardiac function, or who are at risk thereof, such as for instance subjects suffering from congestive heart failure (CHF), left ventricular dysfunction (LVD)and / ora≥10%decreasedLeftVentricularEjectionFraction (LVEF), and / or subjects who have had a myocardial infarction. Antibodies according to the invention that do not significantly affect the survival of cardiomyocytes are, therefore, preferred. In vitro, the function of cardiomyocytes is for instance measured by determining the viability of cardiomyocytes, bydeterminingBNP (B-typenatriuretic peptide,which is a cardiac biomarker), by determining QT prolongation, and / or by determining mitochondrial membrane potential.

[0058] Said antibody according to the invention preferably comprises a first antigen-binding site that binds domain I of ErbB‑2 and a second antigen-binding site that binds domain III of ErbB‑3. One embodiment provides an antibody according to the invention that does not significantly affect the survival of cardiomyocytes, comprising a first antigen- binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein the affinity of said second antigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB‑2 positive cell. The affinity of said second antigen-binding site for an ErbB‑3 positive cell is preferably lower than or equal to2.0nM,morepreferably lower thanor equal to1.39nM,morepreferably lower thanorequal to0.99nM.Theaffinity of said first antigen-binding site for anErbB‑2positive cell is preferably lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM preferably lower than or equal to 4.0 nM.

[0059] In one preferred embodiment said antibody that does not significantly affect the survival of cardiomyocytes comprises: - at least the CDR3 sequence, preferably at least the CDR1, CDR2 and CDR3 sequences, or at least the heavy chain variable region sequence, of an ErbB‑2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913,MF1847,MF3001,MF3003 andMF1898 as depicted in Figure 16A or Figure 16E, or a heavy chain variable region sequence that differs in at most 15 amino acids, preferably in at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably in at most 1, 2, 3, 4 or 5 amino acids, from the recited heavy chain variable region sequences; and / or - at least the CDR3 sequence, preferably at least the CDR1, CDR2 and CDR3 sequences, or at least the heavy chain variable region sequence, of an ErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or a heavy chain variable region sequence that differs in at most 15 amino acids, preferably in at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably in at most1, 2, 3, 4or5aminoacids, from the recitedheavychainvariable regionsequences. Inonepreferredembodiment, said antibody is PB4188.

[0060] Another aspect of the present invention provides an antibody according to the invention, comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said antibody comprises an antigen-binding site that binds at least one amino acid residue of domain I of ErbB‑2 selected from the group consisting of T144, T164, R166, P172, G179, S180 andR181, and surface-exposed amino acid residues that are located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181. The amino acid residue numbering is that of Protein Data Bank (PDB) ID #1S78. As shown in the Examples, antibodies binding this region of domain I of ErbB‑2 exhibit particularly good binding characteristics and they are capable of counteracting the activity of ErbB‑2 positive cells (such as ligand-induced receptor function of ErbB‑3 on a ErbB‑2 and ErbB‑3 positive cell, and / or ligand-induced growth of such cell). Moreover, such antibodies are particularly suitable for combination therapy with currently known anti-ErbB‑2 monoclonal antibodies like trastuzumab (that binds domain IV of ErbB‑2) and pertuzumab (that binds domain II of ErbB‑2) because they bind different domains of ErbB‑2. Hence, these antibodies can be used simultaneouslywithout competition for the sameepitope. The term "surface-exposedaminoacid residues that are located within about 5 amino acid positions fromT144, T164,R166, P172,G179, S180 orR181" refers to amino acid residues that are in the primary amino acid sequence located within about the first five amino acid residues adjacent to the recited residues and that are at least in part exposed to the outside of the protein, so that they can be bound by antibodies (see for instance Figure 21B). Preferably, said amino acid residue located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181 is selected from the group consisting of L139, C140, Y141, Q142, D143, I145, L146, W147, K148, D149, L159, T160, L161, I162, D163, N165, S167, R168, A169, C170, H171, C173, S174, P175, M176, C177,K178,C182,W183,G184,E185andS186.Preferably, saidantibodycomprisesanantigen-bindingsite thatbindsat 10 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 least 2 or at least 3 amino acid residues of domain I of ErbB‑2 selected from the group consisting of T144, T164, R166, P172,G179,S180andR181,andsurface-exposedaminoacid residues that are locatedwithin5aminoacidpositions from T144, T164, R166, P172, G179, S180 or R181.

[0061] In one preferred embodiment, a bispecific antibody according to the invention is provided, wherein said antibody comprises an antigen-binding site that binds at least T144, R166 and R181 of domain I of ErbB‑2. Another embodiment provides a bispecific antibody according to the invention, wherein said antibody comprises an antigen-binding site that binds at least T144, R166, P172, G179 and R181 of domain I of ErbB‑2. Another embodiment provides a bispecific antibody according to the invention, wherein said antibody comprises an antigen-binding site that binds at least T144, T164, R166, P172, G179, S180 and R181 of domain I of ErbB‑2.

[0062] Another aspect of the present invention provides an antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that bindsErbB‑3,wherein said antibody comprises an antigen-binding site that binds at least one amino acid of domain III of ErbB‑3 selected from the group consistingR426and surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB‑3 protein. The amino acid residue numbering is that ofProteinDataBank (PDB) ID#4P59.Asshown in theExamples, antibodiesbinding this regionof domain III ofErbB‑3 exhibit particularly good binding characteristics and they are capable of counteracting the activity of ErbB‑3 positive cells (such as ligand-induced receptor function of ErbB‑3 on aErbB‑2 andErbB‑3 positive cell, and / or ligand-induced growth of such cell). The term "surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB‑3 protein" refers to amino acid residues that are in the tertiary structure of the ErbB‑3 protein spationally positioned within 11.2Å fromR426and that areat least in part exposed to theoutside of the protein, so that they canbeboundbyantibodies. Preferably, saidaminoacid residues that are locatedwithin11.2Å fromR426 in thenativeErbB‑3protein are selected from the group consisting of L423, Y424, N425,G427,G452,R453, Y455, E480, R481, L482,D483 andK485 (see for instance Figure 21C and Table 15). In one preferred embodiment, a bispecific antibody according to the invention is provided, wherein said antibody comprises an antigen-binding site that binds at least R426 of domain III of ErbB‑3. Preferably, said antibody comprises an antigen-binding site that binds at least R426 of domain III of ErbB‑3.

[0063] A bispecific antibody of the invention is preferably afucosylated in order to enhance ADCC activity. A bispecific antibody of the invention preferably comprises a reduced amount of fucosylation of the N-linked carbohydrate structure in the Fc region, when compared to the same antibody produced in a normal CHO cell.

[0064] Abispecific antibodyof thepresent invention is preferably used in humans.To this endabispecific antibodyof the invention is preferably a human or humanized antibody. Tolerance of a human to a polypeptide is governed by many different aspects. Immunity, be it T-cell mediated, B-cell mediated or other is one of the variables that are encompassed in tolerance of the human for a polypeptide. The constant region of a bispecific antibody of the present invention is preferably a human constant region. The constant region may contain one or more, preferably not more than 10, preferably not more than 5 amino-acid differences with the constant region of a naturally occurring human antibody. It is preferred that the constant part is entirely derived from a naturally occurringhumanantibody.Variousantibodiesproducedhereinarederived fromahumanantibodyvariabledomain library. Assuch thesevariabledomainsarehuman.TheuniqueCDRregionsmaybederived fromhumans,besyntheticorderived from another organism. The variable region is considered a human variable region when it has an amino acid sequence that is identical to an amino acid sequence of the variable region of a naturally occurring human antibody, but for the CDR region. The variable region of an ErbB‑2 binding VH, an ErbB‑3 binding VH, or a light chain in an antibody of the invention may contain one ormore, preferably notmore than 10, preferably notmore than 5 amino-acid differenceswith the variable region of a naturally occurring human antibody, not counting possible differences in the amino acid sequence of the CDR regions. Such mutations occur also in nature in the context of somatic hypermutation.

[0065] Antibodiesmay be derived from various animal species, at least with regard to the heavy chain variable region. It is common practice to humanize such e.g. murine heavy chain variable regions. There are various ways in which this can be achieved among which there are CDR-grafting into a human heavy chain variable region with a 3D-structure that matches the 3-D structure of the murine heavy chain variable region; deimmunization of the murine heavy chain variable region, preferably done by removing known or suspected T‑ or B‑ cell epitopes from the murine heavy chain variable region. The removal is typically by substituting one or more of the amino acids in the epitope for another (typically conservative) amino acid, such that the sequence of the epitope is modified such that it is no longer a Tor B-cell epitope. Such deimmunizedmurine heavy chain variable regions are less immunogenic in humans than the original murine heavy chain variable region. Preferably a variable region or domain of the invention is further humanized, such as for instance veneered. By using veneering techniques, exterior residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic or substantially non-immunogenic veneered surface. An animal as used in the invention is preferably a mammal, more preferably a primate, most preferably a human.

[0066] A bispecific antibody according to the invention preferably comprises a constant region of a human antibody. According to differences in their heavy chain constant domains, antibodies are grouped into five classes, or isotypes: IgG, IgA, IgM, IgD, and IgE. These classes or isotypes comprise at least one of said heavy chains that is named with a 11 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 corresponding Greek letter. In a preferred embodiment the invention provides an antibody according to the invention wherein said constant region is selected from the group of IgG, IgA, IgM, IgD, and IgE constant regions, more preferably said constant region comprises an IgG constant region, more preferably an IgG1 constant region, preferably a mutated IgG1 constant region. Some variation in the constant region of IgG1 occurs in nature, such as for instance the allotypes G1m1, 17 andG1m3, and / or is allowedwithout changing the immunological properties of the resulting antibody. Typically between about 1‑10 amino acid insertions, deletions, substitutions or a combination thereof are allowed in the constant region.

[0067] The invention in one embodiment provides an antibody comprising a variable domain that bindsErbB‑2,wherein said antibody comprises at least the CDR3 sequence of an ErbB‑2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031,MF2889,MF2913,MF1847,MF3001,MF3003andMF1898as depicted in Figure 16Aor Figure 16E, orwherein said antibody comprisesaheavy chainCDR3sequence that differs in atmost three, preferably in atmost two, preferably in no more than one amino acid from a CDR3 sequence of a VH selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 as depicted in Figure 16A or Figure 16E. Said antibody preferably comprises at least the CDR3 sequence of MF1849, MF2971, MF3958, MF3004 or MF3991, most preferably at least the CDR3 sequence of MF3958.

[0068] Said antibody preferably comprises at least theCDR1,CDR2 andCDR3 sequences of an ErbB‑2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 andMF1898 as depicted in Figure 16Aor Figure 16E, or heavy chainCDR1,CDR2andCDR3sequences that differ in atmost three, preferably in at most two, preferably in at most one amino acid from the CDR1, CDR2 and CDR3 sequences of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898. Said antibody preferably comprises at least the CDR1, CDR2 and CDR3 sequences of MF1849, MF2971, MF3958, MF3004 or MF3991, most preferably at least the CDR1, CDR2 and CDR3 sequences of MF3958.

[0069] The invention also provides an antibody comprising a variable domain that binds ErbB‑3, wherein said antibody comprises at least the CDR3 sequence of an ErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or wherein said antibody comprises a heavy chain CDR3 sequence that differs in at most three, preferably in at most two, preferably in no more than one amino acid from a CDR3 sequence of a VH selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073andMF6074asdepicted inFigure 16BorFigure 16Eor Figure 37. Said antibody preferably comprises at least the CDR3 sequence of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065, most preferably at least the CDR3 sequence of MF3178.

[0070] Said antibody preferably comprises at least theCDR1,CDR2 andCDR3 sequences of an ErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or heavy chainCDR1,CDR2andCDR3sequences that differ in atmost three, preferably in atmost two, preferably in at most one amino acid from the CDR1, CDR2 and CDR3 sequences of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073orMF6074.Saidantibodypreferably comprisesat least the CDR1, CDR2 andCDR3 sequences of MF3178, MF3176,MF3163, MF6058, MF6061 orMF6065, most preferably at least the CDR1, CDR2 and CDR3 sequence of MF3178.

[0071] The invention in one embodiment provides a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site comprises at least the CDR3 sequence of an ErbB‑2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 andMF1898 as depicted in Figure 16A or Figure 16E, or a heavy chain CDR3 sequence that differs in atmost three, preferably in atmost two, preferably in nomore thanoneaminoacid fromaCDR3sequenceof aVH selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 as depicted in Figure 16A or Figure 16E, and wherein said second antigen-binding site comprises at least the CDR3 sequence of an ErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; 12 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073andMF6074asdepicted inFigure 16BorFigure 16Eor Figure 37, or a heavy chain CDR3 sequence that differs in at most three, preferably in at most two, preferably in no more than one amino acid from a CDR3 sequence of a VH selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 andMF6074 as depicted in Figure 16B or Figure 16E or Figure 37. Said first antigen-binding site preferably comprises at least the CDR3 sequence of MF1849, MF2971,MF3958,MF3004orMF3991,most preferably at least theCDR3sequenceofMF3958and said secondantigen- binding site preferably comprises at least the CDR3 sequence of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065, most preferably at least the CDR3 sequence of MF3178.

[0072] Said first antigen-binding site preferably comprises at least theCDR1,CDR2andCDR3sequences of anErbB‑2 specific heavy chain variable region selected from the group consisting ofMF2926,MF2930,MF1849;MF2973,MF3004, MF3958,MF2971, MF3025,MF2916,MF3991, MF3031,MF2889,MF2913, MF1847,MF3001,MF3003 andMF1898 as depicted in Figure 16A or Figure 16E, or heavy chain CDR1, CDR2 and CDR3 sequences that differ in at most three, preferably in at most two, preferably in at most one amino acid from the CDR1, CDR2 and CDR3 sequences of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898, and said second antigen-binding site preferably comprises at least the CDR1, CDR2 and CDR3 sequences of an ErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or heavy chain CDR1, CDR2 and CDR3 sequences that differ in at most three, preferably in at most two, preferably in at most one amino acid from the CDR1, CDR2 and CDR3 sequences of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074 as depicted in Figure 16B or Figure 16E or Figure 37. Said first antigen-binding site preferably comprises at least the CDR1, CDR2 and CDR3 sequences of MF1849, MF2971, MF3958, MF3004 or MF3991, most preferably at least the CDR1, CDR2 and CDR3 sequences of MF3958, and said second antigen-binding site preferably comprises at least the CDR1, CDR2 andCDR3 sequences of MF3178, MF3176,MF3163,MF6058, MF6061 orMF6065, most preferably at least the CDR1, CDR2 and CDR3 sequence of MF3178.

[0073] One preferred embodiment provides a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site comprises at least the CDR3 sequence of MF3958, or a CDR3 sequence that differs in at most three, preferably in at most two, preferably in no more than one amino acid from theCDR3 sequence ofMF3958, andwherein said second antigen-binding site comprises at least the CDR3 sequence of MF3178, or a CDR3 sequence that differs in at most three, preferably in at most two, preferably in no more than one amino acid from the CDR3 sequence of MF3178.

[0074] The invention in one embodiment provides a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site comprises at least the CDR1, CDR2 and CDR3 sequences of MF3958, or CDR1, CDR2 and CDR3 sequences that differ in at most three, preferably in at most two, preferably in at most one amino acid from the CDR1, CDR2 and CDR3 sequences of MF3958, and wherein said second antigen-binding site comprises at least the CDR1, CDR2 and CDR3 sequence of MF3178, or CDR1, CDR2 and CDR3 sequences that differ in at most three, preferably in at most two, preferably in at most one amino acid from the CDR1, CDR2 and CDR3 sequences of MF3178.

[0075] The invention in one embodiment provides a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site comprises at least the CDR3 sequence of MF3958 and wherein said second antigen-binding site comprises at least the CDR3 sequence of MF3178.

[0076] The invention in one embodiment provides a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site comprises at least the CDR1, CDR2 and CDR3 sequences of MF3958 and wherein said second antigen-binding site comprises at least the CDR1, CDR2 and CDR3 sequence of MF3178.

[0077] CDRsequences are for instance varied for optimization purposes, preferably in order to improve binding efficacy or the stability of the antibody. Optimization is for instance performed bymutagenesis procedures where after the stability and / or binding affinity of the resulting antibodies are preferably tested and an improved ErbB‑2 or ErbB‑3 -specific CDR sequence is preferably selected. A skilled person is well capable of generating antibody variants comprising at least one alteredCDRsequenceaccording to the invention.For instance, conservativeaminoacid substitution isapplied.Examples of conservative amino acid substitution include the substitution of one hydrophobic residue such as isoleucine, valine, leucine ormethionine for another hydrophobic residue, and the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.

[0078] The invention in one embodiment provides an antibody comprising a variable domain that bindsErbB‑2,wherein 13 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 the VH chain of said variable domain comprises the amino acid sequence of VH chain MF2926; MF2930; MF1849; MF2973; MF3004; MF3958 (is humanized MF2971); MF2971; MF3025; MF2916; MF3991 (is humanized MF3004); MF3031;MF2889;MF2913;MF1847;MF3001,MF3003orMF1898asdepicted inFigure16AorFigure16E; or comprises the amino acid sequence of VH chain MF2926; MF2930; MF1849; MF2973; MF3004; MF3958 (is humanized MF2971); MF2971;MF3025;MF2916;MF3991 (is humanizedMF3004);MF3031;MF2889;MF2913;MF1847;MF3001,MF3003or MF1898asdepicted inFigure16AorFigure16Ehavingatmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9or 10morepreferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the above mentioned VH chain sequence of Figure 16A or Figure 16E. The VH chain of the variable domain that binds ErbB‑2 preferably comprises the amino acid sequence of: - MF1849; or - MF2971 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence of MF3958; or - MF3004 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence of MF3991; as depicted inFigure 16A. In oneembodiment, theVHchain of the variable domain that bindsErbB‑2comprises theamino acid sequence of VH chain MF1849; or MF2971 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence of MF3958; or MF3004 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence ofMF3991,wherein the recitedVHsequences have at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the respective sequence depicted in Figure 16A. In a preferred embodiment the VH chain of the variable domain that binds ErbB‑2 comprises the amino acid sequence of MF3958; or comprises theaminoacid sequenceofMF3958depicted in figure16Ahavingatmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain sequence. The antibody comprising a variable domain that binds ErbB‑2 is preferably a bispecific antibody that preferably further comprises a variable domain that binds ErbB‑3. The VH chain of the variable domain that binds Erb-B3 preferably comprises the amino acid sequence of VH chainMF3178;MF3176; MF3163;MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074 as depicted in Figure 16B or Figure 16E or Figure 37; or comprises the amino acid sequence of VH chain MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071;MF6072; MF6073 orMF6074 as depicted in Figure 16B or Figure 16E or Figure 37 having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain sequence of Figure 16B or Figure 16E or Figure 37. The VH chain of the variable domain that binds Erb-B3 preferably comprises the amino acid sequence of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065; or comprises the amino acid sequence of MF3178, MF3176, MF3163,MF6058,MF6061 orMF6065 havingatmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,more preferably in atmost 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the respective VH chain sequence of Figure 16B or Figure 37. In a preferred embodiment the VH chain of the variable domain that binds ErbB‑3 comprises the amino acid sequence of MF3178; or comprises the amino acid sequence of MF3178 depicted in Figure 16B having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to theVHchain sequence. Preferably, the above- mentioned amino acid insertions, deletions and substitutions are not present in the CDR3 region. The above-mentioned amino acid insertions, deletions and substitutions are also preferably not present in the CDR1 and CDR2 regions. The above-mentioned amino acid insertions, deletions and substitutions are also preferably not present in the FR4 region.

[0079] The invention further providesanantibodycomprisingavariable domain that bindsErbB‑3,wherein theVHchain of said variable region comprises the amino acid sequence of VH chain MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or comprises the amino acid sequence of VH chain MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071;MF6072; MF6073 orMF6074 as depicted in Figure 16B or Figure 16E or Figure 37 having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to said VH chain sequence. The VH chain of the variable domain that binds ErbB3 preferably comprises the amino acid sequence of VH chain MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065; or comprises the amino acid sequence of VH chain MF3178, MF3176, MF3163, MF6058, MF6061 orMF6065 having atmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,more preferably atmost 1, 2, 3, 4 or 5, amino 14 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 acid insertions, deletions, substitutions or a combination thereof with respect to said VH chain sequence. In a preferred embodiment the VH chain of the variable domain that binds ErbB‑3 comprises the amino acid sequence of VH chain MF3178depicted inFigure16B;or comprises theaminoacidsequenceofVHchainMF3178depicted inFigure16Bhaving at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain sequence. The antibody comprising a variable domain that bindsErbB‑3, is preferably abispecificantibody that preferably further comprisesa variabledomain that bindsErbB‑2. The VH chain of the variable domain that binds ErbB‑2 preferably comprises the amino acid sequence of a VH chain of Figure 16A or Figure 16E. The VH chain of the variable domain that binds ErbB‑2 preferably comprises the amino acid sequence of MF1849; or MF2971 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence of MF3958; or MF3004 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence ofMF3991 as depicted in Figure 16A. In one embodiment, the recitedErb- B2 binding VH sequences have at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, aminoacid insertions, deletions, substitutionsoracombination thereofwith respect to the respective sequencedepicted in Figure 16A. In one preferred embodiment, said ErbB‑2 binding VH chain of Figure 16A comprises the amino acid sequenceofMF3958; or comprises theaminoacid sequenceofMF3958havingatmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to theVHchain sequence.Preferably, the above-mentionedaminoacid insertions, deletionsand substitutions are not present in the CDR3 region. The above-mentioned amino acid insertions, deletions and substitutions are also preferably not present in the CDR1 and CDR2 regions. The above-mentioned amino acid insertions, deletions and substitutions are also preferably not present in the FR4 region.

[0080] Further provided is an antibody according to the invention, wherein said antibody comprises an ErbB‑2 specific heavy chain variable region sequence selected from the group consisting of the heavy chain variable region sequences of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 as depicted in Figure 16A or Figure 16E, or wherein said antibody comprises a heavy chain variable region sequence that differs in atmost 15, preferably in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,more preferably in at most 1, 2, 3, 4 or 5, amino acids from the heavy chain variable region sequences of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898.

[0081] Further provided is an antibody according to the invention, wherein said antibody comprises an ErbB‑3 specific heavy chain variable region sequence selected from the group consisting of the heavy chain variable region sequences of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or wherein said antibody comprises a heavy chain variable region sequence that differs in atmost 15, preferably in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,more preferably in atmost 1, 2, 3, 4 or 5, amino acids from the heavy chain variable region sequences of MF3178; MF3176;MF3163; MF3099;MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074.

[0082] The invention in one embodiment provides an antibody comprising two antigen-binding sites that bind ErbB‑2, wherein at least one of said antigen-binding sites binds domain I of ErbB‑2. Preferably, both antigen-binding sites bind domain I of ErbB‑2. Such antibody according to the invention is particularly suitable for combination therapy with currently used anti‑ ErbB‑2 binding molecules that do not bind domain I of ErbB‑2, such as trastuzumab that binds domain IV of ErbB‑2 and pertuzumab that binds domain II of ErbB‑2, because then the different bindingmolecules do not compete with each other for the same epitope.

[0083] Further provided is an antibody comprising two antigen-binding sites that bind ErbB‑2, wherein at least one of saidantigen-binding sitesbindsdomain I ofErbB‑2andwherein theaffinity (KD)of saidat least oneantigen-binding site for anErbB‑2positive cell is lower thanor equal to5.0nM,preferably lower thanor equal to4.5nM,morepreferably lower than or equal to3.9nM.Preferably, bothantigen-binding sitesbinddomain I ofErbB‑2. Inonepreferredembodiment, theaffinity of said at least one antigen-binding site for ErbB‑2 onSK-BR‑3 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 4.0 nM, more preferably lower than or equal to 3.5 nM, more preferably lower than or equal to 3.0 nM,more preferably lower than or equal to 2.3 nM. In one embodiment, said affinity is within the rangeof 3.0‑1.6 nM. In onepreferred embodiment, the affinity of said at least one antigen-binding site for ErbB‑2 on BT‑474 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 3.9 nM. In one embodiment, said affinity is within the range of 4.5‑3.3 nM.

[0084] The above-mentioned affinities are preferably as measured using steady state cell affinity measurements, wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell-bound radioactivity is measured, as described in the Examples.

[0085] The invention further provides an antibody comprising two variable domains that bind ErbB‑2, wherein a VH chain of said variable domains comprises the amino acid sequence of theVH chainMF2926;MF2930;MF1849;MF2973; 15 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 MF3004; MF3958 (is humanized MF2971); MF2971; MF3025; MF2916; MF3991 (is humanized MF3004); MF3031; MF2889; MF2913; MF1847; MF3001, MF3003 or MF1898 as depicted in Figure 16A or Figure 16E; or the amino acid sequence of the VH chain MF2926; MF2930; MF1849; MF2973; MF3004; MF3958 (is humanized MF2971); MF2971; MF3025;MF2916;MF3991 (is humanizedMF3004);MF3031;MF2889;MF2913;MF1847;MF3001,MF3003 orMF1898 VH-chains as depicted in Figure 16A or Figure 16E, having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably atmost 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the respective sequence depicted in Figure 16Aor Figure 16E. Said VHpreferably comprises the amino acid sequence of VH chain MF1849; or MF2971 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence ofMF3958; orMF3004 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence of MF3991 as depicted in Figure 16A; or comprises the amino acid sequence of VH chain MF1849; or MF2971 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence ofMF3958; orMF3004 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequenceofMF3991as depicted in Figure 16Ahaving atmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,more preferably atmost 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the respective sequence depicted in Figure 16A. The variable domains of the antibody preferably comprise identical VH chains, preferably having a sequence as depicted in Figure 16A or Figure 16E. An antibody with variable domains with identical VH chains is not a bispecific antibody. VH chains are identical for the present invention if they comprise the same VH chain sequence as depicted in Figure 16A or Figure 16E or Figure 37, or the same VH chain sequence but for 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the respective sequence depicted in Figure 16A or Figure 16E or Figure 37.

[0086] The invention in one embodiment provides an antibody comprising two antigen-binding sites that bind ErbB‑3, wherein at least one of said antigen-binding sites binds domain III of ErbB‑3. Preferably, both antigen-binding sites bind domain III ofErbB‑3.Suchantibodyaccording to the invention isparticularly suitable for combination therapywith currently used anti- ErbB‑3 bindingmolecules that do not bind domain III of ErbB‑3, such asMM‑121 (#Ab6) and RG7116 that bind domain I of ErbB‑3, because then the different binding molecules do not compete with each other for the same epitope.

[0087] Further provided is an antibody comprising two antigen-binding sites that bind ErbB‑3, wherein at least one of said antigen-binding sites binds domain III of ErbB‑3 andwherein the affinity (KD) of said at least one antigen-binding site for an ErbB‑3 positive cell is lower than or equal to 2.0 nM, preferably lower than or equal to 1.5 nM,more preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. Preferably, both antigen-binding sites bind domain III of ErbB‑3. In one preferred embodiment, the affinity of said at least one antigen-binding site for ErbB‑3 on SK- BR‑3 cells is lower than or equal to 2.0 nM, preferably lower than or equal to 1.5 nM,more preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. In one embodiment, said affinity is within the range of 1.39‑0.59 nM. In one preferred embodiment, the affinity of said at least one antigen-binding site for ErbB‑3 on BT‑474 cells is lower than or equal to 2.0 nM,morepreferably lower than or equal to 1.5 nM,morepreferably lower than or equal to 1.0 nM,more preferably lower than or equal to 0.5 nM, more preferably lower than or equal to 0.31 nM, more preferably lower than or equal to 0.23 nM. In one embodiment, said affinity is within the range of 0.31‑0.15 nM.

[0088] Again, the above-mentioned affinities are preferably asmeasured using steady state cell affinitymeasurements, wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell-bound radioactivity is measured, as described in the Examples.

[0089] The invention further provides an antibody comprising two variable domains that each bind ErbB3 wherein a VH of the variable domains comprises the amino acid sequence of VH chain MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074 as depicted in Figure 16B or Figure 16E or Figure 37; or comprises the amino acid sequence of VH chain MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068;MF6069;MF6070;MF6071; MF6072;MF6073 orMF6074 having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to any of said VH chain sequences. Said VHpreferably comprises the amino acid sequence of VH chainMF3178, MF3176, MF3163, MF6058, MF6061 or MF6065; or comprises the amino acid sequence of VH chain MF3178, MF3176, MF3163,MF6058,MF6061orMF6065havingatmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,more preferably atmost 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to any of said VH chain sequences. Said VH preferably comprises the amino acid sequence of VH chain MF3178; or comprises the amino acid sequence of VH chain MF3178 depicted in Figure 16B having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably atmost 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the MF3178 VH chain sequence. The variable domains of the antibody preferably comprise identical VH chains, preferably having a sequence as depicted in Figure 16B or Figure 16E or Figure 37. An antibody with variable domains with identical VH chains is not a bispecific antibody. The VH chains are identical if they comprise the same VH chain sequence as depicted in Figure 16B or Figure 16E or Figure 37, or the same VH chain sequence but for 1, 2, 3, 4 or 5 amino acid 16 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 insertions, deletions, substitutions or a combination thereof with respect to theVHchain sequence of Figure 16Bor Figure 16E or Figure 37.

[0090] Monospecific antibodies according to the present invention that are specific for ErbB‑3 have the advantage that they have a better functional activity against ErbB‑3, as compared to prior art compounds such as for instance MM‑121 (#Ab6),meaning that theseantibodies according to the invention are better capable of counteractingErbB‑3activity (such as a ligand-induced receptor function of ErbB‑3 and / or ligand-induced growth of an ErbB‑2 and ErbB‑3 positive cell). This is for instance shown in Table 7 and Figure 38.

[0091] In a preferred embodiment the invention provides a bispecific antibody comprising a variable domain that binds ErbB‑2, wherein the VH chain of said variable domain comprises - the amino acid sequence of VH chain MF1849; or MF2971 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence of MF3958; or MF3004 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence of MF3991, as depicted in Figure 16A; or comprises - the amino acid sequence of VH chain MF1849 or MF2971 or a humanized version thereof, wherein said humanized version preferably comprises the amino acid sequence of MF3958; or MF3004 or a humanized version thereof, wherein saidhumanizedversionpreferably comprises theaminoacidsequenceofMF3991, asdepicted inFigure16A having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,more preferably atmost 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to said VH. Such bispecific antibody according to this embodiment further preferably comprises a variable domain that binds ErbB‑3. The VH chain of the variable domain that binds ErbB‑3 preferably comprises the amino acid sequence of VH chain MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or most preferably comprises the amino acid sequence of VH chain MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably atmost 1, 2, 3, 4or 5, aminoacid insertions, deletions, substitutionsor a combination thereofwith respect to any of said VH chain sequences of Figure 16B or Figure 16E or Figure 37. The VH chain of the variable domain that binds ErbB‑3 preferably comprises the amino acid sequence of VH chain MF3178 as depicted in Figure 16B or comprises the amino acid sequence of VH chainMF3178 depicted in Figure 16Bhaving atmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain sequence of Figure 16B.

[0092] The invention preferably provides a bispecific antibody comprising a variable domain that binds ErbB‑2 and a variable domain that binds ErbB‑3, wherein the VH chain of the variable domain that binds ErbB‑2 comprises - the amino acid sequence of VH chain MF3958 as depicted in Figure 16A; or - theaminoacid sequenceofVHchainMF3958asdepicted inFigure 16Ahavingatmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect said VH; and wherein the VH chain of the variable domain that binds ErbB‑3 comprises - the amino acid sequence of VH chain MF3178 as depicted in Figure 16B; or - the amino acid sequenceof VHchainMF3178 depicted in Figure 16Bhaving atmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10more preferably atmost 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain sequence of Figure 16B.

[0093] The invention preferably provides a bispecific antibody comprising a variable domain that binds ErbB‑2 and a variable domain that binds ErbB‑3, wherein the VH chain of the variable domain that binds ErbB‑2 comprises - the amino acid sequence of VH chain MF3991 as depicted in Figure 16A; or - theaminoacid sequenceofVHchainMF3991asdepicted inFigure 16Ahavingatmost 15, preferably 1, 2, 3, 4, 5, 17 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect said VH; and wherein the VH chain of the variable domain that binds ErbB‑3 comprises - the amino acid sequence of VH chain MF3178 as depicted in Figure 16B; or - the amino acid sequenceof VHchainMF3178 depicted in Figure 16Bhaving atmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9or 10,morepreferably atmost 1, 2, 3, 4 or 5, aminoacid insertions, deletions, substitutionsor a combination thereof with respect to the VH chain sequence of Figure 16B.

[0094] When compared to the sequence in Figure 16, the behavior of a VH chain typically starts to become noticeably differentwhen it hasmore than15aminoacidchangeswith respect to theaminoacidsequenceofaVHchainasdepicted in Figure 16. A VH chain having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutionsor acombination thereofwith respect to theVHchaindepicted inFigure16, preferablyhas1,2, 3, 4or5amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain depicted in Figure 16, preferably 1, 2, 3 or 4 insertions, deletions, substitutions or a combination thereof, preferably 1, 2 or 3 insertions, deletions, substitutions or a combination thereof, more preferably 1 or 2 insertions, deletions, substitutions or a combination thereof, and preferably 1 insertion, deletion, substitution or a combination thereof with respect to the VH chain depicted in Figure 16.Theoneormoreaminoacid insertions, deletions, substitutionsoracombination thereofarepreferablynot in theCDR1, CDR2andCDR3 regionof theVHchain.Theyarealsopreferably not present in theFR4 region.Anaminoacid substitution is preferably a conservative amino acid substitution.

[0095] In a preferred embodiment the invention provides a bispecific antibody comprising an amino acid sequence as depicted inFigure16D,or abispecificantibodyofFigure16Dhavingatmost 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9or 10,more preferably atmost 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the sequence of Figure 16D, wherein the at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions are preferably conservative amino acid substitutions. The insertions, deletions, substitutions or a combination thereof are preferablynot in theCDR3 regionof theVHchain, preferablynot in theCDR1,CDR2andCDR3regionof theVHchain, and preferably not in the FR4 region.

[0096] Rational methods have evolved toward minimizing the content of non-human residues in the human context. Various methods are available to successfully graft the antigen-binding property of a bispecific antibody onto another antibody. The binding properties of antibodies rest predominantly in the exact sequence of the CDR3 region, often supportedby thesequenceof theCDR1andCDR2 regions in the variable domaincombinedwith theappropriate structure of the variable domain as a whole. Various methods are presently available to graft CDR regions onto a suitable variable domain of another antibody. Some of these methods are reviewed in J.C. Almagro1 and J. Fransson (2008) Frontiers in Bioscience 13, 1619‑1633, which is included by reference herein. The invention therefore further provides a human or humanized bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein the variable domain comprising the ErbB‑2 binding site comprises a VH CDR3 sequence as depicted inFigure 16Aor Figure 16E, andwherein the variable domain comprising theErbB‑3binding site comprises aVH CDR3 region as depicted in Figure 16B or Figure 16E or Figure 37. TheVH variable region comprising the ErbB‑2 binding sitepreferably comprises thesequenceof theCDR1 region,CDR2 regionand theCDR3 regionofaVHchain inFigure16A or Figure 16E. TheVHvariable region comprising theErbB‑3 binding site preferably comprises the sequence of theCDR1 region, CDR2 region and the CDR3 region of a VH chain in Figure 16B or Figure 16E or Figure 37. CDR graftingmay also be used to produce a VH chain with the CDR regions of a VH of Figure 16 or Figure 37, but having a different framework. The different framework may be of another human VH, or a different mammal.

[0097] The mentioned at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions are preferably conservative amino acid substitutions. The insertions, deletions, substitutions or a combination thereof are preferably not in theCDR3 regionof theVHchain, preferably not in theCDR1,CDR2orCDR3 regionof theVHchainandpreferably not in the FR4 region.

[0098] The light chain of a variable domain comprising a variable heavy chain sequence as depicted in Figure 16 or Figure 37, is preferably germline light chain O12, preferably the rearranged germline human kappa light chain IgVκ1‑39*01 / IGJκ1*01 or a fragment or a functional derivative thereof (nomenclature according to the IMGT database worldwide web at imgt.org). The terms rearranged germline human kappa light chain IgVκ1‑39*01 / IGJκ1*01, IGKV1‑39 / IGKJ1, huVκ1‑39 light chain or in short huVκ1‑39 are used. The light chain can have 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof. Thementioned1, 2, 3, 4 or 5 aminoacid substitutions are preferably conservative amino acid substitutions, the insertions, deletions, substitutions or a combination thereof are preferably not in the CDR3 region of the VL chain, preferably not in the CDR1, CDR2 or CDR3 region or FR4 region of the VL chain.

[0099] Various methods are available to produce bispecific antibodies. One method involves the expression of two 18 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 different heavy chains and two different light chains in a cell and collecting antibody that is produced by the cell. Antibody produced in this way will typically contain a collection of antibodies with different combinations of heavy and light chains, someofwhichare thedesiredbispecificantibody.Thebispecificantibodycansubsequently bepurified from thecollection. The ratio of bispecific to other antibodies that are produced by the cell can be increased in various ways. In a preferred embodiment of the invention, the ratio is increased by expressing not two different light chains but two essentially identical light chains in the cell. This concept is in the art also referred to as the "common light chain" method.When the essentially identically light chains work together with the two different heavy chains allowing the formation of variable domains with different antigen-binding sites and concomitant different binding properties, the ratio of bispecific antibody to other antibody that is produced by the cell is significantly improved over the expression of two different light chains. The ratio of bispecific antibody that is produced by the cell can be further improved by stimulating the pairing of two different heavy chains with each other over the pairing of two identical heavy chains. The art describes various ways in which such heterodimerization of heavy chains canbe achieved.Oneway is to generate ’knob into hole’ bispecific antibodies. SeeUS Patent Application 20030078385 (Arathoon et al. - Genentech). Another and preferred method is described in US provisional application 61 / 635,935, which has been followed up by US regular application No. 13 / 866,747 and PCT application No. PCT / NL2013 / 050294 (WO 2013 / 157954 A1), which are incorporated herein by reference. Methods and means are disclosed for producing bispecific antibodies from a single cell, whereby means are provided that favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be favorably employed in the present invention. Thus the invention provides amethod for producing a bispecific antibody according to the invention (froma single cell), wherein said bispecific antibody comprises twoCH3domains that are capable of forming an interface, said method comprising providing in said cell a) a first nucleic acid molecule encoding a 1st CH3 domain comprising heavy chain, b) a second nucleic acidmolecule encoding a 2ndCH3domain comprising heavy chain, wherein said nucleic acid molecules are provided with means for preferential pairing of said 1st and 2nd CH3 domain comprising heavy chains, said method further comprising the step of culturing said host cell and allowing for expression of said two nucleic acid molecules and harvesting said bispecific antibody from the culture. Said first and second nucleic acid molecules may be part of the same nucleic acid molecule, vector or gene delivery vehicle and may be integrated at the same site of the host cell’s genome. Alternatively, said first and second nucleic acid molecules are separately provided to said cell.

[0100] Apreferred embodiment provides amethod for producing a bispecific antibody according to the invention (froma single cell), wherein said bispecific antibody comprises two CH3 domains that are capable of forming an interface, said method comprising providing: - a cell having a) a first nucleic acid molecule encoding a heavy chain comprising an antigen binding site that binds ErbB‑2 and that contains a 1st CH3 domain, and b) a second nucleic acid molecule encoding a heavy chain comprising an antigen-binding site that binds ErbB‑3 and that contains a 2nd CH3 domain, wherein said nucleic acid molecules are provided with means for preferential pairing of said 1st and 2nd CH3 domains, saidmethod further comprising thestepof culturing said cell andallowing for expressionof said twonucleic acidmolecules and harvesting said bispecific IgG antibody from the culture. In a particularly preferred embodiment, said cell also has a thirdnucleicacidmoleculeencodingacommon light chain.Said first, secondand thirdnucleicacidmoleculemaybepart of the same nucleic acid molecule, vector or gene delivery vehicle and may be integrated at the same site of the host cell’s genome. Alternatively, said first, second and third nucleic acid molecules are separately provided to said cell. A preferred common light chain is 012, preferably the rearranged germline human kappa light chain IgVκ1 39*01 / IGJκ1*01, as described above. Means for preferential pairing of said 1st and said 2nd CH3 domain are preferably the corresponding mutations in the CH3 domain of the heavy chain coding regions. The preferred mutations to produce essentially only bispecific antibodies are the amino acid substitutions L351K and T366K (numbering according to Kabat) in the first CH3 domain and the amino acid substitutions L351D and L368E in the second CH3 domain, or vice versa. Further provided is thereforeamethodaccording to the invention for producingabispecificantibody,wherein saidfirstCH3domain comprises the amino acid substitutions L351K and T366K (numbering according to Kabat) and wherein said second CH3 domain comprises the amino acid substitutions L351D and L368E, said method further comprising the step of culturing said cell and allowing for expression of said nucleic acid molecules and harvesting said bispecific antibody from the culture. Also provided is a method according to the invention for producing a bispecific antibody, wherein said first CH3 domain comprises the amino acid substitutions L351D and L368E (numbering according to Kabat) andwherein said secondCH3 domain comprises the amino acid substitutions L351K and T366K, said method further comprising the step of culturing saidcell andallowing forexpressionof saidnucleicacidmoleculesandharvestingsaidbispecificantibody from theculture. Antibodies that can be produced by these methods are also part of the present invention. The CH3 heterodimerization domains are preferably IgG1 heterodimerization domains. The heavy chain constant regions comprising the CH3 heterodimerization domains are preferably IgG1 constant regions.

[0101] In one embodiment the invention provides a nucleic acid molecule encoding an antibody heavy chain variable 19 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 region according to the invention. The nucleic acid molecule (typically an in vitro, isolated or recombinant nucleic acid) preferably encodes a heavy chain variable region as depicted in Figure 16A or Figure 16B or Figure 37, or a heavy chain variable region as depicted in Figure 16A or Figure 16B or Figure 37 having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof. In a preferred embodiment the nucleic acid molecule comprises a sequence as depicted in Figure 16 or Figure 37. In another preferred embodiment the nucleic acid molecule encodes the same amino acid sequence as the nucleic acid depicted in Figure 16 or Figure 37, but has a different sequence because it encodes one ormore different codons. For instance, such nucleic acidmolecule is codon optimized for antibody producer cells, such as for instanceChinesehamster ovary (CHO)cells,NS0cells orPER-C6™cells. The invention further providesanucleic acid sequence encoding a heavy chain of Figure 16D or Figure 37.

[0102] A nucleic acid molecule as used in the invention is typically but not exclusively a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). Alternative nucleic acids are available for a person skilled in theart. A nucleic acid according to the invention is for instance comprised in a cell. When said nucleic acid is expressed in said cell, said cell produces an antibody according to the invention. Therefore, the invention in one embodiment provides a cell comprising an antibody according to the invention and / or a nucleic acid according to the invention. Said cell is preferably an animal cell, more preferably amammal cell,morepreferably aprimate cell,most preferably a humancell. For the purposesof the invention a suitable cell is any cell capable of comprising and preferably of producing an antibody according to the invention and / or a nucleic acid according to the invention.

[0103] The invention further provides a cell comprising an antibody according to the invention. Preferably said cell (typically an in vitro, isolated or recombinant cell) produces said antibody. In a preferred embodiment said cell is a hybridoma cell, a CHO cell, an NS0 cell or a PER-C6™ cell. In a particularly preferred embodiment said cell is a CHO cell. Further provided is a cell culture comprising a cell according to the invention. Various institutions and companies have developed cell lines for the large scale production of antibodies, for instance for clinical use.Non-limiting examples of such cell linesareCHOcells,NS0cellsorPER.C6™cells.Thesecellsarealsoused forotherpurposessuchas theproductionof proteins. Cell lines developed for industrial scale production of proteins and antibodies are herein further referred to as industrial cell lines. Thus in a preferred embodiment the invention provides the use of a cell line developed for the large scale production of antibody for the production of an antibody of the invention.

[0104] The invention further providesamethod for producinganantibody comprising culturinga cell of the inventionand harvesting said antibody from said culture. Preferably said cell is cultured in a serum free medium. Preferably said cell is adapted for suspension growth. Further provided is an antibody obtainable by a method for producing an antibody according to the invention. The antibody is preferably purified from the medium of the culture. Preferably said antibody is affinity purified.

[0105] Acell of the invention is for instanceahybridomacell line, aCHOcell, anNS0cell or another cell typeknown for its suitability for antibody production for clinical purposes. In a particularly preferred embodiment said cell is a human cell. Preferably a cell that is transformed by an adenovirus E1 region or a functional equivalent thereof. A preferred example of such a cell line is thePER.C6™cell line or equivalent thereof. In a particularly preferred embodiment said cell is aCHOcell or a variant thereof.Preferably a variant thatmakesuseof aGlutaminesynthetase (GS) vector system for expressionof an antibody.

[0106] The invention further provides a composition, preferably a pharmaceutical composition, comprising an antibody according to the invention. The pharmaceutical composition preferably comprises a (pharmaceutically acceptable) excipient or carrier. In a preferred embodiment the pharmaceutical composition comprises 5‑50 mM Histidine, 100‑300 mM Trehalose, 0.1‑03 g / L PolySorbate20 or a combination thereof. The pH is preferably set at pH = 5.5 - 6.5. In a preferred embodiment the pharmaceutical composition comprises 25 mM Histidine, 220 mM Trehalose, 0.2 g / L PolySorbate20 or a combination thereof. The pH is preferably set at pH = 5.5 - 6.5, most preferably at pH = 6.

[0107] An antibody of the invention preferably further comprises a label, preferably a label for in vivo imaging. Such a label is typically not necessary for therapeutic applications. In for instance a diagnostic setting, a label can be helpful. For instance in visualizing target cells in the body. Various labels are suited andmany are well known in the art. In a preferred embodiment the label is a radioactive label for detection. In another preferred embodiment, the label is an infrared label. Preferably the infrared label is suited for in vivo imaging.Various infrared labels areavailable to theperson skilled in theart. Preferred infrared labels are for instance, IRDye 800; IRDye 680RD; IRDye 680LT; IRDye 750; IRDye 700DX; IRDye 800RS IRDye 650; IRDye 700 phosphoramidite; IRDye 800 phosphoramidite (LI-COR USA; 4647 Superior Street; Lincoln, Nebraska).

[0108] The invention further provides amethod for the treatment of a subject having aErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor or at risk of having said tumor comprising administering to the subject an antibody or pharmaceutical composition according to the invention. Before start of said treatment, the method preferably comprises determining whether said subject has, or is at risk of, suchErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor. In someembodiments, the subject is classified as [+] or [++] for ErbB‑2. In another embodiment the subject is classified as [+++] for ErbB‑2. The invention further provides an antibody of the invention for use in the treatment of a subject having or at risk of having an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor. Alternatively formulated, the invention provides a use of an antibody 20 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 according to the invention for the manufacture of a medicament or prophylactic agent for the treatment of an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor. As used herein, the term treatment encompasses prophylaxis.

[0109] The tumor is preferably anErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive cancer. Preferably said positive cancer is a breast cancer, suchasearly-stagebreast cancer.However, the invention canbeapplied to awide rangeofErbB‑2,ErbB‑3 or ErbB‑2 / ErbB‑3 positive cancers, like gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivarygland cancer, headandneck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, melanoma, and the like. Said antibody according to the present invention is typically capable of reducing a ligand-induced receptor function, preferably ligand induced growth, of ErbB‑3 on a ErbB‑2 and ErbB‑3 positive cell. Said antibody according to the invention preferably comprises a first antigen-binding site that binds domain I of ErbB‑2 and a second antigen-binding site that binds domain III of ErbB‑3. In one preferred embodiment, the affinity (KD) of said second antigen-binding site for anErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for anErbB‑2positive cell. Further provided is therefore anantibody comprisinga first antigen-binding site that bindsErbB‑2 and a second antigen-binding site that bindsErbB‑3 for use in the treatment of a subject having or at risk of having an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, preferably breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma, wherein the affinity of said second antigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen- binding site for an ErbB‑2 positive cell. The affinity of said second antigen-binding site for an ErbB‑3 positive cell is preferably lower than or equal to 2.0 nM, more preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. The affinity of said first antigen-binding site for anErbB‑2 positive cell is preferably lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM preferably lower than or equal to 4.0 nM. In one preferred embodiment, said antibody is antibody PB4188.

[0110] In one preferred embodiment, said antibody according to the invention comprises an antigen-binding site that binds at least one amino acid of domain I of ErbB‑2 selected from the group consisting of T144, T164, R166, P172, G179, S180andR181, and surface-exposedaminoacid residues that are locatedwithin about 5 aminoacid positions fromT144, T164, R166, P172, G179, S180 or R181.

[0111] In one preferred embodiment, said antibody according to the invention preferably comprises an antigen-binding site that binds at least one amino acid of domain III of ErbB‑3 selected from the group consisting R426 and surface- exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB‑3 protein.

[0112] Further provided is therefore an antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3 for use in the treatment of a subject having or at risk of having an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, preferably breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esopha- geal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma, wherein said antibody according to the invention comprises an antigen-binding site that binds at least one amino acid of domain I of ErbB‑2 selected from the group consisting of T144, T164, R166, P172, G179, S180 andR181, and surface-exposed amino acid residues that are located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181, and / or wherein said antibody according to the invention preferably comprises an antigen-binding site that binds at least one amino acid of domain III of ErbB‑3selected from thegroupconsistingofR426andsurface-exposedaminoacid residues that are locatedwithin11.2Å from R426 in the native ErbB‑3 protein.

[0113] The subject is preferably a human subject. The subject is preferably a subject eligible for monoclonal antibody therapyusinganErbB‑2specificantibodysuchas trastuzumab. Inapreferredembodiment thesubject comprisesa tumor, preferably an ErbB‑2 / ErbB‑3 positive cancer, preferably a tumor / cancer with an ErbB‑2 therapy resistant phenotype and / or a heregulin resistance phenotype, preferably a monoclonal antibody resistant phenotype. A tumor involving such phenotype can escape treatment with a current anti-HER2 regimen, such as (but not limited to) monoclonal antibody therapy against ErbB‑2.

[0114] The amount of antibody according to the invention to be administered to a patient is typically in the therapeutic window, meaning that a sufficient quantity is used for obtaining a therapeutic effect, while the amount does not exceed a threshold value leading to anunacceptable extent of side-effects. The lower theamount of antibodyneeded for obtaining a desired therapeutic effect, the larger the therapeutic window will typically be. An antibody according to the invention exerting sufficient therapeutic effects at low dosage is, therefore, preferred. The dosage can be in the range of the dosing regime for trastuzumab or lower.

[0115] Thepresent invention describes amongothers antibodies that target theErbB‑2andErbB‑3 receptors and result in potent proliferation inhibition of cancer cell lines in vitro and tumor growth inhibition in vivo, even in the presence of an escapemechanismsuchas for instanceupregulationofNRG1-β1.Adiversepanel of humanandmurineFabbindingarms 21 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 specific for either ErbB‑2 or ErbB‑3 were identified. These were produced as bispecific antibodies by cloning them into complementary expression vectors that contain mutations in the CH3 region that drives heterodimerization of heavy chains. More than 500 bispecific antibodies were produced at small scale and tested in binding and functional assays on three different cancer cell lines. Various bispecific antibodies were selected and tested in an orthotopic xenograft model using the BxPC3 cell line. This cell line expresses both the ErbB‑2 and ErbB‑3 receptors and is partially dependent on the ErbB‑3 ligand for growth. BxPC3 models are a robust and stringent screening model. Furthermore, a strong anti-tumor activity in vivo has been confirmed using a xenograft model using the JIMT‑1 cell line. JIMT‑1 cells are derived from a pleural metastasis of a 62-year old patient with breast cancer who was clinically resistant to trastuzumab. JIMT‑1 cells growasanadherentmonolayer and formxenograft tumors innudemice. JIMT‑1cells haveanamplifiedHER‑2oncogene, which showed no identifiable mutations in its coding sequence. JIMT‑1 cells overexpress HER‑2mRNA and protein, and the levels of HER‑1, HER‑3, and HER‑4 mRNA and protein are similar to the trastuzumab-sensitive cell line SKBR‑3 (Tanner et al, Mol Cancer Ther 2004).

[0116] Importantly, a better anti-tumor effect was obtained using an antibody according to the invention as compared to the currently used monoclonal antibodies trastuzumab and pertuzumab, as well as the chemical compound lapatinib.

[0117] Antibodies of the invention can be produced at levels > 50 mg / L after transient transfection in suspension 293F cells. The bispecific antibodies can be purified to greater than 98% purity with yields > 70%. Analytical characterization studies show bispecific lgGl antibody profiles that are comparable to bivalent monospecific IgG1. In terms of functional activity a bispecificantibody of the invention candemonstrate superior potency compared to trastuzumab+pertuzumab in vitro and in vivo.

[0118] Preferredembodimentsof the inventionprovidecombination therapy. Inoneembodiment, anantibodyaccording to the invention is combinedwith trastuzumaborpertuzumab, since theseantibodiesbinddifferentErbB‑2epitopesso that they do not compete for the same epitope with an antibody according to the invention, as shown in the Examples. In another embodiment, an antibody according to the invention is combined with MM‑121 (#Ab6) or RG7116 (Roche), since these antibodies bind different ErbB‑3 epitopes so that they do not compete for the same epitope with an antibody according to the invention, as shown in the Examples.

[0119] In another preferred embodiment, a binding compound that is specific for ErbB‑2andErbB‑3 is combinedwith an inhibitor of a component of the PI3Kinase pathway and / or with an inhibitor of a component of theMAPKpathway, such as for instance with a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, anmTOR inhibitor or an Src inhibitor. In one embodiment a binding compound that is specific for ErbB‑2 and ErbB‑3 is combined with a microtubuli disrupting drug or with an inhibitor of a histone deacetylase (HDAC). Surprisingly, the inventors have found a synergistic effect when these combinations are used. Further provided is therefore a method for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor or at risk of having said tumor, the method comprising administering to the subject: - a binding compound that is specific for ErbB‑2 and ErbB‑3, and - one or more compounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK pathway, a microtubuli disrupting drug, and an inhibitor of a histone deacetylase (HDAC). Said inhibitor preferably comprises a tyrosine kinase inhibitor, a PI3Ka inhibitor, anAkt inhibitor, an mTOR inhibitor or an Src inhibitor. Said tyrosine kinase inhibitor is preferably afatinib, lapatinib and / or neratinib. Said PI3Ka inhibitor is preferably BYL719. In one embodiment, said Akt inhibitor is MK‑2206. In one preferred embodiment, saidmTOR inhibitor is everolimus. In one preferred embodiment, saidSrc inhibitor is saracatinib. In one preferred embodiment, said microtubuli disrupting drug is paclitaxel. In one preferred embodiment, said HDAC inhibitor is vorinostat. In one preferred embodiment, said binding compound that is specific for ErbB‑2 and ErbB‑3 is MM‑111 (Merrimack Pharmaceuticals). In one preferred embodiment, said binding compound that is specific for ErbB‑2 and ErbB‑3 is a bispecific antibody. In one preferred embodiment, said binding compound that is specific for ErbB‑2 and ErbB‑3 is a bispecific antibody according to the invention.

[0120] Further provided is therefore a method for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor or at risk of having said tumor, the method comprising administering to the subject: - a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, and - one or more compounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK pathway, a microtubuli disrupting drug, and an HDAC inhibitor.

[0121] Also provided is a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3 for use in the treatment of a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, wherein said treatment comprises administering said bispecific antibody and at least one compound selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK 22 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 pathway, a microtubuli disrupting drug, and an HDAC inhibitor to a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor. Preferably, a bispecific antibody according to the invention having a first antigen-binding site that binds domain I of ErbB‑2 and a second antigen-binding site that binds domain III of ErbB‑3 is combined with one or more compounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK pathway, a microtubuli disrupting drug, and an HDAC inhibitor. Said inhibitor preferably comprises a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, an mTOR inhibitor or an Src inhibitor. Said tyrosine kinase inhibitor is preferably afatinib, lapatinib and / or neratinib. Said PI3Ka inhibitor is preferably BYL719. In one embodiment, said Akt inhibitor is MK‑2206. In one preferred embodiment, said mTOR inhibitor is everolimus. In one preferred embodiment, said Src inhibitor is saracatinib. In one preferred embodiment, said microtubuli disrupting drug is paclitaxel. In one preferred embodiment, said HDAC inhibitor is vorinostat.

[0122] Said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor is preferably breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma. Most preferably, said tumor is breast cancer. In one embodiment, said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor has less than 1.000.000 ErbB‑2 cell-surface receptors per tumor cell.

[0123] Inoneembodiment, anantibodyaccording to thepresent invention that is combinedwithoneormorecompounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK pathway, a microtubuli disrupting drug and an HDAC inhibitor, preferably with at least one compound selected from the group consisting of a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, an mTOR inhibitor, an Src inhibitor, vorinostat and paclitaxel, more preferably with at least one compound selected from the group consisting of afatinib, lapatinib, neratinib, BYL719, MK‑2206, everolimus, saracatinib, vorinostat and paclitaxel, is typically capable of reducing a ligand-induced receptor function, preferably ligand induced growth, of ErbB‑3 on aErbB‑2 andErbB‑3 positive cell. Said antibodyaccording to the inventionpreferably comprisesafirst antigen-binding site that bindsdomain I ofErbB‑2 and a second antigen-binding site that binds domain III of ErbB‑3. In one preferred embodiment, the affinity (KD) of said secondantigen-binding site for anErbB‑3positivecell is equal to, or higher than, theaffinityof said first antigen-bindingsite for an ErbB‑2 positive cell. The affinity of said second antigen-binding site for an ErbB‑3 positive cell is preferably lower thanorequal to2.0nM,morepreferably lower thanorequal to1.39nM,morepreferably lower thanorequal to0.99nM.The affinity of said first antigen-binding site for an ErbB‑2 positive cell is preferably lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM preferably lower than or equal to 4.0 nM.

[0124] In one preferred embodiment, an antibody according to the invention that is combined with one or more compounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK pathway, a microtubuli disrupting drug and an HDAC inhibitor, preferably with at least one compound selected from the group consisting of a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, an mTOR inhibitor, an Src inhibitor, vorinostat and paclitaxel, more preferably with at least one compound selected from the group consisting of afatinib, lapatinib, neratinib, BYL719, MK‑2206, everolimus, saracatinib, vorinostat and paclitaxel, com- prisesanantigen-binding site that bindsat least oneaminoacid of domain I ofErbB‑2selected from thegroup consisting of T144, T164,R166,P172,G179,S180andR181, and surface-exposedaminoacid residues that are locatedwithin about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181.

[0125] In one preferred embodiment, an antibody according to the invention that is combined with one or more compounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK pathway, a microtubuli disrupting drug and an HDAC inhibitor, preferably with at least one compound selected from the group consisting of a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, an mTOR inhibitor, an Src inhibitor, vorinostat and paclitaxel, more preferably with at least one compound selected from the group consisting of afatinib, lapatinib, neratinib, BYL719, MK‑2206, everolimus, saracatinib, vorinostat and paclitaxel, com- prises an antigen-binding site that binds at least one amino acid of domain III of ErbB‑3 selected from the group consisting of R426 and surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB‑3 protein.

[0126] Preferably, a bispecific antibodyaccording to the invention comprising at least theCDR3sequence, preferably at least the CDR1, CDR2 and CDR3 sequences, of an ErbB‑2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889,MF2913,MF1847,MF3001,MF3003andMF1898asdepicted inFigure 16AorFigure 16E, and / or comprising at least the CDR3 sequence, preferably at least the CDR1, CDR2 and CDR3 sequences, of an ErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37 is combinedwith one ormore compounds selected from the group consisting of an inhibitor of a component of thePI3Kinase pathway,an inhibitor of acomponentof theMAPKpathway,amicrotubuli disruptingdrugandanHDAC inhibitor, preferably with at least one compound selected from the group consisting of a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt 23 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 inhibitor, an mTOR inhibitor, an Src inhibitor, vorinostat and paclitaxel, more preferably with at least one compound selected from the group consisting of afatinib, lapatinib, neratinib, BYL719, MK‑2206, everolimus, saracatinib, vorinostat and paclitaxel

[0127] In one preferred embodiment a bispecific antibody according to the invention comprising: - an ErbB‑2 specific heavy chain variable region sequence selected from the group consisting of the heavy chain variable region sequences of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 as depicted in Figure 16A or Figure 16E, or comprising an ErbB‑2 specific heavy chain variable region sequence that differs in at most 15 amino acids, preferably in atmost 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids,more preferably in atmost 1, 2, 3, 4 or 5 amino acids, from the heavy chain variable region sequences of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898, and - an ErbB‑3 specific heavy chain variable region sequence selected from the group consisting of the heavy chain variable region sequences of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or comprising an ErbB‑3 specific heavy chain variable region sequence that differs in atmost 15 amino acids, preferably in atmost 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids,more preferably in atmost 1, 2, 3, 4 or 5 amino acids, from the heavy chain variable region sequences ofMF3178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074, is combined with one or more compounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK pathway, a microtubuli disrupting drug and an HDAC inhibitor, preferably with at least one compound selected from the group consisting of a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, an mTOR inhibitor, an Src inhibitor, vorinostat and paclitaxel, more preferably with at least one compound selected from the group consisting of afatinib, lapatinib, neratinib, BYL719, MK‑2206, everolimus, saracatinib, vorinostat and paclitaxel. In one preferred embodiment, antibody PB4188 is combined with one or more compounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK pathway, a microtubuli disrupting drug and an HDAC inhibitor, preferably with at least one compound selected from the group consisting of a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, anmTOR inhibitor, an Src inhibitor, vorinostat and paclitaxel, more preferably with at least one compound selected from the group consisting of afatinib, lapatinib, neratinib, BYL719, MK‑2206, everolimus, saracatinib, vorinostat and paclitaxel.

[0128] Preferred embodiments of the invention provide uses of antibodies according to the invention under heregulin stress conditions. Heregulin is a growth factor that is involved in growth of ErbB‑3 positive tumor cells. Typically, when the tumor cells express high levels of heregulin (referred to as heregulin stress), currently known therapies like trastuzumab, pertuzumab and lapatinib are no longer capable of inhibiting tumor growth. This phenomenon is called heregulin resistance. Surprisingly, however, an antibody according to the invention is also capable of counteracting growth of tumor cells that expresshigh levelsof heregulin.Asusedherein, anexpression level of heregulin is consideredhigh if a cell hasaheregulinexpression level that isat least 60%,preferablyat least 70%,morepreferablyat least 80%,morepreferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells. Heregulin expression levels are for instancemeasured using qPCRwith tumorRNA (such as for instance described in Shames et al. PLOSONE,February2013,Vol.8, Issue2, pp1‑10and inYonesakaet al., Sci.transl.Med.,Vol.3, Issue99 (2011); pp1‑11), or using protein detectionmethods, like for instanceELISA, preferably using blood, plasmaor serum samples (such as for instance described in Yonesaka et al., Sci.transl.Med., Vol.3, Issue 99 (2011); pp1‑11). Further provided is therefore an antibody according to the invention for use in the treatment of a subject having or at risk of having an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, wherein said cells of said tumor have a heregulin expression level that is at least 60%, preferablyat least 70%,morepreferablyat least 80%,morepreferablyat least 85%,morepreferablyat least 90%or95%of the heregulin expression level of BXPC3 or MCF7 cells. Said antibody according to the invention preferably comprises a first antigen-binding site that binds domain I of ErbB‑2. Also provided is a method for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, wherein cells of said tumor have a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%or 95%of theheregulin expression level ofBXPC3orMCF7cells, themethodcomprisingadministering to the subject an antibody or pharmaceutical composition according to the invention. One preferred embodiment provides a use of an antibody according to the invention for the preparation of a medicament for the treatment of an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3positive tumor,wherein cells of said tumor haveaheregulin expression level that is at least 60%,preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells. Said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor is preferably 24 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endo- metrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, headandneck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma.Most preferably, said tumor is breast cancer. Further provided is therefore an antibody according to the invention for use in the treatment of a subject havingor at risk of havingbreast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer includingnon-small cell lungcancer, clear cell sarcoma, salivaryglandcancer, headandneckcancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma, preferably breast cancer, wherein cells of said cancer haveaheregulin expression level that is at least 60%,preferably at least 70%,morepreferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3orMCF7cells. Said antibody according to the invention preferably comprises a first antigen-binding site that binds domain I of ErbB‑2.

[0129] Highheregulin levelsare typically present during the formationofmetastases (i.e. themigration, invasion, growth and / or differentiationof tumor cells or tumor initiating cells). Typically, tumor initiating cells are identifiedbasedonstemcell markers suchas for instanceCD44,CD24,CD133and / or ALDH1. Theseprocesses can therefore barely be counteracted with currently known therapies like trastuzumab and pertuzumab. Since an antibody according to the invention is capable of counteracting growth and / or differentiation of tumor cells or tumor initiating cells that express high levels of heregulin, suchantibodyaccording to the invention is alsoparticularly suitable for counteracting the formation ofmetastases. Further provided is therefore a method for counteracting the formation of a metastasis in a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, wherein said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor cell has a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells, comprising administering to the subject a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3. Also provided is a bispecific antibody comprising a first antigen-binding site that bindsErbB‑2 and a second antigen-binding site that bindsErbB‑3 for use in the treatment or prevention of the formation of metastases, wherein said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor cell has a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells. Further provided is a use of a bispecific antibody according to the invention for the preparation of a medicament for the treatment or prevention of the formation of metastases, wherein said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor cell has a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells. Said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor is preferably breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma. Most preferably, said tumor is breast cancer. Further provided is therefore a bispecific antibody according to the invention comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3 for use in the treatment or prevention of the formation of metastases of breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma cells, preferably breast cancer cells, wherein said cells have a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%or 95%of the heregulin expression level of BXPC3orMCF7 cells. Said antibody according to the present invention is typically capable of reducing a ligand-induced receptor function, preferably ligand induced growth, of ErbB‑3 on a ErbB‑2 and ErbB‑3 positive cell. Said antibody according to the invention preferably comprises a first antigen-binding site that binds domain I of ErbB‑2 and a second antigen-binding site that binds domain III of ErbB‑3. In one preferred embodiment, the affinity (KD) of said second antigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB‑2 positive cell. The affinity of said second antigen-binding site for an ErbB‑3 positive cell is preferably lower than or equal to 2.0 nM,more preferably lower than or equal to 1.39 nM,more preferably lower than or equal to 0.99 nM. The affinity of said first antigen-binding site for anErbB‑2 positive cell is preferably lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM preferably lower than or equal to 4.0 nM.

[0130] In one preferred embodiment, said antibody according to the invention comprises an antigen-binding site that binds at least one amino acid of domain I of ErbB‑2 selected from the group consisting of T144, T164, R166, P172, G179, S180andR181, and surface-exposedaminoacid residues that are locatedwithin about 5 aminoacid positions fromT144, T164, R166, P172, G179, S180 or R181.

[0131] In one preferred embodiment, said antibody according to the invention preferably comprises an antigen-binding site that binds at least one amino acid of domain III of ErbB‑3 selected from the group consisting of R426 and surface- 25 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB‑3 protein.

[0132] One preferred embodiment provides amethod according to the invention for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor wherein cells of said tumor have a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%or 95%of the heregulin expression level of BXPC3 orMCF7 cells, or an antibody according to the invention for use in such treatment, wherein said antibody comprises at least the CDR3 sequence, preferably at least the CDR1, CDR2 and CDR3 sequences, or at least the heavy chain variable region sequence, of anErbB‑2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 as depicted in Figure 16A or Figure 16E.

[0133] One preferred embodiment provides amethod according to the invention for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor wherein cells of said tumor have a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%or 95%of the heregulin expression level of BXPC3 orMCF7 cells, or an antibody according to the invention for use in such treatment, wherein said antibody comprises at least the CDR3 sequence, preferably at least the CDR1, CDR2 and CDR3 sequences, or at least the heavy chain variable region sequence, of anErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070;MF6071;MF6072;MF6073andMF6074asdepicted inFigure 16BorFigure 16EorFigure 37.Oneembodiment provides antibody PB4188 for use in the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, wherein cells of said tumor have a heregulin expression level that is at least 60%, preferably at least 70%,more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells.

[0134] As already described, antibodies according to the present invention are particularly suitable for treating ErbB‑2 positive tumor cells with less than 1.000.000 ErbB‑2 receptors on their cell surface. Patients with such tumors, who are typically classified as ErbB‑2 [++] or ErbB‑2 [+], include patients with primary tumors as well as patients with relapsed ErbB‑2positive tumors.Currentlyused therapiessuchas trastuzumab(Herceptin) andpertuzumabareonlyprescribed for patients with malignant ErbB‑2 positive cells that havemore than 1.000.000 ErbB‑2 receptors on their cell surface, which are classifiedasErbB‑2 [+++]. Patients that are classifiedasErbB‑2 [++] orErbB‑2 [+] are therefore preferably treatedwith an antibody according to the present invention. Further provided is therefore amethod or antibody for use according to the invention, wherein said subject has an ErbB‑2 or ErbB‑2 / ErbB‑3 positive tumor that has less than 1.000.000 ErbB‑2 cell- surface receptors per tumor cell. One preferred embodiment provides a bispecific antibody according to the invention comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3 for use in the treatment or prevention of the formation ofmetastases, wherein said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor cell hasaheregulinexpression level that isat least 60%,preferablyat least 70%,morepreferablyat least 80%,morepreferably at least 85%,more preferably at least 90%or 95%of the heregulin expression level of BXPC3 orMCF7 cells, andwherein said tumor cell has less than 1.000.000 ErbB‑2 cell-surface receptors.

[0135] In another preferred embodiment, an antibody according to the invention is used for counteracting an ErbB‑2, ErbB‑3orErbB‑2 / ErbB‑3positive tumor in a subjectwhohasan impaired cardiac function, orwho is at risk thereof.With an impaired cardiac function is meant that the subject has a cardiac function, such as for instance the left ventricular ejection fraction (LVEF), that is lower than 90%, preferably lower than 85% or lower than 80%, preferably lower than 75% or lower than 70%, as compared to a healthy cardiac function. Said healthy cardiac function is, for instance, the average cardiac function (such as for instance the average LVEF) of the healthy population. Alternatively, said healthy cardiac function is the function (such as the LVEF) asmeasured in a patient before the start of anti-tumor therapy with an antibody according to the invention.

[0136] Cardiac function is for instancemonitored by a physical examination of the subject and by an examination of the LVEF, using for instance an echocardiogram or a MUGA scan.

[0137] ErbB‑2 is involved in growth, repair, and survival of adult cardiomyocytes as part of a signalling network that involves the heregulin receptor complex HER2:HER4. As described herein before, cardiotoxicity is a known risk factor in ErbB‑2 targeting therapies and the frequency of complications is increasedwhen trastuzumab is used in conjunction with anthracyclines thereby inducing cardiac stress. For instance, the combination of doxycycline with trastuzumab induces severe cardiac side effects. Despite the increasing number of clinical cases of trastuzumab-induced cardiac dysfunction, its mechanism of action is unknown. In view of the cardiotoxicity of currently known therapies against ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumors, it is of particular advantage to use an antibody according to the invention. As shown in the Examples, antibodies have now been provided that do not, or to a significantly lesser extent as compared to trastuzumab and pertuzumab, affect the survival of cardiomyocytes. This provides an important advantage since cardiotoxicity is reduced. This is already advantageous for people who do not suffer from an impaired cardiac function, and evenmore so for people who do suffer from an impaired cardiac function, such as for instance subjects suffering from congestive heart 26 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 failure (CHF), left ventricular dysfunction (LVD) and / or a decreased Left Ventricular Ejection Fraction (LVEF), and / or subjects who have had a myocardial infarction. Further provided is therefore a bispecific antibody according to the invention foruse in the treatment of a subject havingorat riskof havinganErbB‑2,ErbB‑3orErbB‑2 / ErbB‑3positive tumor, wherein said subject has a cardiac function that is lower than 90%, preferably lower than 85% or lower than 80% or lower than 75% or lower than 70%, as compared to a healthy cardiac function. Said cardiac function preferably includes the LVEF. Said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor is preferably breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer includingnon-small cell lungcancer, clear cell sarcoma, salivaryglandcancer, headandneckcancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, ormelanoma.Most preferably, said tumor is breast cancer. Said antibody according to the invention preferably comprises a first antigen-binding site that binds domain I of ErbB‑2 anda secondantigen-binding site that binds domain III of ErbB‑3.Onepreferred embodiment provides amethodaccording to the invention for the treatment of a subject havingaErbB‑2,ErbB‑3orErbB‑2 / ErbB‑3positive tumor wherein the subject has a cardiac function that is lower than 90%, preferably lower than 85%, preferably lower than 80%, preferably lower than 75% or lower than 70%, as compared to a healthy cardiac function, or an antibody according to the invention for use in such treatment, wherein said antibody comprises: - at least the CDR3 sequence, preferably at least the CDR1, CDR2 and CDR3 sequences, or at least the heavy chain variable region sequence, of an ErbB‑2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913,MF1847,MF3001,MF3003 andMF1898 as depicted in Figure 16A or Figure 16E, or a heavy chain variable region sequence that differs in at most 15 amino acids, preferably in at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably in at most 1, 2, 3, 4 or 5 amino acids, from the recited heavy chain variable region sequences; and / or - at least the CDR3 sequence, preferably at least the CDR1, CDR2 and CDR3 sequences, or at least the heavy chain variable region sequence, of an ErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or a heavy chain variable region sequence that differs in at most 15 amino acids, preferably in at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably in at most1, 2, 3, 4or5aminoacids, from the recitedheavychainvariable regionsequences. Inonepreferredembodiment, said antibody is PB4188.

[0138] In one embodiment, said bispecific antibody is for use in the treatment of a subject under heregulin stress conditions, as explained in more detail elsewhere. Further provided is therefore a bispecific antibody according to the invention foruse in the treatment of a subject havingorat riskof havinganErbB‑2,ErbB‑3orErbB‑2 / ErbB‑3positive tumor, wherein said subject has a cardiac function that is lower than 90%, preferably lower than 85%, preferably lower than 80%, preferably lower than 75% or lower than 70%, as compared to a healthy cardiac function, and wherein said cells of said tumor have a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells. Said cardiac function preferably includes the LVEF. Also provided is a method for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, wherein the subject has a cardiac function that is lower than 90%, preferably lower than 85%, preferably lower than 80%, preferably lower than 75%, preferably lower than 70%, as compared to a healthy cardiac function, and wherein cells of said tumor have a heregulin expression level that is at least 60%, preferably at least 70%,more preferably at least 80%,more preferably at least 85%,more preferably at least 90%or 95% of the heregulin expression level of BXPC3 or MCF7 cells, the method comprising administering to the subject a bispecific antibody or pharmaceutical composition according to the invention. One preferred embodiment provides a use of a bispecific antibody according to the invention for the preparation of a medicament for the treatment of an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor in a subject who has a cardiac function, preferably a LVEF, that is lower than 90%, preferably lower than 85%, preferably lower than 80%, preferably lower than 75% or lower than 70%, as compared to a healthy cardiac function, preferably a healthy LVEF,wherein cells of said tumor have a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells.

[0139] Also provided is a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3 for use in the treatment or prevention of the formation of metastases, wherein said subject has a cardiac function that is lower than 90%, preferably lower than 85%, preferably lower than 80%, preferably lower than 75%, preferably lower than 70% as compared to a healthy cardiac function. Further provided is a use of a bispecific antibody according to the invention for the preparation of a medicament for the treatment or prevention of the formation of metastases, wherein said subject has a cardiac function that is lower than 90%, preferably lower than 85%, preferably lower than 80%, preferably lower than 75%, preferably lower than 70% as compared to a healthy cardiac 27 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 function. Said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor is preferably breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma. Most preferably, said tumor is breast cancer. Said cardiac function preferably includes the LVEF. In one preferred embodiment, said antibody is antibody PB4188.

[0140] Inanotherembodiment, use ismadeofantibodiesaccording to the invention for counteractingphosphorylationof various factors of the prosurvival pathway Akt (also referred to as the PI3 kinase pathway) and the MAP kinase pathway. These are downstream pro-proliferative signaling pathways of HER3. Surprisingly, the inventors have succeeded in significantly inhibiting phosphorylation of Akt, ERK1 / 2 andS6 ribosomal protein (S6-RP)with an antibody according to the present invention, whereas trastuzumabandpertuzumabdonot have these stronganti-phosphorylation effects.Counter- acting phosphorylation of factors of the pro-proliferative PI3 kinase andMAPkinase pathways is advantageous, since this counteracts growth of an ErbB‑3 positive tumor cell. Further provided is therefore a use of an antibody according to the invention for counteracting, preferably inhibiting, phosphorylation of Akt, ERK1 / 2 and / or S6-RP. Importantly, phosphor- ylation of Akt can be significantly reduced or even completely blockedwith an antibody of the invention, both in vitro and in vivo,asshown in theExamples.Apreferredembodiment thereforeprovidesauseofanantibodyaccording to the invention for counteracting, preferably inhibiting, phosphorylation of Akt. Also provided is a use of an antibody according to the invention for counteracting the formation of a HER3-p85 complex. Since the formation of a HER3-p85 complex is the first phase in Akt activation, it is advantageous to counteracting the formation of said HER3-p85 complex. Said antibody according to the invention is preferably a bispecific antibody comprising a first antigen-binding site that binds domain I ErbB‑2 and a second antigen-binding site that binds domain III of ErbB‑3. Said antibody preferably comprises an antigen- binding site that binds at least one amino acid of domain I of ErbB‑2 selected from the group consisting of T144, T164, R166, P172, G179, S180 andR181, and surface-exposed amino acid residues that are located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181. Additionally, or alternatively, said antibody preferably comprises an antigen-binding site that binds at least one amino acid of domain III of ErbB‑3 selected from the group consisting of F409 and R426 and surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB‑3 protein. In one embodiment, said antibody comprises at least oneCDR1, CDR2 andCDR3 sequence, or at least one VH sequence, as depicted in Figure 16 or Figure 37. In one embodiment, said antibody is PB4188.

[0141] For the purpose of clarity and a concise description features are described herein as part of the sameor separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described. Preferred aspects

[0142] Aspect 1. A bispecific antibody comprising a first antigen-binding site that bindsErbB‑2 and a second antigen-binding site that bindsErbB‑3, wherein the antibody can reduce a ligand-induced receptor function of ErbB‑3 on aErbB‑2 and ErbB‑3 positive cell. Aspect 2. A bispecific antibody according to aspect 1, wherein the antibody can reduce ligand-induced growth of an ErbB‑2 and ErbB‑3 positive cell. Aspect 3. A bispecific antibody according to aspect 2, wherein the antibody can reduce ligand-induced growth of an ErbB‑2 and ErbB‑3 positive cell, wherein said cell has at least 100.000 ErbB‑2 cell-surface receptors per cell. Aspect 4.Abispecificantibodyaccording toanyoneof aspects 1‑3,wherein said cell is anMCF‑7cell, anSKBR‑3cell, NCI-N87 cell, an BxPC‑3 cell, an BT‑474 cell or a JIMT‑1 cell. Aspect 5. A bispecific antibody according to any one of aspects 1‑4, wherein the first antigen-binding site binds to domain I or domain IV of ErbB‑2. Aspect 6.Abispecificantibodyaccording toanyoneofaspects 1‑5,wherein thesecondantigen-bindingsite interferes with binding of an ErbB‑3 ligand to ErbB‑3. Aspect 7. A bispecific antibody comprising a first antigen-binding site that bindsErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site binds domain I of ErbB‑2 and said second antigen- binding site binds domain III of ErbB‑3. 28 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Aspect 8. A bispecific antibody comprising a first antigen-binding site that bindsErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein the affinity (KD) of said second antigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB‑2 positive cell. Aspect 9.Abispecificantibodyaccording toanyoneof aspects 1‑8,wherein theantibodycan reducea ligand-induced receptor function of ErbB‑3 on a ErbB‑2 and ErbB‑3 positive cell. Aspect 10. Abispecific antibody according to any oneof aspects 1‑9,wherein theantibody can reduce ligand-induced growth of an ErbB‑2 and ErbB‑3 positive cell. Aspect 11.Abispecificantibodyaccording toanyoneof aspects1‑10,wherein theaffinity (KD)of said secondantigen- binding site for anErbB‑3 positive cell is lower than or equal to 2.0 nM, preferably lower than or equal to 1.39 nM,more preferably lower than or equal to 0.99 nM. Aspect 12. A bispecific antibody according to any one of aspects 1‑11, wherein the affinity (KD) of said first antigen- binding site for an ErbB‑2 positive cell is lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM preferably lower than or equal to 4.0 nM. Aspect 13. A bispecific antibody according to any one of aspects 1‑12, wherein the affinity (KD) of said bispecific antibody for BT‑474 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 4.0 nM, more preferably lower than or equal to 3.2 nM, and / or wherein the affinity of said bispecific antibody for SK-BR‑3 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 3.0 nM, more preferably lower than or equal to 2.0 nM. Aspect 14. An antibody comprising two antigen-binding sites that bind ErbB‑2, wherein at least one of said antigen- binding sites binds domain I of ErbB‑2. Aspect 15. Anantibodyaccording to aspect 14,wherein theaffinity (KD) of at least oneof said antigen-binding sites for anErbB‑2positive cell is lower than or equal to 5.0 nM, preferably lower thanor equal to 4.0 nM,morepreferably lower than or equal to 4.0 nM. Aspect 16. An antibody comprising two antigen-binding sites that bind ErbB‑3, wherein at least one of said antigen- binding sites binds domain III of ErbB‑3. Aspect 17. Anantibodyaccording to aspect 16,wherein theaffinity (KD) of at least oneof said antigen-binding sites for an ErbB‑3 positive cell is lower than or equal to 2.0 nM, preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. Aspect 18. An antibody according to any one of aspects 1‑17, wherein said ErbB‑3 positive cell and / or said ErbB‑2 positive cell is a BT‑474 cell or a SK-BR‑3 cell. Aspect 19. An antibody according to any one of aspects 1‑18, comprising an antigen-binding site that binds at least one amino acid of domain I of ErbB‑2 selected from the group consisting of T144, T164, R166, P172,G179, S180 and R181,andsurface-exposedaminoacid residues that are locatedwithinabout5aminoacidpositions fromT144,T164, R166, P172, G179, S180 or R181. Aspect 20. An antibody according to any one of aspects 1‑19, comprising an antigen-binding site that binds at least one amino acid of domain III of ErbB‑3 selected from the group consisting andR426 and surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB‑3 protein. Aspect 21. An antibody according to any one of aspects 1‑20, wherein said antibody comprises at least the CDR3 sequence of an ErbB‑2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 as depicted in Figure 16A or Figure 16E. Aspect 22. An antibody according to any one of aspects 1‑21, wherein said antibody comprises at least the CDR3 sequence of an ErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as 29 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 depicted in Figure 16B or Figure 16E or Figure 37. Aspect 23. An antibody according to any one of aspects 1‑22, wherein said antibody comprises at least the CDR1, CDR2 and CDR3 sequences of an ErbB‑2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847,MF3001, MF3003 andMF1898 as depicted in Figure 16A or Figure 16E, or wherein said antibody comprises CDR sequences that differ in at most 3 amino acids, preferably in at most 2 amino acids, preferably in at most 1 amino acid from the CDR1, CDR2 and CDR3 sequences of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958,MF2971,MF3025,MF2916,MF3991,MF3031,MF2889,MF2913,MF1847,MF3001,MF3003 orMF1898. Aspect 24. An antibody according to any one of aspects 1‑23, wherein said antibody comprises at least the CDR1, CDR2 and CDR3 sequences of an ErbB‑3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or wherein said antibody comprises CDR sequences that differ in at most 3 amino acids, preferably in at most 2 amino acids, preferably in at most 1 amino acid from the CDR1, CDR2 and CDR3 sequences of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074. Aspect 25. An antibody according to any one of aspects 1‑24, wherein said antibody comprises an ErbB‑2 specific heavy chain variable region sequence selected from the group consisting of the heavy chain variable region sequences of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 as depicted in Figure 16A or Figure 16E, orwherein said antibody comprisesaheavy chain variable region sequence that differs in atmost 15aminoacids from the heavy chain variable region sequences of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898. Aspect 26. An antibody according to any one of aspects 1‑25, wherein said antibody comprises an ErbB‑3 specific heavy chain variable region sequence selected from the group consisting of the heavy chain variable region sequences of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as depicted in Figure 16B or Figure 16E or Figure 37, or wherein said antibodycomprisesaheavychainvariable regionsequence thatdiffers inatmost15aminoacids from theheavychain variable region sequences of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF 6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074. Aspect 27. A bispecific antibody according to aspects 1‑26, which exhibits antibody-dependent cell-mediated cytotoxicity (ADCC). Aspect 28. A bispecific antibody according to any one of aspects 1‑27, which is afucosylated in order to enhance ADCC. Aspect 29. A bispecific antibody according to any one of aspects 1‑28, that is a human or humanized antibody. Aspect 30.Abispecificantibodyaccording toanyoneof aspects1‑29, comprising twodifferent immunoglobulinheavy chains with compatible heterodimerization domains. Aspect 31. A bispecific antibody according to aspect 30, wherein said compatible heterodimerization domains are compatible immunoglobulin heavy chain CH3 heterodimerization domains. Aspect 32. A bispecific antibody according to any one of aspects 1‑31, wherein both arms comprise a common light chain. Aspect 33. A bispecific antibody according to aspect 32, wherein said common light chain is a germline light chain, preferably a rearranged germline human kappa light chain comprising the IgVKl‑39 gene segment, most preferably the rearranged germline human kappa light chain IgVKl‑39*01 / IGJKl*01. 30 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Aspect 34. A bispecific antibody according to any oneof aspects 1‑33, further comprising a label, preferably a label for in vivo imaging. Aspect 35. A pharmaceutical composition comprising a bispecific antibody according to any one of aspects 1‑34. Aspect 36. Amethod for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor or at risk of having said tumor comprising administering to the subject an antibody or pharmaceutical composition according to any one of aspects 1‑35. Aspect 37. An antibody according to any one of aspects 1‑34, for use in the treatment of a subject having or at risk of having an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor. Aspect38.Anantibody foruseaccording toaspect37,wherein saidbispecificantibodydoesnot significantlyaffect the survival of cardiomyocytes. Aspect 39. An antibody for use according to aspect 37 or 38, wherein said bispecific antibody is for use for a subject who has a cardiac function that is lower than 90% as compared to a healthy cardiac function. Aspect 40. Amethod for the treatment of a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor or at risk of having said tumor comprising administering to the subject: - a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, and - one or more compounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of theMAPK pathway, amicrotubuli disrupting drug and an HDAC inhibitor, preferably one or more compounds selected from the group consisting of a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, an mTOR inhibitor, an Src inhibitor, vorinostat and paclitaxel. Aspect 41. A bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen- binding site that binds ErbB‑3 for use in the treatment of a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, wherein said treatment comprises administering said bispecific antibody and at least one compound selected from the group consisting of an inhibitor of a component of thePI3Kinase pathway, an inhibitor of a component of theMAPKpathway, amicrotubuli disrupting drugandanHDAC inhibitor, preferably administering said bispecific antibody andat least one compound selected from the group consisting of a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, an mTOR inhibitor, an Src inhibitor, vorinostat and paclitaxel, to a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor. Aspect 42.Amethodor antibody for useaccording to aspect 40or 41,wherein said tyrosine kinase inhibitor comprises afatinib, lapatinib and / or neratinib. Aspect 43.Amethodor antibody for useaccording to anyoneof aspects40‑42,wherein saidPI3K inhibitor isBYL719. Aspect 44. Amethodor antibody for use according to any oneof aspects 40‑43,wherein saidAkt inhibitor isMK‑2206. Aspect 45. A method or antibody for use according to any one of aspects 40‑44, wherein said mTOR inhibitor is everolimus. Aspect 46. A method or antibody for use according to any one of aspects 40‑45, wherein said Src inhibitor is saracatinib. Aspect 47. A method or antibody for use according to any one of aspects 40‑46, wherein said microtubuli targeting drug is Paclitaxel. Aspect 48. A method or antibody for use according to any one of aspects 40‑47, wherein said HDAC inhibitor is vorinostat. Aspect 49. A method for counteracting the formation of a metastasis in a subject having a ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor, wherein said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor cell has a heregulin 31 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells, comprising administering to the subject a bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3. Aspect 50. A bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen- bindingsite thatbindsErbB‑3 foruse in the treatmentorpreventionof the formationofametastasisof aErbB‑2,ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor cell, wherein said ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor cell has a heregulin expression level that is at least 60%, preferably at least 70%,more preferably at least 80%,more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells. Aspect 51. Amethodor antibody for use according to any oneof aspects 36‑50,wherein said subject hasanErbB‑2or ErbB‑2 / ErbB‑3 positive tumor that has less than 1.000.000 ErbB‑2 cell-surface receptors per cell. Aspect 52. Amethod or antibody for use according to any one of aspects 36‑51, wherein said antibody is an antibody according to any one of aspects 1‑34. Aspect 53. A method or antibody for use according to any one of aspects 36‑52, wherein said tumor cell is a breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, headandneck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma cell. Aspect 54. A method or antibody for use according to any one of aspects 36‑53, wherein said subject has a cardiac function that is lower than 90% as compared to a healthy cardiac function. Aspect 55.Amethodor antibody for useaccording toaspect 39or 54,wherein said cardiac function comprises theLeft Ventricular Ejection Fraction (LVEF). Aspect 56.Amethodor antibody for useaccording to aspect 39or 54‑55,wherein said subject suffers fromcongestive heart failure (CHF), left ventricular dysfunction (LVD) and / or a ≥ 10% decreased Left Ventricular Ejection Fraction (LVEF), and / or wherein said subject has had a myocardial infarction. Aspect 57. Use of an antibody according to any one of aspects 1‑34 for counteracting, preferably inhibiting, phosphorylation of Akt, ERK and / or S6 ribosomal protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0143] Figure 1 : Antigen titration onmonomeric HER2 of a panel of HER2 arms that are also present in active HER2xHER3 bispecificantibodies in combinationwith onearmofPG3178.AllHER2monoclonals of theHER2xHER3panel except for PG3025 were tested on an HER2 antigen titration ELISA. Figure 2: Functional activity of HER2 x HER3 bispecific antibodies on BxPC3 cells with or without ligand stimulation. Dotted lines represent activity of trastuzumab, the reference antibody in this assay, with or without ligand stimulation. Figure 3: Titration curves of HER2 and HER3 monoclonal antibodies (Upper panel) and HER2 x HER3 bispecific antibodies thereof (Lower panel) in the MCF‑7 assay Figure 4: Antibody treatment effect on BxPC3-luc2 tumor size at day 31 in an orthotopic murine model. BLI, tumor growth as measured by bioluminescence. Figure 5: Antibody treatment effect on BxPC3-luc2 tumor size at day 31 in an orthotopic murine model. BLI, tumor growth as measured by bioluminescence. Figure 6: FACS analysis of a bispecific HER2xHER3 antibody and its parental monoclonal antibodies onMCF‑7 and BxPC3-luc2 HER2 expressing cells. MFI, mean fluorescence intensity. 32 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Figure 7 : Analytical characterization by HP-SEC and CIEX-HPLC. PB4188 (upper panel), anti-HER2 parental monoclonal antibody (middle panel), anti-RSV monoclonal reference IgG (lower panel). Figure 8: Inhibition of JIMT‑1 cell proliferation in soft agar by a serial titration of antibody. Figure 9: Inhibition ofBT‑474 (upper panel) andSKBR3 (lower panel) cell proliferation inmatrigel bya serial titration of antibody. Figure 10a: HRG induced proliferation and branching / invasion of SKBR‑3 cells in matrigel. Figure 10b:Inhibition of HRG induced proliferation and branching / invasion of SKBR‑3 cells in matrigel by PB4188 in contrast to the parental monoclonal antibodies. Figure 10c: Inhibition of HRG induced proliferation and branching / invasion of SKBR‑3 cells in matrigel by PB4188 in contrast to anti-HER3 monoclonal antibodies. Figure 10d: Inhibition of HRG induced proliferation and branching / invasion of SKBR‑3 cells in matrigel by PB4188 in contrast to combinations of anti-HER3 monoclonal antibodies with trastuzumab. Figure10e: InhibitionofHRG inducedproliferationandbranching / invasionofSKBR‑3cells inmatrigel byPB4188and the combination PB4188 plus trastuzumab Figure 11: Superior inhibitory activity of PB4188 in HER2+++ N87 cells in the presence of 100 ng / ml HRG. Figure 12: ADCC activity of PB4188 and PB3448 in a dose titration Figure 13: Increased ADCC activity of bispecific antibody compared to monoclonal parental antibodies or a combination thereof Figure 14: ADCC activity of afucosylated PB4188 compared to trastuzumab on low (upper panel) and high (lower panel) HER2 expressing cells Figure 15: ADCC activity of afucosylated PB4188 on SKBR‑3 HER2+++ cells in the presence of reporter cells expressing a high or low FcγR variant Figure 16: Nucleic acid and amino acid sequences of VH-chains, common light chain and heavy chains of antibodies of the invention. Where in this figure a leader sequence is indicated this is not part of the VH chain or antibody, but is typically cleaved of during processing of the protein in the cell that produces the protein. Figure 17: Antibody treatment effect on tumor size in a JIMT‑1 murine xenograft model. Tumor growth measured by tumor volumecalipermeasurement of the different treatment groups. Top, tumorgrowth during60days; bottom tumor growth inhibition (TGI) at the end of treatment period (29 days). Figure 18: Kaplan-Meier survival curves of the different treatment groups in the JIMT‑1 murine xenograft model. Figure 19: Inhibition of N87 ligand driven growth. HRGdriven proliferation of N87 can be overcome over awide range of HRGbyPB4188 in contrast to the parental anti-HER3 antibody. Data shown at antibody concentration of 40 ng / ml. Figure 20: Steady state cell affinity measurements of 125I-labeled IgG HER2xHER3 (PB4188) towards BT‑474 cells (top; three independent assays) and SK-BR‑3 cells (bottom; three independent assays). Non-specific binding was determined using a 100-fold excess of unlabeled HER2xHER3. Figure21A:EpitopemappingHER2.Critical residues identifiedare representedasblacksphereson theHER2crystal structure, secondary critical residues identified are represented as gray spheres (PDB ID #1S78). Figure 21B a) HER2 crystal structure (PDB #1S78) showing verified PG3958 epitope residues as light gray spheres and 33 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 surrounding residues (+ / ‑ five amino acid residues) as dark gray spheres. b) Solvent exposed surface of epitope region showing verified epitope residues in gray and surrounding residues (+ / - five residues) in black. c) Detailed view of epitope region with verified epitope residues in light gray and surrounding residues (+ / - five residues) in dark gray. d) Primary amino acid sequence of HER2 PG3958 epitope region indicating verified epitope residues (gray underlined), surrounding residues (black) and distant residues (gray italic, not shown in a, b and c). Figures and analyses were made with Yasara (www.yasara.org). Figure 21C: a)HER3crystal structure (PDB#4P59) showing epitope residueArg 426 in gray spheres andall surface exposed residueswithin an 11.2 Å radius fromArg 426 in black spheres. b) Solvent exposed surface of epitope regionwith Arg 426 and distant residues shown in gray and all surface exposed residues within a 11.2 Å radius fromArg 426 shown in black. c) Residues in the epitope region Arg 426 in light gray and surrounding residues (all labeled) in dark gray. Figures and analyses were made with Yasara (www.yasara.org). Figure 22:Confirmation of critical binding residues for Fab arm3958 toHER2. Trastuzumabwas includedas a control antibody. Binding was determined in a FACS titration and binding is expressed asAUC in comparison to trastuzumab binding. D143Y is not considered to be part of the 3958 epitope as binding of Trastuzumab to this mutant is also blocked. Figure 23: Critical residues for PG3178 binding represented in the HER3 crystal structure. Critical residues identified for PG3178binding are represented as black spheres on the HER3 crystal structure (PDB ID # 4P59). Figure 24: Confirmation of R426 as a critical binding residue for PG3175 to HER3. Two anti-HER3 antibodies were included as control antibodies. Binding was determined in a FACS titration and binding is expressed as AUC in comparison to binding to WT HER3. Figure 25: Absence of PB4188 toxicity under cardiac stress in vitro. Incubation of cardiomyocytes with PB4188 or monospecific benchmark antibodies in the presence 3 µM of the anthracyclin doxorubicin. Viability of the cardio- myocytes was determined by quantification of ATP and expressed in relative light units (RLU). T, trastuzumab; P, pertuzumab. Figure 26: Binding of PB4188 in comparison to trastuzumab and a HER3 antibody to HER2 amplified cells. FACS titrationswere performed on the indicated cell lines expressing different HER2 levels. Area under the curve ofMedian PE signal values were plotted per cell line. Figure 27: Binding of a serial titration of PB4188FITC to SKBR‑3 cells preincubated with a saturated concentration of PB4188, trastuzumab or a negative control antibody. PB4188FITC binds as effectively to SKBR‑3 in the presence of trastuzumab or control antibody. Figure 28: Inhibitionof cell proliferation underHRGstress conditions byHER2xHER3bispecificantibodies composed of the same HER3 Fab arm and different HER2 arms that are directed against the four HER2 domains. Figure 29: Synergistic combination of PB4188 with lapatinib on the growth and morphology of SKBR‑3 cells. Left, microscopical views of cells treated under different conditions; right morphological changes plotted graphically in relation to the treatment conditions Figure 30A+B: Inhibition of HRG mediated phosphorylation of N87 and SKBR‑3 cells by PB4188 in a time course experiment. Trastuzumab + Pertuzumab and HRG alone were included as controls. Figure 31: Inhibition of HRG mediated phosphorylation of N87 cells by PB4188 in a time course experiment. Trastuzumab + Pertuzumab and lapatinib were included as controls. Figure 32:Changes inAkt levels andAkt phosphorylationwere assessed 4Hafter a twoweekly of fourweekly dose of PB4188. Phosphorylation levels in tumor lysates were assessed by Luminex assays. Analysis were performed in duplicate and five tumors were analyzed per group. Figure 33: In vivomediated effect of PB4188 onHER2:HER3mediated signaling as analyzed byVera Taganalysis on 34 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 JIMT‑1 tumor material. Tumors were analyzed 4H after dosing, tumors derived from PBS treated animals were included as controls. Figure 34: PB4188 reduces cell cycle progression. Cell seeded in assay medium were incubated with titration of antibodies in the presence of a standard (1 ng / ml) or high ( 100 ng / ml) concentration of HRG. 24 hrs later (or 48 hrs for MCF‑7 cells), cells were analyzed for their distribution in the different phases of the cell cycle (G0 / G1, S or G2 / M phases). Proliferation indexwas calculatedas the ratio between thepercentageof cells in theSandG2 / Mphasesand the percentage of cells in the G0 / G1 phase. P+T, pertuzumab + trastruzumab. Figure 35: Internalization of antibodies labelled with pH-sensitive dye in HER2-overexpressing cancer cells. N87 (A, B) and SKBR‑3 (C, D) seeded in assay medium supplemented with 1 ng / ml HRG were incubated for 24 hrs with 100 nM pH-sensitive dye-labelled antibodies. After harvesting, cells were stained with APC-labelled anti-human IgG secondary antibody to detect cell surface-bound antibodies. Cells were analyzed by FACS for fluorescence in the PE (A, C) to determine internalization and APC (B, D) channels to determine surface binding of the antibodies. Figure 36: ADCC activity of Trastuzumab versus Trastuzumab + Pertuzumab with cells derived from two different donors. Figure 37: Amino acid and nucleotide alignments of the F3178 variants. CDR regions are indicated. Figure 38: Titration curves of HER3monoclonal antibodies in the HRGdependent N87 assay. PG6058, PG6061 and PG6065 are variants of PG3178. PG1337 is a negative control specific for tetanus toxoid. Data were normalized to basal proliferation with ligand present on each plate. Figure 39: CIEX-HPLC profiles of HER3 monoclonal antibodies. PG6058, PG6061 and PG6065 are variants of PG3178.Thecalculated iso-electricpoint (pI) of theVH regionand the retention time (tR)of themainpeakaregiven for each antibody. Figure 40: In vitro drug combination isobolograms with PB4188 on HER2 amplified cell lines at HRG stress concentrations (A) or grown in matrigel (B). EXAMPLES Methods, Materials and Screening for Antibodies Cell Lines:

[0144] BxPC‑3-luc2 (Perkin Elmer 125058), N87 (ATCC®CRL‑5822™), SK-BR‑3 (ATCC®HTB‑30™), BT‑474 (ATCC® HTB‑20™), JIMT‑1 (DSMZ ACC 589), L929 (Sigma Aldrich 85011425), K562 (DSMZ ACC10), HEK293T (ATCC® ‑CRL‑11268™), CHO-K1(DSMZ ACC110), MCF‑7 (DSMZ ACC 115), MDA-MB‑468 (#300279‑513, Cell line services) SK-OV‑3 (ATCC ® HTB‑77™), MDA-MB‑175 (ATCC-HTB‑25), MDA-MB‑453 (ATCC-HTB‑131), MDA-MB‑361(ATCC- HTB‑27), ZR‑75‑1 (ATCC-CRL‑1500) and MKN‑45 (DSMZ ACC409) cell lines were purchased from ATCC, DSMZ or Sigma Aldrich and routinely maintained in growth media supplemented with 10% heat inactivated fetal bovine serum (FBS). HEK293F Freestyle cells were obtained from Invitrogen and routinely maintained in 293 FreeStyle medium. Generation of Recombinant Human, Chicken, rat and swapped domain vectors (cloning of HER)

[0145] Human HER2. Full length Human HER2 was amplified by PCR from cDNA derived from RNA isolated from the breast cancer cell line JIMT‑1. The primers used for the amplification of human HER2 were as follows. Forward primer: AAGCTGGCTAGCACCATGGAGCTGGCGGCCTTGTGC Reversed primer: AATAATTCTAGACTGGCACGTCCAGA CCCAGG. The full-length amplified productwas digestedwithNheI andXbaI and subsequently cloned in the correspond- ing sites of pcDNA3.1 (Invitrogen).

[0146] The sequence was verified by comparison with the NCBI Reference Sequence NM_004448.2. To generate constructs solelyexpressing thehumanHER2extracellular domain (ECD) for transfectionand immunizationpurposes the HER2 transmembrane domain and ECDwere PCR amplified and recloned in p Vax 1. For transfection purposes another constructwasgenerated inpDisplaybyamplifying theHER2ECDdomain, in this construct theHER2ECDdomain is fused to the PDGFR transmembrane domain.

[0147] Human HER3. The full length human cDNA clone of HER3 was obtained from Origene. To generate constructs 35 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 solely expressing the human HER3 ECD for transfection and immunization purposes the HER3 transmembrane domain and ECD were PCR amplified and recloned in pVax1. In addition another construct was generated in pVax1 whereby the HER3ECDdomainwas fused to thePDGFR transmembranedomain.All sequenceswere verifiedby comparisonwith the NCBI Reference NM_001982.S

[0148] Cynomolgus HER2 extracellular domain was PCR amplified from cynomolgus cDNA - Monkey) Normal Colon Tissue (Biochain). The primers used for the amplification of cynomolgus HER2 were as follows: Forward primer: AAGCTGGCTAGCACCATGGAGCTGGCGGCCTGGTAC Reversed primer: AATAATTCTAGACTGG CACGTCCAGACCCAGGThe full -length amplified product was digested with NheI-XbaI and subsequently cloned in the corresponding sites of pcDNA3.1. The clone was sequenced and aligned with sequences available of rhesus monkeys (XM_002800451) to check correctness of the ErbB‑2 clone.

[0149] Cynomolgus HER3 extracellular domain was PCR amplified from cynomolgus cDNA - Monkey) Normal Colon Tissue (Biochain). The primers used for the amplification of cynomolgus HER3 were as follows: Forward primer: AAGCTGGCTAGCACCATGAGGGCGAACGGCGCTCTG, Reversed primer: AATAATTCTAGATTAC GTTCTCTGGGCATTAGCThe full -length amplified product was digestedwithNheI-XbaI and subsequently cloned in the corresponding sites of pcDNA3.1. The clone was sequenced and aligned with sequences available of rhesus monkeys (ENSMMUP00000027321) to check correctness of the HER3 clone.

[0150] The chicken HER2 sequence was based on the reference sequence NM_001044661.1. Chimeric swapped domainconstructsweregeneratedbyswappingdomains Iuntil IVof thechickenHER2sequence for thehuman Idomains I until IV. Sequences containing a myc tag were optimized for expression in mammalian cells and synthesized at Geneart.

[0151] The rat HER3 sequence was based on the reference sequence NM_001044661.1. Chimeric swapped domain constructs were generated by swapping domains I until IV of the rat HER3 sequence for the human I domains I until IV. Sequences containing a myc tag were optimized for expression in mammalian cells and synthesized at Geneart. Generation of HER2 and HER3 over-expressing cell lines

[0152] To generate cell lines that express high levels of HER3 on the cell surface a mammalian expression vector was generated by excising the full length HER3 by a NotI and KpnI digestion. Subsequently the fragment was cloned in the corresponding sites of the pcDNA3.1(‑) / hygro vector. A full length HER2 and HER3 expression vector encoding a neomycin resistance gene was used to generate cell lines that express high levels of HER2 on the cell surface. Prior to transfection theplasmidswere linearizedbyaSSpIandFspIdigestion.Bothvectorswere transfectedseparately intoK562 cells and stable poolsweregenerated followingantibiotic selection. The resultant cell lines (K562-HER2andK562-HER3) expressed high levels of HER2 and HER3 on their cell surface. Immunizations

[0153] HER2 immunizations.Four different immunization strategieswereapplied. For cohort #A, sixC57Bl / 6micewere immunized with 2×106 L929 cells transiently transfected with HER2 in 200µl via intraperitoneal injection. Subsequently, mice were boosted with 20 µg Erbb‑2-Fc (RND systems) protein dissolved in 125 µl Titermax Gold via intraperitoneal injection on day 14, followed by boosts with 2×106 L929 cells transiently transfected with HER2 in 200µl on days 28 and 42. For cohort #C, six C57Bl / 6 mice were immunized with 2 ×106 L929 cells transiently transfected with HER2 via intraperitoneal injection.Subsequently,micewereboostedwith2×106L929cells transiently transfectedwithHER2 in200 µl via intraperitoneal injection on day 14, followed by a protein boosts with 20 µg Erbb‑2-Fc protein dissolved in 125 µl TitermaxGold via intraperitoneal injectiononday35andafinal boostwith 20µgErbb‑2-Fcprotein dissolved in 200µl PBS via intraperitoneal injection on day 49. For cohort #E, six C57Bl / 6 mice were immunized with 20 µg Erbb‑2-Fc protein dissolved in 125µl TitermaxGold via intraperitoneal injection. Subsequently, protein boosts with 20µg Erbb‑2-Fc protein dissolved in 125 µl Titermax Gold via intraperitoneal injection were made at day 14 and 28 and a final boost with 20 µg Erbb‑2-Fc protein dissolved in 200 µl PBS via intraperitoneal injection on day 42. For cohort #G, six C57Bl / 6 mice were immunized by DNA vaccination at Genovac (Freiburg, Germany) according to their protocols. The endotoxin-free provided vectors used for the DNA vaccination encoded the transmembrane and extracellular part of HER2 cloned in pVax1. Subsequently, DNA boosts were given at day 14, 28 and 66.

[0154] HER3 immunizations. Four different immunization strategies were applied. For cohort #B, six (C57Bl / 6) mice were immunized with 2 ×106 L929 cells transiently transfected with HER3 in 200 µl via intraperitoneal injection. Subsequently, mice were boosted with 2 ×106 L929 cells transiently transfected with HER3 in 200 µl on days 14, 28, 49 and 63. For cohort #D, six C57Bl / 6 mice were immunized with 2×106 L929 cells transiently transfected with HER3 via intraperitoneal injection on day 0, 14 and 28. Subsequently, mice were boosted with 20µg Erbb‑3-Fc protein dissolved in 125µl TitermaxGold via intraperitoneal injection on day 49 and a final boost with 20µgErbb‑3-Fc protein dissolved in 200 µl PBS via intraperitoneal injection on day 66. For cohort #F, six C57Bl / 6 mice were immunized with 20 µg Erbb‑3-Fc protein dissolved in 125 µl Titermax Gold via intraperitoneal injection. Subsequently, mice were boosted with 20 µg 36 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Erbb‑3-Fc protein dissolved in 125 µl Titermax Gold via intraperitoneal injection at day 14 and 28 and a final boost was given with 20 µg Erbb‑3-Fc protein dissolved in 200 µl PBS via intraperitoneal injection on day 42. For cohort #H, six C57Bl / 6 mice were immunized by DNA vaccination at Genovac (Freiburg, Germany) according to their protocols. The endotoxin-free provided vectors used for the DNA vaccination encoded the transmembrane of PDGFR and extracellular part of HER3 cloned in pVax1. Subsequently, DNA boosts were given at day 14, 28 and 66. Determination of antibody titers.

[0155] Anti-HER2 titers in the serum from immunized C57Bl / 6 mice were determined by ELISA against ECD-Erbb‑2 protein (Bendermedsystems) and FACS analysis on the HER2 negative K562, the HER2 low expressing cell line MCF‑7 and HER2 amplified SKBR‑3 and BT‑474 cells. Anti-HERS titers in the serum from immunized C57Bl / 6 mice were determinedbyELISAagainstErbb‑3-FcproteinandFACSanalysis on theHER3negativeK562, theHER2 lowexpressing cell line MCF‑7 and HER2 amplified SKBR‑3 and BT‑474 cells.

[0156] Serum titers against HER2 and HER3 before sacrificing the animals are described in Table 1 and Table 2 respectively. Animals in all cohorts developed antibody responses against HER2 or HER3. Recovery of lymphoid tissue.

[0157] Spleen and draining lymph nodeswere removed fromall mice vaccinatedwith DNA (cohorts #G and #H). Single cell suspensions were generated from all tissues and subsequently tissues were lysed in Trizol reagent. From cohorts #A until #F spleens were removed from all mice except for one mouse of cohort #C that died after the first boost. Single cell suspensions were generated from all spleens and the total B cell fraction was isolated using the MACS separation procedure either by CD19 enrichment (cohorts # A, E, F) or depletion of non-B cells (cohorts # B, C, D). Generation of phage display libraries from immunized mice

[0158] Onephage librarywasbuilt for eachmouse.To thisend thematerial fromallmicepergroup (5or6micepergroup) was used to prepare phage libraries using the following approach. From each individual mouse RNA was isolated and cDNA was synthesized and VH-family specific PCRs were performed. Subsequently all VH-family PCR products per mouse were purified and the DNA concentration was determined and digested and ligated in a phage-display vector containing the common-light chain to generate amouse-human chimeric phage library. All phage libraries contained>106 clones with an insert frequency of > 85%. Selection of phages carrying Fab fragments specifically binding to HER2 and HER3

[0159] Antibody fragments were selected using antibody phage display libraries. Immunized libraries and synthetic libraries (as described in de Kruif et al. Mol. Biol. (1995), 248, 97‑105) were used for selections. HER2 phage selection and screening

[0160] Phage libraries were rescued with VCS-M13 helper phage (Stratagene) and selected for two rounds in immunotubes (Nunc) coated recombinant protein. In the first round ECD-Erbb‑2 protein (Bendermedsystems) was coated onto immunotubes whereas in the second round Erbb‑2-Fc (RND systems) was coated onto immunotubes. The immunotubes were blocked with 4% non fat drymilk (ELK). Phage antibody libraries were also blockedwith 4%ELK prior to the addition of the phage library to the immunotubes. Incubation with the phage library with the coated protein in the immune tubeswas performed for 2Hat room temperature under rotating conditions. Immunotubeswere thenwashed five to ten timeswith 0.05%Tween‑20 inPBS followed by5 to 10 times inPBS.Boundphageswere eluted using 50mMglycine (pH 2.2) and added to E. coli XL‑1 Blue and incubated at 37°C for phage infection. Subsequently infected bacteria were plated on agar plates containing Ampicillin, tetracyclin and glucose and incubated at 37°C overnight. After the first round, colonieswere scrapedoff theplatesand combinedand thereafter rescuedandamplified toprepareanenriched first round library. The enriched library was then selected onErbb‑2-Fc (RND systems) using the protocol described above. After the second round selection individual clones were picked and rescued to prepare a phage monoclonal miniprep. Positive phageclonesbindingErbb2were then identified inFACS for binding to thebreast cancer cell lineBT‑474.TheVHgenesof all Erbb2 specific clones were sequenced. VH gene rearrangements were established with VBASE2 software to identify unique clones.All unique cloneswere then tested in phage format for binding inFACS toHEK293Tcells (negative control), HEK293T cells transiently transfected with ErbB‑2 and BT‑474 cells. 37 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 HER3 phage selection and screening

[0161] Phage libraries were rescued with VCS-M13 helper phage (Stratagene) and selected for two rounds in immunotubes (Nunc) coated with recombinant protein.

[0162] In both selection rounds round Erbb‑3-Fc (RND systems) was coated onto immunotubes. To overcome a selectionbias towards theFcpart of the fusionprotein, bothselection roundsonErbb‑3-Fcwereperformed in thepresence of 150 µg / ml human IgG. The immunotubes were blocked with 4% ELK. Phage antibody libraries were blocked with 4% ELK prior to the addition of the phage library to the immunotubes. Incubation with the phage library was performed for 2 H under rotating conditions. Immunotubes were then washed five to ten timeswith 0.05%Tween‑20 in PBS followed by 5 to 10 times in PBS.Bound phageswere eluted using 50mMglycine (pH2.2) and added toE. coli XL‑1Blue and incubated for phage infection. Subsequently infected bacteria were plated on agar plates containing Ampicillin, tetracyclin and glucose and incubatedat 37°Covernight. After the first round, colonieswere scrapedoff theplatesandcombinedandphageswere rescuedandamplified toprepareanenrichedfirst round library.Theenriched librarywas thenselectedonErbb‑3-Fc (RND systems) using the protocol described above. After the second round selection individual cloneswere picked and rescued to prepare a phage monoclonal miniprep. Positive phage clones were identified in FACS for binding to the breast cancer cell line BT‑474. The VH genes of all positive clones were sequenced. VH gene rearrangements were established with VBASE2software to identifyuniqueclones.All uniquecloneswere tested inphage format for binding inFACS toK562cells (negative control), stable K562-HER3 cells and BT‑474 cells.

[0163] In total 36 selections were performed on Erbb2 and Erbb3 antigen formats. All selection screening procedures resulted in 89 unique Fab clones directed against HER2 and 137 unique Fab clones directed against HER3. A Fab was considered unique based on its unique HCDR3 sequence, an indication of a unique VDJ recombination event. In some cases clonal variants were obtained, with an identical HCDR3 but differences in the CDR1 and / or CDR2. From the immunizedmice libraries clusters of clonal variants containing substitutions in theVHgene reflecting affinity variantswere selected. Antibody selection / characterization Generation of monoclonal antibodies

[0164] VH genes of unique antibodies, as judged by VH gene sequence and some sequence variants thereof, derived from the immunized mouse phage libraries were cloned in the backbone IgG1 vector. Two different production cell lines were used during the process; HEK293T and 293F Freestyle cells. Adherent HEK293T cells were cultivated in 6-well plates to a confluency of 80%.The cellswere transiently transfectedwith the individual DNA-FUGENEmixture and further cultivated. Seven days after transfection, supernatant was harvested and medium was refreshed. Fourteen days after transfection supernatants were combined and filtrated through 0.22µM (Sartorius). The sterile supernatant was stored at 4°C.Suspension adapted293FFreestyle cellswere cultivated inT125 flasksat a shaker plateauuntil a density of 3.0 x106 cells / ml. Cells were seeded at a density of 0.3‑0.5 x 106 viable cells / ml in eachwell of a 24-deep well plate. The cells were transiently transfected with the individual sterile DNA: PEl mixture and further cultivated. Seven days after transfection, supernatant was harvested and filtrated through 0.22 µM (Sartorius). The sterile supernatant was stored at 4°C. Generation of bispecific antibodies

[0165] Bispecific antibodies were generated using the proprietary CH3 technology to ensure efficient hetero-dimerisa- tion and formation of a bispecific antibody. The CH3 technology uses charge-based point mutations in the CH3 region to allow efficient pairing of two different heavy chainmolecules as previously described (PCT / NL2013 / 050294; published as WO 2013 / 157954 A1). IgG purification for functional screening

[0166] Thepurificationof IgGwasperformedat small scale (<500µg),mediumscale (<10mg) and large scale (>10mg) using affinity chromatography. Small scale purifications were performed under sterile conditions in 24 well filter plates using vacuum filtration. First the pH of the medium was adjusted to pH 8.0 and subsequently the small scale productions were incubatedwith protein ASepharoseCL‑4Bbeads (50%v / v) (Pierce) for 2Hat 25°Cona shaking platformat 600 rpm (Heidolph plate shaker). Next the beadswere harvested by vacuum filtration. Beads were washed twice with PBS pH 7.4. IgG was eluted at pH 3.0 with 0.1 M citrate buffer and the IgG fraction was immediately neutralized by Tris pH 8.0. Buffer exchangewas performed by centrifugation usingmultiscreenUltracel 10multiplates (Millipore). The samples ended up in a final buffer of PBS pH 7.4 38 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Validation of HER2 / HER3 specific IgGs

[0167] Antibodies were tested for binding in FACS to BT‑474, HEK293TandHEK293Toverexpressing HER2 or HER3. Therefore cellswereharvested using trypsin and diluted to 106 cells / ml in FACSbuffer (PBS / 0.5%BSA / 0.5mMEDTA). 1‑2 x105 cells were added to each well in a U-bottom 96 well plate. Cells were centrifuged for 2 minutes at 300 g at 4°C. Supernatant was discarded by inverting plate(s). 50µl of each IgG sample was added at a concentration of 10 µg / ml and incubated for 1H on ice. Cells were centrifuged once, supernatant was removed and cells were washed twice with FACS buffer. 50µl diluted 1:100mouse anti human IgGPE (Invitrogen) was added and incubated for 30‑60minutes on ice in the dark. After adding FACS buffer, cells were centrifuged once, supernatant was removed and cells were washed twice with FACSbuffer. Cells were analysed on a FACSCanto Flow cytometer in aHTSsetting. Binding of the antibodies to cells was assessed by mean fluorescence intensity (MFI).

[0168] To test for non-specific binding reactivity ELISA assays were used. HER2 and HER3 antibodies were tested for reactivity against the antigens fibrinogen, hemoglobulin and tetanus toxin. To test specific binding toHER2andHER3, the antibodies were tested for binding to purified recombinant extracellular domains of EGFR, HER2, HER3 and HER4. Antigenswere coated overnight toMAXISORP™ELISA plates.Wells of the ELISA plateswere blockedwith PBS (pH 7.2) containing 5% BSA for 1 hour at 37°C. Selected antibodies were tested in duplo at a concentration of 10 µg / ml diluted in PBS‑2% BSA and allowed to bind for 2 hours at 25°C. As a control the procedure was performed simultaneously with an antibody specific for the coated antigens and a negative control antibody. The ELISA plates were washed 5 times with PBS-T (PBS‑0.05% v / v Tween 20). Bound IgG was detected with 1:2000 diluted HRP-conjugate (Goat anti-mouse BD) and was allowed to bind for 2 hours at 25°C. The ELISA plates were washed 5 times with PBS-T (PBS‑0.05% Tween 20) and bound IgG was detected by means of OD492nm measurement. Epitope grouping of HER2 / HER3 specific IgGs

[0169] The panel of anti-HER2 antibodies was binned based on their reactivity to the HER2 ECD derived from other species (mouse, chicken) and on their binding to specific domains in the HER2molecule i.e. domains I, II, III and IV using chimeric constructs.

[0170] The panel of anti-HER3 antibodies was binned based on their reactivity to the HER3 ECD derived from other species (cyno, rat) and on their binding to specific domains in theHER3molecule i.e. domains I, II, III and IVusing chimeric constructs.

[0171] For this purpose CHO-K1 cells were transiently transfected with the relevant constructs using lipofectamin / DNA mixes. In the chimeric swapped domain construct, domains of chicken HER2 or rat HER3 are replaced by the human counterpart. Binding of the specific antibodieswasmeasured byFACS. Expression of the constructswas confirmedusing an anti-myc antibody. FACS staining with trastuzumab was included as a control for specific binding to domain IV. Antibodies in each group could be ranked based on the intensity of staining (MFI). TheHER2 panel of 65 antibodies could be mapped into seven bins (Table 3). 1. Domain I specific (25) 2. Domain II specific (2) 3. Domain III specific (23) 4. Domain IV specific (7) 5. Domain IV specific and cross reactive to mouse (2) 6. Reactive to all constructs (2) 7. Only reactive to human HER2 (4) Competition with trastuzumab

[0172] Two antibodies mapped to HER2 domain IV inhibited proliferation of SKBR‑3 cells. Both antibodies shared a similar CDR3 except for one amino acid difference. One antibody, PG1849 was investigated for its capacity to compete with trastuzumab in a competition ELISA. In this ELISA Fc-HER2 was coated and incubated with a concentration of 15 µg / ml IgG antibody. After an incubation of 15 minutes phages were allowed to incubate for another hour. Thereafter, phages were detected. Table 4 demonstrates that PG1849 and trastuzumab could bind simultaneously to HER2 since no loss of signal appeared during the ELISA. True competition only was observed when the same phage and antibody were combined in the assay.

[0173] The HER3 panel of 124 antibodies could be mapped into five bins (Table 5): 1. High Domain III reactivity, rat and mouse reactive and minor reactivity to domain IV (8) 2. High Domain III reactivity, rat, human and cyno reactive, minor reactivity to domain IV (8) 39 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 3. Only reactivity to rat, cyno and human HER3 (43) 4. Only reactive to human HER3 (32) 5. Reactive to all constructs (33) Cell line proliferation assays

[0174] SK-BR‑3 cells were cultured in DMEM-F / 12 supplemented with L-glutamine and 10% heat inactivated FBS. BxPC‑3-luc2 cellswere cultured inRPMI1640supplementedwith 10%heat inactivatedFBS.MCF‑7cellswere cultured in RPMI1640 supplemented with 100 µM, NEAA1 mM sodium pyruvate, 4 µg / ml insulin and 10% heat inactivated FBS.

[0175] For the proliferation assay of SK-BR‑3 cells, subconfluent cell cultures were washed with PBS, trypsinized and trypsin was inactivated by adding culturemedium. Cells were diluted to 6x104 cells / ml in culturemedium. Antibodies were diluted to concentrations of 10 and 1 µg / ml and added in a volume of 100 µl in 96-well black bottom plates (ABgene AB‑0932). Cells were added at density of 6000 cells / well. The cells were cultivated for 3 days at 37°C, 5% CO, in 95% relative humidity. Alamar Blue™ (Invitrogen) was added according to the manufacturer’s instructions and incubated for 6 hours at 37°C, 5%CO, in 95% relative humidity in the dark. Fluorescencewasmeasured at 550 nmexcitation and 590 nm emission wavelength. The extent of growth inhibition was compared to that of the same concentration of trastuzumab (Table 6).

[0176] For the proliferation assay of MCF‑7 and BxPC‑3-luc2 cells, subconfluent cell cultures were washed with PBS, trypsinized and trypsin was inactivated by adding culture medium. Cells were washed twice in large volumes of assay medium (RPMI 1640 medium containing 0.05% BSA and 10 µg / ml Holo Transferrin). MCF‑7 cells were diluted to 5x104 cells / ml in culturemedium. Antibodies were diluted to concentrations of 10 and 1µg / ml and added in a volume of 100µl in 96-well black bottomplates (ABgeneAB‑0932). Cells were added at a density of 5000 cells / well in the presence of 1 ng / ml final concentration human Recombinant Human NRG1-beta 1 / HRG1-beta 1 EGF Domain; (396-HB‑050 RND). Human NRG1-beta 1 / HRG1-beta 1 EGF Domain will hereinafter be referred to as HRG. The cells were cultivated for 5 days at 37°C, 5%CO, in 95%relative humidity. AlamarBlue™ (Invitrogen)wasaddedaccording to themanufacturer’s instructions and incubated for 24 hours at 37°C, 5%CO2, in 95% relative humidity in the dark. Fluorescencewasmeasured at 550 nm excitation with 590 nm emission wave length. The extent of growth inhibition was compared to that of the same concentration of #Ab6 (Table 7).

[0177] BxPC‑3-luc‑2proliferation assayswere used to screen thebispecific antibodies. BxPC‑3-luc‑2 cellswere diluted to 8x104 cells / ml in culturemedium. Antibodies were diluted to concentrations of 10 and 1µg / ml and added in a volume of 100µl in 96-well black bottom plates (ABgene AB‑0932). Cells were added at density of 8000 cells / well in the absence or presence of 10 ng / ml final concentration humanHRG. The cells were cultivated for 4 days at 37°C, 5%CO, in 95% relative humidity. Alamar Blue™ (Invitrogen) was added according to themanufacturer’s instructions and incubated for 4 hours at 37°C, 5%CO, in95%relativehumidity in thedark. Fluorescencewasmeasuredat 550nmexcitationwith590nmemission wave length.

[0178] To minimalize edge effects, the outer wells of the 96 well plates were fully filled with PBS. Affinity ranking of HER2 specific IgGs

[0179] Weused the method described by Devash (PNAS, 1990) to rank the antibodies in a limited antigen-ELISA. The useof decreasedantigen coating concentrationseliminates observed cross-reactivity reactionsand canbeused to detect high-affinity / avidity antibodies. Therefore the antigen concentration on the solid support was gradually decreased to investigate the weak immunoreactivities. A serial titration of ECD-Erbb‑2 protein starting from 2.5µg / ml until 0.019µg / ml was coated overnight toMAXISORP™ELISAplates.Wells of theELISAplateswere blockedwithPBS (pH7.2) containing 5%BSA for 1hour at 37°C.Selectedantibodieswere tested in duplo at a concentration of 10µg / ml diluted inPBS‑2%BSA andallowed tobind for 2hoursat 25°C.Asacontrol theprocedurewasperformedsimultaneouslywith anantibody specific for the coated antigens and a negative control antibody. The ELISA plates were washed 5 times with PBS-T (PBS‑0.05% v / v Tween 20). Bound IgG was detected with 1:2000 diluted HRP-conjugate (Goat anti-mouse IgG, BD Biosciences) and was allowed to bind for 2 hours at 25°C. The ELISA plates were washed 5 times with PBS-T (PBS‑0.05% Tween 20) and bound IgGwas detected bymeans ofOD492nmmeasurement. PG1849, PG2916, PG2926, PG2930, PG2971, PG2973, PG3004 and PG3031 were tested in an HER2 antigen titration ELISA (Fig. 1). Binding of HER2 VH genes with various kappa light chains

[0180] To investigate the binding ofHER2VHsderived fromdifferent phagedisplay libraries a panel of HER2antibodies was cloned and expressed in the context of another VK kappa chain, i.e. the VL of MEHD7945A. Produced IgGs were subjected to FACSanalysis on K562 cells and stable K562-HER2 cells. VH genes derived from the combinatorial libraries and non-combinatorial libraries are listed in Table 8. The VH chains MF2971, MF3958, MF2916, MF2973, MF3004, 40 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 MF3025, MF3031 all could be combined with the MEHD7945A light chain without loosing significant antigen specificity and binding as observed when combined with the common light chain IGKV1‑39. VH chain MF1849 was not able to combine with the variant kappa light chain and retain antigen specificity and binding. Other HER2 and HER3 antibodies

[0181] Antibodies that inhibit the function of HER2 or HER3 are known in the art. Further antibodies were constructed according to published information and expressed in 293F Freestyle cells. The anti-HER2 antibodies pertuzumab and trastuzumab were generated based on the information disclosed in US2006 / 0212956 A1 (Genentech). The anti-HER3 antibody #Ab6, was based on the information disclosed in WO 2008 / 100624 (Merrimack Pharmaceuticals, Inc.) and recloned in a IgG1 back bone vector. The information of the 1‑53 and U1‑59 anti-HER3 antibodies was obtained fromUS 7,705,103B2 (U3PharmaAG). The information of the anti-HER3LJM716antibodywasobtained fromUS2012 / 0107306. The information for the construction of the two-in-one anti-EGFR anti-HER3 antibody MEHD7945A was obtained from WO2010 / 108127. Screening of HER2xHER3 bispecific antibodies

[0182] VH from theHER2 andHER3 antibody panel were recloned into the charged engineered vectors such that upon expression of the antibody heavy chains heterodimerization of heavy chains is forced resulting in the generation of bispecific antibodies after transfection. Three different strategies were used in combining HER2 and HER3 arms in bispecific IgG format: 1. HER2 (blocking ligand independent growth) xHER3 (blocking ligand independent growth) 2. HER2 (blocking ligand independent growth) xHER3 (blocking ligand dependent growth) 3. HER2 from different epitope bins x HER3 (blocking ligand dependent growth)

[0183] In some bispecific combinations, antibodies generated in group 2 and 3 overlapped with group 1.

[0184] A total of 495bispecificantibodieswasproduced in24-well formatandpurified.All antibodieswere tested for their capacity to inhibit the proliferation of the HER2‑ and HER3-expressing pancreatic BxPC‑3-luc‑2 cell line (Caliper). The potency of the antibodies was determined in a HRG-dependent and HRG-independent setting in a black and white screening with antibodies being present at a concentration of 10 and 1 µg / ml. Trastuzumab was included as a reference antibody as well as a negative control antibody at the same concentrations. The functional activity of the top 80 HER2xHER3 bispecifics (based on combined inhibition) at 1 µg / ml is shown in Figure 2.

[0185] Antibodies (40 in total) that showed a higher inhibitory activity compared to the positive control antibody were selected, reproduced and purified in a 24-well format and tested again in the black-and-white BxPC‑3-luc‑2 screen at 10 and 1 µg / ml concentrations. These antibodies were further titrated in HRG-dependent MCF‑7 assay and compared against thecombinationof trastuzumabandpertuzumab (1:1) andanegativecontrol antibody.Figure3showsanexample of titration curves of three bispecific antibodies in comparison to the parental HER3 antibody and the combination of trastuzumab + pertuzumab. The parental monoclonal antibodies are shown in the top panel and the bispecific antibodies are shown in the lower panel. (Figure 3).

[0186] The IC50 for the bispecific antibodies, monoclonals and comparator antibodies was calculated using non-linear regression analysis with Prismsoftware. Graph pad software lists the IC50 values of the bispecific antibodies in theMCF‑7 assay and their inhibitory activity in theBxPC3assay for comparison. A panel of 12HER2xHER3bispecific antibodies had morepotent inhibitingactivity compared to trastuzumab+pertuzumab. Inaddition thebispecificantibodieswereequally or more potent than the parental monoclonal PG3178 (Table 9).

[0187] The bispecific antibodies that inhibited ligand dependent cell growth were composed of HER2 arms in combination with the HER3 arms 3178, 3163, 3099 and 3176. Both the HER2 and HER3 arms of the most potent bispecifics were as a bivalent monoclonal also capable of inhibiting ligand-independent SKBR‑3 proliferation (both the HER2 and HER3 arms) (Table 6) or ligand dependent MCF‑7 proliferation (HER3 arms) (Table 7). The majority of the potent antibodies was composed of a HER2 arm recognizing domain I in combination with anti-HER3 antibody 3178. Inhibition of BxPC‑3-luc2 tumor growth

[0188] The antibodies described in Table 9were tested in a BxPC‑3-luc2 pancreatic xenograft model. TheBxPC‑3-luc2 cell lineexpressesbothHER2andHER3and is consideredaHER2 lowexpressing cell line.CB17SCID femalemice, 8‑10 weeksoldat thebeginningof thestudywereengraftedorthotopically in thepancreaswith1×106 tumor cells in20µl. To this aimmicewereanesthetizedand laid on the right side toexpose the left sideanda0.5 cm incisionwasmadeon the left flank region. The pancreas and spleen were exteriorized and 1×106 tumor cells in 20µl was injected into the sub-capsulary 41 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 space of the pancreas tail. One week after implantation, bioluminescence (BLI) data were generated. 15 minutes prior to the imaging, all of the mice received i.p. injections of 150 mg / kg Luciferin (D-Luciferin-EF Potassium Salt, Cat. #E6552, Promega). BLI imagingwas performed once or twice weekly using the left side view.Outlier animals - based on BLI / tumor volume - were removed and the mice were randomly distributed into groups of 7 mice each. On experimental day 8, the treatmentwasstarted.Theanimals in theantibody treatment groupweredosedweekly for 3 consecutiveweeks (days0, 7, 14 and21)with 30mg / kgof antibody.At day0of the treatment the animals received twice the loadingdose, i.e. 60mg / kg of antibody. The final imaging was carried out at day 31.

[0189] Two BxPC‑3-luc2 xenograft models were run with a different panel of bispecific antibodies and parental antibodies In the first BxPC‑3-luc2 xenograft model (Figure 4), one group received the negative control anti-RSVantibody (Ctrl IgG), one group received the control antibody trastuzumab and one group received the positive control antibody trastuzumab + pertuzumab (1:1 v / v). The seven remaining groups received one of themonoclonal (PG) or bispecific (PB) antibodies PG3004, PG3178, PB3566, PB3710, PB3443, PB3448 and PB3441. Details of the composition of the bispecific antibodies are depicted in Table 9.

[0190] All five bispecific antibodies tested were able to inhibit tumor growth. The mean tumor mass (BLI) of bispecific HER2×HER3 antibody treated animals was similar to that in the animals treated with the combination of trastuzumab + pertuzumab. (Fig. 4)

[0191] In the second BxPC‑3-luc2 xenograft model (Figure 5), one group received the negative control anti-RSV antibody (Ctrl IgG) and one group received the positive control antibody combination trastuzumab+ pertuzumab (1:1 v / v). The five remaining groups received one of the antibodies PG3163, PB3986, PB3990, PB4011 and PB3883. For details about the bispecific PB antibodies: Table 9. These bispecific antibodies contained three different HER3 binding arms combined with the sameHER2 armMF2971 and an additional HER2 arm combined with the HER3 binding armMF3163. In this experiment the tumors in the control group did not show the same level of accelerated growth as in the first experiment complicating interpretation of the results. Nevertheless, in comparison to trastuzumab + pertuzumab the PB3883 and PB3990 HER2xHER3 bispecifics had similar inhibitory activities (Fig. 5).

[0192] Based on the in vivo and in vitro data a bispecific panel of antibodies was selected of which theHER2 armswere composed of MF2971, MF3004, MF1849 and the HER3 arm was composed of MF3178. The MF2971 and MF3004 arm were of mouse origin and were humanized. Binding of bispecific HER2xHER3 antibody compared to parental monoclonal antibodies

[0193] Binding of HER2xHER3 bispecific antibodies as compared to their parental counterparts was determined by FACS analysis. A FACSwas performed on BxPC‑3-luc2 cells andMCF‑7 cells with a serial titration of antibodies ranging from 2,5 µg g / ml - 0, 01 µg g / ml. The tested antibody panel was composed of the bispecific antibody PB3566 and its parental antibodies the anti-HER3antibodyPG3178 and the anti-HER2antibodyPG3004. TheMFI datawere plotted and thegraphsonboth cell lines show that thebispecificPB3566bindsmoreeffectively toboth tumor cell lines compared to the anti-HER3 antibody PG3178 and the anti-HER2 antibody PG3004. (Fig. 6) Humanization of MF2971 and MF3004

[0194] MF2971andMF3004were humanized according to technology known in the art. A total of seven humanised / de- immunised variant sequences of MF2971 were expressed, validated and characterised in vitro as monoclonal and in bispecific format combinationwith theHER3-specific antibodyMF3178. The samewas done for seven variant sequences of MF3004, which were created by replacing the HCDR3 of MF2971 in the seven MF2971 variants with the HCDR3 of MF3004. The expression, integrity, thermal stability and functional activity of all humanized variants was analysed. Based on production, integrity, stability and functionality integrity, a variant of MF2971 (2971-var2) was chosen as the optimal humanized variant of the VH to be used in a bispecific format with MF3178. This 2971-var2 was renamed MF3958. The bispecific HER2xHER3 combination MF3958xMF3178 resulted in PB4188. Large scale production, purification and analytical studies of PB4188

[0195] Suspension adapted 293FFreestyle cellswere cultivated inErlenmeyer flasks at a shaker plateau until a density of 3.0× 106 cells / ml. Cells were seeded in a 4 L erlen flasks at a density of 0.3‑0.5 × 106 viable cells / ml. The cells were transiently transfected with the individual sterile DNA: PEl mixture and further cultivated. Seven days after transfection, conditionedmediumcontainingbispecificantibodywasharvestedby low-speedcentrifugation,5minutes1000g, followed by high speed centrifugation, 5minutes at 4000g. Collected conditionedmediumwas concentrated over a 5 kDaSatorius hydrosart cassette toabout600mlandsubsequently diafiltratedagainst 4LPBS.Antibodieswereboundoncolumn to~35 ml MabSelectSure XL (11°C). A-specifically bound proteins were removed by washing the column in reversed flowmode with150mlPBS,150mlPBScontaining1MNaCl, 100mlPBS.Theboundantibodieswereelutedusing100mMcitratepH 42 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 3.0 in reversed flowmode and 5 ml fractions were collected in 10 ml tubes containing 4 ml 1Tris pH 8.0 for neutralization. The eluted antibodies were further purified by gel-filtration using superdex 200 50 / 1000. Thepurified antibody was filter- sterilized using a 0.22 µm syringe filter. IgG concentration was determined by OD280 measurement and the protein concentration was calculated based on the amino acid sequence. Protein was tested for aggregation (HPSEC), purity (SDS-PAGE, nMS, IEX and IEF). Protein samples were stored at ‑80°C. IgG purification for analytical and xenograft studies.

[0196] MediumscalepurificationswereperformedonanAKTA100Explorer usingHiTrapMabSelect Surecolumnsand HiTrap desalting columns. Sampleswere loaded at 5ml / min. The columnwaswashedwith 2 column volumes ofPBS. IgG was eluted at pH 3.0 with 0.1 M citrate buffer. Next the sample was desalted and ended up in a final buffer of PBS pH 7.4. IgGs were filtered through a 0.45 µM filter (Sartorius). The IgG concentration was measured using Octet with protein A sensors. Protein was tested for aggregation (HPSEC), purity (SDS-PAGE, nMS, IEX and IEF). Protein samples were stored at ‑80°C. Analytical characteristics of PB4188

[0197] The PB4188 (MF3958xMF3178) was subjected to analysis by HP-SEC and CIEX-HPLC (TSK gel-STAT 7 µm column, 4.6 mm ID x10 cm L). The analytical profile of PB4188 was in general consistent with the behavior of normal monospecific IgG1, such as the parental HER2 arm PG3958 and the anti-RSV monoclonal control antibody (Fig. 7). Affinity determination

[0198] The monovalent binding affinity of PB4188 and PB3448 for recombinant HER2 and HER3 was determined by SPR (Biacore T100). Biacore™T100 (GEHealthcare, Uppsala, Sweden) was used to conduct all experiments described. Sensor surface preparation and interaction analyses were performed at 25° C. Buffer and Biacore reagents were purchased from GE Healthcare. ErbB2-Fc and ERbB3-Fc(RND) was coated to the surface of a CM5 sensor chip in potassium acetate buffer (pH5.5) at the target immobilization level of 500 RU. Running buffer was HBS (hepes-buffered saline): 10 mMHEPES pH 7.4, 150 mMNaCl, 0.005% Tween‑20; 0.2µm) filter-sterilized. The bispecific antibodies were diluted to 100, 50, 20, 10, 1 and 0.1 nM in HBSand run at high (30µl / min) flow rate over the antigen-coupled surface of the CM5 sensor chip. With the BIA evaluation software, a curve fitting model for 1:1 monovalent interaction allowed for determination of the HER2 arms affinities (mono-valent interaction), the affinities of theHER2 arms, could be determined. Due to the low-off rate of the HER3 arm the affinity could not be determined. To determine the affinity of the HER3 arm PB4188wascoated toaCM5sensor chipat the target immobilization level of 500RU.Her2-FcandHer3-Fcantigenswere diluted to 100, 50, 20, 10, 1 and 0.1nM in HBS and run at high flow rate (40µl / min) over the PB4188 surface. To determine the kon and koff values, the BIA evaluation software was used in conjunction with a model that takes into account that a monovalent molecule was coated to the sensor chip surface and that the ErbB3-Fc antigen was a bivalent molecule. The affinities of PB4188 and PB3448 are shown in Table 10. PB4188 Affinity determination on cells

[0199] Binding affinities were also determined via steady state cell affinity measurements using BT‑474 and SK-BR‑3 cells. Four IgGwere analyzed: 1) PB4188 (bispecific HER2xHER3), containing anti-HER2 antibody 3958 and anti-HER3 antibody 3178; 2) PB9215 (bispecific HER3xTT), containing anti-HER3 antibody 3178 and anti-TT (tetanus toxoid) antibody 1337; 3) PB9216 (bispecific HER2xTT), containing anti-HER2 antibody 3958 and anti-TT antibody 1337; 4) Herceptin (monospecific HER2). The IgG were radioactively labeled with 125I using IODO-GEN® Precoated Iodonation Tubes (Pierce) andassociated instructions. The labeled IgGwerediluted to anactivity of ~1‑2×108 cpm / ml in 25mMTris- HCl, 0.4 M NaCl, 0.25% BSA, 5 mM EDTA, 0.05% NaN3. Protein concentrations were determined with the BCA Protein Assay Kit (Pierce). Flow cytometry analysis of the labeled and non-labeled IgG using BT‑474 and SK-BR‑3 cells showed no or only minor signs of reduction in binding after labeling. Steady state cell affinity measurements were performed as follows. Cells were seeded in 96-well plates and incubated at 4°C with various concentrations of labeled IgG. Unbound radioactivity was removed after 4 hours and the cell-bound radioactivity wasmeasured using a gammawell counter. Non- specificbindingwasmeasuredbyaddinga receptor-blockingconcentration (100-foldexcess)of unlabeledantibody.Each condition was tested in triplicate and three independent experiments were performed per antibody. KD values were calculated based on a non-linear regression model that compensates for non-specific binding, using Prism 6.0d (GraphPad Software). Graphs including fitted curves are given in Figure 20 for binding of the HER2xHER3 IgG (PB4188) to both cell lines. KD data for all 24 assays, including mean values, are given in Table 12. In summary, the meanKDvalues as determined usingBT‑474 andSK-BR‑3 cells were 3.2 and 2.0 nM forHER2xHER3, 3.7 and 1.3 nM for 43 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Herceptin, 3.9 and 2.3 nM for HER2xTT, and 0.23 and 0.99 nM for HER3xTT, respectively. Thus PB4188 shows a higher affinity forHER3compared toHER2which is in contrast to theHER2xHER3bispecificmoleculeMM‑111 that targetsHER2 with a higher affinity compared to HER3. Anti-proliferative activity on HER2 amplified breast cancer cells JIMT‑1 in soft agar

[0200] PB3448 and PB4188were tested for their potency to inhibit the growth of the trastuzumab resistant JIMT‑1 cells in soft agar. To this aim 96 well suspension cell culture plates were prepared. 100 µL of the soft agar bottom layer (0,6% final concentration in complete medium) was poured and left to solidify. 50 µL of the soft agar top layer (0,4% final concentration) containing 10.000 JIMT‑1 cells / well were then added on top, solidified and such 96 well plates incubated overnight at 37°C, 10% CO2. Next day, a negative control antibody, pertuzumab + trastuzumab (1:1 v / v), PB3448 and PB4188 were added in DMEMmedium in a semi-log titration ranging from 10‑0,003 µg / ml. Subsequently, the assay was incubated in cell culture incubators for 8 days. Finally, the cells were incubated with Alamar Blue for 3‑5 h at 37 °C and fluorescence intensitywasdetermined (excitation: 560nm;emission: 590nm).Anexampleofdosedependent inhibitionof JIMT‑1 proliferation by PB3448 and PB4188 is shown. (Figure 8). BT‑474 and SKBR‑3 in matrigel

[0201] PB3448 and PB4188 were tested for their potency to inhibit the growth of BT‑474 and SKBR‑3 cells. The cells were tested at the company Ocello based in Leiden, the Netherlands that grows cells in three dimensional matrigel and uses principle component analysis to distinguish non-treated cells from treated cells. 2000 SK-BR‑3 or 2250 BT474 cells were seeded in 15µl matrigel per well of a 384 well plate (Greiner 781091 ). The next day a semi-log titration ranging from 10 to 0.003 µg / ml of antibodies were added in culture medium in the absence or presence of 5 ng / ml HRG. The test antibodies included a negative control antibody, pertuzumab + trastuzumab (1:1 v / v), PB3448, PB4188 and the bispecific anti-EGFRxHER3 two-in-one antibody MEHD7945A. In addition a dose-dependent titration of HRG was included as a positive control. Each dosewas tested in quadruplicate. Cells were incubated for 7 days in a cell culture incubator at 37°C, 5%CO2.Next, the cellswere fixedandactin cytoskeletonof the cellswas stainedwith phalloidin and thenuclei are stained with Hoechst. Next, fluorescent images were taken at different levels through the gel (Z-stack) and the images were superimposed. A broad rangeofmorphological featuresweremeasured (800 in total). Only features that differed between mediumandHRG treatments were selected for analysis. Features that were associatedwith growth,mean spheroid area and nuclei per spheroid weremost significantly different betweenmediumandHRG treatments. Bothmultiparameter and single parameter analyses were made. For single parameter measurements, t-tests were performed to compare treatments (HRG or antibody) to medium. P-values for each point were determined. Principal component analysis (PCA), a method for finding low-dimensional combinations of high-dimensional data that capture most of the variability was used in relation to antibody concentration, to plot the data. Figure 9 demonstrates the effect of pertuzumab + trastuzumab (1:1 v / v), PB3448 and PB4188 in the presence of HRG. In both HER2 amplified breast cancer cell lines PB4188 showed superior activity compared to pertuzumab + trastuzumab, PB3448 and the two-in-one antibody MEHD7945A in the presence of HRG. Superior anti-proliferative activity of PB4188 in the presence of HRG on HER2 amplified breast cancer cells

[0202] The activity of PB4188 in the presence of 10 ng / ml HRG on SKBR‑3 and BT‑474 was compared to a panel of HER2, HER3 antibodies and combinations thereof. The assay was performed in matrigel, as described above, and morphological features were analyzed. PCA data plotted in Figure 10a show the HRG -induced proliferation and branching / invasion of SKBR‑3 cells in matrigel. Figure 10b shows that antibody PB4188 can completely revert the HRG induced phenotype, whereas the combination of the parental monoclonal antibodies (PG3958 + PG3178) has no effect. Moreover, PB4188 was far more effective compared to all anti-HER3 antibodies tested (Figure 10c). In addition, combinations of the individual anti‑ HER3 antibodies with trastuzumab (the current standard of care in metastatic breast cancer (mBC)) were not able to revert the HRG induced phenotype (Figure 10d). Adding trastuzumab to PB4188 in the presence of HRG reduced the proliferation and branching / invasion of SK-BR‑3 cells compared to PB4188 alone (Figure 10e). Superior anti-proliferative activity of PB4188 on HER2 amplified gastric cancer cells compared to HER2 and HER3 monoclonal antibodies.

[0203] Upregulation of NRG1-β1 is a key resistance mechanism against HER2 targeted therapies (Wilson, 2012). To 44 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 evaluate whether upregulation of NRG1-β1 would interfere with the anti-proliferative potency of PB4188 a panel of antibodies was tested at 100 ng / ml HRG on the N87 (HER2 amplified) gastric cancer cell line. N87 cells were cultured in RPMI 1640 supplemented with 10% heat inactivated FBS. For the proliferation assay subconfluent cell cultures of N87 cellswerewashedwithPBS trypsinizedand trypsinwas inactivatedbyadding culturemedium.Cellswerewashed twice in large volumes of assay medium (RPMI 1640 medium containing 0.05% BSA and 10 µg / ml Holo Transferrin). Antibodies were diluted in a semi-log titration that varied from 1‑0,0001 ng / ml. Cells were added at a density of 10000 cells / well in the presence of 100 ng / ml final concentration of HRG. The cells were cultivated for 3 days at 37°C, 5% CO2, in 95% relative humidity. Alamar Blue™ (Invitrogen) was added according to themanufacturer’s instructions and incubated for 6 hours at 37°C, 5% CO2, in 95% relative humidity in the dark. Fluorescence was measured at 550 nm excitation with 590 nm emission wavelength. PB4188 showed superior activity over anti-HER2 or anti-HER3monoclonal antibodies (Figure 11). HER2XHER3 bipecific antibodies induce ADCC

[0204] ADCC activity is an important anti-tumour mechanism of action for therapeutic antibodies in cancer. Human monoclonal antibodies directed to the HER family of receptors like cetuximab and trastuzumab induce ADCC. The baseline and enhanced ADCC activity of PB4188 and PB3448 were determined in validated in vitro ADCC assays. Trastuzumab and a negative control antibody were included as control antibodies in the experiment. Whole blood and PBMC fractions were obtained from healthy donors. Each antibody was tested against the HER2 high (SK-BR‑3) and HER2 low (MCF‑7) expressing target cells. Target cells were loaded with 51Cr (Amersham) and opsonized with the indicated concentrations of antibody. Whole-blood or PBMC fraction were used as effector cells in a 200 pL reaction in RPMI 1640 + 10% heat inactivated FCS. Cells were incubated together for 4 h, and lysis was estimated by measuring radioactivity in the supernatant using a Υ-scintillator. Percentage of specific lysis was calculated as follows: (experimental cpm-basal cpm) / (maximal cpm-basal cpm)×100,withmaximal lysisdetermined in thepresenceof 5%TritonX‑100and basal lysis in the absence of antibody and effectors. As shown in Figure 12 bispecific antibody PB3448 showed similar ADCCactivity compared to the combination pertuzumab+ trastuzumab. Bispecific antibodyPB4188was effective at high antibody concentrations (10 µg / ml). HER2XHER3 bipecific antibodies show higher ADCC compared to the combination of parental antibodies

[0205] In a different ADCC setup, the ADCC Reporter Bioassay (Promega) was used. The bioassay uses engineered Jurkat cells stably expressing the FcyRIIIa receptor, V158 (high affinity) or F158 (low affinity) variant, and an NFAT response element driving expression of firefly luciferase. The assay was validated by comparing data obtained with the ADCC Reporter Bioassay to the classical 51Cr release assay. The ADCC assays were performed using the Promega ADCC Bioassay kit using 384 white well plates. In this experimental setup SKBR‑3 cells were plated at a density of 1000 cells / well in 30 µl assay medium (RPMI with 4% low IgG serum) 20‑24H before the bioassay. The next day, the culture medium was removed. Next, a serial dilution of antibodies, PB4188 and its parental anti-HER2 PG3958 and anti-HER3 PG3178 as well as the combination thereof was generated in duplo. 10µl antibody dilutions were added to the wells. The starting concentration of the antibodywas 10µg / ml and a 10 points semi-log fold serial dilutionwas generated to provide a full dose-responsecurve.Finally, 5µl ofADCCBioassayeffector cells (15000cells / well, V158)wereadded.Thecellswere incubated for 6H at 37 °C. Next, 15 µl BIO-Glo luciferase substrate was added and 5 minutes later luminescence was detected in a plate reader. The obtained data are shown in Figure 13. ThePB4188 bispecific anti-HER2xHER3antibodies showed a higher ADCC potentency compared to the parental HER2 and HER3 monoclonals or a combination thereof. ADCC enhancement of PB4188

[0206] ADCC activity can be enhanced by different techniques, one of them being the removal of fucose. Removal of fucose has resulted in increased anti-tumour activity in several in vivo models [Junttila, 2010]. To maximize PB4188 activity, afucosylation technology was applied (Cheng Liu and Andreia Lee. ADCC Enhancement Technologies for Next Generation Therapeutic Antibody. Antibody therapeutics ‑Trends in Bio / Pharmaceutical Industry 2009 [13‑17]) , thereby preventing fucosylation of the N-linked carbohydrate structure in the Fc region. The ADCC potency of afucosylated PB4188 compared to the wildtype PB4188 was determined in an ADCC 51Cr release assay using HER2 low expressing cells (MCF‑7) andHER2amplified cells (SK-BR‑3). Both antibodieswere applied in a serial dilution and a negative control antibody and trastuzumab were included in the assay. Figure 14 shows the increase in ADCC potency of afucosylated PB4188 compared to the wild type version and / or trastuzumab in both high and low HER2 expressing cells. Afucosylated PB4188 shows superior ADCC activity with low affinity FcyRIII receptors

[0207] Afucosylated PB4188 activity was tested on ADCC reporter cells containing either the V158 (high affinity) 45 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 FcyRIIIa receptor variant or the F158 (low affinity) FcyRIIIa receptor variant. A serial titration of antibody, i.e. control antibody, trastuzumab and afucosylated PB4188, was added in combination with ADCC reporter cells harbouring the different FcyRIIIa variants to adherent SK-BR‑3 cells. ADCC activity was measured by measuring luciferase activity. Afucosylated PB4188 showed equal activity compared to trastuzumab in combination with the high affinity V158 FcyRIIIa receptor variant. In contrast afucosylated PB4188 displayed superior ADCC activity compared to trastuzumab in combination with the low affinity F158 FcyRIIIa receptor variant. (Figure 15) JIMT‑1 xenograft study

[0208] JIMT‑1 human breast carcinoma cells were grown in DMEM containing 10% fetal bovine serum, 100 units / mL penicillin G sodium, 100 pg / mL streptomycin sulfate, 25 pg / mL gentamicin, and 2 mM glutamine until the time of implantation. At the day of implantation JIMT‑1 breast cells were harvested during log phase growth and resuspended in coldPBS.FemaleCB.17SCIDmice (CharlesRiver)were8weeksoldonDay1of thestudyandhadabodyweight range of 16.5 to 20.7 g. Each mouse was injected subcutaneously in the right flank with 5 ×106 tumor cells (0.2 mL cell suspension). The tumors were measured with a caliper in two dimensions to monitor size as the mean volume twice per week. Once tumors had reached approximately 100‑150 mm3 in size animals were enrolled in the efficacy study. Outlier animals -tumor volume - were removed and the mice were randomly distributed into groups of 10 mice each. Mice were injected onceweekly (antibody) or daily (lapatinib) for a period of four weeks. Details of the treatment groups are depicted in Table 11.

[0209] Tumor sizes were measured weekly by caliper measurement. The efficacy study revealed that PB4188 at both dosing schedules was equal effective and more potent than lapatinib or the combination pertuzumab and trastuzumab. The data are shown in Figures 17 and 18. PB4188 can overcome HRG mediated resistance

[0210] Upregulation of NRG1-β1 is a key resistance mechanism against HER2 targeted therapies (Wilson, 2012). PB4188 was tested in comparison to its parental anti-HER3 monoclonal antibody PG3178 in a serial titration in the presence of an increasing concentration of HRG (NRG1-β1 EGF). To this aim N87 cells were cultured in RPMI 1640 supplemented with 10% heat inactivated FBS. For the proliferation assay subconfluent cell cultures of N87 cells were washed with PBS trypsinized and trypsin was inactivated by adding culture medium. Cells were washed twice in large volumes of assay medium (RPMI 1640 medium containing 0.05% BSA and 10 µg / ml Holo Transferrin). Antibodies were diluted in a semi-log titration ranging from 1 to 0.0001 µg / ml. Cells were added at a density of 10000 cells / well in the presence an increasing concentration of HRG (0.04‑39,5 nM). The cells were cultivated for 3 days at 37°C, 5% CO2, in 95% relative humidity. Alamar Blue™ (Invitrogen) was added according to the manufacturer’s instructions and incubated for 6 hours at 37°C, 5%CO2, in 95% relative humidity in the dark. Fluorescence wasmeasured at 550 nm excitation with 590nmemissionwavelength. PB4188showedsuperior activity compared to theparental anti-HER3monoclonal antibody (Figure 19).

[0211] Hence, in case of an escape mechanism, such as for instance upregulation of NRG1-β1, a bispecific antibody according to the invention is preferred. Epitope mapping of HER2 / HER3 specific IgGs Shotgun mutagenesis experiments

[0212] Alanine scanningmutagenesiswasused tomap theepitopesofPG3958andPG3178 forHER2and respectively HER3. In the shotgun mutagenesis assay, clones are generated whereby each amino acid residue of the HER2 / HER3 extracellular domain (ECD) is substituted for alanine. Next, a cell array was prepared by reverse transfection (patent US2011 / 0077163A1). Therefore, DNA of each clone was mixed with lipofectamin and the mixture was placed in a dedicated well of a 384 well plate. HEK293Tcells were added to each well and expression of protein was measured 24H later.Subsequently, the reactivity of antibodieswasmeasuredby immunofluorescent staining leading tobindingmapsand identification of critical residues for antibody binding. Expression levels of the HER2 and HER3 ECD constructs were verified by FACS analysis using commercially available monoclonal antibodies (R&D mAb 1129 (HER2) and R&D mAb 66223 (HER3)). HER2

[0213] Binding of monovalent PG3958 Fab to HER2 ECD mutants was tested at a concentration of 0.25 µg / ml in the assay and stringent washing conditions were used (pH 9.0, 350 mM NaCl). This resulted in the identification of three 46 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 ’critical’ residues (T144,R166,R181) inHER2 that showed less than35%residualbindingof thePG3958Fabcompared to WT HER2 while retaining control mAb binding.

[0214] Two residues (P172, G179) that are positioned near the critical residues in the HER2 structure showed significant, but less severe loss of binding and were designated ’secondary critical’ residues (Table 13 and Figure 21A).All thesesurface-exposed residuesare located inDomain I ofHER2and together they formadiscontinuouspatchon the surface of the HER2 molecule. Confirmation experiments HER2 epitope

[0215] Constructs encodingWildtype (WT)HER2ECDand theHER2ECDvariants listed in Table 13were expressed in CHO-K1 cells. Three Domain I residues that are surface exposed and structurally near the determined critical residues were selected for further analysis. T164, S180 and D143 point mutations to tyrosine were generated in the HER2 ECD construct and the resulting constructs were also expressed in CHO-K1. The L159A HER2 ECD variant was expressed in CHO-K1 cells as control sample.

[0216] The bispecific PG3958xTTantibody tested for binding to the ECD variants in a FACS titration experiment. The anti-HER2 antibody trastuzumab which binds domain IV of HER2 was used to verify HER2 ECD expression at the cell surface.MeanMFI valueswere plotted and for each curve theAUCwas calculated usingGraphPadPrism5 software.WT HER2 binding was used to normalize the data. The FACS data showed that in addition to T144A, R166A, R181A, P172A, G179A themutationsT164YandS180Y resulted in significant reduction inbindingof thePG3958xTTantibody (Figure22). The D143Ymutation resulted in severe loss of expression as demonstrated by the decreased binding of the control mAb, so its potential role in the PG3958 epitope could not be determined. HER3

[0217] Binding analysis of PG3178 IgGat 0.25µg / ml toHER3ECDmutants in FACS resulted in the identification of two so-called ’critical’ residues (F409, R426) for whichmutation to alanine caused substantial loss of binding compared toWT HER3, while binding of the control mAb was retained (Table 14 and Figure 23). Both residues are located in Domain III of HER3 and spatially distant. Moreover, F409 is buried in the HER3 hydrophobic core, which makes it unlikely to be part of the PG3178 epitope. Confirmation experiments HER3 epitope

[0218] CHO-K1 cells were transfectedwith HER3ECDmutation constructs (listed in Table 14),WTHER3ECDand two control constructs (H407A and Y424A). PG3178 binding to the HER3 ECD variants was tested in a FACS titration experiment. Two control antibodies, binding Domain I (MM‑121) and Domain III (MEHD7945A) of HER3were included to verifyHER3ECDexpressionon thecell surface.MeanMFI valueswereplottedand foreachcurve theAUCwascalculated usingGraphPad Prism 5 software.WTHER3 binding was used to normalize the data. TheR426Amutation was shown to be critical for PG3178 binding whereas the binding to F409A could not be confirmed due to loss of cell surface expression (Figure 24). PB4188 activity on cardiomyocytes in vitro

[0219] HER2 is involved in growth, repair, and survival of adult cardiomyocytes as part of a signalling network that involves the heregulin receptor complex HER2:HER4. Cardiotoxicity is a known risk factor in HER2 targeting and the frequency of complications is increased when trastuzumab is used in conjunction with anthracyclines thereby inducing cardiac stress.Amodel systembasedonhumanstemcell derived cardiomyocyteswasused to test the potential toxicity of PB4188 and benchmark it against trastuzumab and the combination of trastuzumab and pertuzumab in the presence of the anthracyclin doxorubicin.Human stemcell derived cardiomyocytes (PluriomicsBV)were seededat a concentration of 20.000 well in white flat-bottom assay plates (corning 655098). On day 5 of culture the medium was replaced for glucose andgalactose free culturemediumsupplementedwith 10ng / mlHRG.Onday 7 test antibodieswere added in combination with doxorubicin (3 µM). Cell viability was assayed on day 9 using the Promega Cell titer Glo assay. The monospecific antibodies were tested at single concentrations of 68 nM whereas PB4188 was tested at three concentrations in the presence of 3 µM doxorubicin. Figure 25 shows that the viability of the cardiomyocyte was unaffected by all PB4188 concentrations tested. In contrast, trastuzumab and the combination of trastuzumab and pertuzumab both reduced cardiomyocyte cell viability. 47 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 PB4188 binding to cells with different HER2 levels

[0220] The binding of PB4188 in comparison to trastuzumab and the HER3 antibody U1‑59 was analyzed by FACS on breast and gastric cancer cell lines expressing different levels of HER2. Cells were considered HER2+++ if they express millions of HER2 copies and / or are HER2 gene amplified. The following cell lines were used: MCF‑7 (HER 2 +); MDA- MB‑468 (HER2 +, MKN‑45 (HER2 +), MDA-MB‑175 (HER2+), MDA-MB‑453 (HER2 ++), MDA-MB‑361(HER2 ++), ZR‑75‑1(HER2 ++), JIMT‑1 (HER2+++), BT‑474 (HER2+++), SKBR‑3 (HER2+++), SK-OV‑3 (HER2+++), N87 (HER2++ +). Cells of an exponentially grown culturewere harvested by trypsin and diluted to 106 cells / ml in FACSbuffer (PBS / 0.5% BSA / 0.5mMEDTA). 1‑2 105 cellswere added to eachwell in aU-bottom96well plate. Cellswere centrifuged for 2minutes at 300gat 4°C.Supernatantwasdiscardedby inverting plate(s) above, followedby flickingonce. 50µl of each IgGsample wasadded in a serial dilution from3.16ng‑10µg / ml and incubated for 1Hon ice.Cellswere centrifugedonce, supernatant was removed and cells were washed twice with FACS buffer. 50 µl diluted 1:100 mouse anti human IgG gamma PE (Invitrogen) was added and incubated for 30‑60minutes on ice in the dark. Cells were centrifuged once, supernatant was removed and cells were washed twice with FACS buffer. Cells were analysed on a FACSCanto Flow cytometer in a HTS setting. The quantity of antibody boundwaswas assessed bymedian fluorescence. Datawere plotted and the area under the curve (AUC, a cumulative measurement of the median fluorescence intensity) was determined for each antibody per cell line tested (Figure 26).

[0221] From this experiment it is concluded that PB4188 has a higher binding affinity for HER2+++ cells, HER++ cells and HER+ cells as compared to trastuzumab. Simultaneous binding with trastuzumab PB4188 and trastuzumab do not compete for binding to HER2

[0222] PB4188bindsdomain I of theHER2proteinwhereas thebindingepitopeof trastuzumab is localized in domain IV. To demonstrate that both antibodies do not compete for HER2 binding, a binding assay with HER2 amplified SKBR‑3 breast cellswasperformed.First unlabeledantibodywasallowed tobindSKBR‑3at saturating concentrations.Next FITC- labeled PB4188 was added in a titration range and fluorescence was measured by FACS. Figure 27 demonstrates that PB4188FITC boundaseffectively to cells in the presenceof trastuzumabor the negative control. Pre-incubation of SKBR‑3 cells with PB4188 prevented PB4188FITC from binding. Thus, trastuzumab and PB4188 do not compete for binding to HER2 Targeting domain I of HER2 by a HER2xHER3 bispecific molecule can overcome Heregulin resistance

[0223] To test whether the orientation of PB4188 on theHER2xHER3dimerwas preferred for inhibiting cell proliferation under HRG stress conditions, bispecific antibodies were generated composed of the 3178 HER3 arm and HER2 arms targeting either domain I, II, III or IV. TwoHER2xHER3bispecific antibodiesweregenerated for eachof theHER2domains I-IV. The HER2 arms included: MF3958 and MF3003 targeting domain I; MF2889 and MF2913 targeting domain II; MF1847 and MF3001 targeting domain III and MF1849 and MF1898 targeting domain IV. Each HER2 Fab arm was combined with the 3178 HER3 Fab arm and tested for their potency to inhibit cell proliferation in the presence of high concentrations of heregulin. Antibody titrations were performed on HER2 low expressing MCF‑7 cells and the HER2 overexpresssingN87 andSK-BR‑3 cells. Subconfluent cell cultures of N87, SK-BR‑3, andMCF‑7 cells werewashedwith PBS trypsinizedand trypsinwas inactivatedbyadding culturemedium.Cellswerewashed twice in large volumesof assay medium (RPMI1640mediumcontaining 0.05%BSAand10µg / mlHoloTransferrin). Antibodieswerediluted in a semi-log titration.Cellswereaddedat a density of 10000 cells / well (N87, SKB-BR‑3) and 5000cells / wellMCF‑7 in thepresence the experimentally defined stress concentration of HRG (10nM SK-BR‑3, 100nMN87 andMCF‑7). The cells were cultivated for 3 - 4daysat 37°C,5%CO2, in95%relativehumidity.AlamarBlueTM(Invitrogen)wasadded toassess theproliferation. Absorbancewasmeasured at 550nmexcitationwith 590 nmemissionwave length. In all assays tested, only the bispecific antibodies targeting domain I of HER2 were able to inhibit proliferation in the presence of a high heregulin concentration (Figure 28). Drug combinations with PB4188 in vitro.

[0224] To investigate the possibility to combine PB4188 with small molecule drugs PB4188 was combined with drugs interferingatdifferent levelsof thePI3KorMAPKpathway.Moreover, combinationwith chemotherapeuticdrugsandcyclin inhibitors were tested. Combinations were tested on HER2 overexpressing cells growing in the presence of HRG in matrigel (SK-BR‑3 and BT‑474) or in the presence of HRG stress concentrations (N87 and SK-BR‑3 as described in proliferation assays). The inhibitory effect of drug combinationswas tested by imaging or bymeasuring proliferation using 48 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Alamar Blue as described herein before. First, the EC20 PB4188 and drugs tested was determined. Next, checkerboard titrations were performedwith PB4188 and the drugs. Synergies were observed in all cell lines tested with tyrosine kinase inhibitors (afatinib, lapatinib, neratinib), the PI3Ka inhibitor BYL719, the Akt inhibitor MK‑2206, the mTOR inhibitor everolimus, the Src inhibitor saracatinib, the microtubuli disrupting drug paclitaxel, and the HDAC inhibitor vorinostat (which is misspelled in Figure 40 as "voronistat"). Figure 29 shows an example of the synergistic combination of PB4188 with lapatinib on SKBR‑3 cells grown in matrigel resulting in morphological changes and reduction of cell growth. The extent of growth inhibition obtained with each combination was calculated. Potency shifting can be shown using isobolograms (Greco et al 1995) which shows how much less drug is required in a combination to achieve a desired levelwhencompared to the single agent required to reach that effect. The inhibition valuesof the combinationexperiments were used by CHALICE™ Analyzer software to generate the isobolograms. Isobolograms of the different drug combina- tions on HER2 amplified cells are shown in figure 40. Isobologram analysis indicated that PB4188 displayed synergistic drugcombinationswithafatinib, lapatinib, neratinib,BYL719 ,MK‑2206,everolimus, saracatinib, vorinostat andpaclitaxel.

[0225] These data demonstrate that drugs acting on the PI3K pathway are particular effective in combination with PB4188. In addition, combinations with Tyrosine Kinase Inhibitors are effective. Moreover, a combination with the growth and migration / invasion drug saracatinib can be favourable in the metastatic setting. PB4188 In vitro inhibition of phosphorylation

[0226] Cells of an exponentially grown culture were harvested and seeded in 6 well plates (3.75×106 cells for N87 and 1.5×106 cells for SKBR‑3) in starvation medium (N87 cells: RPMI‑1640, 0.05% BSA, 10µg / ml Holo-transferrin; SKBR‑3 cells: DMEM / F‑12, 2mML-glutamine, 0.05%BSA, 10µg / ml Holo-transferrin) and incubated incubated overnight at 37°C, 5% CO2, in 95% relative humidity. The next day, antibodies were added to a final concentration of 5 nM and cells were incubated for one hour at 37°C, 5% CO2, in 95% relative humidity. HRG was then added to a final concentration of 100 ng / ml. After 1, 3, 6 or 24 hours at 37°C, 5% CO2, in 95% relative humidity, plates were placed on ice, cells were washed twicewith coldPBS.Subsequently 0.3ml ice-cold lysis bufferwasadded (Cell signalingRTK#9803or IC#7018) and cells were lysed for aminimum of 30minutes on ice. Next, protein concentrations weremeasured using BCA (Pierce #23235). Protein concentrations were adjusted to 2 mg / ml with lysis buffer. Next, lysates were applied to PathScan RTK Signaling Antibody Arrays (Cell signaling #7949) or PathScan Intracellular Signaling Antibody Arrays. All incubations were performed with sealed wells on an orbital shaker at room temperature. Lysates (75 µl) were diluted 2 times to 0.8 mg / ml concentration with 75 µl Array Diluent Buffer supplemented with protease inhibitor cocktail and kept on ice. Array wells wereblockedwith100µl Arrayblockbuffer for 15minutes.Blockbufferwas removedandLysateswereapplied to thewells and allowed to incubate for 2 hours. Lysate was aspirated and wells were washed 4 times with 100 µl Wash buffer. Next, 100µl detection antibody cocktail was added per well and incubated for 1 hour. Antibody cocktail was aspirated andwells were washed 4 times with 100 µl Wash buffer. 75 µl Dylight80™ Streptavidin was added to each well. Dylight80™ Streptavidin was aspirated and wells were washed 4 times with 100 µl Wash buffer. The multi-gasket was removed and slides were washed for 10 seconds in 10ml in deionized water. Slides were allowed to dry and processed for imaging on an Odysee®Clx. Spot fluorescence intensity was calculated using Image Studio software.

[0227] InN87andSKBR‑3,PB4188completely blocksAKTphosphorylation during the first 6Hof incubation, in contrast to thecombinationof trastuzumab+pertuzumab. Inadditionastrong inhibition isobserved inERKandS6phosphorylation in contrast to the combinationof trastuzumab+pertuzumab.PB4188doesnot inhibit phosphorylationofHER2 (Figure 30) Western blot analyses

[0228] Toverify thephosphorylation inhibitionobserved in theRTKand intracellularPathscanarraysWesternblotswere performed of cells treated with PB4188, the combination pertuzumab and trastuzumab and a control antibody in the presence of HRG stress concentrations. Cells of an exponentially grown culture were harvested and seeded in 10 cm dishes (20×106 cells for N87 and 7×106 cells for SKBR‑3) in starvation medium (N87 cells: RPMI‑1640, 0.05% BSA, 10µg / mlHolo-transferrin;SKBR‑3cells:DMEM / F‑12, 2mML-glutamine, 0.05%BSA,10µg / mlHolo-transferrin). Thenext day, antibodieswereadded toafinal concentrationof5nMandcellswere incubated foronehour.HRGwas thenadded toa final concentrationof 100ng / ml.After 1, 3, 6or24hours, disheswereplacedon ice, cellswerewashed twicewith coldPBS, transferred to Eppendorf tubes and lysed with 250 µl of RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP‑40, 1% sodium deoxycholate, 0.1% SDS, 2.5 mM sodium pyrophosphate, 1 mM beta- glycerophosphate, 1 mM Na3VO4, 1 µg / ml leupeptin). Lysis was allowed to proceed for 30 minutes on ice. Cell lysates were centrifuged and supernatants were collected in new Eppendorf tubes. Protein concentration was determined using the BCAmethod (Pierce). 30µg of the lysatewas separated on a 4‑12%Bis-Tris NuPage gel (Invitrogen) and proteins on the gel were transferred to a nitrocellulosemembrane. Membranes were blocked for one hour with TBS-Tcontaining 5% BSA and stained with the indicated antibodies according to the manufacturer’s instructions (Cell Signaling Technology). Membranes were then incubated with a HRP-conjugated secondary antibody, incubated with ECL substrate and 49 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 subjected to autoradiography using X-ray films (Amersham). All detection antibodies were from Cell Signaling Technol- ogy: Phospho-Akt (ser 473) #4060, Total Akt #4691, Phospho-HER2 (Tyr 1221 / 1222) #2243, Total HER2 #2242, Phospho-HER3 (Tyr 1289) #4791, Total HER3 #4754, Phospho-ERK1 / 2 (Thr 202 / Tyr 204) #4377, Total ERK1 / 2 #4695, Phospho-S6 RP (Ser 235 / 236) #2211, Total S6 RP #2217, Goat anti- rabbit HRP-linked #7074.

[0229] The results show that PB4188 showsa prolonged inhibition ofHER3phosphorylation resulting in the inhibition of both the MAPK and PI3 kinase pathway with a profound effect on AKT phosphorylation inhibition (Figure 31). PB4188 In vivo pharmacodynamics Phosphoprotein analysis by Luminex

[0230] Tumors (100mm3) of JIMT‑1 transplanted mice treated with 2 doses of PB4188 and 4 doses of PB4188 were removed 24H after dosing. Tumors were flash-frozen and processed to powder. Tumor lysates were prepared to a concentration of 50mg tumor / mLusing coldBioRadLysis Buffer (supplementedwith 0.4%BioRadFactor 1, 0.2%BioRad Factor 2, and 2mMPMSF) to the frozen powder samples, incubated at 4°C on a rocker for 60minutes to ensure complete lysis. The sampleswere centrifugedat 4°C for 10minutes at 16000×g, and aliquoted. Total proteinwas determinedusing theBioradDCProtein Assay reagents according tomanufacturer’s instructions. LuminexAssay: The JIMT‑1 tumor lysate samples were processed and analyzed for: Total AKT AKT(Ser473) and AKT(Thr308using commercially available Luminex kits from Millipore (Cat # 48‑618MAG (Lot No. 2532050), 46‑645MAG (Lot No. 46645M‑1K). Each sample was tested in duplicate. Dilutions were prepared in sample diluent to load a target of approximately 25µg protein per well for all total and phosphorylated analyte determinations. The Millipore kits were used according to the manufacturer’s specifications.

[0231] Tumors treated with PB4188 showed an increase in Akt expression in comparison to untreated tumors. Phosphorylation of AKT was completely inhibited by PB4188 both after a two-weekly dose as after a four-weekly dose (Figure 32). Phosphoprotein analysis by VeraTag assay

[0232] Tumors (100mm3 or 400 mm3) of JIMT‑1 transplanted mice treated with 1 or 2 doses doses of PB4188 were removed and fixed in 10% neutral buffered formalin. Mice bearing 100mm3 tumors were sacrificed 24H after a single PB4188 dose (25mg / kg) whereasmice bearing 400mm3 tumors received 2weekly dosis of 25mg / kg andwere sacrificed 4H after dosing. Next, samples were paraffin-embedded. Sections of 7 um in thickness were sliced with a microtome (LEICA) and placed on positively charged glass slides (VWR) with serial number labeled. Slides were airdried for 30 min and then baked in a heated oven set at 60° C. Next samples were processed for different VeraTag analysis. Total HER2 analysis (HT2) according to U.S. Patent Appl. No. 12 / 340,436, total HER3 analysis (H3T) according to U.S. Patent No. 8,349,574;U.S.Patent Appl. No. 2013 / 0071859and finallyHER2-HER3heterodimer (H23D), HER3pY1289 (H3pY1289) and HER3-PI3 kinase (H3PI3K) according to Int’l Patent Appl. No. PCT / US2014 / 033208. In both dosing regimens a significant PB4188 mediated reduction in HER2:HER3 dimers became apparent in comparison to untreated controls. There was no difference observed in total HER2, HER3 or phosphorylated HER3 between PB4188 treated tumors and controls. Tumors that were analyzed 4H after PB4188 dosing showed a significant reduction in HER3-p85 (PI3K) compared with untreated controls. PB4188 reduces cell cycle progression in HRG-stimulated cancer cells

[0233] The ability of PB4188 to influence cell cycle progressionwas investigated in cancer cell lines expressing various protein levels of HER2.HER2+ (MCF‑7), HER2+++ (JIMT‑1, SK-BR‑3 andN87 cells) cells were seeded in assaymedium (MCF‑7 cells: RPMI‑1640, 0.05% BSA, 10 µg / ml Holo-transferrin, 1 mM sodium pyruvate, MEMNEAA; JIMT‑1: DMEM, 0.05 % BSA, 10 µg / ml Holo-transferrin; SK-BR‑3 cells: DMEM / F‑12, 2 mM L-glutamine, 0.05 % BSA, 10 µg / ml Holo- transferrin; N87 cells: RPMI‑1640, 0.05 % BSA, 10 µg / ml Holo-transferrin). Per well of 24-well plate, 300.000 MCF‑7, or 400.000 N87 or 150.000 SK-BR‑3 or 150.000 JIMT‑1 or cells seeded in 1 ml assay medium and incubated overnight at 37°C, 5%CO2, in 95% relative humidity. The next day, PB4188 or pertuzumab+ trastuzumabor PG3178 or PG1337were added to thecells in thepresenceofafinal concentrationofHRGof1or100ng / ml.After 24hrs (for JIMT‑1,N87orSK-BR‑3 cells) or 48 hrs (forMCF‑7 cells) incubation at 37°C, 5%CO2, in 95% relative humidity, cells were supplementedwith EdU (10 µM final concentration) for 2 hrs before being harvested and stained for EdU incorporation using the Click-iT EdU AlexaFluor488 kit according to the manufacturer instructions (LifeTechnologies, cat.no. C10425). At least 30 min before analyzing the cells by flow cytometry on FACSCanto, cells were incubated with 200 nM FxCycle far red dye (Life- Technologies, cat.no. F10348) and 100 µg / ml RNAse A (LifeTechnologies, cat.no. 12091‑039). Events were acquired in theAlexFluor488 channel (forEdUdetection) and in theAPCchannel (for totalDNAstainwith theFxCycledye).Datawere 50 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 analyzedbygating single cells onaFSC-width vsFSC-height scatter plot, andsubgating theG0 / G1,SandG2Mphasesof the cell cycle on an APC vs AlexaFluor488 scatter plot, as EdUnegAPClow, EdUpos and EdUnegAPChigh populations, respectively.

[0234] Data are represented as the proliferation index calculated by dividing the percentage of cells in the S and G2 / M phases by the percentage of cells in the G0 / G1 phase. Figure 34 shows that PB4188 is consistently more potent than PG3178 or pertuzumab + trastuzumab in inhibiting proliferation induced by a standard (1 ng / ml) or a high (100 ng / ml) concentration of HRG. At high concentrations of HRG PB4188 still inhibits the cell cycle progression. PB4188 induces receptor internalization

[0235] Internalization pattern of antibodieswasmeasuredusing pH-sensitive dyes. This has beendescribed in theart in WO2013134686 A1 where such dyes, when coupled to an antibody, display an increased fluorescence signal when exposed to lower pH. This occurs when the dye-coupled antibodies internalize from the surface of target cells into mildly acidic endosomes (pH 6‑6.5) to acidic lysosomes (pH lower than 5.5). To investigate whether PB4188 internalizes in cancer cells, the antibody was coupled to the pH sensor dye with succinimidyl ester reactive group (Promega, cat.no. CS1783A01) according to the manufacturer’s instructions. As comparators, anti-HER2 (trastuzumab, pertuzumab, PG3958), anti-HER3 (PG3178, #Ab6) and negative control (anti-tetanus toxin, PG1337) dye labeled antibodies were included. HER2-overexpressing SKBR‑3 and N87 cancer cells of an exponentially grown culture were harvested and seeded on 96 well plates (15×103 cells per well) in 100 µl assay medium (N87 cells: RPMI‑1640, 0.05 % BSA, 10 µg / ml Holo-transferrin; SKBR‑3 cells: DMEM / F‑12, 2 mM L-glutamine, 0.05 % BSA, 10 µg / ml Holo-transferrin) containing 1 ng / ml HRG and incubated overnight at 37°C, 5% CO2, in 95% relative humidity. The next day, 20 µl pH-sensitive dye- labelled antibodies were added to reach a final concentration of 100 nM and cells were incubated overnight at 37°C, 5% CO2, in 95% relative humidity. The next day, cells were harvested by collecting non-adherent cells and trypsinizing adherent cells. After washing cells with FACS buffer (PBS 0.5% BSA 0.1% sodium azide), cells were stained with APC- labelled anti-human IgG (Jackson Immunoresearch, cat.no. 109‑136‑098, 1:100 dilution). Cells were analyzed by flow cytometryonFACSCanto (BDBiosciences)measuringmedianfluorescence intensities (MFI) of thePEandAPCchannels to determine internalization and residual surface binding of antibodies, respectively. Data shown in Figure 35 show that PB4188 internalizes to the same extend as trastuzumab whereas the combination trastuzumab + pertuzumab leads to enhanced internalization. The combination of trastuzumab + pertuzumab reduces the ADCC in comparison to trastu- zumab alone (Figure 36). It is therefore anticipated that the level of PB4188 internalization leaves the ADCC potency unaffected. Generation and characterization of anti-HER3 antibody 3178 variants

[0236] Variants of anti-HER3 antibody MF3178 were designed with the aim to improve antibody properties. Mutations were introduced in the VH gene framework region 1 (FR1), complementarity determining region 1 (CDR1), FR2, CDR2 and / or FR3, while CDR3 and FR4 were not modified. The design included, but was not limited to, mutations that were introduced to remove post-translational modification (PTM) motifs (e.g. by changing the deamidation motif NS to NQ), to reduce surface hydrophobicity (e.g. by changing I to T) or to increase the iso-electric point (pI; e.g. by changingQ to K). All 20 variants (See Figure 37) were expressed as bispecific antibody combinedwith a Tetanus Toxoid (TT) arm and tested in theMCF‑7 functional assay and all 20 variants had a similar potency as theMF3178 antibody in this format. All 20 variants were also tested in this format in FACS in a titration for binding to MCF‑7 and all variants had very similar binding profiles suggesting that the affinities of all variants are similar. Three lead variants MF6058, MF6061 and MF6065 were selected for further experiments that contain ten, threeand sevenaminoacidmutations, respectively (see sequences inFigure 16E andFigure37). Thecorrespondingmonospecific IgG1PG6058,PG6061andPG6065wereproducedandpurifiedat large scale. As shown in Figure 38, the inhibitory activity of the three variants in the HRG-dependent N87 cell line proliferation assay is similar to thatofPG3178.TheCIEX-HPLCprofileof the threevariantswassimilar to thatofPG3178with respect to charge heterogeneity aswell as peakwidth and symmetry, as shown in Figure 39. The retention time (tR) of themain peak correlated roughly with the pI of the antibodies, i.e. higher pI resulted in longer retention time. In the design of bispecific antibodies ormixtures of antibodies, selecting antibody variantswith optimal tR is valuable sincepurification of the desired antibody components using CIEX can be facilitated. Serum titers of the different cohorts of immunized mice as determined by FACS. D=day of antibody titer de- termination. Table 1: response against HER2. Table 2: response against HER3. Cell lines used are indicated (MCF7, SKBR3, BT474). The different mice are in the columns

[0237] 51 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 52 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 53 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 54 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Table 9 Functional activity of lead HER2 × HER3 bispecific antibodies (indicated using the PB prefix; each PB comprises an HER2 arm and an HER3 arm as indicated in the table) compared to comparator antibodies in the HRG dependent MCF‑7 and BxPC3 assays. Based on binding profiles using chimeric constructs HER2 and HER3 antibodies could be separated over different bins. For HER2 antibodies the domains all antibodies except PG2926 could be mapped to domains I, III or IV. Name HER2 arm HER2 domain HER3 arm HER3 bin MCF‑7 BxPC3 IC50 (pM) % Inhibition PB3441 2926 NA 3178 5 51,7 ‑24% PB3443 2930 III 3178 5 136 ‑31% PB3448 1849 IV 3178 5 371 ‑22% PB3565 2973 I 3178 5 30,9 ‑19% PB3566 3004 I 3178 5 7,9 ‑20% PB3567 2971 I 3178 5 46,5 ‑17% PB3709 3025 I 3178 5 34,5 ‑19% PB3710 2916 I 3178 5 74,2 ‑19% PB3883 2971 I 3176 5 113 ‑19% PB3986 3025 I 3163 5 30,7 ‑21% PB3990 2971 I 3163 5 13 ‑18% PB4011 2971 I 3099 3 40,2 ND PB3437 3031 I 3178 5 14 ‑10% PG3178 NA NA 3178 5 139 ‑17% #Ab6 504 ‑7% trastuz. + pertuz. 352 ND trastuzumab 500 ‑3% 55 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Table 10 Monovalent binding affinities of PB4188 and PB3448 for HER2 and HER3 as measured in biacore. Both bispecific antibodies share the same HER3 arm. ND, not done. PB KD on Her2 (nM) KD on Her3 (nM) PB3448 5.4* ND PB4188 0.16* 3.9 Table 11 JIMT‑1 xenograft study treatment groups Gr. N Regimen 1 Agent Vehicle mg / kg Route Schedule 1# 10 PBS X - ip qw k × 4 (start on day 1) 2 10 lapatinib - 150 po qd × 28 (start on day 1) 3 10 PB4188 - 2.5 ip qw k × 4 (start on day 1) 4 10 PB4188 - 25 ip qw k × 4 (start on day 1) 5 10 Pertuzumab + Trastuzumab - 2.5 ip qw k × 4 (start on day 1) 6 10 Pertuzumab + Trastuzumab - 25 ip qw k × 4 (start on day 1) Table 12. Affinities of 125I-labeled IgG HER2xHER3 IgG (PB4188), HER3xTT (PB9215), HER2xTT (PB9216) and Herceptin (monospecific for HER2), as determined using steady state cell affinity measurements with BT‑474 cells and SK- BR‑3 cells. Data were obtained from three independent experiments. BT‑474 SK-BR‑3 Herceptin 3.7 ± 0.5 nM 1.3 ± 0.1 nM PB4188 3.2 ± 0.5 nM 2.0 ± 0.4 nM HER2xTT 3.9 ± 0.6 nM 2.3 ± 0.7 nM HER3xTT 0.23 ± 0.08 nM 0.99 ± 0.4 nM Table 13. The mean binding protein reactivities (and ranges) listed for all critical residues identified. Critical residues involved in PG3958Fab binding were identified as those mutated in clones that were negative for PG3958Fab binding (<35% WT) but positive for the control mAb 1129 binding (>80% WT). Two additional critical residues were identified which did not meet the threshold guidelines, but whose mutation reduced antibody binding by a lesser extent. Residue numbering is that of PDB ID #1S78. HER2 Residue Mutation PG3958 Fab binding % of wt binding (range) Control mAb binding % of wt binding (range) Designation 144 T144A 31.9 (11) 82.1 (13) Critical 166 R166A 32.2 (5) 93.7 (17) Critical 181 R181A 10.1 (5) 98.6 (34) Critical 172 P172A 52.5 (2) 94.9 (24) Secondary 179 G179A 41.7 (18) 87.9 (25) Secondary 56 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Table 14. The mean binding protein reactivities (and ranges) are listed for both critical residues. Critical residues involved in PG3178 binding were identified as those mutated in clones that were negative for PG3178 mAb binding (<20% WT) but positive for the control mAb 66223 binding (>70% WT). Residue numbering is that of PDB ID #4P59. HER3 Residue Mutation PG3178 binding % of wt binding (range) Control mAb binding % of wt binding (range) Designation 409 F409A 16.74 (8) 79.63 (0) Critical 426 R426A 3.17 (5) 93.08 (36) Critical Table 15. List of exposed residues within 11.2 Å radius of Arg 426 in HER3: Leu 423 L423 Tyr 424 Y424 Asn 425 N425 Gly 427 G427 Gly 452 G452 Arg 453 R453 Tyr 455 Y455 Glu 480 E480 Arg 481 R481 Leu 482 L482 Asp 483 D483 Lys 485 K485 References

[0238] Arteaga CL, Sliwkowski MX, Osborne CK, Perez EA, Puglisi F, Gianni L. 2011. Treatment of HER2-positive breast cancer: current status and future perspectives. Nat Rev Clin Oncol. 2011 Nov 29;9(1):16‑32. Balko JM, Miller TW, Morrison MM, Hutchinson K, Young C, Rinehart C, Sánchez V, Jee D, Polyak K, Prat A, Perou CM, Arteaga CL, Cook RS. 2012. The receptor tyrosine kinase ErbB3 maintains the balance between luminal and basal breast epithelium. Proc Natl Acad Sci USA. Jan 3;109(1):221‑6. Baselga J, Cortés J, Kim SB, Im SA, Hegg R, Im YH, Roman L, Pedrini JL, Pienkowski T, Knott A, Clark E, Benyunes MC, Ross G, Swain SM. 2012. Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. N Engl J Med. Jan 12;366(2):109‑19. de Kruif et al. Mol. Biol. (1995), 248, 97‑105 Ewer MS, Ewer SM. Cardiotoxicity of anticancer treatments: What the cardiologist needs to know. Nat Rev Cardiol 2010;7:564‑75 Guarneri Jain KK,Casper ES,Geller NL, et al. A prospective randomized comparison of epirubicin and doxorubicin in patients with advanced breast cancer; J Clin Oncol 1985;3:818‑26 Junttila TT, Akita RW, Parsons K, Fields C, Lewis Phillips GD, Friedman LS, Sampath D, Sliwkowski MX. 2009. Ligand-independent HER2 / HER3 / PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC‑0941. Cancer Cell. May 5;15(5):429‑40. Junttila, T. T., K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70(11): 4481‑4489 57 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 Merchant et al. Nature Biotechnology, Vol. 16 July 1998 pp 677‑681 Nissim A, Hoogenboom HR, Tomlinson IM, Flynn G, Midgley C, Lane D, Winter G. 1994. Antibody fragments from a ’single pot’ phage display library as immunochemical reagents. EMBO J. 1994 Feb 1;13(3):692‑8. Ocana A, Vera-Badillo F, Seruga B, Templeton A, Pandiella A, Amir E. 2013. HER3 overexpression and survival in solid tumors: a meta-analysis. J Natl Cancer Inst. Feb 20;105(4):266‑73. Sergina NV, Rausch M,Wang D, Blair J, Hann B, Shokat KM, Moasser MM. 2007. Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3. Nature. Jan 25;445(7126):437‑41. Schaefer et al. Cancer Cell 20, 472‑486, October 2011 Schoeberl B, FaberAC, LiD, LiangMC,CrosbyK,OnsumM,BurenkovaO,PaceE,WaltonZ,NieL,FulghamA,Song Y,NielsenUB,EngelmanJA,WongKK.2010.AnErbB3antibody,MM‑121, is active in cancerswith ligand-dependent activation. Cancer Res. Mar 15;70(6):2485‑94. Shames et al. PLOS ONE, February 2013, Vol.8, Issue 2, pp 1‑10 Tanner M, Kapanen AI, Junttila T, RaheemO, Grenman S, Elo J, Elenius K, Isola J. 2004. Characterization of a novel cell line established fromapatientwithHerceptin-resistant breast cancer.MolCancer Ther. 2004Dec;3(12):1585‑92. Yarden Y, Pines G.2012. The ERBB network: at last, cancer therapy meets systems biology. Nat Rev CancerJul 12;12(8):553‑63. Thery J.‑C. et al., Resistance to human epidermal growth factor receptor type 2-targeted therapies, Eur J Cancer (2014), Vol. 50, Issue 5, pages 892‑901 Wadhwa D, Fallah-Rad N, Grenier D, et al. Trastuzumab mediated cardiotoxicity in the setting of the adjuvant chemotherapy for breast cancer: A retrospective study. Breast Cancer Res Treat 2009;117:357‑64. Wehrman TS, RaabWJ, Casipit CL, Doyonnas R, Pomerantz JH, Blau HM. 2006. A system for quantifying dynamic protein interactions defines a role for Herceptin in modulating ErbB2 interactions. Proc Natl Acad Sci USA. Dec 12;103(50):19063‑8. Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E, Peng J, Lin E,Wang Y, Sosman J, Ribas A, Li J, Moffat J, Sutherlin DP, Koeppen H, Merchant M, Neve R, Settleman J. 2012. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature. Jul 26;487(7408):505‑9. Yonesaka et al., Sci.transl.Med., Vol.3, Issue 99 (2011); pp1‑11 Zhang H, Berezov A, Wang Q, Zhang G, Drebin J, Murali R, Greene MI. 2007. ErbB receptors: from oncogenes to targeted cancer therapies. J Clin Invest. Aug;117(8):2051‑8. Greco, Bravo, Parsons (1995) The search for synergy: a critical review from a response surface perspective. Pharmacol. Rev 47 (2): 331‑85 Claims 1. A bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein said first antigen-binding site binds domain I of ErbB‑2 and said second antigen-binding site binds domain III of ErbB‑3, wherein said first antigen-binding site binds at least one amino acid of domain I of ErbB‑2 selected from the group consisting of T144, T164, R166, P172, G179, S180 and R181, and surface-exposed amino acid residues that are locatedwithin about 5 amino acid positions fromT144, T164, R166, P172,G179, S180 or R181 and wherein said second antigen-binding site binds at least one amino acid of domain III of ErbB‑3 selected from the group consisting of R426 and surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB‑3 protein. 58 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 2. Abispecific antibody according to claim 1, wherein said antibody comprises an antigen-binding site that binds at least 2orat least 3aminoacid residuesofdomain I ofErbB‑2selected from thegroupconsistingofT144,T164,R166,P172, G179, S180 and R181, and surface-exposed amino acid residues that are located within 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181. 3. A bispecific antibody according to claim 1 or 2, wherein said antibody comprises an antigen-binding site that binds at least T144, R166 and R181 of domain I of ErbB‑2, or binds at least T144, R166, P172, G179 and R181 of domain I of ErbB‑2 or binds at least T144, T164, R166, P172, G179, S180 and R181 of domain I of ErbB‑2. 4. A bispecific antibody according to any one of claims 1‑3, wherein said amino acid residues that are located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181 are selected from the group consisting of L139, C140, Y141, Q142, D143, I145, L146, W147, K148, D149, L159, T160, L161, I162, D163, N165, S167, R168, A169, C170, H171, C173, S174, P175, M176, C177, K178, C182, W183, G184, E185 and S186. 5. A bispecific antibody according to any one of claims 1‑4, wherein the amino acid residue numbering for ErbB‑2 is that of Protein Data Bank (PDB) ID #1S78. 6. Abispecific antibody according to any one of claims 1‑5, wherein said amino acid residues that are locatedwithin 11.2 Å from R426 in the native ErbB‑3 protein are selected from the group consisting of L423, Y424, N425, G427, G452, R453, Y455, E480, R481, L482, D483 and K485. 7. Abispecific antibody according to any oneof claims 1‑6,wherein said antibody comprises an antigen-binding site that binds at least R426 of domain III of ErbB‑3. 8. A bispecific antibody comprising a first antigen-binding site that binds ErbB‑2 and a second antigen-binding site that binds ErbB‑3, wherein the affinity (KD) of said second antigen-binding site for an ErbB‑3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB‑2 positive cell. 9. A bispecific antibody according to claim 8, wherein the affinity (KD) of said second antigen-binding site for an ErbB‑3 positive cell is lower than or equal to 2.0 nM, preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. 10. A bispecific antibody according to claim 8 or 9, wherein the affinity (KD) of said first antigen-binding site for an ErbB‑2 positive cell is lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nMpreferably lower than or equal to 4.0 nM. 11. A bispecific antibody according to any one of claims 8‑10, wherein the affinity (KD) of said bispecific antibody for BT‑474 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 4.0 nM, more preferably lower than or equal to3.2nM,and / orwherein theaffinityof saidbispecificantibody forSK-BR‑3cells is lower thanorequal to5.0nM, preferably lower than or equal to 3.0 nM, more preferably lower than or equal to 2.0 nM. 12. Abispecific antibody according to claims1‑11,which exhibits antibody-dependent cell-mediated cytotoxicity (ADCC). 13. A bispecific antibody according to any one of claims 1‑12, which is afucosylated in order to enhance ADCC. 14. A bispecific antibody according to any one of claims 1‑13, that is a human or humanized antibody. 15. A bispecific antibody according to any one of claims 1‑14, wherein both arms comprise a common light chain. 16. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1‑15. 17. An antibody according to any one of claims 1‑15, for use in the treatment of a subject having or at risk of having an ErbB‑2, ErbB‑3 or ErbB‑2 / ErbB‑3 positive tumor. 59 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 60 EP 4 644 425 A1 61 EP 4 644 425 A1 62 EP 4 644 425 A1 63 EP 4 644 425 A1 64 EP 4 644 425 A1 65 EP 4 644 425 A1 66 EP 4 644 425 A1 67 EP 4 644 425 A1 68 EP 4 644 425 A1 69 EP 4 644 425 A1 70 EP 4 644 425 A1 71 EP 4 644 425 A1 72 EP 4 644 425 A1 73 EP 4 644 425 A1 74 EP 4 644 425 A1 75 EP 4 644 425 A1 76 EP 4 644 425 A1 77 EP 4 644 425 A1 78 EP 4 644 425 A1 79 EP 4 644 425 A1 80 EP 4 644 425 A1 81 EP 4 644 425 A1 82 EP 4 644 425 A1 83 EP 4 644 425 A1 84 EP 4 644 425 A1 85 EP 4 644 425 A1 86 EP 4 644 425 A1 87 EP 4 644 425 A1 88 EP 4 644 425 A1 89 EP 4 644 425 A1 90 EP 4 644 425 A1 91 EP 4 644 425 A1 92 EP 4 644 425 A1 93 EP 4 644 425 A1 94 EP 4 644 425 A1 95 EP 4 644 425 A1 96 EP 4 644 425 A1 97 EP 4 644 425 A1 98 EP 4 644 425 A1 99 EP 4 644 425 A1 100 EP 4 644 425 A1 101 EP 4 644 425 A1 102 EP 4 644 425 A1 103 EP 4 644 425 A1 104 EP 4 644 425 A1 105 EP 4 644 425 A1 106 EP 4 644 425 A1 107 EP 4 644 425 A1 108 EP 4 644 425 A1 109 EP 4 644 425 A1 110 EP 4 644 425 A1 111 EP 4 644 425 A1 112 EP 4 644 425 A1 113 EP 4 644 425 A1 114 EP 4 644 425 A1 115 EP 4 644 425 A1 116 EP 4 644 425 A1 117 EP 4 644 425 A1 118 EP 4 644 425 A1 119 EP 4 644 425 A1 120 EP 4 644 425 A1 121 EP 4 644 425 A1 122 EP 4 644 425 A1 123 EP 4 644 425 A1 124 EP 4 644 425 A1 125 EP 4 644 425 A1 126 EP 4 644 425 A1 127 EP 4 644 425 A1 128 EP 4 644 425 A1 129 EP 4 644 425 A1 130 EP 4 644 425 A1 131 EP 4 644 425 A1 132 EP 4 644 425 A1 133 EP 4 644 425 A1 134 EP 4 644 425 A1 135 EP 4 644 425 A1 136 EP 4 644 425 A1 137 EP 4 644 425 A1 138 EP 4 644 425 A1 139 EP 4 644 425 A1 140 EP 4 644 425 A1 141 EP 4 644 425 A1 142 EP 4 644 425 A1 143 EP 4 644 425 A1 144 EP 4 644 425 A1 145 EP 4 644 425 A1 146 EP 4 644 425 A1 147 EP 4 644 425 A1 148 EP 4 644 425 A1 149 EP 4 644 425 A1 150 EP 4 644 425 A1 151 EP 4 644 425 A1 152 EP 4 644 425 A1 153 EP 4 644 425 A1 154 EP 4 644 425 A1 155 EP 4 644 425 A1 156 EP 4 644 425 A1 157 EP 4 644 425 A1 158 EP 4 644 425 A1 159 EP 4 644 425 A1 160 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 161 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 162 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 163 EP 4 644 425 A1 5 10 15 20 25 30 35 40 45 50 55 164 EP 4 644 425 A1 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader’s convenience only. 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Claims

1. A bispecific antibody comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein said first antigen-binding site binds domain I of ErbB-2 and said second antigen-binding site binds domain III of ErbB-3, wherein said first antigen-binding site binds at least one amino acid of domain I of ErbB-2 selected from the group consisting of T144, T164, R166, P172, G179, S180 and R181, and surface-exposed amino acid residues that are located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181 and wherein said second antigen-binding site binds at least one amino acid of domain III of ErbB-3 selected from the group consisting of R426 and surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB-3 protein.

2. A bispecific antibody according to claim 1, wherein said antibody comprises an antigen-binding site that binds at least 2 or at least 3 amino acid residues of domain I of ErbB-2 selected from the group consisting of T144, T164, R166, P172, G179, S180 and R181, and surface-exposed amino acid residues that are located within 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181.

3. A bispecific antibody according to claim 1 or 2, wherein said antibody comprises an antigen-binding site that binds at least T144, R166 and R181 of domain I of ErbB-2, or binds at least T144, R166, P172, G179 and R181 of domain I of ErbB-2 or binds at least T144, T164, R166, P172, G179, S180 and R181 of domain I of ErbB-2.

4. A bispecific antibody according to any one of claims 1-3, wherein said amino acid residues that are located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181 are selected from the group consisting of L139, C140, Y141, Q142, D143, I145, L146, W147, K148, D149, L159, T160, L161, I162, D163, N165, S167, R168, A169, C170, H171, C173, S174, P175, M176, C177, K178, C182, W183, G184, E185 and S186.

5. A bispecific antibody according to any one of claims 1-4, wherein the amino acid residue numbering for ErbB-2 is that of Protein Data Bank (PDB) ID #1S78.

6. A bispecific antibody according to any one of claims 1-5, wherein said amino acid residues that are located within 11.2 Å from R426 in the native ErbB-3 protein are selected from the group consisting of L423, Y424, N425, G427, G452, R453, Y455, E480, R481, L482, D483 and K485.

7. A bispecific antibody according to any one of claims 1-6, wherein said antibody comprises an antigen-binding site that binds at least R426 of domain III of ErbB-3.

8. A bispecific antibody comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein the affinity (KD) of said second antigen-binding site for an ErbB-3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB-2 positive cell.

9. A bispecific antibody according to claim 8, wherein the affinity (KD) of said second antigen-binding site for an ErbB-3 positive cell is lower than or equal to 2.0 nM, preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM.

10. A bispecific antibody according to claim 8 or 9, wherein the affinity (KD) of said first antigen-binding site for an ErbB-2 positive cell is lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM preferably lower than or equal to 4.0 nM.

11. A bispecific antibody according to any one of claims 8-10, wherein the affinity (KD) of said bispecific antibody for BT-474 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 4.0 nM, more preferably lower than or equal to 3.2 nM, and / or wherein the affinity of said bispecific antibody for SK-BR-3 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 3.0 nM, more preferably lower than or equal to 2.0 nM.

12. A bispecific antibody according to claims 1-11, which exhibits antibody-dependent cell-mediated cytotoxicity (ADCC).

13. A bispecific antibody according to any one of claims 1-12, which is afucosylated in order to enhance ADCC.

14. A bispecific antibody according to any one of claims 1-13, that is a human or humanized antibody.

15. A bispecific antibody according to any one of claims 1-14, wherein both arms comprise a common light chain.

16. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1-15.

17. An antibody according to any one of claims 1-15, for use in the treatment of a subject having or at risk of having an ErbB-2, ErbB-3 or ErbB-2 / ErbB-3 positive tumor.

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