CN108239144A - The hinge of transformation and its application in CAR skeletons are built - Google Patents

Abstract

The invention belongs to immunotherapy fields, and in particular to a kind of hinge area of transformation and its application in CAR skeletons are built.Hinge area provided by the invention can extend survival in CAR T cell bodies and/or improve CAR T cells infiltration tumour ability, and the hinge region amino acid sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.Hinge plot structure provided by the invention can extend CAR T cells survival time in vivo, the hinge plot structure combination Chimeric antigen receptor can be more stable be expressed in T lymphocytes, the survival time is longer in vivo, cellular infiltration tumour ability significantly increases by CAR T containing hinge area of the present invention, and fragmentation effect is more preferable.

Description

The hinge of transformation and its application in CAR skeletons are built

Technical field

The invention belongs to immunotherapy field, the hinge area for being related to a kind of transformation and its application in CAR skeletons are built, More particularly to a kind of hinge area that can extend survival in CAR-T cell bodies and/or improve CAR-T cellular infiltration tumour abilities and Its application in CAR skeletons are built.

Background technology

Chimeric antigen receptor (chimeric antigen receptor, CAR) is to simulate the artificial receptors of TCR functions, packet Containing antigen recognition domain, hinge area, transmembrane region and intracellular signal domain.Intracellular signal domain be usually CD3 ζ chains or FcR γ or with one kind Or a variety of costimulatory molecules are connected, such as 4-1BB (CD137), CD28, ICOS (CD278).The antigen (receptor) of tumor cell surface When being combined with the antibody (ligand) of Chimeric antigen receptor, intracellular, intracellular signal are transmitted signals to by hinge area and transmembrane region Domain converts the signal to activation signals, and activation effect cell, effector cell's proliferation, generation cell factor are thin so as to kill tumour Born of the same parents.

In recent years, it is outstanding to present significant therapeutic effect in oncotherapy for Chimeric antigen receptor T lymphocytes (CAR-T) In terms of it is the malignant tumour for treating the CD19 positives.But even for therapeutic effect significantly acute lymphoblastic leukemia (ALL), complete incidence graph median time generally at 8 months or so, still there is a large amount of Patients on Recurrence after treatment, this may and CAR-T There is great meaning in cell survival time in patient's body survival time short correlation, raising CAR-T bodies for CAR-T treatments curative effect Justice.

Although the research that solid tumor is carried out using CAR-T is also continuously available attention, most of CAR-T treat solid tumor Effect it is but and unsatisfactory, on the one hand also in that CAR-T cells survival is poor in vivo, CAR-T cells dead shadow quickly Ring killing validity；On the other hand most of solid tumors can be since it be in metabolism, immunologic escape, the multipotency for organizing the formation of aspect Property, in various aspects limitation immune system control of which and killing, CAR-T cells is caused to be difficult to infiltrate solid tumor cancer tissue Inside, so producing little effect for big solid tumor CAR-T treatments.Extend CAR-T survivals in vivo, promote CAR-T cells It is infiltrated in solid tumor mass, can effectively kill tumour and inhibit the recurrence of tumour cell, but extended there is presently no effective Survival and the method that CAR-T cell tumours is promoted to infiltrate in CAR-T bodies.

Invention content

In view of this, the purpose of the present invention is to provide one kind can extend survival and/or raising in CAR-T cell bodies The hinge area of CAR-T cellular infiltration tumour abilities, the survival time is longer in vivo by the CAR-T containing hinge area of the present invention, cell Infiltration tumour ability significantly increases, and fragmentation effect is more preferable.

To achieve the above object, the technical scheme is that：

The hinge area of survival and/or raising CAR-T cellular infiltration tumour abilities in CAR-T cell bodies can be extended, it is described Hinge region amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.

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

When the antigen (receptor) of tumor cell surface is combined with the antibody (ligand) of the Chimeric antigen receptor, pass through hinge Sequence and transmembrane region transmit signals to intracellular, and intracellular signal domain converts the signal to activation signals, activation effect cell, effect Cell Proliferation generates cell factor and a series of priming reactions so as to killing tumor cell.Applicant passes through to CAR structure hinges The research of area's transformation finds that the internal continuity of hinge plot structure and CAR-T and tumor-infiltrated ability are closely related.

Hinge area is also referred to as spacer region or hinge, connects CAR antigen recognizing districts and transmembrane region.Hinge area should have enough Flexibility allows antigen binding domain to be oriented in different directions, to promote antigen recognizing.The hinge area of different length is for CAR- The stability of T plays different role, applicant by the conjunction of a variety of contrast groups have been surprisingly found that hinge area for survival in CAR-T bodies and It is tumor-infiltrated that there is great influence；By a large amount of screening operations, final obtain can effectively extend survival and promotion in CAR-T bodies Hinge sequence SEQ ID NO.1 and SEQ the ID NO.2 of CAR-T cell tumours infiltration are simultaneously named as：7H and G4HH2H3mt.

Inventor devises 5 kinds of hinge arrangements altogether：7H, G4Hinge, G4HH3, G4HH2H3mt and DH, 7H and G4HH2H3mt amino acid sequences are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2, and nucleotide sequence is respectively such as SEQ ID Shown in NO.20 and SEQ ID NO.19.

Compare currently used hinge arrangement G4h (G4HH2H3) and CAR-T cell CAR positive rates that 8H is respectively combined, Survival and/or the infiltration of CAR-T cell tumours in Cytotoxicity in vitro and CAR-T bodies in CAR-T bodies, find comprising 7H or The survival time is longer in the CAR-T bodies of G4HH2H3mt hinge arrangements CAR, and with the CAR-T containing G4HH2H3 or 8H hinges Comparison, CAR-T cellular infiltration tumour abilities significantly increase, and fragmentation effect is more preferable.

G4HH2H3 amino acid sequences：

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK

8H hinge amino acid sequences：

KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

Extend the remodeling method of the hinge of survival and/or raising CAR-T cellular infiltration tumour abilities in CAR-T cell bodies, Hinge area transformation is carried out in a manner of rite-directed mutagenesis for template by G4HH2H3 sequences.

The second object of the present invention, which is to provide, a kind of obtains the remodeling method of above-mentioned hinge area, the feature of the method Using the method for the rite-directed mutagenesis of PCR mediations, to be transformed using G4HH2H3 amino acid sequences as template, G4HH2H3 amino Acid sequence such as SEQ ID NO.3

Hinge sequence G4HH2H3mt is further obtained according to above-mentioned remodeling method, it is characterised in that G4HH2H3mt is G4HH2H3 amino acid sequences are mutated and/or are lacked at 15-18 and the 79th.

The third object of the present invention is to provide a kind of Chimeric antigen receptor, including above-mentioned hinge area.The hinge area For 7H or G4HH2H3mt.

Further, the Chimeric antigen receptor also includes antigen recognizing district, transmembrane region and intracellular signal domain.

Further, the antigen recognizing district of the Chimeric antigen receptor can with tumor cell express antigen, comprising but not It is limited to PSCA, PSMA, CD19, BCMA, CD123, CD20, CD22, CEA, EGFR, EGFRVIII, GPC3 or mesothelin to resist Original molecule.

The tumor cells expression antigen that the Chimeric antigen receptor can identify is including but not limited to more than antigen molecule.

The present invention also aims to provide a kind of Chimeric antigen receptor for targeting PSCA, the list of anti-human PSCA antigens is included Chain antibody, hinge area, transmembrane region and intracellular signal domain；The hinge area is 7H or G4HH2H3mt, amino acid sequence such as SEQ Shown in ID NO.1 or SEQ ID NO.2.

Further, the amino acid sequence of the anti-human PSCA antigens single-chain antibody such as SEQ ID NO.4 or SEQ ID NO.5 It is shown.

Further, the transmembrane region is CD28TM or CD8TM, the amino acid sequence such as SEQ ID NO of the CD28TM:6, The amino acid sequence of the CD8TM such as SEQ ID NO:7；The intracellular signal domain be CD28 and/or CD137 and/or CD3, institute State the amino acid sequence such as SEQ ID NO of CD28:The amino acid sequence of 8, the CD137 such as SEQ ID NO:9, the CD3's Amino acid sequence such as SEQ ID NO:10.

Further, the amino acid sequence of the Chimeric antigen receptor such as SEQ ID NO.11 or SEQ ID NO.12 or SEQ Shown in ID NO.13 or SEQ ID NO.14 or SEQ ID NO.15 or SEQ ID NO.16.

The present invention also aims to provide a kind of system of the viral vectors of the Chimeric antigen receptor of the targeting PSCA Preparation Method comprises the steps of：

1) nucleotide sequence of the Chimeric antigen receptor of synthesis targeting PSCA：Synthesis includes leader peptide, anti-human PSCA antigens Single-chain antibody, hinge area, transmembrane region and the Chimeric antigen receptor nucleic acid sequence in intracellular signal domain；The leading peptide nucleic acid sequence As shown in SEQ ID NO.21；

2) viral vectors of structure expression Chimeric antigen receptor：Design primer, the nucleotide sequence such as SEQ of forward primer ID NO:Shown in 22, the nucleotide sequence such as SEQ ID NO of reverse primer:Shown in 23, with the gene of the Chimeric antigen receptor Sequence carries out PCR amplification for template, obtains DNA fragmentation；

By the gene order restriction enzymes double zyme cutting of the DNA fragmentation, at the same it is sick with digestion with restriction enzyme Malicious expression vector pCDH-CAG, is then connected the target fragment after digestion and virus expression carrier segment by T4 ligases It connects, obtains the viral vectors of expression Chimeric antigen receptor.The viral vectors including but not limited to adenovirus, retrovirus and Slow virus.

As a preferred embodiment, the restriction enzyme is NheI and SalI.

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

Further, it after step 2), packs and purifies the viral vectors, the viral vectors is slow virus carrier.

The present invention also aims to provide the slow virus carrier obtained by a kind of preparation method.

The slow virus carrier, the positive cell expression of such slow virus carrier transduction are obtained under the method Rate is high, stablizes in patient's cell cultivation process, and CAR positive rates will not be caused to decline over time.With described The cell of slow virus carrier infection, such cell have the function of killing target cell.

The present invention also aims to provide a kind of cell of the slow virus carrier infection, the cell includes but not It is limited to T lymphocytes, NK cells and DC cells.

Further, the cell of the slow virus carrier infection is T cell.

The present invention also aims to provide a kind of CAR-T cells of the slow virus carrier infection be used to prepare it is anti- Application in tumour medicine.

Further, the tumour cell or tissue can express PSCA.

The present invention also aims to provide the amino acid sequence work shown in a kind of SEQ ID NO.1 or SEQ ID NO.2 For application of the hinge area in CAR skeletons are built.

In certain embodiments the hinge area of Chimeric antigen receptor can be G4HH2H3 sequence alterations sequence, such as comprising Lack the Chimeric antigen receptor of the hinge area of the amino acid sequence of the H2H3 sections of G4HH2H3 sequences；In certain embodiments, it is fitted into The hinge legion sequence of antigen receptor is the sequence of H2 sections of G4HH2H3 sequence deletions, and particular sequence is as shown in table 1.

Table 1, the transformation of G4HH2H3 hinge sequences

In certain embodiments, the antigen recognizing district of the Chimeric antigen receptor comprising the hinge arrangement can be it is any can With the polypeptide with PSCA antigen bindings, such as can be with the ligands of PSCA specific bonds, bispecific antibody, scFV, optional crosslinking Fab, F (ab) 2, single domain antibody and the scFV for being connected with His- labels or HA- labels.Specificity is known in certain embodiments The antibody sources of other PSCA are in 7F5；Antigen recognizing district amino acid sequence is as follows in some embodiments：

QVKLQESGGGLVQPGGSLKLSCVASGFTFSSYTMSWVRRTPEKRLEWVAYIHNGGGHTYYPDTIKGRFT ISRDNAKNTLFLEMSSLKSEDTAMYYCTRRMYYGNSHWYFDVWGAGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAPKLLIYYTLKLNSGVPSRFSGSGSGTDFTFTISSLQPEDIATY YCQQSKTLPWTFGGGTKVEIK

The antibody amino acids sequence of specific recognition PSCA is as follows in certain embodiments：

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRAT ISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRV TITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFT FGQGTKVEIK

The antibody amino acids sequence of specific recognition PSCA is as follows in certain embodiments：

DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSC AASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTG GFWGQGTLVTVSS

The antibody amino acids sequence of specific recognition PSCA is as follows in certain embodiments：

DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGGGGSGGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAA SGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGF WGQGTLVTVSS

The beneficial effects of the present invention are：

1) hinge plot structure provided by the invention can extend CAR-T cells survival time in vivo.

2) it is thin that the Chimeric antigen receptor of hinge plot structure combination provided by the invention can be more stable is expressed in T lymphs Born of the same parents have the ability for preferably removing tumour cell, can not only maintain the Chimeric antigen receptor of targeting PSCA in patient's cell Positive rate in incubation and the proliferation of CAR-T can be strengthened and kill the ability of tumour, to the cell of antigen negative without Toxic side effect can be used in the targeted therapy of tumour.

3) ability of the CAR-T cellular infiltration tumor tissues comprising hinge arrangement provided by the invention is strengthened, can be effective Solid tumor is killed.

Description of the drawings

Fig. 1 is the CAR structure charts of different hinge areas and its composition.

Fig. 2 is the CAR expression rates of different hinge areas combination.

Fig. 3 is the CAR-T cell CAR average fluorescent strengths detection of different hinge areas combination.

Fig. 4 is the CAR-T cell long-periods proliferation of different hinge areas combination.

Fig. 5 is the CAR-T cell killing rates of different hinge areas combination.

Fig. 6 is the CAR-T cell CAR expression stabilities detection of different hinge areas combination.

Fig. 7 is fragmentation effect comparison in the CAR-T cell bodies of different hinge areas combination.

Fig. 8 is survival and the research of tumor-infiltrated ability in the CAR-T cell bodies of different hinge areas combination.

Specific embodiment

Hereinafter reference will be made to the drawings, and the preferred embodiment of the present invention is described in detail.Tool is not specified in preferred embodiment The experimental method of concrete conditions in the establishment of a specific crime, usually according to normal condition, such as the Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brookers etc. Write) described in condition or according to the normal condition proposed by manufacturer.Illustrated embodiment is in order to preferably in the present invention Appearance illustrates, but is not that present disclosure is only limitted to illustrated embodiment.So those skilled in the art according to Foregoing invention content carries out nonessential modifications and adaptations to embodiment, still falls within protection scope of the present invention.

1 hinge area of embodiment is transformed

Using the G4HH2H3 hinge areas in IgG4FC sequences source as template, hinge area transformation is carried out in a manner of rite-directed mutagenesis. The results are shown in Figure 1 for transformation：G4HH2H3mt is mutated for G4HH2H3 at 15-17 and the 79th, the 18th missing The transformation sequence of acquisition；G4HH3 is deletes H2 sections of sequences；G4Hinge is to delete the transformation that H2H3 sections of sequences obtain.It obtains simultaneously From people CD7 and people's IgD hinge legion sequences 7H and DH.If Fig. 2 is different hinge legion sequences and transformation site.

The structure of Chimeric antigen receptor virus of the embodiment 2 containing different hinge areas

In order to verify the hinge area of different structure to continuity in CAR-T bodies and tumor-infiltrated influence, with targeting It is tested for the CAR-T of PSCA.Design the CAR of 5 kinds as shown in Figure 1 different hinge arrangements.

The Chimeric antigen receptor gene order of the targeting PSCA of 1 hinge sequence of the synthesis containing above-mentioned design

Single-chain antibody of the synthesis containing leader peptide (also known as signal peptide) (abbreviation LP), anti-human PSCA antigens, 6 groups of difference hinges The CAR structures in area, CD28 transmembrane regions (referred to as TM) and CD28, CD137 and CD3 intracellular signal domain.

The slow virus carrier of 2 structure expression Chimeric antigen receptors

Following primer is designed, and it is synthesized by biotechnology company, specific primer is as follows：

Primer 1：5’-atcgctagcAtggccctgccagtgaccgcc-3 ', underscore are NheI restriction enzymes position Point；

Primer 2：5’-ccaggtcgacTtagcgagggggcagggcctg-3 ', underscore are SalI restriction enzymes Site.

Then using above-mentioned shown sequence as primer, each Chimeric antigen receptor sequence of above-mentioned synthesis carries out PCR expansions for template Increase, reaction system is loaded by KOD FX NEO archaeal dna polymerases (being purchased from TOYOBO companies) specification, after amplified production is identified Carry out DNA fragmentation recycling with QIAquick Gel Extraction Kit (Promega companies), specific method is shown in specification, recycling obtain chimeric antigen by DNA recycling segment is sent biotechnology company to be sequenced by body.

The gene order restriction enzyme NheI and SalI that clone the encoding chimeric antigen receptor obtained (are purchased from Thermo companies) double digestion, while (purchase is extremely with restriction enzyme NheI and SalI digestion Lentiviral pCDH-CAG Addgene Plasmid), endonuclease reaction by specification carries out.Digestion products use agarose after agarose gel electrophoresis detaches Gel DNA fragment QIAquick Gel Extraction Kit carries out DNA fragmentation recycling, and target fragment and carrier segments then are passed through the (purchase of T4 ligases From Promega companies) it is attached, the slow virus carrier of expression Chimeric antigen receptor is obtained, is named as Lv-hinge.It will slow disease Poisonous carrier converts Escherichia coli TOP10, is taken out after picking Colony Culture 12h with plasmid extraction kit (Invitrogen companies) Upgrading grain, specific method are shown in specification.

7 slow virus carriers are built respectively according to as above method：

“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”。

The packaging of 3 slow virus

For the present embodiment packaging slow virus using calcium phosphate method, specific steps are shown in Molecular Cloning:A Laboratory guide (third edition, J. Sas The works such as nurse Brooker).

The purifying of 4 slow virus

Viral supernatants are collected in 50ml centrifuge tubes, centrifugal filtration, filtrate centrifuges 10min to new in 3000r/min 50ml centrifuge tubes；According to viral supernatants amount, it is separately added into PEG6000 the and 4M NaCl that mass fraction is 50%, then with medical salt Water constant volume makes the PEG6000 final concentration of 0.3M of final concentration of 8.5%, NaCl, calmly molten to be stood after 4 DEG C of refrigerators after 4 DEG C, condition It is lower to centrifuge and abandon most supernatant, virus is resuspended with 200 μ l DMEM culture mediums, 1.5mlEP pipes dispense, often 40 μ l of pipe, -80 DEG C of preservations It is spare.

5 slow virus titer determinations

Step 1:Virus infection 293T cells

Bed board 293T cells before infection take purified viral 1 μ l, dilute 10 times, then to every hole with medical saline 1 μ l polybrenes (Polybrene) solution is added in cell, is then added separately in 293T cells, for 24 hours afterwards with containing 10%FBS (wt) DMEM culture mediums change liquid, and infection 72h centrifuges 5min to collect cell under the conditions of 1000r/min, extracts genome.

Step 2:Extract genome

Genome extraction agent box is purchased from Qiagen companies (article No. for QIAamp DNA Blood Mini Kit 511004) it, is operated by kit specification.

Step 3:QRT-PCR measures virus titer

Reaction system is as follows：Premix Ex TaqTM II (2 ×) 10 μ l, 1 μ l of sense primer (GAG up), downstream primer (GAG dn) 1 μ l, 1 μ l, RNase-Free dH2O of genome, the 7 μ l of extracting, each sample, standard items at least three repeating hole. Then it is expanded by following procedure：95 DEG C of pre-degeneration 30s, 95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, reaction After, with analysis software data, virus titer is calculated according to standard curve.

3 CAR of embodiment transfection T lymphocytes ability detections

The separation of 1 human peripheral blood mononuclear cell

Peripheral blood about 60ml is acquired with heparin tube, is sub-packed in 50ml centrifuge tubes, adds in the dilution of 7.5ml hydroxyethyl starch；Room Temperature (18~25 DEG C) natural subsidence about 30min collects upper plasma, is resuspended after the upper plasma of collection is centrifuged with physiological saline Precipitation, is by volume 1:1 is added on lymphocyte separation medium, gradient centrifugation；After centrifugation, centrifuge tube is from top to bottom divided into：The One layer：Plasma layer；The second layer：Cyclic annular milky buffy coat；Third layer：Transparent separation liquid layer；4th layer：Red blood cell layer； It takes second layer white buffy coat, and with brine 2 times, centrifuges 5min, cell is resuspended in physiological saline, and addition contains The 1640 complete medium cultures of RPMI of 10%FBS, obtain human peripheral blood mononuclear cell.

2 slow virus carriers infect T lymphocytes

With 1640 complete medium cultures of the RPMI freshly prepd mononuclearcell PBMC containing 10% fetal calf serum, the 1st day Carry out PBMC activation；Carry out slow-virus infection within 3rd day；The corresponding slow virus carriers of 5MOI are added in, the T lymphocytes being uninfected by are made For blank control；Culture medium is changed to 1640 complete mediums of RPMI containing 500IU/ml recombinant human il-2s afterwards for 24 hours, after Continuous culture 10-20 days.Obtain expression comprising antigen recognizing district, hinge area, transmembrane region and the chimeric antigen in intracellular signal domain by The CAR-T cells of body are named with the hinge area of transformation, are named as：PSCA-CAR-G4HH2H3、PSCA-CAR-G4HH2H3mt、 PSCA-CAR-G4HH3, PSCA-CAR-G4Hinge, PSCA-CAR-7H, PSCA-CAR-8H and PSCA-CAR-DH；In order to make During figure convenient for label, more than CAR-T cells in subsequent embodiment and Figure of description with G4HH2H3, G4HH2H3mt, G4HH3, G4Hinge, 8H, 7H and DH write a Chinese character in simplified form.

1) to cultivating to the T cell of the virus infection of 10 days in incubation, centrifugation abandons most supernatant to collect cell； Cell is resuspended with the PBS solution containing 1% fetal calf serum of volume fraction, and is 1 × 10 by cell adjustment density⁶A/ml；It will receive The cell of collection is dispensed respectively using Flow cytometry Protein-L positive rates, and testing result represents culture the 10th day not It is combined with CAR in T Expressions In Lymphocytes positive rates, while analyzes flow cytometer detection MFI (average fluorescent strength) CAR can be obtained and put down The result of equal fluorescence intensity.

The results are shown in Figure 2, hinge arrangement G4HH2H3mt, G4HH3, G4Hing, 7H and DH group of as a result visible transformation The CAR of conjunction is little in the CAR differences that T cell surface expression positive rate is combined with conventional hinge G4HH2H3 or 8H.As Fig. 3 is represented The CAR-T cell surfaces CAR expression average fluorescent strengths of different hinge arrangements, wherein G4HH2H3, G4HH2H3mt or 7H structure CAR higher can be expressed in T cell surface, surface average fluorescent strength higher, indication CAR molecules are expressed in cell surface More；And the CAR surfaces average fluorescent strength of G4HH3, G4Hinge or DH structure is low.

2) infection is detected not for the 5th day, the 10th day and the 14th day after obtaining CAR-T cells using detection method 1) respectively With the CAR expression of hinge arrangement combination, the stability that CAR-T cells CAR is expressed after long-time is cultivated is detected.

The results are shown in Figure 6：The long-term expression for detecting CAR finds the structure of transformation and non-reconstruction structure G4HH2H3 and 8H Comparison, the CAR expression stabilities of DH hinge arrangements are poor, and CAR positive rates are remarkably decreased and as incubation time prolongs after the 5th day Long decline is more notable, and remainder transformation hinge arrangement is little with the CAR stability differences that hinge combines are not transformed.

4 CAR-T cell long-periods proliferative capacity of embodiment detects

Proliferation of the detection infection PSCA-CAR-T cells under regular culture conditions；The CAR-T cells that embodiment 3 obtains, Using the cultural method culture CAR-T cells 10 days in 3 step 2 of embodiment.24 orifice plate four of PSCA antigen coats spends night, CAR- T cell 1*10^6/ holes bed board cell is in 24 orifice plates of PSCA antigen coats, survival of the observation CAR-T cells after antigenic stimulus Time.Count CAR-T using cell counter within the 3rd day, the 6th day, the 9th day and the 12nd day after stimulation respectively as shown in Figure 4 Cell number simultaneously calculates CAR-T cell Proliferation multiples.According to the experience of experiment in vivo, an antigenic stimulus, Fig. 4 are carried out within every 7 days As a result CAR-T cells are no longer proliferated after antigenic stimulus the 3rd time, so terminating experiment after 3 times the 15th day in stimulation, carry out CAR- T Long-term Proliferations multiple counts, and Long-term Proliferation reacts the survival of CAR-T cells.

Experiment is as shown in figure 4, the CAR-T cell long-periods proliferation phase of G4HH2H3, G4HH2H3mt, 7H and 8H hinge combination To very fast, CAR-T cells in vitro survival abilities are stronger；DH, G4Hinge and G4HH3 long-term proliferative capacity are poor.

5 CAR-T cells against tumor cells of embodiment kill the influence of ability

The CAR-T of different hinge arrangements passes through ACEA xCELLigence RTCA MP instruments to the killing ability of target cell It completes, experimental procedure is carried out according to instrument specification；First day by target cell (tumour cell of expression PSCA) with 2-5*10^4 It is plated in 96 orifice plates of apparatus preparation per hole, the tumour cell for being attached to bottom hole records for every 15 minutes using index of resistance as data Once, spread after 24 hours according to the effect target that is pre-designed than every hole into corresponding CAR-T cells, CAR-T cells spread after every Index of resistance of 15 minutes records judges the proliferation or death condition of adherent target cell by index of resistance.Utilize electricity Hindering index analysis result formula is：CAR-T cell killings rate=baseline electrical resistance index-real time resistance index.

Hela and RT4 is the tumor cell line of PSCA high expression, and T24 is the negative control cell for not expressing PSCA.

Experimental result is as shown in Figure 5：7H has preferably killing ability, this more sensitive to T cell not only for RT4 Tumor cell killing potential it is strong, it is more suitable with RT4 for this tumor cell killing potentials insensitive for T cell of Hela, And negative cells are not killed, high specificity.

Summary is tested, the CAR stability of DH, G4Hinge and G4HH3 hinge arrangement combination, Long-term Proliferation and external Killing ability is poor without subsequent evaluation, subsequently respectively to include G4HH2H3, G4HH2H3mt, 7H and 8H hinge arrangement CAR-T cells carry out experiment in vivo verification.

Antitumous effect verification of the 6 CAR-T cells of embodiment in animal model

The mice-transplanted tumor model of people's PSCA positive tumor cells system is established for verifying the inosculating antibody of expression targeting PSCA Antitumous effect of the T lymphocytes of original receptor in animal model.

Verification in vivo uses mouse as NOD.Cg-PrkdcscidII2rgtm1Sug/JicCrl, abbreviation NOG mouse, by day The Mamoru Ito of this institute of lab animals (CIEA) are cultivated, and are tested most into knurl for correlation in CAR-T bodies in the world Common strain.The expressing luciferase stably used into knurl targeting cell for preliminary in vitro verification that verification in vivo uses PSCA positive cell line Hela (abbreviation Hela-luc).

It finds that hinge G4HH2H3mt and the 7H structure of transformation have by preliminary in vitro experiments preferably to kill and deposit in vitro Continuous ability, therefore using this common G4HH2H3 and 8H of 2 kinds of CAR-T versus as the CAR-T cells of hinge in zoopery The ability of tumour is killed in Mice Body.The effector cell for the treatment of injection is to be cut with scissors containing G4HH2H3mt, G4HH2H3,7H and 8H The CAR-T cells of chain structure compare the PBMC cells for physiological saline group, uninfecting virus.

Tail vein injection effect CAR-T cell 5*10^5 cells/mouse after into knurl.Led to after injecting CAR T cells every 7 days The IVS living imaging systems for crossing PerkinElmer companies are taken pictures imaging, show tumour growth situation.Period observes mouse daily Survival condition simultaneously records, as a result as shown in Fig. 7 A figures：CAR-T cells comprising G4HH2H3mt or 7H hinges are in vivo to tumour Cell has preferable fragmentation effect.Tumor tissues are further taken out after experimental endpoints put to death mouse to be weighed and count knot Fruit, as a result as shown in Fig. 7 B figures：It is similar to 7A figure results, the mouse of the CAR-T cell therapies comprising G4HH2H3mt or 7H hinges Tumor weight is lighter, and the mouse tumor of the especially CAR-T cell therapies of 7H hinges combination is substantially achieved removing.

Survival and the tumor-infiltrated capability study in vivo of 7 CAR-T cells of embodiment

Continue the ability with CAR-T cellular infiltration tumours in CAR-T cell bodies, utilize RT-PCR detection tumor tissues CAR gene copy numbers are detected.

1. design of primers：

BBZ-HF：CAGAAGAAGAAGAAGGAGGATGTG；

BBZ-HR:TACTCCTCTCTTCGTCCTAGATTG。

2. tumor tissues RNA is extracted

First liquid nitrogen is added in mortar, then tumor tissues are cut into small pieces and are clayed into power in liquid nitrogen, with Liquid nitrogen precooler Spoon takes appropriate tissue powder addition to fill in the EP pipes of Trizol liquid, is sufficiently mixed uniformly.It is placed at room temperature for 5min, Ran Houjia Enter the chloroform of 200ml, cover tightly EP and manage and acutely sway 15 seconds.Centrifuging and taking upper strata aqueous phase adds in 500ml in a new EP pipes Isopropanol mildly overturns mixing.It is placed at room temperature for 10min centrifugations.Liquid carefully is discarded supernatant, adds in 75% ethyl alcohol of 1ml, is vortexed mixed Even, centrifugation repetitive operation is primary.Discard supernatant liquid, 5~10min of room temperature or vacuum drying.It will with the processed water of 30ml DEPC RNA dissolves, and is stored in 70% ethyl alcohol and is stored in -70 DEG C.

3.RT-PCR

RT-PCR is carried out, reaction system is as follows：

Reaction system is as follows：0.5 μ l, Reverse primer (10 μM) of Forward primer (10 μM) 0.5 μ l, 2 × 10 μ l, Template 1ul of SYBR Premix Ex Taq II, 1 μ l, RNase-Free dH2O of genome, the 7 μ l of extracting, Each sample, standard items at least three repeating hole.Then it is expanded by following procedure：95 DEG C of 2min, 95 DEG C of 15s, 60 DEG C 1min, 40 cycles, after reaction, with analysis software data, analysis result is as shown in table 3 and Fig. 8.

Table 3 is the CAR copy numbers in the tumor tissues of the CAR-T cell therapies of the different structure detected, and Fig. 8 is foundation The chart that the result of table 3 is drawn.As a result visible 7H or G4HH2H3mt can significantly increase CAR-T infiltration tumours as hinge area Ability.

Table 3:CAR-T cells are fed back after 45 days, the survival time in the CAR-T bodies of different hinge results

Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with The present invention is described in detail in good embodiment, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this In the right of invention.

<110>The accurate Bioisystech Co., Ltd in Chongqing

<120>The hinge of transformation and its application in CAR skeletons are built

<160> 23

<170> PatentIn version 3.3

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<213> Artificial

<220>

<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 (18)

1. the hinge area of survival and/or raising CAR-T cellular infiltration tumour abilities in CAR-T cell bodies, feature can be extended It is, the hinge region amino acid sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.

2. obtain the remodeling method of hinge area described in claim 1, which is characterized in that using the method for rite-directed mutagenesis, with amino Acid sequence such as SEQ ID NO.3 sequences are transformed for template.

3. the hinge sequence that the remodeling method according to claim 2 obtains, it is characterised in that exist for sequence SEQ ID NO.3 15-18 and the 79th are mutated and/or are lacked.

4. a kind of Chimeric antigen receptor, which is characterized in that include hinge arrangement described in claim 1.

5. Chimeric antigen receptor according to claim 4, which is characterized in that the Chimeric antigen receptor is also known comprising antigen Other area, transmembrane region and intracellular signal domain.

6. according to claim 4-5 any one of them Chimeric antigen receptors, which is characterized in that the Chimeric antigen receptor antigen Cog region can with tumor cell express antigen, including but not limited to PSCA, PSMA, CD19, BCMA, CD123, CD20, CD22, CEA, EGFR, EGFRVIII, GPC3 or mesothelin antigen molecule.

7. according to claim 4-6 any one of them Chimeric antigen receptors, it is characterised in that include the list of anti-human PSCA antigens Chain antibody, hinge area, transmembrane region and intracellular signal domain；The amino acid sequence of the hinge area such as SEQ ID NO.1 and SEQ ID Shown in NO.2.

8. Chimeric antigen receptor according to claim 7, which is characterized in that the Chimeric antigen receptor resists for anti-human PSCA Former single-chain antibody, amino acid sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5.

9. Chimeric antigen receptor according to claim 5, which is characterized in that the transmembrane region be CD28TM or CD8TM, institute State the amino acid sequence such as SEQ ID NO of CD28TM:The amino acid sequence of 6, the CD8TM such as SEQ ID NO:7；The intracellular Signal domain is CD28 and/or CD137 and/or CD3, the amino acid sequence such as SEQ ID NO of the CD28:8, the CD137's Amino acid sequence such as SEQ ID NO:The amino acid sequence of 9, the CD3 such as SEQ ID NO:10.

10. according to claim 7-9 any one of them Chimeric antigen receptors, which is characterized in that the Chimeric antigen receptor Amino acid sequence such as SEQ ID NO.11 or SEQ ID NO.12 or SEQ ID NO.13 or SEQ ID NO.14 or SEQ ID Shown in NO.15 or SEQ ID NO.16.

11. the preparation method of the viral vectors of claim 7-9 any one of them Chimeric antigen receptors, which is characterized in that packet Include following steps：

1) nucleotide sequence of the Chimeric antigen receptor of synthesis targeting PSCA：List of the synthesis comprising leader peptide, anti-human PSCA antigens Chain antibody, hinge area, transmembrane region and the Chimeric antigen receptor in intracellular signal domain nucleic acid sequence；The leading peptide nucleic acid sequence is such as Shown in SEQ ID NO.21；The hinge plot structure is as shown in SEQ ID NO.19 or SEQ ID NO.20.

2) viral vectors of structure expression Chimeric antigen receptor：Design primer, the nucleotide sequence such as SEQ ID of forward primer NO:Shown in 22, the nucleotide sequence such as SEQ ID NO of reverse primer:Shown in 23, with the gene order of the Chimeric antigen receptor PCR amplification is carried out for template, obtains DNA fragmentation；

By the gene order restriction enzymes double zyme cutting of the DNA fragmentation, while with digestion with restriction enzyme virus table Up to carrier pCDH-CAG, then the target fragment after digestion and virus expression carrier segment are attached by T4 ligases, Obtain the viral vectors of expression Chimeric antigen receptor, the viral vectors is including but not limited to adenovirus, retrovirus and slow Viral vectors.

12. preparation method according to claim 11, which is characterized in that the nucleotide of the step 1) Chimeric antigen receptor Sequence is as shown in SEQ ID NO.17 or SEQ ID NO.18.

13. preparation method according to claim 11, which is characterized in that after step 2), pack and purify the disease Poisonous carrier, the viral vectors are slow virus carrier.

14. the cell of the slow virus carrier infection described in claim 13, it is characterised in that the cell is thin including but not limited to T Born of the same parents or NK cells or DC cells.

15. the cell described in claim 14, it is characterised in that the cell is T cell.

16. application of the cell in the drug for being used to prepare treatment tumour described in claim 14-15.

17. application according to claim 16, which is characterized in that the tumour cell or tissue can express PSCA.

Amino acid sequence shown in 18.SEQ ID NO.1 or SEQ ID NO.2 as hinge area in CAR skeletons are built should With.