Bispecific antibody targeting EGFR and AXL, drug conjugate thereof, and use thereof

Abstract

Provided are a bispecific antibody targeting EGFR and AXL, a drug conjugate thereof, and use thereof. The provided bispecific antibody comprises an EGFR antigen-binding domain (three CDRs of VH are at positions 270-277, 295-305, and 340-352 of SEQ ID NO: 1; three CDRs of VL are at positions 27-32, 50-51, and 89-97 of SEQ ID NO: 1) and an AXL antigen-binding domain (three CDRs of VH are at positions 275-282, 300-307, and 346-352 of SEQ ID NO: 3; three CDRs of VL are at positions 27-37, 55-56, and 94-102 of SEQ ID NO: 3). The provided bispecific antibody can mediate stronger endocytic activity and ADCC activity than the parental antibodies. The antibody-drug conjugate constructed using the bispecific antibody can be used for anti-tumor purposes.

Description

Bispecific antibodies targeting EGFR and AXL and their drug conjugates and applications Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to bispecific antibodies targeting EGFR and AXL, and drug conjugates and applications thereof. Background Art

[0002] Abnormally high expression or mutation of the human epidermal growth factor receptor (EGFR) is a driving factor in the carcinogenesis of various organs and tissues. A variety of drugs target EGFR. In terms of mechanism and type, some are small molecules that bind covalently or non-covalently, blocking EGFR phosphorylation and thus the downstream transmission of cell proliferation signals, inducing apoptosis of tumor cells. Others are macromolecules that target EGFR, such as monoclonal antibodies, which bind to the extracellular domain of EGFR, blocking the binding of the ligand EGF to EGFR while mediating the killing of target tumor cells by effector cells, thereby inhibiting tumor proliferation.

[0003] Drugs targeting EGFR, particularly tyrosine kinase inhibitors (TYRICs), are first-line treatments for lung cancer. However, these drugs often result in progression-free survival (PFS) of less than two years, indicating rapid development of resistance. The mechanisms of resistance are diverse. First, the EGFR receptor itself often undergoes amplification, generating new mutations that contribute to resistance to TYRICs. Furthermore, mechanisms of resistance independent of EGFR are even more diverse. AXL receptor amplification mediates endogenous TYRICs resistance or insensitivity. High expression of AXL in tumor cells (AXL, derived from the Greek word "Anexelekto (AXL)," meaning "uncontrolled," was first isolated and identified as an oncogene in 1988) is strongly associated with poor prognosis. Evidence suggests that AXL also promotes mutations in the EGFR kinase domain by increasing specific post-translational modifications of transcription factors, accelerating the development of EGFR on-target resistance (referring to resistance caused by mutations in the EGFR molecule itself; i.e., resistance despite targeting the drug).

[0004] The development of bispecific drugs that simultaneously target EGFR and AXL, such as bispecific antibodies and / or antibody-drug conjugates (ADCs), can more effectively inhibit tumor growth and extend the duration of existing drugs, and has great potential for clinical application.

[0005] Invention Disclosure

[0006] The technical problem to be solved by the present invention is to provide a bispecific antibody targeting EGFR and AXL and its drug conjugate and application.

[0007] The present invention claims a bispecific antibody targeting EGFR and AXL.

[0008] The bispecific antibody targeting EGFR and AXL claimed in the present invention contains an EGFR antigen-binding domain and an AXL antigen-binding domain.

[0009] The EGFR antigen-binding domain may be as follows (A1) or (A2):

[0010] (A1) EGFR antigen-binding domain 1; the EGFR antigen-binding domain 1 comprises the heavy chain variable region 1 of an anti-EGFR antibody and the light chain variable region 1 of an anti-EGFR antibody; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region 1 of the anti-EGFR antibody are positions 270-277, 295-305, and 340-352 of SEQ ID No. 1, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region 1 of the anti-EGFR antibody are positions 27-32, 50-51, and 89-97 of SEQ ID No. 1, respectively.

[0011] (A2) EGFR antigen-binding domain 2; the EGFR antigen-binding domain 2 comprises the heavy chain variable region 2 of an anti-EGFR antibody and the light chain variable region 2 of an anti-EGFR antibody; the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region 2 of the anti-EGFR antibody are positions 270-279, 297-303 and 342-352 of SEQ ID No. 2, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region 2 of the anti-EGFR antibody are positions 27-32, 50-51 and 89-97 of SEQ ID No. 2, respectively.

[0012] The AXL antigen-binding domain may comprise the heavy chain variable region of an anti-AXL antibody and the light chain variable region of an anti-AXL antibody; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the anti-AXL antibody are positions 275-282, 300-307, and 346-352 of SEQ ID No. 3, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the anti-AXL antibody are positions 27-37, 55-56, and 94-102 of SEQ ID No. 3, respectively.

[0013] The CDRs described herein are "complementarity determining regions," regions within the antibody variable domain that are highly variable in sequence and form structurally defined "hypervariable loops" and / or contain antigen-contacting residues called "antigen contact sites." CDRs are primarily responsible for binding to antigenic epitopes. A variable region typically contains three CDR regions: CDR1, CDR2, and CDR3, starting from the N-terminus.

[0014] In the present invention, the bispecific antibody is a heterodimer composed of a first peptide chain and a second peptide chain.

[0015] The first peptide chain may include, from the N-terminus to the C-terminus, the light chain variable region, the light chain constant region, the connecting peptide, the heavy chain variable region, the heavy chain constant region CH1, the hinge region (junction), the heavy chain constant region CH2, and the heavy chain constant region CH3 of the anti-EGFR antibody. The second peptide chain may include, from the N-terminus to the C-terminus, the light chain variable region, the light chain constant region, the connecting peptide, the heavy chain variable region, the heavy chain constant region CH1, the hinge region (junction), the heavy chain constant region CH2, and the heavy chain constant region CH3 of the anti-AXL antibody.

[0016] In a specific embodiment of the present invention, the two heavy chain constant regions CH3 of the first peptide chain and the second peptide chain are designed into a knob structure and a hole structure respectively using the knob-into-hole technology.

[0017] Furthermore, in the first peptide chain and the second peptide chain, the light chain constant region may be a kappa chain or a lambda chain. The heavy chain constant region may be an IgG, IgM, IgE, IgA, or IgD, such as any one of IgG1, IgG2, IgG3, and IgG4.

[0018] In one embodiment of the present invention, the light chain constant region is a human Kappa constant region, and the heavy chain constant region is a human IgG1 constant region (CH1+junction+CH2+CH3).

[0019] In the first peptide chain, the amino acid sequence of the light chain variable region of the anti-EGFR antibody may be positions 1-107 of SEQ ID No. 1 or positions 1-107 of SEQ ID No. 2, or may be 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences (the inconsistencies are preferably in the framework region (FR)). And / or, the amino acid sequence of the light chain constant region may be positions 108-214 of SEQ ID No. 1 or positions 108-214 of SEQ ID No. 2, or may be 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences. And / or, the amino acid sequence of the connecting peptide may be SEQ ID No. 1, positions 215-244, or SEQ ID No. 2, positions 215-244, or have 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identity thereto. And / or, the amino acid sequence of the heavy chain variable region of the anti-EGFR antibody may be SEQ ID No. 1, positions 245-363, or SEQ ID No. 2, positions 245-363, or have 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identity thereto (preferably, the inconsistency is in the framework region (FR)). And / or, the amino acid sequence of the heavy chain constant region CH1 may be positions 364-461 of SEQ ID No. 1 or positions 364-461 of SEQ ID No. 2, or may be 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences. And / or, the amino acid sequence of the hinge region may be positions 462-476 of SEQ ID No. 1 or positions 462-476 of SEQ ID No. 2, or may be 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences. And / or, the amino acid sequence of the heavy chain constant region CH2 may be positions 477-586 of SEQ ID No. 1 or positions 477-586 of SEQ ID No. 2, or may have 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identity thereto. And / or, the amino acid sequence of the heavy chain constant region CH3 may be positions 587-693 of SEQ ID No. 1 or positions 587-693 of SEQ ID No. 2.

[0020] In the second peptide chain, the amino acid sequence of the light chain variable region of the anti-AXL antibody may be positions 1-112 of SEQ ID No. 3, or may be 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence (preferably, the difference is in the framework region (FR)). Furthermore, / or, the amino acid sequence of the light chain constant region may be positions 113-219 of SEQ ID No. 3, or may be 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence. Furthermore, / or, the amino acid sequence of the connecting peptide may be positions 220-249 of SEQ ID No. 3, or may be 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence. And / or, the amino acid sequence of the heavy chain variable region of the anti-AXL antibody may be positions 250-363 of SEQ ID No. 3, or may be 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to said sequence (with the exception of positions preferably being in the framework region (FR)). And / or, the amino acid sequence of the heavy chain constant region CH1 may be positions 364-461 of SEQ ID No. 3, or may be 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to said sequence. And / or, the amino acid sequence of the hinge region may be positions 462-476 of SEQ ID No. 3, or may be 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to said sequence. And / or, the amino acid sequence of the heavy chain constant region CH2 may be positions 477-586 of SEQ ID No. 3, or may have 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identity thereto. And / or, the amino acid sequence of the heavy chain constant region CH3 may be positions 587-693 of SEQ ID No. 3.

[0021] Furthermore, the amino acid sequence of the first peptide chain may be SEQ ID No.1 or SEQ ID No.2; and the amino acid sequence of the second peptide chain may be SEQ ID No.3.

[0022] In one embodiment of the present invention, the bispecific antibody is formed by the automatic heterodimerization of the first peptide chain represented by SEQ ID No. 1 and the second peptide chain represented by SEQ ID No. 3 in a Knob-into-Hole manner (corresponding to BSG04 in the embodiment).

[0023] In another embodiment of the present invention, the bispecific antibody is formed by the automatic heterodimerization of the first peptide chain represented by SEQ ID No. 2 and the second peptide chain represented by SEQ ID No. 3 in a Knob-into-Hole manner (corresponding to BSG0402 in the embodiment).

[0024] In the present invention, the Fc region of the bispecific antibody has a typical core glycosylation modification, i.e., an N-linked oligosaccharide modification. In one embodiment of the present invention, the core N-linked glycosylation modification of the Fc region of the bispecific antibody does not contain fucose. In another embodiment of the present invention, the core N-linked glycosylation modification of the Fc region of the bispecific antibody is normal, i.e., the core N-linked glycosylation modification of the Fc region contains fucose. Therefore, the bispecific antibody of the present invention can be in either a normal glycosylation modification form or a fucose-free form.

[0025] In the art, the core glycosylation (i.e., N297 site) of the Fc domain (Fc region) of antibodies expressed in mammalian cells (e.g., 293E, 293F, CHO-S, and CHO-K1 cells) is typically characterized by oligosaccharide modifications. Genetically engineered (or knockout) host cells lack FUT8 (a glycosyltransferase) and are therefore unable to add fucose to oligosaccharide chains. Consequently, antibodies expressed in these host cells lack fucose in the core N-linked glycosylation of the Fc region. These antibodies are referred to as afucose-free (AF) modified antibodies.

[0026] The present invention also claims protection for any of the following biological materials:

[0027] (B1) a nucleic acid molecule encoding the bispecific antibody described above;

[0028] (B2) an expression cassette containing the nucleic acid molecule described in (B1);

[0029] (B3) a recombinant vector containing the nucleic acid molecule described in (B1);

[0030] (B4) a recombinant bacterium containing the nucleic acid molecule described in (B1);

[0031] (B5) A transgenic cell line containing the nucleic acid molecule described in (B1).

[0032] In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2, and CDR3 in the heavy chain variable region 1 of the anti-EGFR antibody in the EGFR antigen-binding domain 1 are positions 808-831, 883-915, and 1018-1056 of SEQ ID No. 4, respectively. And / or, the nucleotide sequences encoding CDR1, CDR2, and CDR3 in the light chain variable region 1 of the anti-EGFR antibody are positions 79-96, 148-153, and 265-291 of SEQ ID No. 4, respectively.

[0033] In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2, and CDR3 in the heavy chain variable region 2 of the anti-EGFR antibody in the EGFR antigen-binding domain 2 are positions 808-837, 889-909, and 1024-1056 of SEQ ID No. 5, respectively. And / or, the nucleotide sequences encoding CDR1, CDR2, and CDR3 in the light chain variable region 2 of the anti-EGFR antibody are positions 79-96, 148-153, and 265-291 of SEQ ID No. 5, respectively.

[0034] In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2, and CDR3 in the heavy chain variable region of the anti-AXL antibody in the AXL antigen-binding domain are positions 823-846, 898-921, and 1036-1056 of SEQ ID No. 6, respectively. And / or, the nucleotide sequences encoding CDR1, CDR2, and CDR3 in the light chain variable region of the anti-AXL antibody are positions 79-111, 163-168, and 280-306 of SEQ ID No. 6, respectively.

[0035] Furthermore, in the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region of the anti-EGFR antibody in the first peptide chain is SEQ ID No. 4, positions 1-321, or SEQ ID No. 5, positions 1-321, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with the aforementioned sequences. And / or, the nucleotide sequence encoding the light chain constant region is SEQ ID No. 4, positions 322-642, or SEQ ID No. 5, positions 322-642, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with the aforementioned sequences. and / or the nucleotide sequence encoding the connecting peptide is SEQ ID No. 4, positions 643-732 or SEQ ID No. 5, positions 643-732, or is 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences. and / or the nucleotide sequence encoding the heavy chain variable region of the anti-EGFR antibody is SEQ ID No. 4, positions 733-1089 or SEQ ID No. 5, positions 733-1089, or is 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences. and / or the nucleotide sequence encoding the heavy chain constant region CH1 is SEQ ID No. 4, positions 1090-1383 or SEQ ID No. 5, positions 1090-1383, or is 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences. and / or the nucleotide sequence encoding the hinge region is SEQ ID No. 4, positions 1384-1428 or SEQ ID No. 5, positions 1384-1428, or is 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences. and / or the nucleotide sequence encoding the heavy chain constant region CH2 is SEQ ID No. 4, positions 1429-1758 or SEQ ID No. 5, positions 1429-1758, or positions 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences. and / or the nucleotide sequence encoding the heavy chain constant region CH3 is SEQ ID No. 4, positions 1759-2079 or SEQ ID No. 5, positions 1759-2079, or positions 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequences.

[0036] Furthermore, in the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region of the anti-AXL antibody in the second peptide chain is SEQ ID No. 6, positions 1-336, or is 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence. And / or, the nucleotide sequence encoding the light chain constant region is SEQ ID No. 6, positions 337-657, or is 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence. And / or, the nucleotide sequence encoding the connecting peptide is SEQ ID No. 6, positions 658-747, or is 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence. and / or the nucleotide sequence encoding the heavy chain variable region of the anti-EGFR antibody is SEQ ID No. 6, positions 748-1089, or is 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence. and / or the nucleotide sequence encoding the heavy chain constant region CH1 is SEQ ID No. 6, positions 1090-1383, or is 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence. and / or the nucleotide sequence encoding the hinge region is SEQ ID No. 6, positions 1384-1428, or is 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence. and / or the nucleotide sequence encoding the heavy chain constant region CH2 is SEQ ID No. 6, positions 1429-1758, or is 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequence. and / or the nucleotide sequence encoding the heavy chain constant region CH3 is SEQ ID No. 6, positions 1759-2079, or is 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to the aforementioned sequence.

[0037] Furthermore, in the nucleic acid molecule, the nucleotide sequence encoding the first peptide chain is SEQ ID No. 4 or SEQ ID No. 5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence; the nucleotide sequence encoding the second peptide chain is SEQ ID No. 6, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

[0038] SEQ ID No.4 encodes the peptide chain shown in SEQ ID No.1; SEQ ID No.5 encodes the peptide chain shown in SEQ ID No.2; and SEQ ID No.6 encodes the peptide chain shown in SEQ ID No.3.

[0039] In one embodiment of the present invention, the recombinant vector consists of a recombinant vector 1 and a recombinant vector 2. The recombinant vector 1 is used to express the first peptide chain; the recombinant vector 2 is used to express the second peptide chain. The recombinant vector 1 is a recombinant vector obtained by replacing a small fragment between the enzyme cleavage sites KpnI and Not I of the pHr-IgG1 vector with a fragment of the gene encoding the first peptide chain in the bispecific antibody (such as SEQ ID No.4 or SEQ ID No.5). The recombinant vector 2 is a recombinant vector obtained by replacing a small fragment between the enzyme cleavage sites KpnI and Not I of the pHr-IgG1 vector with a fragment of the gene encoding the second peptide chain in the bispecific antibody (such as SEQ ID No.6). The vector pHr-IgG1 is described below.

[0040] In one embodiment of the present invention, the transgenic cell line is a recombinant cell obtained by simultaneously introducing the recombinant vector 1 and the recombinant vector 2 into 293F cells.

[0041] The present invention claims an antibody drug conjugate or a pharmaceutically acceptable salt thereof.

[0042] The antibody-drug conjugate claimed in the present invention comprises the bispecific antibody described above and a drug portion coupled to the bispecific antibody; the drug portion contains one, two or more drugs.

[0043] The drug can be selected from at least one of the following: the first category, cytotoxic drugs. (1) Drugs that act on the chemical structure of DNA: alkylating agents, such as nitrogen mustards, nitrosoureas, and methanesulfonates; platinum compounds, such as cisplatin, carboplatin, and oxaliplatin; mitomycin (MMC); (2) Drugs that affect nucleic acid synthesis: dihydrofolate reductase inhibitors, such as methotrexate (MTX) and Alimta; thymidine synthase inhibitors, such as fluorouracil (5FU, FT-207, capecitabine); purine nucleoside synthase inhibitors, such as 6-mercaptopurine (6-MP) and 6-TG; ribonucleotide reductase inhibitors, such as hydroxyurea (HU); DNA polymerase inhibitors, such as cytarabine (Ara-C) and Gemzar (Gemz); (3) Drugs acting on nucleic acid transcription: drugs that selectively act on DNA templates, inhibit DNA-dependent RNA polymerase, and thus inhibit RNA synthesis, such as actinomycin D, daunorubicin, doxorubicin, epirubicin, aclarubicin, mithramycin, etc.; (4) Drugs that mainly act on microtubule synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophyllin, homoharringtonine; (5) Other cytotoxic drugs: asparaginase, mainly inhibiting protein synthesis. The second category is hormones. Antiestrogens: tamoxifen, droloxifene, exemestane, etc.; aromatase inhibitors: aminoglutethimide, lantron, letrozole, arimide, etc.; antiandrogens: flutamide, RH-LH agonists / antagonists: zoledrone, enantone, etc. The third category is biological response modifiers, which primarily suppress tumors through the body's immune system, including interferon, interleukin-2, and thymosin peptides. The fourth category includes other drugs whose mechanisms are currently unknown and require further research, including cell differentiation inducers, such as retinoids, and apoptosis inducers.

[0044] Furthermore, the drug is usually a small molecule drug, such as MMAE that destroys cellular tubulin or Deruxtecan that inhibits topoisomerase I.

[0045] In one embodiment of the present invention, the drug is Dxd (DX-8951 derivative), whose molecular formula is C 26 H 24 FN3O6, the structural formula is as follows:

[0046] The present invention claims a method for preparing the antibody-drug conjugate.

[0047] The drug is coupled to the bispecific antibody to obtain the antibody-drug conjugate. This coupling method is varied, and can be random coupling to the free sulfhydryl group (SH) of the antibody molecule or site-specific coupling.

[0048] In one embodiment of the present invention, TCEP is used to reduce the bispecific antibody described above, thereby converting the cysteine ​​in the bispecific antibody into a sulfhydryl group, and then the maleamide-GGFG-Dxd molecule is coupled to the antibody molecule to obtain the antibody drug conjugate; wherein the maleamide-GGFG-Dxd molecule is Deruxtecan, whose molecular formula is C 52 H 56 FN9O 13 , the structural formula is as follows:

[0049] The coupling is achieved through a thioether bond, specifically, maleamide and thiol react to form a thioether bond, and the coupling is achieved through the thioether bond.

[0050] In one embodiment of the present invention, the drug (Dxd)-antibody coupling ratio (DAR) is 8.

[0051] The present invention claims protection for any of the following applications:

[0052] (C1) Use of the aforementioned biomaterial in the preparation of the aforementioned bispecific antibody or the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof;

[0053] (C2) Use of the aforementioned bispecific antibody in the preparation of the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof;

[0054] (C3) Use of the aforementioned bispecific antibody, the aforementioned biomaterial, or the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0055] Furthermore, the tumor includes but is not limited to lung cancer, glioma, head and neck tumor, colorectal cancer, etc.

[0056] Furthermore, the tumor may be a non-drug-resistant tumor or a drug-resistant tumor. In one embodiment of the present invention, the drug-resistant tumor is specifically a tumor resistant to a third-generation EGFR-targeted tyrosine kinase inhibitor (TKI).

[0057] The present invention also claims protection for any of the following methods:

[0058] Method I: A method for preparing the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, comprising the following steps: using the aforementioned bispecific antibody to prepare the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof;

[0059] Method II: A method for preparing an anti-tumor drug, comprising the following steps: preparing the anti-tumor drug using the aforementioned bispecific antibody, the aforementioned biomaterial, or the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof;

[0060] Method III: An anti-tumor method comprising the following steps: administering the aforementioned bispecific antibody or the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof to a subject.

[0061] In method III, the subject is a tumor patient.

[0062] Furthermore, the tumor includes but is not limited to lung cancer, glioma, head and neck tumor, colorectal cancer, etc.

[0063] Furthermore, the tumor may be a non-drug-resistant tumor or a drug-resistant tumor. In one embodiment of the present invention, the drug-resistant tumor is specifically a tumor resistant to a third-generation EGFR-targeted tyrosine kinase inhibitor (TKI).

[0064] The present invention also claims protection for an anti-AXL antibody and related biological materials and applications.

[0065] The anti-AXL antibody claimed in the present invention comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are positions 26-33, positions 51-58 and positions 97-103 of SEQ ID No. 7, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are positions 27-37, positions 55-56 and positions 94-102 of SEQ ID No. 8, respectively.

[0066] Furthermore, the amino acid sequence of the heavy chain variable region of the anti-AXL antibody is SEQ ID No. 7, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with the above sequence (the inconsistency is preferably in the framework region (FR)).

[0067] Furthermore, the amino acid sequence of the light chain variable region of the anti-AXL antibody is SEQ ID No. 8, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with the above sequence (the inconsistency is preferably in the framework region (FR)).

[0068] Furthermore, in the anti-AXL antibody, the light chain constant region may be of the κ chain or λ chain type; the heavy chain constant region may be of the IgG, IgM, IgE, IgA or IgD type, such as any one of IgG1, IgG2, IgG3 and IgG4.

[0069] In one embodiment of the present invention, the light chain constant region is a human Kappa constant region, and the heavy chain constant region is a human IgG1 constant region (CH1+junction+CH2+CH3).

[0070] The biological material related to the anti-AXL antibody claimed in the present invention may specifically be any of the following:

[0071] (D1) a nucleic acid molecule encoding the anti-AXL antibody described above;

[0072] (D2) an expression cassette containing the nucleic acid molecule described in (D1);

[0073] (D3) a recombinant vector containing the nucleic acid molecule described in (D1);

[0074] (D4) a recombinant bacterium containing the nucleic acid molecule described in (D1);

[0075] (D5) A transgenic cell line containing the nucleic acid molecule described in (D1).

[0076] In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2, and CDR3 in the heavy chain variable region of the anti-AXL antibody are positions 76-99, 151-174, and 289-309 of SEQ ID No. 9, respectively. And / or, the nucleotide sequences encoding CDR1, CDR2, and CDR3 in the light chain variable region are positions 79-111, 163-168, and 280-306 of SEQ ID No. 10, respectively.

[0077] In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the anti-AXL antibody is SEQ ID No. 9, or is 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence. And / or the nucleotide sequence encoding the light chain variable region of the anti-AXL antibody is SEQ ID No. 10, or is 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identical to the aforementioned sequence.

[0078] In one embodiment of the present invention, a recombinant expression vector expressing the heavy chain of the anti-AXL antibody is obtained by replacing the nucleotide sequence encoding the heavy chain variable region of the anti-AXL antibody with a small fragment between two BsmBI enzyme recognition sites of the pHr-IgG1 vector. A recombinant expression vector expressing the light chain of the anti-AXL antibody is obtained by replacing the nucleotide sequence encoding the light chain variable region of the anti-AXL antibody with a small fragment between two BsmBI enzyme recognition sites of the pHr-Kappa vector.

[0079] The pHr-IgG1 vector is a recombinant vector obtained by replacing a small fragment between the KpnI and Not I enzyme recognition sites of the pHR'CMV GFP vector with a target DNA molecule having a nucleic acid sequence of SEQ ID No. 63, while maintaining the other nucleotide sequences on the pHR'CMV GFP vector unchanged. Positions 19-75 of SEQ ID No. 63 are a signal peptide gene, positions 76-123 are a buffer sequence (with BsmBI enzyme recognition sites at both ends of the buffer sequence), and positions 124-1116 are a human IgG1 constant region (CH1+junction+CH2+CH3). The pHr-Kappa vector is a recombinant vector obtained by replacing a small fragment between the KpnI and Not I enzyme recognition sites of the pHR'CMV GFP vector with a target DNA molecule having a nucleic acid sequence as shown in SEQ ID No. 64, while maintaining the other nucleotide sequences on the pHR'CMV GFP vector unchanged. Positions 19-84 of SEQ ID No. 64 are signal peptide genes, positions 85-132 are buffer sequences (both ends of the buffer sequence are BsmBI enzyme recognition sites), and positions 133-456 are human Kappa constant regions (Constant Kappa).

[0080] The recombinant cells are recombinant cells obtained by co-transfecting 293F cells with the two recombinant expression vectors expressing the heavy chain and light chain of the anti-AXL antibody respectively.

[0081] The application of the anti-AXL antibody claimed in the present invention may be any of the following:

[0082] (E1) Use of the nucleic acid molecule described in (D1) above, the expression cassette described in (D2) above, the recombinant vector described in (D3) above, the recombinant bacteria described in (D4) above, or the transgenic cell line described in (D5) above in the preparation of the anti-AXL antibody described above;

[0083] (E2) Use of the anti-AXL antibody described above, or the nucleic acid molecule described above (D1), or the expression cassette described above (D2), or the recombinant vector described above (D3), or the recombinant bacterium described above (D4), or the transgenic cell line described above (D5) in the preparation of a product for preventing and / or treating diseases associated with overexpression of AXL;

[0084] (E3) Use of the anti-AXL antibody described above, or the nucleic acid molecule described above (D1), or the expression cassette described above (D2), or the recombinant vector described above (D3), or the recombinant bacterium described above (D4), or the transgenic cell line described above (D5) in the preparation of a product for detecting AXL;

[0085] (E4) Use of the anti-AXL antibody described above, or the nucleic acid molecule described above (D1), or the expression cassette described above (D2), or the recombinant vector described above (D3), or the recombinant bacterium described above (D4), or the transgenic cell line described above (D5) in the preparation of a product for binding to AXL;

[0086] (E5) Use of the anti-AXL antibody described above, or the nucleic acid molecule described above (D1), or the expression cassette described above (D2), or the recombinant vector described above (D3), or the recombinant bacteria described above (D4), or the transgenic cell line described above (D5) in the preparation of a product for blocking the binding activity of CAS6 to AXL.

[0087] The present invention also claims protection for any of the following methods:

[0088] Method I: A method for preparing the anti-AXL antibody described above, comprising the following steps: preparing the anti-AXL antibody using (D1) the nucleic acid molecule, (D2) the expression cassette, (D3) the recombinant vector, (D4) the recombinant bacteria, or (D5) the transgenic cell line;

[0089] Method II: A method for preventing and / or treating a disease associated with overexpression of AXL, comprising the following steps: administering the anti-AXL antibody described above to a subject;

[0090] Method III: A method for detecting AXL, comprising the following steps: detecting a sample using the anti-AXL antibody described above;

[0091] Method IV: A method for binding to AXL, comprising the following steps: treating a test sample with the anti-AXL antibody described above;

[0092] Method V: A method for blocking the binding activity of CAS6 and AXL, comprising the following steps: treating the test sample with the anti-AXL antibody described above.

[0093] In method II, the subject is a patient with a disease associated with high AXL expression.

[0094] In the present invention, identity refers to amino acid / nucleotide sequence identity. Amino acid / nucleotide sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage.

[0095] In the present invention, the above 99% identity may be at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity. The above 95% identity may be at least 96%, 97% or 98% identity. The above 90% identity may be at least 91%, 92%, 93% or 94% identity. The above 85% identity may be at least 86%, 87%, 88% or 89% identity. The above 80% identity may be at least 81%, 82%, 83% or 84% identity. The above 75% identity may be at least 76%, 77%, 78% or 79% identity. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 shows the enzyme-linked immunosorbent assay (ELISA) method for identifying the activity of homemade CETU antibodies and PANI antibodies.

[0097] FIG2 shows the binding of chimeric antibody 10C1 to AXL-his detected by enzyme-linked immunosorbent assay (ELISA).

[0098] FIG3 shows the activity of chimeric antibody 10C1 in blocking the binding of ligand GAS6 to AXL detected by enzyme-linked immunosorbent assay (ELISA).

[0099] FIG4 is a flow cytometry (FACS) assay showing the binding of different concentrations of chimeric antibody 10C1 to the tumor cell line Calu-1.

[0100] FIG5 shows the activity of chimeric antibody 10C1 in blocking the binding of ligand GAS6 to tumor cell Calu-1 (surface AXL) detected by flow cytometry (FACS).

[0101] FIG6 shows the binding activity of the chimeric antibody 10C1 to AXL of different species (human, monkey, and mouse) detected by enzyme-linked immunosorbent assay (ELISA).

[0102] Figure 7 shows the screening of 10C1 humanized candidate molecules by enzyme-linked immunosorbent assay (ELISA). Xi represents a 10C1 chimeric antibody, and 34, 35, 44, 45, 54, and 55 represent six 10C1 humanized candidate molecules.

[0103] FIG8 shows the binding activity of the preferred humanized molecule 10C1-34 and its parent antibody 10C1 on tumor cells U251 compared by flow cytometry (FACS).

[0104] Figure 9 shows the binding activity of the bispecific antibodies BSG04 and BSG0402, as well as the monospecific antibodies 10C1-34, cetuximab, and panitumumab, to U251 cells as assessed by flow cytometry (FACS). A shows the results of 10C1-34, panitumumab (abbreviated as Pani in the figure), and BSG0402 in the same experiment; B shows the results of 10C1-34, cetuximab (abbreviated as cetu in the figure), and BSG04 in the same experiment.

[0105] FIG10 shows the endocytosis activity of 10C1-34, BSG0402, and panitumumab (abbreviated as Pani in the figure) in U251 cells detected by flow cytometry (FACS).

[0106] FIG11 is a graph showing the detection of the ADCC activity mediated by various bispecific antibody molecules.

[0107] Figure 12 shows the purity of the ADC drug BSG04D after conjugation detected by high performance liquid chromatography (HPLC).

[0108] FIG13 shows the DAR value of BSG04D analyzed by mass spectrometry.

[0109] FIG14 shows the efficacy results of BSG04D in the mouse lung cancer xenograft tumor model H1975.

[0110] FIG15 shows the efficacy results of BSG0402AF and BSG04D in a third-generation tyrosine kinase inhibitor-resistant lung cancer xenograft tumor model in mice.

[0111] Best Mode for Carrying Out the Invention

[0112] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0113] Example 1. Preparation of AXL extracellular segment fusion protein

[0114] According to the NCBI database, sequence number NP_068713.2 (version 2023-12-4), the full-length AXL protein contains 894 amino acids. The mature functional protein does not contain amino acids 1-25, which are cleaved and removed as a signal peptide; amino acids 26-451 form the extracellular domain (ECD), that is, the AXL-ECD sequence is shown in the amino acid sequence 26-451 of NP_068713.2 (version 2023-12-4).

[0115] The plasmid containing the full-length human AXL gene (NM_021913.3) was obtained from Beijing Sino Biological Technology Co., Ltd., with the catalog number HG10279-UT.

[0116] The designed oligonucleotide primers are shown in Table 1.

[0117] Table 1. Primer sequences of this example

[0118] To amplify the genes encoding AXL-hFc, AXL-mFc, and AXL-His fusion proteins (where AXL is the extracellular portion), the corresponding primer pairs (Table 1) were used, respectively, and a plasmid containing the full-length human AXL gene (NM_021913.3) (Beijing Sino Biological Science and Technology Co., Ltd., Cat. No. HG10279-UT) was used as a template. PCR was performed using a 2×Hieff PCR amplification was performed according to the manufacturer's instructions using PCR Master Mix High-Fidelity Enzyme Premix (Yisheng Biotechnology, 10136ES). The resulting PCR fragment (i.e., the coding gene) was subjected to agarose gel electrophoresis (agarose from Beijing Solaibao Biotechnology Co., Ltd.). Agarose was prepared to a 1% (i.e., 1g / 100ml) concentration by weight-to-volume ratio using 1× TAE buffer (Beijing Solaibao Biotechnology Co., Ltd.). The agarose was melted and solidified on a gel cassette. The sample was loaded into the electrophoresis tank, and the gel was run at a constant voltage of 140V. DNA fragments of the correct size were recovered and purified using a gel extraction kit (OMEGA).

[0119] The pHr-hG1Fc (also known as the pHr-Fc vector) and pHr-mG2aFc (also known as the pHr-mFc vector) vectors were modified from the pHR'CMV GFP vector (Addgene, Plasmid #14858). Specifically, the pHr-Fc vector was created by replacing the small fragment between the KpnI and Not I restriction enzyme recognition sites of the pHR'CMV GFP vector with the target DNA molecule represented by SEQ ID No. 17, while maintaining the remaining nucleotide sequences of the pHR'CMV GFP vector unchanged. Positions 19-75 of SEQ ID No. 17 represent the signal peptide gene, positions 76-123 are the buffer sequence (with BsmBI restriction enzyme recognition sites on both ends of the buffer sequence), and positions 124-819 represent the human IgG1 constant region (CH2+CH3). The recombinant pHr-mFc vector was similarly engineered. The target DNA sequence, represented by SEQ ID No. 18, was used to replace the small fragment between the KpnI and NotI restriction enzyme recognition sites of the pHR'CMV GFP vector, while the remaining nucleotide sequence of the pHR'CMV GFP vector remained unchanged. Positions 19-75 of SEQ ID No. 18 represent the signal peptide gene, positions 76-123 are the buffer sequence (with BsmBI restriction enzyme recognition sites on either end), and positions 124-822 represent the murine IgG2a constant region (CH2+CH3).

[0120] To clone the AXL-hFc, AXL-mFc, and AXL-His gene encoding fragments (where AXL is the extracellular domain) obtained by PCR into the pHr-Fc and pHr-mFc vectors, respectively, the multiple cloning sites of the pHr-Fc and pHr-mFc vectors were digested with the BsmBI restriction enzyme to generate linearized vectors. The target DNA fragment (AXL-hFc, AXL-mFc, or AXL-His encoding gene) was flanked by a nucleotide sequence (approximately 30 nt) homologous to the vector. This extended gene and the linearized vector were transformed into Transblue DH5α cells (Solar Bio Products). Transformant colonies were selected, cultured, and plasmids were extracted and sequenced. These expression plasmids, designated pHr-AXL-Fc, pHr-AXL-mFc, and pHr-AXL-His, were then obtained.

[0121] The recombinant vector pHr-AXL-Fc is constructed by replacing the small fragment between the two BsmBI restriction sites of the pHr-Fc vector with the DNA fragment shown in SEQ ID No. 19. The resulting protein is fused to the Fc region of human IgG1, and its full-length sequence is shown in SEQ ID No. 20. Amino acids 1-424 represent the extracellular region of AXL, and amino acids 425-656 represent the human Fc region.

[0122] The recombinant vector pHr-AXL-mFc is constructed by replacing the small fragment between the two BsmBI restriction sites of the pHr-mFc vector with the DNA fragment shown in SEQ ID No. 19. The resulting protein is fused to the Fc region of mouse IgG2a (mG2a), and its full-length sequence is shown in SEQ ID No. 21. Amino acids 1-424 represent the extracellular domain of AXL, and amino acids 425-657 represent the mouse Fc region.

[0123] The recombinant vector pHr-AXL-His is constructed by replacing the small fragment between the two BsmBI restriction sites of the pHr-Fc vector with the DNA fragment shown in SEQ ID No. 22. Positions 1-1272 of this sequence encode the extracellular domain of AXL. Positions 1273-1296 encode nine histidine residues to facilitate protein purification using nickel-chelate gels. The final triplet codon, TAA, serves as a stop codon. The resulting amino acid sequence of AXL-His is shown in SEQ ID No. 23.

[0124] 293F cells were transfected with the prepared pHr-AXL-Fc, pHr-AXL-mFc, and pHr-AXL-His expression plasmids for production and purification of the target protein. Specifically, 100 μg of pHR expression plasmid (i.e., pHr-AXL-Fc, pHr-AXL-mFc, or pHr-AXL-His vector) was mixed with the transfection reagent PEI, allowed to stand for 30 minutes, and then added to the 293F cell culture medium. The cells were cultured with shaking at 37°C and 5% carbon dioxide for 24 hours. Feed A (Zhuhai Kairui, Cat. No. K40001) was added and cultured with shaking for another 3 days. The culture medium was centrifuged at 12,000 rpm for 30 minutes, and the supernatant was collected for later use. For fusion proteins linked to Fc or mFc tags, add 1 ml of Protein A gel filler (Borgron, AA402305) and incubate at room temperature with shaking (145 rpm) for 1-3 hours. Allow the mixture to flow through the gravity chromatography column and collect the flow-through, freezing it for re-testing. Elute the column with 5 ml of PBS, allowing the wash to slowly flow through the gel column. Add 3 ml of glycine eluent (pH 3.2) to elute the captured protein. For the fusion protein connected to the His tag, it was added to the HIS-excel prepacked column (GE, 17-3712-05) equilibrated with PBS and passed through the column. The impurities were washed with PBS-10 imidazole (formula: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, 10mM imidazole) and then eluted with PBS-250 imidazole buffer (formula: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, 250mM imidazole) to collect the target protein.

[0125] All eluates were centrifuged in ultrafiltration concentrator tubes (Millipore) at 3800 × g for 20 minutes and replaced with sterile, pyrogen-free PBS. Ultraviolet absorption at 280 nm was measured using a Nanodrop and divided by the extinction coefficient to obtain the concentration.

[0126] Example 2: Preparation of hybridomas by spleen cell fusion and screening of anti-AXL monoclonal antibodies

[0127] 1. Mouse Immunization

[0128] Ten female BALB / c and SJL mice, weighing approximately 20 grams and aged 6-8 weeks, were selected. Freund's complete adjuvant was used for the first immunization, and 5 BALB / c and 5 SJL mice were immunized with AXL-mFc and AXL-His antigens (prepared in Example 1). Equal volumes of antigen and immune adjuvant were mixed and repeatedly injected through a 5 mL syringe through a three-way stopcock (Aibel, PS-3002) to mix. 50 μg (in terms of antigen amount, the same below) per mouse was injected subcutaneously at multiple points. After a two-week interval, Freund's incomplete adjuvant was used at 30 μg per mouse for two consecutive immunizations. Seven days after the last immunization, the tails were cut and 20 μl of whole blood was collected. The blood was centrifuged at 12,000 g for 2 minutes, and the supernatant was transferred to a PCR tube for titer detection.

[0129] 2. ELISA method to detect mouse immune titer

[0130] Dilute the serum with 1% BSA (w / v, dissolved in PBST (0.05% Tween 20), the same below) to a maximum concentration of 1:500. Dilute the serum collected in step 1 in five-fold dilutions to obtain seven different dilutions. Add the diluted serum solution to the wells of an ELISA plate coated with AXL-His protein (prepared in Example 1, 2 μg / ml, 50 μl / well) at a volume of 50 μl / well. Incubate at 37°C for 1 hour. Discard the supernatant and wash the wells five times with 300 μl / well PBST. Pat the ELISA plate dry and add the secondary antibody. The secondary antibody is HRP-goat anti-mouse IgG (Jackson immunoresearch, catalog #115-035-062). Dilute it 20,000-fold with 1% BSA. Incubate at 37°C for 1 hour. Discard the supernatant and wash the wells five times with 300 μl / well PBST. Pat the ELISA plate dry on absorbent paper, add 50 μl of developer solution, and develop for 2 min at 37°C or room temperature. Add 50 μl / well of stop solution (ready-to-use, solarbio) and read the OD450. Divide the signal value of each well by the background signal at zero concentration to calculate the factor F. A factor greater than 2.1 is considered significantly positive. The serum titer of the positive well with the highest dilution factor is recorded as the serum titer of that animal.

[0131] 3. Cell Fusion, Positive Hybridoma Screening and Cloning

[0132] 1. Hybridoma Preparation

[0133] Logarithmically growing sp2 / 0 cells with an 85% abundance were collected and placed in 50 ml of PBS. The cells were centrifuged at 1000 rpm for 5 minutes, resuspended in 20 ml of BTX fusion buffer (BTX, Catalog No. 47-0001), centrifuged at 1700 rpm for 7 minutes, and the supernatant discarded. The cells were then resuspended in 20 ml of BTX fusion buffer. After counting, the cells were centrifuged at 1700 rpm for 7 minutes, the supernatant discarded, and the cells were resuspended in 2 ml of BTX fusion buffer for later use. Mice immunized with AXL antigen (Step 1) were sacrificed by cervical dislocation and soaked in 75% ethanol for 5 minutes. The spleens of the mice were removed using sterile ophthalmic scissors and forceps, and the surrounding connective tissue was removed. The spleens were ground using a 5 ml syringe plunger on a 200-mesh stainless steel sieve (with the sieve submerged in PBS). The spleens were then rinsed with PBS to remove any splenocytes remaining on the sieve and syringe plunger. The spleens were then passed through a 40 μm sieve into a 50 ml graduated centrifuge tube and centrifuged at 1700 rpm for 7 minutes. Resuspend the cells with 20ml BTX fusion buffer, count them, centrifuge at 1700rpm for 7min, discard the supernatant, and resuspend the cells with 20ml BTX fusion buffer again, centrifuge at 1700rpm for 7min, discard the supernatant, and resuspend the spleen cells with 2ml BTX fusion buffer for later use. Sp2 / 0 cells and spleen cells were mixed at a ratio of 1:2 and 2×10 7 Resuspend cells in BTX fusion buffer at a concentration of 10 cells / mL. Place 1 mL of the mixed cell suspension on a 3 mm fusion electrode and press the Start button on the BEX electrofusion instrument to initiate fusion. 30 seconds after fusion, transfer the cells to complete culture medium (formulation: PRIM1640 medium, Gibco 10% FBS, 1× PS, 1× HAT). Let stand for 10 minutes before aliquoting into 96-well cell culture plates and culturing in a 5% CO2, 37°C incubator.

[0134] The fusion parameters of the BEX fusion instrument are set as follows:

[0135] AC:50V; AC Time:30S; DC:800V

[0136] DC on time:20μS; DC off time:0.5S; DC cycle:1

[0137] Post time:7S;Fade:on;Decay:0%

[0138] 2. Screening of AXL-binding-positive hybridomas

[0139] AXL-His protein (prepared in Example 1) was diluted to 2 μg / ml with PBS and added to an ELISA plate at 50 μl / well. Incubate in a 37°C incubator for 2 hours or in a 4°C refrigerator overnight. Remove the AXL-His protein-coated ELISA plates and place each plate in a PW960 96-well plate washer. Adjust the plate washer parameters to: 1 wash, 300 μl / well wash volume, and wash each plate sequentially. Block the ELISA plate with 200 μl / well of 5% (w / v) skim milk powder dissolved in 0.05% PBST (PBS is from Beijing Solaibao Biotechnology Co., Ltd., product number P1010. Dissolve each package of dry powder in 2 L of pure water and add 1 ml of Tween 20 to obtain PBST). Incubate in a 37°C incubator for 2 hours or in a 4°C refrigerator overnight. Discard the blocking solution from the blocked protein ELISA plate. Add an appropriate amount of PBST buffer to the wash bottle of a Huisong PW960 96-well plate washer. Adjust the plate washer parameters: wash once, wash volume 300 μl / well, and wash each plate sequentially. Store the prepared Axl-His protein-coated 96-well ELISA plate in a -80°C freezer.

[0140] After the hybridoma (prepared in step 1) was cultured for seven days, the Axl-His protein-coated 96-well ELISA plate stored in a -80°C refrigerator was removed and preheated in a 37°C incubator for 10 minutes. 50 μl of the hybridoma supernatant in the 96-well cell culture plate was aspirated using a 300 μl 12-channel pipette and added to the Axl-His protein-coated 96-well ELISA plate. 1% BSA was added to well H11 as a negative control, and 50 μl of 0.5 μg / ml 10G5-mIgG2a antibody (Patent Application Publication No.: CN 000000) was added to well H12. 109311997A. Recombinant chimeric antibodies were prepared based on the sequence of the 10G5 antibody disclosed in this patent. A heavy chain variable region plus a mouse IgG2a hinge region constant region, and a light chain variable region plus a mouse kappa constant region were used as positive controls. Hybridoma supernatants from 40 cell culture plates were transferred to an ELISA plate coated with Axl-His protein and incubated at 37°C for 1 hour. The hybridoma supernatant was discarded from the ELISA plate. The plate was then washed sequentially using a microplate washer with five washes and a wash volume of 300 μl / well. After washing, 50 μl of a 1:10,000 dilution of HRP-conjugated rabbit anti-mouse secondary antibody (Jackson ImmunoResearch, Catalog #115-035-062) was added, and the plates were incubated at 37°C for 1 hour. Use Huisong PW960 model 96-well plate washer for washing, set the plate washer parameters: wash 5 times, wash volume 300μl / well, and wash the single plate in sequence. Pat the washed ELISA plate dry on a hard paper towel, add TMB single-component colorimetric solution at 50μl / well, and place it in a 37°C incubator for 3-10 minutes. Be careful not to over-color. Add ELISA stop solution to the color-developed ELISA plate at 50μl / well. Place the ELISA plate in a thermo scientific enzyme reader (Multiskan) and read the 450nm light absorbance value.

[0141] 50 μl of GAS6-His (Beijing Biopsies, GA6-H5246) was coated at a concentration of 5 μg / mL. Then, 50 μl of the culture supernatant of the hybridoma cells to be tested and 50 μl of AXL-mFc (prepared in Example 1, 2 μg / mL) were added and mixed thoroughly by pipetting. Negative (PBS) and positive control wells (10G5-mIgG2a, 20 μg / mL) were set up. Incubate at 37°C for 1 hour. After washing, add 50 μl of anti-mouse IgG-HRP enzyme-labeled secondary antibody to each well and incubate at 37°C for 1 hour. After washing, add 50 μl of TMB substrate solution and develop at 37°C for 2-15 minutes. The reaction was terminated with 50 μl of 2 mol / L H2SO4 and the OD450nm value was read on a microplate reader. A hybridoma cell line with high blocking efficiency was identified: 10C1. 10C1 was subcloned using the limiting dilution method, and finally a monoclonal clone 10C1 with binding and blocking activities was obtained.

[0142] 3. 10C1 subcloning and sequencing

[0143] The 10C1 hybridomas that were positive in the initial screening were subcloned by limiting dilution to obtain positive monoclonal cell lines. Specifically, the cells in the positive plate wells were aspirated and diluted to 150 cells / 20 ml with complete culture medium. The cell suspension was then dispensed into a 96-well culture plate, with 200 μl per well, resulting in an average of 1.5 cells per well. After culturing in an incubator for 7 days, cell ELISA screening was performed to obtain monoclonal 10C1 clones with binding activity. Positive wells were selected for expansion culture. After expansion to T75 flasks, 1×10 7 Cells were lysed and RNA was extracted (according to the kit instructions, Tiangen Biotechnology, Catalog No. DP430). Using RNA as a template, cDNA synthesis was performed according to the kit instructions (Thermo Fisher Scientific, Catalog No. 4387406) in a 20 μl synthesis volume. The cDNA was then used as a template to amplify the DNA encoding the antibody variable region. The primers used for amplifying the antibody-encoding DNA are shown in Table 2. The reaction systems are shown in Tables 3 and 4.

[0144] Table 2. Primers used to amplify antibody encoding DNA

[0145] Note: The degenerate bases in the table have commonly known meanings in the art, and I represents hypoxanthine.

[0146] Table 3. Reaction system for amplifying heavy chain variable region (VH)

[0147] Table 4. Reaction system for amplifying light chain variable region (VL)

[0148] The amplification program was: 98°C for 3 min; 98°C for 10 s, 55°C for 20 s, and 72°C for 30 s, for 20 cycles.

[0149] The sequencing results were analyzed using the online tool IMGT-V-QUEST, and the variable region coding sequence of the positive monoclonal antibody with clone number 10C1 was determined to be as follows:

[0150] Translated into amino acid sequence:

[0151] Example 3. Preparation of chimeric antibodies and analysis of their activity

[0152] 1. Preparation of Chimeric Antibodies

[0153] Based on the amino acid sequence of the heavy chain variable region of the mouse monoclonal antibody 10C1 in Example 2, a DNA molecule with a nucleotide sequence of SEQ ID No. 61 was designed (codon-optimized, using codons preferred by the host cell 293F). The fragment between the BsmBI enzyme recognition sites of the vector pHr-IgG1 (a small fragment including the BsmBI enzyme recognition site) was replaced to obtain the recombinant expression vector pHr-IgG1-10C1. The recombinant expression vector pHr-IgG1-10C1 can express the heavy chain of the chimeric antibody 10C1.

[0154] Based on the amino acid sequence of the light chain variable region of the murine monoclonal antibody 10C1 in Example 2, a coding nucleotide sequence was designed. This nucleotide sequence is the DNA molecule of SEQ ID No. 62 (codon-optimized, using codons preferred by the host cell 293F). This DNA sequence was used to replace the fragment between the BsmBI enzyme recognition sites of the pHr-Kappa vector (a small fragment including the BsmBI enzyme recognition site) to obtain the recombinant expression vector pHr-Kappa-10C1. The recombinant expression vector pHr-Kappa-10C1 can express the light chain of the chimeric antibody 10C1.

[0155] The pHr-IgG1 and pHr-Kappa vectors were modified from the pHR'CMV GFP vector (Addgene, Plasmid #14858). Using pHr-IgG1 as an example, the recombinant pHr-IgG1 vector was generated by replacing the small fragment between the KpnI and Not I restriction enzyme recognition sites of the pHR'CMV GFP vector with the target DNA molecule represented by SEQ ID No. 63, while maintaining the remaining nucleotide sequence of the pHR'CMV GFP vector unchanged. Positions 19-75 of SEQ ID No. 63 represent the signal peptide gene, positions 76-123 are the buffer sequence (with BsmBI restriction enzyme recognition sites at both ends), and positions 124-1116 represent the human IgG1 constant region (CH1+junction+CH2+CH3).

[0156] The construction of the pHr-Kappa vector was similar to that of pHr-IgG1, with the only difference being that the target DNA was replaced with the DNA fragment shown in SEQ ID No. 64. Positions 19-84 of SEQ ID No. 64 represent the signal peptide gene, positions 85-132 are the buffer sequence (with BsmBI enzyme recognition sites on both ends of the buffer sequence), and positions 133-456 represent the human kappa constant region (Constant Kappa).

[0157] The chimeric antibody heavy chain expression plasmid pHr-IgG1-10C1 and light chain expression plasmid pHr-Kappa-10C1 were co-transfected into 293F cells at a ratio of 1.5:1.

[0158] The target protein was produced by transfecting 293F cells, and the 10C1 chimeric antibody protein was obtained after affinity purification.

[0159] To facilitate the construction of bispecific antibodies and compare them with EGFR monoclonal antibodies, molecular construction and recombinant antibody expression were performed with reference to the clinically used antibodies cetuximab and panitumumab. According to the PDB database, the variable region sequences of the two are as follows:

[0160] According to the amino acid sequence of the above variable region, the coding DNA sequence was synthesized as follows:

[0161] According to the chimeric antibody construction method described above in this example, the DNA encoding the heavy and light chain variable regions of cetuimab and panitumumab were cloned and constructed into pHr-IgG1 and pHr-Kappa vectors, respectively, and transfected into 293F cells according to the transfection method described above to obtain recombinant antibodies. These two antibodies are cetuximab and panitumumab analogs (biosimilars), respectively. For convenience of description, they are referred to as cetuximab, panitumumab, or cetu (abbreviation for cetuximab-biosimilar) and pani (abbreviation for panitumumab-biosimilar) in this case.

[0162] The activity of the homemade Cetu and Pani antibodies was identified by ELISA. The specific steps are as follows:

[0163] Use coating solution (i.e. PBS) to dilute EGFR-His (nearshore protein, product number CI61) and AXL-His protein (prepared in Example 1) to 2 μg / mL, 50 μL / well are added to the ELISA plate wells, and incubate at 37°C for 2 hours. Discard the liquid in the wells and wash once with PBST washing solution using a plate washer (300 μl / time). Add 200 μL of blocking solution to each well and incubate overnight at 4°C. Discard the liquid in the wells and wash twice with washing solution using a plate washer (300 μl / time). Add 50 μL of homemade cetu or pani antibody to each well with an initial concentration of 20 μg / mL, and dilute continuously 5-fold in 7 gradients. At the same time, set up a blank control; incubate at 37°C for 60 minutes; discard the liquid in the wells and wash 5 times with washing solution using a plate washer (200 μl / time). Add 50 μL of Goat anti-human IgG-Fc secondary antibody (1:10,000 dilution) to each well and incubate at 37°C for 60 min. Discard the liquid from the wells and wash five times with a plate washer (200 μL / well). Add 50 μL / well of colorimetric solution and incubate at 37°C for 15 min. Terminate the reaction by adding 50 μL / well of 2 mol / L H₂SO₄, and read the OD₄50 value on a microplate reader. GraphPad Prism 8 software was used to generate the graph, as shown in Figure 1. The figure shows that both cetu and pani antibodies bind well to the EGFR-His-coated plate, with concentration-dependent binding curves. However, these antibodies do not bind to AXL-His, confirming that the homemade cetu and pani antibodies specifically bind to EGFR.

[0164] 2. ELISA-Binding Activity Analysis

[0165] Dilute the AXL-His protein (prepared in Example 1) to 2 μg / mL with coating solution, add 50 μL / well to the ELISA plate wells, and incubate at 37°C for 2 hours. Discard the liquid in the wells and wash once with 300 μL / well of PBST washing solution using a plate washer. Add 200 μL of blocking solution to each well and incubate overnight at 4°C. Discard the liquid in the wells and wash twice with 300 μL / well of washing solution using a plate washer. Add 50 μL of the 10C1 chimeric antibody prepared in step 1 to each well with an initial concentration of 20 μg / mL, and dilute continuously in 5-fold gradients for 7 times. Set up a blank control at the same time; incubate at 37°C for 60 minutes; discard the liquid in the wells and wash 5 times with 200 μL / well of washing solution using a plate washer. Add 50 μL of Goat-anti-human-IgG-Fc-Secondary Antibody (1:10,000 dilution) to each well and incubate at 37°C for 60 minutes. Discard the liquid in the wells and wash five times with a plate washer (200 μL / well of wash buffer). Add 50 μL / well of color development buffer and incubate at 37°C for 15 minutes. Terminate the reaction by adding 50 μL / well of 2 mol / L H₂SO₄, and read the OD₄50 value on a microplate reader. GraphPad Prism 8 software was used to generate the graph, as shown in Figure 2. The graph shows that the 10C1 chimeric antibody binds well to the coated AXL-His antigen, with a very low EC₅50 value indicating high activity.

[0166] 3. ELISA-Blocking Activity Analysis

[0167] Coat an ELISA plate with 50 μl of 2 μg / ml AXL-hFc (prepared in Example 1) and incubate at 37°C for 1 hour. Discard the supernatant, rinse the wells, and wash once with 300 μl / well PBST. Add 300 μl of 5% skim milk and incubate at 37°C for 1 hour. Discard the supernatant and wash once with 300 μl / well PBST. Dilute the 10C1 chimeric antibody prepared in Step 1 with 1% BSA (v / w, dissolved in PBST (0.05% TW20)) to a maximum concentration of 20 μg / ml, and serially dilute seven times in 5-fold increments. Add 25 μl / well of the diluted antibody working solution to the ELISA plate. Add 25 μl of 2 μg / ml GAS6-his to the wells, gently tap to mix, and incubate at 37°C for 2 hours. Discard the supernatant and rinse the wells five times with 200 μl / well PBST. Add 50 μl / well of a 1:5000 dilution of anti-his secondary antibody and incubate at 37°C for 1 hour. Discard the supernatant and wash the wells five times with 200 μl / well of PBST. Add 50 μl of colorimetric solution and develop for 6 minutes at 37°C or room temperature. Terminate the reaction by adding 50 μl / well of 2 mol / L H₂SO₄ and read the OD₄₅₅₀ value on a microplate reader. GraphPad Prism 8 software was used to generate the graph, as shown in Figure 3. The figure shows that the 10C1 chimeric antibody effectively blocks the binding of AXL to its ligand, GAS6.

[0168] IV. FACS-binding activity comparison

[0169] The 10C1 chimeric antibody prepared in step 1 was diluted with 1% BSA (v / w, dissolved in PBST (0.05% TW20)) to a maximum concentration of 20 μg / ml. The antibody was diluted 5-fold in series for 7 times. Calu-1 cells (human lung cancer cells, which are shown in public databases to be highly AXL-expressing cell lines. Link: https: / / www.proteinatlas.org / ENSG00000167601-AXL / cell+line) were counted and 3×10 were added to each well. 5The cells were washed twice with PBS, the supernatant discarded, and 50 μL of serially diluted antibody was added. The cells were incubated at 4°C for 1 hour. The cells were centrifuged at 1000 rpm for 2 minutes, the liquid was discarded, and the cells were washed twice with 200 μL / well of PBS. 50 μL of FITC-anti-human-IgG-Fc-secondary-antibody (1:200 dilution) was added to each well, and the cells were incubated at 4°C for 60 minutes. The cells were centrifuged at 1000 rpm for 2 minutes, the liquid was discarded, the cells were washed with PBS, and the cells were washed twice with 200 μL / well of PBS. 200 μL / well of PBS was added, the cells were resuspended, and the cells were read by flow cytometry. Graph Pad Prism 8 analysis software was used to generate the graph, and the results are shown in Figure 4. The figure shows that the 10C1 chimeric antibody binds well to Calu-1 cells, which overexpress AXL.

[0170] 5. Comparison of FACS blocking activity

[0171] The 10C1 chimeric antibody prepared in step 1 was diluted with 1% BSA (v / w, dissolved in PBST (0.05% TW20)) to a maximum concentration of 40 μg / ml, and diluted 5-fold for 7 times. Calu-1 cells were counted and 3×10 5 After washing the cells twice with PBS, 25 μL of GAS6-his (2 μg / mL) and 25 μL of serially diluted 10C1 chimeric antibody were added. The cells were incubated at 4°C for 60 minutes, centrifuged at 1000 rpm for 2 minutes, and the wells were decanted. The cells were then washed twice with PBS (200 μL / well). 50 μL of 6× His-Monoclonal-Antibody FITC (1:100 dilution) secondary antibody was added to each well. The cells were incubated at 4°C for 60 minutes, centrifuged at 1000 rpm for 2 minutes, and the wells were decanted. The wells were then washed twice with PBS (200 μL / well), and centrifuged at 1000 rpm for 2 minutes. The cells were resuspended in 200 μL / well of PBS and read by flow cytometry. The analysis results are shown in Figure 5, with mean fluorescence signal intensity as the y-axis and antibody concentration (log10 of ng / mL) as the x-axis. The chimeric antibody 10C1 effectively blocked the binding of GAS6 to Calu-1 cells, a cell line that highly expresses the AXL receptor. The blocking activity of 10C1-34, measured by the IC50, was 38.57 ng / ml.

[0172] 6. Species Crossover Experiment

[0173] Mouse AXL (mAXL-His, Sino Biological, 50126-M08H), monkey AXL (cynoAXL-His, ACRO, AXL-C52H3), and human AXL-His protein (prepared in Example 1) were diluted to 2 μg / mL with coating buffer. 50 μL / well of the coating buffer was added to the ELISA plate and incubated at 37°C for 2 hours. Discard the liquid from the wells and wash the plates five times with 200 μL of the washing buffer. Add 200 μL of blocking buffer to each well and incubate overnight at 4°C. Discard the liquid from the wells and wash the plates five times with 200 μL of the washing buffer. Add 50 μL of the 10C1 chimeric antibody dilution prepared in step 1 (diluted in 1% BSA) to each well at a concentration of 20 μg / mL. A seven-step gradient of antibody washer was added, with a blank control. Incubate at 37°C for 60 min. Discard the solution and wash the wells five times with 200 μL of wash buffer using a plate washer. Add 50 μL of horseradish peroxide-conjugated anti-human IgG secondary antibody (Jackson Immunoresearch, Catalog #109-035-190) working solution (diluted 20,000-fold with 1% BSA) to each well and incubate at 37°C for 60 min. Discard the solution and wash the wells five times with 200 μL of wash buffer using a plate washer. Add 50 μL of color development solution per well and incubate at 37°C for 15 min. Terminate the reaction by adding 50 μL of 2 mol / L H₂SO₄ per well, and read the OD₄50 value on a microplate reader. A curve was constructed with OD450 values ​​as the ordinate and the Log10 of concentration values ​​(ng / ml) as the abscissa, as shown in Figure 6. As can be seen from the figure, the 10C1 chimeric antibody can well recognize human and monkey AXL proteins with comparable affinity, with no difference in their EC50 values. However, the 10C1 chimeric antibody does not bind to mouse AXL.

[0174] Example 4: Humanization and screening of 10C1 monoclonal antibody

[0175] 1. Humanization of 10C1 mAb

[0176] Using the CDR grafting method, candidate sequences were designed as shown in Table 5. Three candidate sequences for the humanized heavy chain variable region of 10C1 were designated H3, H4, and H5, respectively; two sequences for the humanized light chain variable region were designated L4 and L5. Six humanized antibodies were obtained by combining two of these humanization approaches, and their corresponding nomenclature is shown in Table 6.

[0177] Table 5 Candidate sequences for humanization of the heavy and light chain variable regions of 10C1

[0178] Table 6. Combinations of 6 humanized antibodies and their corresponding nomenclature

[0179] The following DNA was synthesized by the supplier Shanghai Sangon as the 10C1 humanized heavy chain and light chain variable region encoding genes:

[0180] As described in Example 3, the humanized heavy and light chain variable region genes were ligated into the pHr-IgG1 and pHr-Kappa vectors, respectively, to obtain pHr-IgG1-H3 / 4 / 5 and pHr-Kappa-L4 / 5 recombinant plasmids. Also as described in Example 3, heavy and light chain plasmids were co-transfected into 293F cells in pairs for target protein production. After affinity purification, the 10C1 humanized antibody proteins were obtained: 10C1-34 / 35 / 44 / 45 / 54 / 55 (heavy chain numbering + light chain numbering).

[0181] As described in Example 3, an enzyme-linked immunosorbent assay (ELISA) plate coated with the AXL-His protein (prepared in Example 1) was prepared. Six humanized antibodies were diluted as described in Example 3 and reacted with the AXL-His protein ELISA plate. Absorbance at 450 nm (OD450) was measured on a microplate reader, with the OD450 value plotted on the ordinate and the antibody concentration (Log10, ng / ml) on the abscissa. This indicates the binding strength of the antibodies to the AXL-His protein antigen. The results were plotted using GraphPad Prism 8 software, as shown in Figure 7. 10C1-34 exhibited the lowest EC50 value for AXL-His binding, indicating the highest activity.

[0182] II. FACS Detection of Binding Activity of Humanized Antibody 10C1-34

[0183] Humanized antibody 10C1-34 (step 1) and chimeric antibody 10C1 (Example 3) were diluted with 1% BSA (v / w, dissolved in PBST (0.05% TW20)) to a maximum concentration of 20 μg / ml. The dilutions were repeated 7 times in 5-fold increments. U251 cells (human glioma cells, U251 is a known AXL-high-expressing cell line (doi:10.1007 / s43188-023-00195-z.eCollection 2023Oct)) were counted and 3×10 cells were added to each well. 5Cells were washed twice with PBS, the supernatant discarded, and 50 μL of serially diluted antibody was added. The cells were incubated at 4°C for 1 hour. The cells were centrifuged at 1000 rpm for 2 minutes, the liquid was discarded, and the cells were washed twice with 200 μL / well of PBS. 50 μL of FITC-anti-human-IgG-Fc-secondary antibody (1:200 dilution) was added to each well and the cells were incubated at 4°C for 60 minutes. The cells were centrifuged at 1000 rpm for 2 minutes, the liquid was discarded, the cells were washed with PBS, and the cells were washed twice with 200 μL / well of PBS. 200 μL / well of PBS was added, the cells were resuspended, and the cells were read by flow cytometry. GraphPad Prism 8 software was used to generate the graph, as shown in Figure 8. The figure shows that the activity of the humanized 10C1-34 antibody against the AXL-high-expressing cell line U251 remained unchanged compared to the parental antibody 10C1, demonstrating well-maintained activity.

[0184] Example 5: Construction, expression and purification of EGFR / AXL bispecific antibodies

[0185] The variable region sequences of the anti-EGFR monoclonal antibodies Cetuximab (abbreviated as cetu, see Example 3) and Panitumumab (abbreviated as pani, see Example 3) and the anti-AXL monoclonal antibody 10C1-34 were used to construct scFab-type bispecific antibodies. The antibody arms targeting different targets automatically heterodimerized using a knob-into-hole approach. The resulting antibodies contain two peptide chains that can simultaneously bind to EGFR and AXL. The Cetuximab and Panitumumab antibody variable region encoding genes were synthesized at Shanghai Bioengineering. The upstream and downstream of the encoding genes contain KpnI and Not I restriction sites, respectively. Using Yisheng Bio's endonucleases, according to the instructions, the pHr-IgG1 vector (see Example 3) was double-digested with KpnI (recognition sequence GGTACC, Shanghai Yisheng Bio, catalog number 15015ES70) and Not I (recognition sequence GCGGCCGC, Shanghai Yisheng Bio, catalog number 15021ES50) to prepare a linear vector with corresponding ends. T4 ligase (Shanghai Yisheng Biotechnology, catalog number 10300ES80) was used to connect the gene and vector according to the instructions to obtain scFab-type dual antibody expression vectors pHr-IgG1-cetu-knob, pHr-IgG1-pani-knob, and pHr-IgG1-10C1-34-Hole. The plasmids were extracted after sequencing verification.

[0186] The full sequence of the scFab-hG1 that constitutes the bispecific antibody is as follows:

[0187] Positions 1-107 of SEQ ID No. 1 are the light chain variable region of EGFR monoclonal antibody cetu, positions 108-214 are the light chain constant region (human kappa constant region), positions 215-244 are the linker, positions 245-363 are the heavy chain variable region of EGFR monoclonal antibody cetu, positions 364-461 are the heavy chain constant region CH1, positions 462-476 are the hinge region, positions 477-586 are the heavy chain constant region CH2, and positions 587-693 are the heavy chain constant region CH3.

[0188] Positions 1-107 of SEQ ID No. 2 are the light chain variable region of EGFR monoclonal antibody pani, positions 108-214 are the light chain constant region (human Kappa constant region), positions 215-244 are the linker, positions 245-363 are the heavy chain variable region of EGFR monoclonal antibody pani, positions 364-461 are the heavy chain constant region CH1, positions 462-476 are the hinge region, positions 477-586 are the heavy chain constant region CH2, and positions 587-693 are the heavy chain constant region CH3.

[0189] Positions 1-112 of SEQ ID No. 3 are the light chain variable region of the anti-AXL humanized antibody 10C1-34, positions 113-219 are the light chain constant region (human kappa constant region), positions 220-249 are the linker, positions 250-363 are the heavy chain variable region of the anti-AXL humanized antibody 10C1-34, positions 364-461 are the heavy chain constant region CH1, positions 462-476 are the hinge region, positions 477-586 are the heavy chain constant region CH2, and positions 587-693 are the heavy chain constant region CH3.

[0190] The coding gene sequence of scFab-hG1 constituting the bispecific antibody is as follows:

[0191] The vector combinations and corresponding names used to construct the bispecific antibodies are shown in Table 7.

[0192] Table 7. Vector combinations and corresponding names used to construct bispecific antibodies

[0193] The structure of the recombinant vector pHr-IgG1-cetu-knob is described as follows: the DNA fragment represented by "SEQ ID No. 79 (Kpn I recognition sequence, Kozak sequence, signal peptide sequence from 5' to 3' end) + SEQ ID No. 4 + GCGGCCGC" was double-digested with Kpn I and Not I, followed by ligation with the large pHr-IgG1 vector fragment that had also been double-digested with Kpn I and Not I, resulting in a recombinant plasmid.

[0194] The structure of the recombinant vector pHr-IgG1-pani-knob is described as follows: the DNA fragment represented by "SEQ ID No.79+SEQ ID No.5+GCGGCCGC" was double-digested with Kpn I and Not I, and then ligated with the large pHr-IgG1 vector fragment that had also been double-digested with Kpn I and Not I to obtain a recombinant plasmid.

[0195] The structure of the recombinant vector pHr-IgG1-10C1-34-Hole is described as follows: the DNA fragment represented by "SEQ ID No. 80 (Kpn I recognition sequence, Kozak sequence, signal peptide sequence from 5' to 3' end) + SEQ ID No. 6 + GCGGCCGC" is double-digested with Kpn I and Not I, and then ligated with the large pHr-IgG1 vector fragment that has also been double-digested with Kpn I and Not I to obtain a recombinant plasmid.

[0196] The prepared double-antibody expression vectors were paired and co-transfected into 293F cells as shown in Table 7 to express and purify the target protein. Specifically, 100 μg / species of double-antibody expression vector was mixed with the transfection reagent PEI and allowed to stand for 30 minutes before being added to the 293F cell culture medium. Cultured at 37°C, 5% carbon dioxide with shaking for 24 hours, supplement A (Zhuhai Kairui, item number K40001) was added, and the shaking culture was continued for 3 days. The culture solution was centrifuged at 12000rpm for 30 minutes, the supernatant was collected and added to 1ml ProteinA gel filler (Borgron AA402305), and after incubation at room temperature with shaking (145rpm) for 1-3 hours, it was allowed to flow through the gravity chromatography column, and the effluent was collected and frozen for re-testing. The column material was washed with 5ml of PBS, and the washing solution was allowed to slowly flow through the gel column. 3ml of glycine eluent (pH 3.2) was added to elute the captured protein. All eluates were centrifuged using ultrafiltration concentrator tubes (Millipore) and replaced with sterile, pyrogen-free PBS. The UV absorbance at 280 nm was measured using a Nanodrop and divided by the extinction coefficient to obtain the concentration.

[0197] Example 6: Detection of EGFR / AXL dual-target antibody activity

[0198] 1. FACS detection of EGFR / AXL dual antibody binding activity

[0199] Antibodies cetuximab (prepared in Example 3), panitumumab (prepared in Example 3), 10C1-34, BSG04, and BSG0402 were diluted with 1% BSA (v / w, dissolved in PBST (0.05% TW20)) to a maximum concentration of 20 μg / ml. The dilutions were repeated 7 times in 5-fold increments. U251 cells (human glioma cells) were counted and 3×10 cells were added to each well. 5 Cells were washed twice with PBS, the supernatant discarded, and 50 μL of serially diluted antibody was added. The cells were incubated at 4°C for 1 hour. The cells were centrifuged at 1000 rpm for 2 minutes, the liquid was discarded, and the cells were washed twice with 200 μL / well of PBS. 50 μL of FITC-anti-Human-IgG-Fc-Secondary Antibody (1:200 dilution) was added to each well. The cells were incubated at 4°C for 60 minutes. The cells were centrifuged at 1000 rpm for 2 minutes, the liquid was discarded, and the cells were washed twice with 200 μL / well of PBS. 200 μL / well of PBS was added, the cells were resuspended, and the cells were read by flow cytometry. GraphPad Prism 8 software was used to generate the graphs, and the results are shown in Figure 9. The analysis results show that compared with their parent monoclonal antibodies, the fluorescence signal values ​​of the bispecific antibody molecules BSG0402 (derived from the monoclonal antibody sequences pani and 10C1-34) and BSG04 (derived from the monoclonal antibody sequences cetu and 10C1-34) binding to U251 cells (a tumor cell line that simultaneously expresses EGFR and AXL) are higher, and the maximum value of the binding reaction is significantly higher, indicating better binding.

[0200] 2. Detection of endocytic activity of EGFR / AXL dual antibody

[0201] The cell density of U251 cells was adjusted to 3 × 10 6 / ml. Use a multichannel pipette to take 100μl and add it to the wells of a 96-well PCR plate. Centrifuge at 1500rpm for 5 minutes and discard the supernatant. Use DMEM medium containing 1% FBS to serially dilute the antibodies BSG0402, Panitumumab (prepared in Example 3), and 10C1-34, respectively. The starting concentration of the antibody is 20μg / ml, and the dilution is repeated 7 times. Take 50μl of the diluted antibodies and add them to the wells of a 96-well PCR plate and incubate at 4℃ for 1h. Centrifuge at 1500rpm for 5 minutes at 4℃, discard the supernatant, take 120μl of DMEM medium containing 1% FBS to resuspend the cells, centrifuge again at 1500rpm for 5 minutes, and discard the supernatant. A 1:200 dilution of goat anti-human IgG (H+L) Fab fragments labeled with CypHer5E (Amersham Biosciences, Catalog No. PA15401) were added. The cells were incubated in a dark refrigerator at 4°C for 1 hour. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and 120 μl of the wash buffer was resuspended in the cells. After further centrifugation, the cells were mixed with 100 μl of DMEM medium and incubated in the dark at 37°C for 4 hours. The cells were analyzed using the APC-H channel of a flow cytometer. The mean fluorescence intensity (MFI) was plotted on the vertical axis against the Log10 value of the antibody concentration (ng / ml) on the horizontal axis. The results are shown in Figure 10. This analysis shows that the maximum internalization value (top value) of the bispecific antibody BSG0402 was significantly greater than that of the monoclonal antibody, indicating superior efficiency.

[0202] 3. Detection of EGFR / AXL dual antibody-mediated cytotoxicity (ADCC) activity by reporter gene assay

[0203] Bispecific antibodies simultaneously target EGFR and AXL, and bind to effector cells (such as NK cells) through their Fc regions, thereby mediating the killing of target cells by effector cells. JNJ-372 is known to be a dual-antibody drug targeting EGFR and MET (reference doi:10.1016 / j.jbc.2021.100641.Epub 2021Apr 8.). By comparing the activity of the candidate molecules in the present invention with them, the ADCC efficiency of the molecules of the present invention can be characterized to a certain extent. The glycosylation modification of the JNJ-372 antibody (MCE, HY-P9977) is a low-fucose form. For comparison, the present invention handed over BSG0402 to Sino-Biological for production in special 293F cells to obtain a dual-antibody molecule with afucosylation-free form (AF), namely BSG0402AF.

[0204] Protocol: Target U251 cells were collected (culture conditions were the same as above) and labeled with Calcein AM. Target cells and a gradient of test antibody concentrations were added to a 96-well plate and incubated in a CO2 incubator for 20 minutes. Effector cells (NK92 cells) were collected and added to a 96-well plate at a 10:1 effector-target ratio in a CO2 incubator. After incubation for 4 hours, the 488 (EX) / 515 (EM) fluorescence values ​​were measured with a microplate reader, and the target cell killing rate was calculated. Graphpad Prism nonlinear S-curve regression was used to fit the data to generate dose-effect curves, and EC50 was calculated (8 antibody concentrations were set in triplicate, with a no-antibody control and 5-fold dilution).

[0205] Experimental steps:

[0206] 1) Resuscitate NK92 and U251 cells, passage them 2-3 times, and use them for experiments after recovery.

[0207] 2) Collect U251 cells, centrifuge, discard the supernatant, and resuspend in PBS;

[0208] 3) Calcein AM labeled target cells (U251 cells) were resuspended in complete culture medium to 2×10 5 / mL;

[0209] 4) Target cells were added to a 96-well plate, 50 μL / well;

[0210] 5) Prepare 3× test antibodies at 8 antibody concentrations: 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM.

[0211] 6) Add 50 μL / well of the antibody to be tested at a gradient concentration, set up a no-antibody control, a positive control for maximum target cell release, and a negative control for spontaneous target cell release, three replicates, and incubate in a CO2 incubator for 20 minutes;

[0212] 7) Add 50 μL / well of effector cells (NK92 cells), add culture medium to each control well to make up the volume to 150 μL, and incubate in a CO2 incubator for 4 hours;

[0213] 8) Add 100 μL of lysis buffer to the well with the maximum target cell release, and add 100 μL of PBS to the remaining wells. Shake for 3 minutes, and centrifuge at 2000 rpm for 5 minutes.

[0214] 9) Take the supernatant and measure the 488 (EX) / 515 (EM) fluorescence value using a microplate reader.

[0215] Detection indicators and data analysis:

[0216] The 488(EX) / 515(EM) fluorescence values ​​were detected and the target cell killing rate was calculated. The dose-effect curve was obtained by nonlinear S-curve regression fitting using Graphpad prism, and the EC50 was calculated.

[0217] The results are shown in Figure 11. It can be seen that BSG0402AF has the best ADCC-mediated activity, and its ADCC activity is 7 times that of JNJ-372 calculated by EC50 value.

[0218] Example 7. Preparation and Activity Identification of Bis-Antibody Drug Conjugates (ADCs)

[0219] 1. Preparation of bispecific antibody-drug conjugates (ADCs)

[0220] Antibody-drug conjugates (ADCs) couple small molecule drugs, such as MMAE, which destroys cellular tubulin, or Deruxtecan, which inhibits topoisomerase I, to antibody molecules. These coupling methods vary, ranging from random coupling to the free sulfhydryl groups (SH) of the antibody molecules to site-specific coupling. The present invention uses TCEP to reduce the antibody molecules, thereby converting the cysteine ​​residues in the antibody molecules into sulfhydryl groups. This allows the maleamide-GGFG-Dxd molecule to be coupled to the antibody molecules (maleamide and sulfhydryl groups form a thioether bond, achieving coupling via the thioether bond). The maleamide-GGFG-Dxd molecule is Deruxtecan, and its molecular formula and structural formula are described above.

[0221] The coupling steps are as follows:

[0222] 1. Add 40 mg of BSG04 antibody and 24.78 μl of TCEP to a 1.7 ml reaction system and incubate at 37°C for 2 hours.

[0223] 2. Add 1.3 ml of 2 mM maleamide-GGFG-Dxd and continue the reaction at 37°C for 1 hour.

[0224] 3. Perform chromatography using a PD10 desalting column and elute the ADC molecules with PBS.

[0225] 4. Use HPLC to detect the antibody and ADC molecules to confirm whether the coupling is successful.

[0226] The test results are shown in Figure 12. It can be seen from the figure that the purity of the labeled antibody meets the requirements and the labeling is effective.

[0227] The present invention also used mass spectrometry to analyze the BSG04-based ADC molecule (hereinafter referred to as BSG04D) and confirmed that its drug (Dxd)-antibody coupling ratio (DAR) was 8. As shown in Figure 13 .

[0228] 2. In vivo drug efficacy testing in tumor cell transplantation model mice

[0229] In order to test the efficacy of BSG04D, a mouse transplant tumor model (cell-derived xenograft model, CDX) was constructed. The experiment was completed by Yikang Pharmaceutical. Specifically, tumor cells H1975 (human lung adenocarcinoma cells) were cultured in RPMI-1640 culture medium containing inactivated 10% fetal bovine serum, 100U / ml penicillin and 100μg / ml streptomycin and 2mM glutamine in an incubator at 37°C and 5% CO2. The cells were subcultured every 3 to 4 days after they were fully grown, and the tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo. The tumor cells resuspended in PBS were inoculated subcutaneously on the right flank of Balb / c nude mice (Weitong Lihua, Cat. No. 401). When the tumor grew to 150-200mm 3 The drugs were administered in groups at 14:00 and 18:00, with a total of 3 groups. The specific dosing schedule is shown in Table 8.

[0230] Table 8. Grouping and dosing regimen for in vivo drug efficacy testing in tumor cell transplanted tumor model mice

[0231] Starting from the first dose, the tumor volume of each animal was recorded, and a growth curve of the transplanted tumors for each group was plotted with time as the horizontal axis, as shown in Figure 14. As can be seen, the BSG04D 5 mg / kg dose group effectively inhibited tumor growth, with tumor growth inhibition rates (TGI) repeatedly exceeding 90% (specifically, tumor volume was measured twice weekly and the TGI was calculated. The TGI was consistently greater than 90% for the last six measurements before the end of the experiment).

[0232] To test the efficacy of BSG0402AF and BSG04D against third-generation EGFR-targeted tyrosine kinase inhibitor (TKI)-resistant tumors, a drug-resistant H1975 (H1975 3M) mouse xenograft tumor model was constructed. The model involves replacing the original EGFR double mutation (exon 19del / exon 21L858R) gene with a triple EGFR kinase domain mutation (exon 19del / exon 21L858R / exon20 C797S) using gene editing technology (biallelic substitution). The NOG strain of mice was used. The experimental methods are described above for the H1975 model. The growth curves of the xenograft tumors after inoculation and drug administration are shown in Figure 15 . The growth curves show that BSG04D 5mg / kg, administered twice weekly, effectively inhibited the growth of H1975 3M xenografts, with tumor disappearance observed on day 25. The dual-antibody BSG0402AF 10mg / kg, administered twice weekly for six doses, also resulted in tumor disappearance on day 25.

[0233] Industrial Applications

[0234] The bispecific antibody targeting EGFR and AXL provided by the present invention can mediate stronger endocytosis and ADCC activity than its parental counterpart. Bispecific antibodies and antibody-drug conjugates constructed using 10C1-34 exhibit excellent anti-tumor activity, particularly against drug-resistant tumors. This invention has great potential for clinical application in the field of tumor treatment.

Claims

1. A bispecific antibody targeting EGFR and AXL, comprising an EGFR antigen-binding domain and an AXL antigen-binding domain; The EGFR antigen-binding domain is as follows (A1) or (A2): (A1) EGFR antigen-binding domain 1; the EGFR antigen-binding domain 1 comprises the heavy chain variable region 1 of an anti-EGFR antibody and the light chain variable region 1 of an anti-EGFR antibody; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region 1 of the anti-EGFR antibody are positions 270-277, 295-305, and 340-352 of SEQ ID No. 1, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region 1 of the anti-EGFR antibody are positions 27-32, 50-51, and 89-97 of SEQ ID No. 1, respectively; (A2) EGFR antigen-binding domain 2; the EGFR antigen-binding domain 2 comprises the heavy chain variable region 2 of an anti-EGFR antibody and the light chain variable region 2 of an anti-EGFR antibody; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region 2 of the anti-EGFR antibody are positions 270-279, 297-303, and 342-352 of SEQ ID No. 2, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region 2 of the anti-EGFR antibody are positions 27-32, 50-51, and 89-97 of SEQ ID No. 2, respectively; The AXL antigen-binding domain comprises the heavy chain variable region of an anti-AXL antibody and the light chain variable region of an anti-AXL antibody; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the anti-AXL antibody are positions 275-282, positions 300-307, and positions 346-352 of SEQ ID No. 3, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the anti-AXL antibody are positions 27-37, positions 55-56, and positions 94-102, respectively.

2. The bispecific antibody according to claim 1, wherein: The bispecific antibody is a heterodimer composed of a first peptide chain and a second peptide chain; The first peptide chain includes, from N-terminus to C-terminus, a light chain variable region, a light chain constant region, a connecting peptide, a heavy chain variable region, a heavy chain constant region CH1, a hinge region, a heavy chain constant region CH2, and a heavy chain constant region CH3 of an anti-EGFR antibody; The second peptide chain includes, from N-terminus to C-terminus, the light chain variable region of the anti-AXL antibody, the light chain constant region, a connecting peptide, the heavy chain variable region of the anti-AXL antibody, the heavy chain constant region CH1, the hinge region, the heavy chain constant region CH2 and the heavy chain constant region CH3; Furthermore, the two heavy chain constant regions CH3 of the first peptide chain and the second peptide chain are designed into a knob structure and a hole structure respectively using the knob-into-hole technology.

3. The bispecific antibody according to claim 2, wherein: In the first peptide chain and the second peptide chain, the category of the light chain constant region is κ chain or λ chain; and / or the category of the heavy chain constant region is IgG, IgM, IgE, IgA or IgD.

4. The bispecific antibody according to claim 2, wherein: In the first peptide chain, the amino acid sequence of the light chain variable region of the anti-EGFR antibody is positions 1-107 of SEQ ID No.1 or positions 1-107 of SEQ ID No.2; and / or, the amino acid sequence of the heavy chain variable region of the anti-EGFR antibody is positions 245-363 of SEQ ID No.1 or positions 245-363 of SEQ ID No.

2.

5. The bispecific antibody according to claim 2, wherein: In the first peptide chain, the amino acid sequence of the light chain constant region is positions 108-214 of SEQ ID No. 1 or positions 108-214 of SEQ ID No.

2.

6. The bispecific antibody according to claim 2, wherein: In the first peptide chain, the amino acid sequence of the connecting peptide is positions 215-244 of SEQ ID No. 1 or positions 215-244 of SEQ ID No.

2.

7. The bispecific antibody according to claim 2, wherein: In the first peptide chain, the amino acid sequence of the heavy chain constant region CH1 is positions 364-461 of SEQ ID No. 1 or positions 364-461 of SEQ ID No.

2.

8. The bispecific antibody according to claim 2, wherein: In the first peptide chain, the amino acid sequence of the hinge region is positions 462-476 of SEQ ID No. 1 or positions 462-476 of SEQ ID No.

2.

9. The bispecific antibody according to claim 2, wherein: In the first peptide chain, the amino acid sequence of the heavy chain constant region CH2 is positions 477-586 of SEQ ID No. 1 or positions 477-586 of SEQ ID No.

2.

10. The bispecific antibody according to claim 2, characterized in that: In the first peptide chain, the amino acid sequence of the heavy chain constant region CH3 is positions 587-693 of SEQ ID No. 1 or positions 587-693 of SEQ ID No.

2.

11. The bispecific antibody according to claim 2, characterized in that: In the second peptide chain, the amino acid sequence of the light chain variable region of the anti-AXL antibody is positions 1-112 of SEQ ID No. 3; and / or the amino acid sequence of the heavy chain variable region of the anti-AXL antibody is positions 250-363 of SEQ ID No.

3.

12. The bispecific antibody according to claim 2, characterized in that: In the second peptide chain, the amino acid sequence of the light chain constant region is positions 113-219 of SEQ ID No.

3.

13. The bispecific antibody according to claim 2, wherein: In the second peptide chain, the amino acid sequence of the connecting peptide is positions 220-249 of SEQ ID No.

3.

14. The bispecific antibody according to claim 2, characterized in that: In the second peptide chain, the amino acid sequence of the heavy chain constant region CH1 is positions 364-461 of SEQ ID No.

3.

15. The bispecific antibody according to claim 2, characterized in that: In the second peptide chain, the amino acid sequence of the hinge region is positions 462-476 of SEQ ID No.

3.

16. The bispecific antibody according to claim 2, characterized in that: In the second peptide chain, the amino acid sequence of the heavy chain constant region CH2 is positions 477-586 of SEQ ID No.

3.

17. The bispecific antibody according to claim 2, wherein: In the second peptide chain, the amino acid sequence of the heavy chain constant region CH3 is positions 587-693 of SEQ ID No.

3.

18. The bispecific antibody according to claim 2, characterized in that: The amino acid sequence of the first peptide chain is SEQ ID No. 1 or SEQ ID No.

2.

19. The bispecific antibody according to claim 2, characterized in that: The amino acid sequence of the second peptide chain is SEQ ID No.

3.

20. The bispecific antibody according to claim 2, wherein: The bispecific antibody is modified by glycosylation.

21. Any of the following biological materials: (B1) a nucleic acid molecule encoding the bispecific antibody according to any one of claims 1 to 20; (B2) an expression cassette containing the nucleic acid molecule described in (B1); (B3) a recombinant vector containing the nucleic acid molecule described in (B1); (B4) a recombinant bacterium containing the nucleic acid molecule described in (B1); (B5) A transgenic cell line containing the nucleic acid molecule described in (B1).

22. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the heavy chain variable region 1 of the anti-EGFR antibody in the EGFR antigen-binding domain 1 are positions 808-831, 883-915 and 1018-1056 of SEQ ID No. 4, respectively.

23. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the light chain variable region 1 of the anti-EGFR antibody in the EGFR antigen-binding domain 1 are positions 79-96, 148-153 and 265-291 of SEQ ID No. 4, respectively.

24. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the heavy chain variable region 2 of the anti-EGFR antibody in the EGFR antigen-binding domain 2 are positions 808-837, 889-909 and 1024-1056 of SEQ ID No. 5, respectively.

25. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the light chain variable region 2 of the anti-EGFR antibody in the EGFR antigen-binding domain 2 are positions 79-96, 148-153 and 265-291 of SEQ ID No. 5, respectively.

26. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2, and CDR3 in the heavy chain variable region of the anti-AXL antibody in the AXL antigen-binding domain are positions 823-846, 898-921, and 1036-1056 of SEQ ID No. 6, respectively.

27. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the light chain variable region of the anti-AXL antibody are positions 79-111, 163-168 and 280-306 of SEQ ID No. 6, respectively.

28. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region of the anti-EGFR antibody in the first peptide chain is positions 1-321 of SEQ ID No. 4 or positions 1-321 of SEQ ID No. 5, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more identity with the above sequence.

29. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the light chain constant region in the first peptide chain is positions 322-642 of SEQ ID No.4 or positions 322-642 of SEQ ID No.5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

30. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the connecting peptide in the first peptide chain is positions 643-732 of SEQ ID No.4 or positions 643-732 of SEQ ID No.5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

31. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the anti-EGFR antibody in the first peptide chain is positions 733-1089 of SEQ ID No. 4 or positions 733-1089 of SEQ ID No. 5, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more identity with the above sequence.

32. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain constant region CH1 in the first peptide chain is positions 1090-1383 of SEQ ID No.4 or positions 1090-1383 of SEQ ID No.5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

33. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the hinge region in the first peptide chain is positions 1384-1428 of SEQ ID No.4 or positions 1384-1428 of SEQ ID No.5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

34. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain constant region CH2 in the first peptide chain is positions 1429-1758 of SEQ ID No.4 or positions 1429-1758 of SEQ ID No.5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

35. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain constant region CH3 in the first peptide chain is positions 1759-2079 of SEQ ID No.4 or positions 1759-2079 of SEQ ID No.5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

36. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region of the anti-AXL antibody in the second peptide chain is positions 1-336 of SEQ ID No. 6, or has an identity of greater than 99%, greater than 95%, greater than 90%, greater than 85%, greater than 80% or greater than 75% with the above sequence.

37. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the light chain constant region in the second peptide chain is positions 337-657 of SEQ ID No. 6, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

38. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the connecting peptide in the second peptide chain is positions 658-747 of SEQ ID No. 6, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

39. The biomaterial according to claim 21, wherein: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the anti-AXL antibody in the second peptide chain is positions 748-1089 of SEQ ID No. 6, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more identity with the above sequence.

40. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain constant region CH1 in the second peptide chain is positions 1090-1383 of SEQ ID No. 6, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

41. The biomaterial according to claim 21, wherein: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the hinge region in the second peptide chain is positions 1384-1428 of SEQ ID No. 6, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

42. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain constant region CH2 in the second peptide chain is positions 1429-1758 of SEQ ID No. 6, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

43. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain constant region CH3 in the second peptide chain is positions 1759-2079 of SEQ ID No. 6, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

44. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the first peptide chain is SEQ ID No. 4 or SEQ ID No. 5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

45. The biomaterial according to claim 21, characterized in that: (B1) In the nucleic acid molecule, the nucleotide sequence encoding the second peptide chain is SEQ ID No. 6, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with the above sequence.

46. ​​An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that: The antibody-drug conjugate comprises the bispecific antibody according to any one of claims 1 to 20 and a drug moiety coupled to the bispecific antibody; the drug moiety contains one, two or more drugs.

47. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 46, wherein: The drug is selected from at least one of the following: cytotoxic drugs, hormone drugs and biological response modifier drugs.

48. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 47, wherein: The cytotoxic drug is selected from at least one of the following: a drug that acts on the chemical structure of DNA, a drug that affects nucleic acid synthesis, a drug that acts on nucleic acid transcription, and a drug that mainly acts on tubulin synthesis; The hormone drug is selected from at least one of the following: an anti-estrogen, an aromatase inhibitor, and an anti-androgen; The biological response modifier drug is selected from at least one of the following: interferon, interleukin-2, and thymosin.

49. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 47, wherein: The drug is Dxd.

50. A method for preparing the antibody-drug conjugate according to claim 47, comprising the steps of: reducing the bispecific antibody according to any one of claims 1 to 20 with TCEP to convert cysteine ​​groups in the bispecific antibody into sulfhydryl groups, and then coupling a maleimide-GGFG-Dxd molecule to the antibody molecule to obtain the antibody-drug conjugate; The maleamide-GGFG-Dxd molecule is Deruxtecan.

51. Any of the following applications: The application is any of the following: (C1) Use of the biomaterial of any one of claims 21 to 45 in the preparation of the bispecific antibody of any one of claims 1 to 20 or the antibody-drug conjugate of any one of claims 46 to 49, or a pharmaceutically acceptable salt thereof; (C2) Use of the bispecific antibody of any one of claims 1 to 20 in the preparation of the antibody-drug conjugate of claim 46 or 47, or a pharmaceutically acceptable salt thereof; (C3) Use of the bispecific antibody according to any one of claims 1 to 20, the biomaterial according to any one of claims 21 to 45, or the antibody-drug conjugate according to any one of claims 46 to 49, or a pharmaceutically acceptable salt thereof, in the preparation of an anti-tumor drug.

52. The use according to claim 51, characterized in that: The tumor is selected from at least one of the following: lung cancer, glioma, head and neck tumor, and colorectal cancer.

53. The use according to claim 51, characterized in that: The tumor is a drug-inresistant tumor or a drug-resistant tumor.

54. The use according to claim 53, characterized in that: The drug-resistant tumor is a tumor resistant to third-generation EGFR-targeted tyrosine kinase inhibitors.

55. Either of the following methods: Method I: A method for preparing the antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 46 or 47, comprising the following steps: using the bispecific antibody according to any one of claims 1 to 20 to prepare the antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 46 or 47; Method II: A method for preparing an anti-tumor drug, comprising the following steps: using the bispecific antibody according to any one of claims 1 to 20 or the biological material according to any one of claims 21 to 45 or the Request for preparing an anti-tumor drug using the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of 46-50; Method III: An anti-tumor method comprising the following steps: administering to a subject the bispecific antibody according to any one of claims 1 to 20 or the antibody-drug conjugate according to any one of claims 46 to 50 or a pharmaceutically acceptable salt thereof.

56. The method according to claim 55, characterized in that: The tumor is selected from at least one of the following: lung cancer, glioma, head and neck tumor, and colorectal cancer.

57. The method according to claim 55, wherein: The tumor is a drug-inresistant tumor or a drug-resistant tumor.

58. The method according to claim 57, wherein: The drug-resistant tumor is a tumor resistant to third-generation EGFR-targeted tyrosine kinase inhibitors.

59. An anti-AXL antibody comprising a heavy chain variable region and a light chain variable region; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are positions 26-33, 51-58, and 97-103 of SEQ ID No. 7, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are positions 27-37, 55-56, and 94-102 of SEQ ID No. 8, respectively.

60. The anti-AXL antibody according to claim 59, characterized in that: The amino acid sequence of the heavy chain variable region of the anti-AXL antibody is SEQ ID No. 7, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more identity with the above sequence.

61. The anti-AXL antibody according to claim 59, characterized in that: The amino acid sequence of the light chain variable region of the anti-AXL antibody is SEQ ID No. 8, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more identity with the above sequence.

62. The anti-AXL antibody according to claim 59, characterized in that: In the anti-AXL antibody, the class of the light chain constant region is κ chain or λ chain; and / or the class of the heavy chain constant region is IgG, IgM, IgE, IgA or IgD.

63. The anti-AXL antibody according to claim 62, characterized in that: In the anti-AXL antibody, the light chain constant region is a human Kappa constant region, and the heavy chain constant region is a human IgG1 constant region.

64. Any of the following biological materials: (D1) a nucleic acid molecule encoding the anti-AXL antibody described above; (D2) an expression cassette containing the nucleic acid molecule described in (D1); (D3) a recombinant vector containing the nucleic acid molecule described in (D1); (D4) a recombinant bacterium containing the nucleic acid molecule described in (D1); (D5) A transgenic cell line containing the nucleic acid molecule described in (D1).

65. The biomaterial according to claim 64, characterized in that: In the nucleic acid molecule, the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the heavy chain variable region of the anti-AXL antibody are positions 76-99, 151-174 and 289-309 of SEQ ID No. 9, respectively; and / or, the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the light chain variable region are positions 79-111, 163-168 and 280-306 of SEQ ID No. 10, respectively.

66. The biomaterial according to claim 64, characterized in that: In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the anti-AXL antibody is SEQ ID No. 9, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with the above sequence.

67. The biomaterial according to claim 64, characterized in that: The nucleotide sequence encoding the light chain variable region of the anti-AXL antibody is SEQ ID No. 10, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with the above sequence.

68. Any of the following applications: (E1) Use of the nucleic acid molecule described in (D1) of claim 64, the expression cassette described in (D2) of claim 64, the recombinant vector described in (D3) of claim 64, the recombinant bacterium described in (D4) of claim 64, or the transgenic cell line described in (D5) of claim 64 in the preparation of the anti-AXL antibody described in any one of claims 59 to 63; (E2) Use of the anti-AXL antibody of any one of claims 59 to 63, the nucleic acid molecule of claim 64 (D1), the expression cassette of claim 64 (D2), the recombinant vector of claim 64 (D3), the recombinant bacterium of claim 64 (D4), or the transgenic cell line of claim 64 (D5) in the preparation of a product for preventing and / or treating diseases associated with overexpression of AXL; (E3) Use of the anti-AXL antibody of any one of claims 59 to 63, the nucleic acid molecule of claim 64 (D1), the expression cassette of claim 64 (D2), the recombinant vector of claim 64 (D3), the recombinant bacterium of claim 64 (D4), or the transgenic cell line of claim 64 (D5) in the preparation of a product for detecting AXL; (E4) Use of the anti-AXL antibody according to any one of claims 59 to 63, the nucleic acid molecule according to (D1) according to claim 64, the expression cassette according to (D2) according to claim 64, the recombinant vector according to (D3) according to claim 64, the recombinant bacterium according to (D4) according to claim 64, or the transgenic cell line according to (D5) according to claim 64 in the preparation of a product for binding to AXL; (E5) Use of the anti-AXL antibody according to any one of claims 59 to 63, the nucleic acid molecule according to (D1) according to claim 64, the expression cassette according to (D2) according to claim 64, the recombinant vector according to (D3) according to claim 64, the recombinant bacterium according to (D4) according to claim 64, or the transgenic cell line according to (D5) according to claim 64 in the preparation of a product for blocking the binding activity of CAS6 to AXL.

69. Either of the following methods: Method I: A method for preparing the anti-AXL antibody according to any one of claims 59 to 63, comprising the following steps: preparing the anti-AXL antibody using the nucleic acid molecule according to claim 64 (D1), the expression cassette according to claim 64 (D2), the recombinant vector according to claim 64 (D3), the recombinant bacterium according to claim 64 (D4), or the transgenic cell line according to claim 64 (D5); Method II: A method for preventing and / or treating a disease associated with overexpression of AXL, comprising the steps of: administering to a subject the anti-AXL antibody of any one of claims 59 to 63; Method III: A method for detecting AXL, comprising the following steps: detecting a sample using the anti-AXL antibody according to any one of claims 59 to 63; Method IV: A method for binding to AXL, comprising the steps of: treating a test sample with the anti-AXL antibody according to any one of claims 59 to 63; Method V: A method for blocking the binding activity of CAS6 and AXL, comprising the following steps: treating a test sample with the anti-AXL antibody according to any one of claims 59-63.

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