Claudin18.2-targeting chimeric antigen receptor (CAR) and application thereof

Abstract

The invention relates to the technical field of biology, in particular to CLAUDIN18.2 CART as well as a preparation method and application of the CLAUDIN18.2 CART. The invention firstly provides a single-chain antibody aiming at CLAUDIN18.2, the single-chain antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises a heavy chain variable region complementarity determining region 1 (HCDR1), a heavy chain variable region complementarity determining region 2 (HCDR2) and a heavy chain variable region complementarity determining region 3 (HCDR3); the light chain comprises a light chain variable region, and the light chain comprises a light chain variable region complementarity determining region 1 (LCDR1), a light chain variable region complementarity determining region 2 (LCDR2) and a light chain variable region complementarity determining region 3 (LCDR3). The single-chain antibody can be used for preparing a CLAUDIN18.2-targeted chimeric antigen receptor, a chimeric antigen receptor T cell and a tumor treatment drug, and has an important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a chimeric antigen receptor (CAR) targeting Claudin18.2 and applications thereof. Background Art

[0002] With the continuous deepening of gastric cancer research, targeted therapy is gradually becoming an important technology to improve the overall survival (OS) and progression-free survival of gastric cancer patients. Targeted therapy can target tumor-specific marker molecules, thereby reducing the side effects of drugs. Among them, antibody-mediated immune-guided therapeutic drugs have attracted widespread attention from major pharmaceutical companies because of their easy humanization and low toxicity and side effects. For example, human epidermal growth factor receptor 2 (human EGFR-2, HER-2) drugs targeting epidermal growth factor receptor (EGFR) family proteins, such as trastuzumab and cetuximab, can inhibit the development of about 10% to 30% of gastric cancer patients (HER-2 positive). Drugs targeting vascular endothelial growth factor and vascular endothelial growth factor receptor, such as bevacizumab and apatinib mesylate tablets, are effective for 46.6% of gastric cancer patients, which has brought good news to gastric cancer patients around the world. However, gastric cancer has extremely strong heterogeneity, and the above-mentioned targeted therapeutic drugs are only suitable for some patients. It is of great significance to find new gastric cancer treatment targets with a wider range of applications and develop new therapeutic drugs.

[0003] The claudin family is a class of integrin membrane proteins present in the tight junctions between epithelial and endothelial cells. It is the most critical protein for the formation of tight junctions. There are 27 members of the claudin family in mammals. Claudin-18 splice variant 2 (Claudin-18.2) is a tight junction protein specifically expressed in the stomach and is expressed in many cancers. Claudin-18.2 is abnormally activated and overexpressed in a variety of primary malignant tumors, such as breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, and is particularly prone to malignant tumors of the digestive system. In tumors, Claudin-18.2 is involved in the proliferation, differentiation, and migration of tumor cells. In normal tissues, cells are tightly adhered to each other, and it is extremely difficult for antibody drugs to bind to them, which greatly reduces the impact of Claudin-18.2 drugs on normal tissues. In malignant tumors, tight junctions are destroyed, exposing the Claudin-18.2 epitope on the surface of tumor cells, which can bind to Claudin-18.2 macromolecular drugs. This characteristic and high selectivity of Claudin-18.2 make it an ideal tumor immune target.

[0004] With the development of chimeric antigen receptor T cell (CAR-T) cell therapy technology, Claudin-18.2 target has been gradually applied to CAR-T cell therapy clinical trials. CARsgen Therapeutics' preclinical data showed that CAR-T cells targeting Claudin-18.2 effectively inhibited the growth of Claudin-18.2-positive patient-derived xenograft (PDX) model tumors without toxicity. Currently, CAR-T cell therapy targeting Claudin-18.2 has entered Phase I clinical trials, such as clinical trials on gastric cancer and pancreatic cancer patients in China (NCT04581473) and the United States (NCT04404595).

[0005] However, CAR-T still faces many problems in tumor treatment, such as CAR-T is insensitive to tumor cells and has low killing activity. Since the targeting antibody part has an important influence on the activity of immunotherapy, the skilled person in the art provides a new Claudin-18.2 targeting antibody and its preparation Claudin-18.2 CAR-T to enhance the killing ability of CAR-T. Summary of the invention

[0006] In view of the prior art, the purpose of the present invention is to provide a new CAR-T targeting Claudin-18.2, which has a higher gastric cancer cell killing ability and can significantly inhibit the growth of gastric cancer cells, as well as the preparation and application of the Claudin-18.2 CAR-T.

[0007] In order to achieve the above-mentioned purpose and other related purposes, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a single-chain antibody against Claudin-18.2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain\light chain variable region respectively comprises three complementary determining regions (CDRs), namely HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0009] In some embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0010] In some embodiments, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0011] In some embodiments, the amino acid sequence of the VH is shown in SEQ ID NO:7, and the amino acid sequence of the VL is shown in SEQ ID NO:8.

[0012] In some embodiments, the amino acid sequence of the Claudin-18.2 single-chain antibody obtained by connecting the VH and VL via a connecting peptide is shown in SEQ ID NO:9.

[0013] In another aspect, the present invention provides use of the aforementioned single-chain antibody against Claudin-18.2 in preparing a chimeric antigen receptor targeting Claudin-18.2.

[0014] On the other hand, the present invention provides a chimeric antigen receptor targeting Claudin-18.2 (Claudin-18.2CAR), which includes a signal peptide, the aforementioned single-chain antibody against Claudin-18.2, a hinge region, a transmembrane domain, a co-stimulatory region and a cytoplasmic signaling domain connected in sequence.

[0015] In some embodiments, the transmembrane domain is a CD8 transmembrane domain.

[0016] In some embodiments, the hinge region is a CD8 hinge region.

[0017] In some embodiments, the intracellular signaling domain is the intracellular signaling domain of CD3ζ.

[0018] In some embodiments, the costimulatory signaling domain comprises the costimulatory signaling domain of the following molecules: CD27, CD28, 4-1BB, CD8a, CD40, ICOS, B7-H3 or a variant thereof.

[0019] In some embodiments, the costimulatory signaling domain comprises a 4-1BB costimulatory signaling domain, a CD27 costimulatory signaling domain, or a combination of 4-1BB and CD27 costimulatory signaling domains.

[0020] In some embodiments, the amino acid sequence of the co-stimulatory signaling domain is as shown in SEQ ID NO:10.

[0021] In some embodiments, the signal peptide is a CD8 signal peptide.

[0022] In some embodiments, the chimeric antigen receptor comprises a CD8 signal peptide, the aforementioned single-chain antibody against Claudin-18.2, a CD8 hinge region, a CD8 transmembrane domain, a CD27 co-stimulatory region, a 4-1BB co-stimulatory region, and a CD3ζ cytoplasmic signaling domain, which are sequentially connected.

[0023] In some embodiments, the amino acid sequence of the chimeric antigen receptor targeting Claudin-18.2 (Claudin-18.2CAR) is as shown in SEQ ID NO:11.

[0024] In another aspect, the present invention provides a polynucleotide encoding the chimeric antigen receptor targeting Claudin-18.2.

[0025] In another aspect, the present invention provides an expression vector comprising the polynucleotide.

[0026] In some embodiments, the vector comprises a DNA vector, an RNA vector, a plasmid, a transposon vector, or a viral vector. In some embodiments, the viral vector comprises a lentiviral vector, an adenoviral vector, a herpes virus vector, or a retroviral vector. In some embodiments, the viral vector is a lentiviral vector.

[0027] In another aspect, the present invention provides an engineered cell, wherein the cell comprises or expresses the chimeric antigen receptor of the present invention, the nucleic acid molecule of the present invention or the vector of the present invention.

[0028] In some embodiments, the cell comprises an immune cell. In some embodiments, the immune cell comprises a T cell, a B cell, a NK cell, a NKT cell, a monocyte, a macrophage, a dendritic cell, a myeloid cell, or any combination thereof.

[0029] In some embodiments, the immune cells are T cells, more preferably primary T cells or Jurkat cells.

[0030] In some embodiments, the engineered cells are chimeric antigen receptor T cells targeting Claudin-18.2 (Claudin-18.2CAR-T).

[0031] In another aspect, the present invention provides a method for preparing the cell of the present invention, the method comprising introducing the nucleic acid molecule of the present invention or the vector of the present invention into a cell.

[0032] On the other hand, the present invention provides a pharmaceutical composition comprising a single-chain antibody targeting Claudin-18.2 as described above, or a nucleic acid molecule as described above, or a chimeric antigen receptor targeting Claudin-18.2 as described above, or a vector as described above, or an engineered cell as described above (such as a chimeric antigen receptor T cell targeting Claudin-18.2 (Claudin-18.2CAR-T)).

[0033] On the other hand, the present invention provides the use of the single-chain antibody targeting Claudin-18.2 as described above, or the nucleic acid molecule as described above, or the chimeric antigen receptor targeting Claudin-18.2 as described above, or the vector as described above, or the engineered cells as described above (such as chimeric antigen receptor T cells targeting Claudin-18.2 (Claudin-18.2CAR-T)) in the preparation of drugs for treating cancer.

[0034] In some embodiments, the cancer is selected from breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, esophageal cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, or a combination thereof.

[0035] In some embodiments, the cancer is gastric cancer or ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A-1B As shown, Claudin-18.2CAR-T cells induce interferon gamma ( Figure 1A ) and IL-2( Figure 1B ) released activity.

[0037] Figure 2 As shown, Claudin-18.2CAR-T cells significantly inhibited the growth of NUGC-4 gastric cancer cells in NOG mice. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0039] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0040] As used herein, "treatment" or "treating" is a method of obtaining beneficial or desired results, including clinical results. For the purposes of this application, the beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, alleviating the disease (partially or completely), reducing the dose of one or more other drugs required to treat the disease, delaying disease progression, improving or increasing quality of life, increasing body weight, and / or prolonging survival. At the same time, "treatment" also includes a reduction in the pathological results of the disease (e.g., tumor volume for cancer). The methods of the present application take into account any one or more aspects of these treatments.

[0041] The term "antibody" includes full-length antibodies and antigen-binding fragments thereof. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains usually include three hypervariable loops, known as complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by having α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0042] The "humanized" form of a non-human (such as rodent) antibody is a chimeric antibody that includes minimal sequences from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which the hypervariable region (HVR) residues of the receptor antibody are replaced by residues from a non-human species such as a mouse, rat, rabbit or non-human mammal with an ideal antibody specificity, affinity and performance (donor antibody). In some cases, residues in the framework region of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues that are not present in either the receptor antibody or the donor antibody. These modifications can further improve the performance of the antibody. Typically, a humanized antibody will include substantially all, at least one, and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. Human antibodies optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0043] Example 1. Preparation and screening of anti-Claudin-18.2 single-chain antibodies

[0044] Claudin-18.2 antigen (Bipsis, CL2-H52P7) was mixed with adjuvants and injected into the mouse peritoneal cavity at multiple points. After multiple rounds of immunization, the mouse spleen was taken, RNA was extracted, and cDNA was obtained by reverse transcription. The variable regions of the light and heavy chains of the antibody were amplified using light and heavy chain specific primers to construct a phage display library. Positive clones targeting Claudin 18.2 were obtained by ELISA screening. The positive clones were sequenced to obtain the positive clone sequences. The CDR regions of the positive clones were transplanted into the human germline template with the highest homology, and the VH and VL amino acid sequences of the obtained humanized antibodies were shown in SEQ ID NO:7 and SEQ ID NO:8, respectively. VH and VL were connected by a linker to obtain an anti-Claudin-18.2 single-chain antibody (named G3B), whose amino acid sequence is shown in SEQID NO:9.

[0045] Example 2. Construction of Claudin-18.2CAR lentiviral expression vector

[0046] CD8 signal peptide, single-chain antibody targeting Claudin-18.2, CD8 hinge region, CD8 transmembrane domain, CD27 costimulatory region, 4-1BB costimulatory region and CD3ζ cytoplasmic signaling domain are connected in sequence, which is the CAR structure used in the present invention. The CAR nucleotide sequence having the above structure is prepared by artificial synthesis, and the CAR nucleotide sequence having the above structure is inserted into the multiple cloning site of the pLenti lentiviral expression vector (addgene, #17481) by homologous recombination to construct the Claudin-18.2CAR lentiviral expression plasmid, and the lentiviral expression vector sequence is successfully constructed by electrophoresis and sequencing results, and it is named pLenti-CAR-Claudin18.2-G3B.

[0047] Example 3. Claudin-18.2CAR lentiviral packaging and CAR-T cell preparation

[0048] The recombinant lentiviral vector pLenti-CAR-Claudin18.2-G3B prepared in Example 2 was mixed with pLenti lentiviral packaging plasmids psPAX2 (addgene, #12260) and pMD2.G (addgene, #12259) at a mass ratio of 5:3.2:1.8 and then transfected into 293T cells (about 3×10 6 After mixing, incubate at 37°C with 5% CO 2 Culture, change the medium after 8 hours. Collect the cell culture medium supernatant once every 24 hours, for a total of 3 times. Centrifuge the cell supernatant at 4°C and 50,000g for 2 hours, discard the supernatant, resuspend the precipitate with 100 μL DPBS, obtain the recombinant lentivirus, and store it in a -86°C refrigerator after aliquoting.

[0049] Take 5×10 5 Personal peripheral blood mononuclear cells (peripheral blood donated by volunteers was obtained by separation of lymphocyte separation medium) were added to AIM-V complete medium with 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 at 37°C and 5% CO 2 Culture for 24 h. Adjust the cell density to 2×10 6 / ml and inoculated in a 24-well plate (100 μL / well). Take the concentrated virus solution and infect overnight. The next day, add 1 mL of fresh culture medium. After 7 days of infection, the cells were collected. The prepared Claudin-18.2-targeted CAR-T cells were named Claudin18.2-G3B CAR-T.

[0050] After washing the Claudin18.2-G3B CAR-T cells with PBS, they were resuspended in FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). The positive rate of Claudin-18.2CAR expression on the surface of T cells was determined by FACS method. The results showed that the positive rate of Claudin18.2-G3B CAR-T was 72.1%.

[0051] Example 4. In vitro anti-tumor activity of Claudin-18.2 CAR-T cells

[0052] 4.1 LDH kit to detect CAR-T cell toxicity

[0053] The gastric cancer cell line BGC-823 overexpressing human Claudin18.2 and the gastric cancer cell line NCI-N87 overexpressing Claudin18.2 were constructed and expressed as BGC-823-hClaudin18.2 and NCI-N87hClaudin18.2, respectively. According to the effector-target ratio (E:T) of 3:1 and 1:1, the Claudin18.2-G3B CAR-T prepared in Example 3 was co-cultured with target cells (BGC-823-hClaudin18.2, NCI-N87 hClaudin18.2, BGC-823, NCI-N87) for 18 h (where the target cells were 3×10 4 / well, add the corresponding number of Claudin18.2-G3B CAR-T cells according to the effector-target ratio). At the same time, the group with CLD18A2-175-28BBZ (sequence information, see CN105315375B) added was used as a positive control, and the group with NT cells (untransfected T cells) added was used as a negative control. The killing effect of CAR-T was determined using the LDH detection kit (Shanghai Biyuntian, C0016). According to the instructions for use, the culture medium background well, effector cell spontaneous release well, target cell spontaneous release well, and target cell maximum release well were set respectively. 1 hour before collecting the supernatant, add 15 μL of LDH lysate to the target cell maximum release well, mix well, and place in the incubator for 1 hour. Take the above cell culture plate, centrifuge at 12000rpm for 3 minutes, take 120μL of the supernatant from each well, add 60μL LDH working solution (prepare LDH working solution according to 20μL enzyme solution, 20μL lactic acid solution, 20μL INT solution, and 18μL INT diluent for each reaction), and place it on a horizontal shaker at room temperature and incubate in the dark for 30 minutes. Detect the OD490 value of each well. After subtracting the absorbance of the blank control group from the absorbance of each well, calculate the cytotoxicity according to the following formula: OD value of each experimental well - OD value of the corresponding target cell natural release group - OD value of the corresponding effector cell natural release group) / (OD value of the corresponding target cell maximum release group - OD value of the corresponding effector cell natural release group) × 100%.

[0054] The results are shown in the table below. In vitro, Claudin18.2-G3B CAR-T and CLD18A2-175-28BBZ had significant cytotoxicity against BGC-823-hClaudin18.2 and NCI-N87 hClaudin18.2 cells overexpressing human Claudin18.2, and the cytotoxicity of Claudin18.2-G3B CAR-T was superior to that of CLD18A2-175-28BBZ. However, for Claudin-18.2-negative BGC-823 and NCI-N87 cells, Claudin18.2-G3B CAR-T, CLD18A2-175-28BBZ and NT cells showed no cytotoxicity.

[0055] Table 1

[0056]

[0057] 4.2 ELISA to detect cytokine release

[0058] Claudin18.2-G3B CAR-T, CLD18A2-175-28BBZ, and NT cells prepared in Example 4 were added at 3×10 4 / well) and target cells (NCI-N87 hClaudin18.2, 3×10 4 / well) and then mixed at 37°C and 5% CO 2 The cell culture plate was centrifuged at 800 rpm for 5 min at room temperature, and 50 μL of supernatant was collected to detect the amount of interferon γ and IL-2 released using an interferon γ detection kit (BD Biosciences, 560111) and an IL-2 detection kit (BD Biosciences, 558270).

[0059] The results are as follows Figure 1A-1B As shown, Claudin18.2-G3B CAR-T and CLD18A2-175-28BBZ can induce NCI-N87 hClaudin18.2 to release higher levels of interferon-γ ( Figure 1A ) and IL-2( Figure 1B ); and the activity of Claudin18.2-G3BCAR-T in inducing interferon γ and IL-2 release is better than that of CLD18A2-175-28BBZ.

[0060] Example 5. Detection of anti-tumor activity of Claudin-18.2 CAR-T cells in vivo

[0061] 6×10 6 NUGC-4 cells, when the tumor volume grows to 100-150mm 3 The mice were randomly divided into a control group and different treatment groups, with 6 mice in each group. PBS was injected into the control group mice through the tail vein, and 3×10 6 Claudin18.2-G3B CAR-T or CLD18A2-175-28BBZ. The survival status of each mouse was recorded, and the tumor volume of the mouse was measured twice a week. Tumor volume was calculated as V = [maximum long axis (L) × maximum wide axis (W) of the tumor 2 ] / 2 was calculated, and the tumor growth curve was drawn using Graph Pad.

[0062] The results are as follows Figure 2 The results showed that the tumors of mice injected with PBS continued to grow. The injection of Claudin-18.2-hG18CAR-T and CLD18A2-175-28BBZ could inhibit the growth of NUGC-4 tumor cells, and the tumor inhibition effect of Claudin-18.2-hG18 CAR-T was better than that of CLD18A2-175-28BBZ.

Claims

1. A single-chain antibody against Claudin18.2, characterized in that: include: VH, which comprises HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and VL, which comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

2. The single-chain antibody against Claudin18.2 according to claim 1, characterized in that: include: VH, the amino acid sequence of VH is shown in SEQ ID NO:7; VL, the amino acid sequence of VL is shown in SEQ ID NO:

8.

3. A chimeric antigen receptor targeting Claudin18.2, characterized in that: The chimeric antigen receptor comprises a CD8 signal peptide, a single-chain antibody against Claudin18.2 as described in any one of claims 1-2, a CD8 hinge region, a CD8 transmembrane domain, a CD27 co-stimulatory region, a 4-1BB co-stimulatory region and a CD3ζ cytoplasmic signaling domain, which are connected in sequence.

4. According to the chimeric antigen receptor targeting Claudin18.2 according to claim 3, the amino acid sequence of the chimeric antigen receptor targeting Claudin18.2 (Claudin18.2 CAR) is as shown in SEQ ID NO:

11.

5. A polynucleotide, characterized in that: It encodes the single-chain antibody against Claudin18.2 as described in any one of claims 1-2 or the chimeric antigen receptor targeting Claudin18.2 as described in any one of claims 3-4.

6. A carrier, characterized in that: It contains the polynucleotide as claimed in claim 5.

7. A host cell, characterized in that: It contains or expresses the single-chain antibody against Claudin18.2 as described in any one of claims 1-2, the chimeric antigen receptor targeting Claudin18.2 as described in any one of claims 3-4, the polynucleotide as described in claim 5 or the vector as described in claim 6.

8. A chimeric antigen receptor T cell, characterized in that: The chimeric antigen receptor targeting Claudin18.2 is expressed as described in any one of claims 3 to 4.

9. A pharmaceutical composition comprising the single-chain antibody against Claudin18.2 according to any one of claims 1-2, the chimeric antigen receptor targeting Claudin18.2 according to any one of claims 3-4, or the chimeric antigen receptor T cell according to claim 8.

10. Use of the pharmaceutical composition according to claim 9 in the preparation of a medicament for cancer.

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