ErbB-2 and ErbB3-conjugated bispecific antibodies for use in the treatment of cells containing the NRG1 fusion gene

Abstract

To provide a technique relating to the field of antibodies, in particular a technique relating to the field of therapeutic (human) antibodies for treatment of ErbB-2 / ErbB-3 positive cells, more in particular treatment of cells including an NRG1 fusion gene including at least a portion of the NRG1-gene fused to a sequence derived from a different chromosomal location.SOLUTION: The present invention provides a bispecific antibody that comprises a first antigen-binding site that can bind to an extracellular part of ErbB-2 and a second antigen-binding site that can bind to an extracellular part of ErbB-3 for use in treatment of an individual that has ErbB-2 and ErbB-3 positive cells, the cell including an NRG1 fusion gene including at least a portion of the NRG1 gene fused to a sequence derived from a different chromosomal location.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims priority from European Patent Application No. 17164292.9 filed on March 31, 2017, the content of which is incorporated herein by reference. Background Art

[0002] The present invention relates to the field of antibodies. In particular, the present invention relates to the field of therapeutic (human) antibodies for the treatment of ErbB-2 / ErbB-3 positive cells. More particularly, the present invention relates to the treatment of tumors comprising an NRG1 fusion gene comprising at least a portion of the NRG1 gene fused to sequences derived from different chromosomal positions.

[0003] Neuregrin-1 (NRG1) has been proposed as a candidate oncogene and candidate tumor suppressor gene. Because it encodes a ligand capable of binding to ErbB family receptors, it is highly likely to be involved in epithelial carcinoma. To date, more than 16 soluble and transmembrane proteins have been identified from the NRG1 gene. Proteolytic processing of the extracellular domain of transmembrane NRG1 isoforms releases soluble factors. HRG1-β1 is one of the proteins encoded by this gene. It contains an Ig domain and an EGF-like domain necessary for direct binding to receptor tyrosine kinases ErbB-3 and ErbB-4. The NRG1 gene and its isoforms are known under several different aliases, including: neuregulin-1; pro-NRG1; HRGA; SMDF; HGL; GGF; NDF; NRG1 intron transcript 2 (non-protein coded); heregulin, alpha (45kD, ERBB2 P185-activator); acetylcholine receptor-inducing activity; pro-neuregulin-1, membrane-bound isoform; sensory and motor neuron-derived factor; Neu differentiation factor; glial cell growth factor 2; NRG1-IT2; MSTP131; MST131; ARIA; GGF2; HRG1; and HRG. The external IDs for the NRG1 gene are HGNC:7997; Entrez Gene:3084; Ensembl:ENSG00000157168; OMIM:142445 and UniProtKB:Q02297.

[0004] NRG1 isoforms are formed by alternative splicing and include transmembrane, outer membrane-bound, shedding, secreted, or intracellular forms (Falls, 2003; Hayes and Gullick, 2008). These bind to ErbB-3 or ErbB-4 and likely signal as heterodimers with ErbB-2 (HER2). While the proteins encoded by NRG1 are typically considered mitogens, they can also be potently pro-apoptotic: in particular, NRG1 expression in cells can induce apoptosis in expressing cells (Weinstein et al., 1998).

[0005] The NRG1 gene has been identified as a gene important for potential cancer in two seemingly contradictory contexts. First, it is a leading candidate for a major tumor suppressor gene thought to be located on chromosome 8p, the short arm of chromosome 8. Loss of chromosome 8p is one of the most frequent genomic events in epithelial cancers, including breast, colon, bladder, and prostate cancers. This has been successively demonstrated by loss of heterozygosity, comparative genomic hybridization (CGH), and array-CGH studies (see Birnbaum et al., 2003; Pole et al., 2006 for references). The classic interpretation of this loss of chromosome 8p is the presence of a tumor suppressor gene there. Loss of chromosome 8p in carcinoma cell lines has been mapped using fluorescence in situ hybridization and array-comparative genomic hybridization (array-CGH). Since most of the cleavage was found to be proximal to or virtually within NRG1, NRG1 and genes immediately telomere-side to NRG1 are primary candidates for such tumor suppressors (Pole et al., 2006; Cooke et al., 2008). Secondly, NRG1 appears to be a target of chromosomal translocations in breast cancer, making it a potential oncogene (see Chua et al., 2009 for a review).

[0006] In this invention, it was found that tumors having chromosome 8p modifications exhibit growth inhibition in response to exposure to a bispecific antibody containing a first antigen-binding site that can bind to the extracellular portion of ErbB-2 and a second antigen-binding site that can bind to the extracellular portion of ErbB-3. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2015 / 130173 [Patent Document 2] U.S. Patent Application Publication No. 20030078385 (Arathoon et al., -Genentech) [License 3] PCT application form: PCT / NL2013 / 050294(WO2013 / 157954A1) [Non-licensed literature]

[0008] [Non-licensed Document 1] SchmidtらNature Methods 4, 1051~1057 (2007) doi:10.1038 / nmeth1103 [Non-licensed Document 2] ChernomoridikらMol Imaging. August 2010; 9(4): 192~200 [Non-licensed Document 3] ArdeshirpourTechnol Cancer Res Treat. October 2014; 13(5): 427~434 [Non-licensed Document 4] van der Woning and van Zoelen Biochem Biophys Res Commun. 2009 Jan 9;378(2):285~9 [Non-licensed Document 5] McDonaghらMol Cancer Ther 2012; 11:582 [Non-licensed Document 6] ShamesらPLOS ONE, February 2013, Volume 8, No. 2, Pages 1~10 [Non-licensed Document 7] Yonesaka, Sci.transl.Med., Volume 3, No. 99 (2011); Pages 1~11 [Non-licensed Document 8] Junttila, TT, K. Parsonsら(2010), "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer", Cancer Research 70(11):4481~4489 [Non-licensed Document 9] Figure 1 from Landgraf, R Breast Cancer Res. 2007; 9(1): 202~ [Overview of the project] [Means for solving the problem]

[0009] In one embodiment, a method is provided for treating an individual having ErbB-2 and ErbB-3 positive cells, comprising the step of administering a bispecific antibody to an individual in need, the antibody having a first antigen-binding site capable of binding to the extracellular portion of ErbB-2 and a second antigen-binding site capable of binding to the extracellular portion of ErbB-3, characterized in that the cells contain an NRG1 fusion gene comprising at least a portion of the NRG1 gene fused to a sequence derived from a different chromosomal location. Typically, the cells contain an NRG1 fusion gene comprising at least the 3' end of the NRG1 gene fused to a 5' sequence derived from a different chromosomal location.

[0010] This cell may be a cancer cell. This cancer cell may be a cancer cell associated with the NRG1 fusion gene, for example, a cancer cell driven by an NRG1 fusion.

[0011] In another embodiment, a method is provided for treating an individual having or at risk of having an ErbB-2 and ErbB-3 positive tumor, comprising the step of administering a bispecific antibody to an individual in need, the antibody having a first antigen-binding site capable of binding to the extracellular portion of ErbB-2 and a second antigen-binding site capable of binding to the extracellular portion of ErbB-3, characterized in that the tumor cells contain a portion of an NRG1 fusion fused to a sequence derived from a different chromosomal location, for example, a 5' sequence, for example, an NRG1 fusion gene containing the 3' end of the NRG1 gene.

[0012] Individuals at risk of having ErbB-2 and ErbB-3 positive tumors may be in remission.

[0013] Preferably, the NRG1 fusion gene expresses a protein comprising the NRG1 EGF-like domain. Preferably, the NRG fusion is a fusion of NRG1 and a gene on human chromosome 8. Preferably, the gene on human chromosome 8 encodes an excretory protein or a cell membrane-associated protein. Preferably, the NRG1 fusion gene is a fusion of the 3' end of the NRG1 gene and the 5' sequence of one gene selected from the group consisting of CD74; DOC4; TNFRSF10B; CLU; VAMP2; SLC3A2; RBPMS; WRN; SDC4; KIF13B; SLECA2; PDE7A; ATP1B1; CDK1; BMPR1B; MCPH1 and RAB2IL1.

[0014] Preferably, the cell is an epithelial cell. Preferably, the cell is a breast cancer cell, an ovarian cancer cell, a lung cancer cell such as non-small cell lung cancer, or a metastasis thereof.

[0015] Preferably, the tumor is of epithelial origin. Preferably, the tumor is breast cancer, ovarian cancer, lung cancer, or a metastasis thereof.

[0016] The cell can be, for example, a cancer cell such as an ovarian cancer cell comprising a CLU-NRG1 fusion or RAB2IL1-NRG1.

[0017] The cell can be, for example, a cancer cell such as a breast cancer cell comprising a DOC4-NRG1 fusion.

[0018] The cell can be, for example, a cancer cell such as NSCLC (lung) cancer of a subtype called invasive mucinous adenocarcinoma, comprising VAMP2-NRG1, RBPMS-NRG1, WRN-NRG1, SDC4-NRG1, SLEC3A2-NRG1, KIF13B-NRG1 or CD74-NRG1.

[0019] Preferably, the individual has undergone (has already received) treatment targeting EGFR inhibition, wherein the treatment preferably uses an EGFR-binding antibody, which is preferably cetuximab.

[0020] Preferably, the method further includes the step of determining the density of ErbB-1 cell surface receptors; ErbB-2 cell surface receptors; ErbB-3 cell surface receptors; ErbB-4 cell surface receptors or a combination thereof on tumor cells. Preferably, the cells or tumor have fewer than 400,000 ErbB-1 cell surface receptors per cell, preferably fewer than 200,000 ErbB-1 cell surface receptors per cell.

[0021] Preferably, this method further includes the step of administering an ErbB-1 inhibitor, preferably cetuximab, to the individual.

[0022] Preferably, in the methods disclosed herein, ErbB-2 / ErbB-3 positive cells or tumors have fewer than 50,000 ErbB-3 cell surface receptors per cell.

[0023] Preferably, in the method disclosed herein, the tumor cell(s) have a higher helegulin expression level than the helegulin expression level of MCF7 cells.

[0024] As will be apparent to those skilled in the art, the bispecific antibodies disclosed herein are also intended for use in the preparation of pharmaceuticals and for use in the treatment of medicines, as disclosed herein.

[0025] In particular, a bispecific antibody for use in treating individuals having ErbB-2 and ErbB-3 positive cells, comprising a first antigen-binding site capable of binding to the extracellular portion of ErbB-2 and a second antigen-binding site capable of binding to the extracellular portion of ErbB-3, wherein the cell contains an NRG1 fusion gene comprising at least the 3' end of an NRG1 gene fused to a 5' sequence derived from a different chromosomal location.

[0026] This cell may be a cancer cell. This cancer cell may be a cancer cell associated with an NRG1 fusion, for example, a cancer cell driven by an NRG1 fusion.

[0027] Furthermore, the bispecific antibody is intended for use in the treatment of ErbB-2 / ErbB-3 positive tumors, where tumor cells contain an NRG1 fusion gene that includes the 3' end of the NRG1 gene fused to the 5' sequence derived from a different chromosomal location.

[0028] Preferably, in the methods and uses disclosed herein, the first antigen-binding site binds to domain I of ErbB-2, and the second antigen-binding site binds to domain III of ErbB-3, and preferably, the affinity of the first antigen-binding site to ErbB-2 is lower than the affinity of the second antigen-binding site to ErbB-3. Preferably, this bispecific antibody is i) comprising at least CDR1, CDR2 and CDR3 sequences of the 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, or this antibody comprises MF2926, The CDR1, CDR2, and CDR3 sequences of MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898, and the CDR sequences containing up to 3 amino acids, preferably up to 2 amino acids, preferably up to 1 amino acid different; and / or ii) comprising at least CDR1, CDR2, and CDR3 sequences of the 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;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073, and MF6074, or this antibody contains MF3 The CDR1, CDR2, and CDR3 sequences of 178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059;MF6060;MF6061;MF6062;MF6063;MF6064;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 or MF6074 contain CDR sequences that differ by a maximum of 3 amino acids, preferably a maximum of 2 amino acids, preferably a maximum of 1 amino acid. Preferably, the antibody is i) comprising an ErbB-2 specific heavy chain variable region sequence selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898, or this The antibodies include 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 heavy chain variable region sequences that differ by up to 15 amino acids; and / or ii) comprising an ErbB-3 specific heavy chain variable region sequence selected from the group consisting of the heavy chain variable region sequences MF3178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059;MF6060;MF6061;MF6062;MF6063;MF6064;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 and MF6074, Alternatively, this antibody contains the heavy chain variable region sequences of MF3178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059;MF6060;MF6061;MF6062;MF6063;MF6064;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 or MF6074, and heavy chain variable region sequences that differ by up to 15 amino acids. Preferably, the antibody comprises at least the CDR1, CDR2, and CDR3 sequences of the ErbB-2 specific heavy chain variable region MF3958, and the antibody comprises at least the CDR1, CDR2, and CDR3 sequences of the ErbB-3 specific heavy chain variable region MF3178. Preferably, the bispecific antibody comprises a "heavy chain for erbB-2 binding" as shown in sequence listing part 1D and a "heavy chain for erbB-3 binding" as shown in sequence listing part 1D.

[0029] Preferably, the first antigen-binding site and this second antigen-binding site include a light chain variable region containing the IgVKl-39 gene segment, most preferably a rearranged germline human kappa light chain IgVKl-39*01 / IGJKl*01 or IgVκ1-39*01 / IGJκ5*01. Preferably, the light chain variable region includes CDR1 having the sequence (RASQSISSYLN), CDR2 having the sequence (AASSLQS), and CDR3 having the sequence (QQSYSTPPT). [Modes for carrying out the invention]

[0030] An NRG1 fusion gene comprises at least a portion of the NRG1 gene fused to a sequence derived from a different chromosomal location. “At least a portion” indicates that the entire NRG-1 gene or a portion thereof may be present in the fusion. The fusion preferably has at least the coding sequences of exons 6, 7, and 8. Another way to define the NRG1 portion in an NRG1 fusion gene is that it contains the EGF-like domain of NRG1. At least a portion of the NRG1 gene may be fused to its sequence such that the sequence derived from a different chromosomal location is located 5' or 3' relative to at least a portion of the NRG1 gene.

[0031] Preferably, the 3' end of the NRG1 gene may be fused to a 5' sequence derived from a different chromosomal location. The NRG1 gene encodes various isoforms of NRG1. The various isoforms and their predicted functions are described by Adelaide et al. (2003). The GGF and GGF2 isoforms contain a kringle-like sequence + Ig and EGF-like domains; the SMDF isoform shares only the EGF-like domain with the other isoforms. The EGF-like domain is encoded by the 3' end of its gene. The EGF-like domain is present in all NRG1 fusion genes of the present invention. Fusions have been found in which the 5' from a different chromosomal location, along with at least one extracellular domain, contains a cell membrane protein efflux signal and / or transmembrane domain. One example is the CD74-NRG1 fusion. The 5' sequence from a different chromosomal location may insert a sequence that activates NRG1 transcription, for example, a promoter or enhancer. The 5' sequence is typically a sequence derived from a gene other than NRG1. This sequence may include a coding region, an expression regulatory sequence, such as a promoter or enhancer, or a combination thereof. The NRG fusion includes a 5' sequence of different origins, which may be from a different chromosome or another part of chromosome 8. In a preferred embodiment, the 5' sequence is derived from a gene on human chromosome 8.

[0032] The 3' end of the NRG1 gene in the fusion, for example, the NRG-1 gene, preferably has at least the coding sequence of exons 6, 7, and 8. Another way to define the NRG1 portion in the NRG1 fusion gene is that it contains the EGF-like domain of NRG1. This domain is encoded by the 3' end (exons 6-8) of the NRG1 gene and is required for binding to ErbB-3. The NRG1 fusion holds the coding region of this EGF-like domain in-frame at the 3' end of the fusion. The EGF-like domain is typically a sequence about 30-40 amino acid residues long, and its prototype is found in the sequence of epidermal growth factor (EGF) [PMID:2288911, PMID:6334307, PMID:1522591, PMID:6607417, PMID:3282918, PMID:11498013]. This is known to exist in many other, mostly animal, proteins, in more or less conserved forms. A common feature of EGF-like domains is that they are found in the extracellular domains of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G / H synthase). EGF domains typically contain six cysteine ​​residues (in EGF) that have been shown to be involved in disulfide bonding. Their main structure is a double-stranded beta-sheet followed by a loop to a short C-terminal double-stranded sheet. Conserved cysteine-intervening subdomains vary in length.

[0033] The NRG1 fusion gene is preferably a fusion of the 3' end of the NRG1 gene and the 5' sequence of one gene selected from the group consisting of CD74;DOC4;TNFRSF10B;CLU;VAMP2;SLC3A2;RBPMS;WRN;SDC4;KIF13B;SLECA2;PDE7A;ATP1B1;CDK1;BMPR1B;MCPH1 and RAB2IL1.

[0034] An NRG1 fusion gene may be a fusion of at least a portion of the NRG1 gene with a sequence derived from a different chromosomal location located 3' to it. Such an NRG1 fusion gene may be a fusion of at least a portion of the NRG1 gene with a sequence derived from CD74, STMN2, PMEPA1, PROSC, or PSAP, a different chromosomal location located 3' to it. The receptors for all NRG1 isoforms are tyrosine kinase transmembrane receptors of the ErbB family. This family is also called the human epidermal growth factor (EGF) receptor family (HER). This family has four members: ErbB(Erythroblastoma)-1, ErbB-2, ErbB-3, and ErbB-4. These are epidermal growth factor (EGF) receptors (EGFR, ErbB1, or HER1). These receptors (described in Yarden and Pines, 2012) are widely expressed on epithelial cells. Upregulation of HER receptors or their ligands, such as heregulin (HRG) or epidermal growth factor (EGF), is a frequent event in human cancers (Wilson, Fridlyand et al., 2012). In particular, overexpression of ErbB-1 and ErbB-2 occurs in epithelial tumors and is associated with tumor invasion, metastasis, resistance to chemotherapy, and poor prognosis (Zhang, Berezov et al., 2007). In normal breast tissue, ErbB-3 has been shown to be important in the growth and differentiation of luminal epithelium. For example, loss / inhibition of ErbB-3 results in a selective increase in basal tissue on luminal epithelium (Balko, Miller et al., 2012). Ligand binding to the extracellular domain of RTKs induces receptor dimerization both between the same (homodimerizing) receptor subtypes and between different (heterodimizing) receptor subtypes. Dimerization can activate autophosphorylated intracellular tyrosine kinase domains, which in turn can activate several downstream proliferative signaling pathways, including those mediated by mitogen-activated protein kinases (MAPKs) and the proliferative pathway Akt (as outlined in Yarden and Pines, 2012).Specific endogenous ligands have not been identified for ErbB-2, and therefore it is generally assumed that it signals via heterodimerization (Sergina, Rausch et al., 2007). ErbB-3 can be activated by association with its ligands. These ligands include, but are not limited to, neuregulin (NRG) and heregulin (HRG).

[0035] ErbB-1 is known under various synonyms, the most common being EGFR. EGFR has an extracellular domain (ECD) consisting of four subdomains, two of which are involved in ligand binding, and two of which are involved in homodimerization and heterodimerization. EGFR integrates extracellular signals from various ligands to produce diverse intracellular responses. The major signaling pathway activated by EGFR consists of the Ras-mitogen-activated protein kinase (MAPK) mitotic signaling cascade. Activation of this pathway is initiated by the recruitment of Grb2 to tyrosine-phosphorylated EGFR. This results in Ras activation via Son of Sevenless (SOS), a Grb2-bound Ras-guanine nucleotide exchange factor. Furthermore, the PI3-kinase-Akt signaling pathway is also activated by EGFR, but this activation is considerably stronger in the presence of ErbB-3 (HER3) co-expression. EGFR is associated with several human epithelial malignancies, particularly breast, bladder, non-small cell lung cancer, colon, ovarian, head and neck, and brain cancers. Activating mutations in this gene, as well as overexpression of its receptor and ligand, have been found, resulting in an autocrine activation loop. Therefore, this RTK is widely used as a target for cancer treatment. Small molecule inhibitors targeting the RTK and monoclonal antibodies (mAbs) against the extracellular ligand-binding domain have both been developed and have shown some clinical success, mostly in selected patient populations. The database registry number for the human EGFR protein and the gene encoding it is (GenBank NM_005228.3). This registry number is primarily provided to offer further methods for identifying the EGFR protein as a target, as the actual sequence of the EGFR protein to which the antibody binds may vary due to mutations in the coding gene, such as those present in some cancers.

[0036] Unless specifically noted, the terms cancer and tumor are used herein to refer typically to both cancer.

[0037] Where EGFR is mentioned herein, unless otherwise specified, this refers to human EGFR. The antigen-binding site that binds to EGFR binds to EGFR and its various variants, such as those expressed on certain EGFR-positive tumors.

[0038] As used herein, the term "ErbB-3" refers to the protein encoded by the ERBB3 gene in humans. Alternative names for this gene or protein include HER3;LCCS2;MDA-BF-1;c-ErbB-3;c-ErbB3;ErbB3-S;p180-ErbB3;p45-sErbB3; and p85-sErbB3. When ErbB-3 is referred to herein, this refers to human ErbB-3. Antibodies containing an antigen-binding site that binds to ErbB-3 bind to human ErbB-3. The ErbB-3 antigen-binding site may, but not necessarily, bind to other mammalian orthologues due to the similarity of their sequence and tertiary structure between human orthologues and other mammalian orthologues. The database registration 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). These registration numbers are primarily provided to offer further methods for identifying ErbB-3 as a target, and the actual sequence of the ErbB-3 protein to which the antibody binds may vary due to mutations in the coding gene, such as those present in some cancers. The ErbB-3 antigen-binding site binds to ErbB-3 and its various variants, such as those expressed by some ErbB-3-positive tumor cells. The antigen-binding site that binds to ErbB-3 preferably binds to domain III of ErbB-3.

[0039] The term "ErbB-2," as used herein, refers to the protein encoded by the ERBB-2 gene in humans. Alternative names for this gene or protein include CD340;HER-2;HER-2 / neu;MLN 19;NEU;NGL;TKR1. The ERBB-2 gene is often referred to as HER2 (from human epidermal growth factor receptor 2). When ErbB-2 is referred to herein, this refers to human ErbB-2. Antibodies containing an antigen-binding site that binds to ErbB-2 bind to human ErbB-2. The ErbB-2 antigen-binding site may also bind to other mammalian orthologues, but not necessarily, due to the similarity of sequence and tertiary structure between human orthologues and other mammalian orthologues. The database registration 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). These registration numbers are primarily provided to offer further methods for identifying ErbB-2 as a target, and the actual sequence of the ErbB-2 protein to which the antibody binds may vary due to mutations in the coding gene, such as those present in some cancers. The ErbB-2 antigen-binding site binds to ErbB-2 and its various variants, such as those expressed by some ErbB-2-positive tumor cells. The antigen-binding site that binds to ErbB-2 preferably binds to domain I of ErbB-2.

[0040] CD74 is known under several aliases. Some of these are the CD74 molecule; CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CD74 molecule, major histocompatibility complex, class II invariant chain; HLA-DR antigen-associated invariant chain; gamma chain of class II antigen; Ia-associated invariant chain; MHC HLA-DR gamma chain; HLA-DR-gamma; DHLAG; P33; HLA class II histocompatibility antigen gamma chain; Ia antigen-associated invariant chain; Ia-GAMMA and HLADG. The external IDs for CD74 are HGNC:1697; Entrez Gene:972; Ensembl:ENSG00000019582; OMIM:142790 and UniProtKB:P04233.

[0041] DOC4, or teneurin transmembrane protein 4 (TENM4), is known under several different names, such as protein Odd Oz / Ten-M homolog 4; tenascin-M4; Ten-M4; Ten-4; ODZ4; TNM4; Odz, Odd Oz / Ten-M homolog 4 (Drosophila); Odz, Odd Oz / Ten-M homolog 4; teneurin-4; KIAA1302; Doc4; and ETM5. The external IDs for DOC4 are HGNC:29945; Entrez Gene:26011; Ensembl:ENSG00000149256; OMIM:610084 and UniProtKB:Q6N022.

[0042] TNFRSF10B, or TNF receptor superfamily member 10b, is known under several different names: Tumor Necrosis Factor Receptor Superfamily, Member 10b; TNF-associated Apoptosis-Inducing Ligand Receptor 2; Death Receptor 5; TRAIL-R2; TRAILR2; KILLER; TRICK2; ZTNFR9; DR5; P53-regulated DNA damage-inducible cell death receptor (Killer); Tumor Necrosis Factor Receptor Superfamily Member 10B; Tumor Necrosis Factor Receptor-like Protein ZTNFR9; Death Domain-containing Receptor for TRAIL / Apo-2L; Apoptosis-Inducing Protein TRICK2A / 2B; Apoptosis-Inducing Receptor TRAIL-R2; Cytotoxic TRAIL Receptor-2; Fas-like Protein; TRAIL Receptor 2; CD262 Antigen; KILLER / DR5; TRICK2A; TRICK2B; TRICKB; and CD262. The external IDs for TNFRSF10B are HGNC:11905;Entrez Gene:8795;Ensembl:ENSG00000120889;OMIM:603612; and UniProtKB:O14763.

[0043] The CLU gene, or clusterin, is known under several different names, such as testosterone-suppressive prostate message 2; apolipoprotein J; complement-related proteins SP-40,40; complement cell lysis inhibitor; complement lysis inhibitor; sulfated glycoprotein 2; Ku70-binding protein 1; NA1 / NA2; TRPM-2; APO-J; APOJ; KUB1; CLI; clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-suppressive prostate message 2, apolipoprotein J); aging-related gene 4 protein; aging-related protein 4; SGP-2; SP-40; TRPM2; AAG4; CLU1; CLU2; and SGP2. The external IDs for the CLU are HGNC:2095;Entrez Gene:1191;Ensembl:ENSG00000120885;OMIM:185430; and UniProtKB:P10909.

[0044] VAMP2, or vesicle-binding membrane protein 2, is known under several different names, e.g., synaptobrevin 2; SYB2; vesicle-binding membrane protein 2; and synaptobrevin-2. The external IDs for VAMP2 are HGNC:12643; Entrez Gene:6844; Ensembl:ENSG00000220205; OMIM:185881; and UniProtKB:P63027.

[0045] SLCA3A2, or solute transporter family 3 member 2, is known under several different names, e.g., lymphocyte-activating antigen 4F2 large subunit; solute transporter family 3 (activators of dibasic and neutral amino acid transport), member 2; antigen identified by monoclonal antibody 4F2, TRA1.10, TROP4 and T43; solute transporter family 3 (amino acid transporter heavy chain), member 2; 4F2 cell surface antigen heavy chain; CD98 heavy chain; 4F2HC; MDU1; antigen defined by monoclonal antibody 4F2, heavy chain; antigen defined by monoclonal antibody 4F2; 4F2 heavy chain antigen; 4F2 heavy chain; CD98 antigen; CD98HC; 4T2HC; NACAE; CD98 and 4F2. The external IDs for SLC3A2 are HGNC:11026;Entrez Gene:6520;Ensembl:ENSG00000168003;OMIM:158070; and UniProtKB:P08195.

[0046] RBPMS, or RNA-binding protein with multisplicing, is known by several different names, e.g., RNA-binding protein with multisplicing; cardiac and RRM expression sequence; HERMES; RNA-binding protein with multisplicing; and RBP-MS. The external IDs for RBPMS are HGNC:19097; Entrez Gene:11030; Ensembl:ENSG00000157110; OMIM:601558; and UniProtKB:Q93062.

[0047] WRN, or Werner syndrome RecQ-like helicase, is known under several different names, e.g., Werner syndrome RecQ-like helicase; DNA helicase, RecQ-like type 3; RecQ protein-like 2; exonuclease WRN; RECQL2; RECQ3; Werner syndrome ATP-dependent helicase; Werner syndrome, RecQ helicase-like; Werner syndrome; EC 3.6.4.12; EC 3.1.-.-; EC 3.6.1; and RECQL3. The external IDs for WRN are HGNC:12791; Entrez Gene:7486; Ensembl:ENSG00000165392; OMIM:604611 and UniProtKB:Q14191.

[0048] SDC4, or syndecan 4, is known under several different names, e.g., syndecan 4 (amphiglycan, ryudocan); syndecan proteoglycan 4; ryudocan core protein; amphiglycan; SYND4; ryudocan amphiglycan; and syndecan-4. The external IDs for SDC4 are HGNC:10661; Entrez Gene:6385; Ensembl:ENSG00000124145; OMIM:600017; and UniProtKB:P31431.

[0049] Various NRG1 fusion genes are described by Dhanasekaran et al. (2014).

[0050] The present invention provides a method for treating individuals having ErbB-2 and ErbB-3 positive cells or tumors, or individuals who may be at risk of having tumors. The method comprises administering a bispecific antibody to an individual in need, the antibody comprising a first antigen-binding site capable of binding to the extracellular portion of ErbB-2 and a second antigen-binding site capable of binding to the extracellular portion of ErbB-3. The method is characterized in that the tumor cell(s) contain an NRG1 fusion gene comprising the 3' end of an NRG1 gene fused to a 5' sequence derived from a different chromosomal location.

[0051] The cells may be cancer cells. These cancer cells may be cancer cells associated with NRG1 fusions, for example, cancer cells driven by NRG1 fusions.

[0052] The antigen-binding site in an antibody is typically located within the variable domain. The variable domain includes both the heavy chain variable region and the light chain variable region.

[0053] Preferably, the individual has experienced EGFR-targeting therapy, preferably using an EGFR-binding antibody, preferably cetuximab.

[0054] The treatment method of the present invention preferably further comprises the step of determining the number of ErbB-1 cell surface receptors; ErbB-2 cell surface receptors; ErbB-3 cell surface receptors; ErbB-4 cell surface receptors or combinations thereof on the tumor cell(s).

[0055] The treatment method of the present invention preferably further comprises the step of determining whether cells contain an NRG1 fusion or whether a tumor contains cells having an NRG1 fusion. This can be performed, for example, on cells from a biopsy. Various methods are available, many of which are known in the art. In the case of NRG1 fusions, since the region where chromosomal breaks occur is known, determining whether a tumor contains such an NRG1 fusion is customary to those skilled in the art. One method is by PCR amplification using primers spanning the junction in the NRG1 fusion. This can be readily performed for NRG1 fusions whose existence is known. New fusions can also be readily detected. For example, a suitable method is by junction cloning techniques, for example, used to find integration sites in retroviral genomes. A suitable method is by LAM-PCR. See Schmidt et al. Nature Methods 4, 1051~1057 (2007) doi:10.1038 / nmeth1103 and the specific reference thereto to LAM technology.

[0056] The treatment method of the present invention is preferably characterized in that the cells or tumor have fewer than 400,000 ErbB-1 cell surface receptors per cell, and more preferably fewer than 200,000 ErbB-1 cell surface receptors per cell.

[0057] In a preferred embodiment, the treatment method of the present invention further includes the step of administering an ErbB-1 inhibitor, preferably cetuximab, to an individual.

[0058] The treatment methods defined herein may also be defined as compounds or combinations of compounds for use in treatment. Suitable combinations of compounds are the bispecific antibodies and ErbB-1 inhibitors defined herein.

[0059] To determine whether cells or tumors are positive for ErbB-2 and ErbB-3, a person skilled in the art can, for example, determine the staining in amplification and / or immunohistochemistry for ErbB-2 and ErbB-3. At least 10% of tumor cells in a biopsy should be positive for both ErbB-2 and ErbB-3. The biopsy may also contain 20%, 30%, 40%, 50%, 60%, 70%, or more positive cells. ErbB-1 positive tumors can be identified in a similar manner.

[0060] Preferably, this positive cancer is breast cancer, for example, early breast cancer. However, the present invention can be applied to a wide range of ErbB-2, ErbB-3, or ErbB-2 / ErbB-3 positive cancers, such as gastric cancer, colorectal cancer, colon cancer, gastroesophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, breast 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, melanoma, etc. The cells are preferably epithelial cells. Alternatively, the cells or tumors are preferably cells or tumors of epithelial origin. In preferred embodiments, the cells or tumors are breast cancer, ovarian cancer, lung cancer, or metastases thereof. Preferably, the tumors are of epithelial origin. Preferably, the tumors are breast cancer, ovarian cancer, lung cancer, or metastases thereof.

[0061] Patients with ErbB2-positive cells or tumor cells can be classified based on the number of ErbB-2 receptors on the surface of their tumor cells. Tumors with more than 1,000,000 ErbB-2 receptors on their cell surface are typically classified as ErbB-2[+++], tumors with between 150,000 and 1,000,000 ErbB-2 receptors are classified as ErbB-2[++], and tumors with fewer than 150,000 ErbB-2 receptors are classified as ErbB-2[+]. Preferably, patients are classified as ErbB-2[++] or ErbB-2[+++]. Preferably, ErbB-2 / ErbB-3 positive tumors have at least 1,000,000 ErbB-2 cell surface receptors per cell.

[0062] Preferably, a method is provided in which ErbB-2 / ErbB-3 positive cells or tumors have at least 150,000 ErbB-2 cell surface receptors and less than 50,000 ErbB-3 cell surface receptors per cell. Preferably, a method is provided in which ErbB-2 / ErbB-3 positive cells or tumors have at least 100,000 ErbB-2 cell surface receptors and less than 50,000 ErbB-3 cell surface receptors per cell.

[0063] In some embodiments, the methods disclosed herein are advantageous in that a particular patient population is initially determined based, for example, on the cell surface receptor density of ErbB-1, ErbB-2, and / or ErbB-3. Accordingly, the methods disclosed herein preferably include the step of determining the ErbB-1 cell surface receptor density, ErbB-2 cell surface receptor density, ErbB-3 cell surface receptor density, and / or ErbB-4 cell surface receptor density for the cells or tumor. As used herein, the term cell surface receptor density refers to the number of receptors present on the cell surface of a single cell.

[0064] Preferably, the methods disclosed herein further include the step of determining the ErbB-2 cell surface receptor density for the cells or tumor. Patients may be classified using immunohistochemistry or fluorescence in situ hybridization. Using HercepTest® and / or HER2 FISH (pharm Dx®), both commercially available from Dako Denmark A / S, and / or the HERmark® assay, commercially available from Monogram Biosciences, are examples of suitable assays for determining ErbB-2 or ErbB-3 cell surface receptor density. Other methods for determining ErbB-2 receptor cell density are well known to those skilled in the art. In vivo methods for determining ErbB-2 are also known. See, for example, Chernomoridik et al. Mol Imaging. August 2010; 9(4): 192-200 and Ardeshirpour et al. Technol Cancer Res Treat. October 2014; 13(5): 427-434. Preferably, the methods disclosed herein further include the step of determining the ErbB-2 cell surface receptor density for the cells or tumor. Such methods are known to those skilled in the art (see, for example, van der Woning and van Zoelen Biochem Biophys Res Commun. January 9, 2009; 378(2):285-9). Preferably, the methods disclosed herein further include the step of determining the ErbB-1 cell surface receptor density for the cells or tumor. Such methods are known to those skilled in the art (see, for example, EGFR pharmDx(trademark) Kit(Dako)) and McDonagh et al. Mol Cancer Ther 2012; 11:582). Similar methods can be used to determine the ErbB-4 cell surface receptor density.

[0065] In some embodiments, the density of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 cell surface receptors is determined by FACS analysis of biopsy tumor cells.

[0066] Preferably, ErbB-2 / ErbB-3 positive cells or tumor cells have relatively high levels of heregulin expression. Heregulin is a growth factor involved in the growth of ErbB3 positive cells or tumor cells. Typically, when cells or tumor cells express high levels of heregulin (called heregulin stress), currently known treatments such as trastuzumab, pertuzumab, and lapatinib are no longer able to inhibit the growth of the cells or tumor. This phenomenon is called heregulin resistance. In particular, the heregulin expression level is higher than the heregulin expression level of MCF7 cells. Hereglin expression levels are measured, for example, using qPCR with cells or tumor RNA (as described in Shames et al., PLOS ONE, February 2013, Vol. 8, No. 2, pp. 1-10 and Yonesaka et al., Sci.transl.Med., Vol. 3, No. 99 (2011); pp. 1-11, etc.), or using protein detection methods such as ELISA, preferably using blood, plasma, or serum samples (as described in Yonesaka et al., Sci.transl.Med., Vol. 3, No. 99 (2011); pp. 1-11, etc.).

[0067] High heregulin levels are typically present during metastasis formation (i.e., migration, invasion, growth, and / or differentiation of cells, tumor cells, or tumor-initiating cells). Typically, tumor-initiating cells are identified based on stem cell markers such as CD44, CD24, CD133, and / or ALDH1. These processes can therefore only be adequately counteracted by currently known treatments such as trastuzumab and pertuzumab. The bispecific antibodies disclosed herein are capable of counteracting metastasis formation in subjects with cell tumors containing an NRG1 fusion gene, which includes the 3' end of the NRG1 gene fused to a 5' sequence derived from a different chromosomal location.

[0068] Accordingly, a method is provided for combating the formation of metastasis in a subject having ErbB-2, ErbB-3, or ErbB-2 / ErbB-3 positive cells or tumor, comprising the step of administering a bispecific antibody to the subject having a first antigen-binding site that binds to ErbB-2 and a second antigen-binding site that binds to ErbB-3, wherein the ErbB-2, ErbB-3, or ErbB-2 / ErbB-3 positive cells or tumor cells have a heregulin expression level of 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. Also provided is a bispecific antibody for use in the treatment or prevention of metastasis formation, comprising a first antigen-binding site that binds to ErbB-2 and a second antigen-binding site that binds to ErbB-3, wherein the ErbB-2, ErbB-3, or ErbB-2 / ErbB-3 positive cells or tumor cells have a heregulin expression level of 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, the invention provides for the use of a bispecific antibody according to the present invention for the preparation of a pharmacopoeia for the treatment or prevention of metastasis formation, wherein the ErbB-2, ErbB-3, or ErbB-2 / ErbB-3 positive cells or tumor cells have a heregulin expression level of 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.

[0069] The subjects are preferably human subjects. The subjects are preferably subjects who are eligible for monoclonal antibody therapy using an ErbB-2 specific antibody such as trastuzumab.

[0070] The amount of bispecific antibody administered to a patient is typically within the therapeutic window, meaning that a sufficient amount is used to achieve a therapeutic effect, but that the amount does not exceed a threshold that would result in unacceptable side effects. The lower the amount of antibody required to achieve the desired therapeutic effect, the larger the therapeutic window typically becomes. The chosen dosage level depends on various factors, including the route of administration, timing of administration, the rate of elimination of the specific compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination, the age, sex, weight, condition, overall health, and prior medical history of the treated patient, as well as similar factors well known in the medical field. The dosage may be within or lower than the range of the dosage regime for trastuzumab.

[0071] Bispecific antibodies can be formulated as pharmaceutical compositions comprising pharmaceutically acceptable carriers, diluents or excipients, and further optional active agents. Antibodies and antibody-containing compositions can be administered by any route, including parenteral, enteral, and topical administration. Parenteral administration is usually by injection, which includes, for example, intravenous, intramuscular, intra-arterial, subarachnoid, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebral, intratumoral, and intrasternal injections and infusions.

[0072] In preferred embodiments, the ErbB-1 inhibitor can be combined with treatment with the bispecific antibodies disclosed herein. The ErbB-1 inhibitor can be administered simultaneously with or sequentially with the bispecific antibodies. Treatment with the ErbB-1 inhibitor can be isolated from treatment with the bispecific antibodies for several minutes, several hours, or several days. Preferably, the ErbB-2 / ErbB3 cells or tumors are also positive for ErbB1. Preferably, the combination treatment (combination therapy) is suitable for ErbB-2 / ErbB3 cells or tumors having more than 5,000 surface receptors per cell, preferably at least 20,000 surface receptors per cell, and more preferably more than 50,000 surface receptors per cell.

[0073] A suitable ErbB-1 inhibitor is a compound known in the art that inhibits at least one biological activity of ErbB-1 (EGFR), particularly a compound that reduces ErbB-1 expression or signaling activity. Preferred ErbB-1 inhibitors bind to the extracellular binding site of this tyrosine kinase receptor molecule and block the binding of intrinsic ligands such as EGF. Such inhibitors include antibodies, antibody moieties, and peptides containing epitopes that target this extracellular EGF receptor binding domain. Preferably, the ErbB-1 inhibitor is an anti-ErbB-1 antibody selected from cetuximab, matsuzumab, nesitumumab, nimotuzumab, panitumumab, or zaltumumab. The present invention further relates to ErbB-1 inhibitors that can bind to or interact with the intracellular phosphorylation site or domain of the tyrosine kinase receptor molecule, thereby preventing or reducing phosphorylation by tyrosine kinase. This can be achieved by small (chemical) molecule drugs. Preferred inhibitors include afatinib, erlotinib, gefitinib, lapatinib, osimertinib, and neratinib.

[0074] This disclosure provides bispecific antibodies for use in the methods and procedures described herein. A suitable bispecific antibody comprises a first antigen-binding site that binds to ErbB-2 and a second antigen-binding site that binds to ErbB-3, and this bispecific antibody can reduce or reduce the ligand-inducible receptor function of ErbB-3 on ErbB-2 and ErbB-3 positive cells. Preferred antibodies and their preparations are disclosed in WO2015 / 130173, which is incorporated herein by reference. Examples in WO2015 / 130173 further describe some of the antibody's properties, e.g., ligand binding and epitope mapping.

[0075] As used herein, the term “antigen-binding site” refers to a site derived from a bispecific antibody capable of binding to an antigen, preferably a site located on such a bispecific antibody. An unmodified antigen-binding site is typically formed by and located within the variable domain of an antibody. The variable domain includes the antigen-binding site. An antigen-binding variable domain is a variable domain that includes an antigen-binding site that binds to an antigen.

[0076] In one embodiment, the antibody variable domain includes a heavy chain variable region (VH) and a light chain variable region (VL). The antigen-binding site may be present in the combined VH / VL variable domain, or only in the VH region, or only in the VL region. If the antigen-binding site is present in only one of the two regions of the variable domain, the corresponding variable region may contribute to the folding and / or stability of the binding variable region, but not significantly to the binding of the antigen itself.

[0077] As used herein, antigen binding refers to the typical binding ability of an antibody to that antigen. An antibody containing an antigen-binding site that binds to ErbB-2 will bind to ErbB-2 and, under otherwise identical conditions, to the homologous receptors of the same species, ErbB-1 and ErbB-4, at least 1 / 100th less. An antibody containing an antigen-binding site that binds to ErbB-3 will bind to ErbB-3 and, under otherwise identical conditions, will not bind to the homologous receptors of the same species, ErbB-1 and ErbB-4. Given that the ErbB family is a family of cell surface receptors, binding is typically evaluated on cells expressing the receptors. Antibody binding to an antigen can be evaluated in various ways. One method is to incubate the antibody with the antigen (preferably cells expressing the antigen), remove the unbound antibody (preferably by a washing step), and detect the bound antibody with a labeled antibody that binds to the bound antibody.

[0078] Antibody-antigen binding is typically mediated through the complementary region of the antibody and the specific three-dimensional structure of both the antigen and variable domains, which precisely bind these two structures together, as opposed to random, nonspecific attachment of the antibody (a lock-and-key-like interaction). Antibodies typically recognize the epitope of an antigen, and since such epitopes may also be present in other compounds, antibodies according to the present invention that bind to ErbB-2 and / or ErbB-3 may also recognize other proteins if such other compounds contain the same epitope. Thus, the term “binding” does not exclude the binding of antibodies to other proteins or proteins containing the same epitope. Such other proteins are preferably not human proteins. The ErbB-2 and ErbB-3 antigen-binding sites as defined herein typically do not bind to other proteins on the cell membrane in postnatal, preferably adult, humans. The bispecific antibodies disclosed herein are typically capable of binding to ErbB-2 and ErbB-3 with a binding affinity of at least 1 × 10 e-6 M, as outlined in more detail below.

[0079] The term "interfering with binding," as used herein, means that the antibody is against an epitope on ErbB-3 and competes with the ligand for binding to ErbB-3. The antibody may reduce ligand binding, replace the ligand if it is already bound to ErbB-3, or at least partially prevent the ligand from binding to ErbB-3, for example, through steric hindrance.

[0080] The term “antibody,” as used herein, means a proteinaceous molecule, preferably belonging to the immunoglobulin class of proteins, which include one or more variable domains that bind to an epitope on an antigen, where such domains are derived from the variable domain of the antibody or share sequence homology with such variable domains. Antibodies for therapeutic use are preferably as close as possible to the natural antibody of the target being treated (e.g., a human antibody against a human target). Antibody binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by the binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. Specific binding is defined as binding at an affinity (KD) of at least 1 × 10⁻⁶ M, more preferably 1 × 10⁻⁷ M, and more preferably higher than 1 × 10⁻⁹ M. Typically, antibodies for therapeutic application have an affinity of up to 1 × 10⁻¹⁰ M or higher. Antibodies such as the bispecific antibodies of the present invention include the constant domain (Fc portion) of a natural antibody. The antibodies of the present invention are typically, preferably, bispecific, full-length antibodies of a human IgG subclass. Preferably, the antibodies disclosed herein are antibodies of a human IgG1 subclass. Such antibodies have good ADCC properties, a favorable half-life upon in vivo administration to humans, and there is a CH3 manipulation technology that can provide a modified heavy chain that preferentially forms heterodimers rather than homodimers upon co-expression in clonal cells.

[0081] The antibodies of the invention disclosed herein are preferably “full-length” antibodies. The term “full-length” is defined as including essentially complete antibodies, but such antibodies do not need to possess all the functions of an intact antibody. To avoid misunderstanding, full-length antibodies include two heavy chains and two light chains. Each chain includes a constant (C) region and a variable (V) region, which can be broken down into domains referred to as CH1, CH2, CH3, VH, and CL, VL. The antibody binds to an antigen via the variable domain contained in the Fab portion, and after binding, can interact with molecules and cells of the immune system via the constant domain, mainly via the Fc portion. The terms “variable domain,” “VH / VL pair,” and “VH / VL” are used interchangeably herein. Full-length antibodies according to the present invention include antibodies in which mutations may exist that provide desired characteristics. Such mutations should not be deletions of substantial portions of any of these regions. However, antibodies in which one or more amino acid residues are deleted without essentially altering the binding characteristics of the resulting antibody are included within the term “full-length antibody.” For example, IgG antibodies may have 1 to 20 amino acid insertions, deletions, or combinations thereof in their constant region. For instance, if an antibody itself has low ADCC activity, its ADCC activity can be improved by slightly modifying the constant region of the antibody (Junttila, TT, 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).

[0082] Full-length IgG antibodies are preferred due to their desirable half-lives and the need to be as close to their own (human) molecules as possible due to their immunogenicity. The antibodies disclosed herein are preferably bispecific IgG antibodies, and more preferably bispecific full-length IgG1 antibodies. IgG1 is preferred based on its long circulating half-life in humans. To prevent any immunogenicity in humans, the bispecific IgG antibody is preferably human IgG1.

[0083] The term "bispecificity" (bs) means that one portion of an antibody (as defined above) binds to one epitope on an antigen, while a second portion binds to a different epitope. These different epitopes are typically located on different antigens. These first and second antigens are, in effect, two different proteins. A preferred bispecific antibody is one that contains two different monoclonal antibody portions, and as a result, binds to two different types of antigens. One arm of a 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 a bispecific antibody are typically different from each other, but the light chain variable regions are preferably the same. A bispecific antibody in which different heavy chain variable regions associate with the same or a common light chain is also called a bispecific antibody with a common light chain.

[0084] Preferred bispecific antibodies can be obtained by co-expression of two different heavy chains and one common light chain in a single cell. When a wild-type CH3 domain is used, co-expression of two different heavy chains and one common light chain yields three different species: AA, AB, and BB. To increase the percentage of the desired bispecific product (AB), CH3 manipulation can be used, or in other words, heavy chains having compatible heterodimerizing domains, as defined below herein, can be used.

[0085] The term "compatible heterodimerizing domain," as used herein, refers to a protein domain that is engineered such that engineered domain A' preferentially forms heterodimers with engineered domain B', and vice versa, while homodimerization between A'-A' and B'-B' is reduced.

[0086] The term "common light chain" refers to a light chain that may be identical or have some amino acid sequence differences, but whose binding specificity to the full-length antibody is not affected. For example, it is possible to prepare and find light chains that are not identical but are still functionally equivalent by introducing and testing, for example, conservative amino acid changes or changes in amino acids in regions that do not contribute or only partially contribute to binding specificity when paired with a heavy chain. The terms "common light chain," "common VL," "single light chain," and "single VL" are all used interchangeably herein, with or without the prefix "reorganized."

[0087] The common light chain (variable region) preferably has a germline sequence. The preferred germline sequence is a light chain variable region that is frequently used in the human repertoire and has good thermodynamic stability, yield, and solubility. In a preferred embodiment, the light chain includes a light chain region containing the amino acid sequence of the O12 / IgVκ1-39*01 gene segment shown in sequence 1C "common light chain IGKV1-39 / jk1", which has 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions, or combinations thereof. IgVκ1-39 is an abbreviation for the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin variable kappa 1-39;IGKV139;IGKV1-39;O12a or O12. The external ID of this gene is HGNC:5740;Entrez Gene:28930;Ensembl:ENSG00000242371. The variable regions of IGKV1-39 are listed in sequence 1C. The V region can be combined with one of the five J regions. Sequence 1C describes two preferred sequences of IgVκ1-39 combined with a J region. The concatenated sequences are denoted as IGKV1-39 / jk1 and IGKV1-39 / jk5; alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (nomenclature following the IMGT database World Wide Web at imgt.org).

[0088] The O12 / IgVκ1-39*01 constituting the light chain variable region is preferably a germline sequence. The IGJκ1*01 or / IGJκ5*01 constituting the light chain variable region is even more preferably a germline sequence. In a preferred embodiment, the IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence.

[0089] In a preferred embodiment, the light chain variable region includes germline O12 / IgVκ1-39*01. In a preferred embodiment, the light chain variable region includes kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. In a preferred embodiment, IgVκ1-39*01 / IGJκ1*01. The light chain variable region preferably includes germline kappa light chain IgVκ1-39*01 / IGJκ1*01 or germline kappa light chain IgVκ1-39*01 / IGJκ5*01, preferably germline IgVκ1-39*01 / IGJκ1*01.

[0090] Clearly, those skilled in the art will recognize that “common” also refers to functional equivalents of light chains whose amino acid sequences are not identical. Many variants of the light chains exist in which mutations (deletions, substitutions, additions) exist that do not substantially affect the formation of the functional binding domain. The light chains may also be those specified herein, having 1 to 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0091] Preferably, both the first antigen-binding site and the second antigen-binding site include a light chain variable region comprising CDR1 having the sequence (RASQSISSYLN), CDR2 having the sequence (AASSLQS), and CDR3 having the sequence (QQSYSTPPT).

[0092] As used herein, the term "ErbB-1" refers to the protein encoded by the ERBB-1 gene in humans. Alternative names for this gene or protein include EGFR, ERBB, HER1, and Erb-B2 receptor tyrosine kinase 1. Whenever ErbB-1 is mentioned herein, it refers to human ErbB-1.

[0093] The term "ErbB-2," as used herein, refers to the protein encoded by the ERBB-2 gene in humans. Alternative names for this gene or protein include CD340;HER-2;HER-2 / neu;MLN 19;NEU;NGL;TKR1. The ERBB-2 gene is often referred to as HER2 (from human epidermal growth factor receptor 2). When ErbB-2 is referred to herein, this refers to human ErbB-2. Antibodies containing an antigen-binding site that binds to ErbB-2 bind to human ErbB-2. The ErbB-2 antigen-binding site may also bind to other mammalian orthologues, but not necessarily, due to the similarity of sequence and tertiary structure between human orthologues and other mammalian orthologues. The database registration 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). These registration numbers are primarily provided to offer further methods for identifying ErbB-2 as a target, and the actual sequence of the ErbB-2 protein to which it is bound by antibodies may vary due to mutations in the encoding gene, such as those occurring in some cancers. The ErbB-2 antigen-binding site binds to ErbB-2 and its various variants, such as those expressed by some ErbB-2-positive cells or tumor cells.

[0094] As used herein, the term "ErbB-3" refers to the protein encoded by the ERBB-3 gene in humans. Alternative names for this gene or protein include HER3;LCCS2;MDA-BF-1;c-ErbB-3;c-erbb-3;erbb-3-S;p180-Erbb-3;p45-sErbb-3; and p85-sErbb-3. When ErbB-3 is referred to herein, this refers to human ErbB-3. Antibodies containing an antigen-binding site that binds to ErbB-3 bind to human ErbB-3. Due to the similarity in sequence and tertiary structure between human orthologues and other mammalian orthologues, the ErbB-3 antigen-binding site may also bind to such orthologues, but not necessarily. The database registration 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). These registration numbers are primarily provided to offer further methods for identifying ErbB-3 as a target, and the actual sequence of the ErbB-3 protein bound by the antibody may vary due to mutations in the coding gene, such as those occurring in some cancers. The ErbB-3 antigen-binding site binds to ErbB-3 and its various variants, such as those expressed by some ErbB-2-positive cells or tumor cells.

[0095] As used herein, the term "ErbB-4" refers to the protein encoded by the ERBB-4 gene in humans. Alternative names for this gene or protein include HER4, Erb-B2 receptor tyrosine kinase 4, and human epidermal growth factor receptor 4. When ErbB-1 is mentioned herein, this refers to human ErbB-4.

[0096] The antibodies disclosed herein can reduce the ligand-inducible receptor function of ErbB-3 on ErbB-2 and ErbB-3-positive cells. In the presence of excess ErbB-2, the ErbB-2 / ErbB-3 heterodimer can provide a growth signal to expressing cells in the absence of a detectable ligand for the ErbB-3 chain in the heterodimer. This ErbB-3 receptor function is referred herein to as the ligand-independent receptor function of ErbB-3. This ErbB-2 / ErbB-3 heterodimer also provides a growth signal to expressing cells in the presence of the ErbB-3 ligand. This ErbB-3 receptor function is referred herein to as the ligand-inducible receptor function of ErbB-3.

[0097] The term "ErbB-3 ligand," as used herein, refers to a polypeptide that binds to and activates ErbB-3. Examples of ErbB-3 ligands include, but are not limited to, neuregulin 1 (NRG) and neuregulin 2, beta-cellulin, heparin-binding epidermal growth factor, and epiregulin. This term includes biologically active fragments and / or variants of naturally occurring polypeptides.

[0098] Preferably, the ligand-inducible receptor function of ErbB-3 is the ErbB-3 ligand-inducible growth of ErbB-2 and ErbB-3-positive cells. In one preferred embodiment, these cells are MCF-7 cells (ATCC® HTB-22®); SKBR3 (ATCC® HTB-30®); NCI-87 (ATCC® CRL-5822®); BxPC-3-luc2 cells (Perkin Elmer 125058), BT-474 cells (ATCC® HTB-20®); or JIMT-1 cells (DSMZ number: ACC 589).

[0099] When used herein, ligand-inducible receptor function is reduced by at least 20%, preferably at least 30, 40, 50, 60, or at least 70%, and in one particularly preferred embodiment, this ligand-inducible receptor function is reduced by 80%, more preferably 90%. This reduction is preferably determined by determining the ligand-inducible receptor function in the presence of a bispecific antibody disclosed herein and comparing it to the same function in the absence of the antibody under otherwise identical conditions. These conditions include at least the presence of the ErbB-3 ligand. The amount of ligand present is preferably an amount that induces half of the maximum growth of ErbB-2 and ErbB-3 positive cell lines. The ErbB-2 and ErbB-3 positive cell lines for this test are preferably MCF-7 cell line (ATCC® HTB-22®), SKBR3 cell line (ATCC® HTB-30®), JIMT-1 cell line (DSMZ ACC 589), or NCI-87 cell line (ATCC® CRL-5822®). The test and / or 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.

[0100] The ErbB-2 protein contains several domains (see Figure 1 in Landgraf, R Breast Cancer Res. 2007; 9(1): 202 for reference). These extracellular domains are called domains I to IV. The binding sites of the antigen-binding sites of the antibodies described herein to each domain are mapped. Bispecific antibodies having an antigen-binding site (first antigen-binding site) that binds to domain I or domain IV of ErbB-2 contain a heavy chain variable region that maintains remarkable binding specificity and affinity for ErbB-2 when combined with various light chains. A bispecific antibody having an antigen-binding site (first antigen-binding site) that binds to domain I or domain IV of ErbB-2 and an antigen-binding site (second antigen-binding site) that binds to ErbB-3 has been found to be more effective in reducing the ligand-inducible receptor function of ErbB-3 compared to a bispecific antibody containing an antigen-binding site (first antigen-binding site) that binds to another extracellular domain of ErbB-2. A bispecific antibody containing an antigen-binding site (first antigen-binding site) that binds to ErbB-2 is preferred, wherein this antigen-binding site binds to domain I or domain IV of ErbB-2. Preferably, this antigen-binding site binds to domain IV of ErbB-2. The preferred antibody contains a first antigen-binding site that binds to domain I of ErbB-2 and a second antigen-binding site that binds to domain III of ErbB-3.

[0101] In a preferred embodiment, the antibody includes an antigen-binding site that binds to at least one amino acid of domain I of ErbB-2, selected from the group consisting of surface-exposed amino acid residues located within approximately 5 amino acid positions from T144, T164, R166, P172, G179, S180, and R181.

[0102] In a preferred embodiment, the antibody preferably includes an antigen-binding site that binds to at least one amino acid of domain III of ErbB-3, selected from the group consisting of surface-exposed amino acid residues located within 11.2 Å of R426 in the native ErbB-3 protein.

[0103] Bispecific antibodies that bind to ErbB-2 and have an antigen-binding site (first antigen-binding site) further containing ADCC have been found to be more effective than other ErbB-2 binding antibodies that did not exhibit significant ADCC activity, particularly in vivo. Therefore, bispecific antibodies exhibiting ADCC are preferred. Antibodies in which the first antigen-binding site binds to domain IV of ErbB-2 have been found to have intrinsic ADCC activity. Domain I-binding ErbB-2 binding antibodies with low intrinsic ADCC activity can be engineered to enhance ADCC activity. The Fc region mediates 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, e.g., CD16A (FcγRIIIA) and CD32A (FcγRIIA), activate cells to construct a response to antigens. Other receptors, e.g., CD32B, inhibit the activation of immune cells. By manipulating the Fc region (by introducing amino acid substitutions) to bind to the activating receptor with higher selectivity, antibodies with a greater ability to mediate the desired cytotoxic activity by anti-cancer mabs can be created.

[0104] One technique for enhancing the ADCC of antibodies is non-fucosylation (see, e.g., Junttila, TT, 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). Thus, non-fucosylated bispecific antibodies disclosed herein are further provided. Alternatively, several other strategies, including, for example, glycotechnology (Kyowa Hakko / Biowa, GlycArt (Roche), and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics), may be used to achieve ADCC enhancement, all of which aim to improve Fc binding to low-affinity activated FcγRIIIa and / or reduce binding to low-affinity inhibitory FcγRIIb.

[0105] Several in vitro methods exist to determine the efficacy of antibodies or effector cells in inducing ADCC. These include chromium-51 [Cr51] release assays, europium [Eu] release assays, and sulfur-35 [S35] release assays. Typically, labeled target cell lines expressing a specific antigen exposed on the surface are incubated with antibodies specific to that antigen. After washing, effector cells expressing the Fc receptor CD16 are typically co-incubated with antibody-labeled target cells. Target cell lysis is subsequently measured by the release of the intracellular label, typically by, for example, a scintillation counter or spectrophotometric method.

[0106] In a preferred bispecific antibody, the affinity of this second antigen-binding site for ErbB-3-positive cells is equal to, or preferably higher than, the affinity of this first antigen-binding site for ErbB-2-positive cells. The affinity (KD) of the second antigen-binding site for ErbB-3-positive cells is preferably 2.0 nM or less, more preferably 1.5 nM or less, more preferably 1.39 nM or less, and more preferably 0.99 nM or less. In a preferred embodiment, the affinity of this second antigen-binding site for ErbB-3 on SK-BR-3 cells is 2.0 nM or less, more preferably 1.5 nM or less, more preferably 1.39 nM or less, and more preferably 0.99 nM or less. In one embodiment, this affinity is in the range of 1.39 to 0.59 nM. In one preferred embodiment, the affinity of this second antigen-binding site to ErbB-3 on BT-474 cells is 2.0 nM or less, more preferably 1.5 nM or less, more preferably 1.0 nM or less, more preferably less than 0.5 nM, more preferably 0.31 nM or less, and more preferably 0.23 nM or less. In one embodiment, this affinity is in the range of 0.31 to 0.15 nM. The affinity described above is preferably measured using steady-state cell affinity assay, where cells are incubated at 4°C with a radioactively labeled antibody, as described in the example of WO2015 / 130173, and then the radioactivity bound to the cells is measured.

[0107] The affinity (KD) of the first antigen-binding site to ErbB-2-positive cells is preferably 5.0 nM or less, more preferably 4.5 nM or less, and more preferably 3.9 nM or less. In one preferred embodiment, the affinity of this first antigen-binding site to ErbB-2 on SK-BR-3 cells is 5.0 nM or less, preferably 4.5 nM or less, more preferably 4.0 nM or less, more preferably 3.5 nM or less, more preferably 3.0 nM or less, and more preferably 2.3 nM or less. In one embodiment, this affinity is in the range of 3.0 to 1.6 nM. In one preferred embodiment, the affinity of this first antigen-binding site to ErbB-2 on BT-474 cells is 5.0 nM or less, preferably 4.5 nM or less, and more preferably 3.9 nM or less. In one embodiment, this affinity is in the range of 4.5 to 3.3 nM. The affinity described above is preferably measured using steady-state cell affinity assays, where, as described in the example of WO2015 / 130173, the cells are incubated at 4°C with a radioactively labeled antibody, and then the radioactivity bound to the cells is measured.

[0108] Preferably, the bispecific antibody used in the disclosed method does not significantly affect cardiomyocyte survival. Cardiotoxicity is a known risk factor in ErbB-2 targeted therapy, and the frequency of complications increases when trastuzumab is used in combination with anthracyclines, thereby inducing cardiac load.

[0109] The bispecific antibodies disclosed herein are preferably used in humans. Therefore, preferred antibodies are human or humanized antibodies. Human tolerance to polypeptides is governed by many different aspects. Immunity, such as T cell-mediated or B cell-mediated immunity, is one of the variables encompassed in human tolerance to polypeptides. The constant region of the bispecific antibody is preferably a human constant region. This constant region may contain one or more differences, preferably 10 or fewer, and preferably 5 or fewer amino acids, from the constant region of a naturally occurring human antibody. It is preferable that the constant portion is entirely derived from a naturally occurring human antibody. The various antibodies produced herein are derived from a human antibody variable domain library. Such variable domains are human. The unique CDR region may be human-derived, synthetic, or derived from another organism. A variable region is considered a human variable region if, apart from the CDR region, it has an amino acid sequence identical to the amino acid sequence of a naturally occurring human antibody variable region. The ErbB-2-binding VH, ErbB-3-binding VH, or light chain variable regions in antibodies may contain one or more differences, preferably 10 or fewer, and preferably 5 or fewer amino acids, from the variable regions of naturally occurring human antibodies, without counting potential differences in the amino acid sequence of the CDR region. Such mutations are also naturally present in somatic hypermutations.

[0110] Antibodies can be derived from various animal species, at least with respect to the heavy chain variable region. For example, humanizing the mouse heavy chain variable region is a common practice. Various methods exist to achieve this, including CDR transplantation into a human heavy chain variable region having a 3D structure matching the 3D structure of the mouse heavy chain variable region; and deimmunization of the mouse heavy chain variable region, which is preferably carried out by removing known or suspected T cell epitopes or B cell epitopes from the mouse heavy chain variable region. Removal typically involves substituting one or more amino acids in the epitope with another (typically conserved) amino acid, thereby altering the epitope's sequence so that it is no longer a T cell epitope or a B cell epitope.

[0111] Such deimmunized mouse heavy chain variable regions are less immunogenic in humans than the original mouse heavy chain variable regions. Preferably, the variable region or domain is further humanized, for example, by veneerization. By using veneerization techniques, external residues readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule containing either a weakly immunogenic veneerized surface or a substantially non-immunogenic veneerized surface. The animals used in the present invention are preferably mammals, more preferably primates, and most preferably humans.

[0112] The bispecific antibodies disclosed herein preferably comprise the constant region of a human antibody. According to differences in their heavy chain constant domains, the antibodies are grouped into five classes or isotypes: IgG, IgA, IgM, IgD, and IgE. These classes or isotypes comprise at least one of the heavy chains, named using the corresponding Greek letters. Preferably, the constant region comprises the IgG constant region, more preferably the IgG1 constant region, and preferably a mutated IgG1 constant region. For example, several variations in the constant region of IgG1, such as allotypes G1m1, 17, and G1m3, are naturally occurring and / or acceptable without altering the immunological properties of the resulting antibody. Typically, insertions, deletions, substitutions, or combinations thereof between about 1 and 10 amino acids are acceptable in the constant region.

[0113] Preferred bispecific antibodies disclosed herein include: - At least CDR3 sequences of the 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, preferably at least CDR1, CDR2 and CDR3 sequences, or at least a heavy chain variable region sequence, or a heavy chain variable region sequence that differs from the listed heavy chain variable region sequences by up to 15 amino acids, preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably up to 1, 2, 3, 4 or 5 amino acids; and / or - From the group consisting of MF3178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059;MF6060;MF6061;MF6062;MF6063;MF6064;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 and MF6074 A heavy chain variable region sequence that differs from the selected ErbB-3 specific heavy chain variable region by at least the CDR3 sequence, preferably at least the CDR1, CDR2 and CDR3 sequences, or at least the heavy chain variable region sequence, or by up to 15 amino acids, preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, more preferably up to 1, 2, 3, 4, or 5 amino acids, from the listed heavy chain variable region sequences.

[0114] The CDR sequence is preferably modified for optimization purposes, for example, to improve the antibody binding efficacy or stability. Optimization is carried out, for example, by a mutagenesis procedure, after the stability and / or binding affinity of the resulting antibody is preferably tested and an improved ErbB-2 or ErbB-3 specific CDR sequence is preferably selected. Those skilled in the art can readily produce antibody variants containing at least one modified CDR sequence. For example, conservative amino acid substitutions are applied. Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, and the substitution of one polar residue for another polar residue, for example, arginine to lysine, glutamic acid to aspartic acid, or glutamine to asparagine.

[0115] A preferred antibody contains a variable domain that binds to ErbB-2, and the VH chain of this variable domain contains the amino acid sequence MF2926;MF2930;MF1849;MF2973;MF3004;MF3958 (humanized MF2971);MF2971;MF3025;MF2916;MF3991 (humanized MF3004);MF3031;MF2889;MF2913;MF1847;MF3001, MF3003 or MF1898; or up to 15, preferably 1, 2, with respect to the above VH chain sequence; or The VH chain MF2926;MF2930;MF1849;MF2973;MF3004;MF3958 (humanized MF2971);MF2971;MF3025;MF2916;MF3991 (humanized MF3004);MF3031;MF2889;MF2913;MF1847;MF3001,MF3003, orMF1898 contains the amino acid sequence, having 3, 4, 5, 6, 7, 8, 9, or 10, more preferably up to 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof. The VH chain of the variable domain that binds to ErbB-2 preferably contains the following amino acid sequence: - MF1849; or - MF2971 or a humanized version thereof, wherein the humanized version preferably comprises the amino acid sequence of MF3958; or - MF3004 or a humanized version thereof, wherein the humanized version preferably contains the amino acid sequence of MF3991. In one embodiment, the VH chain of the variable domain bound to ErbB-2 comprises the following amino acid sequences: VH chain MF1849; or MF2971 or its humanized version, where the humanized version preferably comprises the amino acid sequence of MF3958; or MF3004 or its humanized version, where the humanized version preferably comprises the amino acid sequence of MF3991. Hereinafter, the listed VH sequences have, with respect to each sequence, 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 combinations thereof. In one preferred embodiment, the VH chain of the variable domain bound to ErbB-2 comprises the amino acid sequence of MF3958; or comprises the amino acid sequence of MF3958 with respect to this VH chain sequence having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably up to 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0116] The VH chain of the variable domain that binds to Erb-B3 preferably contains the amino acid sequence MF3178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059;MF6060;MF6061;MF6062;MF6063;MF6064;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 or MF6074 with respect to the VH chain sequence, up to 15, preferably 1, 2, 3, 4, 5, 6 The VH chain contains the amino acid sequence of MF3178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059;MF6060;MF6061;MF6062;MF6063;MF6064;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 or MF6074, having 7, 8, 9 or 10, more preferably up to 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof. The VH chain of the variable domain that binds to 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, or MF6065, with respect to each VH chain sequence, having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably up to 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof. In one preferred embodiment, the VH chain of the variable domain bound to ErbB-3 comprises the amino acid sequence of MF3178; or, with respect to this VH chain sequence, comprises the amino acid sequence of MF3178 having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably up to 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof. Preferably, the above-mentioned amino acid insertions, deletions, and substitutions are not present in the CDR3 region.Preferably, the above-mentioned amino acid insertions, deletions, and substitutions are not present in the CDR1 and CDR2 regions. Preferably, the above-mentioned amino acid insertions, deletions, and substitutions are not present in the FR4 region.

[0117] Preferably, the antibody contains 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. This antibody preferably contains at least the CDR1, CDR2, and CDR3 sequences of MF3178, MF3176, MF3163, MF6058, MF6061, or MF6065, most preferably at least the CDR1, CDR2, and CDR3 sequences of MF3178.

[0118] Preferably, the ErbB-2 specific heavy chain variable region includes the amino acid sequence of VH chain MF3958 having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably up to 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof with respect to the VH (preferably, these insertions, deletions, or substitutions are not present in CDR1, CDR2, or CDR3). Preferably, the ErbB-3 specific heavy chain variable region includes the amino acid sequence of VH chain MF3178 having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably up to 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof with respect to the VH. Preferably, one or more amino acid insertions, deletions, substitutions, or combinations thereof are not present in the CDR1, CDR2, and CDR3 regions of the VH chain. These are preferably not present in the FR4 region either. Amino acid substitutions are preferably conservative amino acid substitutions.

[0119] Preferably, the ErbB-2 specific heavy chain variable region includes the amino acid sequence of VH chain MF3991 with respect to this VH, having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably up to 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof (preferably, these insertions, deletions, or substitutions are not present in CDR1, CDR2, or CDR3). Preferably, the ErbB-3 specific heavy chain variable region includes the amino acid sequence of VH chain MF3178 with respect to this VH, having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably up to 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof. One or more amino acid insertions, deletions, substitutions, or combinations thereof are preferably not present in the CDR1, CDR2, and CDR3 regions of the VH chain. They are also preferably not present in the FR4 region. The amino acid substitution is preferably a conservative amino acid substitution.

[0120] Preferably, the first antigen-binding site of the antibody comprises at least the CDR1, CDR2, and CDR3 sequences of MF3958, or the CDR1, CDR2, and CDR3 sequences of MF3958 and CDR1, CDR2, and CDR3 sequences that differ by up to three, preferably up to two, and preferably up to one amino acid; and the second antigen-binding site comprises at least the CDR1, CDR2, and CDR3 sequences of MF3178, or the CDR1, CDR2, and CDR3 sequences of MF3178 and CDR1, CDR2, and CDR3 sequences that differ by up to three, preferably up to two, and preferably up to one amino acid.

[0121] Preferably, the bispecific antibody includes i) a first antigen-binding site including an ErbB-2 specific heavy chain variable region and a light chain variable region containing the CDR1, CDR2, and CDR3 sequences of MF3958, and ii) a second antigen-binding site including an ErbB-3 specific heavy chain variable region and a light chain variable region containing the CDR1, CDR2, and CDR3 sequences of MF3178.

[0122] Preferably, the ErbB-2 specific heavy chain variable region has the MF3958 sequence, and the ErbB-3 specific heavy chain variable region has the MF3178 sequence. This combination is also called the PB4188 antibody. Preferably, the PB4188 antibody is not fucosylated.

[0123] Preferably, the bispecific antibody comprises a "heavy chain for erbB-2 binding" and a "heavy chain for erbB-3 binding" as shown in Part 1D of the sequence listing.

[0124] Preferably, the antigen-binding site of the bispecific antibody comprises germline light chain O12, preferably a rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01 or a fragment or functional derivative thereof (nomenclature according to the IMGT database World Wide Web on imgt.org). Term: rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01, IGKV1-39 / IGKJ1, huVκ1-39 light chain, or more precisely, huVκ1-39, is used. This light chain may have 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof. The above 1, 2, 3, 4, or 5 amino acid substitutions are preferably conservative amino acid substitutions, and the insertions, deletions, substitutions, or combinations thereof are preferably not present in the CDR3 region of the VL chain, and preferably not present in the CDR1, CDR2, or CDR3 region or FR4 region of the VL chain. Preferably, the first antigen-binding site and the second antigen-binding site include the same light chain variable region, and more precisely, include a common light chain. Preferably, the light chain variable region includes CDR1 having the sequence (RASQSISSYLN), CDR2 having the sequence (AASSLQS), and CDR3 having the sequence (QQSYSTPPT). Preferably, the light chain variable region includes a common light chain sequence shown in the sequence listing part 1C.

[0125] Various methods are available for producing bispecific antibodies, as disclosed in WO2015 / 130173. One method involves the expression of two different heavy chains and two different light chains in cells, as well as the collection of antibodies produced by the cells. The antibodies produced by this method typically comprise a group of antibodies having different combinations of heavy and light chains, some of which are the desired bispecific antibodies. These bispecific antibodies can then be purified from this group of antibodies.

[0126] The ratio of bispecific antibodies to other antibodies produced by cells can be increased in various ways. Preferably, this ratio is increased by expressing two essentially identical light chains in cells, rather than two different light chains. This concept is also known in the art as the “common light chain” method. When essentially identical light chains function together with two different heavy chains to enable the formation of variable domains having different antigen-binding sites and associated different binding properties, the ratio of bispecific antibodies to other antibodies produced by cells is significantly improved compared to the expression of two different light chains. The ratio of bispecific antibodies produced by cells can be further improved by stimulating the pairing of two different heavy chains with each other, rather than by pairing two identical heavy chains. The art describes various methods by which such heterodimerization of heavy chains can be achieved. One method is to produce “knob-into-hole” bispecific antibodies. See U.S. Patent Application Publication No. 20030078385 (Arathoon et al., - Genentech). Another and preferred method is described in PCT application PCT / NL2013 / 050294 (WO2013 / 157954A1), which is incorporated herein by reference. Methods and means for producing bispecific antibodies from single cells are disclosed, thereby providing means for which the formation of bispecific antibodies is preferred over the formation of monospecific antibodies.

[0127] The sequences referred to in this disclosure are shown below and in Figure 1.

[0128] Sequence 1A (erbB-2 specific) MF2926: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0129] [ka]

[0130] Amino acid sequence: QVQLQQSGPELVKPGASVMISCKASGYSFTGYHMNWVKQSPEKSLEWIGDINPSIGTTAHNQIFRAKATMTVDKSSNTAYMQLKSLTSEDSGVFYCVRRGDWSFDVWGTGTTVTVSS CDR1: GYHMNWVKQSPEKSLE CDR2: NQIFRA CDR3: RGDWSFDV MF2930: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0131] [ka]

[0132] Amino acid sequence: EVQLQQSGAELVKPGASVMMSCKVSGYTFTSYPIAWMKQVHGKSLEWIGNFHPYSDDTKYNENFKGKATLTVEKSSSTVYLELSRLTSDDSAVYYCARSNPLYYFAMDYWGQGTSVTVSS CDR1: SYPIAWMKQVHGKSLE CDR2: NENFKG CDR3: SNPLYYFAMDY MF1849: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0133] [ka]

[0134] Amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDYGSYSSYAFDYWGQGTLVTVSS CDR1: SYGMH CDR2: VISYDGSNKYYADSVKG CDR3: GDYGSYSSYAFDY MF2973: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0135] [ka]

[0136] Amino acid sequence: QVQLKQSGAELVRPGASVKLSCKASGYIFTGYYINWLRQRPGQGLEWIAKIYPGSGNTYYNEKFRGKATLTAEESSSTAYMQLSSLTSEDSAVYFCARGPHYDYDGPWFVYWGQGTLVTVSS CDR1: GYYINWLRQRPGQGLE CDR2: NEKFRG CDR3: GPHYDYDGPWFVY MF3004: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0137] [ka]

[0138] Amino acid sequence: QVQLKQSGAELVRPGASVKLSCKASGYTFTGYYINWVKQRPGQGLEWIARIYPGSGYTYYNEKFKGKATLTAEESSSTAYMHLSSLTSEDSAVYFCARPHYGYDDWYFGVWGTGTTVTVSS CDR1: GYYINWVKQRPGQGLE CDR2: NEKFKG CDR3: PHYGYDDWYFGV MF2971: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0139] [ka]

[0140] Amino acid sequence: QVQLKQSGAELVRPGASVKLSCKASGYTFTAYYINWVKQRPGQGLEWIARIYPGSGYTYYNEIFKGRATLTADESSSTAYMQLSSLTSEDSAVYFCARPPVYYDSAWFAYWGQGTLVTVSS CDR1: AYYINWVKQRPGQGLE CDR2: NEIFKG CDR3: PPVYYDSAWFAY MF3025: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0141] [ka]

[0142] Amino acid sequence: QVQLKQSGAELVRPGTSVKLSCKASGYTFTGYYINWVKQRPGQGLEWIARIYPGSGYTYYNEKFKGKATLTAEESSNTAYMHLSSLTSEDSAVYFCARPHYGYDDWYFAVWGTGTTVTVSS CDR1: GYYINWVKQRPGQGLE CDR2: NEKFKG CDR3: PHYGYDDWYFAV MF2916: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0143] [ka]

[0144] Amino acid sequence: QVQLQQSGAELVRPGASVKLSCKASGYTFTGYYINWVKQRPGQGLEWIARIYPGSGHTSYNEKFKGKATLTTEKSSSTAYMQLSSLTSEDSAVYFCARPIYFDYAGGYFDVWGTRTSVTVSS CDR1: GYYINWVKQRPGQGLE CDR2: NEKFKG CDR3: PIYFDYAGGYFDV MF3958: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0145] [ka]

[0146] Amino acid sequence: QVQLVQSGAEVKKPGASVKLSCKASGYTFTAYYINWVRQAPGQGLEWIGRIYPGSGYTSYAQKFQGRATLTADESTSTAYMELSSLRSEDTAVYFCARPPVYYDSAWFAYWGQGTLVTVSS CDR1: AYYIN CDR2: RIYPGSGYTSYAQKFQG CDR3: PPVYYDSAWFAY MF3031: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0147] [ka]

[0148] Amino acid sequence: QVQLQQSGAELVRPGASVKLSCKASGYTFTAYYINWVKQRPGQGLEWIAKIYPGSGYTYYNENFRGKATLTAEESSSTAYIQLSSLTSEDSAVYFCARGVYDYDGAWFAYWGQGTLVTVSS CDR1: AYYINWVKQRPGQGLE CDR2: NENFRG CDR3: GVYDYDGAWFAY MF3991: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0149] [ka]

[0150] Amino acid sequence: QVQLVQSGAEVKKPGASVKLSCKASGYTFTAYYINWVRQAPGQGLEWIGRIYPGSGYTSYAQKFQGRATLTADESTSTAYMELSSLRSEDTAVYFCARPHYGYDDWYFGVWGQGTLVTVSS CDR1: AYYIN CDR2: RIYPGSGYTSYAQKFQG CDR3: PHYGYDDWYFGV

[0151] Sequence 1B (erbB-3 specific) MF3178: Heavy chain variable region sequence of erbB-3 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0152] [ka]

[0153] Amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDHGSRHFWSYWGFDYWGQGTLVTVSS CDR1: GYYMH CDR2: WINPNSGGTNYAQKFQG CDR3: DHGSRHFWSYWGFDY MF3176: Heavy chain variable region sequence of erbB-3 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0154] [ka]

[0155] Amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDWWYPPYYWGFDYWGQGTLVTVSS CDR1: SYAMS CDR2: AISGSGGSTYYADSVKG CDR3: DWWYPPYYWGFDY MF3163: Heavy chain variable region sequence of erbB-3 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0156] [ka]

[0157] Amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAKDSYSRHFYSWWAFDYWGQGTLVTVSS CDR1: GYYMH CDR2: WINPNSGGTNYAQKFQG CDR3: DSYSRHFYSWWAFDY MF3099: Heavy chain variable region sequence of erbB-3 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0158] [ka]

[0159] Amino acid sequence: EVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGILDPSDSYTTYNQKFKGKATLTVDTSSSIAYMQLSSLTSEDSALYYCARGGDYDEGGAMDYWGQGTSVTVSS CDR1: SYWMH CDR2: ILDPSDSYTTYNQKFKG CDR3: GGDYDEGGAMDY MF3307: Heavy chain variable region sequence of erbB-3 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0160] [ka]

[0161] Amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGSRKRLSNYFNAFDYWGQGTLVTVSS CDR1: GYYMH CDR2: WINPNSGGTNYAQKFQG CDR3: GSRKRLSNYFNAFDY

[0162] Array 1C Common light chain Variable range of IGKV1-39A DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP CDR 1: RASQSISSYLN CDR 2: AASSLQS CDR 3: QQSYSTPPT IGKV1-39 / jk1 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK Common light chain IGKV1-39 / jk1 (underlined in the steady-state region) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Common light chain variable domains for IGKV1-39 / jk5 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0163] Sequence 1D (erbB-2 specific) Heavy chain for erbB-2 bonding QVQLVQSGAEVKKPGASVKLSCKASGYTFTAYYINWVRQAPGQGLEWIGRIYPGSGYTSYAQKFQGRATLTADESTSTAYMELSSLRSEDTAVYFCARPPVYYDSAWFAYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Heavy chain for erbB-3 binding QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDHGSRHFWSYWGFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0164] Array 1E HER2-specific Ab sequences MF2889: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0165] [ka]

[0166] Amino acid sequence: EVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVIYPEGGGTIYNEKFKGKATLTADKSSSTAYMQLSGLTSEDSAVYFCARGDYDYKYAMDYWGQGTSVTVSS CDR1: NYLIE CDR2: VIYPEGGGTIYNEKFKG CDR3: GDYDYKYAMDY MF2913: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0167] [ka]

[0168] Amino acid sequence: EVKLQQSGPELVKPGASVKISCKASGYSFTDYKMDWVKQSHGKSLEWIGNINPNSGGVIYNQKFRGKVTLTVDRSSSAAYMELRSLTSEDTAVYYCSRGLWDAMDSWGQGTSVTVSS CDR1: DYKMDWVKQSHGKSLE CDR2: NQKFRG CDR3: GLWDAMDS MF1847: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0169] [ka]

[0170] Amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWWHPLLSGFDYWGQGTLVTVSS CDR1: SYGMH CDR2: VISYDGSNKYYADSVKG CDR3: GWWHPLLSGFDY MF3001: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0171] [ka]

[0172] Amino acid sequence: EVQLQQSGAELAKPGASVKLSCKTSGYNFPIYWMHWVKQRPGRGLEWIGYINPSTGYIKNNQKFKDKATLTADKSSNTAYMQLNSLTYEDSAVYYCTREGITGFTYWGQGTLVTVSS CDR1: IYWMHWVKQRPGRGLE CDR2: NQKFKD CDR3: EGITGFTY MF1898: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0173] [ka]

[0174] Amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGFRRTTLSGFDYWGQGTLVTVSS CDR1: SYGMH CDR2: VISYDGSNKYYADSVKG CDR3: DGFRRTTLSGFDY MF3003: Heavy chain variable region sequence of erbB-2 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0175] [ka]

[0176] Amino acid sequence: QVQLKQSGPELVKPGASVKISCKASGDAFSYSWMNWVKQRPGKGLEWIGRIYPGDGDINYNGKFKGKATLTADKSSSTAHLQLNSLTSEDSAVYFCARGQLGLEAWFAYWGQGTLVTVSS CDR1: YSWMNWVKQRPGKGLE CDR2: NGKFKG CDR3: GQLGLEAWFAY HER3-specific Ab sequences MF6058: Heavy chain variable region sequence of erbB-3 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0177] [ka]

[0178] Amino acid sequence: QVQLVQSGADVKKPGASVKVTCKASGYTFTGYYMHWVRQAPGQALEWMGWINPQSGGTNYAKKFQGRVSMTRETSTSTAYMQLSRLRSDDTATYYCARDHGSRHFWSYWGFDYWGQGTLVTVSS CDR1: GYYMH CDR2: WINPQSGGTNYAKKFQG CDR3: DHGSRHFWSYWGFDY MF6061: Heavy chain variable region sequence of erbB-3 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0179] [ka]

[0180] Amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPQSGGTNYAQKFKGRVTMTRDTSTSTAYMELSRLRSDDTAVYYCARDHGSRHFWSYWGFDYWGQGTLVTVSS CDR1: GYYMH CDR2: WINPQSGGTNYAQKFKG CDR3: DHGSRHFWSYWGFDY MF6065: Heavy chain variable region sequence of erbB-3 binding antibody Nucleic acid sequence (underlined sequence codes for the end of the leader peptide):

[0181] [ka]

[0182] Amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPQGGSTNYAQKFQGRVTMTRDTSTSTVYMELSRLRSEDTAVYYCARDHGSRHFWSYWGFDYWGQGTLVTVSS CDR1: SYYMH CDR2: WINPQGGSTNYAQKFQG CDR3: DHGSRHFWSYWGFDY

[0183] For the sake of clarity and conciseness, features are described herein as part of the same or separate embodiments; however, it is understood that the scope of the invention may include embodiments having all or some combinations of the described features. [Brief explanation of the drawing]

[0184] [Figure 1] This figure shows the amino acid alignment of the MF3178 variant. The dots indicate the same amino acids as MF3178 at that position. The CDR1, CDR2, and CDR3 sequences of MF3178 are shown in bold and underlined. [Figure 2A] This figure shows increased in vivo tumor targeting of bispecific antibodies compared to monoclonal antibodies. Micro-PET imaging demonstrates that the PB4188 variant accumulates more efficiently in tumors compared to HER3 monoclonal antibodies. [Figure 2B] This figure shows increased in vivo tumor targeting of the bispecific antibody compared to the monoclonal antibody. Gamma-counter quantification of radioactivity present in the tumor confirmed that the level of the PB4188 variant in the tumor was 2.5 times higher than the level of the parental anti-HER3 antibody. [Figure 2C] This figure shows increased in vivo tumor targeting of bispecific antibodies compared to monoclonal antibodies. Quantitative in vivo distribution of tumor uptake in four mAb groups at 48 hours. Results are shown as percentage of injected dose per gram of tissue (%ID / g), with error bars indicating ±SD. [Figure 3A] This figure shows antibody antagonist-style dose-response curves in EGFR:HER2, HER2:HER3, and HER2:HER4 assays. Reporter cells were seeded at 2.5 K / well for EGFR:HER2, or at 5 K / well for HER2:HER3 and HER2:HER4, at 37°C for 4 hours. Antibodies were serially diluted and incubated at 37°C for 3 hours, followed by stimulation with 10 ng / ml EGF or 30 ng / ml HRG-β2 for 16 hours for EGFR:HER2, HER2:HER3, and HER2:HER4, respectively. Reference stimulation curves for agonists were obtained by incubating ligand-only titrations for 24 hours. Each data point represents the mean and standard deviation of four replicates for one dose. Data were plotted in GraphPad Prism, and curve fitting was performed using log(inhibitor) vs. response variable (4-parameter) fitting to calculate IC50. [Figure 3B] The continuation of Figure 3A is shown. [Figure 4]It is a figure showing the change in body weight of mice in different groups. Body weight change after administration of MCLA-128, PG2863 and PG2869 antibodies to female BALB / c nude mice bearing OV-10-0050 established tumors. Data points represent the mean body weight of the group. Error bars represent the standard error of the mean (SEM). [Figure 5] It is a figure showing the relative change in body weight (%). BW change was calculated based on the body weight of the animals on the first day of dosing. Data points represent the mean percent group change in BW. Error bars represent the standard error of the mean (SEM). [Figure 6] It is a figure showing tumor growth curves. Tumor volume traces after administration of MCLA-128, PG2863 and PG2869 antibodies to female BALB / c nude mice bearing OV-10-0050 established tumors. Data points represent the group mean, and error bars represent the standard error of the mean (SEM). [Figure 7] It is a figure showing growth inhibition of tumor lines MDA-MB-175 and OV-10-0050 in vitro and in vivo. DOC4-NRG1 and CLU-NRG1 gene fusions are expressed in MDA-MB-175 cell line (breast) and OV-10-0050 PDX (ovarian), respectively. Left panel: MCLA-128 treatment in vitro inhibits MDA-MB-175 cell proliferation. Right panel: MCLA-128 treatment in vivo (25 mg / kg twice weekly until day 28) reduced tumor growth and eliminated tumors in 6 / 8 animals.

Examples

[0185] (Example 1) ErbB-2-guided targeting Imaging experiments were conducted comparing a HER2 x HER3 bispecific antibody (PB4188) with a HER3 bivalent monoclonal antibody. A variant of bAb PB4188 and anti-HER3 MF3178 (parent antibody) were labeled with 64Cu and intravenously injected into mice xenografted with HER2 gene-amplified JIMT-1 tumors. Micro-PET imaging demonstrated that the PB4188 variant accumulated more efficiently in the tumor compared to the HER3 monoclonal antibody (Figure 2A). Gamma-counter quantification of radioactivity present in the tumor confirmed that the level of the PB4188 variant in the tumor was 2.5 times higher than that of the parent anti-HER3 antibody (Figure 2B). Overall, in vitro and in vivo data demonstrate that HER2 targeting is responsible for the enhanced binding of PB4188 to tumor cells. Further studies were conducted using an anti-HER2 (MF3958) antibody. Figure 2C summarizes the results of each antibody injected into mice xenografted with JIMT-1 tumors labeled with 64Cu and with HER2 gene amplification (n=4 mice for each antibody treatment).

[0186] method In vivo distribution study. Variants of bAb PB4188, anti-HER2 MF3958 and anti-HER3 MF3178, were conjugated into a bifunctional chelator [Paterson 2014 Dalton Transactions]. The binding characteristics of the conjugated products to their targets were confirmed using flow cytometry-based assays. The proteins were then labeled with 64Cu, and the radiolabeled antibodies were administered to mice with JIMT-1 mammary xenografts via the tail vein (Figures 2A-2B, and "iv" for Figure 2C) or intraperitoneally ("ip" for Figure 2C). MicroPET / CT images were acquired 48 hours after injection, and the tumors were subsequently resected and radioactivity measured with a gamma counter. Results are presented as a percentage of the injected dose per gram of tissue.

[0187] (Example 2) Inhibition of heterodimer formation A heterodimerization assay based on enzyme fragment complementation technology was used. The β-galactosidase enzyme can be artificially split into two inactive fragments, an enzyme donor and an enzyme acceptor, which combine only when in close proximity to form an active enzyme. Each sequence encoding either the enzyme donor or enzyme acceptor is ligated to the extracellular and transmembrane domains of each heterodimerization partner. Both genes are then co-transfected into U2OS cells to express the extracellular domain of an RTK receptor ligated to one domain (ED or EA) of β-galactosidase. Upon agonist stimulation of one of the RTK receptors, both RTK receptors dimerize, inducing the formation of a fully reconstituted active β-galactosidase enzyme. Finally, β-galactosidase activity is measured by adding a substrate that induces luminescence during hybridization.

[0188] Antibodies were tested in EGFR:HER2, HER2:HER3, and HER3:HER4 heterodimerized reporter cell lines. RTK heterodimerization assays were performed using the bispecific antibody MCLA-128 (MF3178 arm and MF3958 arm); anti-HER3 antibodies MF3178 / PG3178 and PG3793 / AMG-888 / patrizumab; and anti-HER2 antibodies MF3958 / PG3958, PG2867 / trastuzumab, PG2869 / pertuzumab, and Perjeta (clinical batch of pertuzumab). EGF and HRG titrations in the EGFR:HER2, HER2:HER3, and HER2:HER4 assays showed dose-dependent agonist responses (Figure 3). MCLA-128 specifically demonstrated complete inhibition of HER2:HER3 dimerization but had no effect on EGFR:HER2 or HER2:HER4 heterodimerization. In contrast, trastuzumab (PG2867) behaved as a partial antagonist in both EGFR:HER2 and HER2:HER3 assays.

[0189] MCLA-128 and PG3178 completely inhibited HRG-induced HER2:HER3 dimerization at the highest intensity (Table 1).

[0190] [Table 1]

[0191] In the HER2:HER3 assay, the potency of trastuzumab was approximately one-quarter that of MCLA-128 or PG3178. Perjeta (clinical pertuzumab) behaved as a complete antagonist in all three assays, producing a similar profile to PG2867 (pertuzumab). In the HER2:HER4 assay, both anti-HER2 PG3958 and PG2867 (pertuzumab) showed a slight decrease in dimerization, which appeared to be dose-dependent. In the EGFR:HER2 assay, small nonspecific responses were observed with high concentrations of PG1337, MCLA-128, PG3178, and PG3958.

[0192] MCLA-128 showed specific inhibition of only the HER2:HER3 heterodimer. This suggests that, upon binding to HER2, MCLA-128 does not sterically impair the interaction between HER2 and EGFR during EGF stimulation, nor impair the heterodimerization of HER2 and HER4 during HRG stimulation.

[0193] The latter is consistent with observations in HRG-induced cell cycle-based proliferation assays of T47D cells. Assays using these cells failed to demonstrate inhibitory activity of either MCLA-128 or PG3178, which was likely due to higher expression of HER4 compared to HER3. It is thought that HRG is preferably signaled via HER2:HER4 rather than HER2:HER3 in T47D cells, which explains the lack of potency of MCLA-128 and indicates the specificity of MCLA-128 to HRG-induced HER2:HER3 dimers rather than to HRG-induced HER2:HER4 dimers.

[0194] In this study, trastuzumab blocked EGF-induced heterodimerization of EGFR:HER2 and HRG-induced heterodimerization of HER2:HER3, respectively. Trastuzumab and pertuzumab behaved as partial and full antagonists, respectively, which is consistent with the generally accepted assertion that trastuzumab blocks ligand-independent activation of HER2, and pertuzumab blocks ligand-dependent signaling. The fact that a trastuzumab inhibitory response was observed in these assays may be due to overexpression of both targets, which could allow for more sensitive readout than traditional immunoprecipitation experiments.

[0195] Finally, PG3793 showed lower binding affinity to MCF-7 than PG3178, but its lower intensity in the HER2:HER3 heterodimerization assay was not as pronounced (a 2.5-fold difference in dimerization assay intensity compared to a 30-fold difference in binding assay affinity). This discrepancy between binding affinity and antagonistic intensity has been previously observed in the cases of MCLA-128 and PG3178. PG3178 binds to MCF-7 with slightly better affinity than MCLA-128, but MCLA-128 is superior to PG3178 in cell cycle-based proliferation assays.

[0196] (Example 3) Test objectives and regulatory compliance The purpose of this study is to evaluate the in vivo antitumor efficacy of MCLA-128, PG2863, and PG2869 antibodies in the treatment of a subcutaneous human ovarian cancer PDX model with OV-10-0050 in BALB / c nude mice.

[0197] Experimental Design The experimental design is shown in table 2 (Table 2). In all groups, blood was sampled from 4 animals on day 2 (24 hours after the first dose), and from the remaining 4 animals on day 6 (5 days after the first dose). At the specified time point, 50 to 100 μl of blood was collected into a sterile collection tube (Microvette CB300Z clot activation factor / serum, Sarstedt B.V. catalog number 16.440.100). The sample was allowed to clot at room temperature for 45 minutes, centrifuged at 3000 rpm for 10 minutes. The aqueous layer (approximately 20 μl of serum) was transferred into another 1.5 mL sterile Eppendorf tube, and immediately stored at -80°C. Samples shall be shipped on dry ice.

[0198] [Table 2]

[0199] All animals were treated on days 1, 8, 15, 22 and 29 (weekly treatment over 5 weeks), and the route of administration was I.P. for all groups.

[0200] Tumor samples were collected 48 hours after the last dose (day 31). Tumors were fixed in neutral buffered formalin (with a tissue:fixative ratio of at least 1:20) for 24 hours, and then processed into FFPE blocks.

[0201] Preparation of neutral buffered formalin: Place one bag of PBS powder into a clean 5 L volumetric flask, add 4.5 L of deionized water, stir to disperse the powder to obtain a clear solution. Then add 500 mL of formaldehyde and stir until a homogeneous solution is obtained.

[0202] Materials Animals: Species: Mouse (Mus musculus); Strain: BALB / c nude; Age: 6 to 8 weeks old; Sex: Female; Body weight: 18 to 22 g; Number of animals: 32 mice + reserve

[0203] Animal supplier: Shanghai Sino-British SIPPR / BK Laboratory Animal Co., LTD.

[0204] Diet: Animals had free access to irradiated, dried granular food throughout the entire study period; Water: Animals had free access to sterile drinking water.

[0205] Antibody packaging and storage conditions: MCLA-128; cryovial, 2.5 mg / ml, 10 x 1.5 ml / vial, store at 4°C. PG2863; cryovial, 2.5 mg / ml, 10 x 1.5 ml / vial, store at 4°C. PG2869; cryovial, 2.5 mg / ml, 10 x 1.5 ml / vial, store at 4°C.

[0206] Generating a PDX Model The OV-10-0050 human ovarian cancer PDX model was originally established from a 48-year-old female patient with grade 3 adenocarcinoma of the ovary. Surgically resected clinical samples were transplanted into nude mice (defined as passage 0, P0), and subsequent serial transplants were defined as P1, P2, etc. P6 tumor tissue was used in this study.

[0207] Tumor transplantation Each mouse was subcutaneously transplanted into its right flank with an OV-10-0050 P6 tumor slice (approximately 30 mm³) that had been cut with scissors to induce tumor development. Treatment began 30 days after tumor transplantation, which was the day when the average tumor size reached approximately 152 mm³. Thirty-two tumor-bearing mice were randomized into four groups using a stratified randomization method, with each group consisting of eight tumor-bearing mice. The day of randomization was designated as day 1, which was the day treatment began. The test material was administered to the mice according to the prescribed regimen as shown in the experimental design sheet (Table 2).

[0208] observation All procedures for handling, caring for, and treating animals in the study were carried out in accordance with the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec. At the time of routine monitoring, animals were checked daily for normal behavior, such as motility, food and water consumption (by visual inspection only), weight gain / loss (weight was measured twice a week), eye / hair matting, any effects of tumor growth and treatment, and any other abnormal effects described in the protocol. Deaths and observed clinical signs were recorded based on the number of animals in each subset.

[0209] Tumor measurement We tested whether tumor growth could be delayed or whether the mice could be cured. Tumor size was measured twice a week in two dimensions using calipers, and the volume was expressed in mm³ using the formula: V = 0.5a × b², where a and b are the long and short diameters of the tumor, respectively. Tumor size was then used to calculate TC, T / C, and TGI values. TC was calculated using T as the median time (days) required for the tumor in the treatment group to reach a given size (e.g., 500 mm³), and C as the median time (days) required for the tumor in the control group to reach the same size. The T / C value (percent) is an indicator of antitumor efficacy; T and C were the mean volumes of the treatment and control groups on a given day, respectively. TGI was calculated for each group using the formula: TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100; where Ti was the mean tumor volume of the treatment group on a given day, T0 was the mean tumor volume of the treatment group on the first day of treatment, Vi was the mean tumor volume of the vehicle control group on the same day as Ti, and V0 was the mean tumor volume of the vehicle group on the first day of treatment.

[0210] statistical analysis Summary statistics, including the mean and standard error of the mean (SEM), are provided for tumor volume in each group at each time point (detailed in Table 3). Statistical analysis of differences in tumor volume between groups and drug interaction analysis were performed on data obtained at the best therapeutic time point after the final dose (29 days after group assignment).

[0211] One-way ANOVA was performed to compare tumor volumes between groups. If a significant F-statistic (P<0.001, ratio of treatment variance to error variance) was obtained, group comparisons were performed using Games-Howell. All data were analyzed using SPSS 17.0. A p<0.05 value was considered statistically significant.

[0212] result Mortality rate, morbidity rate, and weight gain or loss Animal body weight was regularly monitored as an indirect measure of toxicity. No weight loss was observed as a result of administration of the test substance (Figure 4), and no deaths or morbidities were observed. Therefore, there appears to be no apparent toxicity associated with the administration of MCLA-128, PG2863, and PG2869 antibodies to tumor-carrying BALB / c nude mice.

[0213] Figures 4 and 5 show the changes in body weight in female BALB / c nude mice carrying OV-10-0050 xenografts and treated with MCLA-128, PG2863, and PG2869 antibodies. Table 3 shows the mean tumor volume over time in female BALB / c nude mice carrying OV-10-0050 xenografts and treated with MCLA-128, PG2863, and PG2869 antibodies. Figure 6 shows tumor growth.

[0214] [Table 3]

[0215] Results and Discussion This study evaluated the therapeutic efficacy of MCLA-128, PG2863, and PG2869 antibodies as monotherapies in the treatment of the OV-10-0050 human ovarian cancer xenograft model. Tumor size results for different groups at different time points after tumor inoculation are shown in Table 3, Table 4, and Figure 4.

[0216] The average tumor size of vehicle-treated control mice was 1,161 mm² 29 days after group assignment. 3 Treatment with the test items MCLA-128, PG2863, and PG2869 antibodies at 25 mg / kg (QW x 5 weeks) produced significant antitumor activity: their mean tumor sizes at the same time point were 23, 108, and 1 mm³, respectively (T / C values ​​= 1.95%, 9.28%, and 0.06%, respectively; TGI values ​​= 112.78%, 104.37%, and 114.96%; p values ​​= 0.002, 0.003, and 0.002), and all of their tumor growth was 500 mm. 3 In terms of tumor size, it is delayed by more than 14 days compared to the vehicle group. The treatment causes partial or complete regression of the tumor. If the tumor volume is reduced by 50% or more of the volume on day 1 for three consecutive measurements during the course of the study, or if ≥13.5 mm for one or more of these three measurements. 3 If this was the case, the mouse was considered to have partial regression (PR). Furthermore, if the measurement was <13.5 mm in three consecutive measurements during the testing process, 3 If this was the case, the mouse was considered to have achieved complete regression (CR). If no palpable tumor was detected at the end of the study, the mouse was considered to have survived without tumor.

[0217] Treatment with MCLA-128, PG2863, and PG-2869 resulted in different rates of partial response (PR), complete response (CR), and total failure (TFS). The number of mice in each group showing PR, CR, and TFS is shown in Table 5. All test materials were well tolerated by tumor-carrying animals. No weight loss was observed in any of the treatment groups.

[0218] In summary, all three test antibodies, when used as monotherapy agents, demonstrated significant antitumor activity against the OV-10-0050 human ovarian cancer xenograft model in this study. These antibodies were well-tolerated by tumor-bearing animals. These results demonstrate that these antibodies are safe and effective anticancer agents.

[0219] [Table 4]

[0220] [Table 5]

[0221] (Example 4) MCLA-128 is a bispecific antibody that targets HER2 and HER3 receptor tyrosine kinases (RTKs) involved in the proliferation and survival of cancer cells. MCLA-128 has been extensively tested for heregulin (HRG)-induced HER3 signaling and proliferation. MCLA-128 has demonstrated stronger in vitro strength than the following: ligand-dependent and ligand-independent HER2, respectively; the combination of the anti-HER2 antibodies pertuzumab (PG2869) and trastuzumab (PG2867) that can block HER3 signaling [Agus 2002; Juntilla 2009]; and the anti-HER3 antibody MM-121 (PG2863) that blocks HRG-induced HER3 activation [Schoeberl 2010].

[0222] MCLA-128 also exhibits antitumor activity in cells expressing a gene fusion containing the HRG gene. The MDA-MB-175 cell line contains a DOC4-NRG1 gene fusion, which generates a proliferation autocrine loop due to NRG1 expression. This gene fusion has not been found to date in cancer patient settings [Sanchez-Valdivieso 2002].

[0223] From a panel of breast cancer cell lines, MDA-MB-175 cells were sensitive to the monotherapy MCLA-128, demonstrating the importance of the HER3 / HRG signaling axis in this cell line (Figure 7, left panel). HER2 activation in this cell line was also demonstrated in vivo, with a single dose of pertuzumab, rather than trastuzumab, inhibiting orthotopic MDA-MB-175 tumor growth. While the relevance of DOC4-NRG1 gene fusions in breast cancer patients has been discussed [Sanchez-Valdivieso 2002], other gene fusions have recently attracted attention. In particular, the CD74-NRG1 fusion has been reported by an independent group in invasive mucinous adenocarcinoma, a subpopulation of non-small cell lung cancer [Fernandez-Cuesta 2014, Duruisseaux 2016]. Several other NRG1 gene fusions have also been detected, namely VAMP2-NRG1, RBPMS-NRG1, and WRN-NRG1 in lung cancer, and RAB2IL1-NRG1 in ovarian cancer [Jung 2015, Dhanasekaran, 2014]. This diversity of gene fusions may be related to the location of the NRG1 gene on chromosome 8, which is susceptible to translocation [Adelaide 2003].

[0224] OV-10-0050 was found to be HER-dependent. Treatment with afatinib (an irreversible inhibitor of EGFR and HER2 that also inhibits HER3 transphosphorylation) resulted in inhibition of tumor growth. The antitumor efficacy of MCLA-128 was compared to that of PBS (Figure 7, right panel).

[0225] Mice: NOD-SCID, Crl:NU(NCr)-Foxn1nu, and BALB / c nude mice. Antibodies were administered at a dose of 25 mg / kg for 4 weeks. Tumor volume was measured twice a week using calipers.

[0226] (Example 5) Phase I / II trial of MCLA-128, a full-length IgG1 bispecific antibody targeting HER2 and HER3, in patients with solid tumors. Test duration: Enrollment in the dose-escalation phase of the trial (Part 1, first patient administered on February 3, 2015) was completed after supplementing 28 patients. The first patients in Part 2 of the trial, the dose-escalation phase, were administered in Europe on January 15, 2016. The total duration of Part 2 is approximately 25–32 months; however, the actual duration is influenced by several variables, such as the overall patient supplementation rate.

[0227] Number of locations: It is estimated that up to 13 locations will be involved during the trial. Additional locations may be added to ensure that an acceptable enrollment rate exists or to replace non-enrollment / discontinuation locations.

[0228] Number of patients: Twenty-eight patients were enrolled in Part 1. For Part 2, at least 20 and up to approximately 40 evaluable patients may be enrolled in a group of invasive mucinous adenocarcinomas or advanced / metastatic non-small cell lung cancer (NSCLC) with reported NRG1 fusions.

[0229] Patients who fail to complete at least two cycles of the test treatment for reasons other than disease progression are deemed unable to be evaluated for efficacy and will be replaced within their respective groups.

[0230] This example describes Part 2. This example describes the administration of the Erb-2, Erb-3 conjugated bispecific antibody MCLA-128, but this example is not intended to limit the use of this specific embodiment and applies to other bispecific antibodies disclosed herein.

[0231] [Table 6]

[0232] [Table 7]

[0233] Test design: This is a Phase I / II open-label, multicenter, multinational, dose-escalation, single-arm allocation study to evaluate the safety, tolerability, pharmacokinetics, disease progression, immunogenicity, and antitumor activity of MCLA-128.

[0234] This exam is designed in two parts: Part 1 Enrollment for Part 1 of the trial was achieved on November 24, 2015, and as of January 24, 2017, all Part 1 patients had completed the trial. Nine dose levels were investigated: 40 mg, 80 mg, and 160 mg in a single-patient cohort, and 240 mg, 360 mg, 480 mg, 600 mg, 750 mg, and 900 mg in a three-patient cohort. MCLA-128 was initially administered over approximately 60 minutes on day 1 of a three-week treatment cycle. During Part 1, the infusion duration was extended to 2 hours, with the option to increase it to 4 hours to reduce infusion-related reactions (IRRs).

[0235] No dose-limiting toxicity (DLT) was experienced at any of these dose levels. To obtain sufficient PK information, three additional patients were administered to each of the 600 mg and 750 mg cohorts.

[0236] Since the 900 mg dose level did not reach the MTD, the Data Review Committee (DRC) for MCLA-128-CL01 decided to assign a 750 mg dose level as the RP2D for the study, based on cumulative safety, available PK data, and PK simulations.

[0237] Part 2 Part 2 includes further characterization of the safety and tolerability of selected dose levels of MCLA-128, as well as an assessment of the chronicity rate (CBR), defined as the proportion of patients with complete response (CR), partial response (PR), or sustained stable disease (SD with a duration of at least 12 weeks) in the selected patient population.

[0238] In newly supplemented patients, a weekly dose regimen using a 4-week cycle will be evaluated, consisting of a fixed dose of 400 mg per week for the first two cycles, along with an 800 mg loading dose for the initial administration. From cycle 3 onwards, MCLA-128 will be administered at a dose of 400 mg per week for 3 weeks, followed by a 1-week rest period. Mandatory pre-treatment medications will be administered to reduce the IRR. However, corticosteroids are mandatory only before the loading dose on day 1 of cycle 1, and subsequent infusions to manage the IRR should only be used at the discretion of the principal investigator.

[0239] The safety of the weekly schedule will be reviewed during the adjustment period after the first five treated patients have completed at least two treatment cycles. The DRC will review all safety data, focusing on the incidence of grade 3–4 toxicity, the incidence and severity of IRRs, and medication adherence. If the DRC review concludes that toxicity is unacceptable, the trial sponsor will continue patient enrollment with the 3-week cycle dose regimen until a sufficient number of patients per cohort have been enrolled.

[0240] Intra-patient dose escalation is not permitted in Part 2.

[0241] The target patient population to be evaluated in Part 2 of the trial is as follows: • Open only to NSCLC-Asia replenishment with reported NRG1 fusions.

[0242] Each cohort (C-F) can enroll at least 20 and up to approximately 40 patients, with a minimum of 10 patients per cohort, receiving weekly treatment at the recommended dose. Previously closed cohorts may be reopened.

[0243] Duration of treatment Patients in both Part 1 and Part 2 of the trial may continue treatment until disease progression, death, unacceptable toxicity, or discontinuation for any other reason.

[0244] Data Review Committee (DRC): All dose escalation decisions in Part 1 were made by the DRC, which was convened to review all available safety and PK data. DRC participants included the principal investigator (or their representative), the sponsor's medical director, the study medical monitor, the study drug safety surveillance physician, the study project manager, the study statistician, and experts invited as needed (e.g., clinical pharmacology experts).

[0245] In Part 2, the DRC will review data from all subsequent patients after the completion of the safety adjustment period for the weekly dose before increasing the weekly dose regimen.

[0246] Test evaluation: This trial consists of a screening period of up to 4 weeks (28 days) for molecular pre-screening evaluation, followed by sequential treatment cycles until treatment is discontinued or terminated for any reason. The duration of the treatment cycle is 3 weeks (21 days) for patients treated with the initial recommended dose in Part 2, and 4 weeks (28 days) for patients treated with the weekly recommended dose in Part 2. All patients must attend follow-up appointments within one week of treatment discontinuation and at the final trial appointment 30 days after the end of treatment or discontinuation of the trial.

[0247] Patients who do not progress in their disease or withdraw their consent to complete their final trial visit will be followed up every three months for up to two years (approximately) to check their disease progression and / or survival status until the start of the next anti-cancer treatment.

[0248] If ongoing evaluations of safety data during the trial, as well as available PK, PD, and antitumor activity data, suggest that alternative dosing frequencies should be evaluated, or that other patient populations should be evaluated in Part 2, these modifications will be clearly stated in protocol modifications before initiating these evaluations.

[0249] Aqueous pre-screening and screening: Molecular pre-screening will be conducted in a local laboratory qualified to perform molecular screening for NRG1 fusions. To initiate pre-screening, patients must meet one of the following criteria: • Histological diagnosis of IMA and absence of reported EGFR / ALK alterations. Note: IMA patients who did not undergo pre-screening for NRG1 fusions may enter the trial. or • Pathological examination may not lead to a diagnosis of IMA, but the principal investigator may suspect IMA based on symptoms, imaging features (e.g., localized consolidation, multiple bilateral nodules or sclerosis), non-smoker status, and the absence of reported EGFR / ALK alterations.

[0250] Before submitting fresh or archived tumor tissue for analysis to determine NRG1 fusion status, NSCLC patients identified as potential trial participants must sign an Informed Consent Form (ICF) for Molecular Pre-Screening. The trial can be conducted at any point in the natural course of the disease (e.g., at diagnosis, during first-line treatment, during progression, etc.) up to one year prior to day 1 of cycle 1. Fresh tumor samples (formalin-fixed paraffin-embedded; FFPE) or archived tumor samples no older than one year are required for assessment of the presence of NRG1 fusions. Samples should be submitted to a local laboratory qualified to perform molecular profiling (PCR, next-generation sequencing [DNA or RNA], or FISH) for NRG1 fusion status. Patients with positive local NRG1 fusion results are then eligible to sign the Trial ICF if they are willing to and able to enter the trial.

[0251] Main Informed Consent Forms All patients must sign the Study ICF before any screening procedure or evaluation is performed. Screening evaluations should be performed within four weeks prior to day 1 of cycle 1, with the exception of serum pregnancy tests, which should be performed within seven days of day 1 of cycle 1. A baseline mandatory tumor sample, preferably a fresh or archived tissue block, is required for screening consideration. The study sponsor should indicate a preference for fresh tissue. Archives are acceptable and must be taken within two years of screening, except for NSCLC, which must be within one year. It should be noted that for NSCLC patients, a baseline biopsy for screening is still required even if a pre-screening biopsy sample is provided for the regional trial of pre-screening for NRG1. After completion of all required screening evaluations and confirmation of all eligibility criteria, patients may begin medication on day 1 of cycle 1.

[0252] Safety evaluation Concurrent illnesses will be captured at baseline; adverse events (AEs) and concomitant therapies will be monitored throughout the study. Safety assessments will include reviewing Eastern Cooperative Oncology Group (ECOG) activity indices, physical examination (including height and weight), vital signs, and electrocardiogram (ECG). Left ventricular ejection fraction (LVEF) cardiac function tests will also be performed at screening, at the end of cycle 4 (or day 1 of cycle 5), at the end of the study visit, and at any point during the study if clinically necessary. Laboratory assessments will include clinical chemistry, hematology, coagulation tests, urinalysis, and pregnancy tests. Note that cytokine panel analysis was performed until August 1, 2017.

[0253] On all MCLA-128 administration days, patients must remain at the clinic for at least 60 minutes (longer if PK sampling is required) from the end of the infusion for observation and repeated vital sign checks before discharge from the clinic. Further additional safety assessments should be performed where clinically necessary, and the duration of clinic stay should be increased at the discretion of the principal investigator if necessary.

[0254] Immunogenicity evaluation The serum titer of anti-MCLA-128 antibody was measured at the time of pre-medication on day 1 of each cycle (1, 2, 3, and 4), and then measured every four cycles thereafter (cycles 8, 12, 16, etc.), as well as at the end of treatment visits and the final examination visit, within a 3-day window before MCLA-128 administration.

[0255] Pharmacokinetic evaluation Recommended initial dose schedule for Part 1 and Part 2: In Cycle 1, blood samples are collected for PK analysis at the time of pre-medication on Day 1, at the end of infusion (EOI), and 1, 2, 4, 8, and 24 hours after EOI, and then on Day 4 (or Day 3), Day 8, and Day 15. In Cycles 2-4, only blood samples are collected at the time of pre-medication and EOI.

[0256] Part 2 Weekly Recommended Dosage Schedule: In Cycle 1, blood samples are collected for PK analysis at the time of pre-dose on day 1, at EOI, 2, 4, and 24 hours after EOI, then at the time of pre-dose on days 8 and 15, and at the time of pre-dose and EOI on day 22. In Cycles 2 and 3, blood samples from pre-dose and EOI are collected on day 15. In Cycle 4, blood samples are collected at the time of pre-dose on day 1, and at the time of pre-dose and EOI on day 15. Thereafter, pre-dose blood samples are collected every two cycles (cycles 6, 8, 10, etc.) on day 15.

[0257] Tumor evaluation Tumor assessment will be performed according to the local principal investigator's instructions and in accordance with RECIST version 1.1. Images will be taken at screening, at the end of each two-cycle regimen for patients receiving a three-week cycle regimen, and every six weeks for patients receiving a four-week cycle regimen.

[0258] Biomarker and pharmacokinetic evaluation Various biomarker and pharmacokinetic studies are performed on archived and / or fresh tumor sample material and / or blood (liquid biopsy), depending on the availability of archived or existing tumor tissue, consent for further tumor samples, and consent for specific biomarker studies.

[0259] If sufficient samples are available, evaluate the following candidate biomarkers: HER2, HER3, HER2:HER3 dimers, phosphorylated HER2 (pHER2) and HER3 (pHER3), and heregulin; • Use circulating plasma tumor DNA (ctDNA) and tumor sample DNA (availability dependent) to test for mutations in oncogenes, including those associated with HER2 and HER3 signaling. • Phosphorylated molecules in the MAPK and AKT signaling pathways; Fc gamma receptor polymorphism; • Circulating tumor cells related to HER2; • Hereglin gene fusion

[0260] Germline DNA evaluation is not included (excluding Fc gamma receptor polymorphisms).

[0261] At baseline, patients are required to provide mandatory tumor sample tissue, preferably a block that may be derived from fresh or archived tissue. The sponsor will indicate a preference for fresh tissue. Archives are acceptable and must be collected within two years of screening, except for NSCLC which must be within one year. Furthermore, patients are optionally required to submit tumor samples / biopsies at the end of cycle 4 and at the end of treatment visits.

[0262] Blood samples are also collected at these points in time for the purpose of liquid biopsy testing.

[0263] Eligibility criteria: The trial will enroll patients with NSCLC.

[0264] General inclusion criteria for Part 2 1. 18 years of age or older; 2. At least one measurable lesion according to RECIST v1.1; 3. Activity indicators with an ECOG score of 0 or 1; 4. An estimated life expectancy of at least 12 weeks; 5. Toxicity suffered as a result of previous anticancer treatment (as defined by NCI CTCAE v4.03), excluding alopecia, lymphopenia not assessed as clinically significant, and grade 2 sensory neurotoxicity, and resolved to ≤ grade 1; 6. At least four weeks between the last radiotherapy treatment and the first scheduled day of MCLA-128 administration (with the exception of up to 1 × 8 Gy for pain relief); 7. Complete recovery from major surgery (stable and with tolerable toxicity of <Grade 2); 8. Laboratory values ​​at the time of screening: a. Neutrophil absolute count ≥ 1.5 × 10⁻¹⁰ without support of colony-stimulating factors 9 / L; b. Platelets ≧100×10 9 / L; c. Hemoglobin ≥ 9 g / dL or ≥ 2.2 mmol / L (not transfusion-dependent); d. Total bilirubin < 1.5 times the upper limit of normal (ULN) (unless caused by Gilbert's syndrome); e. Patients with advanced solid tumors accompanied by liver metastases, who have confirmed bone metastases and show isolated elevations of >5×ULN in AST (SGOT) ≤ 2.5×ULN; ≤ 5×ULN in ALT (SGPT); are acceptable for the study; f. Based on the Cockcroft-Gault formula, serum creatinine ≤ 1.5 × ULN or estimated glomerular filtration rate (GFR) > 50 mL / min; g. Coagulation function (INR and aPTT ≤ 1.5 × ULN, unless therapeutic anticoagulants are being used) h. Urine protein ≤ 2+ (measured by urine test strip) or ≤ 100 mg / 24 hours in urine; 9. At baseline, mandatory tumor biopsy samples (FFPE), preferably blocks derived from fresh (preferred) or archived tissue, can be provided. Archived tissue must be collected within two years prior to screening, except for NSCLC which must be within one year. 10. Available negative pregnancy test results, as defined by a urine or blood human chorionic gonadotropin (hCG) test, during screening and within 7 days of day 1 of cycle 1, in women of childbearing potential (defined as women under 50 years of age or with a history of amenorrhea for 12 months or less prior to entering the study); 11. Sexually active male and female patients of childbearing potential must consent to the use of effective methods of fertility control (e.g., barrier methods using spermicides, oral or parenteral contraceptives and / or intrauterine devices) for the entire duration of the study and for six months following the last dose of MCLA-128. It should be noted that female infertility should be confirmed in the patient's medical records and should be defined as any of the following: surgical hysterectomy with bilateral oophorectomy, bilateral ductal ligation, spontaneous menopause with last menstruation >1 year prior; radiation-induced oophorectomy with last menstruation >1 year prior; chemotherapy-induced menopause with a one-year interval since last menstruation; 12. The ability to give written informed consent before any trial-specific screening procedure, with an understanding that the patient can withdraw their consent at any point without infringing their rights; 13. The participant is able to understand the mandatory and optional protocol requirements, is willing and able to follow the trial protocol procedures, and has signed the primary informed consent document. Further consent is required for optional biopsy sampling (tissue and / or blood) and long-term sample storage; 14. Patients with metastatic cancer who have experienced disease progression after treatment with all available therapies known to have clinical utility. 15. Unresectable or metastatic NSCLC that meets one of the following conditions: • Invasive mucinous adenocarcinoma (IMA) confirmed by biopsy. Note: IMA patients who did not undergo a pre-screening test for NRG1 fusions are eligible to participate in the study. or NSCLC with reported NRG1 fusions, determined by molecular profiling using methods such as PCR, next-generation sequencing [DNA or RNA], or FISH, in patients who do not have known driver mutations or fusions in the EGFR / ALK gene, in a laboratory in an eligible region. 16. Reported disease progression, as assessed by the principal investigator, to at least one line of standard treatment in locally advanced or metastatic settings.

[0265] Statistical analysis: Part 1 and Part 2 Antitumor and clinical utility variables are described descriptively for each group in Part 2. Where necessary, variables are shown in terms of absolute and relative changes from baseline. Categorical data are presented as percentages and frequencies in tables.

[0266] If necessary, data from patients receiving what is identified as MTD or MRD during Part 1 and patients receiving the same dose in Part 2 can be combined and compiled, or compiled independently.

[0267] The frequency and nature of serious and non-serious adverse events (AEs) will be evaluated using absolute and relative frequencies and coded according to the MedDRA medical terminology glossary.

[0268] Part 1 The data evaluation is descriptive in nature. Patient demographics, disease characteristics, and pharmacokinetic and pharmacodynamic variables are summarized at each dose level. The frequency and nature of DLTs are also summarized at each dose level.

[0269] Part 2 Using N=20 per cohort in Part 2, at least 0.38 observed clinically significant correlation coefficients were discriminable from zero with 95% confidence; smaller, observed clinically insignificant correlations were not discriminable from zero. Therefore, 20 subjects per cohort in Part 2 is considered sufficient to investigate the association between the antitumor activity of MCLA-128 and disease-related biomarkers.

[0270] If signs of clinical activity are observed, up to approximately 40 additional patients may be added in total. Using 40 patients, for example, a true clinical response rate of 10% to 50% can be estimated with a reasonable accuracy of approximately ±5% to ±8%.

[0271] PK parameters are summarized for each cohort in Part 1 and each tumor group in Part 2. Arithmetic and geometric means are provided in addition to median, range, SD, and %CV. AUC is calculated according to the trapezoidal rule. Serum concentration profiles against time are plotted for each group.

[0272] (References) Yarden Y, Pines G.2012. The ERBB network: at last, cancer therapy meets systems biology. Nat Rev CancerJul 12;12(8):553-63. 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. Balko JM, Miller TW, Morrison MM, Hutchinson K, Young C, Rinehart C, Sanchez 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 U S A. Jan 3;109(1):221-6. 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. 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. 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. 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. 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 Merchant et al. Nature Biotechnology, Vol. 16 July 1998 pp 677-681 Adelaide et al. (2003) Genes Chromosome Cancer, 37(4), 333. Agus et al. (2002) Cancer Cell 2(2), 127. Birnbaum et al (2003). Lancet Oncol 4: 639-642. Chua et al (2009). Oncogene 28, 4041-4052 Cooke et al (2008). BMC Cancer 8: 288. Duruisseaux et al. (2016) NRG1 fusion in a French cohort of invasive mucinous lung adenocarcinoma. Canc Med. Falls DL. (2003). Exp Cell Res 284: 14-30. Fernandez-Cuesta et al. (2014) Canc Disc. 4(4), 415. Fernandez-Cuesta and Thomas (2015). Clinical Cancer Research 21(9): 1989-1994. Hayes and Gullick (2008). J Mammary Gland Biol Neoplasia 13:205-214. Jung et al. (2015) J Thor Oncol 10(7), 1107. Juntilla et al. (2009) Cancer Cell 15(5), 429. Pole et al (2006). Oncogene 25:5693-5706. Sanchez-Valdivieso et al. (2002) Br J Canc, 86(8), 1362. Schoeberl et al. (2010) Canc Res 70(6), 2485. Weinstein et al. (1998). Oncogene 17:2107-2113.

Claims

1. A composition comprising a bispecific antibody for treating individuals having ErbB-2 and ErbB-3 positive cells, the antibody comprising a first antigen-binding site capable of binding to the extracellular portion of ErbB-2 and a second antigen-binding site capable of binding to the extracellular portion of ErbB3, wherein the cells comprise an NRG1 fusion gene containing at least a portion of the NRG1 gene fused to sequences derived from different chromosomal locations, the first antigen-binding site capable of binding to domain I of ErbB-2, and the ErbB-2 specific heavy chain variable region MF3958 comprising CDR1 having the amino acid sequence AYYIN, CDR2 having the amino acid sequence RIYPGSGYTSYAQKFQG and the amino acid sequence PPVYYDSAWF A composition comprising a CDR3 sequence containing AY, wherein the second antigen-binding site can bind to domain III of ErbB-3, and the ErbB-3 specific heavy chain variable region MF3178 comprises a CDR1 sequence having the amino acid sequence GYYMH, a CDR2 sequence having the amino acid sequence WINPNSGGTNYAQKFQG, and a CDR3 sequence having the amino acid sequence DHGSRHFWSYWGFDY, and both variable domains comprising a light chain variable region comprising a CDR1 sequence having the amino acid sequence RASQSISSYLN, a CDR2 sequence having the amino acid sequence AASSLQS, and a CDR3 sequence having the amino acid sequence QQSYSTPPT, wherein the bispecific antibody is provided in a dose of 750 mg.

2. The composition according to claim 1, wherein the NRG1 fusion gene comprises at least the 3' end of an NRG1 gene fused to a 5' sequence derived from a different chromosomal location.

3. The composition according to claim 1 or 2, wherein the cells are cancer cells.

4. The composition according to claim 3, wherein the cancer cells are driven by the NRG1 fusion.

5. The composition according to any one of claims 1 to 4, wherein the cells are breast cancer cells, ovarian cancer cells, lung cancer cells such as non-small cell lung cancer cells, pancreatic cancer cells, or metastases thereof.

6. The composition according to any one of claims 1 to 5, wherein the cells are lung cancer cells such as non-small cell lung cancer cells.

7. The composition according to any one of claims 1 to 5, wherein the cells are pancreatic cancer cells.

8. A composition comprising a bispecific antibody for treating individuals with ErbB-2 and ErbB-3 positive tumors, comprising a first antigen-binding site capable of binding to the extracellular portion of ErbB-2 and a second antigen-binding site capable of binding to the extracellular portion of ErbB-3, wherein the tumor cells express an NRG1 fusion gene comprising at least the 3' end of an NRG1 gene fused to a 5' sequence derived from a different chromosomal location, the first antigen-binding site capable of binding to domain I of ErbB-2, and the ErbB-2 specific heavy chain variable region MF3958 comprising CDR1 having the amino acid sequence AYYIN, CDR2 having the amino acid sequence RIYPGSGYTSYAQKFQG and the amino acid sequence PPVYYD A composition comprising a CDR3 sequence containing SAWFAY, wherein the second antigen-binding site can bind to domain III of ErbB-3, and the ErbB-3 specific heavy chain variable region MF3178 comprises a CDR1 sequence having the amino acid sequence GYYMH, a CDR2 sequence having the amino acid sequence WINPNSGGTNYAQKFQG, and a CDR3 sequence having the amino acid sequence DHGSRHFWSYWGFDY, and both variable domains comprise a light chain variable region comprising a CDR1 sequence having the amino acid sequence RASQSISSYLN, a CDR2 sequence having the amino acid sequence AASSLQS, and a CDR3 sequence having the amino acid sequence QQSYSTPPT, wherein the bispecific antibody is provided in a dose of 750 mg.

9. The composition according to claim 8, wherein the tumor is gastric cancer, colorectal cancer, colon cancer, gastroesophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, breast 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.

10. The composition according to claim 8, wherein the tumor is breast cancer, ovarian cancer, lung cancer such as non-small cell lung cancer, or a metastasis thereof.

11. The composition according to claim 8, wherein the tumor is a lung cancer such as non-small cell lung cancer.

12. The composition according to claim 8, wherein the tumor is pancreatic cancer.

13. The composition according to any one of claims 1 to 12, wherein the NRG1 fusion gene expresses a protein containing an NRG1 EGF-like domain.

14. The composition according to any one of claims 1 to 13, wherein the NRG fusion is a fusion of NRG1 and a gene on human chromosome 8.

15. The composition according to claim 14, wherein the gene on human chromosome 8 encodes an efflux protein or a cell membrane-associated protein.

16. The composition according to any one of claims 1 to 15, wherein the NRG1 fusion gene is a fusion of the 3' end of the NRG1 gene and the 5' sequence of one gene selected from the group consisting of CD74, DOC4, TNFRSF10B, CLU, VAMP2, SLC3A2, RBPMS, WRN, SDC4, KIF13B, SLECA2, PDE7A, ATP1B1, CDK1, BMPR1B, MCPH1, and RAB2IL1.

17. The composition according to any one of claims 1 to 16, wherein the cells or tumor are of epithelial origin.

18. The first antigen-binding site includes the heavy chain variable region sequence QVQLVQSGAEVKKPGASVKLSCKASGYTFTAYYINWVRQAPGQGLEWIGRIYPGSGYTSYAQKFQGRATLTADESTSTAYMELSSLRSEDTAVYFCARPPVYYDSAWFAYWGQGTLVTVSS, The second antigen-binding site is the heavy chain variable region sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDHGSRHFWSYWGFDYWGQGTLVTVSS A composition according to any one of claims 1 to 17, comprising:

19. The first antigen-binding site and the second antigen-binding site are located in the light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK A composition according to any one of claims 1 to 18, comprising:

20. The composition according to any one of claims 1 to 19, wherein the bispecific antibody is a full-length antibody of the human IgG subclass.

21. The composition according to any one of claims 1 to 20, wherein the bispecific antibody is not fucosylated.

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