Anti-PSCA antibody 1G9, CAR-NK cell containing same and application

Abstract

The invention provides an anti-PSCA antigen monoclonal antibody 1G9, a PSCA antigen targeting chimeric antigen receptor containing a single-chain antibody derived from a heavy chain variable region and a light chain variable region of the monoclonal antibody, and an NK cell containing the chimeric antigen receptor. The chimeric antigen receptor-NK cell has an excellent killing effect on PSCA positive tumor cells, and has an application prospect in preparation of drugs for treating tumor diseases expressing PSCA antigens.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to prostate stem cell antigens, chimeric antigen receptors, lentiviruses, modified NK cells, and their applications. Background Technology

[0002] Chimeric antigen receptors (CARs) are fusion molecules, typically composed of an extracellular antigen recognition region, a hinge region, a transmembrane region, and an intracellular signaling region. The extracellular antigen recognition region is generally composed of a single-chain variable fragment (scFv). The scFv region determines the specificity of the CAR molecule. After the CAR molecule specifically recognizes the antigen through the scFv, it transduces the signal into the cell, thereby activating effector cells and exerting anti-tumor and other biological effects. As a novel precision targeted therapy, it has shown promising results in the clinical treatment of tumors in recent years, demonstrating a high degree of precision, speed, efficiency, and potential for curing cancer through novel tumor immunotherapy.

[0003] NK cells (Natural Killer, NK) serve as the body's first line of defense against viruses and abnormal cells, exhibiting broad-spectrum anti-tumor effects. As early as 20 years ago, NK cell therapy was considered a safe and effective treatment for advanced leukemia. In recent years, with advancements in antibody technology, gene transduction technology, and in vitro expansion and culture technology, CAR-NK cell drugs have gradually emerged. CAR-NK cells possess the following advantages: no major histocompatibility complex (MHC) restrictions, allowing for the preparation of "universal" products; no cytokine storms or neurotoxicity; anti-tumor effects are achieved through multiple mechanisms, including CAR-dependent and non-CAR-dependent mechanisms, resulting in "all-round, three-dimensional" tumor killing; and NK cells are widely available, with allogeneic NK cells also used in their production. Based on these advantages, CAR-NK cell drugs have enormous potential and are expected to become a powerful tool for overcoming the bottlenecks in solid tumor treatment.

[0004] Prostate stem cell antigen (PSCA) is a small-molecule, glycosylphosphatidylinositol-anchored surface membrane antigen expressed in prostate cells. It belongs to the Thy-1 / Ly-6 protein family and shares 30% homology with surface-labeled stem cell antigen type II. PSCA is upregulated in prostate cancer, bladder cancer, pancreatic cancer, hydatidiform mole, and ovarian mucinous tumors, and downregulated in esophageal and gastric cancers. The positive rate of this antigen in prostate cancer tissues is close to 90%, gradually increasing with disease progression, reaching 100% in bone metastases. Furthermore, its expression level is positively correlated with the clinical grade of the tumor. Meanwhile, PSCA is not expressed or is expressed in only small amounts in normal prostate epithelium, bladder, kidneys, stomach, and other organs. Therefore, CAR-NK cells targeting PSCA have good drug-like potential and hold promise for a major breakthrough in the treatment of PSCA-positive tumors.

[0005] The purpose of this invention is to provide a monoclonal antibody against the PSCA antigen, and further to provide a chimeric antigen receptor targeting the PSCA antigen containing a single-chain antibody derived from the monoclonal antibody, and an NK cell containing the chimeric antigen receptor. Summary of the Invention

[0006] To achieve the above objectives, the present invention first provides a monoclonal antibody against PSCA antigen, wherein the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 and the light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody are shown as positions 26-33, 51-57, and 96-107 of SEQ ID NO.1 and positions 27-33, 51-53, and 90-98 of SEQ ID NO.3, respectively.

[0007] In an optional embodiment, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody against the PSCA antigen are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively. In this invention, the monoclonal antibody having the heavy chain variable region and the light chain variable region is named "1G9".

[0008] Secondly, this invention improves a single-chain antibody against the PSCA antigen, wherein the single-chain antibody against the PSCA antigen is formed by the heavy chain variable region and the light chain variable region of the aforementioned monoclonal antibody against the PSCA antigen in tandem. In a specific embodiment of this invention, the single-chain antibody is formed by the heavy chain variable region with the amino acid sequence shown in SEQ ID NO.1 and the light chain variable region with the amino acid sequence shown in SEQ ID NO.3 in tandem.

[0009] Third, the present invention provides a chimeric antigen receptor targeting PSCA antigen, wherein the chimeric antigen receptor is composed of a CD8α signal peptide, a single-chain antibody targeting PSCA, a CD8α hinge region, a CD8α transmembrane domain, a 2B4 protein co-stimulatory domain, and a CD3ζ intracellular signal transduction domain, wherein the single-chain antibody targeting PSCA is formed by the heavy chain variable region and the light chain variable region of the monoclonal antibody against PSCA antigen in tandem.

[0010] In an optional embodiment, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.5, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.7, the amino acid sequence of the CD8α transmembrane domain is shown in SEQ ID NO.9, the amino acid sequence of the 2B4 protein co-stimulatory domain is shown in SEQ ID NO.11, and the amino acid sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO.13.

[0011] In one specific embodiment of the present invention, the heavy chain variable region and the light chain variable region in the single-chain antibody targeting PSCA are linked by a (G4S)3 linker peptide.

[0012] Fourth, the present invention provides a nucleic acid encoding the above-mentioned chimeric antigen receptor sequence, the sequence of which is shown in SEQ ID NO.15. In the present invention, the nucleic acid is named "1G9-CAR".

[0013] Fifth, this invention provides a lentiviral vector containing the aforementioned nucleic acid encoding a chimeric antigen receptor. This invention clones the nucleic acid into a lentiviral vector, packages the nucleic acid into lentiviral particles using a lentiviral packaging system, and integrates them into the host cell genome. In one specific embodiment of this invention, the lentiviral vector is the lentiviral backbone plasmid pLenti-CMV-mGFP. The recombinant lentiviral plasmid and packaging plasmid can be packaged into lentiviral particles containing the aforementioned nucleic acid.

[0014] Sixth, this invention provides NK cells with a chimeric antigen receptor targeting the PSCA antigen, wherein the NK cells contain the aforementioned chimeric antigen receptor. This invention obtains NK cells expressing a chimeric antigen receptor targeting the PSCA antigen by infecting NK cells with the aforementioned lentiviral particles.

[0015] Seventh, this invention provides the application of the aforementioned anti-PSCA antigen monoclonal antibody in the preparation of PSCA antigen detection reagents. Based on the excellent affinity between the monoclonal antibody provided by this invention and the PSCA antigen, it can be used to detect PSCA antigen and / or cells carrying this marker that may be present in a sample. The detection is based on the immunological principle of specific antigen-antibody binding, and many immunoassay kits in the art, such as Western blot, immunofluorescence, ELISA, chemiluminescent immunoassay, and radioimmunoassay, can be applied to the detection described in this invention.

[0016] Eighth, this invention provides the application of the above-mentioned anti-PSCA antigen monoclonal antibody in the preparation of drugs for treating tumor diseases expressing PSCA antigen. Based on the excellent affinity of the monoclonal antibody provided by this invention for PSCA antigen, it can specifically target PSCA antigen. Therefore, by utilizing these properties of the monoclonal antibody, therapeutic drugs can be specifically targeted to lesions to exert a clinical therapeutic effect. Thus, this invention provides the application of the above-mentioned anti-PSCA antigen monoclonal antibody in the preparation of drugs for treating tumor diseases expressing PSCA antigen.

[0017] Ninth, this invention provides the application of the above-mentioned anti-PSCA antigen single-chain antibody in the preparation of drugs for treating tumor diseases expressing PSCA antigen. Based on the unique advantage of the small molecular weight of single-chain antibodies, these antibodies can be used to specifically target therapeutic drugs to some occult solid lesions that are difficult for monoclonal antibodies to reach, thereby exerting a clinical therapeutic effect. Therefore, this invention provides the application of the above-mentioned anti-PSCA antigen single-chain antibody in the preparation of drugs for treating tumor diseases expressing PSCA antigen.

[0018] Tenth, this invention provides the application of the chimeric antigen receptor described above in the preparation of drugs for treating tumor diseases expressing PSCA antigen. Using the chimeric antigen receptor provided by this invention, cells targeting the PSCA antigen can be prepared, including but not limited to T lymphocytes and NK cells. These cell antigens are used for the treatment of tumor cells expressing PSCA antigen. Therefore, this invention provides the application of the chimeric antigen receptor in the preparation of drugs for treating tumor diseases expressing PSCA antigen.

[0019] Finally, this invention provides the application of the above-mentioned chimeric antigen receptor-NK cells in the preparation of drugs for treating tumor diseases expressing PSCA antigens.

[0020] The monoclonal antibody 1G9 against PSCA antigen provided by this invention possesses unique CDRs in both the heavy chain variable region and the light chain variable region, exhibiting good affinity for both PSCA proteins. D (M) is 7.013 × 10 -9Chimeric antigen receptor-NK cells containing the heavy chain variable region and light chain variable region of the above-mentioned antibodies target PSCA. + Patu8988t tumor cells exhibited excellent killing ability. After co-incubation with tumor cells for 8 hours, the efficiency of 1G9-CAR-NK in killing tumor cells was 34.8%, while that of the control NK group was 6.3%. This demonstrates the application prospect of these antibodies in the preparation of drugs for treating tumor diseases expressing PSCA antigen. Attached Figure Description

[0021] Figure 1 This is a statistical graph of serum titers in mice one week after the second immunization, measured by ELISA. Figure 2 This is an image of hybridoma cells that have clumped together and grown 5 days after cell fusion; Figure 3 This is an immunofluorescence image of hybridoma cell supernatant and Patu8988t cells expressing PSCA protein; Figure 4 This is a graph showing the affinity assay results between monoclonal antibody 1G9 and PSCA protein; Figure 5 This is a schematic diagram of CAR molecule design; Figure 6 This is a graph showing the expression level of CAR molecules. Figure 7 This is an in vitro killing effect diagram of CAR-NK cells on Patu8988t cells; Figure 8 This is a diagram showing the killing effect of CAR-NK cells co-incubated with Patu8988t cells for 8 hours. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0023] Example 1. Screening of PSCA monoclonal antibodies 1.1 Immunized animals Female BALB / c mice aged 6-8 weeks were selected for immunization. 6.4 μL of in vivo-jetPEI (101000040, Polyplus) was mixed with 40 μg of pcDNA3.1(+)-PSCA (nucleic acid sequence shown in SEQ ID NO.16, PSCA inserted into the pcDNA3.1 vector). BamH I and XbaMix thoroughly (I-I) and inject into mice via tail vein. A second immunization is administered 14 days later, using the same dosage and method as the initial immunization. Serum titers are measured one week after the second immunization, and the results are as follows: Figure 1 As shown, the highest serum titer was approximately 1:10000. Mice with the highest serum titer were selected for booster immunization 3 days before fusion.

[0024] 1.2 Cell fusion and cloning Mouse spleens were aseptically harvested, and spleen cell suspensions were prepared. These suspensions were then fused with SP / 20 myeloma cells using the polyethylene glycol method and cultured in HAT selective medium. Five days post-fusion, the medium was partially replaced with HAT medium. Figure 2 The image shows hybridoma cells growing in clusters 5 days after cell fusion. After 10 days, the medium was changed to HT and cultured for another 48 hours. The cell supernatant was collected for indirect ELISA detection to screen for positive clones. Subcloning was performed using the limiting dilution method. Subcloning was repeated until a hybridoma cell line was finally obtained, which was named "1G9".

[0025] 1.3 Immunofluorescence Patu8988t cells (CL-0579, Pronosai) expressing PSCA antigen were adjusted to a concentration of 4 × 10⁻⁶. 5 Cells / mL were seeded into 96-well plates, 100 mL per well, and incubated overnight at 37°C. The next day, when the cell confluence reached 70%-80%, immunofluorescence experiments were performed.

[0026] (1) Discard the culture medium, wash with PBS, and fix with 4% tissue cell fixative for 20 min.

[0027] (2) After washing with PBS, block with 5% BSA at 37°C for 30 min.

[0028] (3) After washing with PBS, the cell supernatant was used as the primary antibody and incubated at 37°C for 1 h.

[0029] (4) After washing with PBS, add FITC-labeled goat anti-mouse at a concentration of 1:500 and incubate at 37°C in the dark for 1 h.

[0030] (5) After washing with PBS, observe the fluorescence under an inverted fluorescence microscope.

[0031] The results are as follows Figure 3 As shown, the monoclonal antibody 1G9 can specifically react with the PSCA protein expressed on the cell surface.

[0032] 1.4 Ascites preparation and subtype identification of monoclonal antibodies 0.5 mL of sterile liquid paraffin was injected intraperitoneally into 10-week-old female BALB / c mice. One week later, each mouse was injected intraperitoneally with 1 × 10⁻⁶ g of paraffin. 6 More than one hybridoma cell line was collected. Approximately one week after injection, ascites fluid was collected from mice. The collected ascites fluid was centrifuged at 5000 r / min for 10 min, and the supernatant was aliquoted and frozen at -80℃ for later use. The subclass (type) of the PSCA monoclonal antibody was identified using the monoclonal antibody subclass identification kit from Beijing Bio-Long Immunotherapy Co., Ltd., following the manufacturer's instructions. The results showed that the heavy chain of monoclonal antibody 1G9 was IgM type, and the light chain was Kappa type.

[0033] 1.5 Purification of Ascites Fluid Add sample diluent (50 mg / mL adipic acid, 4.0 M sodium chloride, pH 5.8) to the ascites fluid at a ratio of 1:4. After filtering through a 0.45 mm filter membrane, load the sample into a purification column (LT-155-1 mL KIT, LigaTrap). Wash the column with wash buffer (10 mg / mL adipic acid, 800 mM sodium chloride, pH 5.8) until the UV line is flat. Elute with elution buffer (500 mM sodium acetate, pH 3.8). Collect the eluent in a collection tube and add 12.5% ​​v / v of neutralizing buffer (3.0 M Tris-Base, pH 11.1).

[0034] 1.6 Affinity Measurement The binding affinity of monoclonal antibodies to PSCA protein was detected using a Sartorius Octet RH16 BLI assay system. All monoclonal antibodies and PSCA protein were diluted in assay buffer (PBS containing 0.02% Tween-20). PSCA protein bound to the ProA biosensor for 120 s. After washing, the sensor-bound PSCA was exposed to a buffer containing a series of serially diluted monoclonal antibodies for 120 s. The sensor was then dissociated in assay buffer for 120 s, and background correction was performed using antibody-free assay buffer. Results were analyzed using DataAnalysis 12.2 software to determine the Ka of the three different antibodies. D Value. Result as follows Figure 4 As shown, monoclonal antibody 1G9 has good affinity for both PSCA protein and its K... D (M) is 7.013 × 10 -9 .

[0035] 1.7 Amplification of the variable region gene of monoclonal antibodies The variable region gene of the monoclonal antibody was amplified using the 5' RACE method. The procedure was performed according to the HiScript-TS 5' / 3' RACE Kit (RA101-01, Novizan) instructions, and 5' GSP primers for the light and heavy chains were designed based on the constant region sequences of the monoclonal antibody. Light chain 5'GSP: 5'-TCAGGACGCCATTTTGTCGTTCACTGCC-3' (SEQ ID NO.17), Heavy chain 5'GSP: 5'-TCGGTTTTGGAGTGAAGTTCGTGGCCTC-3' (SEQ ID NO.18).

[0036] The amplified light and heavy chain fragments were cloned into pCE2 TA / Blunt-Zero Vector (C601-01, Novizan) using TA cloning, and single-clone strains were selected for sequencing. The amino acid sequence of the heavy chain variable region of monoclonal antibody 1G9 is shown in SEQ ID NO.1. The CDR1, CDR2, and CDR3 of the heavy chain variable region are shown at positions 26-33, 51-57, and 96-107 of SEQ ID NO.1, respectively, and the nucleic acid sequence encoding them is shown in SEQ ID NO.2. The amino acid sequence of the light chain variable region is shown in SEQ ID NO.3. The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown at positions 27-33, 51-53, and 90-98 of SEQ ID NO.3, respectively; the nucleic acid sequence encoding them is shown in SEQ ID NO.4.

[0037] Example 2. Design of CAR-NK expression sequences targeting PSCA and construction of lentiviruses 2.1 Design of CAR-NK expression sequences The CAR molecule targeting PSCA in CAR-NK cells is composed of, in sequence, a CD8α signal peptide, a single-chain antibody targeting PSCA, a CD8α hinge region, a CD8α transmembrane domain, a 2B4 protein co-stimulatory domain, and a CD3ζ intracellular signal transduction domain directly linked together (see schematic diagram of CAR molecule structure). Figure 5The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO. 5, and the nucleic acid sequence encoding it is shown in SEQ ID NO. 6; the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO. 7, and the nucleic acid sequence encoding it is shown in SEQ ID NO. 8; the amino acid sequence of the CD8α transmembrane domain is shown in SEQ ID NO. 9, and the nucleic acid sequence encoding it is shown in SEQ ID NO. 10; the amino acid sequence of the 2B4 protein co-stimulatory domain is shown in SEQ ID NO. 11, and the nucleic acid sequence encoding it is shown in SEQ ID NO. 12; the amino acid sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO. 13, and the nucleic acid sequence encoding it is shown in SEQ ID NO. 14. The nucleic acid sequence of the 1G9-CAR molecule obtained in this invention is shown in SEQ ID NO. 15.

[0038] 2.2 Construction of Lentiviral Vectors The designed CAR gene sequence (structural diagram as shown) Figure 5 (As shown) Inserted into the lentiviral backbone plasmid pLenti-CMV-mGFP(100071, Addgene) AsiS I and Not Between the restriction enzyme sites, the recombinant lentiviral plasmid was double-digested with restriction endonucleases at both ends of the designed gene sequence. Nucleic acid electrophoresis was performed, and fragment regions of the same size as the designed digested fragments were selected for gel recovery. Sequencing confirmed the presence of positive clone plasmids. The positive clone plasmids were mixed with Stbl3 competent E. coli, cultured in antibiotic-free LB liquid medium for 1 h, and then plated onto chloramphenicol-resistant LB agar plates and incubated overnight at 37°C. Plasmid extraction was performed using a large-scale extraction kit (P1156-02, Magen) according to the manufacturer's instructions. Plasmid purity and concentration were measured, and the plasmids were stored at -20°C.

[0039] 2.3 Lentiviral Preparation 293T cells were seeded into 10 cm culture dishes. After 24 hours, the culture medium was replaced with fresh medium. The recombinant lentiviral plasmid and packaging plasmids (psPAX2 and pMD2.G) were mixed and co-incubated using PEI MAX (24765-1, Polysciences) according to the manufacturer's instructions. The mixture was then added dropwise to the 293T cell culture dishes, and the dishes were gently shaken to mix thoroughly. The culture dishes were placed in a 37°C, 5% CO2 incubator and cultured for 6 hours, followed by a medium change. After 72 hours of continuous culture, the virus-containing supernatant was collected from the culture dishes, filtered through a 0.45 μm filter membrane, and stored at -80°C.

[0040] Example 3. Preparation of CAR-NK cells Peripheral blood mononuclear cells were isolated and resuspended in MACS buffer (130092747, Miltenyi Biotec). NK cell biotin-antibody cocktail and NK cell microbead cocktail (130092657, Miltenyi Biotec) were added sequentially for cell sorting, and NK cell-rich suspensions were collected. NK cells were resuspended in NK cell culture medium (NE000-NE32, Ekosei) to prepare a cell suspension, seeded in culture flasks, and cultured at 37°C and 5% CO2.

[0041] Lentiviral cells were infected with NK cells. The culture medium was replaced with fresh medium 24 hours after infection, and CAR expression levels were detected by flow cytometry 96 hours later (e.g., ...). Figure 6 (as shown in the figure), followed by expanded culture of CAR-NK cells.

[0042] Example 4. Detection of the CAR-NK cell killing function against tumor cells 5×10 3 PSCA + Patu8988t tumor cells were added to E-Plate 16 plates (756834, Agilent) and cultured for 20 h. CAR-NK cells were then added at a 1:1 effector-to-target ratio, and the Patu8988t cell index was subsequently measured. Results are as follows: Figure 7 As shown: CAR-NK cells on PSCA + The tumor cells exhibit a killing effect, and the killing effect is as follows after co-incubation with tumor cells for 8 hours: Figure 8 As shown, the NK group had a kill rate of 6.3%, while the 1G9-CAR-NK group had a kill rate of 34.8%, indicating that 1G9-CAR-NK showed excellent tumor cell killing rate.

[0043] Unless otherwise defined in this specification, all technical terms herein are used according to conventional definitions commonly used and understood by those skilled in the art. The experimental methods described in the embodiments are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified. The descriptions of the above embodiments are merely for understanding the methods and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and such improvements and modifications will also fall within the scope of protection of the claims of this invention.

Claims

1. A monoclonal antibody against PSCA antigen, characterized in that, The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 and the light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody are shown at positions 26-33, 51-57, and 96-107 of SEQ ID NO.1 and positions 27-33, 51-53, and 90-98 of SEQ ID NO.3, respectively.

2. The monoclonal antibody against PSCA antigen according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody against the PSCA antigen are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively.

3. A single-chain antibody against PSCA antigen, characterized in that, The single-chain antibody against the PSCA antigen is formed by the heavy chain variable region and the light chain variable region of the monoclonal antibody against the PSCA antigen as described in claim 2, in tandem.

4. A chimeric antigen receptor targeting PSCA antigen, characterized in that, The chimeric antigen receptor targeting the PSCA antigen is composed of a CD8α signal peptide, a single-chain antibody against the PSCA antigen as described in claim 3, a CD8α hinge region, a CD8α transmembrane domain, a 2B4 protein co-stimulatory domain, and a CD3ζ intracellular signal transduction domain.

5. The chimeric antigen receptor targeting PSCA antigen according to claim 4, characterized in that, The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.5, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.7, the amino acid sequence of the CD8α transmembrane domain is shown in SEQ ID NO.9, the amino acid sequence of the 2B4 protein co-stimulatory domain is shown in SEQ ID NO.11, and the amino acid sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO.

13.

6. The chimeric antigen receptor targeting PSCA antigen according to claim 5, characterized in that, The heavy chain variable region and light chain variable region in the single-chain antibody targeting PSCA antigen are linked in tandem by a (G4S)3 linker peptide.

7. A nucleic acid encoding a chimeric antigen receptor sequence targeting the PSCA antigen as described in claim 6, characterized in that, The sequence of the nucleic acid is shown in SEQ ID NO.

15.

8. A lentiviral vector containing the nucleic acid of claim 7.

9. A chimeric antigen receptor NK cell targeting PSCA antigen, characterized in that, The NK cells contain a chimeric antigen receptor that targets the PSCA antigen as described in any one of claims 4-6.

10. The use of the monoclonal antibody against PSCA antigen as described in claim 1 or 2 in the preparation of PSCA antigen detection reagents.

11. The use of the monoclonal antibody against PSCA antigen as described in claim 1 or 2 in the preparation of a medicament for treating tumor diseases expressing PSCA antigen.

12. The use of the single-chain antibody against PSCA antigen as described in claim 3 in the preparation of a medicament for treating tumor diseases expressing PSCA antigen.

13. The use of the chimeric antigen receptor targeting PSCA antigen as described in any one of claims 4-6 in the preparation of a medicament for treating tumor diseases expressing PSCA antigen.

14. The use of the chimeric antigen receptor NK cells targeting PSCA antigen as described in claim 9 in the preparation of a medicament for treating tumor diseases expressing PSCA antigen.

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