EGFR / c-Met bispecific antibody and application thereof

Abstract

The present invention discloses bispecific antibodies directed against EGFR and MET. The amino acid sequence of the Fc segment of the binding arm is modified, so that a KIH structure is formed between the EGFR binding arm and the cMet binding arm of the bispecific antibody, and a heterodimer is assembled; the ADCC activity of the bispecific antibody is enhanced, or the inhibition intensity of ligand-mediated antigen phosphorylation is enhanced. The bispecific antibody prepared by the invention is simple to prepare and stable in structure, and is expected to better meet clinical treatment requirements based on better tumor cell selectivity, so that the bispecific antibody has a good market prospect.

Description

[0001] This application claims priority to patent application No. 202310966671.2 filed in China on August 3, 2023. The entire contents of this prior patent application are incorporated herein by reference for all purposes.

[0002] Reference sequence list

[0003] This application incorporates by reference the sequence listing submitted with this application as a text file entitled 'EGFCM-100 Sequence Listing', created on August 3, 2023. Technical Field

[0004] The present disclosure relates to an antibody-drug conjugate containing an anti-EGFR / c-Met antibody or an antigen-binding fragment thereof, and use of the antibody-drug conjugate as an anti-cancer drug. Background Art

[0005] In recent years, bispecific antibodies targeting EGFR and cMet have become an important solution to EGFR-TKI acquired resistance and have the potential to be used in the first-line treatment of 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 corresponding to the bispecific antibodies targeting EGFR and cMet can also interact with each other to produce a synergistic effect, which can not only improve the anti-tumor effect, but also reduce the amount of antibodies used, thereby reducing the risk of adverse reactions.

[0006] Currently, the first approved drug targeting EGFR / cMET 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 CHRYSALISI phase 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, Invitrogen and Hansoh Pharmaceuticals are also deploying 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 or needs to be improved.

[0007] In order to meet the clinical needs of TKI-resistant cancer treatment while taking into account clinical efficacy and safety, more EGFR / cMET dual-target antibody products still need to be developed in the field of cancer treatment. Summary of the invention

[0008] In view of the shortcomings of the prior art described above, the present disclosure provides an EGFR / c-Met bispecific antibody molecule having the desired combination advantages of EGFR and c-Met dual-target selection specificity, anti-tumor efficacy and safety prospects, and an EGFR / c-Met bispecific antibody-drug conjugate (EGFR / c-Met bispecific ADC) formed by coupling the EGFR / c-Met bispecific antibody molecule with a drug-active molecule. 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 meet the needs of tumor selectivity and further improve efficacy while further taking into account safety. It is expected to become a new generation of therapeutic products that make up for the problem that the safety of EGFR and c-Met specific dual-targeted tumor treatment products on the market is not ideal.

[0009] EGFR and c-Met are co-expressed in many cancer types, and antibody molecules targeting these two molecules ("bispecific antibody molecules") offer the prospect of obtaining broad clinical benefits across multiple cancer indications. Compared with the monospecific EGFR or c-Met antibodies of the prior art, the EGFR / c-Met bispecific antibody molecules of the present invention provide improved treatment for cancer.

[0010] In the first aspect, the present invention provides a bispecific antibody, which comprises an antigen binding arm A that can specifically bind to the extracellular part of 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 the extracellular part of 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 light chain A (LCA) and heavy chain A (HCA), and the binding arm B comprises light chain B (LCB) and heavy chain B (HCB).

[0011] 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).

[0012] Wherein, the HCA comprises a heavy chain variable region A (VHA) and a heavy chain A third constant region (HACH3A), and the HCB comprises a heavy chain variable region B (VHB) and a heavy chain B third constant region (HBCH3B);

[0013] The LCA comprises a light chain variable region A (VLA), and the LCB comprises a light chain variable region B (VLB); wherein the VLA and VHA pair to form a binding site that specifically binds to EGFR, and the VLB and VHB pair to form a binding site that specifically binds to c-Met.

[0014] In some embodiments, the HCA comprises at least one amino acid substitution in CH3A of the HCA, the HCB comprises at least one amino acid substitution in CH3B of the HCB, and the substitution in HCA CH3 and the substitution in HCB CH3 occur at different amino acid residue positions when the residues are numbered according to the EU index.

[0015] In some embodiments, the LCA comprises the amino acid sequence shown in SEQ ID NO:1.

[0016] In some embodiments, the HCA comprises the amino acid sequence shown in SEQ ID NO:2.

[0017] In some embodiments, the LCB comprises the amino acid sequence shown in SEQ ID NO:12.

[0018] In some embodiments, the HCB comprises the amino acid sequence shown in SEQ ID NO:13.

[0019] In some embodiments, the LCA, HCA, LCB and HCB comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 12 and 13, respectively.

[0020] In some embodiments, the binding arm A is a fusion peptide having a LCA-linker-HCA structure, and the binding arm B is a fusion peptide having a LCB-linker-HCB structure.

[0021] In some embodiments, the linker is a short peptide structure represented by (GGGS)n.

[0022] In some embodiments, n is any integer between 4-10.

[0023] “-” is a peptide bond.

[0024] In some embodiments, the LCA comprises a light chain variable region A (VLA) and the HCA comprises a heavy chain variable region A (VHA).

[0025] In some embodiments, the VLA comprises the following light chain complementarity determining regions (VLA CDRs):

[0026] LCDR1 having the amino acid sequence of SEQ ID NO:23, LCDR having the amino acid sequence of SEQ ID NO:24, and LCDR3 having the amino acid sequence of SEQ ID NO:25.

[0027] In some embodiments, the VHA comprises the following heavy chain complementarity determining regions (VHA CDRs):

[0028] A HCDR1 having the amino acid sequence of SEQ ID NO:26, a HCDR2 having the amino acid sequence of SEQ ID NO:27, and a HCDR3 having the amino acid sequence of SEQ ID NO:28.

[0029] In some embodiments, the VLA comprises the amino acid sequence shown in SEQ ID NO:5, and the VHA comprises the amino acid sequence shown in SEQ ID NO:7.

[0030] In some embodiments, the LCB comprises a light chain variable region B (VLB), the HCB comprises a heavy chain variable region B (VHB),

[0031] In some embodiments, the antigen binding arm B comprises a light chain B (LCB) and a heavy chain B (HCB), wherein:

[0032] The LCB comprises a light chain variable region B (VLB), and the VLB comprises the following light chain complementarity determining regions (LBCDRs):

[0033] LBCDR1 having the amino acid sequence shown in SEQ ID NO:29, LBCDR2 having the amino acid sequence shown in SEQ ID NO:30, and LBCDR3 having the amino acid sequence shown in SEQ ID NO:31;

[0034] Wherein the HCB comprises a heavy chain variable region B (VHB), and the VHB comprises the following heavy chain complementarity determining regions (HBCDRs):

[0035] HBCDR1 having the amino acid sequence shown in SEQ ID NO:32, HBCDR2 having the amino acid sequence shown in SEQ ID NO:33, and HBCDR3 having the amino acid sequence shown in SEQ ID NO:34.

[0036] In some embodiments, the VLB comprises the amino acid sequence shown in SEQ ID NO:16, and the VHB comprises the amino acid sequence shown in SEQ ID NO:18.

[0037] In a second aspect, the present invention provides a bispecific antibody, which comprises a binding arm A that specifically targets EGFR and a binding arm B that specifically targets C-Met, wherein the antigen binding arm A comprises a light chain variable region A (VLA), a light chain constant region A (CLA), a linker, a heavy chain variable region (VHA) and a heavy chain constant region (CHA) connected from the N-terminus to the C-terminus to form a VLA-CLA-linker-VHA-CHA structure, and the antigen binding arm B comprises a light chain variable region B (VLA), a light chain constant region B (CLB), a linker, a heavy chain variable region (VHB) and a heavy chain constant region (CHB) connected from the N-terminus to the C-terminus to form a VLB-CLB-Linker-VHA-CHB structure; wherein the CHA and the CHB interact to form a KIH structure, and the bispecific antibody is a heterodimer formed by the binding arm A and the binding arm B through the KIH structure.

[0038] In one embodiment, one of the CHA and the CHB comprises an amino acid mutation that forms a hole structure, and the other comprises an amino acid mutation that forms a knob structure. In one embodiment, the CHA comprises an amino acid mutation that forms a knob structure, and the CHB comprises an amino acid mutation that forms a hole structure.

[0039] In a third aspect, the present invention provides a bispecific antibody, comprising an antigen binding arm A specific for human epidermal growth factor receptor (EGFR) and an antigen binding arm B specific for human proto-oncogene receptor tyrosine kinase (c-Met), wherein the antigen binding arm A is a single-chain fusion peptide of Fc (A) composed of ScFab (A) and CH2ACH3A from the N-terminus to the C-terminus, and the antigen binding arm B is a single-chain fusion peptide of Fc (B) composed of ScFab (B) and CH2BCH3B from the N-terminus to the C-terminus, wherein the Fc (A) comprises an amino acid mutation combination forming a Knob structure, and the Fc (B) comprises an amino acid mutation combination forming a hole structure; wherein the Fc (A) and the Fc (B) interact to form a KIH structure, and the binding arm A and the binding arm B form a heterodimer through the KIH structure.

[0040] In a fourth aspect, the present invention provides an antibody-drug conjugate comprising the EGFR / c-Met bispecific antibody molecule described in the preceding aspects.

[0041] In particular, drug conjugates containing the antibody molecule are able to selectively deliver drugs (also called "payloads" or "warheads") to tumors that co-express these targets and treat these tumors with high efficacy. The examples demonstrate that different antibody drug conjugates containing alternative warheads exhibit potent cytotoxic activity in both in vitro cytotoxicity assays and in vivo models of cancer treatment.

[0042] In some embodiments, the drug is a topoisomerase I inhibitor.

[0043] For example, Exatecan, a topoisomerase I inhibitor having the formula (I):

[0044]

[0045] For example, a topoisomerase I inhibitor having the formula (II):

[0046]

[0047] In some embodiments, the drug is an auristatin analog of formula (III), such as methyl auristatin E (MMAE) of formula (III).

[0048]

[0049] In some embodiments, the antibody molecule of the present invention comprises an antigen binding arm A that binds to the human EGFR receptor with a relatively "low affinity" and an antigen binding arm B that binds to the human c-Met receptor with a relatively high affinity. "Low affinity" in the present invention refers to an antigen binding arm A that binds to the human EGFR with a dissociation constant (Kd) equal to or higher than 10 nM. In this article, "higher affinity" or "high affinity" refers to an antigen binding arm A that binds to the human EGFR with a Kd lower than 10 nM.

[0050] As shown in the present invention, the EGR / c-Met bispecific antibodies and bispecific ADCs disclosed herein exhibit reduced on-target toxicity, such as skin toxicity, in normal tissues, and thus exhibit an improved and superior safety profile compared to antibody-drug conjugates comprising the following EGFR antigen-binding arm A: binds to human EGFR with "lower affinity".

[0051] Furthermore, the present disclosure provides a pharmaceutical composition comprising an antibody molecule as defined herein or an antibody-drug conjugate as defined herein.

[0052] The present disclosure provides bispecific antibody molecules, antibody-drug conjugates and pharmaceutical compositions all as defined herein for use in methods of treating the human or animal body, such as methods of treating cancer.

[0053] The present disclosure provides a method for treating cancer, comprising administering an antibody molecule, antibody-drug conjugate or pharmaceutical composition defined in the invention.

[0054] In particular, the present disclosure provides a method of treating a cancer selected from pancreatic cancer, colorectal cancer, non-small cell lung cancer (NSCLC) and squamous head and neck cancer (SQHN), comprising administering an antibody molecule, antibody-drug conjugate or pharmaceutical composition as defined herein.

[0055] In some exemplary embodiments, the cancer to be treated is non-small cell lung cancer (NSCLC).

[0056] The disclosure also provides nucleic acids, vectors and host cells as defined herein.

[0057] Furthermore, the present disclosure provides methods for producing the antibody molecules defined herein.

[0058] The present disclosure includes combinations of described aspects and preferred features unless such combinations are expressly impermissible or explicitly avoided.

[0059] 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 specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they are not listed here.

[0060] The EGFR / c-Met bispecific antibody and ADC disclosed herein have the following functions:

[0061] (1) It can inhibit the autophosphorylation of EGFR mediated by the ligand EGF and the autophosphorylation of C-Met mediated by the ligand HGF;

[0062] (2) It can inhibit the proliferation of EGFR and / or c-Met positive human tumor cells and has a synergistic effect, 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;

[0063] (3) Ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent macrophage phagocytosis (ADCP);

[0064] (4) capable of mediating the internalization and degradation of EGFR and c-MET proteins, wherein the mediated dual-target internalization has a synergistic effect;

[0065] (5) Its binding affinity to c-MET is significantly higher than its binding affinity to EGFR.

[0066] The positive and progressive effects of the anti-EGFR / c-Met bispecific antibody of the present invention are: (1) the killing effect on tumor cells is better than the combination of anti-EGFR monoclonal antibody and anti-c-Met monoclonal antibody; (2) the selection specificity for EGFR and c-Met double-positive tumor cells is high, providing a promising option for the tumor treatment market. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Examples and experiments illustrating the principles of the present disclosure may be discussed with reference to the accompanying drawings, in which:

[0068] Figure 1 BT461 targets EGFR protein (1A) or c-MET ( Figure 1 B) ELISA binding activity analysis results. The horizontal axis represents the antibody concentration (logarithmic value), and the vertical axis represents the antibody binding signal intensity.

[0069] Figure 2 .FACS-binding activity analysis results of BT461 targeting EGFR and c-MET expressed on the cell surface: (A) binding to CHO-K1-HGFR cells, (B) binding to CHO-K1-EGFR cells, (C) binding to MKN45 cells. The horizontal axis represents the antibody concentration (logarithmic value), and the vertical axis represents the antibody binding signal intensity.

[0070] Figure 3 IC50 blocking activity analysis results of BT461 in blocking the binding of ligand protein EGF to antigen protein EGFR (A) and blocking the binding of ligand protein HGF to antigen protein c-MET (B).

[0071] Figure 4 .Binding curves of BT461 with c-MET and EGFR proteins in cynomolgus monkeys.

[0072] Figure 5 Binding and dissociation curves of BT461 and Evanta antibody:

[0073] (A) Binding and dissociation curves of BT461 and EGFR, (B) Binding and dissociation curves of Avanta and EGFR, (C) Binding and dissociation curves of Avanta and c-Met; (D) Binding and dissociation curves of Avanta and c-Met.

[0074] Figure 6 . Molecular interaction instrument detection of epitope competition analysis of BT461 and Avanta: (A) Competition analysis on the EGFR binding epitope and (B) Competition analysis on the c-MET binding epitope.

[0075] Figure 7 . Determination of the analytical curve of the selective binding level of MKN45&HACAT mixed cells:

[0076] (A) The selective binding curve of Taixinsheng, (B) the selective binding curve of Avantuo, (C) the selective binding curve of BT461.

[0077] Figure 8 .Analytical curves for the selection index evaluation of MKN45&BEAS-2B mixed cells:

[0078] (A) The selective binding curve of Taixinsheng, (B) the selective binding curve of Avantuo, (C) the selective binding curve of BT461.

[0079] Fig. 9 .Analysis results of BT461 inhibiting EGFR autophosphorylation and c-Met autophosphorylation: (A) Western blot analysis results, (B) TR-fret technology to detect the level of c-Met phosphorylation (Y1234 / Y1235), (C) TR-fret technology to detect the level of EGFR phosphorylation (Y1068).

[0080] Fig.10 Results of target protein internalization mediated by BT461 and control antibodies: (A) The Live-Cell Analysis System plots the time gradient of target protein internalization curves mediated by BT461 and control antibodies. (B) Western blot analysis was used to detect the internalization and degradation levels of EGFR and c-MET.

[0081] Fig.11 .In vitro proliferation inhibition curves of tumor cells: (A) In vitro proliferation inhibition of HCC827-HGF (expressing HGF) cells, (B) In vitro proliferation inhibition of H1975-HGF (expressing HGF) cells, (C) In vitro proliferation inhibition of KP4 cells. The horizontal axis represents the antibody concentration, and the vertical axis represents the cell viability.

[0082] Fig.12 . Detection analysis curves for evaluating the synergistic efficacy of BT461 in inhibiting the proliferation of MKN45 (A), HCC827-HGF (B), and NCI-H1975-HGF (C) tumor cells in vitro.

[0083] Fig.13 . Evaluation of antibody-dependent ADCC. (A) Antibody-dependent NK cell ADCC on H1975 cells in vitro; (B) Antibody-dependent NK cell ADCC on HCC827 cells in vitro. The vertical axis is the percentage of cell killing toxicity (%), which is calculated by (experimental group TRF value-spontaneous pore TRF value) / (maximum release pore TRF value-spontaneous pore TRF value)*100.

[0084] Fig.14.BT461 antibody mediates macrophage phagocytosis (ADCP) of HCC827 cells.

[0085] Fig.15 . BT461 antibody inhibits the growth of HCC827-HGF (overexpressing HGF) xenograft tumors. (A) HCC827-HGF tumor volume change curve over time; (B) mouse body weight change curve.

[0086] Fig.16 . Inhibition of KP4 tumor cell xenograft tumor growth. (A) KP4 tumor volume change curve over time; (B) mouse body weight change curve.

[0087] Fig.17 .FACS-binding activity analysis curve of BT461-ADC molecule binding to MKN45.

[0088] Fig.18 .The in vitro killing efficacy of BT461-ADC molecules on various tumor cells. (A) MiaPACA2 cells, (B) MKN15 cells, (C) H1975 cells, (D) HCC827 cells. The horizontal axis represents the antibody concentration, and the vertical axis represents the cell viability.

[0089] Fig.19 .Bystandering effect of BT461-ADC molecule: (A) In vitro killing curve of EGFR&c-MET negative cells Jurket, (B) Bystandering effect detection results of the mixed system of EGFR&c-MET positive cells (HCC827 or MKN45) and negative cells (JURKET).

[0090] Fig. 20 .The therapeutic efficacy of BT461-MMAE molecule in HCC827-HGF tumor model.

[0091] Fig.21 .The anti-tumor efficacy of the dual antibodies BT461-DXD and BT461-Ixitectin in the HCC827-HGF tumor model. DETAILED DESCRIPTION

[0092] 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, has a strong selective specificity for binding to double-positive tumor cells, has a synergistic effect in the mediated internalization of the dual targets of EGFR and c-Met, and has a stronger degree of inhibition of cell proliferation than the combination of EGFR monoclonal antibody and c-Met monoclonal antibody, thus having a synergistic effect.

[0093] Unless otherwise defined, scientific and technical terms related to the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature and techniques used in connection with 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, lipofection). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly accomplished in the art or as described herein. The aforementioned 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. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, as well as 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.

[0094] Below, aspects and examples of the present disclosure will be discussed 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.

[0095] Epidermal Growth Factor Receptor (EGFR)

[0096] Human EGFR (also known as proto-oncogene c-ErbB-1) is a transmembrane protein and a receptor for members of the epidermal growth factor family (EGF family). Binding of EGFR to its ligand induces receptor dimerization and autophosphorylation of several tyrosine residues (Y992, Y1045, Y1068, Y1148, and Y1173) in the C-terminal regulatory region of EGFR.

[0097] Aberrant EGFR expression activity is associated with many diseases, including neurological diseases and many cancers.

[0098] In the present specification, "EGFR" refers to EGFR from any species and includes EGFR isoforms, fragments, variants or homologs from any species.

[0099] Tyrosine protein kinase (MET)

[0100] Human c-Met, also known as hepatocyte growth factor receptor (c-MET). Binding to its ligand HGF induces autophosphorylation of several tyrosine residues (Y1234 / Y1235) on the c-Met intracellular domain, providing docking sites for downstream signaling molecules and activating multiple signaling cascades. Overexpression of c-Met has been observed in many human tumors, which 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. In these cancers, co-expression of EGFR and c-Met is often observed.

[0101] Composition of matter

[0102] The present invention provides bispecific antibodies specifically binding to EGFR and c-Met and ADCs thereof, polynucleotides encoding the bispecific agents of the present invention or complementary nucleic acids thereof, vectors, host cells, and methods for preparing and using the same.

[0103] "Antigen binding arm" describes the portion of the molecule in a bispecific antibody that binds to all or part of the corresponding antigen.

[0104] 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, and its affinity for c-Met is significantly higher than its affinity for EGFR, with the difference being greater than or equal to 20 times.

[0105] 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 Table 1 and Table 2, and 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 Table 3 and Table 4.

[0106] Table 1 Exemplary variable region amino acid sequences of binding arm A of the EGFR / c-Met bispecific antibody of the present invention

[0107]

[0108] Table 2 Exemplary CDR sequences of binding arm A

[0109]

[0110]

[0111] Table 3 Sequences of VLB and VHB parent antibodies binding arm B

[0112]

[0113] Table 4 Exemplary CDR sequences included in binding arm B

[0114] Combination arm B VLBCDR1 SVSSSVSSIYLH (SEQ ID NO: 40) VLBCDR2 STSNLAS (SEQ ID NO:41) VLBCDR3 QVYSGYPLT (SEQ ID NO:42) VHBCDR1 DYYMH (SEQ ID NO:43) VHBCDR2 RVNPNRRGTTYNQKFEG(SEQ ID NO:44) VHBCDR3 ANWLDY (SEQ ID NO:45)

[0115] EGFR-specific binding arm A

[0116] In some embodiments, the binding arm A of the bispecific antibody of the present invention that specifically binds to EGFR comprises a light chain A (LCA), a linker, and a heavy chain A (HCA).

[0117] In some embodiments, the LCA of the binding arm A preferably comprises an amino acid sequence derived from SEQ ID NO: 1. In some embodiments, the LCA of the binding arm A preferably comprises an amino acid sequence preferably shown in SEQ ID NO: 12.

[0118] In some embodiments, the LCA of the binding arm A preferably comprises an amino acid sequence preferably as shown in SEQ ID NO:2.

[0119] In some embodiments, the HCA of the binding arm A preferably comprises an amino acid sequence preferably shown in SEQ ID NO:13.

[0120] In some preferred embodiments, the binding arm A comprises light chain A (LCA) from N-terminus to C-terminus, linker, and heavy chain A. In a preferred embodiment, the binding arm A is a LCA-linker-HCA structure, wherein "-" is a peptide bond.

[0121] In some embodiments, the binding arm A comprises ScFab(A) and Fc(A), and a linker connecting the C-terminus of the ScFab(A) and the N-terminus of the Fc(A).

[0122] In some embodiments, the binding arm of the bispecific antibody that specifically binds to the extracellular portion of EGFR is a fusion peptide comprising ScFab (A) and an Fc (A) segment comprising a CH2A segment domain and a CH3A segment domain from the N-terminus to the C-terminus, and the ScFab (A) is connected to the N-terminus of the CH2A segment domain via a hinge region A structure.

[0123] In some embodiments, in the binding arm A, the ScFab(A) and Fc(A), and a linker connecting the C-terminus of the ScFab(A) and the N-terminus of the Fc(A) form a ScFab(A)-linker-Fc(A) structure.

[0124] In some embodiments, the ScFab (A) has an amino acid sequence selected from SEQ ID NO:3.

[0125] In some embodiments, the ScFab (A) has an amino acid sequence selected from SEQ ID NO:14.

[0126] In some embodiments, the amino acid sequence of Fc(A) is the sequence formed by the CH2 plus CH3 domains in the heavy chain A sequence of the amino acid sequence shown in SEQ ID NO:2.

[0127] In some embodiments, the amino acid sequence of Fc(A) is the amino acid sequence shown in SEQ ID NO:11.

[0128] In some embodiments, the linker has an amino acid sequence selected from SEQ ID NO:9.

[0129] In some embodiments, the binding arm A comprises ScFab (A), hinge region A and Fc (A) as shown in SEQ ID NO: 3, 10 and 11, respectively.

[0130] In some embodiments, the ScFab (A) comprises from N-terminus to C-terminus a light chain variable region A (VLA), a light chain constant region A (CLA), a linker, a heavy chain variable region A (VHA) and a first heavy chain constant region A (CH1A).

[0131] In some embodiments, in the binding arm A of the bispecific antibody of the present invention, the LCA comprises a light chain variable region (VLA) and a heavy chain variable region (VHA).

[0132] In some embodiments, the protected VLA and the VHA comprise VHCDRs and VLCDRs as shown in Table 1, respectively.

[0133] In some embodiments, the binding arm A that specifically binds to EGFR has a VLACDR1 with an amino acid sequence as shown in SEQ ID NO:23.

[0134] In some embodiments, the binding arm A that specifically binds to EGFR has a VLACDR2 with an amino acid sequence as shown in SEQ ID NO: 24. In one embodiment, the binding arm A that specifically binds to EGFR has a VLACDR3 with an amino acid sequence as shown in SEQ ID NO: 25.

[0135] In some embodiments, the binding arm A that specifically binds to EGFR has a VLBCDR1 with an amino acid sequence as shown in SEQ ID NO:26.

[0136] In some embodiments, the binding arm A that specifically binds to EGFR has a VLBCDR2 with an amino acid sequence as shown in SEQ ID NO:27.

[0137] In some embodiments, the binding arm A that specifically binds to EGFR has a VLBCDR3 with an amino acid sequence as shown in SEQ ID NO:28.

[0138] 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.

[0139] In some embodiments, the light chain variable region A (VLA) preferably comprises the VLA CDR1, VLA CDR2 and VL CDR3 sequences shown in SEQ ID NOs: 23, 24 and 25, respectively, and the heavy chain variable region A (VHA) preferably comprises the VHA CDR1, VHA CDR2 and VHA CDR3 sequences shown in SEQ ID NOs: 26, 27, 28, respectively.

[0140] In some embodiments, the light chain variable region A (VLA) comprises the amino acid sequence shown in SEQ ID NO:5.

[0141] In some embodiments, the heavy chain variable region A (VHA) comprises the amino acid sequence shown in SEQ ID NO:7.

[0142] In some embodiments, the light chain constant region A (CLA) comprises the amino acid sequence shown in SEQ ID NO:6.

[0143] In some embodiments, the heavy chain constant region A (CHA) comprises the amino acid sequence shown in SEQ ID NO:8.

[0144] In some embodiments, the ScFab of the EGFR-specific binding arm A comprises, from N-terminus to C-terminus, a light chain variable region A (VLA), a light chain constant region A (CLA), a linker, a heavy chain variable region A (VHA) and a first heavy chain constant region A (CH1A) with amino acid sequences as shown in SEQ ID NO: 5, 6, 7, 9 and 40, respectively.

[0145] c-Met-specific binding arm B

[0146] The binding arm B of the bispecific antibody of the present invention binds to C-Met with high affinity and inhibits C-Met signaling, and can provide beneficial effects in terms of specificity and reduced C-Met receptor phosphorylation levels compared to small molecule C-Met inhibitors and 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.

[0147] In some preferred embodiments, the binding arm B comprises light chain B (LCB), linker, and heavy chain B from N-terminus to C-terminus.

[0148] In some preferred embodiments, the binding arm B is a LCB-linker-HCB structure, wherein "-" is a peptide bond.

[0149] In some preferred embodiments, the binding arm B comprises light chain B (LCB), linker, and heavy chain B from N-terminus to C-terminus.

[0150] In some preferred embodiments, the binding arm B is a LCB-linker-HCB structure, wherein "-" is a peptide bond.

[0151] In some embodiments, the LCB of the binding arm B preferably comprises the amino acid sequence shown in SEQ ID NO:12.

[0152] In some embodiments, the HCB of the binding arm B preferably comprises the amino acid sequence shown in SEQ ID NO:23.

[0153] In some embodiments, the linker has an amino acid sequence selected from SEQ ID NO:20.

[0154] In some embodiments, the binding arm B comprises ScFab(B) and Fc(B), and a linker connecting the C-terminus of the ScFab(B) and the N-terminus of the Fc(B).

[0155] In some embodiments, the ScFab (B) has the amino acid sequence described in SEQ ID NO: 14;

[0156] In some embodiments, the amino acid sequence of Fc(B) is the sequence formed by the CH2 plus CH3 domains in the heavy chain B sequence of the amino acid sequence shown in SEQ ID NO:13.

[0157] In some embodiments, the amino acid sequence of Fc(B) is the amino acid sequence shown in SEQ ID NO:22.

[0158] In some embodiments, the ScFBb(B) comprises from N-terminus to C-terminus a light chain variable region B (VLB), a light chain constant region B (CLB), a linker, a heavy chain variable region B (VHB) and a first heavy chain constant region B (CH1B).

[0159] In some embodiments, the ScFab (B) has a structure shown in VLB-CLB-linker-VHB-CH1B

[0160] In some embodiments, the binding arm B comprises a ScFab, a hinge region, and an Fc(B) as shown in SEQ ID NOs: 14, 20, and 22, respectively.

[0161] In some embodiments, the binding arm B that specifically binds to c-Met comprises a light chain variable region B (VLB) and a heavy chain variable region (VHB), wherein the VLB comprises: VLB CDR1, VLB CDR2 and VLB CDR3, and the VHB comprises VHB CDR1, VHB CDR2 and VHB CDR3.

[0162] In some embodiments, the binding arm B that specifically binds to c-Met has a VLBCDR1 with an amino acid sequence as shown in SEQ ID NO:29.

[0163] In some embodiments, the binding arm B that specifically binds to c-Met has a VLBCDR2 with the amino acid sequence shown in SEQ ID NO: 30,

[0164] In some embodiments, the binding arm B that specifically binds to c-Met has a VLBCDR3 with an amino acid sequence as shown in SEQ ID NO:31.

[0165] In some embodiments, the binding arm B that specifically binds to c-Met comprises or has VHB CDR1 with the amino acid sequence shown in SEQ ID NO: 32.

[0166] In some embodiments, the binding arm B that specifically binds to c-Met comprises or has a VHB CDR2 with the amino acid sequence shown in SEQ ID NO:33.

[0167] In some embodiments, the binding arm B that specifically binds to c-Met comprises or has a VHB CDR3 with the amino acid sequence shown in SEQ ID NO: 34.

[0168] In some embodiments, the light chain variable region B (VLB) comprises VLB CDR1, VLB CDR2 and VLB CDR3 sequences shown in SEQ ID NOs: 29, 30 and 31, respectively.

[0169] In one embodiment, the heavy chain variable region B (VHB) comprises the VHB CDR1, VHB CDR2 and VHB CDR3 sequences shown in SEQ ID NOs: 32, 33 and 34, respectively.

[0170] In some embodiments, the c-Met binding arm B comprises VLB CDR1, VLB CDR2 and VLB CDR3 of the amino acid sequences shown in SEQ ID NOs: 29, 30 and 31, respectively, and VHB CDR1, VHB CDR2 and VHB CDR3 of the amino acid sequences shown in SEQ ID NOs: 32, 33 and 34, respectively.

[0171] In some embodiments, the binding arm B that specifically binds to c-Met comprises a VLB having an amino acid sequence as shown in SEQ ID NO:16.

[0172] In some embodiments, the binding arm B that specifically binds to c-Met comprises VHB having an amino acid sequence as shown in SEQ ID NO:18.

[0173] In some embodiments, the binding arm B comprises ScFab (B), linker and Fc (B) with amino acid sequences as shown in SEQ ID NO: 14, 20 and 22, respectively.

[0174] In some embodiments, the light chain variable region B (VLB) comprises the amino acid sequence shown in SEQ ID NO:16.

[0175] In some embodiments, the heavy chain variable region B (VHB) comprises the amino acid sequence shown in SEQ ID NO: 18

[0176] In some embodiments, the light chain constant region B (CLB) comprises the amino acid sequence shown in SEQ ID NO:17.

[0177] In some embodiments, the heavy chain constant region B (CHB) comprises the amino acid sequence shown in SEQ ID NO:19.

[0178] 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.

[0179] In some embodiments, the ScFab (B) of the EGFR-specific binding arm A comprises, from N-terminus to C-terminus, a light chain variable region B (VLB), a light chain constant region B (CLB), a linker, a heavy chain variable region B (VHA) and a first heavy chain constant region B (CH1B) with amino acid sequences as shown in SEQ ID NO: 16, 17, 18, 20 and 40, respectively.

[0180] Structure of a bispecific antibody that specifically binds to EGFR and c-Met

[0181] The bispecific antibody of the present invention that specifically binds to EGFR and C-Met can be composed of a binding arm A that specifically binds to EGFR and a binding arm B that specifically binds to C-Met, which form a KIH structure through heavy chain interaction to form a heterodimer, wherein the EGFR binding arm A and the C-Met binding arm B can be regarded as the antigen binding site of the antibody or the VH / VL pair of the antibody or the light chain / heavy chain pair of the antibody or the fusion peptide single chain composed of the ScFab and Fc of the antibody.

[0182] In some embodiments, the bispecific antibody comprises a binding arm A with a structure as shown in LCA-linker-HCA, and a binding arm B with a structure as shown in LCB-linker-HCB, wherein 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: 12 and 13, respectively.

[0183] In some embodiments, the bispecific antibody comprises a binding arm A with a structure as shown in ScFab(A)-hinge region(A)-Fc(A), and a binding arm B with a structure as shown in ScFab(B)-hinge region(B)-Fc(B), wherein the EGFR-specific binding arm A comprises ScFab(A), hinge region(A) and Fc(A) as shown in SEQ ID NOs:3, 10 and 11, respectively, and the c-Met-specific binding arm B comprises ScFab(B), hinge region(B) and Fc(B) as shown in SEQ ID NOs:14, 21 and 22, respectively.

[0184] In some embodiments, the bispecific antibody comprises a binding arm A with a structure as shown in VLA-CLA-linker (A)-VHA-CH1A, and a binding arm B with a structure as shown in VLB-CLB-linker (B)-VHB-CH1B, the EGFR-specific binding arm A's ScFab (A) comprises a light chain variable region A (VLA), a light chain constant region A (CLA), a linker, a heavy chain variable region A (VHA) and a first heavy chain constant region A (CH1A) as shown in SEQ ID NOs: 5, 6, 9, 7 and 40, respectively, and the c-Met-specific binding arm B's ScFab (B) comprises VLB, CLB, linker, VHB, CH1B as shown in SEQ ID NOs: 16, 17, 20, 18 and 40, respectively.

[0185] The term "linker molecule" or "linker" or linker as used in the present invention preferably refers to a peptide linker. The peptide linker utilized in certain embodiments of the present invention is used to connect antibodies, antigen binding sites, and / or antibody fragments (e.g., scFab, VH domains and / or VL domains) containing different antigen binding sites together to form the bispecific antibody of the present invention. Preferably, the peptide linker is a glycine-rich peptide having at least 5 amino acids, preferably at least 10 amino acids, and more preferably 10-50 amino acids. In some embodiments of the present invention, the glycine-rich peptide linker is (GxS)n, wherein G=glycine, S=serine, (x=3 and n=3, 4, 5 or 6) or (x=4 and n=2, 3, 4 or 5) (i.e., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS ((SEQ ID NO:9) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:20), respectively).

[0186] On the one hand, the present invention provides a bispecific antibody molecule, which comprises an antigen binding arm A that specifically binds to human epidermal growth factor receptor (EGFR) on target tumor cells and an antigen binding arm B that specifically binds to human proto-oncogene receptor tyrosine kinase (c-Met), wherein the heavy chain (HCA) of the binding arm A and the heavy chain (HCB) of the binding arm B interact to form a KIH structure, and the binding arm A and the binding arm B form a heterodimer through the KIH structure.

[0187] In the antibody BT461 according to an exemplary embodiment of the present invention, the numbers of the amino acid sequences of the fragments contained in the binding arm A and the binding arm B are summarized in Table 5.

[0188] Table 5 BT461 binding arm component fragments, ligand amino acids and sequence numbers of encoding nucleic acids

[0189]

[0190]

[0191] The binding target of binding arm A and the amino acid sequence and encoding nucleic acid sequence of ligand HGF and binding arm are as follows:

[0192] SEQ ID NO:35: "EGFR-ECD-HIS (Gene ID: 1956, AA25-645) amino acid

[0193] SEQ ID NO:36: "EGFR-ECD encoding nucleic acid

[0194] SEQ ID NO:37: "C-Met amino acid

[0195] SEQ ID NO:38: "C-Met encoding nucleic acid

[0196] SEQ ID NO:39:"EGF(Asn 971-Arg 1023)

[0197] SEQ ID NO:40: "CH1

[0198] SEQ ID NO:41: "HGF"

[0199] SEQ ID NO:44: Nucleic acid encoding Linker2

[0200] Constant region

[0201] The bispecific antibodies of the present invention disclosed herein are preferably used 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 as disclosed herein are preferably heterodimeric bispecific antibodies formed by the interaction between the binding arm fusion peptide of the light chain-linker-heavy chain structure targeting EGFR and the binding arm fusion peptide of the light chain-linker-heavy chain structure targeting C-Met through the heavy chain at CH3. The light chain and heavy chain of the binding arm A and the binding arm B of the bispecific antibodies of the present invention disclosed herein also include a light chain constant region and a heavy chain constant region, and the constant region is preferably a human constant region.

[0202] Preferably, the binding arm A and binding arm B light chains and heavy chains of the bispecific antibodies according to the invention disclosed herein comprise constant regions of human antibodies.

[0203] In some embodiments, the antibodies of the invention are full length IgG antibodies, preferably bispecific full length IgG1 antibodies, and more preferably human IgG1.

[0204] Such antibodies of the present invention disclosed herein can have good ADCC properties by modifying the heavy chain through heavy chain constant region amino acid mutations. In addition, the antibody industry has CH3 modification technology that can provide modified heavy chains, which preferentially form heterodimers compared to homodimers after co-expression in cloned cells. The ADCC activity of antibodies can also be improved by techniques known to those skilled in the art.

[0205] In some embodiments, the binding arms of the bispecific antibody molecules described herein comprise the heavy chain constant regions (CH) of immunoglobulins, such as the CHA region of binding arm A and the CHB region of binding arm B.

[0206] In some embodiments, the CHA region is or is derived from the amino acid sequence shown in SEQ ID NO:8.

[0207] In some embodiments, the CHB region is or is derived from the amino acid sequence shown in SEQ ID NO:19.

[0208] In some embodiments, the binding arms of the bispecific antibody molecules described herein comprise heavy chains, such as binding arm A comprises heavy chain A (HCA), wherein the HCA comprises a VHA region as described herein and a CHA region as described herein, or heavy chain B (HCB) of binding arm B, wherein the HCB comprises a VHB region as described herein and a CHB region as described herein.

[0209] In some embodiments, the antibody molecules described herein comprise an immunoglobulin light chain constant region or a fragment thereof, such as CLA comprised by antigen binding arm A and CLB comprised by antigen binding arm B.

[0210] In some embodiments, the CLA region is or is derived from the κCLA region of the amino acid sequence shown in SEQ ID NO:6.

[0211] In some embodiments, the CLB region is or is derived from the κCLB region of the amino acid sequence shown in SEQ ID NO:17.

[0212] In some embodiments, the heavy chain constant region or heavy chain of the bispecific antibody molecule described herein may comprise one or more modifications, and the chain and heavy chain constant region that have been modified may be referred to as modified HC and CHA, such as HCA and CHA, HCB and CHB, respectively.

[0213] In some embodiments, the VL region and CL region, VH region and CH1 region of the antibody binding arm together constitute the ScFab region, the rest of the binding arm constitutes the Fc region, and CH2ACH2B constitutes Fc (A), and CH2BCH3B constitutes Fc (B).

[0214] In a preferred embodiment, a KIH structure may be formed between CHA and CHB, or between the Fc(A) region and the Fc(B) region, and the binding arm A and the binding arm B form a heterodimer through the KIH structure.

[0215] For example, as described in more detail below, an asymmetric bispecific antibody molecule has different CHAs and CHBs.

[0216] In some embodiments, the Fc(A) comprises a combination of amino acid mutations that form a knob structure; and the Fc(B) comprises a combination of amino acid mutations that form a hole structure.

[0217] In a preferred embodiment, the Fc(A) of binding arm A comprises the CH2ACH3A 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 the CH2BCH3B segment derived from the constant region CHB of heavy chain B as shown in SEQ ID NO:14.

[0218] In a preferred embodiment, Fc(A) of binding arm A comprises an amino acid sequence derived from heavy chain B as shown in SEQ ID NO:11, and Fc(B) of binding arm B comprises an amino acid sequence derived from heavy chain B as shown in SEQ ID NO:22.

[0219] In some embodiments, the antibody molecules described herein comprise an immunoglobulin light chain constant region or a fragment thereof, such as CLA comprised by antigen binding arm A and CLB comprised by antigen binding arm B.

[0220] In some embodiments, the CLA region is or is derived from the κCLA region of the amino acid sequence shown in SEQ ID NO:6.

[0221] In some embodiments, the CLB region is or is derived from the κCLB region of the amino acid sequence shown in SEQ ID NO:17.

[0222] In some embodiments, the VL region and CL region, VH region and CH1 region of the antibody binding arm together constitute the ScFab region, the rest of the binding arm constitutes the Fc region, and CH2ACH2B constitutes Fc (A), and CH2BCH3B constitutes Fc (B).

[0223] In some embodiments, CH1A contained in ScFab (A) and CH1B contained in ScFab (B) are the amino acid sequence of CH1 of human IgG1 subtype shown in SEQ ID NO:96.

[0224] The Fc(A) region and the Fc(B) region interact with each other to form a KIH structure, and the binding arm A and the binding arm B form a heterodimer through the KIH structure.

[0225] In the method of preparing bispecific antibodies, the probability of mispairing of the light and heavy chains is usually greatly reduced and prevented by modifying the constant region of the heavy chain so that it is more favorable for heterodimerization (i.e., dimerization of the heavy chain with another heavy chain / light chain combination) than homodimerization.

[0226] In a preferred embodiment, the bispecific antibodies of the invention as disclosed herein comprise two different immunoglobulin heavy chains having compatible heterodimerization domains. The compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. In order to increase the percentage of the desired bispecific product (AB), the present invention uses CH3 modification, or, alternatively, a heavy chain having a compatible heterodimerization domain may be used, as defined below. The art describes that one way in which such heterodimerization of heavy chains can be achieved is to generate "bispecific antibodies with a KIH structure."

[0227] In US13 / 866,747 (now authorized as US9,248,181), etc.), methods and means for producing bispecific antibodies using compatible heterodimerization domains are disclosed. These means and methods can also be advantageously used in the present invention. Specifically, the bispecific antibodies of the present invention as disclosed herein.

[0228] In some embodiments, the binding arm A of heavy chain A / light chain A comprising a variable domain that binds to EGFR has an HCA with a knob variant structure, and the heavy chain B / light chain B comprising a variable domain that can bind to c-Met has a heavy chain B with a hole variant structure.

[0229] Preferred heavy chain mutations are amino acid mutations S354C and T366W in the CH3A domain of binding arm A (knob "variant" heavy chain) and amino acid mutations Y349C, T366S, L368A, Y407V (hole variant" heavy chain) in binding arm B, or vice versa. The knob variant heavy chain and the hole variant heavy chain preferentially pair to form a heterodimer, effectively preventing heavy chain mispairing. Further, the light chain mispairing problem of the bispecific antibody is solved by connecting the paired light and heavy chains with linger of 4-10 consecutive groups, preferably 6 consecutive groups of GGGGS sequences. Through the above measures, the EGFR and C-Met bispecific antibodies of the present method can effectively prevent the mispairing of non-target bispecific antibodies caused by light chains and heavy chains with different targets.

[0230] Functional properties of the bispecific antibodies of the invention

[0231] The EGFR / c-Met bispecific antibody of the present invention has the following functions:

[0232] (1) There is epitope competition with Avanta in binding to c-MET;

[0233] (2) being able to inhibit the autophosphorylation of EGFR mediated by the ligand EGF and the autophosphorylation of c-Met mediated by the ligand HGF, wherein the autophosphorylation of EGFR is the phosphorylation of the Y1068 site of EGFR, and the autophosphorylation of c-Met is the phosphorylation of c-Met at the Y1234 / Y1235 site;

[0234] (3) It can inhibit the proliferation of human tumor cells that are both positive for EGFR and c-Met and has a synergistic effect, and the effect of inhibiting the proliferation of tumor cells that are both positive for EGFR and c-Met is stronger than the combination of EGFR monoclonal antibody and c-MET monoclonal antibody;

[0235] (4) Ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP);

[0236] (5) It can mediate the internalization and degradation of dual target proteins of EGFR and c-MET, and the degradation of the target proteins after internalization is stronger than that of Evantor;

[0237] (6) Mediating dual-target internalization has a synergistic effect.

[0238] It has good safety performance selected from the following:

[0239] (1) The affinity for c-MET is significantly higher than that for EGFR;

[0240] (2) Its targeting selectivity for tumor cells is better than that of Evantor.

[0241] Nucleic acids, vectors, host cells, expression and purification

[0242] The present disclosure provides nucleic acid molecules encoding the antibody molecules described herein or their binding arms.

[0243] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence encoding the antigen binding arm A of the antibody molecule disclosed herein, or encoding the antigen binding arm B of the antibody molecule disclosed herein, or a polynucleotide sequence encoding both.

[0244] In one embodiment, the present invention provides a nucleic acid 1# encoding the EGFR / c-Met bispecific antibody described in the aforementioned aspects, wherein the nucleic acid 1# comprises a nucleic acid 2# encoding a binding arm A that specifically binds to EGFR and a nucleic acid 3# encoding a binding arm B that specifically binds to c-Met, wherein the nucleic acid 2# is a nucleic acid comprising a light chain A-linker-heavy chain A structure encoding the binding arm A, and the nucleic acid 3# is a nucleic acid comprising a light chain B-linker-heavy chain B structure encoding the binding arm B respectively; the nucleotide sequences 2# and 3# respectively comprise a CH3 domain mutation (S354C, T366W) of the heavy chain A (HCA) of the binding arm A forming a Knob structure, and a CH3 domain mutation (Y349C, T366S, L368A, Y407V) of the heavy chain B (HCB) of the binding arm B forming a Hole structure.

[0245] In some embodiments, nucleic acid molecules are purified or isolated from other nucleic acids or naturally occurring biological materials. Technicians prepare such nucleic acid molecules using methods well known in the art.

[0246] The one or more nucleic acid molecules may encode the VH domain and / or VL domain of the binding arm A and / or the binding arm B, preferably the VHA domain shown in SEQ ID NO:7 and the VLA domain shown in SEQ ID NO:5, or the VLB domain shown in SEQ ID NO:16 and / or the VHB domain shown in SEQ ID NO:18. The encoding nucleic acids of the VH and VL domain sequences of these antigen binding arms are described herein. When the nucleic acid encodes the VH and VL domains or the heavy and light chains of the antibody molecules of the present disclosure, the two domains or chains may be encoded on two separate nucleic acid molecules or may all be encoded on the same nucleic acid molecule.

[0247] In some embodiments, a nucleic acid molecule may comprise:

[0248] (i) a nucleic acid sequence encoding the LCA domain shown in SEQ ID NO: 1, and / or a nucleic acid sequence encoding the LCB domain shown in SEQ ID NO: 2;

[0249] (ii) encoding the LCB domain nucleic acid sequence shown in SEQ ID NO: 12, and / or the HCB domain nucleic acid sequence shown in SEQ ID NO: 13.

[0250] In some embodiments, the nucleic acid encoding the binding arm A of the amino acid sequence shown in SEQ ID NO:4 comprises the nucleotide sequence shown in SEQ ID NO:42.

[0251] In some embodiments, the nucleic acid encoding the binding arm B of the amino acid sequence shown in SEQ ID NO:15 comprises the nucleotide sequence shown in SEQ ID NO:43.

[0252] The present disclosure also provides vectors comprising nucleic acid molecules encoding the antibody molecules described herein.

[0253] The isolated nucleic acid molecules can be used to express the antibody molecules of the present disclosure or a combination thereof. The nucleic acid is generally provided in the form of a recombinant vector for expression. Therefore, another aspect of the present disclosure provides a vector comprising the nucleic acid as described above. A suitable vector can be selected or constructed, containing suitable 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 a host cell. The vector can be a plasmid, a virus, such as a plasmid. A phage or a phagemid, as the case may be.

[0254] The nucleic acid molecules or vectors described herein can be introduced into a host cell. Techniques for introducing nucleic acids or vectors into host cells are well known in the art and any suitable technique can be used. A range of host cells suitable for producing recombinant antibody molecules are known in the art and include bacteria, yeast, insect or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NSO or HEK cells, such as HEK293 cells.

[0255] Another aspect of the present disclosure provides a method for producing an antibody molecule of the present disclosure, comprising expressing a nucleic acid encoding the antibody molecule in a host cell and optionally isolating and / or purifying the EGFR / c-ME bispecific antibody molecule produced thereby. Methods for culturing host cells are well known in the art. The method may also include a step of isolating and / or purifying the EGFR / c-ME bispecific antibody molecule. Techniques for purifying recombinantly obtained EGFR / c-ME bispecific antibody molecules are well known in the art and include, for example, HPLC, FPLC or affinity chromatography, such as affinity chromatography.

[0256] 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.

[0257] Preparation of bispecific antibodies

[0258] The bispecific EGFR / C-Met antibody of the present invention can be produced by the following method: synthesizing the encoding nucleic acid 2# and the encoding nucleic acid 3# of the binding arm A and the binding arm B, respectively, constructing the recombinant vector of the EGFR binding arm encoding nucleic acid and the recombinant vector of the C-Met binding arm encoding nucleic acid, respectively; generating the recombinant vector carrying the recombinant nucleic acid 1# of the binding arm A and the binding arm B by enzyme cleavage and ligation, transfecting into host cells to culture the host cells, purifying the protein product prepared by the cells to obtain the bispecific EGFR / C-Met antibody of the present invention. The binding arm A of the bispecific antibody can have the characteristics described above for the binding arm that binds to EGFR, and the binding arm B of the bispecific molecule can have the characteristics described above for the binding arm that binds to C-Met. Producing the fermentation culture product of the bispecific bispecific antibody of the present invention.

[0259] The inventors screened the obtained clones and generated bispecific antibody molecules with dual specificity for EGFR-positive tumor cells and / or c-Met-positive tumor cells and with significant differential binding to tumor cells and non-tumor tissues.

[0260] In this way, the inventors identified antibodies that distinguish tumors from non-tumor tissues. The bispecific antibodies were selected to meet the expected characteristics necessary for therapeutic applications, namely, specifically recognizing EGFR targets and c-Met targets present on the surface of cancer cells and being able to effectively trigger internalization after binding to the target, with significant differences in affinity between binding arm A and binding arm B, and c-Met-specific binding arm B having significantly higher affinity than EGFR-specific binding arm B, while conferring tumor killing and safety, such as BT461.

[0261] The antibody shows 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 shows a higher ability to trigger internalization of the antibody complex, as shown in Example 11. Exemplarily, the present invention obtains a bispecific antibody for EGFR / c-Met named BT-461.

[0262] Antibody-drug conjugates

[0263] The present invention relates to an EGFR / c-Met bispecific antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, which comprises the bispecific antibody or antigen-binding fragment as described in any of the preceding aspects coupled to a drug module, and further comprises a drug module and a linker unit that couples the drug module to the antibody molecule.

[0264] Specifically in the form of ADC, wherein the bispecific antibody molecule can be coupled to a drug module. Such bispecific antibody-drug conjugates (bispecific ADCs) can be used to treat and / or diagnose diseases described herein. As used herein, a drug can be referred to as a "payload" or "warhead."

[0265] In some embodiments, the drug moiety comprises a cytotoxin.

[0266] Cytotoxin is a compound that can cause the death of target cells. Generally, in the context of antibody drug conjugates, cytotoxins are delivered to cells targeted by the antibody molecule, released into the cells and induce cell death. The term "cytotoxin" used herein refers to a substance that inhibits or prevents cell function and / or causes cell destruction. The term "cytotoxin" is intended to include chemotherapeutic agents, enzymes, antibiotics and toxins of bacterial, fungal, plant or animal origin such as small molecule toxins or enzymatically active toxins, including fragments and / or variants thereof, and various antitumor agents or anticancer agents disclosed below.

[0267] In some embodiments, the cytotoxin is a microtubule toxin, auristatin, maytansinoid, topoisomerase inhibitor, or pyrrolobenzodiazepine (PBD).

[0268] In some embodiments, the cytotoxic conjugates of the present invention utilize auristatins or dolastatin analogs. Auristatins are analogs of dolastatin, and their mechanism of action is to firmly bind to tubulin to inhibit the formation and polymerization of microtubules, while hindering tubulin-dependent guanosine triphosphate hydrolysis, causing cell mitosis arrest and ultimately leading to cell apoptosis.

[0269] In an exemplary embodiment, the novel auristatin derivative is monomethyl auristatin E (MMAE), and its structural formula is (Formula I):

[0270]

[0271] MonoMethyl auristatin E (MMAE) is a synthetic derivative of dolastatin 10, which has a potent mitotic inhibitory effect by inhibiting tubulin polymerization.

[0272] In other embodiments, the cytotoxic conjugates of the invention utilize DNA topoisomerase I inhibitors.

[0273] In an exemplary embodiment, the DNA topoisomerase I inhibitor is Dxd, and the structural formula is (Formula II):

[0274]

[0275] DxD is a novel exatecan derivative, a DNA topoisomerase I inhibitor, that inhibits DNA replication and transcription, leading to tumor cell death.

[0276] DXd has the effect of inhibiting DNA synthesis, which leads to DNA damage and cell apoptosis in cancer cells by inhibiting the activity of topoisomerase II. It also has good drug tolerance and is suitable for the treatment of chemotherapy-resistant cancers.

[0277] In one embodiment, the DNA topoisomerase I inhibitor is Exatecan, having the structural formula (Formula III):

[0278]

[0279] Exatecan is a water-soluble topoisomerase I inhibitor.

[0280] Examples of such cytotoxic agents and conjugation methods are further given in application WO 2008 / 010101, incorporated herein by reference.

[0281] In some embodiments of the invention, the antibody is covalently attached to at least one cytotoxic molecule, either directly or via a cleavable or non-cleavable linker.

[0282] As used herein, "linker" means a chemical moiety comprising covalent bonds or a chain of atoms that covalently attaches a polypeptide to a drug moiety.

[0283] The techniques for conjugating therapeutic agents to proteins, particularly antibodies, are well known. The therapeutic agent may be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (e.g., by hydrolysis, by proteolytic degradation, or by a cleavage agent).

[0284] In some embodiments, the therapeutic agent may be attached to the antibody via a non-cleavable linker, such as a maleimido-alkylene- or maleimido-aryl linker, which is directly attached to the therapeutic agent and released by proteolytic degradation of the antibody.

[0285] In some aspects, the therapeutic agent is attached to the antibody via a cleavable linker that is sensitive to cleavage in the intracellular environment of a cancer cell expressing ALPP, but substantially insensitive to the extracellular environment, such that when the conjugate is internalized by a cancer cell expressing EGFR and c-Met (e.g., in an endosome, or, for example, by virtue of pH sensitivity or protease sensitivity, in a lysosomal environment or in a caveolae environment), the conjugate is cleaved from the antibody.

[0286] Typically, the ADC comprises a linker region between the therapeutic agent and the anti-EGFR and c-Met bispecific antibody (e.g., anti-BT461). The linker is typically cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., in a lysosome or endosome or cell membrane caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease (including a lysosomal or endosomal protease). The most typical is a peptidyl linker that can be cleaved by an enzyme present in a cell expressing EGFR and c-Met.

[0287] In some preferred embodiments, the linker is a peptidyl linker.

[0288] In some preferred embodiments, the structure is a linker comprising a Phe-Leu or Val-Cit peptide.

[0289] Cleavable linkers can be pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. Other linkers are cleavable under reducing conditions (e.g., disulfide linkers). Disulfide linkers include, but are not limited to, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio) toluene), those formed by SPDB and SMPT. The linker can also be a malonate linker, a maleimidobenzoyl linker, or a 3'-N-amide analog.

[0290] When conjugated to a therapeutic agent (ie, in the context of a linker-therapeutic agent moiety of an ADC or ADC derivative as described herein), the linker can promote cellular internalization.

[0291] In some preferred embodiments, the antibody-drug conjugate is an antibody-drug conjugate based on auristatin, which means that the drug component is an auristatin drug. Auristatin binds to tubulin, has been shown to interfere with microtubule dynamics and nuclear and cell division, and has anti-cancer activity. Typically, an antibody-drug conjugate based on auristatin contains a linker between an auristatin drug and an ABP (e.g., an anti-EGFR / c-Met bispecific antibody). The linker can be, for example, a cleavable linker (e.g., a peptidyl linker) or a non-cleavable linker (e.g., a linker released by antibody degradation). Auristatin can be auristatin E or a derivative thereof. Auristatin can be, for example, an ester formed between auristatin E and a ketoacid. For example, auristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include MMAF and MMAE.

[0292] Exemplarily, the EGFR / c-Met bispecific ADC comprises a new MMAE analog / derivative, and the EGFR / c-Met bispecific ADC is a conjugate of EGFR / c-Met bispecific antibody-MMAE or its derivative, or a pharmaceutically acceptable salt or solvate thereof, wherein the MMAE comprises a structure as shown below:

[0293]

[0294] In an exemplary embodiment, the auristatin-based linker and drug conjugate includes mc-vc-PABC-MMAE, and the antibody drug conjugate is as follows, wherein Ab is an ABP (e.g., an anti-EGFR / c-Met bispecific antibody as described herein) and val-cit (vc) represents a valine-citrulline dipeptide:

[0295]

[0296] In some embodiments, the antibody unit Ab (eg, anti-EGFR / c-Met antibody) is linked to the drug-linker via a cysteine ​​residue of the antibody.

[0297] In some embodiments, the cysteine ​​residue is a cysteine ​​residue engineered into the antibody. In other aspects, the cysteine ​​residue is an interchain disulfide bond cysteine ​​residue.

[0298] An exemplary auristatin-based antibody drug conjugate is named BT461-MMATE.

[0299] In some preferred embodiments, in the bispecific antibody-drug conjugate, the cytotoxin is or comprises a topoisomerase inhibitor. The term "topoisomerase inhibitor" as used herein refers to a cytotoxic agent that inhibits the activity of one or more topoisomerases (topoisomerase I and II), which are enzymes that play an important role in DNA replication and transcription by regulating DNA supercoiling. Therefore, antibody-drug conjugates containing topoisomerase inhibitors as cytotoxins are expected to interfere with normal processes involving DNA, thereby causing cell death. Antibody-drug conjugates containing topoisomerase inhibitors have been shown to be effective against a variety of tumor-containing cell lines and have anti-cancer activity in clinical trials.

[0300] In a preferred embodiment, the antibody molecule is coupled to an inhibitor of topoisomerase I. Representative examples of topoisomerase I inhibitors include, but are not limited to, camptothecin and its analogs topotecan, irinotecan, belotecan, exatecan, derutecan, and cinotecan.

[0301] Exemplarily, the bispecific antibody-drug derivative conjugate or its pharmaceutically acceptable salt or solvate provided by the present disclosure is as shown in the general formula Ab-(LD)p of formula (I): wherein D is a toxin molecule, L is a linker unit; Ab is any anti-EGFR / c-Met antibody or antigen-binding fragment thereof described in the present disclosure.

[0302] In some embodiments, the bispecific ADC of the present disclosure is an EGFR / c-Met bispecific antibody-exitecan derivative or a pharmaceutically acceptable salt or solvate thereof.

[0303] In some embodiments, the EGFR / c-Met bispecific antibody-exitecan derivative disclosed herein, wherein the structural unit LD is

[0304] An exemplary resulting BT461-ADC is a structure shown in formula (V):

[0305]

[0306] In some embodiments, the bispecific ADC of the present disclosure is a conjugate of an EGFR / c-Met bispecific antibody-Dxd derivative or a pharmaceutically acceptable salt or solvate thereof, wherein the structural unit LD- is a structure shown in Formula VI:

[0307]

[0308] The obtained BT-461-ADC has the structure shown in Formula VII:

[0309]

[0310] In some embodiments, p is an integer or decimal between 1 and 5, preferably between 1 and 20, preferably between 1 and 10, or preferably between 1 and 8; for example, an integer or decimal between 1 and 6.

[0311] In some specific embodiments, the present disclosure provides an antibody-isotecan derivative conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide residue of L is an amino acid residue formed by one, two or more amino acids selected from phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), and aspartic acid (N); for example, an amino acid residue formed by one, two or more amino acids selected from phenylalanine and glycine; for example, a tetrapeptide residue; for example, a tetrapeptide residue of GGFG (glycine-glycine-phenylalanine-glycine).

[0312] Preparation method of EGFR / c-Met bispecific ADC

[0313] The present disclosure provides a method for preparing a conjugate such as an EGFR / c-Met bispecific antibody-ADC derivative or a pharmaceutically acceptable salt or solvate thereof.

[0314] Exemplarily, the present invention utilizes a thiol coupling method to link MMAE, Excatecan or DXD to the cysteine ​​of the BT461 molecule.

[0315] In some embodiments, in the method for preparing the EGFR / c-Met bispecific ADC, the thiol coupling method comprises the following steps:

[0316] 1. Antibody thiol reduction,

[0317] 2. Adding the drug-linker and diluting the drug-linker, wherein the dilution is to dilute the drug-linker with a DMSO solution at room temperature,

[0318] 3. Coupling,

[0319] 4. Concentrate,

[0320] 5. Measure the concentration.

[0321] The coupling is to add 5% by volume DMSO solution to the antibody reaction solution to obtain a uniform solution; and then react in a water bath.

[0322] In an exemplary embodiment, the water bath reaction is carried out at 25° C. for 3 hours.

[0323] In an exemplary embodiment, the concentration is gel chromatography filtration and sterile filtration concentration.

[0324] In an exemplary embodiment, the final concentration of the concentrate is about 5 mg / mL, which can meet the requirements of in vivo efficacy and safety evaluation.

[0325] In an exemplary embodiment, the average DAR is measured using an ultraviolet spectrophotometer.

[0326] In an exemplary embodiment, SEC-HPLC is used to measure the purity of the ADC product.

[0327] Different drug-linkers have different absorption peaks. For the first measurement, a full spectrum scan should be performed using the drug-linker solution to obtain the highest absorption peak of the drug-linker.

[0328] In an exemplary embodiment, the average DAR value is measured by ultraviolet spectrophotometer. According to the measurement, the highest absorption peak of MC-GGFG-Dxd drug-linker is at A360, and the highest absorption peak of MC-VC-PAB-Exatecan drug-linker is at A370.

[0329] The antibody concentration and drug concentration are calculated by the following formula, and the ratio of drug to antibody concentration is the DAR value of ADC:

[0330] A280=εD,280CD+εA,280CA

[0331] A360=εD,360CD+εA,360CA;

[0332] Wherein, A280 and A360 represent the absorbance of the antibody-drug conjugate aqueous solution at 280 nm and 360 nm, εA,280 and εA,360 represent the molar absorption coefficients of the antibody at 280 nm and 360 nm, εD,280 and εD,360 represent the molar absorption coefficients of the drug-linker at 280 nm and 360 nm, CA represents the antibody concentration, CD represents the drug-linker concentration,

[0333] After calculation, the coefficients of BT462-Dxd are, εA,280=210600, εA,360=0, εD,280=5280, εD,360=20190;

[0334] The coefficients of BT461-Exatecan are: εA,280=210600, εA,360=0, εD,280=5280, εD,370=19560.

[0335] HIC-HPLC determines the DAR value of ADC products, which can display five peaks: DAR0 (naked antibody), 2, 4, 6, and 8. The ratio of ADCs with different DAR values ​​is calculated. The volume must be above 200 μL and the concentration must be above 1 mg / mL.

[0336] The average amount of drug per antibody in the conjugate preparation from the conjugation reaction can be characterized by conventional methods, such as UV, reverse phase HPLC, HIC, mass spectrometry, ELISA assays, and electrophoresis. The quantitative distribution of the conjugates, represented by n, can also be determined. By ELISA, the average value of p in a particular conjugate preparation can be determined (Hamblett, 2004; Sanderson, 2005). However, the distribution of p (drug) values ​​cannot be discerned by the antibody-antigen binding and detection limits of the ELISA. In addition, the ELISA assay used to detect the conjugate cannot determine the location of the drug moiety attached to the antibody molecule, such as the heavy or light chain fragment, or the specific amino acid residue.

[0337] In some embodiments, separation, purification and characterization of homogeneous conjugates can be achieved by means such as reverse phase HPLC or electrophoresis, where p is a specific value for the conjugate with other drug loads. Such techniques are also applicable to other types of conjugates.

[0338] For some conjugates, p may be limited by the number of attachment sites on the antibody molecule. For example, an antibody molecule may have only one or a few cysteine ​​thiol groups, or may have only one or a few sufficiently reactive thiol groups through which the linker can be coupled.

[0339] Typically, less than the theoretical maximum amount of drug is coupled to the antibody molecule during the coupling reaction. Antibody molecules may contain, for example, many lysine residues that do not react with the linker (L). Only the most reactive lysine groups can react with amine-reactive linking agents. In addition, only the most reactive cysteine ​​thiol groups can react with thiol-reactive linking agents.

[0340] Thus, the conjugate compositions of the present disclosure are mixtures comprising antibody-drug conjugate compounds, wherein the antibody has one or more drug moieties (e.g., tubulysin or a topoisomerase inhibitor) and wherein the drug moieties can be attached to the antibody molecule at multiple amino acid residues.

[0341] The drug loading (p) is the average number of drugs (e.g., tubulin inhibitors or topoisomerase inhibitors) per antibody molecule. In the compositions disclosed herein, the drug loading ranges from 1 to 20 drugs (D) per antibody molecule. For example, the drug loading ranges from 1 to 10 drugs (D) per antibody molecule, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs are covalently attached to the antibody molecule.

[0342] Biological functional characteristics of antibody molecules

[0343] The EGFR / c-Met bispecific antibody molecules described herein and antibody-drug conjugates comprising the same can be characterized by reference to certain functional properties.

[0344] Targeting cell surface EGFR and CMET

[0345] The bispecific antibody of the present invention binds to EGFR with high affinity and specifically binds to c-Met with high affinity.

[0346] In some embodiments, the bispecific antibody of the present invention comprises a light chain variable domain and a heavy chain variable domain having the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 7 herein, and has the function of cross-reactivity to bind to human and cynomolgus monkey EGFR.

[0347] In some exemplary embodiments, such as in BT461, the extracellular EGFR binding level and c-Met binding level of the EGFR / c-Met bispecific antibody are evaluated by measuring the EC50 value of ELISA binding. For example, the binding arm A of the bispecific antibody of the present invention can bind to EGFR at an ELISA binding EC50 of about 780-800 ng / mL, and the binding arm B can bind to c-Met at an ELISA binding EC50 of about 15-20 ng / mL. The determination method of ELISA binding EC50 can refer to the method shown in Example 2.

[0348] In some embodiments such as BT461, the level of binding of the EGFR / c-Met bispecific antibody to EGFR on the cell surface is determined by measuring the FACS binding EC50 value.

[0349] In some embodiments such as BT461, the bispecific antibody BT461 of the present invention can bind to EGFR overexpressed in CHO-K1 cells at a FACS binding EC50 of about 3500-4500 ng / mL and about 100-130 ng / mL, respectively, and bind to c-Met overexpressed in CHO-K1 cells at a FACS binding EC50 of about 100-130 ng / mL. The determination method of FACS binding EC50 can be found in Example 3.

[0350] In some embodiments such as BT461, the bispecific antibody BT461 of the present invention specifically binds to double-positive tumor cells with an EC50 of about 380-420 ng / mL. As in Example 5, the double-positive tumor cells are MNK45. In some embodiments such as BT461, the FACS antagonism IC50 value is used to characterize the level of binding of the EGFR / c-Met bispecific antibody to antagonize the ligands HGF and EGF to the membrane proteins EGFR and c-MET. For example, when measured with EGF-mFC at a concentration of 2 μg / mL, the bispecific antibody BG461 of the present invention can inhibit EGF binding to EGFR with a FACS antagonism IC50 value of about 199500-20500 ng / mL. For example, when measured with HGF-HIS at a concentration of 5 μg / mL, the bispecific antibody BT461 of the present invention can inhibit HGF binding to c-Met with a FACS antagonism IC50 value of about 1030-1100 ng / mL. The method for determining FACS antagonist IC50 can refer to the method shown in Example 5.

[0351] In some embodiments such as BT461, the bispecific antibody of the present invention binds specifically to cynomolgus monkey c-Met and EGFR proteins. For example, when measured with 2 μg / mL cynomolgus monkey c-Met and EGFR proteins, the binding arm B of the bispecific antibody of the present invention can bind to cynomolgus monkey c-Met with an ELISA binding EC50 value of about 10-25 ng / mL, and can bind to cynomolgus monkey EGFR with an ELISA binding EC50 value of about 50-60 ng / mL. The determination method of cross-binding can refer to the method shown in Example 6.

[0352] In some embodiments, when detected by biolayer interferometry (BLI), the bispecific antibody BT461 of the present invention binds to human c-EGFR at a concentration of 5 μg / mL with a dissociation constant (KD) equal to or less than 2.25E-08, less than 2.0E-08, and binds to human c-Met with a dissociation constant (KD) equal to or less than 1.20E-09, less than 1.12E-09. An exemplary BLI detection method is shown in Example 7.

[0353] In some embodiments, such as using the molecular interaction instrument ForteBio's Octet System, 100 nM of the EGFR / c-Met bispecific antibody BT461 is tested with 5 μg / mL of hEGFR-His and c-Met, respectively. The results show that the bispecific antibody of the present invention can compete with HGF for binding to c-Met, as shown in Example 8.

[0354] In some embodiments, 5-fold dilutions of Avantu and Taixinsheng were used as controls at 20 μg / ml in 7 gradients, and the binding level of BT461 to HACAT cells with low CMET expression / high EGFR expression, BEAS-2B cells with low CMET expression / high EGFR expression, and MKN45 cells with high CMET expression / high EGFR expression were determined. The results showed that the rate of BT461 binding to the dual-target positive tumor MKN45 was higher than that of binding to epidermal cells HACAT ( Figure 7 C), with a difference of nearly one order of magnitude (10 times), and a selectivity index of 7.26, which is more specific in selecting and binding tumor cells than Evantor ( Figure 7 B). In some embodiments, BT461 binds to the dual-target positive tumor MKN45 at a higher rate than to the human normal lung bronchial epithelial cell line BEAS-2B cells ( Figure 8 C), the difference is nearly 5 times, and its specificity in selecting and binding tumor cells is stronger than that of Avantol. The test method for selecting specificity is shown in Example 9.

[0355] Selection index = normal cell EC50 / tumor cell EC50*maximum tumor binding / maximum normal cell binding.

[0356] In some embodiments, the changes in the levels of EGFR and c-Met autophosphorylation are determined by Western blotting to explore whether the anti-c-Met / EGFR bispecific antibody can inhibit the c-Met and EGFR signaling pathways. The exemplary development results show that under the culture conditions containing 40ng / mL EGF-mFC+100ng / mL HGF-his, 50μg / mL IgG1, Evantor, BT461, and BT470 cannot induce EGFR and c-Met autophosphorylation and change the protein levels of EGFR and CMET in A549 cells; on this basis, the use of HGF-his (10μg / mL) and EGF-mfc (100μg / mL) stimulation can activate the phosphorylation of EGFR and c-MET in A549 cells; and the addition of BT461 can significantly inhibit the intracellular p-CMET (Y1234 / 1235) phosphorylation level, and slightly inhibit the p-EGFR (Y1173) phosphorylation level.

[0357] In some embodiments, the EGFR autophosphorylation level is detected using an EGFR Phosphorylation (Y1068) Assay Kit.

[0358] In some embodiments, the level of HGF-mediated c-Met autophosphorylation is measured using a c-Met phosphorylation (Y1234 / Y1235) assay kit.

[0359] In an exemplary embodiment, the c-Met-specific ligand HGF mediates the activation of strong c-Met autophosphorylation in HCC827 cells, and BT461 and Avanto can significantly inhibit the level of c-Met autophosphorylation; the EGFR-specific ligand EGF mediates the activation of strong EGFR autophosphorylation in HCC827 cells, and Taixinsheng antibodies can inhibit the level of EGFR phosphorylation activated by EGF, while the antibody BT461 almost completely retains the inhibitory function of its anti-EGFR antibody arm on EGF-activated EGFR phosphorylation.

[0360] In some embodiments, the TR-TRET ratio is calculated after detection using a TR-FRET microplate reader to evaluate the level of EGFR (or CMET) autophosphorylation, wherein the TR-FRET ratio is equal to the absorbance value of 665 divided by the absorbance value of 615 multiplied by 1000, the absorbance value at 665nm represents the total number of EGFR (Y1068) phosphorylation (or CMET (Y1234 / 1235) phosphorylation levels), and the absorbance value at 615nm represents the total number of EGFR (or CMET) total proteins. In some embodiments, the level of extracellular binding of the bispecific antibody of the present invention to human EGFR is detected by biolayer interferoc-Metry (BLI). The binding arm A of the bispecific antibody of the present invention binds to human EGFR with a dissociation constant (KD) (M) of less than about 2.8E-8, such as less than about 2.6E-8, less than about 2.5E-8, less than about 2.4E-8, less than about 2.2E-8, or less than about 2.1E-8.

[0361] In some embodiments, the binding arm A of the bispecific antibody of the present invention binds to human EGFR with a KD (M) between about 3.0E-8 and about 2.0E-9. The affinity of the binding arm A to EGFR can be determined experimentally using any suitable method (such as the method described in the Examples herein). If measured under different conditions (e.g., osmolarity, pH), the affinity of the specific binding arm A-antigen interaction measured may vary. Therefore, the measurement of affinity and other antigen binding parameters (e.g., KD, Kon, Koff) is preferably performed with a standardized solution of the antigen, and a standardized buffer, such as the buffer described herein.

[0362] The bispecific antibodies of the invention as disclosed herein can inhibit EGF-induced growth of EGF-responsive cells without inducing the significant common toxicity associated with high affinity bivalent EGFR antibodies, which makes the antibodies ideally suited for combination with TKIs with their own toxicity profile, the induced growth is preferably measured using an assay as described in the Examples. Some growth also occurs in the case of inhibitory antibodies.

[0363] In some embodiments, the bispecific antibodies of the present invention specifically bind to c-Met with high affinity and inhibit c-Met signaling, and can provide improved specificity and improved inhibition of the autophosphorylation level of c-Met mediated by the ligand HGF and the beneficial effects of the signaling cascade reaction of tumorigenesis and development triggered by such phosphorylation compared with small molecule c-Met inhibitors, as well as synergistic effects in tumor cell growth inhibition and killing compared with conventional c-Met monospecific antibodies and combination therapeutic agents thereof.

[0364] The variable domains that can bind to c-Met of the bispecific antibodies of the present invention are preferably derived from the variable domains of the bivalent monospecific antibodies that can inhibit HGF-induced death of non-small cell lung cancer cells HCC827 by targeting and binding to c-Met, and the inhibition level is equivalent to or close to that of entuzumab or its combination, and the inhibition is usually not 100%.

[0365] In one embodiment, the level of HGF-mediated c-Met autophosphorylation in the presence and absence of the antibody of the present invention can be measured using a c-Met phosphorylation (Y1234 / Y1235) assay kit. The level of HGF-mediated c-Met autophosphorylation in the presence and absence of the antibody of the present invention is measured by calculating the ratio of the absorbance at 665 nm / the absorbance at 615 nm (TR-TRET ratio).

[0366] In some embodiments, the c-Met-specific ligand HGF mediates activation of strong c-Met autophosphorylation in tumors.

[0367] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention inhibits HGF-mediated c-Met autophosphorylation in HCC827 cells, resulting in a decrease in the TR-TRET ratio of at least 30%, and preferably at least 40%. Without being bound by theory, the resulting significant decrease in the TR-TRET ratio can be considered to be that the binding of the antibody to c-Met blocks the binding of HGF to c-Met, thereby inhibiting the mediation of HGF on c-Met phosphorylation.

[0368] In an exemplary embodiment, the bispecific antibodies of the invention as disclosed herein can inhibit the growth of HGF and EGF / HGF-induced EGFR-TKI-resistant tumor cell lines KP4, A549, MKN45 and HCC827-HGF.

[0369] Biological functions of bispecific antibodies

[0370] The binding arm A of the bispecific antibody of the present invention specifically binds to EGFR with high affinity, and the binding arm B thereof specifically binds to c-Met with high affinity.

[0371] In some embodiments, for example, BT461 binds to EGFR with an EC50 of less than 787.1 ng / mL when measured with 2 μg / mL of EGF-HIS; BT461 binds to c-Met with an EC50 of less than 18.93 when measured with 2 μg / mL of c-MET-HIS.

[0372] Inhibits EGFR autophosphorylation and c-Met autophosphorylation

[0373] In some embodiments, Western blotting is used to determine the function of the EGFR / c-Met antibody of the present invention in inhibiting EGFR autophosphorylation and c-Met autophosphorylation.

[0374] In some embodiments, in a culture medium containing 40 ng / mL EGF-mFC + 100 ng / mL HGF-his% serum, 50 μg / mL of the EGFR / c-Met bispecific antibody provided by the present invention was measured to significantly inhibit the level of HGF-mediated c-Met autophosphorylation, and slightly inhibit EGF-mediated EGFR autophosphorylation.

[0375] In some embodiments, the inhibitory effect of the EGFR / c-Met bispecific antibody of the present invention on EGF-stimulated EGFR phosphorylation in non-small cell lung cancer HCC827 is determined using an EGFR phosphorylation (Y1068) assay kit. Exemplarily, using IgG1 as a control, the degree of reduction of the phosphorylation level of EGFR stimulated by 40 ng / mL EGF-mFC by 50 μg / mL of the EGFR / c-Met bispecific antibody of the present invention is determined, and the degree of reduction of the phosphorylation level of EGFR stimulated by 100 ng / mL HGF-his by 50 μg / mL of the EGFR / c-Met bispecific antibody of the present invention is determined. The absorbance value at 665 nm represents the total number of EGFR phosphorylation, and the absorbance value at 615 nm represents the total number of EGFR total protein. The TR-FRET ratio is equal to the absorbance value of 665 divided by the absorbance value of 615 multiplied by 1000. The TR-FRET ratio is used to characterize the level of EGFR autophosphorylation.

[0376] In some embodiments, the binding arm A of the EGFR / c-Met bispecific antibody BT461 of the present invention inhibits EGF-induced phosphorylation of EGFR at residue Y1068 of EGFR at a TR-FRET ratio (665 nm absorbance value*1000 / 615 nm absorbance value) of about 30-40, preferably 33-38, and the results show that the ability to inhibit EGFR autophosphorylation is weak. Exemplarily, using IgG1 as a control, the degree of reduction of the phosphorylation level of c-Met stimulated by 100 ng / mL HGF-his stimulated by 50 μg / mL of the EGFR / c-Met bispecific antibody of the present invention is determined, the absorbance value at 665 nm represents the total number of C-Met phosphorylation, the absorbance value at 615 nm represents the total number of C-Met total protein, the TR-FRET ratio is equal to the absorbance value of 665 divided by the absorbance value of 615 multiplied by 1000, and the TR-FRET ratio is used to represent the level of C-Met autophosphorylation.

[0377] In some exemplary embodiments, the bispecific EGFR / c-Met antibody BT461 inhibits HGF-induced c-Met phosphorylation at c-Met residues Y1234 / Y1235 with a TR-FRET ratio (665 nm absorbance*1000 / 615 nm absorbance) of about 33-38, which is equivalent to the phosphorylation level of c-Met when no HGF induction is added. The level of inhibition of HGF-mediated c-Met autophosphorylation by the EGFR / c-Met bispecific antibody of the present invention is stronger than that of Avanta.

[0378] In some embodiments, the present invention provides EGFR / c-Met bispecific antibodies that can specifically bind to EGFR / c-Met double-positive tumor cells.

[0379] In some embodiments, in the specific binding embodiment of double positive tumor cells, the ELISA binding EC50 value of the EGFR / c-Met bispecific antibody of the present invention binding to EGFR is not less than 1000ng / mL, not less than 830ng / mL, or preferably not less than 730ng / mL, more preferably not less than 680ng / mL, most preferably not less than 630ng / mL, more preferably not less than 6100ng / mL; and the ELISA binding EC50 value of c-MET is not less than 700ng / mL, preferably not less than 600ng / mL, not less than 530ng / mL, more preferably not less than 480ng / mL, preferably not less than 400ng / mL, and most preferably not less than about 370. In some embodiments, the double positive tumor cell is MKN45.

[0380] In some embodiments, the EGFR / c-Met bispecific antibody provided by the present invention blocks the binding of HGF, EGF to MKN45 cells, and can antagonize the binding of EGF to EGFR / c-Met double-positive tumor cells, and can also antagonize the binding of HGF to EGFR / c-Met double-positive tumor cells.

[0381] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention blocks the specific binding of EGF and HGF to EGFR and c-Mett on the surface of double positive tumor cells. Exemplary bispecific antibodies can inhibit EGF binding to EGFR on the surface of double positive cells with a FACS blocking IC50 value of not less than 22000 ng / mL, preferably not less than 21000 ng / mL, preferably not less than 20800 ng / mL, preferably not less than 20500 ng / mL, preferably not less than 20100 ng / mL, and more preferably not less than 20010 ng / mL; binding arm B can be not less than 1700 ng / mL, preferably not less than 1600 ng / mL, not less than 1500 / mL, or not less than 1400 ng / mL, or more preferably not less than 1300 ng / mL, not less than 11200 ng / mL, or most preferably not less than 1100 ng / mL FACS blocking IC50 to inhibit HGF binding to c-Met on the surface of double positive cells. In some embodiments, the double positive tumor cell is MKN45.

[0382] In some embodiments, based on the bio-layer interferometry (BLI) technique to detect the affinity of the bispecific antibody of the present invention to EGFR, the binding arm B in the bispecific antibody of the present invention binds to human c-Met at a value equal to or less than 2.45E-9, less than 2.25E-9, less than 1.95E-9, preferably less than 1.75E-9, less than 1.55E-9, less than 1.40E-9, more preferably less than 1.25E-9, less than 1.20E-9, and most preferably less than 1.15E-9.

[0383] In some embodiments, based on the bio-layer interferometry (BLI) technology to detect the affinity of the bispecific antibody of the present invention for binding to EGFR, the binding arm A in the bispecific antibody of the present invention binds to EGFR with a KD (M) equal to or less than 2.8E-8, less than 2.7E-8, less than 2.6E-8, preferably less than 2.5E-8, less than 2.4E-8, less than 2.3E-8, ​​more preferably less than 2.2E-8, less than 2.1E-8, and most preferably less than 2.0E-8.

[0384] In some embodiments, TR-fret cell signaling pathway analysis was performed by adding 10 ng / mL of EGF-mFC or 100 ng / mL of HGF-his to 50 μg / mL of BT461, IgG1 and Avantol, respectively, and the resulting TR-FRET ratios were used to characterize the level of EGFR (or c-MET) autophosphorylation. The results showed that the antibody BT461 almost completely retained the inhibitory function of its anti-EGFR antibody arm on EGF-activated EGFR phosphorylation, but its inhibitory ability was weaker than that of Avantol.

[0385] In some embodiments, The Live-Cell Analysis System detection curve shows that the internalization efficiency of the bispecific antibody molecule BT461 of the present invention is stronger than that of the monoclonal antibody against EGFR or c-Met, indicating that the dual-target internalization mediated by the bispecific antibody molecule BT461 has a synergistic effect.

[0386] In some embodiments, the internalization and degradation levels of EGFR and c-MET are detected by Western blot analysis. Exemplarily, after treating MKN45 cells with IgG1 at a concentration of 50 μg / mL, the antibody Avantu and BT461 for 48 hours, the chemiluminescence system can significantly detect the downregulation of EGFR total protein level, and the downregulation caused by BT461 and Avantu is comparable; BT461 causes a significant decrease in the c-MET protein level of MKN45 for 24 hours, while Avantu treated MKN45 cells for 48 hours before significant degradation of CMET protein level was observed. The results show that the internalization efficiency mediated by BT461 in MKN45 cells is lower than that of Avantu, but the degradation of the target protein after internalization mediated by BT461 is stronger than that of Avantu.

[0387] In some embodiments, the bispecific antibody of the present invention specifically binds to cynomolgus monkey c-Met and EGFR proteins, and has cross-binding activity of cynomolgus monkey EGFR and c-Met. Exemplarily, the EC50 of the antibody BT461 of the present invention binding to EGFR and c-Met is not less than 60.53 ng / mL and not less than 15.10 ng / mL, respectively.

[0388] Has remarkable selectivity for tumor cells

[0389] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention has a significantly higher selective binding level to EGFR / c-MET high-expressing tumor cells than EGFR monoclonal antibodies and marketed EGFR / c-Met bispecific antibodies.

[0390] In some embodiments, the EGFR / c-Met bispecific antibody BT-461 of the present invention binds to the dual-target positive tumor MKN45 at a higher rate than that of binding to human immortalized keratinocytes HACAT and human normal lung bronchial epithelial cell line BEAS-2B ( Figure 7 C).

[0391] Exemplarily, the selection index of BT-461 for EGFR / c-MET high-expressing tumor cells MKN45 is as high as 7.26, which is significantly higher than the selection index of EGFR binding arm-derived monoclonal antibody Taixinsheng and positive control Avantuo, and is nearly an order of magnitude (10 times) different from the selection index of 1.80 for human immortalized keratinocytes HACAT, and its specificity for selecting and binding tumor cells is stronger than Avantuo. Exemplarily, the selection index of BT461 for EGFR / c-MET high-expressing tumor cells MKN45 is as high as 5.44, which is significantly higher than the selection index of EGFR binding arm-derived monoclonal antibody Taixinsheng and positive control Avantuo, and is nearly 5 times different from the selection index of 0.91 for normal lung bronchial epithelial cell line BEAS-2B cells, and its specificity for selecting and binding tumor cells is stronger than Avantuo.

[0392] In one embodiment, the selection specificity is characterized by a selection index, wherein the selection index is calculated as follows: selection index = normal cell EC50 / tumor cell EC50*maximum tumor binding / maximum normal cell binding.

[0393] Inhibits tumor cell proliferation in vitro

[0394] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention can inhibit tumor cell proliferation in vitro.

[0395] In some embodiments, the tumor cell lines include KP4, A549, MKN45, and HCC827-HGF.

[0396] 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.

[0397] 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 EGFR mutant cells in vitro.

[0398] For example, by using CellCounting- The 3D kit detects the effects of the antibodies of the invention on the proliferation of these cell lines, and compares the levels of reduction in tumor cell viability caused by a mixture comprising an EGFR / c-Met bispecific antibody and two monospecific antibodies (one binding to EGFR and the other binding to c-Met) to evaluate synergistic inhibition.

[0399] In one embodiment, the EGFR / c-Met bispecific antibody of the present invention can inhibit the proliferation of three types of cells: MKN45, HCC827-HGF, and NCI-H1975-HGF. In some exemplary embodiments, when the bispecific molecules of the present invention inhibit tumor cell proliferation, the cell viability of tumor cells is comparable to the measured value under the inhibition of Amivantanab, or even lower than the cell viability under the inhibition of Amivantanab. The ability of BT461 bispecific antibody to inhibit tumor cell activity is also stronger than its related monoclonal antibody combination (Taixinsheng & LY358-B12), which indicates that BT461 has a synergistic effect in the efficacy of inhibiting tumors in vitro.

[0400] In some embodiments, the bispecific antibodies of the present invention as disclosed herein can mediate the cytotoxic effect (ADCC effect) of NK cells on target cells. Exemplarily, BT-461 mediates the ADCC effect of NK cells on HCC827 cells. Exemplarily, BT-461 can mediate the ADCC effect of NK cells on H1975 cells.

[0401] In some embodiments, the EGFR / c-Met bispecific antibodies of the present invention mediate antibody-dependent phagocytosis (ADCP) in tumor cells.

[0402] In an exemplary embodiment, the tumor cells for which the EGFR / c-Met bispecific antibody of the present invention mediates ADCP are preferably HCC827 cells.

[0403] Exemplarily, the cell having phagocytic function is THP-1 (monocytic cell line of human peripheral blood).

[0404] Has the effect of inhibiting tumor growth in vivo

[0405] 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.

[0406] In some exemplary embodiments, the antibody has therapeutic efficacy in inhibiting tumor growth in a mouse xenograft model of human non-small cell lung cancer cells.

[0407] In some exemplary embodiments, the tumor inhibition efficacy %TGI of the antibody BT-461 in the mouse xenograft model of HCC827-HGF cells after 21 days of administration reached 104.2%, which is better than that of Avanta.

[0408] In some exemplary embodiments, the antibody BT-461 has therapeutic efficacy in inhibiting tumor growth in a NSG mouse xenograft model of human pancreatic cancer cells.

[0409] In some exemplary embodiments, the tumor inhibition efficacy %TGI of the antibody BT-461 in the KP4 cell mouse xenograft model after 21 days of administration reached 73.2%, which is slightly better than that of Evanta.

[0410] In some embodiments, the EGFR / c-Met bispecific antibodies of the present invention can bind to tumor cells.

[0411] In some embodiments, the binding of the EGFR / c-Met bispecific antibody of the present invention to tumor cells is dependent on the antibody concentration gradient, and the binding levels are similar.

[0412] In some embodiments, the binding EC50 of BT461, BT461-MMAE, BT461-DXD, BT461-EX and MKN45 are comparable, which are 609.0 ng / mL, 599.8 ng / mL, 525.2 ng / mL, and 816.9 ng / mL, respectively. The binding activity of BT461-ADC is not significantly different from that of BT461 antibody.

[0413] In some embodiments, the EGFR / c-Met bispecific ADC of the present invention is capable of inhibiting the proliferation of human tumor cell lines and killing human tumor cell lines in vitro.

[0414] In some embodiments, the human tumor cell lines include human pancreatic cancer cells, human non-small cell lung cancer EGFR mutant cells, human gastric cancer cells and human pancreatic cancer KRas G12C mutant cell lines.

[0415] In some embodiments, the human tumor cell lines include human pancreatic cancer cells, non-small cell lung cancer EGFR mutant cells, non-small cell lung cancer EGFR mutant cells HCC827, human gastric cancer cells MKN45, and human pancreatic cancer KRas G12C mutant cell line MIAPACA-2.

[0416] Functionality of EGFR / c-Met bispecific ADCs

[0417] In some embodiments, the bispecific ADC of the present invention has the function of killing tumor cells in vitro and in vivo.

[0418] In some exemplary embodiments, in MKN45, HCC827, and H1975 cells, the killing effects of antibody-ADCs are as follows: the strongest killing effect is achieved with MMAE coupling, followed by exotecan coupling>=DXD coupling.

[0419] In some exemplary embodiments, in MIAPACA-2 cells, the killing effect of the antibody-ADC is conjugated with exitecan greater than DXD greater than MMAE greater than BT461.

[0420] In some exemplary embodiments, the ADC formed by coupling BT461 with MMAE, DXD, and exotecan has a much stronger killing effect on tumor cells in vitro than the naked BT461 antibody.

[0421] In some embodiments, the EGFR / c-Met bispecific ADC molecules of the present invention have bystander effect function while killing tumor cells.

[0422] In some exemplary embodiments, the BT461-ADC molecule does not have specific killing effect on the EGFR&c-MET negative cells Jurket. In a mixed system of EGFR&c-MET positive cells (HCC827 or MKN45) and negative cells (JURKET), BT461-ADC not only kills the positive cells, but also has a bystander effect on the negative cells.

[0423] In some exemplary embodiments, the BT461-ADC is BT461-MMAE.

[0424] Exemplarily, the BT461-ADC is BT461-DXD.

[0425] In some embodiments, the dual-antibody ADC of the present invention has an in vivo tumor-suppressing therapeutic effect.

[0426] In some exemplary embodiments, in the human HCC827-HGF tumor model, the tumor inhibition efficacy %TGI measured by BT461-MMAE at 5 mg / Kg body weight 21 days after administration is equivalent to twice the tumor inhibition efficacy (TGI) of BG461 21 days after administration, and is significantly higher than BT461. The tumor inhibition efficacy %TGI has better efficacy in inhibiting tumor growth than BT-46.

[0427] In some exemplary embodiments, in non-small cell lung cancer HCC827-HGF cells, the tumor inhibition efficacy (TGI) measured by 5 mg / Kg body weight of BT461-DXD and BT461-ixitecan 21 days after administration is equivalent to 1.4 times the tumor inhibition efficacy (TGI) of BT461 21 days after administration, which is significantly higher than BT461 and has better efficacy in inhibiting tumor growth than BT461.

[0428] Use in treatment and in the preparation of medicaments

[0429] The present invention also relates to the use of the EGFR / c-Met bispecific antibody of the present invention or an antibody-drug conjugate (ADC) formed by the EGFR / c-Met bispecific antibody and a drug module for preparing medicines for treating diseases.

[0430] 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 disease or specific pathology or alleviate its symptoms.

[0431] 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.

[0432] 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.

[0433] In some embodiments, the method of administering the drug comprises administering a therapeutically effective amount of the EGFR / c-Met bispecific antibody of the present invention to a patient in need thereof for a period of time sufficient to treat cancer.

[0434] In some embodiments, the cancer is resistant to or has acquired resistance to treatment with one or more EGFR inhibitors.

[0435] In some embodiments, the patient may be treated with the anti-EGFR antibody Taxinomab, or may have acquired resistance to such treatment.

[0436] Detection of elevated c-Met expression levels or increased c-Met activity, such as caused by elevated levels of circulating HGF, activating mutations of the c-Met gene, or c-Met gene amplification, can identify patients who are resistant to EGFR inhibitors.

[0437] In some embodiments, the cancer is associated with EGFR mutation, residue deletion, EGFR amplification, or c-Met amplification or mutation.

[0438] The EGFR / c-Met bispecific antibodies of the present invention can be used to treat any disease or disorder characterized by abnormal activation or production of EGFR, c-Met, EGF, soluble EGFR, soluble c-Met or other EGFR ligands or HGF, or a disorder associated with EGFR or c-Met expression, which may or may not involve malignancy or cancer, wherein abnormal 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.

[0439] 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 cancers and benign tumors.

[0440] 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 (

[0441] 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.

[0442] In some embodiments, the increased c-Met activity and / or expression level is caused by, but is not limited to, c-Met activating mutations, c-Met gene amplification, or HGF-mediated c-Met activation, or mutant KRAS

[0443] In some embodiments, the EGFR activating mutation that may be associated with cancer causes 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.

[0444] 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, wherein the increase in the c-Met-related biological activity includes but is not limited to tyrosine kinase activity, the formation of receptor homodimers and heterodimers, enhanced ligand binding, etc.

[0445] In another embodiment of the present invention, the experimental mouse model has a 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.

[0446] In another aspect, the present invention provides a method for treating a cancer patient, the method 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.

[0447] In some embodiments, the cancer is associated with wild-type EGFR and wild-type c-Met.

[0448] In some embodiments, the cancer is associated with wild-type EGFR and c-Met amplification.

[0449] In some embodiments, the patient has NSCLC associated with EGFR amplification and wild-type c-Met.

[0450] In some embodiments, the patient has NSCLC associated with EGFR amplification and c-Met amplification.

[0451] In some embodiments, the patient is treated with the EGFR / c-Met bispecific antibody or its ADC of the present invention. When the tumor is associated with L858R, T790M, del(E746, A750) EGFR, EGFR amplification, wild-type c-Met and / or c-Met amplification, BT461-MMAE, BT461-Dxd and BT461-exitecan of the present invention show efficacy in in vivo tumor animal models.

[0452] In one embodiment, the EGFR / c-Met bispecific antibody or ADC thereof of the present invention can be used to prepare a drug for treating non-small cell lung cancer (NSCLC).

[0453] In some embodiments, the cells of NSCLC have an epithelial phenotype.

[0454] In some embodiments, the NSCLC has acquired resistance to EGFR monoclonal antibodies.

[0455] In one embodiment, the EGFR monoclonal antibody is Taxin.

[0456] 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 suffering from a tumor or at risk of suffering from the 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.

[0457] Prior to initiating said treatment, said method preferably further comprises determining whether said subject suffers from such an EGFR, c-Met or EGFR / c-Met positive tumor.

[0458] 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.

[0459] In some exemplary embodiments, the cancer is non-small cell lung cancer.

[0460] In some exemplary embodiments, the cancer is gastric cancer.

[0461] In some exemplary embodiments, the cancer is pancreatic cancer.

[0462] Pharmaceutical composition

[0463] The present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention or its ADC as disclosed herein and a pharmaceutically acceptable excipient, which is used for treating susceptible people suffering from EGFR, c-Met or EGFR / c-Met positive tumors or at risk of the disease.

[0464] In some preferred embodiments, the pharmaceutical composition of the present invention can be preferably used to treat a patient population comprising a tumor, preferably an EGFR / c-Met positive cancer, preferably having an EGFR monoclonal antibody resistant phenotype or a tumor / cancer. In some preferred embodiments, the subject is preferably a subject suitable for antibody therapy using an EGFR specific antibody such as Nimotuzumab.

[0465] EGFR positive tumors are generally tumors with 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 for the cancerous state of the tumor. The reason why such tumors may be resistant to EGFR targeted therapy is through activation of the HGF / c-Met signaling pathway. The tumor may also be a tumor associated with HGF. Activation of the c-Met / HGF signaling pathway is one of the ways in which EGFR positive tumors may escape treatment with EGFR targeted therapy. The antibodies of the present invention are particularly suitable for treating HGF-related tumors or HGF-dependent tumors, tumors in which activation of the c-Met / HGF signaling pathway is associated with the presence or excess of HGF. Compared with HGF / c-Met signaling negative tumor cells, positive tumor cells in which the HGF / c-Met signaling pathway is activated have a more superior selective growth advantage.

[0466] In some embodiments, the pharmaceutically acceptable carrier or excipient is intended to include, but is not limited to, non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or any type of formulation auxiliary.

[0467] In some embodiments, the pharmaceutical composition is a dry, especially freeze-dried, composition which allows the production of an injectable solution after addition of sterile water or physiological saline.

[0468] In some embodiments, the pharmaceutical composition is prepared by dissolving or dispersing an effective amount of the antibody or immunoconjugate of the present invention in a pharmaceutically acceptable carrier or aqueous medium.

[0469] In one embodiment, the pharmaceutical composition in a pharmaceutical form suitable for injection includes sterile aqueous solutions or dispersions; and sterile powders for the ex situ preparation of sterile injectable solutions or dispersions.

[0470] 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, etc.), or suitable mixtures thereof.

[0471] 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.

[0472] 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, in particular for the treatment of cancer.

[0473] The bispecific antibody molecules, antibody-drug conjugates or pharmaceutical compositions described herein can be used in methods for treating human EGFR and / or c-Met positive related tumors or cancers. Related aspects of the present disclosure provide;

[0474] (i) the bispecific antibody molecule or conjugate described herein is used as a medicament;

[0475] (ii) a bispecific antibody molecule or conjugate as described herein for use in a method for treating a disease or disorder;

[0476] (iii) use of the bispecific antibody molecule or conjugate described herein in the manufacture of a medicament for treating a disease or disorder; and,

[0477] (iv) a method of treating a disease or disorder in an individual, wherein the method comprises administering to the individual a therapeutically effective amount of an antibody molecule or conjugate described herein.

[0478] 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 treatment that reduces the rate of progression, halts the rate of progression, ameliorates the condition of a disorder, cures or alleviates (whether partially or completely), prevents, ameliorates, delays, alleviates or arrests one or more symptoms and / or signs of a disorder, or prolongs the survival of an individual or patient beyond what would be expected in the absence of the disorder.

[0479] In one embodiment, the described treatment methods may include administering at least one additional therapeutic agent to an individual in addition to the bispecific antibody molecule or conjugate of the present invention. In one embodiment, the bispecific antibody molecule or conjugate of the present invention may be administered to an individual alone or in combination with one or more other treatments, wherein the additional treatment may be administered to the individual simultaneously, sequentially or separately with the administration of the antibody molecule or conjugate. When an additional treatment is administered simultaneously with the antibody molecule or conjugate, the antibody molecule and the additional treatment may be administered to the individual as a combined preparation. For example, the additional treatment may be a known therapy or therapeutic agent for the disease to be treated.

[0480] Although the antibody molecule can be administered alone, the antibody molecule or conjugate is usually administered in the form of a pharmaceutical composition, which may contain at least one component other than the antibody molecule or conjugate. Therefore, another aspect of the present disclosure provides a pharmaceutical composition comprising the antibody molecule or conjugate described herein. Also provided is a method comprising formulating the antibody molecule or conjugate into a pharmaceutical composition.

[0481] In addition to the antibody molecule or conjugate, the pharmaceutical composition may also contain a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. The precise nature of the carrier or other materials will depend on the route of administration, which may be by infusion, injection or any other suitable route.

[0482] In preferred embodiments, the antibody molecules or conjugates described herein can be used in methods for treating cancer.

[0483] Cancer can be characterized by the abnormal proliferation of malignant cancer cells.

[0484] In some embodiments, the cancer treated using the antibody molecules or conjugates 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.

[0485] In one embodiment, the cancer to be treated is non-small cell lung cancer (NSCLC).

[0486] In one embodiment, the cancer is gastric cancer.

[0487] In some embodiments, the cancer is pancreatic cancer.

[0488] In some embodiments, the treatment may include inhibiting cancer growth, including complete remission of cancer, and / or inhibiting cancer metastasis, and inhibiting cancer recurrence. Cancer growth generally refers to any of a variety of indicators that indicate changes within the cancer to a more developed form. The features disclosed in the foregoing description, or the attached claims, or the drawings, are expressed in their specific form or in means for performing the disclosed functions, or methods or processes for obtaining the disclosed results (as the case may be). These features may be used alone or in any combination to implement the present disclosure in its different forms.

[0489] Although the present disclosure has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when given the present disclosure. Therefore, the exemplary embodiments disclosed above are considered to be illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the present disclosure.

[0490] To avoid 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.

[0491] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0492] 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 specified integer or step or groups of integers or steps but not the exclusion of any other integer or step or groups of integers or steps.

[0493] 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 one particular value and / or to another 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%. Specific embodiments

[0495] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0496] The experimental methods in the following embodiments where specific conditions are not specified are performed under conventional conditions, or under conditions recommended by raw material or product manufacturers, or according to the experimental methods described in biotechnology textbooks such as Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, Contemporary Molecular Biology Methods, Cell Biology, etc. Reagents where specific sources are not specified are conventional reagents purchased through commercial channels.

[0497] The reagents and raw materials used in the present invention are commercially available.

[0498] Example 1 Combination, construction and expression of anti-c-MET / EGFR bispecific antibodies

[0499] 1.1 Amino acid sequences of anti-EGFR binding arm A and anti-c-MET binding arm B and fragments thereof:

[0500] The amino acid sequences of the heavy and light chains of the anti-EGFR antibody are as shown in Table 5, and the amino acid sequences of each segment of the heavy and light chains are shown in Table 6. The amino acid sequences of the anti-c-MET antibody and its heavy and light chains are as shown in Table 5, and the amino acid sequences of each segment of the heavy and light chains are shown in Table 7.

[0501] The encoding nucleic acids of binding arm A and binding arm B are shown in Table 8.

[0502] Table 6 Amino acid sequence of binding arm A

[0503]

[0504]

[0505] Table 7 Amino acid sequence of binding arm B

[0506]

[0507]

[0508] Table 8 Encoding nucleic acid of binding arm

[0509]

[0510]

[0511]

[0512] 1.2 Construction of plasmids for anti-c-MET / EGFR dual antibody

[0513] The construction of anti-c-MET / EGFR antibody is based on Roche's KIH (knob into hole) technology. The CH3 domain of the heavy chain of the anti-EGFR antibody was mutated using genetic engineering technology (L234A, L235A, S354C, T366W) to form a Knob structure, and the CH3 of the heavy chain of the anti-c-MET antibody was mutated (L234A, L235A, Y349C, T366S, L368A, Y407V) to form a Hole. The formation of heterodimers is guided by spatial complementarity and hydrophobic effects, and the interchain disulfide bonds are increased to reduce homodimer impurities, which can effectively prevent heavy chain mispairing; and the paired light and heavy chains are connected through 6 consecutive groups of GGGGS-linker sequences, solving the light chain mispairing problem of bispecific antibodies. Next, the nucleotide sequences of the two peptide chains 1# and 2# of the anti-c-MET / EGFR bispecific antibody were constructed into the PEE6.4 and PEE12.4 expression vectors, respectively, to obtain anti-c-MET and anti-EGFR single-arm antibody plasmids, respectively. The two plasmids were then ligated with SalI / NotI enzymes (NEB) to obtain a plasmid that co-expressed anti-c-MET and anti-EGFR, and the two nucleotide sequences were constructed into the same expression vector.

[0514] 1.3 Expression and purification of anti-c-MET / EGFR dual antibody

[0515] According to the manufacturer's instructions, the pHR plasmid DNA encoding the dual antibody protein was transiently transfected into expiCHO S cells (Thermo Fisher) using the ExpiFectamine CHO transfection kit (Gibco). The transfected ExpiCHO-S cells were placed in the ExpiCHO-S expression medium and cultured for 10 days, and the cell supernatant was collected. The obtained cell (CHO cells, Chinese hamster ovary cells, ATCC, CRL-2092) supernatant was centrifuged (6000×g, 10mins) and filtered (using a 0.45μm filter) for purification.

[0516] The purification conditions are as follows: affinity chromatography is used in the first step of BT461 purification, which can effectively remove some process-related impurities, such as HCP and residual DNA, and has a good removal effect on endotoxins; hydroxyapatite is used in the second step of fine purification, which can not only remove some process-related impurities, but also has a good removal effect on product-related impurities, and can effectively remove aggregation and degradation fragments in bispecific antibodies. The stock solution obtained after two steps of purification can meet the quality requirements of the stock solution.

[0517] In the first step of affinity chromatography, 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 an eluent at pH 3.8. The specific experimental steps are shown in Table 9.

[0518] Table 9 Affinity chromatography steps selected for the first capture step of purification of the EGFR / c-Met bispecific antibody BT461 of the present invention

[0519] step Buffer Column Volume (CV) balance 20 mM PB, 150 mM NaCl, pH 7.4 5 Loading Cell culture medium / balance 20 mM PB, 150 mM NaCl, pH 7.4 2 Rinse 1 20mM NaAC, 0.5M NaCl, pH 5.0 5 Rinse 2 20 mM NaAC, pH 5.0 4 Elution 20 mM NaAC, pH 3.8 6 CIP 0.2M NaOH 2 balance 20 mM PB, 150 mM NaCl, pH 7.4 2 save 20% ethanol 2

[0520] In the second step, hydroxyapatite (CHT) from Bio-Rad was used for fine purification. The pH and conductivity of the affinity elution solution were adjusted to be consistent with the balance solution of the CHT chromatography, and then the sample was loaded. The retention time was 5 minutes, and the loading capacity was 20 mg of protein per milliliter of filler. After the loading was completed, the column was equilibrated with the balance solution for 5 CV, and linear gradient elution was performed. The elution peaks were collected in segments. The specific experimental steps are shown in Table 10.

[0521] Table 10 Steps of the second fine purification of the EGFR / c-Met bispecific antibody BT461 of the present invention

[0522] step Buffer Column Volume (CV) balance 10mM PB pH7.0 5 Loading Adjust the pH of the loaded protein to be consistent with the equilibrium buffer Loading capacity 20mg / mL balance 10mM PB pH7.0 4 Elution 1 Linear gradient elution 0-50% B 30 Elution 2 20 mM Pb, 1 M NaCl pH 7.0 5 regeneration 400mM PB 4 CIP 0.5M NaOH 4

[0523] The obtained antibody solution was subjected to buffer exchange (buffer exchanged to PBS) and concentration through a 30KD ultrafiltration tube. The concentration of the obtained protein was tested by Nanodrop, and the purity was tested by HPLC-SEC.

[0524] BT461 was expressed and purified using the above-mentioned expression and purification method of the eukaryotic cell expression vector.

[0525] Example 2 BT461 targets and binds to EGFR and c-MET antigen proteins

[0526] 2.1 Targeting c-MET protein

[0527] c-MET-HIS (Bipsys, c-MET-H5227) was diluted to 2 μg / mL with coating solution (take 8 ml of 0.2 mol / L Na2CO3, mix 17 ml of 0.2 mol / L NaHCO3, add 75 ml of distilled water, and adjust the pH to 9.6), and the coating amount of 50 μL / well was added to the wells of the ELISA plate, and incubated at 37°C for 2 hours. Discard the liquid in the wells, and wash 5 times with a plate washer, 200 μL / time of washing solution (PBST, 0.05% Tween-20 added to the PBS diluent). Add 200 μL of blocking solution (PBST + 5% skim milk powder) to each well and incubate at 4°C overnight. Discard the liquid in the wells, and wash 5 times with a plate washer (tecan, model Hydrospeed) with 200 μL / time of washing solution. Add 50 μL of BT461 and Avanto (Johnson, batch number MAS0001) at a concentration of 20 μg / mL, 5-fold dilution, 7 gradient antibodies, and set up a blank control; incubate at 37°C for 60 minutes; discard the liquid in the wells, wash 5 times with a plate washer, 200 μL / time of washing solution. Add 50 μL of Goat-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution) secondary antibody to each well, incubate at 37°C for 60 minutes; discard the liquid in the wells, wash 5 times with a plate washer, 200 μL / time of washing solution. Add 50 μL / well of colorimetric solution and incubate at 37°C for 15 minutes. Add 50 μL / well of 2 mol / L H2SO4 to terminate the reaction, and read the OD450 value on the microplate reader. The results of ELISA-binding activity analysis are shown in Table 11 and Figure 1 As shown in A.

[0528] 2.2 Targeting EGFR protein

[0529] EGFR-HIS (Bipsys, EGR-H5222) was diluted to 2μg / mL with coating solution (take 8ml of 0.2mol / L Na2CO3, mix 17ml of 0.2mol / L NaHCO3, add 75ml of distilled water, and adjust the pH to 9.6), and the coating amount of 50μL / well was added to the wells of the ELISA plate, and incubated at 37°C for 2 hours. Discard the liquid in the wells, and wash 5 times with a plate washer, using 200μL / time of washing solution (PBST, 0.05% Tween-20 added to the PBS diluent). Add 200μL of blocking solution (PBST+5% skimmed milk powder) to each well and incubate at 4°C overnight. Discard the liquid in the wells, and wash 5 times with 200μL / time of washing solution using a plate washer (tecan, model Hydrospeed). Add 50 μL of anti-c-MET / EGFR dual antibody (BT461, Avanto) at a concentration of 20 μg / mL, 5-fold dilution, 7 gradient antibodies to each well, and set up a blank control at the same time; incubate at 37°C for 60 minutes; discard the liquid in the wells, wash 5 times with 200 μL / time of washing solution using a plate washer. Add 50 μL of Goat-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution) secondary antibody to each well, incubate at 37°C for 60 minutes; discard the liquid in the wells, wash 5 times with 200 μL / time of washing solution using a plate washer. Add 50 μL / well of colorimetric solution and incubate at 37°C for 15 minutes. Add 50 μL / well of 2 mol / L H2SO4 to terminate the reaction, and read the OD450 value on the microplate reader. The results of ELISA-binding activity analysis are shown in Table 11 and Figure 1 As shown in B.

[0530] Table 11 EC50 of the binding of the bispecific antibodies of the present invention to c-MET and EGFR proteins

[0531]

[0532]

[0533] From Table 11 and Figure 1 It can be seen that the antibody BT461 can specifically bind to the human c-MET protein, with an EC50 (the lower the EC50, the higher the binding level) of 18.93 ng / mL, which is lower than the binding level of the marketed drug Avantuo (EC50 is 9.291 ng / mL); at the same time, the results also showed that the antibody BT461 can specifically bind to the human EGFR protein, with an EC50 of 787.1 ng / mL, and the binding level to the EGFR protein is much lower than that of the marketed drug Avantuo (EC50 is 31.51 ng / mL).

[0534] Example 3 BT461 targets and binds to CHO-K1 cells that overexpress EGFR or c-MET

[0535] 3.1 BT461 targets CHO-K1 cells overexpressing c-MET membrane protein

[0536] In order to detect the specificity of the antibody of the present invention binding to the c-MET membrane protein on the cell surface, we stably overexpressed the human c-MET membrane protein in CHO-K1 cells (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 with PBS (twice); a blank control group and an experimental group were set up on the 96-well plate, and the antibody BT461 and Evantor (concentration of 20μg / mL) were added to the wells of the experimental group and diluted into 7 gradients with 5-fold concentration by gradient dilution method; the 96-well plate was placed at 4°C for incubation for 30mins, centrifuged (1000rpm, 2mins), and the liquid in the wells was removed. Then wash with PBS (200 μL / time, 2 times); add PE-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution, 50 μL) to each well, incubate at 4°C for 30 mins, centrifuge (1000 rpm, 2 mins), remove the liquid and wash with PBS (200 μL / time, 2 times); add PBS (200 μL / well) to each well, resuspend the cells, and read with a flow cytometer. The results of FACS-binding activity analysis are shown in Table 12 and Figure 2 As shown in A.

[0537] 3.2 BT461 targets CHO-K1 cells overexpressing EGFR membrane protein

[0538] 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 antibody of the present invention binding 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 Avanto (concentration of 20μg / mL) were added to the wells of the experimental group and diluted to 7 gradients with 5-fold concentration by gradient dilution method; the 96-well plate was placed at 4°C for incubation for 30mins, centrifuged (1000rpm, 2mins), and the wells were removed. After the liquid in the well was removed, the cells were washed with PBS (200 μL / time, 2 times); PE-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution, 50 μL) was added to each well, incubated at 4°C for 30 mins, centrifuged (1000 rpm, 2 mins), and the liquid was removed and washed with PBS (200 μL / time, 2 times); PBS (200 μL / well) was added to each well, the cells were resuspended, and the flow cytometer was used for reading. The results of FACS-binding activity analysis are shown in Table 12 and Figure 2 As shown in B.

[0539] Table 12 EC50 of the binding of the bispecific antibodies of the present invention to cells expressing c-MET and / or EGFR membrane proteins

[0540] EC50(ng / mL) Evanta BT461 CHO-K1 c-MET 47.35 117.1 CHO-K1 EGFR 764.1 3977 MKN45 269.7 609.0

[0541] The results showed that the antibody BT461 of the present invention bound to the EGFR or c-MET protein expressed on the surface of CHO-K1 cells, with EC50 of 3977 ng / mL and 117.1 ng / mL, respectively.

[0542] Example 4 Specific binding of BT461 to EGFR / c-Met double positive tumor cells MKN45 cells

[0543] In order to further detect the binding of the antibody of the present invention to tumor cells expressing dual targets, we digested and counted MKN45 (Shine Bio, catalog number SNL-173) cells 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 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 Avanto (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 and incubated for 30 minutes s, centrifuged (1000rpm, 2mins), removed the liquid in the plate wells and washed with PBS (200μL / time, 2 times); added PE-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution, 50μL) to each well, incubated at 4°C for 30mins, centrifuged (1000rpm, 2mins), removed the liquid and washed with PBS (200μL / time, 2 times); added PBS (200μL / well) to each well, resuspended the cells, and read with a flow cytometer. The results of FACS-binding activity analysis are shown in Table 12 and Figure 2 As shown in C.

[0544] The results showed that the binding of antibodies BT461 and Avanto to MKN45 was dependent on the antibody concentration gradient, with EC50 of 609.0 ng / mL and 369.7 ng / mL, respectively.

[0545] Example 5 Antagonizing the binding of ligands HGF and EGF to cell membrane proteins EGFR and c-MET

[0546] 5.1 Antagonize the binding of EGF to membrane protein EGFR:

[0547] MKN45 cells 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 EGF-mFC (concentration of 2 μg / mL, 25 μL) and anti-c-Met / EGFR antibody (concentration of 250 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients according to 5-fold concentration; the 96-well plate was placed at 4°C for 30 minutes, centrifuged (1000 rpm, 2 minutes), and the After the liquid was removed, the cells were washed with PBS (200 μL / time, 2 times); PE-anti-Mouse-IgG-Fc-Secondary-Antibody (50 μL, Jackson, 1:200 dilution) was added to each well, incubated at 4°C for 30 mins, centrifuged (1000 rpm, 2 mins), and the liquid was removed and washed with PBS (200 μL / time, 2 times); PBS (200 μL / well) was added to each well, the cells were resuspended, and the flow cytometer was used for reading. The results of FACS-binding activity analysis are shown in Tables 13 and 13 below. Figure 3 As shown in A.

[0548] 5.2 Antagonizing the binding of HGF to membrane protein c-MET

[0549] MKN45 cells 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 HGF-HIS (concentration of 5 μg / mL, 25 μL) and dual antibody BT461 and Evanta (concentration of 100 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients according to 5-fold concentration; the 96-well plate was placed at 4°C for 30 minutes, centrifuged (1000 rpm, 2 minutes), and the plate was removed. The liquid in the wells was then washed with PBS (200 μL / time, 2 times); 6xHis-Monoclonal-AntibodyFITC (25 μL, Invitrogen, diluted at a ratio of 1:100) was added to each well, incubated at 4°C for 30 mins, centrifuged (1000 rpm, 2 mins), and the liquid was removed and washed with PBS (200 μL / time, 2 times); PBS (200 μL / well) was added to each well, the cells were resuspended, and read using a flow cytometer. The FACS-analysis results are shown in Table 13 and Figure 3 As shown in B.

[0550] Table 13 Blocking HGF, EGF and MKN45 cell binding IC50

[0551] IC50(ng / mL) Evanta BT461 HGF 333.4 1070 EGF 1827 20005

[0552] From Table 13 and Figure 3A It can be seen that antibodies BT461 and Avantu can both block the binding of ligand EGF to receptor EGFR, and the blocking level (the lower the IC50, the higher the blocking ability) is ranked as follows: Avantu>BT461. The IC50 of antibodies BT461 and Avantu are 20005ng / mL and 1827ng / mL, respectively.

[0553] From Table 13 and Figure 3 B shows that antibodies BT461 and Avantu can both block the binding of ligand HGF to receptor c-Met, and the blocking levels (the lower the IC50, the higher the blocking ability) are ranked as follows: Avantu>BT461. The IC50 of antibodies BT461 and Avantu are 1070ng / mL and 333.4ng / mL, respectively.

[0554] Example 6 BT461 antibody specifically binds to cynomolgus monkey c-Met and EGFR proteins

[0555] The cross-species binding reaction was detected by ELISA. The recombinant proteins mouse EGFR-his, mouse c-Met-his, cynomolgus EGFR-his, cynomolgus c-Met-his, rat EGFR-his, and rat c-Met-hFC were diluted to 2 μg / mL with coating solution, and 50 μL / well was added to the wells of the ELISA plate and incubated at 37°C for 2 hours. The liquid in the well was discarded and washed 5 times with 200 μL / well of washing solution using a plate washer. 200 μL of blocking solution was added to each well and incubated overnight at 4°C. The liquid in the well was discarded and washed 5 times with 200 μL / well of washing solution using a plate washer. Add 50 μL of anti-c-Met / EGFR antibodies BT461, BT468, BT470, and control (Evantor, Johnson, batch number MAS0001) at a concentration of 20 μg / mL to each well, 5-fold dilution, 7 gradient antibodies, and set up a blank control at the same time; incubate at 37°C for 60 minutes; discard the liquid in the well, wash 5 times with 200 μL / time of washing solution using a plate washer. Add 50 μL of Goat-anti-human-IgG-Fc-Secondary-Antibody (1:10000 dilution) of the second antibody to each well (the secondary antibody used to detect rat c-Met-hFC cross is Goat-anti-human-Fab-Fc-Secondary-Antibody), incubate at 37°C for 60 minutes; discard the liquid in the well, wash 5 times with 200 μL / time of washing solution using a plate washer. Add 50 μL / well of color development solution and incubate at 37°C for 15 minutes. 50 μL / well 2 mol / L H2SO4 was added to terminate the reaction, and the OD450 value was read on a microplate reader. The summary results are shown in Table 14 below. BT461 did not cross-bind with mouse and rat EGFR&c-MET.

[0556] Table 14: Statistical table of species crossover, X represents no crossover, √ represents crossover.

[0557] name Amivantamab BT461 c-Met monkey cross √ √ EGFR monkey cross √ √ c-Met mouse cross × × EGFR mouse cross × × c-Met rat cross √ × EGFR rat cross × ×

[0558] Table 15 EC50 of the binding of the bispecific antibodies of the present invention to cynomolgus monkey c-Met and EGFR proteins

[0559] EC50(ng / mL) BT461 Cynomolgus monkey c-MET 15.10 Cynomolgus monkey EGFR 55.53

[0560] From Table 15 and Figure 4 It can be seen that the antibody BT461 of the present invention has cross-binding activity to cynomolgus monkey EGFR and c-Met, with EC50 of 55.53 ng / mL and 15.10 ng / mL, respectively.

[0561] Example 7 Detection of the affinity between the antibody of the present invention and the antigen based on biolayer interferometry (BLI)

[0562] 7.1 Binding kinetics of the bispecific antibody of the present invention against recombinant human EGFR-His

[0563] EGFR-His (ACRO, catalog number EGR-H5222) was diluted from 100nM in gradient dilution with PBST, and the gradient dilution method was used to dilute it 2-fold to 7 concentration points, and a zero concentration reference well was set. The antibody c-Met / EGFR bispecific antibody BT461 and the control antibody Avanto were diluted to 5μg / mL respectively. The molecular interaction instrument (Fortebio, manufacturer: Sartorius, model: Octet R8) was set up with the following operating conditions: temperature 30℃, shake speed 1000rpm. The coated AHC2 probe (Sartorius, catalog number 18-5142) was used to capture the antibody, with a capture time of 180s; the gradient diluted EGFR-HIS sample was bound for a binding time of 120s; the dissociation time was 300s; and the regeneration buffer (10mM glycine, PH 1.7) was used to regenerate 3 times, 30s each time. The 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 software to fit the ideal binding and dissociation curves, such as Figure 5 The equilibrium dissociation constant KD (kd / ka) between the antibody and antigen was calculated as shown in A and 5B. The results are shown in Table 16.

[0564] Table 16 EGFR-HIS affinity (AHC2 sensor)

[0565]

[0566] 7.2 Binding kinetics of the bispecific antibody of the present invention against recombinant human c-MET-His

[0567] As in Example 7.1, the binding constant (ka) and dissociation constant (kd) of BT461, Evanta and c-MET-HIS (Bipsys, c-MET-H5227) were analyzed using Octet BLI Analysis software to fit the ideal binding and dissociation curves, as shown in FIG. Figure 5 C and 5D, the equilibrium dissociation constant KD (kd / ka) between the antibody and antigen was calculated. The results are shown in Table 17. The affinity of BT461 to c-MET-his is 1.12nM, while the affinity to EGFR-HIS is 20nM, and the affinity of the two arms differs by nearly 20 times.

[0568] Table 17 c-MET-HIS affinity (AHC2 sensor)

[0569]

[0570] Example 8 Competition analysis of binding epitopes between BT461 and Evanta

[0571] 8.1 EGFR Epitope Competition Assay

[0572] The molecular interaction instrument ForteBio's Octet System (brand: Sartorius, model: Octet R8) was set up with the following operating conditions: temperature 30°C, shake speed 1000rpm. The sensor was placed on the sensor rack, and 200μl of buffer was added to the wells of the black 96-well plate corresponding to the sensor below, and the sensor was soaked for more than 10 minutes. After pre-wetting, the first baseline was balanced with Loading Buffer (1×PBS, pH7.4, with 0.02% Tween-20, 0.1% BSA), and hEGFR-His (ACRO, catalog number EGR-H5222) was diluted to 5μg / mL with Loading Buffer and loaded onto the sensor, solidified for 180s, and the second baseline was run after solidification. 100nM EGFR / c-MET bispecific antibodies 461 and Evant were prepared, and a zero concentration reference well was set at the same time. The coated HIS1K sensor (brand: Sartorius, catalog number: 18-5120) was used to bind the first antibody Avanto and the second antibody (Avanto or BT461) in sequence, with a binding time of 120s and a dissociation time of 300s. The ForteBio's Octet System (brand: Sartorius, model: Octet R8) was used for on-machine detection. The experimental results are shown in Figure 6As shown in A, after Avanto binds to EGFR-HIS, BT461 can still bind to it, indicating that there is no competition between BT461 and Avanto in the EGFR binding epitope.

[0573] Table 18 Analysis of the binding competition epitope between Evantu and BT461 molecules

[0574] Antibody EGFR protein binding epitope c-MET protein binding epitope Evanta and BT461 No competition compete

[0575] Referring to the experimental method of Example 8.1, we detected whether BT461 and Avanto competed on the c-MET binding epitope. The experimental results are as follows: Figure 6 As shown in B.

[0576] 8.2 c-MET epitope competition assay

[0577] From Table 18 and Figure 6 A shows that BT461 and Avanto do not compete for binding epitopes on EGFR; from Table 18 and Figure 6 B shows that BT461 and Avanta compete for binding epitopes on c-MET.

[0578] Example 9 BT461 Antibody Selection Specific Binding Evaluation

[0579] 9.1 FACS detection of EGFR and c-MET expression in cells

[0580] Well-growing tumor cells MKN45, human immortalized keratinocytes HACAT (Wuhan Sean Biotechnology, SNL-163), and human normal lung bronchial epithelial cell line BEAS-2B (Wuhan Sean Biotechnology, SNL-203) 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, an EGFR staining group, and a c-MET staining group were set up on the 96-well plate, 20 μg / ml IgG1 was added to the blank control group, 20 μg / ml anti-EGFR monoclonal antibody (cetuximab) was added to the EGFR staining group, and 20 μg / ml anti-c-MET monoclonal antibody (Evantor) was added to the c-MET staining group. Anti-c-MET arm monoclonal antibody), place the 96-well plate at 4°C for 30 minutes, centrifuge (1000rpm, 2 minutes), remove the liquid in the plate wells and wash with PBS (200μL / time, 2 times); add PE-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution, 50μL) to each well, incubate at 4°C for 30 minutes, centrifuge (1000rpm, 2 minutes), remove the liquid and wash with PBS (200μL / time, 2 times); add PBS (200μL / well) to each well, resuspend the cells, and read with a flow cytometer. The fluorescence intensity results of FACS detection are shown in Table 19 below:

[0581] Table 19 Evaluation of membrane expressed cMet protein level and EGFR protein level

[0582]

[0583]

[0584] The results showed that HACAT cells were c-MET low expression / EGFR high expression cells, BEAS-2B cells were also c-MET low expression / EGFR high expression cells, and tumor cells MKN45 were c-MET high expression / EGFR high expression cells.

[0585] 9.2 Evaluation of the selection index of BT461 for MKN45 & HACAT mixed cells

[0586] In order to further evaluate the selective binding level of the antibody of the present invention to EGFR / c-MET high-expressing tumor cells, we selected well-growing MKN45 and HACAT cells, digested them and counted them, and used the cell intracellular dye PB (Thermo, CellTrace TMViolet, C34571) were used to label MKN45, and then MKN45 and HACAT cells were mixed at a ratio of 1:10, 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 antibodies BT461, Evantor, and Taixinsheng were added to the wells of the experimental group at a concentration of 20 μg / mL 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, 2 minutes). , remove the liquid in the wells and wash with PBS (200 μL / time, 2 times); add PE-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution, 50 μL) to each well, incubate at 4°C for 30 mins, centrifuge (1000 rpm, 2 mins), remove the liquid and wash with PBS (200 μL / time, 2 times); add PBS (200 μL / well) to each well, resuspend the cells, and analyze by flow cytometry. The results of the antibody binding activity analysis with MKN45 and HACAT are shown in Table 20 and Figure 7 shown.

[0587] Table 20 Statistics of the selection index for MKN45 & HACAT mixed cells, selection index = normal cell EC50 / tumor cell EC50 * tumor maximum binding / normal cell maximum binding

[0588] Selection Index Tai Xinsheng Evanta BT461 Tumor & Skin Cell HACAT 0.67 1.80 7.26

[0589] The results showed that BT461 bound to the dual-target positive tumor MKN45 at a higher rate than that of binding to epidermal cell HACAT ( Figure 7 C), which is nearly an order of magnitude (10 times) different, and its specificity in selecting and binding tumor cells is stronger than that of Evantor ( Figure 7 B), the worst is Tai Xinsheng ( Figure 7 A). The selectivity indexes of the three antibodies, Taixinsheng, Avantu, and BT461 (selectivity index = normal cell EC50 / tumor cell EC50*maximum tumor binding / maximum normal cell binding) are 0.67, 1.80, and 7.26, respectively. The larger the index, the better the selectivity for binding to double-positive tumor cells.

[0590] 9.3 Evaluation of the Selective Index of BT461 for MKN45 & BEAS-2B Mixed Cells

[0591] Similarly, referring to Example 9.2, we also selected well-growing MKN45 and human normal lung bronchial epithelial cell line BEAS-2B cells, digested and counted them, and used the cell intracellular dye PB (Thermo, CellTrace TMViolet, C34571) were used to label MKN45, and then MKN45 and BEAS-2B cells were mixed at a ratio of 1:10, 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 antibodies BT461, Evantor, and Tysin were added to the wells of the experimental group at a concentration of 20 μg / mL 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, 2 minutes). ), remove the liquid in the wells and wash with PBS (200 μL / time, 2 times); add PE-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution, 50 μL) to each well, incubate at 4°C for 30 mins, centrifuge (1000 rpm, 2 mins), remove the liquid and wash with PBS (200 μL / time, 2 times); add PBS (200 μL / well) to each well, resuspend the cells, and analyze by flow cytometry. The results of the antibody binding activity analysis with MKN45 and HACAT are shown in Table 21 and Figure 8 shown.

[0592] Table 21 Statistics of the selection index of MKN45 & BEAS-2B mixed cells, selection index = normal cell EC50 / tumor cell EC50 * tumor maximum binding / normal cell maximum binding

[0593]

[0594] The results also showed that BT461 bound to the dual-target positive tumor MKN45 at a higher rate than that bound to the human normal lung bronchial epithelial cell line BEAS-2B cells ( Figure 8 C), with a difference of nearly 5 times, and its specificity in selecting and binding tumor cells is stronger than that of Evantor ( Figure 8 B), the worst is Tai Xinsheng ( Figure 8 A). The selectivity indexes of the three antibodies, Taixinsheng, Avantu, and BT461 (selectivity index = normal cell EC50 / tumor cell EC50*maximum tumor binding / maximum normal cell binding) are 0.91, 3.16, and 5.44, respectively. The larger the index, the better the selectivity for binding to double-positive tumor cells.

[0595] Example 10 Antibody BT461 inhibits EGFR autophosphorylation and c-Met autophosphorylation

[0596] To explore whether the anti-c-Met / EGFR bispecific antibody can inhibit the signaling pathways of c-Met and EGFR, we measured the changes in the phosphorylation levels of EGFR and c-Met, respectively.

[0597] 10.1 Western Blot Analysis

[0598] Non-small cell lung cancer A549 cells were seeded in 6-well plates (4×105 cells / well). After adherence, control and experimental groups were set up on the 6-well plates. In the medium containing 1% serum of 40 ng / mL EGF-mFC (Bipusys) + 100 ng / mL HGF-his (Bipusys), 50 μg / mL IgG1, Evantol, BT461, BT470 were added to the negative group, control group and experimental group (BT461, BT470) 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 reducing agent β-mercaptoethanol (Life Technologies)) was added to each well. Technologies), boiled at 95°C for 10 minutes to denature the protein; used Tris-SDS as the electrophoresis buffer, and subjected the denatured protein to electrophoresis (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 then the PVDF membrane was 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-CET 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). At room temperature, the cells were reacted with ECL substrate (Sangong, l115AA0118) for 1 min and developed using a chemiluminescence system.

[0599] The development results are as follows Fig. 9As shown in A, induction with different antibodies (50 μg / mL) for 15 min will not change the protein levels of EGFR and c-MET in A549 cells; stimulation with HGF-his (10 μg / mL) and EGF-mfc (100 μg / mL) for 15 min can activate the phosphorylation of EGFR and c-MET in A549 cells; on this basis, the addition of Avanto can significantly inhibit the intracellular phosphorylation levels of p-EGFR (Y1173) and pc-MET (Y1234 / 1235); while the addition of BT461 can only significantly inhibit the intracellular phosphorylation level of pc-MET (Y1234 / 1235), and slightly inhibit the phosphorylation level of p-EGFR (Y1173).

[0600] 10.2TR-FRET Cell Signaling Pathway Analysis

[0601] The EGFR phosphorylation (Y1068) assay kit from Bioauxilium (Cat. No. KIT-EGFRP-500, THUNDER TM Phospho-EGFR(Y1068)TR-FRET Cell Signaling Assay Kit) was used to detect the level of EGFR autophosphorylation.

[0602] Similarly, the c-Met phosphorylation (Y1234 / Y1235) assay kit from Bioauxilium (Cat. No. KIT-c-MetP-500, THUNDER TM Phospho-c-Met (Y1234 / Y1235) TR-FRET Cell Signaling Assay Kit) was used to detect the level of c-Met autophosphorylation.

[0603] The non-small cell lung cancer HCC827 cells grown to 80% confluence were washed with PBS, and trypsin / EDTA (0.25%, Gibco, catalog number 25200-072) was added, and the cells were resuspended in complete medium RPMI164010% FBS) after centrifugation. The cells resuspended in the medium (RPMI1640 / 10% FBS) were inoculated in a 96-well plate (costar catalog number 3599) (1×104 cells / well), incubated at 37°C for 6 hours, washed with PBS, and placed in starvation medium (RPMI1640 / 1% FBS) (Gibco, catalog number 72400047) for 6 hours; the 96-well plate containing cells was classified into a negative control group and an experimental group, and IgG1 (Sino-Bio HG1K), Avanto, and BT461 at a concentration of 50 μg / mL were added to the wells of the negative control group and the wells of the experimental group; pre-incubated at 37 degrees for 1 hour s, then add 10 ng / mL EGF-mFC (or 100 ng / mL HGF-his) to stimulate for 15 mins, add the lysis solution in the detection kit (50 μL / well), and shake (400 rpm) for 30 mins; transfer 18 μL of the mixture from each well to a white-bottom opaque plate (Costar, 3917), then add Detection Buffer (6 μL), react for 1 h at room temperature and protected from light, centrifuge (1000 rpm) for 2 mins, and detect using a TR-FRET microplate reader (PerkinElmer, model Envision-2105). The absorbance at 665 nm represents the total number of EGFR (Y1068) phosphorylation (or c-MET (Y1234 / 1235) phosphorylation levels), and the absorbance at 615 nm represents the total number of EGFR (or c-MET) proteins. The TR-FRET ratio is equal to the absorbance at 665 divided by the absorbance at 615 multiplied by 1000. The TR-FRET ratio is used to characterize the level of EGFR (or c-MET) autophosphorylation.

[0604] The results are as follows Fig. 9 As shown in B, the c-Met specific ligand HGF mediated strong c-Met autophosphorylation in HCC827 cells, and BT461 and Avanta significantly inhibited the level of c-Met autophosphorylation. Fig. 9 As shown in C, the EGFR-specific ligand EGF mediated the activation of strong EGFR autophosphorylation in HCC827 cells. The Taixinsheng antibody was able to inhibit the EGF-activated EGFR phosphorylation level, while the antibody BT461 almost completely retained the inhibitory function of its anti-EGFR antibody arm on EGF-activated EGFR phosphorylation, but its inhibitory ability was weaker than that of Avantu.

[0605] Example 11 Antibody BT461 mediates internalization and degradation of target protein

[0606] 11.1 Utilization Live-Cell Analysis System draws the internalization curve of antibody time gradient

[0607] Count the live cells of MKN45, resuspend the cells in 1640 medium containing 10% FBS and adjust the density to 5×104 / ml. Use a spray gun to add 100 μL of cells (5000 cells / well) to each well of a flat-bottomed 96-well culture plate and place in a CO2 incubator overnight to allow the cells to adhere. The next day, add the antibody to be tested and the pH probe ( Human Fabfluor-pH AntibodyLabeling Dye for Antibody Internalization, Sartorius, Cat. No. 4722) was prepared into 20 μg / ml concentration in 10% FBS medium, 30 μL antibody and 30 μL pH probe were mixed in a 1:1 ratio, and incubated in a 37°C incubator in the dark for 30 min. 50 μL of the incubated primary antibody-secondary antibody complex was added to the wells inoculated with cells to avoid the formation of bubbles. Place the cell culture plate in In the Live-Cell Analysis System, photos were taken every hour. Integrated software was used for data analysis, and graphs were drawn with time points as the X-axis and fluorescence intensity as the Y-axis.

[0608] The results are as follows Fig.10 As shown in A, anti-cMet-B12 (a bivalent antibody composed of BT461 anti-cMet monovalent and non-specific antibody B12), BT461, and Avanto promote the internalization of the target protein, and the internalization efficiency mediated by the bivalent antibody molecules is ranked as Avanto>BT461>anti-cMet-B12. At the same time, the results also showed that the internalization efficiency of the bivalent antibody molecule BT461 was stronger than that of the monoclonal antibodies against EGFR or c-Met: Taixinsheng and anti-cMet-B12, which indicates that the dual-target internalization mediated by the bivalent antibody molecule BT461 has a synergistic effect.

[0609] 11.2 Western blot analysis to detect the internalization and degradation levels of EGFR and c-MET

[0610] MKN45 cells were seeded in 24-well plates (2×105 cells / well). After adherence, control and experimental groups were set up on 6-well plates. In the medium with 1% serum, IgG1, Evantor, BT461, and BT470 at a concentration of 50 μg / mL were added to the negative group, control group, and experimental group (BT461, BT470) respectively. After reacting for 24 or 48 hours, RIPA-lysis buffer (150 μL, Bio-Time) was added and lysed at 0°C (on ice) for 30 mins. Loading buffer (containing reducing agent β-mercaptoethanol (Life Technologies)) was added to each well. Technologies), boiled at 95°C for 10 minutes to denature the protein; used Tris-SDS as the electrophoresis buffer, and subjected the denatured protein to electrophoresis (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 then the PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour; anti-c-MET antibody (invitrogen, RJ243364) or anti-EGFR antibody was added. The cells were incubated with anti-GAPDH antibody (ABCAM, ab52894) or anti-GAPDH antibody (Solarbio, k200057m) at 4°C overnight and washed with TBST (3 times, each time for 10 mins). 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, each time for 10 mins). At room temperature, the cells were reacted with ECL substrate (Sangon, l115AA0118) for 1 min and developed using a chemiluminescence system.

[0611] The results are as follows Fig.10 As shown in B, after treating MKN45 cells with antibodies Avantu and BT461 at a concentration of 50 μg / mL for 48 hours, a significant downregulation of EGFR total protein level was detected, and the extent of downregulation was comparable between BT461 and Avantu. However, after treating MKN45 cells with BT461 at a concentration of 50 μg / mL for 24 hours, a significant downregulation of c-MET total protein level was not observed, and a significant decrease in c-MET protein level was observed only after treating MKN45 cells with Avantu for 24 hours.

[0612] In summary, the internalization efficiency mediated by BT461 in MKN45 cells is lower than that of Avanta, but the degradation of target protein after internalization mediated by BT461 is stronger than that of Avanta.

[0613] Example 12 Inhibition of tumor cell line proliferation in vitro

[0614] In this example, multiple human tumor cell lines were selected, including human pancreatic cancer cell KP4 (Nanjing Kebai Biotechnology, catalog number CBP60541), non-small cell lung cancer EGFR mutant cell H1975 (EGFR L858R / T790M mutation, ATCC#CRL-5908), and non-small cell lung cancer EGFR mutant cell HCC827. KP4 can autosecrete HGF ligand, so we overexpressed HGF in H1975 and HCC827 cells (lentiviral transfection) and used CellCounting- The 3D kit (Novozyme, DD1102) was used to detect the effects of the antibodies of the present invention on the proliferation of these cell lines.

[0615] Tumor cells growing in the logarithmic phase were digested and inoculated into 96-well plates (2000 cells / well), and cultured overnight in complete medium. After removing the medium in the 96-well plate, 100 μL of anti-c-Met / EGFR antibody BT461 and control (Evantor) diluted with complete cell medium (initial concentration of 500 μg / mL) were added and diluted into 5 gradients with 2-fold concentration by gradient dilution method. After culturing in an incubator for 5 days in an atmosphere of 5% CO2 and a temperature of 37°C, an equal volume of CellCounting- 3D reagent (Vazyme, Norwegian, catalog number DD1102), oscillate and mix for 5 minutes until the cell clusters are completely lysed. After incubation for 25 minutes, 100 μL of the mixture was transferred to a white opaque plate and detected using a TR-FRET microplate reader (PerkinElmer, model, catalog number). The results are shown in Fig.11 As shown, the antibody BT461 of the present invention can significantly inhibit the proliferation of human tumor cells KP4, H1975-HGF (expressing HGF), and HCC827-HGF (expressing HGF) at multiple gradient concentrations, wherein the inhibition level of KP4 and HCC827-HGF cell proliferation is comparable to that of the marketed drug Avanta, while the efficacy in H1975-HGF is lower than that of Avanta.

[0616] Example 13 Synergistic effect in inhibiting cell proliferation: bispecific antibodies are stronger than combined use

[0617] Similar to the experimental method of "Example 12 In vitro inhibition of tumor cell line proliferation", this example selected multiple human tumor cell lines, including human gastric cancer c-Met gene amplified cells MKN45 (Shine Bio, catalog number SNL-173), non-small cell lung cancer EGFR mutant cells HCC827-HGF (transfected to overexpress human HGF) and NCI-H1975-HGF (transfected to overexpress human HGF), and used CellCounting- 3D kit (Novozyme, DD1102) was used to detect the effect of the antibodies of the present invention on the proliferation of these cell lines. In short, these cells were exposed to therapeutic antibodies diluted with complete cell culture medium: BT461, Taixinsheng & anti-cMet arm-B12 (BT461 anti-cMet monovalent and inactive antibody B12 form a double antibody) with an initial concentration of 500 μg / mL (when used in combination, the equivalent amount of monovalent antibody was converted), and the gradient dilution method was used to dilute the concentration by 2 times into 5 gradients. After culturing in an incubator for 5 days in an atmosphere of 5% CO2 and a temperature of 37°C, an equal volume of CellCounting- 3D reagent (Vazyme, Norwegian, catalog number DD1102) was shaken and mixed 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, catalog number).

[0618] The results are as follows Fig.12 As shown, in the three types of cells, MKN45, HCC827-HGF, and NCI-H1975-HGF, the ability of BT461 dual antibody to inhibit tumor cell activity is also stronger than its related monoclonal antibody combination (Taixinsheng & BT461 anti-cMet arm-B12), which indicates that BT461 has a synergistic effect in inhibiting tumors in vitro.

[0619] Example 14 Evaluation of Antibody-Dependent Cellular Cytotoxicity (ADCC) Mediated by the Antibodies of the Present Invention

[0620] Enriched NK cells (Shanghai Miaoshun Biotechnology) were used to detect whether antibodies BT461 and Avantu mediated antibody-dependent cellular cytotoxicity (ADCC).

[0621] NK cell preparation: NK cells were revived, resuspended in complete medium (1640 + 10% FBS), and then added with hIL2 at a final concentration of 10 ng / mL and cultured overnight (the storage concentration of hIL2 was 400 μg / ml, i.e., 1:40,000);

[0622] Target cell labeling: Use PERKINELMER kit: Lysis buffer, 4005-0010; Eu-solution, C135-100; DELFIA BATDA reagent, C136-100 to label target cells. Collect H1975 cells in the logarithmic growth phase ( Fig.13 A) or HCC827( Fig.13B) Cells were washed once with culture medium, then resuspended to adjust the cell density to 1E6 cells / ml, 1 mL was added with 1.7 μL of BATDA, incubated at 37°C for 30 min, and then washed twice with PBS+20 mM HEPES, each time at 1100 rpm for 4 min.

[0623] Target cell plating: Resuspend the labeled cells in 1640 complete medium (add 1mM sulfinpyrazone), adjust the cell density to 1E5 cells / ml, take the cell suspension, plate, as shown in the table below, add 100μL cells to each well in a V-bottom transparent 96-well plate. Preparation of sulfinpyrazone: Take 404.48mg sulfinpyrazone powder + 5mL acetone, that is, 200mM sulfinpyrazone, filter with a 0.22μM filter, it is a suspension, not completely dissolved.

[0624] Antibody loading: dilute the antibody to the corresponding concentration (1 μg / ml), add 50 μL per well to the corresponding well plate; mix well and incubate at 37°C, 5% CO2 for 30 min;

[0625] NK plating: NK cells were collected and the cell density was adjusted to 2.5E5 cells / mL with 1640 complete medium (1mM sulfinpyrazone added). 100μL was added to each well of G4-G23 group (efficacy-target ratio 2.5:1); 250μL was added to G1 well, 150μL was added to G2 and G3 wells, and 50μL was added to G4 well. 25μL of lysis solution (HBSS+0.5% TritonX-100) was added to the maximum release well (G3). Incubate at 37°C, 5% CO2 for 4h.

[0626] Machine detection: After the incubation time is over, take out the 96-well plate and equilibrate it to room temperature, centrifuge it at 1500rpm for 5 minutes, aspirate 20μl of supernatant into a PE-bottomed transparent white plate, add 200μl of europium, shake it in the dark for 15 minutes, and measure TRF. The detection program is: Delay (μm) 600, Window time 100, Number of flash 200.

[0627] The cell killing toxicity percentage (%) was calculated as follows: (TRF value of the experimental group - TRF value of the spontaneous pore) / (TRF value of the maximum release pore - TRF value of the spontaneous pore)*100.

[0628] The results are as follows Fig.13As shown, in H1975 cells and HCC827 cells, the antibody BT461 of the present invention has the ability to mediate NK cytotoxicity (ADCC effect) on target cells, but the ADCC effect mediated by the BT461 antibody is lower than that of Avanta. The killing rates of BT461 and Avanta mediated NK on H1975 cells are 23.9% and 27.8%, respectively, and the killing rates of BT461 and Avanta mediated NK on HCC827 cells are 25.9% and 31.7%, respectively.

[0629] Example 15 The antibodies of the present invention mediate antibody-dependent phagocytosis (ADCP)

[0630] Enriched human macrophages (Shanghai Miaoshun Biotechnology) were used to detect whether the antibody BT461 mediated the ADCP effect in vitro.

[0631] Prepare human macrophages: Resuscitate human macrophages and inoculate them in T25 culture flasks. Harvest macrophages: Aspirate the cell culture medium, wash twice with PBS, and completely aspirate the remaining culture medium. Add 2 ml of pancreatin containing EDTA, incubate at 37°C for 5 minutes, observe the cells become round under a microscope, use a 1 ml pipette to repeatedly blow for 1 minute, add 4 ml of complete culture medium to terminate digestion, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, wash once with PBS, and centrifuge at 1000 rpm for 5 minutes.

[0632] Macrophage labeling: Use PBS to adjust the density of harvested macrophages to 1E6 cells / ml, add 1μl CTV per ml, incubate at 37°C for 5 minutes, and then use 2 times the volume of complete medium to terminate staining. Macrophage plating: After cell centrifugation, use 1640 complete medium to adjust the density to 2E5 cells / ml, take the cell suspension as shown in the table below, and add 50μl cells per well to a round-bottom transparent 96-well plate.

[0633] Antibody loading: dilute the antibody to the corresponding concentration, from low to high concentration, add 50 μl to each well of the corresponding well plate, add 50 μl 1640 culture medium to the G1 well, and place it in the incubator for pre-incubation for 30 minutes.

[0634] Target cell labeling: H1975 cells were collected and the cell density was adjusted to 1E6 / ml using PBS. 10 μl pHrodo was added per ml. TM The Red AM probe was incubated at 37°C for 30 min, and then 2 volumes of PBS were added at 1000 rpm for 5 min to wash away the free probe.

[0635] Target cell plating: Use 1640 complete medium to adjust the density of labeled H1975 cells to 1E6 / ml

[0636] Take the cell suspension as shown in the table above, add 50 μl cells to each well of a round-bottom transparent 96-well plate, mix well and place at 37°C, 5% CO2 for incubation for 2.5 hours.

[0637] Cell fixation: After the incubation time is over, the cell plate is centrifuged at 1500 rpm for 5 minutes, the supernatant is discarded, 100 μl of cell fixative is added to each well, and the cells are incubated at room temperature in the dark for 20 minutes. The cell plate is then centrifuged at 1500 rpm for 5 minutes, the supernatant is discarded, 100 μl of PBS is added to each well, and the cells are resuspended.

[0638] On-machine detection: Each well needs to be gently blown 3-5 times before being put on the machine. The software is set to shake the plate once every 2 wells and collect 50μl of sample.

[0639] Calculation: The number of PB450 and PE double positive cells was detected by flow cytometry, and the phagocytic percentage was the sum of the number of double positive cells divided by the number of PB450 positive cells.

[0640] The results are as follows Fig.14 As shown, the antibody BT461 of the present invention has the ability to mediate the phagocytosis (ADCP effect) of macrophages on HCC827 cells, but the ADCP effect mediated by the BT461 antibody is lower than that of Avantol.

[0641] Example 16 In vivo tumor efficacy study of bispecific antibodies

[0642] 16.1 NSG mouse xenograft model of HCC827-HGF cells

[0643] The in vivo antitumor activity of the anti-bispecific antibodies was evaluated in a mouse xenograft model of human non-small cell lung cancer HCC827-HGF cells (NSG, Vital River, 6 weeks old, female).

[0644] Human HCC827-HGF cells were propagated as monolayer cultures in vitro using RPM1640 + 10% FBS as complete culture medium. The cells in the logarithmic growth phase were digested and resuspended in serum-free culture medium / Matrigel (1:1) to obtain a suspension. The cells were inoculated at 5×106 / mouse on the right side of the back of the mouse subcutaneously (sc) to form tumors. Ten days after implantation, the mice were divided into three groups according to the tumor volume, and the average tumor volume in each group was about 80-100mm3, and drug administration began. IgG1 or antibody molecules were administered intravenously (IV) twice a week for a total of three weeks.

[0645] The experimental results are as follows Fig.15As shown, in the HCC827-HGF tumor model, BT461 has the therapeutic effect of inhibiting tumor growth, and the therapeutic effect is equivalent to that of Avantuo. The tumor inhibition efficacy % TGI measured 21 days after administration is shown in Table 22 below: The TGI of BT461 and Avantuo are 101.3% and 104.2%, respectively.

[0646] Table 22 Antitumor efficacy of BT461 molecule in tumor model HCC827-HGF %TGI

[0647] Tumor model HCC827-HGF (EGFR exon 19 deletion mutation) Evanta BT461 TGI 101.3％ 104.2％

[0648] 16.2 KP4 cell NSG mouse xenograft model

[0649] KP4 can autosecrete HGF protein. Similar to Example 16.1, the in vivo anti-tumor activity of the anti-bispecific antibody was evaluated in a mouse xenograft model of human pancreatic cancer cells KP4 (NSG, Vital River, 6 weeks old, female).

[0650] The results are as follows Fig.16 As shown, in the KP4 tumor model, BT461 has the therapeutic effect of inhibiting tumor growth, and the therapeutic effect is at least equivalent to that of Avanta (slightly better than that of Avanta). The tumor inhibition efficacy % TGI measured 21 days after administration is shown in Table 23 below: The TGI of 5 mg / kg BT461 and Avanta are 61.4% and 73.2%, respectively.

[0651] Table 23 Tumor inhibition efficacy of BT461 molecule in tumor model KP4 %TGI

[0652] Tumor Model KP4 Evanta 5mg / kg BT461 1mg / kg BT461 5mg / kg TGI 61.4％ 61.1％ 73.2％

[0653] Example 17 ADC molecule construction of BT461

[0654] MMAE, DXD, and ixotecan-linker were connected to the cysteine ​​of the BT461 molecule using the thiol coupling method.

[0655] Thiol coupling method:

[0656] 50mmol / L TCEP: molecular weight 286.65, prepared with pure water, dispensed into 100μL / tube, stored at -20℃ away from light, one tube can be stored at 4℃ to avoid repeated freezing and thawing.

[0657] 10mmol / L drug-linker (Linker-drug), using DMSO solution, is prepared according to the specific drug-linker molecular weight. The molecular weight of drug-linker MC-GGFG-Dxd (Sino-US Huashitong Biopharmaceutical Technology Co., Ltd., batch number WS2434-004-01, purity 99.8%) is 1034.2 ( Fig.17 ), the molecular weight of drug-linker MC-VC-PAB-Exatecan (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., product number HY-145929, purity 99.55%) is 1034.1 ( Fig.18 ), dispense into 100 μL / tube, store at -80℃, take out before use, and avoid repeated freezing and thawing.

[0658] 5 mg / mL antibody solution: dissolved in phosphate buffer, pH about 7.0.

[0659] Phosphate buffer (chromatographic eluent): 6.7mmol / L disodium hydrogen phosphate (molecular weight 141.96), 3.3mmol / L sodium dihydrogen phosphate monohydrate (molecular weight 137.99), 137mmol / L sodium chloride (molecular weight 58.44). pH 7.0, conductivity 15-16mS / cm.

[0660] DMSO solution at room temperature

[0661] 15mL centrifuge tube, 1mL pipette, 200μL pipette, 10μL pipette

[0662] ADC molecule preparation process:

[0663] Antibody thiol reduction: add 3 mL of 5 mg / mL antibody solution to a 15 mL centrifuge tube; add 50 mmol / L TCEP aqueous solution under continuous stirring at room temperature. Here, the TCEP solution can be sucked up with a gun tip, and TCEP is slowly added while stirring with the gun tip for 1 min to obtain a uniform solution. The TCEP: antibody feed ratio is 6.5 equiv, and 0.013 μL of TCEP aqueous solution should be added. Place in a 37°C incubator for reaction for 2 h;

[0664] The feed ratio of drug-linker:antibody is 15 equiv, 150 μL of 10 mmol / L drug-linker is taken out and restored to liquid state; 10 mmol / L drug-linker is diluted to 5 mmol / L with DMSO solution at room temperature;

[0665] Coupling: Take out the antibody reaction solution, add 5% DMSO solution by volume, and continue stirring for 1 min; add the drug-linker solution into the reaction solution in 5 portions, stirring for 1 min to obtain a uniform solution; place in a 25°C water bath and react for 3 hours; after the reaction is completed, take out the reaction solution and filter it by gel chromatography.

[0666] Gel chromatography filtration (applicable to Suzhou Saipu SCG-030): Take out the column, 1M and 10mM NAOH, and eluent; turn on the machine, turn on the computer, and open the software; click manual control, insert the inlet pump, inlet valve (load), column valve (forward), and flow rate (20ml / min); place the selected inlet pump in the 1M NAOH solution, click execute, and start flushing the pipeline; flush about 50mL until the conductivity curve is flat; place the inlet tube in the eluent to balance until the pH becomes the pH of the eluent (7); set the flow rate to 1ml / min, set the pressure alarm to 0.3Mpa, and execute; remove the closed connecting tube, connect the column, first connect the column port, and screw the column head separately; set the flow rate to 5ml / min, and execute until the pH is the eluent pH (7); end the program, click manual control to start a new program, and insert: sample record, inlet Pump, inlet valve (load), column valve (forward), flow rate (2.55ml / min), pressure alarm, planned pause volume (10ml), execute; before loading, pause, mark the sample, put the pump into the sample, start loading (note to pause in time after washing the sample to avoid inhaling bubbles), pause at the end of loading, rinse the tail of the pump, put the pump into the eluent, and continue loading; when UV is 30, pause, mark the start of collection, rinse the tail of the collection pump, put it in the collection tube, continue, if the conductivity starts to drop (less than 17), pause, if the conductivity does not drop, UV drops to 13, pause, after pausing, put the collection pump into the waste liquid bucket, mark the end of collection, continue, balance the column until the conductivity is flat, and collect the next sample; after the sample collection is completed, pause, rinse the loading pump, put in 1M NAOH, continue until the conductivity rises and levels off (about 200), pause, flush the sample pump, put it in 10mM NAOH, continue until the conductivity drops to a level (about 2), you can set the flow rate appropriately, and keep the pressure in front of the column not exceeding 0.3Mpa; end the program, remove the column, insert the sealing tube, flush the sample pump, put it in ethanol, restart a program, insert, inlet pump, inlet valve (load), column position valve (forward), flow rate (20ml / min), execute until the conductivity is 0; exit the software, shut down the instrument, shut down the computer, and take away the column.

[0667] Concentration of ADC: In a sterile clean bench, aseptically filter the chromatographic collection liquid with a 0.22μm sterile filter; concentrate the filtered ADC solution in batches, select concentration tubes according to the chromatographic collection liquid and the final concentrated volume, and select 50mL, 15mL, and 1.5mL concentration tubes. The size of the concentration tube can be 30Kd and 50Kd; centrifuge at 4000rpm for 15min; after each concentration, the waste liquid needs to be retained, and the protein concentration of the filtrate is tested on Nanodrop. The concentration below 0.02mg / mL is normal. If a higher concentration of filtrate appears, it indicates that the filter membrane of the concentration tube is damaged and the filtrate needs to be re-concentrated; repeat the concentration operation until it is concentrated to the target volume and concentration. Generally, a concentration of about 5mg / mL can meet the in vivo efficacy and safety assessment requirements.

[0668] Concentration, purity, DAR value determination:

[0669] Nanodrop was used to measure the concentration, which could obtain the results quickly and easily, but the results fluctuated. The UV method was used to measure the protein concentration, 1.5 μL at a time, 3 times, and if there were any abnormal data, it was repeated. The absorbance coefficient (JH021, 1.5) was input, and the concentration, A280 and A360 values ​​were recorded.

[0670] The concentration is measured by ultraviolet spectrophotometer, which requires a large sample and takes a long time to test, but the result is more accurate. The total volume is 3mL and the absorbance value is more reliable between 0.2-0.8. Therefore, 0.6mg of ADC stock solution is generally the most suitable. The concentration needs to be calculated using the absorbance coefficient.

[0671] SEC-HPLC measures the purity of ADC products. It can detect the purity of aggregates, fragments, and monomers of ADC. The volume needs to be above 100 μL and the total amount needs to be above 0.2 mg.

[0672] The average DAR value is measured by UV spectrophotometer. The antibody has the highest absorption peak at A280. Different drug-linkers have different absorption peaks. For the first measurement, a full spectrum scan is required with the drug-linker solution to obtain the highest absorption peak of the drug-linker. After measurement, the highest absorption peak of the MC-GGFG-Dxd drug-linker is at A360, and the highest absorption peak of the MC-VC-PAB-Exatecan drug-linker is at A370. The next step is to calculate the absorption coefficients of the antibody and drug-linker at A280, A360 or A370 in advance based on the known concentrations of naked antibody and drug-linker to obtain the absorption coefficient. When measuring ADC, the antibody concentration and drug concentration are calculated using the following formula. The ratio of drug to antibody concentration is the DAR value of ADC:

[0673] A280 and A360 represent the absorbance of the antibody-drug conjugate aqueous solution at 280 nm and 360 nm, εA,280 and εA,360 represent the molar absorptivity of the antibody at 280 nm and 360 nm, εD,280 and εD,360 represent the molar absorptivity of the drug-linker at 280 nm and 360 nm, CA represents the antibody concentration, and CD represents the drug-linker concentration.

[0674] A280=εD,280CD+εA,280CA

[0675] A360=εD,360CD+εA,360CA

[0676] After calculation, the coefficients of JH021-Dxd are, εA,280=210600, εA,360=0, εD,280=5280, εD,360=20190;

[0677] The coefficients of JH021-Exatecan are εA,280=210600, εA,360=0, εD,280=5280, εD,370=19560.

[0678] HIC-HPLC determines the DAR value of ADC products, which can display five peaks: DAR 0 (naked antibody), 2, 4, 6, and 8. The ratio of ADCs with different DAR values ​​is calculated. The volume must be above 200 μL and the concentration must be above 1 mg / mL.

[0679] Example 18 Evaluation of ADC molecule binding activity of bispecific antibody BT461

[0680] Similar to the experimental method of Example 4 of the present invention, we digested and counted MKN45 cells 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 with PBS (twice); a blank control group and an experimental group were set up on the 96-well plate, and antibodies BT461, BT461-MMAE, BT461-DXD, and BT461-EX (exitecan) (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 and incubated. Centrifuge (1000rpm, 2mins), remove the liquid in the plate wells and wash with PBS (200μL / time, 2 times); add PE-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution, 50μL) to each well, incubate at 4°C for 30mins, centrifuge (1000rpm, 2mins), remove the liquid and wash with PBS (200μL / time, 2 times); add PBS (200μL / well) to each well, resuspend the cells, and read with a flow cytometer. The results of FACS-binding activity analysis are shown in Table 24 and Fig.17 shown.

[0681] The results showed that the binding of antibodies BT461, BT461-MMAE, BT461-DXD, and BT461-EX to MKN45 was dependent on the antibody concentration gradient, and the binding levels were similar, with EC50 of 609.0 ng / mL, 599.8 ng / mL, 525.2 ng / mL, and 816.9 ng / mL, respectively. In summary, there was no significant difference in the binding activity of BT461-ADC and BT461 antibody.

[0682] Table 24 EC50 of binding of BT461-ADC molecules to cells expressing c-MET and / or EGFR membrane proteins

[0683] EC50(ng / mL) BT461 BT461-MMAE BT461-DXD BT461-EX MKN45 combination 600.2 599.8 525.2 816.9

[0684] Example 19 Study on the in vitro killing efficacy of the ADC molecule of the bispecific antibody BT461

[0685] In this example, multiple human tumor cell lines were selected, including human pancreatic cancer cell non-small cell lung cancer EGFR mutant cell H1975, non-small cell lung cancer EGFR mutant cell HCC827, human gastric cancer cell MKN45, and human pancreatic cancer KRas G12C mutant cell line MIAPACA-2 (Shine Bio, SNL-164). CellCounting- The 3D kit (Novozyme, DD1102) was used to detect the effect of the antibody ADC of the present invention on the proliferation of these cell lines.

[0686] The experimental method is similar to that of Example 12 and Example 13, except that the drug concentration and treatment time are changed. Take tumor cells growing in the logarithmic phase, digest them and inoculate them in a 96-well plate (2000 cells / well), and culture them in complete culture medium overnight; after removing the culture medium in the 96-well plate, add 100 μL of antibodies BT461, BT461-MMAE, BT461-DXD, BT461-EX (initial concentration 150 μg / mL) diluted with complete cell culture medium and use gradient dilution method to dilute them into 7 gradients with 5-fold concentration, culture them in an incubator under the conditions of 5% CO2 atmosphere and 37°C for 3 days, and then add an equal volume of CellCounting- 3D reagent (Vazyme, Norwegian, catalog number DD1102) was shaken and mixed 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, catalog number).

[0687] The results are shown in Figure 18. In MKN45, HCC827, and H1975 cells, the killing effects of antibody-ADC are as follows: MMAE-coupled has the strongest killing effect, followed by exotecan-coupled>=DXD-coupled, and the worst effect is BT461 naked antibody. In MIAPACA-2 cells, the killing effects of antibody-ADC are as follows: exotecan-coupled>DXD>MMAE>BT461. In general, the killing effect of BT461 coupled with MMAE, DXD, and exotecan to form ADC on tumor cells in vitro is much stronger than that of BT461 naked antibody.

[0688] Example 20 Detection of bystander effect of ADC molecule of bispecific antibody BT461

[0689] 20.1 Detection of BT461 ADC molecules killing EGFR&c-MET negative cells Jurket

[0690] Referring to the experimental method of Example 19 of the present invention, except that the drug concentration is different, tumor cells growing in the logarithmic phase are taken, digested and inoculated into a 96-well plate (2000 cells / well), and cultured overnight in complete culture medium; after removing the culture medium in the 96-well plate, 100 μL of antibodies BT461, BT461-MMAE, BT461-DXD (initial concentration 15 μg / mL) diluted with complete cell culture medium are added and diluted into 7 gradients with 5-fold concentration by gradient dilution method, and cultured in an incubator for 3 days in an atmosphere of 5% CO2 and a temperature of 37°C, an equal volume of CellCounting- 3D reagent (Vazyme, Norwegian, catalog number DD1102) was shaken and mixed 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, catalog number).

[0691] The experimental results are as follows Fig.19 As shown in A, the BT461-ADC molecule does not have specific killing effect on the EGFR&c-MET negative cells Jurket.

[0692] 20.2 Detection of Bystander Effects of ADC Molecules

[0693] Positive cell staining: HCC827 cells or MKN45 cells growing in the logarithmic phase were taken, the digested cells were washed once with PBS, and then resuspended in PBS solution at a cell concentration of 2E6 / mL, 50μM CFSE dye (invitrogen, catalog number 65-0850-84) was added, and the cells were stained at 37°C for 5 minutes; then the staining was immediately terminated with 3-5 times the volume of 1640 culture medium + 10FBS, and then washed twice with PBS, and finally resuspended in complete culture medium at a cell concentration of 2.5E5 / mL.

[0694] Negative cell JURKET preparation: Centrifuge JURKET cells growing in the logarithmic phase and resuspend in complete medium at a cell concentration of 1E5 / mL.

[0695] Experimental groups: negative cell group (JURKET), HCC827 & JURKET mixed cell group, MKN45 & JURKET mixed cell group; 2.5E5 HCC827, MKN45 cells and / or 1E5 Jurkat cells were added to each well of a 6-well plate according to the experimental group requirements.

[0696] Drug administration: divided into a drug administration group and a blank control group, BT461-ADC was added to the drug administration group at a drug concentration of 100 nM, and the drug was added immediately after cell plating; treated in a 37°C incubator for 72 hours.

[0697] Detection and calculation: The total number of cells in each well was detected by cellcount instrument, and then the proportion of CFSE-positive cells was detected by flow cytometry, and then the number of CFSE-positive cells and CFSE-negative cells were calculated. The number of CFSE-positive cells corresponds to the number of living cells of HCC827 and MKN45, and the number of CFSE-negative cells corresponds to the number of living cells of JURKET.

[0698] The results are as follows Fig.19As shown in B, in the mixed system of EGFR&c-MET positive cells (HCC827 or MKN45) and negative cells (JURKET), BT461-MMAE and BT461-DXD not only have a killing effect on positive cells, but also have a bystander effect on negative cells.

[0699] Example 21 In vivo tumor treatment effect of dual antibody BT461-MMAE

[0700] The experimental method of this example is the same as that of Example 16, except that the administration method is intraperitoneal administration and the administration frequency is once a week.

[0701] The in vivo antitumor activity of anti-BT461-MMAE was evaluated in a mouse xenograft model of human non-small cell lung cancer HCC827-HGF cells (NSG, Vitallife, 6 weeks old, female).

[0702] Human HCC827-HGF cells were propagated as monolayer cultures in vitro using RPM1640 + 10% FBS as complete culture medium. The cells in the logarithmic growth phase were digested and resuspended in serum-free culture medium / Matrigel (1:1) to obtain a suspension. The cells were inoculated at 5×106 / mouse on the right side of the back of the mouse subcutaneously (sc) to form tumors. Ten days after implantation, the mice were divided into three groups according to the tumor volume, and the average tumor volume in each group was about 80-100mm3, and drug administration began. IgG1 or antibody molecules were administered intraperitoneally (IP) once a week for a total of three weeks.

[0703] The experimental results are as follows Fig. 20 As shown, in the HCC827-HGF tumor model, BT461-MMAE has better efficacy in inhibiting tumor growth than BT461. The tumor inhibition efficacy %TGI measured 21 days after administration is shown in Table 25 below.

[0704] Table 25 Antitumor efficacy of ADC molecules coupled with MMAE in the tumor model HCC827 %TGI

[0705] TGI of tumor model HCC827 (EGFR exon 19 deletion mutation) BT461 BT461-MMAE HCC827 66.2％ 125.0％

[0706] Example 22 In vivo tumor treatment effects of dual antibodies BT461-DXD and BT461-Ixitectin

[0707] The method of this embodiment refers to that of embodiment 16, and the administration method is intravenous administration, except that the administration frequency is once a week.

[0708] The in vivo antitumor activity of anti-BT461-MMAE was evaluated in a mouse xenograft model of human non-small cell lung cancer HCC827-HGF cells (NSG, Vitallife, 6 weeks old, female).

[0709] Human HCC827-HGF cells were propagated as monolayer cultures in vitro using RPM1640 + 10% FBS as complete culture medium. The cells in the logarithmic growth phase were digested and resuspended in serum-free culture medium / Matrigel (1:1) to obtain a suspension. The cells were inoculated at 5×106 / mouse on the right side of the back of the mouse subcutaneously (sc) to form tumors. Ten days after implantation, the mice were divided into three groups according to the tumor volume, and the average tumor volume in each group was about 80-100mm3, and drug administration began. IgG1 or antibody molecules were administered intraperitoneally (IV) once a week for a total of three weeks.

[0710] The experimental results are as follows Fig.21 As shown, in the HCC827-HGF tumor model, BT461-DXD and BT461-ixitecan have better efficacy in inhibiting tumor growth than BT461. The tumor inhibition efficacy %TGI measured 21 days after administration is shown in Table 26 below.

[0711] Table 26 Antitumor efficacy of ADC molecules coupled with DXD or exitecan in the tumor model HCC827 %TGI

[0712]

Claims

1. A bispecific antibody comprising a binding arm A that can specifically bind to human epidermal growth factor receptor (EGFR) and antagonize epidermal growth factor (EGF) binding to EGF and a binding arm B that can specifically bind to the extracellular portion of human proto-oncogene receptor tyrosine kinase (c-Met) and antagonize hepatocyte growth factor (HGF) binding 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 NOs: 1, 2, 12 and 13, 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) It can inhibit the growth and proliferation of EGFR and / or C-Met positive human tumor cells and has a synergistic effect, and 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) and antibody-dependent phagocytosis (ADCP); (4) It can mediate the internalization and degradation of dual-target proteins of EGFR and CMET, and the dual-target internalization mediation has a synergistic effect.

3. The bispecific antibody according to claim 1 and 2, wherein the affinity of the bispecific antibody to CMET is significantly higher than the affinity of the bispecific antibody to EGFR.

4. The bispecific antibody according to any one of claims 1 to 3, wherein The binding arm A is a fusion peptide having a light chain A-linker-heavy chain A (LCA-linker-HCA) structure, the binding arm B is a fusion peptide having a light chain B-linker-heavy chain B (LCB-linker-HCB) structure, the constant region of the heavy chain A (HCA) comprises a combination of amino acid mutations forming a knob structure, and the constant region of the heavy chain B (HCB) comprises a combination of amino acid mutations forming a hole structure; the heavy chain A (HCA) and the heavy chain B (HCB) interact to form a KIH structure, and the binding arm A and the binding arm B form a heterodimer through the KIH structure; Wherein, the linker is a short peptide having a (GGGS)n structure, wherein n is any integer between 4 and 10; Or optionally, the linker comprises an amino acid sequence selected from SEQ ID NO:9, 20.

5. The bispecific antibody according to any one of claims 1 to 4, wherein The binding arm A comprises the amino acid sequence shown in SEQ ID NO:4, and the binding arm B comprises the amino acid sequence shown in SEQ ID NO:

15.

6. The bispecific antibody according to any one of claims 1 to 5, wherein the antigen-binding arm A comprises Fc (A) composed of CH2ACH3A and ScFab (A) as shown in SEQ ID NO: 3, and the antigen-binding arm B comprises Fc (B) composed of CH2BCH3B and ScFab (B) as shown in SEQ ID NO: 14, wherein, The Fc (A) comprises a combination of amino acid mutations that form a Knob structure, the Fc (B) comprises a combination of amino acid mutations that form a hole structure, the Fc (A) and the Fc (B) interact with each other to form a KIH structure, and the binding arm A and the binding arm B form a heterodimer through the KIH structure; Wherein, the Fc(A) comprises the amino acid sequence shown in SEQ ID NO:11, and the Fc(B) comprises the amino acid sequence shown in SEQ ID NO:

22.

7. The bispecific antibody according to claims 1-6, wherein the antigen binding arm A comprises a light chain variable region A (VLA), a light chain constant region A (CLA), a linker, a heavy chain variable region (VHA) and a heavy chain constant region (CHA) connected from the N-terminus to the C-terminus to form a VLA-CLA-Linker-VHA-CHA structure, and the antigen binding arm B comprises a light chain variable region B (VLA), a light chain constant region B (CLB), a linker, a heavy chain variable region (VHB) and a heavy chain constant region (CHB) connected from the N-terminus to the C-terminus to form a VLB-CLB-linker-VHA-CHB structure; in, The VLA, VHA, VLB and VHB comprise the amino acid sequences shown in SEQ ID NO: 5, 7, 16 and 18, respectively, and the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 20 or 31.

8. The bispecific antibody of claim 7, wherein the antigen binding arm A comprises light chain complementary determining regions LACDR1, LACDR2 and LACDR3 and heavy chain complementary determining regions HACDR1, HACDR2 and HACDR3, the antigen binding arm B comprises light chain complementary determining regions LBCDR1, LBCDR2 and LBCDR3, and the VHB comprises heavy chain complementary determining regions HBCDR1, HBCDR2 and HBCDR3; wherein, The antigen binding arm A and the antigen binding arm B interact with each other to form a KIH structure, and the EGFR / C-Met bispecific antibody is a heterodimer formed by the antigen binding arm A and the antigen binding arm B through the KIH structure; Wherein, the LACDR1, LACDR2 and LACDR3, HACDR1, HACDR2 and HACDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:23, 24, 25, 26, 27 and 28, and the LBCDR1, LBCDR2 and LBCDR3, HBCDR1, HBCDR2 and HBCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:29, 30, 31, 32, 33 or 34.

9. The bispecific antibody of any one of claims 1-8, wherein the antigen binding arm that can bind to human EGFR can also bind to cynomolgus monkey EGFR, and the antigen binding arm that can bind to human c-met of the bispecific antibody can also bind to cynomolgus monkey HGFR.

10. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprising the bispecific antibody or antigen-binding fragment of any one of claims 1 to 9 coupled to a drug moiety, and further comprising a drug moiety and a linker coupling the drug moiety to the antibody molecule.

11. The antibody-drug conjugate according to claim 10, characterized in that The drug module is one of a maytansine derivative, auristatin, dolastatin, tubulysin or pyrrolobenzodiazepine, or a topoisomerase I inhibitor; optionally, the drug module is selected from monomethyl auristatin, topoisomerase I or topoisomerase II; further optionally, the drug module is selected from monomethyl auristatin E (MMAE), Excatecan or Dxd.

12. The antibody-drug conjugate of claim 11, characterized in that: (1) The antibody-drug conjugate can bind to EGFR and c-Met simultaneously; (2) the antibody-drug conjugate can be internalized into cells; (3) the antibody-drug conjugate has direct cell killing toxicity or bystander killing toxicity to tumor cells in vitro; (4) the antibody-drug conjugate can inhibit tumor growth in vivo.

13. A pharmaceutical composition comprising the bispecific antigen binding molecule according to any one of claims 1 to 9, or the antibody-drug conjugate according to any one of claims 10 to 12, and a pharmaceutically acceptable carrier or diluent.

14. Use of the bispecific antibody of claims 1 to 9 and the antibody-drug conjugate of claims 10 to 12, or the pharmaceutical composition of claim 13 in the preparation of a therapeutic drug for treating a disease or tumor involving abnormal EGFR and 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 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; Wherein, the cancer comprises one selected from 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; optionally, the cancer is non-small cell lung cancer, pancreatic cancer, gastric cancer, colon cancer, liver cancer.

15. The use according to claim 14, wherein: The second therapeutic agent or treatment regimen includes 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.

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