CN105131126A - Chimeric antigen receptor for treating malignant tumor and preparation method and application of chimeric antigen receptor - Google Patents
Chimeric antigen receptor for treating malignant tumor and preparation method and application of chimeric antigen receptor Download PDFInfo
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Abstract
The invention discloses a chimeric antigen receptor for treating malignant tumor and a preparation method and application of the chimeric antigen receptor. The chimeric antigen receptor is an NY-ESO-1 specificity chimeric antigen receptor; specifically, the chimeric antigen receptor is protein formed by a variable region of an NY-ESO-1 TCR alpha chain, a variable region of an NY-ESO-1 TCR beta chain, a hinge region and a trans-membrane region of CD8, a CD28 intracellular signal structure region, a CD137 intracellular signal structure region and a CD3 zeta intracellular signal structure region in sequence from the amino terminal to the carboxyl terminal. Experiments prove that CAR-CTL cells prepared through NY-ESO-1 antigen peptide have higher tumor cell targeting activity and killing activity, and therefore the chimeric antigen receptor has wide clinical application prospects.
Description
Technical field
The invention belongs to biology and medical art, relate to a kind of Chimeric antigen receptor being used for the treatment of malignant tumour and preparation method thereof and application.
Background technology
Chimeric antigen receptor (chimericantigenreceptor, the T cell (CAR-T) of CAR) modifying to identify that the single-chain antibody of tumor associated antigen combines with T cell activation motif, modified give the stronger tumor-targeting of T cell and killing activity by engineering.
From 1989, since proposing CAR concept first by Eshhar and colleague thereof, three different developmental stage are experienced by.First-generation CAR acceptor, comprise the scFv fragment of the outer specific recognition tumour antigen of born of the same parents, in born of the same parents, activation signal is transmitted by " immunoreceptor tyrosine-based activation motif " ITAM (immunoreceptortyrosine-basedactivationmotifs) signal chains of CD3 ζ or Fc ε RI γ.
" immunoreceptor tyrosine-based activation motif " ITAM (immunoreceptortyrosine-basedactivationmotifs) refer to activated immune cell associated receptor (such as TCR/CD3, FcR γ etc.) cytoplasmic domain common with tyrosine motif (tyrosine, Y) aa sequence motifs based on, can be combined with the signaling molecule in signal transduction pathway downstream after being phosphorylated, cause cell activation.
What identify due to CAR-T cell is the antigen of tumor cell surface, but not is combined with MHC molecule and forms MHC-antigenic compound thus offered to the antigen of cell surface, and the MHC molecule that thus can bypass T cell is restricted.And the first signal of T cell and second signal are coupled in same molecular structure by CAR-T technology usually, make CAR-T cell have stronger autonomy, substantially can play therapeutic action without the need to the auxiliary of other types immunocyte.
But first-generation CAR acceptor is owing to lacking the costimulatory signal needed for T cell activation, cause T cell in vivo the survival time shorter, cytokine secretion is few, can only play instant activation effect.S-generation CAR acceptor is by adding the intracellular domain (such as CD28 molecular structure territory) of a costimulatory signal molecule, provide two kinds of signals of T cell activation, enhance the multiplication capacity of T cell and the secretion capacity of cytokine (such as IL-2, IFN-γ and GM-CSF), thus breach the immunosuppression of tumor microenvironment.Third generation CAR acceptor, on the basis of s-generation CAR, has newly increased the intracellular domain of another costimulatory signal molecule, and then is provided with the survival time in better effector function and longer body.
Summary of the invention
The object of this invention is to provide a kind of Chimeric antigen receptor being used for the treatment of malignant tumour and preparation method thereof and application.
The Chimeric antigen receptor being used for the treatment of malignant tumour provided by the present invention is that development obtains on the basis of third generation CAR receptor technology, be specially NY-ESO-1 specific chimeric antigen receptor, from structure composition angle, described Chimeric antigen receptor is the protein be made up of the variable region of NY-ESO-1TCR α chain, the variable region of NY-ESO-1TCR β chain, the hinge area of CD8 and cross-film district, CD28 intracellular signal structural domain, CD137 intracellular signal structural domain, CD3 ζ intracellular signal structural domain successively to carboxyl terminal from aminoterminal.
Wherein, the aminoacid sequence of the variable region of described NY-ESO-1TCR α chain is as shown in sequence in sequence table 9; The aminoacid sequence of the variable region of described NY-ESO-1TCR β chain is as shown in sequence in sequence table 10; The hinge area of described CD8 and the aminoacid sequence in cross-film district are as shown in sequence in sequence table 11; The aminoacid sequence of described CD28 intracellular signal structural domain is as shown in sequence in sequence table 12; The aminoacid sequence of described CD137 intracellular signal structural domain is as shown in sequence in sequence table 13; The aminoacid sequence of described CD3 ζ intracellular signal structural domain is as shown in sequence in sequence table 14.
Further, the aminoacid sequence of described Chimeric antigen receptor is as shown in sequence in sequence table 8.
In the present invention, described Chimeric antigen receptor is specially and will expresses the protein containing aminoacid sequence shown in sequence 8 in ordered list obtained after recombinant slow virus expression vector pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ transfered cell; Described recombinant slow virus expression vector pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ is for replacing with the small segment between restriction enzyme site XhoI and BamHI of Lentiviral pLVX-IRES-ZsGreen1 the recombinant plasmid obtained after DNA fragmentation shown in sequence 1 in sequence table.
The gene of described Chimeric antigen receptor of encoding also belongs to protection scope of the present invention.
Wherein, the nucleotide sequence of gene of variable region of described NY-ESO-1TCR α chain is encoded as shown in sequence in sequence table 2; Encode the nucleotide sequence of gene of variable region of described NY-ESO-1TCR β chain as shown in sequence in sequence table 3; Encode the nucleotide sequence of the hinge area of described CD8 and the gene in cross-film district as shown in sequence in sequence table 4; Encode the nucleotide sequence of gene of described CD28 intracellular signal structural domain as shown in sequence in sequence table 5; Encode the nucleotide sequence of gene of described CD137 intracellular signal structural domain as shown in sequence in sequence table 6; Encode the nucleotide sequence of gene of described CD3 ζ intracellular signal structural domain as shown in sequence in sequence table 7.
Concrete, the nucleotide sequence of the gene of described Chimeric antigen receptor of encoding is as shown in sequence in sequence table 1.Wherein, encode the variable region of described NY-ESO-1TCR α chain in the 1-279 position of sequence 1,280-501 encodes position the variable region of described NY-ESO-1TCR β chain, 502-699 encodes position the hinge area of described CD8 and cross-film district, 700-822 encodes position described CD28 intracellular signal structural domain, 823-948 encodes position described CD137 intracellular signal structural domain, and 949-1287 encodes position described CD3 ζ intracellular signal structural domain.
Recombinant vectors, expression cassette, recombinant virus or the reconstitution cell of the gene (sequence 1) containing the described Chimeric antigen receptor of coding also belong to protection scope of the present invention.
In the present invention, described recombinant vectors is the recombinant slow virus expression vector can expressing described Chimeric antigen receptor.Small segment between being specially restriction enzyme site XhoI and BamHI of Lentiviral pLVX-IRES-ZsGreen1 replaces with the recombinant plasmid (called after pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ) obtained after DNA fragmentation shown in sequence 1 in sequence table.
Wherein, described reconstitution cell is the immune effector cell can expressing described Chimeric antigen receptor; Described recombinant virus for can express described Chimeric antigen receptor, and can infect the virus of immune effector cell.
Concrete, described immune effector cell can be cytotoxic T lymphocyte, NKT cell, NK cell or helper T cell; Described virus can be slow virus, simplexvirus, cytomegalovirus, Epstein-Barr virus, hepatitis B virus, hepatitis C virus or virus of AIDS.
Present invention also offers a kind of preparation method of NY-ESO-1 specific C AR-T cell.
The preparation method of NY-ESO-1 specific C AR-T cell provided by the present invention, is specifically included in step T cell being expressed described Chimeric antigen receptor.
Further, described " in T cell, expressing described Chimeric antigen receptor " is by realizing as follows: with recombinant expression vector A transfecting T cells, from the cell after transfection, obtain the T cell (CTL cell) expressing described Chimeric antigen receptor, be described NY-ESO-1 specific C AR-T cell (described NY-ESO-1 specific C AR-CTL cell); Described recombinant expression vector A is for replacing with the small segment between restriction enzyme site XhoI and BamHI of Lentiviral pLVX-IRES-ZsGreen1 the recombinant plasmid obtained after DNA fragmentation shown in sequence 1 in sequence table.
Wherein, the source of described T cell can from comprising peripheral blood mononuclear cell, ascites, hydrothorax or tumor tissues.
The application in arbitrary as follows of previously described Chimeric antigen receptor or gene or recombinant vectors or expression cassette or recombinant virus or reconstitution cell also belongs to protection scope of the present invention:
A () is for the preparation of the product for the treatment of treatment malignant tumour;
B () is for the preparation of the product killing and wounding the tumour cell of expressing NY-ESO-1 antigen peptide;
C () is for the preparation of the HLA-A2 killing and wounding load NY-ESO-1 antigen peptide
+the product of T2 cell strain.
Wherein, described malignant tumour is express the malignant tumour of described NY-ESO-1 antigen peptide; Concrete, described malignant tumour is optional arbitrary in following: the tumour of primary colon cancer, carcinoma of the pancreas, cholangiocarcinoma, cancer of the stomach, esophagus cancer, gland cancer, lung cancer, mammary cancer and urinary system.The aminoacid sequence of described NY-ESO-1 antigen peptide is as shown in sequence in sequence table 15.
More concrete, the described load HLA-A2 of NY-ESO-1 antigen peptide
+t2 cell strain is by HLA-A2
+adding final concentration in the nutrient solution of T2 non-loaded cells strain is after the described NY-ESO-1 antigen peptide of 50 μ g/ml, in 37 DEG C, 5%CO
2to hatch 120min under condition and realize load.
Experiment proves, the CAR-CTL cell that the present invention utilizes NY-ESO-1 antigen peptide to prepare has stronger tumour cell targeting and killing activity, thus has wide potential applicability in clinical practice.
Accompanying drawing explanation
Fig. 1 be mouse-anti people CD3-PerCP CD4-FITC CD8-APC NY-ESO-1TCR-PE Identification of the antibodies CAR
nY-ESO-1TCRthe FCM analysis result of the CTL cell of genetic modification.Wherein, CD3
+cD8
+cD4
-cell be CTL cell.
Fig. 2 is CAR
nY-ESO-1TCRthe killing effect in vitro measurement result of the CTL cell of genetic modification.Wherein, the target cell of A is the HLA-A2 of load tumor antigen peptide NY-ESO-1
+t2 cell strain; The target cell of B is HLA-A2
+the strain of T2 non-loaded cells.In A and B, CAR-T represents " embodiment 2 obtain through CAR
nY-ESO-1TCRthe CTL cell of genetic modification "; CTL represents " the CTL cell of unmodified ".
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
HLA-A2
+the strain of T2 non-loaded cells: ATCC, ATCC are numbered "
cRL-1992
tM".
Lentiviral pLVX-IRES-ZsGreen1:Invitrogen Products, article No. Biovector008.
PUC57-NY-ESO-1TCR α β plasmid: the pUC57 plasmid containing NY-ESO-1TCR α and β chain variable region sequence, its structrual description is as follows: the DNA fragmentation (i.e. NY-ESO-1TCR α and β chain variable region sequence) shown in the 1-501 position of sequence in sequence table 1 is inserted into the recombinant plasmid obtained between restriction enzyme site EcoRV and SmaI of pUC57 plasmid, and this recombinant plasmid is synthesized by Hua Da genome company.
PUC57-CD8-CD28-CD137-CD3 ξ plasmid: the pUC57 plasmid containing CD8-CD28-CD137-CD3 ξ sequence, its structrual description is as follows: the DNA fragmentation (i.e. CD8-CD28-CD137-CD3 ξ sequence) shown in the 502-1287 position of sequence in sequence table 1 is inserted into the recombinant plasmid obtained between restriction enzyme site EcoRV and SmaI of pUC57 plasmid, and this recombinant plasmid is synthesized by Hua Da genome company.
The structure of the Lentiviral pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ of embodiment 1, load Chimeric antigen receptor NY-ESO-1TCR and qualification
The acquisition of the DNA fragmentation of the variable region of the variable region one, containing NY-ESO-1TCR α chain and NY-ESO-1TCR β chain
With the plasmid pUC57-NY-ESO-1TCR α β containing NY-ESO-1TCR α and β chain variable region sequence for template, carry out pcr amplification with F1 and R1 for primer, obtain the sequence of 528bp, wherein primers F 1 has XhoI restriction enzyme site; Primer R1 contains overlap, for subsequent overlay PCR.Utilize Primer5 software design primer, completed the synthesis of primer by Hua Da gene limited liability company.
F1:5 '-CCG
cTCGAGcAGAAGGTAACTCAAGCGCAGACT-3 ' (underscore part is the recognition sequence of XhoI, and sequence is thereafter the 1-24 position of sequence 1 in sequence table);
R1:5 '-CGGCGCTGGCGTCGTGGTACTGCTGGCACAGAAGTACACAG-3 ' (reverse complementary sequence of the 479-519 position of sequence 1).
Reaction system:
Reaction conditions: 95 DEG C, 5min; 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35 circulations; 72 DEG C of 10min; 4 DEG C of ∞.
After reaction terminates, carry out agarose gel electrophoresis and cut glue reclaiming object fragment, the nucleotides sequence of object fragment is classified as " 5 '-CCG
cTCGAGthe 1-519 position of+sequence 1 ".
The acquisition of the DAN fragment of the hinge area two, containing CD8 and cross-film district, CD28 intracellular signal structural domain, CD137 intracellular signal structural domain and CD3 ζ intracellular signal structural domain
With the plasmid pUC57-CD8-CD28-CD137-CD3 ξ containing CD8-CD28-CD137-CD3 ξ sequence for template, with F2 and R2 for primer carries out pcr amplification, obtain the sequence of 818bp, wherein primers F 2 is containing overlap, for subsequent overlay PCR; Primer R2 contains BamHI restriction enzyme site.
The 479-519 position of F2:5'-CTGTGTACTTCTGTGCCAGCAGTACCACGACGCCAGCGCCG-3'(sequence 1);
R2:5'-CGC
gGATCCgCGAGGGGGCAGGGCCTG-3'(underscore part is the recognition sequence of BamHI, and sequence is thereafter the reverse complementary sequence of last 18 of sequence 1 in sequence table).
Reaction system:
Reaction conditions: 95 DEG C, 5min; 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s, 35 circulations; 72 DEG C of 10min; 4 DEG C of ∞.
After reaction terminates, carry out agarose gel electrophoresis and cut glue reclaiming object fragment, the nucleotides sequence of object fragment is classified as " the 479-1287 position of sequence 1+
gGATCCgCG ".
Three, the fusion of step one and 2 two DNA fragmentations
Object fragment is obtained respectively for template with step one and step 2, F1 and R2 is primer (sequence is see step one and step 2), carry out over-lap PCR, two object fragment assemblies are got up, obtain the sequence of 1305bp, the restriction enzyme site of XhoI and BamHI is contained at sequence two ends, is NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ by sequence designations.
The nucleotides sequence of NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ is classified as " 5 '-CCG
cTCGAGsequence 1+ in+sequence table
gGATCCgCG-3 ' ".Wherein, the variable region of the NY-ESO-1TCR α chain shown in sequence 9 in 1-279 position (the i.e. sequence 2) polynucleotide of sequence 1, the variable region of the NY-ESO-1TCR β chain shown in sequence 10 in 280-501 position (i.e. sequence 3) polynucleotide, the hinge area of the CD8 shown in sequence 11 and cross-film district in 502-699 position (i.e. sequence 4) polynucleotide, CD28 intracellular signal structural domain shown in sequence 12 in 700-822 position (i.e. sequence 5) polynucleotide, CD137 intracellular signal structural domain shown in sequence 13 in 823-948 position (i.e. sequence 6) polynucleotide, CD3 ζ intracellular signal structural domain in 949-1287 position (i.e. sequence 7) polynucleotide shown in sequence 14.The CAR being specific to NY-ESO-1 shown in sequence 8 in sequence 1 polynucleotide.
Wherein, reaction system is:
Reaction conditions is: 95 DEG C, 5min; 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 35 circulations; 72 DEG C of 10min; 4 DEG C of ∞.
Four, recombinant slow virus expression vector pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ is built
Adopt restriction enzyme XhoI and BamHI (TaKaRa) to carry out double digestion reaction the fusion gene " NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ " that Lentiviral pLVX-IRES-ZsGreen1 and step 3 obtain simultaneously.Digestion products, after agarose gel electrophoresis detects, carries out cutting glue and reclaims, and carries out being connected of object fragment and carrier with T4DNA ligase enzyme (TaKaRa).Recombinant expression vector is carried out DH5 α E. coli competent (TaKaRa) to transform, obtain positive colony by plasmid PCR and double digestion reaction screening, will the correct recombinant plasmid called after pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ of sequence verification be passed through.The structrual description of pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ is as follows: the small segment between restriction enzyme site XhoI and BamHI of Lentiviral pLVX-IRES-ZsGreen1 is replaced with the recombinant plasmid obtained after DNA fragmentation shown in sequence 1 in sequence table.
Double digestion reaction system:
Reaction conditions: 37 DEG C, 4h.
T4DNA ligase enzyme reacts:
Reaction conditions: 16 DEG C, spend the night.
Plasmid PCR reaction system:
Reaction conditions: 95 DEG C, 5min; 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 35 circulations; 72 DEG C of 10min; 4 DEG C of ∞.
The preparation of the CAR-CTL cell that embodiment 2, Chimeric antigen receptor are modified
One, the lymphocytic preparation of T
Get the human peripheral that 60ml is fresh, with lymphocyte separation medium (Mediatech Products) separating peripheral blood mononuclear cells (PBMC), with the RPMI1640 substratum inducing culture 48h of OKT3 and 5% (volume fraction) people AB serum (Invitrogen Products) of IL-2, the 50ng/ml containing 500IU/ml, obtain T lymphocyte.
Two, the preparation of the CAR-CTL cell of Chimeric antigen receptor modification
Recombinant slow virus expression vector pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ embodiment 1 being built acquisition utilize PEI (purchased from sigma company, article No.: 03880) the T lymphocyte of inducing culture in transfection to step one.After transfection 48h, cell is transferred to and have in the RPMI1640 substratum of Liu Suanyan NEOMYCIN SULPHATE, and by limiting dilution assay by Cell-cloned, through the screening of 21 days, obtain and there is neomycin resistance, through CAR
nY-ESO-1TCRthe CTL cell of genetic modification.
Adopt flow cytomery CAR after screening in 21 days
nY-ESO-1TCRthe expression of the CTL cell surface mosaic antigen receptor of genetic modification.Concrete steps are: getting 100 μ l concentration is 1 × 10
6the CAR-CTL cell suspension of individual/ml, add respectively detection mouse-anti people CD3-PerCP CD4-FITC CD8-APC NY-ESO-1TCR-PE antibody and each 5 μ l of Isotype control IgG1 monoclonal antibody, 4 DEG C of lucifuges hatch 30min, after PBS buffer solution 2 times, suspension cell, in 500ul damping fluid PBS, carries out FCM analysis.Wherein, mouse-anti people CD3-PerCP antibody is U.S. company BD product, and its catalog number is 560835; Mouse-anti people CD4-FITC antibody is U.S. company BD product, and its catalog number is 555346; Mouse-anti people CD8-APC antibody is U.S. company BD product, and its catalog number is 561421; Mouse-anti people NY-ESO-1TCR-PE antibody is U.S. company BD sintetics, adopts its catalog number to be 551263.
FCM analysis result as shown in Figure 1.Visible, successfully induce CAR through the post-stimulatory T lymphocyte of recombinant slow virus expression vector pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ expression product
nY-ESO-1TCRspecific CTL.
Embodiment 3, CAR
nY-ESO-1TCRthe IFN-γ secretory volume of the CTL cell of genetic modification measures
Euzymelinked immunosorbent assay (ELISA) detects the ability of secretion of gamma-IFN, IFN-γ people enzyme-linked immunospot assay (ELISPOT) test kit (article No.: ab46551) that concrete employing Abcam company produces detects: adopt ELISPOT detection kit, IFN-γ capture antibody (carrying in test kit) the 100 μ L diluted by 1:100 (volume ratio) with PBS is added in 96 orifice plates, be placed in 4 DEG C of overnight incubation, with adding the effector cell (CAR that embodiment 2 obtains after PBS washing
nY-ESO-1TCRthe CTL cell of genetic modification) 100 μ L, and target cell (the load HLA-A2 of tumor antigen peptide NY-ESO-1
+t2 cell strain) 100 μ L, wherein the number ratio (E/T) of effector cell and target cell is 10, at 37 DEG C, 5%CO
2after hatching 24h under condition, after washing with the PBS containing 0.1% (volume fraction) polysorbas20, add with the PBS containing 1% (1g/100ml) BSA by the biotin labeled anti-IFN-gamma antibodies (carrying in test kit) after 1:100 (volume ratio) dilution, at 37 DEG C, 5%CO
22h is hatched under condition, with alkaline phosphatase (carrying in test kit) the 100 μ L that the PBS containing 1% (1g/100ml) BSA marks by the avidin together after 1:5000 (volume ratio) dilution, hatch 1h, after patting dry culture plate after washing, use distilled water termination reaction, read point after dry.
Wherein, " the load HLA-A2 of tumor antigen peptide NY-ESO-1
+t2 cell strain " be by HLA-A2
+adding final concentration in the nutrient solution of T2 non-loaded cells strain is after the tumor antigen peptide NY-ESO-1 of 50 μ g/ml, in 37 DEG C, 5%CO
2to hatch 120min under condition and realize load.The aminoacid sequence of tumor antigen peptide NY-ESO-1 is as shown in sequence in sequence table 15.
Setup Experiments repeats for 3 times.
Experiment arranges PBS damping fluid simultaneously and substitutes target cell as nonspecific control group.
Result shows: the spot number of experimental group CAR-CTL emiocytosis IFN-γ is (125.2 ± 5.62), obviously more than non specific control group spot number (30.6 ± 4.61), (P<0.05).
Embodiment 4, CAR
nY-ESO-1TCRthe killing effect in vitro of the CTL cell of genetic modification measures
One, target cell prepares
Target cell 1:HLA-A2
+the strain of T2 non-loaded cells;
Target cell 2: the load HLA-A2 of tumor antigen peptide NY-ESO-1
+t2 cell strain (preparation method is see embodiment 3).
Two, pass through
51cr release test, measures CAR
nY-ESO-1TCRthe killing effect in vitro of the CTL cell of genetic modification
The chromium of CTL (effector) activity standard (
51cr) release test is weighed.With embodiment 2 obtain through CAR
nY-ESO-1TCRthe CTL cell of genetic modification and CTL cell action effect cell (E) of unmodified, ready two kinds of target cell Dual culture with step one respectively by two kinds of effector cells, detect the specific killing activity of CTL.Concrete operations are as follows:
Adjustment target cell 2 × 10
6cell/100 μ l is placed in the PBS containing 4% (volume fraction) FCS, with 100 μ l
51cr (PerkinElmer) mark also cultivates 1h in 37 DEG C.The target cell marked finally washes 4 times with 1 × Hank ' s balanced salt solution (Invitrogen) containing 2% (volume fraction) calf serum (Invitrogen), in 4 DEG C of centrifugal 8min of 1200-1500rpm, Eddy diffusion, in the fresh RPMI-1640 substratum of 15ml (GIBCO article No.: 31800-022:31800-022), is 2 × 10 to final concentration
5cell/ml.
(initial concentration is respectively 3 × 10 to the CTL that 100 μ l steps 2 obtain
6cell/ml, 1 × 10
6cell/ml, 3 × 10
5cell/ml), the concentration of each effector cell establishes 3 repetitions.Containing different dilution CTL100 μ l in each hole, add 50 μ l target cells (2 × 10
5cell/ml), 4 kinds of effects target ratio (E/T) are formed altogether, i.e. 30:1,10:1,5:1 and 1:1.Establish Spontaneous release hole (50 μ l target cell+0.1ml complete RPMI-1640 nutrient solution) and maximum release aperture (50 μ l target cell+0.1ml2%SDS) simultaneously.Respectively to process in 37 DEG C of 5%CO
2cultivate 4h under condition, the centrifugal 5min of 900rpm, 100 μ l supernatant liquors are got extremely in every hole
51(cpm value) is counted in Cr counter tube.
3 multiple holes established by each sample, average as detected result.
Cell killing activity calculates as follows:
Cell killing activity (%)=(treating gaging hole fluorescence intensity-Spontaneous release hole fluorescence intensity)/(maximum release aperture fluorescence intensity-Spontaneous release hole fluorescence intensity) × 100%
Result as shown in A in Fig. 2, as seen under each effect target ratio, through CAR
nY-ESO-1TCRthe CTL cell of genetic modification compared with the CTL cell of unmodified, to target cell 2 (the i.e. load HLA-A2 of tumor antigen peptide NY-ESO-1
+t2 cell strain) there is significant killing activity (P<0.01).
Claims (10)
1.NY-ESO-1 specific chimeric antigen receptor is the protein be made up of the variable region of NY-ESO-1TCR α chain, the variable region of NY-ESO-1TCR β chain, the hinge area of CD8 and cross-film district, CD28 intracellular signal structural domain, CD137 intracellular signal structural domain, CD3 ζ intracellular signal structural domain successively to carboxyl terminal from aminoterminal.
2. NY-ESO-1 specific chimeric antigen receptor according to claim 1, is characterized in that: the aminoacid sequence of the variable region of described NY-ESO-1TCR α chain is as shown in sequence in sequence table 9; And/or
The aminoacid sequence of the variable region of described NY-ESO-1TCR β chain is as shown in sequence in sequence table 10; And/or
The hinge area of described CD8 and the aminoacid sequence in cross-film district are as shown in sequence in sequence table 11; And/or
The aminoacid sequence of described CD28 intracellular signal structural domain is as shown in sequence in sequence table 12; And/or
The aminoacid sequence of described CD137 intracellular signal structural domain is as shown in sequence in sequence table 13; And/or
The aminoacid sequence of described CD3 ζ intracellular signal structural domain is as shown in sequence in sequence table 14; And/or
The aminoacid sequence of described Chimeric antigen receptor is as shown in sequence in sequence table 8.
3. the gene of NY-ESO-1 specific chimeric antigen receptor described in coding claim 1 or 2.
4. gene according to claim 3, is characterized in that: the nucleotide sequence of the gene of the variable region of described NY-ESO-1TCR α chain of encoding is as shown in sequence in sequence table 2; And/or
Encode the nucleotide sequence of gene of variable region of described NY-ESO-1TCR β chain as shown in sequence in sequence table 3; And/or
Encode the nucleotide sequence of the hinge area of described CD8 and the gene in cross-film district as shown in sequence in sequence table 4; And/or
Encode the nucleotide sequence of gene of described CD28 intracellular signal structural domain as shown in sequence in sequence table 5; And/or
Encode the nucleotide sequence of gene of described CD137 intracellular signal structural domain as shown in sequence in sequence table 6; And/or
Encode the nucleotide sequence of gene of described CD3 ζ intracellular signal structural domain as shown in sequence in sequence table 7; And/or
The nucleotide sequence of the gene of described Chimeric antigen receptor of encoding is as shown in sequence in sequence table 1.
5. the recombinant vectors containing gene described in claim 3 or 4, expression cassette, recombinant virus or reconstitution cell.
6. recombinant virus according to claim 5 or reconstitution cell, is characterized in that: described reconstitution cell is for expressing the immune effector cell of Chimeric antigen receptor described in claim 1 or 2;
Described recombinant virus for expressing Chimeric antigen receptor described in claim 1 or 2, and can infect the virus of immune effector cell;
Concrete, described immune effector cell is cytotoxic T lymphocyte, NKT cell, NK cell or helper T cell;
Described virus is slow virus, simplexvirus, cytomegalovirus, Epstein-Barr virus, hepatitis B virus, hepatitis C virus or virus of AIDS.
The preparation method of 7.NY-ESO-1 specific C AR-T cell, is included in step T cell being expressed NY-ESO-1 specific chimeric antigen receptor described in claim 1 or 2.
8. method according to claim 7, is characterized in that: described method comprises the steps:, with recombinant expression vector A transfecting T cells, to obtain the T cell expressing described NY-ESO-1 specific chimeric antigen receptor from the cell after transfection; Described recombinant expression vector A is for replacing with the small segment between restriction enzyme site XhoI and BamHI of Lentiviral pLVX-IRES-ZsGreen1 the recombinant plasmid obtained after DNA fragmentation shown in sequence 1 in sequence table.
9. arbitrary described NY-ESO-1 specific chimeric antigen receptor or gene or recombinant vectors or expression cassette or recombinant virus or the reconstitution cell application in arbitrary as follows in claim 1-6:
A () is for the preparation of the product for the treatment of treatment malignant tumour;
B () is for the preparation of the product killing and wounding the tumour cell of expressing NY-ESO-1 antigen peptide;
C () is for the preparation of the HLA-A2 killing and wounding load NY-ESO-1 antigen peptide
+the product of T2 cell strain.
10. application according to claim 9, is characterized in that: described malignant tumour is express the malignant tumour of described NY-ESO-1 antigen peptide;
Concrete, described malignant tumour is selected from as follows arbitrary: the tumour of primary colon cancer, carcinoma of the pancreas, cholangiocarcinoma, cancer of the stomach, esophagus cancer, gland cancer, lung cancer, mammary cancer and urinary system.
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| CN105837693A (en) * | 2016-05-30 | 2016-08-10 | 李斯文 | BCMA-based (B cell maturation antigen-based) chimeric antigen receptor and preparation method and application thereof |
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| WO2019084538A1 (en) * | 2017-10-27 | 2019-05-02 | Board Of Regents, The University Of Texas System | Tumor specific antibodies and t-cell receptors and methods of identifying the same |
| CN110468106A (en) * | 2019-07-09 | 2019-11-19 | 上海宇研生物技术有限公司 | NY-ESO-1 special TCR and preparation method thereof |
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| CN107893055A (en) * | 2017-11-03 | 2018-04-10 | 深圳市默赛尔生物医学科技发展有限公司 | A kind of NK of specific chimeric antigen receptor genetic modification and its production and use |
| CN110526975A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of CEA |
| CN110468106A (en) * | 2019-07-09 | 2019-11-19 | 上海宇研生物技术有限公司 | NY-ESO-1 special TCR and preparation method thereof |
| AU2020316429B2 (en) * | 2019-07-23 | 2022-01-06 | Wen Yang | Composition and method for adoptive immunotherapy |
| CN114249811A (en) * | 2021-12-27 | 2022-03-29 | 北京大学 | T cell receptor for specifically recognizing cancer/testis antigen HCA587/MAGEC2 and application thereof |
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