CN107384870A - A kind of T lymphocytes of targeting PD L1 Chimeric antigen receptors modification and its preparation method and application - Google Patents

Abstract

The invention discloses one kind the present invention relates to biological technical field, a kind of T lymphocytes of targeting PD L1 Chimeric antigen receptors modification and its preparation method and application are specifically disclosed.The T lymphocytes of described targeting PD L1 Chimeric antigen receptors modification, its T lymphocytic cell surface expression targeting PD L1 Chimeric antigen receptor PD L1 CAR.Described targeting PD L1 Chimeric antigen receptor PD L1 CAR amino acid sequence is as shown in SEQ ID NO.2.The T lymphocytes of described targeting PD L1 Chimeric antigen receptors modification not only can mediate the specific killing to PD L1 positive tumors in body by specific recognition tumor cell surface PD L1；PD 1/PD L1 immunosupress signal paths can be prevented simultaneously, eliminate the immunosuppressive action of tumour；So as to reach the purpose of specific killing tumour.

Description

A kind of T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification and preparation method thereof And application

Technical field

The present invention relates to biological technical field, and in particular to a kind of T lymphs of targeting PD-L1 Chimeric antigen receptors modification are thin Born of the same parents and its preparation method and application.

Background technology

With the development of tumour immunity theory and clinical technology, the T lymphocyte therapies of Chimeric antigen receptor modification (Chimeric antigen receptor T-cell immunotherapy, CAR-T) (June CH, Blazar BR, Riley JL.Engineering lymphocyte subsets:tools,trials and tribulations.Nat Rev Immunol.2009；9:704-16) turn into one of tumour immunotherapy most promising at present.So far, CAR-T is thin Born of the same parents' immunotherapy achieves preferable effect on neoplastic hematologic disorder, the treatment of lymthoma and glioma.And for solid tumor, by It is difficult the tumour immunity suppression microenvironment for breaking through solid tumor in CAR-T cells, CAR-T cellular immunotherapies need further excellent Change.

Usually, Chimeric antigen receptor CAR is by a tumor associated antigen land, extracellular hinge area, altogether transmembrane region, thorn Swash area and intracellular signal transduction district's groups into.CAR-T cell therapies by foreign gene rotaring dyeing technology, can specific recognition swell The single-chain antibody (Single chain fragment variable, scFv) of knurl related antigen and melting for T cell activation sequence T cell surface arrive in hop protein expression, make can the scFv of specific recognition tumor associated antigen pass through transmembrane region and T cell intracellular Activation and proliferation signal domain is coupled.The T cell for expressing CAR identifies tumour antigen in a manner of antigen is relied on but non-MHC is limited, and opens Dynamic and activation specific killing tumor response.

During whole immune response, t cell activation needs dual signal path to mediate.Pass through the T on T cell surface first Cell receptor TCR specific recognition antigen presenting cell APC or tumor cell surface MHC molecule antigenic peptide complexes；Secondly Costimulatory molecules on APC or tumour cell is combined with the costimulation acceptor in T cell, activation or suppresses T lymphocytes.T Costimulatory molecules on cell mainly includes co-activation molecule 4-1BB, CD27, CD28, OX40, ICOS, and Co inhibitor CTLA4, PD-1.

Programmed death acceptor 1 (Programmed Death 1, PD-1) is the suppression being mainly expressed on activating T cell Property acceptor, can be combined with its part PD-L, suppress activation and the propagation of T cell, adjust the expression and secretion of cell factor.At present 2 kinds of PD1 cell surface glycoprotein parts, PD-L1 and PD-L2 are identified, both parts are in antigen presenting cell and a variety of High expression on tumour cell be present, and confirm that the part lowers the activation of T cell and the secretion of cell factor after PD1 is combined (Freeman GJ,Long AJ,Iwai Y,et al.Engagement of the PD-1immunoinhibitory receptor by a novel B7family member leads to negative regulation of lymphocyte activation.J Exp Med.2000Oct 2；192(7):1027-34).PD-1/PD-L1 signal paths It is primarily involved in the Organism immunoregulation process such as autoimmunity, tumour immunity, trnasplantion immunity.

PD-L1 is 40KD transmembrane protein, exists in many solid tumors and is overexpressed, and usually with it is poor pre- Relevant (Okazaki T, Honjo T.PD-1and PD-1ligands afterwards:from discovery to clinical application.Int Immunol.2007Jul；19(7):813-24).On the other hand, it is thin with the T lymphs in normal structure Born of the same parents and periphery blood T lymphocyte are compared, the high expression of tumor infiltrating T lymphocytes PD-1.Indicate the PD-1/PD-L1 of high expression The immunosupress of signal path mediate tumor.(Tumor antigen-specific CD8T cells infiltrating the tumor express high levels of PD-1and are functionally impaired)。

So far, by suppressing PD-1/PD-L1 signal paths, also achieved in clinical stage to be used for oncotherapy Preferable effect.The PD- of Bristol Myers Squibb (Bristol-Myers Squibb) and Mo Shadong (Merck Sharp＆Dohme) L1 antibody achieves preferable effect in melanoma, the clinical research of non-small cell lung cancer.Roche Roche, Merck Merck Also obtained in carcinoma of urinary bladder, the clinical research of bladder transitional cell carcinoma with Pfizer Pfizer Pharmaceuticals PD-L1 antibody Preferable effect.At present, FDA approveds Roche PD-L1 inhibitor Tecentriq is used for the treatment of bladder transitional cell carcinoma.Enter One step illustrates to suppress great potential of the PD-1/PD-L1 signal paths on immunotherapy of tumors.

But PD-L1 antibody is integrated into CAR structures, it is expressed in T cell, structure PD-L1-CAR-T cells are carried out CAR-T cellular immunotherapies not yet studies have reported that.

The content of the invention

It is an object of the invention to provide a kind of T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification, described target The T lymphocytes modified to PD-L1 Chimeric antigen receptors are mediated to PD-L1 by specific recognition tumor cell surface PD-L1 The specific killing of positive tumor.

To achieve the above object, the present invention is achieved by following technical solution：

A kind of T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification, the T lymphocytic cell surfaces expression targeting PD- L1 Chimeric antigen receptors PD-L1-CAR.

Preferably, described targeting PD-L1 Chimeric antigen receptors PD-L1-CAR amino acid sequence such as SEQ ID NO.2 It is shown.

Preferably, described targeting PD-L1 Chimeric antigen receptors PD-L1-CAR coding nucleotide sequence such as SEQ ID Shown in NO.1.

Preferably, described targeting PD-L1 Chimeric antigen receptors PD-L1-CAR is by GM-CSF signal peptides, PD-L1 antibody Single-stranded variable region (Anti-PD-L1scFv), CD8 α hinge areas (Hinge), CD8 α transmembrane regions (transmembrane Domain), 4-1BB costimulatory signals domain (co-stimulatory signal domain) and CD3 ζ intracellular signals domain (signal domain) is composed in series；

The single-stranded variable region (Anti-PD-L1scFv) of wherein PD-L1 antibody is by weight chain variabl area sequence (PD- L1heavy chain scFv) and light-chain variable sequence (PD-L1light chain scFv) pass through (Gly4-Ser) n (linker) it is composed in series.

It is further preferred that the amino acid sequence of described GM-CSF signal peptides is as shown in SEQ ID NO.4；Described The amino acid sequence of CD8 α hinge areas (Hinge) is as shown in SEQ ID NO.6；The amino acid sequence of described CD8 α transmembrane regions is such as Shown in SEQ ID NO.8；The amino acid in described 4-1BB costimulatory signals domain (co-stimulatory signal domain) Sequence is as shown in SEQ ID NO.10；The amino acid sequence such as SEQ in described CD3 ζ intracellular signals domain (signal domain) Shown in ID NO.12；

The amino acid sequence such as SEQ ID NO.14 of described light-chain variable sequence (PD-L1light chain scFv) It is shown；The amino acid sequence such as SEQ ID NO.16 institutes of described weight chain variabl area sequence (PD-L1heavy chain scFv) Show；Described (Gly4-Ser) n amino acid sequence is as shown in SEQ ID NO.18.

It is further preferred that the coding nucleotide sequence of described GM-CSF signal peptides is as shown in SEQ ID NO.3；It is described CD8 α hinge areas (Hinge) coding nucleotide sequence as shown in SEQ ID NO.5；The coding core of described CD8 α transmembrane regions Nucleotide sequence is as shown in SEQ ID NO.7；Described 4-1BB costimulatory signals domain (co-stimulatory signal Domain coding nucleotide sequence) is as shown in SEQ ID NO.9；Described CD3 ζ intracellular signals domain (signal domain) Coding nucleotide sequence as shown in SEQ ID NO.11；

The coding nucleotide sequence such as SEQ ID of described light-chain variable sequence (PD-L1light chain scFv) Shown in NO.13；The coding nucleotide sequence such as SEQ ID of described weight chain variabl area sequence (PD-L1heavy chain scFv) Shown in NO.15；Described (Gly4-Ser) n coding nucleotide sequence is as shown in SEQ ID NO.17.

The preparation method of the T lymphocytes of above-mentioned targeting PD-L1 Chimeric antigen receptors modification, it is comprised the following steps：

(1) targeting PD-L1 Chimeric antigen receptors PD-L1-CAR nucleotide sequence is obtained by PCR method；

(2) nucleotide sequence for targetting PD-L1 Chimeric antigen receptors PD-L1-CAR is cloned into expression vector, resisted PD-L1-CAR expression plasmid；

(3) anti-PD-L1-CAR expression plasmid is transfected to 293T cells；Packaging obtains virion, through centrifugal concentrating Slow virus concentrate is obtained afterwards；

(4) slow virus concentrate infects T lymphocytes so as to obtain the T lymphs of targeting PD-L1 Chimeric antigen receptor modifications Cell.

Preferably, the expression vector in step (2) is one kind in slow virus, retrovirus or rna expression carrier.

It is further preferred that the expression vector in step (2) is pCDH-CMV-MCS-EF1-copGFP-T2A-puro (letters Claim pCDH) Lentiviral, obtain anti-PD-L1-CAR expression plasmid pCDH-PD-L1-CAR.

Application of the T lymphocytes of above-mentioned targeting PD-L1 Chimeric antigen receptors modification in antineoplastic is prepared.

The inventive method has the following advantages that：The invention provides a kind of targeting PD-L1 chimeric antigens of brand new by The T lymphocytes (PD-L1-CAR-T cells) of body modification；Described PD-L1-CAR-T cells can not only pass through spy in body Different in nature tumor cell surface PD-L1, mediates the specific killing to PD-L1 positive tumors；PD-1/PD- can be prevented simultaneously L1 immunosupress signal paths, eliminate the immunosuppressive action of tumour.So as to reach the purpose of specific killing tumour.

Brief description of the drawings

Fig. 1 is PD-L1-CAR used in the present invention structural representation.

Fig. 2 is pCDH-CMV-MCS-EF1-copGFP-T2A-puro Lentiviral collection of illustrative plates.

Fig. 3 is the expression of green fluorescent protein in the T cell for infected PD-L1-CAR；A：Any processing is not done T cell；B：The T cell of PD-L1-CAR slow virus is infected.

Fig. 4 is killing activity of the T cell to tumour that PD-L1-CAR has been infected in TOTOX detections.

Fig. 5 is the situation of change of mouse tumor volume after periodic detection has injected PD-L1-CAR-T cells；A：Mouse tail It is injected intravenously the physiological saline of equivalent；B：Mouse tail vein injection 1x10^6pCDH slow virus empty carrier transduction in vitro culture T is thin Born of the same parents；C：Mouse tail vein injection 1x10^6PD-L1-CAR-T cells.

Fig. 6 is the survival condition of mouse after periodic detection has injected PD-L1-CAR-T cells；A：Mouse tail vein injection The physiological saline of equivalent；B：Mouse tail vein injection 1x10^6pCDH slow virus empty carrier transduction culturing T cells in vitro；C：Mouse Tail vein injection 1x10^6PD-L1-CAR-T cells.

Embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.

The determination of the PD-L1-CAR gene orders of embodiment 1

1.1 from U.S. national library of medicine website (http://www.ncbi.nlm.nih.gov/entrez) GM-CSF signal peptide genes, people's CD8 α hinge areas gene, people CD8 α transmembrane regions, 4-1BB is searched in GenBank databases to pierce altogether Energizing signal domain and CD3 ζ intracellular signal domain gene sequences.Anti- PD-L1 single-chain antibodies (anti-PD-L1scFv) gene order comes from patent (patent publication No.：CN105793288A).Each gene sequence information is shown in sequence table.

Said gene sequence is pressed human GM-CSF signal peptide gene, anti-PD-L1scFv, people's CD8 α hinge area bases by 1.2 successively Cause, people CD8 α transmembrane regions, 4-1BB costimulatory signals domain and CD3 ζ intracellular signal domain gene sequences are attached, and are formed final complete Whole PD-L1-CAR gene orders (PD-L1-CAR nucleotide sequence) information.

The structure of the PD-L1-CAR expression plasmids of embodiment 2

2.1 full genomes synthesize：

Full genome synthesizes complete PD-L1-CAR sequences, and adds restriction enzyme site at both ends.

Complete PD-L1-CAR gene orders are entered performing PCR by 2.2 with primer PD-L1-F, PD-L1-R to be expanded, PCR system For：

PCR reaction conditions：94 DEG C 4 minutes；94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 90 seconds, 34 circulation；72 DEG C 5 minutes；

PCR primer is run into Ago-Gel and carries out clip size identification, and carries out glue reclaim, that is, obtains complete PD-L1- CAR gene orders.

Above-mentioned sequence is cloned into pCDH-CMV-MCS-EF1-copGFP-T2A-puro (abbreviation pCDH) slow virus table by 2.3 Up in carrier, anti-PD-L1-CAR expression plasmid is obtained.

PD-L1-CAR and Lentiviral pCDH is subjected to digestion with XbaI/EcoRI respectively, digestion system is as follows, 37 DEG C of water-baths>1 hour.

Digestion products race Ago-Gel is identified, glue reclaim is carried out to correct product.

Corresponding digestion products are attached with T4DNA ligases, linked system is as follows：

16 DEG C of connections overnight, connection product conversion DH5a competent cells.

2.4 have 4 monoclonals of random picking in pCDH-PD-L1-CAR flat board from conversion, are separately added into blue or green containing ammonia benzyl Spread cultivation in the 4ml of mycin (100ug/ml) LB culture mediums, plasmid extraction is carried out after 6-8 hours, and use XbaI/EcoRI Digestion identification is carried out, reclaims the correct endonuclease bamhi of size, the raw work in sea is served and carries out sequencing and further determine that.Sequencing is correct i.e. For pCDH-PD-L1-CAR.PD-L1-CAR structures are shown in Fig. 1.PCDH information is shown in Fig. 2.

Packaging, concentration and the titer determination of the slow virus of embodiment 3

The packaging of 3.1 slow virus：

3.1.1 cell is handled：The 3-10 in exponential phase is collected for 293T cells within 24 hours before transfection, by 5 × 10^6 293T cells are inoculated in 10cm culture plates, and cell grows in the DMEM culture mediums containing 10ml 10%FBS, puts 37 DEG C 5%CO₂Incubator culture 24 hours, it can be transfected during up to 60-80% density.

3.1.2 into cell with 5:2:2:2 ratio cotransfection slow virus carrier plasmid (pCDH-PD-L1-CAR or its sky Carrier pCDH) and its packaging plasmid pLP1, pLP2, pLPVSV-G；Rotaring redyeing system is as follows：

Culture medium in 10cm culture plates is changed into complete medium after 6~8 hours.

3.1.3 24 hours after transfecting, the GFP luciferase expression situations after fluorescence microscopy Microscopic observation 293T cell transfectings.Point 293T culture supernatants are not collected after transfecting 24 hours, 48 hours, it is thin that 3000rpm collects 293T after centrifuging 15 minutes, 72 hours Born of the same parents and supernatant are managed in 15ml, multigelation three breakup cell, supernatant are collected by centrifugation；

3.1.4 with 0.45 μm of filter filter virus supernatant, pCDH- empty carriers and pCDH-PD-L1-CAR viruses are obtained respectively Stoste；- 80 DEG C of preservations.

3.2 slow virus concentrate

3.2.1 cell and culture supernatant are collected respectively, PEG6000 concentrating virus, 1/4 volume are added into viral solution PEG6000/NaCl solution (25%PEG6000+4.4%NaCl), mix, 4 DEG C stand 1 hour；

3.2.2 4 DEG C, 5000rpm is centrifuged 30 minutes；

3.2.3 supernatant is abandoned, adds appropriate virolysis liquid (10mM Tris-HCl (pH8.0), 2mM MgCl2,5% sugarcane Sugar) dissolving slow virus precipitation, and it is sub-packed in -80 DEG C of storages.Obtain slow virus concentrate.

3.3 slow virus titer determinations

3.3.1 inoculation BHK21 cells to 24 orifice plates, 10^5 cells/wells, are placed in 37 DEG C of 5%CO in advance₂Cell culture incubator is trained Support 18 hours；

3.3.2 lytic virus, prepare from 10^-2To 10^-7Do 10 times of gradient dilution viral samples；

3.3.3 cell culture fluid is removed, adds the virocyte nutrient solution containing different gradients, and added in nutrient solution 6ug/ml polybrene (polybrene), it is placed in 37 DEG C of 5%CO2 cell culture incubators cultures 2 hours；

3.3.4 cell culture fluid is removed, adds 10%FBS DMEM nutrient solutions, is placed in 37 DEG C of 5%CO2 cell culture incubators Culture 48 hours；

3.3.5 cell culture fluid is removed, puromycin containing 2ug/ml and 1%FBS DMEM nutrient solutions is added, is placed in 37 DEG C 5%CO2 cell culture incubators culture 72 hours；

3.3.6 cell culture fluid is removed, adds violet staining liquid, counting is colored clone's number, and calculates virus titer.

3.3.7 by viral dilution to 10^7TU/ml, stored in -80 DEG C.

In vitro culture, infection and the amplification of the T cell of embodiment 4

4.1 use CD3+T cell sortings kit (being purchased from Miltenyi Biotec companies) by CD3+T cells from PBMC Sort out.

4.2 are resuspended cell with RPMI-1640 culture mediums, add CD3, CD28 antibody and final concentration of 200U/ml IL-2 Costimulation；

4.3 are separately added into slow virus concentrate into the T cell of culture 6-8 days per hole, and (gradient is set, it is determined that optimal infection Titre), and final concentration of 4 μ g/ml polybrene, mix, be placed in 37 DEG C of 5%CO₂Incubator culture, liquid is not changed in 24h； Carry out infecting for second after 24h after infection；

96h after 4.4 infection, with the expression of fluorescence microscope T cell Green fluorescence, is as a result shown in Fig. 3.

After 4.5 collect cell, 1000rpm centrifugation 10min, PBS washing 1 time, cell is resuspended with appropriate PBS and is placed in streaming pipe In, with flow cytomery GFP positive rates.T lymphocytes (the PD-L1- of PD-L1 Chimeric antigen receptors modification must be targetted CAR-T cells).

The killing activity to tumour of the external PD-L1-CAR-T cells of embodiment 5

By PD-L1-CAR-T cells or control group sky pCDH carrier cells the malignant glioma cell positive with PD-L1 U87 is respectively with 1:1,5:1,10:1 ratio co-cultures, and every group of setting 3 is parallel, after 24-48h, with the non-radioactives of CytoTox 96 Property citotoxicity detection kit (Promega companies) detection PD-L1-CAR-T cells the killing activity to tumour, as a result see Fig. 4.As shown in figure 4, compared to the T cell of in vitro culture, PD-L1-CAR-T cells can effectively kill PD-L1 sun in vitro Property tumour cell, fully confirm the validity of PD-L1-CAR-T cells.

The killing activity to tumour of PD-L1-CAR-T cells in the Mice Body of embodiment 6.

6.1 are subcutaneously implanted the primary U87 of 1.5x10^6 (people's malignant glioma) cell in NSG mouse, and observation and measurement are swollen Knurl growing state, up to tumour growth to 40mm², carry out PD-L1-CAR-T cell therapies.

6.2PD-L1-CAR-T cell therapy glioma mouse model effect measurings：Controlled with PD-L1-CAR-T cells After treatment, observation and the growth of measurement tumour, Fig. 5 is as a result seen.As shown in figure 5, after with PD-L1-CAR-T cell therapies, mouse Gross tumor volume is gradually reduced, until be wholly absent, and the mouse tumor volume treated using the T cell of in vitro culture is persistently increased Greatly.Mouse survival rate is recorded, as a result sees Fig. 6, as shown in fig. 6, survived completely through PD-L1-CAR-T cell therapy group mouse, and The mouse treated using the T cell of in vitro culture all uses euthanasia because gross tumor volume is excessive.

Although above with general explanation and specific embodiment, the present invention is described in detail, at this On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, These modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.

SEQUENCE LISTING

<110>When power biotechnology（Beijing）Co., Ltd

<120>A kind of T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification and its preparation method and application

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Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

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<400> 8

Ala Ala Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr

1 5 10 15

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

20 25 30

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

35 40 45

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

50 55 60

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

65 70 75 80

Tyr Cys Asn His Arg Asn

85

<210> 9

<211> 141

<212> DNA

<213>Artificial sequence

<400> 9

cgtttctctg ttgttaaacg gggcagaaag aagctcctgt atatattcaa acaaccattt 60

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 120

gaagaaggag gatgtgaact g 141

<210> 10

<211> 47

<212> PRT

<213>Artificial sequence

<400> 10

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40 45

<210> 11

<211> 339

<212> DNA

<213>Artificial sequence

<400> 11

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc ycaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339

<210> 12

<211> 112

<212> PRT

<213>Artificial sequence

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa can be any naturally occurring amino acid

<400> 12

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Xaa Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 13

<211> 324

<212> DNA

<213>Artificial sequence

<400> 13

gatattcaaa tgactcaatc tccttcttct ctttctgctt ctgttggtga tagagttact 60

ataacttgta gagcttctca agatgtttct actgctgttg cttggtatca agaaaaacct 120

ggtaaagctc ctaaattatt gatctactcc gcctcatttt tatattcggg tgtaccatcg 180

cgtttttcgg gatcaggttc ggggacggac ttcacattaa cgatttcttc actacaacca 240

gaagacttcg caacatatta ctgtcaacag tatttatacc atccagcaac ttttggtcaa 300

ggcacaaaag tagagatcaa gaga 324

<210> 14

<211> 108

<212> PRT

<213>Artificial sequence

<400> 14

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

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Glu Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 15

<211> 354

<212> DNA

<213>Artificial sequence

<400> 15

gaagttcaat tagttgagtc tggtggcgga ctcgttcagc ctgggggttc ccttcgtctc 60

tcatgtgcag cgtctggttt tactttctct gattcatgga ttcattgggt aagacaagct 120

ccaggtaaag gtcttgaatg ggttgcttgg atttctcctt acggaggttc aacttattac 180

gcagatagtg ttaagggtcg tttcactatt tctgcagata cttctaagaa tactgcttat 240

ttacaaatga attctcttag agctgaagat actgctgttt attactgcgc aagaaggcat 300

tggcctggcg gatttgatta ttggggtcag ggaactcttg ttactgtttc tgca 354

<210> 16

<211> 118

<212> PRT

<213>Artificial sequence

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 17

<211> 45

<212> DNA

<213>Artificial sequence

<400> 17

ggcggaggcg ggtcaggagg tggcggcagc ggaggaggag ggtcc 45

<210> 18

<211> 15

<212> PRT

<213>Artificial sequence

<400> 18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Claims (10)

1. A kind of 1. T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification, it is characterised in that the T lymphocytic cell surfaces table Up to targeting PD-L1 Chimeric antigen receptors PD-L1-CAR.
2. 2. the T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification according to claim 1, it is characterised in that described Targeting PD-L1 Chimeric antigen receptors PD-L1-CAR amino acid sequence as shown in SEQ ID NO.2.
3. 3. the T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification according to claim 1, it is characterised in that described Targeting PD-L1 Chimeric antigen receptors PD-L1-CAR coding nucleotide sequence as shown in SEQ ID NO.1.
4. 4. the T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification according to claim 1, it is characterised in that described Targeting PD-L1 Chimeric antigen receptors PD-L1-CAR by GM-CSF signal peptides, the single-stranded variable region of PD-L1 antibody, CD8 α hinges Area, CD8 α transmembrane regions, 4-1BB costimulatory signals domain and CD3 ζ intracellular signals domain are composed in series；

   The single-stranded variable region of wherein PD-L1 antibody is to pass through (Gly4-Ser) by weight chain variabl area sequence and light-chain variable sequence N is composed in series.
5. 5. the T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification according to claim 4, it is characterised in that institute The amino acid sequence for the GM-CSF signal peptides stated is as shown in SEQ ID NO.4；The amino acid sequence of described CD8 α hinge areas is such as Shown in SEQ ID NO.6；The amino acid sequence of described CD8 α transmembrane regions is as shown in SEQ ID NO.8；Described 4-1BB is pierced altogether The amino acid sequence in energizing signal domain is as shown in SEQ ID NO.10；The amino acid sequence such as SEQ in described CD3 ζ intracellular signals domain Shown in ID NO.12；

   The amino acid sequence of described light-chain variable sequence is as shown in SEQ ID NO.14；Described weight chain variabl area sequence Amino acid sequence is as shown in SEQ ID NO.16；Described (Gly4-Ser) n amino acid sequence is as shown in SEQ ID NO.18.
6. 6. the T lymphocytes of targeting PD-L1 Chimeric antigen receptors modification according to claim 4, it is characterised in that institute The coding nucleotide sequence for the GM-CSF signal peptides stated is as shown in SEQ ID NO.3；The encoding nucleoside of described CD8 α hinge areas Acid sequence is as shown in SEQ ID NO.5；The coding nucleotide sequence of described CD8 α transmembrane regions is as shown in SEQ ID NO.7；Institute The coding nucleotide sequence in the 4-1BB costimulatory signals domain stated is as shown in SEQ ID NO.9；Described CD3 ζ intracellular signals domain Coding nucleotide sequence is as shown in SEQ ID NO.11；

   The nucleic acid sequence encoding of described light-chain variable sequence is as shown in SEQ ID NO.13；Described weight chain variabl area sequence Coding nucleotide sequence as shown in SEQ ID NO.15；Described (Gly4-Ser) n coding nucleotide sequence such as SEQ ID Shown in NO.17.
7. 7. the preparation method of the T lymphocytes of the targeting PD-L1 Chimeric antigen receptors modification described in any one of claim 1~6, Characterized in that, the preparation method comprises the following steps：

   (1) targeting PD-L1 Chimeric antigen receptors PD-L1-CAR nucleotide sequence is obtained by PCR method；

   (2) nucleotide sequence for targetting PD-L1 Chimeric antigen receptors PD-L1-CAR is cloned into expression vector, obtains anti-PD- L1-CAR expression plasmid；

   (3) anti-PD-L1-CAR expression plasmid is transfected to 293T cells；Packaging obtains virion, is obtained after centrifugal concentrating Obtain slow virus concentrate；

   (4) slow virus concentrate infects T lymphocytes so as to obtain the T lymphocytes of targeting PD-L1 Chimeric antigen receptor modifications.
8. 8. preparation method according to claim 7, it is characterised in that the expression vector in step (2) is slow virus, reversed One kind in record virus or rna expression carrier.
9. 9. preparation method according to claim 7, it is characterised in that the expression vector in step (2) is pCDH-CMV- MCS-EF1-copGFP-T2A-puro Lentivirals, obtain anti-PD-L1-CAR expression plasmid pCDH-PD-L1- CAR。
10. 10. the T lymphocytes of the targeting PD-L1 Chimeric antigen receptors modification described in any one of claim 1~6 are preparing anti-swell Application in tumor medicine.