CN108239144B

CN108239144B - Modified hinge and application thereof in constructing CAR framework

Info

Publication number: CN108239144B
Application number: CN201810075905.3A
Authority: CN
Inventors: 张巍; 赵文旭; 陈军; 黄霞; 赵永春; 单娟娟; 徐艳敏; 张茜真
Original assignee: Chongqing Precision Biotech Co ltd
Current assignee: Chongqing Precision Biotech Co ltd
Priority date: 2018-01-26
Filing date: 2018-01-26
Publication date: 2021-05-25
Anticipated expiration: 2038-01-26
Also published as: CN108239144A; CN112125976A; CN112125976B

Abstract

The invention belongs to the field of immunotherapy, and particularly relates to a modified hinge region and application thereof in construction of a CAR framework. The hinge region provided by the invention can prolong the in vivo persistence of the CAR-T cell and/or improve the tumor infiltration capacity of the CAR-T cell, and the amino acid sequence of the hinge region is shown as SEQ ID No.1 and SEQ ID No. 2. The hinge region structure provided by the invention can prolong the in vivo survival time of CAR-T cells, the chimeric antigen receptor combined with the hinge region structure can be more stably expressed in T lymphocytes, the CAR-T containing the hinge region structure has longer in vivo survival time, the ability of the cells to infiltrate tumors is remarkably enhanced, and the killing effect is better.

Description

Modified hinge and application thereof in constructing CAR framework

Technical Field

The invention belongs to the field of immunotherapy, relates to a modified hinge region and application thereof in constructing a CAR skeleton, and particularly relates to a hinge region capable of prolonging the survival of CAR-T cells in vivo and/or improving the tumor infiltration capacity of CAR-T cells and application thereof in constructing a CAR skeleton.

Background

Chimeric Antigen Receptors (CARs) are artificial receptors that mimic TCR function and comprise an antigen recognition domain, a hinge region, a transmembrane region, and an intracellular signal domain. The intracellular signaling domain is typically the CD3 zeta chain or FcR gamma, or is associated with one or more costimulatory molecules, such as 4-1BB (CD137), CD28, ICOS (CD 278). When an antigen (receptor) on the surface of a tumor cell is bound to an antibody (ligand) of a chimeric antigen receptor, a signal is transmitted into the cell through a hinge region and a transmembrane region, an intracellular signal region converts the signal into an activation signal, an effector cell is activated, and the effector cell proliferates to produce a cytokine, thereby killing the tumor cell.

In recent years, chimeric antigen receptor T lymphocytes (CAR-T) have shown significant therapeutic effects in tumor therapy, particularly in the treatment of CD 19-positive malignancies. However, even for Acute Lymphoblastic Leukemia (ALL) with remarkable treatment effect, median time for complete remission after treatment is generally about 8 months, a large number of patients still have relapse, which is probably related to short survival time of CAR-T cells in the patients, and the improvement of the survival time of CAR-T cells in the patients has great significance for the curative effect of CAR-T treatment.

Although the study of solid tumors using CAR-T is also gaining attention, most CAR-T treatments are unsatisfactory in their effectiveness, on the one hand, because CAR-T cells are poorly maintained in vivo and die quickly, affecting killing effectiveness; on the other hand, most solid tumors have little effect on the treatment of the large solid tumors because the CAR-T cells are difficult to infiltrate into the tissues of the solid tumors due to the pluripotency of the solid tumors in metabolism, immune escape and tissue formation and the control and killing of the solid tumors by the immune system are limited in multiple aspects. Prolonging CAR-T survival in vivo, promoting CAR-T cell infiltration in solid tumor tissue, being able to effectively kill tumors and inhibit tumor cell recurrence, but there is currently no method to effectively prolong CAR-T survival in vivo and promote CAR-T cell tumor infiltration.

Disclosure of Invention

In view of the above, the present invention provides a hinge region capable of prolonging the survival of CAR-T cells in vivo and/or improving the tumor infiltration capacity of CAR-T cells, wherein CAR-T cells containing the hinge region have a longer survival time in vivo, significantly enhanced tumor infiltration capacity of cells, and a better killing effect.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a hinge region capable of prolonging the survival of the CAR-T cell in vivo and/or improving the tumor infiltration capacity of the CAR-T cell, wherein the amino acid sequence of the hinge region is shown as SEQ ID No.1 or SEQ ID No. 2.

The nucleotide sequence of the hinge region is shown as SEQ ID NO.20 or SEQ ID NO. 19.

When the antigen (receptor) on the surface of the tumor cell is combined with the antibody (ligand) of the chimeric antigen receptor, signals are transmitted to the inside of the cell through a hinge region and a transmembrane region, an intracellular signal domain converts the signals into activation signals, effector cells are activated, and the effector cells proliferate, generate cytokines and perform a series of activation reactions to kill the tumor cell. Through research on the transformation of the hinge region of the CAR structure, the applicant finds that the hinge region structure is closely related to the in vivo continuity and tumor infiltration capacity of CAR-T.

The hinge region, also known as the spacer or hinge, connects the CAR antigen recognition region and the transmembrane region. The hinge region should be sufficiently flexible to allow the antigen binding region to be oriented in different directions to facilitate antigen recognition. Hinge regions of different lengths play different roles in the stability of CAR-T, and applicants have unexpectedly discovered through various comparative combinations that hinge regions have a significant impact on CAR-T survival and tumor infiltration in vivo; through a large amount of screening work, hinge sequences SEQ ID NO.1 and SEQ ID NO.2 which can effectively prolong the survival of the CAR-T body and promote the tumor infiltration of the CAR-T cells are finally obtained and named as: 7H and G4HH2H3 mt.

The inventor designs 5 hinge structures together: 7H, G4Hinge, G4HH3, G4HH2H3mt and DH, the amino acid sequences of 7H and G4HH2H3mt are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the nucleotide sequences are shown in SEQ ID NO.20 and SEQ ID NO.19, respectively.

Comparing CAR-T CAR positive rates, CAR-T in vitro killing, and CAR-T in vivo persistence and/or CAR-T tumor infiltration of the currently commonly used hinge structures G4H (G4HH2H3) and 8H, respectively, combined, CAR-T is found to have longer in vivo persistence for CAR-T comprising 7H or G4HH2H3mt hinge structure CAR, and significantly enhanced CAR-T tumor infiltration capacity and better killing effect compared to CAR-T containing G4HH2H3 or 8H hinge.

G4HH2H3 amino acid sequence:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

8H hinge amino acid sequence:

KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

a hinge modification method for prolonging the survival of CAR-T cells in vivo and/or improving the tumor infiltration capacity of CAR-T cells comprises the step of performing hinge region modification by using a G4HH2H3 sequence as a template in a site-specific mutation mode.

Another object of the present invention is to provide a method for modifying the hinge region, which comprises modifying the hinge region using the G4HH2H3 amino acid sequence as a template, wherein the G4HH2H3 amino acid sequence is as shown in SEQ ID NO.3, by means of PCR-mediated site-directed mutagenesis

The hinge sequence G4HH2H3mt was further obtained according to the above described engineering method, characterized in that the G4HH2H3mt is G4HH2H3 amino acid sequence mutated and/or deleted at positions 15-18 and 79.

It is a further object of the present invention to provide a chimeric antigen receptor comprising the hinge region described above. The hinge region is 7H or G4HH2H3 mt.

Further, the chimeric antigen receptor further comprises an antigen recognition region, a transmembrane region, and an intracellular signal domain.

Further, the antigen recognition region of the chimeric antigen receptor may recognize tumor cell expressed antigens including, but not limited to, PSCA, PSMA, CD19, BCMA, CD123, CD20, CD22, CEA, EGFR, EGFRVIII, GPC3, or mesothelin antigenic molecules.

The tumor cell expression antigen that can be recognized by the chimeric antigen receptor includes, but is not limited to, the above antigen molecules.

The invention also aims to provide a chimeric antigen receptor targeting PSCA, which comprises a single-chain antibody of anti-human PSCA antigen, a hinge region, a transmembrane region and an intracellular signal region; the hinge region is 7H or G4HH2H3mt, and the amino acid sequence of the hinge region is shown as SEQ ID NO.1 or SEQ ID NO. 2.

Furthermore, the amino acid sequence of the anti-human PSCA antigen single-chain antibody is shown as SEQ ID NO.4 or SEQ ID NO. 5.

Further, the transmembrane region is CD28TM or CD8TM, the amino acid sequence of the CD28TM is shown as SEQ ID NO. 6, and the amino acid sequence of the CD8TM is shown as SEQ ID NO. 7; the intracellular signal domain is CD28 and/or CD137 and/or CD3, the amino acid sequence of the CD28 is shown as SEQ ID NO. 8, the amino acid sequence of the CD137 is shown as SEQ ID NO. 9, and the amino acid sequence of the CD3 is shown as SEQ ID NO. 10.

Furthermore, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO.11 or SEQ ID NO.12 or SEQ ID NO.13 or SEQ ID NO.14 or SEQ ID NO.15 or SEQ ID NO. 16.

The invention also aims to provide a preparation method of the viral vector targeting the PSCA chimeric antigen receptor, which comprises the following steps:

1) nucleotide sequence for the synthesis of chimeric antigen receptor targeting PSCA: synthesizing a chimeric antigen receptor nucleic acid sequence comprising a leader peptide, a single chain antibody to human PSCA antigen, a hinge region, a transmembrane region, and an intracellular signal domain; the leader peptide has a nucleic acid sequence shown as SEQ ID NO. 21;

2) construction of a viral vector expressing a chimeric antigen receptor: designing a primer, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO. 22, the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 23, and performing PCR amplification by taking the gene sequence of the chimeric antigen receptor as a template to obtain a DNA fragment;

and (2) carrying out double enzyme digestion on the gene sequence of the DNA fragment by using restriction enzyme, simultaneously carrying out enzyme digestion on the virus expression vector pCDH-CAG by using the restriction enzyme, and then connecting the enzyme digested target fragment and the virus expression vector fragment by using T4 ligase to obtain the virus vector for expressing the chimeric antigen receptor. Such viral vectors include, but are not limited to, adenoviruses, retroviruses, and lentiviruses.

Preferably, the restriction enzymes are NheI and SalI.

Further, the nucleotide sequence of the chimeric antigen receptor in the step 1) is shown as SEQ ID NO.17 or SEQ ID NO. 18.

Further, after step 2), packaging and purifying the viral vector, which is a lentiviral vector.

The invention also aims to provide a lentivirus vector obtained by the preparation method.

The lentiviral vector is obtained under the method, and the expression rate of positive cells transduced by the lentiviral vector is high, the lentiviral vector is stable in the cell culture process of a patient, and the CAR positive rate is not reduced along with the time. A cell infected with the lentiviral vector, such cell having a function of killing a target cell.

It is also an object of the present invention to provide a cell infected with said lentiviral vector, said cell including but not limited to T lymphocytes, NK cells and DC cells.

Further, the cells infected with the lentiviral vector are T cells.

The invention also aims to provide application of the CAR-T cell infected by the lentiviral vector in preparing antitumor drugs.

Further, the tumor cell or tissue is capable of expressing PSCA.

The invention also aims to provide application of the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 as a hinge region in constructing the CAR skeleton.

In certain embodiments the hinge region of the chimeric antigen receptor may be a sequence engineered from the G4HH2H3 sequence, such as a chimeric antigen receptor comprising a hinge region that lacks the amino acid sequence of segment H2H3 of the G4HH2H3 sequence; in certain embodiments, the hinge region sequence of the chimeric antigen receptor is a sequence lacking H2 stretch from the G4HH2H3 sequence, with the specific sequences shown in table 1.

TABLE 1G 4HH2H3 hinge sequence modifications

In certain embodiments, the antigen recognition region of the chimeric antigen receptor comprising the hinge structure can be any polypeptide that can bind to a PSCA antigen, such as a ligand that specifically binds to PSCA, a bispecific antibody, a scFV, an optionally cross-linked Fab, a F (ab)2, a single domain antibody, and a scFV linked to a His-tag or an HA-tag. In certain embodiments the antibody that specifically recognizes PSCA is derived from 7F 5; in certain embodiments, the amino acid sequence of the antigen recognition region is as follows:

QVKLQESGGGLVQPGGSLKLSCVASGFTFSSYTMSWVRRTPEKRLEWVAYIHNGGGHTYYPDTIKGRFTISRDNAKNTLFLEMSSLKSEDTAMYYCTRRMYYGNSHWYFDVWGAGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAPKLLIYYTLKLNSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSKTLPWTFGGGTKVEIK

in certain embodiments, the amino acid sequence of an antibody that specifically recognizes PSCA is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIK

DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSS

DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGGGGSGGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSS

the invention has the beneficial effects that:

1) the hinge region structure provided by the invention can prolong the survival time of CAR-T cells in vivo.

2) The chimeric antigen receptor combined by the hinge region structure provided by the invention can be more stably expressed in T lymphocytes, has better capacity of removing tumor cells, can maintain the positive rate of the chimeric antigen receptor targeting PSCA in the cell culture process of a patient, can enhance the proliferation and tumor killing capacity of CAR-T, has no toxic or side effect on antigen-negative cells, and can be used for targeted therapy of tumors.

3) The CAR-T cells containing the hinge structure provided by the invention have enhanced ability to infiltrate tumor tissues, and can effectively kill solid tumors.

Drawings

FIG. 1 is a diagram of the structure of a CAR with different hinge regions and their composition.

FIG. 2 is the CAR expression rates for different hinge region combinations.

FIG. 3 is the CAR-T cell CAR mean fluorescence intensity measurements for different hinge region combinations.

FIG. 4 is the long-term proliferation of CAR-T cells with different hinge region combinations.

FIG. 5 is the CAR-T cell killing rate for different hinge region combinations.

Figure 6 CAR-T cell CAR expression stability assays for different hinge region combinations.

FIG. 7 is a comparison of the killing effect of CAR-T cells in vivo with different hinge region combinations.

FIG. 8 is a study of the in vivo persistence and tumor infiltration capacity of CAR-T cells with different hinge region combinations.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental procedures, in the preferred embodiments, which do not specify specific conditions, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers. The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

EXAMPLE 1 hinge region engineering

The hinge region was engineered by site-directed mutagenesis using the G4HH2H3 hinge region derived from IgG4FC sequence as a template. The results of the transformation are shown in fig. 1: g4HH2H3mt is an engineered sequence obtained by mutating G4HH2H3 at the 15 th to 17 th positions and the 79 th position and deleting the 18 th position; g4HH3 is a deletion of H2 sequence; g4Hinge is a modification obtained by deleting H2H3 sequence. The hinge region sequences 7H and DH derived from human CD7 and human IgD were obtained simultaneously. FIG. 2 shows the sequences of the different hinge regions and the sites of modification.

Example 2 construction of chimeric antigen receptor viruses with different hinge regions

To test the effect of different structures of the hinge region on CAR-T in vivo persistence and tumor infiltration, experiments were conducted using PSCA-targeted CAR-T as an example. The CAR was designed for 5 different hinge structures as shown in FIG. 1.

1 Synthesis of PSCA-targeted chimeric antigen receptor Gene sequence containing the hinge sequence designed above

A CAR structure was synthesized containing a leader peptide (also called signal peptide) (abbreviated LP), a single chain antibody against human PSCA antigen, 6 different hinge regions, a CD28 transmembrane region (abbreviated TM), and CD28, CD137 and CD3 intracellular signal domains.

2 construction of a Lentiviral vector expressing a chimeric antigen receptor

The following primers were designed and synthesized by Biotech, Inc., and the specific primers were as follows:

primer 1: 5' -atcgctagcatggccctgccagtgaccgcc-3', the NheI restriction endonuclease site is underlined;

primer 2: 5' -ccaggtcgacttagcgagggggcagggcctg-3', the SalI restriction enzyme site is underlined.

Then, the sequences shown above are used as primers, the sequences of the synthesized chimeric antigen receptors are used as templates for PCR amplification, the reaction system is loaded according to the instructions of KOD FXNEO DNA polymerase (purchased from TOYOBO company), the amplified products are identified and then DNA fragments are recovered by a recovery kit (Promega company), the specific method is shown in the instructions, the chimeric antigen receptors are recovered, and the DNA recovered fragments are sent to biotechnology company for sequencing.

The cloned gene sequence encoding the chimeric antigen receptor was digested simultaneously with restriction enzymes NheI and SalI (from Thermo) and the lentiviral expression vector pCDH-CAG (from Addge Plasmid) with restriction enzymes NheI and SalI, and the digestion was carried out as described in the specification. The enzyme digestion product is separated by agarose gel electrophoresis, DNA fragment recovery is carried out by an agarose gel DNA fragment recovery kit, and then the target fragment and the vector fragment are connected by T4 ligase (purchased from Promega company) to obtain the lentiviral vector expressing the chimeric antigen receptor, which is named as Lv-hinge. The lentiviral vector was transformed into E.coli TOP10, and after 12h of monoclonal culture, plasmids were extracted using a plasmid extraction kit (Invitrogen), the specific procedure being described in the specification.

7 lentiviral vectors were constructed separately as above:

“scFv-8H hinge-CD28TM-CD28-CD137-CD3Z”；

“scFv-7H hinge-CD28TM-CD28-CD137-CD3Z”；

“scFv-DH hinge-CD28TM-CD28-CD137-CD3Z”；

“scFv-G4HH2H3 hinge-CD28TM-CD28-CD137-CD3Z”；

“scFv-G4HH2H3mt hinge-CD28TM-CD28-CD137-CD3Z”；

“scFv-G4HH3 hinge-CD28TM-CD28-CD137-CD3Z”；

“scFv-G4Hinge-CD28TM-CD28-CD137-CD3Z”。

packaging of lentiviruses

The lentivirus packaging in this example was performed using the calcium phosphate method, and the specific procedures are described in the molecular cloning protocols (third edition, sambrook et al).

4 purification of lentiviruses

Collecting the virus supernatant in a 50ml centrifuge tube, centrifuging and filtering, and centrifuging the filtrate at 3000r/min for 10min to obtain a new 50ml centrifuge tube; adding 50% PEG6000 and 4M NaCl according to the virus supernatant, fixing the volume with medical saline to make the final concentration of PEG6000 be 8.5% and the final concentration of NaCl be 0.3M, standing in a refrigerator at 4 deg.C after fixing and dissolving, centrifuging at 4 deg.C, discarding the supernatant, resuspending virus with 200 μ l DMEM culture medium, subpackaging with 1.5ml EP tube, 40 μ l per tube, and storing at-80 deg.C for later use.

5 Lentiviral titre assay

Step 1 Virus infection of 293T cells

293T cells are plated before infection, 1 mu l of purified virus is taken and diluted by 10 times by medical normal saline, 1 mu l of Polybrene (Polybrene) solution is added into each hole of the cells, then the Polybrene solution is respectively added into the 293T cells, 24h later, the solution is replaced by DMEM medium containing 10% FBS (wt), the cells are collected by centrifugation for 5min at 1000r/min after 72h of infection, and the genome is extracted.

Step 2 extraction of genome

The genome extraction Kit was QIAamp DNA Blood Mini Kit purchased from Qiagen (cat # 511004), and was used according to the Kit instructions.

Step 3, qRT-PCR determination of virus titer

The reaction system is as follows: premix Ex TaqTM II (2 ×)10 μ l, upstream primer (GAGup) 1 μ l, downstream primer (GAGdn) 1 μ l, extracted genome 1 μ l, RNase-Free dH2O 7 μ l, at least 3 replicate wells per sample, standard. Amplification was then performed as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, after the reaction is finished, analyzing data by using analysis software, and calculating the virus titer according to a standard curve.

Example 3 CAR transfection of T lymphocytes ability assay

1 isolation of human peripheral blood mononuclear cells

Collecting peripheral blood about 60ml by using a blood collection tube, subpackaging in a 50ml centrifuge tube, and adding 7.5ml hydroxyethyl starch for dilution; naturally settling for about 30min at room temperature (18-25 ℃), collecting upper layer plasma, centrifuging the collected upper layer plasma, then resuspending and precipitating the collected upper layer plasma with physiological saline, adding the supernatant onto lymphocyte separation liquid according to the volume ratio of 1:1, and performing gradient centrifugation; after centrifugation, the centrifuging tube divide into from top to bottom: a first layer: a plasma layer; a second layer: a layer of annular opalescent lymphocytes; and a third layer: a transparent separation liquid layer; a fourth layer: a layer of red blood cells; and taking the second white lymphocyte layer, washing the second white lymphocyte layer with normal saline for 2 times, centrifuging the second white lymphocyte layer for 5min, resuspending the cells by the normal saline, and adding RPMI 1640 containing 10% FBS into the complete culture medium to culture the cells to obtain the human peripheral blood mononuclear cells.

2 Lentiviral vectors infecting T lymphocytes

Freshly prepared mononuclear cell PBMC were cultured in RPMI 1640 complete medium containing 10% fetal bovine serum and PBMC activation was performed on day 1; lentivirus infection was performed on day 3; adding a lentiviral vector corresponding to 5MOI, and taking uninfected T lymphocytes as a blank control; after 24h, the medium was replaced with RPMI 1640 complete medium containing 500IU/ml recombinant human IL-2, and the culture was continued for 10-20 days. CAR-T cells expressing a chimeric antigen receptor comprising an antigen recognition region, a hinge region, a transmembrane region, and an intracellular signal domain were obtained, named after the engineered hinge region, named: PSCA-CAR-G4HH2H3, PSCA-CAR-G4HH2H3mt, PSCA-CAR-G4HH3, PSCA-CAR-G4 hinde, PSCA-CAR-7H, PSCA-CAR-8H, and PSCA-CAR-DH; for ease of labelling when mapping, the above CAR-T cells are abbreviated in the examples and figures of the specification below with G4HH2H3, G4HH2H3mt, G4HH3, G4 hindge, 8H, 7H and DH.

1) Centrifuging the virus-infected T cells cultured for 10 days in the culture process to discard the supernatant so as to collect the cells; the cells were resuspended in PBS containing 1% fetal bovine serum by volume and adjusted to a density of 1X 10⁶Per ml; and (3) respectively subpackaging the collected cells, and detecting the Protein-L positive rate by using flow cytometry, wherein the detection result shows that different CAR combinations express positive rate in T lymphocytes on the 10 th day of culture, and the result of CAR mean fluorescence intensity can be obtained by analyzing flow detection MFI (mean fluorescence intensity).

As a result, as shown in fig. 2, it can be seen that the positive expression rate of CARs of the engineered hinge structures G4HH2H3mt, G4HH3, G4Hing, 7H, and DH combinations on the T cell surface was not much different from CARs of the conventional hinge G4HH2H3 or 8H combinations. FIG. 3 shows that CAR-T cell surface CAR expression mean fluorescence intensity of different hinge structures, where CAR with G4HH2H3, G4HH2H3mt or 7H structure can be more highly expressed on T cell surface and higher surface mean fluorescence intensity indicates that CAR molecule is more expressed on cell surface; the CAR surface mean fluorescence intensity of G4HH3, G4Hinge or DH structure was low.

2) Detecting the CAR expression of different hinge structure combinations by using the detection method of 1) at 5 days, 10 days and 14 days after the infection of the CAR-T cells respectively, and detecting the stability of the CAR expression of the CAR-T cells after long-term culture.

The results are shown in FIG. 6: long-term expression of CAR was examined and it was found that the engineered structures had poor CAR expression stability compared to the non-engineered structures G4HH2H3 and 8H, with a significant decrease in CAR positivity after day 5 and more significant decrease with longer culture time, with little difference in CAR stability for the remaining engineered hinge structures combined with the non-engineered hinges.

Example 4 CAR-T cell Long term proliferation Capacity assay

Detecting proliferation of infected PSCA-CAR-T cells under normal culture conditions; the CAR-T cells obtained in example 3 were cultured for 10 days using the culture method in step 2 of example 3. Four degrees overnight in 24-well plates coated with PSCA antigen, CAR-T cells 1 x 10^ 6/well plated cells in 24-well plates coated with PSCA antigen, and the duration of CAR-T cells after antigen stimulation was observed. CAR-T cell numbers were counted and CAR-T cell proliferation fold calculated using a cytometric instrument at day 3, day 6, day 9 and day 12 post stimulation, respectively, as shown in figure 4. According to the experience of in vivo experiments, antigen stimulation is carried out every 7 days, and the result in figure 4 shows that the CAR-T cells do not proliferate after 3 times of antigen stimulation, so the experiment is stopped after 3 times of stimulation and 15 days, the CAR-T long-term proliferation fold statistics are carried out, and the long-term proliferation responses are the survival of the CAR-T cells.

Experiments as shown in fig. 4, CAR-T cells with the combination of G4HH2H3, G4HH2H3mt, 7H, and 8H hinges proliferated relatively rapidly for long term and were more potent in vitro in CAR-T cells; DH. G4Hinge and G4HH3 have poor long-term proliferation ability.

Example 5 Effect of CAR-T cells on tumor cell killing Capacity

The killing capacity of CAR-T with different hinge structures to target cells is completed by an ACEA xCELLigence RTCA MP instrument, and the experimental steps are carried out according to the instruction of the instrument; the first day, target cells (tumor cells expressing PSCA) are plated in a 96-well plate equipped with the device in a mode of 2-5 x 10^4 per well, the tumor cells attached to the bottom of the well are recorded every 15 minutes by taking the electrical resistance index as data, corresponding CAR-T cells are plated in each well according to the pre-designed effective target ratio after 24 hours, the electrical resistance index is recorded every 15 minutes after the CAR-T cells are plated, and the proliferation or death condition of the attached target cells is judged according to the electrical resistance index. The result formula of the resistance index analysis is as follows: CAR-T cell killing rate-baseline resistance index-real-time resistance index.

HeLa and RT4 are tumor cell lines with high expression of PSCA, and T24 is a negative control cell which does not express PSCA.

The results of the experiment are shown in FIG. 5: the 7H has better killing capability, has strong killing capability to RT4 tumor cells which are sensitive to T cells, has the killing capability to Hela tumor cells which are not sensitive to T cells, is more equivalent to RT4, does not kill negative cells, and has strong specificity.

Combining the above experiments, CAR stability, long-term proliferation and in vitro killing ability of the combination of DH, G4Hinge and G4HH3 Hinge structures were poor without subsequent evaluation, followed by in vivo experimental validation with CAR-T cells comprising G4HH2H3, G4HH2H3mt, 7H and 8H Hinge structures, respectively.

Example 6 validation of antitumor Effect of CAR-T cells in animal models

A mouse transplantation tumor model of a human PSCA positive tumor cell line is established for verifying the anti-tumor effect of T lymphocytes expressing a chimeric antigen receptor of the targeting PSCA in an animal model.

Cg-PrkdcscidiI2rgtm1Sug/Jiccrl, NOG mouse for short, which is bred by Mamoru Ito of the Japanese institute of Experimental animals (CIEA) is the most common strain for CAR-T related tumor formation experiments internationally. The tumor-forming target cells used in vivo verification are PSCA positive cell line Hela (Hela-luc for short) which is used in early in vitro verification and stably expresses firefly luciferase.

The modified hinge G4HH2H3mt and 7H structure has better killing and in vitro survival ability through previous in vitro experiments, so that the 2 CAR-T cells have the tumor killing ability in mice in animal experiments compared with the CAR-T cells taking the common G4HH2H3 and 8H as the hinge. The effector cells injected for treatment were CAR-T cells containing G4HH2H3mt, G4HH2H3, 7H and 8H hinge structures, and the control was normal saline group, PBMC cells not infected with virus.

Posttumoral tail vein injection of effector CAR-T cells 5 x 10^5 cells/mouse. Tumor growth was visualized by imaging every 7 days after CAR T cell injection by IVS in vivo imaging system from PerkinElmer. Mice survival was observed daily during this period and recorded, and the results are shown in FIG. 7A: CAR-T cells comprising the G4HH2H3mt or 7H hinge have a better killing effect on tumor cells in vivo. Tumor tissue was further removed after sacrifice of mice at the end of the experiment and weighed and the results counted, as shown in figure 7B: similar to the results of figure 7A, CAR-T cell treated mice comprising either G4HH2H3mt or the 7H hinge were less tumor-weighted, especially the CAR-T cell treated mice with the 7H hinge combination were substantially cleared of tumor.

Example 7 CAR-T cells survival in vivo and tumor infiltration Capacity Studies

CAR-T cell persistence in vivo and CAR-T cell ability to infiltrate tumor, was tested by detecting CAR gene copy number of tumor tissue using RT-PCR.

1. Designing a primer:

BBZ-HF：CAGAAGAAGAAGAAGGAGGATGTG；

BBZ-HR:TACTCCTCTCTTCGTCCTAGATTG。

2. tumor tissue RNA extraction

Adding liquid nitrogen into a mortar, cutting tumor tissues into small pieces, grinding the small pieces into powder in the liquid nitrogen, taking a proper amount of tissue powder by using a medicine spoon precooled by the liquid nitrogen, adding the tissue powder into an EP tube filled with Trizol liquid, and fully and uniformly mixing. After 5min at room temperature, 200ml of chloroform were added, the EP tube was closed and shaken vigorously for 15 seconds. The upper aqueous phase was centrifuged off in a new EP tube, 500ml of isopropanol were added and mixed by gentle inversion. Centrifuge at room temperature for 10 min. The supernatant was carefully discarded, 1ml of 75% ethanol was added, vortexed, mixed, and centrifuged once again. And discarding the supernatant, and drying at room temperature or in vacuum for 5-10 min. RNA was dissolved in 30ml DEPC treated water, stored in 70% ethanol and stored at-70 ℃.

3.RT-PCR

RT-PCR was performed in the following reaction system:

the reaction system is as follows: forward primer (10. mu.M) 0.5. mu.l, Reverse primer (10. mu.M) 0.5. mu.l, 2 XSSYBR Premix Ex Taq II 10. mu.l, Template 1. mu.l, extracted genome 1. mu.l, RNase-Free dH2O 7. mu.l, at least 3 replicate wells per sample, standard. Amplification was then performed as follows: 2min at 95 ℃, 15s at 95 ℃ and 1min at 60 ℃ for 40 cycles, and after the reaction is finished, analyzing data by using analysis software, wherein the analysis results are shown in table 3 and fig. 8.

Table 3 is the CAR copy number detected in CAR-T cell treated tumor tissues of different structures, and figure 8 is a graph plotted against the results of table 3. The results show that 7H or G4HH2H3mt as a hinge region can significantly enhance the ability of CAR-T to infiltrate tumors.

TABLE 3 CAR-T in vivo persistence of different hinge outcomes 45 days after reinfusion of CAR-T cells

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

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<223> PSCA-CAR4

<400> 16

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg

20 25 30

Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg

35 40 45

Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

80 85 90

Ser Ser Ser Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

95 100 105

Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly

110 115 120

Ser Thr Lys Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

125 130 135

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

140 145 150

Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His Trp Val Arg Gln Ala

155 160 165

Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile Asp Pro Glu Asn

170 175 180

Gly Asp Thr Glu Phe Val Pro Lys Phe Gln Gly Arg Ala Thr Ile

185 190 195

Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser

200 205 210

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Lys Thr Gly Gly

215 220 225

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys

230 235 240

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln

305 310 315

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

320 325 330

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

335 340 345

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

350 355 360

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

365 370 375

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

380 385 390

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

395 400 405

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

410 415 420

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

425 430 435

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

440 445 450

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

455 460 465

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

470 475 480

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys

485 490 495

Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg

500 505 510

Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

515 520 525

Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Phe Ser Arg Ser Ala

530 535 540

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

545 550 555

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

560 565 570

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

575 580 585

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

590 595 600

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

605 610 615

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

620 625 630

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

635 640

<210> 17

<211> 1485

<212> DNA

<213> Artificial

<220>

<223> PSCA-CAR -7H

<400> 17

gatattcagc tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60

attacctgca gcgcgagcag cagcgtgcgc tttattcatt ggtatcagca gaaaccgggc 120

aaagcgccga aacgcctgat ttatgatacc agcaaactgg cgagcggcgt gccgagccgc 180

tttagcggca gcggcagcgg caccgatttt accctgacca ttagcagcct gcagccggaa 240

gattttgcga cctattattg ccagcagtgg agcagcagcc cgtttacctt tggccagggc 300

accaaagtgg aaattaaagg cagcaccagc ggcagcggca aaccgggcag cggcgaaggc 360

agcaccaaag gcagcgaagt gcagctggtg gaaagcggcg gcggcctggt gcagccgggc 420

ggcagcctgc gcctgagctg cgcggcgagc ggctttaaca ttaaagatta ttatattcat 480

tgggtgcgcc aggcgccggg caaaggcctg gaatgggtgg cgtggattga tccggaaaac 540

ggcgataccg aatttgtgcc gaaatttcag ggccgcgcga ccattagcgc ggataccagc 600

aaaaacaccg cgtatctgca gatgaacagc ctgcgcgcgg aagataccgc ggtgtattat 660

tgcaaaaccg gcggcttttg gggccagggc accctggtga ccgtgagcag cgcgccgccg 720

cgcgcgagcg cgctgccggc gccgccgacc ggcagcgcgc tgccggatcc gcagaccgcg 780

agcgcgctgc cggatccgcc ggcggcgagc gcgctgccgt tttgggtgct ggtggtggtg 840

ggcggcgtgc tggcgtgcta tagcctgctg gtgaccgtgg cgtttattat tttttgggtg 900

cgcagcaaac gcagccgcct gctgcatagc gattatatga acatgacccc gcgccgcccg 960

ggcccgaccc gcaaacatta tcagccgtat gcgccgccgc gcgattttgc ggcgtatcgc 1020

agcgtgaaac gcggccgcaa aaaactgctg tatattttta aacagccgtt tatgcgcccg 1080

gtgcagacca cccaggaaga agatggctgc agctgccgct ttccggaaga agaagaaggc 1140

ggctgcgaac tgcgcgtgaa atttagccgc agcgcggatg cgccggcgta tcagcagggc 1200

cagaaccagc tgtataacga actgaacctg ggccgccgcg aagaatatga tgtgctggat 1260

aaacgccgcg gccgcgatcc ggaaatgggc ggcaaaccgc gccgcaaaaa cccgcaggaa 1320

ggcctgtata acgaactgca gaaagataaa atggcggaag cgtatagcga aattggcatg 1380

aaaggcgaac gccgccgcgg caaaggccat gatggcctgt atcagggcct gagcaccgcg 1440

accaaagata cctatgatgc gctgcatatg caggcgctgc cgccgcgc 1488

<210> 18

<211>2061

<212> DNA

<213> Artificial

<220>

<223> PSCA-CAR –G4HH2H3mt

<400> 18

gatattcagc tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60

attacctgca gcgcgagcag cagcgtgcgc tttattcatt ggtatcagca gaaaccgggc 120

aaagcgccga aacgcctgat ttatgatacc agcaaactgg cgagcggcgt gccgagccgc 180

tttagcggca gcggcagcgg caccgatttt accctgacca ttagcagcct gcagccggaa 240

gattttgcga cctattattg ccagcagtgg agcagcagcc cgtttacctt tggccagggc 300

accaaagtgg aaattaaagg cagcaccagc ggcagcggca aaccgggcag cggcgaaggc 360

agcaccaaag gcagcgaagt gcagctggtg gaaagcggcg gcggcctggt gcagccgggc 420

ggcagcctgc gcctgagctg cgcggcgagc ggctttaaca ttaaagatta ttatattcat 480

tgggtgcgcc aggcgccggg caaaggcctg gaatgggtgg cgtggattga tccggaaaac 540

ggcgataccg aatttgtgcc gaaatttcag ggccgcgcga ccattagcgc ggataccagc 600

aaaaacaccg cgtatctgca gatgaacagc ctgcgcgcgg aagataccgc ggtgtattat 660

tgcaaaaccg gcggcttttg gggccagggc accctggtga ccgtgagcag cgaaagcaaa 720

tatggcccgc cgtgcccgcc gtgcccggcg ccgccggtgg cgggcccgag cgtgtttctg 780

tttccgccga aaccgaaaga taccctgatg attagccgca ccccggaagt gacctgcgtg 840

gtggtggatg tgagccagga agatccggaa gtgcagttta actggtatgt ggatggcgtg 900

gaagtgcata acgcgaaaac caaaccgcgc gaagaacagt ttcagagcac ctatcgcgtg 960

gtgagcgtgc tgaccgtgct gcatcaggat tggctgaacg gcaaagaata taaatgcaaa 1020

gtgagcaaca aaggcctgcc gagcagcatt gaaaaaacca ttagcaaagc gaaaggccag 1080

ccgcgcgaac cgcaggtgta taccctgccg ccgagccagg aagaaatgac caaaaaccag 1140

gtgagcctga cctgcctggt gaaaggcttt tatccgagcg atattgcggt ggaatgggaa 1200

agcaacggcc agccggaaaa caactataaa accaccccgc cggtgctgga tagcgatggc 1260

agcttttttc tgtatagccg cctgaccgtg gataaaagcc gctggcagga aggcaacgtg 1320

tttagctgca gcgtgatgca tgaagcgctg cataaccatt atacccagaa aagcctgagc 1380

ctgagcctgg gcaaattttg ggtgctggtg gtggtgggcg gcgtgctggc gtgctatagc 1440

ctgctggtga ccgtggcgtt tattattttt tgggtgcgca gcaaacgcag ccgcctgctg 1500

catagcgatt atatgaacat gaccccgcgc cgcccgggcc cgacccgcaa acattatcag 1560

ccgtatgcgc cgccgcgcga ttttgcggcg tatcgcagcg tgaaacgcgg ccgcaaaaaa 1620

ctgctgtata tttttaaaca gccgtttatg cgcccggtgc agaccaccca ggaagaagat 1680

ggctgcagct gccgctttcc ggaagaagaa gaaggcggct gcgaactgcg cgtgaaattt 1740

agccgcagcg cggatgcgcc ggcgtatcag cagggccaga accagctgta taacgaactg 1800

aacctgggcc gccgcgaaga atatgatgtg ctggataaac gccgcggccg cgatccggaa 1860

atgggcggca aaccgcgccg caaaaacccg caggaaggcc tgtataacga actgcagaaa 1920

gataaaatgg cggaagcgta tagcgaaatt ggcatgaaag gcgaacgccg ccgcggcaaa 1980

ggccatgatg gcctgtatca gggcctgagc accgcgacca aagataccta tgatgcgctg 2040

catatgcagg cgctgccgcc gcgc 2064

<210> 19

<211> 684

<212> DNA

<213> Artificial

<220>

<223> G4HH2H3mt

<400> 19

gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cgccggtggc gggcccgagc 60

gtgtttctgt ttccgccgaa accgaaagat accctgatga ttagccgcac cccggaagtg 120

acctgcgtgg tggtggatgt gagccaggaa gatccggaag tgcagtttaa ctggtatgtg 180

gatggcgtgg aagtgcataa cgcgaaaacc aaaccgcgcg aagaacagtt tcagagcacc 240

tatcgcgtgg tgagcgtgct gaccgtgctg catcaggatt ggctgaacgg caaagaatat 300

aaatgcaaag tgagcaacaa aggcctgccg agcagcattg aaaaaaccat tagcaaagcg 360

aaaggccagc cgcgcgaacc gcaggtgtat accctgccgc cgagccagga agaaatgacc 420

aaaaaccagg tgagcctgac ctgcctggtg aaaggctttt atccgagcga tattgcggtg 480

gaatgggaaa gcaacggcca gccggaaaac aactataaaa ccaccccgcc ggtgctggat 540

agcgatggca gcttttttct gtatagccgc ctgaccgtgg ataaaagccg ctggcaggaa 600

ggcaacgtgt ttagctgcag cgtgatgcat gaagcgctgc ataaccatta tacccagaaa 660

agcctgagcc tgagcctggg caaa 684

<210> 20

<211> 108

<212> DNA

<213> Artificial

<220>

<223> 7H

<400> 20

gcgccgccgc gcgcgagcgc gctgccggcg ccgccgaccg gcagcgcgct gccggatccg 60

cagaccgcga gcgcgctgcc ggatccgccg gcggcgagcg cgctgccg 108

<210> 21

<211> 63

<212> DNA

<213> Artificial

<220>

<223> leader peptide

<400>21

atggcactgc cagtgaccgc cctgctgctg cccctggcac tgctgctgca cgcagctcgg 60

cct 63

<210> 22

<211> 30

<212>DNA

<213> Artificial

<220>

<223> Forward primer

<400>22

atcgctagca tggccctgcc agtgaccgcc 30

<210> 23

<211> 31

<212> DNA

<213> Artificial

<220>

<223> reverse primer

<400> 23

ccaggtcgac ttagcgaggg ggcagggcct g 31

Claims

1. The PSCA-targeted chimeric antigen receptor is characterized in that the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 11.

2. A method for producing the chimeric antigen receptor virus vector according to claim 1, comprising the steps of:

1) nucleotide sequence for the synthesis of chimeric antigen receptor targeting PSCA: synthesizing a nucleic acid sequence of a chimeric antigen receptor comprising a leader peptide, a single chain antibody against human PSCA antigen, a hinge region, a transmembrane region, and an intracellular signal domain; the leader peptide has a nucleic acid sequence shown as SEQ ID NO. 21; the nucleic acid sequence of the hinge region structure is shown as SEQ ID NO. 20;

2) construction of a viral vector expressing a chimeric antigen receptor: designing a primer, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO. 22, the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 23, and carrying out PCR amplification by taking the nucleotide sequence of the chimeric antigen receptor as a template to obtain a DNA fragment;

the gene sequence of the DNA fragment is subjected to double enzyme digestion by using restriction endonuclease, meanwhile, the restriction endonuclease is used for enzyme digestion of a virus expression vector pCDH-CAG, and then the enzyme digestion target fragment and the virus expression vector fragment are connected by T4 ligase to obtain a virus vector for expressing the chimeric antigen receptor, wherein the virus vector comprises but is not limited to adenovirus, retrovirus and lentivirus vectors.

3. The method according to claim 2, wherein the chimeric antigen receptor of step 1) has a nucleotide sequence of a single-chain antibody encoding an anti-human PSCA antigen, a hinge region, a transmembrane region, and an intracellular signal domain as shown in SEQ ID No. 17.

4. The method according to claim 3, wherein the viral vector is packaged and purified after the step 2), and the viral vector is a lentiviral vector.

5. A cell infected with the lentiviral vector of claim 4, wherein: the cells include, but are not limited to, T cells or NK cells or DC cells.

6. The cell of claim 5, wherein: the cell is a T cell.

7. Use of a cell according to any one of claims 5 to 6 for the preparation of a medicament for the treatment of a PSCA-expressing tumour.