CN109136280A - A kind of construction method of LRFFT1 cell - Google Patents

A kind of construction method of LRFFT1 cell Download PDF

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Publication number
CN109136280A
CN109136280A CN201811153239.7A CN201811153239A CN109136280A CN 109136280 A CN109136280 A CN 109136280A CN 201811153239 A CN201811153239 A CN 201811153239A CN 109136280 A CN109136280 A CN 109136280A
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cell
lrfft1
polypeptide
tcr
construction method
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焦顺昌
张嵘
张天赋
周子珊
宋玉洁
陈小彬
吴子明
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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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Priority to CN201910439145.4A priority patent/CN110093376B/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
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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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    • C12N2510/00—Genetically modified cells
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Abstract

The present invention carries out ctDNA sequencing using source of people peripheral blood or tumor tissues carry out full exon sequencing, filters out mutational site and carries out Epitope prediction, connects and synthesize mutant polypeptide expressing gene sequence；Slow virus carrier is constructed simultaneously, pack slow virus, transfect APC cell, complete the transformation of specificity LV cell, the PBMC separated in vitro with from peripheral blood is co-cultured, effective polypeptide is filtered out, by 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 compiles the knockout that technology of seizing carries out immunosupress target spot to it with gene again, accurately protects specific killing T cell from inhibiting in vivo, improves T cell to the lethality of tumour cell, the LRFFT1 cell more being had conditions in both attack and defence.

Description

A kind of construction method of LRFFT1 cell
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of construction method of LRFFT1 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 virus is carrier, Safety, convenience are not so good as polypeptide mode.And the direct stimulation of polypeptide is simply mixed, although simple and convenient, efficiency is lower.It is special The secondary stimulus of anisotropic precisely polypeptide is more direct not as good as the tumour specific antigen of T cell receptor transduction.Existing TCR-T is being controlled In the solution for treating 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.It is accurately and effectively analyzed general lack of also lacking to patient's antigen.
Summary of the invention
The present invention carries out ctDNA sequencing using peripheral blood in patients or tumor tissues carry out full exon sequencing, filters out prominent Displacement point carries out Epitope prediction, connects and synthesizes mutant polypeptide expressing gene sequence；Slow virus carrier is constructed simultaneously, is packed Slow virus transfects APC cell, completes the transformation of specificity LV cell, and the PBMC separated in vitro with from peripheral blood is co-cultured, sieve Effective polypeptide is selected, by the second Secondary Shocks of accurate effectively polypeptide stimulation, general T cell is transformed into have and is more precisely killed Hurt the RFF cell of ability；TCR-T technical principle is recycled to be transformed；Improved T cell seizes technology with gene volume again The knockout of immunosupress target spot is carried out to it, accurately protects specific killing T cell from inhibiting in vivo, it is thin to improve T Born of the same parents are to the lethality of tumour cell, the LRFFT1 cell that is more had conditions in both attack and defence.
LRFFT1 cell provided by the invention can be widely applied to individuation and precisely treat entity tumor.
Explanation for specific term:
L: slow-virus transfection technology
R: accurate polypeptide secondary pulse technology
FF: mixed polypeptide technology
T:TCR-T technology
1: target spot knocks out guard technology
Such as: LRFFT1 cell, i.e., it is final via above-mentioned L, R, FF, T, 1 every technical solution or technological means transformation The cell of acquisition.
LRFFT1 cell modification scheme is summarized as follows:
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 Detection RNA mutation 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；
3) 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, the gene order of composite coding 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, slow virus is packed
After the slow virus expression plasmid for the gene order building expression epitope peptide that upper step is synthesized, slow virus packet is carried out Dress；
5, it transfects antigen presenting cell (APC) and is co-cultured with PBMC
1) using the slow-virus transfection antigen presenting cell of expression epitope peptide, (including but not limited to: peripheral blood is single 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,；
4) the accurate polypeptide secondary pulse T cell to filter out；
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, cell surface inhibitive ability of immunity signaling molecule is knocked out to get LRFFT1 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 LRFFT1 retrofit scheme cell, identify that specific T-cells (TCR+) ratio of tumour antigen is 50% or more；
3.LRFFT1 cell due to being knocked out to the inhibitive abilities of immunity target spot such as PD1, CTLA4, TIM3, LAG3, it is right The killing ability of tumour is unrestricted, and killing-efficiency is higher.
Detailed description of the invention
Fig. 1: slow-virus transfection APC Efficiency testing；Wherein, 1A: control group, 1B: transfection group.
The detection of Fig. 2: LFF cell typing.
Fig. 3: the screening of accurate polypeptide.
Fig. 4: flow cytometer detection specific T-cells ratio；Wherein, 4A: control group, 4B:LRFF scheme.
Fig. 5: TCR distribution frequency.
Fig. 6: the knockout situation of inhibition target spot；Wherein, 6A: before knockout, 6B: after knockout.
Fig. 7: original TCR knockout Efficiency testing；Wherein, 7A: before knockout, 7B: after knockout.
Fig. 8: the expression efficiency of specificity TCR；Wherein, 8A: before transfection, 8B: after transfection 7 days.
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.
Details are as follows for technical solution:
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 can just be considered weak immunogene as IC50 >=1000nM that >=2 software calculates, when It can just be considered strongly immunogenic when the IC50 < 1000nM that >=2 software calculates)；
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, the gene order of composite coding 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 inverted repeat in gene order, directly repetition and mirror image should be avoided to repeat sequence as far as possible 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, slow virus is packed
1) the epitope peptide gene sequence of synthesis is cloned into pCDH-MSCV-MCS-EF1-copGFP plasmid, is constructed Express the slow virus expression plasmid of epitope peptide；
2) slow virus is packed:
A. recovery 293T cell is packed after passing for two generations for slow virus；
B. cell transfecting: (T175 culture bottle)
A 15ml centrifuge tube (labeled as A) is taken, 4mL is lightly added in 400 μ L Lipofectamine 2000 It in DMEM, lightly mixes, is stored at room temperature culture 5min；
Two 15mL centrifuge tubes (labeled as B and C, B is control group, and C is experimental group) is separately taken, following reagent and gently is added Ground mixes, and is stored at room temperature culture 5min；
A pipe liquid is averagely transferred in B pipe and C pipe, is lightly mixed, culture 20min is stored at room temperature.
Culture medium old in T175 is poured out, cell is washed one time using PBS, changes new 25mL DMEM into (without antibiosis Element and serum), A, B or A, C mixed liquor is lightly added, gently shakes up, is placed in 37 DEG C, is cultivated in 5%CO2 incubator；
After transfecting 6h, the culture medium containing transfection composite is sucked, is changed to the fresh culture of 37 DEG C of preheatings；Culture 48h, and collect；
3) slow virus concentration and titer determination:
After slow virus is packed successfully, slow virus supernatant is collected, 4 DEG C, 4000g is centrifuged 10min；It is filtered with 0.45 μm of filter Supernatant removes cell fragment；According to vial supernatant: the mixing of concentrated reagent=5:1 ratio, 4 DEG C of placement 2h or overnight； By the mixed liquor being incubated in 4 DEG C, 4000g is centrifuged 30min, i.e., visible tube bottom has rice white precipitating；Carefully supernatant is removed (to be sure not Touch sediment)；The DMEM or PBS of appropriate volume is added, gently sediment is resuspended in piping and druming；Packing virus on demand, -80 DEG C save (note: slow virus never multigelation, every freeze thawing is primary, and lentivirus titers will decline 10%-20%)；
Measurement the previous day is diluted to 5 × 10 after counting the good 293H cell dissociation of growth conditions496 holes are added in/mL Plate, 100 holes μ L/ prepare 8-10 hole for each virus.37 DEG C are put into, is cultivated in 5%CO2 incubator
It takes a certain amount of virus liquid infection cell: doing 10 times of gradient dilutions in EP pipe.Dilution process is as follows: every kind of virus Prepare 10 1.5mL EP pipes, every pipe is added 90 μ L culture solutions, 10 μ L virus stock solution useds are added into first pipe, are denoted as 100；It is mixed After even, draw 10 μ L and second pipe mixing is added, be denoted as 10-1；And so on (100-10-8), 10 μ are added in corresponding cell hole Virus liquid that L has diluted simultaneously marks, and observes result after cultivating 48-72h；
Titre calculates: fluorescent technique method can be used to measure titre the slow virus with fluorescent marker；In fluorescence microscopy Under the microscope as a result, and count most latter two have fluorescence fluorecyte clone number, it is assumed that be X and Y, then titre (TU/mL)= The content (μ L) of the virus liquid in the hole (X+Y × 10) × 1000/2/X.
5, slow-virus transfection antigen presenting cell (APC) and with PBMC co-culture
1) it is prepared using RPMI-1640 and contains 300U/mL rIL-2, the full cell culture medium of 10%FBS is denoted as RPMI- 10-IL-2；APC cell concentration is adjusted to 1 × 10 using RPMI-10-IL-26/mL；Use the slow disease of expression epitope peptide With MOI=5-20 infection APC, (including but not limited to: peripheral blood mononuclear cells, Dendritic Cells, neutrophil leucocyte, B drench poison Bar cell, macrophage)；It is put into 37 DEG C of culture 72h；
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 into T75 culture bottle；It is put into 30-37 DEG C of cell incubator and cultivates 14 days, i.e. acquisition LFF 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 LFF 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: T cell+1640+10%FBS+200U/ mL IL2；
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) when experimental group is noticeably greater than negative control, illustrate that polypeptide is effective accurate polypeptide.
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. LRFF 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 LRFF 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, the CRISPR carrier that building inhibitive ability of immunity signaling molecule knocks out
1) cell surface inhibition signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160,2B4(CD244)；
2) exon for analyzing inhibition signaling molecule, finds the area CDS of the mRNA of gene on pubmed, respectively will be every A exon knock out the prediction of target spot；
3) forward primer and reverse primer needed for design synthesis sgRNA, forward primer and reverse primer 1:1 are mixed Afterwards, 95 DEG C of 5~60min of processing, then slow cooling is carried out, form the DNA sequence dna of sgRNA；
4) CRISPR Lentiviral is subjected to double digestion, and double-stranded DNA corresponding with sgRNA is attached, turned Enter and clone competent cell, after 12h, picking monoclonal is sequenced, and retains sequencing correctly clone；
5) the CRISPR slow virus carrier plasmid for carrying sgRNA corresponding DNA sequence is extracted, viral packaging is carried out；
11, building knocks out the CRISPR carrier of TCR
1) area CDS of the mRNA of tcr gene is found on pubmed, and analyzes the conserved region of TCR, and conserved region is struck Except the prediction of target spot；
2) according to the step 3) in step 10~5) complete TCR knockout carrier building and virus packaging；
12, building knocks out the TCR-T of inhibitive ability of immunity signaling molecule:
1) virus to be obtained in step 10 and 11, the CD8+T cell that infection step 8 obtains, while carrying out original TCR Knockout and inhibitive ability of immunity signaling molecule knockout；
2) after knocking out, after CD8+T cell cultivates 0-5 days in the medium, preferably 3 days, then it is transferred to the TCR expression load of building Body；
3) metainfective CD8+T cell is resuspended with OKM100+12%FBS, is placed in overlaying for stimulating factor OKM-25 On plate, it is denoted as the 0th day；
4) it observes cell situation and co-cultured cell was transferred in big culture bottle by cell density at the 5th day, mend fresh Culture solution OKM-100+12%FBS；
5) by cell from 75cm2175cm is transferred in bottle2Culture solution is OKM-200+5%FBS after big bottle；
6) when culture was to 14-21 days, the TCR-T of knockout inhibitive ability of immunity signaling molecule, i.e. LRFFT1 cell can be harvested.
13, 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 LRFFT1 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, slow-virus transfection APC Efficiency testing
1) it is prepared using RPMI-1640 and contains 300U/mL rIL-2, the full cell culture medium of 10%FBS is denoted as RPMI- 10-IL-2；APC cell concentration is adjusted to 1 × 10 using RPMI-10-IL-26/mL；Use the slow disease of expression epitope peptide With MOI=5-20 infection APC, (including but not limited to: peripheral blood mononuclear cells, Dendritic Cells, neutrophil leucocyte, B drench poison Bar cell, macrophage)；It is put into 37 DEG C of culture 72h；
2) using GFP positive ratio (as shown in Figure 1) in flow cytomery APC.
3, LFF cell typing detects
After LFF 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 89.1%, CD4+T cell is 8.11%.
4, with the accurate polypeptide of LFF 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 burst size of IFN-γ caused by No. 3 and No. 7 polypeptides, belongs to effectively precisely more Peptide.
5, to the identification and sorting of the T cell of accurate polypeptid specificity
With No. 3 of screening and No. 7 polypeptides, LFF Protocols Cell is stimulated, it is thin with T of the flow cytometer detection to accurate polypeptid specificity Born of the same parents' ratio, as a result as shown in figure 4, FL1+ is specific T-cells: LRFF Protocols Cell, No. 3 and the caused release of No. 7 polypeptides The cell proportion of IFN-γ, hence it is evident that higher than not having irritant cell (control), illustrate, LRFF scheme, can obtain to precisely more The specific T-cells of peptide；The sorting of CD8+IFN- γ+cell (FL1+) 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 20) TCR6 distribution frequency is higher for frequency division cloth preceding, illustrates that this TCR and mutant antigen are closely related, according to TCR6 sequence, to TCR It is expanded, constructs 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:
AGGAAAGCTT ATCTTCGGAC AGGGAACGGA GTTATCTGTG AAACCCAAT
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:
VPSQTSVYF CASSLGGSTPLHF GQGTRLTV V
Horizontal line is the CDR3 sequence of replacement
7, inhibition target spot knocks out the detection of efficiency
Using CRISPR technology, the inhibition target spot PD-1 on PBMC is knocked out, sgRNA sequence is shown in Table 3, inhibition The knockout efficiencies of target spot are as shown in Figure 6: the knockout efficiency highest of sgRNA1 can be effectively blocked inhibition signaling molecule The expression of PD-1；SgRNA preferred sgRNA1, sgRNA2；The method can also be used, to Tim-3, LAG3, CTLA-4, BTLA, The inhibitions signaling molecules such as VISTA, CD160,2B4 (CD244) are knocked out；The table of inhibition signaling molecule can be effectively blocked It reaches.
3 inhibition target spot sgRNA sequence of table
8, 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 7: can effectively reduce The expression of original TCR, at this point, the transfection of expression specificity TCR slow virus can be carried out.
9, 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 8: the TCR of building can be 67.4% with normal expression, the cell proportion of TCR+.
10, lethal effect of the LRFFT1 cell to target cell
Carry out the inspection of killing-efficiency to the target cell in mutant antigen epitope source with control cell and LRFFT1 cell respectively It surveys, using non-treated cell as control (Mock), imitates target ratio and be set as 40:1, as a result as shown in figure 9, compared with the control group, LRFFT1 cell has stronger fragmentation effect to target cell.
11, the detection of LRFFT1 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 LRFFT1 cell and tumour cell 1:1 are co-cultured, the detection of the IFN-γ of release, the results showed that produced with effector cell itself Raw IFN-γ compares (T cells only), and after co-culturing with tumour cell, LRFFT1 cell can produce a large amount of IFN-γ This result is consistent with killing experiments result to be illustrated: the T cell of expression specificity TCR, can be more in conjunction with the knockout of inhibition target spot It is effective to 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 LRFFT1 cells against tumor tumor-bearing mice, which improves to have, significantly affects effect.
13, clinical case:
Certain female: 58 years old
Medical diagnosis on disease: oophoroma and breast cancer；
First course for the treatment of: monthly LRFFT1 cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year LRFFT1 cell, quantity 1 × 109A cell, totally 2 times；
After administration, 22 months Progression free survivals；
Other cases:
Note: containing for " so far " is meant " on the day before the applying date ".

Claims (6)

1. a kind of construction method of LRFFT1 cell, which is characterized in that the step of construction method is as follows: 1) using outside source of people All blood carries out ctDNA sequencing or tumor tissues carry out full exon sequencing, filters out mutational site；2) it is carried out according to mutational site Epitope prediction synthesizes the gene order of mutant polypeptide；3) slow virus carrier of building expression mutant polypeptide packs slow disease Poison；4) it transfects antigen presenting cell and is co-cultured with PBMC, obtain LFF cell；4) mutant polypeptide is as antigenic stimulus institute LFF cell is stated, effective accurate polypeptide is filtered out；5) it using the accurate polypeptide as LFF cell described in antigenic stimulus, filters out The specific cell that can identify the accurate polypeptide is sequenced and obtains the high frequency tcr gene of specific cell；6) periphery is knocked out Original tcr gene in blood T cell is transferred to capable of obtaining with the tcr gene in conjunction with accurate polypeptid specificity for step acquisition TCR-T cell；7) cell surface inhibitive ability of immunity signaling molecule is knocked out to get LRFFT1 cell.
2. the construction method of LRFFT1 cell as described in claim 1, which is characterized in that the source of people peripheral blood is also possible to Commercially available engineering cell system.
3. the construction method of LRFFT1 cell as described in claim 1, which is characterized in that the Epitope prediction is with prominent Centered on the amino acid sites of change, respectively extend 8 amino acid to two sides, using the polypeptide of 17 amino acid of this section as potentially antigenic Epitope；The IC50 that potentially antigenic epitope is analyzed using forecasting software thinks that this potentially antigenic epitope is if IC50 < 1000nM Epitope.
4. the construction method of LRFFT1 cell as described in claim 1, which is characterized in that in the knockout periphery blood T cell The method of original tcr gene and/or cell surface inhibitive ability of immunity signaling molecule is CRISPR technology.
5. the construction method of LRFFT1 cell as described in claim 1, which is characterized in that the cell surface inhibition signal Molecule includes: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160,2B4 (CD244).
6. the construction method of LRFFT1 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.
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