CN110129372A - A kind of construction method of RFFT1 cell - Google Patents

A kind of construction method of RFFT1 cell Download PDF

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CN110129372A
CN110129372A CN201910438798.0A CN201910438798A CN110129372A CN 110129372 A CN110129372 A CN 110129372A CN 201910438798 A CN201910438798 A CN 201910438798A CN 110129372 A CN110129372 A CN 110129372A
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polypeptide
tcr
rfft1
pbmc
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焦顺昌
张嵘
周子珊
解佳森
王海燕
李营营
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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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Abstract

The present invention provides a kind of construction methods of RFFT1 cell, belong to field of biotechnology.The present invention constructs that a kind of attack-defense integrated, accuracy be high, lethal high T cell.The construction method is the following steps are included: PBMC cell loading causes the polypeptide of Tumor mutations, then to the PBMC cell progress polypeptide impact after load polypeptide；Expand culture after impact, obtains FF cell；So that the polypeptide of Tumor mutations directly stimulates FF cell to screen accurate polypeptide as antigen；Culture PBMC cell carries out repeat impact with accurate polypeptide during the cultivation process；Continue to cultivate after impact, obtains RFF cell；Screening obtains the specific cell that can identify the accurate polypeptide；Tcr gene is obtained by specific cell；PBMC cell is cultivated, original tcr gene and surface inhibitive ability of immunity signaling molecule are knocked out, and is transferred to aforementioned acquired tcr gene, RFFT cell is prepared；Inhibitive ability of immunity signaling molecule closing is carried out to aforementioned gained RFFT cell with monoclonal antibody medicine, obtains RFFT1 cell.

Description

A kind of construction method of RFFT1 cell
Technical field
The present invention relates to field of biotechnology more particularly to a kind of construction methods of RFFT1 cell.
Background technique
Tumour cell immunization therapy is a kind of emerging tumor treatment model, it acquires immunocyte from the patient, so In vitro culture and amplification are carried out afterwards, then is fed back in patient body, and the autoimmune function of Lai Jifa and enhancing body is to treat Tumour.Tumour cell immunization therapy is the 4th kind of tumor therapeuticing method after operation, radiation and chemotherapy.
The human body cell transplanting or input patient's body, the cell newly inputted that normal or bioengineering was transformed can substitute Damaged cell or has the function of stronger immunologic cytotoxicity, to achieve the purpose that treat disease.
The cell specific process that bioengineering was transformed is transformed in incubation in vitro, can effectively be killed except patient Interior tumor cell.Such as, it is thin to provide a kind of killing that human cell factor induces by Chinese patent application CN201210194280.5 Born of the same parents.Chinese patent application CN201510034781.0 provides a kind of polyclonal T cell of tumor cell specific.Chinese patent application CN201510013987.5 provides a kind of antitumor T cell and preparation method thereof.Chinese patent application CN201711060030.1 A kind of CAR-T cell and its preparation method and application treated AIDS and merge lymthoma is provided.CN201610824893.0 is mentioned For a kind of double T cells with antigenic specificity and its preparation method and application of antibody regulation.
In the prior art, it is in T cell generally by DC cell delivery, generates the T cell of specific killing, or make of virus For carrier, pass through the specific killing of slow-virus transfection technological guide T cell.But since tumour antigen is unknown and tumour is micro- The immunosuppressive obstacle of environment, causes ineffective；Due to allowing more thin specific cell directly facing complexity Tumor microenvironment, thus cause ineffective or target spot single, it is only effective to individual tumor.
Summary of the invention
The present invention is intended to provide a kind of construction method of RFFT1 cell, (is named as with constructing the new TCR-T cell of one kind RFFT1 cell), it is used for cellular immunotherapy, realizes accuracy, specificity and the safety of killing.
The present invention provides a kind of construction method of RFFT1 cell, the construction method the following steps are included:
S1 full exon sequencing) is carried out to tumour cell；By the genome phase of full exon sequencing result and normal cell Than filtering out the amino acid sites of mutation；Epitope is predicted centered on the amino acid sites of mutation；It is closed using Solid-phase Polypeptide Mutant polypeptide is synthesized at method；PBMC cell loading causes the polypeptide of Tumor mutations, then carries out to the PBMC cell after load polypeptide Polypeptide impact；
S2 expand culture after) impacting, obtain FF cell；
S3) directly stimulate the FF cell to screen accurate polypeptide so that the polypeptide of Tumor mutations is as antigen；
S4) culture PBMC cell carries out multiple polypeptide impact with accurate polypeptide during the cultivation process；
S5 continue to cultivate after) impacting, obtain RFF cell；
S6) the gained RFF cell using accurate polypeptide as antigenic stimulus, screening obtain the spy that can identify the accurate polypeptide Specific cell；
S7 the TCR β chain CDR3 region sequence of specific cell) is obtained by sequencing, is expanded by the TCR β chain CDR3 region sequence Increasing obtains tcr gene；
S8 PBMC cell) is cultivated, original tcr gene and cell surface inhibitive ability of immunity signaling molecule in cell are knocked out, And be transferred to obtained in step S7 RFFT1 cell can be prepared with the tcr gene in conjunction with accurate polypeptid specificity, it is described Inhibitive ability of immunity signaling molecule includes: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4 (CD244)。
Further, the tumour cell derive from commercially available engineering cell system, including H1299, H226, H358, H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL-6323B16F1, CRL-2539 4T1, U14 Mouse Uterus Neck cancer cell, the small glioma cell of BV-2 mouse, G422 mouse glioma cell.
Further, the prediction of the epitope is to extend 10 to two sides centered on the amino acid sites of mutation Amino acid, using the polypeptide of one section of 21 amino acid as potentially antigenic epitope as potentially antigenic epitope.
Further, in step s 2, expand culture after the impact, obtaining FF cell composition includes:
PBMC cell after polypeptide is impacted cultivates 5 in the cell culture apparatus for overlaying cell stimulation factor OKM-25 It；
It is transferred in the cell culture apparatus containing culture solution OKM-100+12%FBS and continues culture to the 10th day；
It is transferred in the cell culture apparatus containing culture solution OKM-200+5%FBS and continues culture to the 14th~21 day.
Further, it in step S4, repeats polypeptide and impacts 3~4 times.
Further, in polypeptide impact, polypeptide impact is carried out with the polypeptide solution that concentration is 10 μ of μ g/mL~100 g/mL.
Further, the attack time 1-4h of polypeptide impact.
In step S8, Cellular immunity suppression signaling molecule and the original TCR base of cell are successively knocked out with CRISPR technology Cause.
In step S8, with the CRISPR slow virus carrier for knocking out original TCR and surface inhibitive ability of immunity signaling molecule is knocked out CRISPR slow virus carrier remove infection of PBMCs cell, while carrying out the knockout and inhibitive ability of immunity signaling molecule of original TCR Removal；It is then transferred to the tcr gene expression vector of expression the obtained tcr gene of step S7, it is obtained to be transferred to step S7 Tcr gene.
The invention has the following beneficial effects:
1) present invention provides a kind of new cell construction method, (is named as constructing the new TCR-T cell of one kind RFFT1 cell), it is a kind of super T cell having conditions in both attack and defence.In the method, by combining mixed polypeptide technology, accurate polypeptide Secondary pulse, TCR-T technology, target spot knock out guard technology, are transformed to T cell.Mixed polypeptide impact stimulation is carried out first, It then filters out effective precisely polypeptide and polypeptide impact again is carried out to PBMC cell with it, and the polypeptide for carrying out dosage impacts thorn Swash.On the basis of stimulating twice, and combines TCR-T technology and carry out third time stimulation, and then general T cell is transform as more have The T cell of specific cytotoxicity, killing-efficiency is high, accuracy is high.
2) in the present invention, while combining that target spot knocks out guard technology, in vitro culture and amplification in vitro realize killing Accuracy, specificity and safety cover more polyoma kind, and improving, there is the T cell of high accuracy lethal effect to swell to complexity The adaptive faculty of tumor microenvironment.
3) with directly with the lethal effect of slow-virus transfection inducing T cell compared with, it is simple and convenient, highly-safe.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art To obtain other drawings based on these drawings.
Fig. 1 shows antigen load Efficiency testing；
Fig. 2 shows accurate polypeptide the selection results；
Fig. 3 shows flow cytometer detection specific T-cells ratio；
Fig. 4 shows the TCR distribution situation of specific cell；
Fig. 5 shows the knockout situation of original TCR on flow cytometer detection cell；
Fig. 6 shows the external encapsulation situations of inhibitive ability of immunity signaling molecule；
Fig. 7 shows the expression of building TCR；
Fig. 8 shows RFFT1 cell described in the embodiment of the present invention to the lethal effect of target cell；
Fig. 9 shows the detection of RFFT1 cell cytokine release described in the embodiment of the present invention；
Figure 10 shows tumor-bearing mice survivorship curve.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.Base Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts it is all its His embodiment, shall fall within the protection scope of the present invention.
Tumour cell immunization therapy is a kind of emerging tumor treatment model.It is existing in terms of tumour cell immunization therapy LAK, DC, CIK, DC-CIK cell be proved to invalid substantially.The present invention provides a kind of new T cell, can be used for cell and exempts from Epidemic disease treatment.The present invention is transformed T cell, provides a kind of TCR-T cell, and lethality is strong, accuracy is high, a variety of tumors of covering Kind.
The embodiment of the present invention provides the construction method of RFFT1 cell, constructs a kind of TCR-T for cellular immunotherapy Cell (is named as RFFT1 cell) herein.The construction method may comprise steps of:
S1 full exon sequencing) is carried out to tumour cell；By the genome phase of full exon sequencing result and normal cell Than filtering out the amino acid sites of mutation；Epitope is predicted centered on the amino acid sites of mutation；It is closed using Solid-phase Polypeptide Mutant polypeptide is synthesized at method；PBMC cell loading causes the polypeptide of Tumor mutations, then carries out to the PBMC cell after load polypeptide Polypeptide impact；
S2 expand culture after) impacting, obtain FF cell；
S3) directly stimulate the FF cell to screen accurate polypeptide so that the polypeptide of Tumor mutations is as antigen；
S4) culture PBMC cell carries out polypeptide impact with accurate polypeptide during the cultivation process；
S5 continue to cultivate after) impacting, obtain RFF cell；
S6) the gained RFF cell using accurate polypeptide as antigenic stimulus, screening obtain the spy that can identify the accurate polypeptide Specific cell；
S7 the TCR β chain CDR3 region sequence of specific cell) is obtained by sequencing, by the TCR β chain CDR3 region sequence Amplification obtains tcr gene；
S8 original tcr gene and surface inhibitive ability of immunity signaling molecule in T cell) are knocked out, and is transferred in step S7 and obtains What is obtained can be prepared RFFT1 cell, the inhibitive ability of immunity signal point with the tcr gene in conjunction with accurate polypeptid specificity Son includes: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4 (CD244).
Herein, it is as follows to define the meaning that " RFFT1 " cell is related to:
The accurate polypeptide secondary pulse technology of R-；
FF-mixed polypeptide technology；
T-TCR-T technology；
1-target spot knocks out guard technology.
That is, " RFFT1 " cell is by combining mixed polypeptide technology, accurate polypeptide secondary pulse technology, TCR-T herein (T cell receptor (TCR) mosaic type T cell) technology, target spot knock out RFFT1 scheme composed by guard technology is prepared one The new T cell of kind, can be used for immunotherapy of tumors.As its name suggests, " FF " cell is the T cell obtained by FF scheme." RFF " is thin Born of the same parents are the T cell obtained by RFF scheme." RFFT " cell is the T cell obtained by RFFT scheme.
The embodiment of the invention provides a kind of TCR-T cells for cellular immunotherapy.The RFFT1 of the embodiment of the present invention In scheme, firstly, directly being impacted to the PBMC cell after load polypeptide with mixed polypeptide, first time stimulation is carried out；Secondly, It screens accurate polypeptide and the accurate polypeptide for using screening to obtain directly stimulates FF cell as antigen, carrying out second stimulates；Then Third time stimulation is carried out by TCR-T technology.T cell is transformed by stimulating three times, improved T cell realizes Accuracy, specificity and the safety of killing.Meanwhile guard technology is knocked out in conjunction in vitro culture and target spot, keep improved T thin Born of the same parents improve the adaptive faculty to internal complicated immune environment.
In step sl, the polypeptide for causing Tumor mutations can be synthesized to obtain by following methods: 1) exon is sequenced；2) Epitope prediction；3) synthesis polypeptide.
1) exon is sequenced
Full exon sequencing is carried out to tumour cell, then sequencing information is analyzed using software, is on the one hand obtained MHC type information；On the other hand, full exon sequencing result is filtered out into the ammonia of mutation compared with the genome of normal cell Base acid site.
In exon sequencing, tumour cell may come from engineering cell system, can be from peripheral blood in patients.
Engineering cell system may include H1299, H226, H358, H1563, H2228, A549, Renca, LLC Mice Bearing Lewis Lung carcinoma cell, CRL-6323 B16F1, CRL-2539 4T1, U14 mouse carcinoma of uterine cervix cell, the small glioma of BV-2 mouse are thin Born of the same parents, G422 mouse glioma cell.Engineering cell system refers to using technique for gene engineering or cell-fusion techniques to host The inhereditary material of cell carries out modification transformation or recombination, obtains the cell line with the unique shape for stablizing heredity.
2) Epitope prediction
In Epitope prediction, centered on the amino acid sites of mutation, extend 10 amino acid to two sides, by this section The polypeptide of 21 amino acid is used as " potentially antigenic epitope ".(recommended soft using the IC50 that forecasting software analyzes potentially antigenic epitope Part: NetMHCpan 3.0, PickPocket, artificial neural networks (ANN)).If IC50 < 1000nM This potentially antigenic epitope is regarded as into " epitope ".
3) synthesis polypeptide
Using polypeptide solid-state reaction method synthetic antigen epitope peptide.
Specifically, PBMC cell loading causes the polypeptide of Tumor mutations, then carries out to the PBMC cell after load polypeptide more Peptide impact is specifically as follows:
1) it prepares polypeptide solution: polypeptide is dissolved with RPMI 1640+10%FBS (fetal calf serum) or OKM100+12%FBS, Final concentration of 10~100 μ g/mL of polypeptide, preferably 50 μ g/mL, it is spare；
2) PBMC shifts to an earlier date 1 day and recovers, and blows and beats cell, draws 15mL, and count, and is centrifuged；
3) PBMC is resuspended with the polypeptide solution of preparation；
4) it is placed in tissue culture plate and is impacted；
5) 37 DEG C of 5%CO2, 1~4h, preferably 4h are impacted, the PBMC cell after being impacted is spare.
In step s 2, expand culture after impact to be specifically as follows to obtain FF cell:
1) the pre- bed board of stimulating factor OKM-25,40 μ L OKM-25+4mL PBS (phosphate buffered saline solution), 2mL/ ware (4.5cm2) room temperature 4h, 4 DEG C are spare；
2) PBMC after impact is transferred in the tissue culture plate or culture bottle for overlaying OMK-25；
3) even, 37 DEG C of 5%CO are shaken2Culture, is denoted as the 0th day；
4) co-cultured cell, at the 5th day, is transferred to big culture bottle according to cell density by observation co-cultured cell situation In, fresh culture solution OKM-100+12%FBS is mended, 20mL is in 75cm2In culture bottle；
5) it co-cultures the 7th day, adds the fresh OKM-100+12%FBS of 20mL；
6) it co-cultures the 10th day, culture solution OKM-200+5%FBS, by co-cultured cell from 75cm2It is transferred in bottle 175cm2In big bottle；
7) 25mL culture solution OKM-200+5%FBS is blown and beaten, then is transferred to big bottle, is repeated 2 times；With culture solution OKM-200+5% FBS complements to 200mL.
8) when culture was to 14-21 days, available FF cell composition.It is thin by being centrifuged the extraction T from FF cell composition Born of the same parents, as FF cell.
In step S3, the separation and Extraction T cell from the FF cell composition, so that the polypeptide of Tumor mutations is as antigen Stimulate the T cell directly to screen accurate polypeptide.
The screening criteria of accurate polypeptide are as follows:
Polypeptide is as antigen using FF cell as baseline；Two independent to repeat, and detected value is high for high baseline, low to be Low baseline；
The difference of two baselines is systematic error, when data are analyzed, to detected value > low baseline, > high baseline and > Gao Ji Line+systematic error, is labeled respectively；Detected value > high baseline+systematic error is effective accurate polypeptide.
In step S3, T cell is directly stimulated to be specifically as follows to screen accurate polypeptide using polypeptide as antigen:
1) it is centrifuged FF cell composition obtained, 1500rpm is centrifuged 5min and collects T cell, and 10mL PBS is added and is resuspended Cell simultaneously counts, 1500rpm be centrifuged 5min, collect T cell with 1640+10%FBS+200U/mL IL2 resuspension, counting adjust to 1×106A/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 × 105It is a；It is separately added into again 3 multiple holes are arranged in mutant polypeptide 10 μ L, the final concentration of 50 μ g/mL of 1mg/mL, every polypeptide；
3) positive control: T cell+100ng/mL OKT3 (CD3 monoclonal antibody) is set；Negative control: RPMI 1640+ 10%FBS+200U/mL IL2 (interleukin 2)；Two T cells are compareed as background release detection, are respectively added for the first time T cell, and last time plus T cell；Using the difference that two backgrounds discharge 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, sample is taken to carry out ELISA (enzyme linked immunosorbent assay (ELISA)) Detection (or saving sample as -80 DEG C)；
6) accurate polypeptide is screened:
Based on above-mentioned accurate polypeptide screening criteria, detected value > high baseline+systematic error is effective accurate polypeptide.
In step S4, PBMC cell is cultivated, during the cultivation process, polypeptide impact is carried out with accurate polypeptide, carries out second Stimulation.
Compared to step S1, impacted in step s 4 using the accurate polypeptide of dosage.That is, in this step to PBMC Cell carries out multiple polypeptide impact stimulation.Polypeptide can be specifically repeated to impact 3~4 times.
In incubation, culture scheme is similar to the expansion culture in step S2 to the PBMC cell after load polypeptide.First A period of time is cultivated in the device for overlaying cell stimulation factor OKM-25, is then successively transferred to OKM-100+12%FBS training Continue to cultivate in feeding base, OKM-200+5%FBS culture medium.
Step S4 can specifically include following steps:
PBMC cell is cultivated in the cell culture apparatus for overlaying cell stimulation factor OKM-25, after cultivating a period of time With precisely polypeptide carries out polypeptide impact obtained by step S3；
Then it is transferred to cell culture apparatus (the culture plate or culture bottle) relaying containing culture solution OKM-100+12%FBS Continuous culture, every 3~4 days respectively with precisely polypeptide carries out polypeptide impact obtained by step S3.
The attack time of each polypeptide impact can be 1-4h.It is stimulated by the accurate polypeptide impact of dosage by general T cell It is transformed into the RFF cell for more accurately killing ability.
In step S5, continue to cultivate after impact, obtains RFF cell.Applicable culture medium can be OKM200+5%FBS. It is transferred to after polypeptide impact and continues to cultivate in the device containing culture solution OKM200+5%FBS, 37 DEG C, 5%CO2Lower culture Obtain the T cell that accurate polypeptide secondary pulse obtains, i.e. RFF cell.
In step S6, using accurate polypeptide as antigenic stimulus gained RFF cell, to post-stimulatory cell carry out CD8, The dyeing of CD137, IFN-γ, are sorted with flow cytometer, and screening obtains the specificity that can identify the accurate polypeptide Cell.
In step S7, the TCR β chain CDR3 region sequence of specific cell is obtained by sequencing.By the area the TCR β chain CDR3 Sequence amplification obtains tcr gene.
1) extraction of genome is carried out by the cell that step S6 is sorted；
2) TCR sequencing analysis is carried out to genome and the TCR sequence of high frequency is determined according to TCR distribution frequency；
3) mRNA of PBMC is extracted, reverse transcription is at from DNA, and according to the sequence of high frequency TCR, design primer, amplification obtains TCR Gene.
In step s 8, CRISPR technology (Clustered regularly interspaced short can be used Palindromic repeats, gene editing technology) knock out the original tcr gene of cell.
In step S8, original tcr gene and surface inhibitive ability of immunity signaling molecule in periphery blood T cell are knocked out, and turn Enter in step S7 gained can with the tcr gene in conjunction with accurate polypeptid specificity, to obtain RFFT1 cell.Pass through TCR-T technology Third time stimulation is carried out, T cell is transformed.
In step s 8, original tcr gene can be knocked out by CRISPR slow-virus transfection and surface inhibitive ability of immunity is believed Number molecule and it is transferred in step S7 that gained can be with the tcr gene in conjunction with accurate polypeptid specificity.
Specifically, 3 slow virus carriers are constructed respectively: being carried based on the obtained tcr gene building tcr gene expression of step S7 Body；Building knocks out the CRISPR slow virus carrier of original TCR；The CRISPR that building knocks out surface inhibitive ability of immunity signaling molecule is slow Viral vectors.Then remove infection cell with constructed slow virus carrier: the CRISPR that above-mentioned gained is knocked out original TCR is sick slowly Poisonous carrier and the CRISPR slow virus carrier for knocking out surface inhibitive ability of immunity signaling molecule remove infection PMBC cell, knock out original TCR simultaneously knocks out inhibitive ability of immunity signaling molecule；Infection cell then is removed with tcr gene expression vector again, step S7 is transferred to and is obtained Obtain tcr gene.Continue to co-culture after infection, RFFT1 cell can be obtained.
The building process for knocking out the CRISPR slow virus carrier of tcr gene can be with are as follows:
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) 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；
3) 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；
4) the CRISPR slow virus carrier plasmid for carrying sgRNA corresponding DNA sequence is extracted, viral packaging is carried out.
The building for knocking out the CRISPR slow virus carrier of inhibitive ability of immunity signaling molecule is referred to knock out tcr gene The building of CRISPR slow virus carrier.
The building process of the tcr gene expression vector of tcr gene determined by step S7 can be expressed are as follows: based on determined by Tcr gene carries out viral packaging.
It is a kind of new for immunotherapy of tumors to construct the embodiment of the invention provides a kind of new cell construction method T cell (RFFT1 cell), knocked out by joint mixed polypeptide technology, accurate polypeptide secondary pulse, TCR-T technology, target spot anti- Shield technology, by T cell transform as accuracy it is good, it is lethal it is high, a variety of tumor kinds can be covered, strong to the immune microenvironment adaptive faculty of complexity TCR-T cell.
Hereafter further the construction method of RFFT1 cell described in the embodiment of the present invention is described in detail.
(1) full exon sequencing
1) full exon sequencing is carried out using engineering cell system；
2) sequencing information is analyzed using software: on the one hand obtains MHC type information；It on the other hand, will be entirely outer aobvious Sub- sequencing result filters out mutational site compared with the genome of normal cell.
(2) Epitope prediction
1) centered on the amino acid sites of mutation, extend 10 amino acid to two sides, by the more of 21 amino acid of this section Peptide is used as " potentially antigenic epitope "；
2) using forecasting software analyze potentially antigenic epitope IC50 (recommend software: NetMHCpan3.0, PickPocket, artificial neural networks (ANN)), this potentially antigenic table is thought if IC50 < 1000nM Position is " epitope ".
(3) synthesis polypeptide
Epitope peptide symthesis method uses polypeptide solid-state reaction method
1) it is anchored: first amino acid is anchored on solid-phase resin；
2) deprotect: the amino acid of protection removes the blocking group of amino using basic solvent；
3) it activates: activating amino acid carboxyl to be connected using activator；
4) bonded: the carboxyl of the activation amino exposed with previous amino acid reacts, and forms peptide；
5) step 2-4 is repeated, whole epitope peptide chain complete synthesis is made.
(4) PBMC load sudden change polypeptide
1) it prepares polypeptide solution: polypeptide being dissolved with 1640+10%FBS or OKM100+12%FBS, the end of every polypeptide is dense Degree is 50 μ g/mL, spare；
2) PBMC shifts to an earlier date 1 day and recovers, and blows and beats cell, draws 15mL, and count, and is centrifuged；
3) PBMC is resuspended with the polypeptide solution of preparation, and as being impacted in tissue culture plate；
4) 37 DEG C of 5%CO2, 4h is impacted, it is spare.
(5) expansion culture of the PBMC after polypeptide impacts
1) the pre- bed board of stimulating factor OKM-25,40 μ L OKM-25+4mL PBS, 2mL/ ware (4.5cm2) room temperature 4h, 4 DEG C are standby With；
2) PBMC after impact is transferred in the culture bottle for overlaying OMK25；
3) even, 37 DEG C of 5%CO are shaken2Culture, is denoted as the 0th day；
4) co-cultured cell situation is observed, cell is transferred in big culture bottle at the 5th day according to cell density, is mended new Fresh culture solution OKM-100+12%FBS, 20mL is in 75cm2In culture bottle；
5) it co-cultures the 7th day, adds the fresh OKM-100+12%FBS of 20mL；
6) it co-cultures the 10th day, culture solution OKM-200+5%FBS, by co-cultured cell from 75cm2That transfer in bottle To 175cm2In big bottle；
7) 25mL culture solution OKM-200+5%FBS is blown and beaten, then is transferred to big bottle, is repeated 2 times；With culture solution OKM-200+5% FBS complements to 200mL.
8) when culture was to 14-21 days, it can be obtained FF cell composition.
(6) polypeptide directly stimulates T cell to screen accurate polypeptide as antigen:
1) T cell in FF cell composition is the T cell (FF cell) that FF scheme obtains.The FF of acquisition is collected by centrifugation Cell composition, 1500rpm are centrifuged 5min and collect FF cell, 10mL PBS are added, cell is resuspended and counts, 1500rpm centrifugation 5min collects T-cells with 1640+10%FBS+200U/mL IL2 resuspension, and counting is adjusted to 1 × 106cells/mL；
2) FF cell is divided into 96 hole flat undersides with the volley of rifle fire, 200 holes μ L/, cell number is 2 × 105cells；Distinguish again The mutant polypeptide of 10 μ L 1mg/mL, final concentration of 50 μ g/mL is added, 3 multiple holes are arranged in every polypeptide；
3) positive control: FF cell+100ng/mL OKT3 is set；Negative control: 1640+10%FBS+200U/mL IL2；Two FF cell controls are discharged as background to be detected, and respectively adds FF cell, and last time plus FF cell for the first time；With The difference of two backgrounds 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).
(7) accurate polypeptide judgment criteria:
1) positive control and negative control are normal, then illustrate that this data is credible；
2) polypeptide is as antigen using T-cells as baseline；
3) every group of experiment is two baselines, high baseline and low baseline, and the difference of two baselines is systematic error, data analysis When, to detected value > low baseline, > high baseline and > high baseline+systematic error, it is labeled respectively；Detected value > high baseline+ Systematic error is effective accurate polypeptide.
(8) RFF cell is prepared with the accurate polypeptide of screening
1) PBMC is with the culture of FF cell composition culture scheme (the pre- bed board of stimulating factor OKM-25,40 μ L OKM-25+4mL PBS, 2mL/ ware (4.5cm2) room temperature 4h, 4 DEG C are spare；37 DEG C of condition of culture, 5%CO2) to the 3rd day, carry out accurate polypeptide again Impact；
2) 2 × 10 are taken7T cell, the polypeptide of final concentration of 50 μ g/mL is added, impacts 4h；
3) after impacting 4h, it is transferred to 25cm2Culture bottle mends OKM100+12%FBS, 37 DEG C of 5%CO2Culture, it is raw according to cell Long situation, is transferred to 75cm2In culture bottle, holding cell density as far as possible is 1 × 106A/mL；
4) it when cultivating the 7th day, the 10th day and the 14th day, repeats the impact of accurate polypeptide and repeats step 2) and 3)；
5) cell enters 175cm2When in culture bottle, culture medium OKM200+5%FBS, culture can be obtained for 10~21 days The T cell that accurate polypeptide secondary pulse obtains, i.e. RFF cell.
(9) culture and separation of mutant antigen specific killing T cell
1) RFF cell is stimulated directly as antigen with accurate polypeptide, it is spare after stimulating 12~72h；
2) dyeing of CD8, CD137, IFN-γ are carried out to post-stimulatory cell, and is sorted with flow cytometer, are selected Select CD8+CD137+ or CD8+IFN- γ+cell.
(10) clone of CD8+T cell TCR frequency detecting and high frequency TCR
1) extraction of genome is carried out by the cell that step 9 sorts；
2) genome carries out TCR sequencing analysis and determines the TCR sequence of high frequency according to TCR distribution frequency；
3) mRNA of PBMC is extracted, reverse transcription is at from DNA, and according to the sequence of high frequency TCR, design primer, amplification obtains TCR Gene；
4) tcr gene expression vector, packaging virus are constructed.
(11) building knocks out the CRISPR slow virus carrier of inhibitive ability of immunity signaling molecule
1) inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT,2B4(CD244)；
2) area CDS of the mRNA of gene is found on pubmed, and each exon knock out the prediction of target spot respectively；
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.
(12) building knocks out the CRISPR slow virus carrier of original 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) 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；
3) 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；
4) the CRISPR slow virus carrier plasmid for carrying sgRNA corresponding DNA sequence is extracted, viral packaging is carried out.
(13) TCR-T
1) recovery PBMC, it is spare with magnetic bead sorting CD8+ cell；
2) with the CRISPR slow virus carrier and step 12 of the knockout immunosupress signaling molecule obtained in step 11 The CRISPR slow virus carrier of the original TCR of knockout of middle acquisition, infects CD8+T cell, while carrying out inhibitive ability of immunity signal point The knockout of sub and original TCR；
3) after infecting, after CD8+T cell cultivates 3 days in the medium, then it is transferred to the tcr gene expression load of step 10 building Body is transferred to the real gained tcr gene of step；
4) 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；
5) 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；
6) by cell from 75cm2That in bottle is transferred to 175cm2Culture solution is OKM-200+5%FBS after big bottle；
7) when culture was to 14-21 days, the TCR-T cell of knockout inhibitive ability of immunity signaling molecule can be harvested, i.e. RFFT1 is thin Born of the same parents.
(14) tumor-bearing mice survival assay
1) it takes engineering cell system to be inoculated with NSG mouse, does dystopy tumor-bearing model.By 5 × 105Expression specificity antigen Tumour cell is suspended from 100 μ L physiological saline, is subcutaneously injected respectively subcutaneous while right to the right side side of body rib portion of 30 NSG mouse Mouse is numbered.
2) 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 mouse, one group is given placebo physiological saline, gives for one group and does not carry out any heredity behaviour The T cell (control group) 1 × 10 of work7, one group is given RFFT1 cell 1 × 107, second, which is carried out, after injecting cell 7 days for the first time infuses It penetrates, third time injection cell, is observed continuously 50 days after 7 days, counts survival data, draws survivorship curve.
Experimental result
1. mutational site and Epitope prediction
Table 1 be the mutational site that detects of sequencing and Epitope prediction as a result, underscore mark be mutation amino Acid.
1 Epitope prediction of table
2. antigen load Efficiency testing
According to the mutant antigen of the synthesis prediction of table 1, and the label of biotin is carried out, after antigen load PBMC, is marked with PE Affine streptomysin detection biotin cell surface distribution situation, with detect PBMC deduction polypeptide antigen efficiency；As a result Such as Fig. 1 --- shown in antigen load Efficiency testing: a is the testing result without loading labeling polypeptide, and b is load biotinyl polypeptide Testing result, the results showed that the load efficiency of PBMC is that 42.7% (FL4-H subset 42.7% indicates that PE is tagged to 42.7%) cell accounts for.
3. screening accurate polypeptide
As shown in Fig. 2, stimulating the FF cell of culture respectively with 12 polypeptides, by detecting the secretion of IFN-γ, detection has The polypeptide of effect, as a result as shown in Figure 2: the burst size of IFN-γ caused by No. 6 polypeptides > high baseline+systematic error belongs to effective essence Quasi- polypeptide.
The detection of 4.RFF cell-specific cell proportion
With No. 6 polypeptides of screening, repeatedly stimulated on FF cell base, after culture, with flow cytometer detection (FACSCaliburTM (BD Biosciences)) to the T cell ratio of accurate polypeptid specificity, as a result as shown in figure 3, box Interior is specific T-cells: compared with non-treated control group, (RFF cell) can discharge IFN- after repeat impact stimulates The cell proportion of γ is apparently higher than the cell (FF cell) of no repeat impact stimulation.Illustrate, accurate polypeptide repeatedly stimulates, can be with Improve specific T-cells ratio；The sorting of CD8+IFN- γ+cell (in box) is carried out with flow cytometer simultaneously.
5. identification and the clone of high frequency TCR
Sorting is obtained into cell and carries out the extraction of genome and the sequencing of TCR, the distribution situation of TCR (high frequency as shown in Figure 4 Distribution it is preceding 20) TCR16 distribution frequency is higher, illustrate, this TCR and mutant antigen are closely related, according to TCR sequence, to TCR It is expanded.
The sequence situation of 2 TCR β chain CDR3 of table
6. the knockout of original TCR
Using CRISPR technology, original TCR on PBMC is knocked out, flow cytometer detection knocks out situation.As a result such as Fig. 5 It is shown: can effectively to knock out original TCR.
Known TCR- β:
Amino acid:
Horizontal line is CDR3 sequence, the sequence for needing to be replaced
Replaced TCR- β:
Horizontal line is the CDR3 sequence of replacement
7. the knockout of inhibitive ability of immunity signaling molecule
Using CRISPR technology, the inhibition signaling molecule on PBMC is knocked out, flow cytometer detection knocks out signaling molecule Knock out situation.As a result as shown in Figure 6: can have the expression for blocking inhibition signaling molecule.
8. specificity TCR expression
With the expression of flow cytometer detection TCR, as a result as shown in Figure 7: the TCR of building can be transfected 3 days with normal expression, The cell proportion of TCR+ is 48.3%；Transfection 7 days, the cell proportion of TCR+ are 48.7%.
9. RFFT1 cell is to the lethal effect of target cell
Killing effect is carried out with the target cell of RFF cell, RFFT cell and RFFT1 cell to mutant antigen epitope source respectively The detection of rate, using non-treated cell as control (Mock), as a result as shown in figure 8, compared with the control group, RFF cell, RFFT Cell and RFFT1 cell have certain fragmentation effect to target cell, and at 20:1 (effector cell: target cell), with Mock group Difference is obvious；Overall trend is killing-efficiency: RFFT1 cell > RFFT cell > RFF cell；Illustrate the T of expression specificity TCR Cell can effectively improve the killing-efficiency to tumour cell by the knockout of inhibition target spot.
10. the detection of RFFT1 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 in antitumor action, and Fig. 9 is difference When the cell and tumour cell of culture scheme co-culture, the detection of the IFN-γ of release, the results showed that produced with effector cell itself Raw IFN-γ compares (only effector cell), after being co-cultured with tumour cell, RFF Protocols Cell, RFFT Protocols Cell and RFFT1 Cell can produce a large amount of IFN-γ, especially RFFT and RFFT1 cell, due to expressing specificity TCR (RFFT), simultaneously Inhibition signal has been knocked (RFFT1), and releasably more IFN-γ, this result are consistent with killing experiments result by effector cell Illustrate: the T cell of expression specificity TCR can more effectively improve anti-tumor capacity by the knockout of inhibition target spot.
11. tumor-bearing mice survival assay
The results are shown in Figure 10, and the RFFT1 cell for feeding back the embodiment of the present invention, which has mouse survival, to be obviously improved.P value Show less than 0.01 (p value=0.0008) with statistical significance.
12. clinical case
Administration process:
First course for the treatment of: monthly RFFT1 cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year RFFT1 cell, quantity 1 × 109A cell, totally 2 times.
Table 3
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain Lid is within protection scope of the present invention.Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (5)

1. a kind of construction method of RFFT1 cell, the construction method includes: S1) full exon sequencing is carried out to tumour cell； By full exon sequencing result compared with the genome of normal cell, the amino acid sites of mutation are filtered out；With the amino of mutation Epitope is predicted centered on sour site；Mutant polypeptide is synthesized using polypeptide solid-state reaction method；It is mutated described in PBMC cell loading Polypeptide then carries out a polypeptide impact to the PBMC cell after load polypeptide；S2 expand culture after) impacting, obtain FF cell； S3) directly stimulate the FF cell to screen accurate polypeptide so that the polypeptide of Tumor mutations is as antigen；S4) culture PBMC is thin Born of the same parents carry out multiple polypeptide impact with accurate polypeptide during the cultivation process；S5 continue to cultivate after) impacting, obtain RFF cell；S6) The gained RFF cell using accurate polypeptide as antigenic stimulus, screening can identify the specific cell of the accurate polypeptide；S7) lead to It crosses sequencing and obtains the TCR β chain CDR3 region sequence of the specific cell, expand to obtain TCR by the TCR β chain CDR3 region sequence Gene；S8 PBMC cell) is cultivated, knocks out original tcr gene and surface inhibitive ability of immunity signaling molecule in cell, and be transferred to step RFFT1 cell, the inhibitive ability of immunity can be prepared with the tcr gene in conjunction with accurate polypeptid specificity in gained in rapid S7 Signaling molecule includes: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4 (CD244).
2. the construction method of RFFT1 cell according to claim 1, which is characterized in that
The tumour cell derive from commercially available engineering cell system, including 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 glioma cell.
3. the construction method of RFFT1 cell according to claim 1, which is characterized in that
The prediction of the epitope is to extend 10 amino acid to two sides, with one section centered on the amino acid sites of mutation The polypeptide of 21 amino acid is as potentially antigenic epitope as potentially antigenic epitope.
4. the construction method of RFFT1 cell according to claim 1, which is characterized in that in step S4, repeat polypeptide impact 3~4 times.
5. the construction method of RFFT1 cell according to claim 1, which is characterized in that be 10 with concentration in polypeptide impact The polypeptide solution of the μ of μ g/mL~100 g/mL carries out polypeptide impact.
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