CN109136281A - A kind of construction method of MRFFT1 cell - Google Patents

A kind of construction method of MRFFT1 cell Download PDF

Info

Publication number
CN109136281A
CN109136281A CN201811153255.6A CN201811153255A CN109136281A CN 109136281 A CN109136281 A CN 109136281A CN 201811153255 A CN201811153255 A CN 201811153255A CN 109136281 A CN109136281 A CN 109136281A
Authority
CN
China
Prior art keywords
cell
polypeptide
mrfft1
tcr
construction method
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN201811153255.6A
Other languages
Chinese (zh)
Inventor
焦顺昌
张嵘
周子珊
宋玉洁
解佳森
张天赋
王海燕
袁翰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Original Assignee
Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Dingcheng Taiyuan Biotechnology Co Ltd filed Critical Beijing Dingcheng Taiyuan Biotechnology Co Ltd
Priority to CN201811153255.6A priority Critical patent/CN109136281A/en
Publication of CN109136281A publication Critical patent/CN109136281A/en
Priority to CN201910439120.4A priority patent/CN110093374B/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/87—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
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
    • 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
    • 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 MRFFT1 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 compiles the knockout that technology of seizing carries out immunosupress target spot to it with gene again, 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 MRFFT1 cell
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of construction method of MRFFT1 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.Specifically 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 uses base again Because volume seizes the knockout that technology carries out immunosupress target spot to it, accurately protect specific killing T cell from inhibiting in vivo, T cell is improved to the lethality of tumour cell.
MRFFT1 cell provided by the invention can be widely applied to individuation and precisely treat entity tumor.
Explanation for specific term:
M:MVA 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: MRFFT1 cell is transformed via above-mentioned M, R, FF, T, 1 every technical solution or technological means and is obtained Cell.
MRFFT1 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 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 respectively 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, 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,；
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 MRFFT1 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 MRFFT1 retrofit scheme cell, identify that specific T-cells (TCR+) ratio of tumour antigen is 70% or more；
3.MRFFT1 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: MVA virus transfection APC Efficiency testing；Wherein, 1A: control group, 1B: transfection group.
The detection of Fig. 2: MFF cell typing.
Fig. 3: the screening of accurate polypeptide.
Fig. 4: flow cytometer detection specific T-cells ratio；Wherein, 4A: control group, 4B:MRFF scheme.
Fig. 5: TCR distribution frequency.
Fig. 6: the knockout situation of inhibition target spot；Wherein, 6A: after knockout, 6B: before knockout.
Fig. 7: original TCR knockout Efficiency testing；Wherein, 7A: after knockout, 7B: before knockout.
Fig. 8: the expression efficiency of specificity TCR；Wherein, 8A: after transfection 7 days, 8B: before transfection.
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, respectively extend 8 amino acid to two sides, by the more of 17 amino acid of this section Peptide is 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, 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: 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. 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, 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. MRFFT1 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 MRFFT1 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 (as shown in Figure 1) 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 burst size of IFN-γ caused by No. 3 polypeptides, belongs to effective accurate polypeptide.
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 TCR17 Sequence expands TCR, 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:
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, 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 25.1% with normal expression, the cell proportion of TCR+
10, lethal effect of the MRFFT1 cell to target cell
Carry out the inspection of killing-efficiency to the target cell in mutant antigen epitope source with control cell and MRFFT1 cell respectively It surveys, using non-treated cell as control (Mock), as a result as shown in figure 9, compared with the control group, MRFFT1 cell is to target cell There is stronger fragmentation effect.
11, the detection of MRFFT1 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 MRFFT1 cell and tumour cell co-culture, the detection of the IFN-γ of release, the results showed that generated with effector cell itself IFN-γ compare (T cells only), with tumour cell co-culture after, MRFFT1 cell can produce a large amount of IFN-γ this As a result consistent with killing experiments result to illustrate: the T cell of expression specificity TCR can more have in conjunction with the knockout of inhibition target spot The raising anti-tumor capacity of effect.
13, 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 MRFFT1 cells against tumor tumor-bearing mice, which improves to have, significantly affects effect.
14, clinical case:
Certain male: 61 years old
Medical diagnosis on disease: double lung poorly differentiated adenocarcinomas；Left pulmonary tuberculosis
First course for the treatment of: monthly MRFFT1 cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year MRFFT1 cell, quantity 1 × 109A cell, totally 2 times；
After administration, 22 months Progression free survivals；
Other cases:
Patient code Medical diagnosis on disease The Progression free survival time
1 Sdenocarcinoma of stomach hepatic metastases 2016.3- so far
2 Gastric cancer 2016.3- so far
3 Lung cancer 2016.4- so far
4 Adenocarcinoma of lung 2016.4- so far
5 Adenocarcinoma of lung 2016.6- so far
6 The cancer of the esophagus 2016.6- so far
Note: containing for " so far " is meant " on the day before the applying date ".

Claims (6)

1. a kind of construction method of MRFFT1 cell, which is characterized in that the construction method includes the following steps: 1) to use source of people Peripheral blood carries out ctDNA sequencing or tumor tissues carry out full exon sequencing, filters out mutational site；2) according to mutational site into Row Epitope prediction synthesizes the gene order of mutant polypeptide；3) the MVA viral vectors of building expression mutant polypeptide, packs MVA Virus；4) it transfects antigen presenting cell and is co-cultured with PBMC, obtain MFF cell；4) mutant polypeptide is as antigenic stimulus The MFF cell filters out effective accurate polypeptide；5) using the accurate polypeptide as MFF cell described in antigenic stimulus, screening The specific cell that can identify the accurate polypeptide out is sequenced and obtains the high frequency tcr gene of specific cell；6) it knocks out outer Original tcr gene in all blood T cells 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 MRFFT1 cell.
2. the construction method of MRFFT1 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 MRFFT1 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 MRFFT1 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 MRFFT1 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 MRFFT1 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.
CN201811153255.6A 2018-09-30 2018-09-30 A kind of construction method of MRFFT1 cell Withdrawn CN109136281A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201811153255.6A CN109136281A (en) 2018-09-30 2018-09-30 A kind of construction method of MRFFT1 cell
CN201910439120.4A CN110093374B (en) 2018-09-30 2019-05-24 Construction method of MRFFT1 cell

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811153255.6A CN109136281A (en) 2018-09-30 2018-09-30 A kind of construction method of MRFFT1 cell

Publications (1)

Publication Number Publication Date
CN109136281A true CN109136281A (en) 2019-01-04

Family

ID=64813896

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201811153255.6A Withdrawn CN109136281A (en) 2018-09-30 2018-09-30 A kind of construction method of MRFFT1 cell
CN201910439120.4A Active CN110093374B (en) 2018-09-30 2019-05-24 Construction method of MRFFT1 cell

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201910439120.4A Active CN110093374B (en) 2018-09-30 2019-05-24 Construction method of MRFFT1 cell

Country Status (1)

Country Link
CN (2) CN109136281A (en)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014012051A1 (en) * 2012-07-12 2014-01-16 Persimmune, Inc. Personalized cancer vaccines and adoptive immune cell therapies
CN103242448B (en) * 2013-05-27 2015-01-14 郑州大学 Full-humanized anti-PD-1 monoclonal antibody and preparation method and application thereof
DK3200818T3 (en) * 2014-10-02 2022-08-22 Us Health METHODS FOR ISOLATING T-CELL RECEPTORS WITH ANTIGEN SPECIFICITY TOWARDS A CANCER-SPECIFIC MUTATION
CN105505873A (en) * 2016-02-29 2016-04-20 时宏珍 Preparation method of HLA-A0201 restriction anti-Her2-neu antigen-specific CTL (cytotoxic lymphocyte)

Also Published As

Publication number Publication date
CN110093374B (en) 2021-01-22
CN110093374A (en) 2019-08-06

Similar Documents

Publication Publication Date Title
CN109152827A (en) Recombinant MVA expresses the MVA Δ E3L of hFL T3L and its purposes of the immunotherapeutic agent as anti-solid tumor
CN110093376A (en) A kind of construction method of LRFFT1 cell
CN109136284A (en) A kind of AFFT2 cell
CN109136278A (en) A kind of MRFFT1 cell
CN109294997A (en) A kind of LRFFT1 cell
CN109679917A (en) A kind of LRFFT2 cell
CN109136281A (en) A kind of construction method of MRFFT1 cell
CN110157745A (en) A kind of construction method of HAFFT1 cell
CN109136279A (en) A kind of construction method of MRFFT2 cell
CN109295097A (en) A kind of MRFFT2 cell
CN110129373A (en) A kind of construction method of RFF1 cell
CN109294982A (en) A kind of RFF2 cell
CN109294998A (en) A kind of RFF1 cell
CN110205341A (en) A kind of construction method of LRFFT2 cell
CN109790224A (en) Tumor-antigen peptide and its application derived from CACNA1H
CN109337930A (en) A kind of AFFT1 cell
CN110408657A (en) A kind of construction method of AFFT1 cell
CN110129372A (en) A kind of construction method of RFFT1 cell
CN109295106A (en) A kind of HAFFT1 cell
CN110093373A (en) A kind of construction method of AFFT2 cell
CN110129374A (en) A kind of construction method of RFFT cell
CN109294995A (en) A kind of RFFT1 cell
CN110129371A (en) A kind of construction method of RFFT2 cell
CN110093371A (en) A kind of construction method of LRFF cell
CN109280643A (en) A kind of RFFT cell

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication

Application publication date: 20190104

WW01 Invention patent application withdrawn after publication