CN110093375A - A kind of construction method of MRFFT2 cell - Google Patents

A kind of construction method of MRFFT2 cell Download PDF

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CN110093375A
CN110093375A CN201910439135.0A CN201910439135A CN110093375A CN 110093375 A CN110093375 A CN 110093375A CN 201910439135 A CN201910439135 A CN 201910439135A CN 110093375 A CN110093375 A CN 110093375A
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cell
polypeptide
mrfft2
construction method
epitope
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CN110093375B (en
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焦顺昌
张嵘
宋玉洁
周子珊
解佳森
袁翰
李营营
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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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    • 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
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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • 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]
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    • C12N2800/00—Nucleic acids vectors
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Abstract

The present invention relates to a kind of construction methods of MRFFT2 cell, filter out mutant polypeptide by Epitope prediction, connect and synthesize mutant polypeptide expressing gene sequence；MVA viral vectors is constructed simultaneously, pack MVA virus, transfect APC cell, complete the transformation of specificity MV cell, the PBMC separated in vitro with from peripheral blood is co-cultured, filter out effective polypeptide, pass through the second Secondary Shocks of accurate effectively polypeptide stimulation, general T cell is transformed into the RFF cell for more precisely killing ability, TCR-T technical principle is recycled to be transformed, improved T cell carries out the closing of inhibitive ability of immunity signal with antibody drug in vitro, accurately protect specific killing T cell from inhibiting in vivo, T cell is improved to the lethality of tumour cell.

Description

A kind of construction method of MRFFT2 cell
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of construction method of MRFFT2 cell.
Background technique
Currently, existing LAK, DC, CIK, DC-CIK cell and method are basic in terms of the specific active immunotherapy of tumour It is proved to be invalid, and the cell technologies such as NK, CAR-NK, TIL need maturation, CAR-T cell is in safety and solid tumor It is also defective in treatment.The prior art generally passes through transformation DC cell, generates specific killing by DC submission T cell.Some experiments It is attempting to carry out transfection submission T cell, the specific killing of inducing T cell as the method for carrier with virus in room.We were also once PBMC, inducing T cell are directly stimulated with mutation mixed polypeptide.There are also laboratories to utilize TCR-T technology, targets submission MAGE A3 Antigen.
The above treatment method is simultaneously immature, especially external evoked DC cell and DC cell loading tumour antigen technical know-how Upper research is more, but there are many more problems in the specific implementation process, lack specific, tumour cell occurrence and development key letter Number conduction path relevant molecule because tumour antigen is unknown and the immunosuppressive obstacle of tumor microenvironment, makes as inducing antigen Realize that specific cell targeting immunization therapy is difficult to smoothly implement.Although not having in addition, what is had has carried out antigen in vitro impact Carry out it is external cultivate altogether and amplification in vitro, allow more thin specific cell directly facing complicated tumour immunity microenvironment, Therefore, it is difficult to play expected effect.Although also have can also external submission and total cultivation, target spot is single (MAGE-3), Only work to individual cancer kinds such as non-small cell lung cancer.Transfection submission is carried out although also having and attempting the method that slow poison is carrier, is pacified Quan Xing, convenience are not so good as polypeptide mode.And the direct stimulation of polypeptide is simply mixed, although simple and convenient, efficiency is lower.Object is different Property accurate polypeptide secondary stimulus it is more direct not as good as the tumour specific antigen of T cell receptor transduction.Existing TCR-T is being treated In the solution of neoplastic hematologic disorder and entity tumor, lack the accurately TCR of covering more polyoma kind.
Above scheme does not account for the self-protection technology of T cell, so that the direct face of specific T-cells that quantity is few To powerful tumour immunity microenvironment.
Summary of the invention
By the present invention in that carrying out ctDNA sequencing or the full exon sequencing of tumor tissues progress, screening with peripheral blood in patients Mutational site carries out Epitope prediction out, connects and synthesizes mutant polypeptide expressing gene sequence；MVA virus load is constructed simultaneously Body, packaging MVA virus, transfects APC cell, completes the transformation of specificity MV cell, the PBMC separated in vitro with from peripheral blood It co-cultures, filters out effective polypeptide, by the second Secondary Shocks of accurate effectively polypeptide stimulation, general T cell is transformed into tool There is the RFF cell of more accurate killing ability, recycles TCR-T technical principle to be transformed, improved T cell is in vitro The closing of inhibitive ability of immunity signal is carried out with antibody drug, is accurately protected specific killing T cell from inhibiting in vivo, is mentioned High lethality of the T cell to tumour cell.
Explanation for specific term:
M:MVA virus transfection technology
R: accurate polypeptide secondary pulse technology
FF: mixed polypeptide technology
T:TCR-T technology
2: antibody drug closes guard technology in vitro
Such as: MRFFT2 cell is transformed via above-mentioned M, R, FF, T, 2 every technical solutions or technological means and is obtained Cell.
MRFFT2 cell modification scheme:
1, Epitope prediction
1) using source of people peripheral blood carry out ctDNA sequencing or commercially available engineering cell system (such as H1299, H226, H358, H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL-6323B16F1, CRL-2539 4T1, U14 are small Mouse cervical cancer cell, the small glioma cell of BV-2 mouse, G422 mouse glioma cell etc.) carry out MHC type detection It is sequenced with full exon；
2) MHC type and gene mutation information prediction epitope are utilized: centered on the amino acid sites of mutation, to two Side extends 8 amino acid, using the polypeptide of 17 amino acid of this section as potentially antigenic epitope；
4) IC50 that potentially antigenic epitope is analyzed using forecasting software, thinks this potentially antigenic table if IC50 < 1000nM Position is epitope；
2, polypeptide connects
1) IC50 that any epitope is connected two-by-two at rear joint is analyzed using aforementioned software, when IC50 >=1000nM recognizes To be weak immunogene, can connect；It is considered strongly immunogenic when IC50 < 1000nM, cannot connects；
2) according to the above results, the epitope of weak immunogene is linked together, joint IC50 is higher than two sides The IC50 of epitope (namely joint, which avoids generating as far as possible, combines by force antigen)；
3, synthesis polypeptide
1) polypeptide after connection is reduced to nucleic acid sequence, and carries out codon optimization；
2) gene order of solid-phase synthesis synthetic antigen epitope peptide is used；Or it is synthesized by technical service company；
4, the MVA virus of building expression epitope peptide
The MVA viral expression plasmids for the gene order building expression epitope peptide that upper step is synthesized, carry out viral packaging；
5, it transfects antigen presenting cell (APC) and is co-cultured with PBMC
1) (including but not limited to: peripheral blood list using the rMVA virus transfection antigen presenting cell of expression epitope peptide A nucleus, Dendritic Cells, neutrophil leucocyte, bone-marrow-derived lymphocyte, macrophage)；
2) APC that processing is completed is collected, is mixed and is co-cultured with the ratio of APC:PBMC=1:5-20, obtain effector cell；
6, effective accurate polypeptide is screened, and stimulates T cell again using accurate polypeptide
1) T cell of above scheme acquisition is collected by centrifugation, polypeptide directly stimulates T cell to screen accurate polypeptide as antigen:
2) positive control: T cell+100ng/mL OKT3 is set；Negative control: T cell+1640+10%FBS+200U/ mL IL2；
3) accurate polypeptide judgment criteria:
A. positive control and negative control are normal, then illustrate that this data is credible；
B. it is effective accurate polypeptide that experimental group, which is noticeably greater than negative control group,；
7, TCR-T cell is constructed
1) dyeing of CD8, CD137, IFN-γ are carried out to post-stimulatory T cell, and are sorted with flow cytometer；
2) specific cell that can identify accurate polypeptide is sub-elected, is sequenced and is determined high frequency TCR sequence and expand；
3) tcr gene expression vector, packaging virus are constructed；
4) it knocks out original tcr gene in the periphery blood T cell, is transferred to the tcr gene of step building, cultivates to obtain the final product TCR-T cell；
8, inhibitive ability of immunity signal is closed in vitro through antibody drug to get MRFFT2 cell
Cell surface inhibition signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4(CD244)；
9, building specific antigen expression target cell and tumor model survival assay.
Beneficial effects of the present invention:
1. tumour antigen is mutant antigen, different from other tissues, target spot specificity is strong, is not susceptible to undershooting-effect, pacifies Quan Xinggao；
2. the specific cell ratio obtained is high, the specific cell of tumour antigen usually can be identified, in point of PBMC Cloth be 0.5% hereinafter, by MRFFT2 retrofit scheme cell, identify that specific T-cells (TCR+) ratio of tumour antigen is 50% or more；
3.MRFFT2 cell is right due to carrying out " locked in " operation to the inhibitive abilities of immunity signal such as PD1, CTLA4, TIM3, LAG3 The killing ability of tumour is unrestricted, and killing-efficiency is higher.
Detailed description of the invention
Fig. 1: MVA virus transfection APC Efficiency testing；Wherein, 1A: control group, 1B: transfection group.
The detection of Fig. 2: MRFF cell typing.
Fig. 3: the screening of accurate polypeptide.
Fig. 4: flow cytometer detection specific T-cells ratio；Wherein, 4A: control, 4B:MRFF scheme.
Fig. 5: TCR distribution frequency.
Fig. 6: original TCR knockout Efficiency testing；Wherein, 6A: after knockout, 6B: before knockout.
Fig. 7: the expression efficiency of specificity TCR；Wherein, 7A: after transfection 7 days, 7B: before transfection.
Fig. 8: the sealing effect of inhibitive ability of immunity signal；Wherein, 8A: before closing, 8B: after closing.
Fig. 9: LDH release detection killing-efficiency.
The release of Figure 10: ELISA detection cell factor IFN-γ.
Figure 11: animal lotus knurl model survivorship curve.
Specific embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments It belongs to the scope of protection of the present invention.
1, Epitope prediction
1) ctDNA sequencing is carried out using peripheral blood from patients with lung cancer and HLA parting detects；
2) sequencing information is analyzed using software: by ctDNA sequencing result compared with the genome of normal cell, sieve Select mutational site；
3) centered on the amino acid sites of mutation, extend 8 amino acid to two sides, by the polypeptide of 17 amino acid of this section As potentially antigenic epitope；
4) using forecasting software analyze potentially antigenic epitope IC50 (recommend software: NetMHCpan 3.0, PickPocket, artificial neural networks (ANN)), this potentially antigenic table is thought if IC50 < 1000nM Position is epitope.
2, polypeptide connects
1) IC50 that any epitope is connected two-by-two at rear joint is analyzed using aforementioned software, when IC50 >=1000nM recognizes To be weak immunogene, can connect；It is considered strongly immunogenic when IC50 < 1000nM, cannot connects and (be considered as 3 herein The IC50 calculated result of forecasting software, when >=2 software calculate IC50 >=1000nM Shi Caineng be considered weak immunogene, when The IC50 < 1000nM Shi Caineng that >=2 software calculates is considered strongly immunogenic)；
2) according to the above results, epitope is linked together, joint IC50 is higher than two sides epitope IC50 (namely joint, which avoids generating as far as possible, combines by force antigen)；Weak immunogene peptide will be exempted from by force as link peptide when necessary Epidemic focus peptide interval；Or patient's self amino acid is added to joint, for reducing a possibility that generating strong antigen.
3, synthesis polypeptide
1) polypeptide after connection is reduced to nucleic acid sequence, and carries out codon optimization；
If the nucleic acid sequence shorter (< 100bp) after the completion of connection can suitably repeat amino acid sequence, but It is, it should be noted that Ying Jinliang avoids inverted repeat in gene order, directly repetition and mirror image from repeating sequence when being reduced into gene order The appearance of column
2) gene order (being synthesized by technical service company) of solid-phase synthesis synthetic antigen epitope peptide is used.
4, the MVA virus of building expression epitope peptide
1) it constructs shuttle plasmid: the epitope peptide gene sequence of synthesis is cloned into pIIIdHR-P7.5 plasmid；
2) recombinant MVA virus constructs: (hamster kidney is at fiber for CEF cell (chicken embryo fibroblasts) or BHK-21 cell Cell) monolayer growth to 70%~90% coverage rate successively infect MVA, transfection shuttle plasmid, culture obtain cell monolayer；It is described Cell monolayer passes through freeze thawing, broken acquisition rMVA solution；The rMVA solution inoculum is obtained to RK-13 cell (rabbit kidney cell) culture RMVA is taken to infect RK-13 accumulation point, the screening purifying of accumulation point described in picking obtains required rMVA；It disposes in the rMVA RMVA is incubated in CEF BHK-21 cell monolayer by wtMVA, and rMVA of the screening without selection gene K1L, purifying obtains RMVA- epitope peptide expands isolated rMVA- epitope peptide；
5, it transfects antigen presenting cell (APC) and is co-cultured with PBMC
1) (including but not limited to: peripheral blood list using the rMVA virus transfection antigen presenting cell of expression epitope peptide A nucleus, Dendritic Cells, neutrophil leucocyte, bone-marrow-derived lymphocyte, macrophage)；
2) by antigen presenting cell (APC) with 1-10 × 106/ mL is spread in 6-24 orifice plate；
3) the rMVA virus for expressing antigen polypeptide is infected into APC with MOI=0.1-2, after infecting 2-24h, every hole is added 0.5-2mL IL-2 containing 100-2000IU/mL, 10-100ng/ml hTNF- α, 1000-5000IU/mL IL-6 and 10- The 1-20%FBS AIM V culture medium of 100ng/mL IL-1 β continues to cultivate 2-48h；
4) APC that processing is completed is collected, is mixed with the ratio of APC:PBMC=1:5-20, PBMC is about 5 × 107, it is added 50mL OKM100 culture medium is put into 30-37 DEG C of cell incubator into T75 Tissue Culture Flask and cultivates 14 days, i.e. acquisition MFF Scheme effector cell.
6, effective accurate polypeptide is screened
Polypeptide is as the direct accurate polypeptide of stimulating effect cell screening of antigen:
1) the above MFF Protocols Cell is collected by centrifugation, 1500rpm is centrifuged 5min and collects T cell, and 10mL PBS is added and is resuspended carefully Born of the same parents simultaneously count, and 1500rpm is centrifuged 5min, collect T cell with 1640+10%FBS+200U/mL IL2 resuspension, counting is adjusted to 1 ×106cells/mL；
2) T cell is divided into 96 hole flat undersides with the volley of rifle fire, 200 holes μ L/, cell number is 2 × 105cells；Add respectively again Enter the mutant polypeptide of 10 μ L 1mg/mL, final concentration of 50 μ g/mL, 3 multiple holes are arranged in every polypeptide；
3) positive control: T cell+100ng/mL OKT3 is set；Negative control: 1640+10%FBS+200U/mL IL2； Two T cell controls are as background release detection, respectively first time plus T cell, and last time plus T cell；With two sheets The difference of bottom release is as systematic error；
4) 37 DEG C, 5%CO2After stimulation for 24 hours, 1500rpm is centrifuged 10min, shifts 140 μ L supernatants into 96 new orifice plates；
5) 96 orifice plates are centrifuged again, 1500rpm 10min, take sample carry out ELISA detection (or by sample as- 80 DEG C of preservations)；
Detect the ELISA system of IFN-γ:
1) testing the ELISA kit that can be used for detecting IFN-γ has Biolegend:LEGEND MAX Human at present IFN-γ ELISA Kit with Pre-coated Plates (article No.: 430107) He Dake are as follows: Human IFN-γ ELISA Kit (article No.: DKW12-1000-096), is please operated in strict accordance with shop instruction；
2) the manual wrapper sheet system of ELISA (15 blocks of plates): Human IFN-gamma DuoSet 15plate (article No.: DY285B) × 2 (article No.: DY008) × 31, DuoSet ELISA Ancillary Reagent Kit；
Accurate polypeptide judgment criteria:
1) positive control and negative control are normal, then illustrate that this data is credible；
2) it is effective accurate polypeptide that experimental group, which is significantly higher than the polypeptide of negative control group,.
7, the accurate polypeptide secondary pulse T cell to filter out
1) when PBMC is cultivated with step 5 to the 2nd~14 day, 2 × 10 are taken7Effector cell, final concentration of 10 μ g/ is added The accurate polypeptide of the μ of mL~100 g/mL impacts 1-4h；
2) it after impacting 4h, is transferred in the 6 orifice plates of the pre- wrapper sheet of OKM25 or T25cm2Culture bottle mends OKM100+12%FBS, 37 DEG C of 5%CO2Culture, according to cell growth status, is transferred in T75 culture bottle, and holding cell density as far as possible is 1 × 106 cells/mL；
3) when entering in T175 culture bottle, culture medium OKM200+5%FBS, culture can be obtained precisely more for 10~14 days The T cell that peptide secondary pulse obtains, i.e. MRFF cell；
8, the culture and separation of mutant antigen specific killing T cell
1) with the accurate polypeptide that filters out directly as antigenic stimulus, the MRFF cell obtained to step 7 is stimulated, and is pierced It is spare after swashing 12~72h；
2) dyeing of CD8, CD137, IFN-γ are carried out to post-stimulatory T cell, and is sorted with flow cytometer, are selected Select CD8+CD137+ or CD8+IFN- γ+cell；
9, the clone of CD8+T cell TCR frequency detecting and high frequency TCR
1) CD8+CD137+ the or CD8+IFN- γ+cell genome for extracting sorting, carries out the detection of TCR frequency, Determine the TCR sequence of high frequency；
2) mRNA of sorting cell is extracted, reverse transcription is at from DNA, and according to the sequence of high frequency TCR, design primer is expanded To tcr gene；
3) tcr gene expression vector, packaging virus are constructed；
10, TCR-T cell is constructed
1) recovery PBMC, it is spare with magnetic bead sorting CD8+ cell；
2) gene knockout is carried out with CRISPR technology TCR original to CD8+T cell, when detection is without TCR expression, then be transferred to The TCR expression vector of building；
3) the CD8+T cell that will be transferred to tcr gene carries out amplification cultivation, when culture was to 10~21 days, i.e. harvest TCR-T Cell.
11, the closing of inhibitive ability of immunity signal
1) inhibitive ability of immunity signaling molecule includes: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160,2B4 (CD244)；
2) 1000rpm is centrifuged 5min, collects cultured TCR-T cell；
3) it being washed once with PBS, 1000rpm is centrifuged 5min, TCR-T is resuspended with OKM-200+5%FBS, and adjust to 1 × 107/mL；
4) the monoclonal antibody medicine (such as PD1/PDL1) of addition inhibition signaling molecule, final concentration of 50~200 μ g/mL, preferably 150 μ g/mL, 0~37 DEG C of 1~4h of closing, preferably 37 DEG C of 1h are to get MRFFT2 cell.
12, building specific antigen expression target cell and tumor model survival assay
1) building can be with the slow virus carrier of the accurate polypeptide (specific antigen) of expression screening.
2) specific antigen expression slow virus carrier is packaged into lentiviral particle, the infection suitable tumour of HLA distribution type is thin Born of the same parents stablize and are overexpressed specific antigen, flow cytometer detection expression and expression intensity.
3) stablize the tumor cell line inoculation NGS mouse for being overexpressed specific antigen peptide, do dystopy tumor-bearing model.It will 5×105The tumour cell of expression specificity antigen is suspended from 100 μ l physiological saline, is subcutaneously injected respectively to 30 NSG mouse Right side side of body rib portion is subcutaneous, while mouse being numbered.
4) in tumour growth to 100-120mm3Grouping feeds back cell when left and right, according to gross tumor volume size, by animal mould Type is randomly divided into three groups, and every group of 5-6 mouse, one group is given placebo physiological saline, gives for one group and does not carry out any heredity The T cell (control group) 1 × 10 of operation7, one group is given MRFFT2 cell 1 × 107, carried out after injecting cell 7 days for the first time second Injection, third time injection cell, is observed continuously 60 days after 7 days, counts survival data, draws survivorship curve.
Test result:
1, mutational site and Epitope prediction
Table 1 is the mutational site and Epitope prediction result that sequencing detects；
1 Epitope prediction of table
2, MVA virus transfection APC Efficiency testing
1) (including but not limited to: peripheral blood list using the rMVA virus transfection antigen presenting cell of expression epitope peptide A nucleus, Dendritic Cells, neutrophil leucocyte, bone-marrow-derived lymphocyte, macrophage)；
2) by antigen presenting cell (APC) with 1-10 × 106/ mL is spread in 6-24 orifice plate；
3) the rMVA virus for expressing antigen polypeptide is infected into APC with MOI=0.1-2, after infecting 2-24h, every hole is added 0.5-2mL IL-2 containing 100-2000IU/mL, 10-100ng/ml hTNF- α, 1000-5000IU/mL IL-6 and 10- The 1-20%FBS AIM V culture medium of 100ng/mL IL-1 β continues to cultivate 2-48h；
4) using GFP positive ratio in flow cytomery APC
3, MFF cell typing detects
After MFF Protocols Cell culture, the parting detection of CD4+ and CD8+ cell is carried out, as a result as shown in Figure 2: CD8+ T cell is that 68%, CD4+T cell is 9.45%.
4, with the accurate polypeptide of MFF cell screening
The T cell for being stimulated culture respectively with 12 polypeptides is detected effective polypeptide, tied by detecting the secretion of IFN-γ Fruit is as shown in Figure 3: burst size > negative control group burst size of IFN-γ caused by No. 3 polypeptides, belongs to effectively precisely more Peptide.
5, to the identification and sorting of the T cell of accurate polypeptid specificity
With No. 3 polypeptides of screening, MFF Protocols Cell is stimulated, with flow cytometer detection to the T cell ratio of accurate polypeptid specificity Example, as a result as shown in figure 4, being specific T-cells: MRFF Protocols Cell in black box, release IFN-γ caused by No. 3 polypeptides Cell proportion, hence it is evident that higher than not having irritant cell (control), illustrate, MRFF scheme can be obtained to the special of accurate polypeptide Property T cell；The sorting of CD8+IFN- γ+cell (in black box) is carried out with flow cytometer simultaneously；
6, the identification of high frequency TCR and clone
Sorting is obtained into cell and carries out the extraction of genome and the sequencing of TCR, the distribution situation of TCR is (high as shown in Figure 5 22) TCR1 and TCR17 distribution frequency is higher for frequency division cloth preceding, illustrates, this TCR and mutant antigen are closely related, according to TCR sequence Column, expand TCR, construct Lentiviral.
The sequence situation of 2 TCR β chain CDR3 of table
Known TCR- α:
Amino acid sequence:
MMKSLRVLLV ILWLQLSWVW SQQKEVEQNS GPLSVPEGAI ASLNCTYSDR
GSQSFFWYRQ YSGKSPELIM FIYSNGDKED GRFTAQLNKA SQYVSLLIRD SQPSDSATYL CAVNFGGGKL IFGQGTELSV KPN
Base sequence:
Known TCR- β:
Amino acid:
MRIRLLCCVA FSLLWAGPVI AGITQAPTSQ ILAAGRRMTL RCTQDMRHNA
MYWYRQDLGL GLRLIHYSNT AGTTGKGEVP DGYSVSRANT DDFPLTLASA
VPSQTSVYFC ASSLSFGTEA FFGQGTRLTV V
Horizontal line is CDR3 sequence, the sequence for needing to be replaced
Replaced TCR- β:
Horizontal line is the CDR3 sequence of replacement
7, original TCR knocks out the detection of efficiency
Using CRISPR technology, original TCR on PBMC is knocked out, as a result as shown in Figure 6: can effectively reduce The expression of original TCR, at this point, the transfection of expression specificity TCR slow virus can be carried out.
8, the detection of specificity TCR expression
To pack the slow-virus transfection PBMC of specificity TCR, at the 7th day, with the expression efficiency of flow cytometer detection TCR, As a result as shown in Figure 7: the TCR of building can be 25.1% with normal expression, the cell proportion of TCR+
9, inhibitive ability of immunity signal sealing effect
The monoclonal antibody medicine Keytruda of the fluorescent marker of 150 μ g/mL is added in PBS buffer system, in conjunction with situation such as Fig. 8 institute Show, 90% cell can be closed effectively.
10, lethal effect of the MRFFT2 cell to target cell
Carry out the inspection of killing-efficiency to the target cell in mutant antigen epitope source with control cell and MRFFT2 cell respectively It surveys, effect target ratio is set as 40:1, using non-treated cell as (Mock) is compareed, as a result as shown in figure 9, compared with the control group, MRFFT2 cell has stronger fragmentation effect to target cell.
11, the detection of MRFFT2 cell cytokine release
When tumour cell and effector cell co-culture, due to effector cell, can with mutant antigen on tumor cell, because This, can generate a series of cell factor, and IFN-γ is one of most important cell factor, Tu10Wei in antitumor action When MRFFT2 cell and tumour cell co-culture, cell proportion 1:1, the detection of the IFN-γ of release, the results showed that with effect The IFN-γ for answering cell itself to generate is compared to (T cells only), and after co-culturing with tumour cell, MRFFT2 cell be can produce This result of a large amount of IFN-γ is consistent with killing experiments result to be illustrated: the T cell of expression specificity TCR, in conjunction with inhibition target spot Knockout can more effectively improve anti-tumor capacity.
12, building specific antigen expression target cell and tumor model survival assay
Specific antigen expression tumour target cell system is successfully constructed, establishes tumor-bearing model, as the result is shown (Figure 11), The existence of MRFFT2 cells against tumor tumor-bearing mice, which improves to have, significantly affects effect.
13, clinical case:
Certain male: 60 years old
Medical diagnosis on disease: double lung poorly differentiated adenocarcinomas；
First course for the treatment of: monthly MRFFT2 cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year MRFFT2 cell, quantity 1 × 109A cell, totally 2 times；
After administration, 20 months Progression free survivals；
Other cases:
Patient code Medical diagnosis on disease The Progression free survival time
1 Intrahepatic cholangiocarcinoma 2016.5- so far
2 Oophoroma 2016.5- so far
3 Oophoroma 2016.7- so far
4 Sdenocarcinoma of stomach hepatic metastases 2016.8- so far
5 Gastric cancer 2016.8- so far
Note: containing for " so far " is meant " on the day before the applying date ".

Claims (8)

1. a kind of construction method of MRFFT2 cell, which is characterized in that the construction method includes the following steps:
1) mutational site is filtered out by the way that tumour cell is sequenced, centered on the amino acid sites of mutation, respectively extends 8 to two sides Amino acid analyzes potentially antigenic epitope using forecasting software using the polypeptide of 17 amino acid of this section as potentially antigenic epitope IC50 thinks that this potentially antigenic epitope is epitope if IC50 < 1000nM；
2) IC50 that any epitope is connected two-by-two at rear joint is analyzed using upper step software, when IC50 >=1000nM is considered Weak immunogene links together the epitope of weak immunogene, the gene order of the mutant polypeptide after synthesis connection；
3) the MVA viral vectors of the mutant polypeptide, packaging MVA virus are expressed in building；
4) it is co-cultured using the MVA virus transfection antigen presenting cell and with PBMC, obtains MFF cell；
5) mutant polypeptide filters out effective essence by detecting the secretion of IFN-γ as MFF cell described in antigenic stimulus Quasi- polypeptide；
5) using the accurate polypeptide as MFF cell described in antigenic stimulus, the special of the accurate polypeptide can be identified by filtering out Property cell, is sequenced and obtains the high frequency tcr gene of specific cell；
6) using the accurate polypeptide as MFF cell described in antigenic stimulus, CD8, CD137, IFN- are carried out to post-stimulatory cell The dyeing of γ, and sorted with flow cytometer, the specific cell that can identify the accurate polypeptide is filtered out, sequencing is simultaneously Obtain the high frequency tcr gene of specific cell；
7) original tcr gene in periphery blood T cell is knocked out using CRISPR technology, is transferred to high frequency tcr gene described in step, obtains Obtain TCR-T cell；
8) cell surface inhibitive ability of immunity signaling molecule is closed in vitro through antibody drug to get MRFFT2 cell.
2. the construction method of MRFFT2 cell as described in claim 1, which is characterized in that in the step 1 sequencing be using Source of people peripheral blood or commercially available engineering cell system carry out ctDNA sequencing, or carry out full exon sequencing using tumor tissues.
3. the construction method of MRFFT2 cell as claimed in claim 2, which is characterized in that the commercially available engineering cell system packet It includes: H1299, H226, H358, H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL-6323 B16F1, CRL-2539 4T1, U14 mouse carcinoma of uterine cervix cell, the small glioma cell of BV-2 mouse, G422 mouse Nerve colloid Oncocyte.
4. the construction method of MRFFT2 cell as described in claim 1, which is characterized in that connect antigen table in the step 2 The joint IC50 of position is higher than the IC50 of two sides epitope.
5. the construction method of MRFFT2 cell as described in claim 1, which is characterized in that antigen submission is thin in the step 5 Born of the same parents and PBMC are mixed with the ratio of 1:5-20 to be co-cultured.
6. the construction method of MRFFT2 cell as described in claim 1, which is characterized in that with fluidic cell in the step 6 Instrument is sorted, CD8+CD137+ CD8+IFN- γ+cell is selected.
7. the construction method of MRFFT2 cell as described in claim 1, which is characterized in that the antigen presenting cell includes: Peripheral blood mononuclear cells, Dendritic Cells, neutrophil leucocyte, bone-marrow-derived lymphocyte, macrophage.
8. the construction method of MRFFT2 cell as described in claim 1, which is characterized in that the cell surface inhibitive ability of immunity Signaling molecule includes: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160,2B4 (CD244).
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