CAR (chimeric antigen receptor) targeting prostatic cancer, recombinant expression vector containing CAR, lentivirus, CAR-T (chimeric antigen receptor T) and application

Abstract

The invention relates to a prostatic cancer targeting CAR (chimeric antigen receptor), a recombinant expression vector containing the same, a lentivirus, CAR-T and application, and belongs to the technical field of tumor immunotherapy. In order to solve the problem that few reports are reported for treating solid tumors by CAR-T cells, the invention provides a CAR for targeting prostatic cancer, the CAR comprises an scFv fragment consisting of VH with an amino acid sequence as shown in SEQ ID NO.2, a hinge region (Gly4Ser) 3 and VL with an amino acid sequence as shown in SEQ ID NO.4, the CAR is connected to a lentiviral expression vector pLV-EF to obtain an expression vector plasmid, and the expression vector plasmid is used for expressing the prostatic cancer. The CAR-T cell is prepared by producing a high-titer lentivirus through a 293T packaging cell line, human primary T lymphocytes infected and activated by the lentivirus are used for preparing the CAR-T cell, in-vivo and in-vitro experiments prove that the CAR-T cell has a specific killing effect on prostatic cancer, and a new treatment thought and means are provided for solid tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor immunotherapy, and specifically relates to a CAR targeting prostate cancer and a recombinant expression vector, a lentivirus, a CAR-T and applications thereof. Background Art

[0002] Under the influence of multiple factors such as genetics, environment, and diet, the incidence and mortality of malignant tumors have been increasing year by year, and it has become one of the most important factors threatening human life and health. In 2015, about 4.3 million people in China were diagnosed with cancer, and 2.8 million people died of cancer. In 2020, there were 5.5 million new cancer cases in China each year, of which 4 million people died. At present, the clinical treatment methods for malignant tumors mainly include surgical resection, chemotherapy, and radiotherapy. These methods have good efficacy for early tumors, can significantly relieve patients' symptoms and prolong their survival. However, for many patients with malignant tumors, especially those with recurrence and metastasis, the treatment effect is not ideal, which seriously affects the patients' quality of life and overall survival (OS). Against this background, tumor immunotherapy came into being. As a new treatment concept, immunotherapy will surely bring good news to the majority of cancer patients due to its unique advantages.

[0003] At present, tumor immunotherapy has entered a new era. It has become another important treatment method after surgery, chemotherapy and radiotherapy. Among them, chimeric antigen receptor (CAR) T cell therapy has developed rapidly in the field of tumor treatment in recent years. CAR-T therapy combines the high affinity of antigen antibodies with the killing effect of T lymphocytes. T lymphocytes are transduced to express this CAR, and recognize tumor cells and kill tumors in a non-major histocompatibility complex (MHC) restricted manner. In recent years, the breakthrough of CAR-T cell therapy in the treatment of B cell blood tumors has provided a new option for the treatment of blood tumors. Among them, CAR-T therapy targeting CD19 has become a model therapy for most clinical research institutions to carry out gene-modified T cell therapy due to its significant effect and extensive research. Based on this significant therapeutic effect, Kymriah (Tisagenlecleucel, formerly known as CTL019), a CD19 CAR-T cell product of Novartis Pharmaceuticals, was first approved by the FDA for the treatment of diffuse large B-cell lymphoma and refractory B-cell acute lymphoblastic leukemia. However, there are currently few reports on the use of CAR-T cells in the treatment of solid tumors. Summary of the invention

[0004] In order to solve the problem that there are few reports on the use of CAR-T cells to treat solid tumors, the present invention provides a CAR targeting prostate cancer and a recombinant expression vector, a lentivirus, a CAR-T and its use in the treatment of prostate cancer.

[0005] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0006] The first object of the present invention is to provide a CAR targeting prostate cancer, wherein the CAR comprises V represented by the amino acid sequence as shown in SEQ ID NO.2 H , hinge region (Gly4Ser) 3 as shown in SEQ ID NO.6 and V as shown in SEQ ID NO.4 L scFv fragment composed of.

[0007] In one embodiment of the present invention, the CAR comprises, from N-terminus to C-terminus: human cell surface glycoprotein T8 signal peptide, scFv fragment, human CD8α hinge region and transmembrane domain, 4-1BB signal transduction domain and CD3ζ signal transduction domain.

[0008] The inventors have published a paper on the V of the anti-bladder cancer monoclonal antibody H and V L The gene sequence was improved and optimized, and the antibody gene sequence was blasted according to the abYsis and IMGT databases to obtain the complete V H and V L The gene sequence of V H and V L A short polypeptide linker, i.e., hinge region (Gly4Ser) 3, which assists in the spatial structure, was added between the sequences to obtain a complete scFv fragment; the mRNA sequences of human cell surface glycoprotein T8 signal peptide (GenBank: M12828.1), human CD8α hinge region and transmembrane domain (GenBank: NM_001768.6), 4-1BB signal transduction domain (GenBank: NM_001561.5), and CD3ζ signal transduction domain (GenBank: NM_000734.3) were retrieved from the NCBI database, and the recombinant chimeric antigen receptor gene scFv-4-1BB-CD3ζ was synthesized using RT-PCR technology to obtain a complete second-generation CAR (APC01-CAR), the nucleotide sequence of which is shown in SEQ ID NO.21, and the amino acid sequence is shown in SEQ ID NO.22.

[0009] Further defining, code V H The nucleotide sequence of V is shown in SEQ ID NO.1, encodingL The nucleotide sequence is shown in SEQ ID NO.3, the sequence encoding the hinge region is three groups of pentapeptide repeating units (Gly4Ser)3 in series, and the nucleotide sequence is shown in SEQ ID NO.5.

[0010] It is further defined that the nucleotide sequence encoding the human cell surface glycoprotein T8 signal peptide is shown in SEQ ID NO.7, and the amino acid sequence of the human cell surface glycoprotein T8 signal peptide is shown in SEQ ID NO.8; the nucleotide sequence encoding the human CD8α hinge region and transmembrane domain is shown in SEQ ID NO.9, and the amino acid sequence of the human CD8α hinge region and transmembrane domain is shown in SEQ ID NO.10; the intracellular signal transduction domain is composed of 4-1BB and CD3ζ connected in sequence, the nucleotide sequence encoding 4-1BB is shown in SEQ ID NO.11, and the amino acid sequence of 4-1BB is shown in SEQ ID NO.12; the nucleotide sequence encoding CD3ζ is shown in SEQ ID NO.13, and the amino acid sequence of CD3ζ is shown in SEQ ID NO.14.

[0011] The second object of the present invention is to provide a lentiviral recombinant expression vector comprising the above-mentioned CAR.

[0012] The third object of the present invention is to provide a method for constructing the above-mentioned lentiviral recombinant expression vector, wherein the construction method is to connect CAR to a linearized third-generation lentiviral expression vector pLV-EF to obtain a complete APC01-CAR expression vector plasmid.

[0013] In one embodiment of the present invention, APC01-CAR containing enhanced green fluorescent protein (eGFP) (GenBank: MK258730.1) or luciferase (Luciferase) reporter gene (GenBank: MK484108.1) is also synthesized [eGFP or Luciferase gene is connected to the CAR gene using T2A sequence (GenBank: KX055828.1)], which is also connected to the linearized lentiviral expression vector pLV-EF, and subsequently used for in vitro and in vivo experiments, respectively.

[0014] It is further defined that the nucleotide sequence encoding eGFP is shown as SEQ ID NO.15, and the amino acid sequence of eGFP is shown as SEQ ID NO.16; the nucleotide sequence encoding Luciferase is shown as SEQ ID NO.17, and the amino acid sequence of Luciferase is shown as SEQ ID NO.18; the nucleotide sequence encoding T2A is shown as SEQ ID NO.19, and the amino acid sequence of T2A is shown as SEQ ID NO.20.

[0015] The fourth object of the present invention is to provide a lentiviral solution containing the above-mentioned CAR.

[0016] The fifth object of the present invention is to provide the use of the above-mentioned CAR, the above-mentioned lentiviral recombinant expression vector or the above-mentioned lentiviral liquid in the preparation of CAR-T cells targeting prostate cancer.

[0017] The sixth object of the present invention is to provide a CAR-T cell targeting prostate cancer, wherein the CAR-T cell is prepared by infecting primary human T lymphocytes with the above-mentioned lentivirus.

[0018] A seventh object of the present invention is to provide a method for preparing the above-mentioned CAR-T cells, the method comprising the following steps:

[0019] S1. The above-mentioned lentiviral recombinant expression vector containing the target gene CAR, packaging plasmid p-gp, and envelope plasmid p-VG are used to form a three-plasmid lentiviral packaging system, which is transfected into 293T cells according to the calcium phosphate precipitation transduction method, and the supernatant is collected after incubation;

[0020] S2, concentrating the supernatant obtained in S1 to obtain a high-titer lentiviral solution;

[0021] S3. Use the lentiviral solution obtained in S2 to infect activated human primary T lymphocytes to obtain CAR-T cells.

[0022] In one embodiment of the present invention, the incubation in S1 is culturing at 37° C. for 48 hours.

[0023] The eighth object of the present invention is to provide a drug containing the above-mentioned CAR-T cells, which also contains a pharmaceutically acceptable carrier.

[0024] The ninth objective of the present invention is to provide the use of the above-mentioned CAR, the above-mentioned lentiviral recombinant expression vector, the above-mentioned lentivirus or the above-mentioned CAR-T cell in the preparation of a cellular drug for the treatment of prostate cancer.

[0025] Beneficial effects of the present invention:

[0026] The present invention is to analyze the heavy chain variable region (V H ) and light chain variable region (V L) gene sequence was improved and optimized, connected with a flexible polypeptide linker, and constructed into a CAR with an antigen binding site composed of a single-chain variable region domain (scFv), which was then connected to the lentiviral expression vector pLV-EF to obtain a complete APC01-CAR expression vector plasmid. High-titer lentivirus was then produced through the 293T packaging cell line, and the lentiviral particles were used to infect activated human primary T lymphocytes to prepare APC01 CAR-T cells.

[0027] The present invention verifies the function of CAR-T cells through in vitro cell and in vivo animal experiments: the proliferation response, cytokine secretion and cytotoxic activity of CAR-T cells under target cell stimulation were detected in vitro, and the results showed that compared with simulated transduced T cells, CAR-T cells have stronger proliferation activity; CAR-T cells can also secrete a large amount of Th1 cytokines IFN-γ, TNF and IL-2, and less IL-4 and IL-6, while simulated transduced T cells hardly secrete any cytokines; compared with simulated transduced T cells, the CAR-T cell group has stronger cytotoxic activity and a higher tumor cell lysis rate. At the same time, B-NDG severely immunodeficient mice were used to establish a human transplant tumor model, and bioluminescence imaging was used to trace CAR-T cells to observe their biological behavior and anti-tumor activity. The tumor volume was measured and calculated at different time points, and the survival of mice in the APC01CAR-T treatment group and the control group was observed. The results showed that APC01 CAR-T had an extremely significant killing effect on prostate cancer. The mice in the treatment group were completely cured and their survival was greatly prolonged. This laid a solid foundation for our subsequent research on CAR-T for solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a schematic diagram of the structure of all CARs in the present invention; wherein, Figure 1 A in the figure is the structural diagram of APC01 CAR. Figure 1 B in the figure is a schematic diagram of the structure of APC01 CAR-T2A-eGFP. Figure 1 C in the figure is a schematic diagram of the structure of APC01 CAR-T2A-Luciferase;

[0029] Figure 2 The results of fluorescence microscopy observation of eGFP expression in 293T cells infected with lentivirus and flow cytometry analysis of the positive rate of eGFP in 293T cells are shown in the figure; Figure 2 A in the figure is the result of 293T cells without virus solution. Figure 2 B in the figure is the result of adding lentivirus concentrate to 293T cells. Figure 2 C in the figure is the result of adding 3-fold dilution of lentiviral concentrate to 293T cells. Figure 2 D in the figure is the result of adding 9-fold dilution of lentiviral concentrate to 293T cells. Figure 2 E in the figure is the result of adding 27-fold dilution of lentiviral concentrate to 293T cells. Figure 2 F in the figure is the result of adding 81-fold dilution of lentiviral concentrate to 293T cells. Figure 2 G in the figure is the result of adding 293T cells with 243-fold dilution of lentiviral concentrate; Figure 2 The data in are representative of 3 independent samples;

[0030] Figure 3 This is the result of flow cytometry detection of CD4+T cells and CD8+T cells purity; Figure 3 A in the figure is the result of flow cytometry detection of CD4+T cell purity. Figure 3 B in the figure is the result of flow cytometry detection of CD8+T cell purity; the data are representative of 3 independent samples;

[0031] Figure 4 The results of the cell proliferation test after CAR-T, CD4+CAR-T, and CD8+CAR-T cells were infected with lentivirus transduced with APC01-CAR; the data are representative of 3 independent samples;

[0032] Figure 5 The results of CD3 phenotype analysis of CD4+CAR-T and CD8+CAR-T cells are shown in Figure 2. The data are representative of 3 independent samples.

[0033] Figure 6 The figure shows the detection results of target gene transduction rate in CD4+CAR-T and CD8+CAR-T cells; the data are representative of 3 independent samples;

[0034] Figure 7 The figure shows the proliferation detection results of CD4+CAR-T and CD8+CAR-T cells after co-culture with inactivated PC-3 cells or Raji cells; Figure 7 A in the figure is the proliferation detection result of CD4+CAR-T cells after co-culture with inactivated PC-3 cells or Raji cells. Figure 7 B in the figure is the proliferation detection result of CD8+CAR-T cells after co-culture with inactivated PC-3 cells or Raji cells; the data are representative of 3 independent samples, ***p<0.001;

[0035] Figure 8The statistical results of cytokine secretion after APC01 CAR-T cells were stimulated by PC-3 or target antigen-negative Raji cells; the results represent 3 independent experiments, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0036] Fig. 9 The results of observing the expression of eGFP in PC-3 cells and H460 cells under a fluorescence microscope;

[0037] Fig.10 This is a fluorescence microscope observation of the killing effect of APC01 CAR-T cells on target cells PC-3-eGFP, H460-eGFP and Raji-eGFP-Luciferase in vitro; PC-3, H460 and Raji cells were all transduced with eGFP;

[0038] Fig.11 This is a flow cytometer-detected killing effect of APC01 CAR-T cells on target cells PC-3 in vitro; Fig.11 A in the figure is the flow cytometry analysis result of a representative experiment at different effector-target ratios after APC01 CAR-T and CD19 CAR-T cells were co-incubated with target cells PC-3. Fig.11 B in the figure is the flow cytometry analysis results of three independent experiments at different effector-target ratios after APC01 CAR-T and CD19 CAR-T cells were co-incubated with target cells PC-3, H460 and Raji, respectively;

[0039] Fig.12 Experimental flow chart for in vivo observation of CAR-T cell distribution;

[0040] Fig.13 This is a result diagram of using a live imaging device to observe the distribution of T cells in tumor-bearing mice;

[0041] Fig.14 This is a statistical result diagram of the luminescence intensity of T cells in tumor-bearing mice observed using a live imaging device;

[0042] Fig.15 Schematic diagram of the anti-tumor experiment conducted in B-NDG mice;

[0043] Fig.16 This is a combined graph of tumor growth curves corresponding to different experimental groups in the anti-tumor experiment;

[0044] Fig.17 These are pictures of transplanted tumors in mice of different experimental groups in the anti-tumor experiment;

[0045] Fig.18These are pictures of liver tissues of mice in different experimental groups in the anti-tumor experiment;

[0046] Fig.19 This is the result of using immunohistochemical staining to verify whether the tumor, liver and lung tissues of mice in different experimental groups are infiltrated by PC-3 tumor cells in the anti-tumor experiment;

[0047] Fig. 20 This is a survival curve of mice in different experimental groups in the anti-tumor experiment. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The embodiments described in the present invention are not all embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0050] The V used in the present invention H and V L The sequence is a V sequence of a monoclonal antibody against bladder cancer in a published paper H and V L The gene sequence ([1] Zhou Lijun, Wang Yan, Bai Yin, et al. Cloning and expression of anti-bladder cancer monoclonal antibody Fab segment gene [J]. Chinese Journal of Tumor Biotherapy, 2002, (02): 117-121.) was improved and optimized, and the antibody gene sequence was obtained by blast analysis based on the abYsis and IMGT databases.

[0051] Example 1: A CAR targeting prostate cancer

[0052] The gene sequence of the monoclonal antibody was blasted and optimized using the abYsis and IMGT databases to obtain the complete V H and V L The gene sequence (V H The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; V L The nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4); in V H and V LA short polypeptide linker, i.e., hinge region (Gly4Ser) 3 (nucleotide sequence as shown in SEQ ID NO.5, amino acid sequence as shown in SEQ ID NO.6) is added between the gene sequences to assist in their spatial structure, thereby obtaining the scFv fragment.

[0053] The gene sequences of human cell surface glycoprotein T8 signal peptide, human CD8α hinge region and transmembrane domain, 4-1BB signal transduction domain and CD3ζ signal transduction domain were searched from the NCBI database, and the entire CAR gene was connected in the order of T8 signal peptide, specific scFv, CD8α hinge and transmembrane domain, 4-1BB signal transduction domain and CD3ζ signal transduction domain to obtain a complete second-generation CAR (APC01-CAR), the structural schematic diagram of which is shown in the figure. Figure 1 As shown in A, wherein the nucleotide sequence of the human cell surface glycoprotein T8 signal peptide is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the human CD8α hinge region and transmembrane domain is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10; the nucleotide sequence of the 4-1BB signal transduction domain is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12; the nucleotide sequence of the CD3ζ signal transduction domain is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14; the nucleotide sequence of APC01-CAR is shown in SEQ ID NO.21, and the amino acid sequence is shown in SEQ ID NO.22. According to Figure 1 B and Figure 1 The structural schematic diagram shown in C in the figure entrusts Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize APC01 CAR containing enhanced green fluorescent protein (eGFP) (GenBank: MK258730.1) and APC01 CAR containing luciferase (Luciferase) reporter gene (GenBank: MK484108.1), and the eGFP or Luciferase gene is connected to the CAR gene with a T2A sequence (GenBank: KX055828.1), wherein the nucleotide sequence of eGFP is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.16; the nucleotide sequence of Luciferase is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.18; the nucleotide sequence of T2A is shown in SEQ ID NO.19, and the amino acid sequence is shown in SEQ ID NO.20.

[0054] Example 2: Construction and identification of lentiviral expression vector carrying CAR

[0055] APC01-CAR was connected to the linearized third-generation lentiviral expression vector pLV-EF preserved in this laboratory, thus completing the construction of the lentiviral recombinant expression vector plasmid carrying CAR.

[0056] Double enzyme digestion identification of vector plasmid:

[0057] Mix 1 μL Xba I, 1 μL Nhe I, 15 μL plasmid DNA, 5 μL CutSmart Buffer, and 28 μL ddH2O, and react at 37°C for 5-15 minutes (incubate for 1 hour for the standard system, and incubate for 5-15 minutes for the time-saving enzyme digestion system. The enzyme digestion can be performed overnight, and no star activity will occur). The results showed that the plasmid was cut into two fragments of 7400 bp and 1467 bp, which was consistent with the expected results.

[0058] Single enzyme digestion identification of vector plasmid:

[0059] Mix 1 μL Sal I, 15 μL plasmid DNA, 5 μL NEBuffer 2.1, and 29 μL ddH2O, and react at 37°C for 5-15 minutes (the standard system is incubated for 1 hour, and the time-saving enzyme digestion system is incubated for 5-15 minutes. The enzyme digestion can be performed overnight, and no star activity will appear). The results show that the plasmid is cut into two fragments of 6087 bp and 2780 bp, which is consistent with the expected results.

[0060] Example 3: Preparation of lentivirus carrying CAR

[0061] 1. Calcium phosphate precipitation transduction

[0062] (1) Preparation of calcium phosphate precipitation transduction system reagents

[0063] CaCl2 (2.5 mol / L): Dissolve 36.76 g CaCl2·2H2O in ddH2O, make up to 100 mL, filter sterilize with a 0.22 μm filter, divide into 1 mL aliquots, and store at 4°C;

[0064] 0.1×TE Buffer (pH 8.1): Dissolve 158 mg Tris-Cl and 29 mg EDTA in 90 mL ddH2O, adjust the pH to 8.1 with NaOH, make up to 100 mL, and dilute to 0.1× with ddH2O. Sterilize by filtering with a 0.22 μm filter, aliquot, and store at 4°C.

[0065] 2×HEPES-buffered saline (2×HBS): Dissolve 800 mg NaCl, 27 mg Na2HPO4·2H2O, and 1.2 g HEPES in 90 mL ddH2O. Adjust pH to 7.05 with NaOH (0.5 N). Make up to 100 mL. Sterilize by filtering with a 0.22 μm filter. Aliquot into 5 mL portions. Store at -20°C. Use within 1 year.

[0066] (2) Cultivation of 293T cells

[0067] The 293T cells were revived and cultured with DMEM complete medium (10% FBS). When the cells were in good condition, the 293T cells were digested with trypsin and accurately counted. Then, the 293T cells were inoculated in a 10 cm cell culture dish at 4×10 6 Each dish was cultured with 10 mL of DMED complete medium. The cell status was observed the next day, and calcium phosphate precipitation transduction was performed when the cell density was about 30%.

[0068] (3) Calcium phosphate precipitation transduction

[0069] Add 900 μL 0.1×TE buffer to the bottom of a 50 mL centrifuge tube; add the three-plasmid system of the lentivirus, packaging plasmid p-gp, envelope plasmid p-VG, and lentiviral recombinant expression vector plasmid containing the target gene CAR to 0.1×TE buffer at a ratio of 3:2:5 (6 μg: 4 μg: 10 μg); add 100 μL Add CaCl2 to a 50mL centrifuge tube and mix on a vortex oscillator; add 1mL of ice-cold 2×HBS dropwise, shaking while adding, and let it stand for 1min; quickly add the mixed solution to a 10cm dish where 293T cells are cultured, 1mL / dish, and be careful not to blow up the 293T cells as much as possible, and shake quickly and gently; observe under an inverted microscope, if there are fine sand-like black calcium phosphate particles in the culture dish, it means that the precipitation is effective, and then place the cells in an incubator with a temperature of 37°C, a CO2 concentration of 5-8%, and a humidity of 95% for culture; after 6 hours, transfer the 293T cell culture dish to an incubator with a temperature of 32°C, a CO2 concentration of 5-8%, and a humidity of 95% for continued culture.

[0070] 2. Collection and concentration of lentivirus

[0071] Lentivirus collection: Observe the transfection efficiency under a fluorescence microscope 24 hours after calcium phosphate precipitation transduction. Observe the 293T cells 48 hours after transduction. If the culture medium turns yellow, the cells are close to 100% confluence. Collect the cell supernatant and centrifuge at 4°C, 3000 rpm for 10 minutes (to remove 293T cells).

[0072] Concentration of lentivirus: Aspirate the cell supernatant, add it to a pre-cooled 20mL centrifuge tube, and centrifuge it in a micro-ultracentrifuge at 4℃, 50000×g for 95min; gently aspirate the culture supernatant, collect the precipitate, resuspend the precipitate with 500μL culture medium, gently blow off the viral white film, transfer it to a 1.5mL EP tube, clearly mark it, and store it at -80℃, avoiding repeated freezing and thawing as much as possible.

[0073] 3. Determination of lentiviral titer

[0074] 24 h in advance, a 48-well cell culture plate was coated with RetroNectin solution at a final concentration of 20 μg / mL; 293T cells were plated in a 48-well plate (10 5 293T cells); the prepared pLV-EF-APC01-CAR-T2A-eGFP lentiviral concentrate was diluted 3 times in a series of gradients, and the virus dilutions were added to the 293T cell wells. After 48 hours of infection, the images were taken under a fluorescence microscope, and then the cells were digested with trypsin to prepare a single cell suspension, and the percentage of infected positive cells was detected by flow cytometry (see Figure 2 ).Depend on Figure 2 It can be seen that the positive cell percentages of 293T cells added with lentivirus concentrate, 293T cells added with 3-fold dilution of lentivirus concentrate, 293T cells added with 9-fold dilution of lentivirus concentrate, 293T cells added with 27-fold dilution of lentivirus concentrate, 293T cells added with 81-fold dilution of lentivirus concentrate, and 293T cells added with 243-fold dilution of lentivirus concentrate are 99.73%, 99.07%, 90.58%, 56.12%, 21.28%, and 6.65%, respectively. The lentivirus titer is calculated based on the positive cell percentages obtained above, and the calculation formula is as follows:

[0075] Titer (TU / mL, i.e. transformation unit) = % positive cells × dilution factor × 10 5 ×20, the percentage of positive cells used was the wells with a positive rate of >5% <20% by flow cytometry.

[0076] Example 4: Preparation of CAR-T cells

[0077] 1. Preparation of CAR-T cells

[0078] (1) Preparation of T cell activation medium

[0079] 1% penicillin / streptomycin, 0.6 μL / mL anti-human CD3 antibody, 0.6 μL / mL anti-human CD28 antibody, and 1000 IU / mL IL-2 were added to the GT-T551 lymphocyte culture medium; in addition, 5-10% heat-inactivated plasma from blood donors can be added.

[0080] (2) Isolation of Peripheral Blood Mononuclear Cells (PBMC)

[0081] Collect peripheral blood from healthy volunteers; dilute the blood with the same volume of serum-free 1640 medium; add lymphocyte separation solution and diluted blood to a 15 or 50 mL centrifuge tube at a ratio of 1:2 (add lymphocyte separation solution to the centrifuge tube first, and then use a Pasteur pipette to carefully add diluted blood on top of the lymphocyte separation solution to form a clear boundary between the two); centrifuge at 800×g without brake for 20 minutes at room temperature; after centrifugation, the liquid in the centrifuge tube forms a clear boundary, from top to bottom, it is a mixture of 1640 medium and volunteer plasma, lymphocyte layer, lymphocyte separation solution layer, spherocytes and other large cells, carefully aspirate the lymphocyte layer with a Pasteur pipette and transfer it to a new centrifuge tube; dilute the lymphocytes with 5 times the volume of serum-free 1640 medium, centrifuge at 800×g for 10 minutes, and discard the supernatant; then wash the lymphocytes 2-3 times with 1640 complete medium, count the cells, and freeze them.

[0082] (3) Sorting and activation of CD4+ T cells and CD8+ T cells

[0083] Resuscitate PBMC, centrifuge at 2000rpm for 10min, and discard the supernatant; prepare 100μg / mL DNase I solution (take 50μLDnase I (1μg / μL) solution and add it to 450μL EasySep Buffer and mix well); resuspend PBMC with the above DNase I solution, incubate at room temperature (15℃-25℃) for at least 15min, then filter the cells with a 40μm cell filter into a 5mL sterile flow tube, centrifuge at 2000rpm for 10min, and discard the supernatant; centrifuge the cells at 5×10 7 / mL density was resuspended in EasySep Buffer, Enrichment Cocktail (50μL / mL sample) was added to the sample, mixed, and incubated at room temperature for 10min; MagneticParticles were vortexed for 30sec to make them evenly distributed; CD4+T cell sorting: MagneticParticles (100μL / mL sample) were added to the sample, mixed, and incubated at room temperature for 5min; CD8+T cell sorting: MagneticParticles (150μL / mL sample) were added to the sample, mixed, and incubated at room temperature for 5min; EasySep Buffer, make the total volume 2.5mL, gently pipette up and down 2-3 times to mix, put it in the magnet without cover, and incubate at room temperature for 5min; invert the flow tube together with the magnet, pour the solution into a new centrifuge tube, count, centrifuge at 2000rpm for 10min, and discard the supernatant; take some cells and stain them with anti-CD4-PE or anti-CD8-PE antibodies, and detect the purity of the sorting by flow cytometry; add 5mL activation medium to the remaining cells and transfer them to 25cm 2 The cell culture flask was placed in an incubator at 37°C, 5% CO2 concentration, and 95% humidity.

[0084] The results of the CD4+T cell and CD8+T cell purity tests are as follows Figure 3 As shown in the figure, it can be seen that the purity of CD4+T cells after enrichment is 92.60%, and the purity of CD8+T cells after enrichment is 93.87%.

[0085] (4) Lentivirus infection of T / CD4+T / CD8+T cells:

[0086] 24h in advance, a 24-well cell culture plate was coated with RetroNectin solution with a final concentration of 20μg / mL; T / CD4+T / CD8+T cells activated 24h in advance were plated on a 24-well plate (1-2×10 6 cells); according to the multiplicity of infection (MOI) of about 5, the prepared APC01-CAR or CD19-CAR or empty vector simulated transduction lentivirus solution was added to the T / CD4+T / CD8+T cell wells for infection, and the cells were placed in an incubator at a temperature of 32°C, a CO2 concentration of 5%, and a humidity of 95%. After 24 hours, they were transferred to an incubator at a temperature of 37°C for culture; GT-T551 lymphocyte culture medium with an IL-2 concentration of 100 IU / mL was supplemented every two days, and the cells were counted every two days and the expansion multiple was calculated (results in Figure 4 ), cells were processed for experiments 10-12 days after infection, and excess cells were frozen. CD4+CAR-T and CD8+CAR-T cells were used for in vitro experiments, and CAR-T cells were used for in vivo animal experiments.

[0087] The expansion multiple of CAR-T cells after lentivirus infection of T cells is as follows Figure 4 As shown, CAR-T cells expanded up to 207.67±13.87 times (n=3) 12 days after transduction. The expansion times of sorted CD4+CAR-T and CD8+CAR-T cells were weaker than that of whole T cells, with expansion times of 53.67±3.51 and 64.67±4.51 times (n=3) 12 days after transduction, respectively.

[0088] 2. CAR-T cell CD3 phenotype analysis and transduction rate determination

[0089] (1) Analysis of CD3 phenotype of CAR-T cells

[0090] CD4+CAR-T and CD8+CAR-T cells were made into single cell suspensions and counted, 1-2×10 6 cells / flow cytometry tube, prepare two tubes for each cell type, and one tube as a negative control; after washing the cells twice with PBS, resuspend the cells with 100 μL Staining Buffer, add 5 μL anti-CD3-APC antibody, incubate in an ice box in the dark for 30 min, and shake once every 5 min; add 2 mL Staining Buffer to wash the cells, centrifuge at 2000 rpm for 5 min, and discard the supernatant; add 500 μL 1% paraformaldehyde to resuspend the cells, store in a 4°C refrigerator, and wait for flow cytometry detection.

[0091] The results of CD3 phenotype analysis of CD4+CAR-T and CD8+CAR-T cells are shown in Figure 5 As shown, the results showed that the percentage of CD3+T cells in both CD4+CAR-T and CD8+CAR-T cells was higher than 90%.

[0092] (2) CAR-T cell transduction rate determination

[0093] After two weeks of in vitro culture of CD4+CAR-T and CD8+CAR-T cells, the expression rate of eGFP in APC01-CAR-eGFP-T cells was detected to indirectly reflect the transduction rate of the target gene. The images were taken under a fluorescence microscope, and then the cells were made into single cell suspensions and counted, 1-2×10 6 The percentage of eGFP-positive cells was detected by flow cytometry, and uninfected T cells were used as negative control.

[0094] The results are as follows Figure 6 As shown, the transduction rates of the target gene were higher than 70% in both CD4+CAR-T and CD8+CAR-T cells.

[0095] Example 5: CAR-T cell proliferation experiment

[0096] This study selected androgen-independent prostate cancer with poor prognosis and few treatment options as the research object, used prostate cancer PC-3 cells as the research cell line, and set CD19-positive Raji cells as the positive control group. The specific operation is as follows:

[0097] Mitomycin (MMC) was used to inactivate target cells PC-3 and Raji, respectively. After washing the cells three times with 1640 medium, the target cells were plated in a 24-well cell culture plate and cultured overnight. CD4+CAR-T or CD8+CAR-T cells were stained with CFSE at a final concentration of 0.25 μM in the dark for 10 minutes. After staining was terminated, the cells were washed three times. CAR-T cells were co-incubated with inactivated target cells at a 1:1 effector-target ratio (E:T) for 5 days (no additional anti-human CD3 antibody, anti-human CD28 antibody, IL-2 and other factors were added to the culture system). CAR-T cells were collected and stained with anti-CD3-APC antibodies, and T cell proliferation was evaluated by flow cytometry. In addition, one tube of CD4+CAR-T or CD8+CAR-T cells was stained with CFSE at a final concentration of 0.25 μM before being put on the machine as a baseline analysis of CFSE.

[0098] The detection results of CD4+CAR-T and CD8+CAR-T cell proliferation after co-culture are as follows Figure 7 As shown, Figure 7 A in the figure shows the proliferation test results of CD4+CAR-T cells. Under the stimulation of target cells PC-3, the cell proliferation percentage of APC01 CAR-T cells can reach 91.67±2.54% (n=3) after 5 days, which is statistically significant compared with the CD19 CAR-T cell control group of 21.54±1.61% (n=3) (P<0.001). Figure 7 Figure B shows the results of CD8+CAR-T cell proliferation detection. Similarly, under the stimulation of target cells PC-3, the cell proliferation percentage of APC01 CAR-T cells was as high as 93.11±1.61% (n=3), which was significantly different from the CD19CAR-T cell control group of 20.70±2.32% (n=3) (P<0.001).

[0099] Example 6: Cytokine secretion experiment of CAR-T cells

[0100] This study used prostate cancer PC-3 cells as the research object, and set up CD19-positive Raji cells as the positive control group. The specific operation is as follows:

[0101] MMC inactivated target cells PC-3 and Raji respectively. After washing the cells three times with 1640 medium, the target cells were plated in a 48-well cell culture plate and cultured overnight. CD4+CAR-T or CD8+CAR-T cells were co-incubated with inactivated target cells at a 1:1 effector-target ratio for 24 hours (no additional anti-human CD3 antibody, anti-human CD28 antibody, IL-2 and other factors were added to the culture system). The supernatant of each well was collected and centrifuged at 3000 rpm for 10 minutes to remove cell debris. The standards and samples were stained according to the instructions of Human Th1 / Th2 Cytokine Kit II:

[0102] 1) Prepare and continuously dilute the standard:

[0103] ①Open a bottle of freeze-dried human Th1 / Th2 standard, transfer the standard pellet to a 15mL conical centrifuge tube, and mark the tube as "Top Standard"; ②Rewarm the standard in the "Top Standard" tube at room temperature for at least 15 minutes, and then dissolve the standard with 2.0mL Assay Diluent. Note that the recombinant protein can only be gently mixed with a pipette, and do not vortex violently; ③Label 8 12×75mm flow tubes and label the flow tubes in the following order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256; ④Pipette 300μL Assay Diluent into each tube in ③; ⑤Complete the serial dilution: a. From "Top Standard", add 100μL Assay Diluent to each tube; ⑤Complete the serial dilution: a. From "Top Standard", add 100μL Assay Diluent to each tube; a. Pipette 300 μL of liquid from the "Standard" tube into the 1:2 tube and mix gently; b. Pipette 300 μL of liquid from the 1:2 tube into the 1:4 tube, and so on until the 1:256 tube; c. Mix gently in the Pasteur tube without vortexing violently; ⑥ Prepare a 12×75 mm flow tube containing only Assay Diluent as a negative control tube of 0 pg / mL.

[0104] 2) Mix the Capture Beads before testing the sample:

[0105] ① Determine the number of assay tubes (including standards and controls) required for the experiment (for example, 8 unknown samples, 9 cytokine standard dilution samples and 1 negative control group, a total of 18 assay tubes); ② Before mixing the Capture Beads, vortex each bottle of Capture Bead liquid vigorously for a few seconds; ③ Add 10 μL of each bottle of Capture Bead to the flow tube marked "mixed Capture Beads" according to the dose of each tube to be analyzed (for example, 10 μL IL-2 Capture Beads × 18 assay tubes = 180 μL IL-2 Capture Beads are required); ④ Vortex the Capture Beads mixture thoroughly.

[0106] 3) Sample preparation and sample testing:

[0107] ① Thoroughly vortex the Capture Beads mixture and add 50μL of the mixture to all the assay tubes; ② Take 50μL of Th1 / Th2 cytokine standard dilution solution and add it to the corresponding tubes; ③ Take 50μL of the collected cell supernatant sample to be tested and put it into the marked sample tube; ④ Add 50μL Th1 / Th2-IIPE detection reagent to all the tubes to be tested; ⑤ Incubate all the tubes to be tested at room temperature in the dark for 3h; ⑥ Set the BD FACS Calibur flow cytometer program during the incubation period; ⑦ Add 1mL Wash Buffer to each assay tube and centrifuge at 200×g for 5min; ⑧ Carefully discard the supernatant in each assay tube and add 300μL Wash Buffer to each assay tube to resuspend the Capture Beads; ⑨ Sample analysis: In order to facilitate the analysis of samples by FCAPArray software, it is recommended to follow the following guidelines: a. From the lowest (0pg / mL) to the highest (Top b. Analyze the standard with the highest concentration, then analyze the sample to be tested; b. If the sample is diluted, start the analysis from the sample with the highest concentration; c. Store all FCS files (standards and samples) in one folder. ⑩ After all samples are analyzed, continue with data analysis, use FCAPArray software to create a standard curve, and calculate the concentration of each cytokine (see Figure 8 ).

[0108] Depend on Figure 8It can be seen that CAR-T cells can secrete high levels of various cytokines when co-incubated with inactivated corresponding target cells in vitro. CD4+APC01 CAR-T cells can secrete a large amount of IFN-γ (P < 0.001), TNF (P < 0.05) and IL-2 (P < 0.0001) under the stimulation of PC-3 cells, which is significantly different from CD4+CD19 CAR-T cells. In addition, we also detected the factor secretion of CD8+CAR-T cells. Compared with CD8+CD19 CAR-T cells, CD8+APC01 CAR-T cells can also secrete high levels of IFN-γ (P < 0.001), TNF (P < 0.01) and IL-2 (P < 0.0001) under the action of PC-3 cells, and the difference is statistically significant. However, CD4+ / CD8+APC01 CAR-T cells secrete less IL-4 and IL-6, and almost no IL-10 cytokines. It can be seen from the figure that APC01 CAR-T cells are activated under the stimulation of positive target cells, activating a series of signaling pathways and secreting a large amount of Th1 cytokines.

[0109] Example 7: Cytotoxic effect of APC01 CAR-T cells on prostate cancer PC-3 cells in vitro

[0110] This study used prostate cancer PC-3 cells (positive cell line) and lung cancer H460 cells (negative cell line) as research objects, and set CD19-positive Raji-eGFP-Luciferase cells as a positive control group. The specific operations are as follows:

[0111] (1) Construction of target cells

[0112] 24h in advance, a 24-well cell culture plate was coated with RetroNectin solution at a final concentration of 20 μg / mL; PC-3 or H460 cells were plated in a 24-well plate (5×10 4 cells); at an MOI of about 5, the prepared lentivirus pLV-EF-eGFP expressing eGFP was added to the PC-3 or H460 cell wells for infection, and the cells were placed in an incubator at 32°C, 5% CO2 concentration, and 95% humidity for culture. After 24 hours, the cells were transferred to an incubator at 37°C for culture; 1640 complete medium was added every two days, and eGFP-positive monoclonal cells were screened under a fluorescence microscope by multiple limiting dilution methods. After amplification and culture, the cells were photographed under a fluorescence microscope for preservation (the results are shown in Fig. 9 ); The newly constructed target cell lines PC-3-eGFP and H460-eGFP cells were frozen and stored in liquid nitrogen;

[0113] (2) Target cells PC-3-eGFP, H460-eGFP, and Raji-eGFP-Luciferase were plated in 24-well cell culture plates and cultured overnight;

[0114] (3) CD8+CAR-T cells were co-cultured with target cells at different effector-target ratios (E:T was 0.16:1, 0.31:1, 0.63:1, 1.25:1, 2.5:1, 5:1, and 10:1) (no additional anti-human CD3 antibody, anti-human CD28 antibody, IL-2, or other factors were added to the culture system);

[0115] (4) Observe the target cell death under a fluorescence microscope at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, and take photos (see the results). Fig.10 ).

[0116] Depend on Fig. 9 It can be seen that PC-3 cells and H460 cells infected with lentivirus pLV-EF-eGFP both expressed eGFP protein, indicating that the target cell lines PC-3-eGFP and H460-eGFP were successfully constructed.

[0117] Depend on Fig.10 It can be seen that in vitro, APC01 CAR-T cells can effectively and specifically kill PC-3 tumor cells.

[0118] Example 8: Quantitative analysis of the killing effect of CAR-T cells by flow cytometry

[0119] This study used prostate cancer PC-3 cells (positive cell line) and lung cancer H460 cells (negative cell line) as research objects, and set CD19-positive Raji cells as a positive control group. The specific operations are as follows:

[0120] The target cells PC-3, H460, and Raji cells were stained in the dark for 10 minutes each with CFSE at a final concentration of 0.5 μM. After the staining was terminated, the cells were washed three times, and the stained target cells were plated in a 24-well cell culture plate and cultured overnight; CD8+CAR-T cells were co-incubated with target cells at different effector-target ratios (E:T was 0.16:1, 0.31:1, 0.63:1, 1.25:1, 2.5:1, 5:1, and 10:1) (no additional anti-human CD3 antibody, anti-human CD28 antibody, IL-2, and other factors were added to the culture system); after 24 hours of co-incubation, the target cells were digested, the cells were washed once with PBS, the cells were resuspended with 1 mL of Staining Buffer, and the target cells were stained with 1 μL of 7-AAD at room temperature in the dark for 15 minutes; the lysis rate of the target cells was quantitatively analyzed by flow cytometry.

[0121] like Fig.11As shown in the figure, compared with CD19 CAR-T, under the action of APC01 CAR-T cells, more than 50% of PC-3 cells were killed when E / T was 0.63:1, and almost 100% of PC-3 cells were killed at 10:1 (P < 0.01), and the difference was statistically significant. In addition, even when the APC01 CAR-T cells had an efficient target ratio of 10:1, the lysis rate of the negative cell line H460 was only less than 10%, which further proved that APC01 CAR-T cells were specific. The experiment was repeated three times and the results were consistent.

[0122] Example 9: In vivo observation of the distribution of CAR-T cells

[0123] By constructing a prostate cancer mouse model, the distribution of APC01 CAR-T cells in vivo was observed. The specific operations are as follows:

[0124] 1. Selection of mice:

[0125] ①Advantages: The mouse model we selected is the severely immunodeficient mouse B- The mouse (NOD-PrkdcscidIL2rgtm1 / Bcgen) has a severe immunodeficiency phenotype. It is an IL2rg gene knockout mouse with a NOD-scid genetic background. It lacks mature T, B and NK cells. It is currently internationally recognized as a tool mouse with a high degree of immunodeficiency and is very suitable for the transplantation and growth of human hematopoietic stem cells and peripheral blood mononuclear cells.

[0126] ② Feed: Use the "SPF Mouse Growth and Reproduction Feed" produced by Keao Xieli, which has a protein content of 22.7%, a fat content of 4.7%, and has been irradiated with Co60.

[0127] ③ Drinking water: According to the standards of Jackson Lab, use acidified water (adjust the pH to 2.5-3.0 with HCl), and then sterilize it under high pressure to effectively prevent contamination by Pseudomonas and Staphylococcus aureus. During the breeding process, replace the water bottle every 3 days regardless of whether the water in the bottle is drunk or not.

[0128] ④ Bedding: Use wood shavings bedding, which is soft and fluffy, highly hygroscopic, dust-free, impurity-free, and odor-free, and is sterilized by high pressure. The bedding should be changed once a week on a clean bench, and sterilized tweezers should be used to transfer mice to new cages to avoid direct contact with the mice with your hands.

[0129] ⑤ Environment: Raise the fish under SPF level, ensure sufficient light intensity and lighting time during the breeding process, adopt a breeding mode of 12h light and 12h dark, maintain the ambient temperature at 20-26℃, humidity at 40%-70%, ventilation frequency at 15 times / h, and noise ≤60 decibels; the breeding cages should be cleaned and disinfected at least once a week.

[0130] ⑥All animal experiments followed the ethical guidelines for animal protection and animal experimentation in my country, and all animal experiment plans were reviewed and approved by the Ethics Committee of Harbin Medical University in accordance with relevant laws and regulations.

[0131] 2. Establishment and grouping of mouse model: 30 B-NDG male mice, aged 4-6 weeks, were selected and PC-3 cells (5×10 6 The subcutaneous transplanted tumor model was established by 30% cells / mouse. When the tumor volume reached about 80-100mm 3 Twenty-five mice with uniform tumor sizes were selected and randomly divided into five groups: APC01 CAR-T cell intratumor injection group, APC01 CAR-T cell tail vein injection group, mock transduced T cell intratumor injection group, mock transduced T cell tail vein injection group, and PBS intratumor injection group, with 5 mice in each group.

[0132] 3. Treatment: 20 days after tumor implantation (tumor volume 80-100mm 3 ) and treatment on day 27 (1×10 7 T cells / cell / time, CAR-T cells and mock transduced T cells were labeled with Luciferase reporter gene, and the PBS intratumoral injection group was given an equal volume of PBS).

[0133] 4. Imaging: Imaging was performed on days 21, 36, 51, and 66 after tumor inoculation. 300 μL of the catalytic substrate D-luciferin working solution (3 mg / mouse) was injected into the tail vein, and the distribution of T cells in tumor-bearing mice was observed using a small animal in vivo imaging device (see Fig.13 and Fig.14 ). The experimental flow chart for in vivo observation of CAR-T cell distribution is shown in Fig.12 .

[0134] Depend on Fig.13 and Fig.14 It can be seen that APC01 CAR-T cells can be localized and proliferated in tumor-bearing mice by intratumoral injection and tail vein injection.

[0135] Example 10: CAR-T cell anti-tumor experiment

[0136] This study established a prostate cancer mouse model to study the function of APC01 CAR-T cells in mice. The specific operations are as follows:

[0137] 1. Establishment and grouping of mouse model: The modeling and grouping methods were the same as those in Example 9, except that there were 7 mice in each group.

[0138] 2. Treatment: 20 days after tumor implantation (tumor volume 80-100mm 3 ) and treatment on day 27 (1×10 7 T cells / mouse / time, and the PBS intratumor group was given an equal volume of PBS).

[0139] 3. Observation indicators:

[0140] ① Measure the tumor size with a vernier caliper every two days and draw a tumor growth curve (see Fig.16 );

[0141] ② After the mice died or at the end of the experiment, they were killed and dissected to observe whether there was distant metastasis. The subcutaneous transplanted tumor, liver and lung tissues were taken and photographed (see Fig.17 and Fig.18 ), then embedded in paraffin, and HE and immunohistochemical staining were used to observe the histological characteristics of the tumor and the presence of tumor infiltration in the liver and lungs (the pathological results were independently reviewed by three pathologists) (see Fig.19 );

[0142] ③ Draw the survival curve of mice (see Fig. 20 );

[0143] ④ If the mice show any adverse symptoms or the tumor volume is greater than 1000mm 3 , will be euthanized. The flowchart of the mouse in vivo experiment is shown in Fig.15 .

[0144] Depend on Fig.16 and Fig.17 It can be seen that compared with the treatment group given mock transduced T cells, APC01 CAR-T cell therapy can significantly reduce tumor volume, and the therapeutic effect of the intratumoral injection group is better than that of the tail vein injection group.

[0145] Fig.18 It is the liver tissue of mice. Pathological confirmation showed that there was one case of liver metastasis in the APC01 CAR-T intratumor group and one case of liver metastasis in the tail vein group (1 / 7), while there were 6 cases of liver metastasis in the mock transduced T cell intratumor group (6 / 7), all cases of liver metastasis in the mock transduced T cell tail vein group (7 / 7), and 6 cases of liver metastasis in the PBS intratumor group (6 / 7).

[0146] Fig.19This is a typical case of HE and immunohistochemical staining of tumor, liver, and lung tissues. The results showed tumor cell metastasis in the liver tissue but not in the lung tissue.

[0147] Depend on Fig. 20 It can be seen that compared with mock-transduced T cells, APC01 CAR-T cells can significantly prolong the survival of mice.

[0148] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A CAR targeting prostate cancer, characterized in that: The CAR comprises the amino acid sequence V shown in SEQ ID NO.2 H , hinge region (Gly4Ser) 3 as shown in SEQ ID NO.6 and V as shown in SEQ ID NO.4 L scFv fragment composed of.

2. The CAR according to claim 1, characterized in that: The CAR comprises, from N-terminus to C-terminus: human cell surface glycoprotein T8 signal peptide, scFv fragment, human CD8α hinge region and transmembrane domain, 4-1BB signal transduction domain and CD3ζ signal transduction domain.

3. A lentiviral recombinant expression vector comprising the CAR described in any one of claims 1 or 2.

4. The method for constructing the lentiviral recombinant expression vector according to claim 3, characterized in that: The construction method is to connect CAR to a linearized third-generation lentiviral expression vector pLV-EF.

5. A lentivirus comprising the CAR described in any one of claims 1 or 2.

6. Use of the CAR according to any one of claims 1 or 2, the lentiviral recombinant expression vector according to claim 3, or the lentivirus according to claim 5 in the preparation of CAR-T cells targeting prostate cancer.

7. A CAR-T cell targeting prostate cancer, characterized in that: The CAR-T cells are prepared by infecting primary human T lymphocytes with the lentivirus according to claim 5.

8. The method for preparing CAR-T cells according to claim 7, characterized in that: The steps include: S1. The lentiviral recombinant expression vector containing the target gene CAR in claim 3, the packaging plasmid p-gp, and the envelope plasmid p-VG are used to form a three-plasmid lentiviral packaging system, and the lentiviral recombinant expression vector containing the target gene CAR in claim 3, the packaging plasmid p-gp, and the envelope plasmid p-VG are transfected into 293T cells according to the calcium phosphate precipitation transduction method, and the supernatant is collected after incubation; S2, concentrating the supernatant obtained in S1 to obtain a high-titer lentiviral solution; S3. Use the lentiviral solution obtained in S2 to infect activated human primary T lymphocytes to obtain CAR-T cells.

9. A drug containing the CAR-T cell according to claim 7, characterized in that: The medicament further comprises a pharmaceutically acceptable carrier.

10. Use of the CAR according to any one of claims 1 or 2, the lentiviral recombinant expression vector according to claim 3, the lentivirus according to claim 5, or the CAR-T cell according to claim 7 in the preparation of a cell medicine for treating prostate cancer.

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