EGFR / c-met bispecific antibody and use thereof

Abstract

A bispecific antibody targeting EGFR and c-Met. By means of modifying the amino acid sequence of an Fc segment of a binding arm, the heterodimeric interaction between an EGFR binding arm and a c-Met binding arm of the bispecific antibody is stronger than the interaction of the respective homodimers, such that a heterodimer can be assembled in vitro. The bispecific antibody is more effective in inhibiting the proliferation of EGFR and c-Met double positive tumor cells than a combination of an EGFR monoclonal antibody and a c-Met monoclonal antibody. The bispecific antibody mediates antibody-dependent cytotoxicity (ADCC), promotes target protein internalization, and inhibits the cell proliferation of the strain with acquired resistance to osimertinib. The prepared bispecific antibody is simple to prepare and stable in structure, a pure bispecific antibody is obtained by means of in-vitro assembly, and the bispecific antibody is expected to better meet clinical treatment needs, and therefore the bispecific antibody has good market prospects.

Description

EGFR / c-Met bispecific antibodies and their applications

[0001] This application claims priority to patent application No. 202310966671.2 filed in China on August 2, 2023. The entire contents of that prior patent application are incorporated herein by reference for all purposes. Technical Field

[0002] The present invention relates to an anti-EGFR / c-Met bispecific antibody or an antigen-binding fragment thereof, and use of the bispecific antibody as an anticancer drug. Background Art

[0003] In the treatment of EGFR-positive tumors, TKI resistance remains a significant issue affecting the effectiveness of EGFR-targeted therapies. In recent years, bispecific antibodies targeting EGFR and c-Met have emerged as an important solution to EGFR-TKI acquired resistance and have the potential to be used as first-line treatment for non-small cell lung cancer (NSCLC). Based on the crosstalk and direct interaction between the EGFR and c-Met signaling pathways, the two anti-tumor mechanisms of bispecific antibodies targeting EGFR and c-Met can interact synergistically, not only enhancing anti-tumor efficacy but also reducing antibody dosage, thereby reducing the risk of adverse reactions.

[0004] Currently, the first approved drug targeting the EGFR / c-MET target worldwide is Johnson & Johnson's bispecific antibody amivantamab (JNJ-372), which was approved for marketing in the United States in May 2021 for the treatment of patients with non-small cell lung cancer with EGFR exon 20 insertion mutations that progressed during or after platinum-based chemotherapy, providing a new strategy for the treatment of NSCLC. According to the results of the CHRYSALIS Phase I study of amivantamab in non-small cell lung cancer with EGFR exon 20 insertion mutations that progressed after platinum-containing chemotherapy, the incidence of infusion-related reactions (IRR) was high. In addition to amivantamab, domestic and foreign pharmaceutical companies such as Merus, Jiahe Bio, Epigenetics and Hansoh Pharmaceutical are also developing EGFR / c-Met target antibodies. In general, the currently applied EGFR / c-Met bispecific antibodies focus on the therapeutic effect on tumors (and / or TKI-resistant patients), and their safety is still unclear and may need to be improved.

[0005] Summary of the Invention

[0006] The present invention provides an EGFR / c-Met bispecific antibody molecule with the desired combined advantages of EGFR and c-Met dual-target selection specificity, anti-tumor efficacy and safety prospects, which can meet the clinical needs of TKI-resistant cancer treatment while taking into account clinical efficacy and safety.

[0007] The research and development of the EGFR / c-Met bispecific antibody of the present invention can not only be used to treat patients who have developed drug resistance after existing treatment methods such as EGFR monoclonal antibodies and c-Met monoclonal antibodies, but also be used to have tumor selectivity and further improve drug efficacy while further taking into account safety and the needs of assembling EGFR binding arms and c-Met binding arms in production. It is expected to become a new generation of therapeutic products that can overcome the problem of less than ideal safety of EGFR and c-Met specific dual-targeted tumor treatment products on the market and have better binding arm assembly.

[0008] EGFR and c-Met are co-expressed in many cancer types, and antibody molecules targeting these two targets ("bispecific antibody molecules") offer the prospect of achieving broad clinical benefit across multiple cancer indications.

[0009] In a first aspect, the present invention provides a bispecific antibody comprising an antigen-binding arm A that can specifically bind to human epidermal growth factor receptor (EGFR) and antagonize the binding of epidermal growth factor (EGF) to EGFR, and an antigen-binding arm B that can specifically bind to human proto-oncogene receptor tyrosine kinase (c-Met) and antagonize the binding of hepatocyte growth factor (HGF) to c-Met; wherein the binding arm A comprises a light chain A (LCA) and a heavy chain A (HCA), and the binding arm B comprises a light chain B (LCB) and a heavy chain B (HCB).

[0010] In some embodiments, the LCA comprises the amino acid sequence shown in SEQ ID NO:1.

[0011] In some embodiments, the HCA comprises the amino acid sequence shown in SEQ ID NO:2.

[0012] In some embodiments, the LCB comprises the amino acid sequence shown in SEQ ID NO:3.

[0013] In some embodiments, the HCB comprises the amino acid sequence shown in SEQ ID NO:4.

[0014] In some embodiments, the LCA, HCA, LCB and HCB comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 3 and 4, respectively.

[0015] In some embodiments, the binding arm A is a half-antibody structure of an IgG-type anti-EGEG antibody, and the binding arm B is a half-antibody structure of an IgG-type anti-c-Met antibody.

[0016] In a second aspect, the present invention provides a bispecific antibody comprising an antigen-binding arm A that specifically targets EGFR and an antigen-binding arm B that specifically targets c-Met, wherein the binding arm A comprises a light chain variable region A (VLA) and a heavy chain variable region A (VHA), and the binding arm B comprises a light chain variable region B (VLB) and a heavy chain variable region B (VHB), wherein the VHA and VLA of the binding arm A are derived from a homodimeric IgG polypeptide A (IgG(A)) comprising a light chain variable region A (VLA) and a heavy chain variable region A (VHA), and the VHB and VLB of the binding arm B are derived from a homodimeric IgG polypeptide B (IgG(B)) comprising a light chain variable region B (VLB) and a heavy chain variable region B (VHB).

[0017] In some embodiments, the VHA comprises VHACDR1, VHACDR2, and VHACDR3 having amino acid sequences as shown in SEQ ID NOs: 19, 20, and 21, respectively.

[0018] In some embodiments, the VLA comprises VLACDR1, VLACDR2, and VLACDR3 having amino acid sequences as shown in SEQ ID NOs: 22, 23, and 24, respectively.

[0019] In some embodiments, the VHB comprises VHBCDR1, VHBCDR2, and VHBCDR having amino acid sequences as shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0020] In some embodiments, the VLB comprises VLBCDR1, VHBCDR2 and VHBCDR3 with amino acid sequences as shown in SEQ ID NOs: 28, 29 and 30, respectively.

[0021] In some embodiments, the VHA comprises the amino acid sequence shown in SEQ ID NO:5.

[0022] In some embodiments, the VLA comprises the amino acid sequence shown in SEQ ID NO:6.

[0023] In some embodiments, the VHB comprises the amino acid sequence shown in SEQ ID NO:7.

[0024] In some embodiments, the VLB comprises the amino acid sequence shown in SEQ ID NO:8.

[0025] In some embodiments, the VHA, VLA, VHB and VLB comprise the amino acid sequences shown in SEQ ID NOs: 5, 6, 7 and 8, respectively.

[0026] In some embodiments, the binding arm A further comprises a heavy chain constant region A (CHA) and a light chain constant region A (CLA), and the binding arm B further comprises a heavy chain constant region B (CHB) and a light chain constant region B (CLB), wherein the CHA and the CHB are both IgG1 isotype heavy chain constant regions.

[0027] In some embodiments, the CHA comprises an Fc(A) region, and the Fc(A) region comprises a CH3(A) region; wherein the CHB comprises an Fc(B) region, and the Fc(B) region comprises a CH3(B) region.

[0028] In some embodiments, the heterodimeric interaction between the CH3(A) region and the CH3(B) region is stronger than the homodimeric interaction between the CH3(A) region and the CH3(B) region.

[0029] In some embodiments, the binding arm A further comprises a heavy chain constant region A (CHA) and a light chain constant region A (CLA), and the binding arm B further comprises a heavy chain constant region B (CHB) and a light chain constant region B (CLB), wherein the CHA and the CHB are both IgG1 isotype heavy chain constant regions.

[0030] In some embodiments, CH3 (A) of the antigen-binding arm A and CH3 (B) of the antigen-binding arm B both comprise mutations that enhance and promote heterodimer formation.

[0031] In some embodiments, the CH3(A) comprises at least one amino acid substitution, the CH3(B) comprises at least one amino acid substitution, and when the residues are numbered according to the EU index, the substitution in CH3(A) and the substitution in CH3(B) occur at different amino acid residue positions.

[0032] In some embodiments, the CH3(A) comprises an amino acid mutation as shown in F405L, and the CH3(B) comprises an amino acid mutation as shown in K409R.

[0033] In some embodiments, both the Fc(A) and the Fc(B) regions comprise amino acid mutations that reduce immunogenicity.

[0034] In some embodiments, both the Fc(A) and Fc(B) regions contain a combination of amino acid site mutations consisting of K214R, D356E, and L358M.

[0035] In some embodiments, the CHA, CLA, CHB and CLB comprise the amino acid sequences shown in SEQ ID NOs: 11, 12, 13 and 14, respectively.

[0036] In a third aspect, the present invention provides a nucleic acid encoding composition comprising polynucleotide sequences as shown in SEQ ID NOs: 15, 16, 17 and 18, respectively, encoding HCA, HCB, LCA and LCB of the bispecific antibody according to the aforementioned aspects.

[0037] In a fourth aspect, the present invention provides a method for preparing the aforementioned anti-EGFR / c-Met bispecific antibody based on Fab exchange technology.

[0038] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof described in the above aspects, and a pharmaceutically acceptable carrier or diluent.

[0039] In a sixth aspect, the present invention provides bispecific antibody molecules or antigen-binding fragments thereof that specifically bind to EGFR and c-Met, as defined herein, and pharmaceutical compositions comprising the bispecific antibody molecules or antigen-binding fragments thereof, for use in preparing medicaments for treating diseases of the human or animal body, such as cancer.

[0040] In a seventh aspect, the present invention provides a method for treating cancer, comprising administering the antibody molecule or pharmaceutical composition defined in the invention.

[0041] In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, colorectal cancer, non-small cell lung cancer (NSCLC), and squamous cell head and neck cancer (SQHN). In some exemplary embodiments, the cancer to be treated is non-small cell lung cancer (NSCLC).

[0042] The present disclosure includes combinations of the described aspects and preferred features unless such combinations are expressly disallowed or explicitly avoided.

[0043] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they are not listed here.

[0044] The EGFR / c-Met bispecific antibody disclosed herein has the following functions:

[0045] (1) It can specifically bind to human c-Met protein and EGFR protein, and the binding activity of binding arm B to c-Met is significantly stronger than that of binding arm A to EGFR, with the difference in binding activity between the two arms exceeding 20 times;

[0046] (2) It can bind to CHOK1-EGFR cells overexpressing human EGFR and CHOK1-HGFR cells overexpressing human c-Met, respectively, and the binding activity of binding arm B to CHOK1-HGFR cells is significantly higher than the binding activity of binding arm A to CHOK1-EGFR;

[0047] (3) Ability to specifically bind to double-positive tumor cells;

[0048] (4) It can antagonize the binding of the ligand EGF to the target protein human EGFR and the binding of the ligand HGF to the target protein human c-Met;

[0049] (5) It has a similar affinity to that of Avanta for Fc receptor proteins;

[0050] (6) It can inhibit the autophosphorylation of EGFR mediated by the ligand EGF and the autophosphorylation of c-Met mediated by the ligand HGF;

[0051] (7) It can mediate the internalization and degradation of EGFR and c-Met target proteins respectively, and the mediated dual-target internalization has a synergistic effect;

[0052] (8) mediate antibody-dependent cellular cytotoxicity (ADCC) against CHOK1-EGFR target cells and CHOK1-cMet target cells and EGFR and c-Met double-positive tumor cells, respectively, and the ADCC mediated against double-positive tumor cells mainly depends on the EGFR antibody arm;

[0053] (9) It can inhibit the proliferation of various human tumor cells in vitro; its inhibitory effect on tumor cell proliferation in vitro is stronger than the combination of anti-EGFR monoclonal antibody and anti-c-Met monoclonal antibody, showing a synergistic effect;

[0054] (10) It can inhibit the proliferation of tumor cells with acquired resistance to third-generation TKI (such as osimertinib) in vitro;

[0055] (11) It can inhibit tumor growth in a mouse xenograft model of human tumor cells expressing HGF.

[0056] The positive progressive effects of the anti-EGFR / c-Met bispecific antibody of the present invention are: based on the selective and specific binding to EGFR and c-Met double-positive tumor cells, the inhibitory efficacy on human tumor cell proliferation in vitro is stronger than the combined use of anti-EGFR monoclonal antibodies and anti-c-Met monoclonal antibodies, and has a synergistic effect; the functional property of inhibiting the growth of HGF autocrine human tumors in vivo is expected to provide the tumor treatment market with a new option for tumor immunotherapy targeted therapy suitable for patients with osimertinib-acquired drug-resistant tumors and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Examples and experiments illustrating the principles of the present disclosure may be discussed with reference to the accompanying drawings, in which:

[0058] Figure 1. Dose-dependent binding curves of BT461-DB targeting c-Met or EGFR proteins as detected by ELISA. A, Dose-dependent binding curves of anti-EGFR / c-Met antibody to human c-Met protein as detected by ELISA. B, Dose-dependent binding curves of anti-EGFR / c-Met antibody to human EGFR protein as detected by ELISA. The abscissa represents antibody concentration (logarithmic value), and the ordinate represents antibody binding signal intensity.

[0059] Figure 2. Dose-dependent binding curves of BT461-DB targeting cell surface EGFR and c-Met detected by FACS. A, Dose-dependent binding curves of the anti-EGFR / cMet antibody to CHO-K1 cells overexpressing human c-Met detected by FACS. B, Dose-dependent binding curves of the anti-EGFR / cMet antibody to CHO-K1 cells overexpressing human EGFR detected by FACS. The abscissa represents the antibody concentration (logarithmic value), and the ordinate represents the antibody binding signal intensity.

[0060] Figure 3. FACS analysis of the binding curves of anti-EGFR / cMet antibodies to EGFR- and HGFR-positive MKN45 cells at varying concentrations. Left: Binding curve; Right: EC50 of the binding curve. The abscissa represents the antibody concentration (logarithmic value), and the ordinate represents the antibody binding signal intensity.

[0061] Figure 4 shows dose-dependent binding curves of BT461-DB blocking the binding of ligands HGF and EGF to HGFR and EGFR target proteins as measured by ELISA. A, Dose-dependent binding curve of anti-EGFR / cMet antibody blocking HGF binding to human HGFR protein as measured by ELISA. B, Dose-dependent binding curve of anti-EGFR / cMet antibody blocking EGF binding to human EGFR protein as measured by ELISA. The abscissa represents antibody concentration (logarithmic value), and the ordinate represents the binding signal intensity of the ligand HGF.

[0062] Figure 5. Binding and dissociation curves of BT461-DB. A, Binding and dissociation curves of BT461-KIH and EGFR-HIS protein. B, Binding and dissociation curves of BT461-DB and EGFR-HIS protein. C, Binding and dissociation curves of BT461-KIH and HGFR-HIS protein. D, Binding and dissociation curves of BT461-DB and HGFR-HIS protein.

[0063] Figure 6 Western-blot detection of the inhibition of BT461-DB on target protein EGFR autophosphorylation and c-Met autophosphorylation.

[0064] Figure 7 Detection of cellular internalization levels induced by the BT461-DB antibody. Left: FACS analysis of internalization of the antibody of the present invention after treatment with a gradient of antibody concentrations. Right: EC50 and TOP (maximum internalization level) of the internalization curve for the gradient of antibody concentration. The abscissa represents the antibody concentration (logarithmic value), and the ordinate represents the internalization level of the fluorescently labeled antibody.

[0065] Figure 8: BT461-DB-mediated ADCC assay against target cells using an ADCC reporter system. A, In vitro ADCC activity against CHOK1 cells overexpressing EGFR (antibody-dependent). B, In vitro ADCC activity against CHOK1 cells overexpressing cMet (antibody-dependent).

[0066] Figure 9: ADCC reporter system-based assay evaluating BT461-DB antibody-mediated ADCC against HCC827 tumor cells. Left: ADCC activity curve. B: EC50 and TOP value (maximum ADCC activity level) of the curve.

[0067] Figure 10: Antibody BT461-DB inhibits the proliferation of the tumor cell line HCC827-HGF in vitro. A, BT461-DB exhibits comparable in vitro efficacy to BT461-KIH. B, BT461-DB exhibits synergistic in vitro efficacy.

[0068] Figure 11 Results of the resistance verification experiment of osimertinib-acquired resistant cells.

[0069] Figure 12. In vitro inhibition curves of osimertinib-resistant cells by the antibody BT461-DB. A, BT461-DB significantly inhibits the proliferation of osimertinib-resistant cells. B, BT461-DB exhibits a synergistic effect on the in vitro efficacy of osimertinib-resistant cells.

[0070] Figure 13: Experimental results of the BT461-DB antibody inhibiting the growth of HCC827-HGF xenograft tumors. A, Tumor volume growth curve. B, Tumor inhibition rate statistics.

[0071] Figure 14 PEE12.4 plasmid.

[0072] Figure 15 PEE6.4 plasmid. DETAILED DESCRIPTION

[0073] Through creative work, the researchers of the present invention have obtained an anti-EGFR / c-Met bispecific antibody that can solve the problem of TKI resistance in the field of EGFR-positive cancer treatment and achieve high-purity assembly. Compared with the combination of EGFR monoclonal antibody and c-Met monoclonal antibody, the bispecific antibody of the present invention has a significantly higher affinity for c-Met than for EGFR binding, and can antagonize the binding of ligands HGF and EGF to the target proteins EGFR and c-Met; it can significantly inhibit the intracellular phosphorylation level of pc-Met (Y1234 / 1235) while slightly inhibiting the phosphorylation level of p-EGFR (Y1173), thereby inhibiting the signaling pathways of c-Met and EGFR; the Fc has a high affinity for receptor proteins and can mediate ADCC of EGFR-positive cells and c-Met-positive cells and EGFR and c-Met double-positive tumor cells, respectively, promoting the internalization of EGFR and c-Met target proteins, and the internalization of EGFR and c-Met dual targets has a synergistic effect; it can inhibit the proliferation of HGFR-positive cells, and the degree of inhibition is stronger than the combination of anti-EGFR monoclonal antibody and anti-c-Met monoclonal antibody, showing a synergistic effect.

[0074] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature and techniques used in connection with the cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, transient transfection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0075] Aspects and examples of the present disclosure will be discussed below with reference to the accompanying drawings. Other aspects and disclosures will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0076] Epidermal growth factor receptor (EGFR)

[0077] Human EGFR (also known as the proto-oncogene c-ErbB-1) is a transmembrane protein and a receptor for members of the epidermal growth factor family (EGF family). Ligand binding to EGFR induces receptor dimerization and autophosphorylation of several tyrosine residues (Y992, Y1045, Y1068, Y1148, and Y1173) in the C-terminal regulatory region of EGFR.

[0078] Aberrant EGFR expression activity is associated with many diseases, including neurological diseases and many cancers.

[0079] In this specification, "EGFR" may refer to EGFR from any species and includes EGFR isoforms, fragments, variants or homologs from any species.

[0080] In an exemplary embodiment, EGFR comprises the amino acid sequence shown in SEQ ID NO:32.

[0081] Tyrosine protein kinase (c-Met)

[0082] Human c-Met, also known as a receptor tyrosine kinase or hepatocyte growth factor receptor (HGFR), binds to its ligand, HGF, inducing autophosphorylation of several tyrosine residues (Y1234 / Y1235) within the c-Met intracellular domain, providing docking sites for downstream signaling molecules and activating multiple signaling cascades. Overexpression of c-Met is observed in many human tumors and is often associated with a metastatic phenotype and poor prognosis. Examples of cancers in which high levels of c-Met expression have been observed include non-small cell lung cancer, pancreatic cancer, colorectal cancer, head and neck squamous cell carcinoma, breast cancer, and esophageal and gastric cancer. Co-expression of EGFR and c-Met is frequently observed in these cancers.

[0083] In the present specification, “c-Met” may be c-Met from any species and includes c-Met isoforms, fragments, variants or homologs from any species.

[0084] In an exemplary embodiment, HGFR comprises the amino acid sequence shown in SEQ ID NO:31.

[0085] As used herein, the term "Fab arm" refers to a heavy chain-light chain pair. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigens. The constant regions of antibodies (Abs) mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component of the classical pathway of complement activation.

[0086] "Full length antibody" when used herein refers to an antibody that contains all of the heavy and light chain constant and variable domains normally found in an antibody of that isotype.

[0087] As used herein, "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG, or IgG4) encoded by the heavy chain constant region genes.

[0088] When used herein, the term "Fc region" refers to the region of an antibody comprising at least the hinge region, CH2 domain, and CH3 domain.

[0089] The term "heterodimeric interaction between the CH3(A) region and the CH3(B) region" refers to the interaction between the CH3(A) region and the CH3(B) region in a CH3(A) / CH3(B) heterodimeric protein.

[0090] The term "tumor cell protein" refers to a protein located on the cell surface of a tumor cell. In the present invention, tumor cell proteins include EGFR and c-Met.

[0091] The term "effector cell" as used herein refers to an immune cell that participates in the immune response effector phase relative to the recognition phase and activation phase of an immune response. Exemplary immune cells include cells of bone marrow or lymphoid origin, such as lymphocytes (such as B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells and basophils. Some effector cells express specific Fc receptors and perform specific immune functions.

[0092] In some embodiments, the effector cells are capable of inducing antibody-dependent cellular cytotoxicity (ADCC), such as natural killer cells, which are capable of inducing ADCC.

[0093] In some embodiments, effector cells can phagocytose target antigens or target cells.

[0094] Composition of matter

[0095] The present invention provides bispecific antibodies that specifically bind to EGFR and c-Met, polynucleotides encoding the bispecific antibodies of the present invention, vectors, host cells, and methods for preparing and using the same.

[0096] "Antigen-binding arm" describes the portion of a bispecific antibody that binds to all or part of the corresponding antigen.

[0097] The bispecific antibody of the present invention that specifically binds to EGFR and c-Met comprises an antigen-binding arm A that specifically binds to EGFR and an antigen-binding arm B that specifically binds to c-Met, and has a significantly higher affinity for c-Met than for EGFR, with the affinity difference being greater than or equal to 20 times.

[0098] The amino acid sequence number combinations of the fragments comprised by the binding arm A and the binding arm B of the exemplary embodiment of the bispecific antibody of the present invention are shown in Table 1. The light chain, heavy chain, variable region and CDR sequences of the EGFR-specific binding arm A are shown in Table 2 and Table 3 of Example 1, respectively. The amino acid sequences of the light chain, heavy chain, variable region and CDR sequences of the c-Met-specific binding arm B are shown in Table 4 and Table 5 of Example 1, respectively.

[0099] EGFR-specific binding arm A

[0100] The binding arm A of the bispecific antibody of the present invention binds to EGFR with high affinity and inhibits EGFR signaling, and can provide beneficial effects in terms of specificity and reduced EGFR receptor phosphorylation levels compared to small molecule EGFR inhibitors and anti-EGFR monoclonal antibodies. The binding arm A of the present invention is monovalent, thus preventing the desired receptor clustering and activation that may occur with other bivalent molecules.

[0101] In some embodiments, the binding arm A that specifically binds to EGFR of the bispecific antibody of the present invention comprises a light chain A (LCA) and a heavy chain A (HCA).

[0102] In some embodiments, the LCA of the binding arm A preferably comprises the amino acid sequence shown in SEQ ID NO:1.

[0103] In some embodiments, the HCA of the binding arm A preferably comprises the amino acid sequence shown in SEQ ID NO:2.

[0104] In some embodiments, the LCA and HCA of the binding arm A preferably comprise the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0105] In some embodiments, the LCA and HCA are derived from IgG polypeptide A (IgG(A)) having a homodimeric structure composed of light chain A (LCA) and heavy chain A (HCA).

[0106] In some embodiments, the LCA and HCA are derived from IgG polypeptide A (IgG(A)) having a homodimeric structure consisting of light chain A (LCA) and heavy chain A (HCA) as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0107] In some embodiments, the binding arm A comprises a light chain variable region A (VLA) and a heavy chain variable region A (VHA); wherein the binding arm A is derived from an IgG polypeptide A (IgG(A)) having a homodimeric structure composed of a light chain variable region A (VLA) and a heavy chain variable region A (VHA).

[0108] In some embodiments, the binding arm A is a half antibody of a homodimeric IgG antibody.

[0109] In some embodiments, the protected VLA and the VHA comprise the VHA CDRs and VLA CDRs, respectively, as shown in Table 3 of Example 1.

[0110] In some embodiments, the binding arm A that specifically binds to EGFR has a VHA CDR1 with the amino acid sequence shown in SEQ ID NO: 19.

[0111] In some embodiments, the binding arm A that specifically binds to EGFR has a VHA CDR2 with the amino acid sequence shown in SEQ ID NO:20.

[0112] In some embodiments, the binding arm A that specifically binds to EGFR has a VHA CDR3 with the amino acid sequence shown in SEQ ID NO: 21.

[0113] In some embodiments, the binding arm A that specifically binds to EGFR has a VLA CDR1 with the amino acid sequence shown in SEQ ID NO:22.

[0114] In some embodiments, the binding arm A that specifically binds to EGFR has a VLA CDR2 with the amino acid sequence shown in SEQ ID NO:23.

[0115] In some embodiments, the binding arm A that specifically binds to EGFR has a VLA CDR3 with the amino acid sequence shown in SEQ ID NO:24.

[0116] The amino acid sequence of the binding arm A of the bispecific antibody embodiment of the present invention and the amino acid sequence of the parent antibody are shown in Example 1.

[0117] In some embodiments, the heavy chain variable region A (VHA) preferably comprises VHA CDR1, VHA CDR2 and VHA CDR3 of the amino acid sequences shown in SEQ ID NOs: 19, 20 and 21, respectively, and the light chain variable region A (VLA) preferably comprises VLA CDR1, VLA CDR2 and VLA CDR3 of the amino acid sequences shown in SEQ ID NOs: 22, 23 and 24, respectively.

[0118] In some embodiments, the heavy chain variable region A (VHA) comprises the amino acid sequence shown in SEQ ID NO:5.

[0119] In some embodiments, the light chain variable region A (VLA) comprises the amino acid sequence shown in SEQ ID NO:6.

[0120] Wherein, the VHA and VLA comprise the amino acid sequences shown in SEQ ID NO: 5 and 6, respectively.

[0121] In some embodiments, the binding arm A is derived from a homodimeric IgG polypeptide A (IgG(A)) composed of VHA having the amino acid sequence shown in SEQ ID NO:5 and VLA having the amino acid sequence shown in SEQ ID NO:6.

[0122] In some embodiments, the binding arm A further comprises a heavy chain constant region A (CHA) and a light chain constant region A (CLA).

[0123] In some embodiments, the CHA is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0124] In some embodiments, the CHA is an IgG1 isotype heavy chain constant region.

[0125] In some embodiments, the antigen binding arm A comprises an Fc(A) region, and the Fc(A) region comprises a CH3(A) region.

[0126] In some embodiments, the CH3(A) region comprises amino acid mutations that reduce the heavy chain mispairing rate.

[0127] In some embodiments, the CH3(A) region comprises an amino acid mutation as shown in F405L.

[0128] In some embodiments, the Fc(A) region comprises a subtype backmutation that reduces immunogenicity.

[0129] In some embodiments, the Fc(A) region comprises amino acid mutations such as K214R, D356E, and L358M.

[0130] In some embodiments, the Fc(A) comprises the amino acid sequence shown in SEQ ID NO:9.

[0131] In some embodiments, the CHA comprises the amino acid sequence shown in SEQ ID NO:11.

[0132] In some embodiments, the LCA is a kappa-type or lambda-type light chain.

[0133] In some embodiments, the LCA is a kappa light chain.

[0134] In some embodiments, the CLA comprises the amino acid sequence shown in SEQ ID NO:12.

[0135] c-Met-specific binding arm B

[0136] The binding arm B of the bispecific antibodies of the present invention binds to c-Met with high affinity and inhibits c-Met signaling, and can provide advantages in terms of specificity and reduced c-Met receptor phosphorylation levels compared to small molecule c-Met inhibitors and anti-c-Met monoclonal antibodies. The binding arm B of the present invention is monovalent, thus preventing the desired receptor clustering and activation that may occur with other bivalent molecules.

[0137] In some embodiments, the binding arm B that specifically binds to c-Met of the bispecific antibody of the present invention comprises a light chain B (LCB) and a heavy chain B (HCB).

[0138] In some embodiments, the LCB of the binding arm A preferably comprises the amino acid sequence shown in SEQ ID NO:3.

[0139] In some embodiments, the HCB of the binding arm A preferably comprises the amino acid sequence shown in SEQ ID NO:4.

[0140] In some embodiments, the light chain B (LCB) and heavy chain B (HCB) comprise the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0141] In some embodiments, the binding arm B is derived from an IgG polypeptide B (IgG(B)) having a homodimeric structure consisting of a light chain B (LCB) and a heavy chain B (HCB).

[0142] In some embodiments, the binding arm B is a half antibody of a homodimeric IgG antibody comprising light chain B (LCB) and heavy chain B (HCB) with the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0143] In some embodiments, the binding arm B comprises a light chain variable region B (VLB) and a heavy chain variable region B (VHB); wherein, the binding arm B is derived from an IgG polypeptide B (IgG(B)) having a homodimeric structure composed of a light chain variable region B (VLB) and a heavy chain variable region B (VHB).

[0144] In some embodiments, the VHB comprises VHB CDR1, VHB CDR2, and VHB CDR3, and the VLB comprises VLB CDR1, VLB CDR2, and VLB CDR3.

[0145] In some embodiments, the binding arm B that specifically binds to c-Met has a VHB CDR1 with the amino acid sequence shown in SEQ ID NO:25.

[0146] In some embodiments, the binding arm B that specifically binds to c-Met has a VHB CDR2 with the amino acid sequence shown in SEQ ID NO:26.

[0147] In some embodiments, the binding arm B that specifically binds to c-Met has a VHB CDR3 with an amino acid sequence as shown in SEQ ID NO: 27.

[0148] In some embodiments, the binding arm B that specifically binds to c-Met has a VLB CDR1 with the amino acid sequence shown in SEQ ID NO: 28.

[0149] In some embodiments, the binding arm B that specifically binds to c-Met has a VLB CDR2 with an amino acid sequence as shown in SEQ ID NO: 29,

[0150] In some embodiments, the binding arm B that specifically binds to c-Met has a VLB CDR3 with the amino acid sequence shown in SEQ ID NO: 30.

[0151] In some embodiments, the heavy chain variable region B (VHB) comprises VHB CDR1, VHB CDR2, and VHB CDR3 with amino acid sequences as shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0152] In some embodiments, the light chain variable region B (VLB) comprises VLB CDR1, VLB CDR2, and VLB CDR3 having amino acid sequences as shown in SEQ ID NOs: 28, 29, and 30, respectively.

[0153] In some embodiments, the c-Met binding arm B comprises VHB CDR1, VHB CDR2 and VHB CDR3 of the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27, respectively, and VLB CDR1, VLB CDR2 and VLB CDR3 of the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30.

[0154] In some embodiments, the heavy chain variable region B (VHB) comprises the amino acid sequence shown in SEQ ID NO:7.

[0155] In some embodiments, the light chain variable region B (VLB) comprises the amino acid sequence shown in SEQ ID NO:8.

[0156] In some embodiments, the VHB and VLB comprise the amino acid sequences shown in SEQ ID NOs: 7 and 8, respectively.

[0157] In some embodiments, the binding arm B is derived from a homodimeric IgG polypeptide B (IgG(B)) composed of VHB having the amino acid sequence shown in SEQ ID NO: 7 and VLB having the amino acid sequence shown in SEQ ID NO: 8.

[0158] In some embodiments, the binding arm B further comprises a heavy chain constant region B (CHB) and a light chain constant region B (CLB).

[0159] In some embodiments, the CHB is of one of the isotypes IgG1, IgG2, IgG3, or IgG4.

[0160] In some embodiments, the CHB is an IgG1 isotype heavy chain constant region.

[0161] The CHB is an IgG1 isotype heavy chain constant region.

[0162] In some embodiments, the CHB comprises an Fc(B) region, and the Fc(B) region comprises a CH3(B) region.

[0163] In some embodiments, the CH3(B) region comprises amino acid mutations that reduce the heavy chain mispairing rate.

[0164] In some embodiments, the CH3(B) region comprises an amino acid mutation as shown in K409R.

[0165] In some embodiments, the CH3(B) region comprises an IgG subtype amino acid backmutation that reduces immunogenicity.

[0166] In some embodiments, the Fc(B) region comprises a combination of amino acid mutations as shown in K214R, D356E, and L358M.

[0167] In some embodiments, the Fc(B) comprises the amino acid sequence shown in SEQ ID NO:10.

[0168] In some embodiments, the CHB comprises the amino acid sequence shown in SEQ ID NO:13.

[0169] In some embodiments, the CLB is one of a kappa-type and a lambda-type light chain constant region.

[0170] In some embodiments, the CLB is a kappa-type light chain constant region.

[0171] In some embodiments, the CLB comprises the amino acid sequence shown in SEQ ID NO:14.

[0172] The amino acid sequence of the binding arm B of the bispecific antibody embodiment of the present invention and the amino acid sequence of the parent antibody are shown in Example 1.

[0173] Structure of a bispecific antibody that specifically binds to EGFR and c-Met

[0174] In a further aspect, the present invention relates to an EGFR / c-Met bispecific heterodimeric protein obtained or obtainable by the method of the present invention.

[0175] In an embodiment of the method of the present invention, the binding arm A and the binding arm A of the bispecific molecule are both Fab structures, the Fab (A) arm binds to EGFR-specific tumor cells, such as the tumor cell surface protein EGFR, and the Fab arm (B) recognizes c-Met-specific tumor cells, such as the tumor cell surface protein c-Met.

[0176] In some embodiments, the binding arm A that binds to EGFR is an antigen binding arm comprising an EGFR-specific VH / VL pair or a light chain / heavy chain pair, and the binding arm B that binds to c-Met is an antigen binding arm comprising a c-Met-specific VH / VL pair or a light chain / heavy chain pair.

[0177] In addition, the EGFR / c-Met bispecific heterodimeric protein of the present invention is an asymmetric molecule, having different features on each Fab arm (e.g., EGFR-specific Fab (A) or c-Met-specific Fab (B)) or each CH3 domain (e.g., CH3 (A) or CH3 (B)), or having different modifications throughout the molecule, such as a molecule having amino acid substitutions for reducing heavy chain mispairing and immunogenicity. Such asymmetric molecules can be produced in any suitable combination. This is further illustrated below by some non-limiting examples.

[0178] The bispecific antibody specifically binding to EGFR and c-Met of the present invention comprises an antigen-binding arm A that specifically binds to EGFR and an antigen-binding arm B that specifically binds to c-Met, wherein the binding arm A is a half-antibody structure polypeptide A derived from a homodimeric IgG antibody with EGFR binding specificity, comprising a variant Fc(A) region, wherein the Fc(A) region comprises a CH3(A) region; and the binding arm B is a half-antibody structure polypeptide B derived from a homodimeric IgG antibody with c-Met binding specificity, comprising a variant Fc(B) region, wherein the Fc(B) region comprises a CH3(B) region; wherein the sequences of the CH3(A) region and the CH3(B) region are different, and the heterodimeric interaction between the CH3(A) region and the CH3(B) region is stronger than the interaction between the CH3 of the homodimers of the CH3(A) region and the CH3(B) region.

[0179] In some embodiments, the EGFR-specific binding arm A comprises LCA and HCA as shown in SEQ ID NOs: 1 and 2, respectively, and the c-Met-specific binding arm B comprises LCB and HCB as shown in SEQ ID NOs: 3 and 4, respectively. The heavy chain (HCA) of the binding arm A and the heavy chain (HCB) of the binding arm B interact to form a heterodimeric structure.

[0180] In some embodiments, the LCA and the HCA comprise a light chain variable region (VLA) and a heavy chain variable region (VHA) specific for EGFR, respectively, and the LCB and the HCB comprise a light chain variable region (VLB) and a heavy chain variable region (VHB) specific for c-Met, respectively.

[0181] In some embodiments, the VHA comprises VHA CDR1, VHA CDR2, and VHA CDR3 having amino acid sequences as shown in SEQ ID NOs: 19, 20, and 21, respectively.

[0182] In some embodiments, the VLA comprises VLA CDR1, VLA CDR2, and VLA CDR3 having amino acid sequences as shown in SEQ ID NOs: 22, 23, and 24, respectively.

[0183] In some embodiments, the VHB comprises VHB CDR1, VHB CDR2, and VHB CDR3 having amino acid sequences as shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0184] In some embodiments, the VLB comprises VLA CDR1, VLA CDR2, and VLA CDR3 having amino acid sequences as shown in SEQ ID NOs: 28, 29, and 30, respectively.

[0185] In some embodiments, the VLA comprises VLA CDR1, VLA CDR2, and VLA CDR3 having amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively; the VHA comprises VHA CDR1, VHA CDR2, and VHA CDR3 having amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively; the VLB comprises VLA CDR1, VLA CDR2, and VLA CDR3 having amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively; and the VHB comprises VHB CDR1, VHB CDR2, and VHB CDR3 having amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.

[0186] In some embodiments, the binding arm A of the EGFR-specific half-antibody structure comprises VHA and VLA as shown in SEQ ID NO: 5 and 6, respectively.

[0187] In some embodiments, the binding arm B of the c-Met-specific half-antibody structure comprises VLB and VHB as shown in SEQ ID NOs: 7 and 8, respectively.

[0188] The light chain and heavy chain of binding arm A and binding arm B of the bispecific antibodies of the present invention disclosed herein further include a light chain constant region and a heavy chain constant region, respectively.

[0189] In other embodiments, each Fab arm present in the bispecific molecule is derived from a different IgG subclass.

[0190] Preferably, the LCA and HCA of binding arm A and the LCB and HCB of binding arm B of the bispecific antibody according to the present invention disclosed herein both comprise the constant regions of a full-length human IgG antibody.

[0191] In some embodiments, the binding arm A and the binding arm B are full-length IgG antibodies, preferably one of full-length IgG1, IgG2, IgG3 or IgG4 antibodies.

[0192] In some preferred embodiments the full-length IgG antibody is an antibody of the human IgG1 isotype.

[0193] In another embodiment of the heterodimeric protein of the present invention, the Fc region of polypeptide A (Fc(A)) is of an isotype selected from IgG1, IgG2, IgG3 and IgG4 (except for the specified mutations), and the Fc region of polypeptide B (Fc(B)) is of an isotype selected from IgG1, IgG2, IgG3 and IgG4 (except for the specified mutations).

[0194] In another embodiment of the heterodimeric protein of the present invention, the Fc regions of both polypeptide A and polypeptide B are of IgG1 isotype.

[0195] In some embodiments, the binding arms of the bispecific antibody molecules described herein comprise heavy chain constant regions (CH) of the IgG1 isotype, such as the CHA region of binding arm A and the CHB region of binding arm B.

[0196] For example, as described in more detail below, asymmetric bispecific antibody molecules have different CHAs and CHBs.

[0197] In some embodiments, the CHA comprises Fc(A) and the CHB comprises Fc(B).

[0198] In some embodiments, the Fc(A) comprises CH3(A), and the Fc(B) comprises CH3(B).

[0199] In some embodiments, the heavy chain constant regions of the bispecific antibody molecules described herein, such as CHA and CHB, may comprise one or more amino acid modifications. In some embodiments, the amino acid modifications are amino acid substitutions.

[0200] In another aspect, the present invention relates to an EGFR / c-Met bispecific heterodimeric protein, which comprises a polypeptide A comprising an immunoglobulin Fc (A) region, wherein the Fc (A) region comprises a CH3 (A) region; and a polypeptide B comprising an immunoglobulin Fc (B) region, wherein the sequences of the CH3 (A) region and the CH3 (B) region are different, and the heterodimeric interaction between the CH3 (A) region and the CH3 (B) region is stronger than the homodimeric interaction between the CH3 (A) region and the CH3 (B) region.

[0201] In some preferred embodiments, the EGFR / c-Met bispecific heterodimeric protein of the present invention is prepared using a Fab exchange technology platform commonly used in the antibody industry that can provide CH3 modifications of modified heavy chains. After the modified half-antibody of the binding arm A and the half-antibody of the binding arm B are co-expressed in clonal cells, the interaction between the heavy chains HCA and HCB, especially between Fc(A) and Fc(B), is stronger than the interaction between homodimers, thereby preferentially forming heterodimers.

[0202] In some embodiments, CH3 (A) of the antigen-binding arm A and CH3 (B) of the antigen-binding arm B both comprise mutations that enhance heterodimer formation.

[0203] In some embodiments, the Fc(A) and the Fc(B) each comprise amino acid mutations that prevent heavy chain mispairing.

[0204] The bispecific antibodies of the present invention disclosed herein are preferably for use in humans. A preferred embodiment of the bispecific antibodies of the present invention is a human or humanized antibody. The bispecific antibodies of the present invention disclosed herein are preferably heterodimeric bispecific antibodies formed by an interaction between a binding arm of a half-antibody structure targeting EGFR and a binding arm of a half-antibody structure targeting c-Met, through the heavy chain CH3 region, which is stronger than that between the respective homodimers.

[0205] In some embodiments, the CH3(A) region has an amino acid other than Phe at position 405, such as an amino acid other than Phe, Arg, or Gly at position 405; in some embodiments, the CH3(B) region has an amino acid other than Lys, Leu, or Met at position 409.

[0206] In some embodiments, the CH3(A) comprises an amino acid mutation as shown in F405L. In some embodiments, the CH3(B) comprises an amino acid mutation as shown in K409R.

[0207] In some embodiments, the CH3(A) and the CH3(B) comprise heavy chain mispairing preventing amino acid mutations F405L and K409R, respectively.

[0208] In some embodiments, both the Fc(A) and the Fc(B) regions further comprise a combination of amino acid site mutations that reduce immunogenicity.

[0209] In some embodiments, both CH3A and CH3B contain a combination of amino acid site mutations consisting of amino acid mutations K214R, D356E, and L358M that reduce immunogenicity.

[0210] In a preferred embodiment, the binding arm A and the binding arm B can form a heterodimer through the interaction between CHA and CHB, more specifically, through the interaction between CH3(A) and CH3(B).

[0211] In some preferred embodiments, the Fc(A) of binding arm A comprises an Fc(A) segment derived from the constant region CHA of heavy chain A as shown in SEQ ID NO:3, and the Fc(B) of binding arm B comprises an Fc(B) segment derived from the constant region CHB of heavy chain B as shown in SEQ ID NO:4.

[0212] In some preferred embodiments, the Fc(A) of binding arm A comprises an amino acid sequence derived from SEQ ID NO:9, and the Fc(B) of binding arm B comprises an amino acid sequence derived from SEQ ID NO:10.

[0213] In some embodiments, the CHA and the CHB are heavy chain constant regions of the IgG1 isotype.

[0214] In some embodiments, the CHA region comprises CH3(A) of the amino acid sequence shown in SEQ ID NO:33.

[0215] In some embodiments, the CHB region comprises CH3(B) of the amino acid sequence shown in SEQ ID NO:34.

[0216] In some embodiments, the CHA region comprises the amino acid sequence shown in SEQ ID NO:11.

[0217] In some embodiments, the CHB region comprises the amino acid sequence shown in SEQ ID NO:13.

[0218] In some embodiments, the CH3(A) and the CH3(B) comprise the amino acid sequences shown in SEQ ID NO: 33 and SEQ ID NO: 34, respectively.

[0219] In some embodiments, the CHA and CHB comprise the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 13, respectively.

[0220] In some embodiments, the bispecific antibody molecules described herein comprise a light chain constant region fragment thereof, such as CLA comprised by antigen-binding arm A and CLB comprised by antigen-binding arm B.

[0221] In some embodiments, the CLA and CLB are selected from one of a kappa-type light chain constant region or a lambda-type light chain constant region.

[0222] In some embodiments, the CLA and CLB are both kappa-type light chain constant regions.

[0223] In some embodiments, the CLA region comprises a κCLA region having an amino acid sequence shown in EQ ID NO:12.

[0224] In some embodiments, the CLB region comprises a κCLB region having an amino acid sequence shown in SEQ ID NO:14.

[0225] In still other embodiments of the heterodimeric protein of the present invention, the polypeptide A may be a full-length heavy chain of an antibody (preferably a human antibody).

[0226] In other embodiments of the heterodimeric protein of the present invention, the polypeptide B may be a full-length heavy chain of an antibody (preferably a human antibody).

[0227] In some further embodiments of the heterodimeric protein of the present invention, polypeptide A and polypeptide B are both full-length heavy chains of two antibodies (preferably two human antibodies that bind to EGFR and c-Met, respectively), so the resulting heterodimeric protein is a bispecific antibody.

[0228] In some embodiments, the resulting heterodimeric protein comprises, in addition to the heavy chain, two full-length light chains derived from an EGFR-specific antibody and a full-length light chain derived from a c-Met-specific antibody, respectively.

[0229] In another embodiment of the heterodimeric protein of the invention, the increased strength of the EGFR / c-Met bispecific heterodimer interaction compared to the individual EGFR homodimer interactions and c-Met homodimer interactions is due to CH3 modification rather than to the introduction of covalent bonds, cysteine ​​residues or charged residues.

[0230] In another embodiment of the EGFR / c-Met heterodimeric protein of the present invention, the heterodimeric interaction between polypeptide A and polypeptide B in the heterodimeric protein is such that Fab arm exchange does not occur at a final concentration of 75 mM 2-MEA under the conditions described in Example 1.

[0231] In the bispecific antibody preparation method of the present invention, the constant region of the heavy chain is generally modified so that the heterodimerization between the two specific binding arms of the bispecific antibody of the present invention, formed by the interaction between the heavy chains, particularly the CH3 portion of the heavy chain, is stronger than the homodimerization between the half antibodies of the respective sources and the same half antibodies, and is more conducive to heterodimerization (i.e., dimerization of the heavy chain with another heavy chain / light chain combination), thereby significantly reducing and preventing the probability of light and heavy chain mispairing.

[0232] In some preferred embodiments, the ADCC activity of the antibody can also be improved by techniques known to those skilled in the art.

[0233] As disclosed herein, the bispecific antibodies of the present invention can have good ADCC properties by modifying the heavy chain through amino acid mutations in the heavy chain constant region.

[0234] In another embodiment, amino acid mutations that improve binding to Fcγ-receptors or FcRn are included on one of the two Fab arms of the bispecific molecule.

[0235] In another embodiment, amino acid mutations that improve binding to C1q are included on one of the two Fab arms of the bispecific molecule.

[0236] In the antibody according to an exemplary embodiment of the present invention, the numbering combinations of the amino acid sequences of the fragments included in the binding arm A and the binding arm B are summarized in Table 1.

[0237] Functional properties of the bispecific antibodies of the present invention

[0238] The EGFR / c-Met bispecific antibody of the present invention has the following functions:

[0239] (1) It can specifically bind to human c-Met protein and EGFR protein in vitro, and the EC50 of binding to human c-Met protein in ELISA assay is significantly lower than the EC50 of binding to EGFR protein, with a difference of more than 20 times.

[0240] (2) have extremely high affinity for c-Met-HIS and high affinity for EGFR-HIS, respectively;

[0241] (3) It can specifically bind to cells that highly express human c-Met protein, cells that highly express EGFR protein, and double-positive tumor cells for c-Met protein and EGFR protein;

[0242] (3) It can block the binding of the ligand HGF to the receptor EGFR and the binding of the ligand EGF to the receptor c-Met;

[0243] (4) The affinity of Fc for the receptor protein is comparable to that of Avanta;

[0244] (5) It can significantly inhibit the level of p-Met (Y1234 / 1235) autophosphorylation mediated by EGF in tumor cells and slightly inhibit the level of p-EGFR (Y1173) autophosphorylation mediated by ligand;

[0245] (6) It can mediate the internalization and degradation of dual-target proteins of EGFR and c-Met, and the internalization level mediated by the anti-c-Met antibody arm is higher than that mediated by the anti-EGFR antibody arm;

[0246] (7) Able to mediate ADCC in cells that highly express EGFR, cells that highly express c-Met, and double-positive tumor cells that highly express EGFR and c-Met; the mediated ADCC mainly depends on the EGFR antibody arm;

[0247] (8) It can inhibit the proliferation of tumor cells in vitro, and the inhibition has a synergistic effect relative to the combination of EGFR monoclonal antibody and c-Met monoclonal antibody;

[0248] (9) It can inhibit the proliferation of TKI-resistant acquired resistant tumor cells in vitro; the inhibitory effect is stronger than the combination of EGFR monoclonal antibody and c-Met monoclonal antibody, and the inhibitory effect has a synergistic effect relative to EGFR monoclonal antibody and c-Met monoclonal antibody;

[0249] (10) It has anti-tumor activity in a mouse model of tumor cells.

[0250] Nucleic acids, vectors, host cells

[0251] In another aspect, the present invention provides a combination of the nucleic acid encoding the binding arm A and the nucleic acid encoding the binding arm B of the EGFR / c-Met heterodimeric bispecific antibody molecule described in the aforementioned aspects.

[0252] In one embodiment, the present invention provides a composition of nucleic acids encoding nucleic acids 1# encoding a binding arm A that specifically binds to EGFR and nucleic acids 2# encoding a binding arm B that specifically binds to c-Met, wherein the nucleic acid 1# is a fusion of nucleic acids 3# encoding heavy chain A and nucleic acids 4# encoding light chain A comprising binding arm A, and the nucleic acid 2# is a fusion of nucleic acids 5# encoding heavy chain B and nucleic acids 6# encoding light chain B comprising binding arm B, respectively.

[0253] In some embodiments, the heavy chain A (HCA) encoded by the encoding nucleic acid 3# comprises a CH3 mutation F405L that promotes interaction between the EGFR binding arm and the c-Met binding arm to avoid heavy chain mispairing.

[0254] In some embodiments, the heavy chain B (HCB) encoded by the encoding nucleic acid 5# comprises a CH3 mutation K409R that promotes interaction between the EGFR binding arm and the c-Met binding arm to avoid heavy chain mispairing.

[0255] In some embodiments, the Fc(A) of the heavy chain A encoded by the encoding nucleic acid 5# comprises the amino acid mutation combination K214R, D356E, and L358M that reduces immunogenicity.

[0256] In some embodiments, the Fc(B) of the heavy chain B encoded by the encoding nucleic acid 7# comprises the amino acid mutation combination K214R, D356E, L358M that reduces immunogenicity.

[0257] In some embodiments, the nucleic acid molecules are purified or isolated from other nucleic acids or naturally occurring biological materials. Those skilled in the art of antibodies can prepare such nucleic acid molecules using methods well known in the art.

[0258] The light chain A (LCA) encoding nucleic acid #4 and the heavy chain A (HCA) encoding nucleic acid #3 of these EGFR-specific binding arm A are described herein.

[0259] The present invention describes the nucleic acid encoding the light chain B (LCB) of these EGFR-specific binding arm B #6 and the nucleic acid encoding the heavy chain B (HCB) #5.

[0260] In some embodiments, the nucleic acid encoding composition of the present invention may comprise:

[0261] (i) a nucleic acid sequence encoding the LCA domain shown in SEQ ID NO: 1, and a nucleic acid sequence encoding the HCA domain shown in SEQ ID NO: 2;

[0262] (ii) a nucleic acid sequence encoding the LCB domain shown in SEQ ID NO: 3, and a nucleic acid sequence encoding the HCB domain shown in SEQ ID NO: 4.

[0263] In yet another aspect, the present invention relates to an expression vector comprising the above-specified EGFR-specific homodimer-encoding nucleic acid construct and the c-Met-specific homodimer-encoding nucleic acid construct.

[0264] In one aspect, the present invention also provides a composition comprising a nucleic acid molecule encoding the binding arm A of the antibody structure or the binding arm B of the antibody structure of the bispecific antibody molecule described herein, or a composition comprising a vector comprising a nucleic acid molecule encoding the binding arm A of the antibody molecule or the binding arm B of the antibody structure, respectively.

[0265] Isolated nucleic acid molecules can be used to express the antibody molecules of the present disclosure or their combinations. The nucleic acid is typically provided in the form of a recombinant vector for expression. A suitable vector can be selected or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other suitable sequences. Preferably, the vector contains appropriate regulatory sequences to drive expression of the nucleic acid in the host cell. The vector can be a plasmid, a virus, such as a plasmid, a phage, or a phagemid, as appropriate.

[0266] In certain embodiments, the vector is a pHR vector.

[0267] In some embodiments, the PHR vector is PEE12.4, as shown in FIG14 .

[0268] In some embodiments, the PHR vector is PEE6.4, as shown in FIG15 .

[0269] In a further aspect, the present invention relates to a host cell comprising the first and second nucleic acid constructs specified above.

[0270] Preparation method

[0271] In another aspect, the present invention provides an in vitro method for producing an EGFR / c-Met heterodimeric bispecific antibody, the method comprising the following steps:

[0272] a) providing a homodimeric protein A specific for EGFR comprising an immunoglobulin Fc(A) region, wherein the Fc(A) region comprises a CH3(A) region,

[0273] b) providing a homodimeric protein B specific for c-Met comprising an immunoglobulin Fc(B) region, said Fc(B) region comprising a CH3(B) region,

[0274] wherein the sequences of the CH3(A) region and the CH3(B) region are different, and the heterodimeric interaction between the CH3(A) region and the CH3(B) region is stronger than the homodimeric interaction between the CH3(A) region and the CH3(B) region,

[0275] c) incubating the EGFR-specific protein A with the c-Met-specific protein B under reducing conditions sufficient to allow the hinge region cysteines to undergo disulfide bond isomerization, and

[0276] d) obtaining the EGFR / c-Met bispecific heterodimeric protein.

[0277] In some embodiments, the homodimeric protein A and homodimeric protein B, in addition to the Fc region, further comprise one or more or all other regions of an antibody, i.e., a CH1 region, a VH region, a CL region, and / or a VL region. Thus, in some embodiments, the homodimeric protein A is a full-length antibody. In another embodiment, the homodimeric protein B is a full-length antibody.

[0278] In one embodiment, the Fc region of homodimeric protein A is an isotype selected from IgG1, IgG2, IgG3 and IgG4, and the Fc region of homodimeric protein B is an isotype selected from IgG1, IgG2, IgG3 and IgG4. In a preferred embodiment, the Fc regions of both homodimeric protein A and homodimeric protein B are of the IgG1 isotype.

[0279] In some embodiments, the homodimeric proteins A and B provided in steps a) and b) are purified.

[0280] As described above, the sequences of the CH3(A) region and the CH3(B) region of the homodimeric initiating protein are different, and the heterodimeric interaction between the CH3(A) region and the CH3(B) region is stronger than the homodimeric interaction between the CH3(A) region and the CH3(B) region.

[0281] In some embodiments, the increased strength of heterodimeric interactions compared to respective homodimeric interactions is due to CH3 modifications rather than to the introduction of covalent bonds, cysteine ​​residues, or charged residues.

[0282] In some embodiments, the products of the invention are highly stable and do not undergo Fab arm exchange under mild reducing conditions in vitro or, importantly, in vivo after administration to humans. Thus, in one embodiment, the heterodimeric interaction between the two proteins A and B in the resulting heterodimeric protein is such that Fab arm exchange cannot occur at a final concentration of 75 mM 2-MEA under the conditions described in Example 1.

[0283] In some embodiments, the method of the present invention can be used to obtain stable heterodimeric proteins in high yield based on two homodimeric starting proteins containing only a few relatively conservative asymmetric mutations in the CH3 region.

[0284] Therefore, in some embodiments, the sequences of the CH3(A) region and the CH3(B) region contain amino acid substitutions at positions that are not identical.

[0285] In some embodiments, the amino acids are natural amino acids.

[0286] In some embodiments, the homodimeric protein A has no more than one amino acid substitution in the CH3(A) region and the homodimeric protein B has no more than one amino acid substitution in the CH3(B) region compared to the wild-type CH region.

[0287] In some embodiments, homodimeric protein A has an amino acid substitution at a position selected from the group consisting of amino acid 405, and homodimeric protein B has an amino acid substitution at a position selected from the group consisting of amino acid 409, wherein homodimeric protein A and homodimeric protein B are not substituted at the same position.

[0288] In some embodiments, homodimeric protein A has an amino acid substitution at position 405 and homodimeric protein B has an amino acid substitution at position 409.

[0289] In some embodiments, the homodimeric protein A has an amino acid at position 405 that is not Phe and the homodimeric protein B has an amino acid at position 409 that is not Lys, Leu, or Met.

[0290] In some embodiments, the homodimeric Protein A has an amino acid at position 405 that is not Phe, Arg, or Gly.

[0291] In one such embodiment, the homodimeric protein B has an amino acid at position 409 that is not Lys, Leu, or Met, and the homodimeric protein A has an amino acid at position 405 that is not Phe.

[0292] In yet another embodiment, the homodimeric protein B has an amino acid at position 409 that is not Lys, Leu, or Met, and the homodimeric protein A has an amino acid at position 405 that is not Phe, Arg, or Gly.

[0293] In even yet another embodiment, the homodimeric protein A has a Leu at position 405 and the second homodimeric protein B has an Arg at position 409.

[0294] In addition to the amino acid substitutions specified above, the homodimeric proteins A and B may contain other amino acid substitutions, deletions or insertions relative to the wild-type Fc sequence.

[0295] In yet another embodiment, the homodimeric protein B and the homodimeric protein A have a combination of amino acid mutations consisting of K214R, D356E, and L358M.

[0296] In yet another embodiment, the CH3(A) region comprises the sequence shown in SEQ ID NO: 33 in addition to the specified mutations.

[0297] In yet another embodiment, the CH3(B) region comprises the sequence shown in SEQ ID NO: 34 in addition to the specified mutations.

[0298] As described above, step c) of the method of the present invention comprises incubating the first protein with the second protein under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide isomerization. Examples of suitable conditions are given herein. The minimum requirements for the cysteines in the hinge region to undergo disulfide isomerization may vary depending on the homodimeric starting protein, particularly depending on the exact sequence in the hinge region. Importantly, the homodimeric interactions between the CH3(A) and CH3(B) regions are sufficiently weak to allow the cysteines in the hinge region to undergo disulfide isomerization under the given conditions.

[0299] In some embodiments, the reducing conditions of step c) include the addition of a reducing agent, which includes but is not limited to: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine ​​and β-mercapto-ethanol, preferably a reducing agent selected from the following: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine.

[0300] In some preferred embodiments, the reducing conditions of step c) include the addition of the reducing agent 2-mercaptoethylamine (2-MEA).

[0301] In yet another embodiment, step c) comprises incubating in the presence of at least 75 mM 2-mercaptoethylamine at a temperature of at least 31° C. for at least 5 minutes. The incubation may be performed at a pH of 5 to 8, such as pH 7.4.

[0302] In yet another embodiment, step d) comprises returning the conditions to non-reducing or less reducing, for example by removing the reducing agent, such as by desalting.

[0303] In some preferred embodiments, step d) comprises returning the conditions to non-reducing or less reducing, for example at pH 11.0, by removing the reducing agent, such as by desalting.

[0304] In some embodiments, the methods of the invention result in an antibody product wherein greater than 80%, such as greater than 90%, such as greater than 95%, such as greater than 99% of the antibody molecules are the desired bispecific antibody.

[0305] The post-production nature of the EGFR / c-Met bispecific antibodies prepared by Fab swapping under reducing conditions (e.g., by addition of 2-MEA) as disclosed herein makes it a very suitable strategy for (high-throughput) screening of EGFR / c-Met bispecific antibodies. Furthermore, the in vitro approach can be performed in libraries, which allows for greater control, higher flexibility, and higher yield of the EGFR / c-Met heterodimeric protein compared to co-expression, while screening can be performed in the final therapeutic form, eliminating the need for engineering after lead selection.

[0306] In another aspect, the method for preparing the EGFR / c-Met bispecific heterodimeric protein of the present invention can be used for "matrix" screening, i.e., generating a large number of combinations of EGFR / c-Met binding specificities based on two groups of antibodies, one group of anti-EGFR antibodies having exactly the same CH3(A) region and the other group of anti-c-Met antibodies having exactly the same CH3(B) region, wherein the sequences of the CH3(A) and CH3(B) regions are different, and the heterodimeric interaction between the CH3(A) and CH3(B) regions is stronger than the homodimeric interaction between the CH3(A) and CH3(B) regions.

[0307] Therefore, in some embodiments the present invention relates to a method for selecting a heterodimeric protein having EGFR / c-Met bispecificity, said method comprising the steps of:

[0308] a) providing a first group of EGFR-specific homodimeric protein A comprising an Fc(A) region, wherein the homodimeric protein A has completely identical CH3(A) regions,

[0309] b) providing a second group of c-Met-specific homodimeric proteins B comprising an Fc(B) region, wherein the homodimeric proteins B have identical CH3(B) regions, wherein the sequences of the CH3(A) region and the CH3(B) region are different, and the heterodimeric interaction between the CH3(A) region and CH3(B) is stronger than the homodimeric interaction between the CH3(A) region and CH3(B),

[0310] c) incubating the combination of said first set of homodimeric proteins A specific for EGFR and said second set of homodimeric proteins B specific for c-Met under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization, thereby generating a set of bispecific antibodies,

[0311] d) optionally restoring the conditions to non-reducing,

[0312] e) assaying the resulting heterodimeric protein set for a given desired EGFR / c-Met bispecific, etc. property, and

[0313] f) Selecting a heterodimeric protein with the desired EGFR / c-Met bispecific properties.

[0314] In some embodiments, the present invention relates to a method for selecting a desired EGFR / c-Met bispecific antibody, the method comprising the steps of:

[0315] a) providing a first group of homodimeric antibodies comprising a variable region with binding specificity for EGFR, wherein said antibodies of said first group comprise a CH3(A) region,

[0316] b) providing a second group of homodimeric antibodies comprising a variable region having binding specificity for c-Met, wherein the antibodies of the second group comprise a CH3(B) region, wherein the sequences of the CH3(A) region and the CH3(B) region are different and such that the heterodimeric interaction between the CH3(A) region and the CH3(B) region is stronger than the homodimeric interaction between the CH3(A) region and the CH3(B) region,

[0317] c) incubating the combination of the first set of antibodies and the second set of antibodies under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization, thereby generating a set of EGFR / c-Met bispecific antibodies,

[0318] d) optionally restoring the conditions to non-reducing,

[0319] e) assaying the resulting panel of bispecific antibodies for a given desired property, and

[0320] f) Selecting a bispecific antibody with the desired EGFR / c-Met bispecificity.

[0321] The screening strategy involves two sets of antibody vectors containing a range of specificities, where one set is cloned into a backbone capable of participating in Fab arm interchange with the backbone of the second set under reducing conditions (e.g., by addition of 2-MEA). For example, the first set is cloned into an IgG1-F405L backbone and the second set is cloned into an IgG1-K409R backbone (see also Example 1 for other possible backbone combinations).

[0322] Each member of the two sets of antibody vectors is then expressed on a small scale. For example, all antibody vectors are transiently transfected into CHO-K1 cells and expressed in culture.

[0323] The expressed antibodies of the two groups of antibodies are then mixed in pairs in an equimolar ratio in a matrix-like manner. For example, all individual antibodies are purified by small-scale protein A chromatography and the antibody concentration is measured by absorbance at a wavelength of 280 nm. Alternatively, other suitable (small-scale) purification methods or methods known in the art for determining protein concentration can be used.

[0324] In another embodiment, the purification step can be omitted if the expression medium does not affect downstream applications. Subsequently, the antibody concentrations are normalized so that appropriate volumes contain equimolar amounts of anti-EGFR antibody and anti-c-Met antibody.

[0325] For example, a set of anti-EGFR antibodies with an F405L backbone (BT461-DB-EGFR) are each mixed with an anti-cMet antibody with a K409R backbone (BT461-DB-CMET) in a 1:1.1 ratio. An appropriate amount of reducing agent is added to the antibody mixture and incubated at a permissive temperature for an appropriate period of time. For example, to a mixture containing a mg of antibody A (F405L) and 1.1 mg of antibody B (K409R), 0.25 ml of 300 mM 2-MEA is added to form an assembly reaction volume of b, resulting in a final concentration of 75 mM 2-MEA (final concentration of 75 mM 2-MEA) and incubated at 31°C for 5 minutes. The reducing agent is then removed from the mixture (now containing bispecific antibodies) to promote disulfide bond oxidation and prevent interference from the reducing agent during screening assays. For example, 2-MEA is removed by performing buffer exchange on the bispecific antibody mixture using a desalting plate. Alternatively, other suitable methods known in the art for removing the reducing agent may be used.

[0326] The bispecific antibodies are then characterized biochemically or functionally to identify EGFR / c-Met bispecific antibody candidates of the present invention.

[0327] In some embodiments, those skilled in the art of antibodies can also obtain the bispecific heterodimeric protein of the present invention by co-expressing constructs encoding EGFR-specific polypeptide A and c-Met-specific polypeptide B in a single cell.

[0328] Therefore, in another aspect, the method for preparing the heterodimeric protein of the present invention by co-expression in a single cell comprises the following steps:

[0329] a) providing a first nucleic acid construct encoding a polypeptide A comprising an immunoglobulin Fc(A) region, wherein the Fc(A) region comprises a CH3(A) region,

[0330] b) providing a second nucleic acid construct encoding a second polypeptide B comprising an immunoglobulin Fc(B) region, wherein the Fc(B) region comprises a CH3(B) region, wherein the sequences of the CH3(A) region and the CH3(B) region are different and such that the heterodimeric interaction between the CH3(A) region and the CH3(B) region is stronger than the homodimeric interaction between the CH3(A) region and the CH3(B) region,

[0331] c) co-expressing the first and second nucleic acid constructs in a host cell, and

[0332] d) obtaining the heterodimeric protein from cell culture.

[0333] In one such embodiment, the homodimeric protein B has an amino acid at position 409 that is not Lys, Leu, or Met, and the homodimeric protein A has an amino acid at position 405 that is not Phe.

[0334] In yet another embodiment, the homodimeric protein A has an amino acid at position 405 that is not Phe, Arg, or Gly, and the homodimeric protein B has an amino acid at position 409 that is not Lys, Leu, or Met.

[0335] In another embodiment, the first homodimeric protein B comprises Arg at position 409 and the homodimeric protein A comprises Leu at position 405.

[0336] Suitable expression vectors (including promoters, enhancers, etc.) and suitable host cells for preparing antibodies are well known in the art. Examples of host cells include yeast, bacteria, and mammalian cells, such as CHO or HEK cells.

[0337] In other embodiments, the co-expression methods of the invention include any of the other features described above under the in vitro methods.

[0338] In some exemplary embodiments, the anti-EGFR / c-Met bispecific antibody molecule of the present invention is prepared by separately constructing a recombinant expression vector 1 for simultaneously expressing the light and heavy chains of the anti-EGFR antibody and a recombinant expression vector 2 for simultaneously expressing the light and heavy chains of the anti-c-Met antibody, transiently transfecting the two recombinant expression vectors 1 and 2 into host cells, culturing the host cells to produce a fermentation culture product of the bispecific antibody of the present invention, purifying the protein product produced by the recombinant host cells in the culture product, and assembling and purifying the protein product in vitro to obtain the bispecific EGFR / c-Met antibody of the present invention. The binding arm A of the produced bispecific antibody may have the properties described above for the binding arm that binds to EGFR, and the binding arm B of the bispecific molecule may have the properties described above for the binding arm that binds to c-Met.

[0339] In some exemplary embodiments, the expression vector construction of the EGFR / c-Met bispecific antibody of the present invention is based on Genmab's bispecific antibody platform technology, and genetic engineering technology is used to design mutations (F405L, K214R, D356E, L358M) for the heavy chain of the anti-EGFR antibody (HCA), and to design mutations (K409R, K214R, D356E, L358M) for the heavy chain of the anti-c-Met antibody (HCB), and to synthesize nucleic acids 3# and 5# encoding HCA and HCB, and nucleic acids 4# and 6# encoding LCA and LCB. Next, the nucleic acids encoding the LCA and HCA containing the above-mentioned amino acid mutations of the anti-c-Met or anti-EGFR antibody, and the nucleic acids encoding the LCB and HCB containing the above-mentioned amino acid mutations of the anti-c-Met antibody, were respectively constructed into four expression vectors to obtain four plasmids expressing the light and heavy chains of the anti-c-Met or anti-EGFR antibody, respectively. Then, the plasmids expressing the light and heavy chains with the same antigen-binding specificity were digested with SalI / NotI and ligated, and two nucleotide sequences encoding the nucleic acids with the same EGFR antigen-binding specificity, LCA and HCA, were constructed into the same expression vector, and two nucleotide sequences encoding the nucleic acids with the same c-Met antigen-binding specificity, LCB and HCB, were constructed into the same expression vector, to obtain recombinant plasmids carrying the anti-c-Met antibody light and heavy chain encoding nucleic acid fusions and recombinant plasmids expressing the anti-EGFR antibody light and heavy chain encoding nucleic acid fusions, respectively.

[0340] In some exemplary embodiments, the method for preparing the bispecific antibody of the present invention comprises the steps of:

[0341] 1. Based on Genmab's bispecific antibody platform technology, we designed and synthesized nucleic acids encoding heavy chain A (HCA) of an EGFR antibody containing amino acid mutations (F405L, K214R, D356E, L358M) and heavy chain B (HCB) of an anti-c-Met antibody containing amino acid mutations (K409R, K214R, D356E, L358M) using genetic engineering techniques. At the same time, we designed nucleic acids encoding light chain A (LCA) of an anti-EGFR antibody containing an unmutated κ-type light chain constant region and VLA, and light chain B (LCB) of a c-Met antibody, encoding nucleic acids (6#).

[0342] 3. 3# and 5# were constructed into expression plasmid vectors A1 and A2, respectively, and 4# and 6# were constructed into expression plasmids B1 and B2, respectively, to obtain recombinant expression plasmids B1-LCA and B1-LCB1- expressing the light chains of anti-EGFR antibody and anti-c-Met antibody, respectively, and recombinant expression plasmids A1-HCA and A1-HCB expressing heavy chain A containing the aforementioned CH3A mutation and Fc(A) mutation, respectively.

[0343] 4. The recombinant expression plasmids A1 and B1, as well as A2 and B2, of the light and heavy chains were ligated using SalI / NotI enzyme (NEB), and the two nucleotide sequences encoding LCA and HCA, respectively, were constructed into the same expression vector. The two nucleotide sequences encoding LCB and HCB, respectively, were constructed into the same expression vector A1, to obtain recombinant expression plasmid C1 carrying the encoding nucleic acid fusion #1 and co-expressing the light and heavy chains of the anti-EGFR antibody, and recombinant expression plasmid C2 carrying the encoding nucleic acid fusion #2 and co-expressing the light and heavy chains of the anti-c-Met antibody, respectively.

[0344] 5. Plasmid C1 encoding the anti-EGFR antibody and plasmid C2 encoding the anti-c-Met antibody protein were transiently transfected into host cells, respectively, to obtain recombinant host cell A expressing the anti-EGFR binding arms HCA and LCA and recombinant host cell B expressing the anti-c-Met binding arms HCB and LCB.

[0345] 6. Collect the cell supernatant, and optionally separate and purify the binding arm A and binding arm B of the anti-EGFR / c-Met bispecific antibody of the present invention thus produced.

[0346] 7. In vitro assembly and purification.

[0347] In some embodiments, the expression plasmids A1 and A2 for the light chain of the EGFR-binding arm and the light chain of the c-Met-binding arm of the anti-EGFR / c-Met bispecific antibody of the present invention are preferably PEE6.4, as shown in FIG15 .

[0348] In some embodiments, the expression plasmids B1 and B2 for the heavy chain of the EGFR-binding arm and the heavy chain of the c-Met-binding arm of the anti-EGFR / c-Met bispecific antibody of the present invention are preferably PEE12.4, as shown in FIG14 .

[0349] In some embodiments, the nucleic acids encoding the light and heavy chains of the EGFR binding arm of the anti-EGFR / c-Met bispecific antibody of the present invention are co-constructed into a PEE expression vector (pHR plasmid) to obtain a recombinant pHR expression vector C1 expressing EGFR-specific IgG (A), and the nucleic acids encoding the light and heavy chains of the c-Met binding arm are co-constructed into a PEE expression vector (pHR plasmid) to obtain a recombinant expression plasmid C2 expressing c-Met-specific IgG (B).

[0350] The nucleic acid molecules or vectors described herein can be introduced into host cells. The techniques for introducing nucleic acids or vectors into host cells are well known in the art and any suitable technique can be used.

[0351] A range of host cells suitable for producing recombinant antibody molecules are known in the art and include bacterial, yeast, insect or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NSO or HEK cells.

[0352] In some embodiments, the host cell is a CHO cell.

[0353] In some embodiments, the host cell is a CHO-K1 cell.

[0354] In some exemplary preferred embodiments, the host cell is a CHO-K1 cell in which the FUT8 gene is knocked out.

[0355] In some embodiments, FUT8 knockout cells can also be used during the production of the bispecific antibodies of the invention, and one or both of the homodimeric proteins can be glycoengineered to reduce fucose, thereby improving ADCC.

[0356] Knocking out the FUT8 gene in host cells is a genetic engineering approach that reduces fucose levels in antibodies. Within mammalian cells, fucose binds to glucosamine on the Fc terminus of antibodies via an α-1,6 glycosidic bond, catalyzed by the α-1,6-fucosyltransferase encoded by the FUT8 gene. Knocking out the FUT8 gene completely eliminates fucose on the antibody Fc terminus, thereby enhancing the ADCC activity of the binding arm of the bispecific antibody of the present invention.

[0357] The inventors further screened the obtained clones by functional activity and produced the bispecific antibody molecules of the present invention that specifically bind to EGFR-positive tumor cells and / or specifically bind to c-Met-positive tumor cells, and have significant differences in binding to tumor cells and non-tumor tissues.

[0358] In this way, the inventors identified antibodies capable of distinguishing tumor from non-tumor tissue. The bispecific antibodies described herein possess the desired properties necessary for therapeutic applications, namely, specific recognition of EGFR and c-Met targets present on the surface of cancer cells, effective internalization upon binding to these targets, significant differences in affinity between binding arms A and B, with the c-Met-specific binding arm B having a significantly higher affinity for c-Met than the EGFR-specific binding arm A for EGFR. Furthermore, these bispecific antibodies possess both tumor-killing potency and safety, and their structures can provide bispecific antibody monomer purity exceeding 99% during in vitro assembly, such as BT461-DB.

[0359] In some exemplary embodiments, the anti-EGFR / c-Met bispecific antibody of the present invention (e.g., BT461-DB) with a Dubody structure exhibits high binding affinity (in the nanomolar range) to c-Met expressed on the cell surface of cancer cells. In addition, this anti-EGFR / c-Met bispecific antibody exhibits a high ability to trigger internalization of the antibody complex, as shown in Example 11. Exemplarily, the present invention obtained an EGFR / c-Met bispecific antibody designated as BT-461-DB.

[0360] Methods for culturing host cells are well known in the art. The method may further comprise the step of isolating and / or purifying the EGFR / c-Met bispecific antibody molecule. Techniques for purifying recombinantly obtained EGFR / c-Met bispecific antibody molecules are well known in the art and include, for example, HPLC, FPLC, or affinity chromatography, such as affinity chromatography.

[0361] In some embodiments, an affinity tag on the antibody molecule can be used for purification. The method can also include formulating the antibody molecule into a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance as described below.

[0362] In some embodiments, the homodimeric proteins A and B provided in steps a) and b) are purified.

[0363] In some preferred embodiments, the preparation of the anti-EGFR / c-Met bispecific antibody molecule with a Dubody structure of the present invention comprises in vitro assembly and purification steps.

[0364] In some preferred embodiments, the assembly is performed using Fab exchange technology on a duobody platform. An anti-EGFR half-antibody (e.g., BT461-DB-EGFR) and an anti-c-Met half-antibody (e.g., BT461-DB-c-Met) are mixed in vitro after affinity chromatography for assembly. Reduction is performed in a reducing agent water bath, followed by desalting to remove the reducing agent and replacing the solution with a buffer solution. The mixture is then allowed to oxidize at room temperature.

[0365] In some preferred embodiments, the purification is performed using a cationic chromatography method.

[0366] In some preferred embodiments, the assembled system comprises an anti-EGFR antibody (exemplary such as BT461-DB-EGFR) and an anti-cMet antibody (exemplary such as BT461-DB-c-Met) in a ratio of about 1:1.

[0367] In some preferred embodiments, the reducing agent is 2-MEA.

[0368] In some preferred embodiments, the concentration of the reducing agent 2-MEA is 50 to 100 mmol / L.

[0369] In some preferred embodiments, the reduction is performed in a water bath.

[0370] In some preferred embodiments, the desalting is performed using G25 chromatography technology.

[0371] Biological functional characteristics of EGFR / c-Met antibody molecules

[0372] The EGFR / c-Met bispecific antibody molecules described herein (exemplified by BT461 DB) can be evaluated and validated by reference to certain functional properties.

[0373] Specific binding to EGFR protein and c-Met protein in vitro

[0374] In some embodiments, the anti-EGFR / c-Met bispecific antibody of the present invention can specifically bind to EGFR protein and specifically bind to c-Met protein in vitro.

[0375] In some embodiments, the bispecific antibodies of the present invention can bind to human EGFR with high affinity and specifically bind to human c-Met with very high affinity.

[0376] In some exemplary embodiments, the present invention evaluates the extracellular EGFR binding level and c-Met binding level of the EGFR / c-Met bispecific antibody by measuring the EC50 value of binding to human EGFR and c-Met in vitro by ELISA.

[0377] In some exemplary embodiments, the binding arm A of the anti-EGFR / c-Met bispecific antibody of the present invention, such as BT461-DB, can bind to EGFR with an ELISA binding EC50 of about 1260-1450 ng / mL, preferably 1290-1350 ng / mL.

[0378] In some exemplary embodiments, the anti-EGFR / c-Met bispecific antibody of the present invention (such as BT461-DB binding arm B) can bind to c-Met with an ELISA binding EC50 of about 2-20 ng / mL, preferably 5-15 ng / mL, more preferably 7-10 ng / mL.

[0379] The ELISA binding EC50 determination method can be found in the method shown in Example 2.

[0380] Binds to EGFR and / or Met on the cell surface

[0381] In some embodiments, the bispecific antibodies of the present invention are capable of binding to cells overexpressing EGGR and cells overexpressing c-Met, respectively.

[0382] In some embodiments, the binding EC50 value of the EGFR / c-Met bispecific antibody is determined by FACS to evaluate the binding level of the EGFR / c-Met bispecific antibody to EGFR expressed on the cell surface and c-Met expressed on the cell surface.

[0383] In some embodiments, the bispecific antibodies of the present invention, such as BT461-DB, can bind to CHO-K1 cells overexpressing EGFR with a FACS binding EC50 of about 1610-1820 ng / mL.

[0384] In some embodiments, the bispecific antibodies of the present invention, such as BT461-DB, can bind to CHO-K1 cells overexpressing c-Met with a FACS binding EC50 of about 245-285 ng / mL.

[0385] The method for determining the in vitro binding EC50 by FACS can be found in Example 3.

[0386] In some embodiments, the bispecific antibody of the present invention, such as BT461-DB, can specifically bind to EGFR / c-Met double-positive tumor cells.

[0387] In some exemplary embodiments, BT461-DB of the present invention can bind to double-positive tumor cells overexpressing EGFR and c-Met with a FACS binding EC50 of about 780-910 ng / mL.

[0388] In some exemplary embodiments, the double-positive tumor cells overexpressing EGFR and c-Met are MKN45 cells, as shown in Example 4.

[0389] In some embodiments, the BT461-DB of the present invention can antagonize the binding of the ligand HGF to the target protein c-Met.

[0390] In some exemplary embodiments, such as BT461, the antagonistic IC50 value determined by ELISA is used to evaluate the level of antagonism of the EGFR / c-Met bispecific antibody against the binding of the ligands HGF and EGF to the membrane proteins EGFR and c-MET. In some embodiments, in embodiments of specific binding to double-positive tumor cells, the ELISA blocking IC50 value of the EGFR / c-Met bispecific antibody of the present invention against HGF and c-Met is no less than 1000 ng / mL, no less than 830 ng / mL, or preferably no less than 730 ng / mL, more preferably no less than 680 ng / mL, most preferably no less than 630 ng / mL, more preferably no less than 610 ng / mL, more preferably no less than 550 ng / mL, and most preferably no less than 460 ng / mL.

[0391] In some exemplary embodiments, for example, when measured using HGF-HIS, the bispecific antibody BT461 of the present invention can inhibit HGF binding to c-Met with a FACS antagonism IC50 value of about 425-500 ng / mL. The method for determining the antagonism IC50 by FACS can be found in Example 5.

[0392] Affinity for target protein

[0393] In some embodiments, the bispecific binding arm A and binding arm B of the present invention have high affinity for EGFR and c-Met antigens, respectively.

[0394] In some embodiments, biolayer interferometry (BLI) can be used to detect the affinity of the bispecific antibodies of the present invention for binding to EGFR and c-Met target proteins.

[0395] In some embodiments, in BLI monitoring, the binding arm A in the bispecific antibody of the present invention binds to EGFR with an affinity of KD (M) equal to or 1.05E-7, or preferably less than 8.45E-8, more preferably less than 6.95E-8, more preferably less than 4.65E-8, more preferably less than 3.35E-8, and most preferably less than 2.15E-8.

[0396] In some embodiments, the affinity assay results obtained by BLI show that the bispecific antibody of the present invention, such as BT461-DB, binds to human c-EGFR at a concentration of 5 μg / mL with a dissociation constant of KD (M) equal to or less than 2.262E-08, or preferably equal to or less than 2.52E-08, or more preferably equal to or less than 2.42E-08, or more preferably equal to or less than 2.35E-08, or most preferably less than 2.20E-08.

[0397] In some embodiments, in a BLI assay, the binding arm B in the bispecific antibody of the invention binds to human c-Met with an affinity equal to or less than 2.45E-9, or less than 2.25E-9, or preferably less than 1.95E-9, or preferably less than 1.75E-9, or preferably less than 1.55E-9, or more preferably less than 1.40E-9, or more preferably less than 9.96E-10, or more preferably less than 8.85E-10, or most preferably less than 7.62E-10.

[0398] In some embodiments, the bispecific antibody BT461 of the present invention binds to human c-Met with a dissociation constant (KD) of equal to or less than 1.20E-09, equal to or less than 8.12E-10, equal to or less than 7.82E-10, or less than 7.62E-10.

[0399] An exemplary BLI detection method is shown in Example 6.

[0400] In some embodiments, the KD (M) of the EGFR-binding arm of the present invention, such as the exemplary bispecific antibody BT461-DB molecule, for binding to EGFR is significantly higher than the KD (M) of the c-Met-binding arm for binding to c-Met.

[0401] In some embodiments, the ratio of the affinity of the c-Met binding arm of the exemplary bispecific antibody BT461-DB molecule of the invention for c-Met to the affinity of the EGFR binding arm for EGFR is more than 20-fold.

[0402] Affinity of FC for receptor protein

[0403] In some embodiments, the Fc of the exemplary bispecific antibody BT461-DB molecule of the present invention has a high binding affinity to the receptor protein comparable to that of Avanta.

[0404] In some embodiments, the receptor proteins include FcγRI (CD64), FcγRIIIA (CD16a-F176), FcγRIIIA (CD16a-V176), FcγRIIIB (CD16b), FcγRⅡA (CD32a), FcγRⅡB / C (CD32b / c), Fc Rn and C1q.

[0405] In some embodiments, the BLI method is used to determine the binding affinity between Fc and receptor protein.

[0406] Bispecific antibodies can also serve as vehicles for retargeting effector mechanisms to disease-associated tissues such as tumors.

[0407] In some embodiments, the binding affinity KD (M) between the Fc of the BT461-DB of the present invention and the receptor protein FFcγRIIIA (CD16a-V176) measured by BLI method is no more than about 2.62E-8.

[0408] In some embodiments, the binding affinity KD (M) between the Fc of the BT461-DB of the present invention and the receptor protein FcγRIIIA (CD16a-F176) measured by BLI method is no more than about 3.26E-7.

[0409] In some embodiments, the binding affinity KD (M) between the Fc of the BT461-DB of the present invention and the receptor protein FFcγRIIIB (CD16b) measured by BLI method is no greater than about 1.416E-5.

[0410] In some embodiments, the binding affinity KD (M) between the Fc of the BT461-DB of the present invention and the receptor protein FcγRIIA (CD32a) measured by BLI method is no more than about 1.50E-6.

[0411] In some embodiments, the binding affinity KD (M) between the Fc of the BT461-DB of the present invention and the receptor protein FcγRIIB / C (CD32b / c) measured by BLI method is approximately no higher than 1.63E-6.

[0412] In some embodiments, the binding affinity KD (M) between the Fc of the BT461-DB of the present invention and the receptor protein FcγRI (CD64) measured by BLI method is no more than about 8.95E-9.

[0413] In some embodiments, the binding affinity KD (M) between the Fc of the BT461-DB of the present invention and the receptor protein Fc Rn measured by BLI method is no more than about 2.25E-7.

[0414] In some embodiments, the binding affinity KD (M) between the Fc of the BT461-DB of the present invention and the receptor protein C1q measured by BLI method is no greater than about 1.09E-8.

[0415] Inhibits EGFR autophosphorylation and c-Met autophosphorylation

[0416] In some embodiments, BT461 of the present invention can inhibit EGFR autophosphorylation and c-Met autophosphorylation.

[0417] In some embodiments, changes in the autophosphorylation levels of EGFR and c-Met can be measured by Western blotting to explore whether the anti-c-Met / EGFR bispecific antibody can inhibit the signaling pathways of c-Met and EGFR.

[0418] For example, the results of chemiluminescence system development after Western blotting analysis showed that under the conditions of 100 ng / mL EGF-mFC + 100 ng / mL HGF-his, after treating MKN45 cells with 250 μg / mL IgG1, antibodies Avantu, BT461-KIH and BT-461DB for a period of time, BT461-KIH, BT461-DB and Avantu were able to significantly inhibit the phosphorylation level of pC-MET (Y1234 / 1235) and slightly inhibit the phosphorylation level of p-EGFR (Y1173).

[0419] Mediates target protein internalization and degradation

[0420] In some embodiments, BT461-DB of the exemplary antibody Duobody structure of the present invention can mediate and promote the internalization and degradation of target proteins EGFR and c-Met.

[0421] In some embodiments, the level of internalization mediated by the anti-c-Met antibody arm of the exemplary antibody BT461-DB of the present invention is higher than the level of internalization mediated by the anti-EGFR antibody arm. In some embodiments, the level of target protein internalization mediated by the anti-c-Met binding arm and the anti-EGFR binding arm of the exemplary antibody BT461-DB of the present invention can be evaluated by incubating a fluorescently labeled secondary antibody with the bispecific antibody of the present invention and detecting it using a flow cytometer (PE fluorescence excitation and reception) and measuring the EC50 (ng / mL).

[0422] In some embodiments, the ability of the exemplary antibody BT461-DB of the present invention to promote target protein internalization is stronger than that of the anti-EGFR binding arm. In some embodiments, the ability of the exemplary antibody BT461-DB of the present invention to promote target protein internalization is slightly weaker than that of the anti-c-Met binding arm.

[0423] In some embodiments, the exemplary antibody BT461-DB of the present invention promotes target protein internalization with an EC50 of about no higher than 700 ng / mL, or preferably about no higher than 650 ng / mL, or most preferably about no higher than 590 ng / mL.

[0424] In some embodiments, the exemplary antibody BT461-DB-c-Met binding arm of the present invention promotes target protein internalization with an EC50 of about 420 ng / mL or less, or preferably about 350 ng / mL or less, or most preferably about 268 ng / mL or less.

[0425] In some embodiments, the EC50 of the exemplary antibody BT461-DB-EGFR binding arm of the present invention for promoting internalization of the target protein EGFR is no higher than about 680 ng / mL, or preferably no higher than about 650 ng / mL, or most preferably no higher than about 590 ng / mL.

[0426] Mediates antibody-dependent cellular cytotoxicity (ADCC)

[0427] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention, such as BT461-DB, can mediate antibody-dependent cellular cytotoxicity (ADCC).

[0428] In some embodiments, the present invention uses ADCC Reporter Bioassay to detect the ADCC biological activity of the antibody of the present invention.

[0429] In some embodiments, the EC50 value of the EGFR / c-Met bispecific antibody of the present invention, such as BT461-DB, in mediating ADCC against CHOK1-EGFR target cells is about no higher than 300 ng / mL, or about no higher than 280 ng / mL, or about no higher than 260 ng / mL, or about no higher than 208 ng / mL, or about no higher than 188 ng / mL.

[0430] In some embodiments, the EC50 value of the EGFR / c-Met bispecific antibody of the present invention, such as BT461-DB, in mediating ADCC against CHOK1-c-Met target cells is about 1290 ng / mL or less, or about 260 ng / mL or less, or about 238 ng / mL or less, or about 208 ng / mL or less, or about 188 ng / mL or less, or about 168 ng / mL or less.

[0431] In some embodiments, the EC50 value of the EGFR / c-Met bispecific antibody of the present invention, such as BT461-DB, in mediating ADCC against EGFR and c-Met double-positive tumor cells is about no higher than 156 ng / mL, preferably about no higher than 124.48 ng / mL, more preferably about no higher than 89.48 ng / mL, even more preferably about no higher than 72.18 ng / mL, and most preferably about no higher than 52.18 ng / mL.

[0432] In some embodiments, the EGFR / c-Met bispecific antibodies of the invention mediate ADCC of double-positive tumor cells at a level similar to that of both binding arms.

[0433] Inhibits tumor cell proliferation in vitro

[0434] In some embodiments, the EGFR / c-Met bispecific antibodies of the present invention can inhibit tumor cell proliferation in vitro.

[0435] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention, such as BT461-DB, can inhibit the proliferation of EGFR-positive tumor cells and / or c-Met-positive tumor cells in vitro.

[0436] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention, such as BT461-DB, can inhibit the proliferation of tumor cells expressing HGF in vitro.

[0437] In some exemplary embodiments, the EGFR / c-Met bispecific antibody of the present invention can inhibit the proliferation of human pancreatic cancer cells in vitro.

[0438] In some exemplary embodiments, the EGFR / c-Met bispecific antibody of the present invention can inhibit the proliferation of human non-small cell lung cancer cells with EGFR mutations in vitro.

[0439] In some embodiments, the tumor cell line is HCC827-HGF.

[0440] In some embodiments, for example, by utilizing The kit is used to detect the effects of the antibodies of the present invention on the proliferation of these cell lines. The level of reduction in tumor cell viability caused by a mixture comprising an EGFR / c-Met bispecific antibody (e.g., BT461-DB and BT461-KIH) and two monospecific antibodies (one that binds to EGFR and the other that binds to c-Met) is compared to assess the level of synergistic inhibition of tumor cell proliferation by the EGFR / c-Met bispecific antibodies of the present invention.

[0441] In some exemplary embodiments, when the bispecific molecules of the present invention inhibit tumor cell proliferation, the cell viability of the tumor cells is comparable to the value measured under the inhibition of Amivantanab, the activity of tumor cells after inhibition by BT461-DB is comparable to the activity of tumor cells after inhibition by BT-461-KIH, and the inhibitory abilities of BT461-DB and BT461-KIH on tumor cell activity are both stronger than the combination of their related EGFR monoclonal antibodies and anti-c-Met monoclonal antibodies (e.g., Taixinsheng & LY358-B12), indicating that BT461-DB has a synergistic effect in inhibiting tumors in vitro.

[0442] Inhibits TKI-acquired resistance cell proliferation in vitro

[0443] In some embodiments, the bispecific molecules of the present invention, such as BT461-DB, can significantly inhibit the proliferation of TIK-resistant tumor cells.

[0444] In some embodiments, the bispecific molecules of the present invention, such as BT461-DB, can significantly inhibit the proliferation of TIK-resistant HGF-expressing tumor cells.

[0445] In some embodiments, the bispecific molecules of the present invention, such as BT461-DB, can significantly inhibit the proliferation of tumor cells HCC827-HGF-TIK resistant cells at multiple gradient concentrations.

[0446] In some embodiments, the bispecific molecules of the present invention, such as BT461-DB, can significantly inhibit the proliferation of tumor cells HCC827-HGF-osimertinib-resistant cells at multiple gradient concentrations, wherein the inhibition level of HCC827-HGF cell proliferation is comparable to that of BT461-KIH and Avanta.

[0447] In some embodiments, the bispecific molecules of the present invention, such as BT461-DB, can significantly inhibit the proliferation of tumor cells HCC827-HGF-osimertinib-resistant cells in the concentration range of 28-550 μg / mL, wherein the inhibition level of HCC827-HGF cell proliferation is comparable to that of BT461-KIH and Avanta.

[0448] In some embodiments, the antibody BT461-DB of the present invention is more potent than the c-Met binding arm of BT461-DB in inhibiting the proliferation of tumor cells HCC827-HGF-osimertinib-resistant cells, and is more potent than the combination of anti-EGFR monoclonal antibody and anti-c-Met monoclonal antibody, indicating that the anti-EGFR / cMet dual-target antibody of the present invention has a synergistic effect in inhibiting the proliferation of osimertinib-resistant tumor cells in vitro.

[0449] Has the effect of inhibiting tumor growth in vivo

[0450] In some embodiments, the bispecific antibodies of the invention as disclosed herein are capable of inhibiting the growth of EGFR-positive tumors and c-Met-positive tumors in vivo.

[0451] In some exemplary embodiments, the exemplary antibody BT461-DB of the present invention has therapeutic efficacy in inhibiting tumor growth in a mouse xenograft model of human non-small cell lung cancer cells.

[0452] In some exemplary embodiments, the antibody BT461-DB is preferably administered for 21 days in a mouse xenograft model of HCC827-HGF cells.

[0453] In some exemplary embodiments, the antibody BT461 has therapeutic efficacy in inhibiting tumor growth in an NSG mouse xenograft model of human pancreatic cancer cells.

[0454] The EGFR / c-Met bispecific antibodies BT461-DB and BT461-KIH of the present invention have the therapeutic effect of inhibiting tumor growth at a dosage of 1.5 to 8 mg / kg.

[0455] In some exemplary embodiments, at a dosage of 2 mg / kg, the TGI (tumor inhibition ratio) of BT461-DB is 67.7%.

[0456] In some exemplary embodiments, at a dosage of 5 mg / kg, the TGI (tumor inhibition ratio) of BT461-DB is 122.9%.

[0457] Pharmaceutical composition

[0458] The present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention as disclosed herein and a pharmaceutically acceptable excipient for treating a susceptible population having or at risk of an EGFR, c-Met or EGFR / c-Met positive tumor.

[0459] In some embodiments, the pharmaceutically acceptable excipient includes, but is not limited to, non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or any type of formulation auxiliary.

[0460] In some embodiments, the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, stabilizers, cryoprotectants, or antioxidants.

[0461] In some embodiments, the pharmaceutical composition comprises a composition of an EGFR / c-Met bispecific antibody, such as BT461-DB, as provided herein, and further comprises one or more antioxidants that will reduce oxidation of the antibody or antigen-binding fragment. This reduction in oxidation will prevent oxidation of the antibody or antigen-binding fragment, extend its shelf life, and / or improve its efficacy, prevent or reduce loss of binding affinity, thereby improving antibody stability and maximizing shelf life. In certain embodiments, the present invention provides a composition comprising one or more antibodies disclosed herein and one or more antioxidants, such as methionine. In certain embodiments, the antioxidant is not methionine.

[0462] In some embodiments, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), or suitable mixtures thereof.

[0463] In some embodiments, the pharmaceutical composition is prepared by dissolving or dispersing an effective amount of the antibody of the present invention in a pharmaceutically acceptable carrier or aqueous medium.

[0464] In some embodiments, the pharmaceutical forms of the pharmaceutical composition suitable for injectable use include sterile aqueous solutions or dispersions; and sterile powders for the ex situ preparation of sterile injectable solutions or dispersions.

[0465] In some embodiments, the pharmaceutical composition is a dry, in particular freeze-dried, composition which allows the production of an injectable solution upon addition of sterile water or physiological saline.

[0466] In some embodiments, the pharmaceutically acceptable carrier can include, for example, an aqueous vehicle such as Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection.

[0467] Injectable pharmaceutical compositions may be prepared in any conventional form, such as liquid solutions, suspensions, or solid forms suitable for producing liquid solutions, suspensions, or emulsions. Injectable formulations may include: (1) sterile and / or pyrogen-free solutions ready for injection; (2) sterile dry soluble products, such as lyophilized powders, to be combined with a solvent just prior to use; (3) sterile suspensions ready for injection; (4) sterile dry insoluble products to be combined with a vehicle just prior to use; and (5) sterile and / or pyrogen-free emulsions. Solutions may be aqueous or non-aqueous.

[0468] In certain embodiments, the parenteral formulations in unit doses can be packaged in ampoules, vials, or syringes with needles. All formulations for parenteral administration should be sterile and pyrogen-free, as known and practiced in the art. In certain embodiments, a sterile lyophilized powder is prepared by dissolving the antibody or antigen-binding fragment as disclosed herein in a suitable solvent.

[0469] In some embodiments, the solvent may contain a buffer, such as citrate, sodium phosphate or potassium phosphate, or other such buffers known to those skilled in the art. In some embodiments, the buffer is approximately neutral pH. The sterile filtered solution is then added and lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In some embodiments, the resulting solution will be dispensed into vials for lyophilization. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. The lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. In some embodiments, for reconstitution, sterile and / or pyrogen-free water or other liquid suitable carrier is added to the lyophilized powder. The exact amount depends on the selected therapy given and can be determined empirically.

[0470] In some embodiments, the pharmaceutical composition of the present invention further comprises one or more additional therapeutic means or therapeutic combination administration in addition to the anti-EGFR / c-Met bispecific antibody of the present invention, for example, another therapeutic agent, such as a chemotherapeutic agent or an anti-cancer drug combination.

[0471] Combination therapy

[0472] In some embodiments, the antibodies disclosed herein can be administered alone or in combination with one or more additional therapeutic means or agents. For example, the antibodies disclosed herein can be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anticancer drug.

[0473] In some embodiments, the present invention provides a treatment method comprising administering to a subject in need thereof a therapeutic composition consisting of any of the aforementioned anti-EGFR / c-Met bispecific antibodies and one or more additional therapeutically active ingredients.

[0474] In certain embodiments, the anti-EGFR / c-Met bispecific antibodies disclosed herein can be administered concurrently with one or more additional therapeutic agents, and the antibodies or antigen-binding fragments and the additional therapeutic agents can be administered as part of the same pharmaceutical composition. In certain embodiments, administration of an antibody or antigen-binding fragment disclosed herein before or after another agent is considered to be administered "in combination" with that agent, even if the anti-EGFR / c-Met bispecific antibodies disclosed herein and the additional agent are administered by different routes. Where possible, the additional therapeutic agent administered in combination with an antibody disclosed herein is administered according to the schedule listed in the product information sheet for the additional therapeutic agent or according to a regimen well known in the art.

[0475] The present invention includes pharmaceutical compositions in which the aforementioned anti-EGFR / -c-Met antibodies or antigen-binding fragments thereof and anti-FGFR2 antibodies ADC are co-formulated with one or more additional therapeutically active ingredients as described elsewhere herein.

[0476] Although the antibody molecule can be administered alone, the anti-EGFR / c-Met bispecific antibody molecule of the present invention is typically administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the anti-EGFR / c-Met bispecific antibody molecule of the present invention. Therefore, another aspect of the present disclosure also provides a method comprising formulating the antibody molecule into a pharmaceutical composition.

[0477] In some embodiments, the anti-EGFR / c-Met bispecific antibodies of the present invention, or antigen-binding fragments thereof, can be co-formulated and / or administered in combination with one or more additional target-specific therapeutic antibody components selected from the group consisting of: another FGFR2 antagonist, an antagonist of another EGFR family member such as ErbB3 or ErbB4, anti-ErbB2, such as an antagonist of ErbB3 or ErbB4, a MET antagonist, an EGFR antagonist, an IGF1R antagonist, an IGF1R antagonist, a DGFR-α inhibitor, a PDGFR-β inhibitor or a small molecule kinase inhibitor, a VEGF-inhibitor, a DLL4 antagonist.

[0478] In some embodiments, the additional therapeutically active ingredient is an antibody of a second specificity.

[0479] In some embodiments, the second specific antibody includes, but is not limited to, an MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplaque protein antagonist (e.g., a uroplaque protein antagonist), an LGR5 antagonist (e.g., an anti-LGR5 antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab), a CD20xCD3 bispecific antibody, a PD-1 blocker (e.g., an anti-PD-1 antibody such as pembrolizumab or nivolumab), and the like.

[0480] In some embodiments, other therapeutic agents that can be advantageously administered in combination with the antibodies provided herein also include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines, such as but not limited to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, or their respective receptors.

[0481] In some embodiments, the additional therapeutic agents that can be used in combination therapy with any of the anti-FGFR2 antibodies or antigen-binding fragments thereof described herein (including anti-FGFR2 antibody ADCs) include one or more chemotherapeutic agents.

[0482] In some embodiments, examples of chemotherapeutic agents that can be used in combination with any of the anti-EGFR\c-Met bispecific antibodies or antigen-binding fragments thereof described herein, such as BT461-DB, include alkylating agents, aziridines, nitrosoureas, antibiotics, antimetabolites, purine analogs, pyrimidine analogs, alkyl sulfonates, folic acid supplements, aldophosphamide glycosides, taxanes, platinum analogs, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0483] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention, including anti-FGFR2 antibody ADCs, can also be administered in combination and / or co-formulated with antiviral agents, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-γ and / or NSAIDs.

[0484] For the treatment of diseases related to EGFR and c-Met overexpression

[0485] The EGFR / c-Met bispecific antibody of the present invention or ADC comprising the same can be used to regulate, treat, alleviate, help prevent the occurrence of cell, tissue, organ, tumor or human diseases or specific lesions or alleviate their symptoms.

[0486] In some embodiments, the method for administering the drug comprises administering to a patient an effective amount of a drug comprising the EGFR / c-Met bispecific antibody of the present invention, such that the growth or metastasis of tumors or cancer cells expressing EGFR and / or c-Met is inhibited.

[0487] In some embodiments, the method of administering the drug comprises administering to a patient in need thereof a therapeutically effective amount of the EGFR / c-Met bispecific antibody of the present invention for a period of time sufficient to treat cancer.

[0488] In some embodiments, the cancer is resistant to or has acquired resistance to treatment with one or more EGFR inhibitors.

[0489] In some embodiments, the patient may be treated with the anti-EGFR antibody Taxinomab, or may have acquired resistance to such treatment.

[0490] Detection of elevated c-Met expression levels or increased c-Met activity, such as caused by elevated levels of circulating HGF, activating mutations in the c-Met gene, or c-Met gene amplification, can identify patients who are resistant to EGFR inhibitors.

[0491] In some embodiments, the cancer is associated with EGFR mutation, residue deletion, EGFR amplification, or c-Met amplification or mutation.

[0492] The EGFR / c-Met bispecific antibodies of the present invention can be used to treat any disease or disorder characterized by aberrant activation or production of EGFR, c-Met, EGF, soluble EGFR, soluble c-Met or other EGFR ligands, or HGF, or disorders associated with EGFR or c-Met expression, which may or may not involve malignancy or cancer, wherein aberrant activation and / or production of EGFR, c-Met, EGF or other EGFR ligands, or HGF occurs in cells or tissues of a subject suffering from or susceptible to the disease or disorder.

[0493] The EGFR / c-Met bispecific antibody of the present invention, comprising a binding arm A and a binding arm B, can be used to treat tumors, including cancer and benign tumors.

[0494] In some embodiments, exemplary cancers suitable for treatment by the EGFR / c-Met bispecific antibodies of the present invention include cancers that overexpress EGFR and / or c-Met and are associated with increased EGFR activity and / or expression levels.

[0495] In some embodiments, the type of increased EGFR activity and / or expression level includes but is not limited to EGFR activating mutation, EGFR gene amplification, or ligand-mediated EGFR activation.

[0496] In some embodiments, the increased c-Met activity and / or expression level is caused by, but not limited to, c-Met activating mutations, c-Met gene amplification, or HGF-mediated c-Met activation, or mutant KRAS.

[0497] In some embodiments, the EGFR activating mutation that may be associated with cancer results in an increase in at least one biological activity associated with EGFR, including but not limited to increased tyrosine kinase activity, formation of receptor homodimers and heterodimers, and enhanced ligand binding.

[0498] In some embodiments, the c-Met activating mutation that may be associated with cancer causes an increase in at least one biological activity associated with c-Met, including but not limited to tyrosine kinase activity, the formation of receptor homodimers and heterodimers, enhanced ligand binding, etc.

[0499] In another embodiment of the present invention, the experimental mouse model has an NSCLC tumor or tumor metastasis with an activating EGFR mutation or EGFR gene amplification, and the method of administering the EGFR / c-Met bispecific antibody of the present invention to treat the tumor disease model mouse comprises administering a therapeutically effective amount of the EGFR / c-Met bispecific antibody of the present invention to the model mouse.

[0500] In another aspect, the present invention provides a method for treating a patient with cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the EGFR / c-Met bispecific antibody of the present invention for a period of time sufficient to treat the cancer, wherein the cancer is associated with EGFR mutation, EGFR amplification, or c-Met amplification.

[0501] In some embodiments, the cancer is associated with wild-type EGFR and wild-type c-Met.

[0502] In some embodiments, the cancer is associated with wild-type EGFR and c-Met amplification.

[0503] In some embodiments, the patient has NSCLC associated with EGFR amplification and wild-type c-Met.

[0504] In some embodiments, the patient has NSCLC associated with EGFR amplification and c-Met amplification.

[0505] In some embodiments, patients are treated with the EGFR / c-Met bispecific antibodies of the present invention or their ADCs. BT461-MMAE, BT461-Dxd, and BT461-Ixitecan of the present invention demonstrate efficacy in in vivo tumor animal models when the tumor is associated with L858R, T790M, del(E746, A750) EGFR, EGFR amplification, wild-type c-Met, and / or c-Met amplification.

[0506] In one embodiment, the EGFR / c-Met bispecific antibody or ADC thereof of the present invention can be used to prepare a medicament for treating non-small cell lung cancer (NSCLC).

[0507] In some embodiments, the cells of NSCLC have an epithelial phenotype.

[0508] In some embodiments, the NSCLC has acquired resistance to an EGFR monoclonal antibody.

[0509] In one embodiment, the EGFR monoclonal antibody is Taxin.

[0510] The present invention also provides use of the EGFR / c-Met bispecific antibody or its ADC of the present invention for preparing a medicament for treating a subject having a tumor or at risk of having such a tumor, wherein the method for administering the medicament comprises administering a therapeutically effective amount of the antibody or pharmaceutical composition as disclosed herein to a subject in need thereof, wherein the tumor is preferably an EGFR, c-Met single-positive or EGFR / c-Met double-positive tumor.

[0511] Prior to initiating the treatment, the method preferably further comprises determining whether the subject has such an EGFR, c-Met or EGFR / c-Met positive tumor.

[0512] The bispecific KANGTIs of the invention are applicable to a wide range of cancers. Exemplary cancers suitable for treatment by the bispecific molecules of the invention, such as the EGFR / c-Met bispecific antibodies of the invention.

[0513] In some exemplary embodiments, the cancer is non-small cell lung cancer.

[0514] In some exemplary embodiments, the cancer is gastric cancer.

[0515] In some exemplary embodiments, the cancer is pancreatic cancer.

[0516] Application in the preparation of medicines

[0517] In one aspect, the present invention provides use of an anti-EGFR / c-Met bispecific antibody or a pharmaceutical composition comprising the same in preparing a medicament for treating cancer or tumor.

[0518] One aspect of the present invention is to provide use of the bispecific antibody of the present invention for preparing a medicament for inhibiting the growth or proliferation of cells expressing EGFR and / or c-Met, wherein the method for administering the medicament comprises contacting patient cells with the EGFR / c-Met bispecific antibody of the present invention.

[0519] In some preferred embodiments, the tumor, preferably an EGFR / c-Met positive cancer, is preferably a patient population with an EGFR monoclonal antibody-resistant phenotype or tumor / cancer.

[0520] In some preferred embodiments, the subject is preferably a subject suitable for antibody therapy using an EGFR-specific antibody such as nimotuzumab.

[0521] Co-expression of EGFR and c-Met is associated with many cancer types, and bispecific antibody molecules targeting these two molecules and conjugates comprising such bispecific antibody molecules offer broad clinical opportunities across multiple indications. Therefore, the antibody molecules or antibody-drug conjugates described herein can be used in therapeutic applications, particularly for the treatment of cancer.

[0522] The bispecific antibody molecules or pharmaceutical compositions described herein can be used in methods for treating human EGFR and / or c-Met positive tumors or cancers.

[0523] Related aspects of the present disclosure provide;

[0524] (i) a bispecific antibody molecule as described herein for use as a medicament;

[0525] (ii) a bispecific antibody molecule as described herein for use in a method of treating a disease or disorder;

[0526] (iii) use of a bispecific antibody molecule described herein in the manufacture of a medicament for treating a disease or disorder; and,

[0527] (iv) a method of treating a disease or condition in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of an antibody molecule or conjugate as described herein.

[0528] In some embodiments, treatment can be any treatment or therapy that achieves some desired therapeutic effect, such as inhibiting or delaying the progression of a disorder, and includes reducing the rate of progression, halting the rate of progression, ameliorating the condition of a disorder, curing or alleviating (whether partial or total), preventing, ameliorating, delaying, alleviating or arresting one or more symptoms and / or signs of a disorder, or prolonging the survival of an individual or patient beyond that which would be expected in the absence of the treatment.

[0529] In some embodiments, the described methods of treatment may comprise administering to the individual at least one additional therapeutic agent in addition to the bispecific antibody molecule of the invention.

[0530] In some embodiments, the anti-EGFR / c-Met bispecific antibody molecules of the present invention can be administered to an individual alone or in combination with one or more other therapies, wherein the additional therapy can be administered to the individual simultaneously, sequentially, or separately with the administration of the antibody molecule or conjugate. When the additional therapy is administered simultaneously with the antibody molecule or conjugate, the antibody molecule and the additional therapy can be administered to the individual as a combined formulation. For example, the additional therapy can be a known therapy or therapeutic agent for the disease to be treated.

[0531] In a preferred embodiment, the antibody molecules described herein or pharmaceutical compositions comprising the same can be used in methods for treating cancer.

[0532] Cancer can be characterized by the abnormal proliferation of malignant cancer cells.

[0533] In some embodiments, the tumor is an abnormally proliferative tumor of EGFR-positive malignant tumor cells or c-Met-positive malignant cancer cells.

[0534] In some embodiments, the tumor is a c-Met-positive malignant tumor cell or an abnormally proliferative tumor.

[0535] In some embodiments, the tumor is EGFR / / c-Met double positive tumor cells.

[0536] EGFR-positive tumors are typically tumors that harbor EGFR-activating mutations. EGFR-activating mutations are EGFR mutations that result in activation of the EGF / EGFR signaling pathway. EGFR-activating mutations may be important in the development of a tumor's cancerous state. Such tumors may be resistant to EGFR-targeted therapies through activation of the HGF / c-Met signaling pathway. These tumors may also be HGF-dependent. Activation of the c-Met / HGF signaling pathway is one way in which EGFR-positive tumors can evade treatment with EGFR-targeted therapies. The antibodies of the present invention are particularly suitable for treating HGF-dependent tumors or HGF-dependent tumors, where activation of the c-Met / HGF signaling pathway is associated with the presence or excess of HGF. Tumor cells that are simultaneously HGF / c-Met signaling-positive have a superior selective growth advantage compared to HGF / c-Met signaling-negative tumor cells.

[0537] In some embodiments, the cancer treated using the antibody molecules described herein can be selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC)), pancreatic cancer, breast cancer, colorectal cancer, renal cancer, gastric cancer, head and neck cancer, ovarian cancer, or glioblastoma.

[0538] In one embodiment, the cancer to be treated is non-small cell lung cancer (NSCLC).

[0539] In one embodiment, the cancer is gastric cancer.

[0540] In some embodiments, the cancer is pancreatic cancer.

[0541] In some embodiments, the treatment may include inhibiting cancer growth, including complete remission of the cancer, and / or inhibiting cancer metastasis, as well as inhibiting cancer recurrence. Cancer growth generally refers to any of a variety of indicators that indicate a change in the cancer's internal structure toward a more advanced form. The features disclosed in the foregoing description, the accompanying claims, or the accompanying drawings are expressed in their specific form or as means for performing the disclosed function, or as methods or processes for obtaining the disclosed results, as the case may be. These features may be used individually or in any combination to implement the present disclosure in its various forms.

[0542] While the present disclosure has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art upon presentation of the present disclosure. Therefore, the exemplary embodiments disclosed above are to be considered illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the present disclosure.

[0543] For the avoidance of any doubt, any theoretical explanations provided herein are provided to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0544] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0545] Throughout this specification, including the appended claims, unless the context requires otherwise, the words "comprise" and "comprising" and variations such as "include", "includes" and "including", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0546] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another example includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about," it will be understood that the particular value forms another example. The term "about" in relation to a numerical value is optional and means, for example, + / - 10%.

[0547] Specific embodiments

[0548] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0549] Experimental methods in the following embodiments where specific conditions are not specified were performed according to conventional conditions or the conditions recommended by the raw material or product manufacturers. Alternatively, experimental methods described in biotechnology textbooks such as Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, Current Methods in Molecular Biology, and Cell Biology were used. Reagents where the source is not specified were purchased from commercial sources.

[0550] The reagents and raw materials used in the present invention are commercially available.

[0551] Example 1 Construction, expression, assembly and purification of the anti-c-Met / EGFR bispecific antibody BT461-DB

[0552] 1.1 Construction of BT461-DB

[0553] The anti-c-Met / EGFR antibody BT461-DB was constructed based on Genmab's dual-antibody platform technology. Using genetic engineering techniques, the heavy chain of the anti-EGFR antibody was mutated (F405L, K214R, D356E, L358M), and the heavy chain of the anti-c-Met antibody was mutated (K409R, K214R, D356E, L358M). Subsequently, the nucleotide sequences of the light and heavy chains of the anti-c-Met or anti-EGFR antibody were constructed into the PEE6.4 and PEE12.4 expression vectors, respectively, to generate the anti-c-Met or anti-EGFR antibody light and heavy chain plasmids. The light and heavy chain plasmids were then ligated using SalI / NotI enzymes (NEB), and the two nucleotide sequences were constructed into the same PEE expression vector (pHR plasmid) to generate plasmids that co-express the anti-c-Met or anti-EGFR antibody light and heavy chains.

[0554] The amino acid sequence of the binding arm A of the bispecific antibody embodiment of the present invention is shown in Example 1, comprising the light chain, heavy chain, variable region and CDR sequences of Tables 2 and 3. The amino acid sequence of the binding arm B is shown in Example 1, comprising the light chain, heavy chain, variable region and CDR sequences of Tables 4 and 5.

[0555] The amino acid sequences of the heavy and light chains of the anti-EGFR antibody are shown in Table 2. The sequence numbers of the heavy and light chains and the amino acid sequences of the segments contained therein are shown in Table 1.

[0556] The amino acid sequences of the heavy and light chains of the anti-c-MET antibody are shown in Table 3. The sequence numbers of the amino acid sequences of each segment of the heavy and light chains are shown in Table 1.

[0557] Table 1. BT461 (BT461 DB for short) binding arm components and ligand amino acid sequence numbers of Dubody structures

[0558] Table 2 Amino acid sequence of binding arm A

[0559] Table 3. Exemplary CDR sequences of binding arm A

[0560] Table 4 Amino acid sequence of binding arm B

[0561] Table 5. Exemplary CDR sequences included in binding arm B

[0562] The nucleic acids encoding binding arm A and binding arm B are shown in Table 6.

[0563] Table 6 Nucleic acid encoding binding arms

[0564] 1.2 Expression and purification of anti-EGFR antibody (BT461-DB-EGFR) and anti-c-Met antibody (BT461-DB-c-Met)

[0565] pHR plasmid DNA encoding anti-EGFR and anti-c-Met antibodies was transiently transfected into FUT8 knockout CHO-K1 cells using the ExpiFectamine CHO Transfection Kit (Gibco) as described by the manufacturer. Transfected cells were cultured in OPM-CD TransCHO (OPM, P83059) expression medium at 37°C and 125 rpm in a shaker for 14 days, and the cell supernatant was collected. The resulting cell suspension (CHO-K1 cells, Chinese hamster ovary cells, ATCC, CCL-61) was centrifuged (5000 rpm, 30 min), and the supernatant was filtered (using a 0.22 μm filter) and purified by affinity chromatography.

[0566] Affinity chromatography capture of EGFR and c-Met arms: Mab Select Sure LX filler (Cytiva) was used with a retention time of 6 minutes. The fermentation supernatant was loaded onto the chromatography column with a loading capacity of 40 mg protein per ml of filler. The target protein was in the eluent at pH 3.8. Specific experimental steps are shown in Table 7.

[0567] Table 7

[0568] The antibody solution was buffer exchanged with PBS and concentrated using a 30KD ultrafiltration tube. The protein concentration was determined using Nanodrop and the purity was determined using HPLC-SEC.

[0569] 1.3 In vitro assembly and purification of BT461-DB

[0570] Assembly system: Using Fab exchange technology, affinity chromatography-derived anti-EGFR antibody (BT461-DB-EGFR) and anti-c-Met antibody (BT461-DB-c-Met) were assembled at a 1:1.1 ratio (e.g., 8 mg of the EGFR antibody arm and 8.8 mg of the c-Met antibody arm). The resulting mixture was added to the reducing agent 2-MEA at a final concentration of 75 mmol / L and reduced in a 31°C water bath for 5 h. The 2-MEA reducing agent was removed by desalting with G-25 buffer and the solution was exchanged to PBS. The resulting mixture was then oxidized at room temperature for 48 h in PBS (pH 7.110, Cond: 16.376 mS / cm).

[0571] The configurations of the two single-arm reduction reaction systems are shown in Table 8.

[0572] Table 8

[0573] The reaction system was placed in a water bath at 31°C for 5 hours to fully reduce the disulfide bonds between the antibodies.

[0574] G25 chromatography has a molecular sieving effect and can separate large molecular antibodies from small molecule reducing agent 2-MEA. The experimental steps are shown in Table 9.

[0575] Table 9

[0576] After loading, samples were collected and the UV280 peak range was 100-100 mAU.

[0577] The purification system employed cationic chromatography using Borgron Diamond SP Mustang. Aggregates present during the expression of both antibodies, as well as excess c-Met during assembly, resulted in product-related impurities in the oxidized sample. The isoelectric points of the two antibodies differed from those of the product-related impurities, allowing them to be removed using cationic chromatography. Mobile phase A consisted of 20 mM NaAc, pH 5.0; mobile phase B consisted of 20 mM NaAc, 1 M NaCl, pH 5.0. The experimental procedures are shown in Table 10.

[0578] Table 10

[0579] During the elution process, the peak range of cationic UV280 is 100-100mAU. When the peak shows an inflection point, the tube is changed for collection. The cationic elution samples are sent for SEC, NR-CE, and CEX.

[0580] The test results showed that the SEC monomer purity of BT461-DB was 99.66%, the aggregates accounted for 0.27%, the fragments accounted for 0.08%, and the NR-CE-SDS (non-reducing capillary gel electrophoresis) monomer purity was 96.2%, which met the requirements of subsequent research.

[0581] 1.3 In vitro assembly and purification of BT461-DB

[0582] Assembly system: Using Fab exchange technology, affinity chromatography-derived anti-EGFR antibody (BT461-DB-EGFR) and anti-c-Met antibody (BT461-DB-c-Met) were assembled at a 1:1.1 ratio (e.g., 8 mg of the EGFR antibody arm and 8.8 mg of the c-Met antibody arm). The resulting mixture was added to the reducing agent 2-MEA at a final concentration of 75 mmol / L and reduced in a 31°C water bath for 5 h. The 2-MEA reducing agent was removed by desalting with G-25 buffer and the solution was exchanged to PBS. The resulting mixture was then oxidized at room temperature for 48 h in PBS (pH 7.110, Cond: 16.376 mS / cm).

[0583] The configurations of the two single-arm reduction reaction systems are shown in Table 11.

[0584] Table 11

[0585] The reaction system was placed in a water bath at 31°C for 5 hours to fully reduce the disulfide bonds between the antibodies.

[0586] G25 chromatography has a molecular sieving effect and can separate large molecular antibodies from small molecule reducing agent 2-MEA. The experimental steps are shown in Table 12.

[0587] Table 12

[0588] After loading, samples were collected and the UV280 peak range was 100-100 mAU.

[0589] The purification system employed cationic chromatography using Borgron Diamond SP Mustang. Aggregates present during the expression of the two antibodies, as well as excess c-Met during assembly, resulted in product-related impurities in the oxidized sample. Differences in isoelectric points between the two antibodies and product-related impurities allowed for their removal using cationic chromatography. Mobile phase A consisted of 20 mM NaAc, pH 5.0; mobile phase B consisted of 20 mM NaAc, 1 M NaCl, pH 5.0. The experimental procedures are shown in Table 13.

[0590] Table 13

[0591] During the elution process, the peak range of cationic UV280 is 100-100mAU. When the peak shows an inflection point, the tube is changed for collection. The cationic elution samples are sent for SEC, NR-CE, and CEX.

[0592] The test results showed that the SEC monomer purity of BT461-DB was 99.66%, the aggregates accounted for 0.27%, the fragments accounted for 0.08%, and the NR-CE-SDS (non-reducing capillary gel electrophoresis) monomer purity was 96.2%, which met the requirements of subsequent research.

[0593] Example 2 Evaluation of the Binding Activity of BT461-DB to EGFR and c-Met Antigen Proteins

[0594] 2.1 Targeting c-Met protein

[0595] C-MET-HIS (Bipsys, C-MET-H5227) was diluted to 2 μg / mL using coating solution (8 ml of 0.2 mol / L Na2CO3 and 17 ml of 0.2 mol / L NaHCO3, then 75 ml of distilled water and adjusted to pH 9.6). 50 μL / well of the coating solution was added to the ELISA plate and incubated at 37°C for 2 hours. The liquid in the wells was discarded and the plates were washed five times using a plate washer with 200 μL of washing solution (PBST, PBS diluted with 0.05% Tween-20). 200 μL of blocking solution (PBST + 5% skim milk powder) was added to each well and incubated overnight at 4°C. The liquid in the wells was discarded and the plates were washed five times using a plate washer (Tecan, Hydrospeed) with 200 μL of washing solution. Add 50 μL of BT461 and Avantol (Johnson, batch number MAS0001) at a 20 μg / mL concentration, diluted 5-fold, in a seven-step gradient, and set up a blank control. Incubate at 37°C for 60 min. Discard the remaining liquid in the wells and wash five times with 200 μL of wash buffer using a plate washer. Add 50 μL of Goat anti-human IgG-Fc secondary antibody (Jackson, 1:10,000 dilution) to each well and incubate at 37°C for 60 min. Discard the remaining liquid in the wells and wash five times with 200 μL of wash buffer using a plate washer. Add 50 μL of color development solution per well and incubate at 37°C for 15 min. Terminate the reaction with 50 μL of 2 mol / L H₂SO₄ and read the OD₄50 value on a microplate reader. ELISA binding activity analysis results are shown in Table 14 and Figure 1A.

[0596] 2.2 Targeting EGFR protein

[0597] EGFR-HIS (Bipsys, EGR-H5222) was diluted to 2 μg / mL with coating solution (8 ml of 0.2 mol / L Na2CO3 and 17 ml of 0.2 mol / L NaHCO3, then 75 ml of distilled water was added to adjust the pH to 9.6). 50 μL / well of the coating solution was added to the ELISA plate wells and incubated at 37°C for 2 hours. The liquid in the wells was discarded and the plates were washed 5 times with 200 μL / well of washing solution (PBST, PBS diluted with 0.05% Tween-20). 200 μL of blocking solution (PBST + 5% skim milk powder) was added to each well and incubated at 4°C overnight. The liquid in the wells was discarded and the plates were washed 5 times with 200 μL / well of washing solution (Tecan, model Hydrospeed). Add 50 μL of 20 μg / mL anti-c-Met / EGFR dual antibody (BT461, Avantol) to each well, diluting 5-fold across seven antibody steps. A blank control was also included. Incubate at 37°C for 60 min. Discard the remaining liquid from the wells and wash five times with 200 μL of wash buffer using a plate washer. Add 50 μL of Goat anti-human IgG-Fc secondary antibody (Jackson, 1:10,000 dilution) to each well and incubate for 60 min at 37°C. Discard the remaining liquid from the wells and wash five times with 200 μL of wash buffer using a plate washer. Add 50 μL of color development solution per well and incubate at 37°C for 15 min. Terminate the reaction with 50 μL of 2 mol / L H₂SO₄ and read the OD₄50 value on a microplate reader. ELISA binding activity analysis results are shown in Table 14 and Figure 1B.

[0598] Table 14 EC50 of the binding of the bispecific antibodies of the present invention to c-Met and EGFR proteins

[0599] As can be seen from Table 14 and Figure 1, the antibody BT461-DB can specifically bind to human c-Met protein and EGFR protein, and its binding activity is consistent with that of the BT461-KIH antibody.

[0600] Example 3 Evaluation of the binding activity of BT461-DB to CHO-K1 cells overexpressing EGFR and c-Met

[0601] 3.1 BT461 Targets CHO-K1 Cells Overexpressing c-Met Membrane Protein

[0602] To test the specificity of the antibody of the present invention in binding to the c-Met membrane protein on the cell surface, we stably overexpressed the human c-Met membrane protein in CHO-K1 cells (a hamster ovary cell substrain, ATCC#CCL-61) by plasmid electroporation and drug addition screening. The CHO-K1-c-Met cells overexpressing human c-Met were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed twice with PBS. A blank control group and an experimental group were set up on the 96-well plate. The antibodies BT461 and Avant (at a concentration of 20 μg / mL) were added to the wells of the experimental group and diluted to 7 gradients with a 5-fold concentration using a gradient dilution method. The 96-well plate was placed at 4°C for 30 minutes and centrifuged (1000 rpm for 2 minutes) to remove the liquid in the wells. The cells were then washed twice with PBS (200 μL / well). 50 μL of PE-anti-human-IgG-Fc-Secondary Antibody (Jackson, 1:10,000 dilution) was added to each well and incubated at 4°C for 30 minutes. The cells were centrifuged at 1000 rpm for 2 minutes, and the liquid was removed. The cells were then washed twice with PBS (200 μL / well). 200 μL of PBS was added to each well, the cells were resuspended, and the cells were analyzed by flow cytometry. The results of FACS binding activity analysis are shown in Table 15 and Figure 2A.

[0603] 3.2 Similarly, we stably overexpressed human EGFR membrane protein in CHO-K1 cells (hamster ovary cell substrain, ATCC#CCL-61) by plasmid electroporation and drug addition screening to detect the specificity of the binding of the antibody of the present invention to the EGFR membrane protein on the cell surface. The CHO-K1-EGFR cells overexpressing human EGFR were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed with PBS (twice); a blank control group and an experimental group were set up on the 96-well plate, and anti-c-Met / EGFR antibody BT461 and Avant (at a concentration of 20 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients with a 5-fold concentration using a gradient dilution method; the 96-well plate was placed at 4°C for 30 minutes, centrifuged (1000 rpm, 2 minutes), and the wells were removed. The cells were washed twice with PBS (200 μL / well). 50 μL of PE-anti-human-IgG-Fc-Secondary Antibody (Jackson, 1:10,000 dilution) was added to each well and incubated at 4°C for 30 minutes. The cells were centrifuged at 1000 rpm for 2 minutes, and the liquid was removed. The cells were washed twice with PBS (200 μL / well). 200 μL of PBS was added to each well, and the cells were resuspended and analyzed by flow cytometry. The results of FACS binding activity analysis are shown in Table 15 and Figure 2B.

[0604] Table 15. EC50 of BT461-DB binding to cells expressing c-Met and / or EGFR membrane protein

[0605] The results showed that the antibody BT461-DB of the present invention bound to the EGFR or c-Met protein expressed on the surface of CHO-K1 cells with EC50 of 1725 ng / mL and 266.2 ng / mL, respectively, which were consistent with the binding activity of the antibody BT461-KIH (1962 ng / mL and 270.4 ng / mL, respectively).

[0606] Example 4 Specific Binding of BT461-DB to EGFR / c-Met Double-Positive Tumor Cells MKN45

[0607] To further detect the binding of the antibodies of the present invention to dual-target-expressing tumor cells, we digested and counted MKN45 cells (Shan En Biotech, Cat. No. SNL-173) that highly expressed EGFR and c-Met membrane proteins, resuspended them in a 96-well plate (3E5 cells were added to each well), and washed twice with PBS; a blank control group and an experimental group were set up on the 96-well plate, and the anti-c-Met / EGFR antibody BT461 and Avant (at a concentration of 20 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients with a 5-fold concentration using a gradient dilution method; the 96-well plate was incubated at 4°C for 30 minutes. s, centrifuged (1000 rpm, 2 mins), removed the liquid from the wells, and washed twice with PBS (200 μL / well). PE-anti-human-IgG-Fc-Secondary Antibody (Jackson, 1:10,000 dilution, 50 μL) was added to each well and incubated at 4°C for 30 min. Centrifuged (1000 rpm, 2 mins), removed the liquid, and washed twice with PBS (200 μL / well). PBS was added to each well (200 μL / well), the cells were resuspended, and read using a flow cytometer. The results of FACS binding activity analysis are shown in Table 16 and Figure 3.

[0608] The results showed that the binding activities of antibodies BT461-DB and BT461-KIH to MKN45 were basically the same, with EC50 of 829.2 ng / mL and 809.5 ng / mL, respectively.

[0609] Table 16

[0610] Example 5 Antagonizing the binding of ligands HGF and EGF to target proteins EGFR and c-Met

[0611] 5.1 Antagonizing the binding of EGF to EGFR

[0612] EGF-mFC (Bepsis, product number EGF-H525b) was diluted to 5 μg / mL with coating solution (8 ml of 0.2 mol / L Na2CO3 and 17 ml of 0.2 mol / L NaHCO3 were mixed, and then 75 ml of distilled water was added to adjust the pH to 9.6). The solution was coated onto the ELISA plate at a volume of 50 μL / well and incubated at 37°C for 2 hours. After removing the liquid, the plate was washed with washing solution (PBST, 0.05% Tween-20 was added to the PBS diluent) (200 μL / time, 5 times); 200 μL of blocking solution was added to each well and incubated at 4°C overnight. After removing the liquid, the plate was washed with washing solution (200 μL / time, 5 times); a blank control group and an experimental group were set up on the ELISA plate, and EGFR-his (25 μL, 4 μg / mL) and anti- c-Met / EGFR antibody was diluted into seven 5-fold gradients using a gradient dilution method and incubated at 37°C for 120 min. After removing the liquid, the wells were washed with washing solution (200 μL / time, 5 times). Goat-anti-HIS-Fc-Secondary-Antibody (Jackson, 1:10,000 dilution) was added to each well. After removing the liquid, the wells were washed with washing solution (200 μL / time, 5 times). Color development solution (50 μL / well) was added to each well. After incubation at 37°C for 15 min, H2SO4 aqueous solution (50 μL / well, 2 mol / L) was added to terminate the reaction, and the OD450 value was read on a microplate reader. The results of the ELISA blocking activity analysis are shown in Table 17 and Figure 4A.

[0613] 5.2 Antagonizing the binding of HGF to c-Met

[0614] HGFR-hfc (Bipsys, catalog number MET-H5256) was coated with a coating solution (0.2 mol / L Na2CO38ml, 0.2mol / LNaHCO317ml mixed, then 75ml distilled water was added to adjust the pH to 9.6) and diluted to 2μg / mL, and coated into the ELISA plate at a volume of 50μL / well, incubated at 37°C for 2hrs, and washed with washing solution (PBST, PBS diluent with 0.05% Tween-20) (200μL / time, 5 times); 200μL blocking solution was added to each well, and incubated at 4°C overnight, and washed with washing solution (200μL / time, 5 times) after removing the liquid; a blank control group and an experimental group were set up on the ELISA plate, and 25μL of HGF-HIS (Bepsais, product number HGF-H52H3) with a concentration of 200ng / mL and 500μg / mL of antibodies BT461-DB and BT461-KIH were diluted 5-fold to form seven gradients using a gradient dilution method. The cells were incubated at 37°C for 120 min. After removal of the liquid, the cells were washed with wash buffer (200 μL / well, five times). Goat anti-human his-secondary antibody (Jackson, Catalog No. 109-005-098, 1:10,000 dilution) was added to each well. After removal of the liquid, the cells were washed with wash buffer (200 μL / well, five times). A color development solution (50 μL / well) was added to each well. After incubation at 37°C for 15 min, the reaction was terminated by addition of aqueous H2SO4 solution (50 μL / well, 2 mol / L), and the OD450 value was read on a microplate reader. The results of the ELISA blocking activity assay are shown in Table 17 and Figure 4B.

[0615] Table 17. IC50 of blocking HGF, EGF binding to proteins EGFR and c-Met

[0616] As shown in Figure 4A, both antibodies BT461-DB and BT461-KIH were able to block the binding of the EGF ligand to the EGFR receptor at consistent levels. As shown in Table 17 and Figure 4B, both antibodies BT461-DB and BT461-KIH were able to block the binding of the HGF ligand to the c-Met receptor at essentially the same levels, with IC50 values ​​of 456.4 ng / mL and 543.3 ng / mL for BT461-DB and BT461-KIH, respectively.

[0617] Example 6 Detection of affinity between antibody BT461-DB and antigen based on biolayer interferometry (BLI)

[0618] 6.1 Binding Kinetics of the Bispecific Antibody of the Present Invention to Recombinant Human EGFR-His

[0619] EGFR-His (ACRO, Catalog No. EGR-H5222) was serially diluted from 100 nM in PBST and serially diluted two-fold to seven concentration points using a zero-concentration reference well. The c-Met / EGFR bispecific antibody BT461-DB and the control antibody BT461-KIH (JH021 / B / 2024015) were diluted to 5 μg / mL. The molecular interaction instrument (Fortebio, Sartorius, Model: Octet R8) was operated at 30°C and a shake speed of 1000 rpm. The antibody was captured using a coated AHC2 probe (Sartorius, Catalog No. 18-5142) with a capture time of 180 s. The antibody was then bound to the serially diluted EGFR-His sample with an association time of 120 s and a dissociation time of 300 s. The sample was then regenerated three times with regeneration buffer (10 mM glycine, pH 1.7) for 30 s each. On-device detection was performed using ForteBio's Octet System. After obtaining sensorgram data, Octet BLI Analysis software was used to analyze the binding constant (ka) and dissociation constant (kd). Ideal binding and dissociation curves were fitted, as shown in Figures 5A and 5B. The equilibrium dissociation constant (KD) between the antibody and antigen was calculated (kd / ka). The results are shown in Table 18.

[0620] 6.2 Binding Kinetics of the Bispecific Antibody of the Present Invention to Recombinant Human c-Met-His

[0621] As in Example 6.1, the binding constant (ka) and dissociation constant (kd) of BT461-DB and BT461-KIH with c-Met-HIS (Biopsy, c-Met-H5227) were analyzed using Octet BLI Analysis software. Ideal binding and dissociation curves were fitted, as shown in Figures 5C and 5D, and the equilibrium dissociation constant KD (kd / ka) between the antibody and antigen was calculated.

[0622] Table 18. c-Met-HIS affinity and EGFR-HIS affinity

[0623] The results are shown in Table 18. The affinities of BT461-DB and BT461-KIH to c-Met-his are 0.766 nM and 0.929 nM, respectively, which are basically consistent. BT461-DB and BT461-KIH also have consistent affinities to EGFR-HIS, which are 21.93 nM and 19.85 nM, respectively. The affinities of the two arms of the BT461-DB antibody differ by more than 20 times.

[0624] Example 7 Evaluation of affinity between FC of antibody BT461-DB and receptor protein

[0625] 7.1 Detection of the binding affinity of BT461-DB antibody to FcγRI (CD64)

[0626] The affinity assay for binding of the JH021 antibody to human FcγRI (CD64) (ACRO, Catalog No. FCA-H52H1, Lot No. RC163P1-2387F1-1GU) was performed using biolayer interferometry (BLI). JH021 antibody was serially diluted in PBST starting at 250 nM and then serially diluted two-fold over seven concentration points. A zero-concentration control well was also included. FcγRI (CD64) receptor protein was diluted to 5 μg / mL in loading buffer (1× PBS, pH 7.4, with 0.02% Tween-20, 0.1% BSA) and immobilized onto a HIS1K sensor (Sartorius, Catalog No. 18-5120, Lot No. 2301012611) with a threshold of 0.5 nm and an immobilization time of 180 s. The molecular interaction instrument, ForteBio's Octet System (Sartorius, Model: Octet R8), was set up under the following operating conditions: temperature 30°C, shake speed 1000 rpm. Immobilized HIS1K probes were used to bind to serially diluted samples with an association time of 120 s and a dissociation time of 300 s. Regeneration was performed with regeneration buffer (10 mM glycine, pH 1.7) for 30 s. On-board detection was performed using ForteBio's Octet System. Sensorgram data were obtained and analyzed using Octet BLI Analysis 12.2 software to determine the association constant (ka) and dissociation constant (kd). Idealized association and dissociation curves were fitted to calculate the affinity constant (kd / ka) between the antibody and antigen. The results are shown in Table 19: The binding affinities of BT461-DB and BT461-KIH for FcγRI (CD64) were 2.28E-07 and 2.33E-07, respectively, showing comparable binding affinity.

[0627] 7.2. Detection of Antibody Binding Affinity to FcγRⅢA (CD16a-F176)

[0628] The affinity test of JH021 antibody binding to Human FcγRⅢA (CD16a-F176) (Brand: ACRO, Catalog No.: CDA-H5220, Lot No.: 2415-226YF1-1J9) was based on biolayer interferometry (BLI) technology. JH021 antibody was serially diluted with PBST, starting at a concentration of 5000nM. The dilutions were then performed in 2-fold serial dilutions through 7 concentration points, and a zero concentration control well was set. The FcγRⅢA (CD16a-F176) receptor protein was diluted to 5 μg / mL in loading buffer (1× PBS, pH 7.4, with 0.02% Tween-20 and 0.1% BSA) and immobilized onto a HIS1K sensor (Sartorius, Catalog No. 18-5120, Lot No. 2301012611) with a threshold of 0.5 nm and an immobilization time of 180 s. The molecular interaction instrument, ForteBio's Octet System (Sartorius, Model: Octet R8), was set to run at 30°C and a shake speed of 1000 rpm. Immobilized HIS1K probes were bound to serially diluted samples with an association time of 120 s and a dissociation time of 300 s. Regeneration was performed with regeneration buffer (10 mM glycine, pH 1.7) for 30 s. Detection was performed using the ForteBio Octet System. After obtaining the sensorgram data, the binding constant (ka) and dissociation constant (kd) were analyzed using Octet BLI Analysis 12.2 software, and the ideal binding and dissociation curves were fitted to calculate the affinity constant KD (kd / ka) between the antibody and antigen. The results are shown in Table 19: The binding affinities of BT461-DB and BT461-KIH to FcγRⅢA (CD16a-F176) were 3.30E-07 and 2.68E-07, respectively, which were not much different.

[0629] 7.3 Detection of Antibody Binding Affinity to FcγRⅢA (CD16a-V176)

[0630] The affinity test of JH021 antibody binding to Human FcγRⅢA (CD16a-V176) (Brand: ACRO, Catalog No.: CD8-H52H4, Lot No.: 1824-21BCF1-196) was based on biolayer interferometry (BLI) technology. JH021 antibody was serially diluted with PBST, starting at a concentration of 2500nM. The dilutions were then performed in 2-fold serial dilutions through 7 concentration points, and a zero concentration control well was set. The FcγRⅢA (CD16a-V176) receptor protein was diluted to 10 μg / mL in loading buffer (1× PBS, pH 7.4, with 0.02% Tween-20 and 0.1% BSA) and immobilized onto a HIS1K sensor (Sartorius, Catalog No. 18-5120, Lot No. 2301012611) with a threshold of 0.5 nm and an immobilization time of 180 s. The molecular interaction instrument, ForteBio's Octet System (Sartorius, Model: Octet R8), was set to operate at 30°C and a shake speed of 1000 rpm. Immobilized HIS1K probes were bound to serially diluted samples with an association time of 120 s and a dissociation time of 300 s. Regeneration was performed with regeneration buffer (10 mM glycine, pH 1.7) for 30 s. Detection was performed using the ForteBio Octet System. After obtaining the sensorgram data, the binding constant (ka) and dissociation constant (kd) were analyzed using Octet BLI Analysis 12.2 software. The ideal binding and dissociation curves were fitted to calculate the affinity constant KD (kd / ka) between the antibody and antigen. The results are shown in Table 19: The binding affinities of BT461-DB and BT461-KIH to FcγRⅢA (CD16a-V176) were 2.65E-08 and 2.26E-08, respectively, which are not much different.

[0631] 7.4 Detection of Antibody Binding Affinity to FcγRⅢB (CD16b)

[0632] The affinity of the JH021 antibody for human FcγRⅢB (CD16b) (ACRO, Catalog No. CDB-H5227, Lot No. 2417-228KF1-17G) was determined using biolayer interferometry (BLI). JH021 antibody was serially diluted in PBST, starting at 5000 nM and followed by a two-fold serial dilution through seven concentration points. A zero-concentration control well was also included. The FcγRⅢB (CD16b) receptor protein was diluted to 5 μg / mL in loading buffer (1× PBS, pH 7.4, with 0.02% Tween-20 and 0.1% BSA) and immobilized onto a HIS1K sensor (Sartorius, Catalog No. 18-5120, Lot No. 2301012611) with a threshold of 0.5 nm and an immobilization time of 180 s. The molecular interaction instrument, ForteBio's Octet System (Sartorius, Model: Octet R8), was set up under the following operating conditions: temperature 30°C, shake speed 1000 rpm. Immobilized HIS1K probes were used to bind to serially diluted samples with an association time of 120 s and a dissociation time of 300 s. Regeneration was performed in regeneration buffer (10 mM glycine, pH 1.7) for 30 s. On-board detection was performed using ForteBio's Octet System. Sensorgram data were obtained and analyzed using Octet BLI Analysis 12.2 software to determine the association constant (ka) and dissociation constant (kd). Idealized association and dissociation curves were fitted to calculate the affinity constant (kd / ka) between the antibody and antigen. The results are shown in Table 19: The binding affinities of BT461-DB and BT461-KIH for FcγRⅢB (CD16b) were 1.44E-05 and 1.42E-05, respectively, which are generally consistent.

[0633] 7.5 Detection of Antibody Binding Affinity to FcγRⅡA (CD32a)

[0634] The affinity of the JH021 antibody for human FcγRIIA (CD32a) (ACRO, Catalog No. CDA-H5221, Lot No. 1822-216TF1-139) was determined using biolayer interferometry (BLI). FcγRIIA (CD32a) was serially diluted in PBST starting at 2000 nM and then serially diluted two-fold over seven concentrations. A zero-concentration control well was also included. The JH021 antibody receptor protein was diluted to 20 μg / mL in loading buffer (1× PBS, pH 7.4, with 0.02% Tween-20 and 0.1% BSA) and immobilized onto a ProA sensor (Sartorius, Catalog No. 18-5010, Lot No. 2302000611) with a threshold of 2.0 nm and an immobilization time of 180 s. The molecular interaction instrument, ForteBio's Octet System (brand: Sartorius, model: Octet R8), was set up under the following operating conditions: temperature 30°C, shake speed 1000 rpm. Immobilized ProA probes were used to bind to serially diluted samples with an association time of 120 s and a dissociation time of 300 s. Regeneration was performed in regeneration buffer (10 mM glycine, pH 1.7) for 30 s. On-board detection was performed using ForteBio's Octet System. Sensorgram data were obtained and analyzed using Octet BLI Analysis 12.2 software to determine the association constant (ka) and dissociation constant (kd). Idealized association and dissociation curves were fitted to calculate the affinity constant (kd / ka) between the antibody and antigen. The results are shown in Table 19: The binding affinities of BT461-DB and BT461-KIH to FcγRⅡA (CD32a) were 1.52E-06 and 1.53E-06, respectively, which are generally consistent.

[0635] 7.6 Detection of Antibody Binding Affinity to FcγRⅡB / C (CD32b / c)

[0636] The affinity of JH021 antibody binding to human FcγRIIB / C (CD32b / c) (ACRO, Catalog No. CDB-H5228, Lot No. 478-234AF1-1AJ) was determined using biolayer interferometry (BLI). JH021 antibody was serially diluted in PBST, starting at 5000 nM and followed by a two-fold serial dilution through seven concentration points. A zero-concentration control well was also included. FcγRIIB / C (CD32b / c) receptor protein was diluted to 5 μg / mL in loading buffer (1× PBS, pH 7.4, with 0.02% Tween-20 and 0.1% BSA) and immobilized onto a HIS1K sensor (Sartorius, Catalog No. 18-5120, Lot No. 2301012611) with a threshold of 0.5 nm and an immobilization time of 180 s. The molecular interaction instrument, ForteBio's Octet System (Sartorius, Model: Octet R8), was set up under the following operating conditions: temperature 30°C, shake speed 1000 rpm. Immobilized HIS1K probes were used to bind to serially diluted samples with an association time of 120 s and a dissociation time of 300 s. Regeneration was performed in regeneration buffer (10 mM glycine, pH 1.7) for 30 s. On-board detection was performed using ForteBio's Octet System. Sensorgram data were obtained and analyzed using Octet BLI Analysis 12.2 software to determine the association constant (ka) and dissociation constant (kd). Idealized association and dissociation curves were fitted to calculate the affinity constant (kd / ka) between the antibody and antigen. The results are shown in Table 19: The binding affinities of BT461-DB and BT461-KIH to FcγRIIB / C (CD32b / c) were 1.65E-06 and 1.16E-06, respectively, which are generally consistent.

[0637] 7.7 Detection of Antibody Binding Affinity to Fc Rn

[0638] The affinity assay for binding of the JH021 antibody to human FcRn (ACRO, Catalog No. FCM-H8286, Lot No. BLA476-21CFF1-12V) was performed using biolayer interferometry (BLI). JH021 antibody was serially diluted in PBST, starting at 1000 nM and followed by a two-fold serial dilution through seven concentration points. A zero-concentration control well was also included. The FcRn receptor protein was diluted to 2 μg / mL in loading buffer (1× PBS, pH 7.4, with 0.02% Tween-20 and 0.1% BSA) and immobilized onto a SA sensor (Sartorius, Catalog No. 18-5019, Lot No. 2309011711) with a threshold of 2.5 nm and an immobilization time of 180 s. The molecular interaction instrument, ForteBio's Octet System (brand: Sartorius, model: Octet R8), was set up under the following operating conditions: temperature 30°C, shake speed 1000 rpm. Immobilized SA probes were used to bind to the gradient diluted sample with an association time of 120 s and a dissociation time of 300 s; regeneration was performed with regeneration buffer (10 mM glycine, pH 1.7) for 30 s. On-machine detection was performed using ForteBio's Octet System. After obtaining the sensorgram data, the binding constant (ka) and dissociation constant (kd) were analyzed using Octet BLI Analysis 12.2 software. Ideal binding and dissociation curves were fitted, and the affinity constant KD (kd / ka) between the antibody and antigen was calculated. The results are shown in Table 19: The binding affinities of BT461-DB and BT461-KIH to Fc Rn were 2.28E-07 and 2.33E-07, respectively, which were basically consistent.

[0639] 7.8 Detection of Antibody Binding Affinity to C1q

[0640] The affinity assay for binding of the JH021 antibody to Human C1q (Abcam, Catalog No. AB282858, Lot No. 1034803-2) was performed using biolayer interferometry (BLI). C1q was serially diluted in PBST, starting at 100 nM and followed by a two-fold serial dilution through seven concentration points. A zero-concentration control well was also included. The JH021 antibody receptor protein was diluted to 20 μg / mL in loading buffer (1× PBS, pH 7.4, with 0.02% Tween-20 and 0.1% BSA) and immobilized onto a FAB2G sensor (Sartorius, Catalog No. 18-5125, Lot No. 2212007211). The threshold was 2.5 nm and the immobilization time was 180 s. The molecular interaction instrument, ForteBio's Octet System (brand: Sartorius, model: Octet R8), was set up under the following operating conditions: temperature 30°C, shake speed 1000 rpm. Immobilized FAB2G probes were used to bind to the gradient diluted samples with an association time of 120 s and a dissociation time of 300 s; regeneration was performed with regeneration buffer (10 mM glycine, pH 1.7) for 30 s. On-board detection was performed using ForteBio's Octet System. After obtaining sensorgram data, Octet BLI Analysis 12.2 software was used to analyze the binding constant (ka) and dissociation constant (kd). Ideal binding and dissociation curves were fitted, and the affinity constant KD (kd / ka) between the antibody and antigen was calculated. The results are shown in Table 19: The binding affinities of BT461-DB and BT461-KIH to C1q were 1.12E-08 and 1.62E-08, respectively, which are generally consistent.

[0641] Table 19. Affinity test results of antibody BT461-DB and FC receptor protein

[0642] Example 8 Antibody BT461-DB inhibits EGFR autophosphorylation and c-Met autophosphorylation

[0643] To explore whether the anti-c-Met / EGFR bispecific antibody can inhibit the c-Met and EGFR signaling pathways, we measured the changes in the EGFR and c-Met autophosphorylation levels, respectively.

[0644] Western blot analysis

[0645] Non-small cell lung cancer A549 cells were seeded in 6-well plates (4 × 10 5After attachment, control and experimental groups were set up on 6-well plates. In the medium containing 100 ng / mL EGF-mFC (Biopsy) + 100 ng / mL HGF-his (Biopsy) and 1% serum, 250 μg / mL IgG1, Evantol, BT461-DB, and BT461-KIH were added to the negative group, control group, and experimental group (BT461-DB and BT461-KIH) respectively. After reacting for 15 minutes, RIPA-lysis buffer (150 μL, Biyuntian) was added and lysed at 0°C (on ice) for 30 minutes. Loading buffer (containing the reducing agent β-mercaptoethanol (Life Technologies)) was added to each well. Technologies) and boiled at 95°C for 10 minutes to denature the protein; the denatured protein was subjected to electrophoresis using Tris-SDS as the running buffer (the denatured protein was loaded into the wells of a 10% PAGE precast gel (Genscript)). After the electrophoresis, the protein was transferred to a PVDF membrane using a transfer buffer containing 20% ​​methanol, and the PVDF membrane was then blocked with 5% skim milk at room temperature for 1 hour; anti-c-Met antibody (Invitrogen, RJ243364) or anti-EGFR antibody (ABCAM, ab52894) or anti-GAPDH antibody (S The cells were incubated with anti-p-EGFR antibody (Cellsignaling, #3077S) or anti-p-EGFR antibody (R&D, AF1095) at 4°C overnight and washed with TBST (3 times, 10 min each time). At room temperature, goat anti-mouse / rabbit IgG (H+L) HRP-conjugated secondary antibody (1:3000 dilution) was added to each well and incubated for 1 h. The cells were washed with TBST (3 times, 10 min each time). The cells were reacted with ECL substrate (Sangon, l115AA0118) at room temperature for 1 min and developed using a chemiluminescence system.

[0646] The development results are shown in Figure 6. Stimulation with HGF-his (100 μg / mL) and EGF-mfc (100 μg / mL) for 15 minutes can activate the phosphorylation of EGFR and c-Met in A549 cells. On this basis, the addition of BT461-DB or BT461-KIH can significantly inhibit the intracellular phosphorylation level of pc-Met (Y1234 / 1235) and slightly inhibit the phosphorylation level of p-EGFR (Y1173).

[0647] Example 9 Antibody BT461 mediates internalization and degradation of target protein

[0648] Using pHAb probe to draw internalization curves of gradient antibody concentrations

[0649] 100 μg of Goat-anti-human-IgG-Fc-Secondary-Antibody was dissolved in buffer using an ultrafiltration tube. 500 μL of 10 mM sodium bicarbonate (pH 8.5) was added and the mixture was centrifuged at 15,000 rpm at 4°C for 10 minutes. Repeat twice. 25 μL of a 1:1 DMSO-water mixture was added to 250 μg of pHAb Reactive Dyes (Promega, stored at -80°C) to obtain a 10 mg / mL dye stock solution. The pHAb-reactive dye was conjugated to the antibody at an antibody-to-dye ratio of 100 μg:1.2 μL and allowed to bind on a shaker at room temperature for 60 minutes. Unreacted dye was removed using a desalting column. The column was centrifuged at 900 g for 2 minutes to equilibrate. The dye-antibody conjugated mixture was then dripped onto the center of the slant side of the desalting column and centrifuged at 900 g for 2 minutes, facing outward. The sample was collected and assayed for antibody concentration and dye-to-antibody ratio.

[0650] MKN45 cells were counted and 3E5 cells were added to each well of a U-bottom 96-well plate. The cells were centrifuged at 1000 rpm for 2 minutes and washed twice with PBS. Antibodies BT461-DB, BT461-KIH, BT461-DB-EGFR, BT461-DB-c-Met, and IgG1 were serially diluted to a starting concentration of 20 μg / mL. Five-fold dilutions were added to each well for seven steps, with 50 μL of each antibody added. A blank control was also included. The cells were incubated at 4°C for 30 minutes. After centrifugation at 1000 rpm for 2 minutes, the wells were discarded, and the plates were washed twice with 200 μL of PBS. The plates were then kept on ice. Fifty μL of dye-conjugated secondary antibody (diluted 1:200) was added to each well. One plate was incubated at 37°C and the other plate was incubated at 4°C. After 4 hours, the plates were washed twice with PBS and resuspended in 200 μL of PBS. Flow cytometry was performed (using PE fluorescence excitation and reception).

[0651] The results are shown in Figure 7 . The antibody BT461-DB promoted the internalization of the target protein. Its internalization efficiency was consistent with that of BT461-KIH, and the internalization level mediated by the anti-c-Met antibody arm was greater than that mediated by the anti-EGFR antibody arm.

[0652] Example 10 Evaluation of ADCC Levels Mediated by BT461-DB Antibody

[0653] The ADCC biological activity of the antibody was detected using the ADCC Reporter Bioassay method. The ADCC reporter system used in this example was ADCC Reporter Bioassay (Suzhou Ruian, RA-CK01), and the fluorescent substrate was the Bio-Turbo-One-Step Firefly Luciferase Assay Kit (Suzhou Ruian, RA-GL03).

[0654] 10.1 Target cells are CHOK1-EGFR

[0655] CHOK1-EGFR cells (overexpressing EGFR membrane protein) in the logarithmic growth phase were used as target cells. After digestion and counting, they were resuspended in culture medium (Gibco, F12K + 10% FBS) at a cell density of 2.5E5 / mL. 100 μL of the resulting suspension was plated onto a 96-well plate (96-well opaque flat-bottom white plate, Costar, 3917) and incubated for 6 hours to allow attachment. After cell attachment, the culture medium was aspirated and the cells were washed once with PBS. The corresponding antibodies were diluted to the corresponding concentrations in 1640+1% FBS culture medium according to the plate distribution for the experimental and control groups and added to the corresponding wells, 50 μL per well. The cells were incubated at 37°C, 5% CO2 for 45 minutes. ADCC reporter cells were harvested and resuspended in phenol red-free 1640+1% FBS culture medium (1E6) at 25,000 cells / well, 25 μL per well, to achieve a 1:1 effector cell:target cell ratio. After 6 hours of incubation in an incubator, the plates were returned to room temperature for equilibrium. 75 μL of luciferase substrate was added to each well and incubated for 3 minutes. CTG readings were measured using a microplate reader (PerkinElmer). The results are shown in Figure 8A and Table 20.

[0656] As can be seen from the data in Figure 8A, both antibodies BT461-DB and BT461-KIH mediated antibody-dependent cellular cytotoxicity (ADCC) against CHOK1-EGFR target cells, and the ADCC levels were similar.

[0657] 10.2 Target cells are CHOK1-c-Met

[0658] CHOK1-c-Met cells (overexpressing c-Met membrane protein) in the logarithmic growth phase were used as target cells. After digestion and counting, they were resuspended in culture medium (Gibco, F12K + 10% FBS) at a cell density of 2.5E5 / mL. 100 μL of the resulting suspension was plated onto a 96-well plate (96-well opaque flat-bottom white plate, Costar, 3917) and incubated for 6 hours to allow attachment. After cell attachment, the culture medium was aspirated and the cells were washed once with PBS. The corresponding antibodies were diluted to the corresponding concentrations in 1640+1% FBS culture medium according to the experimental and control groups and added to the corresponding wells, 50 μL per well. The cells were incubated at 37°C, 5% CO2 for 45 minutes. ADCC reporter cells were harvested and resuspended in phenol red-free 1640+1% FBS culture medium (1E6) at a density of 25,000 cells / well, 25 μL per well, to achieve a 1:1 effector cell:target cell ratio. After 6 hours of incubation in an incubator, the plates were brought to room temperature for equilibrium. 75 μL of luciferase substrate was added to each well and incubated for 3 minutes. CTG readings were measured using a microplate reader (PerkinElmer). The results are shown in Figure 8B and Table 20.

[0659] As can be seen from the data in Figure 8B, both antibodies BT461-DB and BT461-KIH mediated antibody-dependent cellular cytotoxicity (ADCC) against CHOK1-c-Met target cells, and the ADCC levels were similar.

[0660] Table 20. EC50 of antibody-mediated ADCC killing curve

[0661] 10.3 Target cells are non-small cell lung cancer cell line HCC827

[0662] HCC827 tumor cells in the logarithmic growth phase were used as target cells. After digestion and counting, they were resuspended in culture medium (Gibco, RPM1640 + 10% FBS) at a cell density of 2.5E5 / mL. 100 μL of the resulting suspension was plated onto a 96-well plate (96-well opaque flat-bottom white plate, Costar, 3917) and incubated for 6 hours to allow attachment. After cell attachment, the culture medium was aspirated and the cells were washed once with PBS. The corresponding antibodies were diluted to the corresponding concentrations in 1640 + 1% FBS culture medium according to the experimental and control groups and added to the corresponding wells, 50 μL per well. The cells were incubated at 37°C, 5% CO2 for 45 minutes. ADCC reporter cells were harvested and resuspended in phenol red-free 1640 + 1% FBS culture medium (1E6) at 25,000 cells / well, 25 μL per well, to achieve a 1:1 effector cell:target cell ratio. After 6 hours of incubation in an incubator, the plate was brought to room temperature for equilibrium. 75 μL of luciferase substrate was added to each well and incubated for 3 minutes. CTG readings were measured using a microplate reader (PerkinElmer). The results are shown in Figure 9.

[0663] As can be seen from the data in Figure 9, both antibodies BT461-DB and BT461-KIH mediated antibody-dependent cellular cytotoxicity (ADCC) against HCC827 tumor cells, and the ADCC levels were similar, with EC50 of 52.58 ng / mL and 41.67 ng / mL, respectively, and maximum killing fluorescence release of 247.6*10^3 and 255.3*10^3, respectively; in addition, the experimental results also showed that 461-DB-mediated ADCC mainly depends on the BT461-DB-EGFR antibody arm.

[0664] Example 11 BT461-DB Antibody Inhibits Tumor Cell Proliferation in Vitro

[0665] In this example, non-small cell lung cancer EGFR mutant cells HCC827 (Punosai #CL-0094, EGFR exon 19 deletion) were selected, and HGF was overexpressed in HCC827 cells (lentiviral transfection, which can be autocrine after expression). The effects of the antibodies of the present invention on the proliferation of these cell lines were detected using a kit (Novozymes, DD1102).

[0666] Tumor cells growing in the logarithmic phase were obtained, digested, and seeded into 96-well plates (2000 cells / well) and cultured overnight in complete culture medium. After removing the culture medium from the 96-well plates, 100 μL of anti-c-Met / EGFR antibodies BT461-DB, BT461-KIH, and control IgG1 (initial concentration was 500 μg / mL) diluted with complete cell culture medium were added and diluted into 5 gradients with a 2-fold concentration using a gradient dilution method. After culturing in an incubator at a concentration of 5% CO2 and a temperature of 37°C for 5 days, an equal volume of 1% 1% 1% 2% 2% 3% 4% 5% 6% 7% 8% 3% 4% 2% 5% 6% 7% 8% 2% 3% 4% 2% 5% 6% 7% 8% 8% 9% 10 ... Reagent (Vazyme, Novozymes, Cat. No. DD1102) was added and shaken for 5 min until the cell clusters were completely lysed. After incubation for 25 min, 100 μL of the mixture was transferred to an opaque plate with a white bottom and detected using a TR-FRET microplate reader (PerkinElmer, model, cat. no.).

[0667] The results are shown in FIG10A . The antibody BT461-DB of the present invention can significantly inhibit the proliferation of tumor cells HCC827-HGF (expressing HGF) at multiple gradient concentrations. The inhibitory level on HCC827-HGF cell proliferation is comparable to that of BT461-KIH.

[0668] Example 12 The in vitro efficacy of BT461-DB bispecific antibody has a synergistic effect

[0669] The experimental method was the same as in Example 11. HCC827-HGF tumor cells growing in the logarithmic phase were digested and seeded into 96-well plates (2000 cells / well) and cultured overnight in complete culture medium. After removing the culture medium from the 96-well plate, 100 μL of antibodies BT461-DB, BT461-DB-EGFR, BT461-DB-c-Met, BT461-DB-EGFR, and BT461-DB-c-Met diluted in complete cell culture medium were added at equal doses. The initial concentration was 500 μg / mL and the cells were diluted by a gradient dilution method with a 2-fold concentration to form 5 gradients. After culturing in an incubator at a concentration of 5% CO2 and a temperature of 37°C for 5 days, an equal volume of BT461-DB was added to each well. Reagent (Vazyme, Novozymes, Cat. No. DD1102) was added and shaken for 5 min until the cell clusters were completely lysed. After incubation for 25 min, 100 μL of the mixture was transferred to an opaque plate with a white bottom and detected using a TR-FRET microplate reader (PerkinElmer, model, cat. no.).

[0670] The results are shown in FIG10B . The efficacy of the antibody 461-DB of the present invention is greater than that of the combination antibody 461-DB and greater than that of 461-DB-EGFR and 461-DB-c-Met, indicating that the anti-EGFR / c-Met dual-target antibody has a synergistic effect in inhibiting tumor cell proliferation in vitro.

[0671] Example 13 BT461-DB Antibody Inhibits Osimertinib-Resistant Cell Proliferation in Vitro

[0672] 13.1 Construction of the HCC827-HGF-OSI osimertinib-resistant cell line

[0673] In this example, the HCC827-HGF-OSI osimertinib-resistant cell line was obtained by gradient administration.

[0674] HCC827-HGF cells were revived and cultured in 1640+10% FBS+1% double-antibody medium, and stably passaged for 2-3 generations to stabilize the cell properties.

[0675] Osimertinib induction: First, osimertinib at a concentration of 100 nM was administered for 2 months; then, osimertinib at a concentration of 200 nM was administered for 1 month and cell proliferation was detected; then, osimertinib at a concentration of 400 nM was administered for 1 month and cell proliferation was detected; finally, osimertinib at a concentration of 600 nM was administered for 1 month and cell proliferation was detected;

[0676] TKI resistance experiment:

[0677] (1) HCC827-HGF cell lines and non-drug-resistant HCC827-HGF cells in the logarithmic growth phase were obtained, digested, and resuspended in complete culture medium. The cell concentration was adjusted to 4E4 / mL. 50 μL of the cell suspension was added to each well of a 96-well plate (3599, corning), and the cells and osimertinib were co-incubated in an incubator for 3 days.

[0678] (2) After 3 days, the cells were removed from the incubator and allowed to equilibrate to room temperature;

[0679] (3) Thaw the detection solution from -20°C in advance, equilibrate to room temperature, add 100 μL of detection reagent to each well, mix on an oscillator at 300 rpm / min for 5 minutes, and then let it stand at room temperature for 25 minutes;

[0680] (4) Detection: Use a dispenser to pipette and mix the liquid in each well. Take out 100 μL from each well and transfer it to a new OptiPlate standard non-transparent microplate. Select CTG mode for reading.

[0681] The results are shown in FIG11 , showing that HCC827-HGF-OSI acquires resistance to osimertinib at a concentration below 1 μM.

[0682] 13.2 In vitro efficacy of BT461-DB against osimertinib-resistant HCC827-HGF-OSI cell line

[0683] The experimental method is similar to that of Example 11. Tumor cells growing in the logarithmic phase were digested and seeded into 96-well plates (2000 cells / well) and cultured overnight in complete culture medium. After removing the culture medium from the 96-well plate, 100 μL of anti-c-Met / EGFR antibodies BT461-DB, BT461-KIH, and control IgG1 (initial concentration of 500 μg / mL) diluted with complete cell culture medium were added and diluted into 5 gradients of 2-fold concentration using a gradient dilution method. After culturing in an incubator at a concentration of 5% CO2 and a temperature of 37°C for 5 days, an equal volume of 1% ... Reagent (Vazyme, Novozymes, Cat. No. DD1102) was added and shaken for 5 min until the cell clusters were completely lysed. After incubation for 25 min, 100 μL of the mixture was transferred to an opaque plate with a white bottom and detected using a TR-FRET microplate reader (PerkinElmer, model, cat. no.).

[0684] The results are shown in Figure 12A. The antibody BT461-DB of the present invention can significantly inhibit the proliferation of tumor cells HCC827-HGF-osimertinib-resistant cells at multiple gradient concentrations. The inhibitory level on HCC827-HGF cell proliferation is comparable to that of BT461-KIH and Avanta.

[0685] 13.3 Evaluation of the Synergistic Effect of BT461-DB on the Proliferation Inhibition of Osimertinib-Resistant Cells

[0686] The experimental method was the same as in Example 12. HCC827-HGF-OSI tumor cells growing in the logarithmic phase were digested and seeded into 96-well plates (2000 cells / well) and cultured overnight in complete culture medium. After removing the culture medium from the 96-well plate, 100 μL of antibodies BT461-DB, BT461-DB-EGFR, BT461-DB-c-Met, BT461-DB-EGFR, and BT461-DB-c-Met diluted in complete cell culture medium were added at equal doses. The initial concentration was 500 μg / mL and the cells were diluted by a gradient dilution method with a 2-fold concentration to form 5 gradients. After culturing in an incubator at a concentration of 5% CO2 and a temperature of 37°C for 5 days, an equal volume of BT461-DB was added to each well. Reagent (Vazyme, Novozymes, Cat. No. DD1102) was added and shaken for 5 min until the cell clusters were completely lysed. After incubation for 25 min, 100 μL of the mixture was transferred to an opaque plate with a white bottom and detected using a TR-FRET microplate reader (PerkinElmer, model, cat. no.).

[0687] The results are shown in Figure 12B. The 461-EGFR monoclonal antibody has basically no effect on osimertinib-resistant cells, while the efficacy of the antibody 461-DB of the present invention is >461-DB-c-Met>combination>461-DB-EGFR, indicating that the anti-EGFR / c-Met dual-target antibody has a synergistic effect in inhibiting the proliferation of osimertinib-resistant tumor cells in vitro.

[0688] Example 14 In vivo study of the efficacy of bispecific antibodies

[0689] The in vivo anti-tumor activity of the anti-bispecific antibody was evaluated in a mouse xenograft model of human non-small cell lung cancer HCC827-HGF cells (NSG, Vital River, 6 weeks old, female).

[0690] Human HCC827-HGF cells were propagated as monolayer cultures in vitro using RPM1640 + 10% FBS as complete culture medium. Cells in the logarithmic growth phase were digested and resuspended in serum-free culture medium / Matrigel (1:1) to obtain a suspension. Cells were plated at 5×10 6 The mice were inoculated subcutaneously (sc) on the right side of the back to form tumors. Ten days after implantation, the mice were divided into five groups according to the tumor volume. The average tumor volume in each group was 80-100 mm 3 Dosing was started around 14:00 AM. IgG1 or antibody molecules were administered intravenously (IV) once a week for three weeks.

[0691] The experimental results are shown in Figure 13. In the HCC827-HGF tumor model, the tumor inhibitory efficacy of BT461-DB and BT461-KIH is positively correlated with dose. At a dose of 2 mg / kg, the TGI (tumor inhibition ratio) of BT461-DB and BT461-KIH are 67.7% and 64.2%, respectively; at a dose of 5 mg / kg, the TGI (tumor inhibition ratio) of BT461-DB and BT461-KIH are 122.9% and 120.4%, respectively.

Claims

1. A bispecific antibody comprising an antigen binding arm A that can specifically bind to human epidermal growth factor receptor (EGFR) and antagonize the binding of epidermal growth factor (EGF) to EGFR, and an antigen binding arm B that can specifically bind to hepatocyte growth factor receptor (c-Met) and antagonize the binding of hepatocyte growth factor (HGF) to c-Met, wherein: The binding arm A comprises light chain A (LCA) and heavy chain A (HCA), and the binding arm B comprises light chain B (LCB) and heavy chain B (HCB), wherein the LCA, HCA, LCB and HCB comprise the amino acid sequences shown in SEQ ID NO: 1, 2, 3 and 4, respectively.

2. The bispecific antibody according to claim 1, wherein the bispecific antibody has one or more of the following functions: (1) It can inhibit the autophosphorylation of EGFR mediated by the ligand EGF and the autophosphorylation of c-Met mediated by the ligand HGF; (2) capable of inhibiting the growth and proliferation of EGFR and / or c-Met double-positive human tumor cells, wherein: The effect of inhibiting the proliferation of EGFR and c-Met double positive tumor cells is stronger than the combination of EGFR monoclonal antibody and c-Met monoclonal antibody; (3) Ability to mediate antibody-dependent cellular cytotoxicity (ADCC); (4) promoting the internalization of the target protein, and the internalization level mediated by the bispecific antibody is higher than the internalization level mediated by the c-Met-specific binding arm B or the EGFR-specific binding arm A; (5) It can inhibit the proliferation of osimertinib-acquired resistance cells.

3. The bispecific antibody according to claim 1 or 2, wherein: The affinity of the bispecific antibody to c-Met is significantly higher than that to EGFR.

4. The bispecific antibody according to claims 1 to 3, wherein: The binding arm A comprises a light chain variable region A (VLA) and a heavy chain variable region A (VHA), and the binding arm B comprises a light chain variable region B (VLB) and a heavy chain variable region B (VHB), wherein the VHA and VLA of the binding arm A are derived from an IgG polypeptide A (IgG(A)) having a homodimeric structure comprising VHA and VLA, and the VHB and VLB of the binding arm B are derived from an IgG polypeptide B (IgG(B)) having a homodimeric structure comprising VHB and VLB; wherein the VHA comprises VHA CDR1, VHA CDR2 and VHA CDR3 having amino acid sequences as shown in SEQ ID NOs: 19, 20 and 21, respectively, the VLA comprises VLA CDR1, VLA CDR2 and VLA CDR3 having amino acid sequences as shown in SEQ ID NOs: 22, 23 and 24, respectively, the VHB comprises VHB CDR1, VHB CDR2 and VHB CDR3 having amino acid sequences as shown in SEQ ID NOs: 25, 26 and 27, respectively, and the VLB comprises VHB CDR1, VHB CDR2 and VHB CDR3 having amino acid sequences as shown in SEQ ID NOs: 28, 29 and 30, respectively. VLB CDR1, VLB CDR2 and VLB CDR3 of the amino acid sequences shown in NOs:28, 29 and 30.

5. The bispecific antibody according to claim 4, wherein The VHA, VLA, VHB and VLB comprise the amino acid sequences shown in SEQ ID NOs: 5, 6, 7 and 8, respectively.

6. The bispecific antibody according to claims 1 to 5, wherein: The binding arm A further comprises a heavy chain constant region A (CHA) and a light chain constant region A (CLA), and the binding arm B further comprises a heavy chain constant region B (CHB) and a light chain constant region B (CLB), wherein the CHA and the CHB are both IgG1 isotype heavy chain constant regions.

7. The bispecific antibody according to claims 1 to 6, wherein: The antigen CHA contains an Fc(A) region, and the Fc(A) region contains a CH3(A) region; the CHB contains an Fc(B) region, and the Fc(B) region contains a CH3(B) region, and the CH3(A) region and the CH3(B) region can form a heterodimer through interaction, and the heterodimer interaction is stronger than the homodimer interaction between the CH3(A) region and the CH3(B) region, or optionally, the CH3(A) of the antigen binding arm A and the CH3(B) of the antigen binding arm B both contain amino acid mutations that enhance the formation of heterodimers; Or optionally, wherein the CH3(A) comprises an amino acid mutation as shown in F405L, and the CH3(B) comprises an amino acid mutation as shown in K409R; or further optionally, both the Fc(A) and the Fc(B) comprise a combination of amino acid mutations that reduce immunogenicity.

8. The bispecific antibody according to claims 1 to 7, wherein: The Fc(A) comprises the amino acid sequence shown in SEQ ID NO:9, and the Fc(B) comprises the amino acid sequence shown in SEQ ID NO:

10.

9. The bispecific antibody according to claims 1 to 8, wherein: The CHA, CLA, CHB and CLB comprise the amino acid sequences shown in SEQ ID NOs: 11, 12, 13 and 14, respectively.

10. A composition encoding nucleic acids, comprising polynucleotide sequences as shown in SEQ ID NOs: 15, 16, 17 and 18, which can encode HCA, HCB, LCA and LCB of the bispecific antibody of claim 1, respectively. 11 . A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier or diluent.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition further comprises a second therapeutic agent or treatment regimen, the second therapeutic agent or treatment regimen comprising a chemotherapeutic drug, a DNA alkylating agent, an immunomodulator, a proteasome inhibitor, a histone deacetylase inhibitor, radiation therapy, stem cell transplantation, different bispecific antibodies that interact with different tumor cell surface antigens and T cell or immune cell antigens, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a PD-1, PD-L1 or CTLA-4 checkpoint inhibitor, or a combination thereof.

13. Use of the bispecific antibody of claims 1 to 9 or the pharmaceutical composition of claim 11 or 12 in the preparation of a therapeutic drug for treating a disease or tumor involving abnormal EGFR and c-Met cells and a therapeutic drug for inhibiting the growth or metastasis of tumors or cancer cells expressing EGFR and / or c-Met in a patient; in, The EGFR and c-Met abnormal cells include EGFR activating mutations, EGFR gene amplification, up-regulated HGF expression levels, c-Met activating mutations, c-Met gene amplification or mutant KRAS-related.

14. Use of the bispecific antibody according to claims 1 to 9 or the pharmaceutical composition according to claim 11 or 12 in the preparation of a medicament for treating osimertinib-resistant diseases.

15. The use according to claim 13 or 14, wherein: The tumor or cancer is one of epithelial cell cancer, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, or sporadic or hereditary papillary renal cell carcinoma; or optionally, the cancer is one of non-small cell lung cancer, pancreatic cancer, gastric cancer, colon cancer, liver cancer.

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