WO2021115303A1 - Anti-claudin18.2 monoclonal antibody, preparation method therefor and use thereof - Google Patents

Abstract

Disclosed are an anti-Claudin18.2 monoclonal antibody and a use thereof. Also disclosed are a nucleic acid molecule encoding the antibody, an expression vector and a host cell for expressing the antibody, and a pharmaceutical composition containing the antibody. Further disclosed is the use of the antibody alone or in combination with other agents, such as an anti-Her2 antibody and/or IL15, in the preparation of an anti-cancer drug.

Description

An anti-Claudin 18.2 monoclonal antibody, its preparation method and application Technical field

The present invention relates to the field of biotechnology, in particular to an anti-Claudin 18.2 monoclonal antibody, its preparation method and application.

Background technique

The statements herein only provide background information related to the present invention, and do not necessarily constitute prior art.

Humanized antibodies are derived from non-human species, and their sequence similarity with natural antibodies in humans is increased by modifying the protein sequence. The "humanization" process usually involves the development of monoclonal antibodies for use in humans, such as the development of antibodies as anticancer drugs. The humanization process is necessary for the production of specific antibodies in the immune system of non-humans (e.g., mice). The protein sequence of the antibody produced in this way is partially different from the homologous antibody naturally occurring in humans, so it is potentially immunogenic when administered to a human patient.

Targeted therapy is one of the main ways to treat cancer with drugs. Others include hormone therapy and cytotoxic chemotherapy. As a form of molecular medicine, targeted therapy prevents the growth of cancer cells by interfering with specific targeted molecules required for canceration and tumor growth, rather than simply interfering with all rapidly dividing cells (such as traditional chemotherapy). Because most drugs used for targeted therapy are biological drugs, when used for cancer treatment, the term "biological therapy" is sometimes synonymous with targeted therapy, which is different from chemotherapy (ie, cytotoxic therapy). However, these methods can be used in combination. Antibody-drug conjugates combine biological and cytotoxic mechanisms into a targeted therapy.

Bispecific monoclonal antibody (BsMAb, BsAb), also known as bifunctional antibody, is an artificial protein composed of two different antibody fragments. It can recognize and bind two different antigens and epitopes at the same time, and block the two Different signaling pathways to play its role. BsMabs can be manufactured in a variety of structural forms, and applications for cancer immunotherapy and drug delivery have been explored.

Gastric cancer (GC) is one of the most common cancers and serious health problems in the world today. 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 is still poor. Adjuvant chemotherapy and chemotherapy improved overall survival. Researchers have studied a variety of new chemotherapy regimens that have higher remission rates and tolerability, but the 5-year survival rate is frustrating. In recent years, it has been reported that some therapies targeting biomolecules can prolong the survival period of AGC patients. Since trastuzumab (a monoclonal antibody targeting HER2) was established as the standard treatment for unresectable GC in HER2-positive patients, many other targets have been reported as new therapeutic targets. Regardless of whether there is chemotherapy in clinical trials, many molecular targeted therapies (such as HER2, VEGFR or EGFR) have been confirmed as established standard therapies. In addition, the clinical trial data of immunotherapy is promising and it is expected to become an effective therapy. In particular, immune checkpoint inhibitors, such as PD-1/PD-L1 or CTLA-4, have proven to be innovative in the treatment of GC. In addition, ongoing clinical trials including targeted therapies and immunotherapy have shown encouraging results in improving clinical outcomes, safety and tolerability. However, the results of clinical trials of many targeted drugs are mixed. Qi. The emergence of immune checkpoint inhibitors has produced similar hope, and the results of early trials are encouraging.

Developed by Genentech

Trastuzumab is a humanized monoclonal antibody targeting HER2. In 1998, trastuzumab combined with paclitaxel was approved by the US FDA as a first-line treatment for HER2/neu overexpressing metastatic breast cancer, or as a treatment for HER2/neu overexpressing metastatic breast cancer after at least one chemotherapy cycle Single drug. 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 afatin Nylon (tyrosine kinase inhibitor).

About 22% of patients with metastatic gastric cancer have HER2 overexpression or amplification, but the tumor subtype (intestinal vs. diffuse) and tumor location (gastroesophageal junction (GEJ) vs. stomach) are different. Many studies have reported the correlation between HER2-positive gastric cancer and adverse outcomes and more aggressive diseases. There are still some controversies between these reports and other contradictory studies. Trastuzumab plus chemotherapy has been approved as the standard treatment option for patients with gastric cancer in the Her-2(IHC)-3 positive group, but its therapeutic benefits are indeed limited.

Claudin is a family of proteins first discovered by Shorichiro Tsukita and others. It is an important part of forming tight junctions of cells. It establishes a paracellular barrier and controls the flow of molecules between cells. The transmembrane domain of Claudin includes N-terminal and C-terminal in the cytoplasm. Different Claudin proteins are expressed on different tissues, and their altered functions are related to the formation of cancer in their respective tissues. It has been shown that Claudin-1 is expressed in colon cancer, Claudin-18 is in gastric cancer, and Claudin-10 has prognostic value in hepatocellular carcinoma. UgurSahin et al. determined that the tight junction molecule Claudin-18 isoform 2 (CLDN18.2) is a highly selective cell lineage marker, and its expression in normal tissues is strictly limited to epithelial cells differentiated from the gastric mucosa, but in the gastric stem cell area. does not exist. Claudin 18.2 remains in malignant transformation and is 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 has frequent ectopic activation in a variety of human cancers. The correlation between Claudin protein and isotype 2, especially gastric cancer and its metastatic cancer, has led to the development of specific antibodies against Claudin 18.2 as a targeted therapy for gastric cancer and other human solid malignancies.

Claudiximab is a new type of chimeric IgG1 antibody with high specificity to Claudin 18.2. The clinical phase IIa (MONO) study aims to determine the safety and effectiveness of multiple doses of Claudiximab as a monotherapy in patients with metastatic, refractory, and 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 adverse reactions observed were grade 1-3. The most common grade 3 adverse reaction was vomiting, with 31 cases. No grade 4 adverse reactions occurred.

The 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: ≥40% of tumor cells expressing ≥2+ CLDN18.2 (verified by CLAUDETECT ^TM 18.2 kit), Eastern Cooperative Oncology Group (ECOG) score of 0-1 and not suitable for trastuzumab Treated patients. According to the protocol standard, 739 patients were screened for enrollment, of which 352 (48%) were tested positive for CLDN 18.2. Among them, 161 patients (80% of gastric cancer; GEJ, 16%; esophageal cancer, 4%) were randomly assigned to the first-line EOX (epirubicin 50mg/m ² , oxaliplatin 130mg/m ² d1, With capecitabine ^{625mg/m 2} twice a day, d1-21, a cycle every 21 days), with or without Claudiximab (loading dose 800mg/m ² , then 600mg/m ² d1, a cycle every 21 days) . This study has carried out an extended exploration, in the third group (N=85) study high-dose Claudiximab (1000mg/m ² ) combined with EOX. The study reached the primary endpoint of progression-free survival (PFS). Compared with EOX alone, Claudiximab combined 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).

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

Interleukin 15 (IL-15) is a 12-14kD cytokine discovered by Grabstein et al. in 1994. It can play a role in the body's normal immune response, such as promoting T cells, B cells, and natural killer ( NK) cell proliferation.

IL-15 belongs to a member of the four small alpha-helical bundle cytokine families (small four-helix bundle cytokine family). IL-15 requires receptor binding to exert its biological activity. The IL-15 receptor is composed 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, which can bind to IL-15 and is necessary for the combined IL-15 to perform biological functions.

In recent years, it has been discovered that after IL-15 forms a complex with the receptor IL-15Rα, it can significantly enhance the biological activity of IL-15. Studies have shown that the complex formed by IL-15 and its soluble receptor IL-15Rα in stimulating the proliferation of memory CD8+ T lymphocytes and NT/NKT cells is significantly converted to IL-15 alone. The ability of IL-15/IL-15Rα complex to stimulate memory CD8+ T cell proliferation and maintain its survival is more than 10 times stronger than that of IL-15 alone, and its mechanism may be related to delivery.

In order to meet the treatment requirements of cancers, especially breast cancer, gastric cancer, pancreatic cancer and other complex diseases, it is necessary to find new and effective anti-tumor drugs.

Summary of the invention

The first objective of the present invention is to provide a new anti-Claudin 18.2 monoclonal antibody.

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

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

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

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

The sixth objective of the present invention is to provide the application of the anti-Claudin 18.2 monoclonal antibody.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

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

In some embodiments, the anti-Claudin 18.2 monoclonal antibody includes:

(1) The heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the HCDR1 has the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the HCDR2 has the amino acid sequence shown in SEQ ID NO: 3 Sequence, the HCDR3 has an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

(2) The light chain complementarity determining regions LCDR1, LCDR2, LCDR3, the LCDR1 has an amino acid sequence as 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 the LCDR3 has the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.

In some embodiments, the anti-Claudin 18.2 monoclonal antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region has a variable region such as SEQ ID NO: 15, SEQ ID NO: 16, SEQ The amino acid sequence shown in ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, or a sequence with at least 85% homology with the above sequence, such as 85%, 90%, 92%, 94%, 95 %, 96%, 97%, 98%, or 99% homology; the light chain variable region has a sequence such as SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO : The amino acid sequence shown in 23, or a sequence with at least 85% homology with the above sequence, such as 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology Source sequence.

In some embodiments, the anti-Claudin 18.2 monoclonal antibody includes a light chain and a heavy chain, and the heavy chain has such features as SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34, or a sequence with at least 85% homology to the above sequence, such as 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 has an amino acid sequence with the above sequence A sequence with at least 85% homology, such as a sequence with 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

In some embodiments, the anti-Claudin 18.2 monoclonal antibody may be a murine, human, chimeric or humanized antibody, preferably a humanized antibody.

In some embodiments, the anti-Claudin 18.2 monoclonal antibody is preferably a defucosylated antibody.

In some embodiments, the anti-Claudin 18.2 monoclonal antibody further includes a Fab fragment comprising the aforementioned heavy chain variable region and light chain variable region, scFv, and the Fab or the scFv binding to Claudin 18.2 Part of the antigen-binding fragment, bispecific antibody or multispecific antibody.

According to the second aspect of the present invention, there is provided a nucleic acid molecule encoding any of the aforementioned anti-Claudin 18.2 monoclonal antibodies.

The preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the field, and preferably includes the following preparation method: obtain the nucleotide molecule encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR method, or by The method of artificial full-sequence synthesis obtains the nucleotide molecule encoding the above-mentioned monoclonal antibody.

Those skilled in the art know that the nucleotide sequence encoding the amino acid sequence of the monoclonal antibody can be replaced, deleted, changed, inserted or added as appropriate to provide a polynucleotide homolog. The polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the monoclonal antibody gene encoding the monoclonal antibody within the scope of maintaining the activity of the antibody.

According to the third aspect of the present invention, there is provided an expression vector containing the aforementioned nucleic acid molecule.

The expression vector may be a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate Expression vector of the sequence.

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

According to the fifth aspect of the present invention, there is provided a pharmaceutical composition comprising the aforementioned anti-Claudin 18.2 monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.

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

According to the sixth aspect of the present invention, the use of the above-mentioned anti-Claudin 18.2 monoclonal antibody or the above-mentioned pharmaceutical composition in the preparation of a medicine for the treatment of cancer is provided. In one embodiment, the cancer is breast cancer, gastric cancer, and pancreatic cancer.

Correspondingly, the present invention also provides a method for treating cancer using the above-mentioned anti-Claudin 18.2 monoclonal antibody or the above-mentioned pharmaceutical composition containing the antibody. The anti-Claudin 18.2 monoclonal antibody can be used in combination with other cancer treatment methods, including but not limited to: administration of targeted therapeutic agents, radiotherapy, surgery, or hormone removal. In one embodiment, the anti-Claudin 18.2 antibody is used in combination with other targeted therapeutic agents, and the preferred targeted therapeutic agent is anti-Her2 monoclonal antibody, IL-15, IL-15/IL-15Rα complex .

When multiple cancer treatment methods are used in combination, the order of administration of different treatment methods at different time points may be the same or different; when multiple agents are administered, the time and order of administration of different agents may be the same or different, or in any way Combined administration depends on the clinical treatment plan.

The anti-Claudin 18.2 monoclonal antibody of the present invention can specifically bind to human Claudin 18.2, but does not bind to human Claudin 18.1; the present invention finds that the anti-Claudin 18.2 monoclonal antibody can be used in combination with the anti-Her-2 monoclonal antibody. .2 positive and Her-2 positive gastric tumor cells produced more significant killing activity; and it was also found that the addition of IL-15 to the anti-Claudin 18.2 monoclonal antibody can further enhance its ADCC-mediated cytotoxicity on cancer cells.

Brief description of the drawings

Figure 1. Antigen affinity detection of the humanized anti-Claudin 18.2 antibody of the present invention;

Figure 2. ADCC activity detection of the humanized anti-Claudin 18.2 antibody of the present invention (E:T=20:1);

Figure 3. ADCC of human peripheral blood mononuclear cells (PBMC) of the humanized anti-Claudin 18.2 antibody of the present invention (Figure 3A: h20D5, Figure 3B: h20D5-3) and anti-Her2 antibody (trastuzumab) Activity detection (E:T=40:1), NC means anti-VEGF antibody (does not bind Claudin 18.2 and Her2).

Figure 4. The humanized anti-Claudin 18.2 antibodies of the present invention h20D5 (Figure 4A) and h20D5-3mu (Figure 4B) used alone or in combination with IL-15 (Figure 4B) ADCC activity detection of PBMC (E: T ＝40:1), processing time: 20 hours;

Figure 5. ADCC activity detection of the humanized anti-Claudin 18.2 antibody (h20D5, h20D5-3, h20D5-3mu) of the present invention in combination with anti-Her2 antibody (trastuzumab) and IL15 (E:T=40 :1);

Figure 5A shows the combined application of parent antibody h20D5-3 and mutant h20D5-3mu with trastuzumab and IL15;

Figure 5B shows the killing effect of h20D5-3mu, trastuzumab and IL15 in combination;

Figure 6. The results of the tumor suppression test in the first four weeks of the gastric cancer model, the negative control refers to PBS;

Figure 7. The results of the six-week tumor suppression test in a gastric cancer model.

Detailed ways

Definition of Terms:

The "variable region" of an antibody refers to the variable region (VL) of the antibody light chain or the variable region (VH) of the antibody heavy chain, alone or in combination. As is known in the art, the variable regions of the heavy chain and the light chain each consist of 4 framework regions (FR) connected by 3 complementarity determining regions (CDR) (also called hypervariable regions). The CDRs in each chain are held together tightly by 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 two techniques for determining CDRs: (1) Methods based on cross-species sequence variability (ie, Kabat et al. Sequences of Proteins of Immunological Interest (5th edition, 1991, National Institutes of Health, Bethesda MD)); And (2) A method based on the crystallographic study of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. 273:927-948 (1997)). As used herein, CDR may refer to a CDR determined by either method or a combination of the two methods.

The term "antibody framework" or "FR region" refers to a part of a variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.

The terms "complementarity determining region" and "CDR" refer to one of the six hypervariable regions in the variable domain of an antibody that mainly contribute to antigen binding. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering rules (Al-Lazikani et al. (1997), JMB 273: 927-948) and ImmunoGenTics (IMGT) numbering rules (Lefranc MP, Immunologist, 7, 132-136 (1999) ; Lefranc, MP, etc., Dev. Comp. Immunol., 27, 55-77 (2003)) and so on. For example, for the classical format, following the Kabat rule, 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 chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the Chothia rule, the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue numbers in VL are 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, CDR is defined by amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-35 in human VL. 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) constitute. Following the IMGT rules, the CDR amino acid residue numbers in VH are roughly 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are roughly 27-32 (CDR1) ), 50-52 (CDR2) and 89-97 (CDR3). Following IMGT rules, the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.

In the present invention, the term "Claudin 18.2" refers to Claudin 18. The term includes variants, homologs, orthologs and paralogs.

In the present invention, the term "IL-15" is a human cytokine with NK cell proliferation and activating activity, and refers to human interleukin 15 and the functionality of human IL-15 extracellular domain or IL-15 extracellular domain. Variants and IL-15/IL-15Rα complexes that retain IL-15 and enhance the immune response. IL-15 functional variants include human IL-15 truncation, amino acid substitutions, deletions and additions and still retain all or part of IL-15 to enhance the immune response variants, exemplary IL-15 functional variants include But it is not limited to patent publication numbers WO2008143794A1, WO2012040323A2, US8940288B2, WO2012175222A1, WO2016095642A1, WO2015103928A1, WO2019204592, US201902907 34A1, CA3034912A1, US20190209653A1, US20180312560A1, and US20180200366A1 that retain variants of human IL-15 that have enhanced immune responses.

In the present invention, the term "IL-15Rα" refers to the α receptor and its functional variants that can interact with IL-15 to form a complex. After IL-15Rα and IL-15 form a complex, it can enhance the stability of IL-15. To further enhance the immune response effect of IL-15. IL-15Rα functional variant refers to a fragment containing the sushi domain in the extracellular region of IL-15Rα, which retains the interaction with IL-15 and enhances the stability of IL-15. Exemplary IL-15Rα functional variants include, but are not limited to, patent publication numbers WO2008143794A1, WO2012040323A2, US8940288B2, WO2012175222A1, WO2016095642A1, WO2015103928A1, WO2019204592A1, CA3034912A1, US20190290734A1, US20190209653A1, US20180312560A1, and RUS20180200366A1, and other disclosed functional variants of human IL-15 .

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

Specifically, such as expressing the above-mentioned antibodies in the form of full-length monoclonal antibodies in CHO-S cells (Cobioer, China) for further characterization. In brief, the respective heavy chain/light chain was cloned into the EcoRI/BamHI restriction endonuclease sites of pCDNA3.1 (Invitrogen, Carlsbad, USA) to construct an expression vector.

According to the manufacturer's instructions, using PEI transfection, the chimeric human Claudin 18.2 antibody was transiently expressed in CHO-S cells. In short, polyethyleneimine (PEI) was used to transfect CHO-S cells with the resulting vector, and the ratio of DNA:PEI was 1:3. The total DNA used for transfection was 1.5 μg/ml. The transfected CHO-S cells were cultured in a 37°C, 5% CO ₂ incubator at 120 RPM. After 10-12 days, the cell culture supernatant was collected, centrifuged at 3500 rpm for 5 minutes, and filtered through a 0.22 μm capsule to remove cell debris to purify the antibody. After that, the antibody was purified using pre-equilibrated Protein-A (GE; USA; Cat#: 17040501; Lot#: 10252250) and eluted with an elution buffer (20mM citric acid, pH3.0-pH3.5). In addition to the buffer exchange, the antibody is stored in PBS buffer (pH 7.0), and its concentration is determined by a NanoDrop instrument. The purified monoclonal antibody was further characterized.

Antibodies are screened for affinity matured modified antibodies by phage display

In order to further improve the binding affinity, clone 20D5 chose to undergo affinity maturation modification through phage display technology. Simply put, 3D structural modeling simulations were performed to identify residues in the heavy and light chain CDRs of clone 20D5 that may be important for binding affinity. The identified CDR residues are mutated by PCR, using primers specially designed for point mutations and standard method steps. A phage display library was constructed, and as described above, CHO-S cells stably overexpressing human Claudin 18.2 or Claudin 18.1 were used for biological screening. After 3 rounds of biological screening, high-binding clones were selected, collected and infected with bacterial cells. Pick bacterial colonies and grow them on 96-well plates, then use cell ELISA to identify high-binding clones and sequence them. Identify the beneficial mutations in the heavy and light chain CDRs, and combine them into a new phage display library, and then perform 3 rounds of biological screening and sequencing verification. More than 10 clones containing single or multiple mutations and high binding capacity compared with the parent clone 20D5 were identified, and 12 clones were selected to express in CHO-S cells as full-length chimeric human IgG/κ antibodies. The binding affinity of the full-length antibody was tested by FACS using CHO-S cells expressing human Claudin 18.2 or Claudin 18.1.

Hereinafter, the present invention will be further described in detail through examples, but it should not be understood that the present invention is limited to the scope of the described examples. The experimental methods for which specific conditions are not indicated in the following examples are all selected in accordance with conventional methods and conditions, or in accordance with the product specification.

Experimental materials and instruments:

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

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

Anti-Her2 antibody: Trastuzumab, prepared by our company through 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 Amino acid sequence

Human IL-15 (hIL-15): purchased from Peprotech article number 200-15, the sequence is shown in SEQ ID NO: 37;

mIL15Ra-Fc: purchased from biolegend, catalog number 761606;

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

Positive control antibody IMAB362 (disclosed from WO2014/146672A1): It was prepared by our 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.

Example 1. Preparation of Anti-Claudin 18.2 Antibody

1. Animal immunity

Eighteen Balb/c mice were immunized with the plasmid expression vector encoding the fully human Claudin 18.2 and electroporated. 100μg plasmid and 20U bovine hyaluronidase were injected intramuscularly on day 1, 75μg plasmid and 20U bovine hyaluronidase were injected on day 14, and 50μg plasmid and 20U bovine were injected on day 28, 42 and 56 respectively. Hyaluronidase, and finally 5×10 ⁶ Claudin18.2 transfected CHO-S cells were injected on the 65th day to strengthen immunity, and splenectomy was performed two days later to produce monoclonal antibodies. The anti-Claudin 18.2 antibody produced in the mouse serum was monitored by flow cytometry (FACS) on 28, 42, 56 and 65 days, respectively.

2. Preparation and screening of hybridomas

According to the FACS analysis of the sera of immunized mice, mice numbered No. 2, 8, and 12 were used for the production of CRO hybridomas. Based on the high titer of the anti-Claudin 18.2 antibody and the low titer of the anti-Claudin 18.1 antibody, the resulting hybridomas were screened to produce Claudin 18.2 specific IgG.

Method: Dilute the serum of immunized mice 100 times, and incubate them in 2×10 ⁵ Claudin18.1 or Claudin18.2 transfected CHO-S cells for 30 minutes at 2°C, and then wash with 2% FBS+PBS 2 Then, it was incubated with the secondary antibody goat anti-human Fc-FITC antibody at 4°C for 30 minutes, and then washed with 2% FBS+PBS and analyzed by flow cytometry. The positive Claudin 18.2 colony (20D5) was identified in SS320 competent cells.

3. Determination of the amino acid sequence of antibody 20D5

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

Example 2. Preparation of humanized anti-Claudin 18.2 antibody 20D5 (h20D5)

The humanized anti-Claudin 18.2 antibody 20D5 (h20D5) was produced using the human germ cell line variable light chain (IGKV4-1*01) and the human germ cell line heavy chain variable region (IGHV4-4*08). In short, humanization is accomplished by grafting the CDR residues from the light and heavy chains of the chimeric antibody 20D5 to the similar light and heavy chain frameworks of human immunoglobulin.

Humanized antibody libraries grafted with CDRs can be generated for further affinity maturation based on in vitro phage display to enhance the affinity for their antigens.

Finally, multiple humanized antibodies were obtained, in which the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 or SEQ ID NO.19, The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

The heavy chain variable region of each antibody and the antibody heavy chain constant region (SEQ ID NO: 24) are connected to form an antibody full-length heavy chain, and the light chain variable region of each antibody and the antibody light chain constant region (SEQ ID NO: 25) Linked to form the full-length light chain of the antibody.

The amino acid sequence of part of the formed full-length antibody is shown in Table 1 below:

Table 1

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

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

Example 3. Purification of antibodies

Protein A affinity chromatography to extract fusion protein or antibody with Fc tag

First, the supernatant of the cell culture expressing the Fc fusion protein or antibody is centrifuged at a high speed to collect the supernatant. The ProteinA affinity column was washed 3-5 times the column volume with 0.1M NaOH, and then washed 3-5 times the column volume with 1×PBS. Use a buffer system such as 1×PBS (pH7.4) as an equilibration buffer to equilibrate the chromatography column by 3-5 times the column volume. The cell supernatant is loaded and bound at a low flow rate, and the flow rate is controlled so that the retention time is about 1 min or longer. After the binding is completed, the column is washed with 1×PBS (pH 7.4) by 3-5 times the column volume until the UV absorption falls back to the baseline . The sample was eluted with 0.1M glycine sodium chloride (pH3.0-3.5) buffer, and the elution peaks were collected according to UV detection. The eluted product was quickly adjusted to pH 5-6 with 1M Tris-HCl (pH8.0). Save. The eluted product can be replaced by a method well known to those skilled in the art, such as using an ultrafiltration tube for ultrafiltration and concentration and replacing the solution to the required buffer system, or using molecular exclusion such as G-25 desalting to replace it with the required Buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove the aggregate components in the eluted product to improve the purity of the sample.

Example 4. Specific binding of humanized anti-Claudin 18.2 antibody to Claudin 18.2

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

Table 2

Antibody	CHO	Claudin18.1-CHO	Claudin18.2-CHO
h20D5	-	-	++++
h20D5-3	-	-	++++

The results show that h20D5 and h20D5-3 specifically bind Claudin 18.2 on the cell surface but not Claudin 18.1 on the cell surface, indicating that the anti-Claudin 18.2 antibody of the present invention has good binding specificity to the antigen.

Example 5. Affinity detection of anti-Claudin 18.2 antibody

Use flow cytometry to compare the binding affinity difference between parent antibody h20D5 and affinity mature antibody and antigen. The present invention constructs a Fab phage library for affinity maturation, selects 3 clones by affinity selection, and incubates the supernatant of Fab clone 20D5 hybridoma cells or purified antibodies (h20D5, h20D5-3 and IMAB362, concentration 20μg/ ml). The CHO-S cells transfected with Claudin 18.1 or 18.2 were incubated at 4°C for 30 minutes, washed with 2% FBS+PBS buffer, and stained with a secondary antibody FITC-labeled anti-human or mouse Fc antibody.

The results are shown in Figure 1, wherein the EC50 values of the humanized antibodies h20D5, h20D5-1, h20D5-2, h20D5-3, h20D5-3mu, and the positive control antibody IMAB362 of the present invention are 1.37, 1.77, 0.97, 0.89, and 2.42μg/ml. The results showed that the anti-Claudin 18.2 antibodies h20D5, h20D5-1, h20D5-2, h20D5-3, h20D5-3mu and Claudin 18.2 expressed on the cell surface have strong affinity, and the modified affinity maturation antibody h20D5-3mu and The antigen has the highest affinity.

Example 6. ADCC activity detection of anti-Claudin 18.2 antibody

This example analyzes and detects the ability of antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) of NUGC4 (JCRB0834) gastric cancer cells (Claudin 18.2-NUGC4) stably expressing human Claudin 18.2.

The target cells (1.5×10 ⁵ /well) were pre-plated on a 96-well plate in RPMI-1640+2% FBS in a 37°C incubator overnight. On the next day, fresh PBMC (effector cells: target cells=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 hours before the stop of the measurement, the lysis buffer (LDH cytotoxicity detection kit

DOJINDO MOLECULAR TECHNOLOGIES) was added to the wells containing only target cells and incubated at 37°C for another 0.5 hour.

Centrifuge the plate, transfer 50 μl of supernatant to a new plate for measurement, add 50 μl of working solution to all wells, and incubate at 37° C. for about 20 minutes, and measure the absorbance 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 amount is determined by adding lysis buffer to the target cells; the spontaneous release amount is measured in the absence of antibodies and effector cells and only target cells.

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, respectively (Figure 2). In the comparative experiment of antibodies IMAB362, h20D5-3 and h20D5-3mu, the EC50 values of the three were 1.06, 0.047 and 0.025nM, respectively. The results showed that, compared with the control antibody, the anti-Claudin18.2 antibodies h20D5, h20D5-3 and h20D5-3mu of the present invention all showed stronger ADCC activity against Claudin 18.2-NMGC4 target cells.

Example 7. ADCC activity detection of anti-Claudin 18.2 antibody in combination with anti-Her2 antibody

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

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

The ADCC test results are shown in Figures 3A and 3B. The results show that the combination of anti-Claudin 18.2 antibody and trastuzumab can enhance the ADCC lethality mediated by trastuzumab antibody, and even reach the maximum lethality rate.

Example 8. ADCC activity detection of anti-Claudin 18.2 antibody in combination with IL15

In this example, h20D5 was combined with IL15 (Peprotech Catalog No. 200-15) to test its combined benefits. The h20D5 antibody was serially diluted (200μg/ml, 5-fold dilution). The antibody was added to 6 rows of wells. IL15 was Fixed in different concentrations in each row. The results are shown in Table 3 and Figure 4A. In Figure 4A, +IL15 represents the combination of h20D5 antibody and IL15.

In addition, use different concentrations of h20D5-3mu (0.1024ng/ml or 0.512ng/ml) alone or separately with IL15 (5ng/ml) or with IL15 (5ng/ml) + trastuzumab (1.6μg/ml) Combined use to determine the ADCC activity of the drug, the result is shown in Figure 4B.

table 3

Antibody	EC50(nM)
h20D5	0.22
h20D5+0.4ng/ml IL15	0.215
h20D5+2ng/ml IL15	0.163
h20D5+10ng/ml IL15	0.086

The results show that the combination of the anti-Claudin18.2 antibody h20D5 or h20D5-3mu and commercial IL15 in this example can enhance ADCC lethality and show stronger ADCC activity. When h20D5-3mu is combined with IL15 and trastuzumab, it shows stronger ADCC activity.

Example 9. ADCC activity detection of anti-Claudin 18.2 antibody in combination with anti-Her2 antibody and IL15

This example examines whether the h20D5-3 mutant has similar combinatorial advantages compared with the parent antibody h20D5-3. Trastuzumab antibody is serially diluted (200μg/ml, 5-fold dilution), h20D5-3 or its mutant h20D5-3mu antibody is fixed at 0.016μg/ml, and IL15 is fixed at 5ng/ml.

The results are shown in Figure 5A and Figure 5B. It can be seen that the mutant antibody h20D5-3mu of the present invention has a combination advantage comparable to the parent antibody h20D5-3, and the combination of these two antibodies with the anti-Her2 antibody and IL15 can significantly enhance the ADCC lethality. At the same time, Figure 5B shows that the combination of h20D5-3mu, trastuzumab and IL15 shows a stronger cell killing rate than the combination of trastuzumab and IL15.

Example 10. Anti-tumor experiment of antibody drugs

This example evaluates the drug trastuzumab, h20D5-3, hIL15-mIL15Rα in

The anti-tumor effect of gastric cancer GA0006 model Balb/c nude female mouse subcutaneous xenograft tumor model.

Method: BALB/c nude mice were subcutaneously inoculated with GA0006 model tumor masses to establish

Gastric cancer GA0006 tumor model. The experiment is divided into 6 groups, and the drug dosage and administration method are shown in Table 4 below. Except for the 6 rats in the fourth group, 8 rats in each group were administered by intraperitoneal injection, twice a week, for a total of 8 times for 4 weeks. After the dosing cycle is completed (see Figure 6 for the results), select 4 mice in each of group 1, group 3, group 4, and group 6, and extend the administration for 2 weeks, where hIL15-mIL15Rα is changed to BIW administration. Medicine (see Figure 7 for the results). Efficacy was evaluated based on the relative tumor inhibition rate (TGI%), and the safety was evaluated based on changes in animal weight and death.

Table 4

The results show that:

1) Test drug trastuzumab, h20D5-3, hIL15-mIL15Rα, in

Efficacy evaluation in gastric cancer GA0006 model:

After the end of the first dosing cycle, the h20D5-3, hlL15-mIL15Rα, and trastuzumab single-agent groups did not show tumor-inhibiting effects compared with the control group. Compared with the control group, the trastuzumab+h20D5-3 combination group also failed to show tumor suppressive effect. The combination of trastuzumab+h20D5-3+hlL15-mIL15Rα showed a slight anti-tumor effect, but did not reach statistical difference.

After the end of the first dosing cycle, we changed the dosing method of hlL15-mIL15Rα from once a week to twice a week, and the antibody dosing method remained unchanged. After the end of the second dosing cycle, there was no statistical difference in tumor volume between the h20D5-3 single-drug group and the hIL15-mIL15Rα single-drug group (P>0.05). The two drugs and the 2mg/kg dose of trastuzumab When the three drugs are used in combination, the TGI is between 48.53% at the end of the experiment. Compared with the control group and the single drug group, it has a significant tumor-inhibiting effect (P<0.01). The results of flow cytometry showed that ILRα-IL15 single-drug and three-drug combination can significantly increase the content of NK cells in mouse PBMC, and only three-drug combination can increase the content of NK cells in the tumor microenvironment. )

Test drug trastuzumab, h20D5-3, hlL15-mIL15Rα, in

In the gastric cancer GA0006 model, because the model is a cachexia model, individual mice in each group have lost weight, but no animal died in each treatment group, and there was no obvious drug toxicity, and it was well tolerated during the treatment.

Conclusion: The test drug h20D5-3 (20mg/kg), hIL15-mIL15Rα (2μg+9μg/mouse) and trastuzumab (2mg/kg) combined treatment

Gastric cancer GA0006 model has a significant inhibitory effect on tumor growth (P<0.01). Tumor-bearing mice tolerated well to h20D5-3, hIL15-mIL15Rα and trastuzumab.

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. As long as they do not deviate from the spirit and scope of the appended claims of the present invention, other selection forms, modifications and equivalent substitutions shall fall within the protection scope of the present invention.

Claims (16)

1. An anti-Claudin 18.2 monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the heavy chain shown in SEQ ID NO: 15, 16, 17, 18 or 19. The variable regions have HCDR1, HCDR2, and HCDR3 regions with the same sequence, and the light chain variable region includes LCDR1, LCDR2, and LCDR2, which have the same sequence as the light chain variable region shown in SEQ ID NO: 20, 21, 22, or 23. LCDR3 area.
2. The anti-Claudin 18.2 monoclonal antibody of claim 1, wherein the anti-Claudin 18.2 monoclonal antibody comprises:

   (1) The heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the HCDR1 has the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the HCDR2 has the amino acid sequence shown in SEQ ID NO: 3 Sequence, the HCDR3 has an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

   (2) The light chain complementarity determining regions LCDR1, LCDR2, LCDR3, the LCDR1 has an amino acid sequence as 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 the LCDR3 has the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.
3. The anti-Claudin 18.2 monoclonal antibody of claim 2, wherein the anti-Claudin 18.2 monoclonal antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region has SEQ ID NO: 15. The amino acid sequence shown in 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 sequence; the light chain may The variable region has an amino acid sequence as 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 sequence.
4. The anti-Claudin 18.2 monoclonal antibody of claim 2, wherein the anti-Claudin 18.2 monoclonal antibody includes a light chain and a heavy chain, and the heavy chain has the following SEQ ID NO: 26, SEQ ID NO: 28, SEQ The amino acid sequence shown in ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34, or a sequence with at least 85% homology with the above sequence; the light chain has the sequence shown in SEQ ID NO: 27, SEQ ID The amino acid sequence shown in NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33, or a sequence with at least 85% homology with the above sequence.
5. The anti-Claudin 18.2 monoclonal antibody of claim 1, wherein the antibody is a murine, human, chimeric or humanized antibody or the antibody is made of any one of claims 1-3. A Fab fragment or scFv composed of a chain variable region and a light chain variable region, or an antigen-binding fragment, a bispecific antibody or a multispecific antibody in which the Fab or the scFv is the binding part of Claudin 18.2.
6. The anti-Claudin 18.2 monoclonal antibody of claim 1, which is a defucosylated antibody.
7. A nucleic acid molecule encoding the anti-Claudin 18.2 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, which is a CHO-S cell.
11. A pharmaceutical composition comprising the anti-Claudin 18.2 monoclonal antibody according to any one of claims 1-6 and a pharmaceutically acceptable carrier.
12. A pharmaceutical composition comprising one or more of anti-Claudin 18.2 monoclonal antibody, anti-Her2 monoclonal antibody, IL-15, IL-15/IL-15Rα complex, and pharmaceutically Acceptable carrier.
13. The use of the anti-Claudin 18.2 monoclonal antibody according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 11 or 12 in the preparation of a medicament for the treatment of cancer.
14. The use according to claim 13, wherein the cancer is Claudin 18.2 positive and/or Her2 positive tumor cell-related cancer.
15. The use according to claim 13, wherein the anti-Claudin 18.2 monoclonal antibody is combined with an anti-Her2 monoclonal antibody, IL-15 or IL-15/IL-15Rα complex.
16. The use according to claim 13, wherein the cancer is breast cancer, gastric cancer or pancreatic cancer.

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