Erbb-2 and erbb-3 binding bispecific antibodies for use in treatment of cells that have 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 to 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 from different chromosomal locations.

[0003] Neuregulin-1 (NRG1) has been proposed as a candidate oncogene and candidate tumor suppressor gene. Since it encodes a ligand that can bind to receptors of the ErbB family, it is highly likely to be involved in epithelial cancer. To date, there are more than 16 soluble and transmembrane proteins derived 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 the receptor tyrosine kinases ErbB-3 and ErbB-4. The NRG1 gene and isoforms are known under several different aliases as follows: Neuregulin 1; pro-NRG1; HRGA; SMDF; HGL; GGF; NDF; NRG1 intron transcript 2 (non-protein coding); 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 forms that are transmembrane, extracellular membrane-bound, shedding, secreted, or intracellular (Falls, 2003; Hayes and Gullick, 2008). These bind to ErbB-3 or ErbB-4, and they probably signal as heterodimers with ErbB-2 (HER2). The proteins encoded by NRG1 are usually considered mitogens, but they can also be strongly apoptosis-promoting: in particular, expressing NRG1 in cells can cause apoptosis of the expressing cells (Weinstein et al., 1998).

[0005] The NRG1 gene has been identified as a potentially important gene in cancer in two apparently contradictory contexts. First, it is a major candidate for the main 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 continuously shown by loss of heterozygosity, comparative genomic hybridization (CGH), and array-CGH tests (see Birnbaum et al., 2003; Pole et al., 2006 for references). The classical interpretation of this loss of chromosome 8p is that there is a tumor suppressor gene there. Loss of chromosome 8p in cancer cell lines has been mapped using fluorescence in situ hybridization and array-comparative genomic hybridization (array-CGH). Since most breakpoints were found to be proximal to or virtually within NRG1, NRG1 and the genes immediately telomeric to NRG1 are major candidates for such a tumor suppressor (Pole et al., 2006; Cooke et al., 2008). Second, NRG1 has the potential to be an oncogene as it appears to be the target of chromosomal translocations in breast cancer (see Chua et al. 2009 for review).

[0006] In the present invention, it has been found that tumors having a chromosome 8p modification exhibit growth inhibition in response to exposure to a bispecific 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.

Prior Art Documents

Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0009] In one aspect, a method for treating an individual having ErbB-2 and ErbB-3 positive cells, the method comprising administering to the individual in need thereof a bispecific 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, wherein the cells comprise an NRG1 fusion gene comprising at least a portion of the NRG1 gene fused to sequences from different chromosomal locations. Typically, the cells comprise an NRG1 fusion gene comprising at least the 3' end of the NRG1 gene fused to a 5' sequence from different chromosomal locations.

[0010] These cells can be cancer cells. These cancer cells can be cancer cells associated with the NRG1 fusion gene, for example, cancer cells driven by the NRG1 fusion.

[0011] In another aspect, a method for treating an individual having, or at risk of having, an ErbB-2 and ErbB-3 positive tumor, the method comprising administering to the individual in need thereof a bispecific 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, wherein the cells of the tumor comprise an NRG1 fusion gene comprising a portion of an NRG1 fusion, such as the 3' end of the NRG1 gene, fused to sequences from different chromosomal locations, such as a 5' sequence.

[0012] Individuals at risk of having an ErbB-2 and ErbB-3 positive tumor can be individuals in a remission state.

[0013] Preferably, the NRG1 fusion gene expresses a protein containing 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 a secreted 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 of the genes 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 a non-small cell lung cancer cell, or a metastasis thereof.

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

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

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

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

[0019] Preferably, the individual has preferably experienced (already received) a treatment targeting (targeting) EGFR inhibition, preferably using an EGFR-binding antibody that is preferably cetuximab.

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

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

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

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

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

[0025] In particular, a bispecific antibody for use in the treatment of an individual 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 comprises an NRG1 fusion gene comprising at least the 3'-end of the NRG1 gene fused to a 5'-sequence from a different chromosomal location.

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

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

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

[0029] Preferably, the first antigen-binding site and the second antigen-binding site comprise 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 comprises CDR1 having the sequence (RASQSISSYLN), CDR2 having the sequence (AASSLQS), and CDR3 having the sequence (QQSYSTPPT).

Mode for Carrying Out the Invention

[0030] The NRG1 fusion gene contains at least a portion of the NRG1 gene fused to sequences from different chromosomal locations. "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 the NRG1 fusion gene is that it contains the EGF-like domain of NRG1. At least a portion of the NRG1 gene can be fused to a sequence such that the sequence from a different chromosomal location is located 5' or 3' to at least a portion of the NRG1 gene.

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

[0032] The 3' end of the NRG1 gene in the fusion, e.g., the NRG-1 gene, preferably has at least the coding sequences 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 necessary for binding to ErbB-3. The NRG1 fusion retains 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 in length, 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]. It is known to exist in more conserved or less conserved forms in a number of other mostly animal proteins. A common feature of EGF-like domains is that they are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G / H synthase). The EGF domain typically contains six cysteine residues (in EGF) that have been shown to be involved in disulfide bonding. Its main structure is a double-stranded beta-sheet followed by a loop to a short C-terminal double-stranded sheet. The subdomains between conserved cysteines 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 of the genes 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] The NRG1 fusion gene can be a fusion of at least a portion of the NRG1 gene and a sequence derived from a different chromosomal location located 3' to it. Such an NRG1 fusion gene can be a fusion of at least a portion of the NRG1 gene and a sequence derived from a different chromosomal location CD74, STMN2, PMEPA1, PROSC or PSAP 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. The epidermal growth factor (EGF) receptor (EGFR, ErbB1 or HER1). These receptors (reviewed 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, 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 the basal on luminal epithelium (Balko, Miller et al., 2012). Ligand binding to the extracellular domain of RTK induces receptor dimerization both between the same (homo-dimerization) receptor subtypes and between different (hetero-dimerization) receptor subtypes. Dimerization can activate the intracellular tyrosine kinase domain that undergoes autophosphorylation and can then activate several downstream growth-promoting signaling pathways, including those mediated by the mitogen-activated protein kinase (MAPK) and the survival-promoting pathway Akt (reviewed in Yarden and Pines, 2012).Specific endogenous ligands have not been identified for ErbB-2, and thus it is generally assumed to signal through heterodimerization (Sergina, Rausch et al., 2007). ErbB-3 can be activated by the association of its ligands. These ligands include, but are not limited to, neuregulin (NRG) and heregulin (HRG).

[0035] ErbB-1 is known under various aliases, the most common of which is EGFR. EGFR has an extracellular domain (ECD) composed 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) mitogenic signaling cascade. Activation of this pathway is initiated by the recruitment of Grb2 to tyrosine-phosphorylated EGFR. This leads to the activation of Ras via Grb2-bound Ras-guanine nucleotide exchange factor, son of sevenless (SOS). Furthermore, the PI3-kinase-Akt signaling pathway is also activated by EGFR, but this activation is considerably stronger when co-expression of ErbB-3 (HER3) is present. EGFR is associated with several human epithelial malignancies, particularly cancers of the breast, bladder, non-small cell lung, colon, ovary, head and neck, and brain. Activating mutations in this gene, as well as overexpression of the receptor and its ligands, which give rise to an autocrine activation loop, have been found. Thus, this RTK is widely used as a target for cancer therapy. Small molecule inhibitors that target the RTK and monoclonal antibodies (mAbs) against the extracellular ligand-binding domain have both been developed and have shown some clinical success, mostly for selected groups of patients. The database accession number for the human EGFR protein and the gene encoding it is (GenBank NM_005228.3). This accession number is mainly provided to offer an additional means of identifying the EGFR protein as a target, and the actual sequence of the EGFR protein to which the antibody binds can vary due to mutations in the coding gene, such as those present in some cancers.

[0036] The terms cancer and tumor are used herein to refer to cancer, typically both, unless specifically noted otherwise.

[0037] When reference is made to EGFR in this specification, unless otherwise specified, this reference refers to human EGFR. The antigen-binding site that binds to EGFR binds to EGFR and its various variants, for example, those expressed on some EGFR-positive tumors.

[0038] The term "ErbB-3", as used in this specification, refers to the protein encoded by the ERBB3 gene in humans. Alternative names for this gene or protein are HER3; LCCS2; MDA-BF-1; c-ErbB-3; c-ErbB3; ErbB3-S; p180-ErbB3; p45-sErbB3; and p85-sErbB3. When reference is made to ErbB-3 in this specification, this reference refers to human ErbB-3. An antibody containing an antigen-binding site that binds to ErbB-3 binds to human ErbB-3. The ErbB-3 antigen-binding site may also bind to such orthologs, but not necessarily, due to sequence and tertiary structure similarities between the human ortholog and other mammalian orthologs. The database accession numbers for the human ErbB-3 protein and the gene encoding it are (NP_001005915.1, NP_001973.2, NC_000012.11, NC_018923.2, NT_029419.12). These accession numbers are provided primarily to provide additional methods for the identification of 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, for example, 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] As used herein, the term "ErbB-2" 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 reference is made to ErbB-2 herein, such reference refers to human ErbB-2. Antibodies containing antigen-binding sites that bind to ErbB-2 bind to human ErbB-2. Due to sequence and tertiary structure similarities between the human ortholog and other mammalian orthologs, the ErbB-2 antigen-binding site may also bind to such orthologs, but not necessarily. Database accession numbers for the human ErbB-2 protein and the gene encoding it are (NP_001005862.1, NP_004439.2, NC_000017.10, NT_010783.15, NC_018928.2). These accession numbers are provided primarily to provide additional 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 alternative names. Some of these are: CD74 molecule; CD74 antigen (major histocompatibility complex invariant polypeptide, 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; Tenasin - 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 Tumor Necrosis Factor Receptor Superfamily Member 10b, is known under several different names, Tumor Necrosis Factor Receptor Superfamily, Member 10b; TNF-Related Apoptosis-Inducing Ligand Receptor 2; Death Receptor 5; TRAIL-R2; TRAILR2; KILLER; TRICK2; ZTNFR9; DR5; p53-Regulated DNA Damage-Induced 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 Repressed Prostate Message 2; Apolipoprotein J; Complement-Related Protein SP-40,40; Complement Cytolysis 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 Repressed 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 CLU are HGNC:2095; Entrez Gene:1191; Ensembl:ENSG00000120885; OMIM:185430; and UniProtKB:P10909.

[0044] VAMP2, or vesicle-associated membrane protein 2, is known under several different names, such as synaptobrevin 2; SYB2; vesicle-associated 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] SLC3A2, or solute carrier family 3 member 2, is known under several different names, such as lymphocyte activation antigen 4F2 heavy subunit; solute carrier family 3 (activator of dibasic and neutral amino acid transport), member 2; antigen identified by monoclonal antibodies 4F2, TRA1.10, TROP4 and T43; solute carrier 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 as well as 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 multiple splicing, is known under several different names, such as RNA-binding protein with multiple splicing; heart and RRM expressed sequence; HERMES; RNA-binding protein with multiple splicing; 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, such as 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, such as 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 in Dhanasekaran et al. (2014).

[0050] The present invention provides a method of treating an individual having ErbB-2 and ErbB-3 positive cells or tumors. Alternatively, the individual may be at risk of having a tumor. The method comprises administering to an individual in need thereof a bispecific 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) comprise a NRG1 fusion gene comprising the 3' end of the NRG1 gene fused to a 5' sequence from a different chromosomal location.

[0051] The cell can be a cancer cell. This cancer cell can be a cancer cell associated with an NRG1 fusion, for example, a cancer cell driven by an NRG1 fusion.

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

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

[0054] The method of treatment 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 cell(s) of the tumor.

[0055] The method of treatment of the present invention preferably further comprises the step of determining whether the cell contains an NRG1 fusion or whether the 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 an NRG1 fusion, the regions where chromosomal breakage occurs are known, so it is routine for those skilled in the art to determine whether a tumor contains such an NRG1 fusion. One method is by PCR amplification using primers spanning the junction in the NRG1 fusion. This can be readily performed for known NRG1 fusions that are known to exist. New fusions can also be readily detected. For example, a suitable method is, for example, by junction cloning techniques used to find the integration site of the retroviral genome. A suitable method is by LAM-PCR. See Schmidt et al. Nature Methods 4, 1051 - 1057 (2007) doi:10.1038 / nmeth1103 and the specific references to LAM-technology therein.

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

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

[0058] The method of treatment as defined herein can also be defined as a compound or combination of compounds for use in the treatment of. A suitable combination of compounds is the bispecific antibody and ErbB-1 inhibitor as defined herein.

[0059] In order to establish whether the cell or tumor is positive for ErbB-2 and ErbB-3, one of ordinary skill in the art can determine, for example, the amplification of ErbB-2 and ErbB-3 and / or the staining in immunohistochemistry. At least 10% of the tumor cells in the biopsy should be positive for both ErbB-2 and ErbB-3. The biopsy can also contain 20%, 30%, 40%, 50%, 60%, 70% or more positive cells. ErbB-1 positive tumors can be identified in the same way.

[0060] Preferably, this positive cancer is breast cancer, for example, early breast cancer. However, the present invention is applicable 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 cell or tumor is preferably a cell or tumor of epithelial origin. In a preferred embodiment, the cell or tumor is breast cancer, ovarian cancer, lung cancer, or metastases thereof. Preferably, the tumor is of epithelial origin. Preferably, the tumor is breast cancer, ovarian cancer, lung cancer, or metastases thereof.

[0061] Patients having ErbB2-positive cells or tumor cells can be classified based on the number of ErbB-2 receptors on the surface of the tumor cells. Tumors having more than 1,000,000 ErbB-2 receptors on their cell surface are typically classified as ErbB-2[+++], tumors having between 150,000 and 1,000,000 ErbB-2 receptors are classified as ErbB-2[++], and tumors having less than 150,000 ErbB-2 receptors are classified as ErbB-2[+]. Preferably, the patient is 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, provided is a method in which ErbB-2 / ErbB-3 positive cells or tumors have at least 150,000 ErbB-2 cell surface receptors per cell and less than 50,000 ErbB-3 cell surface receptors per cell. Preferably, provided is a method in which ErbB-2 / ErbB-3 positive cells or tumors have at least 1,00,000 ErbB-2 cell surface receptors per cell 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 first determined, for example, based on ErbB-1, ErbB-2, and / or ErbB-3 cell surface receptor density. Accordingly, the methods disclosed herein preferably include the step of determining the ErbB-1 cell surface receptor density, the ErbB-2 cell surface receptor density, the ErbB-3 cell surface receptor density, and / or the ErbB-4 cell surface receptor density for this cell or tumor. As used herein, the term cell surface receptor density refers to the number of receptors present on the cell surface per cell.

[0064] Preferably, the method disclosed herein further comprises determining the ErbB-2 cell surface receptor density for this cell or tumor. Patients can be classified using immunohistochemistry or fluorescence in situ hybridization. The use of 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 of skill 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 method disclosed herein further comprises determining the ErbB-2 cell surface receptor density for this cell or tumor. Such methods are known to those of skill 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 method disclosed herein further comprises determining the ErbB-1 cell surface receptor density for this cell or tumor. Such methods are known to those of skill in the art (see, for example, EGFR pharmDx™ Kit (Dako) and McDonagh et al Mol Cancer Ther 2012; 11:582). Similar methods can be used to determine ErbB-4 cell surface receptor density.

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

[0066] Preferably, ErbB-2 / ErbB-3 positive cells or tumor cells have a relatively high level 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 a high level of heregulin (referred to as heregulin stress), currently known treatments such as trastuzumab, pertuzumab, and lapatinib can no longer inhibit the growth of the cells or tumors. This phenomenon is called heregulin resistance. In particular, the heregulin expression level is higher than that of MCF7 cells. The heregulin expression level can be measured, for example, using qPCR with cell or tumor RNA (as described, for example, 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 a protein detection method such as ELISA, preferably using blood, plasma, or serum samples (as described, for example, in Yonesaka et al., Sci. transl. Med., Vol. 3, No. 99 (2011); pp. 1-11, etc.).

[0067] High heregulin levels typically exist during the formation of metastases (i.e., the migration, invasion, growth, and / or differentiation of cells or 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, etc. Therefore, these processes can be barely countered by currently known treatments such as trastuzumab and pertuzumab. The bispecific antibodies disclosed herein can counter the formation of metastases in a subject having a cell tumor containing an NRG1 fusion gene that includes the 3' end of the NRG1 gene fused to a 5' sequence from a different chromosomal location.

[0068] Accordingly, a method for combating the formation of metastases in a subject having ErbB-2, ErbB-3 or ErbB-2 / ErbB-3 positive cells or tumors, comprising administering to the subject a bispecific antibody 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 that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells, is further provided. A bispecific antibody comprising a first antigen-binding site that binds to ErbB-2 and a second antigen-binding site that binds to ErbB-3 for use in the treatment or prevention of the formation of metastases, wherein the ErbB-2, ErbB-3 or ErbB-2 / ErbB-3 positive cells or tumor cells have a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells, is also provided. Use of a bispecific antibody according to the invention for the preparation of a medicament for the treatment or prevention of the formation of metastases, wherein the ErbB-2, ErbB-3 or ErbB-2 / ErbB-3 positive cells or tumor cells have a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells, is further provided.

[0069] The subject is preferably a human subject. The subject is preferably 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 a therapeutic window, which means that an amount sufficient to obtain a therapeutic effect is used, but that amount does not exceed a threshold that would result in unacceptable side effects. The lower the amount of antibody required to obtain the desired therapeutic effect, the larger the therapeutic window typically is. The selected dosage level depends on a variety of factors including the route of administration, the timing of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds and / or materials used in combination, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts. The dosage can be within the range of the dosing regimen for trastuzumab or can be lower.

[0071] The bispecific antibody can be formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient, and an additional optional active agent. The antibody and compositions containing the antibody can be administered by any route including parenteral, enteral and topical administration. Parenteral administration is usually by injection and includes, for example, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intraspinal, intracerebro spinal, intratumoral and intrasternal injections and infusions.

[0072] In a preferred embodiment, an ErbB-1 inhibitor can be combined with treatment with the bispecific antibodies disclosed herein. The ErbB-1 inhibitor can be administered simultaneously or sequentially with the bispecific antibody. Treatment with the ErbB-1 inhibitor can be separated from treatment with the bispecific antibody by minutes, hours or days. Preferably, the ErbB-2 / ErbB3 cells or tumors are also positive for ErbB1. Preferably, the combination treatment (combination therapy) is appropriate 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, more preferably more than 50,000 surface receptors per cell.

[0073] Suitable ErbB-1 inhibitors are known in the art and refer to compounds that inhibit at least one biological activity of ErbB-1 (EGFR), particularly compounds that reduce the expression or signaling activity of ErbB-1. Preferred ErbB-1 inhibitors bind to the extracellular binding site of this tyrosine kinase receptor molecule and block the binding of natural ligands such as EGF. Such inhibitors include antibodies, antibody portions, and peptides containing epitopes that target this extracellular EGF receptor binding domain. Preferably, the ErbB-1 inhibitor is an anti-ErbB-1 antibody preferably selected from cetuximab, matuzumab, necitumumab, nimotuzumab, panitumumab, or zalutumumab. 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 and prevent or reduce phosphorylation by tyrosine kinase. This can be achieved by small (chemical) molecule drugs. Preferred inhibitors include afatinib, erlotinib, gefitinib, lapatinib, osimertinib, and neratinib.

[0074] The present disclosure provides bispecific antibodies for use in the methods and treatments described herein. Suitable bispecific antibodies include 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 reduces or can reduce the ligand-induced receptor function of ErbB-3 on ErbB-2 and ErbB-3 positive cells. Preferred antibodies and their preparation are disclosed in WO2015 / 130173, which is incorporated herein by reference. The examples in WO2015 / 130173 further describe some properties of the antibodies, such as 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 present on such a bispecific antibody. An unmodified antigen-binding site is typically formed by the variable domains of an antibody and is present in the variable domains of such an antibody. The variable domain includes the antigen-binding site. The variable domain that binds to an antigen is a variable domain that includes an antigen-binding site that binds to the 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 can be present in the combined VH / VL variable domain, or only in the VH region, or only in the VL region. When the antigen-binding site is present only in one of the two regions of the variable domain, the corresponding variable region of the counterpart can contribute to the folding and / or stability of the binding variable region, but does not significantly contribute to the binding of the antigen itself.

[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 binds to ErbB-2 and binds at least 100-fold less to the homologous receptors ErbB-1 and ErbB-4 of the same species under the same other conditions. An antibody containing an antigen-binding site that binds to ErbB-3 binds to ErbB-3 and does not bind to the homologous receptors ErbB-1 and ErbB-4 of the same species under the same other conditions. Considering that the ErbB family is a family of cell surface receptors, binding is typically evaluated on cells expressing the receptor. The binding of an antibody 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] Antigen binding by an antibody is typically mediated through the specific three-dimensional structures of the antibody's complementary regions and both the antigen and variable domains, which precisely bind these two structures together (a lock-and-key-like interaction), as opposed to random non-specific attachment of the antibody. An antibody typically recognizes an epitope of an antigen, and since such an epitope may also be present in other compounds, an antibody according to the present invention that binds 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 binding of the antibody to another protein or a protein containing the same epitope. Such other proteins are preferably not human proteins. The ErbB-2 antigen-binding site and ErbB-3 antigen-binding site 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 1×10e-6M, as described in more detail below.

[0079] As used herein, the term "interfering with binding" means that the antibody is directed against an epitope on ErbB-3 and competes with the ligand for binding to ErbB-3. The antibody may reduce ligand binding, displace 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] As used herein, the term "antibody" means a proteinaceous molecule, preferably belonging to the immunoglobulin class of proteins that contains one or more variable domains that bind to an epitope on an antigen, where such domains are derived from the variable domains of an antibody or share sequence homology with such variable domains. Antibodies for therapeutic use are preferably as close as possible to the natural antibodies of the subject to be treated (e.g., human antibodies for a human subject). 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 with an affinity (KD) that is at least 1×10e-6M, more preferably 1×10e-7M, and more preferably higher than 1×10e-9M. Typically, antibodies for therapeutic applications have an affinity of up to 1×10e-10M or higher. Antibodies such as the bispecific antibodies of the present invention include the constant domains (Fc portion) of natural antibodies. The antibodies of the present invention are typically bispecific full-length antibodies, preferably of the human IgG subclass. Preferably, the antibodies disclosed herein are antibodies of the human IgG1 subclass. Such antibodies have good ADCC properties, a favorable half-life upon in vivo administration to humans, and a modified heavy chain that can provide a heterodimer preferentially over a homodimer upon co-expression in clonal cells, due to the presence of CH3 engineering technology.

[0081] The antibodies of the invention disclosed herein are preferably "full-length" antibodies. The term "full-length" is defined to include essentially complete antibodies, although such antibodies need not have 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 dissected into domains designated CH1, CH2, CH3, VH, and CL, VL. Antibodies bind to antigens via the variable domains contained in the Fab portion and, after binding, can interact with molecules and cells of the immune system via the constant domains, mainly via the Fc portion. The terms "variable domain", "VH / VL pair", "VH / VL" are used interchangeably herein. Full-length antibodies according to the invention include antibodies that may have mutations that provide desired characteristics. Such mutations should not be deletions of substantial portions of any of these regions. However, antibodies in which one or a few amino acid residues are deleted without substantially altering the binding characteristics of the resulting antibody are included within the term "full-length antibody". For example, IgG antibodies may have insertions, deletions, or combinations thereof of 1 to 20 amino acid residues in the constant region. For example, if the antibody itself has low ADCC activity, the ADCC activity of the antibody can be improved by slightly modifying the constant region of the antibody (Junttila, T. T., K. Parsons et al. (2010), "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer", Cancer Research 70(11):4481-4489).

[0082] Full-length IgG antibodies are preferred due to their preferred half-life and the need to be as close as possible to their own (human) molecules for reasons of immunogenicity. The antibodies disclosed herein are preferably bispecific IgG antibodies, 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 "bispecific" (bs) means that one part of the antibody (as defined above) binds to one epitope on an antigen while the second part binds to a different epitope. This different epitope is typically present on a different antigen. These first and second antigens are in effect two different proteins. Preferred bispecific antibodies are antibodies that contain parts of two different monoclonal antibodies and as a result bind 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 bispecific antibodies typically differ from each other, while the light chain variable regions are preferably the same. Bispecific antibodies in which different heavy chain variable regions associate with the same or a common light chain are also called bispecific antibodies having 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 wild-type CH3 domains are used, co-expression of two different heavy chains and one common light chain gives rise to three different species, AA, AB, and BB. To increase the percentage of the desired bispecific product (AB), CH3 engineering can be used, or in other words, heavy chains having a compatible heterodimerization domain as defined below in this specification can be used.

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

[0086] The term "common light chain" refers to light chains that can be identical or can have some amino acid sequence differences, but the binding specificity of the full-length antibody is not affected. For example, conservative amino acid changes, amino acid changes in regions that do not contribute or only partially contribute to binding specificity when paired with the heavy chain, can be introduced and tested, etc., to prepare and find light chains that are not identical but are still functionally equivalent. The terms "common light chain", "common VL", "single light chain", "single VL" are all used interchangeably herein, with or without the addition of the term "rearranged".

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

[0088] O12 / IgVκ1-39*01 that constitutes the light chain variable region is preferably a germline sequence. IGJκ1*01 or / IGJκ5*01 that constitutes the light chain variable region is 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 contains the germline O12 / IgVκ1-39*01. In a preferred embodiment, the light chain variable region contains the 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 contains the germline kappa light chain IgVκ1-39*01 / IGJκ1*01 or the germline kappa light chain IgVκ1-39*01 / IGJκ5*01, preferably the germline IgVκ1-39*01 / IGJκ1*01.

[0090] Obviously, those skilled in the art will recognize that "common" also refers to functional equivalents of light chains whose amino acid sequences are not identical. There are many variants of the above light chains that have mutations (deletions, substitutions, additions) that do not substantially affect the formation of the functional binding region. The light chain can also be the light chain specified herein that has insertions, deletions, substitutions of 1 to 5 amino acids, or combinations thereof.

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

[0092] The term "ErbB-1" as used herein refers to the protein encoded by the ERBB-1 gene in humans. Alternative names for this gene or protein include EGFR, ERBB, HER1, Erb-B2 receptor tyrosine kinase 1. When reference is made to ErbB-1 herein, this reference refers to human ErbB-1.

[0093] As used herein, the term "ErbB-2" 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. This ERBB-2 gene is often referred to as HER2 (from human epidermal growth factor receptor 2). When reference is made to ErbB-2 herein, such reference refers to human ErbB-2. Antibodies containing antigen-binding sites that bind to ErbB-2 bind to human ErbB-2. The ErbB-2 antigen-binding site may also bind, but does not necessarily bind, to such orthologs due to sequence and tertiary structure similarities between the human ortholog and other mammalian orthologs. The database accession numbers for the human ErbB-2 protein and the gene encoding it are (NP_001005862.1, NP_004439.2 NC_000017.10 NT_010783.15 NC_018928.2). These accession numbers are provided primarily to provide additional methods for the identification of ErbB-2 as a target, and the actual sequence of the ErbB-2 protein bound by an antibody may vary due to, for example, mutations in the coding 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 reference is made herein to ErbB-3, such reference refers to human ErbB-3. Antibody binding sites that bind to ErbB-3 bind to human ErbB-3. Due to sequence and tertiary structure similarities between the human ortholog and other mammalian orthologs, the ErbB-3 antigen binding site may also bind to such orthologs, but not necessarily. Database accession numbers for the human ErbB-3 protein and the gene encoding it are (NP_001005915.1 NP_001973.2, NC_000012.11 NC_018923.2 NT_029419.12). These accession numbers are provided primarily to provide additional 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 reference is made herein to ErbB-1, such reference refers to human ErbB-4.

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

[0097] As used herein, the term "ErbB-3 ligand" 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, Betacellulin, Heparin-binding epidermal growth factor, and Epiregulin. This term includes biologically active fragments and / or variants of naturally occurring polypeptides.

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

[0099] As used herein, the ligand-induced receptor function is reduced by at least 20%, preferably at least 30, 40, 50, 60 or at least 70%, and in a particularly preferred embodiment, this ligand-induced receptor function is reduced by 80%, more preferably 90%. This reduction is preferably determined by determining the ligand-induced receptor function in the presence of the 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 the 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 the MCF-7 cell line (ATCC® HTB-22™), SKBR3 cell line (ATCC® HTB-30™) cells, JIMT-1 cell line (DSMZ ACC 589) or NCI-87 cell line (ATCC® CRL-5822™). The test and / or ligand for determining the 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-IV. The binding locations of the antigen-binding sites of the antibodies described herein to each domain have been mapped. Bispecific antibodies having an antigen-binding site (first antigen-binding site) that binds to Domain I or Domain IV of ErbB-2 include a heavy-chain variable region that maintains significant binding specificity and affinity for ErbB-2 when combined with various light chains. Bispecific antibodies having an antigen-binding site (first antigen-binding site) that binds to Domain I or Domain IV of ErbB-2 (first antigen-binding site) and an antigen-binding site (second antigen-binding site) for ErbB-3 are found to be more effective in reducing the ligand-induced receptor function of ErbB-3 when compared to bispecific antibodies that include an antigen-binding site (first antigen-binding site) that binds to another extracellular domain of ErbB-2. Bispecific antibodies that include an antigen-binding site (first antigen-binding site) that binds to ErbB-2, where this antigen-binding site is an antibody that binds to Domain I or Domain IV of ErbB-2, are preferred. Preferably, this antigen-binding site binds to Domain IV of ErbB-2. Preferred antibodies include 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 at T144, T164, R166, P172, G179, S180, and R181, and within about 5 amino acid positions of T144, T164, R166, P172, G179, S180, or R181.

[0102] In a preferred embodiment, the antibody preferably comprises 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 from R426 in R426 and native ErbB-3 protein.

[0103] Bispecific antibodies having an antigen-binding site (a first antigen-binding site) that binds to ErbB-2 and further comprises ADCC have been found to be more effective than other ErbB-2 binding antibodies that did not have significant ADCC activity, particularly in vivo. Thus, bispecific antibodies that exhibit 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 having 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, such as CD16A (FcγRIIIA) and CD32A (FcγRIIA), activate cells to mount a response against an antigen. Other receptors, such as CD32B, inhibit the activation of immune cells. By engineering the Fc region to bind to activating receptors with higher selectivity (by introducing amino acid substitutions), antibodies can be created that have a higher ability to mediate the desired cytotoxic activity by anti-cancer Mab.

[0104] One technique for enhancing the ADCC of antibodies is afucosylation (see, for example, 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). Accordingly, afucosylated bispecific antibodies disclosed herein are further provided. Alternatively, or in addition, a number of other strategies, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics), can be used to achieve ADCC enhancement, all of these strategies attempting to improve Fc binding to low-affinity activating FcγRIIIa and / or reduce binding to low-affinity inhibitory FcγRIIb.

[0105] There are several in vitro methods for determining the efficacy of an antibody or effector cell in inducing ADCC. These include the chromium-51 [Cr51] release assay, the europium [Eu] release assay, and the sulfur-35 [S35] release assay. Typically, a labeled target cell line expressing a particular antigen exposed on the surface is incubated with an antibody specific for that antigen. After washing, effector cells expressing the Fc receptor CD16 are typically co-incubated with the antibody-labeled target cells. Target cell lysis is subsequently typically measured by the release of intracellular label, for example, by a scintillation counter or spectrophotometry.

[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 the 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 preferably 0.99 nM or less. In one embodiment, this affinity is in the range of 1.39 to 0.59 nM. In a preferred embodiment, the affinity of the second antigen-binding site for 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 above-mentioned affinity is preferably measured using equilibrium cell affinity measurement, where, as described in the examples of WO2015 / 130173, the cells are incubated at 4°C using a radioactively labeled antibody, and then the radioactivity bound to the cells is measured.

[0107] The affinity (KD) of the above-mentioned first antigen-binding site for ErbB-2 positive cells is preferably 5.0 nM or less, more preferably 4.5 nM or less, and even more preferably 3.9 nM or less. In a preferred embodiment, the affinity of this first antigen-binding site for 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, even more preferably 3.5 nM or less, even more preferably 3.0 nM or less, and even more preferably 2.3 nM or less. In one embodiment, this affinity is in the range of 3.0 to 1.6 nM. In a preferred embodiment, the affinity of this first antigen-binding site for 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 above-mentioned affinity is preferably measured using steady-state cell affinity measurement, where, as described in the examples of WO2015 / 130173, the cells are incubated at 4 °C using 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 the survival of cardiomyocytes. 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 stress.

[0109] The bispecific antibodies disclosed herein are preferably used in humans. Accordingly, preferred antibodies are human or humanized antibodies. Human tolerance to polypeptides is governed by many different aspects. Immunity, such as T cell-mediated, B cell-mediated, etc., is one of the variables subsumed in human tolerance to polypeptides. The constant region of the bispecific antibody is preferably a human constant region. This constant region may contain differences from the constant region of a naturally occurring human antibody of 1 or more, preferably 10 or less, preferably 5 amino acids or less. It is preferred that the constant portion is entirely derived from a naturally occurring human antibody. The various antibodies produced herein are derived from human antibody variable domain libraries. These variable domains are human. The unique CDR regions can be of human origin, synthetic, or from another organism. The variable region is considered a human variable region if, apart from the CDR regions, it has an amino acid sequence identical to the amino acid sequence of the variable region of a naturally occurring human antibody. The variable regions of the ErbB-2 binding VH, ErbB-3 binding VH, or light chain in the antibody may contain differences of 1 or more, preferably 10 or less, preferably 5 amino acids or less from the variable region of a naturally occurring human antibody, without counting potential differences in the amino acid sequences of the CDR regions. Such mutations also occur naturally with respect to somatic hypermutation.

[0110] Antibodies can be derived from various animal species, at least with respect to the heavy chain variable region. For example, humanizing mouse heavy chain variable regions and the like is common practice. There are various ways by which this can be achieved, which typically involves CDR grafting into a human heavy chain variable region having a 3D structure that matches the 3D structure of the mouse heavy chain variable region; deimmunization of the mouse heavy chain variable region, preferably by removing known or suspected T cell epitopes or B cell epitopes from the mouse heavy chain variable region. The removal is typically by substituting 1 or more of the amino acids in the epitope with another (typically conservative) amino acid, such that the sequence of the epitope is modified 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, veneered, etc. By using veneering techniques, surface residues that are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that includes either a weakly immunogenic veneered surface or a substantially non-immunogenic veneered 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 include the constant regions of human antibodies. According to the differences in their heavy chain constant domains, antibodies are grouped into five classes or isotypes: IgG, IgA, IgM, IgD, and IgE. These classes or isotypes are named using the corresponding Greek letters and include at least one of the above heavy chains. Preferably, the constant region includes an IgG constant region, more preferably an IgG1 constant region, preferably a mutated IgG1 constant region. For example, some variations in the constant region of IgG1, such as allotypes G1m1, 17, and G1m3, occur naturally and / or are tolerated without changing the immunological properties of the obtained antibodies. Typically, insertions, deletions, substitutions, or combinations thereof of about 1 to 10 amino acids are tolerated in the constant region.

[0113] Preferred bispecific antibodies disclosed herein include the following: - At least the CDR3 sequence of an ErbB-2 specific heavy chain variable region selected from the group consisting of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898, preferably at least the CDR1, CDR2 and CDR3 sequences, or at least the heavy chain variable region sequence, or a heavy chain variable region sequence listed and having at most 15 amino acids, preferably at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably at most 1, 2, 3, 4 or 5 amino acids different therefrom; and / or - At least the CDR3 sequence of an ErbB-3 specific heavy chain variable region selected from the group consisting of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074, preferably at least the CDR1, CDR2 and CDR3 sequences, or at least the heavy chain variable region sequence, or a heavy chain variable region sequence listed and having at most 15 amino acids, preferably at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably at most 1, 2, 3, 4 or 5 amino acids different therefrom.

[0114] The CDR sequences are preferably varied, for example for optimization purposes, in order to improve the binding potency or stability of the antibody. Optimization is preferably carried out, for example, by mutagenesis procedures after the stability and / or binding affinity of the resulting antibody has been tested and improved ErbB-2 or ErbB-3 specific CDR sequences have been preferably selected. A person skilled in the art is fully capable of producing antibody variants comprising 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 the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.

[0115] Preferred antibodies comprise a variable domain that binds to ErbB-2, and the VH chain of this variable domain comprises the amino acid sequence of VH chain MF2926; MF2930; MF1849; MF2973; MF3004; MF3958 (which is humanized MF2971); MF2971; MF3025; MF2916; MF3991 (which is humanized MF3004); MF3031; MF2889; MF2913; MF1847; MF3001, MF3003 or MF1898; or has insertions, deletions, substitutions or combinations thereof of 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 acids, with respect to the VH chain sequences described above, and comprises the amino acid sequence of VH chain MF2926; MF2930; MF1849; MF2973; MF3004; MF3958 (which is humanized MF2971); MF2971; MF3025; MF2916; MF3991 (which is humanized MF3004); MF3031; MF2889; MF2913; MF1847; MF3001, MF3003 or MF1898. The VH chain of the variable domain that binds to ErbB-2 preferably comprises the following amino acid sequence: - MF1849; or - MF2971 or a humanized version thereof, where the humanized version preferably includes the amino acid sequence of MF3958; or - MF3004 or a humanized version thereof, where the humanized version preferably includes the amino acid sequence of MF3991. In one embodiment, the VH chain of the variable domain that binds to ErbB-2 includes the following amino acid sequences: VH chain MF1849; or MF2971 or a humanized version thereof, where the humanized version preferably includes the amino acid sequence of MF3958; or MF3004 or a humanized version thereof, where the humanized version preferably includes the amino acid sequence of MF3991. Here, the listed VH sequences each have insertions, deletions, substitutions, or combinations thereof of 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 acids with respect to their respective sequences. In a preferred embodiment, the VH chain of the variable domain that binds to ErbB-2 includes the amino acid sequence of MF3958; or includes the amino acid sequence of MF3958 with insertions, deletions, substitutions, or combinations thereof of 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 acids with respect to this VH chain sequence.

[0116] The VH chain of the variable domain that binds to Erb-B3 preferably comprises the amino acid sequence of VH chain 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; or has insertions, deletions, substitutions or combinations thereof of 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 acids with respect to the VH chain sequence, and comprises the amino acid sequence of VH chain 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. 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 has insertions, deletions, substitutions or combinations thereof of 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 acids with respect to each VH chain sequence, and comprises the amino acid sequence of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065. In a preferred embodiment, the VH chain of the variable domain that binds to ErbB-3 comprises the amino acid sequence of MF3178; or has insertions, deletions, substitutions or combinations thereof of 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 acids with respect to this VH chain sequence, and comprises the amino acid sequence of MF3178. Preferably, the above-mentioned amino acid insertions, deletions and substitutions do not occur in the CDR3 region.The above-mentioned amino acid insertions, deletions, and substitutions preferably do not exist in the CDR1 and CDR2 regions either. The above-mentioned amino acid insertions, deletions, and substitutions preferably do not exist in the FR4 region either.

[0117] Preferably, the antibody comprises at least the CDR1, CDR2, and CDR3 sequences of MF1849, MF2971, MF3958, MF3004, or MF3991, and most preferably, at least the CDR1, CDR2, and CDR3 sequences of MF3958. This antibody preferably comprises at least the CDR1, CDR2, and CDR3 sequences of MF3178, MF3176, MF3163, MF6058, MF6061, or MF6065, and most preferably, at least the CDR1, CDR2, and CDR3 sequences of MF3178.

[0118] Preferably, the ErbB-2 specific heavy chain variable region comprises the amino acid sequence of VH chain MF3958 having, with respect to this VH, 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 (preferably, these insertions, deletions, substitutions do not exist in CDR1, CDR2, or CDR3). Preferably, the ErbB-3 specific heavy chain variable region comprises the amino acid sequence of VH chain MF3178 having, with respect to this VH, 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. The insertions, deletions, substitutions, or combinations of one or more amino acids preferably do not exist in the CDR1, CDR2, and CDR3 regions of the VH chain. These preferably do not exist in the FR4 region either. The amino acid substitutions are preferably conservative amino acid substitutions.

[0119] Preferably, the ErbB-2 specific heavy chain variable region, with respect to this VH, has an amino acid sequence of VH chain MF3991 with insertions, deletions, substitutions or combinations thereof of 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 acids (preferably, these insertions, deletions, substitutions are not present in CDR1, CDR2 or CDR3). Preferably, the ErbB-3 specific heavy chain variable region, with respect to this VH, has an amino acid sequence of VH chain MF3178 with insertions, deletions, substitutions or combinations thereof of 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 acids. 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. These are preferably not present in the FR4 region either. Amino acid substitutions are preferably conservative amino acid substitutions.

[0120] Preferably, the first antigen-binding site of the antibody comprises at least the CDR1, CDR2 and CDR3 sequences of MF3958, or CDR1, CDR2 and CDR3 sequences that differ from the CDR1, CDR2 and CDR3 sequences of MF3958 by up to 3, preferably up to 2, preferably up to 1 amino acid, and this second antigen-binding site comprises at least the CDR1, CDR2 and CDR3 sequences of MF3178, or CDR1, CDR2 and CDR3 sequences that differ from the CDR1, CDR2 and CDR3 sequences of MF3178 by up to 3, preferably up to 2, preferably up to 1 amino acid.

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

[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 non-fucosylated.

[0123] Preferably, the bispecific antibody comprises the "heavy chain for erbB-2 binding" shown in Sequence Listing Part 1D and the "heavy chain for erbB-3 binding" shown in Sequence Listing Part 1D.

[0124] Preferably, the antigen-binding site of the bispecific antibody comprises the germline light chain O12, preferably the 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 at imgt.org). The term, rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01, IGKV1-39 / IGKJ1, huVκ1-39 light chain or, in other words, huVκ1-39 is used. This light chain may have insertions, deletions, substitutions of 1, 2, 3, 4 or 5 amino acids or combinations thereof. The above-described substitutions of 1, 2, 3, 4 or 5 amino acids 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 the FR4 region of the VL chain. Preferably, the first antigen-binding site and the second antigen-binding site comprise the same light chain variable region, and more precisely, a common light chain. Preferably, the light chain variable region comprises CDR1 having the sequence (RASQSISSYLN), CDR2 having the sequence (AASSLQS) and CDR3 having the sequence (QQSYSTPPT). Preferably, the light chain variable region comprises the common light chain sequence shown in Sequence Listing Part 1C.

[0125] A variety of methods are available for producing bispecific antibodies and are disclosed in WO2015 / 130173. One method involves the expression of two different heavy chains and two different light chains in a cell, and the collection of the antibodies produced by the cell. Antibodies produced by this method typically include a group of antibodies having different combinations of heavy and light chains, some of which are the desired bispecific antibodies. This bispecific antibody can subsequently be purified from this group of antibodies.

[0126] The ratio of the bispecific antibody to other antibodies produced by the cell can be increased by various methods. Preferably, this ratio is increased by expressing two essentially identical light chains in the cell without expressing two different light chains. This concept is also referred to in the art as the "common light chain" method. When the 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 the bispecific antibody to other antibodies produced by the cell is significantly improved over the expression of two different light chains. The ratio of the bispecific antibody produced by the cell can be further improved by stimulating the pairing of two different heavy chains with each other rather than the pairing of two identical heavy chains. The art describes various methods by which such heterodimerization of heavy chains can be achieved. One method is to create a "knob-into-hole" bispecific antibody. 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 a single cell are disclosed, thereby providing means by which the formation of bispecific antibodies is favored over the formation of monospecific antibodies.

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

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

[0129]

Chemical formula

[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 encodes the end of the leader peptide):

[0131]

Chemical formula

[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 encodes the end of the leader peptide):

[0133]

Chem.

[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 encodes the end of the leader peptide):

[0135]

Chem.

[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 encodes the end of the leader peptide):

[0137]

Chem.

[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 (the underlined sequence encodes the end of the leader peptide):

[0139]

Chem.

[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 (the underlined sequence encodes the end of the leader peptide):

[0141]

Chem.

[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 encodes the end of the leader peptide):

[0143]

Chem.

[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 encodes the end of the leader peptide):

[0145]

Chem.

[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 encodes the end of the leader peptide):

[0147]

Chem.

[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 encodes the end of the leader peptide):

[0149]

Chem.

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

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

[0152]

Chemical formula

[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 encodes the end of the leader peptide):

[0154]

Chemical formula

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

[0156]

Chem.

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

[0158]

Chem.

[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 (the underlined sequence encodes the end of the leader peptide):

[0160]

Chemical formula

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

[0162] Array 1C Common light chain Variable region of IGKV1-39A DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP CDR 1: RASQSISSYLN CDR 2: AASSLQS CDR 3: QQSYSTPPT IGKV1-39 / jk1 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK Common light chain IGKV1-39 / jk1 (underline the constant region) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Variable domain of the common light chain IGKV1-39 / jk5 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0163] Array 1D (erbB-2 specific) Heavy chain for erbB-2 binding QVQLVQSGAEVKKPGASVKLSCKASGYTFTAYYINWVRQAPGQGLEWIGRIYPGSGYTSYAQKFQGRATLTADESTSTAYMELSSLRSEDTAVYFCARPPVYYDSAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Heavy chain for erbB-3 binding QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDHGSRHFWSYWGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

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

[0165]

Chemical formula

[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 encodes the end of the leader peptide):

[0167]

Chem.

[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 encodes the end of the leader peptide):

[0169]

Chem.

[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 encodes the end of the leader peptide):

[0171] [Chemistry]

[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 encodes the end of the leader peptide):

[0173] [Chemistry]

[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 encodes the end of the leader peptide):

[0175] [Chemistry]

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

[0177]

Chemical formula

[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 encodes the end of the leader peptide):

[0179]

Chemical formula

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

[0181]

Chemical formula

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

[0183] For the purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, but it is understood that the scope of the invention may include embodiments having all or a combination of some of the described features.

Brief Description of the Drawings

[0184]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6

Figure 7

Example

[0185] (Example 1) ErbB-2-guided targeting Imaging experiments were conducted to compare the HER2×HER3 bispecific antibody (PB4188) with a HER3 bivalent monoclonal antibody. Variants 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 tumors compared to the HER3 monoclonal (Figure 2A). Gamma-counter quantification of the radioactivity present in tumors confirmed that the level of the PB4188 variant in tumors was 2.5-fold higher than that of the parental anti-HER3 antibody (Figure 2B). Overall, in vitro and in vivo data have demonstrated that HER2 targeting is responsible for the enhanced binding of PB4188 to tumor cells. Further tests were conducted using the anti-HER2 (MF3958) antibody. Figure 2C summarizes the results of each antibody injected into mice xenografted with HER2 gene-amplified JIMT-1 tumors labeled with 64Cu (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 to a bifunctional chelator [Paterson 2014 Dalton Transactions]. The binding characteristics of the conjugated products to the target were confirmed using a flow cytometry-based assay. The proteins were then labeled with 64Cu and the radiolabeled antibodies were administered to mice with JIMT-1 breast xenografts via the tail vein ("i.v." for Figures 2A - 2B and "i.p." for Figure 2C). MicroPET / CT images were acquired 48 hours after injection, after which the tumors were excised and the radioactivity was measured with a gamma counter. The results were shown as the 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 to form an active enzyme only when in close proximity. Each sequence encoding either the enzyme donor or the enzyme acceptor is ligated to the extracellular and transmembrane domains of each heterodimerization partner. Then, both genes are co-transfected into U2OS cells to express the extracellular domain of the RTK receptor linked to one domain (ED or EA) of β-galactosidase. Upon agonist stimulation of one RTK receptor, 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 produces luminescence upon hybridization.

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

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

[0190]

Table 1

[0191] In the HER2:HER3 assay, the potency of trastuzumab was approximately one-fourth of that of MCLA-128 or PG3178. Perjeta (clinical pertuzumab) behaved as a full antagonist in all three assays, generating a profile similar to that of PG2867 (pertuzumab). In the HER2:HER4 assay, both anti-HER2 PG3958 and PG2867 (pertuzumab) showed a minor decrease in dimerization that appeared to be dose-dependent. In the EGFR:HER2 assay, a small non-specific response was observed at high concentrations of PG1337, MCLA-128, PG3178 and PG3958.

[0192] MCLA-128 showed specific inhibition of only the HER2:HER3 heterodimer. This indicates that upon binding to HER2, MCLA-128 should neither sterically impair the interaction between HER2 and EGFR upon EGF stimulation nor impair the heterodimerization between HER2 and HER4 upon HRG stimulation.

[0193] The latter is consistent with observations in the HRG-induced cell cycle-based proliferation assay in T47D cells. Assays using these cells could not demonstrate the inhibitory activity of either MCLA-128 or PG3178, which was probably due to the higher expression of HER4 compared to HER3. HRG is thought to preferably signal through HER2:HER4 rather than HER2:HER3 in T47D cells, which explains the lack of efficacy of MCLA-128 and demonstrates the specificity of MCLA-128 for HRG-induced HER2:HER3 dimers rather than HRG-induced HER2:HER4 dimers.

[0194] In this study, trastuzumab blocked EGF-induced hetero-dimerization of EGFR:HER2 and HER2:HER3 and HRG-induced hetero-dimerization, respectively. Trastuzumab and pertuzumab behaved as partial antagonist and full antagonist, 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 trastuzumab inhibitory responses were observed in these assays may be due to overexpression of both targets. This may enable a more sensitive readout than traditional immunoprecipitation experiments.

[0195] Finally, PG3793 showed lower binding affinity for MCF-7 than PG3178, but its lower intensity in the HER2:HER3 hetero-dimerization assay was less dramatic (a 2.5-fold difference in dimerization assay intensity versus a 30-fold difference in binding assay affinity). This discrepancy between binding affinity and antagonism intensity has been previously observed for MCLA-128 and PG3178. PG3178 binds to MCF-7 with slightly better affinity than MCLA-128, but MCLA-128 is superior to PG3178 in a cell cycle-based proliferation assay.

[0196] (Example 3) Test Objectives and Regulatory Compliance The objective 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 of OV-10-0050 in BALB / c nude mice.

[0197] Experimental Design The experimental design is shown in Table 2. In all groups, blood was sampled in 4 animals on day 2 (24 hours after the first dose) and in the remaining 4 animals on day 6 (5 days after the first dose). At the designated time points, 50 - 100 μl of blood was collected into a sterile collection tube (Microvette CB300Z coagulation activator / 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, and the aqueous layer (approx. 20 μl of serum) was taken into another sterile 1.5 mL Eppendorf and immediately stored at -80°C. Samples were shipped on dry ice.

[0198]

Table 2

[0199] All animals were treated on days 1, 8, 15, 22, and 29 (weekly treatment for 5 weeks), and the route 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 (at least 1:20 tissue:fixative ratio) for 24 hours and then converted into FFPE blocks.

[0201] Preparation of neutral buffered formalin: Put 1 bag of PBS powder into a clear 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 achieved.

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

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

[0204] Diet: The animals had free access to irradiated and sterilized dry pellets throughout the test period; Water: The animals had free access to sterile drinking water.

[0205] Packaging and storage conditions of antibodies: MCLA-128; Cryovial, 2.5 mg / ml, 10 × 1.5 ml / vial, stored at 4°C PG2863; Cryovial, 2.5 mg / ml, 10 × 1.5 ml / vial, stored at 4°C PG2869; Cryovial, 2.5 mg / ml, 10 × 1.5 ml / vial, stored at 4°C

[0206] Generation of PDX model The human ovarian cancer PDX model of OV-10-0050 was originally established from a 48-year-old female patient presenting 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 For each mouse, an OV-10-0050 P6 tumor slice (about 30 mm3) cut with scissors was subcutaneously transplanted into the right flank for tumor development. Treatment was started 30 days after tumor transplantation, which was the day when the average tumor size reached approximately 152 mm3. 32 tumor-bearing mice were randomized into 4 groups using a stratified randomization method, with each group consisting of 8 tumor-bearing mice. The day of randomization was defined as day 1, which was the start day of treatment. The test article was administered to the mice according to a predetermined regimen as shown in the experimental design table (Table 2).

[0208] Observation All procedures related to the handling, care, and treatment of animals in the study were conducted in accordance with the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the guidelines approved by WuXi AppTec's Institutional Animal Care and Use Committee (IACUC). 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), matting of eyes / hair, any effects on 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 within each subset.

[0209] Tumor measurement It was tested whether tumor growth could be delayed or 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. Then, the tumor size was used for the calculation of T-C, T / C, and TGI values. T-C was calculated using T, the median time (days) for the tumors in the treatment group to reach a given size (e.g., 500 mm3), and C, the median time (days) for the tumors 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, respectively, on a given day. TGI was calculated for each group using the formula: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100; 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 the tumor volumes of each group at each time point (detailed in Table 3). Statistical analysis of the differences in tumor volume between groups and analysis of drug interactions were performed on the data obtained at the best therapeutic time point after the final dose (29 days after randomization).

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

[0212] Results Mortality, morbidity, and weight gain or loss Animal weights were regularly monitored as an indirect measure of toxicity. No group was observed to have a decrease in body weight as a result of test article administration (Figure 4), and no deaths or morbidity were observed. Thus, there does not appear to be any obvious toxicity associated with the administration of MCLA-128, PG2863, and PG2869 antibodies to tumor-bearing BALB / c nude mice.

[0213] Changes in body weight in female BALB / c nude mice bearing OV-10-0050 xenografts dosed with MCLA-128, PG2863, and PG2869 antibodies are shown in Figures 4 and 5. Mean tumor volumes over time in female BALB / c nude mice bearing OV-10-0050 xenografts dosed with MCLA-128, PG2863, and PG2869 antibodies are shown in Table 3. Figure 6 shows tumor growth.

[0214] [Table 3]

[0215] Results and discussion In this study, the therapeutic efficacy of MCLA-128, PG2863, and PG2869 antibodies as single agents in the treatment of the OV-10-0050 human ovarian cancer xenograft model was evaluated. The results of tumor sizes in 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 reached 1,161 mm 3 after 29 days of grouping. Treatment with the test articles MCLA-128, PG2863, and PG2869 antibodies at 25 mg / kg (QW×5 weeks) produced significant anti-tumor activity: their average tumor sizes were 23, 108, and 1 mm3 at the same time point, respectively (T / C values = 1.95%, 9.28%, and 0.06%; TGI values = 112.78%, 104.37%, and 114.96%; p values = 0.002, 0.003, and 0.002), and the growth of all their tumors was delayed by more than 14 days compared to the vehicle group at a tumor size of 500 mm 3 Treatment caused partial or complete regression of the tumors. When the tumor volume was reduced by 50% or more of its volume on day 1 for three consecutive measurements during the course of the study, or when it was ≥13.5 mm for one or more of these three measurements 3 the mice were considered to have partial regression (PR). And when it was <13.5 mm for three consecutive measurements during the course of the study 3 the mice were considered to have complete regression (CR). When no palpable tumor was detected at the end of the study, it was considered tumor-free survival.

[0217] Treatment with MCLA-128, PG2863, and PG-2869 produced different proportions of PR, CR, and TFS. The number of mice in each group showing PR, CR, and TFS is shown in Table 5. All test articles were well tolerated by tumor-bearing animals. No weight loss was observed in any of the treatment groups.

[0218] In summary, all three test antibodies as single agents showed significant antitumor activity against the OV-10-0050 human ovarian cancer xenograft model in this study. This was well tolerated by tumor-bearing animals. These results indicated 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 the HER2 and HER3 receptor tyrosine kinases (RTKs) involved in cancer cell proliferation and survival. MCLA-128 has been extensively tested regarding heregulin (HRG)-induced HER3 signaling and proliferation. MCLA-128 has demonstrated stronger in vitro potency than: the combination of the anti-HER2 antibody pertuzumab (PG2869) + trastuzumab (PG2867), which can block ligand-dependent and ligand-independent HER2:HER3 signaling, respectively [Agus 2002; Juntilla 2009]; the anti-HER3 MM-121 (PG2863), which blocks HRG-induced HER3 activation [Schoeberl 2010].

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

[0223] From a panel of breast cancer cell lines, the MDA-MB-175 cells were sensitive to the single agent MCLA-128, which demonstrated the importance of the HER3 / HRG signaling axis in this cell line (Figure 7, left panel). Activation of HER2 in this cell line has also been demonstrated in vivo, and a single dose of pertuzumab, but not trastuzumab, inhibited orthotopic MDA-MB-175 tumor growth. The relevance of the DOC4-NRG1 gene fusion in breast cancer patients has been debated [Sanchez-Valdivieso 2002], but other gene fusions have recently attracted attention. In particular, the CD74-NRG1 fusion has been reported by independent groups in invasive mucinous adenocarcinoma, a subset of non-small cell lung cancer [Fernandez-Cuesta 2014, Duruisseaux 2016]. Several other NRG1 gene fusions, namely VAMP2-NRG1, RBPMS-NRG1, and WRN-NRG1 in lung cancer, and RAB2IL1-NRG1 in ovarian cancer have also been detected [Jung 2015, Dhanasekaran, 2014]. This diversity of gene fusions may be related to the location of the NRG1 gene on chromosome 8, which is sensitive to translocations [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 the transphosphorylation of HER3, resulted in tumor growth inhibition. The antitumor efficacy of MCLA-128 was compared to PBS (Figure 7, right panel).

[0225] Mice: NOD-SCID, Crl:NU(NCr)-Foxn1nu and BALB / c nude mice. Antibodies are dosed at 25 mg / kg for 4 weeks. Tumor volume is measured twice a week with calipers.

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

[0227] Number of sites: Up to 13 sites are estimated to be involved during the trial. Additional sites may be added to ensure that an acceptable enrollment rate exists or to replace non - enrolling / dropped sites.

[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 with invasive mucinous adenocarcinoma or advanced / metastatic non - small cell lung cancer; NSCLC with reported NRG1 fusions can be enrolled.

[0229] Patients who do not complete at least 2 cycles of the trial treatment for reasons other than disease progression are not evaluable for efficacy and are crossed over in each group.

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

[0231] [Table 6]

[0232] [Table 7]

[0233] Study Design: This is a Phase I / II non-blind, multi-center, multinational, dose-escalating, single-arm allocation study to evaluate the safety, tolerability, PK, PD, immunogenicity, and antitumor activity of MCLA-128.

[0234] This study is designed in two parts: Part 1 Enrollment into Part 1 of the study was completed on November 24, 2015, and as of January 24, 2017, all patients in Part 1 had completed the study. Nine dose levels were investigated: 40 mg in a cohort of 1 patient, 80 mg, 160 mg, 240 mg, 360 mg, 480 mg, 600 mg, 750 mg, and 900 mg in cohorts of 3 patients. MCLA-128 was initially administered over approximately 60 minutes on Day 1 of a 3-week treatment cycle. During Part 1, to mitigate infusion-related reactions (IRR), as an option it was increased up to 4 hours to extend the infusion duration up to 2 hours.

[0235] At none of these dose levels was dose-limiting toxicity (DLT) experienced. To obtain sufficient PK information, 3 additional patients in each of the 600 mg cohort and 750 mg cohort were dosed.

[0236] At the 900 mg dose level, since the MTD was not reached, the Data Review Committee (DRC) for MCLA-128-CL01 decided to assign a dose level of 750 mg as the RP2D of 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, and evaluation of CBR defined as the proportion of patients with CR, PR, or durable SD (SD lasting at least 12 weeks) among an expanded cohort of selected patients.

[0238] Evaluate a weekly dosing regimen using 4-week cycles consisting of a loading dose of 800 mg for the first administration and a uniform dose of 400 mg weekly for the first 2 cycles in newly replenished patients. From cycle 3, administer MCLA-128 at a dose of 400 mg weekly for 3 weeks, followed by a 1-week drug holiday. Administer mandatory premedication to reduce IRR. However, corticosteroids are only mandatory before the loading dose on day 1 of cycle 1 and should only be used at the discretion of the principal investigator of the clinical trial for subsequent infusions to manage IRR.

[0239] Examine the safety of the weekly schedule during the acclimation period after the first 5 treated patients have completed at least 2 treatment cycles. The DRC will examine all safety data with a focus on the incidence of grade 3-4 toxicity, the incidence and severity of IRR, and medication compliance. If the DRC concludes that the toxicity is unacceptable, the sponsor will continue patient enrollment with a 3-week cycle dosing regimen until a sufficient number of patients are enrolled per cohort.

[0240] Intrapatient dose escalation is not allowed in part 2.

[0241] The target patient population to be evaluated in part 2 of the study is as follows: · Open only for replenishment in NSCLC-Asia with reported NRG1 fusions

[0242] Each group (C-F), including at least 10 patients per cohort, can enroll at least 20 and up to approximately 40 patients to be treated at the recommended weekly dose. Previously closed cohorts may be reopened.

[0243] Duration of treatment Patients in both parts 1 and 2 of the study can maintain 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 convened to consider all available safety and PK data. DRC participants included the principal investigator (or their representative), the medical director of the sponsor, the trial medical monitor, the trial drug safety monitoring physician, the trial project manager, the trial statistician, and experts invited as necessary (e.g., clinical pharmacology experts).

[0245] In Part 2, the DRC reviews data after completion of the safety run-in period for each weekly dose before increasing the weekly dosing regimen in all continuing patients.

[0246] Trial evaluation: This trial consists of a screening period of up to 4 weeks (28 days) for molecular pre-screening evaluation and sequential treatment cycles until treatment discontinuation or termination for any reason thereafter. The treatment cycle duration is 3 weeks (21 days) for patients treated at the initial recommended dose in Part 2 and 4 weeks (28 days) for patients treated at the weekly recommended dose in Part 2. All patients must attend the last treatment visit within 1 week after treatment interruption and the final study visit 30 days after the end of treatment or trial discontinuation.

[0247] Patients who did not withdraw consent to continue or to withdraw consent to complete the final study visit were followed up every 3 months for up to 2 years (approx.) to check the patient's disease progression and / or survival status until the start of the next anti-cancer treatment.

[0248] If the ongoing evaluation of safety data during the trial and available PK, PD, and anti-tumor activity data suggest that alternative dosing frequencies should be evaluated, or that other patient populations should be evaluated in Part 2, these modifications are specified in a protocol amendment before these evaluations are initiated.

[0249] Molecular pre-screening and screening: Molecular pre-screening is performed in a laboratory in a region qualified to perform molecular screening for NRG1 fusions. To initiate pre-screening, the patient must meet one of the following criteria: · Histological diagnosis of IMA and reported absence of EGFR / ALK alterations. Note: IMA patients who did not undergo pre-screening tests for NRG1 fusions can enter the study. Or · The pathological test does not result in an IMA diagnosis, but the principal investigator suspects IMA based on symptoms, imaging features (e.g., focal consolidation, multiple bilateral nodules or consolidations), non-smoker, and reported absence of EGFR / ALK alterations.

[0250] Before submitting fresh or archived tumor tissue for analysis to determine the NRG1 fusion status, NSCLC patients identified for potential study participation must sign the molecular pre-screening informed consent form (ICF). The study can be conducted at any point during the natural history of the disease (e.g., at diagnosis, during first-line treatment, at progression, etc.), up to a maximum of 1 year before Day 1 of Cycle 1. Fresh tumor samples (formalin-fixed paraffin-embedded; FFPE) or archived tumor samples no older than 1 year are required for assessment of the presence of NRG1 fusions. Samples should be submitted to a laboratory in a region qualified to perform tests by molecular profiling (PCR, next-generation sequencing [DNA or RNA] or FISH) of the NRG1 fusion status. Patients with a positive local NRG1 fusion result are then eligible to sign the study ICF if they are willing and able to enter the study.

[0251] Principal Informed Consent Form All patients must sign the study ICF before any screening procedure or assessment is conducted. Screening evaluations should be conducted within the 4 weeks prior to Day 1 of Cycle 1, with the exception of the serum pregnancy test which should be conducted within 7 days of Day 1 of Cycle 1. A baseline obligatory oncology sample, preferably a block from fresh or archived tissue, is required for consideration for screening. The sponsor indicates preference for fresh tissue. Archived tissue is acceptable and must be within 1 year for non-NSCLC and within 2 years of collection from screening for NSCLC. Note that for NSCLC patients, a baseline biopsy for screening is still required even if a pre-screening biopsy sample was provided to the NRG1 pre-screening regional trial. After completion of all required screening evaluations and confirmation of all eligibility criteria, patients may initiate dosing on Day 1 of Cycle 1.

[0252] Safety Assessment Concurrent illnesses are captured at baseline; AEs and concomitant therapies are monitored throughout the study participation. Safety assessments include consideration of the Eastern Cooperative Oncology Group (ECOG) performance status, physical examination (including height and weight), vital signs, and electrocardiogram (ECG). Cardiac function tests of left ventricular ejection fraction (LVEF) are also 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 when clinically necessary. Laboratory evaluations include clinical chemistry, hematology, coagulation tests, urinalysis, and pregnancy testing. Note that cytokine panel analysis was performed up to August 1, 2017.

[0253] On all days of MCLA-128 administration, patients must stay at the clinic for at least 60 minutes (longer if PK samples are required) from the end of infusion for observation and repeated vital signs before discharge from the clinic. Additional safety evaluations should be performed if clinically necessary, and the duration of hospitalization at the clinic should be increased as determined by the principal investigator of the clinical trial as needed.

[0254] Immunogenicity assessment The serum titer of anti-MCLA-128 antibody is measured at pre-dose on Day 1 for each of Cycles 1, 2, 3, and 4, and then every 4 cycles (Cycles 8, 12, 16, etc.), as well as at the last treatment visit and the final study visit, within a -3 day window prior to MCLA-128 administration.

[0255] Pharmacokinetic assessment Initial recommended dose schedule for Parts 1 and 2: In Cycle 1, blood samples are collected for PK analysis at pre-dose on Day 1, at the end of infusion (EOI), and at 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 at pre-dose and EOI are collected.

[0256] Weekly recommended dose schedule for Part 2: In Cycle 1, blood samples are collected for PK analysis at pre-dose on Day 1, at EOI, at 2, 4, and 24 hours after EOI, then at pre-dose on Day 8 and Day 15, and at pre-dose and EOI on Day 22. In Cycles 2 and 3, blood samples at pre-dose and EOI are collected on Day 15. In Cycle 4, blood samples are collected at pre-dose on Day 1, and at pre-dose and EOI on Day 15. Thereafter, every 2 cycles (Cycles 6, 8, 10, etc.), pre-dose blood samples are collected on Day 15.

[0257] Tumor assessment Tumor evaluation is performed according to RECIST version 1.1 in accordance with the local principal investigator of the clinical trial. Images are obtained at screening and at the end of each 2 cycles of treatment for patients receiving a 3-week cycle regimen, and every 6 weeks for patients receiving a 4-week cycle regimen.

[0258] Biomarker and Pharmacokinetic Evaluation Various biomarker and pharmacokinetic tests 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 additional tumor samples, and consent for specific biomarker tests.

[0259] If sufficient samples are available, the following candidate biomarkers are evaluated: · HER2, HER3, HER2:HER3 dimerization, phosphorylated HER2 (pHER2) and HER3 (pHER3), and heregulin; · Using circulating plasma tumor DNA (ctDNA) and tumor sample DNA (depending on availability), test for mutations in oncogenes, including those related to HER2 and HER3 signaling. · Phosphorylated molecules in the MAPK and AKT signaling pathways; · Fc gamma receptor polymorphisms; · Circulating tumor cells for HER2; · Heregulin gene fusions

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

[0261] At baseline, patients are required to provide an obligatory tumor sample tissue block, preferably a block that can be derived from fresh or archived tissue. The test requester indicates a preference for fresh tissue. Archived tissue is acceptable and must be collected within 1 year for NSCLC and within 2 years from screening for other cancers. Additionally, patients are optionally required to submit tumor samples / biopsies at the end of cycle 4 and optionally at the end of treatment visits.

[0262] For the purpose of liquid biopsy testing, blood samples are also collected at these time points.

[0263] Eligibility Criteria: The trial enrolls 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. ECOG performance status of 0 or 1; 4. An estimated mean life expectancy of at least 12 weeks; 5. Toxicities suffered as a result of previous anti-cancer treatment that resolved to ≤ grade 1, excluding alopecia, lymphopenia not evaluated as clinically significant, and grade 2 sensory neurotoxicity (defined by NCI CTCAE v4.03); 6. At least a 4-week interval between the last radiotherapy received and the first scheduled day of dosing with MCLA-128 (with a maximum of 1 x 8 Gy for pain palliation being an exception); 7. Complete recovery from major surgery (stability and ≤ grade 2 toxicity are acceptable); 8. Laboratory values at screening: a. Absolute neutrophil count ≥ 1.5 x 10 9 / L without support of colony-stimulating factor; b. Platelets ≥ 100 x 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 due to Gilbert's syndrome); e. For patients with advanced solid tumors with liver metastases, AST (SGOT) ≤ 2.5 × ULN; ALT (SGPT) ≤ 2.5 × ULN; ALP > 5 × ULN with a single elevation is acceptable in patients with confirmed bone metastases; f. Serum creatinine ≤ 1.5 × ULN or estimated glomerular filtration rate (GFR) > 50 mL / min based on the Cockcroft-Gault formula; g. Coagulation function (INR and aPTT ≤ 1.5 × ULN, unless on therapeutic anticoagulants) h. Urine protein ≤ 2+ (measured by urine dipstick) or ≤ 100 mg / 24 hours of urine; 9. At baseline, an obligatory tumor biopsy sample (FFPE), preferably a block from fresh (preferred) or archival tissue, can be provided. For tissues other than NSCLC, archival tissue must have been collected within 2 years prior to screening and within 1 year for NSCLC. 10. A negative pregnancy test result defined by urine or blood human chorionic gonadotropin (hCG) test during screening and within 7 days of Cycle 1, Day 1, in women of childbearing potential (defined as women under 50 years of age or women with a history of amenorrhea for 12 months or less prior to entering the study). 11. Sexually active male and female patients with potential for pregnancy must agree to use an effective method of contraception (e.g., spermicide, oral or parenteral contraceptives and / or barrier methods using intrauterine devices) throughout the duration of the study and for 6 months after the last dose of MCLA-128. Note that female patients' infertility should be confirmed in the patient's medical record and defined as any of the following: surgical hysterectomy with bilateral oophorectomy, bilateral tubal ligation, natural menopause with last menstrual period > 1 year ago; irradiation-induced oophorectomy with last menstrual period > 1 year ago; chemotherapy-induced menopause with 1-year interval since last menstrual period; 12. The ability to give written informed consent prior to any study-specific screening procedures, understanding that the patient may withdraw consent at any time without violating rights; 13. The ability to understand the mandatory and optional protocol requirements, the willingness and ability to follow the study protocol procedures, and the signing of the main informed consent document. Additional consent is required for any optional biopsies (tissue and / or blood) and long-term sample storage; 14. Patients with metastatic cancer who have disease progression after treatment with all available therapies known to have clinical utility. 15. Inoperable or metastatic NSCLC that meets one of the following criteria: · Invasive mucinous adenocarcinoma (IMA) proven by biopsy. Note: IMA patients who have not undergone a pre-screening test for NRG1 fusions may be enrolled 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 without known driver mutations or fusions in the EGFR / ALK genes in a qualified regional laboratory. 16. Reported disease progression based on the evaluation of the investigator in charge of the trial for at least one line of standard treatment in a locally advanced or metastatic setting.

[0265] Statistical analysis: Parts 1 and 2 Summarize the anti-tumor and clinically useful variables descriptively for each group in Part 2. If necessary, the variables are presented in terms of absolute and relative changes from baseline. Categorical data are presented as tabulations of percentages and frequencies.

[0266] If necessary, data from patients who received what was identified as the MTD or MRD during Part 1, and patients who received the same dose in Part 2, can be combined and summarized, as well as summarized independently.

[0267] Evaluate the frequency and nature of severe and non-severe AEs in terms of absolute and relative frequencies and code them according to the MedDRA medical terminology set.

[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, an observed clinically significant correlation coefficient of at least 0.38 can be distinguished from zero with 95% confidence; smaller, observed clinically non-significant correlations cannot be distinguished from zero. Therefore, 20 subjects per cohort in Part 2 are considered sufficient to investigate the association between the anti-tumor activity of MCLA-128 and disease-related biomarkers.

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

[0271] Summarize PK parameters 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. Plot the serum concentration profile against time for each group.

[0272] (References) Yarden Y, Pines G. 2012. The ERBB network: at last, cancer therapy meets systems biology. Nat Rev Cancer Jul 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 bispecific antibody for use in the treatment of an individual having ErbB-2 and ErbB-3 positive cells, the bispecific 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, wherein the cells comprise an NRG1 fusion gene comprising at least a portion of the NRG1 gene fused to a sequence derived from a different chromosomal location.

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

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

4. The antibody according to claim 3, wherein the cancer cells are associated with an NRG1 fusion.

5. The antibody according to claim 3 or 4, wherein the cancer cells are driven by an NRG1 fusion.

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

7. A bispecific antibody for use in the treatment of an individual having or at risk of having an ErbB-2 and ErbB-3 positive tumor, the bispecific 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, wherein the method is characterized in that the cells of the tumor express 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.

8. The antibody according to claim 7, wherein the tumor is breast cancer, ovarian cancer, lung cancer such as non-small cell lung cancer, or metastases thereof.

9. The antibody according to any one of claims 1 to 8, wherein the NRG1 fusion gene expresses a protein comprising the NRG1 EGF-like domain.

10. The antibody according to any one of claims 1 to 9, wherein the NRG fusion is a fusion of NRG1 and a gene on human chromosome 8.

11. The antibody according to claim 10, wherein the gene on human chromosome 8 encodes a secreted protein or a cell membrane-associated protein.

12. The antibody according to any one of claims 1 to 11, 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.

13. The antibody according to any one of claims 1 to 12, wherein the cell or tumor is of epithelial origin.

14. The antibody according to any one of claims 1 to 13, wherein the individual has experienced a treatment targeting EGFR inhibition, preferably using an EGFR-binding antibody that is preferably cetuximab.

15. The antibody according to any one of claims 1 to 14, wherein the ErbB-1 cell surface receptor density; ErbB-2 cell surface receptor density; ErbB-3 cell surface receptor density; ErbB-4 cell surface receptor density or a combination thereof on one or more cells of the tumor is determined.

16. The antibody according to any one of claims 1 to 15, wherein the cell or tumor has less than 400,000 ErbB-1 cell surface receptors per cell, preferably less than 200,000 ErbB-1 cell surface receptors per cell.

17. The antibody according to any one of claims 1 to 16, further comprising administering to the individual an ErbB-1 inhibitor, preferably cetuximab.

18. The antibody according to any one of claims 1 to 17, wherein the first antigen-binding site can bind to domain I of ErbB-2 and the second antigen-binding site can bind to domain III of ErbB-3, and preferably, the affinity of the first antigen-binding site for ErbB-2 is lower than the affinity of the second antigen-binding site for ErbB-3.

19. i) at least comprising the CDR1, CDR2 and CDR3 sequences of the ErbB2-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 comprising CDR sequences that differ from the CDR1, CDR2 and CDR3 sequences of MF2926, MF2930, MF1849, MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898 by at most 3 amino acids, preferably at most 2 amino acids, preferably at most 1 amino acid, and / or ii) at least comprising the 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 comprising CDR sequences that differ from the CDR1, CDR2 and CDR3 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 by at most 3 amino acids, preferably at most 2 amino acids, preferably at most 1 amino acid, preferably, i) An ErbB-2 specific heavy chain variable region sequence selected from the group consisting of the heavy chain variable region sequences of MF2926, MF2930, MF1849, MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898, or a heavy chain variable region sequence that differs from the heavy chain variable region sequence of MF2926, MF2930, MF1849, MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898 by at most 15 amino acids, and / or ii) An ErbB-3 specific heavy chain variable region sequence selected from the group consisting of the heavy chain variable region sequences of MF3178, MF3176, MF3163, MF3099, MF3307, MF6055, MF6056, MF6057, MF6058, MF6059, MF6060, MF6061, MF6062, MF6063, MF6064, MF6065, MF6066, MF6067, MF6068, MF6069, MF6070, MF6071, MF6072, MF6073 and MF6074, or a heavy chain variable region sequence that differs from the heavy chain variable region 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 by at most 15 amino acids, the antibody according to claim 18.

20. The antibody according to claim 18, comprising at least the CDR1, CDR2 and CDR3 sequences of the ErbB2 specific heavy chain variable region MF3958 and at least the CDR1, CDR2 and CDR3 sequences of the ErbB3 specific heavy chain variable region MF3178.

21. The variable domain containing the first antigen-binding site and the variable domain containing the second antigen-binding site comprise 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, and preferably, the variable domain containing the first antigen-binding site and the variable domain containing the second antigen-binding site comprise a light chain variable region containing CDR1 having the sequence (RASQSISSYLN), CDR2 having the sequence (AASSLQS), and CDR3 having the sequence (QQSYSTPPT). The antibody according to any one of claims 1 to 20.

Images