CN105131126B - Chimeric antigen receptor and the preparation method and application thereof for treating malignant tumour - Google Patents
Chimeric antigen receptor and the preparation method and application thereof for treating malignant tumour Download PDFInfo
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Abstract
The invention discloses a kind of Chimeric antigen receptors and the preparation method and application thereof for treating malignant tumour.Chimeric antigen receptor disclosed in this invention is NY-ESO-1 specific chimeric antigen receptor, the protein being specially successively made 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 transmembrane region, CD28 intracellular signal structural domain, CD137 intracellular signal structural domain, CD3 ζ intracellular signal structural domain from aminoterminal to c-terminus.It is demonstrated experimentally that the present invention has stronger tumour cell targeting and killing activity using the CAR-CTL cell of NY-ESO-1 Antigenic Peptide preparation, thus there is wide potential applicability in clinical practice.
Description
Technical field
The invention belongs to biology and pharmaceutical technology field, be related to a kind of Chimeric antigen receptor for treating malignant tumour and
Preparation method and application.
Background technique
The T cell (CAR-T) of Chimeric antigen receptor (chimeric antigen receptor, CAR) modification is will to identify
The single-chain antibody of tumor associated antigen is combined with T cell activation motif, is modified by engineering and is assigned the stronger tumor target of T cell
Tropism and killing activity.
From 1989, since being put forward for the first time CAR concept by Eshhar and its colleague, three different development have been had gone through
Stage.First generation CAR receptor, the scFv segment comprising extracellular specific recognition tumour antigen, activation signal intracellular by CD3 ζ or
" immunoreceptor tyrosine activating motif " ITAM (immunoreceptor tyrosine-based of Fc ε RI γ
Activation motifs) signal chains are transmitted.
" immunoreceptor tyrosine activating motif " ITAM (immunoreceptor tyrosine-based activation
Motifs) refer to common to activated immune cell associated receptor (such as TCR/CD3, FcR γ etc.) cytoplasmic domain with tyrosine-based
Aa sequence motifs based on sequence (tyrosine, Y), can be with the signal in signal transduction pathway downstream point after being phosphorylated
Son combines, and leads to cell activation.
Since what CAR-T cell was identified is the antigen of tumor cell surface, rather than it is anti-that MHC- is formed in conjunction with MHC molecule
Former compound is to be offered antigen to cell surface, thus the MHC molecule that can bypass T cell is restricted.Moreover, CAR-T
First signal of T cell and second signal are usually coupled in same molecular structure by technology, have CAR-T cell stronger
Independence can play therapeutic effect substantially without the auxiliary of other types immunocyte.
But first generation CAR receptor costimulatory signal as needed for lacking T cell activation, causes T cell to be survived in vivo
Time is shorter, and cytokine secretion is few, can only play instant activation effect.Second generation CAR receptor is by increasing a thorn altogether
The intracellular domain (such as CD28 molecular structure domain) of energizing signal molecule, provides two kinds of signals of T cell activation, enhances T
The secretion capacity of the proliferative capacity and cell factor (such as IL-2, IFN-γ and GM-CSF) of cell, so that it is micro- to breach tumour
The immunosupress of environment.Third generation CAR receptor has newly increased another costimulatory signal point on the basis of second generation CAR
The intracellular domain of son, and then it is provided with better effector function and longer internal time-to-live.
Summary of the invention
The object of the present invention is to provide a kind of Chimeric antigen receptor for treating malignant tumour and preparation method thereof with answer
With.
Chimeric antigen receptor provided by the present invention for treating malignant tumour is the base in third generation CAR receptor technology
Acquisition, specially NY-ESO-1 specific chimeric antigen receptor are developed on plinth, are said from structure composition angle, the chimeric antigen
Receptor is from aminoterminal to c-terminus successively by the variable region of NY-ESO-1TCR α chain, the variable region of NY-ESO-1TCR β chain, CD8
Hinge area and transmembrane region, CD28 intracellular signal structural domain, CD137 intracellular signal structural domain, CD3 ζ intracellular signal structural domain group
At protein.
Wherein, the amino acid sequence of the variable region of the NY-ESO-1TCR α chain is as shown in sequence 9 in sequence table；It is described
The amino acid sequence of the variable region of NY-ESO-1TCR β chain is as shown in sequence 10 in sequence table；The hinge area and cross-film of the CD8
The amino acid sequence in area is as shown in sequence 11 in sequence table；The amino acid sequence such as sequence table of the CD28 intracellular signal structural domain
Shown in middle sequence 12；The amino acid sequence of the CD137 intracellular signal structural domain is as shown in sequence 13 in sequence table；The CD3 ζ
The amino acid sequence of intracellular signal structural domain is as shown in sequence 14 in sequence table.
Further, the amino acid sequence of the Chimeric antigen receptor is as shown in sequence 8 in sequence table.
In the present invention, the Chimeric antigen receptor is specially by recombinant slow virus expression vector pLVX-NY-ESO-
1TCR-CD8-CD28-CD137-CD3 ζ import cell after expression obtain containing amino acid sequence shown in sequence 8 in ordered list
Protein；The recombinant slow virus expression vector pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ is by slow virus table
Sequence 1 in sequence table is replaced with up to the small fragment between the restriction enzyme site XhoI and BamHI of carrier pLVX-IRES-ZsGreen1
The recombinant plasmid obtained after shown DNA fragmentation.
The gene for encoding the Chimeric antigen receptor also belongs to protection scope of the present invention.
Wherein, sequence 2 in the nucleotide sequence such as sequence table of the gene of the variable region of the NY-ESO-1TCR α chain is encoded
It is shown；The nucleotide sequence of the gene of the variable region of the NY-ESO-1TCR β chain is encoded as shown in sequence 3 in sequence table；Coding
The nucleotide sequence of the gene of the hinge area and transmembrane region of the CD8 is as shown in sequence 4 in sequence table；It is intracellular to encode the CD28
The nucleotide sequence of the gene of signal domain is as shown in sequence 5 in sequence table；Encode the CD137 intracellular signal structural domain
The nucleotide sequence of gene is as shown in sequence 6 in sequence table；Encode the nucleotide of the gene of the CD3 ζ intracellular signal structural domain
Sequence is as shown in sequence 7 in sequence table.
Specifically, encoding the nucleotide sequence of the gene of the Chimeric antigen receptor as shown in sequence 1 in sequence table.Its
In, the variable region of the 1-279 coding NY-ESO-1TCR α chains of sequence 1, the 280-501 coding NY-ESO-
The variable region of 1TCR β chain, the hinge area and transmembrane region of the 502-699 coding CD8,700-822 encode described in
CD28 intracellular signal structural domain, the 823-948 coding CD137 intracellular signal structural domains, 949-1287 coding institutes
State CD3 ζ intracellular signal structural domain.
Recombinant vector, expression cassette, recombinant virus or recombination containing the gene (sequence 1) for encoding the Chimeric antigen receptor
Cell also belongs to protection scope of the present invention.
In the present invention, the recombinant vector is the recombinant slow virus expression load that can express the Chimeric antigen receptor
Body.Specially by the small fragment between the restriction enzyme site XhoI and BamHI of Lentiviral pLVX-IRES-ZsGreen1
It replaces with the recombinant plasmid obtained after DNA fragmentation shown in sequence 1 in sequence table and (is named as pLVX-NY-ESO-1TCR-CD8-
CD28-CD137-CD3ζ)。
Wherein, the recombinant cell is the immune effector cell that can express the Chimeric antigen receptor；The recombination disease
Poison can infect the virus of immune effector cell for that can express the Chimeric antigen receptor.
Specifically, the immune effector cell can be cytotoxic T lymphocyte, NKT cell, NK cell or complementary T
Cell；The virus can be slow virus, herpesviral, cytomegalovirus, Epstein-Barr virus, hepatitis type B virus, hepatitis C virus
Poison or AIDS virus.
The present invention also provides a kind of preparation methods 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 T cell and expresses
The step of Chimeric antigen receptor.
Further, described " Chimeric antigen receptor is expressed in T cell " is achieved by the following procedure: with recombinant expression
Carrier A transfecting T cells obtain the T cell (CTL cell) for expressing the Chimeric antigen receptor, as from the cell after transfection
The NY-ESO-1 specific C AR-T cell (the NY-ESO-1 specific C AR-CTL cell)；The recombinant expression carrier A
Small fragment between the restriction enzyme site XhoI and BamHI by Lentiviral pLVX-IRES-ZsGreen1 replaces with sequence
The recombinant plasmid obtained after DNA fragmentation shown in sequence 1 in list.
Wherein, the source of the T cell can come from including peripheral blood mononuclear cells, ascites, pleural effusion or tumour
Tissue.
Previously described Chimeric antigen receptor or gene or recombinant vector or expression cassette or recombinant virus or recombinant cell exist
It is following it is any in application also belong to protection scope of the present invention:
(a) preparation is for treating the product for the treatment of malignant tumour；
(b) product of tumour cell of the preparation for killing expression NY-ESO-1 Antigenic Peptide；
(c) preparation is for killing the HLA-A2 for having loaded NY-ESO-1 Antigenic Peptide+The product of T2 cell strain.
Wherein, the malignant tumour is to express the malignant tumour of the NY-ESO-1 Antigenic Peptide；Specifically, described pernicious swollen
Tumor can be selected from any in following: primary colon cancer, cancer of pancreas, cholangiocarcinoma, gastric cancer, cancer of the esophagus, gland cancer, lung cancer, breast cancer and
The tumour of urinary system.The amino acid sequence of the NY-ESO-1 Antigenic Peptide is as shown in sequence 15 in sequence table.
More specifically, the HLA-A2 for having loaded NY-ESO-1 Antigenic Peptide+T2 cell strain is by HLA-A2+T2 is unloaded
After the NY-ESO-1 Antigenic Peptide of final concentration of 50 μ g/ml is added in the culture solution of cell strain, in 37 DEG C, 5%CO2Under the conditions of
It is incubated for 120min and realizes load.
It is demonstrated experimentally that the present invention has stronger tumour cell using the CAR-CTL cell of NY-ESO-1 Antigenic Peptide preparation
Targeting and killing activity, thus there is wide potential applicability in clinical practice.
Detailed description of the invention
Fig. 1 be the anti-human CD3-PerCP of mouse CD4-FITC CD8-APC NY-ESO-1TCR-PE Identification of the antibodies
CARNY-ESO-1TCRThe FCM analysis result of the CTL cell of genetic modification.Wherein, CD3+CD8+CD4-Cell be CTL thin
Born of the same parents.
Fig. 2 is CARNY-ESO-1TCRThe killing effect in vitro measurement result of the CTL cell of genetic modification.Wherein, the target cell of A
For the HLA-A2 for having loaded tumor antigen peptide NY-ESO-1+T2 cell strain；The target cell of B is HLA-A2+T2 non-loaded cells strain.A and
In B, CAR-T indicate " embodiment 2 obtain through CARNY-ESO-1TCRThe CTL cell of genetic modification "；CTL indicates " unmodified CTL
Cell ".
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
HLA-A2+T2 non-loaded cells strain: ATCC, ATCC number be "CRL-1992TM”。
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,
Structure is described as follows: by DNA fragmentation shown in 1-501 of sequence 1 in sequence table, (i.e. NY-ESO-1TCR α and β chain are variable
Region sequence) be inserted into obtained recombinant plasmid between the restriction enzyme site EcoRV and SmaI of pUC57 plasmid, the recombinant plasmid by
Huada gene company synthesis.
PUC57-CD8-CD28-CD137-CD3 ξ plasmid: the pUC57 plasmid containing CD8-CD28-CD137-CD3 ξ sequence,
Its structure is described as follows: by DNA fragmentation (i.e. CD8-CD28-CD137- shown in 502-1287 of sequence 1 in sequence table
CD3 ξ sequence) it is inserted into obtained recombinant plasmid between the restriction enzyme site EcoRV and SmaI of pUC57 plasmid, the recombinant plasmid
It is synthesized by Huada gene company.
The Lentiviral pLVX-NY-ESO-1TCR- of embodiment 1, load Chimeric antigen receptor NY-ESO-1TCR
The building and identification of CD8-CD28-CD137-CD3 ζ
One, the acquisition of the DNA fragmentation of the variable region of variable region and NY-ESO-1TCR β chain containing NY-ESO-1TCR α chain
Using the plasmid pUC57-NY-ESO-1TCR α β containing NY-ESO-1TCR α and β chain variable region sequence as template, with F1
It is that primer carries out PCR amplification with R1, obtains the sequence of 528bp, wherein primers F 1 has XhoI restriction enzyme site；Primer R1 contains weight
Folded area, is used for subsequent overlay PCR.Using Primer5 software Design primers, primer is completed by Hua Da gene Co., Ltd
Synthesis.
F1:5 '-CCGCTCGAG(underscore part is the identification sequence of XhoI to CAGAAGGTAACTCAAGCGCAGACT-3 '
Column, sequence thereafter are 1-24 of sequence 1 in sequence table)；
R1:5 '-CGGCGCTGGCGTCGTGGTACTGCTGGCACAGAAGTACACAG-3 ' (479-519 of sequence 1
Reverse complementary sequence).
Reaction system:
Reaction condition: 95 DEG C, 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃10min；4℃∞.
After reaction, agarose gel electrophoresis and gel extraction target fragment, the nucleotide sequence of target fragment are carried out
For " 5 '-CCGCTCGAG1-519 of+sequence 1 ".
Two, the hinge area containing CD8 and transmembrane region, CD28 intracellular signal structural domain, CD137 intracellular signal structural domain and
The acquisition of the DAN segment of CD3 ζ intracellular signal structural domain
Using the plasmid pUC57-CD8-CD28-CD137-CD3 ξ containing CD8-CD28-CD137-CD3 ξ sequence as template, with
F2 and R2 is that primer carries out PCR amplification, obtains the sequence of 818bp, and wherein primers F 2 contains overlay region, is used for subsequent overlay PCR；
Primer R2 contains BamHI restriction enzyme site.
The 479-519 of F2:5'-CTGTGTACTTCTGTGCCAGCAGTACCACGACGCCAGCGCCG-3'(sequence 1
Position)；
R2:5'-CGCGGATCCGCGAGGGGGCAGGGCCTG-3'(underscore part is the identification sequence of BamHI, thereafter
Sequence be sequence table in sequence 1 last 18 reverse complementary sequences).
Reaction system:
Reaction condition: 95 DEG C, 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s, 35 circulations；72℃10min；4℃∞.
After reaction, agarose gel electrophoresis and gel extraction target fragment, the nucleotide sequence of target fragment are carried out
For " 479-1287 of sequence 1+GGATCCGCG”。
Three, the fusion of step 1 and 2 two DNA fragmentations
Obtain target fragment respectively as template using step 1 and step 2, F1 and R2 be primer (sequence referring to step 1 and
Step 2), over-lap PCR is carried out, two target fragments are stitched together, the sequence of 1305bp is obtained, XhoI is contained at sequence both ends
It is NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ by sequence designations with the restriction enzyme site of BamHI.
The nucleotides sequence of NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ is classified as " 5 '-CCGCTCGAGSequence in+sequence table
Arrange 1+GGATCCGCG-3'".Wherein, NY- shown in sequence 9 in 1-279 (i.e. sequence 2) polynucleotides of sequence 1
The variable region of ESO-1TCR α chain, NY-ESO-1TCR β shown in sequence 10 in 280-501 (i.e. sequence 3) polynucleotides
The variable region of chain, the hinge area and transmembrane region of CD8 shown in sequence 11 in 502-699 (i.e. sequence 4) polynucleotides,
CD28 intracellular signal structural domain shown in sequence 12,823-948 (i.e. sequences in 700-822 (i.e. sequence 5) polynucleotides
Column 6) CD137 intracellular signal structural domain shown in sequence 13,949-1287 (i.e. sequence 7) coded sequences in polynucleotide
CD3 ζ intracellular signal structural domain shown in sequence 14 in table.NY-ESO-1 is specific to shown in sequence 8 in 1 polynucleotide of sequence
CAR.
Wherein, reaction system are as follows:
Reaction condition are as follows: 95 DEG C, 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 35 circulations；72℃10min；4℃
∞。
Four, recombinant slow virus expression vector pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ is constructed
Fusion " the NY-ESO-1TCR- that Lentiviral pLVX-IRES-ZsGreen1 and step 3 are obtained
CD8-CD28-CD137-CD3 ζ " carries out double enzyme digestion reaction using restriction enzyme XhoI and BamHI (TaKaRa) simultaneously.Enzyme
Product is cut after agarose gel electrophoresis detects, carry out gel extraction, with T4DNA ligase (TaKaRa) carries out target fragment with
The connection of carrier.Recombinant expression carrier is subjected to DH5 α E. coli competent (TaKaRa) conversion, passes through plasmid PCR and double enzymes
It cuts reaction screening and obtains positive colony, pLVX-NY-ESO-1TCR- will be named as by the correct recombinant plasmid of sequence verification
CD8-CD28-CD137-CD3ζ.The structure of pLVX-NY-ESO-1TCR-CD8-CD28-CD137-CD3 ζ is described as follows: will slow disease
Small fragment between the restriction enzyme site XhoI and BamHI of malicious expression vector pLVX-IRES-ZsGreen1 replaces with sequence in sequence table
The recombinant plasmid obtained after DNA fragmentation shown in column 1.
Double enzyme digestion reaction system:
Reaction condition: 37 DEG C, 4h.
T4DNA connection enzyme reaction:
Reaction condition: 16 DEG C, overnight.
Plasmid PCR reaction system:
Reaction condition: 95 DEG C, 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 35 circulations；72℃10min；4℃∞.
The preparation for the CAR-CTL cell that embodiment 2, Chimeric antigen receptor are modified
One, the preparation of T lymphocyte
The human peripheral that 60ml is fresh is taken, separates peripheral blood list with lymphocyte separation medium (Mediatech Products)
A nucleus (PBMC), with the OKT3 of IL-2,50ng/ml containing 500IU/ml and 5% (volume fraction) people AB serum
The RPMI1640 culture medium Fiber differentiation 48h of (Invitrogen Products), obtains T lymphocyte.
Two, the preparation of the CAR-CTL cell of Chimeric antigen receptor modification
The recombinant slow virus expression vector pLVX-NY-ESO-1TCR-CD8-CD28-CD137- that the building of embodiment 1 is obtained
CD3 ζ (is purchased from sigma company, article No.: 03880) T lymphocyte of transfection Fiber differentiation into step 1 using PEI.Transfection
Cell is transferred in the RPMI1640 culture medium with neomycin after 48h, and is passed through Cell-cloned by limiting dilution assay
Screening in 21 days, obtaining has neomycin resistance, through CARNY-ESO-1TCRThe CTL cell of genetic modification.
Using flow cytomery through 21 days screening after CARNY-ESO-1TCRThe CTL cell surface mosaic of genetic modification is anti-
The expression of original receptor.Specific steps are as follows: taking 100 μ l concentration is 1 × 106The CAR-CTL cell suspension of a/ml, is separately added into inspection
Survey with the anti-human CD3-PerCP of mouse CD4-FITC CD8-APC NY-ESO-1TCR-PE antibody and each 5 μ of Isotype control IgG1 monoclonal antibody
L, 4 DEG C are protected from light incubation 30min, and after PBS buffer solution is washed 2 times, it is thin to carry out streaming in 500ul buffer PBS for suspension cell
Born of the same parents' detection.Wherein, the anti-human CD3-PerCP antibody of mouse is U.S. company BD product, catalog number 560835；Mouse is anti-human
CD4-FITC antibody is U.S. company BD product, catalog number 555346；The anti-human CD8-APC antibody of mouse is U.S. BD public
Take charge of product, catalog number 561421；The anti-human NY-ESO-1TCR-PE antibody of mouse is U.S. company BD sintetics, is used
Its catalog number is 551263.
FCM analysis result is as shown in Figure 1.As it can be seen that through recombinant slow virus expression vector pLVX-NY-ESO-1TCR-
The post-stimulatory T lymphocyte of CD8-CD28-CD137-CD3 ζ expression product successfully induces CARNY-ESO-1TCRThe CTL of specificity.
Embodiment 3, CARNY-ESO-1TCRThe IFN-γ secretion of the CTL cell of genetic modification measures fixed
Enzyme-linked immunization detects the ability of secretion of gamma-IFN, and the specific IFN-γ people using the production of Abcam company is enzyme-linked to exempt from
Epidemic disease spotting method (ELISPOT) kit (article No.: ab46551) is detected: ELISPOT detection kit is used, in 96 orifice plates
Middle be added (carries) 100 μ L by 1:100 (volume ratio) diluted IFN-γ capture antibody with PBS in kit, be placed in 4 DEG C of incubations
Overnight, the effector cell (CAR that embodiment 2 obtains is added after being washed with PBSNY-ESO-1TCRThe CTL cell of genetic modification) 100 μ L,
(HLA-A2 of tumor antigen peptide NY-ESO-1 has been loaded with target cell+T2 cell strain) 100 μ L, wherein effector cell and target cell
Quantity ratio (E/T) be 10, at 37 DEG C, 5%CO2Under the conditions of be incubated for for 24 hours after, with contain 0.1% (volume fraction) polysorbas20 PBS
After washing, it is added with the PBS containing 1% (1g/100ml) BSA by the anti-of the biotin labeling after 1:100 (volume ratio) dilution
IFN-γ antibody (in kit carry), at 37 DEG C, 5%CO2Under the conditions of be incubated for 2h, with contain 1% (1g/100ml) BSA PBS
100 μ L (are carried) in kit by the alkaline phosphatase of the label of Avidin together after 1:5000 (volume ratio) dilution, are incubated for 1h,
After patting dry culture plate after washing, is terminated and reacted with distilled water, read point after drying.
Wherein, " HLA-A2 of tumor antigen peptide NY-ESO-1 has been loaded+T2 cell strain " is by HLA-A2+T2 non-loaded cells
After the tumor antigen peptide NY-ESO-1 of final concentration of 50 μ g/ml is added in the culture solution of strain, in 37 DEG C, 5%CO2Under the conditions of be incubated for
120min realizes load.The amino acid sequence of tumor antigen peptide NY-ESO-1 is as shown in sequence 15 in sequence table.
3 repetitions of experimental setup.
It tests while PBS buffer solution substitution target cell is set as nonspecific control group.
The result shows that: the spot number of experimental group CAR-CTL cell secretion of gamma-IFN is (125.2 ± 5.62), hence it is evident that is more than
Non specific control group spot number (30.6 ± 4.61), (P < 0.05).
Embodiment 4, CARNY-ESO-1TCRThe killing effect in vitro of the CTL cell of genetic modification measures
One, target cell prepares
Target cell 1:HLA-A2+T2 non-loaded cells strain；
Target cell 2: the HLA-A2 of tumor antigen peptide NY-ESO-1 has been loaded+(preparation method is referring to embodiment for T2 cell strain
3)。
Two, pass through51Cr release test measures CARNY-ESO-1TCRThe killing effect in vitro of the CTL cell of genetic modification
CTL (effector) active standard chromium (51Cr) release test is measured.The warp obtained with embodiment 2
CARNY-ESO-1TCRThe CTL cell of genetic modification and unmodified CTL cell are as effector cell (E), by two kinds of effector cells point
It is not co-cultured with the ready two kinds of target cells of step 1, detects the specific killing activity of CTL.Concrete operations are as follows:
Adjust target cell 2 × 106Cell/100 μ l is placed in the PBS containing 4% (volume fraction) FCS, with 100 μ
l51Cr (Perkin Elmer) is marked and in 37 DEG C of culture 1h.The target cell marked is finally with small containing 2% (volume fraction)
1 × Hank ' s balanced salt solution (Invitrogen) of cow's serum (Invitrogen) is washed 4 times, in 4 DEG C of 1200-1500rpm from
Heart 8min is resuspended in the fresh RPMI-1640 culture medium of 15ml (GIBCO article No.: 31800-022:31800-022), until
Final concentration of 2 × 105cell/ml。
(initial concentration is respectively 3 × 10 to the CTL that 100 μ l steps 2 obtain6cell/ml、1×106cell/ml、3×
105Cell/ml), the concentration of each effector cell sets 3 repetitions.The CTL100 μ l for containing different dilutions in each hole, adds
Enter 50 μ l target cells (2 × 105Cell/ml), 4 kinds of effect target ratios (E/T), i.e. 30:1,10:1,5:1 and 1:1 are formed altogether.It sets simultaneously
Spontaneous release hole (the 50 complete RPMI-1640 culture solution of μ l target cell+0.1ml) and maximum relief hole (50 μ l target cell+0.1ml
2%SDS).Each processing is in 37 DEG C of 5%CO2Under the conditions of cultivate 4h, 900rpm is centrifuged 5min, and every hole takes 100 μ l supernatants extremely51Cr
(cpm value) is counted in counting tube.
Each sample sets 3 multiple holes, is averaged as testing result.
Cell killing activity calculates as follows:
Cell killing activity (%)=(to gaging hole fluorescence intensity-Spontaneous release hole fluorescence intensity)/(maximum relief hole fluorescence
Intensity-Spontaneous release hole fluorescence intensity) × 100%
As a result as shown in A in Fig. 2, it is seen that under each effect target ratio, through CARNY-ESO-1TCRThe CTL cell of genetic modification with do not repair
The CTL cell of decorations is compared, and (has loaded the HLA-A2 of tumor antigen peptide NY-ESO-1 to target cell 2+T2 cell strain) have significantly
Killing activity (P < 0.01).
Claims (22)
1.NY-ESO-1 specific chimeric antigen receptor is from aminoterminal to c-terminus successively by the variable of NY-ESO-1TCR α chain
Area, the variable region of NY-ESO-1TCR β chain, the hinge area of CD8 and transmembrane region, CD28 intracellular signal structural domain, CD137 letter intracellular
The protein of number structural domain, CD3 ζ intracellular signal structural domain composition；
The amino acid sequence of the variable region of the NY-ESO-1TCR α chain is as shown in sequence 9 in sequence table；
The amino acid sequence of the variable region of the NY-ESO-1TCR β chain is as shown in sequence 10 in sequence table；
The hinge area of the CD8 and the amino acid sequence of transmembrane region are as shown in sequence 11 in sequence table；
The amino acid sequence of the CD28 intracellular signal structural domain is as shown in sequence 12 in sequence table；
The amino acid sequence of the CD137 intracellular signal structural domain is as shown in sequence 13 in sequence table；
The amino acid sequence of the CD3 ζ intracellular signal structural domain is as shown in sequence 14 in sequence table.
2. NY-ESO-1 specific chimeric antigen receptor according to claim 1, it is characterised in that: the chimeric antigen by
The amino acid sequence of body is as shown in sequence 8 in sequence table.
3. encoding the gene of NY-ESO-1 specific chimeric antigen receptor as claimed in claim 1 or 2.
4. gene according to claim 3, it is characterised in that: encode the gene of the variable region of the NY-ESO-1TCR α chain
Nucleotide sequence as shown in sequence 2 in sequence table.
5. gene according to claim 3, it is characterised in that: encode the gene of the variable region of the NY-ESO-1TCR β chain
Nucleotide sequence as shown in sequence 3 in sequence table.
6. gene according to claim 3, it is characterised in that: encode the hinge area of the CD8 and the gene of transmembrane region
Nucleotide sequence is as shown in sequence 4 in sequence table.
7. gene according to claim 3, it is characterised in that: encode the core of the gene of the CD28 intracellular signal structural domain
Nucleotide sequence is as shown in sequence 5 in sequence table.
8. gene according to claim 3, it is characterised in that: encode the gene of the CD137 intracellular signal structural domain
Nucleotide sequence is as shown in sequence 6 in sequence table.
9. gene according to claim 3, it is characterised in that: encode the core of the gene of the CD3 ζ intracellular signal structural domain
Nucleotide sequence is as shown in sequence 7 in sequence table.
10. gene according to claim 3, it is characterised in that: encode the nucleotide of the gene of the Chimeric antigen receptor
Sequence is as shown in sequence 1 in sequence table.
11. the recombinant vector containing the gene any in claim 3-10.
12. the expression cassette containing the gene any in claim 3-10.
13. the recombinant cell containing the gene any in claim 3-10.
14. the recombinant virus containing the gene any in claim 3-10.
15. recombinant cell according to claim 13, it is characterised in that: the recombinant cell is that can express claim
The immune effector cell of 1 or 2 Chimeric antigen receptors.
16. recombinant virus according to claim 14, it is characterised in that: the recombinant virus is that can express claim
1 or 2 state Chimeric antigen receptor, and can infect the virus of immune effector cell.
17. recombinant virus according to claim 16, it is characterised in that: the immune effector cell is cytotoxic T leaching
Bar cell, NKT cell, NK cell or helper T lymphocyte；
It is described virus be slow virus, herpesviral, cytomegalovirus, Epstein-Barr virus, hepatitis type B virus, Hepatitis C Virus or
AIDS virus.
The preparation method of 18.NY-ESO-1 specific C AR-T cell, including NY- as claimed in claim 1 or 2 is expressed in T cell
The step of ESO-1 specific chimeric antigen receptor.
19. according to the method for claim 18, it is characterised in that: described method includes following steps: being carried with recombinant expression
Body A transfecting T cells obtain the T cell for expressing the NY-ESO-1 specific chimeric antigen receptor from the cell after transfection；Institute
State recombinant expression carrier A be will be between the restriction enzyme site XhoI and BamHI of Lentiviral pLVX-IRES-ZsGreen1
Small fragment replace with the recombinant plasmid obtained after DNA fragmentation shown in sequence 1 in sequence table.
20. described in any in NY-ESO-1 specific chimeric antigen receptor of any of claims 1 or 2 or claim 3-10
Described in expression cassette described in recombinant vector described in gene or claim 11 or claim 12 or claim 13 or 15
Recombinant virus described in recombinant cell or claim 14 or 16 or 17 it is following it is any in application:
(a) preparation is for treating the product for the treatment of malignant tumour；
(b) product of tumour cell of the preparation for killing expression NY-ESO-1 Antigenic Peptide；
(c) preparation is for killing the HLA-A2 for having loaded NY-ESO-1 Antigenic Peptide+The product of T2 cell strain.
21. application according to claim 20, it is characterised in that: the malignant tumour is to express the NY-ESO-1 antigen
The malignant tumour of peptide.
22. application according to claim 21, it is characterised in that: the malignant tumour is any in following: primary
Colon cancer, cancer of pancreas, cholangiocarcinoma, gastric cancer, cancer of the esophagus, gland cancer, lung cancer, the tumour of breast cancer and urinary system.
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| US20170224733A1 (en) * | 2016-02-05 | 2017-08-10 | City Of Hope | Administration of Engineered T Cells for Treatment of Cancers in the Central Nervous System |
| 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 |
| US20200172615A1 (en) * | 2017-05-08 | 2020-06-04 | Tsinghua University | Novel method for producing antibodies |
| US11866785B2 (en) | 2017-10-27 | 2024-01-09 | Board Of Regents, The University Of Texas System | Tumor specific antibodies and T-cell receptors and methods of identifying the same |
| CN107893055B (en) * | 2017-11-03 | 2020-07-17 | 深圳市默赛尔生物医学科技发展有限公司 | Natural killer cell modified by specific chimeric antigen receptor gene and preparation method and application thereof |
| 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 |
| US20220267405A1 (en) * | 2019-07-23 | 2022-08-25 | Wen Yang | Composition and method for adoptive immunotherapy |
| CN114249811B (en) * | 2021-12-27 | 2024-04-19 | 北京大学 | T cell receptor capable of specifically recognizing cancer/testis antigen HCA587/MAGEC2 and application thereof |
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