Bispecific antibodies against claudin 18A2 and CD3 and their use

Abstract

The present invention relates to bispecific antibodies against claudin 18.2 and CD3, pharmaceutical compositions comprising said bispecific antibodies, and related uses in the treatment of cancer.

Description

[Technical Field]

[0001] This invention belongs to the field of immunology and, more specifically, relates to a bispecific antibody against claudin 18.2 (Claudin18.2, CLDN18A2, CLDN18.2) and CD3. The antibody simultaneously comprises a binding domain that recognizes claudin 18.2 and a CD3 binding domain that specifically binds to T cells. Furthermore, this invention relates to a pharmaceutical composition comprising the bispecific antibody and its related use in the treatment of cancer. [Background technology]

[0002] Claudin 18 (CLDN18) is an intrinsic membrane protein located in the tight junction between the epithelium and endothelium. With a molecular weight of approximately 27.9 kD, it forms tight junctions between cells along with other tight junction proteins, regulating the passage of tissue molecules and ions through the intercellular space and maintaining the stability of the tissue environment. Human claudin 18 is known to exist as two subtypes: splice mutant 1 (CLDN18A1, CLDN18.1) with GenBank registration numbers NP_057453 and NM_016369, and splice mutant 2 (CLDN18A2, CLDN18.2) with GenBank registration numbers NP_001002026 and NM_001002026. In normal cells, CLDN18A1 is selectively expressed in lung epithelium, while CLDN18A2 is specifically expressed in normal gastric epithelial differentiated cells and not expressed in gastric epithelial stem cells with cell division activity. However, in tumor cells, CLDN18A2 is overexpressed in multiple types of cancer. For example, CLDN18A2 is highly expressed in 75% of gastric cancer patients, 50% of pancreatic cancer patients, 30% of esophageal cancer patients, and is also highly expressed in other cancers such as lung cancer. Therefore, finding an antibody that specifically binds to CLDN18A2 but not to CLDN18A1 is of great importance in cancer treatment and diagnosis.

[0003] The conventional CLDN18.2 antibody IMAB362 has already entered clinical research, and clinical results have shown that in gastric cancer patients with high CLDN18.2 expression, chemotherapy + IMAB362 extended progression-free survival from 6.1 months to 9.1 months and overall survival from 9.3 months to 16.6 months compared to simple chemotherapy. In addition to the antibody IMAB362, at least three other CLDN18.2 monoclonal antibody drugs are currently in Phase I clinical research. CAR-T receptor thrombi (CAR-T) therapy targeting CLDN18.2 have also entered clinical research. However, these antibodies that have entered clinical research have weak affinity for CLDN18.2, weak in vivo antitumor activity in the preclinical stage, and significant side effects. Therefore, it is necessary to continue screening and developing CLDN18.2 antibodies with higher activity, lower toxicity, and a wider therapeutic window. This will allow for stronger therapeutic effects at lower doses and a wider range of optimal dosages.

[0004] T-cell therapy has shown clear antitumor effects in many animal models, and recently, many T-cell therapies have made remarkable progress in treating cancer indications. Therefore, it is crucial to develop novel T-cell bispecific antibodies that are highly efficient and low-toxicity by unleashing the potential of T cells. This invention provides a bispecific antibody against CLDN18.2 and CD3 that recruits T cells to tumor sites via a CD3 target, specifically kills tumor cells that highly express CLDN18.2, and enables the targeting of the cytotoxic effect of T cells to cancer cells. Currently, in the prior art, a bispecific binding molecule that simultaneously recognizes CLDN18.2 and CD3ε is known, for example, 1BiMAB disclosed in CN105073776B. However, this molecule has a small molecular weight and lacks components such as an Fc segment, resulting in a short molecular half-life and poor stability. Therefore, it is necessary to construct a bispecific binding molecule that is more stable in vivo and in vitro and has a longer half-life. This is expected to enable intravenous administration once or twice a week, rather than daily, as is the case with 1-BiMAB. [Overview of the project]

[0005] The present invention provides a bispecific antibody comprising an anti-CLDN18.2 antigen-binding domain and an anti-CD3 antigen-binding domain, wherein the first binding domain can bind to the CLDN18.2 protein and the second binding domain can bind to CD3ε.

[0006] In some embodiments, CLDN18.2 has a protein (mRNA:NM_001002026) with GenBank registration number NP_001002026. CLDN18.1 has a protein (mRNA:NM_016369) with GenBank registration number NP_057453.

[0007] In the bispecific antibody according to the present invention, the anti-CLDN18.2 binding domain is derived from antibody 6#AA, and antibody 6#AA or its antigen-binding fragment specifically binds to CLDN18.2 but does not significantly bind to CLDN18.1. In some embodiments, the binding level of antibody 6#AA or its antigen-binding fragment to CLDN18.1 does not exceed 20% of its binding level to CLDN18.2. For example, the binding level may be 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 1% of the binding level of the antibody or antigen-binding fragment to CLDN18.2. In some embodiments, the binding level of antibody 6#AA or its antigen-binding fragment to CLDN18.2 is 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, or more than 10x the binding level to CLDN18.1.

[0008] The antibody 6#AA or its antigen-binding fragment may be specifically bound to CLDN18.2 and include a heavy chain variable region and a light chain variable region. The sequences of the three CDRs in the heavy chain variable region, HCDR1, HCDR2, and HCDR3, are shown in SEQ ID NO: 11, 12, and 13, respectively, and the sequences of the three CDRs in the light chain variable region, LCDR1, LCDR2, and LCDR3, are shown in SEQ ID NO: 14, 15, and 16, respectively.

[0009] Accordingly, in the bispecific antibody according to the present invention, the anti-CLDN18.2 binding domain may also bind specifically to CLDN18.2 and includes a heavy chain variable region and a light chain variable region. The sequences of the three CDRs in the heavy chain variable region, HCDR1, HCDR2, and HCDR3, are shown in SEQ ID NO: 11, 12, and 13, respectively, and the sequences of the three CDRs in the light chain variable region, LCDR1, LCDR2, and LCDR3, are shown in SEQ ID NO: 14, 15, and 16, respectively.

[0010] The antibody 6#AA or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has at least 80-100% sequence identity with SEQ ID NO:23, and the light chain variable region has at least 80-100% sequence identity with SEQ ID NO:24.

[0011] Accordingly, in the bispecific antibody according to the present invention, the anti-CLDN18.2 binding domain also includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has at least 80-100% sequence identity with SEQ ID NO:23, and the light chain variable region has at least 80-100% sequence identity with SEQ ID NO:24.

[0012] In the bispecific antibody according to the present invention, the anti-CD3 binding domain is derived from the antibody h160C9AA, and the antibody h160C9AA or its antigen-binding fragment specifically binds to CD3. h160C9AA includes a heavy chain variable region and a light chain variable region. The sequences of the three CDRs in the heavy chain variable region, HCDR1, HCDR2, and HCDR3, are shown in SEQ ID NO: 17, 18, and 19, respectively, and the sequences of the three CDRs in the light chain variable region, LCDR1, LCDR2, and LCDR3, are shown in SEQ ID NO: 20, 21, and 22, respectively.

[0013] Therefore, in the bispecific antibody according to the present invention, the anti-CD3 binding domain may also specifically bind to CD3, and includes a heavy chain variable region and a light chain variable region. The sequences of HCDR1, HCDR2, and HCDR3, which are the three CDRs in the heavy chain variable region, are shown in SEQ ID NO: 17, 18, and 19, respectively. The sequences of LCDR1, LCDR2, and LCDR3, which are the three CDRs in the light chain variable region, are shown in SEQ ID NO: 20, 21, and 22, respectively.

[0014] The antibody h160C9AA or an antigen-binding fragment thereof includes a heavy chain variable region having at least 80-100% sequence identity with SEQ ID NO: 6 and a light chain variable region having at least 80-100% sequence identity with SEQ ID NO: 7.

[0015] Therefore, in the bispecific antibody according to the present invention, the anti-CD3 binding domain also includes a heavy chain variable region having at least 80-100% sequence identity with SEQ ID NO: 6 and a light chain variable region having at least 80-100% sequence identity with SEQ ID NO: 7.

[0016] In some preferred embodiments, the binding domain included in the bispecific antibody according to the present invention is Fab, Fv, scFv, F(ab’)2, a linear antibody, a single domain antibody, or a full-length antibody.

[0017] In some preferred embodiments, the bispecific antibody according to the present invention further includes a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region includes Fc or a mutant Fc. Preferably, Fc is derived from a human.

[0018] In some more preferred embodiments, the bispecific antibody is configured such that the scFv of CD3 is bound to the C-terminus of one heavy chain in the full-length anti-CLDN18.2 antibody, or the scFv of CD3 is bound to the C-terminus of two light chains in the full-length anti-CLDN18.2 antibody, thereby constructing two bispecific antibodies with different structures. The bispecific antibody molecule can bind to the CD3 antigen to recruit T cells to the target tumor site, while simultaneously recognizing and specifically killing tumor cells that highly express CLDN18.2.

[0019] In some preferred embodiments, in the bispecific antibody according to the present invention, the anti-CLDN18.2 binding domain is a full-length antibody, and the anti-CD3 binding domain is scFv. Here, the sequence of the heavy chain of the full-length antibody is shown in SEQ ID NO:1, and the sequence of the light chain is shown in SEQ ID NO:5. scFv is constructed by linking VH and VL in series with a linker, which may be VH-linker-VL or VL-linker-VH, preferably VL-linker-VH, and the sequence of VL-linker-VH is shown in SEQ ID NO:8.

[0020] In one preferred embodiment, the bispecific antibody according to the present invention has an anti-CD3scFv molecule ligated to the C-terminus of one heavy chain or the C-terminus of two light chains of a full-length anti-CLDN18.2 antibody.

[0021] In one preferred embodiment, when two scFv peptide chains of an anti-CD3 antibody are fused to the C-terminuses of two light chains of a full-length anti-CLDN18.2 antibody, the resulting bispecific antibody comprises two fused homologous light chains and two unmodified homologous heavy chains, the sequence of the fused light chains is shown in SEQ ID NO:2, and the sequence of the heavy chains is shown in SEQ ID NO:1.

[0022] In one preferred embodiment, when one scFv peptide chain of an anti-CD3 antibody is fused to the C-terminus of one heavy chain of a full-length anti-CLDN18.2 antibody, the resulting bispecific antibody contains two homologous light chains and two heterogeneous heavy chains. In the two heterogeneous heavy chains, the heavy chain containing scFv is substituted with several amino acids to form a "knob" structure, and the heavy chain not containing scFv is substituted with several amino acids to form a "hole" structure. Here, the sequence of the homologous light chain after construction is shown in SEQ ID NO:5, the sequence of the heavy chain with the "knob" structure is shown in SEQ ID NO:3, and the sequence of the heavy chain with the "hole" structure is shown in SEQ ID NO:4.

[0023] The present invention further provides a nucleic acid encoding the bispecific antibody and a recombinant vector comprising the nucleic acid, preferably a recombinant expression vector.

[0024] In some embodiments, the present invention further provides a host cell having a genome incorporating the recombinant vector or a nucleic acid encoding the bispecific antibody. In some preferred forms, the host cell may be a prokaryotic cell, such as Escherichia coli, or a eukaryotic cell, such as yeast or a mammalian cell, the mammalian cell being, for example, a CHO cell, HEK293 cell, HEK293E cell, or Expi293 cell.

[0025] In some embodiments, the present invention provides a method for producing the bispecific antibody, comprising culturing the host cells of the present invention under appropriate conditions and obtaining an expression product by purification from the cells.

[0026] In some embodiments, the present invention provides the use of a bispecific antibody for producing a drug that specifically targets tumor cells expressing CLDN18.2, wherein the tumors expressing CLDN18.2 include gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, colon cancer, rectal cancer, liver cancer, head and neck cancer, and gallbladder cancer, and their metastases, where the gastric cancer metastases are, for example, Krukenberg tumors.

[0027] In some embodiments, the present invention provides a pharmaceutical composition comprising an effective amount of a bispecific antibody according to the present invention, or a nucleic acid encoding the bispecific antibody, or a recombinant vector comprising an effective amount of the coding nucleic acid, or a host cell comprising an effective amount of the coding nucleic acid. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0028] In some preferred embodiments, the pharmaceutical composition further comprises one or more additional other therapeutic agents. These additional therapeutic agents include cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor demulsifiers, chemotherapeutic agents, radiotherapeutic agents, targeted anticancer agents, biological response modifiers, cancer vaccines, cytokines, hormones, anti-metastatic agents, and immunotherapeutic agents.

[0029] In some embodiments, the present invention provides a cartridge or kit comprising a container and a pharmaceutical composition of the present invention disposed within the container.

[0030] In some embodiments, the present invention provides a method for inducing cell death of cells expressing CLDN18.2, comprising contacting the cells with a pharmaceutical composition of the present invention. In some embodiments, the cells are contacted with the pharmaceutical composition in vitro. In some embodiments, the cells are contacted with the pharmaceutical composition in vivo. In some embodiments, the cells are cancer cells. In some embodiments, the cells are solid tumor cells. In some embodiments, the cells are selected from gastric cancer cells, esophageal cancer cells, intestinal cancer cells, pancreatic cancer cells, Wilms' tumor cells, lung cancer cells, ovarian cancer cells, colon cancer cells, rectal cancer cells, liver cancer cells, head and neck cancer cells, chronic myeloid leukemia cells, and gallbladder cancer cells.

[0031] In some embodiments, the present invention provides a method for treating a disease associated with the expression of CLDN18.2 in a subject, comprising administering the subject a pharmaceutical composition of the present invention, which is required. In some embodiments, the disease is a tumor. In some embodiments, the tumor is preferably gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, Wilms' tumor, lung cancer, ovarian cancer, colon cancer, rectal cancer, liver cancer, head and neck cancer, chronic myeloid leukemia, or gallbladder cancer. In some embodiments, the method further comprises administering an additional therapeutic agent to the subject.

[0032] The antibodies of the present invention include, but are not limited to, chemotherapeutic agents, cytotoxic agents, radiotherapeutic agents, cancer vaccines, tumor debulking agents, targeted anticancer agents, anti-angiogenic agents, biological response modifiers, cytokines, hormones, anti-metastatic agents, and immunotherapeutic agents. They may be administered in combination with other additional therapeutic agents.

[0033] In some preferred embodiments, chemotherapeutic agents that can be used in combination with the antibodies or antigen-binding fragments of the present invention include: mitotic inhibitors comprising vincristine, vinblastine, vindesine, and navelbine; topoisomerase I inhibitors, such as camptothecin compounds comprising irinotecan, topotecan, camptothecin, and other compounds derived from their analogs; podophyllotoxin derivatives, such as etoposide, teniposide, and midoxizoz; alkylating agents, such as cisplatin, carboplatin, cyclophosphamide, and nitrogen mustard. , but not limited to, other chemotherapeutic agents, including but not limited to, paclitaxel, docetaxel, dacarbazine, carmustine, busulfan, chlorambucil, briquinolizine, uracil mustard, cloprofen, and dacarbazine; antimetabolites, such as cytarabine, 5-fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine; antibiotics, including but not limited to, doxorubicin, bleomycin, actinomycin, daunorubicin, mitomycin, zalcomycin C, actinomycin D, roxithromycin, adriamycin, rapamycin, and their derivatives, and daunomycin; and other chemotherapeutic agents, including but not limited to, paclitaxel, docetaxel, dacarbazine, azacitidine, amsacon, melphalan, ifosfamide, and mitoxantrone. In some preferred embodiments, the additional therapeutic agent is one or more selected from epirubicin, oxaliplatin, and 5-fluorouracil.

[0034] In some embodiments, the targeted anticancer agent includes, but is not limited to, high molecular weight targeted drugs and low molecular weight targeted drugs.

[0035] In some preferred embodiments, the polymeric targeted drugs include, but are not limited to, drugs targeting other targets, including, but not limited to, target sites such as PI3K, PARP, PI3Kα, PKB / AKT, and STAT3.

[0036] In some embodiments, small molecule targeted drugs include, but are not limited to: drugs targeting epidermal growth factor, such as erlotinib or gefitinib; HER-2 or HER-3 signaling pathway inhibitors, such as lapatinib or alfatinib; tyrosine kinase inhibitors, such as imatinib or schunitinib; anti-vascular endothelial growth factor drugs, such as sorafinib, regorafenib, pazopanib, recombinant human endostatin and apatinib; drugs targeting c-Met / ROS1, such as clozotinib; other targeted drugs, such as borlinotat and marimastat; drugs targeting mTOR, such as everolimus; and drugs targeting other targets, such as PI3Kα, PKB / AKT and STAT3.

[0037] In some embodiments, the immunotherapeutic agent includes, but is not limited to, immunosuppressants and agonists, and cell therapies related to immunotherapy, and the target sites include PD-1 / PD-L1, PD-L2, CTLA-4, LAG-3, IDO, TIM3, TIGIT, CD47, SIRPα, 4-1BB, CSF-1 / CSF1R, GITR, OX40, CD40, CD27, CD28, B7H4, B7H3, TGFβ, BTLA, VISTA, ICOS, CD39, CD73, A2AR, KIR, and NKG2A.

[0038] In some embodiments, PD-1 / PD-L1-targeted immune checkpoint inhibitors include, but are not limited to, high molecular weight drugs such as pembrolizumab, nivolumab, atezolizumab, avelumab, and cintilimab, as well as small molecular weight drugs such as cemiplimab and durvalumab.

[0039] In some embodiments, immune checkpoint inhibitors targeting CTLA-4 include, but are not limited to, ipilimumab; cytokines include, but are not limited to, IL-10, IL-15, IL-4, and IL-13; and inhibitors targeting BRAF include, but are not limited to, bimimetinib.

[0040] In some embodiments, other therapeutic agents are selected from oncolytic viruses such as parvovirus, adenovirus, herpesvirus, poxvirus, poliovirus, reovirus, alphavirus, marabavirus, retrovirus, and coxsackievirus, or other therapeutic agents are selected from cancer vaccines or protease inhibitors, such as bortezomib. [Brief explanation of the drawing]

[0041] The drawings further illustrate the new regulations disclosed herein. While these drawings will provide a better understanding of the characteristics and benefits disclosed herein, they are used solely to illustrate specific embodiments of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0042] [Figure 1] The structures of the two bispecific antibodies according to the present invention are shown. [Figure 2] This shows the binding status of either of the two bispecific antibodies or the reference antibody according to the present invention to HEK293 cells that stably express hCLDN18.2. [Figure 3]This shows the binding status of either of the two bispecific antibodies or the reference antibody according to the present invention to Jurkat cells that naturally express hCD3. [Figure 4] This shows the binding status of either of the two bispecific antibodies or the reference antibody according to the present invention to HEK293 cells that stably express hCLDN18.1. [Figure 5] This shows the binding status of either of the two bispecific antibodies or the reference antibody according to the present invention to HEK293 cells that stably express mCLDN18.2. [Figure 6] This shows the binding status of either of the two bispecific antibodies or the reference antibody according to the present invention to HEK293 cells that stably express mCLDN18.1. [Figure 7] This shows the binding status of either of the two bispecific antibodies or the reference antibody according to the present invention to HEK293 cells that stably express cynoCLDN18. [Figure 8] The results of cytotoxicity experiments (TDCC) against HEK293 cells stably expressing hCLDN18.2, mediated by two bispecific antibodies according to the present invention, are shown. [Figure 9] The results of T cell activation in HEK293 cells stably expressing hCLDN18.2 using two bispecific antibodies according to the present invention are shown. [Figure 10] The results of INF-γ secretion from HEK293 cells stably expressing hCLDN18.2, using two bispecific antibodies according to the present invention, are shown. [Figure 11] The results of IL-2 secretion by HEK293 cells stably expressing hCLDN18.2, using two bispecific antibodies according to the present invention, are shown. [Figure 12] The results of IL-6 secretion by HEK293 cells stably expressing hCLDN18.2, using two bispecific antibodies according to the present invention, are shown. [Figure 13] The results of TNF-α secretion by HEK293 cells stably expressing hCLDN18.2, using two bispecific antibodies according to the present invention, are shown. [Figure 14] The results of measuring luciferase expression levels against the Jurkat-NFAT reporter gene using two bispecific antibodies according to the present invention are shown. [Figure 15] The results of cytotoxicity experiments (ADCC) against HEK293 cells that stably express hCLDN18.2, mediated by two bispecific antibodies according to the present invention, are shown. [Figure 16] This study demonstrates the effect of the bispecific antibody 31905-44AA according to the present invention on tumor growth in a humanized HEK293-hCLDN18.2 model. [Figure 17] This study demonstrates the effect of the bispecific antibody 31905-44AA according to the present invention on the body weight of humanized HEK293-hCLDN18.2 model load-bearing mice. [Modes for carrying out the invention]

[0043] All publications, patents, and patent applications described herein are incorporated herein by reference to the same extent that each publication, patent, or patent application is incorporated herein by reference in a specific and individual manner.

[0044] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodologies, solutions, and reagents described herein, and that these may be modified. Furthermore, it should be understood that the terms used herein are solely for the purpose of describing embodiments for carrying out the invention and are not intended to limit the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.

[0045] Some embodiments disclosed herein include numerical ranges, and some aspects of the invention can be described in range form. Unless otherwise stated, numerical ranges or forms described in range form should be understood to be merely for convenience and brevity and should not be considered to strictly limit the scope of the invention. Accordingly, range form descriptions should be considered to specifically disclose all possible subranges and all specific numerical values ​​that can take within those ranges, as already explicitly stated herein. Regardless of the width of the numerical values, the above principles apply equally. When described using ranges, the range includes its endpoints.

[0046] When referring to measurable values ​​such as quantity or duration, the term "approximately" means a change that includes ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0047] The three-letter and one-letter amino acid symbols used herein are described in J. Biol. Chem, 243, p3558 (1968).

[0048] The terms "Claudin18.2," "CLDN18.2," or "CLDN18A2" as used herein have the following meanings: The tight junction protein 18 (also known as claudin 18 and abbreviated as CLDN18) is a membrane-bound protein (a four-transmembrane protein) having four transmembrane hydrophobic regions and two extracellular loops (loop 1 is surrounded by hydrophobic regions 1 and 2, and loop 2 is surrounded by hydrophobic regions 3 and 4). CLDN18 exists as two different splice variants, which have been described in mouse and human (Niimi, Mol. Cell. Biol. 21:7380-90, 2001). The GenBank registry numbers for splice variant 1 (Claudin18.1, CLDN18.1, CLDN18A1) are NP_057453 and NM_016369, respectively, and the GenBank registry numbers for splice variant 2 (Claudin18.2, CLDN18.2, CLDN18A2) are NP_001002026 and NM_001002026. Splice variants CLDN18.1 and CLDN18.2 differ in the N-terminal region, which includes the first transmembrane (TM) region and loop 1, but their C-terminal primary protein sequences are identical.

[0049] As used herein, the terms “anti-Claudin18.2 antibody,” “anti-CLDN18A2 antibody,” “anti-CLDN18.2 antibody,” and “antibody against CLDN18.2” mean an antibody that can bind to the CLDN18.2 protein or a fragment thereof with sufficient affinity, and does not significantly bind to CLDN18.1, so that the antibody can be used as a diagnostic and / or therapeutic agent targeting CLDN18.2. Since the human CLDN18.2 protein is represented as hCLDN18.2, “anti-human Claudin18.2 antibody,” “anti-human CLDN18A2 antibody,” “antibody against anti-hCLDN18.2,” and “antibody against hCLDN18.2” mean, in particular, an antibody that can bind to the human CLDN18.2 protein or a fragment thereof with sufficient affinity, so that the antibody can be used as a diagnostic and / or therapeutic agent targeting human CLDN18.2.

[0050] In addition to the human CLDN18.2 protein, the mouse CLDN18.2 protein is represented as mCLDN18.2, and the cynomolgus monkey CLDN18 protein is represented as cynoCLDN18. Similarly, the human CLDN18.1 protein is represented as hCLDN18.1, and the mouse CLDN18.1 protein is represented as mCLDN18.1.

[0051] In this field, "CD3" is known as a six-chain polyprotein complex (see Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999). In mammals, the complex contains homologous dimers of CD3(γ), CD3(δ), two CD3(ε), and CD3(ζ). The CD3(γ), CD3(δ), and CD3(ε) chains are cell surface proteins highly associated with the immunoglobulin superfamily, which contains a single immunoglobulin domain. The transmembrane regions of the CD3(γ), CD3(δ), and CD3(ε) chains are negatively charged, a feature that allows these chains to bind to positively charged T cell receptor chains. The intracellular tails of the CD3(γ), CD3(δ), and CD3(ε) chains each contain one conserved motif, either an immunoreceptor tyrosine activation motif or an ITAM, and three such motifs are present in each CD3(ζ) chain. While we do not wish to be theoretically limited, ITAMs are considered important for the signaling capacity of the TCR complex. In this specification, when the term "CD3" is used alone, it may originate from various species, including humans, mice, rats, or other mammals.

[0052] As used herein, "anti-CD3 antibody" refers to an antibody that can specifically bind to a single CD3 chain (e.g., a CD3(γ) chain, a CD3(δ) chain, or a CD3(ε) chain) or to a complex of two or more single CD3 chains (e.g., a complex of one or more CD3(ε) chains, a complex of a CD3(γ) chain and a CD3(ε) chain, or a complex of a CD3(δ) chain and a CD3(ε) chain). In some embodiments, the anti-CD3 antibody can specifically bind to CD3(γ), CD3(δ), or CD3(ε) or any combination thereof, more preferably to CD3(ε). Since human CD3 is represented as hCD3, "anti-human CD3 antibody" and "anti-hCD3 antibody" mean an antibody that can specifically bind to human CD3.

[0053] As used herein, the term “antibody” typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains (HC) and two light (L) polypeptide chains (LC) held together by the interaction of covalent disulfide bonds and non-covalent bonds. Natural IgG antibodies have such a structure. Each light chain consists of one variable domain (VL) and one constant domain (CL). Each heavy chain contains one variable domain (VH) and one constant domain (CH).

[0054] In this field, five main types of antibodies are known: IgA, IgD, IgE, IgG, and IgM. Their corresponding heavy chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses; for example, IgG is divided into IgG1, IgG2, IgG3, and IgG4, and IgA is divided into IgA1 and IgA2. The light chains of antibodies derived from any vertebrate species can be classified into two distinct types, called κ and λ, based on the amino acid sequence of their constant domains.

[0055] In the case of IgG, IgA, and IgD antibodies, the constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated into a flexible hinge region, which is a variable-length section rich in proline and cysteine. Antibodies of each class further contain interchain and intrachain disulfide bonds formed by pairs of cysteine ​​residues.

[0056] The term "variable region" or "variable domain" refers to a significant change in amino acid composition from one type of antibody to another, primarily responsible for antigen recognition and binding. The variable region of each light / heavy chain pair forms an antibody binding site, and a complete IgG antibody has two binding sites (i.e., it is bivalent). The heavy chain variable region (VH) and light chain variable region (VL) domains each contain three highly variable regions called hypervariable regions (HVR) or more commonly, complementarity-determining regions (CDRs). VH and VL each have four framework regions FR (or referred to as framework regions), represented as FR1, FR2, FR3, and FR4, respectively. Thus, the CDR and FR sequences generally appear in the heavy chain variable domain (VH) (or light chain variable domain (VL)) as FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0057] The term "Fc" is used herein to define a dimer-forming two polypeptide chain, including the C-terminal region of an immunoglobulin heavy chain, i.e., the C-terminal constant region that can stabilize its binding to the immunoglobulin heavy chain. This term includes the native sequence Fc region and the mutant Fc region. Although the boundary of the Fc region of an IgG heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain; for example, the IgG Fc region includes the IgG CH2 and IgG CH3 constant domains. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0058] However, antibodies produced by host cells may be cleaved after translation, resulting in the removal of one or more amino acids, particularly one or two, from the C-terminus of the heavy chain. By expressing a specific nucleic acid molecule encoding the full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or cleaved variants of the full-length heavy chain. This situation occurs when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447). Thus, the C-terminal lysine (K447), or C-terminal glycine (G446) and lysine (K447), may or may not be present in the Fc region. To prevent the loss of other antigen-binding domains fused thereto due to C-terminal cleavage of the Fc region, in one embodiment of the present invention, K447 needs to be replaced with A by amino acid substitution, i.e., K447A.

[0059] As used herein, the broad definition of "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-grafted antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotype antibodies, and synthetic antibodies (including mutant proteins and their variants).

[0060] The terms "full-length antibody," "complete antibody," and "complete antibody" are interchangeable herein and refer to antibodies whose structure is fundamentally similar to that of a natural antibody or antibodies containing an Fc region.

[0061] The term "monoclonal antibody" (or "mab") refers to an antibody produced from a single cell clone that is substantially homogeneous and targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in this art, including hybridoma techniques, recombinant techniques, phage representation techniques, genetically modified animals, synthetic techniques, or combinations of the above techniques.

[0062] The term "chimeric antibody" refers to a construct in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and this or the remainder of these chains is identical or homologous to a corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass and an antibody belonging to a fragment of such an antibody. In a narrower sense, a chimeric antibody comprises all or most of a selected mouse heavy chain and light chain variable region that is operably linked to the human light chain and heavy chain constant region. The constant region sequence or its variants or derivatives can be operably associated with the disclosed heavy chain and light chain variable region using standard molecular biology techniques, thereby providing a full-length antibody that can be used as is or incorporated into the anti-CLDN18.2 of the present invention.

[0063] The term "humanized antibody" refers to a hybrid immunoglobulin, immunoglobulin chain, or fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. In many cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues of the receptor-derived CDR are replaced with residues of the CDR derived from a non-human species (donor antibody), such as mouse, rat, rabbit, or primate, possessing desired specificity, affinity, and capability. In some cases, framework region residues of the human immunoglobulin are replaced with corresponding non-human residues. In some cases, "reverse mutations" can be introduced into the humanized antibody, replacing one or more FR residues in the variable region of the receptor human antibody with corresponding residues derived from a non-human donor antibody. Such reverse mutations contribute to maintaining the proper three-dimensional structure of one or more grafted CDRs, thereby improving affinity and antibody stability. Antibodies from various donor species can be used, and these donor species include, but are not limited to, mice, rats, rabbits, or non-human primates. Furthermore, humanized antibodies may contain novel residues not found in receptor antibodies or donor antibodies in order to further improve antibody performance.

[0064] The CDR and FR divisions of the monoclonal antibody variable region of the present invention are determined based on Kabat. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, any humanized antibody based on the mab sequence of the present invention and containing one or more CDRs derived from any naming system is clearly within the scope of the present invention.

[0065] The terms “sequence identity,” “sequence similarity,” or “sequence homology” refer to the percentage of amino acid residues in a candidate sequence that are the same as those in a reference polypeptide sequence, obtained by aligning the sequences (introducing gaps where necessary) to obtain the maximum percentage of sequence identity, and excluding any conservative substitutions from being considered part of the sequence identity. The percentage of amino acid sequence identity can be measured by aligning sequences using various methods of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0066] The term "antibody fragment" includes at least a portion of a complete antibody. As used herein, an "antibody fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment in an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its antigenic epitope, or a fusion protein product further derived from such fragment, such as a single-chain antibody or an extracellular binding region in a chimeric antigen receptor. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to, variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments.

[0067] The term "antigen-binding domain" or "binding domain" refers to a domain that specifically binds to, interacts with, or recognizes a predetermined target epitope on a target molecule (antigen). An "antigen-binding domain" may be a single "antigen-binding fragment" or a combination of "antigen-binding fragments," and the term "antigen-binding domain" is a broader concept than "antigen-binding fragment."

[0068] The term "Fab fragment" refers to a monovalent antibody fragment that includes a heavy chain variable region and a light chain variable region, and further includes a light chain constant region and a heavy chain first constant region CH1. The term "F(ab')2 fragment" refers to a bivalent antibody fragment that includes two Fab fragments and a hinge region.

[0069] The term "Fd fragment" generally includes the heavy chain variable region and the constant region CH1, while the term "Fv fragment" includes the antibody heavy chain variable region and the light chain variable region, but lacks the constant region and has the smallest antibody fragments of all antigen-binding sites.

[0070] The term "single-domain antibody," also known as a nanoantibody, refers to a naturally occurring light-chain-deficient antibody found in the peripheral blood of alpacas. This antibody contains only one heavy-chain variable region (VHH) and two normal CH2 and CH3 regions. The VHH structure, when cloned and expressed independently, possesses structural stability and antigen-binding activity comparable to that of the original heavy-chain antibody. Because it is the smallest known unit capable of binding to a target antigen, it is also called a nanobody (Nb).

[0071] The term "scFv" means a fusion protein comprising at least one variable region antibody fragment containing a light chain and at least one variable region antibody fragment containing a heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker), and the scFv can be expressed in the form of a single-chain polypeptide, and the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or VH-linker-VL.

[0072] Naturally occurring antibodies are typically monospecific, binding to a single antigen. The present invention provides a molecule that simultaneously binds to cytotoxic cells (e.g., CD3 on T cells) and target cells (e.g., CLDN18.2 on cancer cells). The molecule binds to at least two different types of antigens and is at least bispecific or multispecific. The binding molecule of the present invention may be at least trivalent. As used herein, "valency" refers to the number of antigen-binding sites within the molecule; for example, a typical natural IgG antibody is bivalent. Antigen-binding sites that bind to the same antigen can recognize the same or different epitopes. Trivalent and tetravalent bispecific antibodies are known in the art.

[0073] The term "multispecific antibody" refers to a novel antibody construct formed by functionally linking an antibody or antibody fragment to one or more other binding molecules (including antibodies or antibody fragments or other molecules capable of binding) (e.g., by chemical coupling, gene fusion, non-covalent bonding, or other methods) that binds to two or more different sites and / or target sites. Therefore, a "bispecific antibody" (or "bispecific antigen-binding molecule" or "bi-antibody") refers to an antibody construct that is specific to two different antigens and / or epitopes in particular. Typically, a bispecific antibody or multispecific antibody contains at least two different antigen (or epitope) binding domains.

[0074] In the context of this invention, the term "recombinant" means "prepared by genetic modification." Generally, recombinants do not exist naturally.

[0075] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), International Publication No. 93 / 08829, and Traunecker, A., et al., EMBO J.10:3655 (1991)) and the "knob-into-hole" operation (see, for example, U.S. Patent No. 5,731,168). Furthermore, methods include manipulating the electrostatic steering effect to produce antibody Fc heterodimer molecules (International Publication No. 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J.Immunol. 148 (5):1547-1553 (1992)); using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Holliger et al., Proc. Natl.Acad.Sci.USA, 90:6444-6448 (1993)); and using single-chain Fv(sFv) dimers (see, e.g., Gruber et al. See al., J.Immunol.152:5368 (1994); and, for example, as described in Tutt et al., J.Immunol.147:60 (1991), multispecific antibodies can also be prepared by preparing tripspecific antibodies.

[0076] The bispecific antibody according to the present invention may use its full-length antibody or complete antibody as a framework for adding multiple scFvs. For example, scFvs may be fused to the C-terminus of the heavy chain CH3 domain or the C-terminus of the light chain CL domain.

[0077] The terms "fusion" or "binding" mean that various elements (e.g., antigen-binding fragments or antigen-binding domains) are joined by peptide bonds, either directly or via one or more peptide linkers.

[0078] As used herein, "homonymy" and "heterogeneity" are relative concepts. They can refer to different elements in a structure having the same or different origins, or they can refer to a situation where, after the construction of a structure is complete, several elements that were originally of the same origin, i.e., "homonymy," undergo modifications to some of them, resulting in a change compared to the other original elements that have not been modified, thus becoming "heterogeneity."

[0079] In one embodiment, the bispecific antibody according to the present invention, when scFv is fused to the C-terminus of the light chain CL domain of a full-length antibody or a complete antibody, comprises four polypeptide chains, each consisting of two homologous light chains and two homologous heavy chains. Here, the homologous light chain polypeptide contains a domain called VL-CL-scFv from the N-terminus to the C-terminus, and the homologous heavy chain polypeptide contains a domain called VH-CH from the N-terminus to the C-terminus.

[0080] In one embodiment, the bispecific antibody according to the present invention comprises four polypeptide chains, each consisting of two homologous light chains and two heterologous heavy chains, when scFv is fused to the C-terminus of a single heavy chain CH3 domain of a full-length antibody or a complete antibody. Here, the homologous light chain polypeptide contains a domain called VL-CL from the N-terminus to the C-terminus, one of the heterologous heavy chain polypeptides contains a domain called VH-CH from the N-terminus to the C-terminus, and the other heterologous heavy chain polypeptide contains a domain called VH-CH-scFv from the N-terminus to the C-terminus.

[0081] The term "linker" refers to any tool used to link two different functional units (e.g., antigen-binding fragments). Linker types include, but are not limited to, chemical linkers and polypeptide linkers. The sequence of a polypeptide linker is not limited. Polypeptide linkers are preferably non-immunogenic and flexible, and for example, contain serine and glycine sequences. The linker is determined by the specific construct and may be long or short.

[0082] According to the present invention, the linker connecting different functional units preferably includes a flexible peptide linker such as a glycine-serine peptide linker. In one embodiment, the linker includes an amino acid sequence (G4S)x, where x is any integer from 1 to 6, and preferably includes an amino acid sequence (G4S)1 or (G4S)3. The linker connecting the VH domain and the VL domain to form a VH-VL or VL-VH scFv domain preferably includes a flexible peptide linker such as a glycine-serine peptide linker. In one embodiment, the linker includes an amino acid sequence (G4S)x, where x is any integer from 1 to 6, and preferably includes an amino acid sequence (G4S)3.

[0083] The term “antigen” refers to a substance that is recognized by an antibody or antibody-binding fragment and specifically binds to the antibody or antibody-binding fragment. In a broad sense, an antigen may include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, or a complete extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides, and lipids, parts thereof, and combinations thereof, can all constitute an antigen. Non-limiting exemplary antigens include tumor antigens or pathogen antigens. An “antigen” may also be a molecule that elicits an immune response. Any form of antigen, or cells or preparations containing such antigen, can be used to produce antibodies specific to an antigenic determinant. An antigen may be an isolated full-length protein, a cell surface protein (e.g., immunized in cells expressing at least a portion of the antigen on its surface), or a soluble protein (e.g., immunized only by the ECD portion of such protein), or a protein construct (e.g., an Fc antigen). Such antigens can be produced in genetically modified cells. The aforementioned antigen may be used alone or in combination with one or more immunogenicity-enhancing adjuvants known in the art. The DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA) and may encode at least a portion of the ECD sufficient to elicit an immunogenic response. Cells expressing the antigen can be transformed using any vector, which includes, but is not limited to, adenovirus vectors, lentiviral vectors, plasmids, and cationic lipids.

[0084] The term "epitope" refers to a site in an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids, or from amino acids that are parallel but not adjacent due to tertiary folding of the protein. Epitopes composed of adjacent amino acids are usually retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes typically contain at least 3 to 15 amino acids in a unique spatial conformation. Methods for determining the epitope to which a particular antibody binds are well known in the art and include immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include, for example, techniques in the art such as X-ray crystallography and two-dimensional nuclear magnetic resonance, as well as techniques described herein.

[0085] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues of any length. The polymers may be linear, cyclic, or branched, and may contain modified amino acids, particularly conservatively modified amino acids, and may be interrupted by non-amino acids. The term further includes modified amino acid polymers, e.g., those modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodization, methylation, oxidation, protein hydrolysis, prenylation, racemization, selenylation, transfer-RNA mediated aminoaddition, e.g., arginineization, ubiquitousization, or any other operation, e.g., compounding with a labeling component. When used herein, the term “amino acid” refers to natural and / or unnatural or synthetic amino acids, including glycine and its D or L optical isomers, and amino acid analogs and peptide mimics. A polypeptide or amino acid sequence “derived to” a specified protein refers to the origin of the polypeptide. The term further includes polypeptides expressed in a specified nucleic acid sequence.

[0086] The term “amino acid modification” (or “modified amino acid”) includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. In this specification, “amino acid substitution” or “substitution” means replacing an amino acid at a specific position in a parent polypeptide sequence with another amino acid. For example, substitution S32A means that serine at position 32 is replaced with alanine.

[0087] The sequence identity or homology between the humanized antibody variable region and the human receptor variable region can be measured as described herein, and such measurement preferably results in a sharing of at least 60% or 65% sequence identity, more preferably at least 70%, 75%, 80%, 85%, or 90%, and even more preferably at least 93%, 95%, 98%, or 99%. Preferably, the positions of different residues differ by conservative amino acid substitutions. A "conservative substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region or framework region of the antibody of the present invention can be replaced with amino acid residues of other similar side chains. If two or more amino acid sequences differ from each other by conservative substitution, the conservative nature of this substitution can be corrected by adjusting the percentage of sequence identity or similarity upwards.

[0088] During antibody production, various physicochemical factors, such as glycosylation, oxidation, glycation, deamidation, isomerization, and terminal cyclization, can easily produce various post-translational modification (PTM) variants. These PTMs can alter the physicochemical properties of the antibody, change its interaction with the antibody-Fc receptor, and potentially affect its binding activity to the target antigen. The occurrence of some PTMs can also reduce antibody stability and potentially cause immunogenicity (JARASCH et al., JOURNAL OF PHARMACEUTICAL SCIENCES, 2015). These adverse effects can be eliminated by amino acid modification of the PTM site, such as conservative substitution. Based on the purpose of PTM modification, amino acid substitution of the antibody CDR is clearly included within the scope of this invention.

[0089] The term "antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to the binding of an antibody to an antigenic epitope on a virus-infected cell or tumor cell. Its Fc segment then binds to Fc receptors (FcRs) on the surface of killer cells (such as NK cells and macrophages), directly killing the target cell via the killer cell.

[0090] The term "complement-dependent cytotoxicity" (CDC) refers to cytotoxic effects involving complement, specifically, where specific antibodies bind to corresponding antigens on the cell membrane surface, forming complexes that activate the complement pathway. These membrane attack complexes then exert a degrading effect on target cells.

[0091] The bispecific antibodies according to the present invention may further include substitutions or modifications of a constant region (e.g., Fc), which include, but are not limited to, substitutions, mutations, and / or modifications of amino acid residues, thereby generating compounds having the following preferred characteristics, which include, but are not limited to, altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased yield, altered binding of the Fc receptor (FcR) to the Fc ligand, enhanced or weakened ADCC or CDC, altered glycosylation and / or disulfide bond, and modified binding specificity. In some embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions (e.g., substitutions at positions 234 and 235 of the Fc region) that weaken FcγR binding. In one embodiment, these substitutions are L234A and L235A.

[0092] The bispecific antibody according to the present invention comprises an scFv fragment fused to one or the other C-terminus of the Fc domain of a full-length antibody, thereby forming two heterogeneous polypeptide chains containing the Fc domain. Recombinant co-expression and subsequent dimerization of these polypeptides result in several possible combinations of the two polypeptides. To improve the yield and purity of bispecific antibodies in recombinant production, it is advantageous to introduce modifications to the Fc domain of the bispecific antibody that promote the binding of the desired polypeptide. Accordingly, in a specific embodiment, the Fc domain of the bispecific antibody according to the present invention includes modifications that promote the binding of the first polypeptide chain and the second polypeptide chain of the Fc domain. The most extensive site of protein-protein interaction between the two polypeptide chains of the human IgG Fc domain is located in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is located in the CH3 domain of the Fc domain.

[0093] There are several ways to modify the CH3 domain of the Fc domain to enhance heterodimerization, which are described in detail, for example, WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954, and WO2013 / 096291. Typically, in all such methods, both the CH3 domain of the first polypeptide chain of the Fc domain and the CH3 domain of the second polypeptide chain of the Fc domain are complementaryly engineered to the extent that each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself but is forced to heterodimerize with other complementaryly engineered CH3 domains (the first and second CH3 domains are heterodimerized, and no homodimer is formed between the two first CH3 domains or the two second CH3 domains).

[0094] In certain embodiments, the modification that facilitates the binding of the first polypeptide chain and the second polypeptide chain of the Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification on one of the two polypeptide chains of the Fc domain and a "hole" modification on the other of the two polypeptide chains of the Fc domain.

[0095] The knob-in-hole technique is described, for example, in US5,731,168, US7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a bump ("knob") at the interface of a first polypeptide chain and a corresponding cavity ("hole") at the interface of a second polypeptide chain, where the bump can be positioned within the cavity to promote heterodimerization and inhibit homologous dimerization. The bump is constructed by substituting a smaller amino acid side chain from the interface of the first polypeptide chain with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of similar or similar size to the bump is constructed at the interface of the second polypeptide chain, and this cavity is formed by substituting a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0096] Accordingly, in one specific embodiment, by substituting one amino acid residue in the CH3 domain of the first polypeptide chain of the Fc domain of the bispecific antibody according to the present invention with an amino acid residue having a larger side chain volume, a bulge may be created within the CH3 domain of the first polypeptide chain, and this bulge may be located in a cavity within the CH3 domain of the second polypeptide chain. Alternatively, by substituting one amino acid residue in the CH3 domain of the second polypeptide chain of the Fc domain with an amino acid residue having a smaller side chain volume, a cavity may be created within the CH3 domain of the second polypeptide chain, and the bulge in the CH3 domain of the first polypeptide chain may be located in this cavity.

[0097] Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0098] The ridges and cavities may be generated by modifying the nucleic acid encoding the polypeptide, for example, by inducing site-directed mutation, or by peptide synthesis.

[0099] In one particular embodiment, the threonine residue at position 366 in the CH3 domain of the first polypeptide chain of the Fc domain ("knob" polypeptide chain) is replaced with a tryptophan residue (T366W), and the tyrosine residue at position 407 in the CH3 domain of the second polypeptide chain of the Fc domain ("whole" polypeptide chain) is replaced with a valine residue (Y407V). In one embodiment, the threonine residue at position 366 in the second polypeptide chain of the Fc domain is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0100] In other embodiments, the serine residue at position 354 of the first polypeptide chain of the Fc domain is further replaced with a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine ​​residue (E356C), while the tyrosine residue at position 349 of the second polypeptide chain of the Fc domain is further replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues forms a disulfide bridge between the two polypeptide chains of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0101] In one specific embodiment, the first polypeptide chain of the Fc domain comprises amino acid substitutions S354C and T366W, and the second polypeptide chain of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V.

[0102] Other substitutions that can be made to the CH3 domain to facilitate the purification of polypeptide chain homodimers with a "hole" from molecules containing the Fc domain of the final bispecific heterodimer include substitutions that affect binding to protein A. Non-exclusive examples of such substitutions include H435R, H435R, and / or Y436F (U.S. Patent No. US8,586,713 B2). The protein A-binding domains of the CH2 and CH3 domains with a "hole" in the polypeptide chain are preferably mutated by an amino acid substitution at position 435 (H435R). Thus, polypeptide chain homologous dimers with a "hole" do not bind to protein A, while the bispecific heterodimer retains the ability to bind to protein A via the protein A-binding domain.

[0103] The term "specificity" means that the binding of an antibody to an antigen is selective and can be distinguished from undesirable or nonspecific interactions.

[0104] The term "affinity" or "binding affinity" refers to the sum of the strength of non-covalent interactions between a single binding site on a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). D "Binding affinity" refers to the dissociation constant of a specific antibody-antigen interaction. Binding affinity can be determined using various techniques known in this field, such as surface plasmon resonance, biolayer interferometry, two-plane polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, ultracentrifugation, and flow cytometry.

[0105] The term "pharmaceutical composition" refers to a preparation that is in a form that allows for the biological activity of the active ingredient contained therein, and that does not contain other ingredients that are unacceptably toxic to the subject to whom the preparation is administered.

[0106] The terms “pharmaceutical carrier” or “pharmaceutically acceptable carrier” mean a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), excipient, or medium administered with a therapeutic agent.

[0107] The term "effective dose" refers to the dosage of a pharmaceutical formulation containing the active ingredient of the present invention, which, after being administered to a patient in one or more doses, produces the expected effect in the treated patient. The effective dose can be easily determined by a physician skilled in the art by considering several factors, such as differences in race, weight, age and health status, the specific disease associated, the severity of the disease, the individual patient's response, the specific antibody administered, the mode of administration, the bioavailability characteristics of the administered formulation, the selected administration scheme, and the use of any associated treatments.

[0108] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells into which exogenous nucleic acids have been introduced, and include the offspring of such cells. Host cells include “transformers” and “transformed cells,” and include primary transformed cells and their offspring, without regard to the number of passages. Offspring may not be exactly identical to the parent cells in nucleic acid content, but may contain mutations. In this specification, offspring of mutants having the same function or biological activity as those screened or selected from the initially transformed cells are included.

[0109] As used herein, the term “transfection” refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolytic guns.

[0110] The term "stable transfection" refers to the introduction and integration of exogenous nucleic acids, DNA, or RNA into the genome of transfected cells. The term "stable transfectant" refers to cells that stably integrate exogenous DNA into their genomic DNA.

[0111] The terms "coding nucleic acid molecule," "coding DNA sequence," and "coding DNA" refer to the order of deoxyribonucleotides along a deoxyribonucleic acid chain. This order of deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Therefore, a nucleic acid sequence codes for an amino acid sequence.

[0112] Methods for producing and purifying antibodies and antigen-binding fragments are well known and discovered in conventional techniques, such as those described in Chapters 5-8 and 15 of the Cold Spring Harbor Laboratory's Guide to Antibody Experimental Techniques. The antibodies or antigen-binding fragments according to the present invention are obtained by adding one or more human FR regions to a non-human CDR region using a genetic engineering method. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from the Journal of Immunoglobulins, (2001) ISBN:012441351.

[0113] The engineered antibodies or their antigen-binding fragments of the present invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombinant into an expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. A more preferred conventional technique is the mammalian expression system, which induces antibody glycosylation, particularly glycosylation of the highly conserved N-terminus in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human-derived antigens. Positive clones are cultured in serum-free medium in a bioreactor to produce antibodies. The culture medium from which the antibodies are secreted is purified and collected using conventional techniques. The antibodies are filtered and concentrated using conventional methods. Soluble mixtures and polymers may also be removed by conventional methods such as molecular sieving and ion exchange.

[0114] As used herein, the terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. The individuals of the present invention include, but are not limited to, humans, non-human primates (e.g., crab-eating macaques, rhesus macaques, or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0115] As used herein, the term “tumor” refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion into other tissues or organs. Tumor growth is generally uncontrolled, progressive, and does not induce or inhibit the proliferation of normal cells. Tumors can affect a variety of cells, tissues, or organs, including, but not limited to, those of the bladder, bone, brain, mammary glands, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestines, kidneys, liver, lungs, lymph nodes, nerve tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, trachea, urethra, ureters, uterus, vaginal organs, or tissues or corresponding cells selected from these. Tumors include, for example, sarcomas, carcinomas, or cancers such as cytoplasmic tumors (malignant tumors of plasma cells). The tumors according to the present invention include leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute granulocytic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, chronic leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, heavy chain disease, solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endosarcoma, lymphangiosarcoma, angiosarcoma, lymphangiosarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma) This includes, but is not limited to, sarcoma, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, bronchial cancer, medullary carcinoma, renal cell carcinoma, liver cancer, Nile duct carcinoma, choriocarcinoma, seminomas, embryonic carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytic tumor, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, auditory neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma, esophageal cancer, gallbladder cancer, kidney cancer, and multiple myeloma. Preferably, the term "tumor" includes, but is not limited to, pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, rectal cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer, and glioma.

[0116] As used herein, terms such as “disease,” “symptom,” or “disorder” refer to any change or disorder that impairs or interferes with the normal functioning of a cell, tissue, or organ. For example, such “disease” includes, but is not limited to, tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or defects in immune tolerance (e.g., transplant rejection).

[0117] As used herein, the term “treatment” refers to a clinical intervention in the process of altering a disease caused by an individual or treated cells, which may be preventive or clinicopathological. Therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of direct or indirect pathological consequences of any disease, prevention of metastasis, slowing of disease progression, improvement or mitigation of symptoms, and improvement or mitigation of prognosis.

[0118] The terms “cartridge” or “kit” include the pharmaceutical composition of the present invention in one or more unit dose forms of an effective amount. In some embodiments, the pharmaceutical kit may include a sterile container, which may be in the form of a box, ampoule, bottle, vial, tube, bag, blister pack, or other suitable container known in the art. Such containers may be made from plastic, glass, laminated paper, metal foil, or other material suitable for holding the drug. The pharmaceutical kit also further includes instructions for administering the pharmaceutical composition of the present invention to an individual. The instructions generally include a method for treating a disease using the pharmaceutical composition of the present invention.

[0119] The present invention will be further described below with reference to specific examples. Please note that these examples are for illustrative purposes only and do not limit the scope of the present invention. Experimental methods in the following examples that do not specify concrete conditions were generally carried out under normal conditions, for example, the conditions described in J. Sambrook et al., Molecular Cloning Experiment Manual, Third Edition, Scientific Press, 2002, or the conditions recommended by the manufacturer.

[0120] Example 1: Construction and expression of a bispecific antibody against CD3-CLDN18.2

[0121] The bispecific antibody according to the present invention has a structure in which the binding domain of the human T cell receptor subunit CD3ε is ligated to the C-terminus of the heavy chain or the C-terminus of the light chain of a full-length anti-human CLDN18.2 antibody. The full-length anti-human CLDN18.2 antibody is 6#AA, having the heavy chain sequence shown in SEQ ID NO:1 and the light chain sequence shown in SEQ ID NO:5. The CD3ε binding domain is derived from the full-length antibody h160C9AA, the heavy chain sequence is shown in SEQ ID NO:9, and the light chain sequence is shown in SEQ ID NO:10. To reduce the ADCC activity of the antibody, the Fc segments of both 6#AA and h160C9AA underwent amino acid substitutions of L234A and L235A, respectively.

[0122] The light chain variable region and heavy chain variable region of h160C9AA were linked via a flexible linker to form a single-chain antibody scFv, whose structure is VL-(G4S)3-VH, and whose sequence is shown in SEQ ID NO:8. The scFv was further fused via (G4S)3 to the C-terminus of one heavy chain or two light chains of the full-length CLDN18.2 antibody 6#AA.

[0123] When scFv is fused to the C-terminus of the two light chains of 6#AA, the resulting bispecific antibody is named 31905-38AA and contains two homologous light chains and two homologous heavy chains. The sequence of the fused light chain is shown in SEQ ID NO:2, and the sequence of the heavy chain is shown in SEQ ID NO:1.

[0124] When scFv is fused to the C-terminus of a single 6#AA heavy chain, the resulting bispecific antibody is named 31905-44AA and contains two homologous light chains and two heterogeneous heavy chains. In the two heterogeneous heavy chains, the heavy chain containing scFv is designed as a "knob" structure with amino acid substitutions at two sites: S354C and T366W. Furthermore, to prevent cleavage of scFv from the C-terminus of the heavy chain, the K at the final position of the C-terminus of the heavy chain was mutated to A, resulting in the K447A amino acid substitution. In the two heterogeneous heavy chains, the heavy chain not containing scFv is designed as a "hole" structure with amino acid substitutions at four sites: Y349C, T366S, L368A, and Y407V. In addition, to facilitate the purification of the bispecific antibody, the "hole" structure heavy chain underwent an H435R substitution. The modified homologous light chain sequence is shown in SEQ ID NO:5, the heavy chain sequence of the "knob" structure is shown in SEQ ID NO:3, and the heavy chain sequence of the "hole" structure is shown in SEQ ID NO:4.

[0125] The structures and associated molecular sequences of the bispecific antibodies according to the present invention are summarized in Table 1 and Table 2, respectively.

[0126] [Table 1]

[0127] [Table 2]

[0128] [Table 3]

[0129] [Table 4]

[0130] Example 2: Construction of a bispecific antibody against CD3-CLDN18.2 and its transient expression in eukaryotic cells.

[0131] The target gene fragments of the heavy and light chains of the aforementioned bispecific antibody molecule were cloned into a PTT5 expression vector, respectively, to prepare transfection-level expression plasmids. The plasmids were inoculated into serum-free medium, and HEK293E or Expi293 cells were cultured in a shaker at 37°C and 8% CO2. After 6 days of cell culture, the supernatant was collected and purified, and the final purified bispecific antibody was subjected to SDS-PAGE purity analysis and A280 concentration measurement.

[0132] Example 3: Affinity detection test of anti-CD3-CLDN18.2 bispecific antibody

[0133] A. Detection of affinity between anti-CD3-CLDN18.2 bispecific antibody and cells expressing hCLDN18.2 and hCD3.

[0134] Using FACS, we detected the binding status of an anti-CD3-CLDN18.2 bispecific antibody to HEK293 cells that stably express hCLDN18.2 (HEK293-hCLDN18.2) and to T lymphocytes that spontaneously express hCD3 (Jurkat).

[0135] HEK293-hCLDN18.2 or Jurkat cells were collected, resuspended in FACS buffer (PBS + 1% BSA + 0.5 mM EDTA), and adjusted for cell concentration. These cells were then added to a 96-well plate at a cell count of 1E5 per well. Antibodies, gradient diluted to pre-set concentrations, were then added. The negative control was human IgG1AA (Negative-IgG1AA), the CLDN18.2 positive control was 6#AA antibody, and the CD3 positive control was h160C9AA antibody. After incubation in a 4°C shaker for 2 hours, the cells were centrifuged with FACS buffer and washed twice. Then, 100 μL / well of fluorescently labeled anti-human IgG secondary antibody was added, incubated in a 4°C shaker for 1 hour, centrifuged with FACS buffer and washed twice. The cells were then filtered into a new 96-well plate, and the prepared sample was detected by flow cytometry. The average fluorescence intensity (MFI) for each concentration was calculated using software, and then the half-binding concentration (EC) was calculated using GraphPad software. 50 The maximum average fluorescence intensity (Top MFI) was calculated (abbreviated as ) and the results are shown in Table 3.

[0136] [Table 5]

[0137] Table 3 and Figures 2-3 show the affinity results for the anti-CD3-CLDN18.2 bispecific antibody and reference antibody (6#AA and h160C9AA) according to the present invention to HEK293-hCLDN18.2 cells and Jurkat cells, respectively. The experimental results showed that the maximum average fluorescence intensity for binding of the bispecific antibody according to the present invention to HEK293-hCLDN18.2 cells was in the range of 24200-31000. The maximum average fluorescence intensity for binding of the anti-CLDN18.2 reference antibody 6#AA to HEK293-hCLDN18.2 under the same reaction conditions was 18600, and for the anti-CD3 reference antibody h160C9AA, the maximum average fluorescence intensity for binding to HEK293-hCLDN18.2 under the same reaction conditions was only 897. The half-binding concentration (EC) for binding of the bispecific antibody according to the present invention to HEK293-hCLDN18.2 was also shown.50 ) is in the range of 0.98 to 1.18, and the half-binding concentration (EC) of the anti-CLDN18.2 reference antibody 6#AA with HEK293-hCLDN18.2 under the same reaction conditions. 50 The cereal concentration (EC2) is 0.95 nM, and in the case of the anti-CD3 reference antibody h160C9AA, the half-binding concentration (EC2) with HEK293-hCLDN18.2 under the same reaction conditions is 0.95 nM. 50 The value was 0 nM.

[0138] The maximum mean fluorescence intensity of the anti-CD3-CLDN18.2 bispecific antibody according to the present invention bound to CD3-highly expressing Jurkat cells was in the range of 1698 to 2025, while the maximum mean fluorescence intensity of the anti-CD3 reference antibody h160C9AA bound to Jurkat under the same reaction conditions was 2020. The half-binding concentration (EC) of the bispecific antibody according to the present invention bound to Jurkat was... 50 ) is in the range of 9.3 to 14.08, and the half-binding concentration (EC) of the anti-CD3 reference antibody h160C9AA with Jurkat under the same reaction conditions. 50 The concentration was 0.13 nM. Thus, the binding ability of the bispecific antibodies according to the present invention to the hCLDN18.2 antigen was substantially the same as that of the CLDN18.2 reference monoclonal antibody 6#AA, with no significant difference, and the binding ability to the hCD3 antigen was weaker than that of the CD3 reference antibody h160C9AA.

[0139] B. In vitro binding affinity and kinetics of anti-CD3-CLDN18.2 bispecific antibodies This experiment used surface plasmon resonance (SPR) to measure and analyze the affinity and kinetic properties of bispecific antibodies and human CD3E&D proteins using a Biacore 8K instrument. Human CD3E&D proteins were immobilized on the experimental channel of a CM5 chip using an amino group binding method. Next, the antibodies to be measured were sequentially diluted to a series of concentrations, then flowed sequentially across the surfaces of the experimental channel and reference channel to bind, followed by dissociation. Binding and dissociation curves for each sample were obtained, and finally, the results were analyzed and evaluated using Biacore Insight Evaluation software.

[0140] The bispecific antibody of the present invention was diluted to 400 nM in a working buffer of 1×HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P2O), and then further diluted to 1.56 nM in the same buffer to obtain antibody solutions at a range of concentrations from 400 nM to 1.56 nM. After each experiment, the chip was rinsed with 3 M MgCl2 solution at a flow rate of 30 μL / min for 30 seconds to remove the captured antibody along with the antigen, thus completing the regeneration of the chip. The raw data was analyzed using Biacore Insight Evaluation Software (version 2.0.15.12933) with a 1:1 fitting model. The affinity and kinetic experimental data of the obtained bispecific antibodies are shown in Table 4. As a result, the bispecific antibody showed very high affinity for human CD3E&D proteins, while the 31905-44AA molecule showed weaker affinity for human CD3E&D proteins than the 31905-38AA molecule.

[0141] [Table 6]

[0142] C. Binding selectivity of anti-CD3-CLDN18.2 bispecific antibodies The binding of the anti-CD3-CLDN18.2 bispecific antibody, anti-CLDN18.2 reference antibody 6#AA, and anti-CD3 reference antibody h160C9AA according to the present invention to HEK293 cells that stably express hCLDN18.1 (HEK293-hCLDN18.1), HEK293 cells that stably express mCLDN18.2 (HEK293-mCLDN18.2), HEK293 cells that stably express mCLDN18.1 (HEK293-mCLDN18.1), and HEK293 cells that stably express cynoCLDN18 (HEK293-cynoCLDN18) was detected by FACS.

[0143] HEK293-hCLDN18.1, HEK293-mCLDN18.2, HEK293-mCLDN18.1, and HEK293-cynoCLDN18 cells were prepared, deuterated in FACS buffer (PBS + 1% BSA + 0.5 mM EDTA) to adjust cell concentration, and then added to a 96-well plate at a cell count of 1E5 per well. Next, antibodies gradient-diluted to pre-set concentrations were added. Here, the negative control was Negative-IgG1AA, the CLDN18.2 positive control was 6#AA antibody, and the CD3 positive control was h160C9AA antibody. After culturing in a 4°C shaker for 2 hours, the cells were centrifuged in FACS buffer and washed twice. Then, 100 μL / well of fluorescently labeled anti-human IgG secondary antibody was added, incubated in a 4°C shaker for 1 hour, centrifuged in FACS buffer and washed twice, and the cells were filtered into a new 96-well plate. Subsequently, the prepared sample was detected by flow cytometry, and the average fluorescence intensity (MFI) for each concentration was calculated using software. Then, the half-binding concentration (EC) was calculated using GraphPad software. 50 The maximum average fluorescence intensity (Top MFI) was calculated (abbreviated as ) and the results are shown in Table 5.

[0144] [Table 7]

[0145] The results of the affinity of the anti-CD3-CLDN18.2 bispecific antibody, anti-CLDN18.2 reference antibody 6#AA, and anti-CD3 reference antibody h160C9AA according to the present invention with HEK293-hCLDN18.1 cells, HEK293-mCLDN18.2 cells, HEK293-mCLDN18.1 cells, and HEK293-cynoCLDN18 cells are shown in Table 5 and Figures 4 to 7. As a result of the experiment, the bispecific antibody according to the present invention binds to both mCLDN18.2 and cynoCLDN18, similar to the reference antibody 6#AA. The highest average fluorescence intensity of the binding between the bispecific antibody and HEK293-mCLDN18.2 is in the range of 37,300 to 51,400. The highest average fluorescence intensity of the binding between the reference antibody 6#AA and HEK293-mCLDN18.2 under the same reaction conditions is 36,000, and the highest average fluorescence intensity of the binding between the reference antibody h160C9AA and HEK293-mCLDN18.2 under the same reaction conditions is only 543. The half-binding concentration (EC 50 ) of the bispecific antibody according to the present invention is in the range of 1.13 to 1.18 nM, and the half-binding concentrations (EC 50 ) of antibodies 6#AA and h160C9AA are 0.90 nM and 0 nM, respectively. Furthermore, as a result of the experiment, the highest average fluorescence intensity of the binding between the bispecific antibody according to the present invention and HEK293-cynoCLDN18 is in the range of 28,600 to 41,800. The highest average fluorescence intensities of the binding between the reference antibodies 6#AA and h160C9AA and HEK293-cynoCLDN18 under the same reaction conditions are 39,500 and 1,045, respectively. The half-binding concentration (EC 50 ) of the bispecific antibody is in the range of 0.58 to 0.84 nM, and the half-binding concentrations (EC 50 ) of the reference antibodies 6#AA and h160C9AA are 0.62 nM and 0 nM, respectively. As a result of further research, it is shown that neither the bispecific antibody nor the reference antibody according to the present invention binds to hCLDN18.1 and mCLDN18.1. Thus, the binding of the bispecific antibody according to the present invention to mCLDN18.2 and cynoCLDN18 is substantially consistent with that of the reference antibody 6#AA, that is, it specifically binds to human and mouse CLDN18.2 and cynoCLDN18 and does not bind to human and mouse CLDN18.1, and has good selectivity.

[0146] Example 4: In vitro functional experiment of anti-CD3-CLDN18.2 bispecific antibody

[0147] AT cell-mediated cytotoxicity experiment (TDCC)

[0148] HEK293-hCLDN18.2 was used as the target cell, and PBMC cells from healthy individuals were used as effector cells. The amount of lactate dehydrogenase (LDH) released from cells was detected using a cytotoxicity detection reagent (Roche), and this was used as an indicator of cell-killing activity.

[0149] HEK293-hCLDN18.2 cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in cell buffer (RMPI1640 + 1% FBS + 1% P / S) to adjust the cell density. The cells were then transferred to 96 wells at a cell density of 1E4 per well, with a volume of 50 μL. After incubating overnight to allow the cells to adhere to the walls, 50 μL of the pre-prepared antibody and control sample working solutions at each concentration gradient were added. Next, 50 μL of pre-prepared PBMC cells in an effector cell:target cell ratio of 20:1 were added to replenish the wells (i.e., 1E5 cells per well). The cells were incubated in a cell incubator (37°C, 5% CO2) for approximately 24 hours. After incubation was complete, the 96-well plate was removed, centrifuged at 1500 rpm for 10 minutes, and 50 μL of supernatant was slowly aspirated and transferred to a new 96-well plate. An equal volume of LDH detection working solution was added, and the plate was incubated at room temperature for 20 minutes. Detection was performed using a microplate reader, and the detection wavelength was 492 nm, with a reference wavelength of 650 nm.

[0150] The rate of cell degradation due to the TDCC effect is calculated using the following formula.

[0151]

number

[0152] In the formula, maximum release is the absorbance value obtained in the well treated with target cells using Triton X-100, target cell / effector cell mixed release is the absorbance value obtained in the well containing a mixture of target cells and effector cells, target cell release is the absorbance value obtained in the well containing only target cells, and sample release is the absorbance value obtained in the well containing a mixture of antibody, target cells, and effector cells. EC by GraphPad software 50 The maximum decomposition was also calculated, and the results are shown in Table 6.

[0153] BT cell activation experiment

[0154] T cell activation experiments primarily use the cell activation marker CD25. + CD69 + Identification was performed by measuring the percentage. Cells after TDCC experiments were collected in 96-well plates, centrifuged and washed twice with FACS buffer (PBS + 1% BSA + 0.5 mM EDTA), then a specific amount of anti-human APC-CD3, anti-human PE-CD25, and anti-human BV421-CD69 mixture was added and incubated at 4°C for 1 hour. After centrifuging and washing twice with FACS buffer, the cells were filtered into a new 96-well plate, and the prepared samples were detected by flow cytometry, and CD25 was identified by software. + CD69 + CD3 + The percentage in T cells is calculated, and then the half-binding concentration (hereinafter referred to as EC) is calculated using GraphPad software. 50 (abbreviated as) and the highest percentage (CD25 + CD69 + The percentage is calculated and the results are shown in Table 6.

[0155] [Table 8]

[0156] The results of the TDCC activity and T cell activation of the anti-CD3-CLDN18.2 bispecific antibody according to the present invention against HEK293-hCLDN18.2 cells are shown in Table 6 and Figures 8-9. The experimental results showed that the maximum TDCC effect of the bispecific antibody according to the present invention against HEK293-hCLDN18.2 cells ranged from 30.03% to 54.62%, while the maximum TDCC effect of the anti-CLDN18.2 reference antibody 6#AA against HEK293-hCLDN18.2 cells under the same reaction conditions was 16.34%. The concentration (EC) that yields a 50% TDCC effect of the bispecific antibody is shown below. 50 The concentration (EC2) is in the range of 0.13-0.54 nM, which is the concentration of reference antibody 6#AA that produces a 50% TDCC effect under the same reaction conditions. 50 The value was 1.45 nM.

[0157] The maximum T cell activation effect of the biantibody according to the present invention on HEK293-hCLDN18.2 cells was in the range of 33.43% to 38.86%, while the maximum T cell activation effects of the reference antibodies 6#AA and h160C9AA on HEK293-hCLDN18.2 cells under the same reaction conditions were only 3.18% and 4.2%, respectively. The concentration (EC) at which the biantibody elicits 50% T cell activation 50 The concentration (EC2) is in the range of 0.022 to 0.049 nM, and is the same as the concentration (EC2) of the reference antibodies 6#AA and h160C9AA that elicits 50% T cell activation under the same reaction conditions. 50 The value of ) is 0. From the above results, it is clear that the biantibody according to the present invention has strong TDCC activity and T cell activation effect, and that such effect is target cell dependent.

[0158] C. Quantitative experiment of cytokines Cytokine quantification experiments (including INF-γ, IL-2, IL-6, and TNF-α) were primarily performed using the Elisa Kit (Biolegend). The supernatant was collected after centrifugation, diluted to a set ratio, and then detected using the Kit. The specific procedure followed the protocol outlined in the Kit's instruction manual. Detection was performed using a microplate reader, with a detection wavelength of 450 nm and a reference wavelength of 650 nm. EC was performed using GraphPad software.50 The maximum concentration was also calculated, and the results are shown in Tables 7 and 8.

[0159] [Table 9]

[0160] Table 7 and Figures 10-11 show the results of inducing INF-γ and IL-2 secretion from T cells by the anti-CD3-CLDN18.2 bispecific antibody according to the present invention under conditions where HEK293-hCLDN18.2 cells are present. The experimental results showed that the maximum INF-γ secretion level produced by inducing T cells with the bispecific antibody according to the present invention ranged from 260.5 pg / mL to 769.5 pg / mL, and the concentration (EC) that induces 50% INF-γ secretion of the bispecific antibody is shown. 50 The concentration was in the range of 0.012 to 0.033 nM. In the case of reference antibodies 6#AA and h160C9AA, no significant INF-γ secretion induction was observed under the same reaction conditions.

[0161] The maximum IL-2 secretion level induced by T cells using the biantibody according to the present invention is in the range of 60.25 pg / mL to 201.9 pg / mL, and the concentration (EC) that induces the production of 50% IL-2 secretion by the biantibody is 50 The concentration was in the range of 0.013 to 0.05 nM. In the case of reference antibodies 6#AA and h160C9AA, no significant IL-2 secretion induction was observed under the same reaction conditions.

[0162] [Table 10]

[0163] Table 8 and Figures 12-13 show the results of inducing IL-6 and TNF-α secretion from T cells using the anti-CD3-CLDN18.2 bispecific antibody according to the present invention under conditions where HEK293-hCLDN18.2 cells are present. The experimental results showed that the maximum IL-6 secretion level induced by T cells using the bispecific antibody according to the present invention was in the range of 12718 pg / mL to 21942 pg / mL, while the maximum IL-6 secretion level induced by T cells using the reference antibody h160C9AA under the same reaction conditions was 84209 pg / mL. The concentration (EC2) at which the bispecific antibody induces 50% IL-6 secretion was determined. 50 The concentration (EC2) is in the range of 0.005 to 0.16 nM, which is the concentration (EC2) that induces the production of 50% IL-6 secretion under the same reaction conditions as the reference antibody h160C9AA. 50 The concentration was 2.9 nM. However, in the case of reference antibody 6#AA, no significant IL-6 secretion induction effect was observed under the same reaction conditions.

[0164] The maximum TNF-α secretion level induced by T cells using the anti-CD3-CLDN18.2 antibody according to the present invention is in the range of 365.9 pg / mL to 664.6 pg / mL, while the maximum TNF-α secretion level induced by T cells using the reference antibody h160C9AA under the same reaction conditions is 3175 pg / mL. The concentration (EC) that induces the production of 50% TNF-α secretion with the biantibody is the concentration (EC) required to induce this secretion. 50 The concentration (EC2) is in the range of 0.01 to 1.3 nM, and is the concentration (EC2) that induces 50% TNF-α secretion production under the same reaction conditions as the reference antibody h160C9AA. 50 The concentration was 5.9 nM. However, in the case of reference antibody 6#AA, no significant TNF-α secretion induction effect was observed under the same reaction conditions.

[0165] From these results, it is clearer that the anti-CD3-CLDN18.2 antibody according to the present invention has a strong T-cell activating effect. Under conditions where target cells are present, the effect of inducing the secretion of INF-γ and IL-2 was stronger than that of the anti-CD3 reference antibody h160C9AA, while the effect of inducing the secretion of IL-6 and TNF-α was weaker than that of h160C9AA.

[0166] Experiments with the D. luciferase reporter gene

[0167] We constructed a Jurkat-NFAT cell line that stably expresses luciferase, and found that the luciferase gene is regulated by the NFAT element. On the other hand, the NFAT reaction element is regulated by the CD3 receptor on the surface of Jurkat cells, and stimulating the CD3 receptor activates the NFAT reaction element, leading to luciferase expression. Using this system, we simulated the activating effect of CD3 antibodies on T cells at the cellular level.

[0168] Jurkat-NFAT cells were diluted in culture medium and homogeneously mixed (RMPI1640 + 1% FBS + 1% P / S). Cells were inoculated into specific well plates at a rate of 5E4 per well, with a volume of 50 μL per well. Next, 50 μL of the test antibody, pre-diluted in culture medium, was added and co-cultured with the cells. The cells were incubated at 37°C in a 5% carbon dioxide incubator for approximately 4-6 hours. 50 μL of the Bright-Glo luciferase detection reagent was added, and the cells were incubated at room temperature and protected from light for 5 minutes. The chemiluminescence signal value was read using PerkinElmer Envision, and signal suppression EC was performed using GraphPad Prism. 50 The values ​​were fitted.

[0169] [Table 11]

[0170] The results for the anti-CD3-CLDN18.2 bispecific antibody according to the present invention on the luciferase expression level of the Jurkat-NFAT reporter gene are shown in Table 9 and Figure 14. The experimental results showed that the maximum luciferase expression of the bispecific antibody according to the present invention for the Jurkat-NFAT reporter gene was in the range of 113700 to 116960, while the maximum luciferase expression of the reference antibody h160C9AA for the Jurkat-NFAT reporter gene was 220000. In the case of reference antibody 6#AA, there was no effect on the maximum luciferase expression of the Jurkat-NFAT reporter gene.

[0171] The concentration (EC) of the biantibody according to the present invention that elicits 50% luciferase expression 50 The concentration (EC2) is in the range of 7.62 to 123.6 nM, which is the concentration (EC2) that results in 50% luciferase expression of the reference antibody h160C9AA antibody. 50 The concentration was 0.32 nM. From the above results, it is clear that the activation effect of the bispecific antibody according to the present invention is lower than that of the CD3 reference antibody, and the activation effect of the CD32-valent antibody (31905-38AA) is higher than that of the CD31-valent antibody (31905-44AA).

[0172] E.NK cell-mediated cytotoxicity experiment (ADCC)

[0173] HEK293-hCLDN18.2 was used as the target cell, and NK cells isolated from healthy human PBMCs were used as effector cells. The amount of lactate dehydrogenase (LDH) released from cells was detected using a cytotoxicity detection reagent (Roche), and this was used as an indicator of cell-killing activity.

[0174] HEK293-hCLDN18.2 cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in ADCC buffer (RMPI1640 + 1% FBS + 1% P / S) to adjust the cell density. The cells were then transferred to 96 wells at a cell count of 1E4 per well, with a volume of 50 μL. After incubating overnight to allow the cells to adhere to the walls, 50 μL of the pre-prepared antibody and control sample working solutions at various concentration gradients were added. Next, 50 μL of pre-isolated NK cells were added in an effector cell:target cell ratio of 5:1 (i.e., 1E5 cells per well). The cells were incubated in a cell incubator (37°C, 5% CO2) for approximately 24 hours. After incubation was complete, the 96-well plate was removed, centrifuged at 1500 rpm for 10 minutes, and 50 μL of supernatant was slowly aspirated and transferred to a new 96-well plate. An equal volume of LDH detection working solution was added, and the plate was incubated at room temperature for 20 minutes. Detection was performed using a microplate reader, and the detection wavelength was 492 nm, with a reference wavelength of 650 nm.

[0175] The rate of cell degradation due to the ADCC effect is calculated using the following formula:

[0176]

number

[0177] In the formula, maximum release is the absorbance value obtained in a well treated with target cells using Triton X-100, target cell / effector cell mixed release is the absorbance value obtained in a well containing a mixture of target cells and effector cells, target cell release is the absorbance value obtained in a well containing only target cells, and sample release is the absorbance value obtained in a well containing a mixture of chimeric antibody, target cells, and effector cells. EC by GraphPad software 50 The maximum decomposition was also calculated, and the results are shown in Table 10.

[0178] [Table 12]

[0179] Table 10 and Figure 15 show the results of the ADCC activity of the anti-CD3-CLDN18.2 bispecific antibody according to the present invention against HEK293-hCLDN18.2 cells. The experimental results showed that the maximum ADCC effect of the bispecific antibody according to the present invention against HEK293-hCLDN18.2 cells ranged from 7.03% to 19.95%, while the ADCC effects of the reference antibodies 6#AA and h160C9AA under the same reaction conditions were 6.92% and 0%, respectively. The concentration (EC) at which the bispecific antibody according to the present invention elicits a 50% ADCC effect was... 50 ) is in the range of 0.501~0.532 nM, and the concentration (EC) of 6#AA that produces a 50% ADCC effect under the same reaction conditions is 50 The concentration was 0.771 nM. From these results, it is clear that the ADCC effect of the anti-CD3-CLDN18.2 bispecific antibody according to the present invention is equivalent to that of the anti-CLDN18.2 reference antibody 6#AA, but the CD3 reference antibody h160C9AA does not show an ADCC effect.

[0180] Example 5: In vivo efficacy experiment of anti-CD3-CLDN18.2 bispecific antibody

[0181] Peripheral blood was collected from healthy individuals, and human PBMCs were isolated and counted by density gradient centrifugation. Subsequently, the isolated PBMCs were added to HEK293-hCLDN18.2 cells treated with Mitomycin C in RPMI-1640 medium (containing IL-2 and 10% FBS) and cultured for 6 days. After 6 days, the PBMCs were harvested, and the PBMC cells were mixed with freshly digested HEK293-hCLDN18.2 cells (Shanghai Qilu Pharmaceutical Research Center). This mixture was then subcutaneously inoculated into NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to construct a humanized HEK293-hCLDN18.2 model. All experimental animals were housed in independent, ventilated chambers with constant temperature and humidity. The chamber temperature was 20.0–26.0°C, humidity was 40–70%, air was replaced 10–20 times / hour, and the day / night cycle was 12 hours / 12 hours.

[0182] The experiment was divided into three groups: a PBS control group, a 0.6 mg / kg 31905-44AA treatment group, and a 3 mg / kg 31905-44AA treatment group (5 mice / group). The drug was administered intraperitoneally twice a week for a total of six injections (see Table 11). After administration, the animals' daily behavior was monitored for a total of 24 days. Throughout the experiment, the longest and widest diameters of the tumors were measured twice a week using calipers, and the tumor volume (mm³) was measured. 3 ) = 0.5 × (Tumor diameter × Tumor diameter) 2 The relative tumor inhibition rate (TGI) was calculated as follows: TGI% = (1 - T / C) × 100%. T / C% is the relative tumor growth rate, which is the percentage of the relative tumor volume or tumor weight between the treatment group and the PBS control group at a given time. T and C are the tumor volume (TV) or tumor weight (TW) of the treatment group and the PBS control group, respectively, at a specific time. All data are expressed as Mean ± SEM, and a student's t-test was used to compare the tumor volume and tumor weight of the treatment group and the control group to determine if there was a significant difference. A p < 0.05 indicates a significant difference.

[0183] As shown in Figures 16 and 17, the biantibody 31905-44AA demonstrated clear antitumor activity in the humanized HEK293-hCLDN18.2 model, with the final mean tumor volume for each group (PBS, 0.6 mg / kg of 31905-44AA, and 3 mg / kg of 31905-44AA) being 230.59 mm². 3 96.98mm 3 97.6mm 3 In the 0.6 mg / kg 31905-44AA group and the 3 mg / kg 31905-44AA group, the tumor growth inhibitory rate (TGI) was 101.8% and 101.2%, respectively. Neither treatment group showed any significant weight loss in NCG mice, indicating that NCG mice have good tolerance to this dose of anti-CD3-CLDN18.2 bispecific antibody.

[0184] [Table 13]

[0185] Example 6: Pharmacokinetic experiment of an anti-CD3-CLDN18.2 bispecific antibody

[0186] In the experiment, one naive female and one naive male cynomolgus monkey (total: 2 monkeys) were used and given free access to water. The bispecific antibody was administered at a dose of 1 mg / kg and intravenously infused over 60 minutes. Blood samples were collected at pre-dose, 1h, 4hr, 6hr, 24hr, 2d, 4d, 7d, 10d, 14d, 21d, 28d, 35d, and 42d. After whole blood collection, the samples were allowed to stand for 0.5 hours, then centrifuged to collect serum (4000 rpm, 10 min, 4°C). The serum was collected, the blood samples were left at room temperature before centrifugation, and after centrifugation, the serum was individually packaged and frozen at -80°C.

[0187] The concentration of bispecific antibodies in serum is detected using the ELISA method, and the detection process is described below.

[0188] A 96-well plate was coated with human CD3 protein to a concentration of 1 μg / mL and 100 μL / well. It was left overnight at 4°C, washed three times with 200 μL / well PBST, added 300 μL of 5% powdered milk (blocking reagent), incubated at 37°C for 1 hour, washed three times with 200 μL / well PBST, added 100 μL of standard, QC, and target sample, incubated at 37°C for 1 hour, washed six times with 300 μL / well PBST, added 100 μL of Anti-Human IgG Fc (HRP), diluted 1:5000, incubated at 37°C for 1 hour, washed six times with 300 μL / well PBST, added 100 μL of TMB, stood in the dark for 10 minutes, and then added 100 μL of stop solution to stop the color development.

[0189] MD's M5 plate reader detected absorbance values ​​at a wavelength of 450 nm, and the data was processed by Softmaxsoft.

[0190] The serum concentrations of the bispecific antibody 31905-44AA were calculated using Phoenix Winnolin 8.2 software, and pharmacokinetic parameters were obtained, which are shown in Table 12.

[0191] [Table 14]

[0192] The concentration units listed in the table above are all in μg / mL, and the BLQ was below the detection limit.

[0193] The bispecific antibody 31905-44AA had a half-life of 59.46 ± 7.0 h, a Cmax of 22.80 ± 2.25 μg / mL, and an AUC in monkeys. 0~336H Since the concentration is 765.85±38.98 day*μg / mL, this bispecific antibody exhibits stable properties in the monkey body, no apparent off-target binding, and good pharmacokinetic properties.

[0194] The embodiments of the present invention described above are merely illustrative, and those skilled in the art can recognize and determine countless equivalents of specific compounds, materials, and operations without the need to perform unconventional experiments. All of these equivalents are within the scope of the present invention and included in the claims.

Claims

1. It contains an anti-CLDN18.2 binding domain and an anti-CD3 binding domain, The first binding domain binds to the CLDN18.2 protein. The second binding domain can bind to CD3, The anti-CLDN18.2 binding domain includes a heavy chain variable region and a light chain variable region. The sequences of the three CDRs in the heavy chain variable region, HCDR1, HCDR2, and HCDR3, are shown in SEQ ID NO: 11, 12, and 13, respectively. The sequences of the three CDRs, LCDR1, LCDR2, and LCDR3, in the aforementioned light chain variable region are shown in SEQ ID NO: 14, 15, and 16, respectively. The anti-CD3 binding domain includes a heavy chain variable region and a light chain variable region. The sequences of the three CDRs in the heavy chain variable region, HCDR1, HCDR2, and HCDR3, are shown in SEQ ID NO: 17, 18, and 19, respectively. The sequences of the three CDRs in the light chain variable region, LCDR1, LCDR2, and LCDR3, are shown in SEQ ID NO: 20, 21, and 22, respectively, representing a bispecific antibody.

2. The anti-CLDN18.2 binding domain includes a heavy chain variable region and a light chain variable region. The heavy chain variable region has the sequence represented by SEQ ID NO: 23, The bispecific antibody according to claim 1, wherein the light chain variable region has a sequence represented by SEQ ID NO:

24.

3. The anti-CD3 binding domain includes a heavy chain variable region and a light chain variable region. The heavy chain variable region has an arrangement represented by SEQ ID NO: 6, The bispecific antibody according to claim 1 or 2, wherein the light chain variable region has a sequence represented by SEQ ID NO:

7.

4. The aforementioned binding domains are Fab, Fv, scFv, F(ab') ２ A bispecific antibody according to any one of claims 1 to 3, which is a linear antibody or a full-length antibody.

5. A bispecific antibody according to any one of claims 1 to 4, further comprising a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region comprises a natural Fc or a mutant Fc.

6. The bispecific antibody according to any one of claims 1 to 5, wherein the anti-CLDN18.2 binding domain is a full-length antibody and the anti-CD3 binding domain is scFv.

7. The anti-CLDN18.2 binding domain is a full-length antibody having the heavy chain sequence shown in SEQ ID NO: 1 and the light chain sequence shown in SEQ ID NO: 5, and the anti-CD3 binding domain is an scFv shown in SEQ ID NO: 8, according to any one of claims 1 to 6.

8. A bispecific antibody according to any one of claims 1 to 7, wherein an anti-CD3 binding domain, scFv, is conjugated to the C-terminus of the heavy chain or the C-terminus of the light chain of a full-length anti-CLDN18.2 antibody.

9. The structure involves fusing the anti-CD3 binding domain, scFv, to the C-terminus of the two light chains of the full-length anti-CLDN18.2 antibody, thereby forming two homologous light chains and two homologous heavy chains. The bispecific antibody according to any one of claims 1 to 8, wherein the sequence of the fused light chain is shown in SEQ ID NO: 2, and the sequence of the heavy chain is shown in SEQ ID NO:

1.

10. The structure involves fusing the anti-CD3 binding domain, scFv, to the C-terminus of one heavy chain of the full-length anti-CLDN18.2 antibody, forming two homologous light chains and two heterologous heavy chains. The heavy chain sequence containing scFv is shown in SEQ ID NO:

3. The heavy chain sequence without scFv is shown in SEQ ID NO:

4. The light chain sequence is shown in SEQ ID NO: 5, a bispecific antibody according to any one of claims 1 to 9.

11. A nucleic acid encoding a bispecific antibody according to any one of claims 1 to 10.

12. A recombinant vector comprising the nucleic acid according to claim 11.

13. A host cell comprising the recombinant vector according to claim 12 or the nucleic acid according to claim 7.

14. The host cells described in claim 13 are cultured under appropriate conditions, A method for producing a bispecific antibody, comprising obtaining an expression product by purifying the cells.

15. A bispecific antibody according to any one of claims 1 to 6, used for therapeutic purposes that specifically target tumor cells expressing CLDN18.2, The bispecific antibody according to any one of claims 1 to 10, wherein the tumor expressing CLDN18.2 includes gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, colon cancer, rectal cancer, liver cancer, head and neck cancer, and gallbladder cancer, as well as metastases thereof.

16. The bispecific antibody according to claim 15, wherein the gastric cancer metastasis is a Krukenberg tumor.

17. Contains an effective amount of the bispecific antibody described in any one of claims 1 to 10, or Containing an effective amount of the nucleic acid described in claim 11, Containing an effective amount of the recombinant vector described in claim 12, A pharmaceutical composition comprising an effective amount of the host cells described in claim 13.

18. The pharmaceutical composition according to claim 17, further comprising a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 17, further comprising one or more additional other therapeutic agents, the additional therapeutic agents comprising cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor demulsifiers, chemotherapeutic agents, radiotherapeutic agents, targeted anticancer agents, biological response modifiers, cancer vaccines, cytokines, hormones, anti-metastatic agents, and immunotherapeutic agents.

20. A pharmaceutical composition according to any one of claims 17 to 19, used for the treatment of inducing cell death of cells expressing CLDN18.2, comprising contacting the cells with the pharmaceutical composition, The aforementioned cells are cancer cells, in this pharmaceutical composition.

21. The pharmaceutical composition according to claim 20, wherein the cancer cells are solid tumor cells.

22. The pharmaceutical composition according to claim 20, wherein the cells are selected from gastric cancer cells, esophageal cancer cells, intestinal cancer cells, pancreatic cancer cells, Wilms' tumor cells, lung cancer cells, ovarian cancer cells, colon cancer cells, rectal cancer cells, liver cancer cells, head and neck cancer cells, chronic myeloid leukemia cells, and gallbladder cancer cells.

23. A pharmaceutical composition according to any one of claims 17 to 19, used for the treatment of a disease associated with the expression of CLDN18.2 in a subject, comprising administering the pharmaceutical composition to a subject as needed.

24. A pharmaceutical composition according to claim 23, used for the treatment of a disease associated with the expression of CLDN18.2 in a subject, wherein the disease is a tumor.

25. A pharmaceutical composition according to claim 23 or 24, used for the treatment of a disease associated with the expression of CLDN18.2 in a subject, wherein the disease is a tumor, and the tumor is gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, Wilms' tumor, lung cancer, ovarian cancer, colon cancer, rectal cancer, liver cancer, head and neck cancer, chronic myeloid leukemia, or gallbladder cancer.

26. A pharmaceutical composition according to any one of claims 23 to 25, used for the treatment of a disease associated with the expression of CLDN18.2 in a subject, wherein the treatment further comprises administering an additional therapeutic agent to the subject.

27. The pharmaceutical composition according to claim 26, wherein the additional therapeutic agent comprises a chemotherapeutic agent, a cytotoxic agent, a cell proliferation inhibitor, a radiotherapy agent, a cancer vaccine, a tumor debulking agent, a targeted anticancer agent, an anti-angiogenic agent, a biological response modifier, a cytokine, a hormone, an anti-metastatic agent, and an immunotherapy agent.

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