Treatment with antibodies that bind EGFR and cMET

Abstract

The present invention relates to treatment with antibodies that bind to EGFR and cMET. 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

[0001] This application is a divisional application of a Chinese patent application with an application date of March 7, 2023, an application number of 202380026033.0, and an invention title of "Treatment with Antibodies that Bind EGFR and cMET". Technical Field

[0002] 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, which therapeutic antibodies include human antibodies. In addition, it relates to antibodies that bind EGFR and cMET, which include multispecific antibodies, and their use in binding EGFR- and cMET-positive cells, particularly tumor cells. Background Art

[0003] 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. This 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, which has 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).

[0004] EGFR is present on the cell surface and can be activated by binding to its specific ligands, including epidermal growth factor and transforming growth factor alpha (TGFα). After being activated by its growth factor ligand, the receptor can undergo a transition from an inactive predominantly monomeric form to an active homodimer. In addition to forming homodimers after ligand binding, EGFR can also pair with another member of the ErbB receptor family, such as ErbB2, to produce an activated heterodimer. Dimers can also form in the absence of ligand binding, and activated EGFR clusters may form after ligand binding.

[0005] EGFR dimerization stimulates the activity of the intrinsic intracellular protein tyrosine kinase (PTK). This activity induces several signal transduction cascades leading 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.

[0006] Mutations involving EGFR have been found in several types of cancer. It is a target for an increasing category of anti-cancer therapies. Such therapies include EGFR tyrosine kinase inhibitors (EGFR-TKIs) for lung cancer such as gefitinib and erlotinib, and antibodies for colon cancer and head and neck cancer such as cetuximab and panitumumab.

[0007] Cetuximab and panitumumab are monoclonal antibodies that inhibit the receptor. Other monoclonal antibodies in clinical development are zalutumumab, nimotuzumab, and matuzumab. Monoclonal antibodies are designed to block extracellular ligand-induced receptor activation, mainly by blocking the binding of the ligand to the receptor. Since the binding site is blocked, signal-inducing molecules may not be able to attach effectively and thus cannot activate downstream signals. Ligand-induced receptor activation can also be inhibited by stabilizing the unactivated receptor conformation (matuzumab).

[0008] To date, EGFR-targeted therapies have been associated with the development of treatment resistance over time. Various mechanisms of resistance to EGFR-TKIs have been described. In patients with advanced non-small cell lung cancer (NSCLC), mechanisms of resistance include the occurrence of secondary or tertiary mutations (e.g., T790M, C797S, L718Q, exon 20 insertion mutations), activation of alternative signaling (e.g., Met, HGF, AXL, Hh, IGF-1R), aberrant downstream pathways (e.g., AKT mutations, PTEN deletion), impaired EGFR-TKIs-mediated apoptosis pathways (e.g., BCL2-like 11 / BIM deletion polymorphisms), and histological type transformation. Although some mechanisms of resistance have been identified, other mechanisms 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, colorectal cancer patients receiving EGFR antibody therapy 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 start or may develop resistance during treatment. Resistance to EGFR-targeted therapy has been found in many EGFR-positive cancers, and there is a need in the art for more effective EGFR cancer therapies that can improve the standard of care and have an advantage in the ability to address EGFR-targeted therapy resistance.

[0009] Dysregulation of the MET proto-oncogene receptor tyrosine kinase (cMET) and hepatocyte growth factor (HGF) has been reported in multiple tumors. Ligand-driven cMET activation has been observed in several cancers. Elevated serum and intratumoral HGF have been observed in lung cancer, breast cancer, and multiple myeloma (J.M. Siegfried et al., Ann Thorac Surg 66, 1915 (1998); P.C. Ma et al., Anticancer Res 23, 49 (2003); B.E. Elliott et al., Can J Physiol Pharmacol 80, 91 (2002); C. Seidel et al., Med Oncol 15, 145 (1998)). Overexpression of cMET, cMET amplification, or mutations have 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)). Expression of HGF has also been 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 has been associated with the emergence of cMET aberration escape mechanisms.

[0010] The cMET receptor is formed by proteolytic processing of a common precursor into a single-pass, 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 that contains the entire α-subunit and part of the β-subunit. The plexin-semaphorin-integrin (PSI) domain lies after the Sema domain and includes four disulfide bonds. This domain is linked to the transmembrane helix via four immunoglobulin-plexin-transcription (IPT) domains that are related to immunoglobulin-like domains. Intracellularly, the cMET receptor contains a tyrosine kinase catalytic domain flanked by unique juxtamembrane 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).

[0011] The ligands of cMET, hepatocyte growth factor (HGF; also known as scatter factor) and its spliced isoforms (NK1, NK2) are known ligands of the cMET receptor. HGF was discovered as a potent mitogen / morphogen in 1991. The HGF / cMET signaling pathway plays an important role in the development and progression of various cancers. Dysregulation and / or overactivation of HGF or cMET in human cancers is associated with 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-related and an HGF-independent manner. HGF-independent activation occurs in cases of cMET overexpression. In the absence of ligand, a large amount of cMET may also trigger (hetero)dimerization and intracellular signaling. Additional ligand does not seem to affect the function of such cMET-overexpressing cells. cMET amplification is associated with cMET overexpression and has emerged as a biomarker for tumor subtypes.

[0012] HGF is widely expressed throughout the body, suggesting that this growth factor is a systemically available cytokine and is derived from the tumor stroma. The positive paracrine and / or autocrine loops of cMET activation can lead to further cMET expression. The HGF-specific antibody rilotumumab (AMG102) was developed for gastric cancer. Phase I and Phase II trials seemed promising, but a Phase III study (RILOMET-2) of cisplatin and capecitabine as first-line treatment for gastric cancer was terminated after a safety review by a pre-planned data monitoring committee for study 20070622.

[0013] The correlation between cMET / HGF signaling and EGFR-targeted therapy resistance has promoted the development of methods to address resistance. To date, antibody-based methods, 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 seems to be effective against colorectal cancer when combined with the EGFR-inhibitor erlotinib. However, these results could not be replicated in randomized Phase III clinical trials. 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).

[0014] To overcome the problems of anti-EGFR, cMET, and HGF immunotherapies, the present invention provides novel bispecific antibodies that comprise a first variable domain that can bind to the extracellular portion of the epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of the cMET proto-oncogene receptor tyrosine kinase (cMET).

[0015] To date, certain bispecific EGFR x cMET antibodies have been described in the art. Castoldi R. et al. (2013) describe a bispecific EGFR x cMET antibody named MetHer1 that has the cMET-binding site of the antibody 5D5 (or MetMab) and the EGFR-binding site of cetuximab. This bispecific antibody has a fixed EGFR and cMET-binding stoichiometry of 2:1 (see Supplementary Figures).

[0016] US20140378664 describes a cMET x EGFR bispecific antibody among various other antibodies. The intact bispecific antibody is made as a single protein, which is then proteolytically cleaved. The two VH / VL domains are made as single-chain Fv fragments. Binding of the antibody induces 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, made 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 shown in vivo activity using a xenograft model of the tumor cell line H1975 that expresses the cMET ligand HGF. This tumor model is known to be dependent on the ADCC activity of the antibody (Ahmed et al., 2015). JNJ-61186372 has been reported to have unbalanced affinities, with an affinity for cMET approximately 40-fold higher than for EGFR (Moores et al. (2016)), and the anti-EGFR arm derived from zalutumumab is known to cause problems such as infusion-related reactions and skin diseases.

[0017] LY3164530 is a bispecific cMET x EGFR antibody that comprises the EGFR-binding domain of cetuximab as a single-chain Fv fragment fused 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 contains two binding sites for each antigen. Data on the inhibition of HGF by this antibody were not provided. The antibody has been reported to bind and internalize cMET and EGFR without cooperative activity. The authors reviewed various cMET, EGFR, and cMET x EGFR target therapies and concluded that to date these inhibitors have not shown significant efficacy in clinical trials.

[0018] Accordingly, there is a current need for novel bispecific cMET x EGFR antibodies, including those that may have superior properties as described herein. SUMMARY OF THE INVENTION

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

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

[0021] In some aspects, the present invention provides a bispecific antibody comprising a first variable domain that binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET) for use in a method of treating cancer in an individual who has previously received iii) a cMET tyrosine kinase inhibitor.

[0022] In some aspects, the present invention provides a bispecific antibody comprising a first variable domain that binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET) for use in a method of treating cancer in an individual who iv) has not received prior anti-cancer treatment. In some aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0023] In some aspects, the present invention provides a method of treating cancer in an individual who has previously received 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 binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0024] In some aspects, the present invention provides a method of treating cancer in an individual who has previously received 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 binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0025] In some aspects, the present invention provides a method for treating cancer in an individual who has received prior treatment with iii) a cMET tyrosine kinase inhibitor, said treatment comprising administering to the individual an effective amount of a bispecific antibody according to the present invention, which comprises a first variable domain that binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0026] In some aspects, the present invention provides a method for treating cancer in an individual who iv) has not received any prior anti-cancer treatment, said treatment comprising administering to the individual an effective amount of a bispecific antibody according to the present invention, which comprises a first variable domain that binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET). In some aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0027] In some aspects, the present invention provides the 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, said bispecific antibody comprising a first variable domain that binds (or binds to) the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0028] In some aspects, the present invention provides the 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, said bispecific antibody comprising a first variable domain that binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds (or binds to) the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0029] In some aspects, the present invention provides the 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 iii) a cMET tyrosine kinase inhibitor, said bispecific antibody comprising a first variable domain that binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds (or binds to) the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0030] In certain aspects, the present invention provides the use of a bispecific antibody according to the present invention in the preparation of a medicament for treating cancer in an individual who iv) has not received any prior anti-cancer treatment, the bispecific antibody comprising a first variable domain that can bind to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that can bind (or binds) to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET). In certain aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0031] In certain aspects, the cancer is an EGFR-positive cancer, a cMET-positive cancer, or an EGFR- and cMET-positive cancer. In some aspects, the cancer comprises an EGFR aberration, a cMET aberration, or an EGFR and cMET aberration.

[0032] In certain aspects, an 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.

[0033] In certain aspects, an individual or cancer that has received prior treatment comprising chemotherapy and an EGFR tyrosine kinase inhibitor according to ii) is resistant to treatment with a first-generation, second-generation, and / or third-generation EGFR tyrosine kinase inhibitor.

[0034] In certain aspects, an individual or cancer that has received prior treatment comprising a cMET tyrosine kinase inhibitor according to iii) is resistant to treatment with a cMET tyrosine kinase inhibitor.

[0035] In certain aspects, the administration of the bispecific antibody according to i) is administered as a second-line treatment after treatment with a third-generation EGFR tyrosine kinase inhibitor.

[0036] In certain aspects, the administration of the bispecific antibody according to ii) is administered as a third-line treatment after treatment with chemotherapy and an EGFR tyrosine kinase inhibitor.

[0037] In certain aspects, the administration of the bispecific antibody according to iii) is administered as a second-line treatment after treatment with a cMET tyrosine kinase inhibitor.

[0038] In some aspects, the cancer or the individual has received prior treatment with a third-generation EGFR tyrosine kinase inhibitor, and / or the cancer or the 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.

[0039] In some aspects, the cancer or the individual has received prior treatment with chemotherapy and a first-generation or second-generation or third-generation tyrosine kinase inhibitor. In some aspects, the cancer or the individual is resistant to treatment with a first-generation or second-generation or third-generation tyrosine kinase inhibitor, or is resistant to first-generation and second-generation tyrosine kinase inhibitors, or is resistant to first-generation, second-generation, 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 inhibitor.

[0040] In some aspects, the cancer or the individual has received prior treatment with a cMET tyrosine kinase inhibitor, and / or the cancer or the 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.

[0041] In some aspects, the individual comprises an EGFR and / or 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.

[0042] In some aspects, the third-generation EGFR tyrosine kinase inhibitors provided as a previous treatment comprise or are osimertinib, lazertinib, alflutinib, rezivertinib, rociletinib, olmutinib, almonertinib, abivertinib, ASK120067, befotertinib (also known as BPI-D0316 or D-0316), SH-1028, nazartinib (EGF816), naquotinib (ASP8273), mavelertinib (PF-0647775), olafertinib (CK-101), keynatinib or ES-072, and in some aspects is osimertinib. In some aspects, the EGFR tyrosine kinase inhibitor is osimertinib, BPI-D0316 / befotertinib, lazertinib or almonertinib.

[0043] In some aspects, the first-generation EGFR tyrosine kinase inhibitors provided as a previous treatment in combination with the chemotherapy comprise or are gefitinib, erlotinib or icotinib.

[0044] In some aspects, the second-generation tyrosine kinase inhibitors provided as a previous treatment comprise or are afatinib, dacomitinib, XL647, AP26113, CO-1686 or neratinib.

[0045] In some aspects, the cMET tyrosine kinase inhibitor provided as prior therapy is or comprises capmatinib, tepotinib, crizotenib, cabozantinib, savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833.

[0046] In some aspects, the previously administered chemotherapy comprises platinum-based chemotherapy, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide or pemetrexed, or any combination thereof, and in some aspects is a composition comprising cisplatin or carboplatin.

[0047] In some aspects, the individual is a human individual.

[0048] In some aspects, the cancer is non-small cell lung cancer (NSCLC), head and neck cancer, particularly head and neck squamous cell carcinoma; gastric cancer, particularly gastric adenocarcinoma; or esophageal cancer, particularly esophageal squamous cell carcinoma; gastroesophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer. In some 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, a cMET mutation, a cMET aberration or increased HGF expression.

[0049] In some aspects, the cancer or individual comprises an in-frame EGFR exon 20 insertion mutation.

[0050] In some aspects, the cancer comprises a cMET aberration, such as cMET amplification, cMET overexpression, enhanced cMET pathway signaling, cMET gene amplification, and / or increased cMET protein activity. In some aspects, the cancer includes increased HGF expression. In some aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0051] In some aspects, the cMET dysregulation includes cMET amplification, cMET overexpression, enhanced cMET pathway signaling, cMET gene amplification, or increased cMET protein activity or cMET dysregulation caused by increased HGF expression. In some aspects, the cMET aberration includes a cMET exon 14 skipping mutation. The cMET dysregulation is or includes a dysregulation of cMET signaling in some aspects.

[0052] In some aspects, the use or treatment includes providing a bispecific antibody at a dose of 1000, 1500 or 2000 mg to an individual. In some aspects, the bispecific antibody is provided once a week or once every two weeks. In some aspects, the use or treatment includes providing a bispecific antibody at a dose of 1500 mg to an individual once every two weeks.

[0053] In some aspects, the bispecific antibody of the present invention exhibits ADCC activity, and in some aspects, the antibody has enhanced ADCC activity. In this aspect, the antibody may have altered ADCC activity through one or more CH2 mutations. Thus, there is further provided a bispecific antibody according to the present invention, which is defucosylated. In some aspects, the antibody of the present invention includes two defucosylated CH2 domains. In some aspects, the antibody of the present invention includes a total of two CH2 domains, both of which are defucosylated. In some aspects, the antibody of the present invention includes two CH2 domains, both of which are defucosylated.

[0054] In some aspects, the bispecific antibody of the present invention exhibits ADCP activity, and in some aspects, the antibody has enhanced ADCP activity. In some aspects, both the EGFR and cMET binding arms, or the two heavy chains containing the EGFR and cMET binding arms, contribute to ADCP. In some aspects, the bispecific antibody of the present invention has or exhibits ADCP activity against NSCLC cells. In some aspects, the bispecific antibody of the present invention induces ADCP of NSCLC cells.

[0055] The bispecific antibody may comprise a common light chain. In some 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 one known to pair well with a variety of human variable region gene segments that have been recombined. In some aspects, the common light chain is a variable region encoded by a germline Vk gene segment, such as the O12 / IgVκ1-39*01 variable region gene segment. Preferred light chain variable regions comprise rearranged IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. In some 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 some aspects, the common light chain is a rearranged kappa (κ) light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01, which is linked 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 binds to EGFR and a variable domain that binds to cMET. In some aspects, the variable domain that binds to human EGFR may also beneficially bind to mouse EGFR and / or cynomolgus EGFR. In some aspects, the variable domain that binds or can bind to human EGFR binds to domain III of human EGFR. The variable domain that binds to cMET may block the binding of antibody 5D5 to cMET. The variable domain that binds to cMET may block the binding of HGF to cMET. The Kd of the antibody for cMET may be at least 10-fold lower than the Kd of the antibody for EGFR. The amino acids at positions 405 and 409 in the CH3 domain may be the same as the amino acids at the corresponding positions in other CH3 domains (EU numbering).

[0056] In some aspects, the antibody of the invention comprises a first variable domain comprising a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYX 1 X 2 NTNYAQKLQG and comprising the sequence X 3 X 4 X 5 X 6 HWWLX 7 for CDR3,

[0057] wherein X 1 = N or S; X 2 = A or G; X 3 = D or G; X 4 = R, S or Y; X 5 = H, L or Y; X 6 = D or W, and X 7 = D or G; in X 1to X 7 has 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof at positions other than 7 .

[0058] In certain aspects, the antibodies of the invention comprise a second variable domain that comprises a heavy chain variable region having 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 combinations thereof.

[0059] Describes bispecific antibodies, wherein

[0060] X 1 = N; X 2 = G; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G;

[0061] X 1 = N; X 2 = A; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G;

[0062] X 1 = S; X 2 = G; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G;

[0063] X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0064] X 1 = N; X 2 = A; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0065] X 1 = S; X 2 = G; X 3= D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0066] X 1 = N; X 2 = G; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G;

[0067] X 1 = N; X 2 = A; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G; or

[0068] X 1 = S; X 2 = G; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G。

[0069] In some aspects, X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; or X 1 = N; X 2 = A; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; or X 1 = S; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D。

[0070] In some aspects, X 3 -X 7 = DRHWD, and X 1 and X 2It is NG; SG or NA.

[0071] Among the bispecific antibodies described, the heavy chain variable region of the second variable domain comprises 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, which has 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof. One advantage of the present invention is that in some aspects, the bispecific antibodies of the present invention exhibit more effective ADCC activity than amivantamab, especially against cells or cancers comprising cMET aberrations.

[0072] The present invention also provides a method of treating an individual suffering from a tumor, the method comprising administering to the individual in need a bispecific antibody as described herein. Generally, the individual suffers from a disease involving abnormal cells, for example, the individual may suffer from a tumor or cancer.

[0073] The present invention also provides a bispecific antibody comprising a first variable domain that can bind to the extracellular portion of the epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of the MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region having the CDR1 sequence SYGIS; the CDR2 sequence WISAYX 1 X 2 NTNYAQKLQG and comprising the sequence X 3 X 4 X 5 X 6 HWWLX 7 A of CDR3, wherein

[0074] X 1 = N or S; X 2 = A or G; X 3 = D or G; X 4 = R, S or Y; X 5 = H, L or Y; X 6 = D or W, and X 7 = D or G, having 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof at positions other than X 1 -X 7 and wherein the second variable domain comprises a heavy chain variable region having the 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 combinations thereof.

[0075] In some 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 in some aspects 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.

[0076] The present invention also provides a bispecific antibody for treating an individual suffering from a disease involving abnormal cells (such as a tumor).

[0077] Also provided is the use of the bispecific antibody of the present 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 been previously treated 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.

[0078] The antibody of the present invention inhibits HGF and EGF / HGF-induced cancer growth in a cancer that has 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.

[0079] In some aspects, the antibody of the present invention inhibits the growth of HGF-responsive cells induced by HGF, and in some aspects inhibits EGFR TKI-resistant or refractory tumors, tumor models or cell lines in a human individual, such as cell lines or models including activating EGFR mutations, approved tyrosine kinase inhibitor-resistant mutations, tertiary tyrosine kinase inhibitor-resistant mutations, mutations that reduce the binding of a third-generation tyrosine kinase inhibitor to EGFR, acquired tyrosine kinase inhibitor-resistant mutations, EGFR gene amplifications, cMET mutations or cMET aberrations, and in some aspects, in-frame exon 20 insertion mutations. In some aspects, the inhibition is exhibited in the presence of HGF.

[0080] In some aspects, the antibody of the present invention inhibits the growth of HGF-responsive cells induced by HGF, and in some aspects inhibits tumors, tumor models or cell lines in a human individual, the tumors, tumor models or cell lines of which contain activating EGFR mutations, approved tyrosine kinase inhibitor-resistant mutations, tertiary tyrosine kinase inhibitor-resistant mutations, mutations that reduce the binding of a third-generation tyrosine kinase inhibitor to EGFR, acquired tyrosine kinase inhibitor-resistant mutations, EGFR gene amplifications, cMET mutations or cMET aberrations, or in-frame exon 20 insertion mutations. In some aspects, the inhibition is exhibited in the presence of HGF.

[0081] The antibody of the present invention inhibits the growth of EGF-responsive cells induced by EGF, but does not induce toxicities associated with high-affinity bivalent EGFR antibodies such as rash and diarrhea. This makes the antibody highly suitable for combination use with TKIs having their own toxicity profiles.

[0082] The antibody of the present invention can be used to treat tumors resistant to treatment with EGFR tyrosine kinase inhibitors, such as resistant or refractory tumors to Osimertinib, erlotinib, gefitinib, or afatinib; analogs of Osimertinib, erlotinib, gefitinib, or afatinib, or combinations of one or more corresponding compounds and / or their analogs.

[0083] The present invention also encompasses nucleic acid molecules or groups of nucleic acid molecules that individually or together encode the heavy chain or heavy chain variable region of the bispecific antibodies or variants thereof disclosed herein. Also provided are nucleic acid molecules or groups of nucleic acid molecules encoding the antibodies disclosed herein.

[0084] In certain aspects, the heavy chain comprises the constant region of an IgG1 antibody, in certain aspects a human IgG1 antibody. The CH2 region of the IgG1 constant region can be engineered to alter the ADCC and / or CDC activity of the antibody, or not to alter the ADCC and / or CDC activity of the antibody. In certain aspects, the alteration results in enhanced ADCC and / or CDC activity. In certain aspects, the CH3-region of the antibody is engineered to promote heterodimerization of the heavy chains, the heavy chains comprising a first heavy chain that binds EGFR and a second heavy chain that binds cMET.

[0085] The present invention also encompasses a cell comprising one or more nucleic acid molecules that individually or jointly encode the bispecific antibodies or variants thereof disclosed herein. Also provided is a method of producing the bispecific antibodies or variants thereof disclosed herein using the above cells, and in certain aspects, harvesting the bispecific antibodies or variants thereof from cell culture.

[0086] The present invention also encompasses a cell system comprising the bispecific antibodies or variants thereof disclosed herein.

[0087] The present invention also provides a cell that expresses the bispecific antibody and / or comprises a nucleic acid molecule encoding the bispecific antibody.

[0088] The present invention also encompasses a bispecific antibody as disclosed herein that further comprises a label, in certain aspects a label for in vivo imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 . Amino acid sequences of the heavy chain variable regions of the variable domains mentioned in this application.

[0090] Figure 2 . MF3370 and its variants. CDR1, CDR2, and CDR3 sequences in MF8226 (underlined from left to right). CDRs in other sequences are in the corresponding positions (according to Kabat).

[0091] Figure 3 and Continuation of Figure 3 . MF4356 and its variants. CDR1, CDR2, and CDR3 sequences in MF4356 (underlined from left to right). CDRs in other sequences are in the corresponding positions (according to Kabat).

[0092] Figures 4A to 4E . Common light chains used in single- and bispecific IgG.

[0093] Figure 4A : Amino acid sequence of the common light chain. Figure 4B : DNA sequence and translation of the common light chain variable domain (IGKV1-39 / jk1). Figure 4C : DNA sequence and translation of the common light chain constant region. Figure 4D : Translation of the IGKV1-39 / jk5 common light chain variable domain. Figure 4E : V region IGKV1-39A.

[0094] Figures 5A to 5F . IgG heavy chains for generating bispecific molecules. Figure 5A : CH1 region. Figure 5B : Hinge region. Figure 5C : CH2 region. Figure 5D : CH2 containing silent substitutions L235G and G236R. Figure 5E : CH3 domain containing substitutions L351K and T366K (KK). Figure 5F : CH3 domain containing substitutions L351D and L368E (DE).

[0095] Figure 6 . Results of Western blot analysis of the LXFE2478 model showing protein levels of EGFR, cMET, and HGF.

[0096] Figure 7 . Overview of the treatment course. Antibodies or carriers were administered as a single therapy to each group over a 5-week period.

[0097] Figure 8. Effect of antibody therapy on tumor volume in NSCLC PDX model LXFE2478 with exon 20 mutation. Tumor volume growth curves representing groups 1 - 5 are shown at different time points (mean TV ± SEM).

[0098] Figure 9 . Overview of treatment duration. Antibodies or vehicle were administered as single therapy to each group over a 3 - week period.

[0099] Figure 10 . Effect of antibody therapy on tumor volume in NSCLC CDX model HCC827 / ER1 with exon 19 mutation. Tumor volume growth curves representing the vehicle and antibody groups are shown at different time points (mean TV ± SEM).

[0100] Figure 11 . Survival curves of animals in the NSCLC CDX model HCC827 / ER1 with exon 19 mutation mice vs vehicle group. Detailed implementation

[0101] EGFR is a member of the four-receptor tyrosine kinase (RTK) family, named Her- or cErbB-1, -2, -3, and -4. EGFR has an extracellular domain (ECD) composed of four subdomains, two of which are involved in ligand binding and 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 titled "Cited References in the Specification", each of which is incorporated herein by reference in its entirety. 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 the gene, as well as overexpression of the receptor and its ligands, have been found to cause autocrine activation loops (for a review, see Robertson et al., 2000). Thus, this RTK has been widely targeted for cancer therapy. Both small molecule inhibitors that target RTKs and monoclonal antibodies (mAbs) that direct to the extracellular ligand-binding domain have been developed and have achieved several clinical successes to date, although mainly for selected patient populations. The database accession number for the human EGFR protein and its encoding gene is (GenBank NM_005228.3). Other database identifiers for this gene and / or protein are HGNC:3236; Entrez Gene:1956; Ensembl:ENSG00000146648; OMIM:131550 and UniProtKB:P00533. The accession numbers are provided mainly to provide a means of further identifying the EGFR protein as a target, and the actual sequence of the EGFR protein bound by the antibody may vary, for example due to mutations in the encoding gene such as those occurring in certain cancers, or the like. Unless otherwise specified, references to EGFR herein refer to human EGFR. The antigen-binding site that binds EGFR binds EGFR and its various variants, such as those expressed on certain EGFR-positive tumors.

[0102] As used herein, the term "EGFR ligand" refers to a polypeptide that binds and activates EGFR. Examples of EGFR ligands include, but are not limited to, EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, and epiregulin (for a review, see Olayioye MA et al.; EMBO J (2000) Vol. 19: pp. 3159-3167). The term includes bioactive fragments and / or variants of naturally occurring polypeptides.

[0103] Aberrant activation forms of EGFR, such as via EGFR mutations or EGFR gene amplifications, are known as oncogenic drivers of non-small cell lung cancer (NSCLC) and are known to occur in therapies 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 received prior anti-cancer treatment, wherein the individual in iv) comprises in some aspects a cMET exon 14 skipping mutation or cancer comprising a cMET exon 14 skipping mutation.

[0104] In some aspects, the treatment comprises treating cancer caused by ligand-independent activation of EGFR and / or ligand-independent activation of cMET. In another aspect, the treatment comprises treating cancer caused by ligand-dependent activation of EGFR and / or ligand-dependent activation of cMET.

[0105] In some aspects, the cancer or individual has previously received treatment with Osimertinib and has acquired or tertiary Osimertinib resistance. The prior Osimertinib treatment is in some aspects a first-line or second-line therapy, and in some aspects, the first-line therapy is followed by treatment with the bispecific antibody of the present invention as a second-line treatment.

[0106] In some 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, a cMET mutation, a cMET aberration, or increased HGF expression.

[0107] In clinical trials conducted for the efficacy of the bispecific antibody of the present invention, clinical efficacy was observed in a variety of cancers with different gene oncogenic backgrounds. In particular, 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 (such as L858R), deletion mutations of EGFR exon 19, or c-MET exon 14 skipping mutations. Thus, in one aspect, the cancer is NSCLC and / or the individual has NSCLC, wherein the cancer or individual comprises an EGFR exon 21 mutation such as L858R, a deletion mutation of EGFR exon 19, a mutation of EGFR exon 18, or a c-MET exon 14 skipping mutation.

[0108] In addition, clinical efficacy has been observed in head and neck cancer, particularly head and neck squamous cell carcinoma (HNSCC). Thus, in one aspect, the cancer is and / or the individual has head and neck cancer, particularly head and neck squamous cell carcinoma.

[0109] In certain aspects, the cancer is gastric adenocarcinoma with c-MET amplification.

[0110] In certain aspects, the treatment results in or comprises depletion of soluble EGFR and / or cMET.

[0111] 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). Here, the term "activating EGFR mutation" refers to a mutation that develops after treatment with a third-generation EGFR tyrosine kinase inhibitor, including an in-frame deletion of exon 19 (del19) and substitution of leucine with arginine in exon 21 (L858R).

[0112] In certain aspects, the cancer comprises an approved tyrosine kinase inhibitor resistance mutation. As used herein, the term "approved tyrosine kinase inhibitor resistance mutation" refers to the development of resistance after treatment with an EGFR tyrosine kinase inhibitor currently approved for the treatment of cancer, such as T790M, which confers resistance to afatinib. In certain aspects, the EGFR tyrosine kinase inhibitor is an approved EGFR tyrosine kinase inhibitor.

[0113] 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). As used herein, the term "tertiary tyrosine kinase inhibitor resistance mutation" refers to the development of resistance after treatment with a third-generation EGFR tyrosine kinase inhibitor (Osimertinib in certain aspects).

[0114] In certain aspects, the cancer comprises a mutation that reduces the binding of a third-generation tyrosine kinase inhibitor to EGFR, such as L792X, L718X.

[0115] In some aspects, the cancer comprises an acquired tyrosine kinase inhibitor resistance mutation (such as T790M, L858R, exon 19 deletion mutation, C797X, L792X, G796X, G724X, S768X, L718X, or exon 20 insertion mutation), and in some aspects, it is a mutation conferring resistance to Osimertinib or occurring after the use of Osimertinib, including G724X (such as G724S), S768X (such as S768I), L792X (such as L792H), C797X (including C797S and C797G), L798X (such as L798I). Here, the term "acquired tyrosine kinase inhibitor resistance mutation" refers to the resistance acquired after treatment with a tyrosine kinase inhibitor (such as after treatment with a third-generation EGFR tyrosine kinase inhibitor).

[0116] In some aspects, the cancer comprises EGFR gene amplification, such as an increase in EGFR mRNA or amplification of the wild-type EGFR allele, and the emergence of the EGFR-ex19del allele after the use of Osimertinib.

[0117] In some aspects, the cancer comprises a cMET mutation, such as a cMET exon 14 skipping mutation.

[0118] In some aspects, the cancer comprises a cMET aberration, such as cMET amplification, cMET overexpression, enhanced cMET pathway signaling, 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 copy numbers or any combination thereof.

[0119] In some aspects, the 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 numbers, such as (≥ 1.8, < 2.2), or (> 2.2, < 5) or (≥ 5).

[0120] In some aspects, the cancer comprises an exon 19 deletion mutation, and in some aspects, it comprises an in-frame exon 19 deletion, an exon 20 missense mutation (such as T790M), or an exon 21 mutation, such as L858R.

[0121] In some aspects, the cancer comprises an EGFR exon 20 mutation, and in some aspects, it comprises an exon 20 insertion mutation, and in some aspects, it comprises an in-frame exon 20 insertion mutation.

[0122] In some aspects, the cancer or individual comprises an exon 20 mutation selected from near-loop insertions (positions 767-772), far-loop insertions (positions 773-775), and in some aspects is V769_D770insASV, D770_N771insSVD, H773_V774insNPH, H773_V774insH, D770_N771insG, D770delinsGY, N771_P772insN, V774_C775insHV, D770_N771insGL, H773_V774insPH, A763_Y764insFQEA, D770_N771delinsEGN, D770_N771insGD, D770_N771insH, D770_N771insP, H773_V774insAH, 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 the mutations T790M, L792X (such as L792H), C796X (such as G796R, G796S, G796D), C797X (such as 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 some aspects, the cancer or the individual comprises two or more of said mutations.

[0123] In some aspects, the cancer or the individual comprises mutations such as L718X (such as L718Q, L718V), G719X (such as G719A), L792X (such as L792H, L792F, L792R, L792Y, L792V and L792P), G796X (such as G796R, G796S, G796D), C797X, C797X (such as C797S, C797G, C797N), C797X, L792X, G796X, G724X, S768X, L718X, M766X (such as M766Q), R776X (such as R776C), or exon 20 insertion mutations. In some aspects, the cancer or the individual comprises two or more of said mutations.

[0124] In some aspects, the cancer or the individual comprises an exon 19 deletion mutation, and 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 the individual comprises two or more of said mutations.

[0125] In some aspects, the cancer or the individual comprises a cancer which comprises an EGFR exon 20 mutation, and in some aspects an exon 20 insertion mutation.

[0126] In some aspects, the cancer or the individual comprises mutations 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 the individual comprises two or more of said mutations.

[0127] In some aspects, the cancer or the individual comprises double mutations L858X / T790X (such as L858R / T790M), T790X / L798X (such as T790M / L798I), T790X / C797X (such as T790M / C797S), G719X / R776X (such as G719A / R776C) or delE746_A750 / T790M.

[0128] In some aspects, the cancer or individual comprises a dual mutation D770insSVD / E762X (such as E762K), D770insSVD / L792X (such as L792I, L792S), D770insSVD / P794X (such as P794S), or D770insSVD / G796X (such as G796D).

[0129] In some aspects, the cancer or individual comprises a dual mutation H773insH / E762X (such as E762K), H773insH / L792X (such as L792I, L792S), H773insH / P794X (such as P794S), or H773insH / G796X (such as G796D).

[0130] In some aspects, the cancer or individual comprises a dual mutation H773insNPH / E762X (such as E762K), H773insNPH / L792X (such as L792I, L792S), H773insNPH / P794X (such as P794S), or H773insNPH / G796X (such as G796D).

[0131] In some aspects, the cancer or individual comprises a dual mutation L858X / L718X (such as L858R / cis-L718Q), L858X / C797X (such as L858R / cis-C797S), exon 19del / C797X (such as exon 19del / cis-C797S).

[0132] In some aspects, the cancer or individual comprises a triple mutation L858X / T790X / C797X (such as L858R / T790M / C797S), L858X / T790X / M766X (such as L858R / T790M / M766Q), L858X / T790X / L718X (such as 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).

[0133] In some aspects, the cancer or individual comprises a cMET exon 14 skipping mutation.

[0134] As used herein, the term "refractory" refers to a disease that does not respond to a particular treatment. A refractory disease may develop resistance to the treatment before or at the start of the treatment, or a refractory disease may become resistant during the treatment.

[0135] As used 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 carrying EGFR activating mutations (such as exon 19 deletions and exon 21 L858R mutations).

[0136] 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 carrying EGFR activating mutations (such as exon 19 deletions and exon 21 L858R mutations).

[0137] 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 the T790M mutation, and have lower inhibitory activity against wild-type EGFR.

[0138] As used herein, the term "resistance" refers to a cancer or patient not responding to a treatment when administered the relevant therapeutic agent at the prescribed dose, including third-generation EGFR tyrosine kinase inhibitors.

[0139] In some aspects, the third-generation EGFR tyrosine kinase inhibitor is Osimertinib (AZD9291). Osimertinib (AZD9291) is a covalent, orally active, irreversible, and mutant-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 80 mg daily.

[0140] In some aspects, the third-generation EGFR tyrosine kinase inhibitor is almonertinib (HS-10296). Almonertinib is an orally available, irreversible third-generation EGFR tyrosine kinase inhibitor that is selective for EGFR-sensitizing 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.

[0141] In some aspects, the third-generation EGFR tyrosine kinase inhibitor is lazertinib. Lazertinib (YH25448) is a potent, mutant-selective, blood-brain barrier-penetrating, orally available, and irreversible third-generation EGFR tyrosine kinase inhibitor that can be used in the study of non-small cell lung cancer. The recommended Phase 2 dose has been established as a daily dose of 240 mg.

[0142] In some aspects, the third-generation EGFR tyrosine kinase inhibitor is befotertinib (or BPI-D0316 or sometimes called D-0316). Befotertinib (Beta Pharmaceuticals, Co., China) is a third-generation EGFR tyrosine kinase inhibitor. Befotertinib can be used in 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, three times daily, orally) and icotinib (125 mg, three times daily, 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 the D-0316 oral suspension. In the Phase II, single-arm study NCT03861156, locally advanced / metastatic non-small cell lung cancer patients received an oral dose of 75 mg for a cycle of 21 days, and the dose was increased to 100 mg if tolerated. Otherwise, the dose was maintained at 75 mg. In the Phase II / III study NCT04206072, the efficacy and safety of D-0316 at 70 mg once daily for 21 days and then increased to 100 mg once daily were evaluated.

[0143] In some aspects, the third-generation EGFR tyrosine kinase inhibitor is alflutinib (AST2818 or furmonertinib). AST2818 is the subject of clinical trial NCT03787992 to study its clinical efficacy in NSCLC.

[0144] In some 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 to study its clinical efficacy in NSCLC.

[0145] In some 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 to study its clinical efficacy in NSCLC.

[0146] In some 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 the study of non-small cell lung cancer (NSCLC). ASK120067 is the subject of a clinical trial (NCT04143607) to study its clinical efficacy in NSCLC.

[0147] In some aspects, the third-generation EGFR tyrosine kinase inhibitor is Oritinib (SH-1028, Nanjing, China, Nanjing Shenghe Pharmaceutical Co., Ltd.). SH-1028 is the subject of clinical trial NCT04239833 to study its clinical efficacy in NSCLC.

[0148] In some aspects, the third-generation EGFR tyrosine kinase inhibitor is Rociletinib (CO-1686), which is an orally delivered kinase inhibitor that specifically targets mutant EGFR forms. Rociletinib is the subject of clinical trial NCT02186301 to study its clinical efficacy in NSCLC.

[0149] In some 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 a cysteine residue near the kinase domain. Olmutinib can be used in the study of NSCLC. Olmutinib is the subject of clinical trial NCT02485652 to study its clinical efficacy in NSCLC.

[0150] In some 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 patients with advanced EGFR-mutated NSCLC. Nazartinib is the subject of clinical trial NCT03529084 to study its clinical efficacy in NSCLC.

[0151] In some aspects, the third-generation EGFR tyrosine kinase inhibitor is naquotinib, which is an oral, irreversible, third-generation, mutant-selective epidermal growth factor receptor (EGFR) inhibitor. Naquotinib is the subject of clinical trial NCT02588261 to study its clinical efficacy in NSCLC.

[0152] In some aspects, the third-generation EGFR tyrosine kinase inhibitor is Mavelertinib (PF-0647775), which is a selective, oral and irreversible EGFR tyrosine kinase inhibitor (EGFR TKI). Mavelertinib is the subject of clinical trial NCT02349633 to study its clinical efficacy in NSCLC.

[0153] cMET, also known as tyrosine-protein kinase MET or hepatocyte growth factor receptor (HGFR), is a protein encoded by the MET gene in humans. This protein has tyrosine kinase activity. The primary single-chain precursor protein is cleaved after translation to produce α and β subunits, which are linked by disulfide bonds to form the mature receptor.

[0154] Dysregulation or abnormal activation of cMET may induce tumor growth, formation of new blood vessels that supply nutrients to the tumor (angiogenesis), and spread of cancer to other organs (metastasis). cMET is dysregulated in many types of human malignancies, including kidney cancer, liver cancer, gastric 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; The external Ids of cMET are HGNC:7029; Entrez Gene:4233; Ensembl:ENSG00000105976; OMIM:164860 and UniProtKB:P08581. The accession numbers are provided mainly to provide a method for further identification of the cMET protein as a target, and the actual sequence of the cMET protein bound by the antibody may be different, for example, due to mutations in the encoding gene, such as those that occur in certain cancers, or similar situations. When referring to cMET in this article, unless otherwise specified, human cMET is referred to. The antigen-binding site that binds cMET binds to cMET and its various variants, such as those expressed on certain cMET-positive tumors. Examples of cMET aberration or dysregulation include cMET mutations (such as exon 14 skipping mutations), cMET amplification, cMET overexpression, enhanced cMET pathway signaling, cMET gene amplification, and / or increased cMET protein activity. cMET dysregulation may also be caused by increased HGF expression. c-MET dysregulation 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 are currently found to be the primary or secondary drivers of tumor growth in principle, 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, Volume 3, 2019).

[0155] In some aspects, the individual under iv) of the present invention has not received prior anti-cancer treatment. In some aspects, the individual has not received prior treatment with a tyrosine kinase inhibitor. In some aspects, the individual has not received prior treatment with a cMET inhibitor. In some aspects, the individual has not received prior treatment with a cMET tyrosine kinase inhibitor. In some aspects, the individual has not received prior treatment with a tyrosine kinase inhibitor (such as capmatinib, tepotinib, or savolitinib). The individual may have received treatment with a chemotherapeutic agent (such as platinum-based chemotherapy) or an immunotherapeutic agent (such as pembrolizumab, nivolumab, cetuximab). In some aspects, the individual has metastatic non-small cell lung cancer. In some aspects, the metastatic NSCLC has a mutation that results in exon 14 skipping of cMET. Exon 14 skipping can be detected by an FDA-approved test.

[0156] In some aspects, the individual has a cMET exon 14 skipping mutation or a cancer comprising a cMET exon 14 skipping mutation. The individual has not previously received anti-cancer treatment for any cancer, such as an EGFR and / or cMET positive cancer, or a cancer comprising an EGFR and / or cMET aberration. Thus, in some aspects, the administration or treatment with the bispecific antibody of the present invention is a first-line treatment. In some aspects, the first-line treatment is to prevent the development of resistance to the EGFR and / or cMET tyrosine kinase treatment mechanism, for example, 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, particularly in individuals with non-small cell lung cancer, such as in metastatic or advanced cancers. Thus, the present invention also provides a bispecific antibody according to the present invention for use in a method of preventing an individual from developing or developing a cancer resistant to an EGFR and / or cMET tyrosine kinase inhibitor.

[0157] In some aspects, the individual of iv) of the present invention is a human individual.

[0158] In certain aspects, the use or treatment comprises providing to the individual of iv) a dose of 1000, 1500 or 2000 mg of the bispecific antibody of the invention. In certain aspects, the bispecific antibody is provided once a week or once every two weeks. In certain aspects, the use or treatment comprises providing to the individual a dose of 1500 mg of the bispecific antibody once every two weeks. The invention also provides a method of treating the individual suffering from a tumor, the method comprising administering to the individual in need a bispecific antibody as described herein. Generally, the individual suffers from a disease involving abnormal cells, for example, the individual may suffer from a tumor or cancer.

[0159] In certain aspects, the individual of iv) suffers from non-small cell lung cancer (NSCLC), head and neck cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer. In certain aspects, the individual of iv) suffers from cancer, which is non-small cell lung cancer (NSCLC).

[0160] In certain aspects, the individual in iv) suffers from advanced or metastatic cancer, such as advanced or metastatic NSCLC.

[0161] In certain aspects, the treatment of the individual in iv) comprises a diagnostic step for evaluating whether the cancer is an EGFR-positive and / or cMET-positive cancer, or for evaluating the presence of EGFR and / or cMET aberrations in the cancer.

[0162] Antibodies generally can only recognize a part of an antigen. An antigen is generally but not necessarily a protein. The recognition or binding site on the antigen bound by an antibody is called an epitope, where the epitope can be a linear or conformational epitope. The binding of an antibody to an antigen is usually specific. The "specificity" of an antibody refers to its selectivity for a specific epitope, while the "affinity" refers to the strength of the interaction between the antigen-binding site of the antibody and the epitope it binds to.

[0163] 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 antibody of the invention binds to EGFR and, under other identical conditions, binds at least 100-fold less than to the receptors ErbB-2 and ErbB-4 of the same species. The EGFR / cMET bispecific antibody of the invention binds to cMET and, under other identical conditions, binds at least 100-fold less than 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 evaluated on cells expressing the receptor. The bispecific antibody of the invention binds to human, cynomolgus EGFR and / or mouse EGFR in certain aspects.

[0164] Antibodies that bind to both EGFR and cMET can also bind to other proteins if such other proteins contain the same epitope. Thus, the term "bind" does not exclude binding of the antibody to another protein or group of proteins containing the same epitope. Such binding is commonly referred to as cross - reactivity. EGFR / cMET bispecific antibodies generally do not bind to other proteins other than EGFR and / or cMET on the cell membrane after birth (in some aspects, adult humans). The antibodies according to the invention can generally bind to EGFR with a binding affinity (i.e., equilibrium dissociation constant Kd) of at least 1x10e - 6M, as pointed out in more detail below.

[0165] As used herein, the term "antibody" refers to a protein molecule that in some aspects belongs to the immunoglobulin class of proteins. 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 invention contain two variable domains in some aspects. Antibodies for therapeutic use are in some aspects as close as possible to the natural antibodies of the individual to be treated (e.g., human antibodies for human individuals). Antibody binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by the binding domain. Generally, antibodies for therapeutic applications can have an affinity of up to 1x10e - 10M or higher. Antibodies such as the bispecific antibodies of the invention contain the constant domain (Fc portion) of a natural antibody in some aspects. The antibodies of the invention are generally full - length bispecific antibodies, and in some aspects are human IgG subclasses. In some aspects, the antibodies of the invention are of the human IgG1 subclass. Such antibodies of the invention can have good ADCC properties, have a favorable half - life after administration in vivo to humans, and currently available CH3 engineering techniques can provide a modified heavy chain that preferentially forms heterodimers rather than homodimers when co - expressed in clonal cells. The ADCC activity of the antibody can also be enhanced by techniques known to those skilled in the art.

[0166] In certain aspects, the antibodies of the invention are "full-length" antibodies. As used herein, the term "full-length" is defined as encompassing an antibody that is substantially complete, although it may not necessarily have all of the functions of a complete antibody. 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 broken down into domains designated CH1, CH2, CH3, VH and CL, VL. Generally, an antibody binds to an antigen via the variable domains contained in the Fab portion and, upon binding, can interact with molecules and cells of the immune system via the constant domains (primarily via the Fc portion). Full-length antibodies according to the invention encompass antibodies in which there may be mutations that provide desired properties. Antibodies in which one or several amino acid residues have been deleted but which substantially do not alter the specificity and / or affinity characteristics of the resulting antibody are included within the term "full-length antibody". For example, an IgG antibody may have 1-20 amino acid residue insertions, deletions or substitutions or combinations thereof in the constant region.

[0167] In certain aspects, the antibodies of the invention are bispecific IgG antibodies, such as bispecific full-length IgG1 antibodies or human IgG1. Preferably, they are full-length IgG antibodies because they generally have a favorable half-life and, for immunogenicity reasons, it is desirable to be as close as possible to a fully autologous (human) molecule. In certain aspects, the antibodies of the invention are full-length IgG1, full-length IgG2, full-length IgG3 or full-length IgG4 antibodies.

[0168] A variable domain that can bind to EGFR and contains the amino acid sequence of MF3370 or its variant as shown herein binds, in certain aspects, to EGFR domain III (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 epitopes recognized by cetuximab and zalutumumab. For example, the variable domain binds to murine EGFR, while cetuximab and zalutumumab do not, indicating that one or more residues that differ between murine and human EGFR domain III play a role in the binding to cetuximab and zalutumumab but not in the antibodies of the invention described herein. The advantage of the bispecific antibodies of the invention with human, murine, and cynomolgus EGFR cross-reactivity is that it allows for xenograft studies in human cancer models, which can be more predictive in terms of efficacy and toxicity because the antibody also binds to normal murine cells that have the receptor and can also be used in toxicology studies in cynomolgus monkeys. In certain aspects, the invention provides a bispecific antibody that comprises a first variable domain that can bind to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain can also bind to murine EGFR, cynomolgus EGFR, or both.

[0169] In some aspects, the cMET variable domain comprises the amino acid sequence of MF4356 or a variant thereof as shown herein, and in some aspects blocks the binding of the antibody MetMab to cMET. The cMET variable domain in some aspects comprises the amino acid sequence of MF8230 or a variant thereof as shown herein, and in some aspects blocks the binding of the antibody MetMab to cMET. The variable domain in some aspects blocks the binding of the ligand HGF to cMET, or competes with the ligand HGF for binding to cMET. When in the presence of a saturating amount of the variable domain, 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 some aspects by at least 60% under semi-maximal binding conditions. The variable domain is provided in the case of a bivalent monospecific antibody in some aspects. The cMET variable domain in some aspects can bind to the sema domain of cMET. The cMET variable domain of the present invention can compete with 5D5 for binding to cMET, or does not compete with reported anti-cMET reference antibodies such as 5D5. See Table 2.

[0170] The variable domain of the present invention can bind to EGFR (the first variable domain) and in some aspects comprises a heavy chain variable region having a CDR1 sequence of SYGIS; a CDR2 sequence of WISAYX 1 X 2 NTNYAQKLQG, and comprises the sequence X 3 X 4 X 5 X 6 HWWLX 7 of CDR3 of A, wherein X 1 = N or S; X 2 = A or G; X 3 = D or G; X 4 = R, S or Y; X 5 = H, L or Y; X 6 = D or W and X 7 = D or G.

[0171] X 1-7 In some aspects is:

[0172] X 1 = N; X 2 = G; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G;

[0173] X 1 = N; X 2 = A; X3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G;

[0174] X 1 = S; X 2 = G; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G;

[0175] X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0176] X 1 = N; X 2 = A; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0177] X 1 = S; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0178] X 1 = N; X 2 = G; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G;

[0179] X 1 = N; X 2 = A; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G; or

[0180] X 1 = S; X2 = G; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G.

[0181] In some aspects,

[0182] X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0183] X 1 = N; X 2 = A; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; or

[0184] X 1 = S; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X7 = D.

[0185] In some aspects, X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D.

[0186] The sequence X in the CDR3 sequence of the first variable domain 3 X 4 X 5 X 6 HWWLX 7 The amino acid after amino acid A in A can vary. The sequence X 3 X 4 X 5 X 6 The amino acid sequence after HWWLX7A can be FDY. The CDR3 of the first variable domain contains the sequence X in some aspects 3 X 4 X 5 X 6HWWLX 7 AF, in some aspects, which includes X 3 X 4 X 5 X 6 HWWLX 7 AFD, in some aspects, which includes X 3 X 4 X 5 X 6 HWWLX 7 AFDY.

[0187] The first variable domain in some aspects includes a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYNGNTNYAQKLQG, and a CDR3 sequence X 3 X 4 X 5 X 6 HWWLX 7 A.

[0188] The first variable domain in some aspects includes a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYNGNTNYAQKLQG, and a CDR3 including the sequence DRHWHWWLDA. The amino acids 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 some aspects includes the sequence DRHWHWWLDAF, in some aspects, which includes DRHWHWWLDAFD, in some aspects, which includes DRHWHWWLDAFDY.

[0189] The first variable domain in some aspects includes a heavy chain variable region having as Figure 2 shown MF3353; MF8229; MF8228; MF3370; MF8233; MF8232; MF3393; MF8227 or MF8226 amino acid sequences, which have at most 10, in some aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in some aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the specified sequences. In some aspects, the first variable domain includes a heavy chain variable region having as Figure 2 shown MF3353; MF8229; MF8228; MF3370; MF8233; MF8232; MF3393; MF8227 or MF8226 amino acid sequences. In some aspects, the first variable domain includes a heavy chain variable region having as Figure 2The CDR1, CDR2, and CDR3 amino acid sequences of MF3353; MF8229; MF8228; MF3370; MF8233; MF8232; MF3393; MF8227 or MF8226 as shown.

[0190] The variable domain that can bind to cMET (the second variable domain) in some aspects comprises a heavy chain variable region that comprises SEQ ID NO: 1-23( Figure 3) of one of the amino acid sequences having from 0 to 10, in some aspects from 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 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, having from 0 to 10, in some aspects from 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 the amino acid sequence of one of the sequences of SEQ ID NO: 2; 7; 8; 10; 13 or 23, having from 0 to 10, in some aspects from 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 the amino acid sequence of the sequence of SEQ ID NO: 13 or SEQ ID NO: 23, having from 0 to 10, in some aspects from 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In some aspects, the second variable domain comprises a heavy chain variable region having the CDR1, CDR2 and CDR3 amino acid sequences of 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), MF8241 (SEQ ID NO: 20), MF8244 (SEQ ID NO: 21), MF8221 (SEQ ID NO: 22), or MF4356 (SEQ ID NO: 23).

[0191] In some 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, where in some aspects the CDR3 comprises 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 chains of the first and second variable domains comprise, in some aspects, the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0192] In some 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, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence TYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG, and a CDR3 comprising the sequence ETYFYDRGGYPFDP. The CDR1, CDR2 and CDR3 of the light chains of the first and second variable domains comprise, in some aspects, the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0193] A bispecific antibody comprising a first variable domain that binds to the extracellular portion of EGFR and a second variable domain that binds to the extracellular portion of cMET, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, 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 chains of the first and second variable domains comprise, in some aspects, the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0194] A bispecific antibody comprising a first variable domain that binds to the extracellular portion of EGFR and a second variable domain that binds 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 chains of the first and second variable domains respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT in certain aspects, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0195] A bispecific antibody comprising a first variable domain that binds to the extracellular portion of EGFR and a second variable domain that binds 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 chains of the first and second variable domains respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT in certain aspects, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0196] A bispecific antibody comprising a first variable domain that binds to the extracellular portion of EGFR and a second variable domain that binds 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 chains of the first and second variable domains respectively comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT in certain aspects, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0197] In some aspects, the cMET-binding variable domain is described as having a CDR2 sequence of "WINTYTGDPTYAQGFTG", which CDR2 sequence can also be "WINTYTGDPTYAQGFT".

[0198] The CDR1, CDR2, and CDR3 of the light chain of the first and second variable domains as described herein in some 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 some aspects is Figure 4B the light chain variable region shown.

[0199] In some 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, the first variable domain comprising the CDR1, CDR2, and CDR3 of the heavy chain variable region of MF3755 as Figure 1 shown, and the second variable domain comprising the CDR1, CDR2, and CDR3 of the heavy chain variable region of MF4297 as Figure 1 shown. The light chain variable regions in the first and second variable domains are in some aspects the common light chain variable region described herein. The CDR1, CDR2, and CDR3 of the light chain of the first and second variable domains in some 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 aspects, the antibody comprises a heavy chain variable region having the amino acid sequence of MF3755 as Figure 1 shown, which has at most 10, in some aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in some aspects 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof relative to the specified sequence. In some aspects, the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF3755 as Figure 1 shown. The variable domain that can bind cMET (the second variable domain) in some aspects comprises a heavy chain variable region that comprises as Figure 1The amino acid sequence of MF4297 as shown, which has 0 - 10, and 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 as Figure 1 shown the amino acid sequence of MF4297.

[0200] In the context of the present invention, the term "bispecific" (bs) refers to an antibody being able to bind two different targets or two epitopes on the same target. For example, where one variable domain of the antibody (as defined above) binds to an epitope on EGFR, while the second variable domain binds to an epitope on cMET. Depending on the expression levels, (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, while the other arm comprises the variable domain of another antibody (i.e., one arm of the bispecific antibody is formed by pairing a heavy chain with a light chain, while the other arm is formed by pairing a different heavy chain with a light chain). Thus, in some aspects, the EGFR:cMET binding stoichiometric ratio of the bispecific antibody of the present invention is 1:1.

[0201] The heavy chain variable regions of the bispecific antibodies of the present invention are generally different from each other, while the light chain variable regions are the same in some aspects. A bispecific antibody in which the different heavy chain variable regions are associated with the same light chain variable region is also referred to as a bispecific antibody having a common light chain variable region (cLcv). Preferably the light chain constant regions are also the same. Such a bispecific antibody is called having a common light chain (cLc). Thus, there is further provided a bispecific antibody according to the present invention, wherein both arms comprise a common light chain.

[0202] According to the present invention, the term "common light chain" refers to two or more light chains in a bispecific antibody, which may be the same 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 are still functionally equivalent, such as by introducing and testing conservative amino acid changes, amino acid changes in regions that do not contribute or only partially contribute to the binding specificity when paired with the heavy chain. The terms "common light chain", "common LC", "cLC", "single light chain", with or without the term "rearranged", are used interchangeably herein. The terms "common light chain variable region", "common VL", "common LCv", "cLCv", "single VL", with or without the term "rearranged", are used interchangeably herein. In some aspects, the bispecific antibodies of the present invention have a common light chain (variable region) that can bind to at least two, and in some aspects multiple heavy chains (variable regions) with different binding specificities to form an antibody with a functional antigen-binding domain (e.g., WO2009 / 157771). 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 contains a light chain encoded by a germline human Vk gene segment in some aspects and is a rearranged germline human kappa (κ) light chain IgVκ1-39*01 / IGJκ1*01( Figure 4A ). The common light chain variable region is the variable region of a rearranged germline human kappa (κ) light chain IgVκ1-39*01 / IGJκ1*01 in some aspects. The common light chain contains a light chain variable region as shown in Figure 4B or 4D in some aspects, which has 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. The common light chain also contains a light chain constant region in some aspects and contains a kappa (κ) light chain constant region in some aspects. 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 the germline nucleic acid sequence.

[0203] In some aspects, the light chain contains a light chain region that contains the amino acid sequence of the O12 / IgVκ1-39*01 gene segment, as shown in Figure 4A , which has 0-10, and in some aspects 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. The phrase "O12 light chain" will be used throughout the specification as "a light chain containing a light chain variable region that contains Figure 4AAmino acid sequence of the O12 / IgVκ1-39*01 gene segment of the portion shown, which has 0-10, in some aspects 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof". IgVκ1-39 is the abbreviation of immunoglobulin variable κ1-39 gene. This gene is also known as immunoglobulin κ variable 1-39; IGKV139; IGKV1-39; O12a or O12. The external Ids of this gene are HGNC:5740; Entrez Gene:28930; Ensembl:ENSG00000242371. In some 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 Describe two preferred sequences of the combination of IgVκ1-39 and J regions. The linked sequences are designated as IGKV1-39 / jk1 and IGKV1-39 / jk5; another name is IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (named according to the IMGT database on the global website imgt.org).

[0204] Preferably, the O12 / IgVκ1-39*01 containing the light chain variable region is a germline sequence. More preferably, the IGJκ1*0 or / IGJκ5*01 containing the light chain variable region is a germline sequence. In some aspects, the IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence.

[0205] In some aspects, the light chain variable region contains the germline O12 / IgVκ1-39*01. In some aspects, the light chain variable region contains the κ light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. In some aspects, IgVκ1-39*01 / IGJκ1*01. The light chain variable region contains the germline κ light chain IgVκ1-39*01 / IGJκ1*01 or germline κ light chain IgVκ1-39*01 / IGJκ5*01 in some aspects, and contains the germline IgVκ1-39*01 / IGJκ1*01 in some aspects.

[0206] Mature B cells that produce antibodies with an O12 light chain typically produce a light chain that has undergone one or more mutations relative to the germline sequence (i.e., the normal sequence in non-lymphocytes of that organism). The process that results in these mutations is commonly referred to as somatic (hyper)mutation. The resulting light chain is called an affinity-matured light chain. Such a light chain (when derived from the O12 germline sequence) is an O12-derived light chain. 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 introduced artificially in the laboratory. Other mutations can also be introduced in the laboratory without affecting the class nature of the light chain, and not necessarily affecting its quantity. If the light chain contains a sequence as shown in Figure 4A and Figure 4B ; Figure 4D or Figure 4E and has 0 - 10, in some aspects 0 - 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof, then the light chain is at least an O12 light chain. In some aspects, the O12 light chain is a light chain that contains a sequence as shown in Figure 4A ; 4b; 4d or 4e and has 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 that contains a sequence as shown in Figure 4A and Figure 4B ; Figure 4D or Figure 4E and has 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 that contains a sequence as shown in Figure 4A and Figure 4B ; Figure 4D or Figure 4E and has 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 that contains a sequence as shown in Figure 4A or Figure 4B and has the above-described amino acid insertions, deletions, substitutions, additions, or combinations thereof. In some aspects, the light chain contains the sequence of Figure 4A . In some aspects, the variable region of the light chain contains the sequence of Figure 4B . The above 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions in some aspects and can be present in the CDR regions 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 some aspects, and in some aspects, not in the CDR1, CDR2, or CDR3 regions or the FR4 region of the VL chain.

[0207] The common light chain may have a lambda (λ) light chain and is thus 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 the constant region of a kappa or lambda light chain. In some aspects, it is the constant region of a kappa light chain. In some aspects, the common light chain is a germline light chain. In some aspects, it is a rearranged germline human kappa light chain comprising the IgVκl-39 gene segment. In some aspects, the rearranged germline human kappa light chain is IgVκl-39*01 / IGJκl*01( Figures 4A to 4E ). The terms rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01, IGKV1-39 / IGKJ1, huVκ1-39 light chain or simply huVκ1-39 or simply 1-39 may be used interchangeably throughout the application.

[0208] Cells that produce the common light chain may 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.

[0209] In some aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVTITCRASQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQPEDFATYYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRASQSIS SYLNW YQQKPGKAPK LLIYAASSLQ SGVPS RFSGS GSGTD FTLTI SSLQPEDFAT YYCQQ SYSTP PITFG QGTRLEIK, which has 0 to 10, in some aspects 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In some aspects, the light chain variable region comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, in some aspects 0 to 3, in some aspects 0 to 2, in some aspects 0 to 1, and in some aspects 0 amino acid insertions, deletions, substitutions, additions, relative to the specified amino acid sequence, or combinations thereof. If the aligned sequences do not differ in more than 5 positions, the combination of insertions, deletions, additions, or substitutions is the claimed combination. In some aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASVGDRVT ITCRASQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGSGSGTDFTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASVGDRVTITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTDFTLTI SSLQP EDFATYYCQQ SYSTP PITFG QGTRL EIK. In some aspects, the light chain variable region comprises the amino acid sequence DIQMTQSPSS LSASV GDRVT ITCRASQSIS SYLNW YQQKP GKAPKLLIYA ASSLQ SGVPS RFSGS GSGTDFTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQGTKVE IK.In another aspect, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVTITCRASQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPSRFSGS GSGTD FTLTISSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK.

[0210] The amino acid insertion, deletion, substitution, addition, or combination thereof is in some aspects not in the CDR3 region of the light chain variable region and in some aspects is not in the CDR1 or CDR2 region of the light chain variable region. In some aspects, the light chain variable region does not contain deletions, additions, or insertions 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. The amino acid substitutions are in some aspects conservative amino acid substitutions. The CDR1, CDR2, and CDR3 of the light chain of the antibody of the present invention comprise in some aspects the amino acid sequences CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e., the CDRs of IGKV1 - 39 (according to IMGT). In some aspects, the CDR3 sequence comprises QQSYSTPPT.

[0211] The bispecific antibody as described herein has in some aspects one 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 VLs in the first and second VH / VL combinations are the same. In some aspects, the bispecific antibody is a full - length antibody that has one heavy chain / light chain (H / L) combination that binds to the extracellular portion of EGFR and an 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.

[0212] Several methods have been disclosed for generating host cells whose expression favors the production of bispecific antibodies and vice versa, i.e., monospecific antibodies. In the present invention, it is preferred that the cellular expression of the antibody molecules favors the production of bispecific antibodies rather than the corresponding monospecific antibodies. This is generally achieved by modifying the constant regions of the heavy chains such that they favor heterodimerization (i.e., dimerization with heavy chains of other heavy chain / light chain combinations) rather than homodimerization. In some aspects, the bispecific antibodies of the present invention comprise two different immunoglobulin heavy chains having compatible heterodimerization domains. A variety of compatible heterodimerization domains have been described in the art. The compatible heterodimerization domain is, in some aspects, a compatible immunoglobulin heavy chain CH3 heterodimerization domain. When using wild-type CH3 domains, co-expression of two different heavy chains (A and B) and a common light chain will result in 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 the bispecific antibody. 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. The art describes various ways to achieve such hetero-dimerization of heavy chains. One method is to generate "knob into hole" bispecific antibodies.

[0213] As used herein, the term "compatible heterodimerization domain" refers to a protein domain that has been engineered such that the engineered domain A' will preferentially form a heterodimer with the engineered domain B' and vice versa, with reduced homodimerization between A'-A' and B'-B'.

[0214] 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 (published as WO2013 / 157954; incorporated herein by reference in its entirety). These means and methods can also be advantageously used in the present invention. Specifically, the bispecific antibodies of the present invention contain mutations in certain aspects to produce a bispecific full-length IgG molecule that is highly 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. The US 9,248,181 and US 9,358,286 patents, and the WO2013 / 157954 PCT application (incorporated herein by reference in its entirety) illustrate that the DE-variant and KK-variant preferentially pair to form a heterodimer (referred to as the "DEKK" bispecific molecule). Due to the repulsion between charged residues at the CH3-CH3 interface between equal heavy chains, homodimerization of the DE-variant heavy chain (DEDE homodimer) is unfavorable.

[0215] Bispecific antibodies can be produced by (transiently) transfecting plasmids encoding a light chain and two different heavy chains, which are engineered in the CH3 to ensure efficient hetero-dimerization and formation of bispecific antibodies. The production of these chains in a single cell results in the formation of bispecific antibodies being superior to the formation of monospecific antibodies. Preferred mutations that essentially 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, or vice versa.

[0216] In one embodiment, the heavy chain / light chain combination comprising a variable domain that binds to EGFR comprises the 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 the KK variant of the heavy chain. The KK variant of the heavy chain that binds cMET does not produce homodimers, thus making the observed effect of the bispecific antibody on inhibiting HGF-induced cMET activation very accurate. This avoids the cMET activation (agonistic effect) sometimes observed with bivalent cMET antibodies.

[0217] The Fc region mediates the effector functions of antibodies, such as complement-dependent cytotoxicity (CDC), antibody-dependent 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 is activated, enhanced, or stimulated as in some embodiments of the present invention, 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 certain aspects, the antibodies of the present invention promote ADCP. In certain aspects, the antibodies of the present invention promote ADCC. In certain aspects, the antibodies of the present invention promote ADCC to a greater extent than amivantamab. In certain aspects, for cells or cancers having 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.

[0218] Antibodies with reduced effector function are, in certain aspects, IgG antibodies comprising a modified CH2 / lower hinge region to, for example, reduce Fc-receptor interaction or reduce Clq binding. In some aspects, the antibodies of the present invention are IgG antibodies having a mutated CH2 and / or lower hinge domain such that the bispecific IgG antibody has a reduced interaction with Fc-gamma receptors. Antibodies comprising a mutated CH2 region are, in certain aspects, IgG1 antibodies. Such mutated IgG1 CH2 and / or lower hinge domains comprise, in certain aspects, amino acid substitutions at positions 235 and / or 236 (EU numbering), and in certain aspects comprise L235G and / or G236R substitutions ( Figure 5D ).

[0219] 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 (see, for review, 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 an antibody or effector cell to induce ADCC. These include the chromium-51 [Cr51] release assay, the europium [Eu] release assay, and the sulfur-35 [S35] release assay. Generally, a labeled target cell line expressing a certain 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 the antibody-labeled target cells. Subsequently, the release of intracellular label is measured by scintillation counter or spectrophotometry to measure lysis of the target cells. In certain aspects, the bispecific antibodies of the present invention exhibit 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. One technique for enhancing the ADCC of an antibody is afucosylation (see, for example, Junttila, T.T., K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70(11): 4481-4489). Accordingly, further provided are bispecific antibodies according to the present invention that are afucosylated. In certain aspects, the antibodies of the present invention comprise two afucosylated CH2 domains. In certain aspects, the antibodies of the present invention comprise a total of two CH2 domains, both of which are afucosylated. In certain aspects, the antibodies of the present invention are full-length antibodies, such as IgG-type antibodies, that have two CH2 domains, both of which are non-fucosylated.Alternatively, or additionally, a variety of other strategies can be used to achieve ADCC enhancement, such as including glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis, all of which attempt to increase the binding of Fc to low-affinity activating FcγRIIIa, and / or decrease the binding to low-affinity inhibitory FcγRIIb. The bispecific antibodies of the present invention are defucosylated in some aspects to enhance ADCC activity. When compared to the same antibody produced in normal CHO cells, the bispecific antibodies of the present invention contain, in some aspects, a reduced amount of fucosylation of the N-linked glycan structures in the Fc region.

[0220] Variants of the antibodies or bispecific antibodies as described herein include functional parts, derivatives, and / or analogs of the antibodies or bispecific antibodies. The variant retains the binding specificity of the (bispecific) antibody. The functional part, derivative, and / or analog retains the binding specificity of the (bispecific) antibody. Binding specificity is defined as the ability to bind to the extracellular portions of the first and second membrane proteins described herein.

[0221] 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, in certain aspects no more than 10, in certain aspects no more than 5 amino acid differences from the constant region of a naturally occurring human antibody. Preferably, the constant portion is fully derived from a naturally occurring human antibody. The various antibodies generated herein are derived from a library of human antibody variable domains. Thus, these variable domains are human variable domains. The unique CDR regions 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, except for the CDR regions, the variable region is considered a humanized variable region. In such an embodiment, compared to the variable region of a naturally occurring human antibody, the VH of the variable domain of the antibody of the present invention that binds to EGFR or cMET may contain one or more, in certain aspects no more than 10, in certain aspects no more than 5 amino acid differences, not counting possible differences in the amino acid sequences of the CDR regions. Compared to the variable region of a naturally occurring human antibody, the light chain variable region of the EGFR binding domain and / or the cMET binding domain in the antibody of the present invention may contain one or more, in certain aspects no more than 10, in certain aspects no more than 5 amino acid differences, not counting possible differences in the amino acid sequences of the CDR regions. Compared to the variable region of a naturally occurring human antibody, the light chain in the antibody of the present invention may contain one or more, in certain aspects no more than 10, in certain aspects no more than 5 amino acid differences, not counting possible differences in the amino acid sequences of the CDR regions. Such mutations also occur in the case of somatic hypermutation in nature.

[0222] Antibodies can be derived from various animal species, at least with respect to the heavy chain variable region. It is common practice, for example, to humanize murine heavy chain variable regions. There are a variety of ways to achieve this, among which are 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; deimmunizing the murine heavy chain variable region, which in certain 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 with 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.

[0223] The immunogenicity of the deimmunized murine heavy chain variable region in humans is lower than that of the original murine heavy chain variable region. In some aspects, the variable regions or domains of the present invention are further humanized, such as veneered. By using veneering technology, the external residues that are easily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule comprising a weakly immunogenic or substantially non-immunogenic veneered surface. The animals used in the present invention are mammals in some aspects, primates in some aspects, and humans in some aspects.

[0224] The bispecific antibodies according to the present invention comprise the constant regions of human antibodies in some aspects. According to the differences in their heavy chain constant regions, antibodies are divided into five classes or isotypes: IgG, IgA, IgM, IgD, and IgE. These classes or isotypes comprise at least one of said heavy chains named with the corresponding Greek letter. One aspect comprises an antibody, wherein the constant region is selected from the group consisting of IgG, IgA, IgM, IgD, and IgE constant regions, and in some aspects, the constant region comprises an IgG constant region, i.e., selected from the group consisting of: IgG1, IgG2, IgG3, and IgG4. In some aspects, the constant region is an IgG1 or IgG4 constant region, and in some 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 can also be introduced artificially to add certain preferred features to the antibody or parts thereof. These features are described, for example, in the content related to CH2 and CH3 herein. Generally, about 1 - 10 amino acid insertions, deletions, substitutions, or combinations are allowed in the constant region.

[0225] Figure 1 、 2 The VH chain of or 3 has at most 15, and in some aspects, has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or combinations thereof, relative to Figure 1 、 2 The VH chain shown in or 3 has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, relative to Figure 1 、 2 The VH chain shown in or 3 has 0, 1, 2, 3, or 4 amino acid insertions, deletions, substitutions, or combinations thereof, and in some aspects, has 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or combinations thereof, and more in some aspects, has 0, 1, or 2 amino acid insertions, deletions, substitutions, or combinations thereof, and in some aspects, has 0 or 1 amino acid insertions, deletions, substitutions, or combinations thereof, relative to Figure 1 、 2the VH chain shown in 3. The one or more amino acid insertions, deletions, substitutions, or combinations thereof are in some aspects not in the CDR1, CDR2, and / or CDR3 regions of the VH chain. In some aspects, they are also not present in the FR4 region. The amino acid substitutions are in some aspects conservative amino acid substitutions.

[0226] Rational approaches have evolved towards minimizing the content of non-human residues in the human environment. There are multiple ways 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, usually supported by the sequences of the CDR1 and CDR2 regions of the variable domain and the appropriate structure of the entire variable domain.

[0227] CDR sequences can be defined using different methods, including but not limited to according to the Kabat numbering scheme (Kabat et al., J. Biol. Chem. 252:6609 - 6616 (1977); and / or Kabat et al., U.S. 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 M.P. et 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)).

[0228] Each of these numbering schemes defines the CDRs based on the predicted contribution of amino acid residues in the variable regions of the heavy or light chains to antigen binding. Thus, each method for identifying CDRs can be used to identify the CDRs of the binding domains of the invention. In certain embodiments, the heavy chain CDRs of the binding domains of the invention are according to Kabat, Chothia, or IMGT. In certain aspects, the heavy chain CDRs of the binding domains of the invention are according to Kabat. In certain aspects, the heavy chain CDRs of the binding domains of the invention are according to Chothia. In certain aspects, the heavy chain CDRs of the binding domains of the invention are according to IMGT. In certain aspects, the light chain CDRs of the binding domains of the invention are according to Kabat. In certain aspects, the light chain CDRs of the binding domains of the invention are according to Chothia. In certain aspects, the light chain CDRs of the binding domains of the invention are according to IMGT. The amino acid sequences of the heavy chain CDR regions as described herein are determined according to the Kabat definition.

[0229] Currently, there are various methods available for grafting CDR regions onto the appropriate variable domains of another antibody. See, for example, J.C. Almagro1 and J. Fransson (2008) Frontiers in Bioscience 13, 1619 - 1633, which is incorporated herein by reference in its entirety. Thus, the invention further provides a humanized bispecific antibody, or in certain aspects a human bispecific antibody, such as those included in the treatment of the invention, which comprises 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 the VH CDR3 sequence as shown by MF3370 in Figure 1 and wherein the variable domain comprising the cMET - binding site comprises the VH CDR3 region as shown by MF4356 in Figure 1 . The VH variable region comprising the EGFR - binding site, in certain aspects, comprises the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown by MF3370 in Figure 1 . The VH variable region comprising the cMET - binding site, in certain aspects, comprises the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown by MF4356 in Figure 1 . CDR grafting can also be used to produce antibodies having Figure 1VH CDR regions as shown but with different frameworks. The different frameworks can be another human VH, or a different mammal. Accordingly, the present invention further provides a humanized bispecific antibody, or in some aspects a human bispecific antibody, such as for inclusion in the treatment of the present invention, the bispecific antibody 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, as Figure 2 shown in MF8233 in Figure 3 , and wherein the variable domain comprising the cMET-binding site comprises, as Figure 2 shown in MF8230 in Figure 3 . The VH variable region comprising the EGFR-binding site in some aspects comprises the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as Figure 2 shown in MF8233 in Figure 3 . The VH variable region comprising the cMET-binding site in some aspects comprises the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown in MF8230 in

[0230] CDR grafting can also be used to produce VH chains with the CDR regions of the VH as Figure 1 shown in MF3370 in Figure 3 Figure 1 and wherein the VH variable region comprising the cMET-binding site in some aspects comprises the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as Figure 3 shown in MF8230 in Figure 2 CDR grafting can also be used to produce VH chains with the CDR regions of the VH as Figure 3 shown but with different frameworks. The different frameworks can be the framework of another human VH, or the framework of a different mammal.

[0231] Accordingly, the present invention further provides a human or humanized bispecific antibody, such as for inclusion in the treatment of the present invention, which comprises 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 the VH CDR3 sequence as shown by MF8233 in Figure 2 , and wherein the variable domain comprising the cMET-binding site comprises the VH CDR3 region as shown by MF4356 in Figure 3 . The VH variable region comprising the EGFR-binding site in certain aspects comprises the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown by MF8233 in Figure 2 . The VH variable region comprising the cMET-binding site in certain aspects comprises the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown by MF4356 in Figure 3 . CDR grafting can also be used to produce VH chains having the CDR regions of the VH as shown in Figure 2 or Figure 3 but having a different framework. The different framework can be the framework of another human VH or the framework of a different mammal.

[0232] Accordingly, the present invention further provides a humanized bispecific antibody, or in certain aspects a human bispecific antibody, such as for inclusion in the treatment of the present invention, which comprises 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 the VH CDR3 sequence as shown by MF8232 in Figure 2 , and wherein the variable domain comprising the cMET-binding site comprises the VH CDR3 region as shown by MF8230 in Figure 3 . The VH variable region comprising the EGFR-binding site in certain aspects comprises the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown by MF8232 in Figure 2 . The VH variable region comprising the cMET-binding site in certain aspects comprises the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown by MF8230 in Figure 3 . CDR grafting can also be used to produce VH chains having the CDR regions of the VH as shown in Figure 2 or Figure 3 but having a different framework. The different framework can be the framework of another human VH or the framework of a different mammal.

[0233] Methods for generating sequence variants are known in the art. Random methods can be used to generate sequence variants, or targeted methods can be employed, such as those aimed at introducing mutations 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. Mutations aimed at introducing mitigation of development risks can also be used, with a view to large-scale manufacture of the binding domain or parts containing such binding domains. Mutations that may not appear to result in loss of binding specificity and / or affect binding affinity can be introduced. Whether amino acid residues in the CDR and / or framework regions can be substituted, for example with 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, for example, alanine scanning (Cunningham BC, Wells JA. Science. 1989;244(4908):1081-5), and deep mutational scanning (Araya CL, Fowler DM. Trends Biotechnol. 2011;29(9):435-42). Computational methods for predicting the effects of amino acid variations have also been developed, such as those 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.

[0234] The present invention further provides any variant anti-human EGFR and c-MET binding domains generated by the above methods; binding portions, such as antibodies, comprising any of the said variant binding domains; pharmaceutical compositions comprising any of the said variant anti-human EGFR and c-MET binding domains or binding portions; nucleic acids encoding any of the said variant binding domains; vectors and cells comprising the nucleic acids; and the use of the said variant binding domains or pharmaceutical compositions for the treatment of cancer.

[0235] Accordingly, the present invention further provides a human or humanized bispecific antibody, such as for inclusion in the treatment of the present invention, which 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 the VH CDR3 sequence as shown in MF8232 in Figure 2 and wherein the variable domain comprising the cMET-binding site comprises as Figure 3the VH CDR3 region shown in MF8230 in. The VH variable region containing this EGFR binding site contains, in certain aspects, the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain shown in Figure 2 MF8232. The VH variable region containing this cMET binding site contains, in certain aspects, the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain shown in Figure 3 MF8230. CDR grafting can also be used to produce VH chains having the CDR regions of the VH shown in Figure 2 or Figure 3 but having a different framework. This different framework can be the framework of another human VH or the framework of a different mammal.

[0236] Accordingly, the present invention further provides a human or humanized bispecific antibody, such as included in the treatment of the present invention, which comprises a first antigen-binding site that binds to EGFR and a second antigen-binding site that binds to cMET, wherein the variable domain containing the EGFR binding site contains the VH CDR3 sequence shown in Figure 2 MF8232, and wherein the variable domain containing the cMET binding site contains the VH CDR3 region shown in Figure 3 MF4356. The VH variable region containing this EGFR binding site contains, in certain aspects, the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain shown in Figure 2 MF8232. The VH variable region containing this cMET binding site contains, in certain aspects, the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain shown in Figure 3 MF4356. CDR grafting can also be used to produce VH chains having the CDR regions of the VH shown in Figure 2 or Figure 3 but having a different framework. This different framework can be the framework of another human VH or the framework of a different mammal.

[0237] Accordingly, the present invention further provides a humanized or, in certain aspects, a human bispecific antibody, such as included in the treatment of the present invention, which comprises a first antigen-binding site that binds to EGFR and a second antigen-binding site that binds to cMET, wherein the variable domain containing the EGFR binding site contains the VH CDR3 sequence shown in Figure 2 MF8232, and wherein the variable domain containing the cMET binding site contains the VH CDR3 region shown in Figure 3 MF8230. The VH variable region containing this EGFR binding site contains, in certain aspects, the sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain shown in Figure 2The sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain shown in MF8232. The VH variable region containing this cMET binding site contains, in certain aspects, as Figure 3 The sequences of the CDR1 region, CDR2 region, and CDR3 region of the VH chain shown in MF8230. CDR grafting can also be used to produce VH chains having the Figure 2 or Figure 3 shown CDR regions of VH but having a different framework. This different framework can be the framework of another human VH or the framework of a different mammal.

[0238] The present invention further provides a human or humanized bispecific antibody, such as included in the treatment of the present invention, which comprises a first variable domain that binds to EGFR and a second variable domain that binds to cMET, wherein the first variable domain comprises a heavy chain variable region having an amino acid sequence as shown in Figure 2 MF3370, having at most 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 an amino acid sequence as shown in Figure 3 MF4356 (SEQ ID NO:23), having 0 - 10, in certain aspects 0 - 5 amino acid insertions, deletions, substitutions, or combinations thereof. The present invention further provides a human or humanized bispecific antibody, such as included in the treatment of the present invention, the bispecific antibody comprising a first variable domain that binds to EGFR and a second variable domain that binds to cMET, wherein the first variable domain comprises a heavy chain variable region having an amino acid sequence as shown in Figure 2 MF8233, having at most 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 an amino acid sequence as shown in Figure 3 MF8230 (SEQ ID NO:13), having 0 - 10, in certain aspects 0 - 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0239] The present invention further provides a human or humanized bispecific antibody, such as included in the treatment of the present invention, which comprises a first variable domain that binds to EGFR and a second variable domain that binds to cMET, wherein the first variable domain comprises a heavy chain variable region having an amino acid sequence as shown in Figure 2The amino acid sequence of MF3370 as shown, which has at most 10, and in some aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in some 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, which comprises as Figure 3 shown in the amino acid sequence of MF8230 (SEQ ID NO:13), which has 0 - 10, and in some aspects 0 - 5 amino acid insertions, deletions, substitutions or combinations thereof.

[0240] The present invention further provides a human or humanized bispecific antibody, such as included in the treatment of the present invention, which comprises a first variable domain that binds to EGFR and a second variable domain that binds to cMET, wherein the first variable domain comprises a heavy chain variable region, which has as Figure 2 shown in the amino acid sequence of MF8233, which has at most 10, and in some aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in some 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, which comprises as Figure 3 shown in the amino acid sequence of MF4356 (SEQ ID NO:23), which has 0 - 10, and in some aspects 0 - 5 amino acid insertions, deletions, substitutions or combinations thereof.

[0241] The present invention further provides a human or humanized bispecific antibody, such as included in the treatment of the present invention, which comprises a first variable domain that binds to EGFR and a second variable domain that binds to cMET, wherein the first variable domain comprises a heavy chain variable region, which has as Figure 2 shown in the amino acid sequence of MF8232, which has at most 10, and in some aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in some 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, which comprises as Figure 3 shown in the amino acid sequence of MF4356 (SEQ ID NO:23), which has 0 - 10, and in some aspects 0 - 5 amino acid insertions, deletions, substitutions or combinations thereof.

[0242] The present invention further provides a human or humanized bispecific antibody, such as included in the treatment of the present invention, which comprises a first variable domain that binds to EGFR and a second variable domain that binds to cMET, wherein the first variable domain comprises a heavy chain variable region, which has as Figure 2The amino acid sequence of MF8232 as shown, which has at most 10, and in some aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in some 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 that comprises the amino acid sequence of MF8230 as shown Figure 3 in (SEQ ID NO:13), which has 0-10, and in some aspects 0-5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0243] The at most 15, and in some aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in some aspects 0, 1, 2, 3, 4, or 5 amino acid substitutions described are in some aspects conservative amino acid substitutions, and the insertions, deletions, substitutions, or combinations thereof are in some aspects not in the CDR3 region of the VH chain, in some aspects not in the CDR1, CDR2, or CDR3 regions of the VH chain, and in some aspects not in the FR4 region.

[0244] There are multiple methods for manufacturing 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 produced in this way typically comprise a collection of antibodies with different combinations of heavy and light chains, some of which are the desired bispecific antibodies. The bispecific antibodies can subsequently be purified from the collection. The ratio of bispecific antibodies produced by the cell to other antibodies can be increased in various ways. In some aspects, the ratio is increased by expressing a common light chain in the cell rather than two different light chains. When the common light chain is expressed together with two different heavy chains, the ratio of bispecific antibodies produced by the cell to other antibodies is significantly increased compared to expressing two different light chains. The ratio of bispecific antibodies produced by the cell can be further increased by stimulating the pairing of the two different heavy chains with each other rather than the pairing of two identical heavy chains. Methods and means for manufacturing bispecific antibodies (from a single cell) are disclosed, thus providing means that favor the formation of bispecific antibodies rather than monospecific antibodies. These methods can also be advantageously used in the present invention. Accordingly, in some aspects, the present invention provides a method for manufacturing 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 of a heavy chain, b) a second nucleic acid molecule encoding a second CH3 domain of a heavy chain, wherein the nucleic acid molecules are provided in a manner that preferentially pairs the first and second CH3 domains of the heavy chains, the method further comprising the steps of culturing the host cell, allowing expression of the two nucleic acid molecules, and harvesting the bispecific antibody from the culture. The first and second nucleic acid molecules can be parts of the same nucleic acid molecule, vectors, or gene delivery vehicles and can be integrated at the same locus in the genome of the host cell. Alternatively, the first and second nucleic acid molecules are provided to the cell separately.

[0245] Certain aspects provide a method for manufacturing a bispecific antibody of the present invention from a single cell, wherein the bispecific antibody comprises two CH3 domains capable of forming an interface, the method comprising providing:

[0246] - 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,

[0247] The method further comprises the steps of culturing the cells, allowing expression of the proteins encoded by the two nucleic acid molecules, and harvesting the bispecific IgG antibody from the culture. In certain aspects, the cells also have a third nucleic acid molecule encoding a common light chain. The first, second, and third nucleic acid molecules can be part of the same nucleic acid molecule, a vector, or a gene delivery vehicle, and can be integrated at the same locus in the genome of the host cell. Alternatively, the first, second, and third nucleic acid molecules are provided to the cells separately. In certain aspects, the common light chain is based on O12, and in certain aspects, it is the rearranged germline human κ light chain IgVκ1 39*01 / IGJκ1*01 as described above. The means for preferential pairing of the first and the second CH3 domains is, in certain aspects, corresponding mutations in the CH3 domains of the heavy chain coding regions. Preferred mutations for the preferentially produced bispecific antibody 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. Accordingly, there is further provided a method for producing a bispecific antibody according to the invention, wherein the first CH3 domain comprises the amino acid substitutions L351K and T366K (EU-numbering), and wherein the second CH3 domain comprises the amino acid substitutions L351D and L368E, the method further comprising culturing the cells and allowing expression of the proteins encoded by the nucleic acid molecules, and harvesting the bispecific antibody from the culture. There is also provided a method for producing a bispecific antibody according to the invention, wherein the first CH3 domain comprises the amino acid substitutions L351D and L368E (EU-numbering), and wherein the second CH3 domain comprises the amino acid substitutions L351K and T366K, the method further comprising culturing the cells and allowing expression of the nucleic acid molecules, and harvesting the bispecific antibody from the culture. Antibodies producible by these methods are also part of the invention. The CH3 hetero-dimerization domain is, in certain aspects, an IgGl hetero-dimerization domain. The heavy chain constant region comprising this CH3 hetero-dimerization domain is, in certain aspects, an IgGl constant region.

[0248] One embodiment of the 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 certain aspects, a heavy chain variable region as shown in Figure 2 or Figure 3 , or a heavy chain variable region having 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof as shown in Figure 2 or Figure 3 . In certain aspects, the nucleic acid molecule comprises a nucleic acid sequence for encoding a heavy chain variable region as shown in Figure 2 or Figure 3A codon-optimized nucleic acid sequence of the amino acid sequence shown. The codon optimization is optimized for the species and / or cell type of the cells that produce 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 an encoding Figure 2 or Figure 3 nucleic acid molecule of the heavy chain.

[0249] The nucleic acid molecules used in the present invention are generally, but not exclusively, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Alternative nucleic acids are available to those skilled in the art. The nucleic acids according to the present invention are, for example, contained in cells. When the nucleic acid is expressed in the cells, the cells can produce the antibody according to the present invention. Accordingly, one embodiment of the present invention includes a cell comprising the antibody according to the present invention and / or the nucleic acid according to the present invention. The cell is, in some aspects, an animal cell, in some aspects a mammalian cell, in some aspects a primate cell, and in some aspects a human cell. Suitable cells are any cells that are capable of containing and, in some aspects, producing the antibody according to the present invention and / or the nucleic acid according to the present invention.

[0250] The present invention also provides a cell comprising the antibody according to the present invention. In some aspects, the cell (usually an in vitro, isolated or recombinant cell) produces the antibody. The cell can also be a stored cell that is capable of producing the antibody when removed from storage and cultured. In some aspects, the cell is a hybridoma cell, a Chinese hamster ovary (CHO) cell, an NS0 cell or a PER-C6 TM cell. In some aspects, the cell is a CHO cell. Also provided is a cell culture comprising the cell according to the present invention. Various research institutions and companies have developed cell lines for the large-scale production of antibodies, for example for clinical use. Non-limiting examples of such cell lines are CHO cells, NS0 cells or PER.C6 TM cells. These cells are also used for other purposes, such as protein production. Cell lines developed for the industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. Accordingly, one aspect includes the use of a cell line developed for the large-scale production of antibodies for the production of the antibody according to the present invention, including, in some aspects, for the production of an antibody comprising a nucleic acid molecule encoding a VH, VL, and / or heavy chain as shown in Figure 2 or Figure 3 shown.

[0251] The present invention also provides a method for manufacturing an antibody, which method comprises culturing the cells of the present invention and harvesting the antibody from the culture. In certain aspects, the cells are cultured in a serum-free medium. In certain aspects, the cells are adapted to suspension growth. Also provided is an antibody obtainable by the antibody manufacturing method according to the present invention. The antibody is, in certain aspects, purified from the culture medium of the culture. In certain aspects, the antibody is affinity purified.

[0252] 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 manufacturing for clinical purposes. In one aspect, the cells are human cells. In certain aspects, the cells are transformed with the adenovirus E1 region or a functional equivalent thereof. In certain aspects, the cell line is the PER.C6TM cell line or an equivalent thereof. In certain aspects, the cells are CHO cells or variant cells thereof. In certain aspects, the variant cells utilize the glutamine synthetase (GS) vector system to express the antibody.

[0253] After transient transfection in suspension 293F cells, the antibody of the present invention can be manufactured at a level >50 mg / L. The bispecific antibody can be purified to a purity greater than 98% with a yield >70%. Analytical characterization studies show that the bispecific IgG1 antibody is comparable to the bivalent monospecific IgG1. In terms of functional activity, the bispecific antibody of the present invention can demonstrate superior potency compared to cetuximab in vitro and in vivo.

[0254] The present invention also provides a pharmaceutical composition comprising the antibody according to the present invention. The pharmaceutical composition, in certain aspects, comprises a pharmaceutically acceptable excipient or carrier.

[0255] The antibody may comprise a label, which in certain aspects is a label for in vivo imaging. Such a label is generally not necessary for therapeutic applications. For example, in a diagnostic setting, the label may be helpful. For example, to visualize target cells in vivo. A variety of labels are suitable and many are known in the art. In certain aspects, the label is a radioactive label for detection. In certain aspects, the label is an infrared label. In certain aspects, the infrared label is suitable for in vivo imaging. A variety of 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).

[0256] The present invention also provides a method for treating an individual suffering from or at risk of suffering from a tumor, comprising administering to an individual in need thereof an antibody or pharmaceutical composition according to the present invention. The tumor is in certain aspects an EGFR, cMET or EGFR / cMET positive tumor. Prior to initiating said treatment, the method also in certain aspects comprises determining whether the individual has such an EGFR, cMET or EGFR / cMET positive tumor. The present invention also provides the 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.

[0257] To confirm whether a tumor is EGFR positive, one skilled in the art can determine using, for example, EGFR amplification and / or immunohistochemical staining. At least 10% of the tumor cells in a biopsy should be positive. The biopsy can also comprise 20%, 30%, 40%, 50%, 60%, 70% or more positive cells. To confirm whether a tumor is cMET positive, one skilled in the art can determine using, for example, cMET amplification and / or immunohistochemical staining. At least 10% of the tumor cells in a biopsy should be positive. The biopsy can also comprise 20%, 30%, 40%, 50%, 60%, 70% or more positive cells.

[0258] The cancer or tumor can be an EGFR, cMET, or EGFR / cMET positive cancer. In a preferred aspect, the present invention provides the treatment of an EGFR, cMET, or EGFR / cMET positive cancer which is 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; gastroesophageal 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 which in certain aspects includes non-small cell lung cancer. The individual is in certain aspects a human individual. The individual is in certain aspects an individual eligible for antibody treatment using an EGFR specific antibody (such as cetuximab). In certain aspects, the present invention can treat an individual comprising a tumor which in certain aspects is an EGFR / cMET positive cancer which in certain aspects is a tumor / cancer having an EGFR RTK resistant phenotype, an EGFR monoclonal antibody resistant phenotype or a combination thereof.

[0259] As used herein, the term "cancer" is equally applicable to the term "tumor", and thus the treatment of a tumor is also applicable to the treatment of cancer.

[0260] The antibody dose to be administered to a patient is typically within a therapeutic window, meaning an amount sufficient to achieve a therapeutic effect is used while the amount does not exceed a threshold that causes unacceptable levels of side effects. The lower the amount of antibody required to achieve the desired therapeutic effect, the larger the therapeutic window typically is. Thus, preferably, the antibodies according to the invention exert a sufficient therapeutic effect at a low dose. The dose can be within the range of the dosing regimen of cetuximab. The dose can also be lower. In certain aspects, the dose is 1000 mg, 1500 mg or 2000 mg. The dosing can be once a week or once every two weeks.

[0261] In certain aspects, the cancer is 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; gastroesophageal 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 a fixed dose of 1000 mg once a week.

[0262] In certain aspects, the cancer is 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; gastroesophageal 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 a fixed dose of 1000 mg once every two weeks.

[0263] In certain aspects, the cancer is 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; gastroesophageal 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 a fixed dose of 1500 mg once every two weeks.

[0264] In certain aspects, the cancer is 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; gastroesophageal 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 a fixed dose of 2000 mg once every two weeks.

[0265] In some aspects, bispecific antibodies that bind EGFR and cMET are provided to an individual at a dose of 1000, 1500 or 2000 mg, particularly using a fixed-dose prescription. Fixed-dose prescriptions have several advantages in terms of body surface or body weight administration, as it reduces preparation time and reduces potential dosing calculation errors. In some aspects, the administration of the bispecific antibody is once a week (Q1W), once every 2 weeks (Q2W) or once every 3 weeks (Q3W). In some aspects, the bispecific antibody is administered once every two weeks. In the art, such a dosing regimen is referred to as Q2W. In some embodiments, the fixed-dose prescriptions disclosed herein are 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 a prescription for an individual in which the individual is scheduled to receive the bispecific antibody at a substantially the same predetermined amount each day, an amount independent of the individual's body weight. According to some aspects, a fixed, once-weekly dose of 1000 mg of the bispecific antibody is provided to the individual. Alternatively, a fixed, once-every-two-weeks dose of 1000 mg of the bispecific antibody is provided to the individual. Alternatively, a fixed, once-every-two-weeks dose of 1500 mg of the bispecific antibody is provided to the individual. Alternatively, a fixed, once-every-two-weeks dose of 2000 mg of the bispecific antibody is provided to the individual.

[0266] Under other similar conditions, compared with cetuximab, the bispecific antibody according to the present invention induces less skin toxicity in some aspects. Compared with cetuximab, under other similar conditions, the bispecific antibody according to the present invention produces less pro-inflammatory chemokines (CXCL14 in some aspects). Compared with cetuximab, under other similar conditions, the bispecific antibody of the present invention induces less damage to antimicrobial RNases (Rnase 7 in some aspects).

[0267] 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 and high efficacy. The antibodies of the present invention can be used for various types and grades of EGFR-targeted therapy. When compared with antibodies that bind the same antigen on both arms, the antibodies of the present invention can have an increased therapeutic window. Compared with cetuximab antibodies, the bispecific antibodies of the present invention can exhibit better growth inhibitory effects in vitro, in vivo or in combination thereof.

[0268] The present invention provides a bispecific antibody for treating an individual who may have one or more of a variety of different types of tumors. The tumor can 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 certain aspects, the EGFR tyrosine kinase inhibitor is a third-generation EGFR tyrosine kinase inhibitor, and in certain aspects is osimertinib or an analogue thereof. In certain aspects, the cMET tyrosine inhibitor is or comprises capmatinib or tepotinib. In this and other embodiments, the tumor can be an HGF-related tumor.

[0269] EGFR-positive tumors are typically tumors having an EGFR activating mutation. An EGFR activating mutation is an EGFR mutation that results in activation of the EGF / EGFR signaling pathway. The EGFR activating mutation 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 can be an HGF-related tumor. Activation of the cMET / HGF signaling pathway is one way for EGFR-positive tumors to evade EGFR-targeted therapy. The cMET / HGF pathway can be activated in a variety of ways. Various activation methods have been described in the art, some of which are detailed 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-related tumors or HGF-dependent tumors. The antibodies of the present invention can also be used to at least partially inhibit this possible escape mechanism in EGFR-positive tumors. Such tumors can escape EGFR-targeted therapy through the selective outgrowth of tumor cells, in addition to which 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 can be a tumor in which the cMET / HGF signaling pathway is activated. The tumor can be a tumor associated with elevated levels of hepatocyte growth factor (HGF) or overexpression of the HGF receptor c-Met. The tumor can be a tumor in which growth is driven by EGF and / or HGF. A tumor is said to be driven by a growth factor if the signaling pathway in the tumor cells is activated in response to the presence of the growth factor and removal of the growth factor results in inhibition of tumor cell growth. The decrease 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 permit tumor growth, then the tumor is an HGF-related tumor.

[0270] EGFR-targeted therapies for various tumors have been reviewed in Vecchione et al., "Experimental cell research" Vol. 317 (2011): 2765-2771. Generally speaking, EGFR-targeted therapy is a therapy that uses molecules that interact with EGFR and inhibit EGFR-mediated signal transduction in cells.

[0271] The treatment methods or antibodies for treatment as shown herein also include, in certain aspects, the step of determining whether the tumor is an HGF-related tumor. In certain aspects, the antibodies of the present invention can inhibit the growth of HGF-related tumors.

[0272] When the range provided herein is between the number 1 and the number 2, the range includes the number 1 and the number 2. For example, the range between 2-5 includes the numbers 2 and 5.

[0273] When it is mentioned herein that one affinity is higher than another affinity, its Kd = lower than the other Kd. To avoid doubt, a Kd of 10e-9M is lower than a Kd of 10e-8M. An antibody with a Kd of 10e-9M has a higher affinity for the target than when the Kd is 10e-8M.

[0274] In certain aspects, at the start of treatment, at least one, more than one, or all of the following inclusion factors IF1-IF7 apply to the treated individual. In certain aspects, the individual includes or meets all inclusion factors IF1-IF8:

[0275] IF1. At least 18 years of age at the time of signing the informed consent form,

[0276] IF2. Having a histologically or cytologically confirmed solid tumor, with evidence of metastatic or locally advanced unresected disease, and being incurable.

[0277] IF3.1. For individuals who have failed previous standard first-line treatment: The individual has deteriorated or is intolerant to a treatment known to provide clinical benefit. The individual has NSCLC, which carries an activated EGFR mutation, including a tyrosine kinase inhibitor (TKI) sensitizing mutation, and / or an approved TKI-resistant mutation, or any activated c-MET mutation / amplification.

[0278] IF3.2. For individuals who have previously received first or higher order anti-cancer treatment: Deterioration or intolerance to a treatment known to provide clinical benefit. The patient must have: NSCLC with an EGFR exon 20 insertion that has deteriorated after platinum-based doublet combination therapy, or second (or higher) line patients with NSCLC with a cMet exon 14 skipping mutation, a population resistant to capmatinib or tepotinib, or who have been treated with a cMET TKI or not treated with a cMET TKI, or selected solid tumors with EGFR or cMet driver mutations or cMET alterations (including GC / GEJ, HNSCC, e.g., HNSCC refractory to approved therapy (regardless of driver mutation) or ESCC), NSCLC patients with rare EGFR mutations (G719X, L861Q, and S768I), or individuals with NSCLC with a c-MET exon 14 skipping mutation who have not received treatment for advanced disease. The individual is fatigued or intolerant to all approved therapies that have been proven to provide clinical benefit, or there are no available, approved treatment options.

[0279] IF4. Archival or fresh tumor tissue samples embedded in FFPE are available after the most recent treatment progression.

[0280] IF5. Have measurable disease as defined by the RECIST v1.1 radiographic method (patients with non-measurable but evaluable disease may be included in the dose escalation part).

[0281] IF6. Have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0282] IF7. Have a life expectancy ≥ 12 weeks.

[0283] IF8. Have adequate organ function, determined by at least one or all of the following:

[0284] IF8.1 Absolute neutrophil count (ANC) ≥ 1.5 X 10 9 / L

[0285] IF8.2 Hemoglobin ≥ 9 g / dL.

[0286] IF8.3 Platelets ≥ 100 x 10 9 / L.

[0287] IF8.4 Corrected total serum calcium within the normal range.

[0288] IF8.5 Serum magnesium within the normal range (or corrected with supplements).

[0289] IF8.6 Serum potassium within the normal range.

[0290] IF8.7 Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are equal to or less than 3.0 x the upper limit of normal (ULN), and total bilirubin is equal to or less than 1.5 x ULN, provided that in the case of liver involvement or malignancy, ALT / AST is equal to or less than 5 x ULN, and total bilirubin is equal to or less than 2 x ULN.

[0291] IF8.8 For patients with Gilbert’s syndrome, the conjugated bilirubin value is within the normal range.

[0292] IF8.9 For patients over 65 years of age, calculated according to the Cockroft and Gault formula or the MDRD formula, serum creatinine is equal to or less than 1.5 x ULN, or creatinine clearance is equal to or higher than 50 mL / min.

[0293] IF8.10 Serum albumin > 3.3 g / dL

[0294] In some aspects, all organ function measurements according to IF8 have upper limits observed in healthy individuals.

[0295] In some aspects, treating an individual includes one or more factors selected from IF1 - IF8. In some aspects, treating an individual includes factors IF2, IF3, IF5, and IF8. In some aspects, treating an individual includes all factors of IF1 - IF8.

[0296] In some aspects, at the start of treatment, at least one, more than one, or all of the following exclusion factors EF1 - EF16 apply to the treated individual:

[0297] EF1. Has central nervous system metastases, which:

[0298] EF1.1: Untreated and symptomatic, and may include individuals with asymptomatic lesions if the condition is considered stable;

[0299] EF1.2 Requires radiation or surgery;

[0300] EF1.3 Requires ongoing steroid treatment (> 10 mg prednisone or equivalent) to control symptoms within 14 days after administration, before the first dose is administered. Individuals with other central nervous system metastases are allowed.

[0301] EF2. Has known leptomeningeal involvement.

[0302] EF3. Participated in another clinical trial or was treated with any investigational drug within 4 weeks before the first dose was administered.

[0303] EF4. Systemic anti-cancer 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, nitrosourea), a 6-week washout period is required.

[0304] EF5. Have undergone major surgery or radiotherapy within 3 weeks after the first dose administration. Individuals who have previously received marrow radiotherapy equal to or greater than 25% at any time are ineligible.

[0305] EF6. Have clinically significant toxicity with a persistent grade greater than grade 1, related to previous anti-tumor therapy (except alopecia); provided that stable sensory neuropathy with NCI-CTCAE v5.0 grade equal to or less than 2 and hypothyroidism with grade equal to or less than 2, which is stable with hormone replacement therapy, are not excluded.

[0306] EF7. Have a history of allergic reactions or any toxicity attributable to human proteins or any excipients that require permanent discontinuation of these agents.

[0307] EF8. Have a history of clinically significant cardiovascular diseases, including but not limited to:

[0308] EF8.1 Have been diagnosed with deep vein thrombosis or pulmonary embolism within 1 month before the first dose of the investigational drug, or have been diagnosed with any of the following within 6 months before the first dose of the investigational drug: myocardial infarction, unstable angina, stroke, transient ischemic attack, coronary / peripheral artery bypass graft, or any acute coronary syndrome.

[0309] EF8.2 QT interval prolongation > 480 msec or clinically significant arrhythmia or electrophysiological disease (i.e., implanted implantable cardioverter defibrillator, or atrial fibrillation with uncontrolled heart rate). Clinically stable patients with a cardiac pacemaker are eligible.

[0310] EF8.3 Uncontrolled (persistent) arterial hypertension: systolic blood pressure > 180 mmHg and / or diastolic blood pressure > 100 mmHg.

[0311] EF8.4 Congestive heart failure (CHF), defined as New York Heart Association (NYHA) class III-IV, or hospitalization due to CHF within 6 months after the first dose of the investigational drug.

[0312] EF8.5 Clinically significant pericardial effusion.

[0313] EF8.6 Myocarditis.

[0314] EF9. Have a history of interstitial lung disease, including drug-induced interstitial lung disease, radiation pneumonitis, which requires long-term use of steroids or other immunosuppressive agents for treatment within 1 year.

[0315] EF10. Have a previous or concurrent malignancy, excluding non-basal cell skin cancer or cervical carcinoma in situ, unless the tumor is treated for curative or palliative purposes and the previous or concurrent malignancy does not affect the safety and efficacy assessment of the investigational drug.

[0316] EF11. Currently have a serious illness or medical condition, including but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic or psychiatric diseases.

[0317] EF12. Have active hepatitis B infection (HBsAg positive) without antiviral treatment. Individuals with active hepatitis B (HbsAg positive) must receive antiviral treatment with lamivudine, tenofovir, entecavir or other antiviral agents, starting at least 7 days or more before the first dose is administered. Individuals with a history of hepatitis B (anti-HBc positive, HbsAg and HBV-DNA negative) are eligible.

[0318] EF13. Have a positive HCV RNA test; individuals with spontaneous resolution of HCV infection (positive HCV antibody but no detectable HCV-RNA), or those who have achieved a sustained virological response after antiviral treatment and show no detectable HCV RNA for 6 months or more (using IFN-free regimens) or 12 months or more (using IFN-based regimens) after stopping antiviral treatment are eligible.

[0319] EF14. Have a known history of HIV (HIV 1 / 2 antibody). HIV patients with undetectable viral loads are allowed. HIV testing is not required unless mandated by local health authorities or regulations.

[0320] EF15. For sexually active male and female patients of childbearing potential, agree to use one of the following contraceptive methods throughout the trial and for 6 months after the final dosing of PB19478:

[0321] · Combined (estrogen and progestin) hormonal contraceptives (oral, vaginal, transdermal) associated with ovulation suppression

[0322] · Progestin-only hormonal contraceptives (oral, injectable, implantable) associated with ovulation suppression

[0323] · Intrauterine device (IUD)

[0324] · Intrauterine hormone-releasing system (IUS)

[0325] · Bilateral tubal occlusion

[0326] · Partner vasectomy

[0327] · Abstinence

[0328] EF16. Being pregnant or breastfeeding.

[0329] In some aspects, the treated individual meets one or more factors selected from the group consisting of EF1-EF16. In some aspects, the treated individual meets all of the factors EF1-EF16.

[0330] The patent documents or other matters cited herein should not be taken as an admission that such document or matter was known on the priority date of any claim, or that the information contained therein is part of common general knowledge.

[0331] The conjunction "and / or" between multiple recited elements is understood to include the options of separately and in combination. For example, when two elements are joined 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 one of these options is understood to fall within this meaning, and thus meets 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, and thus meets the requirements of the term "and / or".

[0332] For clarity and conciseness of description, each feature is described herein as part of the same or separate embodiments. However, it should be understood that the scope of the invention may include embodiments having combinations of all or some of the described features.

[0333] Item

[0334] 1. A bispecific antibody comprising a first variable domain that binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the 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 the sequence X3X4X5X6HWWLX7A, where 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; 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 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 combinations 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) an individual who has not received any prior anti-cancer treatment.

[0335] 2. 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 an EGFR tyrosine kinase inhibitor, iii) a cMET tyrosine kinase inhibitor or iv) an individual who has not received any prior anti-cancer treatment, said 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 the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the 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 the sequence X3X4X5X6HWWLX7A, where 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; having 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations 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 combinations thereof

[0336] 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) an individual who has not received any prior anti-cancer treatment, the bispecific antibody comprising a first variable domain capable of binding to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain capable of binding (or binding to) the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain 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; having 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations 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 combinations thereof.

[0337] 4. Use or method as described in any one of the preceding items, wherein the individual or cancer that has received prior treatment according to i) is resistant to treatment with a third-generation EGFR tyrosine kinase inhibitor.

[0338] 5. Use or method as described in any one of items 1 to 3, wherein the individual or cancer that has received prior treatment according to ii) is resistant to treatment with first-generation, second-generation and / or third-generation tyrosine kinase inhibitors.

[0339] 6. Use or method as described in any one of items 1 to 3, wherein the individual or cancer that has received prior treatment according to iii) is resistant to treatment with a cMET tyrosine kinase inhibitor.

[0340] 7. Use or method as described in any one of the preceding items, wherein the administration of the bispecific antibody according to i) comprises second-line treatment, and the administration according to ii) or iii) comprises third-line treatment.

[0341] 8. Use or method as described in any one of the preceding items, wherein the first-generation EGFR tyrosine kinase inhibitor comprises or is gefitinib, erlotinib or icotinib.

[0342] 9. The use or method according to any one of the foregoing items, wherein the second-generation EGFR tyrosine kinase inhibitor comprises or is afatinib, dacomitinib, XL647, AP26113, CO-1686 or neratinib

[0343] 10. The use or method according to any one of the foregoing items, wherein the third-generation EGFR tyrosine kinase inhibitor comprises Osimertinib, Lazertinib, Alflutinib, Rezivertinib, Rociletinib, Olmutinib, Almonertinib, Abivertinib, ASK120067, Befotertinib, Olmutinib, Rociletinib or SH-1028 (Nazartinib (EGF816), Naquotinib (ASP8273), Mavelertinib (PF-0647775), Olafertinib (CK-101), Keynatinib or ES-072, preferably Osimertinib).

[0344] 11. The use or method according to any one of the foregoing 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.

[0345] 12. The use or method as described in any one of the preceding items, wherein the chemotherapy includes platinum-based chemotherapy, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide, or pemetrexed, or any combination thereof, preferably a composition containing cisplatin or carboplatin.

[0346] 13. The use or method as described in 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.

[0347] 14. The use or method as described in any one of the preceding items, wherein the cancer comprises an EGFR aberration, a cMET aberration, and / or an EGFR and cMET aberration.

[0348] 15. The use or method as described in any one 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.

[0349] 16. The use or method as described in any one of the preceding items, wherein the cancer comprises a deletion mutation in exon 19, preferably an in-frame deletion of exon 19, a missense mutation in exon 20 (such as T790M), or a mutation in exon 21, such as L858R.

[0350] 17. The use or method as described in each of the preceding claims, wherein the cancer comprises an EGFR exon 20 mutation, preferably an exon 20 insertion mutation.

[0351] 18. The use or method according to any one of the foregoing 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).

[0352] 19. The use or method according to any one of the foregoing 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_N771insGL, H773_V774insPH, A763_Y764insFQEA, D770_N771delinsEGN, D770_N771insGD, D770_N771insH, D770_N771insP, H773_V774insAH, 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 the mutations T790M, L792X (such as L792H), C796X (such as G796R, G796S, G796D), C797X (such as 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_A7671nsA, 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_V774InsX1-7, or V774_C775 InsX1-7.

[0353] 20. The use or method according to any one of the preceding items, wherein the cancer includes cMET aberration, such as cMET amplification, cMET overexpression, enhanced signal of the cMET pathway, cMET gene amplification, increased cMET protein activity, and / or increased HGF expression.

[0354] 21. The use or method according to any one of the preceding items, wherein the cancer comprises a cMET exon 14 skipping mutation.

[0355] 22. The use or method according to any one of the preceding items, wherein the cancer is non-small cell lung cancer (NSCLC), head and neck cancer, particularly head and neck squamous cell carcinoma; gastric cancer, particularly gastric adenocarcinoma; esophageal cancer, particularly esophageal squamous cell carcinoma; gastroesophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer.

[0356] 23. The use or method according to any one of the preceding items, wherein the cancer is NSCLC, which comprises an activated EGFR mutation, an EGFR tyrosine kinase inhibitor sensitizing mutation (such as exon 19 deletion and L858X), an acquired EGFR tyrosine kinase inhibitor resistant mutation (such as T790X, C797X, L792X, L798X), and an approved EGFR tyrosine kinase inhibitor resistant mutation, an EGFR exon 20 insertion mutation, an activated 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.

[0357] 24. The use or method according to any one of the preceding items, wherein the cancer is advanced or metastatic cancer.

[0358] 25. The use or method according to any one of the preceding items, wherein the cancer or patient deteriorates after receiving prior treatment for the advanced or metastatic cancer.

[0359] 26. The use or method according to any one of the preceding items, wherein the treatment comprises a diagnostic step for evaluating whether the cancer is an EGFR-positive and / or cMET-positive cancer, or for evaluating the presence of EGFR and / or cMET aberration in the cancer.

[0360] 27. The use or method according to any one of the preceding items, wherein the treatment comprises diagnostic steps 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.

[0361] 28. The use or method according to any one of the preceding items, wherein the individual is a human individual.

[0362] 29. The use or method according to any one of the preceding items, wherein the antibody is a human antibody.

[0363] 30. The use or method according to any one of the preceding items, wherein the antibody is enhanced by ADCC.

[0364] 31. The use or method according to any one of the preceding items, wherein the antibody is an IgG1-form antibody having an anti-EGFR and anti-cMET stoichiometric ratio of 1:1.

[0365] 32. The use or method according to any one of the preceding items, wherein the antibody has a variable domain that can bind to EGFR and a variable domain that can bind to cMET.

[0366] 33. The use or method according to any one of the preceding items, wherein the variable domain that can bind to human EGFR can also bind to cynomolgus and mouse EGFR.

[0367] 34. The use or method according to any one of the preceding items, wherein the variable domain that can bind to human EGFR binds to domain III of human EGFR.

[0368] 35. The use or method according to any one of the preceding items, wherein the variable domain that can bind to cMET blocks the binding of antibody 5D5 to cMET.

[0369] 36. The use or method according to any one of the preceding claims, wherein the variable domain that can bind to cMET blocks the binding of HGF to cMET.

[0370] 37. The use or method according to any one of the preceding items, wherein the amino acids at positions 405 and 409 in one CH3 domain are the same as the amino acids at the corresponding positions in the other CH3 domain (EU numbering).

[0371] 38. The use or method according to any one of the preceding items, wherein

[0372] X1 = N; X2 = G; X3 = D; X4 = S; X5 = Y; X6 = W and X7 = G;

[0373] X1 = N; X2 = A; X3 = D; X4 = S; X5 = Y; X6 = W and X7 = G;

[0374] X1 = S; X2 = G; X3 = D; X4 = S; X5 = Y; X6 = W and X7 = G;

[0375] X1 = N; X2 = G; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D;

[0376] X1 = N; X2 = A; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D;

[0377] X1 = S; X2 = G; X3 = D; X4 = R; X5 = H; X6 = W and X7 = D;

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

[0379] X1 = N; X2 = A; X3 = G; X4 = Y; X5 = L; X6 = D and X7 = G; or

[0380] X1 = S; X2 = G; X3 = G; X4 = Y; X5 = L; X6 = D and X7 = G

[0381] 39. The use or method according to any one 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.

[0382] 40. The use or method according to any one 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.

[0383] 41. The use or method according to any one 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, which has 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof.

[0384] 42. The use or method according to any one 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, which has 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof.

[0385] 43. The use or method according to any one of the preceding items, wherein 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, 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.

[0386] 44. The use or method according to any one of the preceding items, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence TYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG, and a CDR3 comprising the sequence ETYFYDRGGYPFDP.

[0387] 45. The use or method according to any one of the preceding items, wherein the first and second variable domains comprise a common light chain, preferably the light chain in Figure 4B the

[0388] 46. The use or method according to any one of the preceding items, wherein the first and second variable domains comprise light chains having CDR1, CDR2 and CDR3 amino acid sequences QSISSY, AAS and QQSYSTP (according to IMGT), respectively.

[0389] 47. The use or method according to any one of the preceding items, wherein the antibody inhibits the growth of HGF-induced HGF-growth responsive cells.

[0390] 48. The use or method according to any one of the preceding items, wherein the antibody inhibits the growth of EGF-induced EGF-growth responsive cells.

[0391] 49. The use or method according to any one of the preceding items, wherein 1000 mg of the bispecific antibody is administered to an individual, especially using a fixed dose of 1000 mg.

[0392] 50. The use or method according to item 49, wherein the bispecific antibody is administered once a week.

[0393] 51. The use or method according to item 49, wherein the bispecific antibody is administered once every two weeks.

[0394] 52. The use or method according to any one of the preceding items 1 - 48, wherein 1500 mg of the bispecific antibody is administered to the individual, in particular using a fixed dose of 1500 mg.

[0395] 53. The use or method according to item 52, wherein the bispecific antibody is administered once every two weeks.

[0396] 54. The use or method according to any one of the preceding items 1 - 48, wherein 2000 mg of the bispecific antibody is administered to the individual, in particular using a fixed dose of 2000 mg.

[0397] 55. The use or method according to item 54, wherein the bispecific antibody is administered once every two weeks.

[0398] 56. The use or method according to any one of the preceding items, wherein the individual in iv) has not received any prior anti - cancer treatment using a cMET inhibitor.

[0399] 57. The use or method according to any one of the preceding items, wherein the individual in iv) has not received any prior anti - cancer treatment using a cMETTKI inhibitor.

[0400] 58. The use or method according to any one of the preceding items, wherein the individual in iv) has not received any prior anti - cancer treatment using capmatinib.

[0401] 59. The use or method according to any one of the preceding items, wherein the individual in iv) has not received any prior anti - cancer treatment using tepotinib.

[0402] 60. The use or method according to any one of the preceding items, wherein the individual in iv) has not received any prior anti - cancer treatment using savolitinib.

[0403] Examples

[0404] As used herein, "MFXXXX", where X is independently a digit from 0 - 9, refers to a Fab comprising a variable domain, wherein VH has an amino acid sequence identified by a 4 - digit number. Unless otherwise specified, the light - chain variable region of the variable domain generally has Figure 4A, a sequence that is typically 4b. "MFXXXX VH" refers to the amino acid sequence of VH identified by 4 digits. MF also contains the light chain constant region and the heavy chain constant region that usually interacts with the light chain constant region. PG refers to a monospecific antibody containing the same heavy chain and light chain. PB refers to a bispecific antibody having two different heavy chains. The VH variable regions of the heavy chains are different and are usually also the CH3 regions, with a KK mutation in the CH3 domain of one heavy chain and a complementary DE mutation in the CH3 domain of the other heavy chain (see reference document PCT / NL2013 / 050294 (publication number WO2013 / 157954)).

[0405] Please refer to PCTNL / 2018 / 050537 (publication number WO2019 / 031965) for detailed information on the production of the antibodies of the present invention. Bispecific antibodies that bind EGFR and cMET and are applicable to the attached examples and the methods of the present invention include those listed in Tables 3, 4, 5, and 6. Specifically, the bispecific antibody PB19478 is applicable to the attached examples.

[0406] Each bispecific antibody contains two VH designated by MF numbers, which can bind EGFR and cMET respectively, and also contains an Fc tail with a KK / DE CH3 heterodimerization domain, as shown in Figure 5E and Figure 5F respectively, a CH2 domain as shown in Figure 5D respectively, a hinge domain as shown in Figure 5B respectively, a CH1 domain as shown in Figure 5A respectively, and a common light chain as shown in Figures 4A to 4E respectively. For example, the bispecific antibody designated as MF8233 x MF8230 has the above general sequence, as well as the variable regions of VH with the sequence of MF8233 and the variable regions of VH with the sequence of MF8230, and is used in the attached examples in some aspects.

[0407] Example 1: Materials and Methods

[0408] Cell line:

[0409] 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-1555TM The 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.

[0410] cDNA construct:

[0411] Generate cMET and EGFR expression vectors for generating stable cell lines (cMET and EGFR) and for immunization (cMET)

[0412] 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 the target cDNA), 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 inserted sequence was verified by comparison with the NCBI reference amino acid sequence.

[0413] Amino acid sequence of the full-length human EGFR insert fragment for cell surface expression (equivalent to GenBank: NP_00533):

[0414]

[0415] Wherein:

[0416] -MRPSGTAGAALLALLAALCPASR: Signal peptide.

[0417] -ALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS: ECD of human EGFR.

[0418] -IATGMVGALLLLLVVALGIGLFM: Predicted TM region.

[0419] -RRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA: Intracellular tail.

[0420] Amino acid sequence of the extracellular domain of human EGFRvarIII, a naturally occurring EGFR variant VAR_066493 [Ji H., Zhao X; PNAS 103:7817-7822 (2006)], caused by an in-frame deletion of exons 2-7. The following _ indicates the absence of amino acid positions 30 - 297.

[0421] MRPSGTAGAALLALLAALCPASRALEEKK_GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[0422] Among them:

[0423] -MRPSGTAGAALLALLAALCPASR: Signal peptide.

[0424] -ALEEKK_GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS: ECD of EGFRvarIII

[0425] Amino acid sequence of the extracellular EGFR domain of a chimeric rhesus macaque (Macaca mulatta) that hybridizes with the human EGFR transmembrane and intracellular domains, for expression on the cell surface (equivalent to GenBank: XP_014988922.1). In the following examples, the human EGFR sequence is underlined.

[0426] MGPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSEFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDTLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSSQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCQNVSRGRECVDKCNILEGEPREFVENSECIQCHPECLPQVMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCARNGPKIPS IATGMLGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQE RELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDE AYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLA ARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYD GIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPT DSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYS SDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCV NSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA

[0427] Among them:

[0428] - MGPSGTAGAALLALLAALCPASR: Signal peptide.

[0429] -LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS: ECD of cyEGFR

[0430] Amino acid sequence of the full-length human cMET insert for cell surface expression (identical to GenBank: P08581-2). This sequence differs from the reference sequence by an insertion at position 755-755: S→

[0431] STWWKEPLNIVSFLFCFAS

[0432]

[0433] Wherein:

[0434] -MKAPAVLAPGILVLLFTLVQRSNG: signal peptide

[0435] -ECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFT: ECD of human cMET

[0436] -GLIAGVVSISTALLLLLGFFLWL: Transmembrane region

[0437] -KKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS: Intracellular region

[0438] Reference antibody

[0439] Anti-cMET antibodies are known in the art (Table 1). Monospecific bivalent cMET antibodies were constructed based on publicly available information and expressed in 293F Freestyle cells. Table 1 shows the relevant disclosure information. Monospecific bivalent antibodies against cMET were constructed based on the disclosed information and expressed in 293F Freestyle cells. For the assay of HGF ligand blockade, the VH- and VL-encoding gene fragments of the patent-derived anti-cMET antibody were recloned into a phage display vector for display on filamentous phage.

[0440] The reference antibody cetuximab (Erbitux) was used as the reference antibody for the EGFR Fab group.

[0441] The 2994 Fab protein was generated by digesting purified PG2994 IgG with papain. Thus, PG2994 was incubated with papain (Pierce #44985) conjugated to beads and allowed to cleave at 37 °C for 5.5 hours under rotating conditions. The Fab fragment was purified from the digestion mixture by filtration through MabSelectSure LX. The fraction containing the Fab protein in the effluent was concentrated to 3 ml using a vivaspin20 10 kDa and further purified by gel filtration using a superdex75 16 / 600 column (in PBS solution).

[0442] Example 2

[0443] Generation of Bivalent Monoclonal Antibodies and Antibody Characterization

[0444] Based on the VH gene sequence and some of its sequence variants, the VH gene of the unique antibody was cloned into a backbone IgG1 vector. Suspension-adapted 293F Freestyle cells were cultured in a T125 flask on a shaker until a density of 3.0x10 6 cells / ml. The cells were seeded into each well of a 24-deep well plate at a density of 0.3 - 0.5x10 6 viable cells / ml. The cells were transiently transfected with a separate 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 beads using batch purification, after which the buffer was changed to PBS.

[0445] Cross-Blocking Assay of cMET Antibodies

[0446] The competition between cMET-specific phages and a cMET reference antibody was tested in an ELISA. Thus, 2.5 μg / ml of cMET-Fc fusion protein was coated onto a MAXISORPTM ELISA plate overnight at 4 °C. Each well on the ELISA plate was blocked with PBS (pH 7.2) containing 2% ELK by shaking (700 rpm) at room temperature for 1 hour. The next reference or negative control IgG was added at a concentration of 5 μg / ml and allowed to bind at 700 rpm at room temperature for 15 minutes. Thereafter, 5 μl of PEG-precipitated phage was added and allowed to bind at 700 rpm at room temperature for 1 hour. The bound phage was detected with an HRP-labeled anti-M13 antibody at 700 rpm at room temperature for 1 hour. As a control, the procedure was carried out simultaneously with an antibody specific to the coated antigen and a negative control phage. The bound secondary antibody was detected by TMB / H 2 O2 Visualize the staining and quantify the staining by OD 450nm Table 2 shows that MF4040 and MF4356 exhibit competition with the 5D5 reference antibody. MF4297 shows less competition with 13.3.2 and C8H241. The positive control phages all show complete competition with the corresponding IgG, while the control group without antibody does not affect the competition assay.

[0447] Generation of bispecific antibodies

[0448] Bispecific antibodies are generated by transient co - transfection of two plasmids encoding IgGs with different VH domains, using proprietary CH3 engineering techniques 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 generating bispecific antibodies from a single cell, thereby providing means that favor the formation of bispecific antibodies over monospecific antibodies. These methods can also be advantageously used in the present invention. Specifically, the preferred mutations that essentially only generate 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 the "KK - variant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, such as L351D and L368E (referred to as the "DE - variant" heavy chain), and vice versa. It has been previously demonstrated in our co - pending applications that the negatively charged DE - variant heavy chain and the positively charged KK - variant heavy chain preferentially pair to form heterodimers (referred to as the "DEKK" bispecific molecule). Homodimerization of the DE - variant heavy chain (DE - DE homodimer) or homodimerization of the KK - variant heavy chain (KK - KK homodimer) is less favorable due to the strong repulsion between the charged residues at the CH3 - CH3 interface between the same heavy chains.

[0449] Table 3 shows which cMET and EGFR Fab arms were cloned into the appropriate KK and DE vectors. After manufacture, the bispecific IgG was purified by protein-A batch purification and the buffer was changed to PBS. Successful manufacture results in an IgG1 full-length antibody at a minimum concentration of 0.1 mg / ml, which is assigned a unique code (PBnnnnn; where nnnnn represents a randomly generated number) to identify the specific combination of Fab fragments that bind to two different targets. The successfully manufactured bispecific IgG was tested for binding to its respective targets in ELISA. For more details on bispecific antibody manufacture, reference is made herein to PCTNL / 2018 / 050537 (published as WO2019 / 031965).

[0450] Example 3

[0451] Screening of c-MET x EGFR bispecific antibodies in EGF / HGF and HGF and EGF proliferation assays

[0452] The potency of a panel of cMET x EGFR bispecific antibodies was tested in N87 cells using HGF / EGF, HGF, and EGF assays. The N87 cell line, official name 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-logarithmic titration from 10 μg / ml to 3.16 ng / ml. Each antibody was tested in duplicate. The anti-RSV-G antibody PG2708 was used as a negative control. The reference antibody 2994Fab was used as a positive control for the HGF assay and the reference antibody cetuximab was used as a positive control for the EGF assay.

[0453] Equimolar 1:1 cetuximab / 5D5 Fab was used as a positive control for the EGF, HGF, and EGF / HGF assays.

[0454] Wells with a ligand or ligand composition and a media control are included to define the assay window. Antibodies are diluted in chemically defined starvation media (CDS: RPMI 1640 media containing 80 U penicillin and 80 μg streptomycin per milliliter, 0.05% (w / v) BSA, and 10 μg / ml holo-transferrin), and 50 μl of the diluted antibody is added to each well of a 96-well black clear bottom plate (Costar). Ligands are 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, catalog numbers 396-HB and 236-EG). N87 cells are trypsinized, harvested, and counted, and 8000 cells in 100 μl of CDS are added to each well of the plate. To avoid edge effects, the plate is 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, Alamarblue (Invitrogen, #DAL1100) (20 μl per well) is added, and the fluorescence is measured after incubating with Alamar blue for 6 hours (37 °C), using 560 nm excitation and 590 nm read on a Biotek Synergy 2 multimode microplate reader. The fluorescence values are normalized to uninhibited growth (antibody not added but both ligands added).

[0455] Table 4 lists the results of various experiments. In the N87 HGF / EGF assay, 14 different cMETxEGFR bispecific antibodies were identified that had potencies comparable to the reference monospecific antibodies (an equimolar mixture of cetuximab and 5D5 Fab): PB7679, PB7686, PB8218, PB8244, PB8292, PB8316, PB8340, PB8364, PB8388, PB8511, PB8535, PB8583, PB8607, and PB8640.

[0456] In the N87 EGF assay, 11 different cMETxEGFR bispecific antibodies were identified with potencies comparable to that of 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 potencies higher than that of the monospecific 5D5 Fab reference antibody: PB8218, PB8388, PB8511, PB8532, PB8535, PB8545, PB8583, PB8639, and PB8640. They contain six different cMET Fab arms: MF4040, MF4297, MF4301, MF4356, MF4491, and MF4506.

[0457] ADCC activity

[0458] The ADCC activities of 24 cMetxEGFR bispecific antibodies were tested against the tumor cell lines N87 (EGFR-high, cMET-low) and MKN-45 (EGFR-low, cMET-amplified). The ADCC assay was performed in 384-well plates using the Promega ADCC Biotest Kit. Antibodies were tested in duplicate at 9 different concentrations in a semi-logarithmic serial dilution ranging from 10 μg / ml to 1 ng / ml.

[0459] The reference cetuximab antibody was included as a positive control for the assay, and PG2708 was used as a negative control antibody. Antibodies or assay medium controls (no IgG) were incubated with ADCC effector cells and target cells (N87 or MKN-45) for 6 hours at 37°C to induce. Luciferase activity was quantified using the Bio-Glo luciferase reagent.

[0460] The cMETxEGFR bispecific antibodies 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.

[0461] Five bispecific antibodies composed of EGFR and cMet arms, which showed high efficacy and high sequence diversity in the N87 HGF / EGF assay (Table 5), were selected for further analysis. Two of the five bispecific antibodies contain MF4356, which competes with 5D5 for binding to cMET (Table 2). Table 5 summarizes the characteristics of the selected candidates.

[0462] Example 4

[0463] Figure 2 Shows the sequences of another variable region of the heavy chain of the EGFR-binding variable domain as disclosed herein. Figure 3 Shows the sequences of another variable region of the heavy chain of the cMET-binding variable domain as disclosed herein. The heavy chain variable regions are used to create a number of different cMET x EGFR bispecific antibodies. The light chains in these antibodies have sequences as Figure 4B shown. The bispecific antibodies are manufactured as described in Example 1. The antibodies are also manufactured in an ADCC-enhanced form. The ADCC-enhanced form is manufactured by adding DNA encoding a reductase to the co-transfection of the antibody construct, which removes fucose residues from the Fc region of IgG1. For a list of the bispecific antibodies used and their PB codes, see Table 6.

[0464] Example 5

[0465] The heavy chain variable region (VH) of the cMET variable domain of PB8532 contains the amino acids of MF4356, as Figure 3 shown. 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 contains the amino acids of MF3370, as Figure 2 shown. The VH of the EGFR variable domain of PB19748 contains the amino acid sequence of MF8233 as Figure 2 shown. The light chains in PB8532 and PB19748 are the same and are shown in Figure 4B . The cMET antibody LY2875358 antibody is described in Kim and Kim 2017.

[0466] Example 6

[0467] Efficacy of bispecific antibody PB19478 in an EGFR exon 20 insertion model

[0468] The purpose of this experiment was to evaluate the anti-tumor efficacy of PB19478 in patient-derived xenograft (PDX) non-small cell lung cancer (NSCLC) models 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, which 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.

[0469] Materials

[0470] PB19748 is produced by Merus, and the control material is the PB19748 vehicle, which consists of 12% of the PB19748 formulation buffer without antibodies. Although other negative controls, such as physiological saline or PBS, can also be used.

[0471] PDX model characteristics

[0472] The LXFE2478 model was generated in nude mice with functional Fc effector cells at Charles River.

[0473] 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.

[0474] 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- and KRAS and PTEN genes (whole exome sequencing of patient tumors and xenografts was performed and the results were consistent).

[0475] Expression of EGFR, c-MET and HGF in the LXFE2478 PDX model

[0476] The expression of EGFR and c-MET receptors and the ligand huHGF in the model LXFE2478 was analyzed to investigate its applicability to in vivo experiments.

[0477] The expression of EGFR and c-MET receptors and the ligand huHGF in two tumor masses was evaluated by western blot. huHGF was included in the analysis. Tumor samples obtained from the PDX model were evaluated by the SimpleWesternSize technology (SWS) to confirm the expression of EGFR, HGF and c-MET.

[0478] Western blotting showed that the LXFE2478 model expressed EGFR, HGF and MET at the protein level ( Figure 6 and Table 7).

[0479] Statistical methods:

[0480] The anti-tumor 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 groups with that of the control group and expressed as the minimum T / C percentage value. To evaluate the statistical significance of the anti-tumor efficacy, a non-parametric Kruskal-Wallis test was performed, followed by multiple comparisons using the Dunn method. The RTVs of the test group and the control group were 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 made on the same day. All p-values < 0.05 were considered statistically significant. Statistical calculations were performed using GraphPad Prism biostatistical software (version 9.10 for Microsoft Windows, GraphPad Software, San Diego, California, USA).

[0481] Treatment schedule and method:

[0482] Figure 7 Schematic diagram showing the treatment schedule.

[0483] Tumors were harvested from donor PDX mice, cut into fragments (LXFE2478: edge length 3 - 4 mm), and implanted subcutaneously (SC) in the flank of recipient nude mice. When the tumor implants reached approximately 80 - 200 mm 3 in a sufficient number of animals, the mice were divided into 5 groups of 5 mice each. Randomization was performed based on the "stratified distribution" method. Treatment started on the same day as randomization (day 0).

[0484] 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. The 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.

[0485] After randomization, the morbidity and mortality of the animals were routinely monitored, the animals were weighed twice a week, and the tumor volume (TV) was measured twice a week using calipers. The relative body weight (RBW) was calculated by dividing the absolute weight or volume on a given day by the absolute weight on day 0 and multiplying by 100. The 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 and multiplying by 100.

[0486] For mice with a body weight loss > 10%, the body weight was measured daily, and the animals had easy access to food and water and received DietGel. For mice with a body weight loss > 15%, treatment was suspended until their RBW recovered to ≥ 90%.

[0487] Monotherapy with the bispecific antibody PB19478 showed dose-dependent antitumor efficacy( Figure 8 ). Monotherapy at an antibody dose of 25 mg / kg led to tumor regression (minimum T / C value 2.7%), and was significantly more effective than treatment with vehicle and at doses of 0.5 mg / kg or 2.5 mg / kg. Treatment at 0.5 mg / kg and 2.5 mg / kg doses showed partial tumor regression.

[0488] Monotherapy was associated with low toxicity in mice, with negligible weight loss and an adjusted survival rate of 100% in all groups.

[0489] EGFR exon 20ins-mutated cancers are generally refractory to EGFR tyrosine kinase inhibitors and are associated with poor prognosis.

[0490] Example 7

[0491] Efficacy of the bispecific antibody PB19478 in an EGFR exon 19 deletion NSCLC model. The purpose of this experiment was to evaluate the antitumor efficacy of 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 (deletion of E746-A750) in exon 19 and is resistant to approved EGFR TKIs such as erlotinib.

[0492] Materials:

[0493] PB19748 was produced by Merus as described in Example 5.

[0494] CDX model characteristics:

[0495] The CC827-ER1 model was generated in BALB / c nude mice. It carries an EGFR mutation (deletion of E746-A750) in exon 19 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 in vitro exposure of the wild-type cell line to increasing concentrations of erlotinib.

[0496] Experimental procedure:

[0497] A mixture of HCC827-ER1 tumor cells and Matrigel was inoculated into the right anterior flank area of each mouse for tumor development. When the average tumor size reached approximately 125 (75 - 175) mm 3Random grouping began at that time. 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 random grouping day was designated as Day 0, and drug administration started from Day 0. The 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. Mice with a tumor volume exceeding 1500 mm 3 , or a body weight loss of more than 20% relative to the body weight on the first day of treatment, were terminated during the treatment period. The antibody was administered intraperitoneally (i.p.) twice a week at a dose of 25 mg / kg for 7 doses within 21 days. The eight mice in Group 1 were treated with a vehicle control (placebo buffer). A schematic overview of the treatment plan is shown as Figure 9 . Tumor volume (mm 3 ) was measured twice a week in two dimensions using calipers. The tumor growth of the animals and any effects of the treatment on behavior were examined, such as mobility, food and water consumption, weight gain / loss, eye / hair entanglement, and any other abnormalities. Body weight was measured twice a week after random grouping. None of the mice had a weight loss of more than 15%, and there was no suspension of the administration of the treatment reagent in any treatment group. Figure 10 Shows the effect of the bispecific antibody PB19478 on the tumor volume of the NSCLC CDX model during the entire observation period and indicates the cessation of treatment on Day 21. Antibody treatment showed tumor regression compared to the vehicle control group. All treatment groups were well tolerated. No mice in the trial showed a weight loss of >10% from the starting weight. In fact, on Day 21, the last treatment day, there was no statistically significant difference in the absolute body weight between Group 1 and Group 2. Compared with Group 1 and Group 2, the absolute body weight of mice in the further treatment group (i.e., 25 mg / kg osimertinib, orally administered once a day) was lower (P = 0.0167 compared with Group 2, data not shown). No adverse events were observed except for tumor crusting or tumor ulceration in some mice in Group 1 and Group 2. The animal survival curve is shown as Figure 11 . The significance of the log-rank (Mantel-Cox) test for the combination compared with each corresponding single treatment group was as follows: antibody 25 mg / kg vs. vehicle P = 0.0001.

[0498] In summary, these results show the preclinical anti-tumor activity of the antibody under study against the NSCLC CDX model carrying exon 19 mutations (E746-A750 deletion).

[0499] Example 8

[0500] In the dose escalation phase, the bispecific antibody PB19478 will be administered at increasing doses to NSCLC patients with activated EGFR mutations (TKI-sensitizing mutations and / or approved TKI-resistant mutations) or activated c-MET mutations (exon 14 skipping) / amplification (MET / CEP7 > 5 or cfDNA ≥ 2 copies), GC / GEJ adenocarcinoma patients with activated 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, all of whom have deteriorated after previous treatment for advanced / metastatic disease in all cases.

[0501] The allometric scaling of the preclinical PK model is used to predict antibody exposure in humans. The starting dose of the antibody is 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.

[0502] The patient population will receive antibody treatment until the MTD is reached or a lower recommended dose is determined.

[0503] Taking into account the available data on PK, pharmacodynamic activity, and preliminary anti-tumor activity, the RP2D (recommended Phase 2 dose) is defined as the dose equal to or lower than the MTD.

[0504] Dose expansion

[0505] Using the RP2D of 1500 mg, an expansion cohort in the Phase 2 plan can be initiated. The safety of the RP2D will be confirmed during the dose expansion in the first 12 patients treated with the antibody alone for at least 2 cycles (recruitment will continue simultaneously). The anti-tumor activity of the antibody (alone or in combination with Osimertinib) will be evaluated based on the ORR, and other efficacy parameters, safety, tolerability, PK, immunogenicity, and biomarkers will be evaluated.

[0506] The following locally advanced unresectable / metastatic solid tumor cohorts may be opened:

[0507] Group A: PB19478 monotherapy: NSCLC patients may carry EGFR exon 20 insertion mutations (first line [1L] and ≥ second line [2L], deteriorated after first-line chemotherapy). The results from NSCLC patients are described in Example 9.

[0508] Group B: PB19478 monotherapy: NSCLC patients may carry cMet exon 14 mutations in the ≥ 2L, capmatinib- and tepotinib-resistant populations, deteriorated after approved c-Met TKI inhibitors.

[0509] Cohort: PB19478 monotherapy: patients with GC / GEJ and HNSCC who may carry EGFR or cMet driver mutations. Results from patients with HNSCC are described in Example 10.

[0510] Trial Population

[0511] Inclusion Criteria

[0512] Patients must meet all of the following requirements to enter the study:

[0513] 1. Sign the informed consent form before starting any trial procedures.

[0514] 2. Be ≥ 18 years of age at the time of signing the informed consent form.

[0515] 3. Histologically or cytologically confirmed solid tumor with evidence of metastatic or locally advanced unresectable disease and incurable.

[0516] 1. Dose Escalation Part - Patients who have failed prior standard first-line therapy. Patients must have deteriorated or be intolerant to a therapy known to provide clinical benefit. There is no limit to the number of prior treatment regimens. Patients must have:

[0517] · Non-small cell lung cancer (NSCLC) carrying an activated EGFR mutation, including tyrosine kinase inhibitor (TKI) sensitizing mutations (e.g., 19del and L858R) and / or approved TKI-resistant mutations (e.g., acquired TKI-resistant mutations, i.e., T790M, C797S, L792, L798I, exon 20 insertion), or any activated 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).

[0518] * Note: Patient identification will be based on the previous treatment history with EGFR tyrosine kinase inhibitors and local testing in a CLIA-certified laboratory.

[0519] 2. Cohort Expansion Part - For patients with ≥ 2L, they must have deteriorated or be intolerant to a therapy known to provide clinical benefit. There is no limit to the number of prior treatment regimens. Patients must have:

[0520] Group A: NSCLC with EGFR exon 20 insertion that has deteriorated after platinum doublet combination therapy (may include a limited number of first-line patients).

[0521] Group B: NSCLC patients with cMet exon 14 skipping mutations ≥ 2L, resistant populations to capmatinib and tepotinib, or those who have been treated with cMET TKI or not.

[0522] Group C: Selected solid tumors (including GC / GEJ, HNSCC such as HNSCC refractory to approved therapies regardless of driver mutations, or ESCC), which carry EGFR or cMet driver mutations or cMET alterations. NSCLC patients with rare EGFR mutations (G719X, L861Q, and S768I) are not included in other groups. Patients are exhausted or intolerant to all approved therapies with proven clinical benefits, or there are no available, approved treatment options.

[0523] Group F: NSCLC with c-MET exon 14 skipping mutations, advanced disease not previously treated.

[0524] 4. Archived or fresh tumor tissue samples embedded in FFPE can be obtained after recent treatment progress.

[0525] 5. Have measurable disease as defined by the RECIST version 1.1 radiological method (patients with non-measurable but evaluable disease may be included in the dose escalation part).

[0526] 6. Eastern Cooperative Oncology Group (ECOG) performance status is 0 or 1.

[0527] 7. Have a life expectancy ≥ 12 weeks, as judged by the researcher.

[0528] 8. Have sufficient organ function, as judged by the researcher:

[0529] · Absolute neutrophil count (ANC) ≥ 1.5 X 10 9 / L

[0530] · Hemoglobin ≥ 9 g / dL

[0531] · Platelets ≥ 100 x 10 9 / L

[0532] · Corrected total serum calcium within the normal range

[0533] · Serum magnesium within the normal range (or corrected with supplements)

[0534] · Serum potassium within the normal range

[0535] · Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 3 x upper limit of normal (ULN), and total bilirubin ≤ 1.5 x ULN (for patients with Gilbert’s syndrome, eligibility is met if the conjugated bilirubin value is within the normal range); in the case of liver injury, ALT / AST ≤ 5 x ULN and total bilirubin ≤ 2 x ULN will be permitted.

[0536] · For patients aged 65 and above, calculated according to the Cockroft and Gault formula or the MDRD formula, serum creatinine ≤ 1.5 x ULN or creatinine clearance rate ≥ 50 mL / min

[0537] · Serum albumin > 3.3 g / dL

[0538] Exclusion criteria

[0539] The presence of any of the following criteria will exclude the patient from participating in the study:

[0540] 1. Central nervous system metastasis (not excluded during up-titration, mandatory during expansion):

[0541] · Untreated and symptomatic (patients with untreated, asymptomatic lesions may be included if the investigator determines that the condition is stable)

[0542] · Requiring radiotherapy or surgery.

[0543] · Requiring ongoing steroid treatment (> 10 mg prednisone or equivalent) to control symptoms within 14 days after entering the trial.

[0544] 2. Known leptomeningeal involvement.

[0545] 3. Participated in another clinical trial or received treatment with any investigational drug within 4 weeks before entering the trial.

[0546] 4. Systemic anti-cancer treatment 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 a long half-life, inclusion before the fifth half-life requires sponsor approval.

[0547] 5. Underwent major surgery or radiotherapy within 3 weeks after the first dose of the investigational drug. Individuals who have received ≥ 25% prior radiotherapy to the bone marrow at any time are ineligible.

[0548] 6. The investigator determines clinically significant toxicity with a persistent grade >1, related to previous anti-tumor treatment (except alopecia); stable sensory neuropathy ≤ grade 2 according to NCI-CTCAE v5.0 and hypothyroidism ≤ grade 2, which is stable with hormone replacement therapy.

[0549] 7. A history of allergic reactions or any toxicity attributable to human proteins or any excipients, which requires permanent discontinuation of these reagents.

[0550] 8. A history of clinically significant cardiovascular diseases, including but not limited to:

[0551] ·QT interval prolongation >480 msec, obtained from 3 electrocardiograms (ECGs), or clinically significant arrhythmia or electrophysiological diseases (i.e., implanted implantable cardioverter defibrillator or atrial fibrillation with uncontrolled heart rate), or any factor increasing the risk of QTc prolongation or arrhythmia events (such as electrolyte abnormalities). Clinically stable patients with a cardiac pacemaker are eligible.

[0552] ·Heart failure, congenital long QT syndrome, family history of long QT syndrome, sudden unexplained death in a first-degree relative under 40 years old, or any concomitant medications known to prolong the QT interval and cause polymorphic ventricular tachycardia (Torsades de Pointes).

[0553] ·Uncontrolled (persistent) arterial hypertension: systolic blood pressure >180 mmHg and / or diastolic blood pressure >100 mmHg.

[0554] ·Congestive heart failure (CHF) defined as New York Heart Association (NYHA) class III-IV or hospitalization due to CHF within 6 months after the first dose of the investigational drug.

[0555] 9. A history of interstitial lung diseases, including drug-induced interstitial lung disease, radiation pneumonitis, which requires long-term steroid or other immunosuppressive treatment within 1 year.

[0556] 10. Having a previous or concurrent malignancy, excluding non-basal cell skin cancer or carcinoma in situ of the cervix, unless the tumor was treated for curative or palliative purposes and the investigator believes that, with the sponsor's consent, the previous or concurrent malignancy does not affect the safety and efficacy assessment of the investigational drug.

[0557] 11. Currently suffering from severe diseases or medical conditions, including but not limited to uncontrolled active infections, clinically significant pulmonary, metabolic or mental diseases.

[0558] 12. Having active hepatitis B infection (HBsAg positive) without antiviral treatment. Note: Individuals with active hepatitis B (HbsAg positive) must receive antiviral treatment with lamivudine, tenofovir, entecavir, or other antiviral agents, 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.

[0559] 13. Positive HCV RNA test; Note: Patients with spontaneous resolution of HCV infection (positive HCV antibody but no detectable HCV-RNA), or those who have achieved a sustained virological response after antiviral treatment and have shown no detectable HCV RNA for ≥6 months (using IFN-free regimens), or ≥12 months after stopping antiviral treatment (using IFN-based regimens) are eligible.

[0560] 14. Known history of HIV (HIV 1 / 2 antibody). HIV patients with undetectable viral loads are permitted. HIV testing is not required unless mandated by local health authorities or regulations.

[0561] 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:

[0562] · Combined (estrogen and progestin) hormonal contraceptives associated with ovulation inhibition (oral, intravaginal, transdermal)

[0563] · Progestin-only hormonal contraceptives associated with ovulation inhibition (oral, injectable, implantable)

[0564] · Intrauterine contraceptive device (IUD)

[0565] · Intrauterine hormone-releasing system (IUS)

[0566] · Bilateral tubal occlusion

[0567] · Partner vasectomy 1

[0568] · Abstinence 2

[0569] 16. Pregnant or lactating women are excluded from this trial.

[0570] The ECOG performance status score levels are defined as follows in the art, with the meanings being: Grade 0: Fully active, able to perform all pre-illness activities without restriction. Grade 1: Limited physical activity, but able to walk and engage in light or sedentary work, such as simple housework, office work. Grade 2: Able to walk and take care of oneself, but unable to perform any work activities. Up to and about more than 50% of waking hours. Grade 3: Only able to perform limited self-care; more than 50% of waking hours are restricted to bed or chair. Grade 4: Completely disabled; unable to perform any self-care; completely confined to bed or chair. Grade 5: Dead.

[0571] Study treatment and prescription

[0572] 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), for a cycle of 4 weeks. Dose levels between 100 - 3000 mg (fixed dose) may be explored. Once the RP2D is reached, dose escalation will be stopped. The administered dose, dose increment, and dosing frequency for each patient (including the expansion cohort) may change based on patient safety, PK and pharmacodynamic data, and sponsor recommendations. The sponsor may recommend an alternative weekly dosing schedule for Cycle 1.

[0573] During treatment

[0574] The trial treatment will be administered until disease progression (per RECIST v1.1) is confirmed, unacceptable toxicity occurs, consent is withdrawn, the patient is non-compliant, the investigator decides (e.g., clinical deterioration), or the antibody is interrupted > continuously for 6 weeks.

[0575] Patients will be followed for safety at least 30 days after the last antibody infusion until all relevant toxicities have recovered or stabilized, and disease progression and survival status will be followed every 3 months for up to 1 year.

[0576] Example 9

[0577] In the clinical trial of Example 8, clinical efficacy was observed in patients with non-small cell lung cancer having an EGFR mutation, including EGFR exon 20 mutation / insertion, EGFR exon 21 mutation such as L858R, and EGFR exon 19 deletion mutation; and c-MET mutation, including c-MET exon 14 skipping mutation. More specifically, a reduction in target lesions was observed in several patients, including confirmed partial responses, two cases of stable disease, 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. The patients had previously received various different prior treatments, including platinum-based chemotherapy, Osimertinib, pembrolizumab, nivolumab, cetuximab, crizotinib, capmatinib, or tepotinib.

[0578] Example 10

[0579] 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 received cisplatin and cemiplimab treatment. More specifically, a 20% reduction in target lesions was observed in the patients who only experienced grade 1 / 2 adverse events.

[0580] Example 11

[0581] In the clinical trial of Example 8, after administration of the bispecific antibody PB19478 at 1500 mg Q2W, clinically relevant efficacy was observed in many patients with non-small cell lung cancer carrying a cMET exon 14 skipping mutation. Clinically relevant efficacy included confirmed partial responses (reviewed by the investigator according to RECIST v1.1 criteria) and stable disease. Several patients had previously received anti-cancer treatment, while several other patients had not received treatment, indicating that they received the bispecific antibody of the clinical trial protocol of Example 8 as the first anti-cancer treatment for cMET exon 14 skipping NSCLC.

[0582] Example 12

[0583] In the clinical trial of Example 8, after administration of the bispecific antibody PB19478 at 1500 mg Q2W, clinically relevant efficacy was observed in many patients with non-small cell lung cancer carrying an EGFR exon 20 insertion mutation. Clinically relevant efficacy included confirmed partial responses (reviewed by the investigator according to RECIST v1.1 criteria) and stable disease. Several patients had previously received anti-cancer treatment, such as platinum-based chemotherapy.

[0584] Cited References

[0585] 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.

[0586] Castoldi, R. et al., "A novel bispecific EGFR / Met antibody blocks tumor - promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity." Oncogene 32.50 (2013): 5593 - 5601.

[0587] Eisenhauer et al., New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). European Journal of Cancer 45 (2009) 228 - 247)

[0588] Ferguson KM. Structure - based view of epidermal growth factor receptor regulation. Annu Rev Biophys 2008; 37: 353 - –73.

[0589] Kim, George P. and Axel Grothey. "Targeting colorectal cancer with human anti - EGFR monoclonal antibodies: focus on panitumumab." Biologics 2.2 (2008): 223 - 228.

[0590] 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)

[0591] 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.

[0592] 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.

[0593] Robertson SC, Tynan J, Donoghue DJ. RTK mutations and human syndromes: when good receptors turn bad. Trends Genet 2000; 16: 368.

[0594] 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.

[0595] Table 1. Reference antibodies with specificity reported against the cMET extracellular domain.

[0596]

[0597] Table 2. Competition between cMet reference antibody and cMET cLC antibody. OD450 values are shown. The OD450 values indicate the presence or absence of competition with the antibody. MF4506 was not tested.

[0598]

[0599] Table 3. List of 24 cMETxEGFR bispecific antibodies selected after dose-dependent titration experiments in the N87 HGF / EGF proliferation assay. The MF numbers of the EGFR and cMET arms and their HCDR3 sequences in each individual PB are shown.

[0600]

[0601] Table 4. Summary of antibody titration experiments performed using 24 cMETxEGFR bispecific antibodies in the N87 HGF / EGF, HGF, and EGF proliferation assays. The bispecifics are labeled PBXXXX and the different Fab arms are labeled MGXXXX. The bispecific antibody activity in each assay is represented as: - no effect; + proliferation inhibition lower than the positive control group; ++ = proliferation inhibition equivalent to the positive control antibody 5D5 Fab; +++ = proliferation inhibition higher than the positive control antibody 5D5 Fab.

[0602]

[0603] Table 5. Composition of the most potent EGFRxcMET bispecific antibodies and their competition with reference antibodies.

[0604]

[0605] Table 6. Composition of bispecific antibodies. The pXX numbers represent the production run numbers and can be used to identify whether the antibody was produced in the ADCC version.

[0606] Bispecific antibody 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

[0607] Table 7: Relevant characteristics of model LXFE2478. AUC = area under the curve.

[0608]

[0609] Table 8 Treatment plan for the PDX model LXFE2478.

[0610]

[0611] Table 9|Treatment plan for the CDX model HCC827-ER1. BIW = twice a week, i.p. = intraperitoneal.

[0612]

[0613] The following content corresponds to the original claim in the parent application and is hereby incorporated herein as part of the specification:

[0614] 1. A bispecific antibody comprising a first variable domain that binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the 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 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; 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 an amino acid sequence that is one of the sequences of SEQ ID NO: 1-23 with 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof, the bispecific antibody for use in a method of treating cancer in an individual who has previously been 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.

[0615] 2. A method of treating cancer in an individual who has previously been treated 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 the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the 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 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; having from 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations 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 with from 0 to 10, preferably from 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof.

[0616] 3. Use of a bispecific antibody in the manufacture of a medicament for treating cancer in an individual who has previously been treated 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 the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the 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 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; having from 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations 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 with from 0 to 10, preferably from 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof.

[0617] 4. The use or method according to any one 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.

[0618] 5. The use or method according to 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-generation, second-generation, and / or third-generation tyrosine kinase inhibitor.

[0619] 6. The use or method according to 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.

[0620] 7. The use or method according to any one of the preceding items, wherein the administration of the bispecific antibody and the EGFR tyrosine kinase inhibitor according to i) and iii) is administered as a second-line treatment, and the administration according to ii) is administered as a third-line treatment.

[0621] 8. The use or method according to any one of the preceding items, wherein the first-generation EGFR tyrosine kinase inhibitor comprises gefitinib, erlotinib, or icotinib.

[0622] 9. The use or method according to any one of the preceding items, wherein the second-generation EGFR tyrosine kinase inhibitor comprises afatinib, dacomitinib, XL647, AP26113, CO-1686, or neratinib.

[0623] 10. The use or method according to any one of the preceding items, 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.

[0624] 11. The use or method according to any one 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.

[0625] 12. The use or method according to any one 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.

[0626] 13. The use or method according to any one of the preceding items, wherein the cancer is an EGFR-positive and / or cMET-positive cancer.

[0627] 14. The use or method according to any one of the preceding items, wherein the cancer comprises EGFR and / or cMET aberration.

[0628] 15. The use or method according to any one of the preceding items, wherein the cancer comprises an activated 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, a cMET mutation or a cMET aberration.

[0629] 16. The use or method according to any one 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 or an exon 21 mutation.

[0630] 17. The use or method according to any one of the preceding items, wherein the cancer comprises an EGFR exon 20 mutation, preferably an exon 20 insertion mutation.

[0631] 18. The use or method according to any one of the preceding items, wherein the cancer comprises an acquired tyrosine kinase inhibitor resistance mutation, such as a mutation that confers resistance to Osimertinib.

[0632] 19. The use or method according to any one of the preceding items, 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, V774_C775insHV, D770_N771insGL, H773_V774insPH, A763_Y764insFQEA, D770_N771delinsEGN, D770_N771insGD, D770_N771insH, D770_N771insP, H773_V774insAH, 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 the mutations T790M, L792X (such as L792H), C796X (such as G796R, G796S, G796D), C797X (such as 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.

[0633] 20. The use or method according to any one of the preceding items, wherein the cancer comprises cMET aberration, such as cMET amplification, cMET overexpression, enhanced signaling of the cMET pathway, cMET gene amplification, increased HGF expression, and / or increased cMET protein activity.

[0634] 21. The use or method according to any one of the preceding items, wherein the cancer comprises a cMET exon 14 skipping mutation.

[0635] 22. The use or method according to any one of the preceding items, wherein the cancer is non-small cell lung cancer (NSCLC), head and neck cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer.

[0636] 23. The use or method according to any one of the preceding items, wherein the cancer is advanced or metastatic cancer.

[0637] 24. The use or method according to any one of the preceding items, wherein the cancer or the patient deteriorates after receiving prior treatment for the advanced or metastatic cancer.

[0638] 25. The use or method according to any one of the preceding items, wherein the treatment comprises a diagnostic step for evaluating whether the cancer is an EGFR-positive and / or cMET-positive cancer, or for evaluating the presence of EGFR and / or cMET aberration in the cancer.

[0639] 26. The use or method according to any one of the preceding items, wherein the individual is a human individual.

[0640] 27. The use or method according to any one of the preceding items, wherein the antibody is a human antibody.

[0641] 28. The use or method according to any one of the preceding items, wherein the antibody is an ADCC-enhanced antibody.

[0642] 29. The use or method according to any one of the preceding items, wherein the antibody is an IgG1-form antibody having an anti-EGFR and anti-cMET stoichiometric ratio of 1:1.

[0643] 30. The use or method according to any one of the preceding items, wherein the antibody has a variable domain capable of binding EGFR and a variable domain capable of binding cMET.

[0644] 31. The use or method according to any one of the preceding items, wherein the variable domain capable of binding human EGFR can also bind macaque and mouse EGFR.

[0645] 32. The use or method according to any one of the preceding items, wherein the variable domain capable of binding to human EGFR binds to domain III of human EGFR.

[0646] 33. The use or method according to any one of the preceding items, wherein the variable domain blocking antibody 5D5 capable of binding to cMET blocks the binding of cMET.

[0647] 34. The use or method according to any one of the preceding items, wherein the variable domain capable of binding to cMET blocks the binding of HGF to cMET.

[0648] 35. The use or method according to any one of the preceding items, wherein the amino acids at positions 405 and 409 in one CH3 domain are the same as the amino acids at the corresponding positions in the other CH3 domain (EU numbering).

[0649] 36. The use or method according to any one of the preceding items, wherein

[0650] X 1 = N; X 2 = G; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G;

[0651] X 1 = N; X 2 = A; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G;

[0652] X 1 = S; X 2 = G; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G;

[0653] X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0654] X 1 = N; X 2 = A; X3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0655] X 1 = S; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D;

[0656] X 1 = N; X 2 = G; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G;

[0657] X 1 = N; X 2 = A; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G; or

[0658] X 1 = S; X 2 = G; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G。

[0659] 37. Use or method as described in any of the preceding items, wherein X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; or X 1 = N; X 2 = A; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; or X 1 = S; X 2 = G; X 3= D; X 4 = R; X 5 = H; X 6 = W and X 7 = D.

[0660] 38. The use or method according to any one of the preceding items, wherein X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; or X 1 = N; X 2 = A; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D.

[0661] 39. The use or method according to any one 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, 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 combinations thereof.

[0662] 40. The use or method according to any one 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, 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 combinations thereof.

[0663] 41. The use or method according to any one of the preceding items, wherein 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, 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.

[0664] 42. The use or method according to any one of the preceding items, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence TYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG, and a CDR3 comprising the sequence ETYFYDRGGYPFDP.

[0665] 43. The use or method according to any one of the preceding items, wherein the first variable domain and the second variable domain comprise a common light chain, preferably the Figure 4B light chain variable domain in

[0666] 44. The use or method according to any one of the preceding items, wherein the antibody inhibits the growth of HGF-responsive cells induced by HGF.

[0667] 45. The use or method according to any one of the preceding items, wherein the antibody inhibits the growth of EGF-responsive cells induced by EGF.

Claims

1. A bispecific antibody comprising a first variable domain that binds to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, where 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; 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, 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 with 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof, the bispecific antibody for use in a method of treating cancer in an individual who has previously been 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.

2. A method of treating cancer in an individual who has previously been treated 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 the human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, where 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; having 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations 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 with 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof.

3. Use of a bispecific antibody in the preparation of a medicament for treating cancer in an individual who has previously been treated 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, said bispecific antibody comprising a first variable domain capable of binding to the extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain capable of binding to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET), wherein said first variable domain 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; having from 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof at positions other than X1-X7, and wherein said second variable domain comprises a heavy chain variable region, said heavy chain variable region of said second variable domain having an amino acid sequence of one of the sequences of SEQ ID NO: 1-23 with from 0 to 10, preferably from 0 to 5 amino acid insertions, deletions, substitutions, additions or combinations thereof.

4. The use or method according to any one of the preceding claims, wherein the individual or cancer that has previously been treated according to i) is resistant to treatment with a third-generation EGFR tyrosine kinase inhibitor.

5. The use or method according to any one of claims 1 to 3, wherein the individual or cancer that has previously been treated according to ii) is resistant to treatment with first-generation, second-generation and / or third-generation tyrosine kinase inhibitors.

6. The use or method according to any one of claims 1 to 3, wherein the individual or cancer that has previously been treated according to iii) is resistant to treatment with a cMET tyrosine kinase inhibitor.

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

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

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

10. The use or method according to any one 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 according to any one 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 according to any one of the preceding claims, 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.

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

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

15. The use or method according to any one 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 the 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 according to 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 according to any one of the preceding claims, wherein the cancer comprises an EGFR exon 20 mutation, preferably an exon 20 insertion mutation.

18. The use or method according to any one of the preceding claims, 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 according to any one 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, V774_C775insHV, D770_N771insGL, H773_V774insPH, A763_Y764insFQEA, D770_N771delinsEGN, D770_N771insGD, D770_N771insH, D770_N771insP, H773_V774insAH, 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 the mutations T790M, L792X (such as L792H), C796X (such as G796R, G796S, G796D), C797X (such as 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 according to any one of the preceding claims, wherein the cancer comprises a cMET aberration, such as cMET amplification, cMET overexpression, enhanced signaling of the cMET pathway, cMET gene amplification, increased HGF expression, and / or increased cMET protein activity.

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

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

23. The use or method according to any one of the preceding claims, wherein the cancer is advanced or metastatic cancer.

24. The use or method according to any one of the preceding claims, wherein the cancer or patient has deteriorated after receiving prior treatment for the advanced or metastatic cancer.

25. The use or method according to any one 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 for assessing the presence of EGFR and / or cMET aberrations in the cancer.

26. The use or method according to any one of the preceding claims, wherein the individual is a human individual.

27. The use or method according to any one of the preceding claims, wherein the antibody is a human antibody.

28. The use or method according to any one of the preceding claims, wherein the antibody is an ADCC-enhanced antibody.

29. The use or method according to any one of the preceding claims, wherein the antibody is an IgG1-form antibody having an anti-EGFR and anti-cMET stoichiometric ratio of 1:

1.

30. The use or method according to any one of the preceding claims, wherein the antibody has a variable domain that can bind to EGFR and a variable domain that can bind to cMET.

31. The use or method according to any one of the preceding claims, wherein the variable domain that can bind to human EGFR can also bind to macaque and mouse EGFR.

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

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

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

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

36. The use or method according to any one of the preceding claims, wherein X 1 = N; X 2 = G; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G; X 1 = N; X 2 = A; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G; X 1 = S; X 2 = G; X 3 = D; X 4 = S; X 5 = Y; X 6 = W and X 7 = G; X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; X 1 = N; X 2 = A; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; X 1 = S; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; X 1 = N; X 2 = G; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G; X 1 = N; X 2 = A; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G; or X 1 = S; X 2 = G; X 3 = G; X 4 = Y; X 5 = L; X 6 = D and X 7 = G.

37. The use or method according to any one of the preceding claims, wherein X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; or X 1 = N; X 2 = A; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; or X 1 = S; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D.

38. The use or method according to any one of the preceding claims, wherein X 1 = N; X 2 = G; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D; or X 1 = N; X 2 = A; X 3 = D; X 4 = R; X 5 = H; X 6 = W and X 7 = D.

39. The use or method according to any one of the preceding claims, wherein the heavy chain variable region of the second variable domain comprises 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, 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 combinations thereof.

40. The use or method according to any one of the preceding claims, wherein the heavy chain variable region of the second variable domain comprises the 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 combinations thereof.

41. The use or method according to any one of the preceding claims, wherein 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, 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.

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

43. The use or method according to any one 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 according to any one of the preceding claims, wherein the antibody inhibits the growth of HGF-induced HGF-growth responsive cells.

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

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