CN110468106A - NY-ESO-1 special TCR and preparation method thereof - Google Patents

NY-ESO-1 special TCR and preparation method thereof Download PDF

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Publication number
CN110468106A
CN110468106A CN201910614365.6A CN201910614365A CN110468106A CN 110468106 A CN110468106 A CN 110468106A CN 201910614365 A CN201910614365 A CN 201910614365A CN 110468106 A CN110468106 A CN 110468106A
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tcr
cell
eso
gene
chain
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Inventor
施炜星
杨春霞
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Jiangsu Xidier Biological Technology Co Ltd
SHANGHAI YUYAN BIOTECHNOLOGY Co Ltd
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Jiangsu Xidier Biological Technology Co Ltd
SHANGHAI YUYAN BIOTECHNOLOGY Co Ltd
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Priority to CN201910614365.6A priority Critical patent/CN110468106A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Abstract

The present invention relates to the special TCR and preparation method thereof of NY-ESO-1, manually drosophila antigen presenting cell loads NY-ESO-1 Antigenic Peptide, CD8+T cell is activated to generate CTL cell, the cytotoxicity CTL cell of generation is through flow sorter HLA-A*02:01 NY-ESO-1 Tetramer-SLLMWITQC-APC(MBL, ) and CD8-FITC double fluorescent staining TS-M011-2, carry out flow cytometer detection, double positive CTL cells are collected in screening, the CTL cell that sorting is obtained carries out unicellular tcr gene sequencing, measure TCR α and β variable region gene sequence, tcr gene is optimized and designed, the constant region of tcr gene is changed to source of mouse, and add signal peptide, The complete genome sequence of TCR α and β chain is obtained, gene chemical synthesis TCR α and TCR β chain connects TCR α and TCR β chain with IRES sequence, co-expresses TCR α and TCR β chain in a plasmid, obtain tcr gene, tcr gene is carried out eukaryotic expression.

Description

NY-ESO-1 special TCR and preparation method thereof
Technical field
The present invention relates to the special TCR and preparation method thereof of NY-ESO-1.
Background technique
TCR-T is that the most promising treatment of solid tumors method after CAR-T cell therapy, this method mainly will at present T cell receptor (Tcell receptor, TCR) gene of tomour specific is transferred to self or allogeneic lymph by various carriers In cell, T cell is made to express antigen specific T CR, can identify tumour antigen, secretes such as IFN-γ, IL-2 cell factor, It plays cytotoxicity [1], therefore the acquisition of tcr gene is the key that TCR-T cell therapy.Traditional tcr gene acquisition side Method mainly passes through tumor infiltrating lymphocyte (Tumor infiltrating lymphocyte, TIL), in recent years, with The development of sequencing technologies, unicellular examining order is also simpler, makes it possible to obtain tcr gene from unicellular sequencing.
NY-ESO-1 full name New York esophageal squamous cell carcinoma 1, is tumor-testis One of antigen (cancer-testis antigens, CTA) family important member.NY-ESO-1 has induction body fluid immune response Ability, it may have activation CD4+and CD8+T lymphocyte ability, be the immunogenicity that is up to the present found most Powerful tumour specific antigen.NY-ESO-1 has expression, including melanoma, cancer of the esophagus, wing in kinds of tumor cells Guang cancer.But NY-ESO-1 is not expressed in the normal tissue in addition to testis tissue, testis tissue do not express MHC I class and II class molecule, so will not be killed by T cell recognition.Just because of this feature, NY-ESO-1 becomes tumour immunity The hot spot of Therapy study.NY-ESO-1 epitope can be divided into cd8 t cell epitope and cd4 t cell epitope, there is 2 HLA-A* at present The restricted cd8 t cell epitope (p157-167, p157-165) of 02:01 can induce immune response.
Summary of the invention
The purpose of the present invention is to provide a kind of TCR and preparation method thereof that new NY-ESO-1 is special.
The present invention includes the preparation method of TCR that NY-ESO-1 is special a kind of first, comprising the following steps:
Manually drosophila antigen presenting cell loads NY-ESO-1 Antigenic Peptide, and activation CD8+T cell, which generates, has specific cell Toxicity CTL cell,
The cytotoxicity CTL cell of generation is through flow sorter HLA-A*02:01 NY-ESO-1 Tetramer- SLLMWITQC-APC(MBL, TS-M011-2) and CD8-FITC double fluorescent staining, progress flow cytometer detection, the double positives of screening collection CTL cell,
The CTL cell that screening is obtained carries out unicellular tcr gene sequencing, measures TCR α and β variable region gene sequence,
Tcr gene is optimized and designed, the constant region of tcr gene source of mouse is changed to, and add signal peptide, obtains TCR α With the complete genome sequence of TCR β chain,
Gene chemical synthesis TCR α and TCR β chain connects TCR α and TCR β chain with IRES sequence, makes TCR α and TCR β chain at one It is co-expressed in plasmid, obtains tcr gene,
Tcr gene is subjected to eukaryotic expression.
The invention also includes the special TCR of NY-ESO-1 is made using above-mentioned preparation method.
The DNA sequence dna of TCR α chain variable region is as shown in SEQ ID No.1.
The DNA sequence dna of TCR β chain variable region is as shown in SEQ ID No.2.
TCR α chain DNA sequence is as shown in SEQ ID No.3.
TCR β chain DNA sequence is as shown in SEQ ID No.4.
The DNA sequence dna of TCR is as shown in SEQ ID No.5.
Detailed description of the invention
Phenotypic analysis after Fig. 1 drosophila APC induction.
CD14 developed by molecule is analyzed in the HLA-A2 developed by molecule and non-cd8 t cell of Fig. 2 .PBMC.
Cell amplification curve diagram in Fig. 3 CTL incubation.
Fig. 4 CTL analyzes the killing of different target cells (T2/T2-peptide) under different effect targets.T2 load SLLMWITQC Antigenic Peptide is positive target cell (T2-pep), and T2 is negative control target cells, and CTL is effector cell, and the two is trained altogether Support 20h.Supernatant is collected by centrifugation, ELISA detects IFN-r secretory volume.
Fig. 5: specific T-cells TCR FITC-CD8+ SLLMWITQC- Tetramer-APC bis- dyes, UR%=0.35%.
Fig. 6: SLLMWITQC Specific CTL Cells airflow classification.The CTL cell that SLLMWITQC is stimulated carries out The bis- dyes of FITC-CD8/SLLMWITQC-Tetramer-APC are drawn a circle to approve and sort double positive cells (region P4), UR%=0.3- 0.5%。
Fig. 7: TCR- pcDNA3.1 plasmid infects the TCR-beta-PE staining analysis of 293T cell, R2=52.36%.
Fig. 8: TCR- pcDNA3.1 plasmid infects the holoprotein expression analysis of 293T cell, right figure HLA-A*02:01 NY-ESO-1 Tetramer-SLLMWITQC-APC dyeing, positive rate 19.38%.
Specific embodiment
Hereinafter, be described further with attached drawing for the present invention in conjunction with the embodiments, embodiment and attached drawing are only used for explaining It is bright rather than limit the scope of protection of the present invention.
The present invention is based on efficient artificial drosophila antigen presenting cell, high-efficient carrier NY-ESO-1 Antigenic Peptide in vitro (SLLMWITQC), activation CD8+T cell generates cytotoxic T lymphocyte (cytotoxic lymphocyte, CTL), produces Raw CTL is sorted through flow sorter, obtains high expression HLA-A*02:01 NY-ESO-1(SLLMWITQC) Tetramer CTL cell, the unicellular tcr gene sequencing (variable region) of further progress, tcr gene is optimized and is designed, be transferred to 293T The expression of cell detection TCR.
The present invention relates to a kind of cloning process of new tcr gene, i.e. specific antigen peptide stimulation generates CTL, streaming point Menu cell clone, is sequenced single cell clone, obtains the variable region sequences of tcr gene.
First part: the CTL cell under opposite antigenic stimulus obtains
Antigenic Peptide SLLMWITQC stimulation generates Specific CTL Cells.Test process is 21 days, including cd8 t cell separation, drosophila APC stimulation, nonCD8 is stimulated again and final toxicity killing verifying, the specific steps are as follows:
Day 1: extracting health people's fresh peripheral blood, and appropriate Ficoll lymphocyte separation medium is added in peripheral blood bottom, centrifugation Buffy coat is sucked out in 30 min, obtains PBMC by centrifuge washing.PBMC is counted and is resuspended, and is incubated altogether with CD8+ T cell magnetic bead It educates, collects cd8 t cell under magnet, freeze non-cd8 cell.Drosophila cell 10 after collecting induction7/ ml is resuspended, with 1 μ g/ml Antigenic Peptide SLLMWITQC and 5 μ g/ml β 2M room temperatures are incubated for 2 ~ 3h, drosophila cell and T cell altogether with the mixing of 1:10 ratio, culture Case stationary culture.
Day 5: 30 U/ml of refinement intracellular cytokine IL-2 and IL-7.
Day 8: counting cd8 cell, adjusts cell concentration 2 × 106/ ml, 1/3 replacement culture medium, calculates non-CD8 PBMC Required number are as follows: the CD14 in effector cell's number × [0.1 ~ 0.2]/non-CD8 PBMC.Non- cd8 cell recovery of recovering is washed, weight Outstanding 5 × 1065 μ g/ml and β 2M of Antigenic Peptide, 5 μ g/ is added in a/ml, 10 37 DEG C of μ g/ml mitomycins, 20min killed cells Ml, room temperature continue to be incubated for 2 hours.Cd8 t cell is mixed with non-cd8 t cell, 30 U/ml of refinement intracellular cytokine IL-2 and IL-7.
Day 10: counting cell, adjusts cell concentration 2 × 106/Cell factor is added in ml.
Day 13: counting cell, adjusts cell concentration 2 × 106/Cell factor is added in ml.
Day 15: counting cd8 cell, adjusts cell concentration 2 × 106/ ml replaces culture medium, calculates non-CD8 PBMC institute Need number.Non- cd8 cell recovery of recovering is washed, and is resuspended 5 × 106A/ml, 10 37 DEG C of μ g/ml mitomycins, 20 min inactivation Cell, is added 5 μ g/ml and β 2M of Antigenic Peptide, 5 μ g/ml, and room temperature continues to be incubated for 2 hours.Cd8 t cell and non-cd8 t cell are mixed It closes, refinement intracellular cytokine IL-2 and IL-7 30U/ml.
Day 17: counting cell, adjusts cell concentration 2 × 106/Cell factor is added in ml.
Day 19: counting cell, adjusts cell concentration 2 × 106/Cell factor is added in ml.
It is positive target cell (T2-peptide) that Day 21:T2, which loads SLLMWITQC Antigenic Peptide, and T2 is that negative control target is thin Born of the same parents, CTL are effector cell, and the two co-cultures 20h.Supernatant is collected by centrifugation, ELISA detects IFN-r secretory volume, verifies preparation CTL cell-specific Mortaility results.
As a result:
Drosophila cell 1120 as APC during the experiment Loading peptides participate in cd8 t cell antigenic stimulus, drosophila cell HLA-A2 expression quantity has direct relation to the validity of Loading peptides with itself, and CD54 and CD80 are straight as costimulatory molecules Connect the stimulation growth for participating in CTL.Fig. 1 is drosophila APC molecular phenotype testing result, it will thus be seen that HLA-A2, CD54, CD80's Developed by molecule all 50% or more, meets experiment needs.
The antigen-presenting function of non-cd8 t cell is completed by wherein CD14+T cell in fact, and in Fig. 2, a figure is PBMC's HLA-A2 detection, in entire CTL complete process, either Antigenic Peptide or APC, molecular mechanism requirement lymphocyte are The HLA-A2 positive participates in, so the higher the better for the HLA-A2 positive ratio of PBMC.B figure is the table of CD14 molecule in non-cd8 t cell Up to analysis.
Experiment respectively counts cell number in the initial D1/D8/D10/D13/D15/D17/D19 of cell culture.Fig. 3 is not The amplification data of Interphase cells simultaneously, as seen from the figure, at experiment initial stage, effector cell takes 5 × 106Resisted as initial cell Former peptide stimulation induction.Before second stimulates, cell the comparison of the growth is slow, and later period the comparison of the growth is quick, during entire experiment, carefully Born of the same parents increase nearly 6 times.
The killing analysis of CTL toxicity:
Whether the CTL cell that verifying Antigenic Peptide stimulation generates has the killing toxicity of specificity, is later antigens peptide specific TCR It obtains and foundation is provided.It is detected by the ELISA of the IFN-r to effect target cell supernatant, analysis is obtained such as Fig. 4.In different effect targets There is biggish gaps for the IFN-r amount that cell and different effect targets discharge during the killing than under.Wherein to Antigenic Peptide sun The killing rate of property target cell T2-pep. will be far longer than negative targets T2 group, between differ nearly 30-40 times.It follows that In The lower CTL cell generated of corresponding antigens peptide stimulation, which has, kills toxicity than stronger specific cell.
Second part: the special CTL positive cell screening of SLLMWITQC Antigenic Peptide
The Specific CTL Cells that first part verifies are collected, with HLA-A*02:01 NY-ESO-1 Tetramer- SLLMWITQC-APC(MBL, TS-M011-2) and CD8-FITC double fluorescent staining, progress flow cytometer detection, it is double positive thin as the result is shown Born of the same parents are obvious, account for about 0.35% or so (Fig. 5).APC-tetramer positive cell is both present in CD8+T cell mass, this is certain The CD8+ immune response attribute of Antigenic Peptide SLLMWITQC is also illustrated in degree.
The screening of SLLMWITQC Antigenic Peptide specific CTL positive cell is main to be carried out using flow sorter, through airflow classification Instrument measurement, delineation and collection double positive cells (region Fig. 6, P4).Take about 104Cell is in complete medium, by centrifugation weight It is suspended from 500 μ l RNA protect cell reagent(Qiagen, 76526) in, carry out unicellular sequencing.
Part III: the sequencing of SLLMWITQC high-affinity tcr gene and construction of recombinant vector
Unicellular gene survey is carried out to the sample (region P4) of positive SLLMWITQC-CTL cell obtained in second part experiment Sequence.Specific method includes:
1. full-automatic flow cell sorter (Sony, SH800) carries out the unicellular sabot of 96 orifice plates.
2. Illumina sequenator (miseq reagent kit v2 Nano, MS-102-2001) carries out unicellular survey Sequence.
3. TCR analyzes software VDJtool (https: //github.com/mikessh/vdjtools) and TCR α, β Chain matches software iPair Analyzer (iRepertoire Inc. https: //www.irepertoire.com/ Ipair the tcr gene that sequencing obtains) is analyzed and matched jointly.
After obtaining specificity TCR α and the β chain gene of SLLMWITQC high-affinity CTL cell, this research is mainly used Following TCR α and β chain gene sequence carry out later experiments.
The obtained α chain variable region TCR is sequenced as shown in SEQ ID NO.1.
The obtained β chain variable region TCR is sequenced as shown in SEQ ID NO.2.
In order to improve the expression rate of tcr gene, and in view of the introducing of external source TCR can cause itself TCR sequence and outer The mispairing of source TCR, therefore the constant region of external source tcr gene is changed to source of mouse.
α chain variable region obtains α chain full genome as shown in SEQ ID NO.3 plus signal peptide and source of mouse constant region.
β chain variable region obtains β chain full genome as shown in SEQ ID NO.4 plus signal peptide and source of mouse constant region.
Gene chemical synthesis TCR α and β chain makes double-strand in a plasmid with α the and β chain of IRES sequence (link peptide) connection TCR Middle co-expression.
TCR full genome optimization is as follows, and link peptide IRES is added between α chain and β chain as shown in SEQ ID NO.5.
Part IV: the eukaryotic expression detection of tcr gene
Tcr gene is constructed to mammalian vector pcDNA3.1, it is errorless through digestion, sequencing confirmation.Recovery culture 293T is thin Born of the same parents carry out plating cells for 24 hours before transfection, be resuspended 4 ~ 5 × 106Cell is in 10cm Tissue Culture Dish.Transfection same day replacement is without blood Clear culture medium, dilute respectively 2000 transfection reagent of 15 μ gDNA and 15 μ l Lipofectamine (invitrogen, 11668027) it in 0.25ml opti-EME(gibco, 31985070) in culture medium and mixes, room temperature 20min.It drops evenly Continue in incubator in culture dish cultivate 48h after flow cytometer detection.
TCR- β chain antibody (TCR-beta-PE, invitrogen, 4327729) coloration result is as shown in fig. 7, β chain is positive Expression rate is 52.36%.TCR holoprotein antibody HLA-A*02:01 NY-ESO-1 Tetramer-SLLMWITQC-APC dyeing knot Fruit is as shown in figure 8, negative control HIV Tetramer(HLA-A*02:01 HIV gag Tetramer-SLYNTVATL- APC, MBL, TS-M027-2) dyeing, TCR positive expression rate is 19.95%.The success in eukaryocyte of the provable tcr gene Expression.
Sequence table
<110>Shanghai Yu Yan Bioisystech Co., Ltd
The western Deere Bioisystech Co., Ltd in Jiangsu
<120>NY-ESO-1 special TCR and preparation method thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 324
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atccaggtgg agcagagccc ccccgacctg atcctgcagg agggcgccaa cagcaccctg 60
agatgcaact tcagcgacag cgtgaacaac ctgcagtggt tccaccagaa cccctggggc 120
cagctgatca acctgttcta catccccagc ggcaccaagc agaacggcag actgagcgcc 180
accaccgtgg ccaccgagag atacagcctg ctgtacatca gcagcagcca gaccaccgac 240
agcggcgtgt acttctgcgc cgtggacggc aacaccggca agctgatctt cggccagggc 300
accaccctgc aggtgaagcc cgac 324
<210> 2
<211> 324
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggcgtgaccc agacccccag acacctggtg atgggcatga ccaacaagaa gagcctgaag 60
tgcgagcagc acctgggcca caacgccatg tactggtaca agcagagcgc caagaagccc 120
ctggagctga tgttcgtgta caacttcaag gagcagaccg agaacaacag cgtgcccagc 180
agattcagcc ccgagtgccc caacagcagc cacctgttcc tgcacctgca caccctgcag 240
cccgaggaca gcgccctgta cctgtgcgcc agcagccagg gccaggagca gtacttcggc 300
cccggcacca gactgaccgt gacc 324
<210> 3
<211> 798
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgaagagaa tcctgggcgc cctgctgggc ctgctgagcg cccaggtgtg ctgcgtgaga 60
ggcatccagg tggagcagag cccccccgac ctgatcctgc aggagggcgc caacagcacc 120
ctgagatgca acttcagcga cagcgtgaac aacctgcagt ggttccacca gaacccctgg 180
ggccagctga tcaacctgtt ctacatcccc agcggcacca agcagaacgg cagactgagc 240
gccaccaccg tggccaccga gagatacagc ctgctgtaca tcagcagcag ccagaccacc 300
gacagcggcg tgtacttctg cgccgtggac ggcaacaccg gcaagctgat cttcggccag 360
ggcaccaccc tgcaggtgaa gcccgacatc cagaaccccg agcccgccgt gtaccagctg 420
aaggacccca gaagccagga cagcaccctg tgcctgttca ccgacttcga cagccagatc 480
aacgtgccca agaccatgga gagcggcacc ttcatcaccg acaagaccgt gctggacatg 540
aaggccatgg acagcaagag caacggcgcc atcgcctgga gcaaccagac cagcttcacc 600
tgccaggaca tcttcaagga gaccaacgcc acctacccca gcagcgacgt gccctgcgac 660
gccaccctga ccgagaagag cttcgagacc gacatgaacc tgaacttcca gaacctgagc 720
gtgatgggcc tgagaatcct gctgctgaag gtggccggct tcaacctgct gatgaccctg 780
agactgtgga gcagctga 798
<210> 4
<211> 909
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atgggctgca gactgctgtg ctgcgccgtg ctgtgcctgc tgggcgccgt gcccatggag 60
accggcgtga cccagacccc cagacacctg gtgatgggca tgaccaacaa gaagagcctg 120
aagtgcgagc agcacctggg ccacaacgcc atgtactggt acaagcagag cgccaagaag 180
cccctggagc tgatgttcgt gtacaacttc aaggagcaga ccgagaacaa cagcgtgccc 240
agcagattca gccccgagtg ccccaacagc agccacctgt tcctgcacct gcacaccctg 300
cagcccgagg acagcgccct gtacctgtgc gccagcagcc agggccagga gcagtacttc 360
ggccccggca ccagactgac cgtgaccgag gacctgagaa acgtgacccc ccccaaggtg 420
agcctgttcg agcccagcaa ggccgagatc gccaacaagc agaaggccac cctggtgtgc 480
ctggccagag gcttcttccc cgaccacgtg gagctgagct ggtgggtgaa cggcaaggag 540
gtgcacagcg gcgtgagcac cgacccccag gcctacaagg agagcaacta cagctactgc 600
ctgagcagca gactgagagt gagcgccacc ttctggcaca accccagaaa ccacttcaga 660
tgccaggtgc agttccacgg cctgagcgag gaggacaagt ggcccgaggg cagccccaag 720
cccgtgaccc agaacatcag cgccgaggcc tggggcagag ccgactgcgg catcaccagc 780
gccagctacc accagggcgt gctgagcgcc accatcctgt acgagatcct gctgggcaag 840
gccaccctgt acgccgtgct ggtgagcggc ctggtgctga tggccatggt gaagaagaag 900
aacagctga 909
<210> 5
<211> 2345
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gctagcgcca ccatgaagag aatcctgggc gccctgctgg gcctgctgag cgcccaggtg 60
tgctgcgtga gaggcatcca ggtggagcag agcccccccg acctgatcct gcaggagggc 120
gccaacagca ccctgagatg caacttcagc gacagcgtga acaacctgca gtggttccac 180
cagaacccct ggggccagct gatcaacctg ttctacatcc ccagcggcac caagcagaac 240
ggcagactga gcgccaccac cgtggccacc gagagataca gcctgctgta catcagcagc 300
agccagacca ccgacagcgg cgtgtacttc tgcgccgtgg acggcaacac cggcaagctg 360
atcttcggcc agggcaccac cctgcaggtg aagcccgaca tccagaaccc cgagcccgcc 420
gtgtaccagc tgaaggaccc cagaagccag gacagcaccc tgtgcctgtt caccgacttc 480
gacagccaga tcaacgtgcc caagaccatg gagagcggca ccttcatcac cgacaagacc 540
gtgctggaca tgaaggccat ggacagcaag agcaacggcg ccatcgcctg gagcaaccag 600
accagcttca cctgccagga catcttcaag gagaccaacg ccacctaccc cagcagcgac 660
gtgccctgcg acgccaccct gaccgagaag agcttcgaga ccgacatgaa cctgaacttc 720
cagaacctga gcgtgatggg cctgagaatc ctgctgctga aggtggccgg cttcaacctg 780
ctgatgaccc tgagactgtg gagcagctga gaattctgta cagccgcccc tctccctccc 840
ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata 900
tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg 960
tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt 1020
tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag 1080
cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc 1140
cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga 1200
tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg 1260
cccagaaggt accccattgt atgggatctg atctggggcc tcggtacaca tgctttacat 1320
gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc 1380
tttgaaaaac acgatgataa tatggccaca agatctggat ccgccaccat gggctgcaga 1440
ctgctgtgct gcgccgtgct gtgcctgctg ggcgccgtgc ccatggagac cggcgtgacc 1500
cagaccccca gacacctggt gatgggcatg accaacaaga agagcctgaa gtgcgagcag 1560
cacctgggcc acaacgccat gtactggtac aagcagagcg ccaagaagcc cctggagctg 1620
atgttcgtgt acaacttcaa ggagcagacc gagaacaaca gcgtgcccag cagattcagc 1680
cccgagtgcc ccaacagcag ccacctgttc ctgcacctgc acaccctgca gcccgaggac 1740
agcgccctgt acctgtgcgc cagcagccag ggccaggagc agtacttcgg ccccggcacc 1800
agactgaccg tgaccgagga cctgagaaac gtgacccccc ccaaggtgag cctgttcgag 1860
cccagcaagg ccgagatcgc caacaagcag aaggccaccc tggtgtgcct ggccagaggc 1920
ttcttccccg accacgtgga gctgagctgg tgggtgaacg gcaaggaggt gcacagcggc 1980
gtgagcaccg acccccaggc ctacaaggag agcaactaca gctactgcct gagcagcaga 2040
ctgagagtga gcgccacctt ctggcacaac cccagaaacc acttcagatg ccaggtgcag 2100
ttccacggcc tgagcgagga ggacaagtgg cccgagggca gccccaagcc cgtgacccag 2160
aacatcagcg ccgaggcctg gggcagagcc gactgcggca tcaccagcgc cagctaccac 2220
cagggcgtgc tgagcgccac catcctgtac gagatcctgc tgggcaaggc caccctgtac 2280
gccgtgctgg tgagcggcct ggtgctgatg gccatggtga agaagaagaa cagctgagcg 2340
gccgc 2345

Claims (7)

1. a kind of preparation method for the TCR that NY-ESO-1 is special, it is characterised in that the following steps are included:
Manually drosophila antigen presenting cell loads NY-ESO-1 Antigenic Peptide, and activation CD8+T cell, which generates, has specific cell Toxicity CTL cell,
The cytotoxicity CTL cell of generation is through flow sorter HLA-A*02:01 NY-ESO-1 Tetramer- SLLMWITQC-APC(MBL, TS-M011-2) and CD8-FITC double fluorescent staining, progress flow cytometer detection, the double positives of screening collection CTL cell,
The CTL cell that screening is obtained carries out unicellular tcr gene sequencing, measures TCR α and β variable region gene sequence,
Tcr gene is optimized and designed, the constant region of tcr gene source of mouse is changed to, and add signal peptide, obtains TCR α With the complete genome sequence of TCR β chain,
Gene chemical synthesis TCR α and TCR β chain connects TCR α and TCR β chain with IRES sequence, makes TCR α and TCR β chain at one It is co-expressed in plasmid, obtains tcr gene,
Tcr gene is subjected to eukaryotic expression.
2. a kind of TCR that NY-ESO-1 is special, it is characterised in that be made using the preparation method of claim 1.
3. NY-ESO-1 as claimed in claim 2 special TCR, it is characterised in that the DNA sequence dna of TCR α chain variable region is such as Shown in SEQ ID No.1.
4. NY-ESO-1 as claimed in claim 2 special TCR, it is characterised in that the DNA sequence dna of TCR β chain variable region is such as Shown in SEQ ID No.2.
5. NY-ESO-1 as claimed in claim 2 special TCR, it is characterised in that TCR α chain DNA sequence such as SEQ ID Shown in No.3.
6. NY-ESO-1 as claimed in claim 2 special TCR, it is characterised in that TCR β chain DNA sequence such as SEQ ID Shown in No.4.
7. NY-ESO-1 as claimed in claim 2 special TCR, it is characterised in that the DNA sequence dna of TCR such as SEQ ID No.5 It is shown.
CN201910614365.6A 2019-07-09 2019-07-09 NY-ESO-1 special TCR and preparation method thereof Pending CN110468106A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103667189A (en) * 2013-09-24 2014-03-26 上海宇研生物技术有限公司 CD8 cytotoxic T-lymphocyte for treating lung cancer and preparation method thereof
CN105131126A (en) * 2015-10-10 2015-12-09 北京康爱瑞浩生物科技股份有限公司 Chimeric antigen receptor for treating malignant tumor and preparation method and application of chimeric antigen receptor
CN106188275A (en) * 2015-05-06 2016-12-07 广州市香雪制药股份有限公司 Identify the φt cell receptor of NY-ESO-1 antigen small peptide

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN103667189A (en) * 2013-09-24 2014-03-26 上海宇研生物技术有限公司 CD8 cytotoxic T-lymphocyte for treating lung cancer and preparation method thereof
CN106188275A (en) * 2015-05-06 2016-12-07 广州市香雪制药股份有限公司 Identify the φt cell receptor of NY-ESO-1 antigen small peptide
CN105131126A (en) * 2015-10-10 2015-12-09 北京康爱瑞浩生物科技股份有限公司 Chimeric antigen receptor for treating malignant tumor and preparation method and application of chimeric antigen receptor

Non-Patent Citations (2)

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Title
刘静维等: "负载NY-ESO-1多肽的树突状细胞激发特异性细胞毒性T淋巴细胞反应", 《北京大学学报(医学版)》 *
施炜星等: "T细胞受体工程T细胞疗法:战略和挑战", 《生物产业技术》 *

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