CN118974088A - Combination therapy comprising an antibody that binds EGFR to cMET - Google Patents

Abstract

The present invention relates to a composition of a third generation EGFR tyrosine kinase inhibitor and a bispecific antibody comprising a first variable domain that can bind to the extracellular portion of a human Epidermal Growth Factor Receptor (EGFR) and a second variable domain that can bind to the extracellular portion of a human MET oncogene receptor tyrosine kinase (cMET), for use in a method of treating cancer in an individual.

Description

Combination therapy comprising an antibody that binds EGFR to cMET

Technical Field

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

Background Art

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

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

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

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

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

To date, EGFR-targeted therapies are associated with resistance to treatment that develops over time. Various mechanisms of resistance to EGFR-TKIs have been described. In patients with advanced non-small cell lung cancer (NSCLC), resistance mechanisms include the occurrence of secondary or tertiary mutations (e.g., T790M, C797S, L718Q, exon 20 insertion mutations), activation of alternative signaling (e.g., Met, HGF, AXL, Hh, IGF-1R), aberrant downstream pathways (e.g., AKT mutations, PTEN deletions), impaired EGFR-TKIs-mediated apoptotic pathways (e.g., 11/BIM deletion polymorphisms like BCL2), and histological transformation. Although some resistance mechanisms have been identified, others remain to be identified. In addition, with respect to third-generation TKI resistance mechanisms, the molecular heterogeneity of NSCLC influences its contribution to the broad spectrum of resistance aberrations found so far. Similarly, patients with colorectal cancer who receive EGFR antibody therapy also develop resistance over time. This may occur through the emergence of KRAS mutations. For those patients 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 has been found in many EGFR-positive cancers, and it has been shown that there is a need in the art for more effective EGFR cancer treatments that can improve the standard of care and have advantages in addressing resistance to EGFR-targeted therapy.

Deregulation 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 have been observed in lung cancer, breast cancer, and multiple myeloma (J. M. Siegfried et al., Ann Thorac Surg 66, 1915 (1998); P. C. Ma et al., Anticancer Res 23, 49 (2003); B. E. Elliott et al., Can J Physiol Pharmacol 80, 91 (2002); C. Seidel et al., Med Oncol 15, 145 (1998)). Overexpression, amplification or mutation of cMET has been reported in various cancers such as colorectal cancer, lung cancer, gastric cancer and renal cancer, and can drive ligand-independent receptor activation (C. Birchmeier et al., Nat Rev Mol Cell Biol 4, 915 (2003); G. Maulik et al., Cytokine Growth Factor Rev 13, 41 (2002)). HGF expression is also associated with activation of the HGF/cMET signaling pathway, which is also one of the tumor escape mechanisms under EGFR targeted therapy screening. In addition, treatment with cMET tyrosine kinase inhibitors (such as Capmatinib or Tepotinib) is associated with the emergence of cMET aberration escape mechanisms.

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

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

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

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

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

To date, the prior art has described some bispecific EGFR×cMET antibodies. Castoldi R. et al. (2013) described a bispecific EGFR×cMET antibody named MetHer1, which has the cMET binding site of antibody 5D5 (or MetMab) and the EGFR binding site of cetuximab. The EGFR and cMET binding stoichiometry of the bispecific antibody is fixed at 2:1 (see Supplementary Figure).

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

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

Thus, there is a need for novel bispecific cMET×EGFR antibodies, including antibodies that may have the superior properties as described herein.

Summary of the invention

In certain aspects, the present invention provides a composition of a bispecific antibody of the present invention and a third-generation EGFR tyrosine kinase inhibitor, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), and the composition is used in a method for treating cancer in an individual.

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

In certain aspects, the present invention provides a composition of a bispecific antibody and a third-generation EGFR tyrosine kinase inhibitor for use in the preparation of a medicament for treating cancer, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET).

In certain aspects, the present invention provides a pharmaceutical composition comprising a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET).

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

In some aspects, the cancer or individual has been previously treated with an EGFR tyrosine kinase inhibitor, and/or the cancer or individual is resistant to treatment with a tyrosine kinase inhibitor, in some aspects, resistant to EGFR tyrosine kinase inhibitors and/or cMET tyrosine kinase inhibitors. In some aspects, the individual has been previously treated with a third-generation EGFR tyrosine kinase inhibitor (e.g., osimertinib). The EGFR tyrosine kinase inhibitor resistance includes first-generation, second-generation and/or third-generation tyrosine kinase inhibitors. In other aspects, the administration or treatment of the composition of the bispecific antibody and EGFR tyrosine kinase inhibitor includes second-line treatment of individuals with EGFR and/or cMET tyrosine kinase inhibitor resistance. In other aspects, the administration or treatment of the composition of the bispecific antibody and EGFR tyrosine kinase inhibitor includes second-line treatment of individuals previously treated with a third-generation EGFR tyrosine kinase inhibitor. In some aspects, in some aspects, the individual includes EGFR and/or cMET aberrations that confer resistance to the third-generation EGFR tyrosine kinase inhibitor. In certain aspects, the individual or cancer is resistant or refractory to a third generation EGFR tyrosine kinase inhibitor, and in certain aspects, is resistant or refractory to Osimertinib.

In some aspects, the use or treatment comprises providing a bispecific antibody at a dosage of 1000, 1500 or 2000 mg to the individual. In some aspects, the bispecific antibody is provided once a week or once every two weeks. In a certain aspect, the third generation EGFR tyrosine kinase inhibitor provides a daily dosage of 50 mg to about 400 mg, such as 70 mg, 75 mg, 80 mg, 100 mg, 110 mg or 240 mg.

In certain aspects, the use or treatment comprises providing the individual with a dose of 1500 mg of the bispecific antibody once every two weeks.

In certain aspects, the EGFR tyrosine kinase inhibitor administered in the combination therapy is or comprises Osimertinib, Lazertinib, Alflutinib, Rezivertinib, Rociletinib, Olmutinib, Almonertinib, Abivertinib, ASK120067, Befotertinib (also known as BPI-D0316 or D-0316), SH-1028, Nazartinib (EGF816), naquotinib (ASP8273), Mavelertinib (PF-0647775), Olafertinib (CK-101), Keynatinib, ES-072. In certain aspects, the EGFR tyrosine kinase inhibitor is preferably Osimertinib, BPI-D0316/Befotertinib, Lazertinib, or Almonertinib.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is osimertinib (AZD9291). Osimertinib (AZD9291) is a covalent, orally active, irreversible and mutation-selective EGFR inhibitor with an IC50 of 12 nM for L858R and 1 nM for L858R/T790M. The recommended Phase 2 dose is determined to be 80 mg per day.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Almonertinib (HS-10296). Almonertinib is an oral, irreversible third generation EGFR tyrosine kinase inhibitor that is selective for EGFR-sensitizing and T790M resistance mutations. Almonertinib is used in the study of non-small cell lung cancer. The recommended Phase 2 dose is determined to be 110 mg per day.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Lazertinib. Lazertinib (YH25448) is a potent, mutation-selective, blood-brain barrier-penetrating, oral, and irreversible third generation EGFR tyrosine kinase inhibitor that can be used in the study of non-small cell lung cancer. The recommended Phase 2 dose is determined to be 240 mg per day.

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

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

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

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

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

In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is aricinib (SH-1028, Nanjing Sanhome Pharmaceutical Co., Ltd., Nanjing, China). SH-1028 is the subject of clinical trial NCT04239833, which studies its clinical efficacy in NSCLC.

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

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

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

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

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

In certain aspects, the individual or cancer is resistant to treatment with a first generation, second generation, third generation EGFR tyrosine kinase inhibitor or a cMET inhibitor.

In certain aspects, the first generation tyrosine kinase resistance is or comprises resistance to Gefitinib, Erlotinib or Icotinib.

In certain aspects, the second generation tyrosine kinase resistance is or comprises resistance to Afatinib, Dacomitinib, XL647, AP26113, CO-1686 or Neratinib.

In some aspects, the cMET tyrosine kinase resistance is or comprises resistance to Capmatinib, Tepotinib, Crizotenib, Cabozantinib, Savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833. In some aspects, the cMET tyrosine kinase resistance is or comprises resistance to Tepotinib. In some aspects, the cMET tyrosine kinase resistance is or comprises resistance to Capmatinib.

In some aspects, the resistance of the third generation EGFR tyrosine kinase is or includes resistance to Osimertinib, Lazertinib, Alflutinib, Rezivertinib, Olmutinib, Almonertinib, Abivertinib, ASK120067, Befotertinib, Rociletinib, Oritinib, Nazartinib, Naquotinib, Mavelertinib. In some aspects, the resistance of the third generation EGFR tyrosine kinase is or includes resistance to Osimertinib. In some aspects, the resistance of the third generation EGFR tyrosine kinase is or includes resistance to Lazertinib. In certain aspects, the resistance to the third generation EGFR tyrosine kinase is or comprises resistance to Befotertinib. In certain aspects, the resistance to the third generation EGFR tyrosine kinase is or comprises resistance to Almonertinib.

In some aspects, the individual has not previously received prior anticancer treatment for EGFR and/or cMET positive cancers, or for cancers comprising EGFR and/or cMET aberrations. In some aspects, the individual has not previously received chemotherapy, or treatment comprising anti-EGFR antibodies or anti-cMET antibodies. In some aspects, the individual has not received EGFR tyrosine kinase inhibitor treatment or cetuximab treatment. In some aspects, the combined administration or treatment of the bispecific antibody and EGFR tyrosine kinase inhibitor is a first-line treatment. In some aspects, the first-line treatment is to prevent the development of resistance to EGFR and/or cMET tyrosine kinase mechanisms, such as in lung cancer patients or lung cancer, specifically in non-small cell lung cancer. Resistance mechanisms to EGFR tyrosine kinase inhibitors and/or cMET tyrosine kinase inhibitors are known to develop in individuals with, in particular, non-small cell lung cancer, such as metastatic or advanced cancer. Therefore, the present invention also provides a combination of the third-generation tyrosine kinase inhibitor and the bispecific antibody for use in a method for preventing the individual from developing or developing cancer that is resistant to an EGFR tyrosine kinase inhibitor and/or a cMET tyrosine kinase inhibitor.

In certain aspects, the subject is a human subject.

In certain aspects, the cancer is lung cancer.

In certain aspects, the cancer is non-small cell lung cancer (NSCLC).

In certain aspects, the cancer or individual comprises an activating EGFR mutation, a confirmed tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation, a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, EGFR gene amplification, a cMET mutation, or a cMET aberration.

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

In certain aspects, the cMET dysregulation comprises cMET amplification, cMET overexpression, increased signaling of the cMET pathway, cMET gene amplification and/or increased cMET protein activity, or cMET exon 14 skipping mutation. In one aspect, the cMET dysregulation is caused by increased expression of HGF.

In certain aspects, the present invention provides a pharmaceutical composition comprising a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET).

In certain aspects, the pharmaceutical composition comprises instructions for use.

In certain aspects, the bispecific antibodies of the present invention express ADCC activity, and in certain aspects, the antibodies have enhanced ADCC activity. In such aspects, the antibodies may have altered ADCC activity by mutating one or more CH2 domains relative to a complete human CH2 domain. Therefore, a bispecific antibody according to the present invention is further provided, which is afucosylated. In certain aspects, the antibodies of the present invention comprise two afucosylated CH2 domains. In certain aspects, the antibodies of the present invention comprise a total of two CH2 domains, both of which are afucosylated. In certain aspects, the antibodies of the present invention comprise two CH2 domains, both of which are afucosylated.

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

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

In certain aspects, an antibody of the invention comprises a _first variable domain comprising a _heavy chain variable region having _a CDR1 sequence _of SYGIS, a CDR2 sequence of _{WISAYX1X2NTNYAQKLQG} and a CDR3 comprising the sequence _{X3X4X5X6HWWLX7A ,}

wherein _X1 = N or S; _X2 = A or G; _X3 = D or G; _X4 = R, S or Y; _X5 = H, L or Y; _X6 = D or W and _X7 = D or G; and there are 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than _X1 - _X7 .

In certain aspects, an antibody of the invention comprises a second variable domain comprising a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1-23 ( FIG. 3 ) with 0 to 10, in certain aspects 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof.

Now, a bispecific antibody is described, wherein

X ₁ =N _; X ₂ = _{G ;} X ₃ =D; X ₄ =S;

X ₁ =N _; X ₂ = _{A ;} X ₃ =D; X ₄ =S;

X ₁ = _S ; X ₂ = _{G ;} X ₃ =D; X ₄ =S;

_X1 = N; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D;

X ₁ =N; X ₂ = _{A ;} X ₃ = _D ; X ₄ =R;

_X1 = S; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D;

X ₁ =N _; X ₂ = _{G; X 3 =G; X 4 = Y ;}

_X1 = N; _X2 = A; _X3 = G; _X4 = Y; _X5 = L; _X6 = D and _X7 = G; or

X ₁ =S; _{X 2} = _{G ;} X ₃ =G; X ₄ =Y;

In certain aspects, _X1 =N; _X2 =G; _X3 =D; _X4 =R; _X5 =H; _X6 =W and _X7 =D; or _X1 =N; _X2 =A; _X3 =D; _X4 =R; _X5 =H; _X6 =W and _X7 =D; or _X1 =S; _X2 =G; _X3 =D; _X4 =R; _X5 =H; _X6 =W and _X7 =D.

In certain aspects, X ₃ -X ₇ =DRHWD, and X ₁ and X ₂ are NG, SG or NA.

Now described are bispecific antibodies in which the heavy chain variable region of the second variable domain comprises an amino acid sequence having one of the sequences of SEQ ID NOs: 1-3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23 resulting in 0 to 10, in certain aspects 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof.

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

The present invention also provides a bispecific antibody included in the treatment of the present invention, the bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase ( _cMET ), wherein the first variable domain comprises a heavy chain variable region having _a CDR1 sequence _of SYGIS, _{a CDR2} sequence of _{WISAYX1X2NTNYAQKLQG} and a CDR3 comprising the sequence _{X3X4X5X6HWWLX7A} , wherein

_X1 =N or S; _X2 =A or G; X3 = _D or G; _X4 =R, S or Y; _X5 =H, L or Y; _X6 =D or W and _X7 =D or G, and there are 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than _X1 - _X7 , and wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1-23 (Figure 3) resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The first variable domain, in some aspects, comprises a heavy chain variable region having a CDR1 sequence of SYGIS, a CDR2 sequence of WISAYNGNTNYAQKLQG, and a CDR3 comprising the sequence DRHWHWWLDAFDY, and the second variable domain, in some aspects, comprises a heavy chain variable region having a CDR1 sequence of SYSMN, a CDR2 sequence of WINTYTGDPTYAQGFTG, and a CDR3 sequence of ETYYYDRGGYPFDP.

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

Also provided is the use of the bispecific antibody of the present invention and a third-generation tyrosine kinase inhibitor in the preparation of a medicament for treating a disease involving abnormal cells, such as a tumor or cancer.

Also provided is a method of treating an individual having a tumor, in certain aspects, the tumor is an EGFR-positive tumor, a cMET-positive tumor, or an EGFR and cMET-positive tumor, the method comprising administering the bispecific antibody and a third generation tyrosine kinase inhibitor to the individual in need thereof.

When used in combination with a third generation TKI, the antibodies of the invention inhibit the growth of EGFR TKI-resistant tumor cell lines induced by HGF and EGF/HGF. In some aspects, the TKI is Osimertinib. In some aspects, the TKI is Lazertinib. In some aspects, the TKI is Almonertinib. In some aspects, the TKI is Befotertinib.

In certain aspects, the combination of the antibody of the present invention and the third generation tyrosine inhibitor inhibits the growth of HGF-responsive cells induced by HGF, in certain aspects, in EGFR TKI-resistant or refractory tumors, tumor models or cell lines of human subjects, such as cell lines or models including activating EGFR mutations, confirmed tyrosine kinase inhibitor resistance mutations, tertiary tyrosine kinase inhibitor resistance mutations, mutations that reduce the binding of third generation tyrosine kinase inhibitors to EGFR, acquired tyrosine kinase inhibitor resistance mutations, EGFR gene amplification, cMET mutations or cMET aberrations, in certain aspects, in-frame exon 20 insertion mutations. In certain aspects, the inhibitory effect is shown in the presence of HGF.

Combinations of antibodies of the invention and third generation TKIs in certain aspects inhibit HGF-induced growth of HGF-responsive cells, in certain aspects EGFR TKI-resistant tumors, tumor models or cell lines, such as cell lines or models comprising an in-frame exon 20 insertion mutation.

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

As used herein, the term "resistant" refers to a cancer or patient that does not respond to treatment when administered with a prescribed dose of the relevant therapeutic agent.

Here, “first-generation EGFR tyrosine kinase inhibitors” (first-generation TKIs) refer to reversible EGFR inhibitors such as gefitinib and erlotinib, which are effective for the first-line treatment of NSCLC carrying EGFR activating mutations (such as exon 19 deletions and exon 21 L858R mutations).

Herein, the term "second-generation EGFR tyrosine kinase inhibitor" (second-generation TKI) refers to covalent irreversible EGFR inhibitors such as afatinib and dacomitinib, which are effective for the first-line treatment of NSCLC carrying EGFR activating mutations (such as exon 19 deletions and exon 21 L858R mutations).

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

The antibodies of the invention inhibit EGF-induced growth of EGF-responsive cells without inducing toxicities associated with high-affinity bivalent EGFR antibodies, such as rash and diarrhea. This makes the antibodies very suitable for use in combination with TKIs that have their own toxicity profile.

The present invention also includes a pharmaceutical composition or a kit as a component, which comprises a combination of a bispecific antibody disclosed herein and a third-generation EGFR tyrosine kinase inhibitor. The pharmaceutical composition is not physically connected in some aspects and comprises a container containing an antibody of the present invention and a third-generation EGFR tyrosine kinase inhibitor. The pharmaceutical composition is accompanied by instructions for use in some aspects. The instructions for use include clinically relevant information, such as instructions for intravenous administration. In some aspects, the third-generation EGFR tyrosine kinase, such as Osimertinib, BPID-0316/Befotertinib, or Almonertinib, is administered in accordance with the instructions for use after approval by relevant departments. In some aspects, the dosage of Osimertinib is 80 mg once a day. In some aspects, the dosage of Almonertinib is 110 mg once a day. In some aspects, the dosage of Lazertinib is 240 mg once a day. In certain aspects, the dose of Befotertinib is 70 mg, 75 mg or 100 mg once daily. The 75 mg daily dose of Befotertinib can be divided into 3 oral administrations, each dose of 25 mg is provided.

In some aspects, the bispecific antibody of the present invention is administered at 1000 mg, particularly at a fixed dose of 1000 mg. In some aspects, the bispecific antibody is provided at a dose of 1000 mg once a week. In some aspects, the bispecific antibody is provided at a dose of 1000 mg once every two weeks.

In some aspects, the bispecific antibody of the present invention is administered at 1500 mg, particularly at a fixed dose of 1500 mg. In some aspects, the bispecific antibody is provided at a dose of 1500 mg once a week. In some aspects, the bispecific antibody is provided at a dose of 1500 mg once every two weeks.

In some aspects, the bispecific antibody of the present invention is administered at 2000 mg, particularly at a fixed dose of 2000 mg. In some aspects, the bispecific antibody is provided at a dose of 2000 mg once a week. In some aspects, the bispecific antibody is provided at a dose of 2000 mg once every two weeks.

The antibodies of the invention can be used to treat tumors that are resistant or have reduced sensitivity to treatment with EGFR tyrosine kinase inhibitors, for example, tumors that are resistant to osimertinib, erlotinib, gefitinib or afatinib, or analogs of osimertinib, erlotinib, gefitinib or afatinib, or a combination of one or more corresponding compounds and/or their analogs.

Therefore, the bispecific antibody of the present invention can be administered simultaneously, sequentially or separately with the EGFR tyrosine kinase inhibitor of the present invention.In certain aspects, the EGFR tyrosine kinase inhibitor comprises or is the third generation EGFR tyrosine kinase inhibitor.

The present invention also comprises a nucleic acid molecule or a group of nucleic acid molecules, which alone or together encode the heavy chain or heavy chain variable region of the bispecific antibody or variant thereof disclosed herein. Also provided are nucleic acid molecules or groups of nucleic acid molecules encoding the antibodies disclosed herein.

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

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

The present invention also includes a cell system comprising the bispecific antibody or variant thereof disclosed herein.

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

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

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the amino acid sequence of the heavy chain variable region of the variable domains mentioned in this application.

Figure 2 shows 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 located at corresponding positions (according to Kabat numbering).

Figure 3 shows 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 located at corresponding positions (according to Kabat numbering).

Figure 4 shows the common light chain used in monospecific and bispecific IgGs.

Fig. 4A shows the amino acid sequence of the common light chain. Fig. 4B shows the common light chain of the variable domain DNA sequence and translation (IGKV1-39/jk1). Fig. 4C shows the common light chain of the constant region DNA sequence and translation. Fig. 4D shows the variable domain translation of the common light chain of IGKV1-39/jk5. Fig. 4E shows the V region IGKV1-39A.

Figure 5 shows IgG heavy chains used to generate bispecific molecules. Figure 5A shows the CH1 region. Figure 5B shows the hinge region. Figure 5C shows the CH2 region. Figure 5D shows a CH2 comprising the silent substitutions of L235G and G236R. Figure 5E shows a CH3 domain comprising the substitutions L351K and T366K (KK). Figure 5F shows a CH3 domain comprising the substitutions L351D and L368E (DE).

Figure 6 shows an overview of the treatment plan. Antibodies, osimertinib, and combinations of antibodies and osimertinib, or vehicle, were administered over the specified period. At the end of treatment (day 31), tumor samples were collected and snap-frozen for target expression analysis. Mice were monitored for the duration of response (DoR) and relapse after the end of treatment.

Figure 7 shows the survival curves for 750 ^mm3 : the log-rank (Mantel-Cox) test for the combination versus each single treatment group was significant.

Figure 8 shows individual tumor volumes on day 28. Statistical analysis was performed using mixed models with Tukey post-test in Graphpad Prism.

Figure 9 shows the mean tumor volume ± SEM during the complete treatment period. The observation period ended at day 100.

An overview of the treatment schedule is shown in Figure 10. Groups were administered over a 3 week period.

Figure 11 shows the effect of treatment with groups containing antibodies, Osimertinib, combination therapy and vehicle alone on tumor volume of NSCLC CDX model HCC827/ER1 carrying exon 19 mutations. Tumor volume growth curves representing vehicle and treatment groups are shown at different time points (mean TV ± SEM).

FIG. 12 shows the animal survival curves of mice carrying the NSCLC CDX model HCC827/ER1 with exon 19 mutation in different treatment groups and the vehicle group.

DETAILED DESCRIPTION

In certain aspects, the present invention provides a composition of a bispecific antibody of the present invention and a third-generation EGFR tyrosine kinase inhibitor, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), and the composition is used in a method for treating cancer.

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

In certain aspects, the present invention provides a use of a composition of a bispecific antibody of the present invention and a third-generation EGFR tyrosine kinase inhibitor for the preparation of a medicament for treating cancer, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET).

In some aspects, the bispecific antibody of the present invention can be used simultaneously, sequentially or separately with the EGFR tyrosine kinase inhibitor of the present invention. Therefore, the combination of the bispecific antibody of the present invention and the third generation EGFR tyrosine kinase inhibitor encompasses simultaneous, sequential or separate use. In some aspects, the EGFR tyrosine kinase inhibitor comprises or is the third generation EGFR tyrosine kinase inhibitor.

Therefore, in certain aspects, the present invention provides a bispecific antibody of the present invention, comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), for use in a method for treating cancer, wherein the treatment further comprises administering a third-generation EGFR tyrosine kinase inhibitor, wherein the bispecific antibody of the present invention and the third-generation EGFR tyrosine kinase inhibitor of the present invention are administered simultaneously, sequentially or separately as appropriate.

Therefore, in certain aspects, the present invention provides a method for treating an individual suffering from cancer, comprising administering to the individual an effective amount of a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody of the present invention, wherein the bispecific antibody of the present invention and the third-generation EGFR tyrosine kinase inhibitor of the present invention are administered simultaneously, sequentially or separately, as the case may be.

Therefore, in certain aspects, the present invention provides a use of a combination of a bispecific antibody of the present invention and a third-generation EGFR tyrosine kinase inhibitor of the present invention for preparing a medicament for treating cancer, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the bispecific antibody of the present invention and the third-generation EGFR tyrosine kinase inhibitor of the present invention are administered simultaneously, sequentially or separately as appropriate.

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

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

Abnormally activated forms of EGFR, such as by EGFR mutations or EGFR gene amplification, are known to be oncogenic drivers of non-small cell lung cancer (NSCLC) and are known to occur in EGFR tyrosine kinase inhibitor therapy. The present invention provides a combination therapy in which an antibody of the present invention is administered in combination with a third generation EGFR tyrosine kinase inhibitor to treat the EGFR oncogenic driver. In certain aspects, the tyrosine kinase inhibitor resistance comprises resistance to first, second and/or third generation tyrosine kinase inhibitors.

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

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

In certain aspects, the cancer or individual comprises an activating EGFR mutation, a confirmed 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 aberration, or increased HGF expression.

In some aspects, the cancer or individual comprises an activating EGFR mutation, such as an in-frame exon 19 deletion mutation or an exon 21 mutation (L858R in some aspects). Here, the term "activating EGFR mutation" refers to a mutation that develops after the use of a third-generation EGFR tyrosine kinase inhibitor. In NSCLC, the most common activating mutations are in-frame deletions of exon 19 and substitutions of leucine by arginine (L858R) in exon 21, which together account for 85-90% of EGFR mutations in NSCLC.

In clinical trials conducted for the efficacy of the bispecific antibodies of the present invention, clinical efficacy was observed in a variety of cancers with different genetic carcinogenic backgrounds. Specifically, clinical efficacy was observed in NSCLC. For example, clinical efficacy was observed in patients with EGFR exon 20 mutations, EGFR exon 21 mutations (e.g., L858R), EGFR exon 19 deletion mutations, c-MET exon 14 skipping mutations, cMET amplification, and EGFR amplification mutations. Therefore, in one aspect, the cancer is NSCLC and/or the individual suffers from NSCLC, which comprises EGFR exon 21 mutations such as L858R, EGFR exon 19 deletion mutations, or c-MET exon 14 skipping mutations.

In certain aspects, the cancer or individual comprises a confirmed tyrosine kinase inhibitor resistance mutation. Herein, the term "confirmed tyrosine kinase inhibitor resistance mutation" refers to the development of resistance after using an EGFR tyrosine kinase inhibitor currently approved for the treatment of cancer, such as T790M. Examples of confirmed tyrosine kinase inhibitors are osimertinib and ametinib.

In certain aspects, the cancer or individual 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). Here, the term "tertiary tyrosine kinase inhibitor resistance mutation" refers to resistance developed after use of a third-generation EGFR tyrosine kinase inhibitor.

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

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

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

In certain aspects, the cancer or individual comprises a cMET mutation, such as a cMET exon 14 skipping mutation.

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

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

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

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

In some aspects, the cancer or individual comprises an exon 20 mutation selected from a proximal loop insertion (positions 767-772), a distal loop insertion (positions 773-775), in some aspects 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, H773delins SNPY, N771_P772insH, N771_P772in sVDN, N771delinsGY, N771delinsKH, N771delinsRD, P772_H773delinsHNPY, P772_H773insGT, P772_H773insPNP, P772_H773insT, V769_D770insA, V769_D770insGG, V769_D770insGSV, V769_D770insGVV, and V769_D770ins MASV; or mutations T790M, L792X (e.g., L792H), C796X (e.g., G796R, G796S, G796D), C797X (e.g., C797S, C797G), L798I, or in-frame exon 20 insertions, such as M766_A767insASV or H773-V774insNPH, Ins761(EAFQ), Ins770(ASV), Ins771(G), Ins774(NPH), M766_A7671ns In some aspects, the cancer or individual comprises two or more of the mutations.

In some aspects, the cancer or individual comprises an in-frame exon 19 deletion mutation, an exon 21 mutation (in some aspects L858R) or an in-frame deletion of exon 19 (del19), or a substitution of leucine by arginine (L858R), a mutation L861X (e.g., L861Q) or L844X (e.g., L844V) in exon 21. In some aspects, the cancer or individual comprises two or more of the mutations.

In certain aspects, the cancer or individual comprises the EGFR mutation T790M.

In some aspects, the cancer or individuality comprises an EGFR mutation selected from 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 (e.g., C797S, C797G, C797N), M766X (e.g., M766Q), R776X (e.g., R776C). In some aspects, the cancer or individuality comprises two or more of the mutations.

In some aspects, the cancer or individuality comprises EGFR mutation, selected from T790M, L858R, exon 19 deletion mutation, C797X, L792X, G796X, G724X, S768X, L718X, exon 20 insertion mutation, mutation G724X (such as G724S), S768X (such as S768I), L792X (such as L792H), C797X (including C797S and C797G), L798X (such as L798I), I941X (such as I941R), V948X (such as V948R). In some aspects, the cancer or individuality comprises two or more of the mutations.

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

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

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

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

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

In certain aspects, the cancer or individual comprises the triple mutations L858X/T790X/C797X (e.g., L858R/T790M/C797S), L858X/T790X/M766X (e.g., L858R/T790M/M766Q), L858X/T790X/L718X (e.g., L858R/T790M/cis-L718Q, L858R/T790M/L718Q), L858X/T790X/C797S (e.g., L858R/T790M/C797S), L858X/T790X/M766X (e.g., L858R/T790M/M766Q), L858X/T790X/L718X (e.g., L858R/T790M/C797S), L858X/T790X/M766X (e.g., L858R/T790M/C797S), L858X/T790X/L718X (e.g., L858R/T790M/C718Q), L858X/T790X /C797X (such as L858R/T790M/cis-C797S), exon 19del/T790X/C797X (such as exon 19del/T790M/cis-C797S), L858X/T790X/C941X (such as L858R/T790M/I941R), delE746_A750/T790X/C797X (such as delE746_A750/T790M/C797S).

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

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

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

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

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

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

Antibodies that bind to EGFR and cMET can also bind to other proteins if such other proteins contain the same epitope. Therefore, the term "binding" does not exclude the binding of an antibody to another protein or proteins containing the same epitope. Such binding is generally referred to as cross-reactivity. EGFR/cMET bispecific antibodies generally do not bind to other proteins other than EGFR and/or cMET on postnatal (in some aspects adult human) cell membranes. The antibodies of the present invention are generally capable of binding to EGFR with a binding affinity (i.e., equilibrium dissociation constant _Kd ) of at least 1 ^{× 10-6} M, as indicated in more detail below.

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

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

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

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

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

The variable domain of the invention (the first variable domain) can bind to EGFR and in certain aspects comprises a heavy chain variable region having a CDR1 sequence of SYGIS, a _CDR2 sequence of _{WISAYX1X2NTNYAQKLQG} and _{a CDR3 comprising the sequence X3X4X5X6HWWLX7A , wherein X1} = _N or S; _X2 =A or G; _X3 =D or G; _X4 =R, S or Y; _X5 =H, L or Y; _X6 =D or W and _{X7 =} D or G.

X _1-7 in some respects are:

X ₁ =N _; X ₂ = _{G ;} X ₃ =D; X ₄ =S;

X ₁ =N _; X ₂ = _{A ;} X ₃ =D; X ₄ =S;

X ₁ = _S ; X ₂ = _{G ;} X ₃ =D; X ₄ =S;

_X1 = N; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D;

X ₁ =N; X ₂ = _{A ;} X ₃ = _D ; X ₄ =R;

_X1 = S; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D;

X ₁ =N _; X ₂ = _{G; X 3 =G; X 4 = Y ;}

_X1 = N; _X2 = A; _X3 = G; _X4 = Y; _X5 = L; _X6 = D and _X7 = G; or

X ₁ =S; _{X 2} = _{G ;} X ₃ =G; X ₄ =Y;

In some ways,

_X1 = N; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D;

_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.

In certain aspects, _X1 =N; _X2 =G; _X3 =D; _X4 =R; _X5 =H; _X6 =W and _X7 =D.

The amino acid after the amino acid A in the sequence X ₃ X ₄ X ₅ X ₆ HWWLX ₇ A in the CDR3 sequence of the first variable domain may vary. The amino acid sequence after the sequence X ₃ X ₄ X ₅ X ₆ HWWLX ₇ A may be FDY. The CDR3 of the first variable domain in certain aspects comprises the sequence X ₃ X ₄ X 5 _{X 6} HWWLX ₇ AF, in certain aspects, it comprises X ₃ X ₄ X ₅ X ₆ HWWLX ₇ AFD, and in certain aspects, it comprises X ₃ X ₄ X ₅ X ₆ HWWLX ₇ AFDY.

In certain aspects, the first variable domain comprises a _heavy chain variable region having _a CDR1 sequence of SYGIS, a CDR2 sequence _of WISAYNGNTNYAQKLQG, and a CDR3 sequence _{of X3X4X5X6HWWLX7A} .

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

The first variable domain in some aspects comprises a heavy chain variable region having an amino acid sequence of MF3353, MF8229, MF8228, MF3370, MF8233, MF8232, MF3393, MF8227 or MF8226 as shown in Figure 2, which produces up to 10, in some aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in some aspects 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the specified sequence. In some aspects, the first variable domain comprises a heavy chain variable region having an 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 FIG. 2 .

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

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

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

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

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

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

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

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

The CDR1, CDR2 and CDR3 of the light chain of the first variable domain and the second variable domain as described herein in some aspects respectively 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, the CDR3 comprises the amino acid sequence QQSYSTP. In some embodiments of the bispecific antibodies described herein, the first variable domain and the second variable domain comprise a common light chain, which in some aspects is the light chain variable region shown in Figure 4B.

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

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

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

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

In some aspects, the light chain comprises a light chain region comprising the amino acid sequence of the O12/IgVκ1-39*01 gene segment, as shown in FIG4A , which has 0-10, in some aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. The phrase "O 12 light chain" will be used throughout the specification as an abbreviation for "a light chain comprising a light chain variable region, the light chain variable region comprising the amino acid sequence of the O12/IgVκ1-39*01 gene segment of the portion shown in FIG4A and having 0-10, in some aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof." IgVκ1-39 is an abbreviation for the immunoglobulin variable κ1-39 gene. The gene is also referred to as immunoglobulin κ variable 1-39; IGKV139; IGKV1-39; O 12a or O12. The external ID of the gene is HGNC:5740; Entrez Gene:28930; Ensembl:ENSG00000242371. The preferred amino acid sequence of IgVκ1-39 is provided in Figure 4E. It lists the sequence of the V region. The V region can be combined with one of the five J regions. Figures 4B and 4D illustrate two preferred sequences of IgVκ1-39 combined with the J region. The connected sequences are represented as IGKV1-39/jk1 and IGKV1-39/jk5; another name is IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01 (named according to the IMGT database on the global information network imgt.org).

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

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

Mature B cells that produce antibodies with O12 light chains typically produce light chains that have undergone one or more mutations relative to the germline sequence (i.e., the normal sequence in the non-lymphocytes of the organism). The process that leads to these mutations is generally referred to as somatic (hyper) mutation. The resulting light chain is called an affinity-matured light chain. Such light chains (when derived from the O12 germline sequence) are O12-derived light chains. In this specification, the phrase "common light chain" will include "common light chain-derived light chain", and the phrase "O 12 light chain" will include O12-derived light chains. Mutations introduced by somatic hypermutation can also be artificially introduced in the laboratory. Other mutations can also be introduced in the laboratory without affecting the properties of the light chain, not necessarily its amount. If the light chain comprises a sequence as shown in Figures 4A, 4B; 4D or 4E and has 0-10, in some aspects 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof, it is at least an O12 light chain. In some aspects, the O12 light chain comprises a light chain of a sequence as shown in Figure 4A; 4B; 4D or 4E, which has 0-9, 0-8, 0-7, 0-6, 0-5, 0-4 amino acid insertions, deletions, substitutions, additions or combinations thereof. In some aspects, the O 12 light chain comprises a sequence as shown in Figure 4A, Figure 4B; Figure 4D or Figure 4E, which has 0-5, in some aspects 0-4, in some aspects 0-3 amino acid insertions, deletions, substitutions, additions or combinations thereof. In some aspects, the O12 light chain comprises a sequence as shown in Figure 4A, Figure 4B; Figure 4D or Figure 4E, which has 0-2, in some aspects 0-1, in some aspects 0 amino acid insertions, deletions, substitutions, additions or combinations thereof. In some aspects, the O 12 light chain comprises a sequence as shown in Figure 4A or Figure 4B, which has the above-mentioned amino acid insertions, deletions, substitutions, additions or combinations thereof. In some 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 above 1, 2, 3, 4 or 5 amino acid substitutions are conservative amino acid substitutions in certain aspects and may be present in the CDR regions of the heavy chain and/or light chain; the insertion, deletion, substitution or combination thereof is not in the CDR3 region of the VL chain in certain aspects, and is not in the CDR1, CDR2 or CDR3 region or FR4 region of the VL chain in certain aspects.

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

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

In certain aspects, the light chain variable region comprises an amino acid sequence having 0-10, in certain aspects 0-5, amino acid insertions, deletions, substitutions, additions, or a combination thereof, in the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRASQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQSYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPKLLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK. In some aspects, the light chain variable region comprises 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, in some aspects 0-3, in some aspects 0-2, in some aspects 0-1, and in some aspects 0 amino acid insertions, deletions, substitutions, additions, relative to the specified amino acid sequence, or a combination thereof. If the aligned sequences differ at more than 5 sites, the combination of insertions, deletions, additions or substitutions is the claimed combination. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGSGSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVTITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFATYYCQQ SYSTP PITFG QGTRL EIK. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMTQSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTDFTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQSGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK.

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

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

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

As used herein, the term "compatible hetero-dimerization domains" refers to a protein domain that has been engineered such that the engineered domain A' will preferentially form a hetero-dimer with the engineered domain B', and vice versa, the homodimerization between A'-A' and B'-B' is reduced.

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

Bispecific antibodies can be produced by (transient) transfection of plasmids encoding light chains and two different heavy chains, which are transformed after CH3 to ensure effective hetero-dimerization and form bispecific antibodies. The production of these chains in single cells causes the formation of bispecific antibodies to be superior to monospecific antibodies. The preferred mutations that substantially only produce bispecific full-length IgG1 molecules are amino acid substitutions at sites 351 and 366 in the first CH3 domain ("KK variant" heavy chain), such as L351K and T366K (numbered according to EU numbering), and amino acid substitutions at sites 351 and 368 in the second CH3 domain ("DE variant" heavy chain), such as L351D and L368E, and vice versa (see, e.g., Figures 5E and 5F).

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

The Fc region mediates the effector functions of antibodies, such as complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Depending on the application of the therapeutic antibody or Fc fusion protein, it may be necessary to weaken or enhance the effector function. When the immune response as in some aspects of the present invention is activated, enhanced or stimulated, it may be desirable to weaken the effector function. Antibodies with weakened effector functions can be used for targeting cell surface molecules of immune cells, etc. In some aspects, the antibodies of the present invention promote antibody dependent cellular phagocytosis (ADCP). In some aspects, the antibodies of the present invention promote antibody dependent cellular cytotoxicity (ADCC). In some aspects, an advantage of the present invention is that the bispecific antibodies of the present invention show more effective ADCC activity than Amivantamab, especially for cells or cancers containing cMET aberrations.

The antibody with reduced effector function is in some aspects an IgG antibody comprising a modified CH2/lower hinge region to, for example, reduce Fc-receptor interactions or reduce C1q binding. In some aspects, the antibody of the present invention is an IgG antibody with a mutant CH2 and/or lower hinge domain, such that the interaction of the bispecific IgG antibody with the Fc-γ receptor is reduced. The antibody comprising a mutant CH2 region is in some aspects an IgG1 antibody. Such mutant IgG1 CH2 and/or lower hinge domains comprise amino acid substitutions (EU numbering) at sites 235 and/or 236 in some aspects, and comprise L235G and/or G236R substitutions (FIG. 5D) in some aspects.

The antibodies of the present invention have effector functions in some aspects. The bispecific antibodies of the present invention include antibody-dependent cell-mediated cytotoxicity (ADCC) in some aspects. The antibodies can be modified to enhance ADCC activity (for review, please 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 currently several in vitro methods to determine the efficacy of antibodies or effector cells in inducing ADCC. These include chromium-51 [Cr51] release assays, europium [Eu] release assays, and sulfur-35 [S35] release assays. Typically, a target cell line expressing a surface-exposed antigen is placed in a static state together with an antibody specific for the antigen. After washing, the effector cells expressing the Fc receptor CD16 are placed in a static state together with the target cells through the antibody-marker. The target cell lysis is then measured, and the release of the intracellular marker is measured by a scintillation counter or spectrophotometry. In a certain aspect, the bispecific antibody of the present invention exhibits ADCC activity. In such aspects, the bispecific antibody may have improved ADCC activity. In such aspects, the antibody may have altered ADCC activity, by one or more CH2 mutations as described elsewhere herein, and by techniques known in the art. A technique for enhancing the ADCC of an antibody is afucosylation (see, e.g., Junttila, T.T., K.Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70 (11): 4481-4489). Therefore, a bispecific antibody afucosylated of the present invention is further provided. In certain aspects, the antibody of the present invention comprises two afucosylated CH2 domains. In certain aspects, the antibodies of the present invention comprise a total of two CH2 domains, both of which are non-fucosylated. In certain aspects, the antibodies of the present invention are full-length antibodies, such as IgG-type antibodies, which have two CH2 domains, both of which are non-fucosylated. Alternatively, various other strategies can be used to achieve ADCC enhancement, such as glycoengineering (Kyowa Hakko/Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutations, all of which attempt to increase the binding of Fc to low-affinity activating FcγRIIIa, and/or weaken the binding to low-affinity inhibitory FcγRIIb. The bispecific antibodies of the present invention are in certain aspects non-fucosylated to enhance ADCC activity. When compared to the same antibody made in normal CHO cells, the bispecific antibodies of the present invention have a reduced amount of fucosylation of N-linked sugar chain structures in the Fc region in certain aspects.

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

The bispecific antibodies of the present invention are used in humans in some aspects. The preferred antibodies of the present invention are humanized antibodies, or in some aspects human antibodies. The constant region of the bispecific antibodies of the present invention is a human constant region in some aspects. The constant region may contain one or more, in some aspects no more than 10, in some aspects no more than 5 amino acid differences with the constant region of a naturally occurring human antibody. Preferably, the constant portion is completely derived from a naturally occurring human antibody. The various antibodies produced herein are derived from a human antibody variable domain library. Therefore, these variable domains are human variable domains. The unique CDR region may be derived from humans, synthesized or derived from another organism. When the amino acid sequence of the variable region is identical to the amino acid sequence of the naturally occurring human antibody variable region, excluding the CDR region, the variable region is considered to be a humanized variable region. In such aspects, the VH of the variable domain of the antibody that binds to EGFR or cMET of the present invention may contain one or more, in some aspects no more than 10, in some aspects no more than 5 amino acid differences with the variable region of a naturally occurring human antibody, without calculating possible differences in the amino acid sequence of the CDR region. The light chain variable region of the EGFR binding domain and/or cMET binding domain in the antibody of the present invention may contain one or more, in some aspects no more than 10, in some aspects no more than 5 amino acid differences with the variable region of a naturally occurring human antibody, excluding possible differences in the amino acid sequence of the CDR region. The light chain in the antibody of the present invention may contain one or more, in some aspects no more than 10, in some aspects no more than 5 amino acid differences with the variable region of a naturally occurring human antibody, excluding possible differences in the amino acid sequence of the CDR region. Such mutations also occur in the case of somatic hypermutation in nature.

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

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

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

The VH chains of Figures 1, 2 or 3 in certain aspects have up to 15, in certain aspects have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH chains shown in Figures 1, 2 or 3, in certain aspects have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, relative to the VH chains shown in Figures 1, 2 or 3, in certain aspects have 0, 1, 2, 3 or 4 amino acid insertions, deletions, substitutions or combinations thereof, in certain aspects, have 0, 1, 2, or 3 amino acid insertions, deletions, substitutions or combinations thereof, more in certain aspects, have 0, 1 or 2 amino acid insertions, deletions, substitutions or combinations thereof, and in certain aspects, have 0 or 1 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH chains shown in Figures 1, 2 or 3. The one or more amino acid insertions, deletions, substitutions or combinations thereof are in certain aspects not in the CDR1, CDR2 and/or CDR3 regions of the VH chain. In certain aspects, they are also absent from the FR4 region. The amino acid substitutions in certain aspects are conservative amino acid substitutions.

Rational approaches have been developed towards minimizing non-human residues in a human context. There are a variety of methods to successfully transplant the antigen binding properties of an antibody onto another antibody. The binding properties of an antibody may depend primarily on the precise sequence of the CDR3 region, which is usually supported by the sequences of the CDR1 and CDR2 regions of the variable domain and combined with the variable domain of the appropriate structure as a whole.

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

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

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

Thus, the present invention further provides a humanized, or in some aspects human, bispecific antibody, such as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as shown in MF3370 in Figure 1, and wherein the variable domain comprising the cMET binding site comprises a VH CDR3 region as shown in MF8230 in Figure 3. The VH variable region comprising the EGFR binding site is in some aspects a sequence comprising the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown in MF3370 in Figure 1. The VH variable region comprising the cMET binding site is in some aspects a sequence comprising the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown in MF8230 in Figure 3. CDR grafting can also be used to make a VH chain having the CDR regions of the VH shown in Figure 2 or Figure 3, but with a different framework. The different framework can be the framework of another human VH, or the framework of a different mammalian VH.

Thus, the present invention further provides a humanized, or in some aspects human, bispecific antibody, such as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as shown in MF8233 in Figure 2, and wherein the variable domain comprising the cMET binding site comprises a VH CDR3 region as shown in MF4356 in Figure 3. The VH variable region comprising the EGFR binding site in some aspects comprises the sequence of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown in MF8233 in Figure 2. The VH variable region comprising the cMET binding site in some aspects comprises the sequence of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown in MF4356 in Figure 3. CDR grafting can also be used to make a VH chain having the CDR regions of the VH shown in Figure 2 or Figure 3, but with a different framework. The different framework can be the framework of another human VH, or the framework of a different mammalian VH.

Thus, the present invention further provides a humanized, or in some aspects human, bispecific antibody, such as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises the VH CDR3 sequence as shown in MF8232 in Figure 2, and wherein the variable domain comprising the cMET binding site comprises the VH CDR3 region as shown in MF8230 in Figure 3. The VH variable region comprising the EGFR binding site in some aspects comprises the sequence of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown in MF8232 in Figure 2. The VH variable region comprising the cMET binding site in some aspects comprises the sequence of the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown in MF8230 in Figure 3. CDR grafting can also be used to make a VH chain having the CDR regions of the VH shown in Figure 2 or Figure 3, but with a different framework. The different framework can be the framework of another human VH, or the framework of a different mammalian VH.

Thus, the present invention further provides a humanized, or in some aspects human, bispecific antibody, such as included in the treatment of the present invention, comprising a first antigen binding site that binds EGFR and a second antigen binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises the VH CDR3 sequence as shown in MF8232 in Figure 2, and wherein the variable domain comprising the cMET binding site comprises the VH CDR3 region as shown in MF4356 in Figure 3. The VH variable region comprising the EGFR binding site is in some aspects a sequence comprising the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown in MF8232 in Figure 2. The VH variable region comprising the cMET binding site is in some aspects a sequence comprising the CDR1 region, CDR2 region, and CDR3 region of the VH chain as shown in MF4356 in Figure 3. CDR grafting can also be used to make a VH chain having the CDR regions of the VH shown in Figure 2 or Figure 3 but with a different framework. The different framework can be the framework of another human VH, or a framework of a different mammal.

Methods for generating sequence variants are known in the art. Sequence variants can be generated by random methods, or by targeted methods, such as those intended to introduce variations that may enhance or reduce binding affinity. Conventional methods for affinity maturation of antibody binding domains are well known in the art, see, for example, Tabasinezhad M. et al., Immunol Lett. 2019; 212: 106-113. Variants intended to introduce reduced risk of deterioration may also be used in order to manufacture binding domains or portions comprising such binding domains on a large scale. Variations that may not appear to cause loss of binding specificity and/or affect binding affinity may be introduced. Whether amino acid residues in CDR and/or framework regions can be substituted, for example, with conservative amino acid residues, with no or substantially no loss of binding specificity and/or affinity can be determined by methods well known in the art. Experimental examples include, but are not limited to, alanine scanning (Cunningham BC, Wells JA. Science. 1989; 244(4908): 1081-5), and deep mutation scanning (Araya CL, Fowler DM. Trends Biotechnol. 2011; 29(9): 435-42). Computational methods that can predict the effects of amino acid variations have also been developed, such as 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.

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

The present invention further provides a humanized, or in some aspects human, bispecific antibody, as included in the treatment of the present invention, comprising a first variable domain that binds EGFR and a second variable domain that binds cMET, wherein the first variable domain comprises a heavy chain variable region having an amino acid sequence of up to 10, in some aspects having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in some aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, from the MF3370 amino acid sequence as shown in Figure 2, and wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of 0-10, in some aspects having 0-5 amino acid insertions, deletions, substitutions or a combination thereof, from the MF4356 amino acid sequence as shown in Figure 3 (SEQ ID NO: 23). The present invention further provides a humanized, or in some aspects human, bispecific antibody comprising a first variable domain that binds to EGFR and a second variable domain that binds to cMET, wherein the first variable domain comprises a heavy chain variable region having an amino acid sequence of up to 10, in some aspects having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in some aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, from the MF8233 amino acid sequence as shown in Figure 2, and wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of 0-10, in some aspects having 0-5 amino acid insertions, deletions, substitutions or a combination thereof, from the MF8230 amino acid sequence as shown in Figure 3 (SEQ ID NO: 13).

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

The present invention further provides a humanized bispecific antibody, or in certain aspects, a human bispecific antibody as included in the treatment of the present invention, comprising a first 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 an amino acid sequence of up to 10, in certain aspects having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and in certain aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, from the MF8233 amino acid sequence as shown in Figure 2, and wherein the second variable domain comprises a heavy chain variable region comprising an amino acid sequence of 0-10, in certain aspects having 0-5 amino acid insertions, deletions, substitutions or combinations thereof, from the MF4356 amino acid sequence as shown in Figure 3 (SEQ ID NO: 23).

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

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

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

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

In one aspect, a method for producing a bispecific antibody of the present invention from a single cell is provided, wherein the bispecific antibody comprises 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, the heavy chain comprising an antigen binding site that binds to EGFR and comprises a first CH3 domain, and b) a second nucleic acid molecule encoding a heavy chain, the heavy chain comprising an antigen binding site that binds to ErbB-3 and comprises a second CH3 domain, wherein the nucleic acid molecule is provided in a method of preferentially pairing the first and second CH3 domains, the method further comprising culturing the cell, allowing expression of proteins encoded by the two nucleic acid molecules, and harvesting the bispecific IgG antibody from the culture. In one aspect, the cell also has a third nucleic acid molecule encoding a common light chain. The first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule can be part of the same nucleic acid molecule, a vector, or a gene delivery vehicle, and can be integrated at the same site of the host cell genome. Alternatively, the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule are provided to the cell separately. The preferred common light chain is based on O12, and in some aspects, it is a rearranged germline human κ light chain IgVκ1 39*01/IGJκ1*01, as described above. The method of preferentially pairing the first CH3 domain and the second CH3 domain is in some aspects a corresponding mutation in the CH3 domain of the heavy chain coding region. Preferred mutations for preferentially manufacturing bispecific antibodies are amino acid substitutions L351K and T366K (EU-numbering) in the first CH3 domain and amino acid substitutions L351D and L368E in the second CH3 domain, and vice versa. Therefore, a method for manufacturing a bispecific antibody of the present invention is further provided, wherein the first CH3 domain comprises amino acid substitutions L351K and T366K (EU-numbering), and wherein the second CH3 domain comprises amino acid substitutions L351D and L368E, the method further comprising culturing the cell and allowing expression of a protein encoded by the nucleic acid molecule, and the step of harvesting the bispecific antibody from the culture. Also provided is a method for producing a bispecific antibody of the present invention, wherein the first CH3 domain comprises amino acid substitutions L351D and L368E (EU-numbering), and wherein the second CH3 domain comprises amino acid substitutions L351K and T366K, the method further comprising culturing the cells and allowing expression of the protein encoded by the nucleic acid molecule, and harvesting the bispecific antibody from the culture. Antibodies that can be manufactured by these methods are also part of the present invention. The CH3 hetero-dimerization domain is an IgG1 hetero-dimerization domain in some aspects. The heavy chain constant region comprising the CH3 hetero-dimerization domain is an IgG1 constant region in some aspects.

One aspect of the present invention includes nucleic acid molecules encoding antibody heavy chain variable region.Described nucleic acid molecule (typically in vitro separated or recombinant nucleic acid molecule) is to encode heavy chain variable region as shown in Figure 2 or Figure 3 in some aspects, or heavy chain variable region as shown in Figure 2 or Figure 3, and this heavy chain variable region has 1,2,3,4 or 5 amino acid insertions, deletions, substitutions or its combination.In some aspects, described nucleic acid molecule comprises the nucleic acid sequence by codon optimization of the amino acid sequence as shown in Figure 2 or Figure 3 encoding.Codon optimization is optimized for the species and/or cell type of the cell making antibody.For example, for CHO manufacturing, the nucleic acid sequence of described molecule is to carry out codon optimization for Chinese hamster cell.The present invention further provides the nucleic acid molecule of the heavy chain of coding Figure 2 or Figure 3.

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

The present invention also provides cells comprising antibodies of the present invention. In some aspects, the cells (typically in vitro, isolated or recombinant cells) produce the antibodies. The cells may also be a storage cell that can produce the antibodies when taken out of storage and cultured. In some aspects, the cells are hybridoma cells, Chinese hamster ovary (CHO) cells, NSO cells or PER-C6 ^TM cells. In a particular aspect, the cells are CHO cells. Cell cultures comprising cells of the present invention are also provided. Various research institutions and companies have developed cell strains for large-scale production of antibodies, such as for clinical use. Non-limiting examples of such cell strains are CHO cells, NSO cells or PER.C6 ^TM cells. These cells are also used for other purposes, such as making proteins. Cell strains developed for industrial-scale production of proteins and antibodies are further referred to herein as industrial cell strains. Therefore, one aspect includes the use of cell strains developed for large-scale production of antibodies for the manufacture of antibodies of the present invention, including cells for the manufacture of antibodies comprising nucleic acid molecules encoding VH, VL and/or heavy chains as shown in Figures 2 or 3 in some aspects.

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

The cell of the present invention is, for example, a hybridoma cell line, a CHO cell, a 293F cell, a NS0 cell, or another cell type known to be suitable for antibody production for clinical purposes. In one aspect, the cell is a human cell. In some aspects, the cell is transformed by the adenovirus E1 region or its functional equivalent. A preferred example of this cell line is a PER.C6TM cell line or its equivalent. In one aspect, the cell is a CHO cell or a variant thereof. In some aspects, the variant cell expresses the antibody using a glutamine synthetase (GS) vector system.

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

The present invention also provides a pharmaceutical composition comprising a combination of the antibody of the present invention and the EGFR tyrosine kinase inhibitor. The pharmaceutical composition in certain aspects comprises a pharmaceutically acceptable excipient or carrier.

Antibodies may include a label, which in some aspects is a label for in vivo imaging. Such labels are generally not necessary for therapeutic applications. For example, in a diagnostic setting, a label may be helpful. For example, to visualize target cells in vivo. Various labels are suitable, and many are known in the art. In some aspects, the label is a radioactive label for detection. In another aspect, the label is an infrared label. In some aspects, the infrared label is suitable for in vivo imaging. Various infrared labels are available to those skilled in the art. Preferred infrared labels are, for example, IRDye 800, IRDye 680RD, IRDye680LT, IRDye 750, IRDye 700DX, IRDye 800RS IRDye 650, IRDye 700 phosphoramidite, IRDye 800 phosphoramidite (LI-COR USA, 4647 Superior Street, Lincoln, Nebraska).

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

In certain aspects, the treatment comprises administering to the individual an effective amount of the bispecific antibody and the third generation tyrosine kinase inhibitor.

In some aspects, the bispecific antibody that binds EGFR to cMET is provided to an individual at a dose of 1000, 1500 or 2000 mg, specifically using a fixed dose prescription. Fixed dose prescriptions have multiple advantages in terms of surface or body weight administration because they reduce preparation time and reduce potential dose calculation errors. In some aspects, the bispecific antibody is administered once a week (Q1W), once every 2 weeks (Q2W) or once every 3 weeks (Q3W). In some aspects, the bispecific antibody is administered once every two weeks. In the art, this dosing process is referred to as Q2W. In certain embodiments, the fixed dose prescription disclosed herein is suitable for adults and/or individuals weighing at least 35 kg. As understood by those skilled in the art, the dose can be administered over time. As understood by those skilled in the art, the term "fixed dose" or "fixed dose prescription" refers to an individual taking a medication regimen, wherein the individual is arranged to receive the bispecific antibody or third-generation EGFR TKI at a dose substantially the same as a predetermined amount every day, and the amount is independent of the individual's weight. According to certain aspects, a fixed, weekly dose of 1000mg bispecific antibody is provided to an individual. Alternatively, a fixed, biweekly dose of 1000mg bispecific antibody is provided to an individual. Alternatively, a fixed, biweekly dose of 1500mg bispecific antibody is provided to an individual. Alternatively, a fixed, biweekly dose of 2000mg bispecific antibody is provided to an individual. In addition, a daily dose of a third-generation EGFR tyrosine kinase inhibitor is also generally provided to an individual. In certain aspects, a daily dose of 80mg osimertinib, a daily dose of 110mg ametinib, a daily dose of 240mg lazertinib, a daily dose of 70mg D-0316, or a daily dose of 75mg D-0316 is provided to an individual. In certain aspects, the daily dose is a fixed daily dose. In certain aspects, the individual is provided with a daily dose of 70 mg of D-0316 for 21 days, followed by a daily dose of 100 mg or a daily dose of 75 mg of D-0316 for 21 days, followed by a daily dose of 100 mg. The daily dose of 75 mg of D-0316 for 21 days can be divided into 3 oral doses of 25 mg each.

Therefore, in some aspects, the bispecific antibody of the present invention is administered or applied at 1000 mg, specifically a fixed dose of 1000 mg. In some aspects, the bispecific antibody is administered once a week at a dose of 1000 mg. In some aspects, the bispecific antibody is administered once every two weeks at a dose of 1000 mg. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to an individual, including a daily dose of 80 mg of osimertinib.

Therefore, in some aspects, the bispecific antibody of the present invention is administered or applied at 1500 mg, specifically using a fixed dose of 1500 mg. In some aspects, the bispecific antibody is administered once every two weeks at a dose of 1500 mg. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to the individual, including a daily dose of 80 mg of osimertinib.

Therefore, in some aspects, the bispecific antibody of the present invention is administered or applied at 2000 mg, specifically using a fixed dose of 2000 mg. In some aspects, the bispecific antibody is administered once every two weeks at a dose of 2000 mg. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to the individual, including a daily dose of 80 mg of osimertinib.

In certain other aspects, the bispecific antibody of the present invention is administered or applied with 1000 mg, specifically a fixed dose of 1000 mg. In certain aspects, the bispecific antibody is administered once a week with a 1000 mg dosage. In certain aspects, the bispecific antibody is administered once every two weeks with a 1000 mg dosage. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to an individual, including a daily dose of 110 mg of Almonertinib.

Therefore, in some aspects, the bispecific antibody of the present invention is administered or applied at 1500 mg, specifically a fixed dose of 1500 mg. In some aspects, the bispecific antibody is administered once every two weeks at a dosage of 1500 mg. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to an individual, including a daily dose of 110 mg of Almonertinib.

Therefore, in certain other aspects, the bispecific antibody of the present invention is administered or applied at 2000 mg, specifically using a fixed dose of 2000 mg. In certain aspects, the bispecific antibody is administered once every two weeks at a dosage of 2000 mg. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to an individual, including a daily dose of 110 mg of Almonertinib.

In certain other aspects, the bispecific antibody of the present invention is administered or applied at 1000 mg, specifically a fixed dose of 1000 mg. In certain aspects, the bispecific antibody is administered once a week at a dosage of 1000 mg. In certain aspects, the bispecific antibody is administered once every two weeks at a dosage of 1000 mg. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to an individual, including a daily dose of 240 mg of Lazertinib.

Therefore, in some aspects, the bispecific antibody of the present invention is administered or applied at 1500 mg, specifically using a fixed dose of 1500 mg. In some aspects, the bispecific antibody is administered once every two weeks at a dose of 1500 mg. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to an individual, including a daily dose of 240 mg of Lazertinib.

Therefore, in some aspects, the bispecific antibody of the present invention is administered or applied at 2000 mg, specifically using a fixed dose of 2000 mg. In some aspects, the bispecific antibody is administered once every two weeks at a dose of 2000 mg. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to an individual, including a daily dose of 240 mg of Lazertinib.

In certain other aspects, the bispecific antibody of the present invention is administered or applied at 1000 mg, specifically using a fixed dose of 1000 mg. In certain aspects, the bispecific antibody is administered once a week at a dose of 1000 mg. In certain aspects, the bispecific antibody is administered once every two weeks at a dose of 1000 mg. In addition, an approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to an individual, including a daily dose of 70 mg, 75 or 100 mg of Befotertinib. In certain aspects, a daily dose of 70 mg of Befotertinib or a daily dose of 75 mg of Befotertinib is provided. In certain aspects, the daily dose is a fixed daily dose. In certain aspects, a daily dose of 70 mg of Befotertinib is provided to an individual for 21 days, followed by a daily dose of 100 mg, or a daily dose of 75 mg of Befotertinib for 21 days, followed by a daily dose of 100 mg. A daily dose of 75 mg of Befotertinib may be provided as three oral administrations of 25 mg each.

Therefore, in some aspects, the bispecific antibody of the present invention is administered or applied at 1500 mg, specifically using a fixed dose of 1500 mg. In some aspects, the bispecific antibody is administered once every two weeks at a dose of 1500 mg. In addition, the approved dose of a third-generation EGFR tyrosine kinase inhibitor is provided to the individual, including a daily dose of 70 mg, 75 or 100 mg of Befotertinib. In some aspects, a daily dose of 70 mg of Befotertinib or a daily dose of 75 mg of Befotertinib is provided. In some aspects, the daily dose is a fixed daily dose. In some aspects, a daily dose of 70 mg of Befotertinib is provided to the individual for 21 days, followed by a daily dose of 100 mg, or a daily dose of 75 mg of Befotertinib is provided for 21 days, followed by a daily dose of 100 mg. A daily dose of 75 mg of Befotertinib may be provided as three oral administrations of 25 mg each.

Therefore, in some aspects, the bispecific antibody of the present invention is administered or applied at 2000 mg, specifically using a fixed dose of 2000 mg. In some aspects, the bispecific antibody is administered once every two weeks at a dose of 2000 mg. In addition, the approved dose of the third-generation EGFR tyrosine kinase inhibitor is provided to the individual, including a daily dose of 70 mg, 75 or 100 mg of Befotertinib. In some aspects, a daily dose of 70 mg of Befotertinib or a daily dose of 75 mg of Befotertinib is provided. In some aspects, the daily dose is a fixed daily dose. In some aspects, a daily dose of 70 mg of Befotertinib is provided to the individual for 21 days, followed by a daily dose of 100 mg, or a daily dose of 75 mg of Befotertinib is provided for 21 days, followed by a daily dose of 100 mg. A daily dose of 75 mg of Befotertinib may be provided as three oral administrations of 25 mg each.

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

The cancer or tumor may be EGFR, cMET or EGFR/cMET positive cancer. In one aspect, the present invention provides treatment of positive cancer, the cancer is EGFR, cMET or EGFR/cMET positive cancer, which is lung cancer, in some aspects non-small cell lung cancer. The individual is a human individual in some aspects. The individual is an individual who is eligible for antibody therapy with an EGFR-specific antibody (e.g., Cetuximab) in some aspects. In some aspects, the present invention can treat an individual comprising a tumor, in some aspects, the tumor is EGFR/cMET positive cancer, in some aspects the tumor/cancer is an EGFR RTK resistance phenotype, an EGFR monoclonal antibody resistance phenotype, or a combination thereof.

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

The dosage of the antibody administered to the patient is usually within the therapeutic window, which means that a sufficient amount is used to obtain a therapeutic effect while the amount does not exceed the threshold that causes an unacceptable degree of side effects. The lower the amount of antibody required to obtain the desired therapeutic effect, the larger the therapeutic window is generally. Therefore, it is preferred that the antibodies of the present invention exert a sufficient therapeutic effect at a low dose. The dosage may be within the range of the Cetuximab administration prescription. The dosage may also be lower. In certain aspects, the dosage is 1000 mg, 1500 mg, or 2000 mg. Administration may be once a week or once every two weeks.

Osimertinib is administered according to the dosage confirmed by the regulatory authorities. Typically, the dosage is 80 mg once a day.

Almonertinib is administered according to the dosage confirmed by the regulatory authorities. Typically, the dosage is 110 mg once a day.

Lazertinib is administered according to the dosage confirmed by the regulatory authorities. Typically, the dosage is 240 mg once a day.

Typically, the dose of Befotertinib is 75 mg per day, administered orally in 3 divided doses of 25 mg each time, for 21 days, followed by a daily dose of 100 mg if tolerated. Alternatively, the dose is 70 mg Befotertinib for 21 days, followed by a daily dose of 100 mg if tolerated.

Under other similar conditions, the bispecific antibodies of the present invention induce less skin toxicity in some aspects compared to Cetuximab. The bispecific antibodies of the present invention produce less pro-inflammatory chemokines (CXCL14 in some aspects) in some aspects, compared to Cetuximab, under other similar conditions. Compared to Cetuximab, under other similar conditions, the bispecific antibodies of the present invention induce less damage to antimicrobial RNA enzymes (in some aspects, RNase 7) in some aspects.

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

The present invention provides bispecific antibodies as disclosed herein for treating individuals who may suffer from one or more of a variety of different types of tumors. The tumor may be an EGFR-positive tumor, a cMET-positive tumor, or an EGFR and cMET-positive tumor. The tumor may be lung cancer, including non-small cell lung cancer. The tumor may be resistant to treatment with an EGFR tyrosine kinase inhibitor. In certain aspects, the EGFR tyrosine kinase inhibitor is a third-generation EGFR tyrosine kinase inhibitor, in certain aspects osimertinib or its analogs. The treatment comprises treatment with the tyrosine kinase inhibitor. When co-treated with the tyrosine kinase inhibitor, the tumor may become resistant to treatment with the EGFR tyrosine kinase inhibitor. The co-treatment at least partially restores the sensitivity of the tumor to the tyrosine kinase inhibitor. The EGFR tyrosine kinase inhibitor is a third-generation EGFR tyrosine kinase inhibitor in certain aspects. Examples of clinically relevant third generation EGFR tyrosine kinase inhibitors are Osimertinib, Lazertinib, Alflutinib, Rezivertinib, Rociletinib, Olmutinib, Almonertinib, Abivertinib, ASK120067, Befotertinib, Olmutinib, Rociletinib or SH-1028, Nazartinib (EGF816), naquotinib (ASP8273), Mavelertinib (PF-0647775), Olafertinib (CK-101), Keynatinib or ES-072. In some aspects, the tyrosine kinase inhibitor is osimertinib. In some aspects, the tyrosine kinase inhibitor is lazertinib. In some aspects, the tyrosine kinase inhibitor is almonertinib. In some aspects, the tyrosine kinase inhibitor is befotertinib. In this and other aspects, the tumor may be an HGF-associated tumor.

In certain aspects, the third-generation EGFR tyrosine kinase inhibitor is an irreversible inhibitor, such as osimertinib, olmutinib or almonertinib.

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

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

As used herein, the term "treatment" includes prevention. The prevention aspect of the present invention is specifically provided as follows.

The present invention also provides a composition of a third-generation tyrosine kinase inhibitor and a bispecific antibody of the present invention, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), for use in a method for preventing the occurrence of cancer with tyrosine kinase inhibitor resistance in an individual. In certain aspects, the first variable domain comprises a heavy chain variable region having a _CDR1 sequence of SYGIS, a CDR2 sequence of _{WISAYX1X2NTNYAQKLQG} and _a CDR3 comprising the sequence _{X3X4X5X6HWWLX7A} , wherein _X1 = _N or _S ; _X2 =A or G; _X3 =D or G; _X4 =R, S or Y; _X5 =H, L or Y; _X6 =D or W, and _X7 =D or G; and having 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than X1 to X7, and wherein the second variable domain comprises _a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1-23 resulting in an amino acid sequence of 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The prevention aspect of the present invention also relates to providing a method for preventing the occurrence of a cancer that is resistant to a tyrosine kinase inhibitor in an individual, comprising administering to the individual an effective amount of a third-generation tyrosine kinase inhibitor in combination with a bispecific antibody of the present invention, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET).

In particular, the method of preventing the occurrence of cancer with tyrosine kinase inhibitor resistance in an individual of the present invention is related to mutations that occur after treatment with first-generation, second-generation and/or third-generation EGFR tyrosine kinase inhibitors. In a certain aspect, the cancer is NSCLC. Treatment of such cancers with first-generation tyrosine inhibitors, second-generation tyrosine inhibitors or third-generation tyrosine inhibitors has been reported to result in several mutations, such as acquired resistance mutations T790M or exon 20 insertion mutations.

In certain aspects, the preventing a cancer with tyrosine kinase inhibitor resistance from occurring in an individual comprises preventing the development or occurrence of a cancer with an EGFR acquired resistance mutation, such as T790M or exon 20 insertion mutation.

In certain aspects, the prevention comprises treating a cancer or patient that comprises an activating EGFR mutation, such as an exon 19 deletion, an exon 21 L858R point substitution, or an EGFR amplification.

In some aspects, benefit from the individuality of preventing the occurrence of cancer with tyrosine kinase inhibitor resistance, diagnosed as the presence of EGFR activating mutations. In some aspects, this individual is eligible for treatment according to the clinically relevant criteria of the first-line care standard, including platinum chemotherapy or one of the tyrosine kinase inhibitors. Such standards of NSCLC or other cancer types are known to practitioners with professional qualifications. In some aspects, such standards include solid tumors confirmed by histology or cytology, with evidence of metastatic or locally advanced unresectable disease, which are incurable, measurable diseases, such as RECIST version 1.1, defined in radiological methods (c.f.Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247), the Eastern Coast Cancer Clinical Research Collaborative Organization (ECOG) performance status is 0 or 1, including or excluding life expectancy ≥ 12 weeks, according to researcher judgment.

Here, the ECOG performance status scale indicating grade and performance status is as follows:

Grade 0: Fully active, able to perform all pre-disease expressions without limitation. Grade 1: Limited physical activity, but ambulatory and able to perform light or sedentary work, such as simple housework, office work. Grade 2: Ambulatory, able to take care of oneself, but unable to perform any work activities; up to and approximately more than 50% of waking time. Grade 3: Can only perform limited self-care; confined to bed or chair for more than 50% of waking time. Grade 4: Total disability; unable to perform any self-care; completely confined to bed or chair. Grade 5: Death.

In some cases, the antibody for said prevention comprises a first variable domain comprising a _heavy chain variable region having a CDR1 sequence of SYGIS, a CDR2 sequence of _{WISAYX1X2NTNYAQKLQG} and _{a CDR3 comprising the sequence X3X4X5X6HWWLX7A ,} wherein _X1 = _{N or} S; _X2 =A or G; _X3 =D or G; _X4 =R, S or Y; _X5 =H, L or Y; _X6 =D or W, and _X7 =D or G; and having 0 _to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than _X1 to _X7 , and wherein said second variable domain comprises a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1-23 resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The therapeutic methods or antibodies for use in treatment as described herein in certain aspects further comprise the step of determining whether the tumor is an HGF-associated tumor.

The antibodies of the present invention can inhibit the growth of HGF-related tumors.

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

When an affinity is referred to herein as being higher than another affinity, its _Kd = lower than the other _Kd . For the avoidance of doubt, a _Kd of ^10-9 M is lower than a _Kd of ^10-8 M. An antibody with _{a Kd} of 10e-9 M has a higher affinity for the target than one with a _Kd of ^10-8 M.

In some aspects, at the beginning of treatment, at least one, more than one or all of the following inclusion factors IF1-IF8 are applicable to treating individuals. In some aspects, the individual comprises or meets all inclusion factors IF1-IF8.

IF1. Aged 18 or above when signing the informed consent form.

IF2. Patients with histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced unresectable disease that is incurable.

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

IF3.2. For individuals who have previously received first or higher-order anticancer therapy: Deterioration on or intolerance to treatment known to provide clinical benefit. Patients with NSCLC EGFR sensitizing mutations (e.g., Del19, L858R) who have not received anticancer therapy (i.e., first-line treatment), or with osimertinib-resistant NSCLC who have not received chemotherapy.

IF4. Archived or fresh tumor tissue samples embedded in FFPE are available after recent treatment progression.

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

IF6. The status shown by the Eastern Cooperative Oncology Group (ECOG) is 0 or 1.

IF7. Has a life expectancy of ≥12 weeks.

IF8. Adequate organ function as determined 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 ≥100×10 ⁹ /L.

IF8.4 corrects total serum calcium within the normal range.

IF8.5 Serum magnesium is within normal range (or corrected with supplementation).

IF8.6 Serum potassium was within normal range.

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

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

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

IF8.10 serum albumin>3.3g/dL.

In certain aspects, all organ function measures based on IF8 have an upper limit observed in healthy individuals.

In some aspects, the treatment individual comprises one or more factors selected from IF1-IF8. In some cases, the treatment individual comprises factors IF2, IF3 (such as IF3.1, IF3.2), IF5 and IF8. In some aspects, the treatment individual comprises all factors of IF1-IF8.

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

EF1. There are central nervous system metastases, which:

EF1.1: untreated and symptomatic, may include individuals with asymptomatic disease if considered disease-stable;

EF1.2 requires radiation therapy or surgery;

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

EF2. With known leptomeningeal involvement.

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

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

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

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

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

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

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

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

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

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

EF8.5 has clinically significant pericardial effusion.

EF8.6 myocarditis.

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

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

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

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

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

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

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

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

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

Intrauterine device (IUD)

Intrauterine hormone-releasing system (IUS)

Bilateral fallopian tube obstruction

Partner vasectomy

Abstinence

EF16. Pregnant or breastfeeding.

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

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

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

For clarity and simplicity of description, various features are described herein as being part of the same or separate embodiments, however, it is to be understood that the scope of the present invention may include embodiments having a combination of all or some of the described features.

Terms

1. A composition of a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody, the bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain _that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence _of SYGIS, a _CDR2 sequence of _{WISAYX1X2NTNYAQKLQG} , _and a CDR3 comprising the _{sequence X3X4X5X6HWWLX7A ,} wherein _X1 =N or S; _X2 =A or G; X3=D or G; _X4 =R, S or Y; _X5 =H, L or Y; _X6 =D or W, and _X7 =D or G; and _X1 to X7 _are selected from the group consisting of: _The present invention relates to a method for treating cancer in an individual wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1-23, resulting in 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof at positions other than 7, and wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1-23, resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

2. A method for treating an individual suffering from cancer, the treatment comprising administering to the individual an effective amount of a third generation EGFR tyrosine kinase inhibitor in combination with a bispecific antibody, the bispecific antibody comprising a first variable domain that binds to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that binds to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a _heavy chain variable region having a CDR1 sequence of SYGIS, a CDR2 sequence of _{WISAYX1X2NTNYAQKLQG} , and a CDR3 comprising the sequence _{X3X4X5X6HWWLX7A} , wherein _X1 =N or _S ; _X2 =A or G; X3=D or G; _X4 =R, S or Y; _X5 =H, L or Y; _X6 =D or W, and _X7 =D or G; and between _X1 and _X7 , _{the first variable} domain comprises a heavy chain variable region having a _CDR1 sequence of SYGIS, a CDR2 sequence of WISAYX1X2NTNYAQKLQG _, and a CDR3 comprising the sequence X3X4X5X6HWWLX7A. The positions other than ₇ have 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof, and wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1-23 sequences resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

3. A use of a composition of a bispecific antibody and a third generation EGFR tyrosine kinase inhibitor for the preparation of a medicament for treating cancer in an individual, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a _heavy chain variable region having a CDR1 sequence of SYGIS, a _CDR2 sequence of _{WISAYX1X2NTNYAQKLQG} and a CDR3 comprising the sequence _{X3X4X5X6HWWLX7A, wherein X1=N or S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G; and between X1 and X7 , the first variable domain comprises a heavy chain variable} region having _{a CDR1 sequence} of SYGIS, a CDR2 sequence of WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A. The positions other than ₇ have 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof, and wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1-23 sequences resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

4. The use or method according to any one of the preceding items, wherein the cancer is EGFR-positive cancer, cMET-positive cancer, or EGFR and cMET-positive cancer.

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

6. The use or method according to any one of the preceding items, wherein the cancer is resistant to treatment with a tyrosine kinase inhibitor.

7. The use or method according to any one of the preceding items, wherein the cancer is resistant to an EGFR tyrosine kinase inhibitor and/or a cMET tyrosine kinase inhibitor.

8. The use or method according to item 7, wherein the EGFR tyrosine kinase inhibitor resistance comprises resistance to first-generation tyrosine kinase inhibitors, second-generation tyrosine kinase inhibitors and/or third-generation tyrosine kinase inhibitors, preferably resistance to third-generation EGFR tyrosine kinase inhibitors.

9. The use or method according to item 7, wherein the cMET tyrosine kinase inhibitor resistance comprises resistance to Capmatinib, Tepotinib, Crizotenib, Cabozantinib, Savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833, preferably Capmatinib or Tepotinib.

10. The use or method according to any of the preceding clauses, wherein the subject has been previously treated with a tyrosine kinase inhibitor, preferably an EGFR tyrosine kinase inhibitor and/or a cMET tyrosine kinase inhibitor.

11. The use or method according to any one of the preceding items, wherein the subject has been previously treated with a first generation EGFR tyrosine kinase inhibitor, a second generation EGFR tyrosine kinase inhibitor or a third generation EGFR tyrosine kinase inhibitor.

12. The use or method according to any one of the preceding clauses, wherein the subject has been previously treated with a cMET tyrosine kinase inhibitor.

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

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

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

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

17. The use or method according to any one of the preceding items, wherein the cancer comprises an exon 20 (762-823) mutation selected from a proximal loop insertion (site 767-772), a distal loop insertion (site 773-775), preferably V769_D770insASV, D770_N771insSVD, H773_V774insNPH, H773_V774insH, D770_N771insG, D770delinsGY, N771_P772insN, V774_C775insHV, D770_N771ins sGL、H773_V774insPH、A763_Y764insFQEA、D770_N771delinsEGN、D770_N771insGD、D770_N771insH、D770_N771insP、H773_V774insAH、H773_V774insGNPH、H773delinsSNPY、N771_P7 72insH、N771_P772insVDN、N771delinsGY、N771delinsKH、N771delinsRD、P 772_H773delinsHNPY, P772_H773insGT, P772_H773insPNP, P772_H773insT, V769_D770insA, V769_D770insGG, V769_D770insGSV, V769_D770insGVV and V769_D770insMASV; or mutations T790M, L792X (such as L792H, C796X (such as G796R, G796S, G796D), C797X (such as C797S, C797G), L798 I, or in-frame exon 20 insertion, such as M766_A767insASV or H773-V774insNPH, Ins761 (EAFQ), Ins770 (ASV), Ins771 (G), Ins774 (NPH), M766_A7671nsA, S768_V769InsSVA, P772_H773InsNS, D761_E762InsX1-7, A763_Y764InsX1-7, Y764_Y765InsX1-7, M766_A767InsX1-7, A767_V768 InsX1-7, S768_V769 InsX1-7>V769_D770InsX1-7>D770_N771 InsX1-7>N771_P772 InsX1-7>P772_H773 InsX1-7, H773_V774 InsX1-7, or V774_C775 InsX1-7.

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

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

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

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

22. The use or method according to any of the preceding items, wherein the third generation EGFR tyrosine kinase comprises or is osimertinib, lazertinib, afatinib, rezivertinib, rociletinib, olmutinib, almonertinib, abivertinib, ASK120067, befotertinib, SH-1028, nazartinib (EGF816), naquotinib (ASP8273), maveritinib (PF-0647775), olafertinib (CK-101), keynatinib or ES-072, preferably osimertinib.

23. The use or method according to any of the preceding items, 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.

24. The use or method according to any of the preceding items, wherein the treatment comprises administering a composition of the bispecific antibody and the tyrosine kinase inhibitor to an individual in need thereof, and wherein the bispecific antibody and the third-generation tyrosine kinase inhibitor are administered simultaneously, sequentially or separately.

25. The use or method according to any one of items 1 to 5 above, wherein the individual has not received anti-cancer treatment before, such as an individual who has not received tyrosine kinase inhibitor treatment or anti-EGFR treatment.

26. The use or method according to any one of items 1 to 5, wherein the administration of the bispecific antibody and the TKI inhibitor is as first-line treatment.

27. The use or method of any one of items 1 to 5, wherein the individual or cancer comprises an EGFR and/or cMET activating mutation, such as an exon 19 deletion mutation or an exon 21 mutation (eg, L858R).

28. The use or method according to any one of the preceding clauses, wherein the subject is a human subject.

29. The use or method according to any one of the preceding items, wherein the cancer is lung cancer, in particular non-small cell lung cancer, preferably metastatic or advanced non-small cell lung cancer.

30. The use or method according to any of the preceding items, wherein the cancer is NSCLC comprising activating EGFR mutations, EGFR tyrosine kinase inhibitor sensitizing mutations (e.g., exon 19 deletions and L858X), acquired EGFR tyrosine kinase inhibitor resistance mutations (e.g., T790X, C797X, L792X, L798X), and confirmed EGFR tyrosine kinase inhibitor resistance mutations, EGFR exon 20 insertion mutations, activating c-MET mutations, preferably exon 14 skipping mutations, or cMET amplification, preferably comprising MET/CEP7＞5 or cfDNA≥2 copies or any combination thereof.

31. The use or method according to any one of the preceding items, wherein the cancer is an advanced or metastatic cancer.

32. A pharmaceutical composition comprising a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS, a CDR2 sequence of _{WISAYX1X2NTNYAQKLQG} , and a CDR3 comprising the sequence _{X3X4X5X6HWWLX7A} , wherein _X1 =N or S; _X2 =A or G; _X3 =D or G; _X4 =R, S or Y; _X5 =H, L or Y; _X6 =D or W, and _X7 =D or G; and _{between X1} and _X7 , _X7 =H, L or Y is selected from the group consisting of: The positions other than ₇ have 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof, and wherein the second variable domain comprises a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 1-23 sequences resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

33. The use, method or pharmaceutical composition according to any one of the preceding items, wherein the antibody is a human antibody.

34. The use, method or pharmaceutical composition according to any one of the preceding clauses, wherein the antibody is ADCC enhancing.

35. The use, method or pharmaceutical composition according to any one of the preceding items, wherein the antibody is an IgG1 format antibody with a stoichiometric ratio of 1:1 between anti-EGFR and anti-cMET.

36. The use, method or pharmaceutical composition according to any of the preceding items, wherein the antibody has one variable domain that can bind to EGFR and one variable domain that can bind to cMET.

37. The use, method or pharmaceutical composition according to any one of the preceding items, wherein the variable domain that can bind to human EGFR can also bind to cynomolgus monkey and mouse EGFR.

38. The use, method or pharmaceutical composition according to any one of the preceding items, wherein the variable domain that can bind to human EGFR is domain III that binds to human EGFR.

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

40. The use, method or pharmaceutical composition according to any one of the preceding items, wherein the variable domain that can bind to cMET blocks the binding of HGF to cMET.

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

42. The use, method or pharmaceutical composition according to any one of the preceding items, wherein

X ₁ =N _; X ₂ = _{G ;} X ₃ =D; X ₄ =S;

X ₁ =N _; X ₂ = _{A ;} X ₃ =D; X ₄ =S;

X ₁ = _S ; X ₂ = _{G ;} X ₃ =D; X ₄ =S;

_X1 = N; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D;

X ₁ =N; X ₂ = _{A ;} X ₃ = _D ; X ₄ =R;

_X1 = S; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D;

X ₁ =N _; X ₂ = _{G; X 3 =G; X 4 = Y ;}

_X1 = N; _X2 = A; _X3 = G; _X4 = Y; _X5 = L; _X6 = D and _X7 = G; or

X ₁ =S; _{X 2} = _{G ;} X ₃ =G; X ₄ =Y;

43. The use, method or pharmaceutical composition according to any of the preceding clauses, wherein _X1 = N; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D; or _X1 = N; _X2 = A; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D; or _X1 = S; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D.

44. The use, method or pharmaceutical composition according to any of the preceding clauses, wherein _X1 = N; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D; or _X1 = N; _X2 = A; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D.

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

46. The use, method or pharmaceutical composition according to any of the preceding items, wherein the heavy chain variable region of the second variable domain has an amino acid sequence of one of the sequences of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13 or SEQ ID NO:23, resulting in an amino acid sequence with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof.

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

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

49. The use, method or pharmaceutical composition of any of the foregoing items, wherein the first variable domain and the second variable domain comprise a common light chain, preferably the light chain variable domain in Figure 4B.

50. The use, method or pharmaceutical composition according to any one of the preceding items, wherein the first variable domain and the second variable domain comprise a light chain, wherein the light chain has CDR1, CDR2 and CDR3, respectively, and according to the International Immunogenetics Information System (IMGT), the amino acid sequence of the CDR1 is QSISSY, the amino acid sequence of the CDR2 is AAS, and the amino acid sequence of the CDR3 is QQSYSTP.

51. The use, method or pharmaceutical composition according to any one of the preceding items, wherein the antibody inhibits HGF from inducing the growth of HGF growth-responsive cells.

52. The use, method or pharmaceutical composition of any one of the preceding items, wherein the antibody inhibits EGF-induced growth of EGF growth-responsive cells.

53. The use, method or pharmaceutical composition according to any of the preceding items, wherein 1000 mg of the bispecific antibody is administered to the individual, in particular using a fixed dose of 1000 mg.

54. The use, method or pharmaceutical composition of item 53, wherein the bispecific antibody is administered once a week.

55. The use, method or pharmaceutical composition of item 53 or 54, wherein Osimertinib is also administered to the individual, preferably comprising a daily dose of 80 mg.

56. The use, method or pharmaceutical composition of item 53 or 54, wherein Almonertinib is also administered to the individual, preferably comprising a daily dose of 110 mg.

57. The use, method or pharmaceutical composition of item 53 or 54, wherein Lazertinib is also administered to the individual, preferably comprising a daily dose of 240 mg.

58. The use, method or pharmaceutical composition of item 53 or 54, wherein Befotertinib is also administered to the individual, preferably comprising a daily dose of 75 mg.

59. The use, method or pharmaceutical composition of item 53, wherein the bispecific antibody is administered once every two weeks.

60. The use, method or pharmaceutical composition of item 53 or 59, wherein Osimertinib is also administered to the individual, preferably comprising a daily dose of 80 mg.

61. The use, method or pharmaceutical composition of item 53 or 59, wherein Almonertinib is also administered to the individual, preferably comprising a daily dose of 110 mg.

62. The use, method or pharmaceutical composition of item 53 or 59, wherein Lazertinib is also administered to the individual, preferably comprising a daily dose of 240 mg.

63. The use, method or pharmaceutical composition of item 53 or 59, wherein Befotertinib is also administered to the individual, preferably comprising a daily dose of 75 mg.

64. The use, method or pharmaceutical composition of any one of items 1 to 52, wherein 1500 mg of the bispecific antibody is administered to the individual, specifically using a fixed dose of 1500 mg.

65. The use, method or pharmaceutical composition of item 64, wherein the bispecific antibody is administered once every two weeks.

66. The use, method or pharmaceutical composition of item 64 or 65, wherein Osimertinib is also administered to the individual, preferably comprising a daily dose of 80 mg.

67. The use, method or pharmaceutical composition of item 64 or 65, wherein Almonertinib is also administered to the individual, preferably comprising a daily dose of 110 mg.

68. The use, method or pharmaceutical composition of item 64 or 65, wherein Lazertinib is also administered to the individual, preferably comprising a daily dose of 240 mg.

69. The use, method or pharmaceutical composition of item 64 or 65, wherein Befotertinib is also administered to the individual, preferably comprising a daily dose of 75 mg.

70. The use, method or pharmaceutical composition of any one of items 1 to 51, wherein 2000 mg of the bispecific antibody is administered to the individual, specifically using a fixed dose of 2000 mg.

71. The use, method or pharmaceutical composition of item 70, wherein the bispecific antibody is administered once every two weeks.

72. The use, method or pharmaceutical composition of item 70 or 71, wherein Osimertinib is also administered to the individual, preferably comprising a daily dose of 80 mg.

73. The use, method or pharmaceutical composition of item 70 or 71, wherein Almonertinib is also administered to the individual, preferably comprising a daily dose of 110 mg.

74. The use, method or pharmaceutical composition of item 70 or 71, wherein Lazertinib is also administered to the individual, preferably comprising a daily dose of 240 mg.

75. The use, method or pharmaceutical composition of item 70 or 71, wherein Befotertinib is also administered to the individual, preferably comprising a daily dose of 75 mg.

Example

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

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

Each bispecific antibody comprises two VHs designated by MF numbers, each capable of binding EGFR to cMET, and an Fc tail sequence having a KK/DE CH3 heterodimerization domain, as shown in Figures 5E and 5F, respectively, a CH2 domain according to Figure 5D, a hinge domain according to Figure 5B, a CH1 domain according to Figure 5A, and a common light chain as shown in Figures 4A-E. For example, a bispecific antibody represented as MF8233×MF8230 has the above general sequence, a variable region of a VH whose sequence is MF8233, and a variable region of a VH whose sequence is MF8230, and is preferably used in the attached examples.

Example 1: Materials and Methods

Cell lines:

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

cDNA constructs:

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

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

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

MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQ CSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVT DHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEP RDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNT LVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGM NYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWEL MTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPS RDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA

in:

-MRPSGTAGAALLALLAALCPASR: signal peptide.

-ALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYV QRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSSGRCRGKSPSDCCHN QCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCT SISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLHPNCTYGCTGPGLEGCPTNGPKIPS: ECD of human EGFR.

-IATGMVGALLLLLVVALGIGLFM: predicted TM area.

-RRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPI KWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDV YMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPA HWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA: intracellular tail sequence.

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

MRPSGTAGAALLALLAALCPASRALEEKK_GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKT KIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

in:

-MRPSGTAGAALLALLAALCPASR: signal peptide.

-ALEEKK_GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVC HALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS: ECD of EGFRvarIII

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

IATGMLGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQE RELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDE AYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLA ARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYD GIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPT DSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYS SDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCV NSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA

in:

-MGPSGTAGAALLALLAALCPASR: signal peptide.

-LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQ RNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHN QCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCT SISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLHPNCTYGCTGPGLEGCPTNGPKIPS: ECD of cyEGFR

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

MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPL HSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPD SAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYK VFPNSAPLEGGTRLTICGWDFGFRRNNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSN SILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISTWWKEPLNIVSFLFCFASGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLK LNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGVVSISTALLLLLGFFLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSG DSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPV KWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS

in:

-MKAPAVLAPGILVLLFTLVQRSNG: signal peptide

-ECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYV LNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTD QSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLH SYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVI TG KKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEF AVKLK IDLANRETSIFSYREDPIVYEIHPTIKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFT: Human cMET ECD

-GLIAGVVSISTALLLLLGFFLWL: transmembrane region

-KKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVD YRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYASKKFVHRDLAARNCML DEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS: extracellular domain

Reference Antibodies

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

Reference antibody Cetuximab (Erbitux) was used as the reference antibody for the EGFR Fab panel.

The 2994Fab protein was produced by cleaving the purified PG2994 IgG with papain. Therefore, PG2994 was allowed to stand with papain (Pierce #44985) coupled to microbeads and allowed to rotate and cleave for 5.5 hours at 37°C. The Fab fragment was purified from the cleavage mixture by filtration through MabSelectSure LX. The fraction containing the Fab protein was concentrated to 3 ml using vivaspin20 10kDa and further purified by gel filtration using a superdex7516/600 column (in PBS solution).

Example 2

Bivalent monoclonal antibody generation and antibody recognition

Based on the VH gene sequence and some of its sequence variants, the VH genes of the unique antibodies were cloned into the backbone IgG1 vector. Suspension-adapted 293F Freestyle cells were cultured in a T125 flask on a shaker until the density was 3.0×10 ⁶ cells/ml. The cells were seeded into each well of a 24-deep well plate at a density of 0.3-0.5×10 ⁶ viable cells/ml. The cells were transiently transfected with a separate sterile DNA:PE1 mixture and further cultured. Seven days after transfection, the supernatant was collected and filtered through 0.22 μM (Sartorius) and purified on protein A microbeads using a batch purification method, after which the buffer was exchanged for PBS.

Cross-blocking detection of cMET antibodies

Competition of cMET-specific phages with cMET reference antibodies was tested in ELISA. Therefore, 2.5 μg/ml of cMET-Fc fusion protein was coated on MAXISORPTM ELISA plates at 4°C overnight. The wells on the ELISA plates were blocked with PBS (pH 7.2) containing 2% ELK, shaking (700 rpm) for 1 hour at room temperature. The next reference or negative control IgG was added at a concentration of 5 μg/ml and allowed to bind at 700 rpm for 15 minutes at room temperature. Afterwards, 5 μl of PEG-precipitated phages were added and allowed to bind at 700 rpm for 1 hour at room temperature. Bound phages were detected with anti-M13 antibodies labeled with HRP at room temperature, 700 rpm, for 1 hour. As a control, the process was performed simultaneously with antibodies specific for the coated antigen and negative control phages. The bound secondary antibody was visualized by TMB/H ₂ O ₂ staining, and the staining was quantified by OD _450nm measurement. Table 2 shows that MF4040 and MF4356 exhibited competition with the 5D5 reference antibody. MF4297 competed to a lesser extent with 13.3.2 and C8H241. The positive control phages all showed complete competition with the corresponding IgG, while the control group without antibody did not affect the competition assay.

Generation of bispecific antibodies

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

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

Example 3

Screening of c-MET×EGFR bispecific antibodies in EGF/HGF and HGF and EGF proliferation assays

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

An equimolar 1:1 Cetuximab/5D5 Fab was used as a positive control for the EGF, HGF, and EGF/HGF assays.

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

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

In the N87 EGF assay, 11 different cMET×EGFR bispecific antibodies were identified with comparable potency to the monospecific Cetuximab: PB7679, PB8244, PB8292, PB8340, PB8364, PB8388, PB8511, PB8535, PB8583, PB8607, and PB8640. They all contain the EGFR Fab arm MF3755. In the HGF N87 assay, 9 bispecific antibodies were identified with higher potency than the monospecific 5D5 Fab reference antibody: PB8218, PB8388, PB8511, PB8532, PB8535, PB8545, PB8583, PB8639, and PB8640. They contain six different cMET Fab arms: MF4040, MF4297, MF4301, MF4356, MF4491 and MF4506.

ADCC activity

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

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

Figure 15 shows an example of one of the ADCC assays. The cMETxEGFR bispecific antibody did not show significant ADCC activity in either cell line. The positive control reference Cetuximab antibody showed dose-dependent ADCC activity against both cell lines.

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

Example 4

Figure 2 shows the sequences of another variable region of the heavy chain of the EGFR binding variable domain as disclosed herein. Figure 3 shows the sequences of another variable region of the heavy chain of the cMET binding variable domain as disclosed herein. The heavy chain variable region was used to create many different cMET×EGFR bispecific antibodies. The light chain in these antibodies had the sequence shown in Figure 4B. The bispecific antibodies were made as described in Example 1. The antibodies were generated as ADCC enhanced versions. The ADCC enhanced version was made by co-transfecting the antibody construct with DNA encoding a reductase that removes fucose residues from the Fc region of IgG1. See Table 6 for a list of the bispecific antibodies used and their PB encodings.

Example 5

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

Example 6

Efficacy of bispecific antibody PB19478 and osimertinib in NSCLC models with EGFR exon 20 insertions

The purpose of this trial is to evaluate the anti-tumor efficacy of antibody PB19478 alone and in combination with osimertinib in a patient-derived xenograft (PDX) non-small cell lung cancer (NSCLC) model with EGFR exon 20 insertions. EGFR exon 0 insertions ("EGFRex20ins") represent a class of coding mutants with amino acid insertion sites concentrated between positions 762 and 774, which result in constitutive activation of EGFR. EGFR exon 20 insertions confer resistance to proven EGFR TKIs in human individuals with cancers carrying such mutations and are associated with poor prognosis.

Material:

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

PDX model characteristics:

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

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

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

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

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

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

Western blotting showed that the LXFE2478 model expressed protein levels of EGFR, HGF, and MET (Table 7).

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

Treatment duration and methods:

The anti-tumor efficacy of the combination of antibody and Osimertinib was evaluated in the LXFE2478 NSCLC PDX tumor model. Figure 6 shows a schematic diagram of the treatment schedule.

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

Mice were treated with antibody, Osimertinib, or a combination of both compounds for 4 weeks, followed by an observation period of up to 100 days without administration. Treatment started on the same day of enrollment (Day 0). Mice were terminated during the observation period if tumor volume was >1000 mm ³ .

The antibody was administered intraperitoneally (IP) at a dose of 5 mg/kg and 25 mg/kg once a week for 5 weeks. Osimertinib was administered daily (QD) at 5 mg/kg, 25 mg/kg per oral dose (PO) for 30 days. Mice in Group 1 were treated with Vehicle 1 (antibody buffer) once a week for 5 weeks or Vehicle 2 (Osimertinib buffer) daily for 30 days. The treatment schedule is shown in Table 8.

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

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

An observation period without administration was included to compare the response period and relapse (tumor regrowth) after the end of treatment. In addition, a low dose of antibody was included to create a window for comparison of tumor growth inhibition.

Since significant weight loss (>10%) was observed with osimertinib 25 mg/kg treatment (Group 2 and Group 6), these two groups were not administered the therapeutic compound between days 1-14. Weight was restored during this non-administration period. On day 28, weight loss (>10%) was observed again in these groups, after which it was decided to stop osimertinib 25 mg/kg administration on day 28. The combination of 25 mg/kg osimertinib and bispecific antibody PB19478 did not enhance any adverse reactions observed during 25 mg/kg osimertinib treatment.

The animal survival curve of this experiment is shown in Figure 7. The significance of the log-rank (Mantel-Cox) test of the combination vs. each corresponding single treatment group is as follows:

Osimertinib 25 mg/kg vs. antibody + osimertinib 25 mg/kg P = 0.0102

Antibody 25 mg/kg vs. Antibody + Osimertinib 25 mg/kg P = 0.0344

Osimertinib 5 mg/kg vs. antibody + osimertinib 5 mg/kg P = 0.0006

Antibody 5 mg/kg vs. Antibody + Osimertinib 5 mg/kg P = 0.0162

The individual tumor volumes on day 28 are shown in Figure 8. Statistical analysis was performed using mixed models and Tukey post-tests (GraphpadPrism). All 25 mg/kg groups induced tumor stasis or regression, so the comparison of the combination group with the corresponding single treatment group was significant only in the 5 mg/kg group. On day 28, all groups were significantly different from the vehicle group.

FIG. 9 shows the effects of bispecific antibody PB19478 and osimertinib monotherapy and combination therapy on tumor volume in NSCLC PDX models over the full observation period with indication for treatment cessation on day 28.

For monotherapy, dose-dependent antitumor efficacy of the bispecific antibody PB19478 and osimertinib was observed. At 5 mg/kg, the model showed reduced sensitivity to osimertinib treatment. In addition, the combination of the antibody and osimertinib enhanced tumor growth inhibition compared to the two corresponding monotherapy groups.

After treatment cessation (day 28), the combination therapy of antibody and osimertinib significantly prolonged progression-free survival (defined as tumor size <750 mm ³ ) compared with antibody or osimertinib alone. The log-rank (Mantel-Cox) test showed that each combination treatment group was compared with the corresponding single treatment group at P < 0.05. At 11 weeks after treatment, only 1 of 9 mice (11%) in the 25 mg/kg antibody group showed xenograft progression, while 5/9 and 6/9 xenografts in the 25 mg/kg antibody and 25 mg/kg osimertinib arms had progression to 750 mm ³ (Figure 9).

In summary, these results show preclinical antitumor activity of the antibody against a NSCLC PDX model carrying an EGFR exon 20 insertion mutation (H773-V774insNPH). Combination of the antibody with osimertinib enhanced tumor growth inhibition in this model and prolonged progression-free survival.

Example 7

Efficacy of the bispecific antibody PB19478 and osimertinib in the setting of EGFR exon 19 deletion

NSCLC Model. The purpose of this trial is to evaluate the antitumor efficacy of antibody PB19478 alone and in combination with osimertinib in a cell line-derived xenograft (CDX) non-small cell lung cancer (NSCLC) model with EGFR exon 19 deletion. This adenocarcinoma cell line, HCC827-ER1, has an activating EGFR mutation in exon 19 (deletion E746-A750) and is resistant to proven EGFR TKIs such as erlotinib.

Material:

PB19748 was produced from Merus as described in Example 5.

CDX Model Features:

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

Experimental process:

Each mouse was inoculated with a mixture of HCC827-ER1 tumor cells and Matrigel in the right anterior flank region for tumor development. Randomization began when the average tumor size reached approximately 125 (75-175) mm ^3. A total of 32 tumor-bearing mice were included in the tumor efficacy trial and randomly divided into 4 groups, as shown in Table 9, with 8 mice in each group. The randomization day is represented as day 0, and dosing began on day 0. Mice were treated with antibodies, osimertinib, or a combination of the two compounds for 3 weeks, followed by a non-administered observation period of up to 74 days. The non-administered observation period was included to compare the response period and recurrence (tumor regrowth) after the end of treatment. Mice with tumor volumes exceeding 1500 mm ³ or body weight loss exceeding 20% relative to the first day of treatment were terminated during the treatment period. The antibody was administered intraperitoneally (ip) twice a week at a dose of 25 mg/kg for 21 days for a total of 7 doses. Osimertinib was administered daily (QD) at 25 mg/kg per oral dose (po) for 21 days for a total of 22 doses. Eight mice in Group 1 were treated with a vehicle control group (placebo buffer). Figure 10 shows a schematic diagram of the treatment schedule. Tumor volume (mm ³ ) was measured twice a week in two dimensions using a caliper. Animals were examined for tumor growth and any effects of treatment on behavior, such as mobility, food and water consumption, weight gain/loss, eye/hair tangling, and any other abnormalities. Body weight was measured twice a week after randomization. No mouse lost more than 15% of its body weight, and administration of the therapeutic agent was not suspended in any treatment group.

Figure 11 shows the effects of monotherapy and combination therapy of bispecific antibody PB19478 and osimertinib on tumor volume in the NSCLC CDX model throughout the observation period, and treatment was stopped on day 21. Monotherapy with antibody or osimertinib showed tumor regression compared with the vehicle control group. However, the combination therapy of antibody and osimertinib showed strong synergy and complete tumor regression compared with any other treatment group. All treatment groups were well tolerated. No mouse in the study showed a weight loss of >10% from the starting weight. No adverse events were observed except for tumor scabs or tumor ulcers in some mice in Groups 1, 2 and 3. No adverse events were reported in Group 4. Only mild weight loss (between 5-10%) was observed in one mouse in Group 4.

The animal survival curve is shown in Figure 12. The log-rank (Mantel-Cox) test significance of the combination and each corresponding single treatment group is as follows:

Antibody 25 mg/kg + Osimertinib 25 mg/kg vs. Antibody 25 mg/kg P = 0.0009

Antibody 25 mg/kg + Osimertinib 25 mg/kg vs. Osimertinib 25 mg/kg P < 0.0001

In summary, these results show preclinical antitumor activity of the investigational antibody in a NSCLC CDX model carrying an exon 19 mutation (E746-A750 deletion). Combination of the antibody with osimertinib enhanced tumor growth inhibition in this model and prolonged progression-free survival.

Example 8

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

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

Patient cohorts will receive the antibody treatment until the MTD is reached or a lower recommended dose is determined.

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

Dose expansion

The planned expansion cohort in Phase 2 will be initiated with the RP2D of 1500 mg in combination with 80 mg of osimertinib orally daily. The safety of the RP2D will be confirmed during the dose expansion of the first 12 patients treated with at least 2 cycles of antibody alone (recruitment will continue in the meantime). The anti-tumor activity of the antibody (alone or in combination with osimertinib) will be evaluated based on ORR, and other efficacy parameters, safety, tolerability, PK, immunogenicity and biomarkers will be evaluated.

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

Extension cohort 1: PB19478 + osimertinib as first-line treatment for NSCLC

Expansion Cohort 2: PB19478 + osimertinib as second-line treatment for NSCLC in the osimertinib-resistant population

Osimertinib will be administered once daily at a dose of 80 mg. Administration may be with or without food. It is best to take the dose in the morning (i.e., upon waking up). On the day of the antibody infusion, the dose must be taken before the infusion. If the dose is missed, it should not be replaced and the patient should wait until the next scheduled dose.

Experimental group

Inclusion criteria

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

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

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

3. Solid tumors confirmed by histology or cytology with evidence of metastatic or locally advanced unresectable disease that is incurable.

1. Dose escalation portion - patients who have failed prior standard first-line therapy. Patients must have deteriorated on or been intolerant of therapy known to provide clinical benefit. There is no limit on 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 proven TKI-resistance mutations (e.g., acquired TKI-resistance mutations, i.e., T790M, C797S, L792, L798I, exon 20 insertions), or any activating c-MET mutation/amplification (e.g., high-level c-MET amplification [MET/CEP7>5 or cfDNA ≥2 copies], or c-MET exon 4 skipping mutations).

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

2. Cohort expansion: Patients with ≥2L disease must have deteriorated on or been intolerant to therapy known to provide clinical benefit. There is no limit on the number of prior treatment regimens.

Patients must have:

Group 1: First-line treatment for NSCLC with EGFR sensitizing mutations (such as Del19, L858R) or advanced disease with EGFR sensitizing mutations who have not received NSCLC treatment.

Cohort 2: Patients with NSCLC osimertinib-resistant/chemotherapy-naive, or NSCLC osimertinib-resistant and worsening after 3 months of osimertinib treatment, with at least stable disease reported.

4. Availability of archived or fresh tumor tissue samples (preferred for dose escalation and mandatory for dose expansion).

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

6. The Eastern Cooperative Oncology Group (ECOG) expression status is 0 or 1.

7. Life expectancy ≥ 12 weeks, based on the researcher's judgment.

8. Have adequate organ function, at the discretion of the researcher

Absolute neutrophil count (ANC) ≥ 1.5 × 10 ⁹ /L

Hemoglobin ≥ 9 g/dL

Platelet count ≥100×10 ⁹ /L

Correct total serum calcium within normal range

Serum potassium within normal range

Serum magnesium within normal range (or corrected with supplementation)

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 3x upper limit of normal (ULN), and total bilirubin ≤ 1.5x ULN (Gilbert syndrome patients are eligible if conjugated bilirubin values are within the normal range); provided that in the case of liver involvement, ALT/AST ≤ 5x ULN and total bilirubin ≤ 2x ULN are permitted.

For patients aged ≥65 years, serum creatinine ≤1.5x ULN or creatinine clearance ≥50 mL/min, calculated according to the Cockroft and Gault formula or the MDRD formula

Serum albumin >3.3 g/dL

Exclusion criteria

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

1. Central nervous system metastasis (not excluded during dose escalation, mandatory during dose expansion):

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

·Need radiation therapy or surgery.

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

2. With known leptomeningeal involvement.

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

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

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

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

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

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

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

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

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

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

9. Patients with a history of interstitial lung disease, including drug-induced interstitial lung disease and radiation pneumonitis, who require long-term treatment with steroids or other immunosuppressants within 1 year.

10. Patients with previous or concurrent malignancies, excluding non-basal cell skin cancer or carcinoma in situ of the cervix, unless the tumor is treated for curative or palliative purposes and the investigator believes, with the sponsor's consent, that the previous or concurrent malignancy does not affect the safety and efficacy evaluation of the investigational drug.

11. Current serious illness or medical condition, including but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic or psychiatric disease.

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

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

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

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

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

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

Intrauterine device (IUD)

Intrauterine hormone-releasing system (IUS)

Bilateral fallopian tube obstruction

Partner vasectomy

Abstinence

16. Pregnant or breastfeeding women are excluded from this trial.

Research treatment and prescription

The antibody is administered by IV infusion at a dose level of 1500 mg (fixed dose) at the RP2D level. The dose, dose increment, and frequency of administration for each patient (including the expansion group) may vary based on patient safety, PK, and pharmacodynamic data and based on the sponsor's recommendations. The sponsor may recommend an alternative weekly dosing schedule for cycle 1.

During treatment

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

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

Example 9

A 54-year-old female patient with non-small cell lung cancer carrying EGFR 19 deletion and EGFR mutation received a combination of osimertinib and 1500mg q2w of bispecific antibody PB19478 according to the clinical trial process described in Example 8. The patient had previously received treatment with osimertinib and an experimental anti-tumor drug. After 6 cycles of the antibody and 6 cycles of osimertinib, the patient showed clinically relevant effects in the form of confirmed partial responses (PR). No other adverse reactions were observed except for grade 1 or grade 2 reactions.

Example 10

A 61-year-old female patient with non-small cell lung cancer with EGFR 19 deletion, EGFR amplification and cMET amplification received a combination of osimertinib (80 mg q2w) and bispecific antibody (1500 mg q2w) PB19478 according to the clinical trial process described in Example 8. The patient had previously received treatment with osimertinib. After 3 cycles of the antibody and 4 cycles of osimertinib, the patient showed clinically relevant effects in the form of confirmed PR. The most serious adverse reaction observed was hypokalemia. No other adverse reactions were observed except for grade 1 or grade 2 reactions.

Citations

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 haspotent antitumor activity." Oncogene 32.50(2013):5593-5601.

Eisenhauer et al., New response evaluation criteria in solid tumors:Revised RECIST guideline(version 1.1).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 theHGF-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 andcMet is effective against EGFR inhibitor-resistant lung tumors." Cancerresearch 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 JClin 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.

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

Table 2. Competition of cMet reference antibodies with cMET cLC antibodies. Shown are OD ₄₅₀ values. OD ₄₅₀ values indicate the presence or absence of competition with the antibody. MF4506 failed the test.

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

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

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

Table 6. Composition of bispecific antibodies. The pXX numbers indicate the number of the manufacturing round and can be used to identify whether the antibody was produced as an ADCC version.

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

Table 8 Treatment plan for PDX model LXFE2478.

Table 9 | Treatment schedule for CDX model HCC827-ER1. QD = once daily, BIW = twice weekly, i.p. = intraperitoneal, p.o. = oral.

Claims (50)

1. A composition of a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody, the bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region, the heavy _chain variable region of the first variable domain has a CDR1 sequence of SYGIS, a CDR2 sequence of _{WISAYX1X2NTNYAQKLQG} and a CDR3 comprising the sequence _{X3X4X5X6HWWLX7A} , wherein _X1 = _N or _S ; _X2 =A or G; _X3 =D or G; _X4 =R, _S or Y; _X5 =H, L or Y; _X6 =D or W, and _X7 =D or G; and between _X1 and X7, _X7 =H, L or Y is selected from the group consisting of: The present invention relates to a method for treating cancer in an individual wherein the second variable domain comprises a heavy chain variable region, the heavy chain variable region of the second variable domain has an amino acid sequence of one of SEQ ID NOs: _1-23 , resulting in 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof. 2. A method for treating an individual suffering from cancer, the treatment comprising administering to the individual an effective amount of a composition of a third generation EGFR tyrosine kinase inhibitor and a bispecific antibody, the bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region, the heavy chain variable region of _the first _{variable domain having a CDR1 sequence of SYGIS, a CDR2 sequence of WISAYX1X2NTNYAQKLQG, and a CDR3 comprising the sequence X3X4X5X6HWWLX7A , wherein X1 =} N or S; _{X2 =} A or G; _X3 =D or G; _X4 =R, S or Y; _X5 =H, L or Y _{; X6} =D or W, and _X7 =D or G; and within the _{range of X1} to X The positions other than ₇ have 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof, and wherein the second variable domain comprises a heavy chain variable region, and the heavy chain variable region of the second variable domain has an amino acid sequence of one of SEQ ID NOs: 1-23 sequences resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof. 3. A use of a composition of a bispecific antibody and a third generation EGFR tyrosine kinase inhibitor for the preparation of a medicament for treating cancer in an individual, the bispecific antibody comprising a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region, the heavy chain variable region of the first variable domain has a CDR1 sequence of SYGIS, a CDR2 sequence of WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence _{X3X4X5X6HWWLX7A} , wherein _X1 =N or _{S; X2=A or G; X3=D or G; X4=R, S or Y; X5=H, L or Y; X6=D or W, and X7=D or G; and between X1 and X7 , the first variable domain comprises a heavy chain} variable region having a CDR1 sequence of SYGIS, a _CDR2 sequence of WISAYX1X2NTNYAQKLQG and a _CDR3 comprising the sequence X3X4X5X6HWWLX7A. The positions other than ₇ have 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof, and wherein the second variable domain comprises a heavy chain variable region, and the heavy chain variable region of the second variable domain has an amino acid sequence of one of SEQ ID NOs: 1-23 sequences resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof. 4. The use or method according to any one of the preceding claims, wherein the cancer is an EGFR-positive and/or cMET-positive cancer. 5. The use or method of any preceding claim, wherein the cancer comprises EGFR and/or cMET aberrations. 6. The use or method according to any one of the preceding claims, wherein the cancer is resistant to treatment with a tyrosine kinase inhibitor. 7. The use or method according to any one of the preceding claims, wherein the cancer is resistant to an EGFR tyrosine kinase inhibitor and/or a cMET tyrosine kinase inhibitor. 8. The use or method according to claim 7, wherein the EGFR tyrosine kinase inhibitor resistance comprises resistance to first-generation tyrosine kinase inhibitors, second-generation tyrosine kinase inhibitors and/or third-generation tyrosine kinase inhibitors, preferably resistance to third-generation EGFR tyrosine kinase inhibitors. 9. The use or method according to claim 7, wherein the cMET tyrosine kinase inhibitor resistance comprises resistance to Capmatinib, Tepotinib, Crizotenib, Cabozantinib, Savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833, preferably Capmatinib or Tepotinib. 10. The use or method according to any one of the preceding claims, wherein the individual has been previously treated with a tyrosine kinase inhibitor, preferably an EGFR tyrosine kinase inhibitor and/or a cMET tyrosine kinase inhibitor. 11. The use or method of any preceding claim, wherein the subject has been previously treated with a first generation EGFR tyrosine kinase inhibitor, a second generation EGFR tyrosine kinase inhibitor, or a third generation EGFR tyrosine kinase inhibitor. 12. The use or method according to any one of the preceding claims, wherein the subject has been previously treated with a cMET tyrosine kinase inhibitor. 13. The use or method of any of the preceding claims, wherein the cancer comprises an activating EGFR mutation, a confirmed tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation, a mutation that reduces the binding of a third generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, EGFR gene amplification, a cMET mutation, or a cMET aberration. 14. 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, such as L858R. 15. 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. 16. The use or method according to any one of the preceding claims, wherein the cancer comprises an acquired tyrosine kinase inhibitor resistance mutation, such as a mutation that confers resistance to Osimertinib. 17. The use or method according to any one of the preceding claims, wherein the cancer comprises an exon 20 mutation selected from a proximal loop insertion, a distal loop insertion, preferably V769_D770insASV, D770_N771insSVD, H773_V774insNPH, H773_V774insH, D770_N771insG, D770delinsGY, N771_P772insN, V774_C775insHV, D770_N771insVD, 771insGL、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_D770insMAS V; or mutations T790M, L792X (e.g., L792H), C796X (e.g., G796R, G796S, G796D), C797X (e.g., C797S, C797G), L798I, or 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_V769 InsX1- 7>V769_D770InsX1-7>D770_N771 InsX1-7>N771_P772 InsX1-7>P772_H773 InsX1-7, H773_V774 InsX1-7, or V774_C775 InsX1-7. 18. The use or method of any of the preceding claims, wherein the cancer comprises a cMET aberration, such as cMET amplification, cMET overexpression, increased signaling of the cMET pathway, cMET gene amplification, increased HGF expression, and/or increased cMET protein activity. 19. The use or method of any preceding claim, wherein the cancer comprises a cMET exon 14 skipping mutation. 20. The use or method according to any one of the preceding claims, wherein the first generation EGFR tyrosine kinase inhibitor comprises or is Gefitinib, Erlotinib or Icotinib. 21. The use or method according to any one of the preceding claims, wherein the second generation EGFR tyrosine kinase inhibitor comprises or is Afatinib, Dacomitinib, XL647, AP26113, CO-1686 or Neratinib. 22. The use or method according to any one of the preceding claims, wherein the third generation EGFR tyrosine kinase comprises or is osimertinib, lazertinib, afatinib, rezivertinib, rociletinib, olmutinib, almonertinib, abivertinib, ASK120067, befotertinib, SH-1028, nazartinib EGF816, naquotinib ASP8273, mavelertinib PF-0647775, olafertinib CK-101, keynatinib or ES-072, preferably osimertinib. 23. The use or method of any of the preceding claims, 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. 24. The use or method according to any one of the preceding claims, wherein the treatment comprises administering the composition of the bispecific antibody and the tyrosine kinase inhibitor to an individual in need thereof, and wherein the bispecific antibody and the third generation tyrosine kinase inhibitor are administered simultaneously, sequentially or separately. 25. The use or method according to any one of the preceding claims 1 to 5, wherein the subject has not previously received anti-cancer therapy, such as a subject who has not received tyrosine kinase inhibitor therapy or anti-EGFR therapy. 26. The use or method according to any one of the preceding claims 1 to 5, wherein the administration of the bispecific antibody and TKI inhibitor is as first-line treatment. 27. The use or method of any one of the preceding claims 1 to 5, wherein the individual or cancer comprises an EGFR and/or cMET activating mutation, such as an exon 19 deletion mutation or an exon 21 mutation, such as L858R. 28. The use or method of any preceding claim, wherein the subject is a human subject. 29. The use or method according to any one of the preceding claims, wherein the cancer is lung cancer, in particular non-small cell lung cancer, preferably metastatic or advanced non-small cell lung cancer. 30. The use or method of any preceding claim, wherein the cancer is an advanced or metastatic cancer. 31. A pharmaceutical composition comprising a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody, wherein the bispecific antibody comprises a first variable domain that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind to the extracellular portion of human MET proto-oncogene receptor tyrosine kinase (cMET), wherein the first variable domain comprises a heavy chain variable region, the heavy chain variable region of the first variable domain has _a CDR1 sequence SYGIS, a CDR2 sequence _{WISAYX1X2NTNYAQKLQG} and a CDR3 comprising the sequence _{X3X4X5X6HWWLX7A} , wherein _X1 =N or S; _X2 =A or _G ; _X3 =D or G; _X4 =R, S or Y; _X5 =H, L or Y; _X6 =D or W, and _X7 =D or G; and between _X1 and X7, _X7 =H, L or Y is selected from the group consisting of: The positions other than ₇ have 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof, and wherein the second variable domain comprises a heavy chain variable region, and the heavy chain variable region of the second variable domain has an amino acid sequence of one of SEQ ID NOs: 1-23 sequences resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof. 32. The use, method or pharmaceutical composition of any preceding claim, wherein the antibody is a human antibody. 33. The use, method or pharmaceutical composition of any preceding claim, wherein the antibody is ADCC enhancing. 34. The use, method or pharmaceutical composition according to any one of the preceding claims, wherein the antibody is an IgG1 format antibody with a stoichiometric ratio of 1:1 between anti-EGFR and anti-cMET. 35. The use, method or pharmaceutical composition of any preceding claim, wherein the antibody has one variable domain that binds to EGFR and one variable domain that binds to cMET. 36. The use, method or pharmaceutical composition of any preceding claim, wherein the variable domain that binds to human EGFR also binds to cynomolgus monkey and mouse EGFR. 37. The use, method or pharmaceutical composition of any preceding claim, wherein the variable domain that binds to human EGFR is domain III that binds to human EGFR. 38. The use, method or pharmaceutical composition of any preceding claim, wherein the variable domain that binds to cMET blocks the binding of antibody 5D5 to cMET. 39. The use, method or pharmaceutical composition of any preceding claim, wherein the variable domain that binds to cMET blocks the binding of HGF to cMET. 40. The use, method or pharmaceutical composition of any preceding claim, wherein the amino acids at positions 405 and 409 in one CH3 domain are identical to the amino acids at the corresponding positions in the other CH3 domain according to the EU numbering method. 41. The use, method or pharmaceutical composition according to any preceding claim, wherein _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; _X1 = S; _X2 = G; X3 = D; _X4 = S; _X5 = Y; _X6 = W and _X7 = G; _X1 = N; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and X7 = D; _X1 = N; _X2 = A; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D; =G; _X3 =G; _X4 = _{Y ; X5} = _L ; _X6 =D and _X7 =G; _X1 =N; _X2 =A; _X3 =G; _X4 =Y; _X5 =L; _X6 = _D and _X7 =G; or _X1 = _S ; _X2 = _G ; _X3 =G _{; X4 =} Y; _{X5 =} L; _X6 =D and _{X7 = G} . 42. The use, method or pharmaceutical composition of any preceding claim, wherein _Xi = N; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D; or _Xi = N; _X2 = A; _X3 = D; X4 = R; _X5 = H; _X6 = _W and _X7 = D; or _Xi = S; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D. 43. The use, method or pharmaceutical composition according to any preceding claim, wherein _Xi = N; _X2 = G; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D; or _Xi = N; _X2 = A; _X3 = D; _X4 = R; _X5 = H; _X6 = W and _X7 = D. 44. The use, method or pharmaceutical composition of any of the preceding claims, wherein the heavy chain variable region of the second variable domain has an amino acid sequence of one of the sequences of SEQ ID NO: 1-3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23 resulting in 0 to 10, preferably 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof. 45. The use, method or pharmaceutical composition of any of the preceding claims, wherein the heavy chain variable region of the second variable domain has an amino acid sequence of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13 or SEQ ID NO: 23 resulting in 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof. 46. The use, method or pharmaceutical composition of any preceding claim, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS, a CDR2 sequence of WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, and wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of SYSMN, a CDR2 sequence of WINTYTGDPTYAQGFTG and a CDR3 sequence of ETYYYDRGGYPFDP. 47. The use, method or pharmaceutical composition of any preceding claim, wherein the first variable domain comprises a heavy chain variable region having a CDR1 sequence of SYGIS, a CDR2 sequence of WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, wherein the second variable domain comprises a heavy chain variable region having a CDR1 sequence of TYSMN, a CDR2 sequence of WINTYTGDPTYAQGFTG and a CDR3 sequence of ETYFYDRGGYPFDP. 48. The use, method or pharmaceutical composition of any preceding claim, wherein the first variable domain and the second variable domain comprise a common light chain, preferably the light chain variable domain in Figure 4B. 49. The use, method or pharmaceutical composition of any preceding claim, wherein the antibody inhibits HGF-induced growth of HGF growth-responsive cells. 50. The use, method or pharmaceutical composition of any preceding claim, wherein the antibody inhibits EGF-induced growth of EGF growth-responsive cells.