Anti-EGFR / MET Antibody and Its Use

Abstract

The present disclosure relates to antigen-binding protein constructs (e.g., bispecific antibodies, antigen-binding fragments thereof, or ADCs), which specifically bind to two different antigens (e.g., EGFR and MET).

Description

Technical Field

[0001] <Claims of Priority> This application claims the benefit of PCT Application No. PCT / CN2022 / 102231, filed on June 29, 2022; PCT Application No. PCT / CN2022 / 108838, filed on July 29, 2022; PCT Application No. PCT / CN2022 / 116678, filed on September 2, 2022; and PCT Application No. PCT / CN2022 / 141416, filed on December 23, 2022. The entire above content is incorporated herein by reference.

[0002] The present disclosure relates to antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof).

Background Art

[0003] A bispecific antibody is an artificial protein capable of simultaneously binding two different types of antigens or two different epitopes. This bispecificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting of different disease mediators, and delivery of payloads to targeted sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) has been a major milestone in the development of bispecific antibodies. Since bispecific antibodies have various applications, there is a need to continue developing various therapeutic agents based on bispecific antibodies.

Summary of the Invention

[0004] The present disclosure relates to antigen-binding protein constructs that specifically bind to two different antigens (e.g., EGFR and MET). In some embodiments, the multispecific antibody (e.g., bispecific antibody) has the same light chain variable region. In some embodiments, the multispecific antibody (e.g., bispecific antibody) has a common light chain. In some embodiments, the multispecific antibody (e.g., bispecific antibody) forms part of an antibody-drug conjugate. In one aspect, the present disclosure relates to an antigen-binding protein construct comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to MET. In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0005] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity-determining regions (CDRs) 1, 2, and 3. In some embodiments, the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence. The first light chain variable region (VL1) comprises CDRs 1, 2, and 3. In some embodiments, the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence. In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences, and the selected VL1 CDR1, 2, and 3 amino acid sequences are as follows: (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6 respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3 respectively; (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9 respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3 respectively; (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18 respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3 respectively; and (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21 respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3 respectively; is one of the following.

[0006] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3. In some embodiments, the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR2 amino acid sequence, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR3 amino acid sequence. The second light chain variable region (VL2) comprises CDR1, 2, and 3. In some embodiments, the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR2 amino acid sequence, the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR3 amino acid sequence. In some embodiments, the selected VH2 CDR1, 2, and 3 amino acid sequences, and the selected VL2 CDR1, 2, and 3 amino acid sequences are as follows: (1) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10-12 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (2) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13-15 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (3) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22-24 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; and (4) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25-27 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; is one of the above.

[0007] In some embodiments, the antigen-binding protein constructs described herein are as follows: (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4-6 respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10-12 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7-9 respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10-12 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7-9 respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13-15 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16-18 respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22-24 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (5) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19-21 respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22-24 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; and (6) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19-21 respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25-27 respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; has one of the features of

[0008] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 28, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 30, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32.

[0009] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 30, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32. In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 31, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32.

[0010] In some embodiments, VH1 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, VL1 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, and the selected VH sequence and the selected VL sequence are as follows: (1) the selected VH sequence is SEQ ID NO: 28 and the selected VL sequence is SEQ ID NO: 32; and (2) the selected VH sequence is SEQ ID NO: 29 and the selected VL sequence is SEQ ID NO: 32; is one of.

[0011] In some embodiments, VH1 comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence, VL1 comprises VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, and the selected VH sequence and the selected VL sequence are as follows: (1) the selected VH sequence is SEQ ID NO: 28 and the selected VL sequence is SEQ ID NO: 32; and (2) the selected VH sequence is SEQ ID NO: 29 and the selected VL sequence is SEQ ID NO: 32; is one of.

[0012] In some embodiments, VH2 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, VL2 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, and the selected VH sequence and the selected VL sequence are as follows: (1) the selected VH sequence is SEQ ID NO: 30 and the selected VL sequence is SEQ ID NO: 32; and (2) the selected VH sequence is SEQ ID NO: 31 and the selected VL sequence is SEQ ID NO: 32; is one of.

[0013] In some embodiments, VH2 comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence, VL2 comprises VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, and the selected VH sequence and the selected VL sequence are as follows: (2) the selected VH sequence is SEQ ID NO: 30 and the selected VL sequence is SEQ ID NO: 32; and (2) The selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 32; is one of them.

[0014] In some embodiments, VH1 comprises the sequence of SEQ ID NO: 28, and VL1 comprises the sequence of SEQ ID NO: 32.

[0015] In some embodiments, VH1 comprises the sequence of SEQ ID NO: 30, and VL1 comprises the sequence of SEQ ID NO: 32.

[0016] In some embodiments, VH2 comprises the sequence of SEQ ID NO: 31, and VL2 comprises the sequence of SEQ ID NO: 32.

[0017] In some embodiments, the first antigen-binding domain specifically binds to human or monkey EGFR, and / or the second antigen-binding domain specifically binds to human or monkey MET.

[0018] In some embodiments, the first antigen-binding domain is human or humanized, and / or the second antigen-binding domain is human or humanized.

[0019] In some embodiments, the antigen-binding protein construct is a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the first antigen-binding domain is a single-chain variable fragment (scFv), and / or the second antigen-binding domain is an scFv. In some embodiments, the first light chain variable region and the second light chain variable region are identical.

[0020] In one aspect, the present disclosure provides a nucleic acid comprising a polynucleotide encoding the antigen-binding protein construct described herein.

[0021] In one aspect, the present disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0022] In one aspect, the present disclosure relates to a cell comprising a vector described herein. In some embodiments, the cell is a CHO cell.

[0023] In one aspect, the present disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0024] In one aspect, the present disclosure relates to a method for producing an antibody or an antigen-binding fragment thereof, or an antigen-binding protein construct, the method comprising: (a) culturing a cell described herein under conditions sufficient for the cell to produce the antigen-binding protein construct; and (b) collecting the antigen-binding protein construct produced by the cell.

[0025] In one aspect, the present disclosure relates to an antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to an antigen-binding protein construct described herein. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent. In some embodiments, the therapeutic agent is MMAE or MMAF.

[0026] In some embodiments, the therapeutic agent is

Chemical formula

[0027] In some embodiments, the therapeutic agent is linked to the antigen-binding protein construct via a linker. In some embodiments, the linker has the following structure:

Chemical formula

[0028] In some embodiments, the antibody-drug conjugate has the following structure:

Chemical formula

[0029] In one aspect, the present disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a composition comprising a therapeutically effective amount of an antigen-binding protein construct described herein or an antibody-drug conjugate described herein. In some embodiments, the subject has cancer that expresses EGFR and / or MET. In some embodiments, the cancer is a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung cancer), gastric cancer (e.g., gastric carcinoma), skin cancer (e.g., cutaneous carcinoma), colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal cancer, or brain tumor. In some embodiments, the subject is human. In some embodiments, the method further comprises administering an anti-PD1 antibody to the subject. In some embodiments, the method further comprises administering chemotherapy to the subject.

[0030] In one aspect, the present disclosure relates to a method of reducing tumor growth rate, the method comprising contacting tumor cells with a composition comprising an effective amount of an antigen-binding protein construct described herein or an antibody-drug conjugate described herein.

[0031] In one aspect, the present disclosure relates to a method of killing tumor cells, the method comprising contacting tumor cells with a composition comprising an effective amount of an antigen-binding protein construct described herein or an antibody-drug conjugate described herein.

[0032] In one aspect, the present disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) an antigen-binding protein construct described herein, and / or (b) an antibody-drug conjugate described herein.

[0033] As used herein, the term "antigen-binding protein construct" refers to (i) a single polypeptide comprising at least two different antigen-binding domains, or (ii) a complex of two or more polypeptides (e.g., identical or different polypeptides) that together form at least two different antigen-binding domains. Non-limiting examples and embodiments of antigen-binding protein constructs are described herein. Further examples and embodiments of antigen-binding protein constructs are well known in the art.

[0034] As used herein, the term "antigen-binding domain" refers to one or more protein domains (e.g., formed from amino acids derived from a single polypeptide or from amino acids derived from two or more polypeptides (e.g., identical or different polypeptides) that are specifically bindable to one or more different antigens (e.g., effector antigens or control antigens)). In some embodiments, the antigen-binding domain can bind an antigen or epitope with specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. An example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. In some embodiments, the antigen-binding domain can include an alternative scaffold. In some embodiments, the antigen-binding domain is a VHH. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are well known in the art. In some embodiments, the antigen-binding domain can bind a single antigen (e.g., one of the effector and control antigens). In other embodiments, the antigen-binding domain can bind two different antigens (e.g., an effector antigen and a control antigen).

[0035] As used herein, the term "antibody" is used in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Specific examples of antibodies include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. An example of an antibody is a protein complex that includes two heavy chains and two light chains. Further examples of antibodies are described herein.

[0036] As used herein, the term "multispecific antigen-binding protein construct" is an antigen-binding protein construct that includes two or more different antigen-binding domains that collectively and specifically bind to two or more different epitopes. The two or more different epitopes can be epitopes on the same antigen (e.g., a single polypeptide present on the surface of a cell) or on different antigens (e.g., different proteins present on the surface of the same cell or on different cells). In some embodiments, the multispecific antigen-binding protein construct binds to two different epitopes (i.e., a "bispecific antigen-binding protein construct"). In some embodiments, the multispecific antigen-binding protein construct binds to three different epitopes (i.e., a "trispecific antigen-binding protein construct"). In some embodiments, the multispecific antigen-binding protein construct binds to four different epitopes (i.e., a "tetraspecific antigen-binding protein construct"). In some embodiments, the multispecific antigen-binding protein construct binds to five different epitopes (i.e., a "pentaspecific antigen-binding protein construct"). Each binding specificity can be present in any suitable valency. Non-limiting examples of multispecific antigen-binding protein constructs are described herein.

[0037] As used herein, the term "bispecific antibody" means an antibody that binds to two different epitopes. The epitopes can be present on the same antigen or on different antigens.

[0038] As used herein, the term "common light chain" means a single light chain that can interact with two or more different heavy chains that form different antigen-binding sites, and these different antigen-binding sites can specifically bind to different antigens or epitopes. Similarly, the term "common light chain variable region" means a single light chain variable region that can interact with two or more different heavy chain variable regions that form different antigen-binding sites, and these different antigen-binding sites can specifically bind to different antigens or epitopes. In some embodiments, the antigen-binding construct can have a common light chain. In some embodiments, the antigen-binding construct can have a common light chain variable region.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, this specification, including definitions, will control. Other features and advantages of the present invention will become apparent from the following detailed description, drawings, and claims.

Brief Description of the Drawings

[0040]

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Mode for Carrying Out the Invention

[0041] A bispecific antibody or an antigen-binding fragment thereof is an artificial protein capable of simultaneously binding to two different epitopes (e.g., on two different antigens). In some embodiments, the bispecific antibody or an antigen-binding fragment thereof can have two arms. Each arm can have one heavy chain variable region and one light chain variable region, forming an antigen-binding domain (or antigen-binding region). In some embodiments, the bispecific antibody has a common light chain.

[0042] The present disclosure relates to two different antigens (e.g., EGFR and MET), and an antigen-binding protein construct (e.g., a bispecific antibody or an antigen-binding fragment thereof) that specifically binds to an antibody-drug conjugate.

[0043] Anti-EGFR / MET antigen-binding protein construct The epidermal growth factor receptor (EGFR, ErbBI, or HER1) is a 170 kDa type I transmembrane glycoprotein encoded by the c-erbBl proto-oncogene. The epidermal growth factor receptor is a member of the ErbB family of receptors, a subfamily of four closely related receptor-type tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). In many types of cancer, mutations that affect the expression or activity of EGFR can lead to cancer. EGFR signaling is initiated by ligand binding followed by a conformational change, homodimerization or heterodimerization of the receptor with other ErbB family members, and trans-autophosphorylation of the receptor, which in turn initiates a signal transduction cascade that ultimately affects a wide variety of cellular functions including cell proliferation and survival. Increased expression or kinase activity of EGFR has been associated with a range of human cancers, making EGFR an attractive target for therapeutic intervention. Increases in both EGFR gene copy number and protein expression are associated with a favorable response to IRESSA TM (gefitinib).

[0044] MET, also known as c-Met, 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 major single-chain precursor protein is cleaved after translation to produce the α and β subunits, which disulfide bond to form the mature receptor. When MET is activated by its ligand, hepatocyte growth factor (HGF), numerous cellular processes are stimulated, including proliferation, motility, invasion, metastasis, epithelial-mesenchymal transition, angiogenesis / wound healing, and tissue regeneration. The exact stoichiometry of HGF:MET binding is unclear, but it is generally thought that when two HGF molecules bind to two MET molecules, it results in dimerization and autophosphorylation of the receptor at tyrosine 1230, 1234, and 1235. MET autophosphorylation independent of ligand can also occur due to gene amplification, mutation, or overexpression of the receptor.

[0045] In many types of cancer, including gastric, lung, colorectal, breast, bladder, head and neck, ovarian, prostate, thyroid, pancreatic, and CNS cancers, MET is frequently amplified, mutated, or overexpressed. Missense mutations typically localized to the kinase domain are commonly found in hereditary papillary renal cell carcinoma (PRCC) and in 13% of sporadic PRCC (Schmidt et al, Oncogene 18:2343 - 2350, 1999), and MET mutations localized to the semaphorin or juxtamembrane domains of MET are frequently found in gastric, head and neck, liver, ovarian, NSCLC, and thyroid cancers. MET amplification has been detected in brain, colorectal, gastric, and lung cancers and often correlates with disease progression. MET amplification is shown in up to 4% and up to 20% of non-small cell lung cancer (NSCLC) and gastric cancers, respectively. Overexpression of MET is also frequently observed in lung cancer. Furthermore, in clinical samples, nearly half of lung adenocarcinomas showed high levels of both MET and HGF, and both were correlated with increased tumor growth, metastasis, and poor prognosis.

[0046] Nearly 60% of all tumors that have become resistant to EGFR tyrosine kinase inhibitors increase MET expression, amplify MET, or increase HGF, the only known ligand of MET, suggesting the presence of a compensatory pathway for EGFR via MET. MET amplification was first identified in cultured cells that had become resistant to gefitinib, an EGFR kinase inhibitor, and demonstrated improved survival via the Her3 pathway. This was further confirmed in clinical samples, with 9 out of 43 patients who had acquired resistance to either erlotinib or gefitinib showing MET amplification.

[0047] Aberrant MET signaling has been implicated in the development / progression of many human cancers. This is due to overexpression of MET, activating mutations in MET, transactivation, autocrine or paracrine signaling, or amplification of the MET gene. A significant relationship between EGFR and MET signaling has been recognized by studies on cancer treatment outcomes. MET is an important player in the development of resistance to targeted therapies, including those directed at EGFR. Similarly, activation of alternative pathways, including EGFR and downstream gene mutations such as KRAS, histological transformation, and the MET signaling pathway, has been identified as a mechanism of resistance to EGFR-targeted therapies. As a result, blocking one receptor tends to upregulate other receptors, leading to resistance to monotherapy. Amplification of MET and / or high-level HGF ligand expression have been observed in NSCLC patients with natural or acquired resistance to EGFR tyrosine kinase inhibitors, including erlotinib and gefitinib. Conversely, MET-amplified lung cancer cells that have been exposed to MET inhibitors for a long time develop resistance via the EGFR pathway. The signal transduction crosstalk between EGFR and Met may result in improved outcomes for patients with cancers induced by both receptors, through combined inhibition of both receptors. Furthermore, compared to blocking just one pathway, simultaneous inhibition may overcome or delay treatment resistance.

[0048] When ligands such as EGF bind to EGFR, the dimerization, autophosphorylation of the receptor, activation of the cytoplasmic tyrosine kinase domain inside the receptor, and initiation of multiple signal transduction and transactivation pathways involved in DNA synthesis (gene activation) and progression or division of the cell cycle are stimulated. Inhibition of EGFR signaling can result in the inhibition of one or more EGFRs. In some embodiments, EGFR ligands include EGF, TGFα, heparin-binding EGF (HB-EGF), amphiregulin (AR), and epiregulin (EPI).

[0049] When HGF binds to MET, the dimerization, autophosphorylation of the receptor, activation of the cytoplasmic tyrosine kinase domain inside the receptor, and initiation of multiple signal transduction and transactivation pathways involved in DNA synthesis (gene activation) and progression or division of the cell cycle are stimulated, and inhibition of MET signaling can result in the inhibition of one or more MET downstream signaling pathways. Therefore, neutralization of MET can have various effects including inhibition of cell proliferation and differentiation, angiogenesis, cell motility, and metastasis.

[0050] The roles of EGFR and MET in cancer are described, for example, in WO2014081954A1, WO2008 / 127710, WO2009 / 111691, WO2009 / 126834, WO2010 / 039248, WO2010 / 115551, and US2009 / 0042906, Engelman et al., "MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling." science 316.5827 (2007): 1039-1043, Bean et al., "MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib." Proceedings of the National Academy of Sciences 104.52 (2007): 20932-20937, which are hereby incorporated by reference in their entirety.

[0051] In the present disclosure, the anti-EGFR antibodies (e.g., E-1G11 ("1G11") and E-6C4 ("6C4")), and the anti-MET antibodies (e.g., M-2F11 ("2F11") and M-2G10 ("2G10")) are human antibodies produced in RenLite(R) mice. Since these antibodies have the same fully humanized common light chain, they produce an anti-EGFR / MET bispecific antibody having a heavy chain variable region targeting EGFR (e.g., any one of the VHs targeting EGFR described herein), a heavy chain variable region targeting MET (e.g., any one of the VHs targeting MET described herein), and two identical common light chain variable regions. In some embodiments, the anti-EGFR antigen-binding domain comprises the CDRs of the anti-EGFR antibody 1G11 or 6C4. In some embodiments, the anti-MET antigen-binding domain comprises the CDRs of the anti-MET antibody 2F11 or 2G10. In some embodiments, the anti-EGFR antigen-binding domain comprises the VH and VL of the anti-EGFR antibody 1G11 or 6C4. In some embodiments, the anti-MET antigen-binding domain comprises the VH and VL of the anti-MET antibody 2F11 or 2G10. For example, E-1G11-M-2F11 means a bispecific anti-EGFR / MET antibody containing an anti-EGFR antigen-binding domain derived from E-1G11 and an anti-MET antigen-binding domain derived from M-2F11. In some embodiments, the anti-EGFR antigen-binding domain comprises the CDRs of E-1G11. In some embodiments, the anti-MET antigen-binding domain comprises the CDRs of M-2F11. In some embodiments, the anti-EGFR antigen-binding domain comprises the VH and VL of E-1G11. In some embodiments, the anti-MET antigen-binding domain comprises the VH and VL of M-2F11.

[0052] The bispecific antibodies described herein can be designed to have an IgG1 subtype structure with a knobs-into-holes (KIH) mutation, thereby promoting heterodimerization and avoiding incorrect pairing between the two heavy chains.

[0053] In some embodiments, the bispecific antibody has a higher endocytosis rate than the corresponding monoclonal antibody or a control bispecific antibody.

[0054] In some embodiments, the bispecific antibodies described herein can be conjugated to a therapeutic agent to form an antibody-drug conjugate (ADC). In some embodiments, the DAR of the ADCs described herein is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In some embodiments, the DAR of the ADCs described herein is about 3.5 to about 4.5, about 3.6 to about 4.5, about 3.7 to about 4.5, about 3.8 to about 4.5, about 3.9 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.5, about 4.2 to about 4.5, about 4.3 to about 4.5, about 4.4 to about 4.5, about 3.5 to about 4.4, about 3.6 to about 4.4, about 3.7 to about 4.4, about 3.8 to about 4.4, about 3.9 to about 4.4, about 4.0 to about 4.4, about 4.1 to about 4.4, about 4.2 to about 4.4, about 4.3 to about 4.4, about 3.5 to about 4.3, about 3.6 to about 4.3, about 3.7 to about 4.3, about 3.8 to about 4.3, about 3.9 to about 4.3, about 4.0 to about 4.3, about 4.1 to about 4.3, about 4.2 to about 4.3, about 3.5 to about 4.2, about 3.6 to about 4.2, about 3.7 to about 4.2, about 3.8 to about 4.2, about 3.9 to about 4.2, about 4.0 to about 4.2, about 4.1 to about 4.2, about 3.5 to about 4.1, about 3.6 to about 4.1, about 3.7 to about 4.1, about 3.8 to about 4.1, about 3.9 to about 4.1, about 4.0 to about 4.1, about 3.5 to about 4.0, about 3.6 to about 4.0, about 3.7 to about 4.0, about 3.8 to about 4.0, about 3.9 to about 4.0, about 3.5 to about 3.9, about 3.6 to about 3.9, about 3.7 to about 3.9, about 3.8 to about 3.9, about 3.5 to about 3.8, about 3.6 to about 3.8, about 3.7 to about 3.8, about 3.5 to about 3.7, about 3.6 to about 3.7, or about 3.5 to about 3.6. In some embodiments, the DAR of the ADCs described herein is from about 7.5 to about 8.5, from about 7.6 to about 8.5, from about 7.7 to about 8.5, from about 7.8 to about 8.5, from about 7.9 to about 8.5, from about 8.0 to about 8.5, from about 8.1 to about 8.5, from about 8.2 to about 8.5, from about 8.3 to about 8.5, from about 8.4 to about 8.5, from about 7.5 to about 8.4, from about 7.6 to about 8.4, from about 7.7 to about 8.4, from about 7.8 to about 8.4, from about 7.9 to about 8.4, from about 8.0 to about 8.4, from about 8.1 to about 8.4, from about 8.2 to about 8.4, from about 8.3 to about 8.4, from about 7.5 to about 8.3, from about 7.6 to about 8.3, from about 7.7 to about 8.3, from about 7.8 to about 8.3, from about 7.9 to about 8.3, from about 8.0 to about 8.3, from about 8.1 to about 8.3, from about 8.2 to about 8.3, from about 7.5 to about 8.2, from about 7.6 to about 8.2, from about 7.7 to about 8.2, from about 7.8 to about 8.2, from about 7.9 to about 8.2, from about 8.0 to about 8.2, from about 8.1 to about 8.2, from about 7.5 to about 8.1, from about 7.6 to about 8.1, from about 7.7 to about 8.1, from about 7.8 to about 8.1, from about 7.9 to about 8.1, from about 8.0 to about 8.1, from about 7.5 to about 8.0, from about 7.6 to about 8.0, from about 7.7 to about 8.0, from about 7.8 to about 8.0, from about 7.9 to about 8.0, from about 7.5 to about 7.9, from about 7.6 to about 7.9, from about 7.7 to about 7.9, from about 7.8 to about 7.9, from about 7.5 to about 7.8, from about 7.6 to about 7.8, from about 7.7 to about 7.8, from about 7.5 to about 7.7, from about 7.6 to about 7.7, or from about 7.5 to about 7.6.

[0055] In some embodiments, the anti-EGFR / MET ADCs described herein can effectively inhibit in vitro cancer cell proliferation at a concentration of less than 10 μg / mL, less than 3.33 μg / mL, less than 1.11 μg / mL, less than 0.37 μg / mL, less than 0.12 μg / mL, less than 0.04 μg / mL, or less than 0.01 μg / mL. In some embodiments, the anti-EGFR / MET ADCs described herein can inhibit in vivo cancer cell proliferation (e.g., lung cancer, gastric cancer, or skin cancer) at a dose level of less than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg in a xenograft mouse model.

[0056] In some embodiments, the bispecific antibodies or antigen-binding fragments thereof described herein have a common light chain.

[0057] The anti-EGFR / MET antigen-binding protein construct (e.g., bispecific antibody) can include an anti-EGFR antigen-binding domain (e.g., E-1G11 (“1G11”), E-6C4 (“6C4”)), or an anti-MET antigen-binding domain (e.g., M-2F11 (“2F11”), M-2G10 (“2G10”)). In some embodiments, the anti-EGFR / MET antigen-binding protein construct has a heavy chain variable region targeting EGFR (e.g., any one of the VH targeting EGFR described herein), a heavy chain variable region targeting MET (e.g., any one of the VH targeting MET described herein), and two identical common light chain variable regions.

[0058] The CDR sequences for 1G11 and antibodies derived from 1G11 (e.g., human antibodies) include the CDRs of the heavy chain variable domain, SEQ ID NOs: 4-6, and the CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat numbering. The CDRs can also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 16-18, and the CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region for 1G11 are shown in SEQ ID NOs: 32 and 28, respectively.

[0059] The CDR sequences for 6C4 and antibodies derived from 6C4 (e.g., human antibodies) include the CDRs of the heavy chain variable domain, SEQ ID NOs: 7-9, and the CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat numbering. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are described in SEQ ID NOs: 19-21, and the CDR sequences of the light chain variable domain are described in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region for 6C4 are shown in SEQ ID NOs: 32 and 29, respectively.

[0060] The CDR sequences for 2F11 and antibodies derived from 2F11 (e.g., human antibodies) include the CDRs of the heavy chain variable domain, SEQ ID NOs: 10-12, and the CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat numbering. The CDRs can also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 22-24, and the CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region for 2F11 are shown in SEQ ID NO: 32 and SEQ ID NO: 30, respectively.

[0061] The CDR sequences for 2G10 and antibodies derived from 2G10 include the CDR sequences of the heavy chain variable domain, SEQ ID NOs: 13-15, and the CDR sequences of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat numbering. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are described in SEQ ID NOs: 25-27, and the CDR sequences of the light chain variable domain are described in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region for 2G10 are shown in SEQ ID NO: 32 and SEQ ID NO: 31, respectively.

[0062] Furthermore, in some embodiments, the anti-EGFR / MET antigen-binding protein constructs described herein can also contain 1, 2, or 3 heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 4-6, SEQ ID NOs: 7-9, SEQ ID NOs: 10-12, SEQ ID NOs: 13-15, SEQ ID NOs: 16-18, SEQ ID NOs: 19-21, SEQ ID NOs: 22-24, and SEQ ID NOs: 25-27, and / or 1, 2, or 3 light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1-3.

[0063] In some embodiments, the anti-EGFR / MET antigen-binding protein construct is a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the CDR1 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, 3 amino acid sequences, and the selected VL CDR1, 2, 3 amino acid sequences are shown in FIGS. 8 and 10 (Kabat CDR), and FIGS. 9 and 10 (Chothia CDR).

[0064] In some embodiments, the anti-EGFR / MET antigen-binding protein construct described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0065] In some embodiments, the anti-EGFR / MET antigen-binding protein construct described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0066] In some embodiments, the anti-EGFR / MET antigen-binding protein construct described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0067] In some embodiments, the anti-EGFR / MET antigen-binding protein construct described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0068] In some embodiments, the anti-EGFR / MET antigen-binding protein construct described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0069] In some embodiments, the anti-EGFR / MET antigen-binding protein construct described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0070] In some embodiments, the anti-EGFR / MET antigen-binding protein construct described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0071] In some embodiments, the anti-EGFR / MET antigen-binding protein construct described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0072] In some embodiments, the anti-EGFR / MET antigen-binding protein construct described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0073] Insertions, deletions, and substitutions can be present within the CDR sequence or at one or both ends of the CDR sequence.

[0074] In some embodiments, the anti-EGFR / MET antigen-binding protein construct contains, or consists of, a VH amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a VL amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 28, 29, 30, or 31, and the selected VL sequence is SEQ ID NO: 32.

[0075] In some embodiments, the anti-EGFR / MET antigen-binding protein construct can have three VH CDRs that are identical to the CDRs of any VH sequence described herein. In some embodiments, the anti-EGFR / MET antigen-binding protein construct can have three VL CDRs that are identical to the CDRs of any VL sequence described herein.

[0076] The present disclosure also provides a nucleic acid comprising a polynucleotide encoding an anti-EGFR / MET antigen-binding protein construct that includes an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain includes the CDRs shown in FIG. 8, FIG. 9, or FIG. 10, or has the sequence shown in FIG. 11.

[0077] The anti-EGFR / MET antigen-binding protein construct can also be an antibody variant (including derivatives and conjugates) of the anti-EGFR / MET antigen-binding protein construct. The additional antibodies provided herein are polyclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, antibodies produced intracellularly (i.e., intrabodies), and antigen-binding fragments thereof. The anti-EGFR / MET antigen-binding protein construct can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In some embodiments, the anti-EGFR / MET antigen-binding protein construct is an IgG (e.g., IgG1) antibody or an antigen-binding fragment thereof.

[0078] Fragments of the anti-EGFR / MET antigen-binding protein construct are suitable for use in the methods provided so long as they retain the desired affinity and specificity for both EGFR and MET. Thus, fragments of the anti-EGFR / MET antigen-binding protein construct retain the ability to bind to EGFR and MET.

[0079] Antigen-binding protein construct The present disclosure provides antigen-binding protein constructs (e.g., bispecific antibodies). The antigen-binding protein constructs (e.g., bispecific antibodies) can comprise an anti-EGFR antibody or an antigen-binding fragment thereof, and an anti-MET antibody or an antigen-binding fragment thereof. These antigen-binding protein constructs (e.g., bispecific antibodies) can take various forms.

[0080] Generally, an antibody (also referred to as an immunoglobulin) can be composed of two classes of polypeptide chains, a light chain, and a heavy chain. The non-limiting antigen-binding protein constructs of the present disclosure can be intact four-immunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype including IgM, IgG, IgE, IgA, or IgD, or a sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a κ light chain or a λ light chain.

[0081] Hypervariable regions known as complementarity-determining regions (CDRs) form loops that contain the principal antigen-binding surface of the antibody. The four framework regions mostly adopt a β-sheet structure, and the CDRs form connecting loops and in some cases part of the β-sheet structure. The CDRs of each chain are held in proximity to the framework region and, together with the CDRs of the other chains, contribute to the formation of the antigen-binding region.

[0082] Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and several CDR definitions are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the position of structural loop regions. These methods and definitions are described, for example, in Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439, Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839, Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250, Martin et al., Methods Enzymol. 203: 121-53 (1991), Morea et al., Biophys Chem. 68(1-3): 9-16 (Oct. 1997), Morea et al., J Mol Biol. 275(2): 269-94 (Jan. 1998), Chothia et al., Nature 342(6252): 877-83 (Dec. 1989), Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007), and the entirety of each of these is incorporated herein by reference.

[0083] CDRs are important for recognizing epitopes of antigens. As used herein, an "epitope" is the minimal portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimal size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but because epitopes can depend on the three-dimensional structure of the antigen based on its secondary and tertiary structure, these amino acids need not be in a continuous linear sequence of the primary structure of the antigen.

[0084] In some embodiments, the anti-EGFR / MET antigen-binding protein construct is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014), Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182, Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference in its entirety.

[0085] The anti-EGFR / MET antigen-binding protein construct can also be an immunoglobulin molecule derived from any species (e.g., human, rodent, mouse, rat, camel). Examples of the anti-EGFR / MET antigen-binding protein constructs disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies containing immunoglobulin binding domains fused to another polypeptide. The antigen-binding domain or antigen-binding fragment is a part of an antibody that retains the specific binding activity of the intact antibody, i.e., any part of the antibody that can specifically bind to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab’, F(ab’)2, and variants of these fragments. Thus, in some embodiments, the anti-EGFR / MET antigen-binding protein construct or its antigen-binding fragment can be, for example, an scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is an antibody-binding domain or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0086] In some embodiments, the scFv in the anti-EGFR / MET antigen-binding protein construct has two heavy chain variable domains and two light chain variable domains. In some embodiments, the scFv has two antigen-binding regions (antigen-binding regions: A and B), and the two antigen-binding regions can bind to their respective target antigens with different affinities.

[0087] In some embodiments, the anti-EGFR / MET antigen-binding protein construct or antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of the single-chain variable fragment (scFv) described herein fused to the CD3ζ transmembrane and endodomains. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, e.g., CD3z-CD28-41BB, or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors described herein.

[0088] In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) can bind two different antigens, or two different epitopes.

[0089] In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) can include 1, 2, or 3 heavy chain variable region CDRs selected from FIGS. 8 and 9. In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) can include 1, 2, or 3 light chain variable region CDRs selected from FIG. 10.

[0090] Antibody multimerization can be achieved by natural aggregation of the antibody or by chemical or recombinant conjugation techniques well known in the art. For example, some percentage of a purified antibody preparation (e.g., purified IgG1 molecules) will naturally form protein aggregates that contain antibody dimers and other higher order antibody multimers.

[0091] In some embodiments, the antigen-binding protein construct is a bispecific antibody. By engineering the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture, bispecific antibodies can be produced. For example, the interface can comprise at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0092] Any of the antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of an antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life of an antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human), or can extend its biological activity.

[0093] Antigen-binding protein constructs (e.g., bispecific antibodies) can also take various forms. Many different formats of antigen-binding constructs are well known in the art and are described, for example, in Suurs, et al. A review of bispecific antibodies and antibody constructs in oncology and clinical challenges, Pharmacology&therapeutics (2019), which is hereby incorporated by reference in its entirety.

[0094] In some embodiments, the antigen-binding protein construct is a BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. In some embodiments, the antigen-binding protein construct is a VHH-scAb, VHH-Fab, Dual scFab, F(ab’)2, diabody, crossMab, DAF(2in1), DAF(4in1), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, Intrabody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0095] In some embodiments, the antigen-binding protein construct can be a TrioMab. In a TrioMab, the two heavy chains are from different species, and the different sequences restrict heavy chain-light chain pairing.

[0096] In some embodiments, the antigen-binding protein construct has two different heavy chains and one common light chain. Heavy chain heterodimerization can be based on knob-into-hole or some other heavy chain pairing technology.

[0097] In some embodiments, bispecific antibodies can be generated using CrossMAb technology. Using CrossMAb technology, correct light chain pairing in bispecific heterodimeric IgG antibodies can be enhanced, and this technology enables the generation of various bispecific antibody formats, including bivalent (1+1), trivalent (2+1), and tetravalent (2+2) bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab)-based antibodies. These formats can be derived from any existing antibody pair without the need to use domain crossover for common light chain identification, post-translational processing / in vitro chemical assembly, or introduction of a series of mutations to enhance correct light chain pairing. This method is described in Klein et al., "The use of CrossMAb technology for the generation of bi-and multispecific antibodies." MAbs. Vol. 8. No. 6. Taylor & Francis, 2016, which is hereby incorporated by reference in its entirety. In some embodiments, the CH1 within the heavy chain and the CL domain within the light chain are exchanged.

[0098] The antigen-binding protein construct can be a Duobody. The Fab exchange mechanism that naturally exists within IgG4 antibodies is mimicked in a controlled manner in IgG1 antibodies, which is a mechanism called controlled Fab exchange. This format can ensure specific pairing between the heavy and light chains.

[0099] In a dual variable domain antibody (DVD-Ig), additional VH and variable light chain (VL) domains are added to each N-terminus for bispecific targeting. This format is similar to IgG-scFv, but the added binding domains bind individually to the corresponding N-termini instead of scFv to each heavy chain N-terminus.

[0100] In scFv-IgG, the two scFvs are linked to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two different bivalent binding sites and is consequently also referred to as tetravalent. There is no problem with the pairing of the heavy and light chains in scFv-IgG.

[0101] In some embodiments, the antigen-binding protein construct can have an IgG-IgG format. Two intact IgG antibodies are conjugated by chemically bonding the C-termini of the heavy chains.

[0102] The antigen-binding protein construct can also have a Fab-scFv-Fc format. In the Fab-scFv-Fc format, a third chain containing the light chain, heavy chain, and the Fc region and scFv is assembled. This can ensure efficient production and purification.

[0103] In some embodiments, the antigen-binding protein construct can be a TF. Three Fab fragments are linked by disulfide bridges. Two fragments target tumor-associated antigens (TAAs) and one fragment targets a hapten. The TF format does not have an Fc region.

[0104] ADAPTIR has two scFvs bound to both sides of a certain Fc region. It discards the intact IgG that is the basis for its construct, but preserves the Fc region, extending the half-life and facilitating purification.

[0105] Bispecific T cell engagers (“BiTEs”) are composed of two scFvs, VLA, VHA, and VHB VLB on one peptide chain. BiTEs have binding domains and no Fc region.

[0106] In BiTE-Fc, the Fc region fuses to the BiTE construct. Adding the Fc region extends the half-life, results in a longer effective concentration, and avoids continuous IV.

[0107] Dual-affinity retargeting (DART) has two peptide chains that link opposing fragments (i.e., VLA with VHB and VLB with VHA), and a disulfide bond that fuses them to each other at the C-terminus. In DART, the disulfide bond can improve stability compared to BiTE.

[0108] In DART-Fc, the Fc region is attached to the DART structure. This can be generated by assembling three chains (similar to DART, two via disulfide bonds). One chain contains half of the Fc region, which dimerizes with the third chain to express only the Fc region. Adding the Fc region extends the half-life, provides a longer effective concentration, and avoids continuous IV.

[0109] In tetravalent DART, four peptide chains are assembled. Basically, two DART molecules are made with half of the Fc region and dimerize. This format has bivalent binding to both targets and is thus a tetravalent molecule. Tandem diabody (TandAb) contains two diabodies. Each diabody is composed of a VHA and a VLB fragment, and a VHA and a VLB fragment, which are associated by a covalent bond. The two diabodies are linked by a peptide chain. This can improve stability compared to a diabody composed of two scFvs. It has two bivalent binding sites.

[0110] scFv-scFv-toxin contains a toxin and two scFvs with a stabilizing linker. This can be used for specific delivery of the payload. In the molecular scFv-scFv-scFv, the scFv directed against the TAA is tagged with a short recognizable peptide that is assembled against a bsAb composed of two scFvs (one directed against CD3 and one directed against a recognizable peptide).

[0111] In ImmTAC, the stabilized and soluble T cell receptor is fused to an scFv that recognizes CD3. By using the TCR, ImmTAC is suitable for targets to be treated, such as intracellular proteins.

[0112] The trispecific nanobody has two single variable domains (nanobodies) that include additional molecules for half-life extension. Additional modules are added to extend the half-life. In Trispecific Killer Engager (TriKE), the two scFvs are linked via a polypeptide linker that incorporates human IL-15. A linker to IL-15 is added to increase NK survival and proliferation.

[0113] In some embodiments, the antigen-binding protein construct is a bispecific antibody. In some embodiments, the bispecific antibodies in the present disclosure are designed to be 1+1 (monovalent for each target) and have an IgG1 subtype structure. Thereby, the binding activity to cells having low expression levels of EGFR and MET can be reduced, the binding activity to cells co-expressing EGFR and MET can be increased, and an improvement in the targeting function can be achieved.

[0114] In some embodiments, the anti-EGFR / MET antigen-binding protein construct (e.g., an antibody, bispecific antibody, or antibody fragment thereof) comprises a KIH mutation. In some embodiments, the antigen-binding protein construct comprises a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to MET. In some embodiments, the first antigen-binding domain comprises a heavy chain (knob heavy chain) comprising one or more knob mutations, and the second antigen-binding domain comprises a heavy chain (hole heavy chain) comprising one or more hole mutations. In some embodiments, the first antigen-binding domain comprises a heavy chain (hole heavy chain) comprising one or more hole mutations, and the second antigen-binding domain comprises a heavy chain (knob heavy chain) comprising one or more knob mutations. In some embodiments, the anti-EGFR / MET antigen-binding protein construct comprises a knob heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33. In some embodiments, the anti-EGFR / MET antigen-binding protein construct comprises a hole heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. In some embodiments, the bispecific antibody or antigen-binding fragment thereof described herein has a common light chain.

[0115] Antibody-drug conjugate (ADC) In some embodiments, the antigen-binding protein constructs described herein (e.g., bispecific antibodies) can be conjugated to a therapeutic agent, optionally via a linker, to form an antibody-drug conjugate. The antibody-drug conjugate, which includes an antibody or an antigen-binding fragment thereof, can bind to the therapeutic agent covalently or non-covalently. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, camptothecin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracene, maytansinoids (such as DM-1 and DM-4), dion, mitoxantrone, mitomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs thereof). In some embodiments, the therapeutic agent is MMAE or MMAF.

[0116] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition (back reference), and specific functional groups are generally defined as described therein. Further, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Organic Chemizstry, Thomas Sorrell, University Science Books, Sausalito, 1999, Smith and March, March’s Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001, Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, Carruthers, Some Modem Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0117] All ranges recited herein are inclusive whether or not explicitly stated. When a range of values is recited, that range is intended to include each value and sub-range within the range. For example, "C1-6" is intended to include C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6.

[0118] A compound, or any formula that illustrates and describes the compounds of the present disclosure, can have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomers of the compounds of the present invention, or any formula that illustrates and describes the compounds of the present invention. The asymmetric centers present in a compound, or any formula that illustrates and describes the compounds of the present invention, can each independently have an (R) or (S) configuration relative to each other. When a bond to a chiral carbon is depicted as a straight line in a structural formula, or when a compound name is listed without (R) or (S) chiral notation for a chiral carbon, both the (R) and (S) configurations of each such chiral carbon, and thus each enantiomer or diastereomer, and mixtures thereof, are understood to be included within the formula or by its name.

[0119] The present disclosure includes all possible enantiomers and diastereomers, as well as mixtures of two or more stereoisomers, for example, mixtures of enantiomers and / or diastereomers in any ratio. Thus, enantiomers in enantiomerically pure form, in the form of racemic compounds as both levorotatory and dextrorotatory enantiomers, and in the form of mixtures of two enantiomers in any ratio are the subject of the present disclosure. In the case of cis / trans isomerism, the present disclosure includes both the cis form and the trans form, as well as mixtures of these forms in any ratio. If desired, the preparation of individual stereoisomers can be carried out, as necessary, by separation of the mixture by conventional methods, for example, by chromatography or crystallization, by using a stereochemically homogeneous starting material for synthesis, or by stereoselective synthesis. Optionally, derivatization can be carried out prior to the separation of the stereoisomers. The separation step of the stereoisomer mixture can be carried out in an intermediate step during the synthesis of the compound or can be carried out on the final racemic product. The absolute stereochemistry can be measured, if necessary, by X-ray crystallographic analysis of the crystalline product or of the derivatized crystalline intermediate using a reagent containing a known calibrated stereocenter. Alternatively, the absolute stereochemistry can be measured by Vibrational Circular Dichroism (VCD) spectroscopy.

[0120] Unless otherwise specified, the structures described herein also include compounds that differ only in the presence of one or more isotopically enriched atoms, i.e., compounds in which one or more atoms have been replaced by atoms having the same atomic number but a different atomic mass or mass number than the preponderant atomic mass or mass number found in nature. Such compounds are referred to as “isotope variants.” This disclosure is intended to include all pharmaceutically acceptable isotope variants of the compounds of the invention, or of any formula that illustrates or describes the compounds of the invention. Examples of isotopes suitable for inclusion in the compounds of the invention include, but are not limited to, isotopes of hydrogen such as 2H (i.e., D) and 3H; carbon such as 11C, 13C, and 14C; chlorine such as 36Cl; fluorine such as 18F; iodine such as 123I and 125I; nitrogen such as 13N and 15N; oxygen such as 15O, 17O, and 18O; phosphorus such as 32P; and sulfur such as 35S. Compounds of the invention, or of any formula that illustrates or describes the compounds of the invention, that incorporate a particular isotope variant, e.g., a radioisotope, may be useful in drug and / or substrate tissue distribution studies. Specifically, compounds having the indicated structures that differ only in the substitution of a heavier isotope, e.g., substitution of hydrogen with deuterium (2H, or D), can afford certain therapeutic advantages resulting from, for example, greater metabolic stability, increased in vivo half-life, or reduced dosage requirements, and can therefore be utilized in some particular situations. Isotope variants of the compounds of the disclosure, or of any formula that illustrates or describes the compounds of the disclosure, can generally be prepared by procedures similar to those described in the appended examples and syntheses, using appropriately isotopically labeled reagents by techniques well known to those of ordinary skill in the art or by using isotopically labeled reagents in place of the previously used unlabeled reagents.

[0121] The compounds provided herein are described with reference to both general formulas and specific compounds. Further, the compounds of the present disclosure can exist in a number of different forms or derivatives, all of which are within the scope of the present disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, positional isomers, prodrugs, solvated forms, different crystalline forms or polymorphs, and activated metabolites.

[0122] As used herein, unless otherwise specified, the term "pharmaceutically acceptable salt" includes salts that retain the biological effectiveness of the free acid / base form of the specified compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts can include salts formed with inorganic bases or acids, and organic bases or acids. When the compounds of the present disclosure contain one or more acidic or basic groups, the present disclosure also includes their corresponding pharmaceutically acceptable salts. Thus, the compounds of the present invention containing acidic groups such as carboxyl groups can exist in salt form and can be used, according to the present invention, for example, as alkali metal salts, alkaline earth metal salts, aluminum salts, or ammonium salts. Further non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts with organic amines such as ammonia, ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids. These salts can be readily obtained, for example, by reacting a compound having an acidic group with a suitable base such as lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide. Other base salts of the compounds of the present disclosure include, but are not limited to, copper(I), copper(II), iron(II), iron(III), manganese(II), and zinc salts. The compounds of the present disclosure containing one or more basic groups, for example, groups that can be protonated, can exist in salt form and can be used in the form of addition salts with inorganic or organic acids according to the present disclosure.Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, embonic acid, mandelic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, and other acids well known to those skilled in the art. The salts formed include, inter alia, hydrochloride, chloride, hydrobromide, bromide, iodide, sulfate, phosphate, methanesulfonate (mesylate), tosylate, carbonate, bicarbonate, formate, acetate, sulfoacetate, triflate, oxalate, malonate, maleate, succinate, tartrate, malate, embonate, mandelate, fumarate, lactate, citrate, glutarate, stearate, aspartate, and glutamate. The stoichiometry of the salts formed from the compounds of the present disclosure may further be an integer or non-integer multiple of 1.

[0123] The compounds of the present disclosure containing a basic nitrogen-containing group can be quaternized using a C1-4 haloalkyl, for example, an agent such as methyl, ethyl, isopropyl, and tert-butyl chloride, bromide, and iodide; a di-C1-4 alkyl sulfate, for example, dimethyl, diethyl, and diamyl sulfate; a C10-18 haloalkyl, for example, decyl, dodecyl, lauryl, myristyl, and stearyl chloride, bromide, and iodide; and an aryl C1-4 haloalkyl, for example, benzyl chloride and phenethyl bromide.

[0124] When the compounds of the present disclosure contain both acidic and basic groups within the molecule, the present disclosure also includes inner salts or betaines (zwitterions) in addition to the described salt forms. The corresponding salts can be obtained by conventional methods well known to those skilled in the art, for example, by contacting them with an organic or inorganic acid in a solvent or dispersant, or by anion exchange or cation exchange with another salt. The present disclosure includes all salts of the compounds of the present disclosure which, due to their low physiological compatibility, are not directly suitable for use as drugs but can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts. For a review of more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002).

[0125] Any formula that illustrates and describes the compounds of the present disclosure, or the compounds of the present disclosure and their pharmaceutically acceptable salts thereof, can exist in unsolvated and solvated forms. As used herein, the term "solvate" means a molecular complex comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules. For example, the term "hydrate" is used when the solvent is water. Pharmaceutically acceptable solvates according to the present disclosure can include those in which the crystallization solvent can be isotope-substituted, for example, D2O, d6-acetone, d6-DMSO.

[0126] Linker (binding agent compound) In some embodiments, the therapeutic agent is conjugated via a linker (or a linker compound). As used herein, the terms "linker" or "linker compound" refer to a ligand (e.g., an antigen-binding protein construct described herein (e.g., a bispecific antibody)) and a therapeutic agent (e.g., any of the therapeutic agents described herein), for example, by a coupling reaction, by reacting a group of the ligand compound with the therapeutic agent compound respectively to connect and form a ligand-drug conjugate.

[0127] In some embodiments, the linker described herein is a compound having the following formula:

Chemical formula

[0128] In some embodiments, the joining moiety (Q in formula (I)) has the following structure:

Chemical formula

[0129] In some embodiments, the linker moiety (L in formula (I)) has the following formula:

Chemical formula

Chemical formula

[0130] In some embodiments, the polypeptide residue L1 is NH-Glu-Val-Ala-COOH. In some embodiments, the hydrophilic group L2 has the following structure:

Chemical formula

[0131] In some embodiments, the linker described herein is a compound having the following structure:

Chemical formula

[0132] In some embodiments, the linker is a VC linker. Details of linkers that can be used for ADCs can be found, for example, in Su, Z. et al. "Antibody-drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), which is hereby incorporated by reference in its entirety.

[0133] Therapeutic agent In some embodiments, the therapeutic agent conjugated to the antigen-binding protein constructs (e.g., bispecific antibodies) described herein is discussed as follows.

[0134] In some embodiments, the therapeutic agent described herein is a cytotoxic agent. In some embodiments, the cytotoxic agent is a camptothecin compound, an analog or derivative thereof. In some preferred embodiments, the camptothecin compound is a compound having the following structure:

Chemical formula

[0135] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[3’,4’:6,7]indolizino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione) (CPT-1). The structure of CPT-1 is shown below:

Chemical formula

[0136] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(2H)-dione (CPT-2). The structure of CPT-2 is shown below:

Chemical formula

[0137] In some embodiments, the therapeutic agent is CPT3. The structure of CPT-3 is shown below:

Chemical formula

[0138] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(2H)-dione (CPT-4). The structure of CPT-4 is shown below:

Chemical formula

[0139] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also well-known in the art as a derivative of dolastatin-10), or a derivative thereof. The auristatin can be, for example, an ester formed from auristatin E and a keto acid. For example, auristatin E can react with para-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. Exemplary syntheses and structures of auristatins are described in U.S. Patent Publication No. 2003-0083263; International Patent Publication Nos. WO 04 / 010957, WO 02 / 088172, and U.S. Patents Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated herein by reference in its entirety for all purposes.

[0140] Auristatins have been shown to interfere with microtubule dynamics and with nuclear and cell division, and have been shown to have anti-cancer activity. Auristatins can bind to tubulin and exert a cytotoxic or cytostatic effect in cancer cells. A number of different assays well-known in the art exist and can be used to measure whether an auristatin or an antibody-drug conjugate obtained therefrom exerts a cytostatic or cytotoxic effect in the desired cells.

[0141] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include thiotepa and cyclophosphamide (CYTOXAN TMalkylating agents such as; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; nitrogen mustards such as chlorambucil, chloronaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobenbitin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chloroozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, calminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodrubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;Bestrabucil; Bisantrene; Edatrexate; Defofamine; Demecortin; Diacquone; Elfomithine; Elliptinium acetate; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK7; Razoxane; Schizophyllan; Spirogermanium; Tenuazonic acid; Triaziquone; 2’,2’,2’-Trichlorotriethylamine; Urethane; Vincdesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacitabine; Arabinoside (“Ara-C”); Cyclophosphamide; Taxanes, such as paclitaxel (TAXOL(R), Bristol-Myers Squibb Oncology, Princeton, N.J.), docetaxel (TAXOTERE(R), Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-Thioguanine, platinum analogs such as cisplatin and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, ZERODA, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO); Retinoic acid; Esperamicin; Capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. This definition includes, for example, tamoxifen, raloxifene, aromatase inhibitor 4(5)imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;Also included are antihormonal agents that have the effect of regulating or inhibiting the hormonal action in tumors, such as antiestrogen agents containing any of the above pharmaceutically acceptable salts, acids, or derivatives. A detailed description of the chemotherapeutic agents can be found, for example, in US20180193477A1, which is incorporated herein by reference in its entirety.;

[0142] Linker-therapeutic agent compound In some embodiments, a linker (e.g., any of the linkers described herein) and a therapeutic agent (e.g., any of the therapeutic agents described herein) can be linked to form a "linker-therapeutic agent" compound.;

[0143] In some embodiments, the linker-therapeutic agent compound has the following structure: [Chemical formula]

[0144] In some embodiments, the linker-therapeutic agent compound has the following structure: [Chemical formula]

[0145] In some embodiments, an antibody ("Ab"), e.g., any of the antigen-binding protein constructs (e.g., bispecific antibodies) described herein, can be linked to a linker-therapeutic agent compound (e.g., any of the linker-therapeutic agent compounds described herein) to generate an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate has the following structure: [Chemical formula] wherein n = 1, 2, 3, 4, 5, 6, 7, or 8.;

[0146] Antibody and ADC properties An anti-EGFR / MET antigen-binding protein construct (e.g., an antibody, bispecific antibody, or antibody fragment thereof) can include an antigen-binding region derived from any anti-EGFR antibody described herein, or any antigen-binding fragment thereof.

[0147] The present disclosure provides anti-EGFR / MET antigen-binding protein constructs that specifically bind to EGFR. These antigen-binding protein constructs can be agonists or antagonists. The antigen-binding protein constructs described herein can bind to EGFR and block the binding of EGFR to EGF and / or the binding of EGFR to TGFα. By blocking the binding of EGFR to EGF and / or the binding of EGFR to TGFα, the antigen-binding protein constructs can inhibit the EGFR-related signaling pathway, and thus treat cancer (e.g., NSCLC). In some embodiments, the antigen-binding protein constructs can initiate CDC or ADCC.

[0148] General techniques that can be used to measure the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). The affinity can be estimated from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, an antigen-binding protein construct (e.g., a bispecific antibody) can bind to EGFR (e.g., human EGFR, monkey EGFR, mouse EGFR, and / or chimeric EGFR) with a dissociation rate (koff) of less than 0.1 s-1, less than 0.01 s-1, less than 0.001 s-1, less than 0.0001 s-1, or less than 0.00001 s-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s-1, greater than 0.001 s-1, greater than 0.0001 s-1, greater than 0.00001 s-1, or greater than 0.000001 s-1.

[0149] In some embodiments, the association rate (kon) is greater than 1×102 / Ms, greater than 1×103 / Ms, greater than 1×104 / Ms, greater than 1×105 / Ms, or greater than 1×106 / Ms. In some embodiments, the association rate (kon) is less than 1×105 / Ms, less than 1×106 / Ms, or less than 1×107 / Ms.

[0150] In some embodiments, an antigen-binding protein construct (e.g., a bispecific antibody) can bind to EGFR (e.g., human EGFR, monkey EGFR, mouse EGFR, and / or chimeric EGFR) with a KD less than 1×10−6M, less than 1×10−7M, less than 1×10−8M, less than 1×10−9M, or less than 1×10−10M. In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1×10−7M, greater than 1×10−8M, greater than 1×10−9M, or greater than 1×10−10M.

[0151] The anti-EGFR / MET antigen-binding protein construct (e.g., a bispecific antibody) can also include an antigen-binding region derived from any anti-MET antibody or antigen-binding fragment thereof described herein. The anti-MET antibody or antigen-binding fragment thereof described herein can block the binding of MET to HGF. In some embodiments, by binding to MET, the antigen-binding protein construct can also inhibit the MET-related signaling pathway, thereby inhibiting cell proliferation, differentiation, and / or metastasis. Thus, in some embodiments, the antigen-binding protein construct described herein is a MET agonist. In some embodiments, the antigen-binding protein construct is a MET antagonist.

[0152] In some embodiments, an antigen-binding protein construct (e.g., a bispecific antibody) can bind to MET (e.g., human MET, monkey MET, mouse MET, and / or chimeric MET) with a dissociation rate (koff) of less than 0.1 s-1, less than 0.01 s-1, less than 0.001 s-1, less than 0.0001 s-1, or less than 0.00001 s-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s-1, greater than 0.001 s-1, greater than 0.0001 s-1, greater than 0.00001 s-1, or greater than 0.000001 s-1.

[0153] In some embodiments, the association rate (kon) is greater than 1×102 / Ms, greater than 1×103 / Ms, greater than 1×104 / Ms, greater than 1×105 / Ms, or greater than 1×106 / Ms. In some embodiments, the association rate (kon) is less than 1×105 / Ms, less than 1×106 / Ms, or less than 1×107 / Ms.

[0154] Affinity can be estimated from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10-6 M, less than 1×10-7 M, less than 1×10-8 M, less than 1×10-9 M, or less than 1×10-10 M. In some embodiments, KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1×10-7 M, greater than 1×10-8 M, greater than 1×10-9 M, or greater than 1×10-10 M.

[0155] Since the antigen-binding protein construct (e.g., a bispecific antibody) binds to both MET and EGFR, with respect to cells that express both MET and EGFR, the antigen-binding protein construct has a high binding affinity for these cells. Using the binding activity, the binding affinity of the antigen-binding protein construct for these cells can be measured. Binding activity is the cumulative strength of the affinities of individual non-covalent interactions.

[0156] Thermal stability can also be measured. The antigen-binding protein constructs (e.g., bispecific antibodies) described herein can have a Tm above 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. Since IgG can be described as a multi-domain protein, the melting curve may, in some cases, show two transitions, with the first denaturation temperature being Tm D1 and the second denaturation temperature being Tm D2. The presence of these two peaks often indicates the denaturation of the Fc domain (Tm D1) and the Fab domain (Tm D2), respectively. When two peaks are present, Tm usually refers to Tm D2. Thus, in some embodiments, the antibodies or antigen-binding fragments described herein can have a Tm D1 above 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the antibodies or antigen-binding fragments described herein can have a Tm D2 above 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, Tm, Tm D1, and Tm D2 are below 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0157] In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) can bind to human EGFR or monkey EGFR. In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) cannot bind to human EGFR or monkey EGFR. In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) can bind to human MET or monkey MET. In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) cannot bind to human MET or monkey MET.

[0158] In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) has a purity of greater than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as measured by, for example, HPLC. In some embodiments, the purity is less than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as measured by, for example, HPLC.

[0159] In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) has a tumor growth inhibition rate or percentage (TGI%) of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or more than 200%. In some embodiments, the antibody has a tumor growth inhibition percentage (%) of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. TGI(%) can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days after the start of treatment. As used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%

[0160] Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day 0. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day 0.

[0161] In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0162] In some embodiments, the antigen-binding protein construct (e.g., bispecific antibody) does not have a functional Fc region. For example, the protein construct is a Fab, Fab’, F(ab’)2, and Fv fragment. In some embodiments, the protein constructs described herein have an Fc region that does not contain effector functions. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (L234A and L235A mutations in EU numbering), or an LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

[0163] Several other modifications can be made to the Fc region. For example, cysteine residues can be introduced into the Fc region to allow for the formation of inter-chain disulfide bonds within this region. The homodimeric fusion protein thus generated may have some increased in vitro and / or in vivo half-life.

[0164] In some embodiments, IgG4 has an S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab-arm exchange in vivo and in vitro.

[0165] In some embodiments, there is provided an Fc region having a carbohydrate structure lacking fucose (directly or indirectly) linked to the Fc region. For example, the amount of fucose in such an Fc region composition may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined, for example, by calculating the average amount of fucose in the sugar chain at Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn297, as measured by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 within the Fc region (Eu numbering of Fc region residues, or position 314 in Kabat numbering), however, Asn297 may also be located upstream or downstream by about ±3 amino acids from position 297, i.e., between positions 294 and 300, due to minor sequence variations in the Fc region sequence. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce the heterogeneity of the glycan, the Fc region is further engineered to substitute the asparagine at position 297 with alanine (N297A).

[0166] In some embodiments, the main peak of HPLC-SEC accounts for at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the protein complexes described herein after purification by protein A-based affinity chromatography and / or size exclusion chromatography.

[0167] In some embodiments, the ADCs described herein have an IC50 for in vitro killing of cancer cells (e.g., lung cancer cell line NCI-H1975) of less than 2 μg / mL, less than 1.5 μg / mL, less than 1 μg / mL, less than 0.9 μg / mL, less than 0.8 μg / mL, less than 0.7 μg / mL, less than 0.6 μg / mL, less than 0.5 μg / mL, less than 0.4 μg / mL, less than 0.3 μg / mL, less than 0.2 μg / mL, or less than 0.1 μg / mL.

[0168] In some embodiments, the bispecific antibodies described herein have an endocytosis rate that is higher than that of the corresponding monoclonal antibodies and / or control bispecific antibodies described herein. In some embodiments, the anti-EGFR antibodies described herein have an endocytosis rate that is higher than that of cetuximab analogs. In some embodiments, the anti-MET antibodies described herein have an endocytosis rate that is higher than that of teprotumumab analogs. In some embodiments, the bispecific antibodies described herein have an endocytosis rate that is higher than that of amivantamab analogs.

[0169] Method for producing an antigen-binding protein construct Isolated fragments of human proteins can be used as immunogens, and antibodies can be generated using standard techniques for the preparation of polyclonal and monoclonal antibodies. Polyclonal antibodies can be raised in animals by injecting the antigenic peptide or protein multiple times (e.g., subcutaneously or intraperitoneally). In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species being immunized. The antigenic peptide or protein can be injected into the animal two or more times (e.g., two, three, or four times).

[0170] Full-length polypeptides or proteins can be used, or antigenic peptide fragments thereof can be used as immunogens. The antigenic peptides of the protein comprise at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of the protein and encompass the epitopes of the protein such that the antibodies generated against the peptide form specific immune complexes with the protein.

[0171] Immunogens are typically used for the preparation of antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations can contain, for example, recombinantly expressed or chemically synthesized polypeptides. The preparations can further contain adjuvants such as Freund's complete or incomplete adjuvant, or similar immunostimulants.

[0172] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide or its antigenic peptide (e.g., a part of a protein) as an immunogen. Standard techniques such as enzyme-linked immunosorbent assay (ELISA) using an immobilized polypeptide or peptide can be used to monitor the antibody titer in the immunized subject over time. If desired, antibody molecules can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques such as protein G or protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, when the titer of specific antibodies is at its maximum, antibody-producing cells are obtained from the subject and used to prepare monoclonal antibodies by standard techniques such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), and EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma technique. Techniques for producing hybridomas are well known (generally, see Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). For example, monoclonal antibodies can be detected by screening hybridoma culture supernatants for antibodies that bind to the polypeptide or epitope of interest using a standard ELISA assay.

[0173] Appropriate nucleotide changes can be introduced into DNA encoding human, humanized, or chimeric antibodies, or the antibodies or antigen-binding fragments thereof described herein, or variants of the antigen-binding protein constructs described herein can be prepared by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids of the sequence that produces the antigen-binding site or antigen-binding domain of an antibody. Among such a population of variants, some antibodies or antigen-binding fragments have an increased affinity for the target protein. Any combination of deletions, insertions, and / or substitutions can be achieved in an antibody or its antigen-binding fragment with an increased binding affinity for the target. Changes in the number of glycosylation sites (e.g., increase or decrease), changes in the type of glycosylation sites (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in the cell), or introduction of new glycosylation sites, etc., can change the antibody or antigen-binding fragment or introduce new post-translational modifications into the antibody or antigen-binding fragment by the amino acid changes introduced into the antibody or antigen-binding fragment.

[0174] The antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates such as monkeys and apes, cows, pigs, horses, sheep, camels (e.g., dromedaries and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

[0175] Antibody sequences with desired binding affinities can be optimized using phage display (panning). In this technique, a gene encoding a single-chain Fv (including VH or VL) is inserted into the phage coat protein gene, allowing the phage to "display" the scFv on its outer surface while containing the gene for the protein inside, resulting in the linkage of genotype and phenotype. To detect the interaction between the displayed antigen-binding site and the target antigen, the phage displaying the antigen can then be screened against the target antigen. Thus, a large library of proteins can be screened and amplified in a process called in vitro selection, and antibody sequences with desired binding affinities can be obtained.

[0176] Human and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences (or having the same amino acid sequence as those derived therefrom). Examples of human antibodies can include amino acid residues encoded by human germline immunoglobulin sequences (such as mutations introduced by in vitro random or site-directed mutagenesis, or somatic mutations in vivo) that are not present within the CDRs.

[0177] Humanized antibodies typically have a human framework (FR) onto which non-human CDRs are grafted. Thus, humanized antibodies have one or more amino acid sequences introduced from a non-human source into a human. Therefore, a "humanized" antibody is a chimeric antibody in which a considerably smaller portion than an intact human V domain is replaced by the corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues, as well as some FR residues, are replaced by residues derived from similar sites within the human antibody.

[0178] Furthermore, it is important to humanize antibodies while retaining high specificity and affinity for the antigen, as well as other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by an analytical process of parental and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that can illustrate and display the possible three-dimensional conformations of selected candidate immunoglobulin sequences. By observing these displays, it becomes possible to analyze the possible roles of residues in the functionalization of candidate immunoglobulin sequences, i.e., to analyze the residues of candidate immunoglobulins that affect their ability to bind to the antigen. In this way, FR residues can be selected and combined from recipient and import sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0179] In some embodiments, a mouse having a humanized heavy-chain immunoglobulin locus and a humanized κ-chain immunoglobulin locus (e.g., RenMab TM mouse) is used to generate antibodies. A heavy-chain immunoglobulin locus is a region on a chromosome that contains genes for the heavy chain of an antibody. The locus can include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy-chain constant domain genes. A κ-chain immunoglobulin locus is a region on a chromosome that contains genes encoding the light chain (κ-chain) of an antibody. Examples of κ-chain immunoglobulin loci include human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light-chain constant domain genes. RenMab TM A detailed description of the RenMab mouse can be found in PCT / CN2020 / 075698 or US20200390073A1, which are hereby incorporated by reference in their entirety. In some embodiments, a mouse having a humanized heavy-chain immunoglobulin locus and a humanized κ-chain immunoglobulin locus (e.g., RenLite TMUsing a mouse, antibodies are generated. The heavy chain immunoglobulin locus is a region on a chromosome that contains genes for the heavy chains of antibodies. The locus can include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes. The κ-chain immunoglobulin locus is a region on a chromosome that contains genes encoding a common light chain. Examples of the κ-chain immunoglobulin locus can include human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes. RenLite TM Detailed descriptions regarding the mouse can be found in PCT / CN2021 / 097652, which is hereby incorporated by reference in its entirety.

[0180] Identity or homology to the original sequence typically refers to the percentage of amino acid residues present in a candidate sequence that is identical to the sequence present in a human, humanized, or chimeric antibody or fragment, where the sequences are aligned, gaps are introduced if necessary to achieve maximum percent sequence identity, and conservative substitutions as part of sequence identity are not considered.

[0181] In some embodiments, a covalent modification can be added to an antibody, its antigen-binding fragment, or an antigen-binding protein construct (e.g., a bispecific antibody). These covalent modifications can be added by chemical or enzymatic synthesis or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting the targeted amino acid residues of the antibody or fragment with an organic derivatizing agent capable of reacting with a selected side chain or N- or C-terminal residue.

[0182] In some embodiments, provided are antibody variants having a carbohydrate structure lacking fucose (directly or indirectly) linked to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined, for example, by calculating the average amount of fucose within the sugar chain at Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn297, as measured by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 within the Fc region (EU numbering of Fc region residues, or position 314 in Kabat numbering), however, Asn297 may also be located upstream or downstream by about ±3 amino acids from position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody is further recombinantly engineered to substitute the asparagine at position 297 with alanine (N297A).

[0183] In some embodiments, to promote production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further recombinantly engineered to substitute the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is provided, for example, in Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9 (2015):5462 - 5469, which is incorporated herein by reference in its entirety.

[0184] In some embodiments, the methods described herein are designed to make bispecific antibodies. By engineering the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture, bispecific antibodies can be produced. For example, the interface can comprise at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers. The method is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0185] In some embodiments, Knobs-into-Holes (KIH) technology can be used, which involves engineering the CH3 domain to create either a "knob" or a "hole" in each heavy chain, facilitating heterodimerization. The KIH technology is described, for example, in Xu, Yiren, et al. "Production of bispecific antibodies in 'knobs-into-holes' using a cell-free expression system." MAbs. Vol. 7. No. 1. Taylor & Francis, 2015, which is incorporated herein by reference in its entirety. In some embodiments, one heavy chain has T366W and / or S354C (knob) substitutions (EU numbering), and the other heavy chain has Y349C, T366S, L368A, and / or Y407V (hole) substitutions (EU numbering). In some embodiments, one heavy chain has one or more of the substitutions Y349C and T366W (EU numbering). The other heavy chain can have one or more of the substitutions E356C, T366S, L368A, and Y407V (EU numbering). Additionally, the substitution (-ppcpScp-->-ppcpPcp-) can be introduced into the hinge region of both substituted IgGs. Bispecific antibodies can also include, for example, cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies within the heteroconjugate can be conjugated to avidin and the other to biotin. Heteroconjugate antibodies can also be produced using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0186] Methods for generating bispecific antibodies from antibody fragments are also well known in the art. For example, bispecific antibodies can be prepared using chemical linkages. Brennan et al. (Science 229:81, 1985) describe procedures by which intact antibodies are cleaved by proteolysis to generate F(ab’)2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize the neighboring dithiols and prevent intermolecular disulfide formation. The resulting Fab’ fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, mixed with an equimolar amount of another Fab’ TNB derivative, and a bispecific antibody is formed.

[0187] Recombinant vector The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cells contain the polynucleotide and / or a vector containing the polynucleotide), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0188] As used herein, a "vector" is any construct that can deliver one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" can deliver one or more polynucleotides of interest as a polypeptide encoded therein and expressed in a host cell into which the expression vector has been introduced. Thus, within the expression vector, the polynucleotide of interest is operably linked to control elements such as a promoter, an enhancer, and / or a polyA tail at, near, or adjacent to the integration site of the polynucleotide of interest within the vector or in the genome of the host cell, such that the polynucleotide of interest is positioned for expression within the vector.

[0189] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant virus). Thus, non-limiting examples of vectors include viral vectors (those that can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents. In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of a non-pathogenic (defective) replicable virus, or may involve the use of a replication-incompetent virus. In the latter case, viral growth generally occurs only in complementary virus packaging cells. For example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317 - 321, Flexner et al., 1989, Ann. N.Y. Acad Sci. 569:86 - 103, Flexner et al., 1990, Vaccine, 8:17 - 21, U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487, WO 89 / 01973, U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242, WO 91 / 02805, Berkner - Biotechniques, 6:616 - 627, 1988; Rosenfeld et al., 1991, Science, 252:431 - 434, Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215 - 219; Kass - Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498 - 11502; Guzman et al., 1993, Circulation, 88:2838 - 2848; and Guzman et al., 1993, Cir. Res., 73:1202 - 1207 disclose suitable systems. Techniques for incorporating DNA into such expression systems are well known to those of skill in the art. DNA may also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745 - 1749, and Cohen, 1993, Science, 259:1691 - 1692. Incorporation of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells. For expression, a DNA insert containing a polynucleotide encoding an antibody or a polypeptide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage λPL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and the promoter of the retroviral LTR. Other suitable promoters are known to those skilled in the art. The expression construct can further contain sites for transcription initiation and termination, and within the transcription region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct can include a translation initiation at the beginning and a stop codon (UAA, UGA, or UAG) approximately located at the end of the translated polypeptide.

[0190] As shown, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase for eukaryotic cell culture, or neomycin resistance, and the tetracycline or ampicillin resistance genes for E. coli and other bacterial cultures. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Suitable culture media and conditions for the host cells described herein are well known in the art.

[0191] Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9 available from Qiagen, pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic cell vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0192] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic cell promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral type LTR such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0193] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters such as the alpha factor, alcohol oxidase, and PGH may be used. See Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y and Grant et al., Methods Enzymol., 153:516-544 (1997) for reviews.

[0194] Introduction of the construct into host cells can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), the entire contents of which are incorporated herein by reference.

[0195] Transcription of the DNA encoding the antibodies of the present disclosure by more eukaryotes can be increased by inserting enhancer sequences into the vector. Enhancers are typically cis-acting elements of DNA, about 10 - 300 bp, that serve to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located downstream of the origin of replication at base pairs 100 - 270, the cytomegalovirus immediate early promoter enhancer, the polyoma enhancer downstream of the origin of replication, and the adenovirus enhancer.

[0196] To secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. The signal can be endogenous to the polypeptide, or the signal can be a heterologous signal.

[0197] A polypeptide (e.g., an antibody) can be expressed in a modified form such as a fusion protein (e.g., a GST fusion), or with a histidine tag, and can also contain not only a secretion signal but also additional heterologous functional regions. For example, regions of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and durability in the host cell during purification or subsequent handling and storage. Also, a peptide moiety can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. Adding a peptide moiety to a polypeptide to, inter alia, effect secretion or excretion, improve stability, and facilitate purification is well-known and routine in the art.

[0198] The present disclosure also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence described herein, and amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence described herein.

[0199] The present disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any nucleotide sequence described herein, and amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any amino acid sequence described herein.

[0200] In some embodiments, the present disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0201] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, and the amino acid sequence is any one of the sequences described herein.

[0202] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, and the nucleic acid sequence is any one of the sequences described herein.

[0203] To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences to optimize alignment for comparison, and non-homologous sequences may be disregarded). Subsequently, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are considered identical at that position (as used herein, "identity" of an amino acid or nucleic acid corresponds to "homology" of an amino acid or nucleic acid). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparison and determination of percent identity between two sequences can be performed using the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0204] The percentage of sequence identity (e.g., amino acid sequence identity or nucleic acid identity) can also be measured. Methods for measuring the percentage of sequence identity are well known in the art. In some embodiments, sequence similarity can be measured using amino acid residues conserved with similar physicochemical properties (% identity), such as leucine and isoleucine. Families of amino acid residues with similar physicochemical properties are defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Often, the percentage of homology is higher than the percentage of identity.

[0205] The present disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some embodiments, the nucleic acid (e.g., cDNA) comprises a polynucleotide encoding a polypeptide of the heavy chain described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide of the light chain described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding an scFv polypeptide described herein.

[0206] In some embodiments, the vector can have two of the nucleic acids described herein, and the vector encodes a VL region and a VH region that both bind to EGFR. In some embodiments, a pair of vectors is provided, each vector comprising one of the nucleic acids described herein, and the pair of vectors together encodes a VL region and a VH region that both bind to EGFR.

[0207] In some embodiments, the vector comprises two of the nucleic acids described herein, and the vectors together encode a VL region and a VH region that both bind to MET. In some embodiments, a pair of vectors is provided, each vector comprising one of the nucleic acids described herein, and the pair of vectors together encodes a VL region and a VH region that both bind to MET.

[0208] Vectors can also be constructed to express specific antibodies or polypeptides. In some embodiments, a vector can be constructed to co-express the light chain (EGFR-K) and heavy chain (EGFR-H) of an anti-EGFR antibody. In some embodiments, the vector can contain the sequences of the cytomegalovirus promoter (CMV), EGFR-K, polyadenylation (polyA), CMV, EGFR-H, polyA, simian vacuolar virus 40 terminator (SV40), and glutamine synthetase marker (GS) in the direction from the 5'-end to the 3'-end. In some embodiments, a vector can be constructed to co-express the anti-MET antibody light chain (MET-K) and the anti-MET antibody heavy chain (MET-H). In some embodiments, the vector can contain the sequences of CMV, MET-K, polyA, CMV, MET-H, SV40, and GS in the direction from the 5'-end to the 3'-end. In some embodiments, the vector can be constructed to express the anti-MET antibody scFv polypeptide chain. In some embodiments, cells (e.g., CHO cells) are co-transfected using a first vector that expresses an antibody heavy chain (e.g., any of the heavy chains described herein) and a second vector that expresses an antibody light chain (e.g., any of the light chains described herein) to produce the monoclonal antibody or antigen-binding fragment thereof described herein. In some embodiments, cells (e.g., CHO cells) are co-transfected using a first vector that expresses an anti-EGFR antibody heavy chain (e.g., any of the anti-EGFR antibody heavy chains described herein), a second vector that expresses an anti-MET antibody heavy chain (e.g., any of the anti-MET antibody heavy chains described herein), and a third vector that expresses a common light chain (e.g., any of the common light chains described herein) to produce the antigen-binding protein construct described herein (e.g., any of the anti-EGFR / MET bispecific antibodies described herein).

[0209] Treatment method The methods described herein include methods of treating disorders associated with cancer. Generally, the methods include administering to a subject in need of such treatment, or in whom such treatment need has been measured, a therapeutically effective amount of an antigen-binding protein construct (e.g., a bispecific antibody) described herein.

[0210] As used in this context, "treatment" means alleviating at least one symptom of a disorder associated with cancer. Often, cancer results in death. Thus, treatment can result in an increase in mean life expectancy (e.g., of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). Treatment of a condition associated with cancer results in a decrease in the number of cancer cells and / or alleviation of symptoms by administering a therapeutically effective amount of an agent described herein.

[0211] As used herein, the term "cancer" means an abnormal condition or state characterized by cells having the ability of autonomous growth, i.e., rapidly proliferating cell growth. This term means to include all types of cancerous growths or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. As used herein, the term "tumor" means cancerous cells, e.g., a mass of cancerous cells. Cancers treatable or diagnosable using the methods described herein include malignant tumors of various organ systems such as those affecting the lung, breast, thyroid, lymphatic, gastrointestinal, and urogenital systems, in addition to adenocarcinomas including most colorectal cancers, renal cell cancers, prostate cancers and / or testicular tumors, non-small cell lung cancers, small intestine cancers, and malignant tumors such as esophageal cancers. In some embodiments, the agents described herein are designed to treat or diagnose cancerous tumors in a subject. The term "cancerous tumor" is recognized in the art and means a malignant tumor of epithelial or endocrine gland tissue, including respiratory system cancer, digestive system cancer, urogenital system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancerous tumors include those formed from tissues of the cervix, lung, prostate, chest, head and neck, colon, and ovary. This term also includes carcinosarcomas, which include, for example, malignant tumors composed of cancerous and sarcomatous tissues. "Adenocarcinoma" means a cancerous tumor derived from glandular tissue, or a cancerous tumor in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and means a malignant tumor of mesenchymal origin.

[0212] In some embodiments, the cancer is a chemotherapy-resistant cancer. In one aspect, the present disclosure also provides a method for treating cancer in a subject, a method for decreasing the rate of increase of tumor volume over time in a subject, a method for reducing the risk of metastasis, or a method for reducing the risk of further metastasis in a subject. In some embodiments, treatment can interrupt, slow down, arrest, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0213] In one aspect, the present disclosure relates to a method comprising administering a therapeutically effective amount of an antigen-binding protein construct (e.g., a bispecific antibody) or an antibody-drug conjugate disclosed herein to a subject in need thereof, such as a subject having or identified or diagnosed as having cancer, such as a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung cancer), gastric cancer (e.g., gastric carcinoma), skin cancer (e.g., cutaneous carcinoma), colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal cancer, carcinoma of the ampulla, or a brain tumor.

[0214] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to an animal, human, or non-human to whom treatment according to the methods of the invention is provided. Veterinary and non-veterinary uses are contemplated by the present invention. A human patient can be an adult human or a juvenile human (e.g., a human less than 18 years old). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, suids (e.g., pigs, mini-pigs), equids, canids, felids, bovids, and other domestic, livestock, and zoo animals.

[0215] In some embodiments, the compositions and methods disclosed herein can be used for the treatment of patients at risk of cancer. Patients suffering from cancer can be identified by various methods well known in the art.

[0216] As used herein, "effective amount" means an amount or dosage sufficient to produce a beneficial or desired result, including interrupting, delaying, preventing, or inhibiting the progression of a disease, such as cancer. The effective amount can vary, for example, depending on the age and weight of the subject to whom the antibody, antigen-binding fragment, antibody-drug conjugate, polynucleotide encoding the antibody, vector containing the polynucleotide, and / or composition thereof is administered, the severity of the symptoms, and the route of administration, and thus, the administration can be determined individually.

[0217] The effective amount can be administered in one or more administrations. For example, an effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate is an amount sufficient to alleviate, arrest, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or, in vitro, an amount sufficient to alleviate, arrest, stabilize, reverse, slow, and / or delay the growth of a cell (e.g., a biopsy cell, any of the cancer cells described herein, or a cell line (e.g., a cancer cell line)). As will be understood in the art, the effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate can vary, inter alia, depending on other factors, including the patient's medical history, as well as the type (and / or dosage) of the antibody used.

[0218] The effective amount and schedule for administering the antigen-binding protein constructs, polynucleotides encoding antibodies, antibody-drug conjugates, and / or compositions disclosed herein can be determined experimentally, and making such determinations is within the purview of those of ordinary skill in the art. Those of ordinary skill in the art will understand that the dosage that needs to be administered will vary depending on, for example, the mammal receiving the antibody, polynucleotide encoding the antibody, antibody-drug conjugate, and / or composition disclosed herein, the route of administration, the specific type of antigen-binding protein construct, polynucleotide encoding the antibody, antigen-binding fragment, antibody-drug conjugate, and / or composition used, and other agents administered to the mammal. Guidance for selecting appropriate dosages for antibodies or antigen-binding fragments can be found in the literature regarding the therapeutic use of antibodies and antigen-binding fragments, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0219] The typical daily dose of an effective amount of an antibody, its antigen-binding fragment, or an antigen-binding protein construct (e.g., a bispecific antibody) is from 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can be less than 100 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose can be more than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about, or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0220] In any of the methods described herein, at least one antigen-binding protein construct (e.g., bispecific antibody), antibody-drug conjugate, or pharmaceutical composition (e.g., any of the protein constructs, antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions described herein), and optionally, at least one additional therapeutic agent, can be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one protein construct, antigen-binding fragment, antigen-binding protein construct (e.g., bispecific antibody), or antibody-drug conjugate, and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment, and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment, and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered as a sustained-release oral formulation.

[0221] In some embodiments, one or more additional therapeutic agents can be administered to a subject before, or after administering, at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents, and the at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap between the bioactive period of the one or more additional therapeutic agents and the bioactive period of the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) within the subject.

[0222] In some embodiments, a subject can be administered at least one protein construct, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the protein constructs, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over a long period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of the treatment period using any of the methods described herein to diagnose or monitor the effectiveness of the treatment (e.g., observe at least one symptom of cancer). As described herein, a skilled medical professional can also vary (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments, antibody-drug conjugates (and / or one or more additional therapeutic agents) administered to the subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of the at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and well known in the art).

[0223] In some embodiments, one or more additional therapeutic agents can be administered to a subject. The additional therapeutic agent can include one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of phosphatidylinositol 3-kinase (PI3K), an inhibitor of Akt, an inhibitor of mTOR, a dual PI3K / mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK), and an inhibitor of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

[0224] In some embodiments, the additional therapeutic agent can include one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of an activated hedgehog signaling pathway, and an agent that selectively degrades an estrogen receptor. In some embodiments, the additional therapeutic agent can include one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leolysin, alimta, dicaida, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0225] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0226] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0227] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-4-1BB antibody, an anti-TIM3 antibody, or an anti-GITR antibody.

[0228] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions comprising at least one (e.g., 1, 2, 3, or 4) of the antigen-binding protein constructs, antibodies (e.g., bispecific antibodies), antigen-binding fragments, or antibody-drug conjugates described herein. Any two or more (e.g., 2, 3, or 4) of the antigen-binding protein constructs, antibodies, antigen-binding fragments, or antibody-drug conjugates described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical composition can be formulated in any manner well known in the art.

[0229] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition can include a sterile diluent (e.g., sterile water or saline), a nonvolatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (such as ascorbic acid or sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). Preparations of the composition can be formulated and enclosed in ampoules, disposable syringes, or multi-dose vials. If necessary (e.g., as in injectable formulations), appropriate fluidity can be maintained, for example, by using a coating such as lecithin or a surfactant. The absorption of the antibody or its antigen-binding fragment can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implant and microencapsulation delivery systems, including biodegradable and biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.). A composition containing one or more of the antigen-binding protein constructs, antibodies, antigen-binding fragments, and antibody-drug conjugates described herein can be formulated in unit dosage forms (i.e., physically discrete units containing a predetermined amount of the active compound, which facilitate administration and provide a uniform dosage) for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration.

[0230] The toxicity and therapeutic effect of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). The LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Agents with a high therapeutic index are preferred. If the agent exhibits undesirable side effects, care should be taken to minimize the potential for harm (i.e., reduce the undesirable side effects). Toxicity and therapeutic effect can be determined by other standard pharmaceutical procedures.

[0231] Data obtained from cell culture assays and animal studies can be used in formulating the appropriate dosage of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., 1, 2, 3, or 4) antigen-binding protein constructs, antibodies, or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) is an amount that treats (e.g., kills cancer cells) the subject's disease in a subject (e.g., a human subject identified as having cancer), or a subject identified as being at risk of developing a disease (e.g., a subject who has previously had cancer but is currently in remission), and reduces the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., a human). The effectiveness and administration of any of the antigen-binding protein constructs, antibodies, or antigen-binding fragments described herein can be determined by a healthcare professional or a veterinary professional using methods well known in the art, and additionally by observing one or more symptoms of the disease in the subject (e.g., a human). Certain factors can affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and the presence of other diseases).

[0232] Exemplary dosages include the amount (in milligrams or micrograms) of any of the antigen-binding protein constructs, antibodies, antigen-binding fragments, or antibody-drug conjugates described herein per kilogram of the subject's body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg). Although these dosages cover a wide range, those skilled in the art will understand that therapeutic agents, including antigen-binding protein constructs, antibodies, and antigen-binding fragments thereof, can determine these potencies and effective amounts by methods well known in the art. Typically, a relatively low dosage is first administered, and the responsible health care professional or veterinary professional (in the case of therapeutic use), or researcher (if still working in the development stage), can subsequently and gradually increase the dosage until an appropriate response is obtained. In addition, the specific dosage level for any particular subject is understood to depend on various factors including the activity of the specific compound used, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.

[0233] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for use in administration. The present disclosure also provides methods for manufacturing anti-EGFR / MET antigen-binding protein constructs or antibody-drug conjugates for various uses described herein.

Example

[0234] The present invention will be further illustrated by the following examples, which do not limit the scope of the present invention described in the claims.

[0235] Example 1. Preparation and Analysis of Anti-EGFR / MET Bispecific Antibody Provided herein are bispecific antigen-binding molecules that target EGFR and MET. These antigen-binding molecules are referred to herein as anti-EGFR / MET bispecific antibodies hereinafter.

[0236] Preparation of anti-EGFR / MET bispecific antibodies Anti-EGFR antibodies (E-1G11, VH SEQ ID NO: 28, VL SEQ ID NO: 32, and E-6C4, VH SEQ ID NO: 29, VL SEQ ID NO: 32) and anti-MET antibodies (M-2F11, VH SEQ ID NO: 30, VL SEQ ID NO: 32, and M-2G10, VH SEQ ID NO: 31, VL SEQ ID NO: 32) can be paired to form various bispecific antibodies. Vectors encoding the light and heavy chains of the antibodies were constructed. CHO-S cells were co-transfected with three vectors including a first vector encoding the heavy chain of the anti-EGFR antibody, a second vector encoding the heavy chain of the anti-MET antibody, and a third vector encoding a common light chain. After culturing for 14 days, the cell supernatant was collected and purified by protein A affinity chromatography.

[0237] Various methods can be used to reduce the possibility of incorrect pairing between the two heavy chains. For example, knob-into-hole mutations were introduced into the Fc region of the heavy chain of the anti-EGFR arm and the heavy chain of the anti-MET arm. Exemplary bispecific antibodies obtained include E-1G11-M-2F11, E-6C4-M-2F11, and E-6C4-M-2G10. To verify the binding affinity of the bispecific antibodies, anti-EGFR or anti-MET control bispecific antibodies were also generated in which one arm of the control bispecific antibody recognizes EGFR or MET and the other arm recognizes CD28. These control bispecific antibodies were generated using a similar method, for example, RenLite TM VH sequences were obtained by immunizing mice. Exemplary control bispecific antibodies were named E-1G11-CD28, E-6C4-CD28, CD28-M-2G10, and CD28-M-2F11.

[0238] The knob-into-hole mutation was introduced into all bispecific antibodies. For example, in E-1G11-M-2F11, the heavy chain constant region of E-1G11 contains the knob mutation, and the heavy chain constant region of M-2F11 contains the hole mutation. In E-6C4-M-2F11, the heavy chain constant region of E-6C4 contains the knob mutation, and the heavy chain constant region of M-2F11 contains the hole mutation. Exemplary antibody structures are shown in Figure 1, where Target 1 and Target 2 can be EGFR and MET, respectively; MET and EGFR, respectively; EGFR and CD28, respectively; or CD28 and MET, respectively.

[0239] The sequences of the light chain constant region, the heavy chain constant region with the knob mutation, and the heavy chain constant region with the hole mutation are shown in SEQ ID NO: 35, SEQ ID NO: 33, and SEQ ID NO: 34, respectively.

[0240] Internalization of Antibodies Targeting EGFR and MET Anti-EGFR / MET bispecific antibodies were added to NCI-H1975 cells (ATCC, accession number: CRL-5908), HCC827 cells (ATCC, accession number: CRL-2868), and NCI-H292 (ATCC, accession number: CRL-1848) cells together with pHAb-goat anti-human IgG secondary antibody, respectively, and incubated for 1 hour. The cells were centrifuged and washed with FACS buffer. MFI was measured using a flow cytometer. The endocytosis rate of the antibody was calculated. For the isotype control (ISO), an antibody targeting an irrelevant target protein was used. The results are shown in the table below.

[0241]

Table 1

[0242] The results showed that the endocytosis rate of the bispecific antibody E-6C4-M-2F11 was higher than that of E-6C4-M-2G10. These two bispecific antibodies also showed higher endocytosis rates than the corresponding monoclonal antibodies M-2F11 or M-2G10.

[0243] Purity Analysis of Antibody The purified anti-EGFR / MET bispecific antibody was analyzed by non-reducing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and SEC-UPLC (size exclusion chromatography-high performance liquid chromatography).

[0244] Non-reducing SDS-PAGE was performed using a 4-12% acrylamide gel. The protein samples were prepared as follows. First, 2.4 μL of the protein sample was mixed with 6 μL of Tris-glycine SDS sample buffer (2×) (Invitrogen, catalog number: LC2676) and 3.6 μL of distilled water. Next, the mixture was boiled for 2 minutes and immediately centrifuged before loading. 4 μg of each sample was loaded onto the gel.

[0245] In the SEC-UPLC method, the antibody sample was diluted to 1 mg / mL with purified water and analyzed using an Agilent 1290 chromatography system (equipped with an XBridge TM Protein BEH SEC column (200 Å, Waters Corporation)). The following parameters were used: mobile phase: 100 mmol / L phosphate buffer (PB) (pH 7.4) + 0.2 mmol / L NaCl + 10% acetonitrile; flow rate: 1.8 mL / min; column temperature: 25°C; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: 6°C; and effective time: 7 minutes. The results are summarized in the table below.

[0246]

Table 2

[0247] Binding Activity of Anti-EGFR / MET Bispecific Antibody The binding activities of the anti-EGFR / MET bispecific antibody against human EGFR, human MET, monkey EGFR, and monkey MET were determined using a Biacore equipped with a pre-immobilized Protein A sensor chip. TM(Biacore, Inc., Piscataway, N.J.) It was verified by surface plasmon resonance (SPR) using an 8K biosensor. Specifically, hEGFR-His (ACROBiosystems Inc., catalog number: EGR-H5222), hMET-His (ACROBiosystems Inc., catalog number: MET-H5227), fasEGFR-His (ACROBiosystems Inc., catalog number: EGR-C52H5), and fasMET-His (Sino Biological Inc., catalog number: 90304-C08H) were diluted with 1×HBS-EP+ buffer (pH 7.4) to 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 6.25 nM, 3.125 nM, and 1.5626 nM, and then injected into the Biacore TM 8K biosensor at 10 μL / min for about 50 seconds to achieve the desired protein density (e.g., about 100 response units (RU)). Next, a purified antibody at a concentration of 1 μg / mL in 1×HBS-EP+ buffer (pH 7.4) was injected at 10 μL / min for 50 seconds. Dissociation was monitored for 400 seconds. After the last injection of each titrant, the chip was regenerated with a glycine solution (pH 1.5) at 30 μL / min for 30 seconds.

[0248] Biacore TM Using Biacore 8K Evaluation software 3.0, the entire data was fitted to a 1:1 Langmuir binding model (Karlsson, R., Roos, H., Fagerstam, L., Petersson, B., 1994. Methods Enzymology 6.99~110) to simultaneously obtain the association rate (kon) and dissociation rate (koff). Affinity was estimated from the quotient of the kinetic rate constants (KD = koff / kon).

[0249] As will be understood by those skilled in the art, the same method with appropriate adjustment of parameters (e.g., antibody concentration) was performed for each test antibody. The results for the test antibodies are summarized in the table below.

[0250]

Table 3

[0251] Cetuximab was originally developed by ImClone Systems and was first introduced in Switzerland in 2003 by Merck KGaA as ErbituxTM for the treatment of metastatic colorectal cancer refractory to irinotecan, as a monotherapy and in combination with irinotecan, and is a chimeric monoclonal IgG1 antibody targeting EGFR, the heavy and light chain sequences of which are shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively.

[0252] Telisotuzumab is a humanized IgG1 monoclonal antibody targeting MET and is in early clinical development at AbbVie for the treatment of progressive solid tumors due to MET gene amplification, the heavy and light chain sequences of which are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

[0253] The results showed that both E-6C4-M-2F11 and E-6C4-M-2G10 of the anti-EGFR / MET bispecific antibodies can bind to human EGFR, human MET, monkey EGFR, and monkey MET.

[0254] The binding activity of E-6C4-M-2F11 of the anti-EGFR / MET bispecific antibody to other human EGF family proteins was also verified, and the results showed that E-6C4-M-2F11 cannot bind to human HER2, HER3, and HER4 (data not shown).

[0255] Stability of anti-EGFR / MET bispecific antibodies The anti-EGFR / MET bispecific antibodies E-1G11-M-2F11, E-6C4-M-2F11, and E-6C4-M-2G10 were diluted to 5 mg / mL using a buffer at pH 6.0 (3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL Tween(R) 80). The diluted antibodies were held in sealed Eppendorf tubes at 4 ± 3 °C (hereinafter referred to as 4 °C) for 7 days or at 40 ± 3 °C (hereinafter referred to as 40 °C) for 7 days to evaluate their thermal stability. Alternatively, the bispecific antibodies were frozen at -80 °C and then thawed at room temperature. The freeze-thaw experiment was repeated 10 times (over 5 days), and antibody samples were detected after the final thawing at room temperature. The bispecific antibodies were also incubated under low pH conditions. Specifically, the antibodies were incubated in 1 mol / L acetic acid (pH 3.5) for 0 hours or 6 hours.

[0256] After the above treatment, the following analyses were performed: (1) Observation of the appearance of the solution and the presence of visible insoluble objects; (2) Detection of changes in antibody purity by size exclusion ultra-high performance liquid chromatography (SEC-UPLC) (shown as the percentage of the main peak area to the total peak area (purity, %)); (3) Detection of changes in the nominal hydrophobicity of the antibody using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) method (shown as the retention time of the main peak (HIC, min)); (4) Detection of the purity ratio of the antibody by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) under non-reducing (CE-SDS (NR) conditions) (shown as the percentage of the main peak area to the total peak area (purity, %)); (5) Detection of charge variants in the antibody by capillary isoelectric focusing electrophoresis (cIEF) method (shown as the percentages of the main component, acidic component, and alkaline component).

[0257] In the SEC-UPLC experiment, the antibody sample was diluted to 1 mg / mL with purified water and analyzed using an Agilent 1290 chromatograph system (XBridge TMA Protein BEH SEC column (200 Å, Waters Corporation) was connected. The following parameters were used: mobile phase: 100 mmol / L phosphate buffer (pH 7.4) + 0.2 mmol / L NaCl + 10% acetonitrile; flow rate: 1.8 mL / min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: approximately 6 °C; and effective time: 7 minutes.

[0258] In the HIC-HPLC experiment, an Agilent 1260 chromatograph system (ProPac TM HIC-10 column (4.6 × 250 mm, Thermo Scientific) was connected, and the sample was diluted to 0.5 mg / mL using mobile phase A. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB), 10% acetonitrile (pH 6.5); mobile phase B: 0.1 M PB, 10% acetonitrile (pH 6.5); flow rate: 0.8 mL / min; gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; column temperature: 30 °C; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: approximately 6 °C; and run time: 45 minutes.

[0259] In the cIEF experiment, for sample preparation, the Maurice cIEF method development kit (Protein Simple, catalog number: PS-MDK01-C) was used. Specifically, 40 μg of protein sample was mixed in the kit with the following reagents: 1 μL of Maurice cIEF pI Marker-4.05, 1 μL of Maurice cIEF pI Marker-9.99, 35 μL of 1% methylcellulose solution, 2 μL of Maurice cIEF 500 mM arginine, 4 μL of ampholyte (Pharmalyte pH range 3 - 10), and water (added to make a final volume of 100 μL). Using a Maurice analyzer (Protein Simple, Santa Clara, CA) with a Maurice cIEF cartridge (PS-MC02-C), an imaging capillary isoelectric focusing electrophoresis spectrum was generated. The sample was focused for a total of 10 minutes. Using the analysis software installed on the instrument, the absorbance of the protein focused at 280 nm was integrated.

[0260] In the CE-SDS (NR) experiment, Maurice (Protein simple, Maurice TM ) and the Maurice CE-SDS size application kit (Protein simple, catalog number: PS-MAK02-S) were used. 54 μL of sample buffer, 6 μL of antibody sample, 2.4 μL of 25x internal standard, 3 μL of 250 nM iodoacetamide (SIGMA, catalog number: 16125) were added to a microcentrifuge tube, followed by centrifugation at 3000 rpm for 1 minute and heating in a 70 °C water bath for 10 minutes. Next, the sample was cooled to room temperature and then centrifuged at 10000 rpm for 3 minutes. Next, the supernatant sample preparation was transferred to a 96-well plate and tested with Maurice. The following parameters were used: injection voltage 4.6 kV, injection time 20 seconds, separation voltage 5.75 kV, and separation time 40 minutes.

[0261] The detailed results of the anti-EGFR / MET bispecific antibodies are shown in the table below. The results showed that E-6C4-M-2F11 and E-6C4-M-2G10 have better stability, in addition to physical and chemical properties, than the other test antibodies.

[0262]

Table 4

[0263] Example 2. Antibody-Drug Conjugate After protein A purification, the bispecific antibodies E-1G11-M-2F11, E-6C4-M-2F11, and E-6C4-M-2G10 were dialyzed and concentrated in PBS buffer by ultrafiltration. The concentration was measured by UV absorption. These antibodies were used in subsequent antibody-drug coupling reactions.

[0264] Coupling of the Antibody with the Drug Molecule Each purified antibody was coupled with MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F) by a maleimidocaproyl-valine-citrulline-p-aminobenzyl oxycarbonyl (VC) linker.

[0265] For the name of the antibody-drug conjugate, add "ADC" immediately after the antibody name. For example, when E-1G11-M-2F11 is coupled to MMAE, it is named E-1G11-M-2F11-ADC.

[0266] HIC-HPLC was used to detect the coupling of the antibody with the drug molecule. In the HIC-HPLC experiment, an Agilent 1260 chromatography system (ProPac TMAn HIC-10 column (4.6 × 250 mm, Thermo Scientific) was connected, and the sample was diluted to 0.5 mg / mL using mobile phase A. The following parameters were used: Mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB), 10% acetonitrile (pH 6.5); Mobile phase B: 0.1 M PB, 10% acetonitrile (pH 6.5); Flow rate: 0.8 mL / min; Gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; Column temperature: 30 °C; Detection wavelength: 280 nm; Injection volume: 10 μL; Sample tray temperature: approximately 6 °C; and Run time: 45 minutes.

[0267] For the isotype control, a human IgG1 isotype control was coupled to MMAE to form an isotype-ADC (ISO-ADC). The results of HIC-HPLC detection are shown in the table below. The results indicate that the drug-to-antibody ratio (DAR) of the ADC is approximately 4. Here, the average DAR is measured by multiplying the corresponding drug load of 0, 2, 4, 6, or 8 by the PA% (PA% is the percentage (%) of the peak area measured by the original area of the 280 nm peak) and dividing by 100. For example, the average DAR of E-1G11-M-2F11-ADC can be calculated as follows: [(6.69×0)+(23.45×2)+(41.60×4)+(18.60×6)+(9.67×8)] / 100 = 4.0.

[0268]

Table 5

[0269] In vitro killing activity Purified antibodies at different concentrations (10 μg / mL, 3.33 μg / mL, 1.11 μg / mL, 0.37 μg / mL, 0.123 μg / mL, 0.041 μg / mL, 0.013 μg / mL, and 0.004 μg / mL), and the corresponding ADCs were used to treat human lung cancer cell line NCI-H1975 (5×103) cultured in a cell culture plate. After incubation for 72 hours with IncuCyte (Sartorius AG, IncuCyte(R) S3), the killing activity was detected. The results are shown in the following table.

[0270]

Table 6

[0271] Amivantamab is a fully human bispecific antibody targeting EGFR and MET, developed by Janssen. It was approved in the United States and the EU in 2021 for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion mutations that progressed during or after platinum-based chemotherapy. The heavy and light chain sequences of amivantamab are shown in SEQ ID NOs: 40-43.

[0272] Some of the data, for example, the cancer cell killing results of the bispecific antibody E-6C4-M-2F11, were further analyzed. The corresponding ADC E-6C4-M-2F11-ADC at a concentration of 10 μg / mL is shown in Figure 2. The detailed killing results of E-6C4-M-2F11-ADC at different concentrations and different times are shown in the following table.

[0273]

Table 7

[0274] The above results indicate that neither E-6C4-M-2F11 nor the amivantamab analog has the ability to kill NCI-H1975 at a maximum concentration of 10 μg / mL. However, E-6C4-M-2F11-ADC was able to efficiently inhibit the growth of tumor cells in a dose-dependent manner at various concentrations.

[0275] Example 3. Antitumor Activity in the NCI-H1975 Xenograft Model In a model of lung adenocarcinoma, antibodies were tested for their effect on in vivo tumor growth. Specifically, approximately 5×106 NCI-H1975 cells were subcutaneously injected into B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., catalog number B-CM-002). When the tumor volume in the mice reached approximately 300 mm3, the mice were randomly divided into different groups based on tumor volume. Next, the mice were injected intravenously (i.v.) with phosphate-buffered saline (PBS) or the antibody. The dosing frequency was once a week (for a total of 2 administrations). Details are shown in the table below.

[0276] The lengths of the major and minor axes of the tumor were measured, and the tumor volume was calculated as 0.5×(major axis)×(minor axis)2. Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)]×100%. Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day 0. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day 0. A t-test was performed for statistical analysis. TGI exceeding 60% indicates a clear inhibition of tumor growth. P < 0.05 is the threshold for indicating a significant difference.

[0277]

Table 8

[0278] The body weight of the mice was also measured twice a week. On the day of grouping (day 0), the average body weight of each group ranged from 22.0 g to 24.3 g. At the end of the experiment (day 21), the average body weight of each group ranged from 20.9 g to 24.9 g. Therefore, the average body weight change of each group ranged from 91.1% to 103.7%. The results showed that all the tested antibodies were well tolerated and had no obvious toxicity to the mice. The following table summarizes the results of this experiment, including the tumor volume on the day of grouping (day 0), 11 days after grouping (day 11), and at the end of the experiment (day 21); the survival rate of the mice; TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0279]

Table 9

[0280] The tumor volume in all treatment groups (G3 - G6) was smaller than that in the control groups (G1 and G2). The treatment groups had various tumor inhibitory effects. All of the anti-EGFR / MET bispecific antibody ADCs (G3 - G5) at a dose level of 3 mg / kg showed a continuous and potent tumor suppression effect with a TGI exceeding 100%. In particular, E-6C4-M-2F11-ADC (G4) had a maximum TGI of 110.5%. All TGI values of the tested ADCs were higher than those of the positive control amivantamab analog (TGI: 98.4%) at 10 mg / kg.

[0281] In another experiment, approximately 5×105 NCI-H1975 cells were subcutaneously injected into B-NDG mice, and the antitumor activities of E-6C4-M-2F11 and E-6C4-M-2F11-ADC were measured. When the tumor volume reached approximately 400 mm3, the mice were randomly assigned to the control group and different treatment groups based on tumor size. The details of grouping and administration are shown in the following table.

[0282]

Table 10

[0283] During the experimental period, almost no difference was observed in the body weights of the mice in each group. Tumor volume and body weight were measured twice a week. The following table summarizes the results of this experiment, including the tumor volume on the day of grouping (day 0), 12 days after grouping (day 12), and at the end of the experiment (day 22); the survival rate of the mice; TGI (%); and the statistical difference in tumor volume (P-value) between the treatment group and the control group.

[0284]

Table 11

[0285] The tumor volumes of mice in different groups treated with antibody, ADC, or PBS are shown in Figure 3, where the ADC E-6C4-M-2F11-ADC (G2 and G3) of the anti-EGFR / MET bispecific antibody showed a better tumor inhibitory effect compared to PBS (G1) in the control group and the corresponding anti-EGFR / MET bispecific antibody E-6C4-M-2F11 in groups G4 and G5.

[0286] In another experiment, NCI-H1975 cells were subcutaneously injected into Balb / c nude mice, and the antitumor activity of the ADC was measured. When the tumor volume reached approximately 400 mm3, the mice were randomly assigned to a control group and different treatment groups based on tumor size. Next, the mice were injected intravenously (i.v.) with PBS, E-6C4-M-2F11-ADC (1.5 mg / kg, 3 mg / kg, or 6 mg / kg, QW, administered twice in total), or amivantamab (Janssen, reference number LFS0L03) (10 mg / kg, BIW, administered four times in total). The results showed that all three antitumor activities of E-6C4-M-2F11-ADC (97.5%, 110.3%, and 110.8% TGI, respectively) were more potent than the antitumor activity of amivantamab at a dose of 10 mg / kg (12.9% TGI).

[0287] Example 4. Antitumor Activity in NCI-H292 Xenograft Model In a model of lung cancer tumors, antibodies were tested for their effect on in vivo tumor growth. Approximately 1×107 NCI-H292 cells were subcutaneously injected into B-NDG mice. When the tumor volume in the mice reached approximately 300 mm3, the mice were randomly divided into different groups based on tumor volume. Next, the mice were injected with PBS or the antibody by intravenous (i.v.) administration. The dosing frequency was once a week (a total of 2 administrations). Details are shown in the table below.

[0288] [Table 12]

[0289] During the experimental period, almost no difference was observed in the body weights of the mice in each group. The tumor sizes in the groups treated with the antibody are shown in Figure 4. The following table summarizes the results of this experiment, including the tumor volume at the day of grouping (day 0), 14 days after grouping (day 14), and at the end of the experiment (day 24); the survival rate of the mice; TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0290] [Table 13]

[0291] During the experimental period, the body weights of the mice in each group were maintained. The body weight of the mice treated with 10 mg / kg of E-6C4-M-2F11-ADC (G2) showed an increasing trend from 21.1 g on day 0 to 21.8 g on day 24, and the average body weight change was 103.4%. Under the same dose level, the anti-EGFR / MET bispecific antibody ADC showed better antitumor activity than the positive control amivantamab analog in a dose-dependent manner.

[0292] In another experiment, approximately 5×106 NCI-H292 cells were subcutaneously injected into B-NDG mice to measure the antitumor activities of E-6C4-M-2F11 and E-6C4-M-2F11-ADC. When the tumor volume reached approximately 200 mm3, the mice were randomly assigned to a control group and different treatment groups based on tumor size. Details of the grouping and administration are shown in the table below.

[0293]

Table 14

[0294] During the experimental period, almost no difference was observed in the body weights of the mice in each group. Tumor volume and body weight were measured twice a week. The following table summarizes the results of this experiment, including the tumor volume on the day of grouping (day 0), 11 days after grouping (day 11), and at the end of the experiment (day 21); the survival rate of the mice; TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0295]

Table 15

[0296] The tumor sizes in the group treated with the antibody are shown in Figure 5. The treatment groups showed various tumor inhibitory effects. Generally, the antitumor activity of the bispecific antibody ADC was more potent than that of the monoclonal antibody ADC and more potent than that of the control bispecific antibody ADC.

[0297] In another experiment, approximately 2×106 NCI-H292 cells were subcutaneously injected into Balb / c nude mice to measure the antitumor activity of the ADC. When the tumor volume of the ADC reached approximately 300 mm3, the mice were randomly assigned to a control group and different treatment groups based on tumor size. Next, the mice were injected with PBS, the ADC, or amivantamab (Janssen, reference number: LFS0L03) by intravenous (i.v.) administration. Details of the grouping and administration are shown in the table below.

[0298]

Table 16

[0299] MRG003 is an antibody-drug conjugate composed of a fully human IgG1 monoclonal antibody that targets EGFR conjugated to monomethyl auristatin E (MMAE) for the treatment of solid tumors. It is in early clinical development at Shanghai Miracogen, and its heavy and light chain sequences are shown in SEQ ID NO: 44 and SEQ ID NO: 45, respectively.

[0300] Tumor volume and body weight were measured twice a week. The tumor sizes in the group treated with ADC are shown in Figure 15. The antitumor activity of E-6C4-M-2F11-ADC (G3) was more potent than that of the positive control (G5 - G6) at a dose of 3 mg / kg, and the results showed that the antitumor activity of E-6C4-M-2F11-ADC (G2) at 6 mg / kg was more potent than that of amivantamab at a dose of 10 mg / kg. Furthermore, the bispecific antibody ADC E-6C4-M-2F11-ADC showed a dose-dependent tumor inhibitory effect.

[0301] Example 5. Antitumor Activity in the SNU-5 Xenograft Model In a gastric cancer tumor model, the antibodies were tested for their effect on in vivo tumor growth. Approximately 1 × 107 SNU-5 cells were subcutaneously injected into B-NDG mice. When the tumor volume in the mice reached approximately 200 mm3, the mice were randomly divided into different groups based on tumor volume. Next, the mice were injected with PBS or the antibody by intravenous (i.v.) administration. The dosing frequency was once a week (a total of 2 administrations). Details are shown in the table below.

[0302]

Table 17

[0303] During the experimental period, almost no difference was observed in the body weights of the mice in each group. The tumor sizes in the group treated with ADC are shown in Figure 6. The following table summarizes the results of this experiment, including the tumor volume at the day of grouping (day 0), 10 days after grouping (day 10), and at the end of the experiment (day 24); the survival rate of the mice; TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0304]

Table 18

[0305] In the gastric cancer tumor model of SNU-5, both ADC E-6C4-M-2F11-ADC and E-6C4-M-2G10-ADC showed strong antitumor activity at dose levels of 10 mg / kg or 3 mg / kg. These tumor inhibitory effects were greater than those of the corresponding bispecific antibodies E-6C4-M-2F11 and E-6C4-M-2G10, and also greater than the tumor inhibitory effect of the positive control amivantamab analog.

[0306] Example 6. Antitumor Activity in the A431 Xenograft Model In a skin cancer tumor model, the antibodies were tested for their effects on in vivo tumor growth. Approximately 5 × 106 A431 cells (ATCC, reference number: CRL-1555) were subcutaneously administered to B-NDG mice. When the tumor volume in the mice reached approximately 300 mm3, the mice were randomly divided into different groups based on tumor volume. Next, the mice were injected with PBS or the antibody by intravenous (i.v.) administration. The dosing frequency was once a week (a total of 2 doses). The details are shown in the following table.

[0307]

Table 19

[0308] During the experimental period, almost no difference was observed in the body weights of the mice in each group. The tumor sizes in the group treated with the antibody are shown in Figure 7. Similar to the above experimental results, in the A431 skin cancer in vivo model, E-6C4-M-2F11-ADC showed a greater tumor inhibitory effect than the corresponding bispecific antibody E-6C4-M-2F11, and also had a greater tumor inhibitory effect than the positive control amivantamab analog.

[0309] Example 7. In Vivo Efficacy in a Patient-Derived Xenograft (PDX) Model of Human Lung Cancer / Tumor The effects of the antibodies were tested in two human lung PDX (PDX001 and PDX002) models. Immunofluorescence staining of patient-derived lung tumor fragments was performed and the images were analyzed with HALO 3.2 version. The results showed that the percentages of EGFR-positive cells and MET-positive cells in PDX001 were 24.28% and 25.71% respectively. In PDX002, the percentages of EGFR-positive cells and MET-positive cells were 12.88% and 105.93% respectively.

[0310] In the PDX001 model, patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. When the tumor volume in the mice reached approximately 250 - 300 mm3, the mice were randomly divided into different groups based on tumor volume. Next, the mice were injected with PBS, an amivantamab analog, or E-6C4-M-2F11-ADC by intravenous (i.v.) administration. The details of the dosing scheme are shown in the table below.

[0311]

Table 20

[0312] The tumor volume was measured twice a week, and the results are shown in Table 26. This shows that treatment with E-6C4-M-2F11-ADC at 10 mg / kg (G2) and 3 mg / kg (G3) resulted in potent tumor growth inhibition in the EGFR / MET co-expressing human lung PDX model compared to the control group (G1) and the amivantamab analog treatment groups (G5, G6, and G7), with TGI% being 109.3% and 38.3% respectively on day 23 (23 days after randomization).

[0313]

Table 21

[0314] In the PDX002 model, when the tumors in B-NDG mice reached a volume of approximately 250 - 300 mm3, the cells were randomized into a control group and different treatment groups (6 mice per group) based on tumor size. Treatment groups were randomly selected for treatment with E-6C4-M-2F11-ADC at 3 mg / kg (G2) and 1 mg / kg (G3), or with an amivantamab analog at 3 mg / kg (G4) and 1 mg / kg (G5). Mice in the control group were injected with PBS (G1). The dosing frequency was once a week (for a total of 2 doses).

[0315] The tumor sizes in each group are shown in Table 22. Treatment with E-6C4-M-2F11-ADC at 3 mg / kg (G2) showed a better tumor suppression effect in the MET-high expressing human lung PDX model compared to the control group (G1) and the amivantamab analog treatment groups (G4 and G5).

[0316]

Table 22

[0317] Example 8. In Vivo Efficacy in Human Pancreatic PDX Model The effects of the antibodies were tested in two human pancreatic PDX (PDX003 and PDX004) models. Immunofluorescence staining of patient-derived pancreatic tumor fragments was performed and the images were analyzed using the HALO 3.2 version. The results showed that the percentages of EGFR-positive cells and MET-positive cells in PDX003 were 7.98% and 32.44%, respectively. In PDX004, the percentages of EGFR-positive cells and MET-positive cells were 54.65% and 36.63%, respectively.

[0318] In the PDX003 model, patient-derived pancreatic tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. When the tumor volume in the mice reached approximately 300 - 400 mm3, the mice were randomly divided into different groups based on the tumor volume. Next, the mice were injected with PBS, an amivantamab analog, or E-6C4-M-2F11-ADC by intravenous (i.v.) administration. The details of the dosing scheme are shown in the table below.

[0319]

Table 23

[0320] The tumor volume was measured twice a week and the results are shown in Table 24. This indicates that treatment with E-6C4-M-2F11-ADC at 10 mg / kg, 3 mg / kg, and 1 mg / kg resulted in significant tumor growth inhibition in the human pancreatic PDX model compared to the control group, and the TGI% was 129.2%, 127.7%, and 45.8% on day 21, respectively.

[0321]

Table 24

[0322] In the PDX004 model, when the tumors in the mice reached a volume of approximately 250 - 300 mm3, the cells were divided into a control group and different treatment groups (6 mice per group) based on the tumor size. The antibodies tested and the dosing frequency were the same as in Table 28.

[0323] The E-6C4-M-2F11-ADC treatment groups (including doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg) all showed results of substantial antitumor activity in the human pancreatic PDX model, with the TGI% being 46.9%, 109.4%, and 110.9% respectively on day 35 (35 days after grouping), which were higher than the antitumor activities of the amivantamab analog treatment groups (-13.3%, -0.6%, and 4.1% respectively).

[0324] Example 9. Pharmacokinetic Profile The pharmacokinetic clearance rate of the anti-EGFR / MET bispecific antibody ADC was measured in C57BL / 6 mice. Specifically, the mice were divided into four groups (5 mice per group), and E-6C4-M-2F11-ADC (G1, 3 mg / kg; G2, 10 mg / kg), or E-6C4-M-2F11 (G3, 3 mg / kg; G4, 10 mg / kg) was administered by intravenous injection. Blood samples were collected 3 days before administration, and at 15 minutes, 6 hours, 1 day, 2 days, 5 days, 10 days, 14 days, and 21 days after administration.

[0325] The serum levels of the antibody and ADC were measured by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch Inc., catalog number: 109-005-088), or anti-MMAE mIgG (ACRO Biosystems Inc., catalog number: MME-M5252) was diluted to a final concentration of 2000 ng / mL and added to a 96-well plate (ELISA plate) at 100 μL / well, and then incubated overnight at 2 - 8°C. After incubation, the plate was washed with PBS-T buffer (Tween TMIt was washed 4 times with 20 mM PBS (pH 7.4) supplemented with 0.05% Tween 20. The regions where the antibody was not bound were blocked with 2% BSA (bovine serum albumin) at 37 °C for 2 hours. Thereafter, the plate was washed 4 times with PBS-T buffer. After washing, 100 μL of blocking buffer (2% BSA) was added to each well. The wells were sealed and incubated at 37 °C for 1 hour. After the plate was washed with a plate washer, Peroxidase AffiniPure F(ab’)2 fragment goat anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch Inc., catalog number: 109-036-098) was added to each well of the plate at 100 μL / well and incubated at 37 °C for 1 hour. After the plate was washed, tetramethylbenzidine (TMB) solution was added as a substrate to the 96-well plate at 100 μL / well. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, catalog number: P0215) was added to each well. The luminescence signal of the plate was measured at 450 nm and 630 nm, and the concentration was calculated. Using the absorbance values and the corresponding concentrations of the calibration samples prepared with each test product, a standard curve with four parameters (i.e., T1 / 2, Cmax, AUC0-21 days, and CL) was produced. Using the standard curve, the antibody or ADC concentration of each serum sample was calculated. A drug concentration-time curve was produced using the calculated sample concentrations at each time point. Phoenix TM The pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.3. The results are shown in the table below, which indicate that the conjugation of the drug molecule does not affect the in vivo pharmacokinetic clearance rate of the bispecific antibody.

[0326]

Table 25

[0327] Example 10. In Vivo Efficacy of ADC Combinations In another experiment, approximately 5×105 NCI-H1975 cells were subcutaneously injected into B-NDG mice, and the in vivo efficacy of the combination of E-6C4-ADC and M-2F11-ADC was measured. When the tumor volume reached approximately 300 mm3, the mice were randomly assigned to a control group and different treatment groups based on tumor size. Details of the grouping and administration are shown in the table below.

[0328]

Table 26

[0329] During the experimental period, almost no difference was observed in the body weights of the mice in each group. Tumor volume and body weight were measured twice a week. The following table summarizes the results of this experiment, including the tumor volume on the day of grouping (day 0), 14 days after grouping (day 14), 21 days after grouping (day 21), and 39 days after grouping (day 39, if applicable); the survival rate of the mice; TGI (%); and the P value of the tumor volume between the treatment group and the control group on day 21.

[0330]

Table 27

[0331] The tumor volumes of the mice in different groups treated with ADC or PBS are shown in Figure 13. Compared with the PBS group (G1), the monoclonal antibody ADC groups of E-6C4-ADC (G3 and G5), and M-2F11-ADC (G2 and G4), the bispecific antibody ADC groups (G11 and G12), and the combined groups of monoclonal antibody ADC (G6, G7, and G8) all showed significant anti-tumor activity on day 21.

[0332] In particular, the TGI% of M-2F11-ADC(G4) and E-6C4-ADC(G5) at 1.5 mg / kg was comparable to the TGI% of the combination of M-2F11-ADC and E-6C4-ADC(G8) at 0.75 mg / kg, but lower than the TGI% of E-6C4-M-2F11-ADC(G11) at 1.5 mg / kg.

[0333] Similar to the above results, the TGI% of M-2F11-ADC(G2) and E-6C4-ADC(G3) at 3 mg / kg was the same as the TGI% of the combination of M-2F11-ADC and E-6C4-ADC(G7) at 1.5 mg / kg, but lower than the TGI% of E-6C4-M-2F11-ADC(G12) at 3 mg / kg.

[0334] Mouse tumor volume and survival were continuously monitored even after day 21. At the end of the experiment on day 39, all mice died in groups G1, G4, G5, G8, G9, and G10. In contrast, all mice survived in groups G2, G3, G6, G7, G11, and G12. The tumor volume results on day 39 indicated that at a dose level of 3 mg / kg, E-6C4-M-2F11-ADC(G12) treatment had a better antitumor effect compared to M-2F11-ADC(G2), E-6C4-ADC(G3), or their combination (G7).

[0335] The results also show that the anti-EGFR / MET bispecific antibody has a synergistic effect on tumor suppression.

[0336] Example 11. In Vivo Efficacy in a Human Gastric Cancer PDX Model The effect of ADC was tested in a human gastric cancer PDX model. Immunofluorescence staining of patient-derived gastric tumor fragments was performed and the images were analyzed with HALO 3.2 version. The results showed that the EGFR-positive cells and MET-positive cells were 94.03% and 0.25%, respectively.

[0337] Patient-derived tumor fragments (2 mm × 2 mm × 2 mm) of the gastric cancer were transplanted into the right flank of B-NDG mice. When the tumor volume in the mice reached approximately 250 - 300 mm3, the mice were randomly divided into different groups based on the tumor volume. Next, the mice were injected with PBS, ADC, or amivantamab (Janssen, catalog number: LFS0L03) by intravenous (i.v.) administration. The details of the dosing scheme are shown in the table below.

[0338]

Table 28

[0339] The following table summarizes the results for this experiment, including the tumor volume; TGI (%); and the statistical difference in tumor volume (P-value) between the treatment group and the control group on the day of grouping (day 0), 14 days after grouping (day 14), and 21 days after grouping (day 21).

[0340]

Table 29

[0341] The tumor volumes of the mice in different groups are shown in Figure 16. The results showed that the treatment groups exhibited different tumor inhibitory effects compared to the control group G1. Among the treatment groups, at the same dose, E-6C4-M-2F11-ADC (G2 and G3) showed the best tumor inhibitory effect, followed by the corresponding anti-EGFR antibody ADC E-6C4-ADC, and finally the positive control cetuximab analog-ADC. E-6C4-M-2F11-ADC showed a dose-dependent tumor inhibitory effect. In addition, the experiment was then continued until 39 days after grouping (day 39). E-6C4-M-2F11-ADC (G2 - G3) still showed a better tumor inhibitory effect (108.6% and 83.7% TGI respectively) than E-6C4-ADC (G5) (71.0% TGI) and the cetuximab analog-ADC (G6 - G7) (78.7% and 57.5% TGI respectively).

[0342] Example 12. In Vivo Efficacy in a Human Gastric Cancer PDX Model Immunofluorescence staining was performed on patient-derived gastric cancer tumor fragments, and the images were analyzed using the HALO 3.2 version. The results showed that in the gastric PDX model, the percentages of EGFR-positive cells and MET-positive cells were 80.53% and 3.10%, respectively.

[0343] Patient-derived gastric cancer tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. When the tumor volume in the mice reached approximately 250 - 300 mm3, the mice were randomly divided into different groups based on tumor volume. Next, the mice were injected with PBS, ADC, or amivantamab (Janssen, reference number: LFS0L03) by intravenous (i.v.) administration. The details of the dosing scheme are shown in the table below.

[0344] [Table 30]

[0345] The tumor volume was measured twice a week, and the results are shown in the table below and Figure 14. This indicates that in the treatment groups with E-6C4-M-2F11-ADC (G2 - G4), compared with the control group (G1), strong inhibition of tumor growth was maintained in the human gastric PDX model, and 6 mg / kg of E-6C4-M-2F11-ADC showed a higher tumor inhibitory effect compared with 10 mg / kg of amivantamab. Monitoring of the tumor volume was continued until 77 days after grouping (day 77). E-6C4-M-2F11-ADC (G2 - G4) still showed a better tumor inhibitory effect compared with amivantamab (10 mg / kg).

[0346] [Table 31]

[0347] Example 13. Antibody-Drug Conjugate The purified antibody was conjugated to CPT1, CPT2, CPT3, or CPT4 via a CPT-L linker. For the name of the antibody-drug conjugate, add CPTx (x = 1, 2, 3, or 4) immediately after the antibody name. For example, when E-6C4-M-2F11 is conjugated to CPT1, it is named E-6C4-M-2F11-CPT1. As another example, when E-6C4-M-2F11 is conjugated to CPT2, it is named E-6C4-M-2F11-CPT2.

[0348] HIC-HPLC was used to detect the coupling of the antibody with the drug molecule. A human IgG1 molecule was conjugated to CPT2 to form an isotype control, isotype-CPT2 (ISO-CPT2). The detection results of HIC-HPLC indicated that the drug-antibody ratio (DAR) of the ADC was approximately 4 or 8. Regarding the ADC name, when the DAR of E-6C4-M-2F11-CPT1 is 4, the ADC is named E-6C4-M-2F11-CPT1 (DAR4). When the DAR of E-6C4-M-2F11-CPT1 is 8, the ADC is named E-6C4-M-2F11-CPT1 (DAR8).

[0349] Example 14. Anti-tumor Activity of ADC in a Lung Cancer Model The inhibitory effect of ADC on tumor growth in vivo was tested in a lung cancer tumor model. Specifically, approximately 2 × 106 NCI-H292 cells were subcutaneously injected into Balb / c nude mice. When the tumor volume in the mice reached approximately 300 mm3, the mice were randomly divided into different groups based on the tumor volume. Next, the mice were injected with PBS, ADC, or amivantamab by intravenous (i.v.) administration. The administration frequency of ADC was once a week (a total of one administration). The details of the administration schedule, route, and frequency are shown in the following table.

[0350]

Table 32

[0351] The results showed that both E-6C4-M-2F11-CPT2 with DAR4 and DAR8 exhibited good tumor inhibitory effects.

[0352] In a similar experiment, approximately 2×106 NCI-H1975 cells were subcutaneously injected into Balb / c nude mice. When the tumor volume in the mice reached approximately 300 mm3, the mice were randomly divided into different groups based on the tumor volume. Next, the mice were injected with PBS, ADC, or amivantamab intravenously (i.v.). The details of the administration schedule, route, and frequency are shown in the table below.

[0353]

Table 33

[0354] Similar to the above results, both E-6C4-M-2F11-CPT2 with DAR4 and DAR8 showed good inhibitory effects on tumor growth.

[0355] Example 15. Antitumor activity of ADC in a pancreatic cancer model Tumor tissue fragments (2 mm × 2 mm × 2 mm) derived from pancreatic cancer patients were transplanted into the right flank of B-NDG mice. When the tumor volume in the mice reached approximately 200 - 300 mm3, the mice were randomly divided into different groups based on the tumor volume. Next, the mice were injected with PBS, ADC, or amivantamab by i.v. administration. The details of the administration schedule, route, and frequency are shown in the table below.

[0356]

Table 34

[0357] The results showed that both E-6C4-M-2F11-CPT2 with DAR4 and DAR8 exhibited excellent efficacy in the treatment of pancreatic cancer.

[0358] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the above description is for illustrative purposes only and does not limit the scope of the present invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antigen-binding protein construct comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to MET.

2. The first antigen-binding domain comprises a first heavy-chain variable region (VH1) and a first light-chain variable region (VL1), and the second antigen-binding domain comprises a second heavy-chain variable region (VH2) and a second light-chain variable region (VL2). The antigen-binding protein construct according to claim 1.

3. The first heavy-chain variable region (VH1) comprises complementarity-determining regions (CDR) 1, 2, and 3, the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence, and The first light-chain variable region (VL1) comprises CDR1, 2, and 3, the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence, The selected VH1 CDR1, 2, and 3 amino acid sequences, and the selected VL1 CDR1, 2, and 3 amino acid sequences are as follows: (1) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (2) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (3) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16-18, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; and ​ (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; being one of The antigen-binding protein construct according to claim 2.

4. The second heavy chain variable region (VH2) comprises CDR1, 2, and 3, the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR2 amino acid sequence, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR3 amino acid sequence, and The second light chain variable region (VL2) comprises CDR1, 2, and 3, the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR2 amino acid sequence, the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR3 amino acid sequence, The selected VH2 CDR1, 2, and 3 amino acid sequences, and the selected VL2 CDR1, 2, and 3 amino acid sequences are as follows: (1) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (3) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22 to 24, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; and (4) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25 to 27, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; being one of The antigen-binding protein construct according to claim 2 or 3.

5. (1) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4-6 respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10-12 respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (2) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7-9 respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10-12 respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (3) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7-9 respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13-15 respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (4) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16-18 respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22-24 respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; (5) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 19-21 respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22-24 respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3 respectively; and (6) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21 respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3 respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 25 to 27 respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3 respectively. The antigen-binding protein construct according to any one of claims 2 to 4.

6. The first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 28, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 30, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:

32. The antigen-binding protein construct according to any one of claims 2 to 5.

7. The first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 30, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:

32. The antigen-binding protein construct according to any one of claims 2 to 5.

8. The first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 31, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:

32. The antigen-binding protein construct according to any one of claims 2 to 5.

9. The VH1 comprises an amino acid sequence that is at least 90% identical to the selected VH sequence, the VL1 comprises an amino acid sequence that is at least 90% identical to the selected VL sequence, and the selected VH sequence and the selected VL sequence are as follows: (1) The selected VH sequence is SEQ ID NO: 28 and the selected VL sequence is SEQ ID NO: 32; and (2) The selected VH sequence is SEQ ID NO: 29 and the selected VL sequence is SEQ ID NO: 32; being one of the following: The antigen-binding protein construct according to any one of claims 2 to 8.

10. The VH1 comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, the VL1 comprises VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are as follows: (1) The selected VH sequence is SEQ ID NO: 28 and the selected VL sequence is SEQ ID NO: 32; and (2) The selected VH sequence is SEQ ID NO: 29 and the selected VL sequence is SEQ ID NO: 32; being one of the following: The antigen-binding protein construct according to any one of claims 2 to 9.

11. The VH2 comprises an amino acid sequence that is at least 90% identical to the selected VH sequence, the VL2 comprises an amino acid sequence that is at least 90% identical to the selected VL sequence, and the selected VH sequence and the selected VL sequence are as follows: (1) The selected VH sequence is SEQ ID NO: 30 and the selected VL sequence is SEQ ID NO: 32; and (2) The selected VH sequence is SEQ ID NO: 31 and the selected VL sequence is SEQ ID NO: 32; being one of the following: The antigen-binding protein construct according to any one of claims 2 to 10.

12. The VH2 includes VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, the VL2 includes VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are as follows: (1) The selected VH sequence is SEQ ID NO: 30 and the selected VL sequence is SEQ ID NO: 32; and (2) The selected VH sequence is SEQ ID NO: 31 and the selected VL sequence is SEQ ID NO: 32; one of which is The antigen-binding protein construct according to any one of claims 2 to 11.

13. The VH1 includes the sequence of SEQ ID NO: 28 and the VL1 includes the sequence of SEQ ID NO: 32, The antigen-binding protein construct according to any one of claims 2 to 12.

14. The VH1 includes the sequence of SEQ ID NO: 29 and the VL1 includes the sequence of SEQ ID NO: 32, The antigen-binding protein construct according to any one of claims 2 to 13.

15. The VH2 includes the sequence of SEQ ID NO: 30 and the VL2 includes the sequence of SEQ ID NO: 32, The antigen-binding protein construct according to any one of claims 2 to 14.

16. The VH2 includes the sequence of SEQ ID NO: 31 and the VL2 includes the sequence of SEQ ID NO: 32, The antigen-binding protein construct according to any one of claims 2 to 15.

17. The first antigen-binding domain specifically binds to human or monkey EGFR, and / or the second antigen-binding domain specifically binds to human or monkey MET, The antigen-binding protein construct according to any one of claims 1 to 16.

18. The first antigen-binding domain is human or humanized, and / or the second antigen-binding domain is human or humanized, The antigen-binding protein construct according to any one of claims 1 to 17.

19. The antigen-binding protein construct is a multispecific antibody (for example, a bispecific antibody), The antigen-binding protein construct according to any one of claims 1 to 18.

20. The first antigen-binding domain is a single-chain variable fragment (scFv), and / or the second antigen-binding domain is an scFv, An antigen-binding protein construct according to any one of claims 1 to 19.

21. The first light chain variable region and the second light chain variable region are identical, An antigen-binding protein construct according to any one of claims 1 to 20.

22. A nucleic acid comprising a polynucleotide encoding an antigen-binding protein construct according to any one of claims 1 to 21.

23. A vector comprising one or more of the nucleic acids according to claim 22.

24. A cell comprising the vector according to claim 23.

25. The cell is a CHO cell, The cell according to claim 24.

26. A cell comprising one or more of the nucleic acids according to claim 22.

27. A method for producing an antigen-binding protein construct, comprising: (a) culturing a cell according to any one of claims 24 to 26 under conditions sufficient for the cell to produce the antibody or its antigen-binding fragment, or the antigen-binding protein construct; and (b) collecting the antibody or its antigen-binding fragment, or the antigen-binding protein construct produced by the cell. A method comprising the above steps.

28. An antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to an antigen-binding protein construct according to any one of claims 1 to 21.

29. The therapeutic agent is a cytotoxic agent or a cell growth inhibitor, The antibody-drug conjugate according to claim 28.

30. The therapeutic agent is MMAE or MMAF, The antibody-drug conjugate according to claim 28 or 29.

31. The therapeutic agent is 【Chemical 1】 selected from The antibody-drug conjugate according to claim 28.

32. The therapeutic agent is linked to the antibody or its antigen-binding fragment, or the antigen-binding protein construct via a linker, The antibody-drug conjugate according to claim 28 or 31.

33. The linker has the following structure: [Chemical 2] having The antibody-drug conjugate according to any one of claims 28, 31, and 32.

34. The antibody-drug conjugate has the following structure: [Chemical Formula 3] wherein n = 1, 2, 3, 4, 5, 6, 7, or 8, and "Ab" represents the antibody or its antigen-binding fragment, or the antigen-binding protein construct. The antibody-drug conjugate according to any one of claims 28, and 31 to 33.

35. A method for treating a subject having cancer, the method comprising administering to the subject a composition comprising a therapeutically effective amount of an antigen-binding protein construct according to any one of claims 1 to 21 and an antibody-drug conjugate according to any one of claims 28 to 34.

36. The subject has cancer that expresses EGFR and / or MET. The method according to claim 35.

37. The cancer is a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung cancer), gastric cancer (e.g., gastric carcinoma), skin cancer (e.g., cutaneous carcinoma), carcinoma of the dilated part (e.g., carcinoma of the dilated part), colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal cancer, or brain tumor. The method according to claim 35.

38. The subject is human. The method according to any one of claims 35 to 37.

39. The method further comprises administering an anti-PD1 antibody to the subject. The method according to any one of claims 35 to 38.

40. The method further comprises administering chemotherapy to the subject. The method according to any one of claims 35 to 39.

41. A method for reducing tumor growth rate, the method comprising: contacting tumor cells with a composition comprising an effective amount of an antigen-binding protein construct according to any one of claims 1 to 21 and an antibody-drug conjugate according to any one of claims 28 to 34.

42. A method for killing tumor cells, the method comprising: contacting tumor cells with a composition comprising an effective amount of an antigen-binding protein construct according to any one of claims 1 to 21 and an antibody-drug conjugate according to any one of claims 28 to 34.

43. A pharmaceutically acceptable carrier, and (a) an antigen-binding protein construct according to any one of claims 1 to 21, and / or (b) an antibody-drug conjugate according to any one of claims 28 to 34, A pharmaceutical composition comprising.

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