An anti-Claudin18.2 monoclonal antibody, its preparation method and uses

Abstract

An anti-Claudin18.2 monoclonal antibody and its uses. A nucleic acid molecule encoding the antibody, an expression vector expressing the antibody, a host cell, and a pharmaceutical composition containing the antibody. The use of the antibody alone or in combination with other agents such as anti-Her2 antibody and / or IL15 for preparing an anti-cancer drug.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an anti-Claudin18.2 monoclonal antibody, a preparation method thereof, and uses thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Humanized antibodies are derived from non-human species and have their protein sequences modified to increase their sequence similarity to natural antibodies in the human body. The "humanization" process is typically for developing monoclonal antibodies for use in humans, such as antibodies developed as anti-cancer drugs. The humanization process is necessary for producing specific antibodies in a non-human (e.g., mouse) immune system. The protein sequences of the antibodies produced in this way are partially different from the homologous antibodies naturally present in humans, and thus they have potential immunogenicity when administered to human patients.

[0004] Targeted therapy is one of the main ways of drug treatment for cancer, and the others include hormone therapy and cytotoxic chemotherapy. As a form of molecular medicine, targeted therapy blocks the growth of cancer cells by interfering with specific target molecules required for carcinogenesis and tumor growth, rather than simply interfering with all rapidly dividing cells (such as traditional chemotherapy). Since most drugs used for targeted therapy are biopharmaceuticals, the term "biotherapy" is sometimes synonymous with targeted therapy when used for cancer treatment, which is different from chemotherapy (i.e., cytotoxic therapy). However, these methods can be used in combination. Antibody-drug conjugates combine biological and cytotoxic mechanisms into one targeted therapy.

[0005] Bispecific monoclonal antibodies (BsMAb, BsAb), also known as bifunctional antibodies, are artificial proteins composed of two different antibody fragments, which can simultaneously recognize and bind two different antigens and epitopes, and block two different signal pathways to exert their functions. BsMabs can be manufactured in various structural forms, and their applications in cancer immunotherapy and drug delivery have been explored.

[0006] Gastric cancer (GC) is one of the most common cancers and serious health problems worldwide. For unresectable or metastatic advanced gastric cancer, chemotherapy is the first choice. Although chemotherapy can improve the survival rate of patients with advanced gastric cancer (AGC), the prognosis of these patients remains poor. Adjuvant chemotherapy and chemoradiotherapy have improved the overall survival. Researchers have studied a variety of new chemotherapy regimens, which have higher response rates and tolerances, but the 5-year survival rate is disappointing. In recent years, some therapies targeting biomolecules have been reported to extend the survival of AGC patients. Since trastuzumab, a monoclonal antibody targeting HER2, has been established as the standard treatment for unresectable GC in HER2-positive patients, many other targets have been reported as new treatment targets. Whether there is chemotherapy in clinical trials or not, many molecular targeted therapies (such as HER2, VEGFR or EGFR) have been recognized as established standard therapies. In addition, the clinical trial data of immunotherapy are promising and are expected to become an effective therapy. In particular, immune checkpoint inhibitors, such as PD-1 / PD-L1 or CTLA-4, have demonstrated innovative progress in the treatment of GC. In addition, ongoing clinical trials including targeted therapy and immunotherapy have shown encouraging results in improving clinical outcomes, safety and tolerance, however, the clinical trial results of numerous targeted drugs are uneven. The emergence of immune checkpoint inhibitors has also generated similar hopes, and the results of early trials are encouraging.

[0007] Developed by Genentech Trastuzumab is a humanized monoclonal antibody targeting HER2. In 1998, trastuzumab combined with paclitaxel was approved by the US FDA as the first-line treatment for metastatic breast cancer with HER2 / neu overexpression, or as a single drug for metastatic breast cancer with HER2 / neu overexpression after at least one chemotherapy cycle. So far, several HER2-directed therapies for HER2-positive breast cancer and non-small cell lung cancer have been approved, including trastuzumab, pertuzumab, T-DM1, lapatinib and afatinib (tyrosine kinase inhibitor).

[0008] Approximately 22% of patients with metastatic gastric cancer will have HER2 overexpression or amplification, with differences only in tumor subtype (intestinal vs diffuse) and tumor location (gastroesophageal junction (GEJ) vs stomach). Many studies have reported the correlation between HER2-positive gastric cancer and poor outcomes and more aggressive diseases, and there are still some controversies with other conflicting studies. Trastuzumab plus chemotherapy has been approved as the standard treatment option for gastric cancer patients in the Her-2 (IHC)-3 positive group, but its therapeutic benefit is indeed limited.

[0009] Claudin is a family of proteins first discovered by Shorichiro Tsukita et al. It is an important component of the formation of cell tight junctions, which establish a paracellular barrier and control the flow of molecules between cells. The transmembrane domain of Claudin includes the N-terminal and C-terminal in the cytoplasm. Different Claudin proteins are expressed in different tissues, and their altered functions are related to the formation of cancers in their respective tissues. It has been shown that Claudin-1 expression has prognostic value in colon cancer, Claudin-18 in gastric cancer, and Claudin-10 in hepatocellular carcinoma. Ugur Sahin et al. identified the isoform 2 (CLDN18.2) of the tight junction molecule Claudin-18 as a highly selective cell lineage marker, whose expression in normal tissues is strictly limited to the epithelial cells differentiated from the gastric mucosa and absent in the gastric stem cell area. Claudin 18.2 is retained in malignant transformation and expressed in most primary gastric cancers and their metastatic cancer types. In addition, ectopic activation of Claudin 18.2 is often observed in pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer. Studies have shown that CLDN18.2 has a highly restricted expression pattern in normal tissues and frequent ectopic activation in various human cancers. The correlation of Claudin proteins with isoform 2, especially gastric cancer and its metastatic cancers, has led to the development of anti-Claudin 18.2 specific antibodies as targeted therapies for gastric cancer and other human solid malignancies.

[0010] Claudiximab is a novel chimeric IgG1 antibody highly specific for Claudin 18.2. The clinical phase IIa (MONO) study aimed to determine the safety and efficacy of multiple doses of Claudiximab as a single therapy in patients with metastatic, refractory, recurrent gastric or lower esophageal adenocarcinoma. The response rate was 10%, and the disease control rate was 30% (best observed response: PR, n = 4, SD, n = 8). The median PFS was 102 days (95% CI, 70 - 146 days). All observed adverse reactions were grade 1 - 3. The most common grade 3 adverse reaction was vomiting, in 31 cases. No grade 4 adverse reactions occurred.

[0011] Subsequent clinical phase IIb (FAST) study evaluated Claudiximab as a first-line drug in patients with advanced / recurrent gastroesophageal cancer. The patients included in the study were those with ≥40% of tumor cells expressing ≥2+ CLDN18.2 (by CLAUDETECT TM18.2 Kit Verification), patients with an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1 and ineligible for trastuzumab treatment. According to the protocol criteria, 739 patients were screened for enrollment, of which 352 (48%) were tested positive for CLDN18.2. Among them, 161 patients (80% gastric cancer; 16% GEJ; 4% esophageal cancer) were randomly assigned 1:1 to first-line EOX (epirubicin 50 mg / m 2 , oxaliplatin 130 mg / m 2 on day 1, and capecitabine 625 mg / m 2 twice daily, days 1-21, every 21 days a cycle), with or without Claudiximab (loading dose 800 mg / m 2 , then 600 mg / m 2 on day 1, every 21 days a cycle). An exploratory extension of the study was conducted in a third group (N = 85) to investigate high-dose Claudiximab (1000 mg / m 2 ) in combination with EOX. The study met the primary endpoint of progression-free survival (PFS). Compared with EOX alone, Claudiximab in combination with EOX significantly improved PFS (median 7.9 vs 4.8 months; HR 0.47; p = 0.0001) and OS (median 13.3 vs 8.4 months; HR 0.51; p < 0.001).

[0012] For the subgroup analysis of patients with high CLDN18.2 expression (≥2+ intensity in ≥70% of tumor cells), the efficacy was more obvious (PFS, 7.2 vs 5.6 months; HR 0.36; p = 0.0005; OS, 9.0 vs 16.7 months; P = 0.0005; OS: 9.0 vs 16.7 months; HR 0.45, p < 0.0005). The objective response rate (ORR) of patients receiving Claudiximab treatment was also higher, at 39%, compared with 25% in the EOX group. In the Claudiximab group, 8 patients (10.4%) achieved a complete response (CR), 22 patients (28.6%) achieved a partial response (PR), and 34 patients (44.2%) had stable disease (SD). Among the patients receiving chemotherapy, 3 patients (3.6%) achieved CR, 18 patients (21.4%) achieved PR, and 43 patients (51.2%) achieved SD. The proportions of patients with disease progression in the Claudiximab treatment and chemotherapy groups were 5.2% and 11.9%, respectively. The treatment was well tolerated, mostly with grade 1 / 2-related adverse reactions, including vomiting, neutropenia, and anemia. There was no significant increase in grade 3 / 4 adverse reactions among patients receiving Claudiximab. Overall, 55.8% of the patients in the study group had grade 1 / 2 vomiting, and 10.4% had grade 3 / 4 vomiting; in the chemotherapy group, 34.5% of the patients had grade 1 / 2 vomiting, and 3.6% had grade 3 / 4 adverse reactions. The incidence and severity of vomiting seemed to be dose-related. The researchers concluded that Claudiximab combined with first-line chemotherapy had clinically relevant benefits for PFS and OS in patients with CLDN18.2-positive gastric cancer and GEJ adenocarcinoma.

[0013] Interleukin-15 (IL-15) is a cytokine of approximately 12 - 14 kD discovered by Grabstein et al. in 1994 and can play a role in the normal immune response of the body, such as promoting the proliferation of T cells, B cells, and natural killer (NK) cells.

[0014] IL-15 belongs to the members of the four small α-helical bundle cytokine family (the small four α-helical bundle cytokine family). IL-15 needs to bind to receptors to exert its biological activity. The IL-15 receptor consists of three receptor subunits: IL-15 receptor α (IL-15Rα), IL-2 receptor β (IL-2Rβ, also known as IL-15Rβ or CD122), and γc (also known as CD132). IL-15Rα contains a Sushi domain that can bind to IL-15 and is necessary for the bound IL-15 to exert its biological function.

[0015] In recent years, it has been found that after IL-15 forms a complex with its receptor IL-15Rα, the biological activity of IL-15 can be significantly enhanced. Studies have shown that the complex formed by IL-15 and its soluble receptor IL-15Rα significantly transforms the effect of stimulating the proliferation of memory CD8+ T lymphocytes and NT / NKT cells into the effect of IL-15 alone. The ability of the IL-15 / IL-15Rα complex to stimulate the proliferation and maintain the survival of memory CD8+ T cells is more than 10 times stronger than that of IL-15 alone, and its mechanism may be related to antigen presentation.

[0016] To meet the treatment requirements of cancers, especially complex diseases such as breast cancer, gastric cancer, and pancreatic cancer, it is necessary to search for new and effective anti-tumor drugs. Summary of the Invention

[0017] The first object of the present invention is to provide a new anti-Claudin 18.2 monoclonal antibody.

[0018] The second object of the present invention is to provide a nucleic acid molecule encoding the anti-Claudin 18.2 monoclonal antibody.

[0019] The third object of the present invention is to provide an expression vector containing the nucleic acid molecule.

[0020] The fourth object of the present invention is to provide a host cell containing the expression vector.

[0021] The fifth object of the present invention is to provide a composition containing the anti-Claudin 18.2 monoclonal antibody.

[0022] The sixth object of the present invention is to provide the application of the anti-Claudin 18.2 monoclonal antibody.

[0023] To achieve the above objects, the present invention adopts the following technical solutions:

[0024] According to the first aspect of the present invention, there is provided an anti-Claudin 18.2 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 regions having the same sequences as the heavy chain variable regions shown in SEQ ID NO.15, 16, 17, 18, or 19, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 regions having the same sequences as the light chain variable regions shown in SEQ ID NO.20, 21, 22, or 23.

[0025] In some embodiments, the anti-Claudin 18.2 monoclonal antibody comprises:

[0026] (1) Heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, wherein HCDR1 has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, HCDR2 has the amino acid sequence shown in SEQ ID NO:3, and HCDR3 has the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6;

[0027] (2) Light chain complementarity determining regions LCDR1, LCDR2, LCDR3, wherein LCDR1 has the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, LCDR2 has the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12, and LCDR3 has the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0028] In some embodiments, the anti-Claudin18.2 monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19, or a sequence having at least 85% homology with the above sequences, such as a derivative sequence having 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology; the light chain variable region has the amino acid sequence shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23, or a sequence having at least 85% homology with the above sequences, such as a sequence having 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0029] In some embodiments, the anti-Claudin18.2 monoclonal antibody comprises a light chain and a heavy chain, and the heavy chain has an amino acid sequence as shown in SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34, or a sequence having at least 85% homology with the above sequences, such as sequences having 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology; the light chain has an amino acid sequence as shown in SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33, or a sequence having at least 85% homology with the above sequences, such as sequences having 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0030] In some embodiments, the anti-Claudin18.2 monoclonal antibody can be a murine, human, chimeric or humanized antibody, preferably a humanized antibody.

[0031] In some embodiments, the anti-Claudin18.2 monoclonal antibody is preferably a defucosylated antibody.

[0032] In some embodiments, the anti-Claudin18.2 monoclonal antibody further comprises a Fab fragment, scFv, which comprises the aforementioned heavy chain variable region and light chain variable region, and an antigen-binding fragment, bispecific antibody or multispecific antibody with the Fab or the scFv as the Claudin18.2 binding moiety.

[0033] According to a second aspect of the present invention, there is provided a nucleic acid molecule encoding any of the above anti-Claudin18.2 monoclonal antibodies.

[0034] The preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the art, preferably including the following preparation methods: obtaining a nucleotide molecule encoding the above monoclonal antibody by gene cloning techniques such as PCR methods, etc., or obtaining a nucleotide molecule encoding the above monoclonal antibody by the method of artificial total sequence synthesis.

[0035] Those skilled in the art know that the nucleotide sequence encoding the amino acid sequence of the above monoclonal antibody can be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a homolog of the polynucleotide. The homolog of the polynucleotide in the present invention can be prepared by substituting, deleting or adding one or more bases of the gene encoding the monoclonal antibody within the range of maintaining the antibody activity.

[0036] According to a third aspect of the present invention, there is provided an expression vector, which contains the above-mentioned nucleic acid molecule.

[0037] The expression vector may be a conventional expression vector in the art, which refers to an expression vector containing appropriate regulatory sequences, such as a promoter sequence, a terminator sequence, a polyadenylation sequence, an enhancer sequence, a marker gene and / or sequence, and other appropriate sequences.

[0038] According to a fourth aspect of the present invention, there is provided a host cell, which contains the above-mentioned expression vector. In one embodiment, the host cell is a CHO-S cell.

[0039] According to a fifth aspect of the present invention, there is provided a pharmaceutical composition, which comprises the above-mentioned anti-Claudin18.2 monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.

[0040] In one embodiment, the above composition may further comprise other agents, such as an anti-Her2 monoclonal antibody, IL-15, or an IL-15 / IL-15Rα complex.

[0041] According to a sixth aspect of the present invention, there is provided the use of the above-mentioned anti-Claudin18.2 monoclonal antibody or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating cancer. In one embodiment, the cancer is breast cancer, gastric cancer, and pancreatic cancer.

[0042] Correspondingly, the present invention also provides a method for treating cancer using the above-mentioned anti-Claudin18.2 monoclonal antibody or the above-mentioned pharmaceutical composition containing the antibody. The anti-Claudin18.2 monoclonal antibody can be used in combination with other cancer treatment methods, including but not limited to: administering a targeted therapeutic agent, radiotherapy, surgery, or hormone ablation, etc. In one embodiment, the anti-Claudin18.2 antibody is used in combination with other targeted therapeutic agents, and the preferred targeted therapeutic agents are an anti-Her2 monoclonal antibody, IL-15, and an IL-15 / IL-15Rα complex.

[0043] When multiple cancer treatment methods are used in combination, the administration order of different treatment methods at different time points can be the same or different; when multiple agents are administered, the administration time and order of different agents can be the same or different, or can be administered in any combination, depending on the clinical treatment plan.

[0044] The anti-Claudin 18.2 monoclonal antibody of the present invention can specifically bind to human Claudin 18.2, but not to human Claudin 18.1; the present invention discovers that the anti-Claudin 18.2 monoclonal antibody can be used in combination with an anti-Her-2 monoclonal antibody, and this combination has a more significant killing activity against Claudin18.2-positive and Her-2-positive gastric tumor cells; and it is also discovered that adding IL-15 to the anti-Claudin 18.2 monoclonal antibody can further enhance its ADCC-mediated cytotoxic effect on cancer cells.

[0045] Brief Description of the Drawings

[0046] Figure 1 . Detection of the antigen affinity of the humanized anti-Claudin18.2 antibody of the present inventor;

[0047] Figure 2 . Detection of the ADCC activity of the humanized anti-Claudin18.2 antibody of the present inventor (E:T = 20:1);

[0048] Figure 3. Detection of the ADCC activity (E:T = 40:1) of the humanized anti-Claudin18.2 antibody ( Figure 3A : h20D5, Figure 3B : h20D5-3) of the present invention and anti-Her2 antibody (trastuzumab) on human peripheral blood mononuclear cells (PBMCs), NC represents anti-VEGF antibody (does not bind to Claudin18.2 and Her2).

[0049] Figure 4. Detection of the ADCC activity (E:T = 40:1) of the humanized anti-Claudin18.2 antibodies h20D5 ( Figure 4A ) and h20D5-3mu ( Figure 4B ) of the present invention alone or in combination with IL-15 ( Figure 4B ) on PBMCs, treatment time: 20 hours;

[0050] Figure 5. Detection of the ADCC activity (E:T = 40:1) of the humanized anti-Claudin18.2 antibodies (h20D5, h20D5-3, h20D5-3mu) of the present invention in combination with anti-Her2 antibody (trastuzumab) and IL15;

[0051] Figure 5A Showing the combined application of the parental antibody h20D5-3 and the mutant h20D5-3mu with trastuzumab and IL15;

[0052] Figure 5B Showing the killing effect of the combination of h20D5-3mu, trastuzumab and IL15;

[0053] Figure 6 . Tumor inhibition test results graph for the first four weeks of the gastric cancer model. The negative control refers to PBS;

[0054] Figure 7 . Tumor inhibition test results graph for six weeks of the gastric cancer model. Detailed implementation methods

[0055] Term definitions:

[0056] The "variable region" of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of 4 framework regions (FRs) connected by 3 complementarity-determining regions (CDRs) (also known as hypervariable regions). The CDRs in each chain are held tightly together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least 2 techniques for determining CDRs: (1) a method based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991, National Institutes of Health, Bethesda, MD)); and (2) a method based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. 273:927-948 (1997)). As used herein, CDR can refer to CDRs determined by either method or by a combination of the two methods.

[0057] The term "antibody framework" or "FR region" refers to a part of the variable domain VL or VH that serves as a scaffold for the antigen-binding loops (CDRs) of that variable domain. In essence, it is the variable domain without CDRs.

[0058] The terms "complementary determining region" and "CDR" refer to one of six hypervariable regions within the variable domains of an antibody that are primarily responsible for antigen binding. Typically, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (Al-Lazikani et al. (1997), JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), among others. For example, for the classical format, following the Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the Chothia rules, the CDR amino acid numbering in VH is 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT rules, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT / DomainGapAlign.

[0059] In the present invention, the term "Claudin 18.2" refers to Claudin 18 type 2. This term includes variants, homologs, orthologs, and paralogs.

[0060] In the present invention, the term "IL-15" is a human cytokine with NK cell proliferation and activation activities, referring to human interleukin 15 and the IL-15 / IL-15Rα complex that contains the extracellular domain of human IL-15 or a functional variant of the extracellular domain of IL-15 and retains the ability of IL-15 to enhance the immune response. Functional variants of IL-15 include variants that still retain all or part of the immune response-enhancing effect of IL-15 after truncation, amino acid substitution, deletion, and addition to human IL-15. Exemplary functional variants of IL-15 include, but are not limited to, the human IL-15 variants that retain the immune response-enhancing effect and are disclosed in Patent Publication Nos. WO2008143794A1, WO2012040323A2, US8940288B2, WO2012175222A1, WO2016095642A1, WO2015103928A1, WO2019204592, US20190290734A1, CA3034912A1, US20190209653A1, US20180312560A1, and US20180200366A1, etc.

[0061] In the present invention, the term "IL-15Rα" refers to the α receptor that can interact with IL-15 to form a complex and its functional variants. After IL-15Rα forms a complex with IL-15, it can enhance the stability of IL-15 to further enhance the immune response effect of IL-15. Functional variants of IL-15Rα refer to fragments containing the sushi domain in the extracellular region of IL-15Rα, which retain the interaction with IL-15 and enhance the stability of IL-15. Exemplary functional variants of IL-15Rα include, but are not limited to, the human IL-15Rα functional variants disclosed in Patent Publication Nos. WO2008143794A1, WO2012040323A2, US8940288B2, WO2012175222A1, WO2016095642A1, WO2015103928A1, WO2019204592A1, CA3034912A1, US20190290734A1, US20190209653A1, US20180312560A1, and US20180200366A1, etc.

[0062] For the different antibodies or antibody fragments in Table 1 below, in addition to the above preparation methods, corresponding antibodies can also be prepared by conventional gene cloning and recombinant techniques in the art based on their amino acid sequences.

[0063] Specifically, for example, the above antibodies were expressed in CHO-S cells (Cobioer, China) in the form of full-length monoclonal antibodies for further characterization. Briefly, the respective heavy chains / light chains were cloned into the EcoRI / BamHI restriction enzyme sites of pCDNA3.1 (Invitrogen, Carlsbad, USA) to construct an expression vector.

[0064] According to the manufacturer's instructions, transient expression of chimeric human Claudin18.2 antibodies in CHO-S cells was carried out using PEI transfection. Briefly, polyethyleneimine (PEI) was used to transfect CHO-S cells with the resulting vector at a DNA:PEI ratio of 1:3. The total DNA used for transfection was 1.5 μg / ml. The transfected CHO-S cells were cultured in an incubator at 37 °C and 5% CO2 at a rotation speed of 120 RPM. After 10 - 12 days, the cell culture supernatant was collected, centrifuged at 3500 rpm for 5 minutes, and cell debris was removed by filtration through a 0.22 μm capsule to purify the antibody. Subsequently, the antibody was purified using pre-equilibrated Protein-A (GE; USA; Cat#: 17040501; Lot#: 10252250) and eluted with elution buffer (20 mM citric acid, pH 3.0 - pH 3.5). Except for buffer exchange, the antibody was stored in PBS buffer (pH 7.0), and its concentration was determined using a NanoDrop instrument. The purified monoclonal antibodies were further characterized.

[0065] Antibodies were screened for affinity-matured engineered antibodies by phage display

[0066] To further improve the binding affinity, 20D5 was selected for affinity maturation by phage display technology. Briefly, 3D structural modeling simulations were performed to identify residues in the heavy and light chain CDRs of clone 20D5 that might be important for binding affinity. The identified CDR residues were mutated by PCR using primers specifically designed for point mutations and standard method steps. A phage display library was constructed and, as described above, biological screening was performed using CHO-S cells stably overexpressing human Claudin 18.2 or Claudin 18.1. After 3 rounds of biological screening, high-binding clones were selected, collected, and used to infect bacterial cells. Bacterial colonies were picked and grown in 96-well plates, and then cell ELISA was used to identify high-binding clones and sequence them. Beneficial mutations in the heavy and light chain CDRs were identified and incorporated into a new phage display library, followed by 3 rounds of biological screening and sequencing verification. More than 10 clones containing single or multiple mutations and exhibiting high binding affinity compared to the parental clone 20D5 were identified, and 12 were selected for expression as full-length chimeric human IgG / κ antibodies in CHO-S cells. The binding affinity of the full-length antibodies was detected by FACS using CHO-S cells expressing human Claudin 18.2 or Claudin 18.1.

[0067] The present invention will be further described in detail by the following examples, but should not be construed as being limited to the scope of the described examples. The experimental methods without specific conditions noted in the following examples were all carried out according to conventional methods and conditions, or according to the product specifications.

[0068] Experimental materials and instruments:

[0069] Balb / c mice: female, 8 weeks old, weighing about 20 g, purchased from Shanghai SLAC Laboratory Animal Co., Ltd.;

[0070] Bovine hyaluronidase: Sigma H3506; CHO-S cell line: invitrogen;

[0071] Anti-Her2 antibody: Trastuzumab, prepared by the company by cloning and synthesis according to the amino acid sequence, the heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the light chain has the amino acid sequence shown in SEQ ID NO: 36;

[0072] Human IL-15 (hIL-15): purchased from Peprotech, cat. no. 200-15, sequence shown in SEQ ID NO: 37;

[0073] mIL15Ra-Fc: purchased from biolegend, cat. no. 761606;

[0074] Human IL-15Rα: The sequence of hIL15Rα (human IL15Rα sushi domain) is shown in SEQ ID NO:38;

[0075] Positive control antibody IMAB362 (disclosed in WO2014 / 146672A1): Prepared by the company through cloning and synthesis according to the amino acid sequence. The heavy chain amino acid sequence is shown in SEQ ID NO:39, and the light chain amino acid sequence is shown in SEQ ID NO:40.

[0076] Example 1. Preparation of anti-Claudin18.2 antibody

[0077] 1. Animal immunization

[0078] Immunize 18 Balb / c mice with a plasmid expression vector encoding full-length human Claudin18.2 and perform electroporation. Inject 100 μg of plasmid and 20 U of bovine hyaluronidase intramuscularly on day 1, 75 μg of plasmid and 20 U of bovine hyaluronidase on day 14, and 50 μg of plasmid and 20 U of bovine hyaluronidase on days 28, 42, and 56 respectively. Finally, inject 5×10 6 Claudin18.2-transfected CHO-S cells to enhance immunity. Splenectomy was performed two days later for monoclonal antibody production. The anti-Claudin18.2 antibodies produced in the mouse sera were monitored by flow cytometry (FACS) on days 28, 42, 56, and 65 respectively.

[0079] 2. Preparation and screening of hybridomas

[0080] According to the FACS analysis of the immunized mouse sera, mice numbered NO.2, 8, and 12 were used for the production of CRO hybridomas. Based on the high titer of anti-Claudin18.2 antibody and the low titer of anti-Claudin18.1 antibody, the resulting hybridomas were screened to produce Claudin18.2-specific IgG.

[0081] Method: Dilute the sera of the immunized mice 100-fold and incubate them in 2×10 5 Claudin18.1- or Claudin18.2-transfected CHO-S cells for 30 minutes at 2°C, then wash twice with 2% FBS + PBS, and then incubate with the secondary antibody goat anti-human Fc-FITC antibody for 30 minutes at 4°C. After washing with 2% FBS + PBS, analyze by flow cytometry. Positive Claudin18.2 colonies (20D5) were identified in SS320 competent cells.

[0082] 3. Determination of the amino acid sequence of antibody 20D5

[0083] Antibody 20D5 comprises: heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:1 or SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; light chain complementarity determining regions LCDR1, LCDR2 or LCDR3, wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11 or SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13 or SEQ ID NO:14.

[0084] Example 2. Preparation of humanized anti-Claudin18.2 antibody 20D5 (h20D5)

[0085] The humanized anti-Claudin18.2 antibody 20D5 (h20D5) is generated using the human germline light chain variable region (IGKV4-1*01) and the human germline heavy chain variable region (IGHV4-4*08). Briefly, humanization is accomplished by transplanting the CDR residues from the light and heavy chains of the chimeric antibody 20D5 into the analogous light and heavy chain frameworks of human immunoglobulins.

[0086] A library of humanized antibodies with transplanted CDRs can be generated for further affinity maturation based on in vitro phage display to enhance the affinity for its antigen.

[0087] Finally, multiple humanized antibodies are obtained, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 or SEQ ID NO.19, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23.

[0088] The heavy chain variable region of each antibody is linked to the antibody heavy chain constant region (SEQ ID NO:24) to form the full-length antibody heavy chain, and the light chain variable region of each antibody is linked to the antibody light chain constant region (SEQ ID NO:25) to form the full-length antibody light chain.

[0089] The amino acid sequences of some of the full-length antibodies formed are shown in Table 1 below:

[0090] Table 1

[0091] antibody heavy chain variable region light chain variable region heavy chain light chain h20D5 SEQ ID NO:15 SEQ ID NO:20 SEQ ID NO:26 SEQ ID NO:27 h20D5-1 SEQ ID NO:16 SEQ ID NO:21 SEQ ID NO:28 SEQ ID NO:29 h20D5-2 SEQ ID NO:17 SEQ ID NO:22 SEQ ID NO:30 SEQ ID NO:31

[0092] h20D5-3 SEQ ID NO:18 SEQ ID NO:23 SEQ ID NO:32 SEQ ID NO:33 h20D5-3mu SEQ ID NO:19 SEQ ID NO:23 SEQ ID NO:34 SEQ ID NO:33

[0093] Example 3. Purification of Antibodies

[0094] Extraction of Fc-tagged fusion proteins or antibodies by Protein A affinity chromatography

[0095] First, the cell culture supernatant expressing the Fc fusion protein or antibody was subjected to high-speed centrifugation to collect the supernatant. The Protein A affinity column was washed with 0.1 M NaOH for 3 - 5 column volumes, and then washed with 1×PBS for 3 - 5 column volumes. The chromatography column was equilibrated with a buffer system such as 1×PBS (pH 7.4) for 3 - 5 column volumes. The cell supernatant was loaded onto the column at a low flow rate for binding, and the flow rate was controlled to ensure a retention time of about 1 min or longer. After binding, the chromatography column was washed with 1×PBS (pH 7.4) for 3 - 5 column volumes until the UV absorption returned to the baseline. The sample was eluted with 0.1 M glycine - sodium chloride (pH 3.0 - 3.5) buffer, and the elution peak was collected according to UV detection. The pH of the eluted product was quickly adjusted to 5 - 6 with 1 M Tris - HCl (pH 8.0) for temporary storage. For the eluted product, methods well-known to those skilled in the art can be used for solution replacement, such as ultrafiltration concentration and solution replacement to the required buffer system using an ultrafiltration tube, or desalting and replacement to the required buffer system using size exclusion such as G - 25, or removing the aggregate components in the eluted product using a high-resolution size exclusion column such as Superdex 200 to improve the sample purity.

[0096] Example 4. Specific Binding of Humanized Anti-Claudin18.2 Antibody to Claudin18.2

[0097] Flow cytometry was used to detect the presence of anti-CLD18 antibody in the serum of immunized mice or the binding of monoclonal antibodies to live cells expressing CLD18. The supernatant of clone 20D5 hybridoma cells or purified antibodies (h20D5, h20D5 - 3, concentration 20 μg / ml) were incubated with CHO - S cells transfected with Claudin18.1 or Claudin18.2 at 4°C for 30 minutes. After washing with 2% FBS + PBS buffer, staining was performed using a secondary antibody, FITC-labeled human or mouse Fc antibody. The results are shown in Table 2, where the binding strength is represented as ++++, +++, ++, +, - from strong to weak.

[0098] Table 2

[0099] antibody CHO Claudin18.1-CHO Claudin18.2-CHO h20D5 - - ++++ h20D5-3 - - ++++

[0100] The results showed that h20D5 and h20D5-3 specifically bound to Claudin18.2 on the cell surface but not to Claudin18.1 on the cell surface, indicating that the anti-Claudin18.2 antibody of the present invention had good binding specificity to the antigen.

[0101] Example 5. Detection of the affinity of the anti-Claudin18.2 antibody

[0102] Flow cytometry was used to compare the difference in the binding affinity between the parental antibody h20D5 and the affinity matured antibody with the antigen. The present invention constructed a Fab phage library for affinity maturation, and 3 clones were selected by affinity selection method. The supernatants of Fab clone 20D5 hybridoma cells or purified antibodies (h20D5, h20D5-3 and IMAB362, concentration 20 μg / ml) were incubated. CHO-S cells transfected with Claudin18.1 or 18.2 were incubated at 4 °C for 30 minutes, and after washing with 2% FBS + PBS buffer, they were stained with a secondary antibody, FITC-labeled anti-human or mouse Fc antibody.

[0103] The results were as Figure 1 shown, in which the EC50 values of the humanized antibodies h20D5, h20D5-1, h20D5-2, h20D5-3, h20D5-3mu of the present invention and the positive control antibody IMAB362 were 1.37, 1.77, 0.97, 0.89 and 2.42 μg / ml, respectively. The results indicated that the anti-Claudin18.2 antibodies h20D5, h20D5-1, h20D5-2, h20D5-3, h20D5-3mu had strong affinity for Claudin18.2 expressed on the cell surface, and among them, the affinity matured antibody h20D5-3mu after modification had the highest affinity for the antigen.

[0104] Example 6. Detection of the ADCC activity of the anti-Claudin18.2 antibody

[0105] In this example, the ability of the antibody to induce antibody-dependent cell cytotoxicity (ADCC) against NUGC4 (JCRB0834) gastric cancer cells (Claudin18.2-NUGC4) stably expressing human Claudin18.2 was analyzed and detected.

[0106] The target cells (1.5×10 5Pre-coated on a 96-well plate (at 1.5×10 / well) and incubated overnight in a 37°C incubator with RPMI-1640 + 2% FBS. The next day, fresh PBMCs (effector cell:target cell = 20 or 40:1) and serially diluted antibodies were added to the 96-well plate containing the target cells and incubated at 37°C for 5 hours. 0.5 hour before the assay was stopped, lysis buffer (LDH cytotoxicity detection kit

[0107] DOJINDO MOLECULAR TECHNOLOGIES) was added to the wells containing only target cells and incubated at 37°C for an additional 0.5 hour.

[0108] The plate was centrifuged, 50 μl of the supernatant was transferred to a new plate for measurement. 50 μl of the working solution was added to all wells and incubated at 37°C for approximately 20 minutes, and the absorbance was measured at 490 nm. The cell killing rate was calculated using the following formula: Specific lysis = (experimental release - spontaneous release) / (maximum release - spontaneous release) × 100. The maximum release was determined by adding lysis buffer to the target cells; the spontaneous release was measured in the absence of antibodies and effector cells, with only target cells. Figure 2 ) In the comparative experiment of antibodies h20D5, IMAB362 and trastuzumab, the EC50 values of the three were 0.15, 1.78 and 0.23 nM (

[0109] Example 7. Detection of ADCC activity of the combination of anti-Claudin18.2 antibody and anti-Her2 antibody

[0110] The target cells (1.5×10 5 / well) were pre-coated on a 96-well plate and incubated overnight in a 37°C incubator with RPMI-1640 + 2% FBS. The next day, fresh PBMCs (effector cell:target cell = 40:1) and serially diluted antibodies were added to the 96-well plate containing the target cells and incubated at 37°C for 5 hours.

[0111] In this example, the combined benefits of the combination of antibodies h20D5 and h20D5-3 with trastuzumab were tested. Trastuzumab was serially diluted (200 μg / ml, 5-fold dilution) and added to 6 rows of wells, and antibodies h20D5 and h20D5-3 were fixed at different concentrations in each row of wells.

[0112] The ADCC detection results are as Figure 3A and 3B shown. The results show that the combination of anti-Claudin18.2 antibody and trastuzumab can enhance the antibody-mediated ADCC killing ability of trastuzumab, even reaching the maximum killing rate.

[0113] Example 8. Detection of ADCC activity of the combination of anti-Claudin18.2 antibody and IL15

[0114] In this example, h20D5 was combined with IL15 (Peprotech catalog number 200-15) to test their combined benefits. The h20D5 antibody was serially diluted (200 μg / ml, 5-fold dilution), and the antibody was added to 6-well plates. IL15 was fixed at different concentrations in each row. The results are shown in Table 3 and Figure 4A shown, where Figure 4A in, +IL15 indicates the combination of h20D5 antibody and IL15.

[0115] In addition, the ADCC activities of different concentrations of h20D5-3mu (0.1024 ng / ml or 0.512 ng / ml) alone or in combination with IL15 (5 ng / ml) or with IL15 (5 ng / ml) + trastuzumab (1.6 μg / ml) were measured, and the results are shown in Figure 4B .

[0116] Table 3

[0117] antibody EC50 (nM) h20D5 0.22 h20D5 + 0.4 ng / ml IL15 0.215 h20D5 + 2 ng / ml IL15 0.163 h20D5 + 10 ng / ml IL15 0.086

[0118] The results show that the anti-Claudin18.2 antibody h20D5 or h20D5-3mu in this example combined with commercial IL15 can enhance the ADCC killing ability and show stronger ADCC activity. And when h20D5-3mu, IL15, and trastuzumab are combined, even stronger ADCC activity is shown.

[0119] Example 9. Detection of ADCC activity of the combination of anti-Claudin18.2 antibody, anti-Her2 antibody and IL15

[0120] In this example, it was investigated whether the h20D5-3 mutant has similar combined advantages compared with the parental antibody h20D5-3. The trastuzumab antibody was serially diluted (200 μg / ml, 5-fold dilution), the h20D5-3 or its mutant h20D5-3mu antibody was fixed at 0.016 μg / ml, and IL15 was fixed at 5 ng / ml.

[0121] The results are as Figure 5A and Figure 5BAs shown, it can be seen that the mutant antibody h20D5-3mu of the present invention has comparable combined advantages to the parental antibody h20D5-3. The combination of these two antibodies with anti-Her2 antibody and IL15 can significantly enhance the ADCC killing ability. At the same time, Figure 5B It shows that the combination of h20D5-3mu, trastuzumab and IL15 shows a stronger cell killing rate than the combination of trastuzumab and IL15.

[0122] Example 10. Tumor Inhibition Experiment of Antibody Drugs

[0123] This example evaluates the anti-tumor effects of the drugs trastuzumab, h20D5-3, and hlL15-mIL15Rα in a subcutaneous xenograft tumor model of female Balb / c nude mice with gastric cancer GA0006 model.

[0124] Method: BALB / c nude mice were subcutaneously inoculated with GA0006 model tumor masses to establish a gastric cancer GA0006 tumor model. The experiment was divided into 6 groups, and the drug doses and administration methods are shown in Table 4 below. Except for 6 in the 4th group, each of the other groups had 8 mice. The drugs were administered by intraperitoneal injection, twice a week for 4 weeks, a total of 8 times. After the administration cycle was completed (the results are shown in Figure 6 ), 4 mice were selected from each of the 1st, 3rd, 4th, and 6th groups and given extended administration for 2 weeks, in which hIL15-mIL15Rα was changed to BIW administration (the results are shown in Figure 7 ). The efficacy was evaluated according to the relative tumor inhibition rate (TGI%), and the safety was evaluated according to the changes in animal body weight and death conditions.

[0125] Table 4

[0126]

[0127]

[0128] The results showed that:

[0129] 1) The test drugs trastuzumab, h20D5-3, and hIL15-mIL15Rα, in the efficacy evaluation in the gastric cancer GA0006 model:

[0130] After the end of the first administration cycle, the single-drug groups of h20D5-3, hlL15-mIL15Rα, and trastuzumab did not show tumor inhibition effects compared with the control group. The combined administration group of trastuzumab + h20D5-3 also failed to show tumor inhibition effects compared with the control group. When trastuzumab + h20D5-3 + hlL15-mIL15Rα were combined for three drugs, a slight tumor inhibition effect was shown, but it did not reach statistical significance.

[0131] After the end of the first dosing cycle, we changed the dosing regimen of hlL15-mIL15Rα from once a week to twice a week, while the dosing regimen of the antibody remained unchanged. After the end of the second dosing cycle, there was no significant difference in tumor volume between the h20D5-3 monotherapy group and the hIL15-mIL15Rα monotherapy group (P>0.05). When the two drugs were combined with trastuzumab at a dose of 2 mg / kg, the TGI was between 48.53% at the end of the experiment, and it had a significant tumor inhibitory effect compared with the control group and the single-drug treatment group (P<0.01). Flow cytometry analysis results showed that ILRα-IL15 monotherapy and triple combination therapy could significantly increase the content of NK cells in mouse PBMC, while only triple combination therapy could increase the content of NK cells in the tumor microenvironment.)

[0132] The test drugs trastuzumab, h20D5-3, hlL15-mIL15Rα, in In the gastric cancer GA0006 model, since the model is a cachexia model, individual mice in each group showed weight loss, but there were no animal deaths in each treatment group, no obvious drug toxicity was shown, and they were well tolerated during the treatment.)

[0133] Conclusion: The triple combination therapy of the test drug h20D5-3 (20 mg / kg), hIL15-mIL15Rα (2 μg + 9 μg / mouse) and trastuzumab (2 mg / kg) has a significant effect on inhibiting tumor growth in the gastric cancer GA0006 model (P<0.01). Tumor-bearing mice were well tolerated to h20D5-3, hIL15-mIL15Rα and trastuzumab.)

[0134] Although the present invention has been described in conjunction with multiple embodiments, it should be understood that the present invention is not limited to these embodiments. Other alternative forms, modifications and equivalent substitutions made without departing from the spirit and scope of the appended claims of the present invention all fall within the scope of protection of the present invention.)

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

Claims

1. An anti-Claudin18.2 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein: (1) The heavy chain variable region contains heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3. The HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, the HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:3, and the HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:4; (2) The light chain variable region contains light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. The LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:8, the LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:11, and the LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:

13.

2. The anti-Claudin18.2 monoclonal antibody according to claim 1, wherein the anti-Claudin18.2 monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:19, or a sequence having at least 85% homology with the above sequences; the light chain variable region has the amino acid sequence shown in SEQ ID NO:23, or a sequence having at least 85% homology with the above sequences.

3. The anti-Claudin18.2 monoclonal antibody according to claim 1, wherein the anti-Claudin18.2 monoclonal antibody comprises a light chain and a heavy chain. The heavy chain has the amino acid sequence shown in SEQ ID NO:32 or SEQ ID NO:34, or a sequence having at least 85% homology with the above sequences; the light chain has the amino acid sequence shown in SEQ ID NO:33, or a sequence having at least 85% homology with the above sequences.

4. The anti-Claudin18.2 monoclonal antibody according to claim 1, wherein the antibody is a murine, human, chimeric, or humanized antibody, or the antibody is a Fab fragment or scFv composed of the heavy chain variable region and the light chain variable region according to claim 1 or 2.

5. The anti-Claudin18.@ monoclonal antibody according to claim 4, wherein the antibody is an antigen-binding fragment with the Fab fragment or the scFv as the Claudin18.2 binding portion.

6. The anti-Claudin18.2 monoclonal antibody according to claim 1, wherein the antibody is a defucosylated antibody.

7. A nucleic acid molecule encoding the anti-Claudin18.2 monoclonal antibody according to any one of claims 1-6.

8. An expression vector containing the nucleic acid molecule according to claim 7.

9. A host cell containing the expression vector according to claim 8.

10. The host cell according to claim 9, wherein the host cell is a CHO-S cell.

11. A pharmaceutical composition, which contains the anti-Claudin18.2 monoclonal antibody according to any one of claims 1-6 and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition, which includes the anti-Claudin18.2 monoclonal antibody according to any one of claims 1-6, an anti-Her2 monoclonal antibody, and optionally IL-15 or an IL-15 / IL-15Rα complex, and a pharmaceutically acceptable carrier.

13. Use of the anti-Claudin18.2 monoclonal antibody according to any one of claims 1-6 or the pharmaceutical composition according to claim 11 or 12 in the preparation of a medicament for treating Claudin18.2-positive and / or Her2-positive gastric cancer.

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