Treatment with antibodies that bind EGFR and cMET

Abstract

The present invention relates to a bispecific antibody comprising a first variable domain that can bind to an extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to an extracellular portion of human MET prooncogene receptor tyrosine kinase (cMET) for use in a method of treatment of cancer in an individual, the subject has received a previous treatment with i) a third generation EGFR tyrosine kinase inhibitor, or ii) chemotherapy and tyrosine kinase inhibitor, or iii) a cMET tyrosine kinase inhibitor.

Description

Technical Field

[0001] The present invention relates to the field of antibodies. In particular, it relates to the field of therapeutic antibodies for treating diseases involving abnormal cells, including human antibodies. In addition, it relates to antibodies that bind to EGFR and cMET, including multispecific antibodies, and their use in binding to EGFR and cMET positive cells, especially tumor cells. Background Art

[0002] The epidermal growth factor (EGF) receptor (EGFR) is a cell surface receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. EGFR is also known as the ErbB-1 receptor. The receptor has been given various names in the past (EGFR; ERBB; ERBB1; HER1; PIG61; mENA). In the present invention, its names ErbB-1, EGFR or HER1 in humans will be used interchangeably. EGFR is a member of the ErbB receptor family, with the following four closely related receptor tyrosine kinase subfamilies: ErbB-1 (EGFR), ErbB-2 (HER2 / c-neu; Her2), ErbB-3 (Her 3) and ErbB-4 (Her 4).

[0003] EGFR is present on the cell surface and can be activated by binding to its specific ligands, including epidermal growth factor and type-transforming growth factor α (TGFα). After being activated by its growth factor ligand, the receptor can undergo a transition from an inactive, mainly monomeric form to an active homodimer. In addition to forming homodimers after binding to a ligand, EGFR can also be paired with another member of the ErbB receptor family (e.g., ErbB2) to produce activated heterodimers. Dimers can also be formed without ligand binding, and activated EGFR clusters may be formed after binding to a ligand.

[0004] EGFR dimerization stimulates the activity of intrinsic intracellular protein tyrosine kinases (PTKs). This activity induces several signal transduction cascades that lead to cell proliferation and differentiation. The kinase domain of EGFR can cross-phosphorylate tyrosine residues of other receptors complexed with it and can itself be activated in this way.

[0005] Mutations involving EGFR have been found in several types of cancer. It is the target of a growing class of anticancer therapies. Such therapies include EGFR tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib and erlotinib for lung cancer, and antibodies such as cetuximab and panitumumab for colon cancer and head and neck cancer.

[0006] Cetuximab and panitumumab are monoclonal antibodies that inhibit receptors. Other monoclonal antibodies in clinical development are zalutumumab, nimotuzumab, and matuzumab. Monoclonal antibodies are designed to block extracellular ligand-induced receptor activation, primarily by blocking the binding of the ligand to the receptor. Since the binding site is blocked, the signal-inducing molecule may not be able to attach effectively and therefore cannot activate downstream signaling. Ligand-induced receptor activation can also be inhibited by stabilizing the inactivated receptor conformation (matuzumab).

[0007] To date, EGFR-targeted therapy has been associated with treatment resistance that develops over time. Various mechanisms of resistance to EGFR-TKIs have been described. In patients with advanced non-small cell lung cancer (NSCLC), resistance mechanisms include the occurrence of secondary or tertiary mutations (e.g., T790M, C797S, L718Q, exon 20 insertion mutations), activation of alternative signals (e.g., Met, HGF, AXL, Hh, IGF-1R), abnormal downstream pathways (e.g., AKT mutations, PTEN deletions), EGFR-TKIs-mediated impaired apoptosis pathways (e.g., BCL2-like 11 / BIM deletion polymorphisms), and histological type transitions. Although some resistance mechanisms have been identified, others remain to be identified. In addition, with respect to third-generation TKI resistance mechanisms, the molecular heterogeneity of NSCLC affects the contribution of the broad-spectrum resistance aberrations currently found. Similarly, patients with colorectal cancer who receive EGFR antibody therapy will also develop resistance over time. This may occur through the emergence of KRAS mutations. For those without KRAS mutations, amplification of the MET proto-oncogene may be associated with acquired resistance during anti-EGFR therapy (Bardelli et al., 2013; Cancer Discov. Jun; 3(6):658-73. doi:10.1158 / 2159-8290.CD-12-0558). Tumors may be resistant from the outset or may develop resistance during treatment. Resistance to EGFR-targeted therapies has been found in many EGFR-positive cancers and has demonstrated a need in the art for more effective EGFR cancer treatments that can improve the standard of care and have advantages in the ability to address resistance to EGFR-targeted therapies.

[0008] The dysregulation of MET proto-oncogene receptor tyrosine kinase (cMET) and hepatocyte growth factor (HGF) has been reported in a variety of tumors. Ligand-driven cMET activation has been observed in several cancers. Elevated serum and intratumoral HGF were observed in lung cancer, breast cancer, and multiple myeloma (JM Siegfried et al., Ann Thorac Surg 66, 1915 (1998); PC Ma et al., Anticancer Res 23, 49 (2003); BE Elliott et al. Can J Physiol Pharmacol 80, 91 (2002); C. Seidel et al., Med Oncol 15, 145 (1998)). Overexpression, amplification or mutation of cMET has been reported in various cancers such as colorectal cancer, lung cancer, gastric cancer and renal cancer, and can drive ligand-independent receptor activation (C. Birchmeier et al., Nat Rev Mol Cell Biol 4, 915 (2003); G. Maulik et al., Cytokine Growth Factor Rev 13, 41 (2002)). HGF expression is also associated with activation of the HGF / cMET signaling pathway and is also one of the tumor escape mechanisms under EGFR targeted therapy screening. In addition, treatment with cMET tyrosine kinase inhibitors (such as capmatinib or tepotinib) is associated with the emergence of cMET aberration escape mechanisms.

[0009] The cMET receptor is formed by proteolytic processing of a common precursor into a single-channel, disulfide-linked α / β heterodimer. The extracellular portion of cMET consists of three domain types. The N-terminal region folds to form a large semaphoring (Sema) domain, which contains the entire α-subunit and part of the β-subunit. The plexin-semaphoring-integrin (PSI) domain is located after the Sema domain and includes four disulfide bonds. This domain is connected to the transmembrane helix via four immunoglobulin-plexin-transcription (IPT) domains (which are associated with immunoglobulin-like domains). In the cell, the cMET receptor includes a tyrosine kinase catalytic domain flanked by unique near-membrane and carboxyl-terminal sequences (Organ and Tsao. Therapeutic advances in medical oncology 3.1_Supplement (2011): S7-S19, which is incorporated herein by reference in its entirety).

[0010] The ligand of cMET, hepatocyte growth factor (HGF; also known as scatter factor) and its splice isoforms (NK1, NK2) are known ligands for the cMET receptor. HGF was discovered in 1991 as a potent mitogen / morphogen. The HGF / cMET signaling pathway plays an important role in the development and progression of many cancers. Dysregulation and / or overactivation of HGF or cMET in human cancers is associated with a poor prognosis. cMET can be activated via overexpression, amplification or mutation. Activation can promote cancer development, progression, invasive growth and metastasis. cMET can be activated in an HGF-dependent and HGF-independent manner. HGF-independent activation occurs in the case of cMET overexpression. In the absence of ligand, large amounts of cMET may also trigger (hetero)dimerization and intracellular signaling. Additional ligands do not seem to affect the function of such cMET overexpressing cells. cMET amplification is associated with cMET overexpression and has become a biomarker for tumor subtypes.

[0011] HGF is widely expressed throughout the body, suggesting that this growth factor is a systemically available cytokine and is derived from the tumor stroma. Positive paracrine and / or autocrine loops of cMET activation may lead to further cMET expression. The HGF-specific antibody rilotumumab (AMG102) is in development for gastric cancer. Phase I and II trials appeared promising, but a phase III study (RILOMET-2) of cisplatin and capecitabine as first-line treatment for gastric cancer was terminated following a safety review of Study 20070622 by a pre-planned data monitoring committee.

[0012] The relevance of cMET / HGF signaling to resistance to EGFR-targeted therapies has prompted the development of approaches to address resistance. To date, antibody-based approaches, including anti-HGF antibodies; anti-cMET or cMET antibodies and cMET / EGFR (reviewed in Lee et al., 2015; Immunotargets and Therapy 4:35–44), have not been clinically effective. The cMET antibody onartuzumab (MetMab TM) and Emibetuzumab (LY-2875358) have been evaluated in Phase II clinical trials. Among them, Onartuzumab appears to be effective for colorectal cancer when combined with the EGFR-inhibitor erlotinib. However, these results could not be replicated in a randomized Phase III clinical trial. MetMAb is a monovalent monoclonal antibody (mAb) against cMET that blocks the binding of HGF to cMET and subsequent pathway activation (Jin et al., 2008 Cancer Research Vol. 68: pp. 4360-68).

[0013] To overcome the problems of anti-EGFR, cMET and HGF immunotherapy, the present invention provides novel bispecific antibodies comprising a first variable domain that can bind to the extracellular portion of epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of cMET proto-oncogene receptor tyrosine kinase (cMET).

[0014] To date, some bispecific EGFR x cMET antibodies have been described in the art. Castoldi R. et al. (2013) described a bispecific EGFR x cMET antibody named MetHer1, which has the cMET binding site of antibody 5D5 (or MetMab) and the EGFR binding site of cetuximab. The bispecific antibody has a fixed EGFR and cMET binding stoichiometry of 2:1 (see Supplementary Figure).

[0015] US20140378664 describes a cMET x EGFR bispecific antibody, among other antibodies. The complete bispecific antibody was produced as a single protein, which was then proteolytically cleaved. The two VH / VL domains were produced as single chain Fv fragments. Binding of the antibody induced degradation of cMET and phosphorylation of Akt in gastric cancer cell lines. Moores et al. (2016) describe a bispecific cMET x EGFR antibody named JNJ-61186372, produced by controlled Fab-arm exchange (cFAE), with mutations at positions 405 and 409 (according to EU numbering), which may have the potential for immunogenicity. JNJ-61186372 has been shown to have in vivo activity using a xenograft model of the tumor cell line H1975 expressing the cMET ligand HGF. This tumor model is known to rely on the ADCC activity of the antibody (Ahmed et al., 2015). JNJ-61186372 has been reported to have an unbalanced affinity, with an affinity for cMET approximately 40-fold higher than EGFR (Moores et al. (2016)), and the anti-EGFR arm derived from zalutumumab is known to cause infusion-related reactions, skin disorders and other issues.

[0016] LY3164530 is a bispecific cMET x EGFR antibody comprising the EGFR binding domain of cetuximab fused as a single chain Fv fragment to the heavy chain variable domain of the cMET binding antibody LY2875358 (Emibetuzumab; Kim and Kim 2017). It is also known as a dual variable domain antibody, which comprises two binding sites for each antigen. No data for HGF inhibition were provided for this antibody. The antibody was reported to bind and internalize cMET and EGFR without synergistic activity. The authors reviewed various cMET, EGFR, and cMET x EGFR targeted therapies and concluded that these inhibitors have not shown significant efficacy in clinical trials to date.

[0017] Therefore, there is a need for novel bispecific cMET x EGFR antibodies, including such antibodies that may have the superior properties as described herein. Summary of the invention

[0018] In certain aspects, the present invention provides a bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), for use in a method of treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor.

[0019] In certain aspects, the present invention provides a bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), for use in a method of treating cancer in an individual who has received prior treatment with ii) chemotherapy and a tyrosine kinase inhibitor.

[0020] In certain aspects, the present invention provides a bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), for use in a method of treating cancer in an individual who has received prior treatment with iii) a cMET tyrosine kinase inhibitor.

[0021] In certain aspects, the present invention provides a bispecific antibody comprising a first variable domain that binds to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), for use in a method of treating cancer in an individual, the individual iv) not having received prior anti-cancer therapy. In certain aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0022] In certain aspects, the present invention provides a method of treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor, the treatment comprising administering to the individual an effective amount of a bispecific antibody according to the present invention, comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET).

[0023] In certain aspects, the present invention provides a method of treating cancer in an individual who has received prior treatment with ii) chemotherapy and an EGFR tyrosine kinase inhibitor, the treatment comprising administering to the individual an effective amount of a bispecific antibody according to the present invention, comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET).

[0024] In certain aspects, the present invention provides a method of treating cancer in an individual who has received prior treatment with iii) a cMET tyrosine kinase inhibitor, the treatment comprising administering to the individual an effective amount of a bispecific antibody according to the present invention, comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET).

[0025] In certain aspects, the present invention provides a method of treating cancer in an individual, the individual iv) not having received any prior anti-cancer treatment, the treatment comprising administering to the individual an effective amount of a bispecific antibody according to the present invention, comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET). In certain aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0026] In certain aspects, the present invention provides a use of a bispecific antibody according to the present invention in the preparation of a medicament for treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor, the bispecific antibody comprising a first variable domain that can bind (or binds) to the extracellular part of the human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular part of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0027] In certain aspects, the present invention provides a use of a bispecific antibody according to the present invention in the preparation of a medicament for treating cancer in an individual who has received prior treatment with ii) chemotherapy and an EGFR tyrosine kinase inhibitor, the bispecific antibody comprising a first variable domain that can bind to the extracellular part of the human epidermal growth factor receptor (EGFR) and a second variable domain that can bind (or binds) to the extracellular part of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0028] In certain aspects, the present invention provides a use of a bispecific antibody according to the present invention in the preparation of a medicament for treating cancer in an individual who has received previous treatment with iii) a cMET tyrosine kinase inhibitor, the bispecific antibody comprising a first variable domain that can bind to the extracellular part of the human epidermal growth factor receptor (EGFR) and a second variable domain that can bind (or binds) to the extracellular part of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0029] In certain aspects, the present invention provides a use of a bispecific antibody according to the present invention in the preparation of a medicament for treating cancer in an individual, the individual iv) not having received any previous anti-cancer treatment, the bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind (or binds) to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET). In certain aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0030] In certain aspects, the cancer is an EGFR-positive cancer, a cMET-positive cancer, or an EGFR and cMET-positive cancer. In certain aspects, the cancer comprises an EGFR aberration, a cMET aberration, or an EGFR and cMET aberration.

[0031] In certain aspects, the individual or cancer that has received prior treatment comprising a third generation EGFR tyrosine kinase inhibitor according to i) is resistant to treatment with a third generation EGFR tyrosine kinase inhibitor.

[0032] In certain aspects, the individual or cancer that has received prior treatment comprising chemotherapy according to ii) and an EGFR tyrosine kinase inhibitor is resistant to treatment with first, second, and / or third generation EGFR tyrosine kinase inhibitors.

[0033] In certain aspects, the individual or cancer that has received prior treatment comprising a cMET tyrosine kinase inhibitor according to iii) is resistant to treatment with the cMET tyrosine kinase inhibitor.

[0034] In certain aspects, administration of the bispecific antibody according to i) is as a second line of treatment following treatment with a third generation EGFR tyrosine kinase inhibitor.

[0035] In certain aspects, the administration of the bispecific antibody according to ii) is as a third line of treatment following treatment with chemotherapy and with an EGFR tyrosine kinase inhibitor.

[0036] In certain aspects, the administration of the bispecific antibody according to iii) is as a second line of treatment following treatment with a cMET tyrosine kinase inhibitor.

[0037] In some aspects, the cancer or individual has been previously treated with a third generation EGFR tyrosine kinase inhibitor, and / or the cancer or individual is resistant to treatment with a third generation EGFR tyrosine kinase inhibitor. Preferably, the individual comprises an EGFR aberration, a cMET aberration, or an EGFR and cMET aberration that confers resistance to the third generation EGFR tyrosine kinase inhibitor. In some aspects, the third generation EGFR tyrosine kinase inhibitor comprises or is osimertinib.

[0038] In some aspects, the cancer or individual has been previously treated with chemotherapy and first, second or third generation tyrosine kinase inhibitors. In some aspects, the cancer or individual is resistant to treatment with first, second or third generation tyrosine kinase inhibitors, or is resistant to first and second generation tyrosine kinase inhibitors, or is resistant to first, second and third generation tyrosine kinase inhibitors. In some aspects, the individual comprises an EGFR aberration, or a cMET aberration, or an EGFR and cMET aberration that confers resistance to the first, second or third generation EGFR tyrosine kinase inhibitors.

[0039] In some aspects, the cancer or individual has been previously treated with a cMET tyrosine kinase inhibitor, and / or the cancer or individual is resistant to treatment with the cMET tyrosine kinase inhibitor. In some aspects, the individual comprises a cMET aberration that confers resistance to the cMET tyrosine kinase inhibitor. In some aspects, the cMET inhibitor comprises capmatinib or tepotinib.

[0040] In certain aspects, the individual comprises an EGFR and / or cMET aberration that confers resistance to the third generation EGFR tyrosine kinase inhibitor. In certain aspects, the third generation EGFR tyrosine kinase inhibitor comprises or is osimertinib.

[0041] In certain aspects, the third generation EGFR tyrosine kinase inhibitor provided as prior treatment comprises or is osimertinib, lazertinib, alflutinib, rezivertinib, rociletinib, olmutinib, almonertinib, abivertinib, ASK120067, befotinib, or In some aspects, the EGFR tyrosine kinase inhibitor is osimertinib, BPI-D0316 / Befotertinib, lazertinib or almonertinib.

[0042] In certain aspects, the first generation EGFR tyrosine kinase inhibitor provided as prior treatment with the chemotherapy comprises or is gefitinib, erlotinib, or icotinib.

[0043] In certain aspects, the second generation tyrosine kinase inhibitor provided as prior treatment comprises or is afatinib, dacomitinib, XL647, AP26113, CO-1686, or neratinib.

[0044] In certain aspects, the cMET tyrosine kinase inhibitor provided as a prior treatment is or comprises capmatinib, tepotinib, crizotenib, cabozantinib, savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833.

[0045] In certain aspects, the prior chemotherapy comprises platinum-based chemotherapy, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide or pemetrexed, or any combination thereof, and in certain aspects is a composition comprising cisplatin or carboplatin.

[0046] In certain aspects, the subject is a human subject.

[0047] In certain aspects, the cancer is non-small cell lung cancer (NSCLC), head and neck cancer, in particular head and neck squamous cell carcinoma; gastric cancer, in particular gastric adenocarcinoma; or esophageal cancer, in particular esophageal squamous cell carcinoma; gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer. In certain aspects, the cancer or individual comprises an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation, a mutation that reduces the binding of a third-generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, EGFR gene amplification, cMET mutation, cMET aberration or increased HGF expression.

[0048] In certain aspects, the cancer or individual comprises an in-frame EGFR exon 20 insertion mutation.

[0049] In some aspects, the cancer comprises a cMET aberration, such as cMET amplification, cMET overexpression, cMET pathway signaling enhancement, cMET gene amplification, and / or increased cMET protein activity. In some aspects, the cancer comprises increased HGF expression. In some aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0050] In certain aspects, the cMET dysregulation comprises cMET amplification, cMET overexpression, cMET pathway signal enhancement, cMET gene amplification, or cMET protein activity increase or cMET dysregulation caused by increased HGF expression. In certain aspects, the cMET aberration comprises a cMET exon 14 skipping mutation. cMET dysregulation in certain aspects is or comprises dysregulation of cMET signaling.

[0051] In some aspects, the use or treatment comprises providing a bispecific antibody to an individual at a dose of 1000, 1500 or 2000 mg. In some aspects, the bispecific antibody is provided once a week or once every two weeks. In some aspects, the use or treatment comprises providing a bispecific antibody to an individual at a dose of 1500 mg once every two weeks.

[0052] In certain aspects, the bispecific antibodies of the present invention exhibit ADCC activity, and in certain aspects, the antibodies have improved ADCC activity. In this aspect, the antibodies may have altered ADCC activity through one or more CH2 mutations. Therefore, a bispecific antibody according to the present invention is further provided, which is defucosylated. In certain aspects, the antibodies of the present invention comprise two defucosylated CH2 domains. In certain aspects, the antibodies of the present invention comprise a total of two CH2 domains, both of which are defucosylated. In certain aspects, the antibodies of the present invention comprise two CH2 domains, both of which are defucosylated.

[0053] In some aspects, the bispecific antibodies of the present invention exhibit ADCP activity, and in some aspects, the antibodies have improved ADCP activity. In some aspects, both EGFR and cMET binding arms, or two heavy chains comprising EGFR and cMET binding arms, contribute to ADCP. In some aspects, the bispecific antibodies of the present invention have or exhibit ADCP activity against NSCLC cells. In some aspects, the bispecific antibodies of the present invention induce ADCP of NSCLC cells.

[0054] The bispecific antibody may comprise a common light chain. In certain aspects, the first and second variable domains comprise the same or substantially the same (common) light chain variable region. The common light chain variable region may be a known good match with a variety of human variable region gene segments that have been recombined. In certain aspects, the common light chain is a variable region encoded by a germline Vk gene segment, such as an O12 / IgVκ1-39*01 variable region gene segment. The preferred light chain variable region comprises rearranged IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. In certain aspects, the light chain of the cMET binding arm and the light chain of the EGFR binding arm are the same (common) light chain. In certain aspects, the common light chain is a rearranged kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01, which is connected to a human light chain constant region. The bispecific antibody may be a human antibody. The bispecific antibody may be a full-length antibody. It may have a variable domain that can bind to EGFR and a variable domain that can bind to cMET. In certain aspects, the variable domain that can bind to human EGFR may also beneficially bind to mouse EGFR and / or macaque EGFR. In a certain aspect, the variable domain that binds or can bind to human EGFR binds to domain III of human EGFR. The variable domain that can bind to cMET can block the binding of antibody 5D5 to cMET. The variable domain that can bind to cMET can block the binding of HGF to cMET. The Kd of the antibody for cMET may be at least 10 times lower than the Kd of the antibody for EGFR. The amino acids at positions 405 and 409 in the CH3 domain may be identical to the amino acids at corresponding positions in other CH3 domains (EU numbering).

[0055] In certain aspects, an antibody of the invention comprises a first variable domain comprising a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A,

[0056] wherein X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G; and there are 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than X1 to X7.

[0057] In certain aspects, an antibody of the invention comprises a second variable domain comprising a heavy chain variable region having an amino acid sequence of one of the sequences of SEQ ID NOs: 1-23, having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

[0058] A bispecific antibody is described wherein

[0059] X1=N; X2=G; X3=D; X4=S;

[0060] X1=N; X2=A; X3=D; X4=S;

[0061] X1=S; X2=G; X3=D; X4=S;

[0062] X1=N; X2=G; X3=D; X4=R;

[0063] X1=N; X2=A; X3=D; X4=R;

[0064] X1=S; X2=G; X3=D; X4=R;

[0065] X1=N; X2=G; X3=G; X4=Y;

[0066] X1=N;X2=A;X3=G;X4=Y;X5=L;X6=D and X7=G;or

[0067] X1=S; X2=G; X3=G; X4=Y; X5=L; X6=D and X7=G.

[0068] In certain aspects, X1=N; X2=G; X3=D; X4=R; X5=H; X6=W and X7=D; or X1=N; X2=A; X3=D; X4=R; X5=H; X6=W and X7=D; or X1=S; X2=G; X3=D; X4=R; X5=H; X6=W and X7=D.

[0069] In certain aspects, X3-X7 =DRHWD, and X1 and X2 are NG; SG or NA.

[0070] In the described bispecific antibodies, the heavy chain variable region of the second variable domain comprises an amino acid sequence of one of the sequences of SEQ ID NO: 1-3; 7; 8; 10; 13; 15; 16; 17; 21; 22 or 23, which has 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof. An advantage of the present invention is that in certain aspects, the bispecific antibodies of the present invention show more potent ADCC activity than amivantamab, especially against cells or cancers containing cMET aberrations.

[0071] The present invention also provides a method of treating an individual suffering from a tumor, the method comprising administering a bispecific antibody as described herein to an individual in need thereof. Typically, the individual suffers from a disease involving abnormal cells, for example, the individual may suffer from a tumor or cancer.

[0072] The present invention also provides a bispecific antibody comprising a first variable domain that can bind to the extracellular portion of an epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of a MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYX1X2NTNYAQKLQG and a CDR3 comprising a sequence of X3X4X5X6HWWLX7A, wherein

[0073] X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G, and there are 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than X1-X7, and wherein the second variable domain comprises a heavy chain variable region, which has an amino acid sequence of one of the sequences of SEQ ID NO:1-23, which has 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

[0074] In certain aspects, the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDAFDY, and the second variable domain in certain aspects comprises a heavy chain variable region having a CDR1 sequence of SYSMN; a CDR2 sequence of WINTYTGDPTYAQGFTG, and a CDR3 sequence of ETYYYDRGGYPFDP.

[0075] The present invention also provides a bispecific antibody for use in treating an individual suffering from a disease involving abnormal cells, such as a tumor.

[0076] Also provided is the use of a bispecific antibody of the invention in the preparation of a medicament for treating a disease involving abnormal cells (such as a tumor or cancer) in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor or ii) chemotherapy and an EGFR tyrosine kinase inhibitor or iii) a cMET tyrosine kinase inhibitor.

[0077] The antibodies of the invention inhibit HGF and EGF / HGF-induced growth of cancers that have been previously treated with i) a third generation EGFR tyrosine kinase inhibitor, or ii) chemotherapy and a tyrosine kinase inhibitor, or iii) a cMET tyrosine kinase inhibitor.

[0078] In certain aspects, the antibodies of the invention inhibit the growth of HGF-responsive cells induced by HGF, in certain aspects, EGFR TKI-resistant or refractory tumors, tumor models or cell lines in human subjects, such as cell lines or models comprising activating EGFR mutations, approved tyrosine kinase inhibitor resistance mutations, third-generation tyrosine kinase inhibitor resistance mutations, mutations that reduce the binding of third-generation tyrosine kinase inhibitors to EGFR, acquired tyrosine kinase inhibitor resistance mutations, EGFR gene amplification, cMET mutations or cMET aberrations, in certain aspects, in-frame exon 20 insertion mutations. In certain aspects, the inhibitory effect is exhibited in the presence of HGF.

[0079] In certain aspects, the antibodies of the invention inhibit HGF-induced growth of HGF-responsive cells, in certain aspects, inhibit tumors in human subjects, tumor models or cell lines, wherein the tumors in human subjects, tumor models or cell lines contain activating EGFR mutations, approved tyrosine kinase inhibitor resistance mutations, third-generation tyrosine kinase inhibitor resistance mutations, mutations that reduce the binding of third-generation tyrosine kinase inhibitors to EGFR, acquired tyrosine kinase inhibitor resistance mutations, EGFR gene amplification, cMET mutations or cMET aberrations, or in-frame exon 20 insertion mutations. In certain aspects, the inhibitory effect is exhibited in the presence of HGF.

[0080] The antibodies of the invention inhibit EGF-induced growth of EGF-responsive cells, but do not induce toxicities associated with high-affinity bivalent EGFR antibodies, such as rash and diarrhea. This makes the antibodies very suitable for use in combination with TKIs that have their own toxicity profile.

[0081] The antibodies of the invention can be used to treat tumors that are resistant to treatment with EGFR tyrosine kinase inhibitors, such as tumors that are resistant or refractory to osimertinib, erlotinib, gefitinib or afatinib; analogs of osimertinib, erlotinib, gefitinib or afatinib, or a combination of one or more corresponding compounds and / or their analogs.

[0082] The present invention also includes a nucleic acid molecule or a set of nucleic acid molecules, which alone or together encode the heavy chain or heavy chain variable region of the bispecific antibody or variant thereof disclosed herein. A nucleic acid molecule or a set of nucleic acid molecules encoding the antibody disclosed herein is also provided.

[0083] In some aspects, the heavy chain comprises the constant region of an IgG1 antibody, in some aspects a human IgG1 antibody. The CH2 region of the IgG1 constant region may be modified to change the ADCC and / or CDC activity of the antibody, or not to change the ADCC and / or CDC activity of the antibody. In some aspects, the modification may result in enhanced ADCC and / or CDC activity. In some aspects, the CH3-region of the antibody is modified to promote heterodimerization of a heavy chain comprising a first heavy chain that binds to EGFR and a second heavy chain that binds to cMET.

[0084] The present invention also includes a cell comprising one or more nucleic acid molecules that encode, alone or in combination, the bispecific antibodies or variants thereof disclosed herein. Methods of using the above-mentioned cells to produce the bispecific antibodies or variants thereof disclosed herein are also provided, in some aspects, in combination with harvesting the bispecific antibodies or variants thereof from cell culture.

[0085] The present invention also comprises a cell system comprising the bispecific antibody or variant thereof disclosed herein.

[0086] The present invention also provides a cell, which expresses the bispecific antibody and / or contains a nucleic acid molecule encoding the bispecific antibody.

[0087] The present invention also comprises a bispecific antibody as disclosed herein, further comprising a label, which in certain aspects is a label for in vivo imaging. DETAILED DESCRIPTION

[0088] EGFR is a member of the family of four receptor tyrosine kinases (RTKs), named Her- or cErbB-1, -2, -3 and -4. EGFR has an extracellular domain (ECD) consisting of four subdomains, two of which are involved in ligand binding, one of which is involved in homodimerization and heterodimerization, Ferguson (2008). The reference numbers used in this section are the reference numbers in the list entitled "Citations in the Specification", each of which is incorporated herein by reference as a whole. EGFR integrates extracellular signals from various ligands to produce divergent intracellular responses (Yarden et al., 2001; and Jorrisen et al., 2003). EGFR is involved in several human epithelial malignancies, notably breast cancer, bladder cancer, non-small cell lung cancer, lung cancer, colon cancer, ovarian cancer, head and neck cancer, and brain cancer. Activating mutations in genes and overexpression of receptors and their ligands have been found to cause autocrine activation loops (see Robertson et al., 2000 for review). Therefore, this RTK has been widely used as a target for cancer therapy. Small molecule inhibitors targeting RTK and monoclonal antibodies (mAbs) directed to the extracellular ligand-binding domain have both been developed and have achieved a number of clinical successes to date, although mainly for selected patient populations. The database accession number for human EGFR protein and its encoding gene is (GenBank NM_005228.3). Other database identifiers for the gene and / or protein are HGNC:3236; Entrez Gene:1956; Ensembl:ENSG00000146648; OMIM:131550 and UniProtKB:P00533. The accession number is provided mainly to provide a method for further identifying the EGFR protein as a target, and the actual sequence of the EGFR protein bound by the antibody may be different, for example, due to mutations in the encoding gene, such as occurring in certain cancers, or similar situations. Unless otherwise specified, when referring to EGFR herein, it refers to human EGFR. The antigen binding site binding to EGFR binds to EGFR and its various variants, such as those expressed on certain EGFR-positive tumors.

[0089] As used herein, the term "EGFR ligand" refers to a polypeptide that binds to and activates EGFR. Examples of EGFR ligands include, but are not limited to, EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, and epiregulin (for review, see Olayioye MA et al.; EMBO J (2000) Vol. 19: pp. 3159-3167). The term includes biologically active fragments and / or variants of naturally occurring polypeptides.

[0090] Abnormally activated forms of EGFR, such as through EGFR mutations or EGFR gene amplification, are known to be oncogenic drivers of non-small cell lung cancer (NSCLC) and are known to occur in therapy with EGFR tyrosine kinase inhibitors. The present invention provides a bispecific antibody for use in a method of treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor, or ii) chemotherapy and a tyrosine kinase inhibitor, or iii) a cMET tyrosine kinase inhibitor, or iv) has not previously received anti-cancer therapy, wherein the individual of iv) comprises a cMET exon 14 skipping mutation or a cancer comprising a cMET exon 14 skipping mutation in certain aspects.

[0091] In certain aspects, the treatment comprises treating a cancer caused by ligand-independent activation of EGFR and / or ligand-independent activation of cMET. In another aspect, the treatment comprises treating a cancer caused by ligand-dependent activation of EGFR and / or ligand-dependent activation of cMET.

[0092] In certain aspects, the cancer or individual has previously been treated with osimertinib and has acquired or tertiary osimertinib resistance. The prior osimertinib treatment is in certain aspects a first-line or second-line therapy, and in certain aspects, the first-line therapy is followed by treatment with a bispecific antibody of the invention as a second-line therapy.

[0093] In certain aspects, the cancer or individual comprises an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a third-generation tyrosine kinase inhibitor resistance mutation, a mutation that reduces binding of a third-generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, EGFR gene amplification, cMET mutation, cMET aberration, or increased HGF expression.

[0094] In clinical trials conducted for the efficacy of the bispecific antibodies of the present invention, clinical efficacy was observed in a variety of cancers with different genetic carcinogenic backgrounds. Clinical efficacy was observed in lung cancer, especially non-small cell lung cancer (NSCLC). For example, clinical efficacy was observed in patients with various EGFR mutations, including EGFR exon 20 mutations / insertions, EGFR exon 21 mutations (e.g., L858R), deletion mutations of EGFR exon 19, or c-MET exon 14 skipping mutations. Therefore, in one aspect, the cancer is NSCLC and / or the individual suffers from NSCLC, wherein the cancer or individual comprises EGFR exon 21 mutations, such as L858R, deletion mutations of EGFR exon 19, mutations of EGFR exon 18, or c-MET exon 14 skipping mutations.

[0095] Furthermore, clinical efficacy was observed in head and neck cancer, in particular head and neck squamous cell carcinoma (HNSCC).Thus, in a certain aspect, the cancer is, and / or the individual suffers from, head and neck cancer, in particular head and neck squamous cell carcinoma.

[0096] In certain aspects, the cancer is gastric adenocarcinoma with c-MET amplification.

[0097] In certain aspects, the treatment results in or comprises depletion of soluble EGFR and / or cMET.

[0098] In certain aspects, the cancer comprises an activating EGFR mutation, such as an in-frame exon 19 deletion mutation or an exon 21 mutation (L858R in certain aspects). Herein, the term "activating EGFR mutation" refers to a mutation developed after the use of a third-generation EGFR tyrosine kinase inhibitor, including an in-frame deletion of exon 19 (del19) and a substitution of leucine by arginine in exon 21 (L858R).

[0099] In some aspects, the cancer comprises an approved tyrosine kinase inhibitor resistance mutation. Herein, the term "approved tyrosine kinase inhibitor resistance mutation" refers to the development of resistance after using an EGFR tyrosine kinase inhibitor currently approved for the treatment of cancer, such as T790M that confers resistance to afatinib. In some aspects, the EGFR tyrosine kinase inhibitor is an approved EGFR tyrosine kinase inhibitor.

[0100] In certain aspects, the cancer comprises a tertiary tyrosine kinase inhibitor resistance mutation, such as L718X (e.g., L718Q), G719X (e.g., G719A), L792X (e.g., L792H), G796X (e.g., G796R, G796S, G796D), C797X, C797X (e.g., C797S, C797G). Herein, the term "tertiary tyrosine kinase inhibitor resistance mutation" refers to resistance developed after the use of a third-generation EGFR tyrosine kinase inhibitor (in certain aspects, osimertinib).

[0101] In certain aspects, the cancer comprises a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, e.g., L792X, L718X.

[0102] In certain aspects, the cancer comprises an acquired tyrosine kinase inhibitor resistance mutation (e.g., T790M, L858R, exon 19 deletion mutation, C797X, L792X, G796X, G724X, S768X, L718X, or exon 20 insertion mutation), which in certain aspects is a mutation that confers resistance to osimertinib or occurs after the use of osimertinib, including G724X (e.g., G724S), S768X (e.g., S768I), L792X (e.g., L792H), C797X (including C797S and C797G), L798X (e.g., L798I). Here, the term "acquired tyrosine kinase inhibitor resistance mutation" refers to resistance acquired after treatment with a tyrosine kinase inhibitor (e.g., after the use of a third-generation EGFR tyrosine kinase inhibitor).

[0103] In certain aspects, the cancer comprises EGFR gene amplification, such as an increase in EGFR mRNA or amplification of a wild-type EGFR allele, and the emergence of an EGFR-ex19del allele after use of osimertinib.

[0104] In certain aspects, the cancer comprises a cMET mutation, such as a cMET exon 14 skipping mutation.

[0105] In some aspects, the cancer comprises a cMET aberration, such as cMET amplification, cMET overexpression, cMET pathway signaling enhancement, cMET gene amplification, increased HGF expression, and / or increased cMET protein activity. In some aspects, the cancer is NSCLC, and the cMET amplification is characterized by MET / CEP7>5 or cfDNA≥2 copies or any combination thereof.

[0106] In certain aspects, cMET amplification is characterized by MET / CEP7≥3, MET / CEP7≥4, or MET / CEP7≥5 (up to 15 or 20 or less), or cfDNA≥1.8 cMET copy number, such as (≥1.8, <2.2), or (>2.2, <5) or (≥5).

[0107] In certain aspects, the cancer comprises an exon 19 deletion mutation, in certain aspects comprises an in-frame exon 19 deletion, an exon 20 missense mutation (eg, T790M), or an exon 21 mutation, eg, L858R.

[0108] In certain aspects, the cancer comprises an EGFR exon 20 mutation, in certain aspects, an exon 20 insertion mutation, in certain aspects, an in-frame exon 20 insertion mutation.

[0109] In some aspects, the cancer or individual comprises an exon 20 mutation selected from near-loop insertion (positions 767-772), far-loop insertion (positions 773-775), in some aspects V769_D770insASV, D770_N771insSVD, H773_V774insNPH, H773_V774insH, D770_N771insG, D770delinsGY, N771_P772insN, V774_C775ins HV, D770_N771insGL, H773_V774insPH, A763_Y764insFQEA, D770_N771delinsEGN, D770_N771insGD, D770_N7 71insH、D770_N771insP、H773_V774insAH、H773_V774insGNPH、H773delinsSNPY、N771_P772insH、N771_P772i nsVDN, N771delinsGY, N771delinsKH, N771delinsRD, P772_H773delinsHNPY, P772_H773insGT, P772_H773insPNP, P772_H773insT, V769_D770insA, V769_D770insGG, V769_D770insGSV, V769_D770insGVV, and V769_D770ins MASV; or mutations T790M, L792X (e.g., L792H), C796X (e.g., G796R, G796S, G796D), C797X (e.g., C797S, C797G), L798I, or in-frame exon 20 insertions, such as M766_A767insASV or H773-V774insNPH, Ins761(EAFQ), Ins770(ASV), Ins771(G), Ins774(NPH), M766_A7671ns A. S768_V769InsSVA, P772_H773InsNS, D761_E762InsX1-7, A763_Y764InsX1-7, Y764_Y765 InsX1-7, M766_A767InsX1-7, A767_V768 InsX1-7, S768_V769 InsX1-7>V769_D770 InsX1-7>D770_N771 InsX1-7>N771_P772 InsX1-7>P772_H773 InsX1-7, H773_V774 InsX1-7, or V774_C775 InsX1-7.In certain aspects, the cancer or individual comprises two or more of said mutations.

[0110] In certain aspects, the cancer or individual comprises a mutation such as L718X (e.g., L718Q, L718V), G719X (e.g., G719A), L792X (e.g., L792H, L792F, L792R, L792Y, L792V, and L792P), G796X (e.g., G796R, G796S, G796D), C797X, C797X (e.g., C797S, C797G, C797N), C797X, L792X, G796X, G724X, S768X, L718X, M766X (e.g., M766Q), R776X (e.g., R776C), or an exon 20 insertion mutation. In certain aspects, the cancer or individual comprises two or more of the mutations.

[0111] In some aspects, the cancer or individual comprises an exon 19 deletion mutation, in some aspects an in-frame exon 19 deletion mutation, an exon 21 missense mutation (such as T790M), or an exon 21 mutation such as L858R, L861X (such as L861Q) or L844X (such as L844V). In some aspects, the cancer or individual comprises two or more of the mutations.

[0112] In certain aspects, the cancer or individual comprises a cancer comprising an EGFR exon 20 mutation, in certain aspects comprising an exon 20 insertion mutation.

[0113] In some aspects, the cancer or individual comprises a mutation selected from G724X (such as G724S), S768X (such as S768I), T790X (such as T790M), L792X (such as L792H), C797X (including C797S and C797G), L798X (such as L798I), I941X (such as I941R), V948X (such as V948R). In some aspects, the cancer or individual comprises two or more of the mutations.

[0114] In certain aspects, the cancer or individual comprises the double mutation L858X / T790X (e.g., L858R / T790M), T790X / L798X (e.g., T790M / L798I), T790X / C797X (e.g., T790M / C797S), G719X / R776X (e.g., G719A / R776C), or delE746_A750 / T790M.

[0115] In certain aspects, the cancer or individual comprises the double mutation D770insSVD / E762X (eg, E762K), D770insSVD / L792X (eg, L792I, L792S), D770insSVD / P794X (eg, P794S), or D770insSVD / G796X (eg, G796D).

[0116] In certain aspects, the cancer or individual comprises a double mutation H773insH / E762X (eg, E762K), H773insH / L792X (eg, L792I, L792S), H773insH / P794X (eg, P794S), or H773insH / G796X (eg, G796D).

[0117] In certain aspects, the cancer or individual comprises a double mutation H773insNPH / E762X (eg, E762K), H773insNPH / L792X (eg, L792I, L792S), H773insNPH / P794X (eg, P794S), or H773insNPH / G796X (eg, G796D).

[0118] In certain aspects, the cancer or individual comprises the double mutation L858X / L718X (eg, L858R / cis-L718Q), L858X / C797X (eg, L858R / cis-C797S), exon 19del / C797X (eg, exon 19del / cis-C797S).

[0119] In certain aspects, the cancer or individual comprises the triple mutations L858X / T790X / C797X (e.g., L858R / T790M / C797S), L858X / T790X / M766X (e.g., L858R / T790M / M766Q), L858X / T790X / L718X (e.g., L858R / T790M / cis-L718Q, L858R / T790M / L718Q), L858X / T790X / C797X (such as L858R / T790M / cis-C797S), exon 19del / T790X / C797X (such as exon 19del / T790M / cis-C797S), L858X / T790X / C941X (such as L858R / T790M / I941R), delE746_A750 / T790X / C797X (such as delE746_A750 / T790M / C797S).

[0120] In certain aspects, the cancer or individual comprises a cMET exon 14 skipping mutation.

[0121] As used herein, the term "refractory" refers to a disease that does not respond to a treatment. A refractory disease may develop resistance to the treatment before or at the start of treatment, or a refractory disease may become resistant during treatment.

[0122] Herein, “first-generation EGFR tyrosine kinase inhibitors” (first-generation TKIs) refer to reversible EGFR inhibitors such as gefitinib and erlotinib, which are effective first-line treatments for NSCLC harboring EGFR activating mutations such as exon 19 deletions and exon 21 L858R mutations.

[0123] As used herein, the term “second-generation EGFR tyrosine kinase inhibitors” (second-generation TKIs) refers to covalent irreversible EGFR inhibitors such as afatinib and dacomitinib, which are effective first-line treatments for NSCLC harboring EGFR activating mutations (such as exon 19 deletions and exon 21 L858R mutations).

[0124] As used herein, the term "third-generation EGFR tyrosine kinase inhibitors" (third-generation TKIs) refers to covalent irreversible EGFR inhibitors, such as osimertinib and lazertinib, which are selective for EGFR activating mutations, such as exon 19 deletions and exon 21 L858R mutations, alone or in combination with T790M mutations, and have lower inhibitory activity against wild-type EGFR.

[0125] As used herein, the term "resistance" refers to a cancer or patient that does not respond to treatment when administered with a prescribed dose of a relevant therapeutic agent, including a third generation EGFR tyrosine kinase inhibitor.

[0126] In certain aspects, the third generation EGFR tyrosine kinase inhibitor is osimertinib (AZD9291). Osimertinib (AZD9291) is a covalent, orally active, irreversible and mutation-selective EGFR inhibitor with an apparent IC50 of 12 nM for L858R and 1 nM for L858R / T790M. The recommended Phase 2 dose has been established as a daily dose of 80 mg.

[0127] In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Almonertinib (HS-10296). Almonertinib is an oral, irreversible third generation EGFR tyrosine kinase inhibitor that is selective for EGFR-sensitization and T790M resistance mutations. Almonertinib is used in the study of non-small cell lung cancer. The recommended Phase 2 dose has been established as a daily dose of 110 mg.

[0128] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is lazertinib. Lazertinib (YH25448) is a potent, mutation-selective, blood-brain barrier-penetrating, orally available, irreversible third-generation EGFR tyrosine kinase inhibitor that can be used in studies of non-small cell lung cancer. The recommended Phase 2 dose has been established as a daily dose of 240 mg

[0129] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is Befotertinib (or BPI-D0316 or sometimes referred to as D-0316). Befotertinib (Beta Pharmaceuticals, Co., China) is a third-generation EGFR tyrosine kinase inhibitor. Befotertinib can be used for EGFR-positive non-small cell lung cancer (NSCLC). In the Phase II, single-arm study NCT05007938, the safety and efficacy of the combination of Befotertinib (25 mg, 3 times a day, orally) and icotinib (125 mg, 3 times a day, orally) were evaluated in patients with locally advanced or metastatic NSCLC. In the Phase I study NCT04464551, individuals received a single oral dose of 75 mg of D-0316 oral suspension. In the Phase II, single-arm study NCT03861156, patients with locally advanced / metastatic non-small cell lung cancer received an oral dose of 75 mg for 21 days, and if tolerated, the dose was increased to 100 mg. Otherwise, the dose was maintained at 75 mg. In the Phase II / III study NCT04206072, the efficacy and safety of D-0316 were evaluated at 70 mg once daily for 21 days, followed by an increase to 100 mg once daily.

[0130] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is Alflutinib (AST2818 or Furmonertinib). AST2818 is the subject of clinical trial NCT03787992, which studies its clinical efficacy in NSCLC.

[0131] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is Rezivertinib (BPI-7711), which is an orally active, selective and irreversible third-generation EGFR tyrosine kinase inhibitor (TKI). Rezivertinib is the subject of clinical trial NCT03866499, studying its clinical efficacy in NSCLC.

[0132] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is Avitinib (Abivertinib / AC0010), which is a pyrrolopyrimidine-based irreversible epidermal growth factor receptor (EGFR) inhibitor with an IC50 of 7.68 nM. Avitinib is the subject of clinical trial NCT03856697, studying its clinical efficacy in NSCLC.

[0133] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is ASK120067, which is an effective, orally active EGFR inhibitor. ASK120067 is a third-generation EGFR-TKI for non-small cell lung cancer (NSCLC) research. ASK120067 is the subject of a clinical trial (NCT04143607) to study its clinical efficacy in NSCLC.

[0134] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is Oritinib (SH-1028, Nanjing Shenghe Pharmaceutical Co., Ltd., Nanjing, China). SH-1028 is the subject of clinical trial NCT04239833, studying its clinical efficacy in NSCLC.

[0135] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is Rociletinib (CO-1686), an orally delivered kinase inhibitor that specifically targets mutated EGFR forms. Rociletinib is the subject of clinical trial NCT02186301, studying its clinical efficacy in NSCLC.

[0136] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is Olmutinib (HM61713; BI-1482694), which is an orally active and irreversible third-generation EGFR tyrosine kinase inhibitor that binds to cysteine ​​residues near the kinase domain. Olmutinib can be used for research in NSCLC. Olmutinib is the subject of clinical trial NCT02485652, which studies its clinical efficacy in NSCLC.

[0137] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is Nazartinib (EGF816), which is a third-generation EGFR TKI that selectively inhibits EGFR activating mutations in NSCLC patients with advanced EGFR-mutations. Nazartinib is the subject of clinical trial NCT03529084, studying its clinical efficacy in NSCLC.

[0138] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is naquotinib, which is an oral, irreversible, third-generation, mutation-selective epidermal growth factor receptor (EGFR) inhibitor. Naquotinib is the subject of clinical trial NCT02588261, studying its clinical efficacy in NSCLC.

[0139] In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is Maveritinib (PF-0647775), which is a selective, oral and irreversible EGFR tyrosine kinase inhibitor (EGFR TKI). Maveritinib is the subject of clinical trial NCT02349633, studying its clinical efficacy in NSCLC.

[0140] cMET, also known as tyrosine protein kinase MET or hepatocyte growth factor receptor (HGFR), is a protein encoded by the MET gene in humans. The protein has tyrosine kinase activity. The primary single-chain precursor protein is post-translationally cleaved to produce α and β subunits, which are linked via disulfide bonds to form the mature receptor.

[0141] Dysregulation or abnormal activation of cMET may induce tumor growth, the formation of new blood vessels that supply nutrients to the tumor (angiogenesis), and the spread of cancer to other organs (metastasis). cMET is dysregulated in many types of human malignancies, including kidney cancer, liver cancer, stomach cancer, breast cancer, and brain cancer. The cMET gene has many different names, such as MET proto-oncogene receptor tyrosine kinase; hepatocyte growth factor receptor; tyrosine protein kinase Met; scatter factor receptor; proto-oncogene C-Met; HGF / SF receptor; HGF receptor; SF receptor; EC 2.7.10.1; Met proto-oncogene; EC 2.7.10; DFNB97; AUTS9; RCCP2; C-Met; MET; HGFR; cMET external IDs are HGNC:7029; Entrez Gene:4233; Ensembl:ENSG00000105976; OMIM:164860 and UniProtKB:P08581. Accession numbers are provided primarily to provide a means of further identifying cMET proteins as targets, and the actual sequence of cMET proteins bound by antibodies may vary, for example, due to mutations in the encoding gene, such as those that occur in certain cancers, or the like. When cMET is referred to herein, unless otherwise indicated, reference is made to human cMET. An antigen binding site that binds to cMET binds to cMET and its various variants, such as variants expressed on certain cMET-positive tumors. Examples of cMET aberrations or disorders include cMET mutations (e.g., exon 14 skipping mutations), cMET amplification, cMET overexpression, cMET pathway signaling enhancement, cMET gene amplification, and / or increased cMET protein activity. cMET disorder may also be caused by increased HGF expression. c-MET disorder is a known driver of tumor invasion, angiogenesis, and metastasis (Birchmeier et al., 2003). The following three types of biological changes in c-MET can lead to tumorigenesis: amplification, mutation, and fusion. These genomic changes have been found to be, in principle, major or minor drivers of tumor growth, and such aberrations have been reported to occur after treatment of cancer patients with EGFR tyrosine kinase inhibitors (see Suzawa et al., DOI: 10.1200 / PO.19.00011 JCO Precision Oncology - May 10, Vol. 3, 2019).

[0142] In some aspects, the individual under iv) of the present invention has not received previous anticancer treatment. In some aspects, the individual has not received previous treatment with a tyrosine kinase inhibitor. In some aspects, the individual has not received previous treatment with a cMET inhibitor. In some aspects, the individual has not received previous treatment with a cMET tyrosine kinase inhibitor. In some aspects, the individual has not received previous treatment with a tyrosine kinase inhibitor (such as capmatinib, tepotinib or savolitinib). The individual may have received treatment with a chemotherapeutic agent (e.g., platinum chemotherapy), or an immunotherapeutic agent (e.g., pembrolizumab, nivolumab, cetuximab). In some aspects, the individual suffers from metastatic non-small cell lung cancer. In some aspects, the metastatic NSCLC has a mutation that causes cMET exon 14 skipping. Exon 14 skipping can be detected by an FDA-approved test.

[0143] In certain aspects, the individual comprises a cMET exon 14 skipping mutation or a cancer comprising a cMET exon 14 skipping mutation. The individual has not previously received any anti-cancer treatment for cancer, such as an EGFR and / or cMET positive cancer, or a cancer comprising an EGFR and / or cMET aberration. Therefore, in certain aspects, the administration or treatment of the bispecific antibody of the present invention is a first-line treatment. In certain aspects, the first-line treatment is to prevent the development of resistance to EGFR and / or cMET tyrosine kinase treatment mechanisms, such as in lung cancer patients or lung cancer, particularly in non-small cell lung cancer. It is well known that resistance mechanisms to EGFR and / or cMET tyrosine kinase inhibitors develop in, especially in individuals with non-small cell lung cancer, such as in metastatic or advanced cancers. Therefore, the present invention also provides a bispecific antibody according to the present invention, for use in a method for preventing an individual from developing or developing a cancer that is resistant to an EGFR and / or cMET tyrosine kinase inhibitor.

[0144] In certain aspects, the individual of iv) of the present invention is a human individual.

[0145] In some aspects, the use or treatment comprises providing iv) the individual with a 1000, 1500 or 2000 mg dose of the bispecific antibody of the invention. In some aspects, the bispecific antibody is provided once a week or once every two weeks. In some aspects, the use or treatment comprises providing the bispecific antibody to the individual at a dose of 1500 mg once every two weeks. The present invention also provides a method for treating the individual with a tumor, the method comprising administering a bispecific antibody as described herein to the individual in need. Typically, the individual suffers from a disease involving abnormal cells, for example, the individual may suffer from a tumor or cancer.

[0146] In certain aspects, the individual of iv) has non-small cell lung cancer (NSCLC), head and neck cancer, stomach cancer, esophageal cancer, stomach / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer. In certain aspects, the individual of iv) has cancer, which is non-small cell lung cancer (NSCLC).

[0147] In certain aspects, the individual in iv) has advanced or metastatic cancer, such as advanced or metastatic NSCLC.

[0148] In certain aspects, the treatment of the individual in iv) comprises a diagnostic step for assessing whether the cancer is an EGFR-positive and / or cMET-positive cancer, or assessing the presence of EGFR and / or cMET aberrations in the cancer.

[0149] Antibodies usually recognize only a portion of an antigen. Antigens are usually, but not necessarily, proteins. The recognition or binding site on an antigen that is bound by an antibody is called an epitope, which can be either a linear or a conformational epitope. The binding of an antibody to an antigen is usually specific. The "specificity" of an antibody refers to its selectivity for a particular epitope, while "affinity" refers to the strength of the interaction between the antigen binding site of an antibody and the epitope to which it binds.

[0150] Exemplary antibodies of the invention bind to EGFR and cMET, and in certain aspects bind to human EGFR and human cMET. The EGFR / cMET bispecific antibodies of the invention bind to EGFR, and under other identical conditions, the binding is at least 100-fold lower than the binding to the receptors ErbB-2 and ErbB-4 of the same species. The EGFR / cMET bispecific antibodies of the invention bind to cMET, and under other identical conditions, the binding is at least 100-fold lower than the binding to the receptors ErbB-2 and ErbB-4 of the same species. Considering that the receptor is a cell surface receptor, the binding can be assessed on cells expressing the receptor. The bispecific antibodies of the invention bind to human, macaque EGFR and / or mouse EGFR in certain aspects.

[0151] Antibodies that bind to EGFR and cMET may also bind to other proteins if such other proteins contain the same epitope. Therefore, the term "binding" does not exclude the binding of an antibody to another protein or protein group containing the same epitope. Such binding is generally referred to as cross-reaction. EGFR / cMET bispecific antibodies generally do not bind to other proteins other than EGFR and / or cMET on postnatal (in some aspects adult humans) cell membranes. Antibodies according to the present invention are generally capable of binding to EGFR with a binding affinity (i.e., equilibrium dissociation constant Kd) of at least 1x10e-6M, as described in more detail below.

[0152] As used herein, the term "antibody" refers to a protein molecule that belongs to the class of immunoglobulin proteins in certain aspects. Antibodies generally contain two variable domains that can bind to an epitope on an antigen. Such domains are derived from or share sequence homology with the variable domains of an antibody. The bispecific antibodies of the present invention comprise two variable domains in certain aspects. Antibodies for therapeutic use are as close as possible to the natural antibodies of the individual to be treated (e.g., human antibodies for human individuals) in certain aspects. Antibody binding can be represented by specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by the binding domain. Typically, antibodies for therapeutic applications can have an affinity of up to 1x10e-10M or higher. Antibodies such as the bispecific antibodies of the present invention comprise the constant domains (Fc parts) of natural antibodies in certain aspects. Antibodies of the present invention are generally full-length bispecific antibodies, which are human IgG subclasses in certain aspects. In certain aspects, antibodies of the present invention are human IgG1 subclasses. Such antibodies of the present invention may have good ADCC properties, have a favorable half-life after being administered to humans in vivo, and the currently existing CH3 modification technology can provide a modified heavy chain that preferentially forms heterodimers rather than homodimers when co-expressed in cloned cells. The ADCC activity of antibodies may also be improved by techniques known to those skilled in the art.

[0153] In certain aspects, the antibodies of the present invention are "full-length" antibodies. The term "full-length" disclosed herein is defined as comprising substantially complete antibodies, but it does not necessarily have all the functions of complete antibodies. For the avoidance of doubt, a full-length antibody comprises two heavy chains and two light chains. Each chain comprises a constant region (C) and a variable region (V), which can be disassembled into domains named CH1, CH2, CH3, VH and CL, VL. Typically, 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 through the constant domain (mainly through the Fc portion). Full-length antibodies according to the present invention encompass antibodies in which mutations providing desired properties may be present. Antibodies in which one or more amino acid residues are deleted but do not substantially change the specificity and / or affinity characteristics of the resulting antibody are included in the term "full-length antibody". For example, an IgG antibody may have 1-20 amino acid residues inserted, deleted or substituted or a combination thereof in the constant region.

[0154] In certain aspects, the antibodies of the present invention are bispecific IgG antibodies, such as bispecific full-length IgG1 antibodies or human IgG1. Full-length IgG antibodies are preferred because they generally have a favorable half-life and, for immunogenic reasons, it is desirable to be as close to a complete autologous (human) molecule as possible. In certain aspects, the antibodies of the present invention are full-length IgG1, full-length IgG2, full-length IgG3 or full-length IgG4 antibodies.

[0155] A variable domain that can bind to EGFR and comprises the amino acid sequence of MF3370 or a variant thereof as shown herein, binds to EGFR domain III in certain aspects (see International Patent Application No. PCT / NL2015 / 050124; Table 4 of WO2015 / 130172, which is incorporated herein by reference in its entirety). In certain aspects, the variable domain blocks the binding of the ligand EGF to EGFR, or competes with the EGF ligand for binding to EGFR. The binding of the variable domain to EGFR can be inhibited by cetuximab. The variable domain binds to an epitope that is different from the epitope recognized by cetuximab and zalutumumab. For example, the variable domain binds to mouse EGFR, while cetuximab and zalutumumab do not, indicating that one or more residues that differ between mouse and human EGFR domain III play a role in binding to cetuximab and zalutumumab, but not in the antibodies of the invention described herein. An advantage of the bispecific antibodies of the invention having cross-reactivity to human, mouse, and macaque EGFR is that it allows for use in xenograft studies in human cancer models, which can be more predictive in terms of efficacy and toxicity, because the antibodies also bind to normal mouse cells with the receptor, and can also be used for toxicology studies in macaques. In certain aspects, the invention provides a bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain can also bind to mouse EGFR, macaque EGFR, or both.

[0156] In certain aspects, the cMET variable domain comprises the amino acid sequence of MF4356 or a variant thereof as set forth herein, and in certain aspects blocks the binding of the antibody MetMab to cMET. The cMET variable domain comprises the amino acid sequence of MF8230 or a variant thereof as set forth herein, and in certain aspects blocks the binding of the antibody MetMab to cMET. The variable domain blocks the binding of the ligand HGF to cMET in certain aspects, or competes with the ligand HGF for binding to cMET. The variable domain blocks the binding of the antibody MetMab to cMET when the binding of MetMab to cMET is reduced by at least 40% and in certain aspects by at least 60% under half-maximal binding conditions in the presence of a saturating amount of the variable domain. The variable domain is provided in certain aspects in the context of a bivalent monospecific antibody. The cMET variable domain may bind to the sema domain of cMET in certain aspects. The cMET variable domains of the present invention can compete with 5D5 for cMET binding, or do not compete with reported anti-cMET reference antibodies such as 5D5. See Table 2.

[0157] The variable domain of the present invention can bind to EGFR (first variable domain) and in certain aspects comprises a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG, and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, wherein X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W and X7=D or G.

[0158] X 1-7 In some aspects:

[0159] X1=N; X2=G; X3=D; X4=S;

[0160] X1=N; X2=A; X3=D; X4=S;

[0161] X1=S; X2=G; X3=D; X4=S;

[0162] X1=N; X2=G; X3=D; X4=R;

[0163] X1=N; X2=A; X3=D; X4=R;

[0164] X1=S; X2=G; X3=D; X4=R;

[0165] X1=N; X2=G; X3=G; X4=Y;

[0166] X1=N;X2=A;X3=G;X4=Y;X5=L;X6=D and X7=G;or

[0167] X1=S; X2=G; X3=G; X4=Y; X5=L; X6=D and X7=G.

[0168] In some aspects,

[0169] X1=N; X2=G; X3=D; X4=R;

[0170] X1=N; X2=A; X3=D; X4=R; X5=H; X6=W and X7=D; or

[0171] X1=S; X2=G; X3=D; X4=R; X5=H; X6=W and X7=D.

[0172] In certain aspects, X1 = N; X2 = G; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D.

[0173] The amino acid after the amino acid A in the sequence X3X4X5X6HWWLX7A in the CDR3 sequence of the first variable domain may vary. The amino acid sequence after the sequence X3X4X5X6HWWLX7A may be FDY. The CDR3 of the first variable domain comprises the sequence X3X4X5X6HWWLX7AF in some aspects, comprises X3X4X5X6HWWLX7AFD in some aspects, comprises X3X4X5X6HWWLX7AFDY in some aspects.

[0174] The first variable domain in certain aspects comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYNGNTNYAQKLQG; and a CDR3 sequence of X3X4X5X6HWWLX7A.

[0175] The first variable domain in certain aspects comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYNGNTNYAQKLQG, and a CDR3 comprising the sequence DRHWHWWLDA. The amino acid after the LDA sequence in the CDR3 sequence of the first variable domain may vary. The amino acid sequence after the LDA sequence may be FDY. The CDR3 of the first variable domain in certain aspects comprises the sequence DRHWHWWLDAF, in certain aspects, it comprises DRHWHWWLDAFD, in certain aspects, it comprises DRHWHWWLDAFDY.

[0176] The first variable domain in certain aspects comprises a heavy chain variable region having Figure 2 The amino acid sequence of MF3353; MF8229; MF8228; MF3370; MF8233; MF8232; MF3393; MF8227 or MF8226 shown in the specification has up to 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the specified sequence. In certain aspects, the first variable domain comprises a heavy chain variable region having Figure 2 In certain aspects, the first variable domain comprises a heavy chain variable region having an amino acid sequence of MF3353; MF8229; MF8228; MF3370; MF8233; MF8232; MF3393; MF8227 or MF8226. Figure 2 The CDR1, CDR2, and CDR3 amino acid sequences of MF3353; MF8229; MF8228; MF3370; MF8233; MF8232; MF3393; MF8227 or MF8226 are shown.

[0177] The variable domain that can bind to cMET (the second variable domain) in certain aspects comprises a heavy chain variable region comprising SEQ ID NO: 1-23 ( Figure 3) with 0 to 10, in some aspects 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. The heavy chain variable region of the second variable domain in some aspects comprises an amino acid sequence of one of the sequences of SEQ ID NO: 1-3; 7; 8; 10; 13; 15; 16; 17; 21; 22 or 23 with 0 to 10, in some aspects 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. The heavy chain variable region of the second variable domain in some aspects comprises an amino acid sequence of one of the sequences of SEQ ID NO: 2; 7; 8; 10; 13 or 23 with 0 to 10, in some aspects 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. The heavy chain variable region of the second variable domain in some aspects comprises an amino acid sequence of the sequence of SEQ ID NO: 13 or SEQ ID NO: 23 with 0 to 10, in some aspects 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In certain aspects, the second variable domain comprises a heavy chain variable region having MF8225 (SEQ ID NO: 1), MF8243 (SEQ ID NO: 2), MF8224 (SEQ ID NO: 3), MF8239 (SEQ ID NO: 4), MF8242 (SEQ ID NO: 5), MF8237 (SEQ ID NO: 6), MF8240 (SEQ ID NO: 7), MF8234 (SEQ ID NO: 8), MF8245 (SEQ ID NO: 9), MF8231 (SEQ ID NO: 10), MF8247 (SEQ ID NO: 11), MF8238 (SEQ ID NO: 12), MF8230 (SEQ ID NO: 13), MF8248 (SEQ ID NO: 14), MF8246 (SEQ ID NO: 15), MF8223 (SEQ ID NO: 16), MF8222 (SEQ ID NO: 17), MF8235 (SEQ ID NO: 18), MF8236 (SEQ ID NO: 19), MF8237 (SEQ ID NO: 20), MF8238 (SEQ ID NO: 21), MF8230 (SEQ ID NO: 22), MF8236 (SEQ ID NO: 23), MF8237 (SEQ ID NO: 24), MF8238 (SEQ ID NO: 25), NO: 18), MF8236 (SEQ ID NO: 19), MF8241 (SEQ ID NO: 20), MF8244 (SEQ ID NO: 21), MF8221 (SEQ ID NO: 22), or MF4356 (SEQ ID NO: 23).

[0178] In certain aspects, the first variable domain comprises a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYNGNTNYAQKLQG, and a CDR3 comprising the sequence DRHWHWWLDA, in certain aspects CDR3 comprising DRHWHWWLDAFDY, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence SYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 sequence ETYYYDRGGYPFDP. The CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT) in certain aspects.

[0179] In certain aspects, the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of TYSMN; a CDR2 sequence of WINTYTGDPTYAQGFTG, and a CDR3 comprising the sequence ETYFYDRGGYPFDP. The CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains in certain aspects respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0180] A bispecific antibody comprising a first variable domain that can bind to the extracellular portion of EGFR and a second variable domain that can bind to the extracellular portion of cMET, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of SYSMN; a CDR2 sequence of WINTYTGDPTYAQGFTG and a CDR3 sequence of ETYYYDRGGYPFDP. The CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT) in certain aspects.

[0181] A bispecific antibody comprising a first variable domain that can bind to the extracellular portion of EGFR and a second variable domain that can bind to the extracellular portion of cMET, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of TYSMN; a CDR2 sequence of WINTYTGDPTYAQGFTG, and a CDR3 comprising the sequence ETYFYDRGGYPFDP. The CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT) in certain aspects.

[0182] A bispecific antibody comprising a first variable domain that can bind to the extracellular portion of EGFR and a second variable domain that can bind to the extracellular portion of cMET, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYSGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of SYSMN; a CDR2 sequence of WINTYTGDPTYAQGFTG and a CDR3 sequence of ETYYYDRGGYPFDP. The CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT) in certain aspects.

[0183] A bispecific antibody comprising a first variable domain that can bind to the extracellular portion of EGFR and a second variable domain that can bind to the extracellular portion of cMET, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYSGNTNYAQKLQG, and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of TYSMN; a CDR2 sequence of WINTYTGDPTYAQGFTG, and a CDR3 comprising the sequence ETYFYDRGGYPFDP. The CDR1, CDR2, and CDR3 of the light chain of the first and second variable domains respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT) in certain aspects.

[0184] In certain aspects, where the cMET binding variable domain is described as having a CDR2 sequence of "WINTYTGDPTYAQGFTG," the CDR2 sequence may also be "WINTYTGDPTYAQGFT."

[0185] The CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains as described herein in certain aspects respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT). In some such aspects, the CDR3 comprises the amino acid sequence QQSYSTP. In some aspects of the bispecific antibodies described herein, the first and second variable domains comprise a common light chain, which in certain aspects is Figure 4B The light chain variable region is shown.

[0186] In certain aspects, the EGFR / cMET bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human EGFR and a second variable domain that can bind to the extracellular portion of human cMET, wherein the first variable domain comprises Figure 1 The CDR1, CDR2 and CDR3 of the heavy chain variable region of MF3755 shown in FIG. 1 and the second variable domain comprises Figure 1 The CDR1, CDR2 and CDR3 of the heavy chain variable region of MF4297 shown in the figure. The light chain variable region in the first and second variable domains is in some aspects a common light chain variable region as described herein. The CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT) in some aspects. In some aspects, the antibody comprises a heavy chain variable region having: Figure 1 The amino acid sequence of MF3755 shown in the embodiment of the present invention has up to 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the specified sequence. In certain aspects, the first variable domain comprises a heavy chain variable region having Figure 1 The variable domain (second variable domain) that can bind to cMET in certain aspects comprises a heavy chain variable region, which comprises Figure 1The amino acid sequence of MF4297 shown in FIG. 1 has 0-10, in some aspects 0-5, amino acid insertions, deletions, substitutions or combinations thereof. The heavy chain variable region of the second variable domain in some aspects comprises Figure 1 The amino acid sequence of MF4297 is shown.

[0187] In the context of the present invention, the term "bispecific" (bs) refers to an antibody capable of binding to two different targets or two epitopes on the same target, for example, wherein one variable domain of the antibody (as defined above) binds to an epitope on EGFR and the second variable domain binds to an epitope on cMET. Depending on the expression level, (sub)cellular localization and stoichiometry of the two antigens recognized by the bispecific antibody, the two Fab arms of the antibody may or may not bind their epitopes simultaneously. One arm of the bispecific antibody typically comprises the variable domain of one antibody and the other arm comprises the variable domain of the other antibody (i.e. one arm of the bispecific antibody is formed by a heavy chain paired with a light chain and the other arm is formed by a different heavy chain paired with a light chain). Thus, in certain aspects, the stoichiometric ratio of EGFR:cMET binding of the bispecific antibodies of the present invention is 1:1.

[0188] The heavy chain variable regions of the bispecific antibodies of the present invention are usually different from each other, while the light chain variable regions are identical in some aspects. The bispecific antibodies in which the different heavy chain variable regions are associated with the same light chain variable regions are also referred to as bispecific antibodies with a common light chain variable region (cLcv). Preferably, the light chain constant regions are also identical. Such bispecific antibodies are referred to as having a common light chain (cLc). Therefore, a bispecific antibody according to the present invention is further provided, wherein both arms comprise a common light chain.

[0189] According to the present invention, the term "common light chain" refers to two or more light chains in a bispecific antibody, which may be identical or have some amino acid sequence differences without affecting the binding specificity of the full-length antibody. For example, within the scope of the common light chain definition used herein, it is possible to prepare or find light chains that are not identical but still functionally equivalent, such as by introducing and testing conservative amino acid changes, amino acid changes in regions that do not contribute to binding specificity or only partially contribute when paired with a heavy chain. The terms "common light chain", "common LC", "cLC", "single light chain" are all used interchangeably herein with or without the term "rearrangement". The terms "common light chain variable region", "common VL", "common LCv", "cLCv", "single VL" are all used interchangeably herein with or without the term "rearrangement". In some aspects, the bispecific antibody of the present invention has a common light chain (variable region) that can be combined with at least two, and in some aspects a plurality of heavy chains (variable regions) with different binding specificities to form an antibody (e.g., WO2009 / 157771) with a functional antigen binding domain. The common light chain (variable region) is a human light chain (variable region) in some aspects. The common light chain (variable region) has a germline sequence in some aspects. In some aspects, the germline sequence is a light chain variable region with good thermodynamic stability, yield and solubility. In some aspects, the germline light chain is O12. The common light chain includes a light chain encoded by a germline human Vk gene fragment in some aspects, and is a rearranged germline human κ (kappa) light chain IgVκ1-39*01 / IGJκ1*01 (Figure 4A) in some aspects. The common light chain variable region is a rearranged germline human κ (kappa) light chain IgVκ1-39*01 / IGJκ1*01 variable region in some aspects. The common light chain includes such as Figure 4B or a light chain variable region as shown in 4D, which has 0-5 amino acid insertions, deletions, substitutions, additions or combinations thereof. The common light chain in some aspects also comprises a light chain constant region, and in some aspects comprises a kappa light chain constant region. The nucleic acid encoding the common light chain can be codon optimized for a cell system for expressing the common light chain protein. The encoding nucleic acid can deviate from a germline nucleic acid sequence.

[0190] In some aspects, the light chain comprises a light chain region comprising the amino acid sequence of the O12 / IgVκ1-39*01 gene fragment, as shown in Figure 4A, which has 0-10, in some aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. The phrase "O12 light chain" will be used throughout the specification as an abbreviation for "a light chain comprising a light chain variable region, which comprises the amino acid sequence of the O12 / IgVκ1-39*01 gene fragment of the portion shown in Figure 4A, which has 0-10, in some aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof." IgVκ1-39 is the abbreviation for the immunoglobulin variable κ1-39 gene. This gene is also referred to as immunoglobulin κ variable 1-39; IGKV139; IGKV1-39; O12a or O12. The external ID of this gene is HGNC:5740; Entrez Gene:28930; Ensembl:ENSG00000242371. In certain aspects, the amino acid sequence of IgVκ1-39 is provided in Figure 4E It lists the sequence of the V region. This V region can be combined with one of the five J regions. Figure 4B and 4D Two preferred sequences for the combination of IgVκ1-39 and J region are described. The linked sequences are denoted IGKV1-39 / jk1 and IGKV1-39 / jk5; alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (according to the IMGT database on the global webpage imgt.org).

[0191] Preferably, O12 / IgVκ1-39*01 comprising the light chain variable region is a germline sequence. More preferably, IGJκ1*0 or / IGJκ5*01 comprising the light chain variable region is a germline sequence. In certain aspects, the IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence.

[0192] In certain aspects, the light chain variable region comprises germline O12 / IgVκ1-39*01. In certain aspects, the light chain variable region comprises a kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. In certain aspects, IgVκ1-39*01 / IGJκ1*01. The light chain variable region comprises in certain aspects a germline kappa light chain IgVκ1-39*01 / IGJκ1*01 or a germline kappa light chain IgVκ1-39*01 / IGJκ5*01, and in certain aspects comprises a germline IgVκ1-39*01 / IGJκ1*01.

[0193] Mature B cells that produce antibodies with O12 light chains typically produce light chains that have undergone one or more mutations relative to the germline sequence (i.e., the normal sequence in the non-lymphocytes of the organism). The process that leads to these mutations is often called somatic (hyper)mutation. The resulting light chains are called affinity-matured light chains. Such light chains (when derived from the O12 germline sequence) are O12-derived light chains. In this specification, the phrase "common light chain" will include "common light chain-derived light chain", and the phrase "O12 light chain" will include O12-derived light chains. Mutations introduced by somatic hypermutation can also be artificially introduced in the laboratory. Other mutations can also be introduced in the laboratory without affecting the type and nature of the light chain, and not necessarily its amount. If the light chain contains as shown in Figure 4A, Figure 4B ; Figure 4D or Figure 4E 4A; 4b; 4d or 4e and having 0-9, 0-8, 0-7, 0-6, 0-5, 0-4 amino acid insertions, deletions, substitutions, additions or combinations thereof. In some aspects, the O12 light chain is a light chain comprising the sequence as shown in FIG. 4A; 4b; 4d or 4e and having 0-9, 0-8, 0-7, 0-6, 0-5, 0-4 amino acid insertions, deletions, substitutions, additions or combinations thereof. In some aspects, the O12 light chain is a light chain comprising the sequence as shown in FIG. 4A; 4b; 4d or 4e and having 0-9, 0-8, 0-7, 0-6, 0-5, 0-4 amino acid insertions, deletions, substitutions, additions or combinations thereof. Figure 4B ; Figure 4D or Figure 4E 4A, 4B and 4C, and having 0-5, in some aspects 0-4, in some aspects 0-3 amino acid insertions, deletions, substitutions, additions or combinations thereof. In some aspects, the O12 light chain is a light chain comprising the sequence shown in FIG. 4A, Figure 4B ; Figure 4D or Figure 4E 4A or 4B, and having 0-2, in some aspects 0-1, in some aspects 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In some aspects, the O12 light chain is a light chain comprising the sequence shown in FIG. 4A or Figure 4B In some aspects, the light chain comprises the sequence of Figure 4A. In some aspects, the light chain variable region comprises Figure 4B The above 1, 2, 3, 4 or 5 amino acid substitutions are conservative amino acid substitutions in certain aspects and may be present in the CDR region of the heavy chain and / or light chain; the insertion, deletion, substitution or combination thereof is not in the CDR3 region of the VL chain in certain aspects, and in certain aspects, is not in the CDR1, CDR2 or CDR3 region or FR4 region of the VL chain.

[0194] The common light chain may have a lambda light chain, and is therefore also provided in the context of the present invention, but is preferably a kappa light chain. The constant portion of the common light chain of the present invention may be a constant region of a kappa or lambda light chain. It is a constant region of a kappa light chain in certain aspects, in certain aspects, the common light chain is a germline light chain, in certain aspects, a rearranged germline human kappa light chain comprising an IgVK1-39 gene fragment, in certain aspects, the rearranged germline human kappa light chain is IgVK1-39 * 01 / IGJK1 * 01 (Fig. 4). The terms rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01, IGKV1-39 / IGKJ1, huVκ1-39 light chain or huVκ1-39 or 1-39 for short are used interchangeably throughout the application.

[0195] Common light chain producing cells can produce, for example, the rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01, and a light chain comprising the variable region of said light chain fused to a lambda constant region.

[0196] In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRASQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQSYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPKLLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK having 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain aspects, the light chain variable region comprises 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, in certain aspects 0-3, in certain aspects 0-2, in certain aspects 0-1, and in certain aspects 0 amino acid insertions, deletions, substitutions, additions, relative to the specified amino acid sequence, or a combination thereof. If the aligned sequences do not differ at more than 5 positions, the combination of insertions, deletions, additions or substitutions is the claimed combination. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGSGSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVTITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFATYYCQQ SYSTP PITFG QGTRL EIK. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMTQSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTDFTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK.In another aspect, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQSGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK.

[0197] The amino acid insertion, deletion, substitution, addition or combination thereof is not in the CDR3 region of the light chain variable region in some aspects, and is not in the CDR1 or CDR2 region of the light chain variable region in some aspects. In some aspects, the light chain variable region does not comprise deletion, addition or insertion relative to the specified sequence. In this embodiment, the heavy chain variable region may have 0-5 amino acid substitutions relative to the specified amino acid sequence. Amino acid substitutions are conservative amino acid substitutions in some aspects. The CDR1, CDR2 and CDR3 of the light chain of the antibody of the present invention respectively comprise the amino acid sequence CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDR of IGKV1-39 (according to IMGT) in some aspects. In some aspects, the CDR3 sequence comprises QQSYSTPPT.

[0198] The bispecific antibodies as described herein have in some aspects a heavy chain variable region / light chain variable region (VH / VL) combination that binds to the extracellular portion of EGFR, and a second VH / VL combination that binds to the extracellular portion of cMET. In some aspects, the VL in the first VH / VL combination is similar to the VL in the second VH / VL combination. In some aspects, the VL in the first and second VH / VL combinations are the same. In some aspects, the bispecific antibody is a full-length antibody having a heavy chain / light chain (H / L) combination that binds to the extracellular portion of EGFR, and a H / L chain combination that binds to the extracellular portion of cMET. In some aspects, the light chain in the first H / L chain combination is similar to the light chain in the second H / L chain combination. In some aspects, the light chains in the first and second H / L chain combinations are the same.

[0199] Several methods have been disclosed to produce host cells whose expression is conducive to the production of bispecific antibodies, and vice versa, i.e., monospecific antibodies. In the present invention, it is preferred that the cell expression of the antibody molecule is conducive to the production of bispecific antibodies, rather than the production of corresponding monospecific antibodies. This is usually achieved by modifying the constant region of the heavy chain so that they are conducive to heterodimerization (i.e., the heavy chain combined with other heavy chains / light chains is dimerized), rather than homodimerization. In some aspects, the bispecific antibody of the present invention comprises two different immunoglobulin heavy chains with compatible heterodimerization domains. A variety of compatible heterodimerization domains have been described in the art. The compatible heterodimerization domains are compatible immunoglobulin heavy chain CH3 heterodimerization domains in some aspects. When using wild-type CH3 domains, the co-expression of two different heavy chains (A and B) and a common light chain will produce three different antibody species, AA, AB and BB. AA and BB are the names of two monospecific, bivalent antibodies, and AB is the name of a bispecific antibody. In order to increase the percentage of the desired bispecific product (AB), CH3 engineering can be employed, or in other words, heavy chains with compatible hetero-dimerization domains can be used, as defined below. Various ways in which such hetero-dimerization of heavy chains can be achieved are described in the art. One such approach is to generate a "knob into hole" bispecific antibody.

[0200] As used herein, the term "compatible hetero-dimerization domains" refers to protein domains that are engineered such that the engineered domain A' will preferentially form hetero-dimers with the engineered domain B', and vice versa, homo-dimerization between A'-A' and B'-B' is reduced.

[0201] Methods and means for making bispecific antibodies using compatible heterodimerization domains are disclosed in US13 / 866,747 (now issued as US 9,248,181), US14 / 081,848 (now issued as US9,358,286) and PCT / NL2013 / 050294 (publication number WO2013 / 157954; incorporated herein by reference in its entirety). These means and methods may also be advantageously used in the present invention. In particular, the bispecific antibodies of the present invention comprise mutations in certain aspects to produce large amounts of bispecific full-length IgG molecules expressed in host cells. Preferred mutations are amino acid substitutions L351K and T366K in the first CH3 domain ("KK variant" heavy chain), and amino acid substitutions L351D and L368E in the second domain ("DE variant" heavy chain), or vice versa. US 9,248,181 and US 9,358,286 patents, and WO2013 / 157954 PCT application (incorporated herein by reference in their entirety) illustrate that DE-variants and KK-variants preferentially pair to form heterodimers (referred to as "DEKK" bispecific molecules). Due to the repulsion between charged residues in the CH3-CH3 interface between equal heavy chains, homodimerization of the DE-variant heavy chains (DEDE homodimers) is unfavorable.

[0202] Bispecific antibodies can be produced by (transient) transfection of plasmids encoding light chains and two different heavy chains, which are engineered to ensure effective hetero-dimerization and form bispecific antibodies. The production of these chains in single cells results in the formation of bispecific antibodies that is superior to the formation of monospecific antibodies. The preferred mutations that substantially only produce bispecific full-length IgG1 molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain ("KK variant" heavy chain), such as L351K and T366K (numbered according to EU numbering), and amino acid substitutions at positions 351 and 368 in the second CH3 domain ("DE variant" heavy chain), such as L351D and L368E, and vice versa.

[0203] In one embodiment, the heavy chain / light chain combination comprising a variable domain that binds to EGFR comprises a DE variant of the heavy chain. In this embodiment, the heavy chain / light chain combination comprising a variable domain that can bind to cMET comprises a KK variant of the heavy chain. The KK variant of the heavy chain that binds to cMET does not produce homodimers, thereby making the observed effect of the bispecific antibody on the inhibition of HGF-induced cMET activation very accurate. This avoids the cMET activation effect (agonism) sometimes observed with bivalent cMET antibodies.

[0204] The Fc region mediates the effector functions of antibodies, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Depending on the application of the therapeutic antibody or Fc fusion protein, it may be necessary to reduce or increase the effector function. When the immune response in some embodiments of the present invention is activated, enhanced, or stimulated, it may be desirable to reduce the effector function. Antibodies with reduced effector function can be used to target cell surface molecules of immune cells, etc. In some aspects, the antibodies of the present invention promote ADCP. In some aspects, the antibodies of the present invention promote ADCC. In some aspects, the antibodies of the present invention promote ADCC to a greater extent than amivantamab. In some aspects, for cells or cancers with cMET aberrations (including cMET amplification and / or cMET exon 14 skipping mutations), the antibodies of the present invention promote ADCC to a greater extent than amivantamab.

[0205] The antibody with reduced effector function is in some aspects an IgG antibody comprising a modified CH2 / lower hinge region, for example to reduce Fc-receptor interactions or reduce Clq binding. In some aspects, the antibody of the invention is an IgG antibody with a mutant CH2 and / or lower hinge domain, such that the interaction of the bispecific IgG antibody with the Fc-γ receptor is reduced. The antibody comprising a mutant CH2 region is in some aspects an IgG1 antibody. Such mutant IgG1 CH2 and / or lower hinge domains in some aspects comprise amino acid substitutions at positions 235 and / or 236 (EU numbering), and in some aspects comprise L235G and / or G236R substitutions ( Figure 5D ).

[0206] The antibodies of the present invention have effector functions in certain aspects. The bispecific antibodies of the present invention comprise antibody-dependent cell-mediated cytotoxicity (ADCC) in certain aspects. The antibody can be engineered to enhance ADCC activity (for review, see Cancer Sci. 2009 Sep; 100 (9): 1566-72. Engineered therapeutic antibodies with improved effector functions. Kubota T, Niwa R, Satoh M, Akinaga S, Shitara K, Hanai N). There are several in vitro methods for determining the efficacy of antibodies or effector cells in inducing ADCC. These include chromium-51 [Cr51] release assays, europium [Eu] release assays, and sulfur-35 [S35] release assays. Typically, a labeled target cell line expressing a surface-exposed antigen is incubated with an antibody specific for the antigen. After washing, effector cells expressing the Fc receptor CD16 are co-incubated with antibody-labeled target cells. The release of the intracellular marker is then measured by a scintillation counter or spectrophotometry to measure the lysis of the target cell. In some aspects, the bispecific antibody of the present invention exhibits ADCC activity. In this aspect, the bispecific antibody may have improved ADCC activity. In this aspect, the antibody may have altered ADCC activity, which is achieved by one or more CH2 mutations as described elsewhere herein, and by techniques known in the art. A technique for enhancing the ADCC of an antibody is defucosylation (see, e.g., Junttila, TT, K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumabin the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70(11): 4481-4489). Therefore, a bispecific antibody according to the present invention is further provided, which is defucosylated. In some aspects, the antibody of the present invention comprises two defucosylated CH2 domains. In certain aspects, the antibodies of the invention comprise a total of two CH2 domains, both of which are defucosylated. In certain aspects, the antibodies of the invention are full-length antibodies, such as IgG-type antibodies, having two CH2 domains, both of which are non-fucosylated.Alternatively, or additionally, ADCC enhancement can be achieved using a variety of other strategies, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis, all of which attempt to increase Fc binding to the low affinity activating FcγRIIIa, and / or reduce binding to the low affinity inhibitory FcγRIIb. The bispecific antibodies of the invention are defucosylated in certain aspects to enhance ADCC activity. The bispecific antibodies of the invention comprise, in certain aspects, a reduced amount of fucosylation of N-linked sugar chain structures in the Fc region when compared to the same antibody produced in normal CHO cells.

[0207] Variants of antibodies or bispecific antibodies as described herein include functional parts, derivatives and / or analogs of antibodies or bispecific antibodies. The variants retain the binding specificity of the (bispecific) antibody. The functional parts, derivatives and / or analogs retain the binding specificity of the (bispecific) antibody. Binding specificity is defined as the ability to bind to the extracellular portion of the first membrane protein and the second membrane protein as described herein.

[0208] The bispecific antibodies of the present invention are used in humans in certain aspects. In certain aspects, the antibodies of the present invention are humanized antibodies, or in certain aspects are human antibodies. The constant region of the bispecific antibodies of the present invention is a human constant region in certain aspects. The constant region may contain one or more, no more than 10, and no more than 5 amino acid differences with the constant region of a naturally occurring human antibody. Preferably, the constant portion is completely derived from a naturally occurring human antibody. The various antibodies produced herein are derived from a human antibody variable domain library. Therefore, these variable domains are human variable domains. The unique CDR region may be derived from humans, synthesized or derived from another organism. When the amino acid sequence of the variable region is the same as the amino acid sequence of the variable region of a naturally occurring human antibody, but excluding the CDR region, the variable region is considered to be a humanized variable region. In this embodiment, compared with the variable region of a naturally occurring human antibody, the VH of the variable domain of the antibody binding to EGFR or cMET of the present invention may contain one or more, no more than 10, and no more than 5 amino acid differences in certain aspects, without calculating the possible differences in the amino acid sequence of the CDR region. Compared to the variable regions of naturally occurring human antibodies, the light chain variable regions of the EGFR binding domain and / or cMET binding domain in the antibodies of the present invention may contain one or more, in some aspects no more than 10, in some aspects no more than 5 amino acid differences, not counting possible differences in the amino acid sequence of the CDR regions. Compared to the variable regions of naturally occurring human antibodies, the light chains in the antibodies of the present invention may contain one or more, in some aspects no more than 10, in some aspects no more than 5 amino acid differences, not counting possible differences in the amino acid sequence of the CDR regions. Such mutations also occur in the context of somatic hypermutations in nature.

[0209] Antibodies can be derived from a variety of animal species, at least with respect to the heavy chain variable region. It is common practice to humanize, for example, a murine heavy chain variable region. There are a variety of ways to achieve this, including transplanting the CDRs into a human heavy chain variable region that has a 3-D structure that matches the 3-D structure of the murine heavy chain variable region; deimmunization of the murine heavy chain variable region, which in some aspects is achieved by removing known or suspected T- or B-cell epitopes from the murine heavy chain variable region. This removal is typically accomplished by substituting one or more amino acids in the epitope for another (usually conservative) amino acid, thereby modifying the sequence of the epitope so that it is no longer a T cell or B cell epitope.

[0210] The immunogenicity of the murine heavy chain variable region of deimmunization in humans is lower than that of the original murine heavy chain variable region. In some aspects, the variable region or domain of the present invention is further humanized, such as veneered. By using veneering technology, external residues that are easily encountered by the immune system are selectively replaced with human residues to provide hybrid molecules comprising a weakly immunogenic or substantially non-immunogenic veneering surface. The animal used in the present invention is a mammal in some aspects, a primate in some aspects, and a human in some aspects.

[0211] The bispecific antibodies according to the present invention comprise the constant region of human antibodies in certain aspects. According to the difference of their heavy chain constant region, antibodies are divided into five categories or isotypes: IgG, IgA, IgM, IgD and IgE. These categories or isotypes comprise at least one heavy chain named with corresponding Greek letters. A certain aspect comprises antibodies, wherein the constant region is selected from the group of IgG, IgA, IgM, IgD and IgE constant regions, and in certain aspects the constant region comprises an IgG constant region, i.e. selected from the group consisting of: IgG1, IgG2, IgG3 and IgG4. In certain aspects, the constant region is an IgG1 or IgG4 constant region, and in certain aspects, it is a mutated IgG1 constant region. Some variations of the IgG1 constant region occur in nature and / or are allowed without changing the immunological properties of the resulting antibody. Variations may also be artificially introduced to add certain preferred features to antibodies or portions thereof. These features are, for example, the contents of CH2 and CH3 described herein. Generally, about 1-10 amino acid insertions, deletions, substitutions or combinations are allowed in the constant region.

[0212] Figure 1 , 2 The VH chain of SEQ ID NO: 1 has up to 15, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or combinations thereof relative to SEQ ID NO: 1. Figure 1 , 2 or the VH chain shown in 3, in certain aspects, has 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to Figure 1 , 2 or the VH chain shown in 3, in certain aspects, having 0, 1, 2, 3 or 4 amino acid insertions, deletions, substitutions or combinations thereof, in certain aspects, having 0, 1, 2, or 3 amino acid insertions, deletions, substitutions or combinations thereof, and in certain aspects, having 0, 1 or 2 amino acid insertions, deletions, substitutions or combinations thereof, and in certain aspects, having 0 or 1 amino acid insertions, deletions, substitutions or combinations thereof, relative to Figure 1 , 2or the VH chain shown in 3. The one or more amino acid insertions, deletions, substitutions or combinations thereof are not in the CDR1, CDR2 and / or CDR3 regions of the VH chain in certain aspects. In certain aspects, they are also not present in the FR4 region. The amino acid substitutions are conservative amino acid substitutions in certain aspects.

[0213] Rational approaches have been developed towards minimizing the amount of non-human residues in the human environment. There are a number of approaches to successfully transfer the antigen-binding properties of one antibody to another. The binding properties of an antibody may depend primarily on the exact sequence of the CDR3 region, which is usually supported by the sequence of the CDR1 and CDR2 regions of the variable domain and the appropriate structure of the entire variable domain.

[0214] CDR sequences can be defined using different methods, including but not limited to the Kabat numbering scheme (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); and / or Kabat et al., US Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991)), the Chothia numbering scheme (Chothia et al., J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:877-883, 1989; and / or Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997)); and / or the Honegger and Plukthun numbering system (Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001)), the MacCallum numbering system (MacCallum et al., J. Mol. Biol. 262:732-745 (1996); and / or Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008)), the Lefranc numbering system (Lefranc MPet al., Dev. Comp. Immunol., 27:55-77 (2003); and / or Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001)), or according to IMGT (discussed in Giudicelli et al., Nucleic Acids Res. 25:206-211 (1997)).

[0215] Each of these numbering schemes defines CDRs based on the predicted contribution of amino acid residues in the heavy or light chain variable region to antigen binding. Therefore, each method of identifying CDRs can be used to identify the CDRs of the binding domains of the present invention. In certain embodiments, the heavy chain CDRs of the binding domains of the present invention are according to Kabat, Chothia or IMGT. In certain aspects, the heavy chain CDRs of the binding domains of the present invention are according to Kabat. In certain aspects, the heavy chain CDRs of the binding domains of the present invention are according to Chothia. In certain aspects, the heavy chain CDRs of the binding domains of the present invention are according to IMGT. In certain aspects, the light chain CDRs of the binding domains of the present invention are according to Kabat. In certain aspects, the light chain CDRs of the binding domains of the present invention are according to Chothia. In certain aspects, the light chain CDRs of the binding domains of the present invention are according to IMGT. The amino acid sequence in the heavy chain CDR region as described herein is determined according to the Kabat definition.

[0216] There are currently a variety of methods for transplanting CDR regions onto a suitable variable domain of another antibody. These methods are described in JC Almagro1 and J. Fransson (2008) Frontiers in Bioscience 13, 1619-1633, which is incorporated herein by reference in its entirety. Therefore, the present invention further provides a humanized bispecific antibody, or in some aspects a human bispecific antibody, as included in the treatment of the present invention, comprising a first antigen binding site that binds to EGFR and a second antigen binding site that binds to cMET, wherein the variable domain comprising the EGFR binding site comprises Figure 1 The VH CDR3 sequence of MF3370 shown in FIG. 1 , and the variable domain comprising the cMET binding site comprises Figure 1 The VH CDR3 region shown in MF4356 in FIG. The VH variable region comprising the EGFR binding site in certain aspects comprises Figure 1 The VH variable region comprising the cMET binding site comprises in some aspects the sequence of the CDR1 region, CDR2 region and CDR3 region of the VH chain shown in MF3370 in FIG. Figure 1 The sequences of the CDR1, CDR2 and CDR3 regions of the VH chain shown in MF4356 in FIG. CDR transplantation can also be used to produce a Figure 1The CDR regions of the VH shown in the embodiment of the present invention but with a VH chain of a different framework. The different framework can be another human VH, or a different mammal. Therefore, the present invention further provides a humanized bispecific antibody, or in some aspects a human bispecific antibody, as included in the treatment of the present invention, the bispecific antibody comprises a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises as Figure 2 The VH CDR3 sequence shown in MF8233 in , and wherein the variable domain comprising the cMET binding site comprises Figure 3 The VH CDR3 region shown in MF8230 in FIG. The VH variable region comprising the EGFR binding site in certain aspects comprises Figure 2 The VH variable region comprising the cMET binding site comprises in some aspects the sequence of the CDR1 region, CDR2 region and CDR3 region of the VH chain shown in MF8233 of FIG. Figure 3 The sequences of the CDR1, CDR2 and CDR3 regions of the VH chain shown in MF8230 in FIG. CDR transplantation can also be used to produce a Figure 2 or Figure 3 The CDR regions of the VH shown but with a VH chain of a different framework. The different framework can be the framework of another human VH, or the framework of a different mammal.

[0217] Thus, the present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises Figure 1 The VH CDR3 sequence of MF3370 shown in FIG. 1 and the variable domain comprising the cMET binding site therein comprises Figure 3 The VH CDR3 region shown in MF8230 in FIG. The VH variable region comprising the EGFR binding site in certain aspects comprises Figure 1 The VH variable region comprising the cMET binding site comprises the following in certain aspects: Figure 3 The sequences of the CDR1, CDR2 and CDR3 regions of the VH chain shown in MF8230 in FIG. CDR transplantation can also be used to produce a Figure 2 or Figure 3 The CDR regions of the VH shown but with a VH chain of a different framework. The different framework can be the framework of another human VH, or the framework of a different mammal.

[0218] Thus, the present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises Figure 2 The VH CDR3 sequence of MF8233 shown in FIG. 1 and the variable domain comprising the cMET binding site therein comprises Figure 3 The VH CDR3 region shown in MF4356 in FIG. The VH variable region comprising the EGFR binding site in certain aspects comprises Figure 2 The VH variable region comprising the cMET binding site comprises the following in certain aspects: Figure 3 The sequences of the CDR1, CDR2 and CDR3 regions of the VH chain shown in MF4356 in FIG. CDR transplantation can also be used to produce a Figure 2 or Figure 3 The CDR regions of the VH shown but with a VH chain of a different framework. The different framework can be the framework of another human VH, or the framework of a different mammal.

[0219] Thus, the present invention further provides a humanized bispecific antibody, or in certain aspects a human bispecific antibody, as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises Figure 2 The VHCDR3 sequence shown in MF8232 in the embodiment of the present invention, and the variable domain comprising the cMET binding site comprises Figure 3 The VH CDR3 region shown in MF8230 in FIG. The VH variable region comprising the EGFR binding site in certain aspects comprises Figure 2 The VH variable region comprising the cMET binding site comprises the following in certain aspects: Figure 3 The sequences of the CDR1, CDR2 and CDR3 regions of the VH chain shown in MF8230 in FIG. CDR transplantation can also be used to produce a Figure 2 or Figure 3 The CDR regions of the VH shown but with a VH chain of a different framework. The different framework can be the framework of another human VH, or the framework of a different mammal.

[0220] Methods for generating sequence variants are known in the art. Sequence variants can be generated by random methods, or by targeted methods, such as those intended to introduce variations that may increase or decrease binding affinity. Conventional methods for affinity maturation of antibody binding domains are well known in the art, see, for example, Tabasinezhad M. et al., Immunol Lett. 2019; 212: 106-113. Variants intended to introduce developmental risk mitigation may also be used in order to manufacture binding domains or portions comprising such binding domains on a large scale. Variants that may not appear to cause loss of binding specificity and / or affect binding affinity may be introduced. Whether amino acid residues in CDRs and / or framework regions can be substituted, for example, by conservative amino acid residues, with no or substantially no loss of binding specificity and / or affinity can be determined by methods well known in the art. Experimental examples include, but are not limited to, alanine scanning (Cunningham BC, Wells JA. Science. 1989; 244(4908): 1081-5), and deep mutation scanning (Araya CL, Fowler DM. Trends Biotechnol. 2011; 29(9): 435-42). Computational methods that can predict the effects of amino acid variations have also been developed, such as described in Sruthi CK, Prakash M. PLoS One. 2020; 15(1): e0227621, Choi Y. et al., PLoS One. 2012; 7(10): e46688, and Munro D, Singh M. Bioinformatics. 2020; 36(22-23): 5322–9.

[0221] Further provided herein are any variant anti-human EGFR and c-MET binding domains produced by the above methods; binding portions, such as antibodies, comprising any of the variant binding domains; pharmaceutical compositions comprising any of the variant anti-human EGFR and c-MET binding domains or binding portions; nucleic acids encoding any of the variant binding domains; vectors and cells comprising the nucleic acids; and uses of the variant binding domains or pharmaceutical compositions for treating cancer.

[0222] Thus, the present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises Figure 2 The VH CDR3 sequence of MF8232 shown in FIG. 1 and the variable domain comprising the cMET binding site therein comprises Figure 3The VH CDR3 region shown in MF8230 in FIG. The VH variable region comprising the EGFR binding site in certain aspects comprises Figure 2 The VH variable region comprising the cMET binding site comprises the following in certain aspects: Figure 3 The sequences of the CDR1, CDR2 and CDR3 regions of the VH chain shown in MF8230 in FIG. CDR transplantation can also be used to produce a Figure 2 or Figure 3 The CDR regions of the VH shown but with a VH chain of a different framework. The different framework can be the framework of another human VH, or a framework of a different mammal.

[0223] Thus, the present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises Figure 2 The VH CDR3 sequence of MF8232 shown in FIG. 1 and the variable domain comprising the cMET binding site therein comprises Figure 3 The VH CDR3 region shown in MF4356 in FIG. The VH variable region comprising the EGFR binding site in certain aspects comprises Figure 2 The VH variable region comprising the cMET binding site comprises the following in certain aspects: Figure 3 The sequences of the CDR1, CDR2 and CDR3 regions of the VH chain shown in MF4356 in FIG. CDR transplantation can also be used to produce a Figure 2 or Figure 3 The CDR regions of the VH shown but with a VH chain of a different framework. The different framework can be the framework of another human VH, or a framework of a different mammal.

[0224] Thus, the present invention further provides a humanized or in certain aspects human bispecific antibody, as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises Figure 2 The VH CDR3 sequence of MF8232 shown in FIG. 1 and the variable domain comprising the cMET binding site therein comprises Figure 3 The VH CDR3 region shown in MF8230 in FIG. The VH variable region comprising the EGFR binding site in certain aspects comprises Figure 2The VH variable region comprising the cMET binding site comprises the following in certain aspects: Figure 3 The sequences of the CDR1, CDR2 and CDR3 regions of the VH chain shown in MF8230 in FIG. CDR transplantation can also be used to produce a Figure 2 or Figure 3 The CDR regions of the VH shown but with a VH chain of a different framework. The different framework can be the framework of another human VH, or the framework of a different mammal.

[0225] The present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having Figure 2 , which has up to 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, and wherein the second variable domain comprises a heavy chain variable region having Figure 3 The amino acid sequence of MF4356 shown in (SEQ ID NO: 23), which has 0-10, in some aspects 0-5 amino acid insertions, deletions, substitutions or combinations thereof. The present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, the bispecific antibody comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having Figure 2 , having up to 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, and wherein the second variable domain comprises a heavy chain variable region comprising Figure 3 The amino acid sequence of MF8230 shown in (SEQ ID NO: 13), which has 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions or a combination thereof.

[0226] The present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having Figure 2, having up to 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, and wherein the second variable domain comprises a heavy chain variable region comprising Figure 3 The amino acid sequence of MF8230 shown in (SEQ ID NO: 13), which has 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions or a combination thereof.

[0227] The present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having Figure 2 , having up to 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, and wherein the second variable domain comprises a heavy chain variable region comprising Figure 3 The amino acid sequence of MF4356 shown in (SEQ ID NO: 23) has 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions or a combination thereof.

[0228] The present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having Figure 2 , having up to 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, and wherein the second variable domain comprises a heavy chain variable region comprising Figure 3 The amino acid sequence of MF4356 shown in (SEQ ID NO: 23) has 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions or a combination thereof.

[0229] The present invention further provides a human or humanized bispecific antibody, as included in the treatment of the present invention, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having Figure 2, having up to 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, and wherein the second variable domain comprises a heavy chain variable region comprising Figure 3 The amino acid sequence of MF8230 shown in (SEQ ID NO: 13), which has 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions or a combination thereof.

[0230] The up to 15, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid substitutions are in certain aspects conservative amino acid substitutions, while the insertions, deletions, substitutions or combinations thereof are in certain aspects not in the CDR3 region of the VH chain, in certain aspects not in the CDR1, CDR2 or CDR3 regions of the VH chain, and in certain aspects not in the FR4 region.

[0231] There are a variety of methods for making bispecific antibodies. One method involves expressing two different heavy chains and two different light chains in a cell, and collecting the antibodies produced by the cell. Antibodies made in this way generally include a collection of antibodies with different combinations of heavy chains and light chains, some of which are desired bispecific antibodies. The bispecific antibodies can then be purified from the collection. The ratio of bispecific antibodies and other antibodies made by cells can be increased in various ways. In some aspects, the ratio is increased by expressing not two different light chains in the cell, but a common light chain. When a common light chain is expressed together with two different heavy chains, the ratio of bispecific antibodies made by cells to other antibodies will be significantly increased compared to expressing two different light chains. The ratio of bispecific antibodies made by cells can be further increased by stimulating the pairing of two different heavy chains with each other, rather than the pairing of two identical heavy chains. Methods and means for making bispecific antibodies (from a single cell) are disclosed, thereby providing means that are conducive to forming bispecific antibodies, rather than forming monospecific antibodies. These methods can also be advantageously used in the present invention. Therefore, in certain aspects, the present invention provides a method for making a bispecific antibody from a single cell, wherein the bispecific antibody comprises two CH3 domains capable of forming an interface, the method comprising providing in the cell a) a first nucleic acid molecule encoding a first CH3 domain comprising a heavy chain, b) a second nucleic acid molecule encoding a second CH3 domain comprising a heavy chain, wherein the nucleic acid molecule is provided in a method of preferentially pairing the first and second CH3 domains comprising a heavy chain, the method further comprising culturing the host cell, allowing the expression of the two nucleic acid molecules, and harvesting the bispecific antibody from the culture. The first and second nucleic acid molecules may be parts of the same nucleic acid molecule, vectors or gene delivery vehicles, and may be integrated at the same site of the genome of the host cell. Alternatively, the first and second nucleic acid molecules are provided to the cell separately.

[0232] Certain aspects provide a method for producing a bispecific antibody of the invention from a single cell, wherein the bispecific antibody comprises two CH3 domains capable of forming an interface, the method comprising providing:

[0233] - a cell having a) a first nucleic acid molecule encoding a heavy chain comprising an antigen binding site that binds to EGFR and comprising a first CH3 domain, and b) a second nucleic acid molecule encoding a heavy chain comprising an antigen binding site that binds to ErbB-3 and comprising a second CH3 domain, wherein the nucleic acid molecules are provided in a manner that preferentially pairs the first and second CH3 domains,

[0234] The method also includes culturing the cell, allowing expression of proteins encoded by the two nucleic acid molecules, and harvesting the bispecific IgG antibody from the culture. In some aspects, the cell also has a third nucleic acid molecule encoding a common light chain. The first, second and third nucleic acid molecules can be a part of the same nucleic acid molecule, a vector or a gene delivery vehicle, and can be integrated in the same site of the genome of the host cell. Alternatively, the first, second and third nucleic acid molecules are provided to the cell respectively. In some aspects, the common light chain is based on O12, and in some aspects, it is a rearranged germline human κ light chain IgVκ1 39*01 / IGJκ1*01 as described above. The means for preferential pairing of the first and second CH3 domains are corresponding mutations in the CH3 domain of the heavy chain coding region in some aspects. The preferred mutations of the bispecific antibodies preferentially manufactured are amino acid substitutions L351K and T366K (EU-numbering) in the first CH3 domain and amino acid substitutions L351D and L368E in the second CH3 domain, and vice versa. Therefore, a method for making a bispecific antibody according to the present invention is further provided, wherein the first CH3 domain comprises amino acid substitutions L351K and T366K (EU-numbering), and wherein the second CH3 domain comprises amino acid substitutions L351D and L368E, and the method further comprises culturing the cell and allowing expression of the protein encoded by the nucleic acid molecule, and the step of harvesting the bispecific antibody from the culture. A method for producing a bispecific antibody according to the present invention is also provided, wherein the first CH3 domain comprises amino acid substitutions L351D and L368E (EU-numbering), and wherein the second CH3 domain comprises amino acid substitutions L351K and T366K, and the method further comprises culturing the cell and allowing expression of the nucleic acid molecule, and the step of harvesting the bispecific antibody from the culture. Antibodies that can be manufactured by these methods are also part of the present invention. The CH3 hetero-dimerization domain is an IgG1 hetero-dimerization domain in some aspects. The heavy chain constant region comprising the CH3 hetero-dimerization domain is an IgG1 constant region in some aspects.

[0235] One embodiment of the present invention includes a nucleic acid molecule encoding an antibody heavy chain variable region. The nucleic acid molecule (usually an in vitro isolated or recombinant nucleic acid molecule) encodes in some aspects such as Figure 2 or Figure 3 The heavy chain variable region shown, or Figure 2 or Figure 3 In some aspects, the nucleic acid molecule comprises a heavy chain variable region encoding a polypeptide having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof. Figure 2 or Figure 3The codon-optimized nucleic acid sequence of the amino acid sequence shown in the embodiment of the present invention is optimized for the species and / or cell type of the cell producing the antibody. For example, for CHO production, the nucleic acid sequence of the molecule is codon-optimized for Chinese hamster cells. The present invention further provides a nucleic acid sequence encoding Figure 2 or Figure 3 A nucleic acid molecule of the heavy chain.

[0236] The nucleic acid molecules used in the present invention are usually, but not exclusively, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Alternative nucleic acids are available to those skilled in the art. The nucleic acid according to the present invention is, for example, contained in a cell. When the nucleic acid is expressed in the cell, the cell can produce an antibody according to the present invention. Therefore, one embodiment of the present invention includes a cell comprising an antibody according to the present invention and / or a nucleic acid according to the present invention. The cell is an animal cell in some aspects, a mammalian cell in some aspects, a primate cell in some aspects, and a human cell in some aspects. A suitable cell is any cell that can contain and produce an antibody according to the present invention and / or a nucleic acid according to the present invention in some aspects.

[0237] The present invention also provides cells comprising antibodies according to the present invention. In some aspects, the cell (typically an in vitro, isolated or recombinant cell) produces the antibody. The cell may also be a stored cell that, when removed from storage and cultured, is capable of producing the antibody. In some aspects, the cell is a hybridoma cell, a Chinese hamster ovary (CHO) cell, a NS0 cell or a PER-C6 TM Cells. In certain aspects, the cells are CHO cells. Cell cultures comprising cells according to the invention are also provided. Various research institutions and companies have developed cell strains for large-scale production of antibodies, for example for clinical use. Non-limiting examples of such cell strains are CHO cells, NS0 cells or PER.C6 TM Cells. These cells are also used for other purposes, such as producing proteins. Cell lines developed for industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. Thus, one aspect includes the use of cell lines developed for large-scale production of antibodies for use in producing antibodies of the invention, including in certain aspects for use in producing a cell line comprising a protein encoding a protein such as Figure 2 or Figure 3 Cells expressing antibodies containing nucleic acid molecules encoding the VH, VL, and / or heavy chains shown.

[0238] The present invention also provides a method for making an antibody, the method comprising culturing the cells of the present invention and harvesting the antibody from the culture. In some aspects, the cells are cultured in a serum-free medium. In some aspects, the cells are suitable for suspension growth. An antibody that can be obtained by an antibody manufacturing method according to the present invention is also provided. The antibody is purified from the culture medium of the culture in some aspects. In some aspects, the antibody is affinity purified.

[0239] The cells of the present invention are, for example, hybridoma cell lines, CHO cells, 293F cells, NS0 cells, or another cell type known to be suitable for antibody production for clinical purposes. In one aspect, the cells are human cells. In some aspects, the cells are transformed by adenovirus E1 region or a functional equivalent thereof. In some aspects, the cell line is a PER.C6TM cell line or an equivalent thereof. In some aspects, the cells are CHO cells or variants thereof. In some aspects, the variant cells express antibodies using a glutamine synthetase (GS) vector system.

[0240] After transient transfection in suspended 293F cells, the antibodies of the present invention can be manufactured at a level of >50 mg / L. The bispecific antibodies can be purified to a purity greater than 98% with a yield of >70%. Analytical identification studies have shown that the bispecific IgG1 antibody situation is comparable to the bivalent monospecific IgG1. In terms of functional activity, the bispecific antibodies of the present invention can demonstrate superior efficacy compared to cetuximab in vitro and in vivo.

[0241] The present invention also provides a pharmaceutical composition comprising the antibody according to the present invention. In certain aspects, the pharmaceutical composition comprises a pharmaceutically acceptable excipient or carrier.

[0242] The antibody may comprise a label, which in some aspects is a label for in vivo imaging. Such a label is generally not necessary for therapeutic applications. For example, in a diagnostic setting, a label may be helpful. For example, to visualize target cells in vivo. Various labels are suitable, many of which are known in the art. In some aspects, the label is a radioactive label for detection. In some aspects, the label is an infrared label. In some aspects, the infrared label is suitable for in vivo imaging. Various infrared labels are available to those skilled in the art. Preferred infrared labels are, for example, IRDye 800; IRDye 680RD; IRDye 680LT; IRDye 750; IRDye 700DX; IRDye 800RS IRDye 650; IRDye 700 phosphoramidite; IRDye 800 phosphoramidite (LI-COR USA; 4647 Superior Street; Lincoln, Nebraska).

[0243] The present invention also provides a method for treating an individual suffering from a tumor or at risk of suffering from the tumor, comprising administering an antibody or pharmaceutical composition according to the present invention to an individual in need thereof. The tumor is an EGFR, cMET or EGFR / cMET positive tumor in certain aspects. Prior to starting the treatment, the method in certain aspects also comprises determining whether the individual suffers from such EGFR, cMET or EGFR / cMET positive tumor. The present invention also provides an antibody or pharmaceutical composition of the present invention for treating an individual suffering from or at risk of suffering from an EGFR, cMET or EGFR / cMET positive tumor.

[0244] To confirm whether a tumor is EGFR positive, a technician can determine, for example, using EGFR amplification and / or immunohistochemical staining. At least 10% of the tumor cells in the biopsy should be positive. The biopsy may also contain 20%, 30%, 40%, 50%, 60%, 70% or more positive cells. To confirm whether a tumor is cMET positive, a technician can determine, for example, using cMET amplification and / or immunohistochemical staining. At least 10% of the tumor cells in the biopsy should be positive. The biopsy may also contain 20%, 30%, 40%, 50%, 60%, 70% or more positive cells.

[0245] The cancer or tumor may be an EGFR, cMET, or EGFR / cMET positive cancer. In a preferred aspect, the present invention provides treatment of EGFR, cMET, or EGFR / cMET positive cancer, the cancer being lung cancer, particularly non-small cell lung cancer, head and neck cancer, particularly head and neck squamous cell carcinoma; gastric cancer, particularly gastric adenocarcinoma; esophageal cancer, particularly esophageal squamous cell carcinoma; gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer, or bladder cancer. The present invention can be applied to a wide range of EGFR, cMET, or EGFR / cMET positive cancers, such as lung cancer, including non-small cell lung cancer in certain aspects. The individual is a human individual in certain aspects. The individual is an individual who is eligible for antibody therapy using an EGFR specific antibody (e.g., cetuximab) in certain aspects. In certain aspects, the invention can treat an individual comprising a tumor, in certain aspects, the tumor is an EGFR / cMET positive cancer, in certain aspects the tumor / cancer has an EGFR RTK resistant phenotype, an EGFR monoclonal antibody resistant phenotype, or a combination thereof.

[0246] As used herein, the term "cancer" applies equally to the term "tumor" and thus treatment of a tumor also applies to treatment of cancer.

[0247] The antibody dosage to be administered to the patient is usually within the therapeutic window, which means that a sufficient amount is used to obtain a therapeutic effect while the amount does not exceed the threshold value that causes an unacceptable degree of side effects. The lower the amount of antibody required to obtain the desired therapeutic effect, the larger the therapeutic window is usually. Therefore, preferably, the antibody according to the present invention exerts a sufficient therapeutic effect at a low dose. The dosage can be within the scope of the medication regimen of cetuximab. The dosage can also be lower. In some aspects, the dosage is 1000mg, 1500mg or 2000mg. Medication can be once a week or once every two weeks.

[0248] In certain aspects, the cancer is lung cancer, in particular non-small cell lung cancer; head and neck cancer, in particular head and neck squamous cell carcinoma; gastric cancer, in particular gastric adenocarcinoma; esophageal cancer, in particular esophageal squamous cell carcinoma; gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer, and the antibody of the invention is administered at a dose of 1000 mg, typically as a fixed dose of 1000 mg once a week.

[0249] In certain aspects, the cancer is lung cancer, in particular non-small cell lung cancer; head and neck cancer, in particular head and neck squamous cell carcinoma; gastric cancer, in particular gastric adenocarcinoma; esophageal cancer, in particular esophageal squamous cell carcinoma; gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer, and the antibody of the invention is administered at a dose of 1000 mg, typically as a fixed dose of 1000 mg once every two weeks.

[0250] In certain aspects, the cancer is lung cancer, in particular non-small cell lung cancer; head and neck cancer, in particular head and neck squamous cell carcinoma; gastric cancer, in particular gastric adenocarcinoma; esophageal cancer, in particular esophageal squamous cell carcinoma; gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer, and the antibody of the invention is administered at a dose of 1500 mg, typically as a fixed dose of 1500 mg once every two weeks.

[0251] In certain aspects, the cancer is lung cancer, in particular non-small cell lung cancer; head and neck cancer, in particular head and neck squamous cell carcinoma; gastric cancer, in particular gastric adenocarcinoma; esophageal cancer, in particular esophageal squamous cell carcinoma; gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer, and the antibody of the invention is administered at a dose of 2000 mg, typically as a fixed dose of 2000 mg once every two weeks.

[0252] In certain aspects, the bispecific antibody binding to EGFR and cMET is provided to an individual at a dosage of 1000, 1500 or 2000 mg, particularly using a fixed dose prescription. Fixed dose prescriptions have multiple advantages in terms of body surface or body weight administration because they reduce preparation time and reduce potential dosage calculation errors. In certain aspects, the bispecific antibody is administered once a week (Q1W), once every 2 weeks (Q2W) or once every 3 weeks (Q3W). In certain aspects, the bispecific antibody is administered once every two weeks. In the art, such a dosing regimen is referred to as Q2W. In certain embodiments, the fixed dose prescription disclosed herein is suitable for adults and / or individuals weighing at least 35 kg. As understood by those skilled in the art, the dose can be administered over time. As understood by those skilled in the art, the term "fixed dose" or "fixed dose prescription" refers to an individual who is prescribed for administration, wherein the individual is arranged to receive the bispecific antibody in substantially the same predetermined amount every day, and the amount is independent of the individual's weight. According to certain aspects, a fixed, once-weekly dose of 1000 mg of bispecific antibody is provided to an individual. Alternatively, the individual is provided with a fixed, biweekly dose of 1000 mg of the bispecific antibody. Alternatively, the individual is provided with a fixed, biweekly dose of 1500 mg of the bispecific antibody. Alternatively, the individual is provided with a fixed, biweekly dose of 2000 mg of the bispecific antibody.

[0253] Under other similar conditions, compared with cetuximab, the bispecific antibodies according to the present invention induce less skin toxicity in some aspects. Compared with cetuximab, under other similar conditions, the bispecific antibodies according to the present invention produce less proinflammatory chemokines (CXCL14 in some aspects) in some aspects. Compared with cetuximab, under other similar conditions, the bispecific antibodies of the present invention induce less damage to antimicrobial RNA enzymes (RNase 7 in some aspects) in some aspects.

[0254] The present invention describes antibodies that target EGFR and cMET receptors and result in effective in vitro cancer cell line proliferation inhibition and in vivo tumor growth inhibition. The bispecific antibodies of the present invention can combine low toxicity characteristics with high efficacy. The antibodies of the present invention can be used for various types and grades of EGFR targeted therapies. When compared with antibodies that both arms bind to the same antigen, the antibodies of the present invention can have an increased therapeutic window. Compared with cetuximab antibodies, the bispecific antibodies of the present invention can show better growth inhibition in vitro, in vivo, or a combination thereof.

[0255] The present invention provides a bispecific antibody for treating an individual who may suffer from one or more of a variety of different types of tumors. The tumor may be an EGFR-positive tumor, a cMET-positive tumor, or an EGFR and cMET-positive tumor. The tumor may be resistant to treatment with an EGFR or cMET tyrosine kinase inhibitor. In some aspects, the EGFR tyrosine kinase inhibitor is a third-generation EGFR tyrosine kinase inhibitor, in some aspects osimertinib or its analogs. In some aspects, the cMET tyrosine inhibitor is or comprises capmatinib or tepotinib. In this and other embodiments, the tumor may be an HGF-related tumor.

[0256] EGFR-positive tumors are typically tumors with EGFR activating mutations. EGFR activating mutations are EGFR mutations that lead to activation of the EGF / EGFR signaling pathway. EGFR activating mutations may be important for the cancerous state of the tumor. One way such tumors become insensitive to EGFR targeted therapy is by activating the HGF / cMET signaling pathway. The tumor may be an HGF-associated tumor. Activation of the cMET / HGF signaling pathway is one of the ways that EGFR-positive tumors escape EGFR targeted therapy. The cMET / HGF pathway can be activated in a variety of ways. Various activation methods are described in the art, some of which are described in detail herein. The antibodies of the present invention are particularly suitable for treating tumors in which activation of the cMET / HGF signaling pathway is associated with the presence or excess of HGF. Such cMET-positive tumors are referred to as HGF-associated tumors or HGF-dependent tumors. The antibodies of the present invention may also be used to at least partially inhibit this possible escape mechanism of EGFR-positive tumors. Such tumors can escape EGFR-targeted therapy by selective outgrowth of tumor cells, in addition, wherein the cMET / HGF signaling pathway is activated. Such cells may be present at the start of EGFR targeted therapy. Such cells have a selective growth advantage over HGF / cMET signaling negative tumor cells. The tumor may be a tumor in which the HGF / cMET signaling pathway is activated. The tumor may be a tumor associated with elevated hepatocyte growth factor (HGF) levels or overexpression of the HGF receptor c-Met. The tumor may be a tumor in which growth is driven by EGF and / or HGF. If the signaling pathway in the tumor cell is activated in response to the presence of a growth factor, and the removal of the growth factor results in inhibition of tumor cell growth, the tumor is said to be driven by a growth factor. The reduction can be measured by reduced cell division and / or induced cell killing, such as apoptosis. If the tumor grows or grows faster in the presence of HGF under conditions that would otherwise allow tumor growth, the tumor is an HGF-related tumor.

[0257] EGFR-targeted therapy for various tumors has been reviewed in Vecchione et al., EGFR-targeted therapy." Experimental cell research vol. 317 (2011): 2765-2771. In general, EGFR-targeted therapy is a therapy that uses molecules that interact with EGFR and inhibit EGFR-mediated-signaling in cells.

[0258] The therapeutic methods or antibodies for use in treatment as described herein in certain aspects further comprise the step of determining whether the tumor is an HGF-associated tumor. In certain aspects, the antibodies of the invention can inhibit the growth of HGF-associated tumors.

[0259] Where a range is provided herein between a number 1 and a number 2, the range includes the number 1 and the number 2. For example, a range between 2-5 includes the numbers 2 and 5.

[0260] When one affinity is mentioned herein as being higher than another affinity, its Kd = lower than the other Kd. For the avoidance of doubt, a Kd of 10e-9 M is lower than a Kd of 10e-8 M. An antibody with a Kd of 10e-9 M has a higher affinity for the target than one with a Kd of 10e-8 M.

[0261] In certain aspects, at the start of treatment, at least one, more than one, or all of the following inclusion factors IF1-IF7 are applicable to treating the individual. In certain aspects, the individual contains or meets all inclusion factors IF1-IF8:

[0262] IF1. The age is equal to or above 18 years old when signing the informed consent form,

[0263] IF2. Patients with histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced unresectable disease and incurable disease.

[0264] IF3.1. For individuals who have failed prior standard first-line therapy: The individual has deteriorated on or is intolerant to treatment known to provide clinical benefit. The individual has NSCLC that carries an activating EGFR mutation, including a tyrosine kinase inhibitor (TKI) sensitizing mutation, and / or an approved TKI-resistance mutation, or any activating c-MET mutation / amplification.

[0265] IF3.2. For individuals who have received first or higher anticancer therapy: progression on or intolerance to therapy known to provide clinical benefit. Patients must have: NSCLC with EGFR exon 20 insertions that have progressed after platinum-based doublet combination therapy, or second (or higher) line patients with NSCLC with cMet exon 14 skipping mutations, capmatinib or tepotinib resistant populations, or cMET TKI-treated or naive, or selected solid tumors with EGFR or cMet driver mutations or cMET alterations (including GC / GEJ, HNSCC, such as HNSCC refractory to approved therapy (regardless of driver mutation), or ESCC), NSCLC patients with uncommon EGFR mutations (G719X, L861Q, and S768I), or individuals with NSCLC with c-MET exon 14 skipping mutations who have not received prior treatment for advanced disease. The individual is exhausted or intolerant of all approved therapies with demonstrated clinical benefit or has no available, approved treatment options.

[0266] IF4. FFPE-embedded archived or fresh tumor tissue samples are available after recent treatment progression.

[0267] IF5. Have measurable disease as defined radiologically by RECIST version 1.1 (patients with non-measurable but evaluable disease may be included in the dose escalation portion).

[0268] IF6. Has Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0269] IF7. Has a life expectancy of ≥12 weeks.

[0270] IF8. Adequate organ function as determined by at least one or all of the following:

[0271] IF8.1 absolute neutrophil count (ANC) ≥ 1.5X 10 9 / L

[0272] IF8.2 hemoglobin ≥9g / dL.

[0273] IF8.3 Platelets ≥ 100 x 10 9 / L.

[0274] IF8.4 corrects total serum calcium within the normal range.

[0275] IF8.5 Serum magnesium is within normal range (or corrected with supplements).

[0276] IF8.6 Serum potassium was within normal range.

[0277] IF8.7 Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) equal to or less than 3.0x upper limit of normal (ULN), and total bilirubin equal to or less than 1.5x ULN, provided that in the case of liver involvement or malignancy, ALT / AST equal to or less than 5x ULN and total bilirubin equal to or less than 2x ULN.

[0278] IF8.8 For patients with Gilbert's syndrome, the conjugated bilirubin value is within the normal range.

[0279] IF8.9 is for patients aged 65 years and older, with serum creatinine equal to or less than 1.5x ULN, or creatinine clearance equal to or higher than 50 mL / min, calculated according to the Cockroft and Gault formula or the MDRD formula.

[0280] IF8.10 Serum albumin >3.3 g / dL

[0281] In certain aspects, all organ function measurements based on IF8 have an upper limit observed in healthy individuals.

[0282] In some aspects, the treatment individual comprises one or more factors selected from IF1-IF8. In some aspects, the treatment individual comprises factors IF2, IF3, IF5 and IF8. In some aspects, the treatment individual comprises all factors of IF1-IF8.

[0283] In certain aspects, at the start of treatment, at least one, more than one, or all of the following exclusion factors EF1-EF16 are applicable to treating the individual:

[0284] EF1. There are central nervous system metastases, which:

[0285] EF1.1: untreated and symptomatic, may include individuals with asymptomatic lesions if considered to be in stable condition;

[0286] EF1.2 requires radiation or surgery;

[0287] EF1.3 requires continued steroid therapy (>10 mg prednisone or equivalent) to control symptoms for 14 days after administration prior to the first dose. Subjects with other central nervous system metastases are permitted.

[0288] EF2. With known leptomeningeal lesions.

[0289] EF3. Participation in another clinical trial or treatment with any investigational drug within 4 weeks prior to the first dose.

[0290] EF4. Systemic anticancer therapy or immunotherapy is administered within 4 weeks or 5 half-lives (whichever is shorter) after the first dose of the investigational drug. For cytotoxic agents with major delayed toxicity (e.g., mitomycin C, nitrosoureas), a 6-week washout period is required.

[0291] EF5. Major surgery or radiation therapy within 3 weeks of first dose administration. Individuals who have previously received radiation therapy equal to or greater than 25% of the bone marrow at any time are not eligible.

[0292] EF6. Clinically significant toxicity with persistent grade greater than grade 1 related to prior antineoplastic therapy (except alopecia); conditions not excluding stable sensory neuropathy with NCI-CTCAE v5.0 grade equal to or less than 2, and hypothyroidism grade equal to or less than 2 that is stable on hormone replacement therapy.

[0293] EF7. History of allergic reaction or any toxicity attributable to human proteins or any excipients requiring permanent discontinuation of these agents.

[0294] EF8. History of clinically significant cardiovascular disease, including but not limited to:

[0295] EF8.1 Deep vein thrombosis or pulmonary embolism diagnosed within 1 month before the first dose of trial drug, or any of the following diagnosed within 6 months before the first dose of trial drug: myocardial infarction, unstable angina, stroke, transient ischemic attack, coronary artery / peripheral artery bypass graft, or any acute coronary syndrome.

[0296] EF8.2 QT prolongation >480 msec or clinically significant arrhythmia or electrophysiological disease (i.e., implantable cardioverter-defibrillator or uncontrolled atrial fibrillation). Clinically stable patients with pacemakers are eligible.

[0297] EF8.3 Uncontrolled (persistent) arterial hypertension: systolic blood pressure >180 mm Hg and / or diastolic blood pressure >100 mm Hg.

[0298] EF8.4 Congestive heart failure (CHF), defined as New York Heart Association (NYHA) class III-IV, or hospitalization for CHF within 6 months of the first dose of trial drug.

[0299] EF8.5 has clinically significant pericardial effusion.

[0300] EF8.6 myocarditis.

[0301] EF9. History of interstitial lung disease, including drug-induced interstitial lung disease, radiation pneumonitis, requiring long-term treatment with steroids or other immunosuppressants within 1 year.

[0302] EF10. Prior or concurrent malignancy, excluding non-basal cell skin cancer or carcinoma in situ of the cervix, unless the tumor is being treated for curative or palliative purposes and the prior or concurrent malignancy does not affect the safety and efficacy evaluation of the investigational drug.

[0303] EF11. Current serious illness or medical condition, including but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic, or psychiatric disorder.

[0304] EF12. Individuals with active hepatitis B infection (HBsAg positive) not receiving antiviral treatment. Individuals with active hepatitis B (HbsAg positive) must be receiving antiviral treatment with amivudine, tenofovir, entecavir, or other antiviral drugs, started at least 7 days or more before the first dose. Individuals with a history of hepatitis B (anti-HBc positive, HbsAg and HBV-DNA negative) are eligible.

[0305] EF13. Hepatitis C RNA (HCV RNA) test positive; individuals with spontaneous resolution of HCV infection (HCV antibody positive but no detectable HCV-RNA), or who achieved a sustained virological response after antiviral therapy and showed no detectable HCV RNA for equal to or greater than 6 months (on an IFN-free prescription), or equal to or greater than 12 months (on an IFN-based prescription) after stopping antiviral therapy are eligible.

[0306] EF14. Known history of HIV (HIV 1 / 2 antibodies). HIV patients with undetectable viral load are allowed. HIV testing is not required unless mandated by local health authorities or regulations.

[0307] EF15. Sexually active male and female patients of reproductive potential agree to use one of the following contraceptive methods throughout the trial and for 6 months after the final dose of PB19478:

[0308] Combination (estrogen and progestin) hormonal contraceptives (oral, vaginal, transdermal) associated with ovulation inhibition

[0309] Progestin-only hormonal contraceptives (oral, injectable, implantable) associated with ovulation inhibition

[0310] Intrauterine device (IUD)

[0311] Intrauterine hormone-releasing system (IUS)

[0312] Bilateral fallopian tube obstruction

[0313] Partner vasectomy

[0314] Abstinence

[0315] EF16. Are pregnant or breastfeeding.

[0316] In certain aspects, the treated individual meets one or more factors selected from the group consisting of EF1-EF 16. In certain aspects, the treated individual meets all factors of EF1-EF16.

[0317] The reference to a patent document or other matter herein is not to be taken as an admission that the document or matter was known as at the priority date of any claim, or that the information it contains was part of the common general knowledge.

[0318] The conjunction "and / or" between multiple quoted elements is understood to include separate and combined options. For example, when two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within this meaning, thus satisfying the requirements of the term "and / or" used herein. The simultaneous applicability of more than one option is also understood to fall within this meaning, thus satisfying the requirements of the term "and / or".

[0319] For clarity and simplicity of description, various features are described herein as being part of the same or separate embodiments, however, it should be understood that the scope of the present invention may include embodiments having a combination of all or some of the described features. BRIEF DESCRIPTION OF THE DRAWINGS

[0320] Figure 1 .The amino acid sequence of the heavy chain variable region of the variable domain mentioned in this application.

[0321] Figure 2 MF3370 and its variants. CDR1, CDR2 and CDR3 sequences in MF8226 (underlined from left to right). CDRs in other sequences are located in corresponding positions (according to Kabat).

[0322] Figure 3 MF4356 and its variants. CDR1, CDR2 and CDR3 sequences in MF4356 (underlined from left to right). CDRs in other sequences are located in corresponding positions (according to Kabat).

[0323] Figure 4. Common light chains used in mono- and bispecific IgGs.

[0324] Figure 4A: Consensus light chain amino acid sequence. Figure 4B : Common light chain variable domain DNA sequence and translation (IGKV1-39 / jk1). Figure 4C : Common light chain constant region DNA sequence and translation. Figure 4D : Translation of the common light chain variable domain of IGKV1-39 / jk5. Figure 4E : V zone IGKV1-39A.

[0325] Figure 5. IgG heavy chains used to generate bispecific molecules. Figure 5A : CH1 area. Figure 5B : Hinge region. Figure 5C : CH2 region. Figure 5D : CH2 containing L235G and G236R silent substitutions. Figure 5E : CH3 domain comprising substitutions L351K and T366K (KK). Fig. 5F : CH3 domain comprising substitutions L351D and L368E (DE).

[0326] Figure 6 . Western blot analysis results of the LXFE2478 model showing protein levels of EGFR, cMET, and HGF.

[0327] Figure 7 . Overview of treatment schedule. Each group was administered antibody or vehicle as a monotherapy over a 5-week period.

[0328] Figure 8 Effect of antibody therapy on tumor volume in LXFE2478, a NSCLC PDX model carrying exon 20 mutations. Tumor volume growth curves representing groups 1-5 are shown at different time points (mean TV ± SEM).

[0329] Fig. 9 . Overview of treatment schedule. Each group was administered antibody or vehicle as a monotherapy over a 3-week period.

[0330] Fig.10 Effect of antibody therapy on tumor volume in the NSCLC CDX model HCC827 / ER1 carrying exon 19 mutations. Tumor volume growth curves representing vehicle and antibody groups are shown at different time points (mean TV ± SEM).

[0331] Fig.11 Animal survival curves of mice carrying the NSCLC CDX model HCC827 / ER1 with exon 19 mutation vs. vehicle group.

[0332] project

[0333] 1. A bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYX1X2NTNYAQKLQG and a CDR3 comprising a sequence of X3X4X5X6HWWLX7A, wherein X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G; and having 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than X1 to X7, and wherein the second variable domain comprises a heavy chain variable region having SEQ ID An amino acid sequence of one of the sequences of NO:1-23 having 0 to 10, in some aspects 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof, for use in a method of treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor or ii) chemotherapy and a tyrosine kinase inhibitor, iii) a cMET tyrosine kinase inhibitor or iv) treatment of cancer in an individual who has not received any prior anti-cancer treatment.

[0334] 2. A method for treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor or ii) chemotherapy and an EGFR tyrosine kinase inhibitor, iii) a cMET tyrosine kinase inhibitor or iv) treatment of cancer in an individual who has not received any prior anti-cancer treatment, the treatment comprising administering to the individual an effective amount of a bispecific antibody comprising a first variable domain that binds to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET). The invention relates to a first variable domain comprising a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYX1X2NTNYAQKLQG and a CDR3 comprising a sequence of X3X4X5X6HWWLX7A, wherein X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G; and having 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof at positions other than X1-X7, and wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of one of the sequences of SEQ ID NOs: 1-23, having 0 to 10, in some aspects 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0335] 3. Use of a bispecific antibody in the preparation of a medicament for treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor or ii) chemotherapy and an EGFR tyrosine kinase inhibitor, iii) a cMET tyrosine kinase inhibitor or iv) treatment of cancer in an individual who has not received any prior anti-cancer treatment, the bispecific antibody comprising a first variable domain that binds to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that binds to (or binds to) the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET). Variable domains, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, wherein X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G; and there are 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than X1-X7, and wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of one of the sequences of SEQ ID NO:1-23, having 0 to 10, in some aspects 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

[0336] 4. The use or method of any of the preceding items, wherein the individual or cancer that has received a previous treatment according to i) is resistant to treatment with a third generation EGFR tyrosine kinase inhibitor.

[0337] 5. The use or method of any one of items 1 to 3, wherein the individual or cancer that has received a previous treatment according to ii) is resistant to treatment with a first, second and / or third generation tyrosine kinase inhibitor.

[0338] 6. The use or method of any one of items 1 to 3, wherein the individual or cancer that has received a previous treatment according to iii) is resistant to treatment with a cMET tyrosine kinase inhibitor.

[0339] 7. The use or method as described in any of the preceding items, wherein the administration of the bispecific antibody according to i) comprises a second line of treatment and the administration according to ii) or iii) comprises a third line of treatment.

[0340] 8. The use or method according to any one of the preceding items, wherein the first-generation EGFR tyrosine kinase inhibitor comprises or is gefitinib, erlotinib or icotinib.

[0341] 9. The use or method of any of the preceding items, wherein the second-generation EGFR tyrosine kinase inhibitor comprises or is afatinib, dacomitinib, XL647, AP26113, CO-1686 or neratinib.

[0342] 10. The use or method as described in any of the preceding items, wherein the third-generation EGFR tyrosine kinase inhibitor comprises osimertinib, lazertinib, afatinib, rezivertinib, rociletinib, olmutinib, almonertinib, abivertinib, ASK120067, befotinib, ertinib), Olmutinib, Rociletinib or SH-1028 (Nazartinib (EGF816), Naquotinib (ASP8273), Mavelertinib (PF-0647775), Olafertinib (CK-101), Keynatinib or ES-072, preferably Osimertinib.

[0343] 11. The use or method according to any of the preceding items, wherein the cMET tyrosine kinase inhibitor is or comprises capmatinib, tepotinib, crizotenib, cabozantinib, savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833, preferably capmatinib or tepotinib.

[0344] 12. The use or method of any of the preceding items, wherein the chemotherapy comprises platinum-based chemotherapy, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide or pemetrexed, or any combination thereof, preferably a composition comprising cisplatin or carboplatin.

[0345] 13. The use or method of any one of the preceding claims, wherein the cancer is an EGFR-positive cancer, a cMET-positive cancer, and / or an EGFR and cMET-positive cancer.

[0346] 14. The use or method of any of the preceding items, wherein the cancer comprises EGFR aberration, cMET aberration, and / or EGFR and cMET aberration.

[0347] 15. The use or method of any of the preceding items, wherein the cancer comprises an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation (such as L718X (such as L718Q), G719X (such as G719A), L792X (such as L792H), G796X (such as G796R, G796S, G796D), C797X, C797X (such as C797S, C797G), a mutation that reduces the binding of a third-generation tyrosine kinase inhibitor to EGFR (such as L792X, L718X), an acquired tyrosine kinase inhibitor resistance mutation (such as C797X, L792X, G796X, G724X, S768X, L718X or an exon 20 insertion mutation), EGFR gene amplification, a cMET mutation or a cMET aberration.

[0348] 16. The use or method of any of the preceding items, wherein the cancer comprises an exon 19 deletion mutation, preferably an in-frame exon 19 deletion, an exon 20 missense mutation (such as T790M), or an exon 21 mutation, such as L858R.

[0349] 17. The use or method according to any of the preceding claims, wherein the cancer comprises an EGFR exon 20 mutation, preferably an exon 20 insertion mutation.

[0350] 18. The use or method of any of the preceding items, wherein the cancer comprises an acquired tyrosine kinase inhibitor resistance mutation, such as a mutation conferring resistance to osimertinib, including G724X (such as G724S), S768X (such as S768I), T790X (such as T790M), L792X (such as L792H), C797X (including C797S and C797G), L798X (such as L798I).

[0351] 19. The use or method of any of the preceding items, wherein the cancer comprises an exon 20 (762-823) mutation selected from: near-loop insertion (positions 767-772), far-loop insertion (positions 773-775), preferably V769_D770insASV, D770_N771insSVD, H773_V774insNPH, H773_V774insH, D770_N771insG, D770delinsGY, N771_P772insN, V774_C775insHV, D770_N771ins nsGL、H773_V774insPH、A763_Y764insFQEA、D770_N771delinsEGN、D770_N771insGD、D770_N771insH、D770_N771insP、H773_V77 4insAH、H773_V774insGNPH、H773delinsSNPY、N771_P772insH、N771_P772insVDN、N771delinsGY、N771delinsKH、N771delinsRD、 P772_H773delinsHNPY, P772_H773insGT, P772_H773insPNP, P772_H773insT, V769_D770insA, V769_D770insGG, V769_D770insGSV, V769_D770insGVV and V769_D770insMASV; or mutations T790M, L792X (e.g., L792H, C796X (e.g., G796R, G796S, G796D), C797X (e.g., C797S, C797G), L798 I, or in-frame exon 20 insertion, such as M766_A767insASV or H773-V774insNPH, Ins761 (EAFQ), Ins770 (ASV), Ins771 (G), Ins774 (NPH), M766_A7671nsA, S768_V769InsSVA, P772_H773InsNS, D761_E762InsX1-7, A763_Y764InsX1-7, Y764_Y765InsX1-7, M766_A767InsX1-7, A767_V768 InsX1-7, S768_V769 InsX1-7>V769_D770 InsX1-7>D770_N771 InsX1-7>N771_P772 InsX1-7>P772_H773 InsX1-7, H773_V774 InsX1-7, or V774_C775 InsX1-7.

[0352] 20. The use or method of any of the preceding items, wherein the cancer comprises a cMET aberration, such as cMET amplification, cMET overexpression, increased signaling of the cMET pathway, cMET gene amplification, increased cMET protein activity, and / or increased HGF expression.

[0353] 21. The use or method of any of the preceding items, wherein the cancer comprises a cMET exon 14 skipping mutation.

[0354] 22. The use or method of any of the preceding items, wherein the cancer is non-small cell lung cancer (NSCLC), head and neck cancer, in particular head and neck squamous cell carcinoma; gastric cancer, in particular gastric adenocarcinoma; esophageal cancer, in particular esophageal squamous cell carcinoma; gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer.

[0355] 23. The use or method of any of the preceding items, wherein the cancer is NSCLC comprising an activating EGFR mutation, an EGFR tyrosine kinase inhibitor sensitizing mutation (e.g., exon 19 deletion and L858X), an acquired EGFR tyrosine kinase inhibitor resistance mutation (e.g., T790X, C797X, L792X, L798X), and an approved EGFR tyrosine kinase inhibitor resistance mutation, an EGFR exon 20 insertion mutation, an activating c-MET mutation, preferably an exon 14 skipping mutation, or cMET amplification, preferably comprising MET / CEP7>5 or cfDNA≥2 copies, increased HGF expression, or any combination thereof.

[0356] 24. The use or method of any of the preceding items, wherein the cancer is advanced or metastatic cancer.

[0357] 25. The use or method of any of the preceding items, wherein the cancer or patient has progressed following previous treatment for the advanced or metastatic cancer.

[0358] 26. The use or method of any of the preceding items, wherein the treatment comprises a diagnostic step for assessing whether the cancer is an EGFR-positive and / or cMET-positive cancer, or assessing the presence of EGFR and / or cMET aberrations in the cancer.

[0359] 27. The use or method of any of the preceding items, wherein the treatment comprises a diagnostic step of determining EGFR amplification, immunohistochemical staining, determining the amount of one or more biomarkers in tumor tissue, blood or serum, such as the amount of EGFR / c-MET signaling pathway activity, measuring soluble EGFR and soluble c-MET in blood or serum, and / or measuring the expression of EGFR and c-MET targets in tumor cells.

[0360] 28. The use or method of any of the preceding items, wherein the subject is a human subject.

[0361] 29. The use or method of any of the preceding items, wherein the antibody is a human antibody.

[0362] 30. The use or method of any of the preceding items, wherein the antibody is enhanced by ADCC.

[0363] 31. The use or method of any of the preceding items, wherein the antibody is an IgG1 antibody having a stoichiometric ratio of anti-EGFR to anti-cMET of 1:1.

[0364] 32. The use or method of any of the preceding items, wherein the antibody has one variable domain that binds to EGFR and one variable domain that binds to cMET.

[0365] 33. The use or method of any of the preceding items, wherein the variable domain that can bind to human EGFR can also bind to macaque and mouse EGFR.

[0366] 34. The use or method of any of the preceding items, wherein the variable domain that can bind to human EGFR binds to domain III of human EGFR.

[0367] 35. The use or method as described in any of the preceding items, wherein the variable domain that can bind to cMET blocks the binding of antibody 5D5 to cMET.

[0368] 36. The use or method of any preceding claim, wherein the cMET-binding variable domain blocks the binding of HGF to cMET.

[0369] 37. The use or method of any of the preceding items, wherein the amino acids at positions 405 and 409 in one CH3 domain are identical to the amino acids at the corresponding positions in the other CH3 domain (EU numbering).

[0370] 38. The use or method according to any one of the preceding items, wherein

[0371] X1=N; X2=G; X3=D; X4=S;

[0372] X1=N; X2=A; X3=D; X4=S;

[0373] X1=S; X2=G; X3=D; X4=S;

[0374] X1=N; X2=G; X3=D; X4=R;

[0375] X1=N; X2=A; X3=D; X4=R;

[0376] X1=S; X2=G; X3=D; X4=R;

[0377] X1=N; X2=G; X3=G; X4=Y;

[0378] X1=N;X2=A;X3=G;X4=Y;X5=L;X6=D and X7=G;or

[0379] X1=S; X2=G; X3=G; X4=Y;

[0380] 39. The use or method of any of the preceding items, wherein X1=N; X2=G; X3=D; X4=R; X5=H; X6=W and X7=D; or X1=N; X2=A; X3=D; X4=R; X5=H; X6=W and X7=D; or X1=S; X2=G; X3=D; X4=R; X5=H; X6=W and X7=D.

[0381] 40. The use or method of any of the preceding items, wherein X1 = N; X2 = G; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D; or X1 = N; X2 = A; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D.

[0382] 41. The use or method of any of the preceding items, wherein the heavy chain variable region of the second variable domain comprises an amino acid sequence of one of the sequences of SEQ ID NO: 1-3; 7; 8; 10; 13; 15; 16; 17; 21; 22 or 23, having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

[0383] 42. The use or method of any of the preceding items, wherein the heavy chain variable region of the second variable domain comprises an amino acid sequence of one of the sequences of SEQ ID NO: 2; 7; 8; 10; 13 or 23, having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

[0384] 43. The use or method of any of the preceding items, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of SYSMN; a CDR2 sequence of WINTYTGDPTYAQGFTG and a CDR3 sequence of ETYYYDRGGYPFDP.

[0385] 44. The use or method of any of the preceding items, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of TYSMN; a CDR2 sequence of WINTYTGDPTYAQGFTG, and a CDR3 comprising the sequence ETYFYDRGGYPFDP.

[0386] 45. The use or method of any of the preceding items, wherein the first and second variable domains comprise a common light chain, preferably Figure 4B Light chain in.

[0387] 46. ​​The use or method of any of the preceding items, wherein the first and second variable domains comprise a light chain having CDR1, CDR2 and CDR3 amino acid sequences QSISSY, AAS and QQSYSTP, respectively (according to IMGT).

[0388] 47. The use or method of any of the preceding items, wherein the antibody inhibits HGF-induced growth of HGF-growth responsive cells.

[0389] 48. The use or method of any of the preceding items, wherein the antibody inhibits EGF-induced growth of EGF-growth responsive cells.

[0390] 49. The use or method of any of the preceding items, wherein 1000 mg of the bispecific antibody is administered to the individual, in particular using a fixed dose of 1000 mg.

[0391] 50. The use or method of item 49, wherein the bispecific antibody is administered once a week.

[0392] 51. The use or method of item 49, wherein the bispecific antibody is administered once every two weeks.

[0393] 52. The use or method according to any one of items 1 to 48 above, wherein 1500 mg of the bispecific antibody is administered to the individual, in particular using a fixed dose of 1500 mg.

[0394] 53. The use or method of item 52, wherein the bispecific antibody is administered once every two weeks.

[0395] 54. The use or method according to any one of items 1 to 48 above, wherein 2000 mg of the bispecific antibody is administered to the individual, in particular using a fixed dose of 2000 mg.

[0396] 55. The use or method of item 54, wherein the bispecific antibody is administered once every two weeks.

[0397] 56. The use or method of any of the preceding items, wherein the individual of iv) has not received any previous anti-cancer treatment with a cMET inhibitor.

[0398] 57. The use or method of any of the preceding items, wherein the individual of iv) has not received any previous anti-cancer treatment with a cMET TKI inhibitor.

[0399] 58. The use or method of any of the preceding items, wherein the individual of iv) has not received any previous anti-cancer treatment with capmatinib.

[0400] 59. The use or method of any of the preceding items, wherein the individual of iv) has not received any previous anti-cancer treatment with tepotinib.

[0401] 60. The use or method of any of the preceding items, wherein the individual of iv) has not received any previous anti-cancer treatment with savolitinib.

[0402] Example

[0403] As used herein, "MFXXXX", wherein X is independently a number 0-9, refers to a Fab comprising a variable domain, wherein the VH has an amino acid sequence identified by a 4-digit number. Unless otherwise indicated, the light chain variable region of the variable domain generally has the sequence of Figure 4A, generally 4b. "MFXXXX VH" refers to the amino acid sequence of the VH identified by a 4-digit number. MF also comprises a light chain constant region and a heavy chain constant region that generally interacts with the light chain constant region. PG refers to a monospecific antibody comprising the same heavy chain and light chain. PB refers to a bispecific antibody having two different heavy chains. The VH variable region of the heavy chain is different and is generally also a CH3 region, wherein the CH3 domain of one heavy chain has a KK mutation, and the CH3 domain of the other heavy chain has a complementary DE mutation (see reference PCT / NL2013 / 050294 (published as WO2013 / 157954)).

[0404] Please refer to PCTNL / 2018 / 050537 (published as WO2019 / 031965) for detailed information on the production of the antibodies of the present invention. Bispecific antibodies that bind to EGFR and cMET suitable for use in the attached examples and methods of the present invention include those listed in Tables 3, 4, 5, and 6. Specifically, the bispecific antibody PB19478 is suitable for use in the attached examples.

[0405] Each bispecific antibody comprises two VHs designated by MF numbers, capable of binding to EGFR and cMET, respectively, and an Fc tail having a KK / DE CH3 heterodimerization domain, as shown in Figures 5e and 5f, respectively, a CH2 domain as shown in Figure 5d, a hinge domain as shown in Figure 5b, a CH1 domain as shown in Figure 5a, and a common light chain as shown in Figures 4a-e. For example, a bispecific antibody represented as MF8233 x MF8230 has the general sequence described above, as well as a variable region of a VH having the sequence of MF8233, and a variable region of a VH having the sequence of MF8230, and is used in the accompanying examples in certain aspects.

[0406] Example 1: Materials and Methods

[0407] Cell lines:

[0408] EBC-1[JCRB0820], PC-9[RCB0446], H358[ CRL-5807 TM ]、HCC827[ CRL-2868 TM ]、MKN-45[DSMZ ACC 409]N87[ CRL-5822 TM] and A431[ CRL-1555 TM ] Cell lines were purchased and routinely maintained in growth medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). HEK293F Freestyle cells were obtained from Invitrogen and routinely maintained in 293 FreeStyle medium.

[0409] cDNA constructs:

[0410] Generation of cMET and EGFR expression vectors for stable cell line generation (cMET and EGFR) and for immunization (cMET)

[0411] The full-length cDNA of each target, including unique restriction sites for cloning and kozak consensus sequences for efficient translation, can be synthesized or obtained by PCR amplification on commercially available expression constructs (containing target cDNAs) using specific primers introduced with unique restriction sites for cloning and kozak consensus sequences for efficient translation. The full-length cDNA of each target was cloned into eukaryotic expression constructs, such as pcDNA3.1, while the extracellular domain was cloned into pVAX1 and pDisplay. The insert sequence was verified by comparison with the NCBI reference amino acid sequence.

[0412] Amino acid sequence of the full-length human EGFR insert for cell surface expression (equivalent to GenBank: NP_00533):

[0413] MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCE

[0414] VVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALA

[0415] VLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDF

[0416] QNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSSGRCRGKSPSDCCHNQCAAG

[0417] CTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRN

[0418] YVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKH

[0419] FKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFE

[0420] NLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTS

[0421] GQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE

[0422] PREFERREDSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGEN

[0423] NTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVAL

[0424] GIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLL DYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPPYDGIPASEISSILEKGERLPQP PICTIDVYMIMVKCWMIDASRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDP TGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA

[0425] Among them:

[0426] -MRPSGTAGAALLALLAALCPASR: signal

[0427] -ALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS: ECD of human EGFR.

[0428] -IATGMVGALLLLLVVALGIGLFM: Predicted TM region.

[0429] -: intracellular tail.

[0430] The amino acid sequence of the extracellular domain of human EGFR varIII, a naturally occurring EGFR variant VAR_066493 [Ji H., Zhao X; PNAS 103:7817-7822 (2006)], is caused by an in-frame deletion of exons 2-7. The following _ indicates the absence of amino acid positions 30-297.

[0431] MRPSGTAGAALLALLAALCPASRALEEKK_GNYVVTDHGSCVRACGADSYEMEED GVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[0432] Wherein:

[0433] -MRPSGTAGAALLALLAALCPASR: Signal peptide.

[0434] -ALEEKK_GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGE FKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS:

[0435] ECD of EGFRvarIII

[0436] Amino acid sequence of a chimeric macaque (Macaca mulatta) extracellular EGFR domain hybridized to the human EGFR transmembrane and intracellular domains for cell surface expression (equivalent to GenBank: XP_014988922.1). In the following examples, the human EGFR sequence is underlined.

[0437] MGPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCE

[0438] VVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALA

[0439] VLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSEFLSNMSMDF

[0440] QNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSSGRCRGKSPSDCCHNQCAAG

[0441] CTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRN

[0442] YVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDTLSINATNIK

[0443] HFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAF

[0444] ENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFG

[0445] TSSQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCQNVSRGRECVDKCNIL

[0446] EGEPREFVENSECIQCHPECLPQVMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGE

[0447] NNTLVWKYADAGHVCHLCPNCTYGCTGPGLEGCARNGPKIPS IATGMLGALLLLLVVA

[0448] LGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAF

[0449] GTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTS

[0450] TVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARN

[0451] VLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYG

[0452] VTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRE

[0453] LIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGF

[0454] FSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFL

[0455] PVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCV

[0456] NSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFI

[0457] GA

[0458] in:

[0459] -MGPSGTAGAALLALLAALCPASR: signal peptide.

[0460] -LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKT: ECD of cyEGFR

[0461] Amino acid sequence of the full-length human cMET insert for cell surface expression (equivalent to GenBank: P08581-2). This sequence differs from the reference sequence by the insertion at positions 755-755: S→

[0462] STWWKEPLNIVSFLFCFAS

[0463] MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEH

[0464] HIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMAL

[0465] VVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALG

[0466] AKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRD

[0467] SYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKK

[0468] RSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPI

[0469] KYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRV

[0470] DLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPE

[0471] VIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEE

[0472] CLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCT

[0473] LTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGG

[0474] TLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIF

[0475] SYREDPIVYEIHPTKSFISTWWKEPLNIVSFLFCFASGGSTITGVGKNLNSVSVPRMVINVH

[0476] EAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFK

[0477] PFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLK

[0478] LNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGVVSISTALLLLLGFFLWLKKRKQIKDL

[0479] GSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQV

[0480] QYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRG

[0481] HFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRS

[0482] EGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAAR

[0483] NCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSD

[0484] VWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKA

[0485] EMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASF

[0486] WETS

[0487] Among them:

[0488] -MKAPAVLAPGILVLLFTLVQRSNG: Signal peptide

[0489] -ECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFT: Human cMET ECD

[0490] -GLIAGVVSISTALLLLLGFFLWL: Transmembrane region

[0491] -KKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQF PNSSQNGSCRQVQYPLTDMSPILTSGDSDISPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYASKKFVHRD LAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS: Intracellular domain

[0492] Reference Antibodies

[0493] Anti-cMET antibodies are known in the art (Table 1). Monospecific bivalent cMET antibodies were constructed according to public information and expressed in 293F Freestyle cells. Table 1 shows the relevant disclosed information. Monospecific bivalent antibodies against cMET were constructed according to public information and expressed in 293F Freestyle cells. For the assay of HGF ligand blocking, the VH- and VL-encoding gene fragments of the patent-derived anti-cMET antibodies were recloned into a phage display vector for display on filamentous phage.

[0494] The reference antibody cetuximab (Erbitux) was used as the reference antibody for the EGFR Fab group.

[0495] The 2994 Fab protein was produced by digestion of purified PG2994 IgG with papain. Therefore, PG2994 was incubated with papain (Pierce #44985) coupled to microbeads and allowed to cleave at 37°C for 5.5 hours under rotation. The Fab fragment was purified from the digestion mixture by filtration through MabSelectSure LX. The separation fraction containing the Fab protein was concentrated to 3 ml using vivaspin20 10 kDa and further purified by gel filtration using a superdex75 16 / 600 column (in PBS solution).

[0496] Example 2

[0497] Bivalent monoclonal antibody generation and antibody characterization

[0498] The VH genes of unique antibodies, as judged by the VH gene sequences and some of their sequence variants, were cloned into the backbone IgG1 vector. Suspension-adapted 293F Freestyle cells were cultured in T125 flasks on a shaker until the density reached 3.0x10 6 Cells were cultured at 0.3-0.5 x 10 6 The cells were transiently transfected with a single sterile DNA:PE1 mixture and further cultured. Seven days after transfection, the supernatant was collected and filtered through 0.22 μM (Sartorius) and purified on protein A microbeads using a batch purification method, after which the buffer was exchanged for PBS.

[0499] Cross-blocking assay for cMET antibodies

[0500] Competition of cMET-specific phage with cMET reference antibodies was tested in ELISA. Therefore, 2.5 μg / ml of cMET-Fc fusion protein was coated on MAXISORPTM ELISA plates overnight at 4°C. The wells on the ELISA plates were blocked with PBS (pH 7.2) containing 2% ELK, shaking (700 rpm) for 1 hour at room temperature. The next reference or negative control IgG was added at a concentration of 5 μg / ml and allowed to bind for 15 minutes at 700 rpm at room temperature. Afterwards, 5 μl of PEG-precipitated phage were added and allowed to bind for 1 hour at 700 rpm at room temperature. The bound phages were detected with HRP-labeled anti-M13 antibodies at room temperature, 700 rpm, for 1 hour. As a control, the process was performed simultaneously with antibodies specific for the coated antigen and negative control phages. The bound secondary antibody was visualized by TMB / H2O2 staining and by OD450nm The staining was quantified by measurement. Table 2 illustrates that MF4040 and MF4356 exhibited competition with the 5D5 reference antibody. MF4297 competed to a lesser extent with 13.3.2 and C8H241. The positive control phages all showed complete competition with the corresponding IgG, while the control group without antibody did not affect the competition assay.

[0501] Generation of bispecific antibodies

[0502] Bispecific antibodies are produced by transient co-transfection of two plasmids encoding IgG with different VH domains, using proprietary CH3 engineering technology to ensure efficient heterodimerization and formation of bispecific antibodies. The common light chain is also co-transfected in the same cell, either on the same plasmid or on another plasmid. In our co-pending applications (e.g., WO2013 / 157954 and WO2013 / 157953; incorporated herein by reference in their entirety), we disclose methods and means for producing bispecific antibodies from a single cell, thereby providing means that facilitate the formation of bispecific antibodies rather than monospecific antibodies. These methods may also be advantageously used in the present invention. Specifically, preferred mutations that substantially only produce bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, such as L351K and T366K (numbered according to the EU numbering system) (referred to as "KK-variant" heavy chains), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, such as L351D and 10L368E (referred to as "DE-variant" heavy chains), and vice versa. It has been previously demonstrated in our co-pending applications that negatively charged DE-variant heavy chains and positively charged KK-variant heavy chains preferentially pair to form heterodimers (referred to as "DEKK" bispecific molecules). Homodimerization of DE-variant heavy chains (DE-DE homodimers) or homodimerization of KK-variant heavy chains (KK-KK homodimers) is less favorable due to the strong repulsion between charged residues in the CH3-CH3 interface between the same heavy chains.

[0503] Table 3 shows which cMET and EGFR Fab arms are cloned into the appropriate KK and DE vectors. After manufacturing, the bispecific IgG is purified by protein-A batch purification and the buffer is exchanged for PBS. Successful manufacturing will produce a minimum concentration of 0.1 mg / ml IgG1 full-length antibody, which is assigned a unique code (PBnnnnn; where nnnnn represents a randomly generated number) to identify a specific combination of Fab fragments that bind to 2 different targets. The successfully manufactured bispecific IgG is tested in ELISA for binding to its respective target. For more details on the manufacture of bispecific antibodies, reference is made to PCTNL / 2018 / 050537 (publication number WO2019 / 031965) herein.

[0504] Example 3

[0505] Screening of c-MET x EGFR Bispecific Antibodies in EGF / HGF and HGF and EGF Proliferation Assays

[0506] A panel of cMET x EGFR bispecific antibodies was tested for potency in N87 cells using HGF / EGF, HGF, and EGF assays. The N87 cell line, officially known as NCI-N87, is a gastric cancer cell line derived from a metastatic site with high EGFR expression levels and moderate cMET expression levels (Zhang et al., 2010). Antibodies were tested in an 8-step semi-log titration from 10 μg / ml to 3.16 ng / ml. Each antibody was tested in duplicate. The anti-RSV-G antibody PG2708 served as a negative control. The reference antibody 2994 Fab served as a positive control for the HGF assay and the reference antibody cetuximab served as a positive control for the EGF assay.

[0507] An equimolar 1:1 mixture of cetuximab / 5D5 Fab served as a positive control for the EGF, HGF, and EGF / HGF assays.

[0508] Wells with one ligand or ligand combination and medium controls were included to define the detection window. Antibodies were diluted in chemically defined starvation medium (CDS: RPMI1640 medium containing 80 U penicillin and 80 μg streptomycin per ml, 0.05% (w / v) BSA and 10 μg / ml holo-transferrin) and 50 μl of diluted antibody was added to each well of a 96-well black transparent bottom plate (Costar). Ligands were added (50 μl per well of a stock solution containing 400 ng / ml HGF and 4 ng / ml EGF, and an EGF / HGF concentrate of 4 ng / ml EGF / 400 ng / ml HGF diluted in CDS: R&D Systems, Cat. Nos. 396-HB and 236-EG). N87 cells were trypsinized, harvested and counted, and 8000 cells in 100 μl CDS were added to each well of the plate. To avoid edge effects, the plate was placed at room temperature for one hour and then placed in a container in a 37°C cell culture incubator for three days. On the fourth day, Alamar blue (Invitrogen, #DAL1100) (20 μl per well) was added and the fluorescence value was measured after incubation with Alamar blue for 6 hours (37°C) using 560nm excitation and 590nm reading on the Biotek Synergy 2 multi-function microplate reader. The fluorescence value was normalized to uninhibited growth (no antibody was added but both ligands were added).

[0509] The results of the various experiments are listed in Table 4. In the N87 HGF / EGF assay, 14 different cMETxEGFR bispecific antibodies were identified with potency comparable to the reference monospecific antibody (an equimolar mixture of cetuximab and 5D5 Fab): PB7679, PB7686, PB8218, PB8244, PB8292, PB8316, PB8340, PB8364, PB8388, PB8511, PB8535, PB8583, PB8607, and PB8640.

[0510] In the N87 EGF assay, 11 different cMETxEGFR bispecific antibodies were identified with comparable potency to the monospecific cetuximab: PB7679, PB8244, PB8292, PB8340, PB8364, PB8388, PB8511, PB8535, PB8583, PB8607, and PB8640. They all contain the EGFR Fab arm MF3755. In the HGF N87 assay, 9 bispecific antibodies were identified with higher potency than the monospecific 5D5 Fab reference antibody: PB8218, PB8388, PB8511, PB8532, PB8535, PB8545, PB8583, PB8639, and PB8640.

[0511] They contain six different cMET Fab arms: MF4040, MF4297, MF4301, MF4356, MF4491 and MF4506.

[0512] ADCC activity

[0513] ADCC activity of 24 cMetxEGFR bispecific antibodies was tested against tumor cell lines N87 (EGFR-high, cMET-low) and MKN-45 (EGFR-low, cMET-amplified). ADCC assays were performed in 384-well plates using the Promega ADCC bioassay kit. Antibodies were tested in duplicate at 9 different concentrations of half-log serial dilutions ranging from 10 μg / ml to 1 ng / ml.

[0514] Reference cetuximab antibody was included as a positive control for the assay, and PG2708 was used as a negative control antibody. Antibody or assay medium controls (no IgG) were incubated with ADCC effector cells and target cells (N87 or MKN-45) and induced for 6 hours at 37°C. Luciferase activity was quantified using the Bio-Glo luciferase reagent.

[0515] The cMETxEGFR bispecific antibody did not show significant ADCC activity in either cell line. The positive control reference cetuximab antibody showed dose-dependent ADCC activity against both cell lines.

[0516] Five bispecific antibodies consisting of EGFR and cMet arms, which showed high efficacy in the N87 HGF / EGF assay and showed high sequence diversity (Table 5), were selected for further analysis. Two of the five bispecific antibodies contained MF4356, which competes with 5D5 for binding to cMET (Table 2). Table 5 summarizes the characteristics of the selected candidates.

[0517] Example 4

[0518] Figure 2 Shown are respective sequences of another variable region of the heavy chain of the EGFR binding variable domain as disclosed herein. Figure 3 The sequences of the other variable regions of the heavy chains of the cMET binding variable domains disclosed herein are shown. The heavy chain variable regions were used to create a number of different cMET x EGFR bispecific antibodies. The light chains in these antibodies have Figure 4B The bispecific antibodies were made as described in Example 1. The antibodies were also made in an ADCC-enhanced form. The ADCC-enhanced form was made by adding DNA encoding a reductase that removes fucose residues from the Fc region of IgG1 during co-transfection of the antibody construct. See Table 6 for a list of the bispecific antibodies used and their PB codes.

[0519] Example 5

[0520] The heavy chain variable region (VH) of the cMET variable domain of PB8532 comprises the amino acids of MF4356, such as Figure 3 The VH of the cMET variable domain of PB19748 contains the amino acid sequence of MF8230 (see Figure 3 ). The VH of the EGFR variable domain of PB8532 comprises the amino acids of MF3370, such as Figure 2 The VH of the EGFR variable domain of PB19748 comprises Figure 2The amino acid sequence of MF8233 is shown in FIG. The light chains in PB8532 and PB19748 are identical and Figure 4B cMET antibody LY2875358 antibody is described in Kim and Kim 2017.

[0521] Example 6

[0522] Efficiency of the bispecific antibody PB19478 in the EGFR exon 20 insertion model

[0523] The purpose of this trial is to evaluate the anti-tumor efficacy of PB19478 in a patient-derived xenograft (PDX) non-small cell lung cancer (NSCLC) model with EGFR exon 20 insertions. EGFR exon 20 insertions ("EGFRex20ins") represent a class of coding mutants with amino acid insertion sites concentrated between positions 762 and 774 that result in constitutive activation of EGFR. EGFR exon 20 insertions confer resistance to approved EGFR TKIs in human individuals with cancers carrying such mutations and are associated with poor prognosis.

[0524] Material

[0525] PB19748 is produced by Merus and the control material is PB19748 vehicle, which consists of 12% PB19748 formulation buffer without antibody, although other negative controls such as saline or PBS may also be used.

[0526] PDX Model Characteristics

[0527] The LXFE2478 model was generated at Charles River in nude mice with functional Fc effector cells.

[0528] This model carries the mutant EGFRex20ins (M766_A767insASV). It also carries a point mutation (E168D) in the SEMA domain of c-MET, which is located in the ligand binding site of c-MET.

[0529] The insertion of 9 nucleotides in exon 20 of this model affects the EGFR tyrosine kinase domain (M766X) and confers resistance to small molecule EGFR inhibitors. This model does not carry mutations in the B-RAF, H- / N-, KRAS, and PTEN genes (whole exome sequencing of patient tumors and transplanted tumors was performed, and the results were consistent).

[0530] Expression of EGFR, c-MET, and HGF in LXFE2478 PDX Model

[0531] The expression of EGFR and c-MET receptors and the ligand huHGF in the model LXFE2478 was analyzed to investigate its suitability for in vivo experiments.

[0532] The expression of EGFR and c-MET receptors and the ligand huHGF was evaluated by western blot in both tumor masses. huHGF was included in the analysis. Tumor samples obtained from PDX models were evaluated by Simple Western Size technology (SWS) to confirm the expression of EGFR, HGF, and c-MET.

[0533] Western blotting showed that the LXFE2478 model expressed EGFR, HGF, and MET at the protein level. Figure 6 and Table 7).

[0534] Statistical methods:

[0535] The antitumor efficacy of all groups was evaluated using the control vehicle / placebo buffer group as a reference. Tumor growth inhibition was determined by comparing the RTV of the test group with the control group and expressed as the minimum T / C percentage value. In order to evaluate the statistical significance of the antitumor efficacy, a nonparametric Kruskal-Wallis test was performed, followed by the Dunn method for multiple comparisons. The RTV of the test group and the control group was compared on the day when the minimum T / C value was reached in the test group. Statistical analysis was performed only when at least 50% of the initially randomly assigned animals remained in the relevant groups. Comparisons between the test groups were performed on the same day. All p values ​​< 0.05 were considered statistically significant. Statistical calculations were performed using GraphPad Prism Bioanalysis Software (Version 9.10, for Microsoft Windows, GraphPad Software, San Diego, California, USA).

[0536] Treatment duration and methods:

[0537] Figure 7 Schematic diagram showing the time course of treatment.

[0538] Tumors were harvested from donor PDX mice, cut into fragments (LXFE2478: 3-4 mm edge length), and inoculated subcutaneously (SC) into recipient nude mice in the flank. When tumor implants reached approximately 80-200 mm in a sufficient number of animals, 3 At 1 hr, mice were divided into 5 groups with 5 mice in each group. Randomization was performed based on the "stratified distribution" method. Treatment started on the same day of randomization (day 0).

[0539] The antibody was administered intraperitoneally (IP) once a week at doses of 0.5 mg / kg, 2.5 mg / kg, 8 mg / kg and 25 mg / kg for 5 weeks. Mice in Group 1 were treated with Vehicle 1 once a week for 5 weeks and once a day for 30 days. The treatment schedule is shown in Table 8.

[0540] After randomization, animals were routinely monitored for morbidity and mortality, weighed twice weekly, and tumor volume (TV) was measured twice weekly with calipers. Relative body weight (RBW) was calculated by dividing the absolute weight or volume on a given day by the absolute weight on day 0 multiplied by 100. Relative tumor volume (RTV) was calculated by dividing the absolute individual tumor volume on a given day by the absolute tumor volume on day 0 multiplied by 100.

[0541] For mice with >10% body weight loss, body weight was measured daily and animals had easy access to feed and water and DietGel. For mice with >15% body weight loss, treatment was withheld until their RBW recovered ≥90%.

[0542] Monotherapy with the bispecific antibody PB19478 showed dose-dependent antitumor efficacy ( Figure 8 ). Monotherapy with the antibody dose of 25 mg / kg resulted in tumor remission (minimum T / C value 2.7%) and was significantly more effective than treatment with vehicle and treatment with 0.5 mg / kg or 2.5 mg / kg doses. Treatment with 0.5 mg / kg and 2.5 mg / kg doses showed partial tumor regression.

[0543] Monotherapy was associated with low toxicity in mice, with negligible weight loss and an adjusted survival of 100% for all groups.

[0544] Cancers with EGFR exon 20ins-mutations are often refractory to EGFR tyrosine kinase inhibitors and are associated with a poor prognosis.

[0545] Example 7

[0546] Efficacy of the bispecific antibody PB19478 in an EGFR exon 19 deleted NSCLC model. The aim of this study was to evaluate the antitumor efficacy of the antibody PB19478 in a cell line-derived xenograft (CDX) non-small cell lung cancer (NSCLC) model with EGFR exon 19 deletion. This adenocarcinoma cell line, HCC827-ER1, carries an activating EGFR mutation in exon 19 (deletion E746-A750) and is resistant to approved EGFR TKIs such as erlotinib.

[0547] Material:

[0548] PB19748 was produced from Merus as described in Example 5.

[0549] CDX Model Features:

[0550] The CC827-ER1 model was generated in BALB / c nude mice. It carries an EGFR mutation in exon 19 (E746-A750 deletion) and has amplified c-MET copy number and Axl expression compared to the wild-type HCC827 cell line. The HCC827-ER1 cell line is resistant to the EGFR TKI erlotinib and was generated by repeated exposure of the wild-type cell line to increasing concentrations of erlotinib in vitro.

[0551] Experimental process:

[0552] Each mouse was inoculated with a mixture of HCC827-ER1 tumor cells and Matrigel in the right anterior flank region for tumor development. When the average tumor size reached approximately 125 (75-175) mm 3 Randomization began at 1:00 p.m. A total of 16 tumor-bearing mice were included in the tumor efficacy trial and randomly divided into 2 different groups, as shown in Table 9, with 8 mice in each group. The day of randomization is denoted as day 0, and dosing began on day 0. Mice were treated with the antibody for 3 weeks, followed by a dose-free observation period of up to 74 days. The dose-free observation period was included to compare the response period and recurrence time (tumor regrowth) after the end of treatment. Tumor volume exceeds 1500mm 3 The treatment period was terminated for mice that lost more than 20% of their body weight relative to the first day of treatment. The antibody was administered intraperitoneally (ip) twice a week for 21 days at a dose of 25 mg / kg for a total of 7 doses. The eight mice in Group 1 were treated with vehicle control (placebo buffer). A schematic overview of the treatment plan is shown in Fig. 9 Tumor volume (mm) was measured twice a week in two dimensions using a caliper. 3 Animals were examined for tumor growth and any effects of treatment on behavior, such as activity, food and water consumption, weight gain / loss, eye / hair tangling, and any other abnormalities. Body weights were measured twice a week after randomization. No mouse lost more than 15% of its body weight, and no treatment was suspended in any treatment group. Fig.10The effect of the bispecific antibody PB19478 on tumor volume in the NSCLC CDX model was shown throughout the observation period, with treatment cessation indicated at day 21. Antibody treatment showed tumor regression compared to the vehicle control group. All treatment groups were well tolerated. No mouse in the trial showed a body weight loss of >10% from the starting body weight. In fact, on day 21, the last treatment day, there was no statistically significant difference in absolute body weight between Group 1 and Group 2. The absolute body weight of mice in the further treatment group (i.e., 25 mg / kg Osimertinib, administered orally once a day) was lower compared to Groups 1 and 2 (P=0.0167 compared to Group 2, data not shown). No adverse events were observed except for tumor scabs or tumor ulcers in some mice in Groups 1 and 2. Animal survival curves are shown in Figure 2. Fig.11 The log-rank (Mantel-Cox) test significance of the combination versus each corresponding single treatment group was as follows: antibody 25 mg / kg vs. vehicle P=0.0001.

[0553] In summary, these results show preclinical antitumor activity of the investigational antibody in NSCLC CDX models carrying exon 19 mutations (E746-A750 deletions).

[0554] Example 8

[0555] In the dose-escalation phase, the bispecific antibody PB19478 will be administered at increasing doses to NSCLC patients harboring activating EGFR mutations (TKI-sensitizing mutations and / or approved TKI-resistance mutations) or activating c-MET mutations (exon 14 skipping) / amplification (MET / CEP7>5 or cfDNA≥2 copies), GC / GEJ adenocarcinoma patients with activating EGFR amplification (EGFR / CEP7≥2 or cfDNA≥8 copies) or c-MET amplification (MET / CEP7>5 or cfDNA≥2 copies), and patients with HNSCC or ESCC, in all cases, who have progressed after receiving prior treatment for advanced / metastatic disease.

[0556] Allometric scaling of the preclinical PK model was used to predict antibody exposure in humans. The starting dose of the antibody was 100 mg (fixed dose, intravenous), once every 2 weeks (q2w), with a cycle of 4 weeks (28 days). Five dose levels are planned to be studied between 100-3000 mg.

[0557] The patient cohort will receive the antibody treatment until the MTD is reached or a lower recommended dose is determined.

[0558] Taking into account the available data on PK, pharmacodynamic activity, and preliminary antitumor activity, the RP2D (recommended phase 2 dose) was defined as a dose equal to or lower than the MTD.

[0559] Dose expansion

[0560] With the RP2D of 1500 mg, expansion cohorts in the Phase 2 program can be initiated. Safety at the RP2D will be confirmed during dose expansion in the first 12 patients treated with at least 2 cycles of antibody alone (recruitment will continue in the meantime). The anti-tumor activity of the antibody (alone or in combination with osimertinib) will be evaluated based on ORR, and other efficacy parameters, safety, tolerability, PK, immunogenicity, and biomarkers will be evaluated.

[0561] The following locally advanced unresectable / metastatic solid tumor groups are open:

[0562] Group A: PB19478 monotherapy: NSCLC patients may carry EGFR exon 20 insertion mutations (first-line [1L] and ≥ second-line [2L] worsened after first-line chemotherapy). Results from NSCLC patients are described in Example 9.

[0563] Arm B: PB19478 monotherapy: NSCLC patients may harbor cMet exon 14 mutations in ≥2L, capmatinib- and tepotinib-resistant populations, who have progressed after approved c-Met TKI inhibitors.

[0564] Group C: PB19478 monotherapy: GC / GEJ and HNSCC patients who may carry EGFR or cMet driver mutations. Results from patients with HNSCC are described in Example 10.

[0565] Experimental group

[0566] Inclusion criteria

[0567] Patients must meet all of the following requirements to enter the study:

[0568] 1. Sign the informed consent form before starting any trial procedures.

[0569] 2. Aged ≥ 18 years when signing the informed consent form.

[0570] 3. Histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced unresectable disease that is incurable.

[0571] 1. Dose escalation part – patients who have failed previous standard first-line therapy. Patients must have deteriorated on or been intolerant to therapy known to provide clinical benefit. There is no restriction on the number of prior treatment regimens. Patients must have:

[0572] Non-small cell lung cancer (NSCLC) harboring activating EGFR mutations, including tyrosine kinase inhibitor (TKI)-sensitizing mutations (e.g., 19del and L858R) and / or approved TKI-resistance mutations (e.g., acquired TKI-resistance mutations, i.e., T790M, C797S, L792, L798I, exon 20 insertions), or any activating c-MET mutation / amplification (e.g., high-level c-MET amplification [MET / CEP7>5 or cfDNA ≥2 copies], or c-MET exon 4 skipping mutation).

[0573] *Note: Patient identification will be based on prior treatment history with EGFR tyrosine kinase inhibitors and topical testing performed in a CLIA-certified laboratory.

[0574] 2. Cohort Expansion - For patients with ≥2L disease, they must have deteriorated on or been intolerant to therapy known to provide clinical benefit. There is no limit on the number of prior treatment regimens. Patients must have:

[0575] Group A: NSCLC harboring EGFR exon 20 insertions that have progressed after platinum-based doublet combination therapy (may include a limited number of first-line patients).

[0576] Group B: NSCLC patients with cMet exon 14 skipping mutation ≥ 2L, resistant to capmatinib and tepotinib, or treated with cMET TKI or not treated with cMET TKI.

[0577] Group C: Selected solid tumors (including GC / GEJ, HNSCC such as HNSCC refractory to approved therapy (regardless of driver mutation), or ESCC) carrying EGFR or cMet driver mutations or cMET alterations, NSCLC patients with uncommon EGFR mutations (G719X, L861Q, and S768I) not included in other groups. Patients are exhausted or intolerant of all approved therapies with demonstrated clinical benefit, or have no available, approved treatment options.

[0578] Group F: NSCLC with c-MET exon 14 skipping mutations and untreated advanced disease.

[0579] 4. FFPE-embedded archived or fresh tumor tissue samples are available after recent treatment progression.

[0580] 5. With measurable disease as defined by RECIST version 1.1 radiologically (patients with non-measurable but evaluable disease may be included in the dose escalation portion).

[0581] 6. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0582] 7. Have a life expectancy ≥ 12 weeks, as judged by the researchers.

[0583] 8. Have adequate organ function, as determined by the researcher:

[0584] Absolute neutrophil count (ANC) ≥ 1.5X 10 9 / L

[0585] Hemoglobin ≥ 9 g / dL

[0586] ·Platelets ≥100x 10 9 / L

[0587] Correct total serum calcium within normal range

[0588] Serum magnesium within normal range (or corrected with supplementation)

[0589] Serum potassium within normal range

[0590] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 3x upper limit of normal (ULN), and total bilirubin ≤ 1.5x ULN (for patients with Gilbert's syndrome, conjugated bilirubin values ​​are within the normal range, they are eligible); in the case of liver impairment, ALT / AST ≤ 5x ULN and total bilirubin ≤ 2x ULN will be allowed.

[0591] For patients aged 65 years and older, serum creatinine ≤1.5x ULN or creatinine clearance ≥50 mL / min calculated by Cockroft and Gault formula or MDRD formula

[0592] Serum albumin > 3.3 g / dL

[0593] Exclusion criteria

[0594] The presence of any of the following criteria excluded the patient from participating in the study:

[0595] 1. There is central nervous system metastasis (not excluded when increasing, mandatory when expanding):

[0596] Untreated and symptomatic (patients with untreated, asymptomatic disease may be included if the investigator judges the disease to be stable)

[0597] Need radiation therapy or surgery.

[0598] • Requires continued steroid treatment (>10 mg prednisone or equivalent) to control symptoms within 14 days of trial entry.

[0599] 2. With known leptomeningeal damage.

[0600] 3. Participated in another clinical trial or received any investigational drug treatment within 4 weeks prior to entering the trial.

[0601] 4. Systemic anticancer therapy or immunotherapy within 4 weeks or 5 half-lives (whichever is shorter) after the first dose of the investigational drug. For cytotoxic agents with major delayed toxicity (e.g., mitomycin C, nitrosoureas), a 6-week washout period is required. Note: For agents with longer half-lives, inclusion before the fifth half-life requires sponsor approval.

[0602] 5. Underwent major surgery or radiation therapy within 3 weeks of the first dose of trial drug. Individuals who received prior radiation therapy to ≥25% of the bone marrow at any time are not eligible.

[0603] 6. Persistent clinically significant toxicity of grade >1 as judged by the investigator, related to prior antineoplastic therapy (except alopecia); stable sensory neuropathy ≤ grade 2 NCI-CTCAE v5.0 and hypothyroidism ≤ grade 2, which is stable on hormone replacement therapy.

[0604] 7. A history of allergic reaction or any toxicity attributable to human proteins or any excipients requiring permanent discontinuation of these agents.

[0605] 8. History of clinically significant cardiovascular disease, including but not limited to:

[0606] QT prolongation >480 msec, obtained from 3 electrocardiograms (ECGs), or clinically significant arrhythmias or electrophysiological disease (i.e., implantable cardioverter-defibrillator or uncontrolled atrial fibrillation), or any factor that increases the risk of QTc prolongation or arrhythmic events (e.g., electrolyte abnormalities). Clinically stable patients with pacemakers are eligible.

[0607] Heart failure, congenital long QT syndrome, family history of long QT syndrome, unexplained sudden death under 40 years of age in a first-degree relative, or any concomitant medication known to prolong the QT interval and cause polymorphic ventricular tachycardia (Torsades de Pointes).

[0608] Uncontrolled (persistent) arterial hypertension: systolic blood pressure >180 mm Hg and / or diastolic blood pressure >100 mm Hg.

[0609] Congestive heart failure (CHF) was defined as New York Heart Association (NYHA) class III-IV or hospitalization for CHF within 6 months after the first dose of trial drug.

[0610] 9. History of interstitial lung disease, including drug-induced interstitial lung disease and radiation pneumonitis, which requires long-term treatment with steroids or other immunosuppressants within 1 year.

[0611] 10. Patients with previous or concurrent malignant tumors, excluding non-basal cell skin cancer or carcinoma in situ of the cervix, unless the tumor is treated for the purpose of cure or palliation, and the investigator believes, with the consent of the sponsor, that the previous or concurrent malignant tumor does not affect the safety and efficacy evaluation of the investigational drug.

[0612] 11. Currently suffering from a serious illness or medical condition, including but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic or psychiatric disease.

[0613] 12. Have active hepatitis B infection (HBsAg positive) not receiving antiviral treatment. Note: Individuals with active hepatitis B (HbsAg positive) must be receiving antiviral treatment with lamivudine, tenofovir, entecavir, or other antiviral drugs, starting at least ≥7 days before starting study treatment. Individuals with a history of hepatitis B (anti-HBc positive, HbsAg and HBV-DNA negative) are eligible.

[0614] 13. Positive hepatitis C ribonucleic acid (HCV RNA) test; Note: Patients with spontaneous resolution of HCV infection (positive HCV antibodies but undetectable HCV-RNA), or who achieve sustained virological response after antiviral treatment and show no detectable HCV RNA for ≥6 months (using IFN-free prescriptions), or ≥12 months after stopping antiviral treatment (using IFN-based prescriptions) are eligible.

[0615] 14. Have a known history of HIV (HIV 1 / 2 antibodies). HIV patients with undetectable viral load are allowed. HIV testing is not required unless mandated by local health authorities or regulations.

[0616] 15. Sexually active male and female patients of reproductive potential must agree to use one of the following highly effective contraceptive methods throughout the trial and for 6 months after the final administration of PB19478:

[0617] Combination (estrogen and progestin) hormonal contraceptives (oral, vaginal, transdermal) associated with ovulation inhibition

[0618] Progestin-only hormonal contraceptives (oral, injectable, implantable) associated with ovulation inhibition

[0619] Intrauterine device (IUD)

[0620] Intrauterine hormone-releasing system (IUS)

[0621] Bilateral fallopian tube obstruction

[0622] Partner vasectomy 1

[0623] Abstinence 2

[0624] 16. Pregnant or breastfeeding women are excluded from this trial.

[0625] The ECOG performance status score levels are defined as follows in accordance with the definition in the field, and the meaning is: Grade 0: Fully active, able to perform all pre-disease performance without restriction. Grade 1: Limited physical activity, but can walk and can perform light or sedentary work, such as simple housework and office work. Grade 2: Can walk and take care of oneself, but cannot perform any work activities. Up to and about more than 50% of waking time. Grade 3: Can only perform limited self-care; more than 50% of waking time is confined to bed or chair. Grade 4: Total disability; unable to perform any self-care; completely confined to bed or chair. Grade 5: Death.

[0626] Research treatment and prescription

[0627] The antibody is administered by IV infusion at a dose level of 1500 mg (fixed dose) at the RP2D level, once every 2 weeks (q2w), with a cycle of 4 weeks. Dose levels between 100-3000 mg (fixed dose) can be explored. Once the RP2D is reached, dose escalation will be stopped. The dose, dose increment, and dosing frequency for each patient (including the expansion group) may change based on patient safety, PK and pharmacodynamic data, and the sponsor's advice. The sponsor may recommend an alternative weekly dosing schedule for cycle 1.

[0628] During treatment

[0629] Trial treatment will be administered until confirmed disease progression (per RECIST v1.1), unacceptable toxicity, withdrawal of consent, patient noncompliance, investigator decision (e.g., clinical worsening), or antibody discontinuation for >6 consecutive weeks.

[0630] Patients will be followed for safety for at least 30 days after the last antibody infusion, until all relevant toxicities have resolved or stabilized, and for disease progression and survival every 3 months for up to 1 year.

[0631] Example 9

[0632] In the clinical trial of Example 8, clinical efficacy was observed in patients with non-small cell lung cancer with EGFR mutations, including EGFR exon 20 mutations / insertions, EGFR exon 21 mutations, such as L858R and EGFR exon 19 deletion mutations; and c-MET mutations, including c-MET exon 14 skipping mutations. In more detail, a reduction in target lesions was observed in several patients, including confirmed partial responses, two cases of disease stabilization and lesion reduction (including one patient with a c-MET exon 14 mutation), one patient with an exon 19 mutation, and one patient with an EGFR amplification mutation. Patients had previously received a variety of prior treatments, including platinum chemotherapy, osimertinib, pembrolizumab, nivolumab, cetuximab, crizotinib, capmatinib, or tepotinib.

[0633] Example 10

[0634] In the clinical trial of Example 8, clinical efficacy was observed in a 68-year-old female patient with head and neck squamous cell carcinoma (HNSCC) who had previously been treated with cisplatin and cemiplimab. In more detail, a 20% reduction in target lesions was observed in the patient who experienced only grade 1 / 2 adverse events.

[0635] Embodiment 11

[0636] In the clinical trial of Example 8, clinically relevant efficacy was observed in many patients with non-small cell lung cancer carrying cMET exon 14 skipping mutations after administration of the bispecific antibody PB19478 at 1500 mg Q2W. Clinically relevant efficacy included confirmed partial responses (according to investigator review according to RECIST criteria 1.1) and stable disease. Several patients had previously received anticancer treatment, while several other patients had not received treatment, which means that they received the bispecific antibody of the clinical trial protocol of Example 8 as the first anticancer treatment for cMET exon 14 skipping NSCLC.

[0637] Example 12

[0638] In the clinical trial of Example 8, clinically relevant efficacy was observed in many patients with non-small cell lung cancer carrying EGFR exon 20 insertion mutations after administration of bispecific antibody PB19478 at 1500 mg Q2W. Clinically relevant efficacy included confirmed partial responses (according to investigator review according to RECIST criteria 1.1) and stable disease. Several patients had previously received anticancer treatment, such as platinum chemotherapy.

[0639] Citations

[0640] Ahmed, M et al., Lack of in Vivo Antibody Dependent Cellular Cytotoxicity with Antibody containing gold particles / Bioconjugate chemistry(2015):26 812-816.DOI:10.1021 / acs.bioconjchem.5b00139.

[0641] Castoldi, R. et al., "A novel bispecific EGFR / Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and haspotent antitumor activity." Oncogene 32.50(2013):5593-5601.

[0642] Eisenhauer et al., New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1). European Journal of Cancer 45 (2009) 228-247)

[0643] Ferguson KM. Structure-based view of epidermal growth factor receptor regulation. Annu Rev Biophys 2008;37:353–73.

[0644] Kim, George P. and Axel Grothey. "Targeting colorectal cancer with human anti-EGFR monoclonal antibodies: focus on panitumumab." Biologics 2.2 (2008): 223-228.

[0645] Kim, Ki-Hyun and Kim, Hyori. Progress of antibody-based inhibitors of the HGF-cMET axis in cancer therapy. Experimental & Molecular Medicine (2017), e307; doi:10.1038 / emm.2017.17)

[0646] Moores, Sheri L. et al., "A novel bispecific antibody targeting EGFR and cMet is effective against EGFR inhibitor–resistant lung tumors." Cancer research 76.13 (2016): 3942-3953.

[0647] Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, Carbone PP. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982 Dec; 5(6): 649-655. PMID: 7165009.

[0648] Robertson SC, Tynan J, Donoghue DJ. RTK mutations and human syndromes: when good receptors turn bad. Trends Genet 2000; 16: 368.

[0649] Yarden Y. The EGFR family and its ligands in human cancer. Signalling mechanisms and therapeutic opportunities. Eur J Cancer 2001;37(Suppl 4):S3–S8.

[0650] Table 1. Reference antibodies with reported specificity against the cMET extracellular domain.

[0651]

[0652]

[0653] Table 2. Competition of cMet reference antibodies with cMET cLC antibodies. Shown are OD450 values. OD450 values ​​indicate the presence or absence of competition with the antibody. MF4506 was not tested.

[0654]

[0655] Table 3. List of 24 cMETxEGFR bispecific antibodies selected after dose-dependent titration experiments in N87 HGF / EGF proliferation assay. The MF numbers of the EGFR and cMET arms in each individual PB and their HCDR3 sequences are shown.

[0656]

[0657] Table 4. Summary of antibody titration experiments performed with 24 cMETxEGFR bispecific antibodies using the N87 HGF / EGF, HGF and EGF proliferation assays. The bispecifics are labeled PBXXXX and the different Fab arms are labeled MGXXXX. The activity of the bispecific antibodies in each assay is represented as: - no effect; + proliferation inhibition is lower than the positive control; ++ = proliferation inhibition is comparable to the positive control antibody 5D5 Fab; +++ = proliferation inhibition is higher than the positive control antibody 5D5 Fab.

[0658]

[0659] Table 5. Composition of the most potent EGFRxcMET bispecific antibodies and their competition with reference antibodies.

[0660]

[0661] Table 6. Composition of bispecific antibodies. The pXX numbers represent the number of the production run and can be used to identify whether the antibody was produced as an ADCC version.

[0662] Bispecific Antibodies cME Arm EGFR arm ADCC Enhancement Cetuximab - - - PB8532p05 MF4356 MF3370 no PB19474p01 MF4356 MF8232 yes PB19475p01 MF4356 MF8233 yes PB19476p01 MF8230 MF3370 yes PB19477p01 MF8230 MF8232 yes PB19478p01 MF8230 MF8233 yes PB8532p06 MF4356 MF3370 yes PB8532p04 MF4356 MF3370 no HER-3 arm EGFR arm PB4522p34 MF3178 MF4280 yes PB4522p25 MF3178 MF4280 no TT arm TT arm PG1337p218 MF1337 MF1337 no

[0663] Table 7: Relevant properties of model LXFE2478. AUC = Area Under the Curve.

[0664]

[0665] Table 8 Treatment plan for PDX model LXFE2478.

[0666]

[0667] Table 9 | Treatment schedule for CDX model HCC827-ER1. BIW = twice weekly, ip = intraperitoneal.

[0668]

Claims

1. A bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYX1X2NTNYAQKLQG and a CDR3 comprising a sequence of X3X4X5X6HWWLX7A, wherein X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G; and having 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof at positions other than X1 to X7, and wherein the second variable domain comprises a heavy chain variable region having a sequence of SEQ ID The amino acid sequence of one of the NO:1-23 sequences produces an amino acid sequence with 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof, and the bispecific antibody is for use in a method of treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor or ii) chemotherapy and a tyrosine kinase inhibitor or iii) a cMET tyrosine kinase inhibitor.

2. A method for treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor or ii) chemotherapy and an EGFR tyrosine kinase inhibitor or iii) a cMET tyrosine kinase inhibitor, the treatment comprising administering to the individual an effective amount of a bispecific antibody comprising a first variable domain that binds to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region , which has a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, wherein X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G; and has 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than X1-X7, and wherein the second variable domain comprises a heavy chain variable region, the heavy chain variable region of the second variable domain having an amino acid sequence of one of the sequences of SEQ ID NO:1-23 resulting in an amino acid sequence of 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

3. Use of a bispecific antibody in the preparation of a medicament for treating cancer in an individual who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor or ii) chemotherapy and an EGFR tyrosine kinase inhibitor or iii) a cMET tyrosine kinase inhibitor, the bispecific antibody comprising a first variable domain that binds to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region, It has a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, wherein X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G; and there are 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than X1-X7, and wherein the second variable domain comprises a heavy chain variable region, the heavy chain variable region of the second variable domain having an amino acid sequence of one of the sequences of SEQ ID NO:1-23 resulting in an amino acid sequence of 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

4. The use or method of any one of the preceding claims, wherein the individual or cancer having received a previous treatment according to i) is resistant to treatment with a third generation EGFR tyrosine kinase inhibitor.

5. The use or method of any one of claims 1 to 3, wherein the individual or cancer that has received a previous treatment according to ii) is resistant to treatment with a first, second and / or third generation tyrosine kinase inhibitor.

6. The use or method of any one of claims 1 to 3, wherein the individual or cancer having received a previous treatment according to iii) is resistant to treatment with a cMET tyrosine kinase inhibitor.

7. The use or method of any of the preceding claims, wherein the administration of the bispecific antibody and the EGFR tyrosine kinase inhibitor according to i) and iii) is administered as a second line of treatment and the administration according to ii) is administered as a third line of treatment.

8. The use or method of any one of the preceding claims, wherein the first generation EGFR tyrosine kinase inhibitor comprises gefitinib, erlotinib or icotinib.

9. The use or method of any one of the preceding claims, wherein the second generation EGFR tyrosine kinase inhibitor comprises afatinib, dacomitinib, XL647, AP26113, CO-1686 or neratinib.

10. The use or method of any of the preceding claims, wherein the third generation EGFR tyrosine kinase inhibitor comprises Osimertinib, Lazertinib, Alflutinib, Rezivertinib, Rociletinib, Olmutinib, Almonertinib, Abivertinib, ASK120067, Befotertinib, or SH-1028, Nazartinib (EGF816), Naquotinib (ASP8273), Mavelertinib (PF-0647775), Olafertinib (CK-101), Keynatinib, ES-072, preferably Osimertinib.

11. The use or method of any of the preceding claims, wherein the cMET tyrosine kinase inhibitor is or comprises capmatinib, tepotinib, crizotenib, cabozantinib, savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833.

12. The use or method of any of the preceding claims, wherein the chemotherapy comprises platinum chemotherapy, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide or pemetrexed, or any combination thereof, preferably a composition comprising cisplatin or carboplatin.

13. The use or method of any one of the preceding claims, wherein the cancer is an EGFR-positive and / or cMET-positive cancer.

14. The use or method of any one of the preceding claims, wherein the cancer comprises EGFR and / or cMET aberrations.

15. The use or method of any of the preceding claims, wherein the cancer comprises an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation, a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, EGFR gene amplification, a cMET mutation, or a cMET aberration.

16. The use or method of any one of the preceding claims, wherein the cancer comprises an exon 19 deletion mutation, preferably an in-frame exon 19 deletion, an exon 20 missense mutation or an exon 21 mutation.

17. The use or method of any preceding claim, wherein the cancer comprises an EGFR exon 20 mutation, preferably an exon 20 insertion mutation.

18. The use or method of any preceding claim, wherein the cancer comprises an acquired tyrosine kinase inhibitor resistance mutation, such as a mutation that confers resistance to Osimertinib.

19. The use or method of any of the preceding claims, wherein the cancer comprises an exon 20 mutation selected from: near-loop insertion (positions 767-772), far-loop insertion (positions 773-775), preferably V769_D770insASV, D770_N771insSVD, H773_V774insNPH, H773_V774insH, D770_N771insG, D770delinsGY, N771_P772insN, V773_V774insNPH, 74_C775insHV、D770_N771insGL、H773_V774insPH、A763_Y764insFQEA、D770_N771delinsEGN、D770_N771insGD , D770_N771insH, D770_N771insP, H773_V774insAH, H773_V774insGNPH, H773delinsSNPY, N771_P772insH, N77 1_P772insVDN, N771delinsGY, N771delinsKH, N771delinsRD, P772_H773delinsHNPY, P772_H773insGT, P772_H773insPNP, P772_H773insT, V769_D770insA, V769_D770insGG, V769_D770insGSV, V769_D770insGVV, and V769_D77 0insMASV; or mutations T790M, L792X (e.g., L792H, C796X (e.g., G796R, G796S, G796D), C797X (e.g., C797S, C797G), L798I, or in-frame exon 20 insertions such as M766_A767insASV or H773-V774insNPH, Ins761(EAFQ), Ins770(ASV), Ins771(G), Ins774(NPH), M766_A7671ns A. S768_V769InsSVA, P772_H773InsNS, D761_E762InsX1-7, A763_Y764InsX1-7, Y764_Y765 InsX1-7, M766_A767InsX1-7, A767_V768 InsX1-7, S768_V769 InsX1-7>V769_D770 InsX1-7>D770_N771 InsX1-7>N771_P772 InsX1-7>P772_H773 InsX1-7, H773_V774 InsX1-7, or V774_C775 InsX1-7.

20. The use or method of any of the preceding claims, wherein the cancer comprises a cMET aberration, such as cMET amplification, cMET overexpression, increased signaling of the cMET pathway, cMET gene amplification, increased HGF expression, and / or increased cMET protein activity.

21. The use or method of any one of the preceding claims, wherein the cancer comprises a cMET exon 14 skipping mutation.

22. The use or method of any one of the preceding claims, wherein the cancer is non-small cell lung cancer (NSCLC), head and neck cancer, gastric cancer, esophageal cancer, gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer, or bladder cancer.

23. The use or method of any preceding claim, wherein the cancer is an advanced or metastatic cancer.

24. The use or method of any preceding claim, wherein the cancer or patient has progressed following prior treatment for the advanced or metastatic cancer.

25. The use or method of any of the preceding claims, wherein the treatment comprises a diagnostic step for assessing whether the cancer is an EGFR-positive and / or cMET-positive cancer, or assessing the presence of EGFR and / or cMET aberrations in the cancer.

26. The use or method of any preceding claim, wherein the subject is a human subject.

27. The use or method of any preceding claim, wherein the antibody is a human antibody.

28. The use or method of any preceding claim, wherein the antibody is an ADCC enhancing antibody.

29. The use or method of any preceding claim, wherein the antibody is in the IgG1 format with a stoichiometric ratio of anti-EGFR to anti-cMET of 1:

1.

30. The use or method of any preceding claim, wherein the antibody has one variable domain that binds EGFR and one variable domain that binds cMET.

31. The use or method of any of the preceding claims, wherein the variable domain that binds to human EGFR also binds to cynomolgus monkey and mouse EGFR.

32. The use or method of any of the preceding claims, wherein the variable domain that binds to human EGFR binds to domain III of human EGFR.

33. The use or method of any of the preceding claims, wherein the variable domain that binds to cMET blocks the binding of antibody 5D5 to cMET.

34. The use or method of any of the preceding claims, wherein the variable domain that binds to cMET blocks the binding of HGF to cMET.

35. The use or method of any preceding claim, wherein the amino acids at positions 405 and 409 in one CH3 domain are identical to the amino acids at the corresponding positions in the other CH3 domain (EU numbering).

36. The use or method as claimed in any one of the preceding claims, wherein X1=N; X2=G; X3=D; X4=S; X1=N; X2=A; X3=D; X4=S; X1=S; X2=G; X3=D; X4=S; X1=N; X2=G; X3=D; X4=R; X1=N; X2=A; X3=D; X4=R; X1=S; X2=G; X3=D; X4=R; X1=N; X2=G; X3=G; X4=Y; X1=N;X2=A;X3=G;X4=Y;X5=L;X6=D and X7=G;or X1=S; X2=G; X3=G; X4=Y; X5=L; X6=D and X7=G.

37. The use or method of any of the preceding claims, wherein X1 = N; X2 = G; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D; or X1 = N; X2 = A; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D; or X1 = S; X2 = G; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D.

38. The use or method of any of the preceding claims, wherein X1 = N; X2 = G; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D; or X1 = N; X2 = A; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D.

39. The use or method of any of the preceding claims, wherein the heavy chain variable region of the second variable domain comprises an amino acid sequence of one of the sequences of SEQ ID NO: 1-3; 7; 8; 10; 13; 15; 16; 17; 21; 22 or 23, and the amino acid sequence of one of the sequences of SEQ ID NO: 1-3; 7; 8; 10; 13; 15; 16; 17; 21; 22 or 23 has 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

40. The use or method of any of the preceding claims, wherein the heavy chain variable region of the second variable domain comprises an amino acid sequence of one of the sequences of SEQ ID NO: 2; 7; 8; 10; 13 or 23, and the amino acid sequence of one of the sequences of SEQ ID NO: 2; 7; 8; 10; 13 or 23 has 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

41. The use or method of any of the preceding claims, wherein the first variable domain comprises a heavy chain variable region having the CDR1 sequence SYGIS; the CDR2 sequence WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having the CDR1 sequence SYSMN; the CDR2 sequence WINTYTGDPTYAQGFTG and the CDR3 sequence ETYYYDRGGYPFDP.

42. The use or method of any of the preceding claims, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of TYSMN; a CDR2 sequence of WINTYTGDPTYAQGFTG, and a CDR3 comprising the sequence ETYFYDRGGYPFDP.

43. The use or method of any of the preceding claims, wherein the first variable domain and the second variable domain comprise a common light chain, preferably the light chain variable domain in Figure 4B.

44. The use or method of any preceding claim, wherein the antibody inhibits HGF-induced growth of HGF-growth responsive cells.

45. The use or method of any one of the preceding claims, wherein the antibody inhibits EGF-induced growth of EGF-growth responsive cells.

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