Treatment with antibodies that bind to EGFR and CMET

Abstract

The invention disclosed herein relates to a 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), for use in a method of treatment of cancer in a subject who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor, or ii) chemotherapy and a tyrosine kinase 10 inhibitor, or iii) a cMET tyrosine kinase inhibitor.

Description

[Technical field]

[0001] This disclosure relates to the field of antibodies. In particular, this disclosure relates to the field of therapeutic antibodies, including human antibodies, for the treatment of diseases involving abnormal cells. Furthermore, this disclosure relates to antibodies that bind to EGFR and cMET, including multispecific antibodies, and their use in binding EGFR and cMET positive cells, particularly tumor cells. [Background technology]

[0002] The epidermal growth factor (EGF) receptor (EGFR) is a cell surface receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. EGFR is also known as the ErbB-1 receptor. The receptor has been given various names in the past (EGFR; ERBB; ERBB1; HER1; PIG61; mENA). In this disclosure, the human names ErbB-1, EGFR or HER1 are used interchangeably. EGFR is a member of the ErbB family of receptors, which are four closely related receptor tyrosine kinases: ErbB-1 (EGFR), ErbB-2 (HER2 / c-neu; Her2), ErbB-3 (Her3), and ErbB-4 (Her4).

[0003] EGFR is present on the cell surface and can be activated by the binding of its specific ligands, including epidermal growth factor and transforming growth factor alpha (TGFα). Upon activation by its growth factor ligand, the receptor can undergo a transition from an inactive, mostly monomeric form to an active homodimer. In addition to forming homodimers after ligand binding, EGFR can pair with another member of the ErbB receptor family, such as ErbB2, to create an activated heterodimer. Dimers can also form in the absence of ligand binding, and clusters of activated EGFR can form after ligand binding.

[0004] EGFR dimerization stimulates intrinsic intracellular protein-tyrosine kinase (PTK) activity, which induces several signal transduction cascades that lead to cell proliferation and differentiation. The kinase domain of EGFR can cross-phosphorylate tyrosine residues of other receptors to which it is complexed and thus can itself be activated.

[0005] Mutations involving EGFR have been identified in several types of cancer. It is the target of an expanding class of anti-cancer therapies, including EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib for lung cancer, and antibodies, such as cetuximab and panitumumab for colon and head and neck cancers.

[0006] Cetuximab and panitumumab are monoclonal antibodies that inhibit receptors. Other monoclonals in clinical development are zalutumumab, nimotuzumab and matuzumab. Monoclonal antibodies are primarily intended to block extracellular ligand-induced receptor activation by blocking ligand binding to the receptor. When the binding site is blocked, signal-inducing molecules may not bind effectively and thereby not activate downstream signaling. Ligand-induced receptor activation can also be inhibited by stabilization of the inactive receptor conformation (matuzumab).

[0007] Thus far, EGFR-targeted therapies have been associated with the development of treatment resistance over time. Various mechanisms for 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 reduction), impaired EGFR-TKIs-mediated apoptosis pathways (e.g., BCL2-like 11 / BIM deletion polymorphism), and histological transformation. Some mechanisms of resistance have been identified, while others remain to be identified. Moreover, in the case of third-generation TKI resistance mechanisms, the molecular heterogeneity of NSCLC influences its contribution to the wide range of resistance abnormalities discovered so far. Similarly, colorectal cancer patients treated with EGFR antibodies also develop resistance over time. This may occur due to the emergence of KRAS mutations. Among 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 beginning or may develop resistance during treatment. Resistance to EGFR-targeted therapy is seen in many EGFR-positive cancers, demonstrating the need in the art for more effective EGFR cancer treatments that improve the standard of care and are superior in terms of their ability to address EGFR-targeted therapy resistance.

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

[0009] The cMET receptor is formed into a single-pass disulfide-linked α / β heterodimer by proteolytic processing of a common precursor. The extracellular portion of cMET is composed of three domain types. The N-terminal region folds to form a large semaphorin (Sema) domain that encompasses the entire α subunit and part of the β subunit. A plexin-semaphorin-integrin (PSI) domain follows the Sema domain and contains four disulfide bonds. This domain is connected to the transmembrane helices through 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 carboxy-terminal sequences (Organ and Tsao. Therapeutic advances in medical oncology 3.1_suppl (2011): S7-S19, which are incorporated herein by reference in their entirety).

[0010] Ligands of cMET, hepatocyte growth factor (HGF; also known as scatter factor), and its splicing isoforms (NK1, NK2) are known ligands of the cMET receptor. HGF was identified in 1991 as a potent mitogen / morphogen. 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-associated and HGF-independent manner. HGF-independent activation occurs in the case of cMET overexpression. Abundant cMET can also induce (hetero)dimerization and intracellular signaling in the absence of ligand. Additional ligands do not appear to affect the function of such cMET-overexpressing cells. cMET amplification has been associated with cMET overexpression and has emerged as a biomarker for tumor subtypes.

[0011] HGF is expressed ubiquitously throughout the body, indicating that this growth factor is a systemically available cytokine and is also from the tumor stroma. Positive paracrine and / or autocrine loops of cMET activation may result in further cMET expression. The HGF-specific antibody rilotumumab (AMG102) has been developed for gastric cancer. Phase I and II trials were promising, but a phase III study (RILOMET-2) with cisplatin and capecitabine as first-line therapy in gastric cancer was terminated after a preplanned Data Monitoring Committee safety review of Study 20070622.

[0012] The relevance of cMET / HGF signaling in resistance to EGFR-targeted therapy has stimulated the development of approaches to address resistance. So far, antibody-based approaches, including anti-HGF antibodies, anti-cMET or cMET antibodies and cMET / EGFR (reviewed in Lee et al., 2015; Immunotargets and Therapy 4:35-44), have not been clinically effective. The cMET antibodies onartuzumab (MetMab™) and emibetuzumab (LY-2875358) have been evaluated in phase II clinical trials. Of these, onartuzumab appeared to be effective against colorectal cancer in combination treatment with the EGFR inhibitor erlotinib. However, these results could not be repeated in a randomized phase III clinical trial. MetMAb is a monovalent monoclonal antibody (mAb) against cMET that blocks HGF binding to cMET and subsequent pathway activation (Jin et al., 2008 Cancer Research Vol. 68: pp 4360-68).

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

[0014] So far, certain bispecific EGFR x cMET antibodies have been described in the art. Castoldi R. et al. (2013) describe a bispecific EGFR x cMET antibody called MetHer1, which has the cMET binding site of antibody 5D5 (or MetMab) and the EGFR binding site of cetuximab. The bispecific antibody has a fixed EGFR and cMET binding stoichiometry of 2:1 (see supplementary figure).

[0015] US2014 / 0378664 describes a cMETxEGFR bispecific antibody, among other bispecific antibodies. The complete bispecific antibody is produced as a single protein that is subsequently proteolytically cleaved. The two VH / VL domains are produced as single chain Fv fragments. Binding of the antibody induces cMET degradation and Akt phosphorylation in gastric cancer cell lines. Moores et al (2016) describe a bispecific cMETxEGFR antibody, named JNJ-61186372, produced by controlled Fab arm exchange (cFAE) with mutations at positions 405 and 409 according to EU numbering, which may have immunogenic potential. JNJ-61186372 was shown to be active in vivo using a xenograft model with the tumor cell line H1975 expressing 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 an affinity imbalance of approximately 40-fold higher affinity for cMET than EGFR (Moores et al. (2016)), and the anti-EGFR arm derived from zalutumumab is known to cause infusion-related reactions, skin disorders, among other issues.

[0016] LY3164530 is a bispecific cMETxEGFR antibody that contains 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 a so-called dual variable domain antibody that contains two binding sites for each of the antigens. No data on HGF inhibition are provided for the antibody. The antibody is reported to bind and internalize cMET and EGFR without agonistic activity. The authors review various cMET, EGFR, and cMETxEGFR targeted therapies and draw the conclusion that so far, none of these inhibitors have shown significant efficacy in clinical trials. Summary of the Invention [Problem to be solved by the invention]

[0017] Thus, there is a need for novel bispecific cMETxEGFR antibodies, including those that may have the superior properties as described herein. [Means for solving the problem]

[0018] In certain aspects, the disclosure provides i) a 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), for use in a method of treating cancer in a subject that has undergone prior treatment with a third generation EGFR tyrosine kinase inhibitor.

[0019] In certain aspects, the disclosure provides ii) a 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), for use in a method of treating cancer in a subject that has received prior treatment with chemotherapy and a tyrosine kinase inhibitor.

[0020] In certain aspects, the disclosure provides iii) a 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), for use in a method of treating cancer in a subject that has undergone prior treatment with a cMET tyrosine kinase inhibitor.

[0021] In certain aspects, the disclosure provides iv) a bispecific antibody comprising a first variable domain capable of binding to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain capable of binding to the extracellular portion of human MET proto-oncogene, receptor tyrosine kinase (cMET), for use in a method of treating cancer in a subject who has not received prior anti-cancer treatment. In certain aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0022] In certain aspects, the disclosure provides i) a treatment of cancer in a subject who has undergone prior treatment with a third generation EGFR tyrosine kinase inhibitor, the treatment comprising administering to the subject an effective amount of a bispecific antibody according to the disclosure, 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 to the extracellular portion of the human MET proto-oncogene, receptor tyrosine kinase (cMET).

[0023] In certain aspects, the disclosure provides ii) a treatment of cancer in a subject who has undergone prior treatment with chemotherapy and an EGFR tyrosine kinase inhibitor, the treatment comprising administering to the subject an effective amount of a bispecific antibody according to the disclosure, 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 to the extracellular portion of the human MET proto-oncogene, receptor tyrosine kinase (cMET).

[0024] In certain aspects, the disclosure provides iii) a treatment of cancer in a subject who has previously been treated with a cMET tyrosine kinase inhibitor, the treatment comprising administering to the subject an effective amount of a bispecific antibody according to the disclosure, 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 to the extracellular portion of the human MET proto-oncogene receptor tyrosine kinase (cMET).

[0025] In certain aspects, the disclosure provides iv) a treatment of cancer in a subject who has not received prior anti-cancer therapy, the treatment comprising administering to the subject an effective amount of a bispecific antibody according to the disclosure, the bispecific antibody comprising a first variable domain capable of binding to an extracellular portion of the human epidermal growth factor receptor (EGFR) and a second variable domain capable of binding to an extracellular portion of the human MET proto-oncogene, receptor tyrosine kinase (cMET). In certain aspects, the cancer comprises a cMET exon 14 skipping mutation.

[0026] In certain aspects, the disclosure provides i) the use of a bispecific antibody according to the disclosure 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), in the manufacture of a medicament for the treatment of cancer in a subject that has received prior treatment with a third generation EGFR tyrosine kinase inhibitor.

[0027] In certain aspects, the disclosure provides ii) the use of a bispecific antibody according to the disclosure comprising a first variable domain capable of binding to the extracellular part of the human epidermal growth factor receptor (EGFR) and a second variable domain capable of binding to the extracellular part of the human MET proto-oncogene, receptor tyrosine kinase (cMET), in the manufacture of a medicament for the treatment of cancer in a subject that has received prior treatment with chemotherapy and an EGFR tyrosine kinase inhibitor.

[0028] In certain aspects, the disclosure provides iii) the use of a bispecific antibody according to the disclosure 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), in the manufacture of a medicament for the treatment of cancer in a subject that has undergone prior treatment with a cMET tyrosine kinase inhibitor.

[0029] In certain aspects, the disclosure provides iv) the use of a bispecific antibody according to the disclosure comprising a first variable domain capable of binding to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain capable of binding to the extracellular portion of human MET proto-oncogene, receptor tyrosine kinase (cMET), in the manufacture of a medicament for the treatment of cancer in a subject who has not received prior anti-cancer treatment. In certain aspects, the cancer comprises a cMET exon 14 skipping mutation.

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

[0031] In certain embodiments, the subject or cancer that has undergone prior treatment involving a third generation EGFR tyrosine kinase inhibitor according to i) is resistant to treatment with a third generation EGFR tyrosine kinase inhibitor.

[0032] In certain embodiments, the subject or cancer that has undergone prior treatment including chemotherapy according to ii) and an EGFR tyrosine kinase inhibitor is resistant to treatment with a first, second and / or third generation tyrosine kinase inhibitor.

[0033] In certain embodiments, the subject or cancer that has had a prior treatment involving a cMET tyrosine kinase inhibitor according to iii) is resistant to treatment with a cMET tyrosine kinase inhibitor.

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

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

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

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

[0038] In certain embodiments, the cancer or subject has been previously treated with chemotherapy and a first, second or third generation tyrosine kinase inhibitor. In certain embodiments, the cancer or subject is resistant to treatment with a first, second or third generation tyrosine kinase inhibitor, or is resistant to a first and second generation tyrosine kinase inhibitor, or is resistant to a first, second and third generation tyrosine kinase inhibitor. In certain embodiments, the subject comprises an EGFR abnormality, or a cMET abnormality, or an EGFR and a cMET abnormality, that confers resistance to the first, second or third generation EGFR tyrosine kinase inhibitor.

[0039] In certain embodiments, the cancer or subject has undergone prior treatment with a cMET tyrosine kinase inhibitor and / or the cancer or subject is resistant to treatment with the cMET tyrosine kinase inhibitor. In certain embodiments, the subject comprises a cMET abnormality that confers resistance to the cMET tyrosine kinase inhibitor. In certain embodiments, the cMET inhibitor comprises capmatinib or tepotinib.

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

[0041] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor provided as a prior treatment includes or is osimertinib, lazertinib, alfurtiniib, resibertinib, rociletinib, olmutinib, almonertinib, abivertinib, ASK120067, befotertinib (also referred to as BPI-D0316 or D-0316), SH-1028, nazartinib (EGF816), nacotinib (ASP8273), mavereltinib (PF-0647775), orafeltinib (CK-101), kenatinib, ES-072, and in certain embodiments includes or is osimertinib. In certain embodiments, the EGFR tyrosine kinase inhibitor is osimertinib, BPI-D0316 / befotertinib, lazertinib, or almonertinib.

[0042] In certain embodiments, the first generation EGFR tyrosine kinase inhibitor provided as a pre-treatment with said chemotherapy comprises or is gefitinib, erlotinib, or icotinib.

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

[0044] In certain embodiments, the cMET tyrosine kinase inhibitor provided as a prior treatment is or comprises capmatinib, tepotinib, crizotenib, cabozantinib, savolitinib, glesatinib, sitravatinib, BMS-777607, merestinib, tivantinib, golvatinib, foretinib, AMG-337, or BMS-794833.

[0045] In certain embodiments, the prior chemotherapy includes platinum-based chemotherapy, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide, or pemetrexed, or any combination thereof, in certain embodiments, a combination including cisplatin or carboplatin.

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

[0047] In certain embodiments, 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, gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer, or bladder cancer. In certain embodiments, the cancer or subject comprises an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation, a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, an EGFR gene amplification, a cMET mutation, a cMET abnormality, or increased HGF expression.

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

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

[0050] In certain embodiments, the cMET dysregulation comprises cMET dysregulation caused by cMET amplification, cMET overexpression, increased cMET pathway signaling, cMET gene amplification, or increased cMET protein activity, or increased HGF expression.In certain embodiments, the cMET abnormality comprises cMET exon 14 skipping mutation.In certain embodiments, the cMET dysregulation is or comprises dysregulation of cMET signaling.

[0051] In certain embodiments, the use or treatment comprises providing to a subject a dose of 1000, 1500 or 2000 mg of the bispecific antibody. In certain embodiments, the bispecific antibody is provided once every week or once every two weeks. In certain embodiments, the use or treatment comprises providing to a subject a dose of 1500 mg of the bispecific antibody once every two weeks.

[0052] In certain embodiments, the bispecific antibodies of the present disclosure exhibit ADCC activity, and in certain embodiments, the antibodies have improved ADCC activity. In such embodiments, the antibodies may have altered ADCC activity by one or more CH2 mutations. Thus, bispecific antibodies according to the present disclosure are further provided that are afucosylated. In certain embodiments, the antibodies of the present disclosure comprise two afucosylated CH2 domains. In certain embodiments, the antibodies of the present disclosure comprise a total of two CH2 domains, both of which are afucosylated. In certain embodiments, the antibodies of the present disclosure comprise two CH2 domains, both of which are afucosylated.

[0053] In certain aspects, the bispecific antibodies of the present disclosure exhibit ADCP activity, and in certain aspects, the antibodies have improved ADCP activity. In certain aspects, both the EGFR binding arm and the cMET binding arm, or both the heavy chains containing the EGFR binding arm and the cMET binding arm, contribute to ADCP. In certain aspects, the bispecific antibodies of the present disclosure have or exhibit ADCP activity against NSCLC cells. In certain aspects, the bispecific antibodies of the present disclosure induce ADCP of NSCLC cells.

[0054] The bispecific antibody may comprise a common light chain. The first and second variable domains, in certain embodiments, comprise the same or substantially the same (common) light chain variable region. The common light chain variable region may be one that is known to match well with the diversity of recombined human variable region gene segments. In certain embodiments, the common light chain may be one that is known to match well with the diversity of recombined human variable region gene segments. In certain embodiments, the common light chain may be one that is known to match well with the diversity of recombined human variable region gene segments. * A preferred light chain variable region is a variable region encoded by a germline Vk gene segment, such as a rearranged IgVkappa 1-39 variable region gene segment. * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01. The light chain of the cMET binding arm and the light chain of the EGFR binding arm are, in certain embodiments, the same (common) light chain. In certain embodiments, the common light chain is a rearranged kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 bound to a human light chain constant region. The bispecific antibody can be a human antibody. The bispecific antibody can be a full-length antibody. It can have one variable domain capable of binding to EGFR and one variable domain capable of binding to cMET. In certain embodiments, the variable domain capable of binding to human EGFR can also beneficially bind to mouse EGFR and / or cynomolgus EGFR. In certain embodiments, the variable domain that binds or can bind to human EGFR binds to domain III of human EGFR. The variable domain capable of binding to cMET can block the binding of antibody 5D5 to cMET. The variable domain capable of binding to cMET can block the binding of HGF to cMET. The Kd of the antibody for cMET may be at least 10-fold less than the Kd of the antibody for EGFR. The amino acids at positions 405 and 409 in one CH3 domain may be the same as the amino acids at the corresponding positions in the other CH3 domain (EU numbering).

[0055] In certain aspects, the antibodies of the disclosure have the CDR1 sequence SYGIS and the CDR2 sequence WISAYX. 1 X 2 NTNYAQKLQG, and sequence X 3 X 4 X 5 X 6 HWWLX 7 a first variable domain comprising a heavy chain variable region having a CDR3 comprising A, 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, and X 1 ~X 7 At positions other than the above, there are 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof.

[0056] In certain aspects, an antibody of the present disclosure comprises a second variable domain comprising a heavy chain variable region having an amino acid sequence of one of the sequences of SEQ ID NOs: 1 to 23, having 0 to 10 or 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0057] X 1 =N,X 2 =G,X 3 =D,X 4 =S,X 5 =Y,X 6 =W and X 7 = G or X 1 =N,X 2 =A,X 3 =D,X 4 =S,X 5 =Y,X 6 =W and X 7 = G or X 1 =S,X 2 =G,X 3 =D,X 4 =S,X 5 =Y,X 6 =W and X7 = G or 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 or X 1 =N,X 2 =G,X 3 =G,X 4 =Y,X 5 =L,X 6 =D and X 7 = G or 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, Bispecific antibodies have been described.

[0058] In certain embodiments, X 1 =N,X 2 =G,X 3 =D,X 4 =R,X5 =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.

[0059] In certain embodiments, X 3 ~X 7 =DRHWD,X 1 and X 2 is NG, SG or NA.

[0060] Bispecific antibodies are described, in which the heavy chain variable region of the second variable domain comprises one of the amino acid sequences of SEQ ID NOs: 1-3, 7, 8, 10, 13, 15, 16, 17, 21, 22, or 23, having 0-10 or 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. One advantage of the present disclosure is that in certain embodiments, the bispecific antibodies of the present disclosure exhibit more potent ADCC activity than amivantamab, particularly against cells or cancers containing cMET abnormalities.

[0061] The present disclosure also provides a method of treating a subject having a tumor, the method comprising administering to an individual in need of such treatment a bispecific antibody as described herein. Typically, the individual is suffering from a disease involving abnormal cells, for example, the individual may be suffering from a tumor or cancer.

[0062] The present disclosure also provides a bispecific antibody comprising a first variable domain capable of binding to an extracellular portion of the epidermal growth factor receptor (EGFR) and a second variable domain capable of binding to an extracellular portion of the MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain has the CDR1 sequence SYGIS, CDR2 sequence WISAYX, 1 X 2 NTNYAQKLQG, and sequence X 3 X 4 X 5 X 6 HWWLX 7 a heavy chain variable region having a CDR3 comprising A, 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, and X 1 ~X 7 and a second variable domain comprising a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1 to 23, the heavy chain variable region having 0 to 10 or 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof at a position other than SEQ ID NO: 1, SEQ ID NO: 2, or a combination thereof.

[0063] In one particular aspect, 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 DRHWHWWLDAFDY, and the second variable domain, in one particular aspect, comprises a heavy chain variable region having a CDR1 sequence SYSMN, a CDR2 sequence WINTYTGDPTYAQGFTG, and a CDR3 sequence ETYYYDRGGYPFDP.

[0064] The present disclosure also provides bispecific antibodies for use in treating a subject having a disease involving abnormal cells, such as a tumor.

[0065] Also provided is the use of a bispecific antibody of the disclosure in the manufacture of a medicament for the treatment of a disease involving abnormal cells, such as a tumor or cancer, in a subject who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor, or ii) chemotherapy and an EGFR tyrosine kinase inhibitor, or iii) a cMET tyrosine kinase inhibitor.

[0066] The antibodies of the disclosure inhibit HGF and EGF / HGF induced growth of cancers that have been previously treated with i) a third generation EGFR tyrosine kinase inhibitor, or ii) chemotherapy and an EGFR tyrosine kinase inhibitor, or iii) a cMET tyrosine kinase inhibitor.

[0067] In certain embodiments, the antibodies of the present disclosure inhibit HGF-induced growth of HGF-responsive cells, in certain embodiments, EGFR TKI-resistant or refractory tumors in human subjects, tumor models, or cell lines, such as cell lines or models that contain activating EGFR mutations, approved tyrosine kinase inhibitor-resistant mutations, tertiary tyrosine kinase inhibitor-resistant mutations, mutations that reduce binding of third generation tyrosine kinase inhibitors to EGFR, acquired tyrosine kinase inhibitor-resistant mutations, EGFR gene amplification, cMET mutations, or cMET aberrations, and in certain embodiments, in-frame exon 20 insertion mutations. In certain embodiments, inhibition is demonstrated in the presence of HGF.

[0068] In certain embodiments, the antibodies of the disclosure inhibit HGF-induced growth of HGF-responsive cells, in certain embodiments, tumors in human subjects, tumor models, or cell lines, that contain activating EGFR mutations, approved tyrosine kinase inhibitor resistance mutations, tertiary tyrosine kinase inhibitor resistance mutations, mutations that reduce binding of third generation tyrosine kinase inhibitors to EGFR, acquired tyrosine kinase inhibitor resistance mutations, EGFR gene amplifications, cMET mutations, cMET aberrations, or in-frame exon 20 insertion mutations. In certain embodiments, inhibition is demonstrated in the presence of HGF.

[0069] The antibodies of the present disclosure inhibit EGF-induced growth of EGF-responsive cells without inducing the toxicities, such as rash and diarrhea, associated with high affinity bivalent EGFR antibodies, making them ideally suited for combination with TKIs, which have their own toxicity profiles.

[0070] The antibodies of the disclosure may be used to treat tumors that are resistant to treatment with an EGFR tyrosine kinase inhibitor, for example, resistant or refractory to osimertinib, erlotinib, gefitinib or afatinib, analogs of osimertinib, erlotinib, gefitinib or afatinib, or combinations of one or more of each of these compounds and / or analogs.

[0071] The present disclosure further includes a nucleic acid molecule or collection of nucleic acid molecules that encode, either alone or together, the heavy chain or heavy chain variable regions of the bispecific antibodies or variants thereof disclosed herein. Also provided is a nucleic acid molecule or collection of nucleic acid molecules that encode the antibodies disclosed herein.

[0072] In certain embodiments, the heavy chain comprises a constant region of an IgG1 antibody, in certain embodiments a human IgG1 antibody. The CH2 region of the IgG1 constant region may or may not be engineered to alter ADCC and / or CDC activity of the antibody. In certain embodiments, the alteration results in enhanced ADCC and / or CDC activity. In certain embodiments, the CH3 region of the antibody is engineered to facilitate heterodimerization of the heavy chains, including a first heavy chain that binds EGFR and a second heavy chain that binds cMET.

[0073] The disclosure further includes cells comprising one or more nucleic acid molecules encoding, alone or together, a bispecific antibody or variant thereof as disclosed herein. Also provided in certain aspects are methods of producing a bispecific antibody or variant thereof as disclosed herein using cells as described, with recovery of the bispecific antibody or variant thereof from the culture of the cells.

[0074] The present disclosure further includes cell lines comprising the bispecific antibodies or variants thereof disclosed herein.

[0075] The present disclosure further provides cells that express and / or contain nucleic acid molecules encoding the bispecific antibodies.

[0076] The present disclosure further includes bispecific antibodies as disclosed herein, which in certain aspects further comprise a label for in vivo imaging.

[0077] EGFR is a member of a family of four receptor tyrosine kinases (RTKs), designated 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)). Reference numbers used in this section refer to the numbering of reference numbers in the column headed "Cited in the specification," each of which is incorporated by reference. EGFR integrates extracellular signals from various ligands, resulting in diverse intracellular responses (Yarden at al. 2001, and Jorrisen et al. 2003). EGFR has been implicated in several human epithelial malignancies, particularly breast, bladder, non-small cell lung, colon, ovarian, head and neck, and brain cancers. Activating mutations in the gene and overexpression of the receptor and its ligands are found, resulting in an autocrine activation loop (for review, see Robertson et al. 2000). Therefore, this RTK is widely used as a target for cancer therapy. Both small molecule inhibitors targeting the RTK and monoclonal antibodies (mAbs) directed to the extracellular ligand-binding domain have been developed and have shown some clinical success so far, but mostly in selected patient groups. The database accession number for the human EGFR protein and its encoding gene is (GenBank NM_005228.3). Other database identifiers for the gene and / or protein are HGNC: 3236, Entrez Gene: 1956, Ensembl: ENSG00000146648, OMIM: 131550 and UniProtKB: P00533. The accession numbers are provided primarily to provide further methods of identifying the EGFR protein as a target, and the actual sequence of the EGFR protein bound by the antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers, etc. When EGFR is referred to herein, reference is made to human EGFR, unless otherwise stated.The antigen-binding site that binds to EGFR binds to EGFR and its various variants, including those expressed on some EGFR-positive tumors.

[0078] The term "EGFR ligand" as used herein refers to a polypeptide that binds to and activates EGFR. Examples of EGFR ligands include, but are not limited to, EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, and epiregulin (for review, see Olayioye MA et al.; EMBO J (2000) Vol 19: pp3159-3167). The term includes biologically active fragments and / or variants of naturally occurring polypeptides.

[0079] For example, aberrantly activated forms of EGFR via mutations in EGFR or EGFR gene amplification are known to be oncogenic drivers in non-small cell lung cancer (NSCLC) and are known to occur upon treatment with EGFR tyrosine kinase inhibitors. The present disclosure provides a bispecific antibody for use in a method of treating cancer in a subject 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) no prior anti-cancer treatment, wherein the subject in iv) in certain embodiments has a cMET exon 14 skipping mutation or a cancer comprising a cMET exon 14 skipping mutation.

[0080] In certain embodiments, the treatment includes treatment of cancer resulting from ligand-independent activation of EGFR and / or ligand-independent activation of cMET, hi other embodiments, the treatment includes treatment of cancer resulting from ligand-dependent activation of EGFR and / or ligand-dependent activation of cMET.

[0081] In certain embodiments, the cancer or subject has been previously treated with osimertinib and has acquired or third-line osimertinib resistance. The prior osimertinib treatment is, in certain embodiments, a first-line or second-line therapy. In certain embodiments, the first-line therapy is followed by treatment with a bispecific antibody of the present disclosure as a second treatment.

[0082] In certain aspects, the cancer comprises an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation, a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, EGFR gene amplification, a cMET mutation, a cMET abnormality, or increased HGF expression.

[0083] In clinical studies of the efficacy of the bispecific antibodies of the present disclosure, clinical efficacy was observed in various cancers with different genetic oncogenic backgrounds. Specifically, clinical efficacy was observed in lung cancer, particularly 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, EGFR exon 19 deletion mutations, or c-MET exon 14 skipping mutations. Thus, in certain embodiments, the cancer is NSCLC and / or the subject suffers from NSCLC, where the cancer or subject includes EGFR exon 21 mutations such as L858R, EGFR exon 19 deletion mutations, EGFR exon 18 mutations, or c-MET exon 14 skipping mutations.

[0084] Also, clinical efficacy has been observed in head and neck cancer, particularly head and neck squamous cell carcinoma (HNSCC).Thus, in certain embodiments, the cancer is head and neck cancer, particularly head and neck squamous cell carcinoma, and / or the subject suffers from head and neck cancer, particularly head and neck squamous cell carcinoma.

[0085] In certain aspects, the cancer is gastric adenocarcinoma with c-MET amplification.

[0086] In certain aspects, the treatment results in or involves depletion of soluble EGFR and / or cMET.

[0087] In certain embodiments, the cancer comprises an activating EGFR mutation, such as an in-frame exon 19 deletion mutation or an exon 21 mutation (in certain embodiments, L858R). As used herein, the term "activating EGFR mutation" refers to mutations that develop after progression to third generation EGFR tyrosine kinase inhibitors, including an in-frame deletion in exon 19 (del19) and a substitution of leucine for arginine in exon 21 (L858R).

[0088] In certain embodiments, the cancer comprises an approved tyrosine kinase inhibitor resistance mutation. As used herein, the term "approved tyrosine kinase inhibitor resistance mutation" refers to resistance that develops after progression to an EGFR tyrosine kinase inhibitor currently approved for the treatment of cancer, such as T790M, which confers resistance to afatinib. In certain embodiments, the EGFR tyrosine kinase inhibitor is an approved EGFR tyrosine kinase inhibitor.

[0089] In certain embodiments, 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 resistance that develops after progression to a third generation EGFR tyrosine kinase inhibitor, in certain embodiments, osimertinib.

[0090] In certain embodiments, the cancer contains a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, such as L792X, L718X.

[0091] In certain embodiments, 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, in certain embodiments, including G724X (e.g., G724S), S768X (e.g., S768I), L792X (e.g., L792H), C797X (including C797S and C797G), L798X (e.g., L798I), that confers resistance to osimertinib or that has arisen following progression on osimertinib. As used herein, the term "acquired tyrosine kinase inhibitor resistance mutation" refers to resistance acquired following progression on treatment with a tyrosine kinase inhibitor, such as following progression on a third generation EGFR tyrosine kinase inhibitor.

[0092] In certain aspects, the cancer comprises EGFR gene amplification, e.g., after progression on osimertinib, an increase in EGFR mRNA, or amplification of the wild-type EGFR allele in combination with the presence of the EGFR-ex19del allele.

[0093] In certain embodiments, the cancer comprises a cMET mutation, such as a cMET exon 14 skipping mutation.

[0094] In certain embodiments, the cancer comprises a cMET aberration, such as cMET amplification, cMET overexpression, increased cMET pathway signaling, cMET gene amplification, increased HGF expression, and / or increased cMET protein activity. In certain embodiments, the cancer is NSCLC, and the cMET amplification is characterized by MET / CEP7>5 or cfDNA≧2 copies, or any combination thereof.

[0095] In certain aspects, cMET amplification is characterized by MET / CEP7≧3, MET / CEP7≧4, or MET / CEP7≧5 (and up to 15 or 20 or less), or by cfDNA≧1.8 cMET copies, e.g., (≧1.8,<2.2) or (>2.2,<5) or (≧5).

[0096] In certain embodiments, the cancer comprises an exon 19 deletion mutation, and in certain embodiments, an in-frame exon 19 deletion, an exon 20 missense mutation (e.g., T790M), or an exon 21 mutation such as L858R.

[0097] In certain embodiments, the cancer comprises an EGFR exon 20 mutation, in certain embodiments an exon 20 insertion mutation, in certain embodiments an in-frame exon 20 insertion mutation.

[0098] In certain embodiments, the cancer or subject has a proximal loop insertion (positions 767-772), a distal loop insertion (positions 773-775), and in certain embodiments, 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 (e.g., L792H), C796X (e.g., G796R, G796S, G796D), C797X (e.g., C797S, C797G), L798I or an in-frame exon 20 insertion, e.g., 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_V769InsX1-7>V769_D770 InsX1-7>D770_N771 InsX1-7>N771_P772 InsX1-7>P772_H773 InsX1-7, H773_V774 InsX1-7, or V774_C775 InsX1-7. In certain embodiments, the cancer or subject comprises two or more of the mutations.

[0099] In certain aspects, the cancer or subject comprises a mutation such as L718X (e.g., L718Q, L718V), G719X (e.g., G719A), L792X (e.g., L792H, L792F, L792R, L792Y, L792V and L792P), G796X (e.g., G796R, G796S, G796D), C797X, C797X (e.g., C797S, C797G, C797N), C797X, L792X, G796X, G724X, S768X, L718X, M766X (e.g., M766Q), R776X (e.g., R776C) or an exon 20 insertion mutation. In certain aspects, the cancer or subject comprises two or more of the mutations.

[0100] In certain embodiments, the cancer or subject comprises an exon 19 deletion mutation, in certain embodiments, an in-frame exon 19 deletion, an exon 20 missense mutation (e.g., T790M), or an exon 21 mutation such as L858R, L861X (e.g., L861Q), or L844X (e.g., L844V). In certain embodiments, the cancer or subject comprises two or more of such mutations.

[0101] In certain embodiments, the cancer or subject comprises a cancer that comprises an EGFR exon 20 mutation, in certain embodiments, an exon 20 insertion mutation.

[0102] In certain aspects, the cancer or subject comprises a mutation selected from G724X (e.g., G724S), S768X (e.g., S768I), T790X (e.g., T790M), L792X (e.g., L792H), C797X (including C797S and C797G), L798X (e.g., L798I), I941X (e.g., I941R), V948X (e.g., V948R). In certain aspects, the cancer or subject comprises two or more of the mutations.

[0103] In certain aspects, the cancer or subject comprises a double mutation L858X / T790X (e.g., L858R / T790M), T790X / L798X (e.g., T790M / L798I), T790X / C797X (e.g., T790M / C797S), G719X / R776X (e.g., G719A / R776C), or delE746_A750 / T790M.

[0104] In certain aspects, the cancer or subject comprises a double mutation D770insSVD / E762X (e.g., E762K), D770insSVD / L792X (e.g., L792I, L792S), D770insSVD / P794X (e.g., P794S), or D770insSVD / G796X (e.g., G796D).

[0105] In certain aspects, the cancer or subject comprises the double mutation H773insH / E762X (e.g., E762K), H773insH / L792X (e.g., L792I, L792S), H773insH / P794X (e.g., P794S), or H773insH / G796X (e.g., G796D).

[0106] In certain aspects, the cancer or subject comprises the double mutation H773insNPH / E762X (e.g., E762K), H773insNPH / L792X (e.g., L792I, L792S), H773insNPH / P794X (e.g., P794S), or H773insNPH / G796X (e.g., G796D).

[0107] In certain aspects, the cancer or subject comprises a double mutation L858X / L718X (e.g., L858R / cis-L718Q), L858X / C797X (e.g., L858R / cis-C797S), exon 19del / C797X (e.g., exon 19del / cis-C797S).

[0108] In certain aspects, the cancer or subject has a triple mutation L858X / T790X / C797X (e.g., L858R / T790M / C797S), L858X / T790X / M766X (e.g., L858R / T790M / M766Q), L858X / T790X / L718X (e.g., L858R / T790M / cis-L718Q, L858R / T790M / L718Q), L858X / T790X / C79 7X (e.g., L858R / T790M / cis-C797S), exon 19del / T790X / C797X (e.g., exon 19del / T790M / cis-C797S), L858X / T790X / C941X (e.g., L858R / T790M / I941R), delE746_A750 / T790X / C797X (e.g., delE746_A750 / T790M / C797S).

[0109] In certain aspects, the cancer or subject comprises a cMET exon 14 skipping mutation.

[0110] As used herein, the term "refractory" refers to a disease that does not respond to treatment. A refractory disease may be resistant to treatment prior to or at the start of treatment, or a refractory disease may become refractory during treatment.

[0111] As used herein, the term "first-generation EGFR tyrosine kinase inhibitors" (first-generation TKIs) refers to reversible EGFR inhibitors, such as gefitinib and erlotinib, which are effective in the first-line treatment of NSCLC harboring EGFR activating mutations, such as deletions in exon 19 and the L858R mutation in exon 21.

[0112] As used herein, the term "second-generation EGFR tyrosine kinase inhibitors" (second-generation TKIs) refers to covalent irreversible EGFR inhibitors, such as afatinib and dacomitib, which are effective in the first-line treatment of NSCLC harboring EGFR activating mutations, such as deletions in exon 19 and the L858R mutation in exon 21.

[0113] 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 deletions in exon 19 and exon 21 L858R, alone or in combination with the T790M mutation, and have low inhibitory activity against wild-type EGFR.

[0114] As used herein, the term "resistant" refers to a cancer or patient that does not respond to treatment when administered prescribed doses of the therapeutic agent involved, including third generation EGFR tyrosine kinase inhibitors.

[0115] In certain embodiments, 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, respectively. The recommended Phase 2 dosing was established at a daily dose of 80 mg.

[0116] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is almonertinib (HS-10296). Almonertinib is an orally available, irreversible, third generation EGFR tyrosine kinase inhibitor with EGFR sensitization and selectivity for T790M resistance mutation. Almonertinib has been used in non-small cell lung cancer research. The recommended phase 2 dosing has been established at a daily dose of 110 mg.

[0117] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is lazertinib.Lazertinib (YH25448) is a potent, mutant-selective, blood-brain barrier permeable, orally available and irreversible third generation EGFR tyrosine kinase inhibitor, and can be used in the study of non-small cell lung cancer.The recommended Phase II dosing is established at a daily dose of 240mg.

[0118] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is befotertinib (or BPI-D0316 or optionally D-0316). Befotertinib (Beta Pharmaceuticals, Co., China) is a third generation EGFR tyrosine kinase inhibitor. Befotertinib can be used for EGFR-positive non-small cell lung cancer (NSCLC). In a phase II single-arm study NCT05007938, the safety and efficacy of befotertinib was evaluated in patients with locally advanced or metastatic NSCLC at 25 mg orally three times a day in combination with icotinib (125 mg orally three times a day). In a phase I study NCT04464551, subjects received a single oral dose of 75 mg D-0316 as an oral suspension. In the phase II, single-arm study NCT03861156, patients with locally advanced / metastatic non-small cell lung cancer received an oral dose of 75 mg in 21-day cycles, which 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 was evaluated at 70 mg once daily for 21 days, then increased to 100 mg once daily.

[0119] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is alflutinib (AST2818 or flumonertinib). AST2818 is the subject of clinical trials NCT03787992 for its clinical efficacy in NSCLC.

[0120] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is resibertinib (BPI-7711), an orally active, selective and irreversible third generation EGFR tyrosine kinase inhibitor (TKI).Resibertinib is the subject of a clinical trial NCT03866499 for its clinical efficacy in NSCLC.

[0121] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is avitinib (Abivertinib / AC0010), a pyrrolopyrimidine-based irreversible epidermal growth factor receptor (EGFR) inhibitor with an IC50 of 7.68 nM. Avitinib is the subject of clinical trials NCT03856697 for its clinical efficacy in NSCLC.

[0122] In certain embodiments, the third-generation EGFR tyrosine kinase inhibitor is ASK120067, which is a potent orally active inhibitor of EGFR.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) for its clinical efficacy in NSCLC.

[0123] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is oritinib (SH-1028, Nanjing Sanhome Pharmaceutical Co., Ltd., Nanjing, China). SH-1028 is the subject of clinical trials NCT04239833 for its clinical efficacy in NSCLC.

[0124] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is rociletinib (CO-1686), an orally delivered kinase inhibitor that specifically targets mutant EGFR forms. Rociletinib has been the subject of clinical trials NCT02186301 for its clinical efficacy in NSCLC.

[0125] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is olmutinib (HM61713; BI-1482694), which is an orally active, irreversible third generation EGFR tyrosine kinase inhibitor that binds to the cysteine ​​residue near the kinase domain.Olmutinib can be used in the study of NSCLC.Olmutinib has been the subject of clinical trial NCT02485652 for its clinical efficacy in NSCLC.

[0126] In certain embodiments, the third-generation EGFR tyrosine kinase inhibitor is nazatinib (EGF816), a third-generation EGFR TKI that selectively inhibits EGFR activating mutations in patients with advanced EGFR mutant NSCLC. Nazatinib has been the subject of clinical trials NCT03529084 for its clinical efficacy in NSCLC.

[0127] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is nacotinib, an orally available, irreversible, third generation, mutant-selective, epidermal growth factor receptor (EGFR) inhibitor. Nacotinib has been the subject of clinical trials NCT02588261 for its clinical efficacy in NSCLC.

[0128] In certain embodiments, the third generation EGFR tyrosine kinase inhibitor is mavereltinib (PF-0647775), a selective, orally available, and irreversible EGFR tyrosine kinase inhibitor (EGFR TKI). Mavereltinib has been the subject of a clinical trial NCT02349633 for its clinical efficacy in NSCLC.

[0129] cMET, also called tyrosine protein kinase MET or hepatocyte growth factor receptor (HGFR), is a protein encoded by the MET gene in humans. The protein has tyrosine kinase activity. A primary single-chain precursor protein is post-translationally cleaved to produce alpha and beta subunits that are disulfide-linked to form the mature receptor.

[0130] Dysregulation or abnormally activated cMET can induce tumor growth, the formation of new blood vessels to supply nutrients to tumors (angiogenesis), and the spread of cancer to other organs (metastasis). cMET is dysregulated in many types of human malignancies, including kidney, liver, stomach, breast, and brain cancer. The cMET gene is known by many different names, including 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, EC2.7.10.1, MET proto-oncogene, EC2.7.10, DFNB97, AUTS9, RCCP2, C-MET, MET, HGFR, and the external Ids for cMET are HGNC:7029, Entrez Gene:4233; Ensembl:ENSG00000105976, OMIM:164860, and UniProtKB:P08581. The accession numbers are provided primarily to provide further methods of identifying the cMET protein as a target, and the actual sequence of the cMET protein bound by the antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers. When cMET is mentioned herein, reference is made to human cMET unless otherwise stated. Antigen binding sites that bind cMET bind to cMET and its various variants, such as those expressed on some cMET-positive tumors. Examples of cMET abnormalities or dysregulation include cMET mutations (such as exon 14 skipping mutations), cMET amplification, cMET overexpression, increased cMET pathway signaling, cMET gene amplification, and / or increased cMET protein activity. cMET abnormalities may also be caused by increased HGF expression. Dysregulation of c-MET is an established driver of tumor invasion, angiogenesis, and metastasis (Birchmeier et al., 2003). Three types of biological changes in c-MET can lead to carcinogenesis: amplification, mutation, and fusion.These genomic alterations are primarily found as either primary or secondary drivers of tumor growth, and such abnormalities have been reported to occur following treatment of cancer patients with EGFR tyrosine kinase inhibitors (see Suzawa et al., DOI:10.1200 / PO.19.00011 JCO Precision Oncology-May10,Vol3,2019).

[0131] In certain embodiments, the subject of the present disclosure in iv) has not undergone prior anti-cancer treatment. In certain embodiments, the subject has not undergone prior treatment with a tyrosine kinase inhibitor. In certain embodiments, the subject has not undergone prior treatment with a cMET inhibitor. In certain embodiments, the subject has not undergone prior treatment with a cMET tyrosine kinase inhibitor. In certain embodiments, the subject has not undergone prior treatment with a tyrosine kinase inhibitor, such as capmatinib, tepotinib, or savolitinib. The subject may have undergone treatment with a chemotherapeutic agent (such as platinum-based chemotherapy) or an immunotherapeutic agent (such as pembrolizumab, nivolumab, cetuximab, etc.). In certain embodiments, the subject has metastatic non-small lung cancer. In certain embodiments, the metastatic NSCLC has a mutation that results in cMET exon 14 skipping. Exon 14 skipping can be detected by an FDA-approved test.

[0132] In certain embodiments, the subject has a cMET exon 14 skipping mutation or a cancer comprising a cMET exon 14 skipping mutation. The subject has not received prior anti-cancer treatment for cancer, such as EGFR and / or cMET positive cancer, or for cancer comprising EGFR and / or cMET aberrations. Thus, in certain embodiments, administration of or treatment with a bispecific antibody of the present disclosure is a first-line treatment. In certain embodiments, the first-line treatment is to prevent the development of resistance to EGFR and / or cMET tyrosine kinase treatment mechanisms in lung cancer patients or lung cancer, such as non-small cell lung cancer, in particular. Resistance mechanisms to EGFR and / or cMET tyrosine kinase inhibitors are well known to develop, particularly in subjects with non-small cell lung cancer, such as metastatic or advanced cancers. Thus, the present disclosure also provides a bispecific antibody of the present disclosure for use in a method for preventing the development or occurrence of a cancer with EGFR and / or cMET tyrosine kinase inhibitor resistance in a subject.

[0133] In certain aspects, the subject of the present disclosure in iv) is a human subject.

[0134] In certain embodiments, the use or treatment comprises providing to the subject, iv), a dose of 1000, 1500 or 2000 mg of the bispecific antibody of the present disclosure. In certain embodiments, the bispecific antibody is provided once every week or once every two weeks. In certain embodiments, the use or treatment comprises providing to the subject a dose of 1500 mg of the bispecific antibody once every two weeks. The present disclosure also relates to a method of treating a subject having a tumor, the method comprising administering to the subject, as an individual in need of the treatment, a bispecific antibody as described herein. Typically, the individual is an individual suffering from a disease involving abnormal cells, for example, the individual may suffer from a tumor or cancer.

[0135] In certain embodiments, the subject in iv) has a cancer that 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. In certain embodiments, the subject in iv) has a cancer that is non-small cell lung cancer (NSCLC).

[0136] In certain embodiments, the subject in iv) has advanced or metastatic cancer, such as advanced or metastatic NSCLC.

[0137] In certain aspects, treating the subject in iv) includes a diagnostic step to assess whether the cancer is an EGFR-positive and / or cMET-positive cancer, or to assess the presence of EGFR and / or cMET aberrations in the cancer.

[0138] An antibody typically recognizes only a portion of an antigen. An antigen is typically, but not necessarily, a protein. The recognition or binding site on an antigen bound by an antibody is called an epitope, which may be linear or conformational. The binding of an antibody to an antigen is typically specific. The "specificity" of an antibody refers to its selectivity for a particular epitope, whereas "affinity" refers to the strength of interaction between the antigen-binding site of an antibody and the epitope it binds.

[0139] Exemplary antibodies of the disclosure bind to EGFR and cMET, in certain embodiments to human EGFR and human cMET. EGFR / cMET bispecific antibodies of the disclosure bind to EGFR and, under otherwise identical conditions, bind to the homologous receptors ErbB-2 and ErbB-4 of the same species at least 100-fold lower. EGFR / cMET bispecific antibodies of the disclosure bind to cMET and, under otherwise identical conditions, bind to the receptors ErbB-2 and ErbB-4 of the same species at least 100-fold lower. Given that the receptors are cell surface receptors, binding can be assessed on cells expressing the receptor. In certain embodiments, bispecific antibodies of the disclosure bind to human, cynomolgus EGFR and / or mouse EGFR.

[0140] Antibodies that bind EGFR and cMET may also bind other proteins if such other proteins contain the same epitope. Thus, the term "binds" does not exclude the binding of the antibody to another protein or proteins that contain the same epitope. Such binding is typically referred to as cross-reactivity. EGFR / cMET bispecific antibodies typically do not bind to other proteins other than EGFR and / or cMET on the membrane of cells after birth, and in certain embodiments in adult humans. Antibodies according to the present disclosure can typically bind to EGFR with a binding affinity (i.e., equilibrium dissociation constant Kd) of at least 1×10e-6 M, as outlined in more detail below.

[0141] The term "antibody" as used herein refers to a proteinaceous molecule in certain embodiments belonging to the immunoglobulin class of proteins. Antibodies typically contain two variable domains that bind to an epitope on an antigen. Such domains are derived from or share sequence homology with the variable domains of an antibody. The bispecific antibodies of the present disclosure in certain embodiments comprise two variable domains. Antibodies for therapeutic use are in certain embodiments as close as possible to the natural antibody of the subject to be treated (e.g., human antibodies for human subjects). 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. Typically, antibodies for therapeutic use can have an affinity of up to 1×10e-10 M or higher. Antibodies such as bispecific antibodies of the present disclosure in certain embodiments comprise the constant domains (Fc portion) of natural antibodies. Antibodies of the present disclosure are typically bispecific full-length antibodies in certain embodiments of the human IgG subclass. In certain embodiments, antibodies of the present disclosure are of the human IgG1 subclass. Such antibodies of the present disclosure may have favorable half-lives upon in vivo administration to humans and may have excellent ADCC properties, with CH3 engineering that may provide modified heavy chains that preferentially form heterodimers over homodimers upon co-expression in clonal cells. The ADCC activity of antibodies may also be improved by techniques known to those of skill in the art.

[0142] In certain aspects, the antibodies of the present disclosure are "full-length" antibodies. As disclosed herein, the term "full-length" is defined to include an essentially complete antibody, but not necessarily having all the functions of an intact antibody. For the avoidance of doubt, a full-length antibody contains two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which may be classified into domains designated CH1, CH2, CH3, VH, and CL, VL. Typically, an antibody binds to an antigen via the variable domain contained in the Fab portion, and after binding, can interact with molecules and cells of the immune system via the constant domain, primarily via the Fc portion. Full-length antibodies according to the present disclosure encompass antibodies in which mutations may be present that provide desired characteristics. Antibodies in which one or more amino acid residues have been deleted without essentially changing the specificity and / or affinity properties of the resulting antibody are encompassed within the term "full-length antibody." For example, an IgG antibody may have an insertion, deletion or substitution of 1-20 amino acid residues, or a combination thereof, in the constant region.

[0143] In certain embodiments, the antibody of the present disclosure is a bispecific IgG antibody, such as a bispecific full-length IgG1 antibody or a human IgG1. Full-length IgG antibodies are preferred due to their typically favorable half-life for immunogenicity reasons, and because it is desirable to remain close to the completely self (human) molecule. In certain embodiments, the antibody of the present disclosure is a full-length IgG1, full-length IgG2, full-length IgG3 or full-length IgG4 antibody.

[0144] The variable domain, which can bind to EGFR and comprises the amino acid sequence of MF3370 or its variants as set forth herein, in certain embodiments binds to EGFR domain III (see Table 4 of International Patent Application PCT / NL2015 / 050124; WO2015 / 130172, incorporated herein by reference). In certain embodiments, the variable domain blocks the binding of the ligand EGF to EGFR or competes with the EGF ligand for binding to EGFR. The binding of the variable domain to EGFR can be inhibited by cetuximab. The variable domain binds to an epitope that is different from the epitope recognized by cetuximab and zalutumumab. For example, the variable domain binds to mouse EGFR, but cetuximab and zalutumumab do not, indicating that one or more of the residues that differ between mouse and human EGFR domain III play a role in the binding of cetuximab and zalutumumab, but not in the antibodies of the present disclosure described herein. An advantage of the disclosed bispecific antibodies with human, mouse and cynomolgus EGFR cross-reactivity is that they allow the use of xenograft studies with human cancer models, which may be more predictive in terms of efficacy and toxicity, since the antibodies also bind to normal mouse cells bearing the receptor, while also allowing use in cynomolgus toxicology studies. In certain aspects, the disclosure provides a 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 the first variable domain can also bind to mouse EGFR, cynomolgus EGFR or both.

[0145] In certain embodiments, the cMET variable domain comprises the amino acid sequence of MF4356 or a variant thereof as set forth herein, and in certain embodiments, blocks the binding of the antibody MetMab to cMET. In certain embodiments, the cMET variable domain comprises the amino acid sequence of MF8230 or a variant thereof as set forth herein, and in certain embodiments, blocks the binding of the antibody MetMab to cMET. In certain embodiments, the variable domain blocks the binding of the ligand HGF to cMET or competes with the ligand HGF for binding to cMET. The variable domain blocks the binding of the antibody MetMab to cMET when the binding of MetMab to cMET under half-maximal binding conditions is reduced by at least 40%, and in certain embodiments by at least 60%, in the presence of a saturating amount of the variable domain. The variable domain is provided in certain embodiments in the context of a bivalent monospecific antibody. In certain embodiments, the cMET variable domain can bind to the sema domain of cMET. The cMET variable domains of the disclosure may or may not compete with 5D5 for binding to cMET, or may not compete with a reported anti-cMET reference antibody, such as 5D5. See Table 2.

[0146] The variable domain of the present disclosure can bind to EGFR (first variable domain), and in one particular embodiment, has CDR1 sequence SYGIS, CDR2 sequence WISAYX. 1 X 2 NTNYAQKLQG, and sequence X 3 X 4 X 5 X 6 HWWLX 7 A, and 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.

[0147] X 1~7 In certain embodiments, X 1 =N,X 2 =G,X 3 =D,X 4 =S,X 5 =Y,X 6 =W and X 7 = G or X 1 =N,X 2 =A,X 3 =D,X 4 =S,X 5 =Y,X 6 =W and X 7 = G or X 1 =S,X 2 =G,X 3 =D,X 4 =S,X 5 =Y,X 6 =W and X 7 = G or 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 or X 1 =N,X 2 =G,X 3 =G,X 4 =Y,X 5 =L,X 6 =D and X 7 = G or X 1 =N,X2 =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.

[0148] In certain embodiments, 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.

[0149] In certain embodiments, X 1 =N,X 2 =G,X 3 =D,X 4 =R,X 5 =H,X 6 =W and X 7 =D.

[0150] Sequence X in the CDR3 sequence of the first variable domain 3 X 4 X 5 X 6HWWLX 7 The amino acid following the amino acid A in A can be varied. 3 X 4 X 5 X 6 HWWLX 7 The amino acid sequence following A can be FDY. In certain embodiments, the CDR3 of the first variable domain has the sequence X 3 X 4 X 5 X 6 HWWLX 7 AF, in certain embodiments X 3 X 4 X 5 X 6 HWWLX 7 AFD, in certain embodiments X 3 X 4 X 5 X 6 HWWLX 7 Including AFDY.

[0151] In certain embodiments, the first variable domain has the CDR1 sequence SYGIS, the CDR2 sequence WISAYNGNTNYAQKLQG, and the CDR3 sequence X 3 X 4 X 5 X 6 HWWLX 7 The heavy chain variable region comprises

[0152] In certain embodiments, 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. The amino acids following the sequence LDA in the CDR3 sequence of the first variable domain can vary. The amino acid sequence following the sequence LDA can be FDY. In certain embodiments, the CDR3 of the first variable domain comprises the sequence DRHWHWWLDAF, in certain embodiments DRHWHWWLDAFD, and in certain embodiments DRHWHWWLDAFDY.

[0153] In certain aspects, the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF3353, MF8229, MF8228, MF3370, MF8233, MF8232, MF3393, MF8227 or MF8226 as shown in Figure 2, with up to 10, and 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 with respect to the sequence shown. In certain aspects, the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF3353, MF8229, MF8228, MF3370, MF8233, MF8232, MF3393, MF8227 or MF8226 as shown in Figure 2. In certain aspects, the first variable domain comprises a heavy chain variable region having the CDR1, CDR2 and CDR3 amino acid sequences of MF3353, MF8229, MF8228, MF3370, MF8233, MF8232, MF3393, MF8227 or MF8226, as shown in Figure 2.

[0154] In a particular embodiment, the variable domain capable of binding to cMET (second variable domain) comprises a heavy chain variable region comprising one of the amino acid sequences of SEQ ID NOs: 1 to 23 ( FIG. 3 ) having 0 to 10, and in a particular embodiment, 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a particular embodiment, the heavy chain variable region of the second variable domain comprises one of the amino acid sequences of SEQ ID NOs: 1 to 3, 7, 8, 10, 13, 15, 16, 17, 21, 22, or 23 having 0 to 10, and in a particular embodiment, 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a particular embodiment, the heavy chain variable region of the second variable domain comprises one of the amino acid sequences of SEQ ID NOs: 2, 7, 8, 10, 13, or 23 having 0 to 10, and in a particular embodiment, 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain embodiments, the heavy chain variable region of the second variable domain comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 23, with 0 to 10, and in certain embodiments 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain embodiments, the second variable domain comprises 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), MF82 MF8221 (SEQ ID NO:22) or MF4356 (SEQ ID NO:23).

[0155] In one particular embodiment, the first variable domain comprises a heavy chain variable region having the CDR1 sequence SYGIS, the CDR2 sequence WISAYNGNTNYAQKLQG, and the CDR3 comprising the sequence DRHWHWWLDA, in one particular embodiment the sequence DRHWHWWLDAFDY, and the second variable domain comprises a heavy chain variable region having the CDR1 sequence SYSMN, the CDR2 sequence WINTYTGDPTYAQGFTG, and the CDR3 sequence ETYYYDRGGYPFDP. In one particular embodiment, the light chain CDR1, CDR2 and CDR3 of the first and second variable domains each comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0156] In certain embodiments, the first variable domain comprises a heavy chain variable region having CDR1 sequence SYGIS, CDR2 sequence WISAYNGNTNYAQKLQG, and CDR3 comprising the sequence DRHWHWWLDA, and the second variable domain comprises a heavy chain variable region having CDR1 sequence TYSMN, CDR2 sequence WINTYTGDPTYAQGFTG, and CDR3 comprising the sequence ETYFYDRGGYPFDP. In certain embodiments, the light chain CDR1, CDR2, and CDR3 of the first and second variable domains each comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0157] A bispecific antibody comprising a first variable domain capable of binding to the extracellular portion of EGFR and a second variable domain capable of binding to the extracellular portion of cMET, wherein the first variable domain comprises a heavy chain variable region having the CDR1 sequence SYGIS, the CDR2 sequence WISAYNANTNYAQKLQG, and a CDR3 comprising the sequence DRHWHWWLDA, and the second variable domain comprises a heavy chain variable region having the CDR1 sequence SYSMN, the CDR2 sequence WINTYTGDPTYAQGFTG, and the CDR3 sequence ETYYYDRGGYPFDP. In certain embodiments, the light chain CDR1, CDR2 and CDR3 of the first and second variable domains each comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0158] A bispecific antibody comprising a first variable domain capable of binding to the extracellular portion of EGFR and a second variable domain capable of binding to the extracellular portion of cMET, wherein the first variable domain comprises a heavy chain variable region having CDR1 sequence SYGIS, CDR2 sequence WISAYNANTNYAQKLQG, and CDR3 comprising the sequence DRHWHWWLDA, and the second variable domain comprises a heavy chain variable region having CDR1 sequence TYSMN, CDR2 sequence WINTYTGDPTYAQGFTG, and CDR3 comprising the sequence ETYFYDRGGYPFDP. In certain embodiments, the light chain CDR1, CDR2 and CDR3 of the first and second variable domains each comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0159] A bispecific antibody comprising a first variable domain capable of binding to the extracellular portion of EGFR and a second variable domain capable of binding to the extracellular portion of cMET, wherein the first variable domain comprises a heavy chain variable region having the CDR1 sequence SYGIS, the CDR2 sequence WISAYSGNTNYAQKLQG, and a CDR3 comprising the sequence DRHWHWWLDA, and the second variable domain comprises a heavy chain variable region having the CDR1 sequence SYSMN, the CDR2 sequence WINTYTGDPTYAQGFTG, and the CDR3 sequence ETYYYDRGGYPFDP. In certain embodiments, the CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT), respectively.

[0160] A bispecific antibody comprising a first variable domain capable of binding to the extracellular portion of EGFR and a second variable domain capable of binding to the extracellular portion of cMET, wherein the first variable domain comprises a heavy chain variable region having CDR1 sequence SYGIS, CDR2 sequence WISAYSGNTNYAQKLQG, and CDR3 comprising the sequence DRHWHWWLDA, and the second variable domain comprises a heavy chain variable region having CDR1 sequence TYSMN, CDR2 sequence WINTYTGDPTYAQGFTG, and CDR3 comprising the sequence ETYFYDRGGYPFDP. In certain embodiments, the CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains each comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0161] In certain embodiments where a cMET-binding variable domain is described as having the CDR2 sequence "WINTYTGDPTYAQGFTG," the CDR2 sequence can also be "WINTYTGDPTYAQGFT."

[0162] In certain embodiments, the CDR1, CDR2 and CDR3 of the light chain of the first and second variable domains described herein each comprise the amino acid sequence CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT). In some such embodiments, CDR3 comprises the amino acid sequence QQSYSTP. In certain embodiments of a bispecific antibody as described herein, the first and second variable domains comprise a common light chain, in certain embodiments the light chain variable domain of Figure 4B.

[0163] In certain embodiments, the EGFR / cMET bispecific antibody comprises a first variable domain capable of binding to the extracellular portion of human EGFR comprising CDR1, CDR2 and CDR3 of the heavy chain variable region of MF3755 as shown in Figure 1, and a second variable domain capable of binding to the extracellular portion of human cMET comprising CDR1, CDR2 and CDR3 of the heavy chain variable region of MF4297 as shown in Figure 1. The light chain variable regions in the first and second variable domains are in certain embodiments a common light chain variable region as described herein. In certain embodiments, the light chain CDR1, CDR2 and CDR3 of the first and second variable domains comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT), respectively. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence of MF3755 as shown in FIG. 1 with up to 10, and in certain embodiments 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain embodiments 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof with respect to the sequence shown. In certain embodiments, the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF3755 as shown in FIG. 1. In certain embodiments, the variable domain capable of binding to cMET (second variable domain) comprises a heavy chain variable region comprising the amino acid sequence of MF4297 as shown in FIG. 1 with 0-10, and in certain embodiments 0-5, amino acid insertions, deletions, substitutions, additions, or combinations thereof. In certain embodiments, the heavy chain variable region of the second variable domain comprises the amino acid sequence of MF4297 as shown in FIG. 1.

[0164] The term "bispecific" (bs) in the context of the present disclosure means that an antibody can bind to two different targets or to two epitopes on the same target, for example, one variable domain (as defined above) of the antibody binds to an epitope on EGFR and 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, both Fab arms of the antibody may or may not simultaneously bind to their epitopes. One arm of the bispecific antibody typically contains the variable domain of one antibody and the other arm contains the variable domain of another antibody (i.e., one arm of the bispecific antibody is formed by one heavy chain paired with one light chain and the other arm is formed by a different heavy chain paired with a light chain). Thus, in certain aspects, the stoichiometry of the bispecific antibody of the present disclosure is 1:1 EGFR:cMET binding.

[0165] The heavy chain variable regions of the bispecific antibodies of the present disclosure are typically different from each other, while the light chain variable regions are the same in certain embodiments. Bispecific antibodies in which different heavy chain variable regions are associated with the same light chain variable region are also referred to as bispecific antibodies with a common light chain variable region (cLcv). The light chain constant regions are also preferably the same. Such bispecific antibodies are referred to as having a common light chain (cLc). Thus, there is further provided a bispecific antibody according to the present disclosure in which both arms comprise a common light chain.

[0166] The term "common light chain" according to the present disclosure refers to two or more light chains in a bispecific antibody that may be identical or may have some amino acid sequence differences, but the binding specificity of the full-length antibody is not affected. It is possible, for example, within the definition of common light chain used herein, to prepare or find light chains that are not identical but are still functionally equivalent, for example, by introducing and testing conservative amino acid changes, changes in amino acids in regions that do not contribute or only partially contribute to the binding specificity when paired with a heavy chain, etc. The terms "common light chain", "common LC", "cLC", "single light chain", whether or not with the addition of the term "rearranged", are all used interchangeably herein. The terms "common light chain variable region", "common VL", "common LCv", "cLCv", "single VL", whether or not with the addition of the term "rearranged", are all used interchangeably herein. In certain embodiments, the multispecific antibodies of the present disclosure have a common light chain (variable region) that can be combined with at least two, and in certain embodiments, multiple, heavy chains (variable regions) of different binding specificities to form an antibody with a functional antigen binding domain (e.g., WO2009 / 157771). The common light chain (variable region) is, in certain embodiments, a human light chain (variable region). The common light chain (variable region) in certain embodiments has a germline sequence. In certain embodiments, the germline sequence is a light chain variable region with good thermodynamic stability, yield and solubility. In certain embodiments, the germline light chain is O12. The common light chain in certain embodiments comprises a light chain encoded by a germline human Vk gene segment, which in certain embodiments is a rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01 (FIG. 4A). The common light chain variable region, in certain embodiments, is the variable region of rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01. In certain embodiments, the common light chain comprises a light chain variable region as shown in Figure 4B or 4d, having 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain embodiments, the common light chain further comprises a light chain constant region, in certain embodiments a kappa light chain constant region.The nucleic acid encoding the common light chain may be codon optimized for the cell system used to express the common light chain protein. The encoding nucleic acid may deviate from the germline nucleic acid sequence.

[0167] In certain embodiments, the light chain comprises a light chain region comprising the amino acid sequence of the O12 / IgVκ1-39*01 gene segment as depicted in FIG. 4A, with 0-10, and in certain embodiments, 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. The phrase "O12 light chain" refers to the O12 / IgVκ1-39*01 gene segment as depicted in FIG. 4A, with 0-10, and in certain embodiments, 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. * IgVκ1-39 is used throughout the specification as an abbreviation for a light chain comprising a light chain variable region comprising the amino acid sequence of the 01 gene segment. IgVκ1-39 is an abbreviation for Immunoglobulin variable kappa 1-39 gene. This gene is also known as Immunoglobulin kappa variable 1-39, IGKV139, IGKV1-39, O12a or O12. The external Ids for this gene are HGNC:5740, Entrez Gene:28930, Ensembl:ENSG00000242371. In one particular embodiment, the amino acid sequence for IgVκ1-39 is given in FIG. 4E. This figure lists the sequence of the V region. The V region can be combined with one of five J regions. Figures 4B and 4d describe two preferred sequences of IgVκ1-39 combined with a J region. The combined sequences are designated IGKV1-39 / jk1 and IGKV1-39 / jk5, with alternative designations IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (designation according to the IMGT database world wide web at imgt.org).

[0168] It is preferred that the O12 / IgVκ1-39*01 comprising the light chain variable region is a germline sequence. It is even more preferred that the IGJκ1*01 or / IGJκ5*01 comprising the light chain variable region is a germline sequence. In certain embodiments, the IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence.

[0169] In certain embodiments, the light chain variable region is selected from the group consisting of germline O12 / IgVκ1-39 * 01. In certain aspects, the light chain variable region comprises the kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. In certain aspects, IgVκ1-39*01 / IGJκ1*01. In certain aspects, the light chain variable region comprises the germline kappa light chain IgVκ1-39*01 / IGJκ1*01 or the germline kappa light chain IgVκ1-39*01 / IGJκ5*01, in certain aspects the germline IgVκ1-39*01 / IGJκ1*01.

[0170] Mature B cells producing antibodies with O12 light chains often produce light chains that have undergone one or more mutations relative to the germline sequence, i.e., the normal sequence in non-lymphoid cells of the organism. The process involved in these mutations is often referred to as somatic (hyper)mutation. The resulting light chain is referred to as an affinity matured light chain. Such a light chain is an O12-derived light chain if it is derived from an O12 germline sequence. As used herein, the phrase "common light chain" includes "light chain derived from common light chain" and the phrase "O12 light chain" includes light chain derived from O12. Mutations introduced by somatic hypermutation can also be artificially introduced in the laboratory. In the laboratory, other mutations can also be introduced without necessarily affecting the properties of the light chain in terms of type but not amount. A light chain is at least an O12 light chain if it comprises a sequence as shown in Figure 4A, Figure 4B, Figure 4D or Figure 4E with 0-10, and in certain embodiments 0-5, amino acid insertions, deletions, substitutions, additions, or combinations thereof. In certain embodiments, the O12 light chain is a light chain comprising a sequence as shown in FIG. 4A, 4b, 4d, or 4e with 0-9, 0-8, 0-7, 0-6, 0-5, or 0-4 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain embodiments, the O12 light chain is a light chain comprising a sequence as shown in FIG. 4A, 4B, 4D, or 4E with 0-5, and in certain embodiments 0-4, and in certain embodiments 0-3 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain embodiments, the O12 light chain is a light chain comprising a sequence as shown in FIG. 4A, 4B, 4D, or 4E with 0-2, and in certain embodiments 0-1, and in certain embodiments 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain aspects, the O12 light chain is a light chain comprising a sequence as shown in Figure 4A or Figure 4B with the amino acid insertions, deletions, substitutions, additions, or combinations thereof noted. In certain aspects, the light chain comprises the sequence of Figure 4A. In certain aspects, the light chain variable region comprises the sequence of Figure 4B.The 1, 2, 3, 4 or 5 amino acid substitutions referred to are in certain embodiments conservative amino acid substitutions and may be present in the CDR regions of the heavy and / or light chain, and the insertions, deletions, substitutions or combinations thereof are in certain embodiments not present in the CDR3 region of the VL chain, and in certain embodiments not present in the CDR1, CDR2 or CDR3 regions or the FR4 region of the VL chain.

[0171] The common light chain may have a lambda light chain, and thus is also provided in the context of this disclosure, however, a kappa light chain is preferred. The constant portion of the common light chain of this disclosure may be a kappa or lambda light chain constant region. In certain embodiments, it is a kappa light chain constant region, and in certain embodiments, the common light chain is a germline light chain, and in certain embodiments, a rearranged germline human kappa light chain comprising the IgVKl-39 gene segment, and in certain embodiments, the rearranged germline human kappa light chain IgVKl-39*01 / IGJKl*01 (Figure 4). Rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * The terms 01, IGKV1-39 / IGKJ1, huVκ1-39 light chain or abbreviated huVκ1-39 or simply 1-39 are used interchangeably throughout this application.

[0172] A cell producing a common light chain can, for example, produce a light chain comprising the rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01 and the variable region of the referenced light chain fused to a lambda constant region.

[0173] In certain embodiments, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK having 0-5, and in certain embodiments 0-10, amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain embodiments, the light chain variable region comprises 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, and in certain embodiments 0-3, and in certain embodiments 0-2, and in certain embodiments 0-1, and in certain embodiments 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to the amino acid sequence shown. A combination of insertions, deletions, additions, or substitutions is a claimed combination where the aligned sequences do not differ at more than 5 positions. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK.In another embodiment, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK.

[0174] Amino acid insertions, deletions, substitutions, additions, or combinations thereof are absent in the CDR3 region of the light chain variable region in certain aspects, and absent in the CDR1 or CDR2 regions of the light chain variable region in certain aspects. In certain aspects, the light chain variable region does not contain deletions, additions, or insertions with respect to the sequence shown. In this embodiment, the heavy chain variable region may have 0-5 amino acid substitutions with respect to the amino acid sequence shown. The amino acid substitutions are, in certain aspects, conservative amino acid substitutions. In certain aspects, the CDR1, CDR2, and CDR3 of the light chain of the antibody of the present disclosure each comprise the amino acid sequence CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTPPT, i.e., the CDRs of IGKV1-39 (according to IMGT). In certain aspects, the CDR3 sequence comprises QQSYSTPPT.

[0175] In certain embodiments, a bispecific antibody as described herein has 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 certain embodiments, the VL in the first VH / VL combination is similar to the VL in the second VH / VL combination. In certain embodiments, the VL in the first and second VH / VL combinations are identical. In certain embodiments, a bispecific antibody is a full-length antibody having one heavy / light (H / L) chain combination that binds to the extracellular portion of EGFR and one H / L chain combination that binds to the extracellular portion of cMET. In certain embodiments, the light chain in the first H / L chain combination is similar to the light chain in the second H / L chain combination. In certain embodiments, the light chains in the first and second H / L chain combinations are identical.

[0176] Several methods have been published to produce host cells with expression that favors the production of bispecific antibodies or vice versa monospecific antibodies. In the present disclosure, it is preferred that the cellular expression of antibody molecules favors the production of bispecific antibodies over the production of each monospecific antibody. This is typically achieved by modifying the constant regions of the heavy chains such that they favor heterodimerization (i.e., dimerization with the heavy chains of other heavy / light chain combinations) over homodimerization. In certain embodiments, the bispecific antibodies of the present disclosure comprise two different immunoglobulin heavy chains with compatible heterodimerization domains. A variety of compatible heterodimerization domains have been described in the art. The compatible heterodimerization domain is, in certain embodiments, 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 symbols for two monospecific bivalent antibodies, and AB is the symbol for a bispecific antibody. To increase the percentage of the desired bispecific product (AB), CH3 engineering can be used, or in other words, it can use heavy chains with compatible heterodimerization domains as defined herein. In the art, various methods are described that can achieve such heterodimerization of heavy chains. One approach is to generate "knob into hole" bispecific antibodies.

[0177] The term "compatible heterodimerization domain" as used herein refers to a protein domain that has been engineered such that engineered domain A' preferentially forms heterodimers with engineered domain B', and vice versa, with reduced homodimerization between A'-A' and B'-B'.

[0178] Methods and procedures in US13 / 866,747 (now published as US9,248,181), US14 / 081,848 (now published as US9,358,286) and PCT / NL2013 / 050294 (published as WO2013 / 157954, incorporated herein by reference) have been disclosed for producing bispecific antibodies using compatible heterodimerization domains. These procedures and procedures can also be advantageously used in the present disclosure. In particular, the bispecific antibodies of the present disclosure in certain embodiments contain mutations that produce substantial expression of bispecific full-length IgG molecules in host cells. Preferred mutations are the amino acid substitutions L351K and T366K in the first CH3 domain ("KK variant" heavy chain) and the amino acid substitutions L351D and L368E in the second domain ("DE variant" heavy chain), or vice versa. The US9,248,181 and US9,358,286 patents, as well as the WO2013 / 157954 PCT application, which are incorporated herein by reference, demonstrate that DE and KK variants preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE variant heavy chains (DEDE homodimers) is unfavorable due to repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0179] Bispecific antibodies can be produced by (transient) transfection of plasmids encoding two different heavy chains and a light chain that has been CH3 engineered to ensure efficient heterodimerization and bispecific antibody formation. The production of these chains in a single cell favors the formation of bispecific antibodies over monospecific antibodies. Preferred mutations that produce essentially only bispecific full-length IgG1 molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, e.g., L351K and T366K (numbering according to EU numbering) ("KK variant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, e.g., L351D and L368E ("DE variant" heavy chain), or vice versa.

[0180] In one embodiment, the heavy / light chain combination that comprises variable domain that binds to EGFR comprises DE variant of heavy chain.In this embodiment, the heavy / light chain combination that comprises variable domain that can bind to cMET comprises KK variant of heavy chain.The KK variant of heavy chain that binds to cMET does not produce homodimer, thereby making the observed effect of HGF-induced cMET activation inhibition by bispecific antibody very accurate.This avoids the activation (agonism) of cMET that is sometimes observed with bivalent cMET antibody.

[0181] The Fc region mediates antibody effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Depending on the use of the therapeutic antibody or Fc fusion protein, it may be desirable to reduce or increase effector function. Reduced effector function may be desired when the immune response is activated, enhanced or stimulated, as in some of the embodiments of the present disclosure. Antibodies with reduced effector function can be used to target cell surface molecules of immune cells, among others. In certain aspects, the antibodies of the present disclosure promote ACDP. In certain aspects, the antibodies of the present disclosure promote ADCC. In certain aspects, the antibodies of the present disclosure promote ADCC to a greater extent than amivantamab. In certain aspects, the antibodies of the present disclosure promote ADCC to a greater extent than amivantamab for cells or cancers with cMET abnormalities, including cMET amplification and / or cMET exon 14 skipping mutations.

[0182] The antibody with reduced effector function is, in certain embodiments, an IgG antibody comprising a modified CH2 / lower hinge region, e.g., to reduce Fc receptor interaction or to reduce C1q binding. In certain embodiments, the antibody of the present disclosure is an IgG antibody with a mutant CH2 and / or lower hinge domain such that the interaction of the bispecific IgG antibody to the Fc-gamma receptor is reduced. The antibody comprising a mutant CH2 region is, in certain embodiments, an IgG1 antibody. Such mutant IgG1 CH2 and / or lower hinge domain in certain embodiments comprises an amino substitution at position 235 and / or position 236 (EU numbering), in certain embodiments comprising a L235G and / or G236R substitution (Figure 5D).

[0183] In certain embodiments, the antibodies of the present disclosure have effector functions. In certain embodiments, the bispecific antibodies of the present disclosure include antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies can be engineered to enhance ADCC activity (for review, see Cancer Sci. 2009 Sep; 100(9): 1566-72. Engineered therapeutic antibodies with improved effector functions. Kubota T, Niwa R, Satoh M, Akinaga S, Shitara K, Hanai N). There are several in vitro methods to determine the effectiveness of antibodies or effector cells in inducing ADCC. Among them are chromium-51 [Cr51] release assay, europium [Eu] release assay, and sulfur-35 [S35] release assay. Typically, a labeled target cell line expressing a particular surface-exposed antigen is incubated with an antibody specific for that antigen. After washing, effector cells expressing the Fc receptor CD16 are co-incubated with the antibody-labeled target cells. Target cell lysis is then measured by intracellular label release by scintillation counting or spectrophotometry. In certain embodiments, the bispecific antibodies of the present disclosure exhibit ADCC activity. In such embodiments, the bispecific antibodies may have improved ADCC activity. In such embodiments, the antibodies may have altered ADCC activity 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, e.g., Junttila, TT, K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70(11):4481-4489). Thus, further provided are bispecific antibodies according to the present disclosure that are afucosylated.In certain embodiments, the antibody of the present disclosure comprises two afucosylated CH2 domains. In certain embodiments, the antibody of the present disclosure comprises a total of two CH2 domains, both of which are afucosylated. In certain embodiments, the antibody of the present disclosure is a full-length antibody, such as an IgG type, with two CH2 domains, both of which are afucosylated. Alternatively or additionally, several other strategies can be used to achieve enhanced ADCC, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis, all of which aim to improve Fc binding to low affinity activating FcγRIIIa and / or reduce binding to low affinity inhibitory FcγRIIb. The bispecific antibody of the present disclosure is, in certain embodiments, afucosylated to enhance ADCC activity. In certain embodiments, a bispecific antibody of the present disclosure comprises a reduced amount of fucosylation of N-linked carbohydrate structures in the Fc region when compared to the same antibody produced in normal CHO cells.

[0184] Variants of an antibody or bispecific antibody as described herein include functional parts, derivatives and / or analogues of the antibody or bispecific antibody. The variants maintain the binding specificity of the (bispecific) antibody. The functional parts, derivatives and / or analogues maintain the binding specificity of the (bispecific) antibody. The binding specificity is defined by the ability to bind to the extracellular portion of the first membrane protein and the second membrane protein as described herein.

[0185] The bispecific antibodies of the present disclosure are used in humans in certain embodiments. In certain aspects, the antibodies of the present disclosure are human or humanized antibodies, in certain aspects human. The constant regions of the bispecific antibodies of the present disclosure are in certain aspects human constant regions. The constant regions 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 regions of naturally occurring human antibodies. It is preferred that the constant portion is completely derived from a naturally occurring human antibody. The various antibodies produced herein are derived from a human antibody variable domain library. Thus, these variable domains are human. The unique CDR regions may be derived from humans, may be synthetic, or may be derived from another organism. A variable region is considered a humanized variable region if it has an amino acid sequence identical to the amino acid sequence of a variable region of a naturally occurring human antibody, except for the CDR regions. In such embodiments, the VH of the variable domain of an antibody that binds EGFR or cMET of the present disclosure may comprise one or more, and in certain embodiments no more than 10, and in certain embodiments no more than 5 amino acid differences, from the variable region of a naturally occurring human antibody, not counting possible differences in the amino acid sequence of the CDR regions. The light chain variable region of the EGFR binding domain and / or the cMET binding domain of an antibody of the present disclosure may comprise one or more, and in certain embodiments no more than 10, and in certain embodiments no more than 5 amino acid differences, from the variable region of a naturally occurring human antibody, not counting possible differences in the amino acid sequence of the CDR regions. The light chain of an antibody of the present disclosure may comprise one or more, and in certain embodiments no more than 10, and in certain embodiments no more than 5 amino acid differences, from the variable region of a naturally occurring human antibody, not counting possible differences in the amino acid sequence of the CDR regions. Such mutations also occur naturally in the context of somatic hypermutation.

[0186] Antibodies can be derived from various animal species, at least for the heavy chain variable region. For example, it is common practice to humanize mouse heavy chain variable regions. There are various ways in which this can be achieved, including CDR grafting to a human heavy chain variable region with a 3D structure that matches that of the mouse heavy chain variable region; in certain embodiments, deimmunization of the mouse heavy chain variable region, which is performed by removing known or suspected T-cell or B-cell epitopes from the mouse heavy chain variable region. This removal is typically by replacing one or more of the amino acids in the epitope with another (typically conservative) amino acid, such that the sequence of the epitope is modified so that it is no longer a T-cell or B-cell epitope.

[0187] Deimmunized mouse heavy chain variable regions are less immunogenic in humans than the original mouse heavy chain variable regions. In certain aspects, the variable regions or domains of the present disclosure are further humanized, e.g., veneering. Using veneering techniques, exterior residues that are easily encountered by the immune system are selectively replaced with human residues to provide hybrid molecules that contain either weakly immunogenic or substantially non-immunogenic veneering surfaces. The animals used in the present disclosure are mammals in certain aspects, primates in certain aspects, and humans in certain aspects.

[0188] In certain embodiments, the bispecific antibody according to the present disclosure comprises the constant region of a human antibody. Depending on the differences in the heavy chain constant domain, antibodies are grouped into five classes or isotypes: IgG, IgA, IgM, IgD and IgE. These classes or isotypes comprise at least one of the heavy chains named with the corresponding Greek letter. Certain embodiments comprise antibodies in which the constant region is selected from the group of IgG, IgA, IgM, IgD and IgE constant regions, in certain embodiments the constant region comprises an IgG constant region, i.e. an IgG constant region selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. In certain embodiments, the constant region is an IgG1 or IgG4 constant region, in certain embodiments a mutated IgG1 constant region. Some mutations of the IgG1 constant region occur naturally and / or are tolerated without changing the immunological properties of the resulting antibody. Mutations can also be artificially introduced to attach certain preferred characteristics to an antibody or part thereof. Such features are described herein, for example, in the context of CH2 and CH3. Typically, about 1-10 amino acid insertions, deletions, substitutions, or combinations thereof are tolerated within the constant region.

[0189] In certain embodiments, the VH chain of Figure 1, 2 or 3 has up to 15, and in certain embodiments 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain shown in Figure 1, 2 or 3; in certain embodiments 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain shown in Figure 1, 2 or 3; in certain embodiments 0, 1, 2, 3 or 4 insertions, deletions, substitutions or a combination thereof; in certain embodiments 0, 1, 2 or 3 insertions, deletions, substitutions or a combination thereof; and further in certain embodiments 0, 1 or 2 insertions, deletions, substitutions or a combination thereof; and in certain embodiments 0 or 1 deletion, substitution or a combination thereof with respect to the VH chain shown in Figure 1, 2 or 3. The one or more amino acid insertions, deletions, substitutions or combinations thereof are in certain embodiments not present in the CDR1, CDR2 and / or CDR3 regions of the VH chain, and in certain embodiments not present in the FR4 region. The amino acid substitutions are in certain embodiments conservative amino acid substitutions.

[0190] Rational methods have been evolved towards minimizing the content of non-human residues in the human context. A variety of methods are available for successfully grafting the antigen-binding properties of an antibody onto another antibody. The binding properties of an antibody can be primarily based on the correct sequence of the CDR3 region, which is often supported by the sequences of the CDR1 and CDR2 regions in the variable domain combined with the proper structure of the entire variable domain.

[0191] CDR sequences may be numbered according to the Kabat numbering scheme (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977), and / or Kabat et al., USDept. 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., Nature 342:877-883, 1989), and / or Al-Lazikani B. et al., Nature 342:877-883, 1989). al., J. Mol. Biol., 273:927-948 (1997)), the numbering system of Honegger and Plukthun (Honegger and Plukthun, J. Mol. Biol., 309:657-670 (2001)), the numbering system of MacCallum (MacCallum et al., J. Mol. Biol. 262:732-745 (1996); and / or Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008)), the numbering system of Lefranc (Lefranc MPet al., Dev. Comp. Immunol., 27:55-77 (2003); and / or Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001)), or by IMGT (discussed in Giudicelli et al., Nucleic Acids Res. 25:206-21 1 (1997)).

[0192] Each of these numbering schemes defines CDRs based on the predicted contribution of amino acid residues in the heavy or light chain variable region to antigen binding. Thus, each method for identifying CDRs can be used to identify the CDRs of the binding domains of the present disclosure. In certain embodiments, the heavy chain CDRs of the binding domains of the present disclosure are according to Kabat, Chothia, or IMGT. In certain aspects, the heavy chain CDRs of the binding domains of the present disclosure are according to Kabat. In certain embodiments, the heavy chain CDRs of the binding domains of the present disclosure are according to Chothia. In certain embodiments, the heavy chain CDRs of the binding domains of the present disclosure are according to IMGT. In certain embodiments, the light chain CDRs of the binding domains of the present disclosure are according to Kabat. In certain embodiments, the light chain CDRs of the binding domains of the present disclosure are according to Chothia. In certain aspects, the light chain CDRs of the binding domains of the present disclosure are according to IMGT. The amino acid sequences of the heavy chain CDR regions as set forth herein are determined by the Kabat definition.

[0193] Currently, various methods are available for grafting CDR regions onto suitable variable domains of another antibody. Some of these methods are reviewed in JC Almagro1 and J. Fransson (2008) Frontiers in Bioscience 13, 1619-1633, which are incorporated herein by reference. Thus, the present disclosure further provides a humanized or, in certain embodiments, human bispecific antibody as included in the treatment of the present disclosure, 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 a VH CDR3 sequence as shown in MF3370 in Figure 1, and the variable domain comprising the cMET-binding site comprises a VH CDR3 region as shown in MF4356 in Figure 1. In certain embodiments, the VH variable region comprising the EGFR-binding site comprises the sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF3370 in Figure 1. In certain embodiments, the VH variable region comprising a cMET binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF4356 in Figure 1. CDR grafting can also be used to generate a VH chain having the CDR regions of the VH of Figure 1 but a different framework. The different framework can be that of another human VH, or that of a different mammal. Thus, the present disclosure further provides a humanized or, in certain embodiments, human bispecific antibody as included in the treatment of the present disclosure, 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 a VH CDR3 sequence as shown for MF8233 in Figure 2, and the variable domain comprising the cMET binding site comprises a VH CDR3 region as shown for MF8230 in Figure 3. In certain embodiments, the VH variable region comprising an EGFR binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8233 in Figure 2. In one particular embodiment, a VH variable region comprising a cMET binding site comprises the sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8230 in FIG.CDR grafting can also be used to generate a VH chain having the CDR regions of the VH of Figure 2 or Figure 3 but with a different framework. The different framework can be that of another human VH, or that of a different mammal.

[0194] Thus, the present disclosure further provides a human or humanized bispecific antibody as included in the treatment of the present disclosure, 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 a VH CDR3 sequence as shown for MF3370 in FIG. 1, and the variable domain comprising the cMET binding site comprises a VH CDR3 region as shown for MF8230 in FIG. 3. In a particular embodiment, the VH variable region comprising the EGFR binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF3370 in FIG. 1. In a particular embodiment, the VH variable region comprising the cMET binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8230 in FIG. 3. CDR grafting can also be used to generate a VH chain having the CDR regions of the VH of FIG. 2 or FIG. 3 but with a different framework. The different framework can be that of another human VH, or that of a different mammal.

[0195] Thus, the present disclosure further provides a human or humanized bispecific antibody as included in the treatment of the present disclosure, comprising a second antigen binding site that binds EGFR and a first antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as shown for MF8233 in FIG. 2, and the variable domain comprising the cMET binding site comprises a VH CDR3 region as shown for MF4356 in FIG. 3. In certain embodiments, the VH variable region comprising the EGFR binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8233 in FIG. 2. In certain embodiments, the VH variable region comprising the cMET binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF4356 in FIG. 3. CDR grafting can also be used to generate a VH chain having the CDR regions of the VH of FIG. 2 or FIG. 3 but with a different framework. The different framework can be that of another human VH, or that of a different mammal.

[0196] Thus, the present disclosure further provides a humanized or, in certain embodiments, human bispecific antibody as included in the treatment of the present disclosure, 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 a VH CDR3 sequence as shown for MF8233 in Figure 2, and the variable domain comprising the cMET binding site comprises a VH CDR3 region as shown for MF8230 in Figure 3. In certain embodiments, the VH variable region comprising the EGFR binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8233 in Figure 2. In certain embodiments, the VH variable region comprising the cMET binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8230 in Figure 3. CDR grafting can also be used to generate VH chains having the CDR regions of the VH of Figure 2 or Figure 3 but with different frameworks. The different frameworks may be from another human VH or from a different mammal.

[0197] Methods for generating sequence variants are well known in the art. A random or targeted approach can be taken in the generation of sequence variants, for example, aiming to introduce mutations that are likely to increase or decrease binding affinity. Routine methods for affinity maturation of antibody binding domains are widely known in the art, see, for example, Tabasinezhad M.et al.Immunol Lett.2019;212:106-113. Also, the aim can be to introduce mutations that reduce developability risk in terms of large-scale production of binding domains, or portions that include such binding domains. Mutations that are likely not to cause a decrease in binding specificity and / or not to affect binding affinity can be introduced. Whether amino acid residues in the CDRs and / or framework regions can be substituted, for example, with conservative amino acid residues, and whether they can be substituted without or substantially without loss of binding specificity and / or affinity can be determined by methods well known in the art. Exemplary experiments include, but are not limited to, 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 have also been developed that can predict the effect of amino acid mutations, such as those described in, for example, 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.

[0198] Further provided herein are any of the variant anti-human EGFR and c-MET binding domains produced by the above methods, binding moieties such as antibodies comprising any of the variant binding domains, pharmaceutical compositions comprising any of the variant anti-human EGFR and c-MET binding domains or binding moieties, nucleic acids encoding any of the variant binding domains, vectors and cells comprising such nucleic acids, and uses of the variant binding domains or pharmaceutical compositions for the treatment of cancer.

[0199] Thus, the present disclosure further provides a human or humanized bispecific antibody as included in the treatment of the present disclosure, comprising a second antigen binding site that binds EGFR and a first antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as shown for MF8232 in FIG. 2, and the variable domain comprising the cMET binding site comprises a VH CDR3 region as shown for MF8230 in FIG. 3. In a particular embodiment, the VH variable region comprising the EGFR binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8232 in FIG. 2. In a particular embodiment, the VH variable region comprising the cMET binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8230 in FIG. 3. CDR grafting can also be used to generate a VH chain having the CDR regions of the VH of FIG. 2 or FIG. 3 but with a different framework. The different framework can be that of another human VH, or that of a different mammal.

[0200] Thus, the present disclosure further provides a human or humanized bispecific antibody as included in the treatment of the present disclosure, comprising a second antigen binding site that binds EGFR and a first antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as shown for MF8232 in FIG. 2, and the variable domain comprising the cMET binding site comprises a VH CDR3 region as shown for MF4356 in FIG. 3. In certain embodiments, the VH variable region comprising the EGFR binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8232 in FIG. 2. In certain embodiments, the VH variable region comprising the cMET binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF4356 in FIG. 3. CDR grafting can also be used to generate a VH chain having the CDR regions of the VH of FIG. 2 or FIG. 3 but with a different framework. The different framework can be that of another human VH, or that of a different mammal.

[0201] Thus, the present disclosure further provides a humanized or, in certain embodiments, human bispecific antibody as included in the treatment of the present disclosure, 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 a VH CDR3 sequence as shown for MF8232 in Figure 2, and the variable domain comprising the cMET binding site comprises a VH CDR3 region as shown for MF8230 in Figure 3. In certain embodiments, the VH variable region comprising the EGFR binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8232 in Figure 2. In certain embodiments, the VH variable region comprising the cMET binding site comprises sequences of the CDR1, CDR2 and CDR3 regions of the VH chain as shown for MF8230 in Figure 3. CDR grafting can also be used to generate VH chains having the CDR regions of the VH of Figure 2 or Figure 3 but with different frameworks. The different frameworks may be from another human VH or from a different mammal.

[0202] The present disclosure further provides a human or humanized bispecific antibody as included in a therapy of the present disclosure, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF3370 as shown in FIG. 2 with up to 10, and in certain embodiments, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain embodiments, 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, and the second variable domain comprises a heavy chain variable region having the amino acid sequence of MF4356 as shown in FIG. 3 (SEQ ID NO: 23) with 0-10, and in certain embodiments, 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. The present disclosure further provides a human or humanized bispecific antibody as included in a therapy of the present disclosure, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF8233 as shown in FIG. 2 with up to 10, and in certain embodiments, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain embodiments, 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, and the second variable domain comprises a heavy chain variable region having the amino acid sequence of MF8230 as shown in FIG. 3 (SEQ ID NO: 13) with 0-10, and in certain embodiments, 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof.

[0203] The present disclosure further provides a human or humanized bispecific antibody as included in a therapy of the present disclosure, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF3370 as shown in FIG. 2 with up to 10, and in certain embodiments, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain embodiments, 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, and the second variable domain comprises a heavy chain variable region having the amino acid sequence of MF8230 as shown in FIG. 3 (SEQ ID NO: 13) with 0-10, and in certain embodiments, 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof.

[0204] The present disclosure further provides a human or humanized bispecific antibody as included in a therapy of the present disclosure, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF8233 as shown in FIG. 2 with up to 10, and in certain embodiments, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain embodiments, 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, and the second variable domain comprises a heavy chain variable region having the amino acid sequence of MF4356 as shown in FIG. 3 (SEQ ID NO: 23) with 0-10, and in certain embodiments, 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof.

[0205] The present disclosure further provides a human or humanized bispecific antibody as included in a therapy of the present disclosure, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF8232 as shown in FIG. 2 with up to 10, and in certain embodiments, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain embodiments, 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, and the second variable domain comprises a heavy chain variable region having the amino acid sequence of MF4356 as shown in FIG. 3 (SEQ ID NO: 23) with 0-10, and in certain embodiments, 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof.

[0206] The present disclosure further provides a human or humanized bispecific antibody as included in a therapy of the present disclosure, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having the amino acid sequence of MF8232 as shown in FIG. 2 with up to 10, and in certain embodiments, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain embodiments, 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, and the second variable domain comprises a heavy chain variable region having the amino acid sequence of MF8230 as shown in FIG. 3 (SEQ ID NO: 13) with 0-10, and in certain embodiments, 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof.

[0207] Up to 15, and in certain embodiments 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain embodiments 0, 1, 2, 3, 4 or 5 amino acid substitutions, which in certain embodiments are conservative amino acid substitutions, and which insertions, deletions, substitutions or combinations thereof, in certain embodiments, are not present in the CDR3 region of the VH chain, and in certain embodiments, are not present in the CDR1, CDR2 or CDR3 regions of the VH chain, and in certain embodiments, are not present in the FR4 region.

[0208] Various methods are available for producing bispecific antibodies. One method involves the expression of two different heavy chains and two different light chains in a cell and collecting the antibodies produced by the cells. The antibodies produced in this way typically contain a collection of antibodies with different combinations of heavy and light chains, some of which are the desired bispecific antibodies. The bispecific antibodies can then be purified from the collection. The ratio of bispecific to other antibodies produced by the cell can be increased in various ways. In certain embodiments, the ratio is increased by expressing a common light chain in the cell rather than two different light chains. When a common light chain is expressed with two different heavy chains, the ratio of bispecific to other antibodies produced by the cell is significantly improved over the expression of two different light chains. The ratio of bispecific antibodies produced by the cell can be further improved by stimulating the pairing of two different heavy chains with each other versus the pairing of two identical heavy chains. Methods and means for producing bispecific antibodies (from a single cell) are disclosed, thereby providing a means to favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be advantageously used in the present disclosure. Thus, the present disclosure in a particular embodiment provides a method for producing a bispecific antibody from a single cell, the bispecific antibody comprising two CH3 domains capable of forming an interface, the method comprising providing to the cell a) a first nucleic acid molecule encoding a first CH3 domain comprising a heavy chain, b) a second nucleic acid molecule encoding a second CH3 domain comprising a heavy chain, the nucleic acid molecule being provided with a means for preferentially pairing the first and second CH3 domains comprising a heavy chain, the method further comprising culturing the host cell to allow expression of the two nucleic acid molecules, and harvesting the bispecific antibody from the culture. The first and second nucleic acid molecules may be part of the same nucleic acid molecule, vector or gene delivery vehicle, or may be integrated into the same site of the genome of the host cell. Alternatively, the first and second nucleic acid molecules are provided separately to the cell.

[0209] Certain embodiments are methods for producing a bispecific antibody according to the present disclosure from a single cell, the bispecific antibody comprising two CH3 domains capable of forming an interface, the method comprising: - providing a cell having a) a first nucleic acid molecule encoding a heavy chain that binds to EGFR and comprises an antigen binding site that contains a first CH3 domain, and b) a second nucleic acid molecule encoding a heavy chain that binds to ErbB-3 and comprises an antigen binding site that contains a second CH3 domain, said cell being provided with a means for preferentially pairing said first and second CH3 domains, 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 embodiments, the cells also have a third nucleic acid molecule encoding a common light chain. The first, second and third nucleic acid molecules may be part of the same nucleic acid molecule, vector or gene delivery vehicle and may be integrated into the same site of the genome of the host cell. Alternatively, the first, second and third nucleic acid molecules are provided separately to the cells. In certain embodiments, the common light chain is based on O12, which in certain embodiments is a rearranged germline human kappa light chain IgVκ1 39*01 / IGJκ1*01, as described above. The means for preferential pairing of the first and second CH3 domains in certain embodiments is a corresponding mutation in the CH3 domain of the heavy chain coding region. Preferred mutations for preferentially producing bispecific antibodies are the amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain and L351D and L368E in the second CH3 domain, or vice versa. Thus, there is further provided a method according to the present disclosure for producing a bispecific antibody, wherein the first CH3 domain comprises the amino acid substitutions L351K and T366K (EU numbering) and the second CH3 domain comprises the amino acid substitutions L351D and L368E, the method further comprising the steps of culturing the cells to allow expression of the protein encoded by the nucleic acid molecule and harvesting the bispecific antibody from the culture. Also provided is a method according to the present disclosure for producing a bispecific antibody, wherein the first CH3 domain comprises amino acid substitutions L351D and L368E (EU numbering) and the second CH3 domain comprises amino acid substitutions L351K and T366K, the method further comprising culturing the cells to allow expression of the nucleic acid molecule and harvesting the bispecific antibody from the culture. Antibodies that can be produced by these methods are also part of the present disclosure. The CH3 heterodimerization domain is, in one particular embodiment, an IgG1 heterodimerization domain.The heavy chain constant region containing the CH3 heterodimerization domain is, in one particular embodiment, an IgG1 constant region.

[0210] In one embodiment of the disclosure, the nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region. In certain aspects, the nucleic acid molecule (typically an in vitro, isolated, or recombinant nucleic acid molecule) encodes a heavy chain variable region as shown in FIG. 2 or FIG. 3, or a heavy chain variable region as shown in FIG. 2 or FIG. 3 with 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof. In certain aspects, the nucleic acid molecule comprises a codon-optimized nucleic acid sequence encoding an amino acid sequence as shown in FIG. 2 or FIG. 3. The codon optimization is optimized for the species and / or cell type of the antibody producing cell. For example, for CHO production, the nucleic acid sequence of the molecule is codon optimized for Chinese Hamster cells. The disclosure further provides a nucleic acid molecule encoding the heavy chain of FIG. 2 or FIG. 3.

[0211] The nucleic acid molecules used in the present disclosure are typically, but not limited to, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Alternative nucleic acids are available to those of skill in the art. The nucleic acid according to the present disclosure is, for example, contained in a cell. When the nucleic acid is expressed in the cell, the cell can produce an antibody according to the present disclosure. Thus, an embodiment of the present disclosure includes a cell that contains an antibody according to the present disclosure and / or a nucleic acid according to the present disclosure. The cell is, in certain aspects, an animal cell, in certain aspects a mammalian cell, in certain aspects a primate cell, and in certain aspects a human cell. A suitable cell is any cell that can contain and, in certain aspects, can produce an antibody according to the present disclosure and / or a nucleic acid according to the present disclosure.

[0212] The present disclosure further provides a cell comprising an antibody according to the present disclosure. In certain aspects, the cell (typically an in vitro, isolated, or recombinant cell) produces the antibody. The cell can also be a stored cell that can produce the antibody when removed from storage and cultured. In certain aspects, the cell is a hybridoma cell, a Chinese Hamster Ovary (CHO) cell, an NS0 cell, or a PER-C6™ cell. In certain aspects, the cell is a CHO cell. A cell culture comprising a cell according to the present disclosure is further provided. Various institutions and companies are developing cell lines for 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™ cells. These cells are also used for other purposes, such as production of proteins. Cell lines developed for industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. Thus, certain embodiments include the use of cell lines developed for large scale production of antibodies for the production of the antibodies of the present disclosure, which in certain embodiments include cells for producing antibodies comprising nucleic acid molecules encoding the VH, VL and / or heavy chains as shown in FIG. 2 or FIG.

[0213] The present disclosure further provides a method for producing an antibody, comprising culturing a cell of the present disclosure and harvesting the antibody from the culture. In certain aspects, the cell is cultured in serum-free medium. In certain aspects, the cell is suitable for suspension growth. Further provided is an antibody obtainable by the method for producing an antibody according to the present disclosure. The antibody is purified from the culture medium in certain aspects. In certain aspects, the antibody is affinity purified.

[0214] The cell of the present disclosure is, for example, a hybridoma cell line, a CHO cell, a 293F cell, an NS0 cell, or another cell type known for its suitability for antibody production for clinical purposes. In certain embodiments, the cell is a human cell. In certain embodiments, a cell transformed by an adenovirus E1 region or a functional equivalent thereof. In certain embodiments, such a cell line is the PER.C6™ cell line or an equivalent thereof. In certain embodiments, the cell is a CHO cell or a variant thereof. In certain embodiments, a variant using the glutamine synthetase (GS) vector system for the expression of the antibody.

[0215] The antibodies of the present disclosure can be produced at levels of >50 mg / L after transient transfection in suspension 293F cells. The bispecific antibodies can be purified to >98% purity with >70% yield. Analytical characterization studies show bispecific IgG1 antibody profiles comparable to bivalent monospecific IgG1. In terms of functional activity, the bispecific antibodies of the present disclosure can demonstrate superior potency compared to cetuximab in vitro and in vivo.

[0216] The present disclosure further provides a pharmaceutical composition comprising an antibody according to the present disclosure. The pharmaceutical composition in certain embodiments comprises a pharma- ceutically acceptable excipient or carrier.

[0217] The antibody may include a label, in certain embodiments, a label for in vivo imaging. Such a label is typically not necessary for therapeutic applications. For example, in diagnostic settings, the label may be useful, for example, in visualizing target cells in the body. A variety of labels are suitable, and many labels are known in the art. In certain embodiments, the label is a radioactive label for detection. In certain embodiments, the label is an infrared label. In certain embodiments, 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).

[0218] The present disclosure further provides a method for the treatment of a subject having or at risk of having a tumor, comprising administering to a subject in need of said treatment an antibody or pharmaceutical composition according to the present disclosure. The tumor, in certain embodiments, is an EGFR, cMET or EGFR / cMET positive tumor. Prior to initiating said treatment, the method in certain embodiments further comprises determining whether said subject has such an EGFR, cMET or EGFR / cMET positive tumor. The present disclosure further provides an antibody or pharmaceutical composition of the present disclosure for use in the treatment of a subject having or at risk of having an EGFR, cMET or EGFR / cMET positive tumor.

[0219] To confirm whether a tumor is positive for EGFR, a person skilled in the art can determine, for example, EGFR amplification and / or immunohistochemical staining. At least 10% of the tumor cells in the biopsy should be positive. The biopsy may also include 20%, 30%, 40%, 50%, 60%, 70% or more positive cells. To confirm whether a tumor is positive for cMET, a person skilled in the art can determine, for example, cMET amplification and / or staining in immunohistochemistry. At least 10% of the tumor cells in the biopsy should be positive. The biopsy may also include 20%, 30%, 40%, 50%, 60%, 70% or more positive cells.

[0220] The cancer or tumor may be an EGFR, cMET or EGFR / cMET positive cancer. In one preferred embodiment, the present disclosure provides for the treatment of 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, gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer. The present disclosure can be applied to a wide range of EGFR, cMET or EGFR / cMET positive cancers, such as lung cancer, including in certain embodiments non-small cell lung cancer. The subject, in certain embodiments, is a human subject. The subject, in certain embodiments, is a subject eligible for antibody therapy using an EGFR specific antibody, such as cetuximab. In certain aspects, the present disclosure may treat a subject comprising a tumor, in certain aspects, an EGFR / cMET positive cancer, in certain aspects, a tumor / cancer having an EGFR RTK resistance phenotype, an EGFR monoclonal antibody resistance phenotype, or a combination thereof.

[0221] As used herein, the term "cancer" applies synonymously to the term "tumor," such that treatment of a tumor also applies to treatment of cancer.

[0222] The amount of antibody administered to a patient is typically within a therapeutic window, meaning that a sufficient amount is used to obtain a therapeutic effect, but the amount does not exceed a threshold that results in an unacceptable degree of side effects. The smaller the amount of antibody required to obtain a desired therapeutic effect, the larger the therapeutic window is typically. Thus, antibodies according to the present disclosure that exert sufficient therapeutic effect at low dosages are preferred. Dosages can be within the range of the dosing regimen of cetuximab. Dosages can be lower. In certain embodiments, the dose is 1000 mg, 1500 mg, or 2000 mg. Dosage can be once every week or once every two weeks.

[0223] 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, gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer, and the antibody of the disclosure is administered once weekly in an amount of 1000 mg, typically in a flat dose of 1000 mg.

[0224] 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, gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer, and the antibody of the disclosure is administered in an amount of 1000 mg, typically a flat dose of 1000 mg, once every two weeks.

[0225] 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, gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer, and the antibody of the disclosure is administered in an amount of 1500 mg, typically a flat dose of 1500 mg, once every two weeks.

[0226] 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, gastric / esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer, and the antibody of the disclosure is administered in an amount of 2000 mg, typically a flat dose of 2000 mg, once every two weeks.

[0227] In certain aspects, the bispecific antibody that binds EGFR and cMET is provided to a subject at a dose of 1000, 1500 or 2000 mg, particularly using a flat dose regimen. A flat dose regimen offers several advantages over surface or body weight dosing, as it reduces preparation time and reduces potential dose calculation errors. In certain aspects, the bispecific antibody is administered once every week (Q1W), once every two weeks (Q2W) or once every three weeks (Q3W). In certain aspects, the bispecific antibody is administered once every two weeks. In the art, such a dosing scheme is described as Q2W. In certain embodiments, the flat dose regimen disclosed herein is suitable for use in adults and / or subjects weighing at least 35 kg. As will be appreciated by those skilled in the art, this dosage can be administered over time. As will be understood by those skilled in the art, the term "flat dose" or "flat dose regimen" means that a subject receives a dosing regimen in which the subject is scheduled to receive substantially the same predetermined amount of bispecific antibody every day, regardless of the subject's body weight. According to certain embodiments, the subject is provided with a flat weekly dose of 1000 mg of the bispecific antibody. Alternatively, the subject is provided with a flat biweekly dose of 1000 mg of the bispecific antibody. Alternatively, the subject is provided with a flat biweekly dose of 1500 mg of the bispecific antibody. Alternatively, the subject is provided with a flat biweekly dose of 2000 mg of the bispecific antibody.

[0228] In certain embodiments, bispecific antibodies according to the present disclosure induce less skin toxicity compared to cetuximab under otherwise similar conditions. In certain embodiments, bispecific antibodies according to the present disclosure produce less proinflammatory chemokines, in certain embodiments CXCL14, compared to cetuximab under otherwise similar conditions. In certain embodiments, bispecific antibodies according to the present disclosure induce less damage to antimicrobial RNAses, in certain embodiments Rnase 7, compared to cetuximab under otherwise similar conditions.

[0229] The present disclosure describes, inter alia, antibodies that target EGFR and cMET receptors, resulting in potent proliferation inhibition of cancer cell lines in vitro and tumor growth inhibition in vivo. Bispecific antibodies can combine low toxicity profiles with high efficacy. The antibodies of the present disclosure may be useful for various types and lines of EGFR-targeted therapy. The antibodies of the present disclosure may have an increased therapeutic window compared to antibodies that bind to the same antigen in both arms. The bispecific antibodies of the present disclosure may exhibit better growth inhibition effects in vitro, in vivo, or a combination thereof when compared to cetuximab antibodies.

[0230] The present disclosure provides a bispecific antibody for use in treating a subject that may have one or more of a variety of different types of tumors. The tumor may be an EGFR positive tumor, a cMET positive tumor, or an EGFR and cMET positive tumor. The tumor may be resistant to treatment with an EGFR or cMET tyrosine kinase inhibitor. In certain aspects, the EGFR tyrosine kinase inhibitor is a third generation EGFR tyrosine kinase inhibitor, in certain aspects, osimertinib or an analog thereof. In certain aspects, the cMET tyrosine inhibitor is or includes capmatinib or tepotinib. In this and other embodiments, the tumor may be an HGF-associated tumor.

[0231] EGFR-positive tumors are typically tumors that have EGFR-activating mutations. EGFR-activating mutations are mutations in EGFR that result in activation of the EGF / EGFR signaling pathway. EGFR-activating mutations may be important for the cancerous state of the tumor. One of the ways in which such tumors may become insensitive to EGFR-targeted therapy is through activation of the HGF / cMET signaling pathway. The tumor may be an HGF-associated tumor. Activation of the cMET / HGF signaling pathway is one of the ways in which EGFR-positive tumors can escape treatment with EGFR-targeted therapy. The cMET / HGF pathway may be activated in various ways. Various methods of activation have been described in the art, some of which are detailed herein. The antibodies of the present disclosure are particularly suitable for treating tumors in which activation of the cMET / HGF signaling pathway is associated with the presence or excess of HGF. Such cMET-positive tumors are referred to as HGF-associated or HGF-dependent tumors. The antibodies of the present disclosure may also be used to at least partially inhibit this possible escape mechanism of EGFR-positive tumors. Such tumors may also escape EGFR-targeted therapy due to the selective development of tumor cells in which the cMET / HGF signaling pathway is activated. Such cells may be present at the onset of EGFR-targeted therapy. Such cells have a selective growth advantage over HGF / cMET signaling negative tumor cells. The tumor may be a tumor in which the HGF / cMET signaling pathway is activated. The tumor may be a tumor associated with elevated levels of hepatocyte growth factor (HGF) or overexpression of the HGF receptor c-Met. The tumor may be a tumor whose growth is driven by EGF and / or HGF. A tumor is said to be driven by a particular growth factor if a signaling pathway is activated in the cells of the tumor in response to the presence of the growth factor and removal of the growth factor results in inhibition of growth of the cells of the tumor. This reduction can be measured by reduced cell division and / or induced cell death, e.g., apoptosis. A tumor is an HGF-associated tumor if the tumor grows or grows faster in the presence of HGF under conditions that would otherwise permit tumor growth.

[0232] EGFR-targeted therapy for various tumors was reviewed by Vecchione et al., EGFR-targeted therapy.” Experimental cell research Vol 317 (2011): 2765-2771. In general, EGFR-targeted therapy is a therapy with a molecule that interacts with EGFR and inhibits EGFR-mediated signaling in cells.

[0233] In certain aspects, the methods of treatment or antibodies for use in treatment as provided herein further comprise the step of determining whether the tumor is an HGF-associated tumor. In certain aspects, the antibodies of the present disclosure are capable of inhibiting the growth of HGF-associated tumors.

[0234] When a range is given herein between the number 1 and the number 2, the range includes the number 1 and the number 2. For example, the range from 2 to 5 includes the numbers 2 and 5.

[0235] When referring to an affinity higher than another affinity herein, Kd = lower than the other Kd. For the avoidance of doubt, a Kd of 10e-9M is lower than a Kd of 10e-8M. An antibody with a Kd of 10e-9M for a target has a higher affinity than one with a Kd of 10e-8M.

[0236] In certain embodiments, at the start of treatment, at least one, more than one, or all of the following inclusion factors IF1-IF7 apply to the subject of treatment. In certain embodiments, the subject includes or complies with all of inclusion factors IF1-IF8: IF1. Be 18 years of age or older at the time of signing the informed consent. IF2. Histologically or cytologically confirmed solid tumor with evidence of incurable metastatic or locally advanced unresected disease. IF3.1. For subjects who have failed prior standard first-line therapy: Subjects have progressed on or are intolerant to a therapy known to provide clinical benefit. Subjects have either NSCLC harboring activating EGFR mutations, including tyrosine kinase inhibitor (TKI)-sensitizing mutations and / or approved TKI-resistant mutations, or any activating c-MET mutation / amplification. IF3.2. For subjects who have received prior first line or higher anti-cancer therapy: have progressed on or are intolerant to therapy known to provide clinical benefit. Patients must have: NSCLC harboring EGFR exon 20 insertions that have progressed after platinum doublets, or second line (or higher) patients, NSCLC harboring cMet exon 14 skipping mutations in capmatinib and tepotinib resistant populations, or those treated with cMET TKI or cMET TKI naive, or selected solid tumors harboring EGFR or cMET driving mutations or cMET alterations (including GC / GEJ, HNSCC, e.g., HNSCC refractory to approved therapies (regardless of driving mutation), or ESCC), NSCLC patients with rare EGFR mutations (G719X, L861Q and S768I) or NSCLC patients harboring c-MET exon 14 skipping mutations, and NSCLC with treatment naive for progressive disease. Subjects have exhausted or are intolerant to all approved therapies with proven clinical benefit, or have no approved therapeutic options available. IF4. Have availability of FFPE embedded after progression of current therapy, archival or fresh tumor tissue samples. IF5. Have measurable disease as defined by RECIST version 1.1 by radiological methods (patients with non-measurable but evaluable disease may be included in the dose escalation portion). Have an Eastern Cooperative Oncology Group (ECOG) performance status of IF6.0 or 1. IF7. Life expectancy of 12 weeks or more. IF8. Have adequate organ function as established by at least one or all of the following: IF8.1 Absolute neutrophil count (ANC) ≥ 1.5 × 10 9 / L. IF8.2 Hemoglobin ≥ 9g / dL. IF8.3 Platelets ≥100×109 / L. IF8.4 Corrected total serum calcium within normal range. IF8.5 Serum magnesium within normal range (or corrected with supplements). IF8.6 Serum potassium within normal range. IF8.7 Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 3.0 times the upper limit of normal (ULN) and total bilirubin ≤ 1.5 x ULN except in cases of hepatic involvement or malignancy, when ALT / AST ≤ 5 x ULN and total bilirubin ≤ 2 x ULN. IF8.8 In patients with Gilbert's syndrome, conjugated bilirubin has values ​​within the normal range. IF8.9 Serum creatinine less than or equal to 1.5 × ULN or creatinine clearance greater than or equal to 50 mL / min, calculated according to the Cockroft and Gault formula or the MDRD formula for patients over 65 years of age. IF8.10 Serum albumin > 3.3 g / dL

[0237] In certain embodiments, all values ​​for IF8 organ function measurements have upper limits observed in healthy subjects.

[0238] In certain embodiments, the subject of treatment includes one or more factors selected from the group consisting of IF1-IF8. In certain embodiments, the subject of treatment includes factors IF2, IF3, IF5, and IF8. In certain embodiments, the subject of treatment includes all of factors IF1-IF8.

[0239] In certain embodiments, at the start of treatment, at least one, more than one, or all of the following exclusion factors EF1 to EF16 apply to a patient who is a candidate for treatment.

[0240] EF1. Have central nervous system metastases to: EF1.1: Subjects with untreated symptomatic CNS metastases, while those with asymptomatic disease, may be included if considered stable; EF1.2 Central nervous system metastases requiring radiation or surgery; EF1.3 Central nervous system metastases requiring ongoing steroid therapy (>10 mg prednisone or equivalent) to control symptoms within 14 days of administration prior to administration of the first dose. Subjects with other central nervous system metastases are permitted. EF2. Known leptomeningeal involvement. EF3. Participation in another clinical study or treatment with any investigational drug within 4 weeks prior to administration of the first dose. Administration of systemic anticancer therapy or immunotherapy within 4 weeks or 5 half-lives of the first dose of investigational drug, whichever is shorter. For cytotoxic agents with significant delayed toxicity (e.g., mitomycin C, nitrosoureas), a 6-week washout period is required. EF5. Major surgery or radiation therapy within 3 weeks of receiving the first dose. Subjects with prior radiation therapy to 25% or more of the bone marrow at any time are not eligible. EF6. Have persistent clinically significant toxicity Grade ≥1 related to prior antineoplastic therapy (excluding alopecia); except for stable sensory neuropathy Grade ≤2 per NCI-CTCAE v5.0 and hypothyroidism Grade ≤2 stable on hormone replacement. EF7. History of hypersensitivity reactions or any toxicity due to either human proteins or excipients that justified permanent discontinuation of these agents. EF8. Have a history of clinically significant cardiovascular disease, including but not limited to: EF8.1 Diagnosis of deep vein thrombosis or pulmonary embolism within 1 month prior to the first dose of investigational product, or diagnosis of any of the following within 6 months prior to the first dose of investigational product: myocardial infarction, unstable angina, stroke, transient ischemic attack, coronary artery / peripheral artery bypass graft, or acute coronary syndrome. EF8.2 Prolonged QT interval >480 ms or clinically significant arrhythmia or electrophysiologic disease (i.e., placement of an implantable cardioverter defibrillator or uncontrolled atrial fibrillation at a rate). Patients with a clinically stable cardiac pacemaker are eligible. EF8.3 Uncontrolled (sustained) arterial hypertension: systolic blood pressure >180mmHg and / or diastolic blood pressure >100mmHg. EF8.4 Congestive heart failure (CHF) defined as New York Heart Association (NYHA) class III-IV or hospitalization for CHF within 6 months of first dose of study drug. EF8.5 Clinically significant pericardial effusion. EF8.6 Myocarditis. EF9. History of interstitial lung disease, including drug-induced interstitial lung disease or radiation pneumonitis requiring long-term treatment with steroids or other immunosuppressants within the past year. EF10. Previous or concurrent malignancy, except for non-basal cell carcinoma of the skin or in situ carcinoma of the cervix, as long as the tumor is treated with curative or palliative intent and the previous or concurrent malignancy does not affect the evaluation of the safety and efficacy of the investigational product. EF11. Have a current serious illness or psychiatric disorder, including but not limited to, active uncontrolled infection, clinically significant pulmonary, metabolic, or psychiatric disease. EF12. Active hepatitis B infection (HBsAg positive) without antiviral therapy. Subjects with active hepatitis B (HbsAg positive) must be on antiviral treatment with lamivudine, tenofovir, entecavir or other antiviral agents starting at least 7 days prior to administration of the first dose. Subjects with prior hepatitis B (anti-HBc positive, HbsAg and HBV-DNA negative) are eligible. EF13. Have a positive test for Hepatitis C ribonucleic acid (HCV RNA); Subjects whose HCV infection has spontaneously resolved (positive HCV antibodies without detectable HCV-RNA) or who have achieved a sustained virologic response after antiviral treatment and demonstrate an absence of detectable HCV RNA for more than 6 months (using IFN-free regimens) or more than 12 months (using IFN-based regimens) after cessation of antiviral treatment are eligible. EF14. Have a history of HIV (HIV1 / 2 antibodies). Patients with HIV with an undetectable viral load are acceptable. HIV testing is not required unless mandated by local health authorities or regulations. EF15. For sexually active male and female patients of childbearing potential, agree to use one of the following methods of contraception for the entire duration of the study and for 6 months after the last dose of PB19478: Combined (estrogen- and progestogen-containing) hormonal contraception associated with the inhibition of ovulation (oral, intravaginal, transdermal) Progestogen-only hormonal contraception (oral, injectable, implantable) associated with inhibition of ovulation Intrauterine device (IUD) Intrauterine hormone releasing system (IUS) Bilateral tubal obstruction Vasectomized partner ·Sexual abstinence EF16. Pregnant or breastfeeding.

[0241] In certain embodiments, the subject of treatment complies with one or more factors selected from the group consisting of EF1-EF16. In certain embodiments, the subject of treatment complies with all of factors EF1-EF16.

[0242] Reference to a cited patent document or other matter in this specification should not be considered an admission that the document or matter was known or that the information contained therein was part of the common general knowledge as of the priority date of any of the claims.

[0243] The connector term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, if 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 be within this meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one of the options is also understood to be within this meaning and thus meets the requirements of the term "and / or."

[0244] Although for clarity and conciseness of description, features are described herein as part of the same or separate embodiments, it will be understood that the scope of the disclosure may include embodiments having all or any combination of the described features. [Brief description of the drawings]

[0245] [Figure 1] The amino acid sequences of the heavy chain variable regions of the variable domains referred to in this application. [Diagram 2] MF3370 and its variants. The CDR1, CDR2 and CDR3 sequences in MF8226 are underlined from left to right. The CDRs in the other sequences are in the corresponding positions (according to Kabat). [Figure 3-1] MF4356 and its variants. The CDR1, CDR2 and CDR3 sequences in MF4356 are underlined from left to right. The CDRs in the other sequences are in the corresponding positions (according to Kabat). [Figure 3-2] MF4356 and its variants. The CDR1, CDR2 and CDR3 sequences in MF4356 are underlined from left to right. The CDRs in the other sequences are in the corresponding positions (according to Kabat). [Figure 4A] Common light chain used in mono- and bispecific IgG. Common light chain amino acid sequence. [Figure 4B] Common light chain used in mono- and bispecific IgG. Common light chain variable domain DNA sequence and translation (IGKV1-39 / jk1). [Figure 4C] Common light chain used in mono- and bispecific IgG. Common light chain constant region DNA sequence and translation. [Figure 4D] Common light chain used in mono- and bispecific IgG. IGKV1-39 / jk5 common light chain variable domain translation. [Figure 4E] Common light chain used in mono- and bispecific IgG. V region IGKV1-39A. [Figure 5A] IgG heavy chain for the generation of bispecific molecules. CH1 region. [Figure 5B] IgG heavy chain for the generation of bispecific molecules. Hinge region. [Figure 5C] IgG heavy chain for the generation of bispecific molecules. CH2 region. [Figure 5D] IgG heavy chain for the generation of bispecific molecules. CH2 containing L235G and G236R silencing substitutions. [Figure 5E] IgG heavy chain for the generation of bispecific molecules. CH3 domain containing substitutions L351K and T366K (KK). [Figure 5F] IgG heavy chain for the generation of bispecific molecules. CH3 domain containing substitutions L351D and L368E(DE). [Figure 6] Western blot analysis results of model LXFE2478 showing EGFR, cMET and HGF protein levels. [Figure 7] Summary of treatment schedule. Antibody or vehicle was administered as monotherapy over a 5 week period. [Figure 8] Effect of antibody therapy on tumor volume in NSCLC PDX model LXFE2478 harboring a mutation in exon 20. Tumor volume growth curves (mean TV ± SEM) representing groups 1–5 shown at different time points. [Figure 9]Summary of treatment schedule. Antibody or vehicle was administered as monotherapy over a 3 week period. [Figure 10] Effect of antibody therapy on tumor volume in the NSCLC CDX model HCC827 / ER1 harboring a mutation in exon 19. Tumor volume growth curves (mean TV±SEM) representing vehicle and antibody groups shown at different time points. [Figure 11] Animal survival curves of mice harboring the NSCLC CDX model HCC827 / ER1 harboring a mutation in exon 19 versus vehicle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0246] Terms 1. A bispecific antibody comprising a first variable domain capable of binding to the extracellular part of the human epidermal growth factor receptor (EGFR) and a second variable domain capable of binding to the extracellular part of the human MET proto-oncogene receptor tyrosine kinase (cMET), for use in a method for the treatment of cancer in a subject 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) a method for the treatment of cancer in a subject who has not received prior anti-cancer treatment, wherein the first variable domain has the CDR1 sequence SYGIS, CDR2 sequence SYGIS, CDR3 sequence SYGIS, CDR4 sequence SYGIS, CDR5 sequence SYGIS, CDR6 sequence SYGIS, CDR7 sequence SYGIS, CDR8 sequence SYGIS, CDR9 sequence SYGIS, CDR10 sequence SYGIS, CDR11 sequence SYGIS, CDR12 sequence SYGIS, CDR13 sequence SYGIS, CDR14 sequence SYGIS, CDR15 sequence SYGIS, CDR16 sequence SYGIS, CDR17 sequence SYGIS, CDR18 sequence SYGIS, CDR19 ...9 sequence SYGIS, CDR11 sequence SYGIS, CDR12 sequence SYGIS, CDR13 sequence SYGIS, CDR14 sequence SYGIS, CDR15 sequence SYGIS, CDR16 sequence SYGIS, CDR17 sequence SYGIS, A bispecific antibody comprising a heavy chain variable region having the 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, and having 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof at positions other than X1 to X7, and a second variable domain having one of the amino acid sequences of SEQ ID NOs: 1 to 23, having 0 to 10, and in a specific aspect, 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0247] 2. A method of treating cancer in a subject 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) a cancer in a subject who has not received prior anti-cancer treatment, the treatment 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), the first variable domain having the CDR1 sequence SYGIS, the CDR2 sequence WISAYX1X2NTN YAQKLQG, 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, and having 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof at positions other than X1 to X7, and a second variable domain having one of the amino acid sequences of SEQ ID NOs: 1 to 23, having 0 to 10, and in a particular aspect 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0248] 3. A 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 the first variable domain has the CDR1 sequence SYGIS, CDR2 sequence SYGIS, CDR3 sequence SYGIS, CDR4 sequence SYGIS, CDR5 sequence SYGIS, CDR6 sequence SYGIS, CDR7 sequence SYGIS, CDR8 sequence SYGIS, CDR9 sequence SYGIS, CDR10 sequence SYGIS, CDR11 sequence SYGIS, CDR12 sequence SYGIS, CDR13 sequence SYGIS, CDR14 sequence SYGIS, CDR15 sequence SYGIS, CDR16 sequence SYGIS, CDR17 sequence SYGIS, CDR18 sequence SYGIS, CDR19 sequence SYGIS, CDR1 ...9 sequence SYGIS, CDR11 sequence SYGIS, CDR11 sequence SYGIS, CDR12 sequence SYGIS, CDR13 sequence SYGIS, CDR14 sequence 2. Use of a bispecific antibody comprising a heavy chain variable region having the CDR2 sequence WISAYX1X2NTNYAQKLQG, and the 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, and having 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof at positions other than X1 to X7, and a second variable domain comprising a heavy chain variable region having one of the amino acid sequences of SEQ ID NOs: 1 to 23, having 0 to 10, and in a certain embodiment 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0249] 4. The use or method of any one of the preceding clauses, wherein the subject or cancer having undergone prior treatment with i) is resistant to treatment with a third generation EGFR tyrosine kinase inhibitor.

[0250] 5.ii) The use or method of any one of clauses 1 to 3, wherein the subject or cancer having undergone prior treatment with is resistant to treatment with a first, second and / or third generation tyrosine kinase inhibitor.

[0251] 6.iii) The use or method of any one of clauses 1 to 3, wherein the subject or cancer having undergone prior treatment with a cMET tyrosine kinase inhibitor is resistant to treatment with a cMET tyrosine kinase inhibitor.

[0252] 7. The use or method according to any one of the preceding clauses, wherein administration of the bispecific antibody according to i) comprises a second line treatment and administration according to ii) or iii) comprises a third line treatment.

[0253] 8. The use or method of any one of the preceding clauses, wherein said first generation EGFR tyrosine kinase inhibitor comprises or is gefitinib, erlotinib, gefitinib or icotinib.

[0254] 9. The use or method of any one of the preceding clauses, wherein the second generation EGFR tyrosine kinase inhibitor comprises or is afatinib, dacomitinib, XL647, AP26113, CO-1686 or neratinib.

[0255] 10. The use or method according to any one of the preceding clauses, wherein said third generation EGFR tyrosine kinase inhibitor comprises osimertinib, lazertinib, alflutinib, resibertinib, rociletinib, olmutinib, almonertinib, abivertinib, ASK120067, befotertinib, olmutinib, rociletinib or SH-1028 (nazartinib (EGF816), nacotinib (ASP8273), mavereltinib (PF-0647775), orafeltinib (CK-101), kenatinib or ES-072, preferably osimertinib.

[0256] 11. The use or method according to any one of the preceding clauses, wherein the cMET tyrosine kinase inhibitor comprises or is capmatinib, tepotinib, crizotenib, cabozantinib, savolitinib, glesatinib, sitravatinib, BMS-777607, merestinib, tivantinib, golvatinib, foretinib, AMG-337 or BMS-794833, preferably capmatinib or tepotinib.

[0257] 12. The use or method according to any one of the preceding clauses, wherein the chemotherapy comprises platinum-based chemotherapy, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide or pemetrexed or any combination thereof, preferably a combination comprising cisplatin or carboplatin.

[0258] 13. The use or method of any one of the preceding clauses, wherein the cancer is EGFR positive cancer, cMET positive cancer, or EGFR and cMET positive cancer.

[0259] 14. The use or method of any one of the preceding clauses, wherein the cancer comprises EGFR aberrations, cMET aberrations, or EGRF and cMET aberrations.

[0260] 15. The use or method of any one of the preceding clauses, wherein the cancer comprises activating EGFR mutations, approved tyrosine kinase inhibitor resistance mutations, tertiary tyrosine kinase inhibitor resistance mutations (L718X (e.g., L718Q), G719X (e.g., G719A), L792X (e.g., L792H), G796X (e.g., G796R, G796S, G796D), C797X, C797X (e.g., C797S, C797G), mutations that reduce binding of third generation tyrosine kinase inhibitors to EGFR (e.g., L792X, L718X), acquired tyrosine kinase inhibitor resistance mutations (such as C797X, L792X, G796X, G724X, S768X, L718X or exon 20 insertion mutations), EGFR gene amplification, cMET mutations or cMET aberrations.

[0261] 16. The use or method according to any one of the preceding clauses, wherein the cancer comprises an exon 19 deletion mutation, preferably an in-frame exon 19 deletion, an exon 20 missense mutation (e.g., T790M), or an exon 21 mutation such as L858R.

[0262] 17. The use or method according to any one of the preceding clauses, wherein the cancer comprises an EGFR exon 20 mutation, preferably an exon 20 insertion mutation.

[0263] 18. The use or method of any one of the preceding clauses, wherein the cancer comprises an acquired tyrosine kinase inhibitor resistance mutation, such as a mutation conferring resistance to osimertinib, including G724X (e.g., G724S), S768X (e.g., S768I), T790X (e.g., T790M), L792X (e.g., L792H), C797X (including C797S and C797G), L798X (e.g., L798I).

[0264] 19. The cancer has a proximal loop insertion (positions 767-772), a distal 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_N 771insP, 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 (e.g., L792H), C796X (e.g., G796R, G796S, G796D), C797X (e.g., C797S, C797G), L798I or an in-frame exon 20 insertion, e.g., 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_Y765InsX1-7, M766_A767InsX1-7, A767_V768InsX1-7, S768_V769InsX1-7>V769_D770 InsX1-7>D770_N771 InsX1-7>N771_P772 InsX1-7>P772_H773 InsX1-7, H773_V774 InsX1-7 or V774_C775 The use or method of any one of the preceding clauses, comprising a mutation in exon 20 (762-823) selected from InsX1-7;

[0265] 20. The use or method of any one of the preceding clauses, wherein the cancer comprises a cMET abnormality, such as cMET amplification, cMET overexpression, increased cMET pathway signaling, cMET gene amplification, increased cMET protein activity, and / or increased HGF expression.

[0266] 21. The use or method of any one of the preceding clauses, wherein the cancer comprises a cMET exon 14 skipping mutation.

[0267] 22. The use or method according to any one of the preceding clauses, 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.

[0268] 23. The use or method of any one of the preceding clauses, wherein the cancer is NSCLC comprising activating EGFR mutations, EGFR tyrosine kinase inhibitor sensitizing mutations (such as exon 19 deletions and L858X), acquired EGFR tyrosine kinase inhibitor resistance mutations (such as T790X, C797X, L792X, L798X), approved EGFR tyrosine kinase inhibitor resistance mutations, EGFR exon 20 insertion mutations, activating c-MET mutations, preferably exon 14 skipping mutations, or cMET amplification, preferably including MET / CEP7>5 or cfDNA≧2 copies, increased HGF expression, or any combination thereof.

[0269] 24. The use or method of any one of the preceding clauses, wherein the cancer is advanced or metastatic cancer.

[0270] 25. The use or method of any one of the preceding clauses, wherein the cancer or patient has progressed after receiving said prior therapy for advanced or metastatic cancer.

[0271] 26. The use or method of any one of the preceding clauses, wherein treatment includes 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.

[0272] 27. The use or method according to any one of the preceding clauses, wherein the treatment comprises a diagnostic step of determining the amount of one or more biomarkers in the tumor tissue, blood or serum, such as determining EGFR amplification, immunohistochemical staining, amount of EGFR / c-MET signaling pathway activity, measuring soluble EGFR and soluble c-MET in blood or serum, and / or measuring EGFR and c-MET target expression in tumor cells.

[0273] 28. The use or method of any one of the preceding clauses, wherein the subject is a human subject.

[0274] 29. The use or method of any one of the preceding clauses, wherein the antibody is a human antibody.

[0275] 30. The use or method of any one of the preceding clauses, wherein the antibody is ADCC enhanced.

[0276] 31. The use or method of any one of the preceding clauses, wherein the antibody is an IgG1 format antibody with a 1:1 anti-EGFR, anti-cMET stoichiometry.

[0277] 32. The use or method of any one of the preceding clauses, wherein the antibody has one variable domain capable of binding to EGFR and one variable domain capable of binding to cMET.

[0278] 33. The use or method according to any one of the preceding clauses, wherein the variable domain capable of binding to human EGFR is also capable of binding to cynomolgus monkey and mouse EGFR.

[0279] 34. The use or method according to any one of the preceding clauses, wherein the variable domain capable of binding to human EGFR binds to domain III of human EGFR.

[0280] 35. The use or method according to any one of the preceding clauses, wherein the variable domain capable of binding to cMET blocks binding of the antibody 5D5 to cMET.

[0281] 36. The use or method according to any one of the preceding clauses, wherein the variable domain capable of binding to cMET blocks binding of HGF to cMET.

[0282] 37. The use or method of any one of the preceding clauses, 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).

[0283] 38. Is X1=N, X2=G, X3=D, X4=S, X5=Y, X6=W, and X7=G? X1=N, X2=A, X3=D, X4=S, X5=Y, X6=W, and X7=G, or X1=S, X2=G, X3=D, X4=S, X5=Y, X6=W, and X7=G, or 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, or X1=N, X2=G, X3=G, X4=Y, X5=L, X6=D, and X7=G, or X1=N, X2=A, X3=G, X4=Y, X5=L, X6=D, and X7=G; or 2. The use or method of any one of the preceding clauses, wherein X1=S, X2=G, X3=G, X4=Y, X5=L, X6=D and X7=G.

[0284] 39. The use or method according to any one of the preceding clauses, wherein X1=N, X2=G, X3=D, X4=R, X5=H, X6=W and X7=D, or wherein X1=N, X2=A, X3=D, X4=R, X5=H, X6=W and X7=D, or wherein X1=S, X2=G, X3=D, X4=R, X5=H, X6=W and X7=D.

[0285] 40. The use or method according to any one of the preceding clauses, wherein X1=N, X2=G, X3=D, X4=R, X5=H, X6=W and X7=D, or wherein X1=N, X2=A, X3=D, X4=R, X5=H, X6=W and X7=D.

[0286] 41. The use or method according to any one of the preceding clauses, wherein the heavy chain variable region of the second variable domain comprises the amino acid sequence of one of the sequences of SEQ ID NOs: 1-3, 7, 8, 10, 13, 15, 16, 17, 21, 22 or 23 having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

[0287] 42. The use or method according to any one of the preceding clauses, 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 having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

[0288] 43. The use or method of any one of the preceding clauses, wherein the first variable domain comprises a heavy chain variable region having the CDR1 sequence SYGIS, the CDR2 sequence WISAYNGNTNYAQKLQG, and the CDR3 comprising the sequence DRHWHWWLDA, and the second variable domain comprises a heavy chain variable region having the CDR1 sequence SYSMN, the CDR2 sequence WINTYTGDPTYAQGFTG, and the CDR3 sequence ETYYYDRGGYPFDP.

[0289] 44. The use or method according to any one of the preceding clauses, wherein the first variable domain comprises a heavy chain variable region having CDR1 sequence SYGIS, CDR2 sequence WISAYNANTNYAQKLQG, and CDR3 comprising the sequence DRHWHWWLDA, and the second variable domain comprises a heavy chain variable region having CDR1 sequence TYSMN, CDR2 sequence WINTYTGDPTYAQGFTG, and CDR3 comprising the sequence ETYFYDRGGYPFDP.

[0290] 45. The use or method according to any one of the preceding clauses, wherein the first and second variable domains comprise a common light chain, preferably the light chain of Figure 4B.

[0291] 46. ​​The use or method according to any one of the preceding clauses, wherein the first and second variable domains comprise a light chain having the CDR1, CDR2 and CDR3 amino acid sequences of QSISSY, AAS and QQSYSTP, respectively (according to IMGT).

[0292] 47. The use or method of any one of the preceding clauses, wherein the antibody inhibits HGF-induced growth of HGF growth-responsive cells.

[0293] 48. The use or method of any one of the preceding clauses, wherein the antibody inhibits EGF-induced growth of EGF growth-responsive cells.

[0294] 49. The use or method according to any one of the preceding clauses, wherein the subject is administered 1000 mg of the bispecific antibody, in particular using a flat dose of 1000 mg.

[0295] 50. The use or method according to clause 49, wherein the bispecific antibody is administered once weekly.

[0296] 51. The use or method according to clause 49, wherein the bispecific antibody is administered once every two weeks.

[0297] 52. The use or method according to any one of clauses 1 to 48, wherein the subject is administered 1500 mg of the bispecific antibody, in particular using a flat dose of 1500 mg.

[0298] 53. The use or method according to clause 52, wherein the bispecific antibody is administered once every two weeks.

[0299] 54. The use or method according to any one of clauses 1 to 48, wherein the subject is administered 2000 mg of the bispecific antibody, in particular using a flat dose of 2000 mg.

[0300] 55. The use or method according to clause 54, wherein the bispecific antibody is administered once every two weeks.

[0301] 56. The use or method of any one of the preceding clauses, wherein the subject in iv) has not received prior anti-cancer treatment with a cMET inhibitor.

[0302] 57. The use or method of any one of the preceding clauses, wherein the subject in iv) has not received prior anti-cancer treatment with a cMET TKI inhibitor.

[0303] 58. The use or method of any one of the preceding clauses, wherein the subject in iv) has not received prior anti-cancer treatment with capmatinib.

[0304] 59. The use or method of any one of the preceding clauses, wherein the subject in iv) has not received prior anti-cancer treatment with tepotinib.

[0305] 60. The use or method of any one of the preceding clauses, wherein the subject in iv) has not received prior anti-cancer treatment with savolitinib. EXAMPLES

[0306] As used herein, "MFXXXX", where X is independently a number 0-9, refers to a Fab comprising a variable domain, and VH has an amino acid sequence identified by four digits. Unless otherwise indicated, the light chain variable region of the variable domain has the sequence of FIG. 4A, typically 4b. "MFXXXX VH" refers to the amino acid sequence of VH, identified by four digits. MF further comprises a constant region of the light chain and a constant region of the heavy chain that typically interacts with the constant region of the light chain. PG refers to a monospecific antibody comprising identical heavy and light chains. PB refers to a bispecific antibody having two different heavy chains. The VH variable regions of the heavy chains are different, and typically the CH3 regions are also different, with one of the heavy chains having a KK mutation in its CH3 domain and the other having a complementary DE mutation in its CH3 domain (see PCT / NL2013 / 050294 (published as WO2013 / 157954) for reference).

[0307] For details on the production of the antibodies of the present disclosure, see PCTNL / 2018 / 050537 (published as WO2019 / 031965). Bispecific antibodies that bind EGFR and cMET suitable for use in the appended examples and in the methods of the invention include those in Tables 3, 4, 5 and 6. In particular, bispecific antibody PB19478 is suitable for use in the appended examples.

[0308] Each bispecific antibody comprises two VHs designated by MF numbers capable of binding to EGFR and cMET, respectively, and further comprises a KK / DE CH3 heterodimerization domain as shown in Figures 5e and 5f, respectively, a CH2 domain as shown in Figure 5d, a hinge domain as shown in Figure 5b, a CH1 domain as shown in Figure 5a, and an Fc tail with a common light chain as shown in Figures 4a-e. For example, the bispecific antibody represented by MF8233xMF8230 has a variable domain with the general sequence described above, a VH with the sequence of MF8233, and a VH with the sequence of MF8230, and in certain embodiments is used in the appended examples.

[0309] Example 1: Materials and Methods Cell line: EBC-1 [JCRB0820], PC-9 [RCB0446], H358 [ATCC® CRL-5807™], HCC827 [ATCC® CRL-2868™], MKN-45 [DSMZ ACC 409] N87 [ATCC® CRL-5822™] and A431 [ATCC® CRL-1555™] 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.

[0310] cDNA construct: Generation of cMET and EGFR expression vectors for generation of stable cell lines (cMET and EGFR) and for immunization (cMET) Full-length cDNA of each target, containing unique restriction sites for cloning and Kozak consensus sequences for efficient translation, was synthesized or obtained via PCR amplification on a commercially available expression construct containing the target cDNA, using specific primers that introduced unique restriction sites for cloning and Kozak consensus sequences for efficient translation. Full-length cDNA of each target was cloned into a eukaryotic expression construct such as pcDNA3.1, while the extracellular domain was cloned into pVAX1 and pDisplay. Insert sequences were verified by comparison with the NCBI reference amino acid sequence.

[0311] Amino acid sequence for expression on the cell surface Full length human EGFR insert (same as GenBank: NP_00533):

[0312] Of which: -MRPSGTAGAALLALLAALCPASR: signal peptide. -: ECD of human EGFR. -IATGMVGALLLLLVVALGIGLFM: predicted TM region. -: Intracellular tail.

[0313] Amino acid sequence of the extracellular domain of human EGFR varIII, a naturally occurring EGFR variant VAR_066493 caused by an in-frame deletion of exons 2-7 [Ji H., Zhao X; PNAS103:7817-7822(2006)]. The _ below indicates where amino acids 30-297 are missing. MRPSGTAGAALLALLAALCPASRALEEKK_GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKE ISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[0314] Of which: -MRPSGTAGAALLALLAALCPASR: signal peptide. -ALEEKK_GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANT INWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS:EGFRvarIII ECD

[0315] Amino acid sequence chimeric Macaca mulatta extracellular EGFR domain hybrid (GenBank: XP_014988922.1) with human EGFR transmembrane and intracellular domains for expression on the cell surface. Human EGFR sequence underlined in examples below. [ka]

[0316] Of which: -MGPSGTAGAALLALLAALCPASR: signal peptide. -:ECD of cyEGFR

[0317] Amino acid sequence of full-length human cMET insert for expression on the cell surface (GenBank: P08581-2). This sequence differs from the reference sequence at position 755 with an insertion at: S→STWWKEPLNIVSFLFCFAS

[0318] Of which: -MKAPAVLAPGILVLLFTLVQRSNG: signal peptide -ECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFT: Human cMET ECD -GLIAGVVSISTALLLLLGFFLWL: Transmembrane region -KKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISPLLQNTVHIDLSALNPELVQ AVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETH NPTVKDLIGFGLQVAKGMKYASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYP DVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS: Intracellular region

[0319] Reference antibody Anti-cMET antibodies are known in the art (Table 1). Monospecific bivalent cMET antibodies were constructed according to published information and expressed in 293F Freestyle cells. Table 1 shows the relevant disclosure information. Monospecific bivalent antibodies directed against cMET were constructed according to published information and expressed in 293F Freestyle cells. For HGF ligand blocking assays, the VH and VL coding gene segments of patent-derived anti-cMET antibodies were recloned into a phage display vector for display on filamentous bacteriophage.

[0320] The reference antibody cetuximab (Erbitux) was used as the reference antibody for the EGFR Fab panel.

[0321] 2994 Fab protein was generated from purified PG2994 IgG by papain digestion. Thus, PG2994 was incubated with papain coupled on beads (Pierce#44985) and digested for 5.5 hours at 37°C with rotation. Fab fragments were purified from the digestion mixture by filtration on MabSelectSure LX. The fraction containing Fab protein was flow-through and concentrated to 3ml using vivaspin 20 10kDa and further purified by gel filtration using a superdex75 16 / 600 column in PBS.

[0322] Example 2 Generation of bivalent monoclonal antibodies and characterization of the antibodies The VH genes of the unique antibodies were cloned in a backbone IgG1 vector, as judged by the VH gene sequence and several sequence variants thereof. Suspension-adapted 293F Freestyle cells were cultured at 3.0 × 10 6 Cells were cultured in T125 flasks on a shaker plateau until they reached a density of 0.3–0.5 × 10 cells / ml. 6 Viable cells / ml were seeded into each well of a 24-deep-well plate. Cells were transiently transfected with the individual sterile DNA:PEI mixtures and further cultured. Seven days after transfection, supernatants were harvested, filtered through 0.22 μM (Sartorius), and purified on Protein A beads using batch purification followed by buffer exchange into PBS.

[0323] Reciprocal Blocking Assay cMET Antibody The cMET-specific phages were tested for competition with the cMET reference antibody in an ELISA. Therefore, 2.5 μg / ml of cMET-Fc fusion protein was coated overnight at 4° C. on a MAXISORP™ ELISA plate. The wells of the ELISA plate were blocked with 2% ELK in PBS (pH 7.2) for 1 h at room temperature with shaking (700 rpm). The following control or negative control IgG was added at a concentration of 5 μg / ml and allowed to bind at room temperature for 15 min at 700 rpm. Then, 5 μl of PEG-precipitated phages were added and allowed to bind at room temperature for 1 h at 700 rpm. Bound phages were detected with an HRP-labeled anti-M13 antibody at room temperature for 1 h at 700 rpm. As a control, this procedure was performed simultaneously with the antibody specific for the coated antigen and the negative control phages. Bound secondary antibodies were detected with TMB / H. 2 O 2 Visualize by staining and measure the staining by OD 450nm The competition was quantified by measurement. Table 2 demonstrates that MF4040 and MF4356 show competition with the 5D5 reference antibody. MF4297 competes to a lesser extent with 13.3.2 and C8H241. All positive control phages show perfect competition with the corresponding IgG, while the no-antibody control has no effect on the competition assay.

[0324] Generation of bispecific antibodies Bispecific antibodies were generated by transient co-transfection of two plasmids encoding IgG with different VH domains using a proprietary CH3 engineering technique that ensures efficient heterodimerization and bispecific antibody formation. A common light chain is also co-transfected in the same cell, either on the same plasmid or on a separate plasmid. In our co-pending applications (e.g. WO2013 / 157954 and WO2013 / 157953, incorporated herein by reference), methods and means for producing bispecific antibodies from a single cell are disclosed, thereby providing a means to favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be advantageously used in the present invention. In particular, preferred mutations that produce essentially only bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, e.g., L351K and T366K (numbering according to EU numbering) ("KK variant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, e.g., L351D and 10L368E ("DE variant" heavy chain), or vice versa. It was previously demonstrated in our co-pending application that negatively charged DE variant heavy chains and positively charged KK variant heavy chains preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE variant heavy chains (DE-DE homodimers) or KK variant heavy chains (KK-KK homodimers) is unfavorable due to the strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0325] Table 3 shows which cMET and EGFR Fab arms were cloned in the appropriate KK and DE vectors. After production, the bispecific IgG was purified by Protein-A batch purification and buffer exchanged into PBS. Successful production resulted in IgG1 full-length antibodies at a minimum concentration of 0.1 mg / ml, which were assigned a unique code (PBnnnnn, where nnnnn represents a randomly generated number) to identify the specific combination of two different target-binding Fab fragments. Successfully produced bispecific IgGs were tested for binding to their respective targets in ELISA. For further details of the production of bispecific antibodies, see PCTNL / 2018 / 050537 (published as WO2019 / 031965) herein.

[0326] Example 3 Screening of c-MET×EGFR bispecific antibodies in EGF / HGF and HGF and EGF proliferation assays The efficacy 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 and has high EGFR expression levels and intermediate cMET expression levels (Zhang et al., 2010). Antibodies were tested in eight half-log titrations ranging from 10 μg / ml to 3.16 ng / ml. Each antibody was tested in duplicate. Anti-RSV-G antibody PG2708 was used as a negative control. Reference antibody 2994 Fab was used as a positive control for the HGF assay and reference antibody cetuximab was used as a positive control for the EGF assay.

[0327] Equimolar 1:1 cetuximab / 5D5 Fab was used as a positive control for the EGF, HGF and EGF / HGF assays.

[0328] Wells with either one or their combinations of ligands and media controls were included to determine the assay window. Antibodies were diluted in chemically defined starvation medium (CDS: RPMI1640 medium containing 80 U penicillin and 80 μg streptomycin per ml, 0.05% (w / v) BSA and 10 μg / ml holotransferrin) and 50 μl of diluted antibody was added to wells of a 96-well black well clear bottom plate (Costar). Ligands were added (50 μl per well of stock solution containing 400 ng / ml HGF and 4 ng / ml EGF and 4 ng / ml EGF / 400 ng / ml EGF / HGF concentrations diluted in CDS: R&D systems, Cat. Nos. 396-HB and 236-EG). N87 cells were trypsinized, harvested, counted and 8000 cells in 100 μl CDS were added to each well of the plate. To avoid edge effects, plates were left at room temperature for 1 hour and then placed in a container in a cell culture incubator at 37°C for 3 days. On day 4, Alamar blue (Invitrogen, #DAL1100) was added (20 μl per well) and fluorescence was measured after 6 hours of incubation (37°C) with Alamar blue using an excitation of 560 nm and a readout of 590 nm on a Biotek Synagy 2 multimode microplate reader. Fluorescence values ​​were normalized to uninhibited growth (no antibody but both ligands added).

[0329] Table 4 lists the results of various experiments. In the N87 HGF / EGF assay, 14 different cMETxEGFR bispecifics were identified with potency comparable to that of a reference monospecific antibody (an equimolar mixture of cetuximab and 5D5 Fab): PB7679, PB7686, PB8218, PB8244, PB8292, PB8316, PB8340, PB8364, PB8388, PB8511, PB8535, PB8583, PB8607 and PB8640.

[0330] In the N87 EGF assay, 11 different cMETxEGFR bispecifics were identified with potency comparable to the monospecific cetuximab: PB7679, PB8244, PB8292, PB8340, PB8364, PB8388, PB8511, PB8535, PB8583, PB8607 and PB8640, all of which contain the EGFR Fab arm MF3755. In the HGF N87 assay, 9 bispecifics were identified that showed higher potency compared to the monospecific 5D5 Fab reference antibody: PB8218, PB8388, PB8511, PB8532, PB8535, PB8545, PB8583, PB8639 and PB8640. These contain six different cMET Fab arms MF4040, MF4297, MF4301, MF4356, MF4491 and MF4506.

[0331] ADCC activity The ADCC activity of 24 cMetxEGFR bispecifics was tested against the tumor cell lines N87 (EGFR-high, cMET-low) and MKN-45 (EGFR-low, cMET-amplified). ADCC assays were performed using a Promega ADCC bioassay kit in a 384-well plate format. Antibodies were tested in duplicate at nine different concentrations in half-log serial dilutions ranging from 10 μg / ml to 1 ng / ml.

[0332] A reference cetuximab antibody was included as a positive control in the assay, and PG2708 was used as a negative control antibody. Antibodies or assay media controls (no IgG) were incubated with ADCC effector cells and target cells (N87 or MKN-45) for 6 hours of induction at 37° C. Luciferase activity was quantified using Bio-Glo luciferase reagent.

[0333] None of the cMETxEGFR bispecifics showed significant ADCC activity in both cell lines. The positive control reference cetuximab antibody showed dose-dependent ADCC activity against both cell lines.

[0334] Five bispecifics consisting of EGFR and cMet arms that showed high efficacy in the N87 HGF / EGF assay and high sequence diversity were selected for further analysis (Table 5). Two of the five bispecifics contain MF4356, which competes with 5D5 for binding to cMET (Table 2). Table 5 summarizes the properties of the selected candidates.

[0335] Example 4 FIG. 2 shows various sequences of alternative variable regions of the heavy chain of the EGFR-binding variable domain as disclosed herein. FIG. 3 shows various sequences of alternative variable regions of the heavy chain of the cMET-binding variable domain as disclosed herein. The heavy chain variable regions were used to generate a number of different cMETxEGFR bispecific antibodies. The light chain in these antibodies has the sequence shown in FIG. 4B. The bispecific antibodies were produced as described in Example 1. The antibodies were also produced as ADCC-enhanced versions. The ADCC-enhanced versions were produced by including in the co-transfection of the antibody construct DNA encoding a reductase enzyme that removes fucose residues from the Fc region of IgG1. See Table 6 for a list of the bispecific antibodies used and their PB codes.

[0336] Example 5 The heavy chain variable region (VH) of the cMET variable domain of PB8532 comprises the amino acid sequence of MF4356, for example, as shown in Figure 3. The VH of the cMET variable domain of PB19748 comprises the amino acid sequence of MF8230 (see Figure 3). The VH of the EGFR variable domain of PB8532 comprises the amino acid sequence of MF3370, for example, as shown in Figure 2. The VH of the EGFR variable domain of PB19748 comprises the amino acid sequence of MF8233 in Figure 2. The light chains in PB8532 and PB19748 are the same and are shown in Figure 4B. The cMET antibody LY2875358 antibody is described, inter alia, in Kim and Kim 2017.

[0337] Example 6 Efficacy of bispecific antibody PB19748 in an EGFR exon 20 insertion model The objective of this study was to evaluate the antitumor efficacy of PB19748 in patient-derived tumor xenograft (PDX) non-small cell lung cancer (NSCLC) models harboring EGFR exon 20 insertions. EGFR exon 20 insertions ("EGFRex20ins") represent a class of encoding variants with amino acid insertions clustered between positions 762 and 774 that result in constitutive activation of EGFR. EGFR exon 20 insertions confer resistance to approved EGFR TKIs and are associated with poor prognosis in human subjects with cancer harboring such mutations.

[0338] material PB19748 is produced by Merus and the control material is PB19748 vehicle, consisting of 12% PB19748 formulation buffer without antibody, although other negative controls such as saline or PBS can also be used.

[0339] PDX model characteristics Model LXFE2478 was generated at Charles River in nude mice with functional Fc effector cells.

[0340] This model harbors the mutant EGFRex20ins (M766_A767insASV), which also harbors a point mutation (E168D) in the SEMA domain of c-MET, which is located in the ligand-binding site of c-MET.

[0341] The nine nucleotide insertion in exon 20 in this model affects the EGFR tyrosine kinase domain (M766X) and confers resistance to small molecule EGFR inhibitors. This model does not have mutations in the B-RAF, H- / N-, KRAS and PTEN genes (whole exome sequencing was performed on both patient tumors and tumor xenografts with concordant results).

[0342] EGFR, c-MET and HGF expression in LXFE2478 PDX model The model LXFE2478 was analyzed for expression of EGFR and c-MET receptors and the ligand huHGF to examine its suitability for in vivo studies.

[0343] Two tumor pieces were assessed for expression of the EGFR and c-MET receptors, and expression of the ligand huHGF was assessed via Western blot. huHGF was included in the analysis. Tumor samples obtained from PDX models were assessed by simplified Western size technology (SWS) to confirm expression of EGFR, HGF, and c-MET.

[0344] Western blotting revealed that the LXFE2478 model expressed EGFR, HGF and MET at the protein level (Figure 6 and Table 7).

[0345] Statistical methods: The antitumor efficacy of all groups was evaluated using the control vehicle / placebo buffer group as a reference. Tumor growth inhibition was determined by comparing the RTV of the test group with the control group and expressed as a percentage as the minimum T / C value. A non-parametric Kruskal-Wallis test followed by Dunn's method for multiple comparisons was performed to evaluate the statistical significance of the antitumor efficacy. The individual RTVs of the test and control groups were compared on the day the minimum T / C value was achieved in the test group. Statistical analysis was performed only if at least 50% of the initially randomized animals still remained in the relevant group. Comparisons between the test groups were performed on the same day. All p values ​​less than 0.05 were considered statistically significant. Statistical calculations were performed using GraphPad Prism bioanalysis software (version 9.10 for Microsoft Windows, GraphPad Software, San Diego, California, USA).

[0346] Treatment schedule and method: FIG. 7 shows a schematic overview of the treatment schedule. Tumors were harvested from donor PDX mice, cut into pieces (LXFE2478: 3-4 mm edge length) and inoculated into recipient nude mice (SC) in the flank. When tumor implants reached approximately 80-200 mm3 in a sufficient number of animals, mice were assigned to five groups with five mice per group. Randomization was performed based on the "stratified distribution" method. Treatment was started on the same day as randomization (day 0).

[0347] Antibodies were administered intraperitoneally (IP) at doses of 0.5 mg / kg, 2.5 mg / kg, 8 mg / kg, and 25 mg / kg once a week for 5 weeks. Mice in Group 1 were treated with Vehicle 1 once a week for 5 weeks and daily for 30 days. The treatment schedule is shown in Table 8.

[0348] After randomization, animals were routinely monitored for morbidity and mortality, weighed twice weekly, and tumor volumes (TV) were determined twice weekly with a caliper. Relative body weights (RBW) were calculated by dividing the absolute body weight or volume on a given day by the absolute body weight at day 0 and multiplying by 100. Relative tumor volumes (RTV) were calculated by dividing the individual absolute tumor volumes on a given day by the absolute tumor volume on day 0 and multiplying by 100.

[0349] For mice that lost more than 10% body weight, body weight was measured daily and animals were given ready access to food and water, as well as DietGel. For mice that lost more than 15% body weight, therapy was discontinued until they had regained 90% or more RBW.

[0350] Monotherapy with the bispecific antibody PB19478 demonstrated dose-dependent antitumor efficacy (Figure 8). Monotherapy with the antibody at a dose of 25 mg / kg resulted in tumor remission (min. T / C value 2.7%) and was significantly more effective than treatment with vehicle and treatment with doses of 0.5 mg / kg or 2.5 mg / kg. Treatment with doses of 0.5 mg / kg and 2.5 mg / kg showed partial regression of tumors.

[0351] Monotherapy correlated with low toxicity in mice, demonstrated by negligible weight loss and 100% adjusted survival in all groups.

[0352] EGFR exon 20-mutated cancers are generally refractory to EGFR tyrosine kinase inhibitors and are associated with a poor prognosis.

[0353] Example 7 Efficacy of bispecific antibody PB19478 in an EGFR exon 19 deletion NSCLC model. The aim of this study was to evaluate the antitumor efficacy of antibody PB19478 in a cell line-derived tumor xenograft (CDX) non-small cell lung cancer (NSCLC) model with an EGFR exon 19 deletion. This adenocarcinoma cell line, HCC827-ER1, harbors an activating EGFR mutation in exon 19 (deletion E746-A750) and is resistant to approved EGFR TKIs such as erlotinib.

[0354] material: PB19748 was produced by Merus, as mentioned in Example 5.

[0355] CDX Model Characteristics: The model HCC827-ER1 was generated in BALB / c nude mice, which harbors an EGFR mutation in exon 19 (deletion E746-A750) 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 via repeated in vitro exposure of the wild-type cell line to increasing concentrations of erlotinib.

[0356] Experimental Procedure: Each mouse was inoculated with HCC827-ER1 tumor cells mixed with Matrigel for tumor development in the right anterior flank region. Randomization was performed to determine the mean tumor size of approximately 125 (75–175) mm 3A total of 16 tumor-bearing mice were enrolled in the tumor efficacy study and randomly assigned to two different groups with 8 mice per group as shown in Table 9. The date of randomization is indicated as day 0, and dosing began on day 0. Mice were treated with antibody for 3 weeks, followed by a dose-free observation period of up to 74 days. Duration of response and recurrence (tumor regrowth) after treatment termination were compared, including the dose-free observation period. 3 Mice with tumor volumes exceeding 100 mg / kg or with a weight loss of more than 20% relative to the weight on the first day of treatment were terminated during the treatment period. Antibodies were administered intraperitoneally (ip) twice weekly for a total of seven doses at a dose of 25 mg / kg over a period of 21 days. Eight mice in group 1 were treated with vehicle control (placebo buffer). A schematic overview of the treatment schedule is shown in Figure 9. Tumor volumes (mm 3) were measured twice weekly in two dimensions using calipers. Animals were checked for the effects of tumor growth and any of the treatments on behavior such as mobility, food and water consumption, weight gain / loss, eye / hair matting and other abnormalities. Body weight was measured twice weekly after randomization. None of these mice lost more than 15% body weight, precluding the need to discontinue administration of therapeutic agents in any of the treatment groups. Figure 10 shows the effect of bispecific antibody PB19478 on tumor volume in the NSCLC CDX model during the full observation period, which indicated treatment cessation on day 21. The antibody treatment showed tumor regression compared to the vehicle control group. All treatments were well tolerated. Mice in the study did not show weight loss of more than 10% from the starting weight. Indeed, no statistically significant difference in absolute body weight was observed between groups 1 and 2 on day 21, which was the last treatment day. The absolute body weight of mice in the additional treatment group (i.e., 25 mg / kg osimertinib, administered once daily per oral gage) was lower compared to groups 1 and 2 (P=0.0167 vs. group 2, data not shown). No adverse events were observed, except for tumor crusting or tumor ulceration in some mice in groups 1 and 2. Animal survival curves are shown in FIG. 11. Log-rank (Mantel-Cox) tests of the combination vs. each corresponding monotherapy group are significant as follows: antibody 25 mg / kg vs. vehicle P=0.0001.

[0357] In summary, these results demonstrate preclinical antitumor activity of the investigational antibody against an NSCLC CDX model harboring a mutation in exon 19 (deletion E746-A750).

[0358] Example 8 In the dose escalation phase, the bispecific antibody PB19478 will be administered at increasing doses to patients with NSCLC harboring activating EGFR mutations (TKI-sensitizing mutations and / or approved TKI-resistant mutations) or activating c-MET mutations (exon 14 skipping) / amplification (MET / CEP7>5 or cfDNA>2 copies), patients with GC / GEJ adenocarcinoma with activating EGFR amplification (EGFR / CEP7>2 or cfDNA>8 copies) or c-MET amplification (MET / CEP7>5 or cfDNA>2 copies), or patients with HNSCC or ESCC who have progressed in all cases after prior therapy for advanced / metastatic disease.

[0359] Allometric scaling of preclinical PK models was used to predict antibody exposure in humans. The starting dose of antibody is 100 mg (flat dose, intravenous) once every 2 weeks (q2w) in a 4-week cycle (28 days). Five dose levels between 100 and 3000 mg are planned to be investigated.

[0360] Cohorts of patients will be treated with the antibody until the MTD is reached or a lower recommended dose is established.

[0361] The RP2D (recommended phase 2 dosing) is defined as a dose below the MTD, taking into account available data on PK, pharmacodynamic activity, and preliminary antitumor activity.

[0362] Dose expansion Planned expansion cohorts in the Phase 2 part can be initiated using 1500 mg of RP2D. Safety of RP2D will be confirmed during dose expansion in the first 12 patients treated with antibody alone for at least 2 cycles (recruitment will continue in the meantime). Antitumor activity of the antibody (alone or in combination with osimertinib) will be evaluated in terms of ORR, and other efficacy parameters, safety, tolerability, PK, immunogenicity and biomarker evaluations will be performed.

[0363] The following locally advanced unresectable / metastatic solid tumor cohorts are open: Cohort A: PB19478 monotherapy: NSCLC patients may harbor EGFR exon 20 insertion mutations (first-line [1L] and second-line [2L] or higher progressed after first-line chemotherapy). Results from patients with NSCLC are described in Example 9. Cohort B: PB19478 monotherapy: NSCLC patients may have ≥2L cMet exon 14 mutations, capmatinib- and tepotinib-resistant populations who have progressed after approved c-Met TKI inhibitors. Cohort C: PB19478 monotherapy: Patients with GC / GEJ and HNSCC who may harbor EGFR or cMet driven mutations. Results from patients with HNSCC are described in Example 10.

[0364] Study population Selection Criteria Patients must meet all of the following requirements to participate in the study: 1. Signed informed consent prior to the start of any study procedures. 2.Age 18 or older at the time of signing the informed consent. 3. Histologically or cytologically confirmed solid tumors with evidence of incurable metastatic or locally advanced unresected disease.

[0365] 1. Dose Escalation Part - Patients who have failed previous standard first-line therapy. Patients must have progressed on 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: Non-small cell lung cancer (NSCLC) harboring activating EGFR mutations, including tyrosine kinase inhibitor (TKI)-sensitizing mutations (e.g., 19del and L858R) and / or approved TKI resistance mutations (e.g., acquired TKI resistance mutations, i.e., T790M, C797S, L792, L798I, exon 20 insertions), or any activating c-MET mutation / amplification (e.g., high-level c-MET amplification [MET / CEP7>5 or cfDNA ≥2 copies], or c-MET exon 14 skipping mutation). · *Note: Patient identification was based on prior treatment with an EGFR tyrosine kinase inhibitor and local testing performed in a CLIA-certified laboratory. 2. Cohort Expansion - Patients at 2L or higher must be progressing on or intolerant to a therapy known to provide clinical benefit. There is no limit to the number of prior treatment regimens. Patients must have: Cohort A: NSCLC with EGFR exon 20 insertions that have progressed after platinum doublet (may include a limited number of first-line patients). Cohort B: NSCLC harboring ≥2L cMet exon 14 skipping mutations, capmatinib and tepotinib resistant populations, or treated with cMET TKI or cMET TKI naive. Cohort C: Selected solid tumors harboring EGFR or cMET driving mutations or cMET alterations not included in other cohorts (including GC / GEJ, HNSCC, e.g., HNSCC refractory to approved therapies (regardless of driving mutation), or ESCC), NSCLC patients with rare EGFR mutations (G719X, L861Q and S768I). Patients have exhausted or are intolerant to all approved therapies with proven clinical benefit or have no approved therapeutic options available. Cohort F: treatment-naive, c-MET exon 14 skipping mutated NSCLC with progressive disease. 4. Having availability of FFPE embedded after progression of current therapy, archival or fresh tumor tissue samples. 5. Measurable disease as defined by RECIST version 1.1 by radiological methods (patients with non-measurable but evaluable disease may be included in the dose escalation portion). Eastern Cooperative Oncology Group (ECOG) performance status of 6.0 or 1. 7. Life expectancy is 12 weeks or more according to the investigator 8. Adequate organ function as determined by the investigator Absolute neutrophil count (ANC) ≥ 1.5 × 10 9 / L Hemoglobin ≥ 9g / dL Platelets ≥ 100 × 10 9 / L Corrected total serum calcium within normal range Serum magnesium within normal range (or corrected with supplements) Serum potassium within normal range Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤ 3 x upper limit of normal (ULN) and total bilirubin ≤ 1.5 x ULN (patients with Gilbert's syndrome are eligible if conjugated bilirubin levels are within the normal range); in cases of hepatic involvement, ALT / AST ≤ 5 x ULN and total bilirubin ≤ 2 x ULN are acceptable Serum creatinine less than or equal to 1.5 × ULN or creatinine clearance greater than or equal to 50 mL / min, calculated according to the Cockroft and Gault formula or the MDRD formula for patients over 65 years of age. Serum albumin > 3.3g / dL

[0366] Exclusion criteria Patients will be excluded from participating in the study if any of the following criteria are present: 1. Central nervous system metastasis (not exclusively in recruitment, but essential in expansion), Untreated or symptomatic (may include patients with untreated asymptomatic disease if deemed stable by the investigator) -Those requiring radiation or surgery Requiring ongoing steroid therapy (>10 mg prednisone or equivalent) to control symptoms within 14 days of study enrollment. 2. Known leptomeningeal involvement. 3. Participation in another clinical investigation or treatment with any investigational drug within 4 weeks prior to study enrollment. 4. Systemic anticancer therapy or immunotherapy within 4 weeks or 5 half-lives of the first dose of investigational drug, whichever is shorter. For cytotoxic agents with significant delayed toxicity (e.g., mitomycin C, nitrosoureas), a 6-week washout period is required. Note: For agents with long half-lives, enrollment prior to the 5th half-life requires sponsor approval. 5. Major surgery or radiation therapy within 3 weeks of first dose of study drug. Patients who have had prior radiation therapy to more than 25% of the bone marrow at any time are ineligible. 6. Persistent grade >1 clinically significant toxicity (excluding alopecia), in the investigator's judgment, related to existing antineoplastic therapy; stable sensory neuropathy ≤ grade 2 NCI-CTCAEv5.0, and hypothyroidism ≤ grade 2 stable on hormone replacement are acceptable. 7. History of hypersensitivity reactions or any toxicity due to either human proteins or excipients that justified permanent discontinuation of these agents. 8. History of clinically significant cardiovascular disease, including but not limited to: A prolonged QT interval >480 ms from three electrocardiograms (ECGs), or clinically significant arrhythmia or electrophysiologic disease (i.e., placement of an implantable cardioverter defibrillator or uncontrolled rate of atrial fibrillation), or any factor that increases the risk of QTc prolongation or the risk of arrhythmic events, e.g., electrolyte abnormalities. Patients with a clinically stable cardiac pacemaker are eligible. Heart failure, congenital long QT syndrome, family history of long QT syndrome or sudden unexplained death under age 40 in a first degree relative, or any concomitant medications known to prolong the QT interval and cause torsades de pointes. · Uncontrolled (persistent) arterial hypertension: systolic blood pressure >180mmHg and / or diastolic blood pressure >100mmHg. Congestive heart failure (CHF) defined as New York Heart Association (NYHA) class III-IV or hospitalization for CHF within 6 months of first dose of study drug. 9. History of interstitial lung disease, including drug-induced interstitial lung disease, radiation pneumonitis requiring treatment with long-term steroids or other immunosuppressants within 1 year. 10. Prior or concurrent malignancy, except for non-basal cell carcinoma of the skin or carcinoma in situ of the cervix, provided that the tumor is treated with curative or palliative intent and, in the investigator's opinion, with the sponsor's consent, the prior or concurrent malignancy does not affect the evaluation of the safety and efficacy of the investigational product. 11. Current serious illness or psychiatric disorder, including but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic or psychiatric disease. 12. Active hepatitis B infection (HBsAg positive) not receiving antiviral treatment. Note: Patients with active hepatitis B (HbsAg positive) must be receiving antiviral treatment with lamivudine, tenofovir, entecavir or other antiviral agents starting at least 7 days prior to the start of study treatment. Patients with prior hepatitis B (anti-HBc positive, HbsAg and HBV-DNA negative) are eligible. 13. Positive test for Hepatitis C ribonucleic acid (HCV RNA); Note: Patients with spontaneously resolved HCV infection (positive HCV antibodies without detectable HCV-RNA) or who have achieved a sustained virologic response after antiviral treatment and demonstrate the absence of detectable HCV RNA for ≥6 months (use of IFN-free regimens) or ≥12 months (use of IFN-based regimens) after cessation of antiviral treatment are eligible. 14. Known history of HIV (HIV1 / 2 antibodies). Patients with HIV with an undetectable viral load are accepted. HIV testing is not required unless mandated by local health authorities or regulations. 15. Sexually active male and female patients of childbearing potential must agree to use one of the following highly effective methods of contraception for the entire duration of the study and for 6 months after the last dose of PB19478: Combined (estrogen- and progestogen-containing) hormonal contraception associated with the inhibition of ovulation (oral, intravaginal, transdermal) Progestogen-only hormonal contraception (oral, injectable, implantable) associated with inhibition of ovulation Intrauterine device (IUD) Intrauterine hormone releasing system (IUS) Bilateral tubal obstruction 1 vasectomized partner ·Sexual abstinence 2 16. Pregnant or lactating women are excluded from this study.

[0367] Definitions of ECOG performance status scoring grades are known in the art as follows: 0. Fully active, able to perform all pre-disease functions without limitations; 1. Limited physically strenuous activity, but ambulatory, able to perform light or sedentary tasks such as light housework, office work, etc.; 2. Ambulatory, able to care for all self, but unable to perform work activities; mobile more than 50% of waking hours; 3. Only able to care for self limited, confined to bed or chair more than 50% of waking hours; 4. Totally disabled, unable to care for self; completely confined to bed and chair; 5. Death.

[0368] Investigational Treatments and Regimens The antibody will be administered as an IV infusion once every 2 weeks (q2w) in a 4-week cycle with a dose level of 1500 mg (flat dose) as the RP2D level. Dose levels between 100 and 3000 mg (flat dose) may be explored. After reaching the RP2D, dose escalation will stop. The administered dose, dose escalation, and dosing frequency for each patient (including expansion cohorts) will be subject to change based on patient safety, PK, and pharmacodynamic data, as well as based on sponsor recommendations. The sponsor may recommend the use of a biweekly dosing schedule for cycle 1.

[0369] Duration of treatment Study treatment will be administered until confirmed progressive disease (by RECIST v1.1), unacceptable toxicity, withdrawal of consent, patient noncompliance, investigator decision (e.g., clinical deterioration), or antibody discontinuation for >6 consecutive weeks.

[0370] Patients will be followed for safety for at least 30 days after the last antibody infusion and until resolution or stabilization of all associated toxicities, as well as for disease progression and survival status every 3 months for up to one year.

[0371] Example 9 In the clinical study of Example 8, clinical efficacy was observed in non-small cell lung cancer patients with EGFR mutations, including EGFR exon 20 mutations / insertions, EGFR exon 21 mutations such as L858R, and EGFR exon 19 deletion mutations, as well as c-MET mutations, including c-MET exon 14 skipping mutations. More specifically, reductions in target lesions were observed in some patients, including confirmed partial responses, two cases of stable disease and reductions in lesions (including a patient with c-MET exon 14 mutations), a patient with exon 19 mutations, and a patient with EGFR amplification mutations. The patients had been pretreated with a variety of different therapies, including platinum-based chemotherapy, osimertinib, pembrolizumab, nivolumab, cetuximab, crizotinib, capmatinib, and tepotinib.

[0372] Example 10 In the clinical study of Example 8, clinical efficacy was observed in a 68-year-old female patient with head and neck squamous cell carcinoma (HNSCC) who had previously been treated with cisplatin and cemiplimab. More specifically, a 20% reduction in target lesions was observed in the patient who experienced only grade 1 / 2 adverse events.

[0373] Example 11 In the clinical study of Example 8, clinically relevant responses were observed in a number of patients with non-small cell lung cancer harboring cMET exon 14 skipping mutations after dosing with bispecific antibody PB19478 at 1500 mg Q2W. Clinically relevant responses included confirmed partial responses and stable disease according to investigator review based on RECIST criteria 1.1. Some patients had received prior anti-cancer treatment, while some other patients were treatment naive, i.e., 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.

[0374] Example 12 In the clinical study of Example 8, clinically relevant responses were observed in a number of patients with non-small cell lung cancer harboring EGFR exon 20 insertion mutations after dosing with bispecific antibody PB19478 at 1500 mg Q2W. Clinically relevant responses included confirmed partial responses and stable disease according to investigator review based on RECIST 1.1. Some patients had received prior anticancer treatment, such as platinum-based chemotherapy.

[0375] References 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. 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. Eisenhauer et al.,New response evaluation criteria in solid tumours:Revised RECIST guideline(version1.1).European Journal of Cancer 45(2009)228-247) Ferguson KM.Structure-based view of epidermal growth factor receptor regulation.Annu Rev Biophys 2008;37:353-73. Kim,George P.,and Axel Grothey.“Targeting colorectal cancer with human anti-EGFR monoclonal antibodies:focus on panitumumab.”Biologics 2.2(2008):223-228. 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) 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. 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. Robertson SC, Tynan J, Donoghue DJ. RTK mutations and human syndromes: when good receptors turn bad. Trends Genet 2000;16:368. 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.

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Claims

1. 1. A bispecific antibody comprising a first variable domain capable of binding to the extracellular part of the human epidermal growth factor receptor (EGFR) and a second variable domain capable of binding to the extracellular part of the human MET proto-oncogene, receptor tyrosine kinase (cMET), for use in a method of treating cancer in a subject 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, wherein said first variable domain has the CDR1 sequence SYGIS, CDR2 sequence WISAYX1X2NTN YAQKLQG, and 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, and having 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than X1 to X7, and said second variable domain comprising a heavy chain variable region having the amino acid sequence of one of the sequences of SEQ ID NOs: 1 to 23 with 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

2. 1. A method of treating cancer in a subject who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor, or ii) chemotherapy and an EGFR tyrosine kinase inhibitor, or iii) a cMET tyrosine kinase inhibitor, said treatment comprising a first variable domain capable of binding to an 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), said first variable domain having the CDR1 sequence SYGIS, CDR2 sequence WISAYX1X2NTNYAQKLQG, and the sequence X3X 4X5X6HWWLX7A, 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, and at positions other than X1 to X7, 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof, and said second variable domain comprises a heavy chain variable region having the amino acid sequence of one of the sequences of SEQ ID NOs: 1-23 with 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

3. A 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 has the CDR1 sequence SYGIS, CDR2 sequence WISAYX1X2NTN, in the manufacture of a medicament for the treatment of cancer in a subject who has received prior treatment with i) a third generation EGFR tyrosine kinase inhibitor, or ii) chemotherapy and an EGFR tyrosine kinase inhibitor, or iii) a cMET tyrosine kinase inhibitor. YAQKLQG, and 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, and having 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than X1 to X7, and said second variable domain comprising a heavy chain variable region having the amino acid sequence of one of the sequences of SEQ ID NOs: 1 to 23 with 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

4. 13. The use or method of any one of the preceding claims, wherein the subject or cancer having undergone prior treatment with i) is resistant to treatment with a third generation EGFR tyrosine kinase inhibitor.

5. 4. The use or method of any one of claims 1 to 3, wherein the subject or cancer having undergone prior treatment with ii) is resistant to treatment with a first, second and / or third generation tyrosine kinase inhibitor.

6. 4. The use or method of any one of claims 1 to 3, wherein the subject or cancer having undergone prior treatment with iii) is resistant to treatment with a cMET tyrosine kinase inhibitor.

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

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

9. 10. 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. 20. The use or method according to any one of the preceding claims, wherein said third generation EGFR tyrosine kinase comprises osimertinib, lazertinib, alflutinib, resibertinib, rociletinib, olmutinib, almonertinib, abivertinib, ASK120067, befotertinib, SH-1028, nazartinib (EGF816), nacotinib (ASP8273), mavereltinib (PF-0647775), orafeltinib (CK-101), kenatinib, ES-072, preferably osimertinib.

11. 13. The use or method of 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. 10. The use or method according to any one of the preceding claims, wherein said chemotherapy comprises platinum-based chemotherapy, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide or pemetrexed, or any combination thereof, preferably a combination comprising cisplatin or carboplatin.

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

15. 2. The use or method of 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 binding of third generation tyrosine kinase inhibitors to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, EGFR gene amplification, a cMET mutation or a cMET abnormality.

16. 13. 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. 10. 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. 2. The use or method of 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 cancer is characterized in that the proximal loop insertion (positions 767-772), the distal 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_N7 71insP, 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 (e.g., L792H), C796X (e.g., G796R, G796S, G796D), C797X (e.g., C797S, C797G), L798I or an in-frame exon 20 insertion, e.g., 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 2. The use or method of any one of the preceding claims, comprising an exon 20 mutation selected from: 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. 2. The use or method of any one of the preceding claims, wherein the cancer comprises a cMET abnormality, such as cMET amplification, cMET overexpression, increased cMET pathway signaling, cMET gene amplification, increased HGF expression and / or increased cMET protein activity.

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

22. 13. 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, gastro-esophageal junction cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, colorectal cancer or bladder cancer.

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

24. 13. The use or method of any one of the preceding claims, wherein the cancer or patient has progressed after receiving said prior therapy for advanced or metastatic cancer.

25. 13. The use or method of any one of the preceding claims, wherein treatment comprises a diagnostic step to assess whether the cancer is an EGFR positive and / or cMET positive cancer, or to assess the presence of EGFR and / or cMET abnormalities in the cancer.

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

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

28. 2. The use or method of any one of the preceding claims, wherein the antibody has enhanced ADCC.

29. 2. The use or method of any one of the preceding claims, wherein the antibody is an IgG1 format antibody with a 1:1 anti-EGFR, anti-cMET stoichiometry.

30. 2. The use or method of any one of the preceding claims, wherein the antibody has one variable domain capable of binding to EGFR and one variable domain capable of binding to cMET.

31. 10. The use or method according to any one of the preceding claims, wherein said variable domain capable of binding to human EGFR is also capable of binding to cynomolgus monkey and mouse EGFR.

32. 2. The use or method according to any one of the preceding claims, wherein said variable domain capable of binding to human EGFR binds to domain III of human EGFR.

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

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

35. 10. 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. X １ = N, X ２ = G, X ３ = D, X ４ = S, X ５ = Y, X ６ = W and X ７ = G or X １ = N, X ２ = A, X ３ = D, X ４ = S, X ５ = Y, X ６ = W and X ７ = G or X １ = S, X ２ = G, X ３ = D, X ４ = S, X ５ = Y, X ６ = W and X ７ = G or 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 or X １ = N, X ２ = G, X ３ = G, X ４ = Y, X ５ = L, X ６ = D and X ７ = G or 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, 10. The use or method according to any one of the preceding claims.

37. 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 2. The use or method according to any one of the preceding claims, wherein =D.

38. 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 2. The use or method according to any one of the preceding claims, wherein =D.

39. 10. 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 NOs: 1-3, 7, 8, 10, 13, 15, 16, 17, 21, 22 or 23 having 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

40. 10. 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 having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

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

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

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

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

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

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