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CN109136276A - A kind of construction method of RFFT2 cell - Google Patents

A kind of construction method of RFFT2 cell Download PDF

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CN109136276A
CN109136276A CN201811153181.6A CN201811153181A CN109136276A CN 109136276 A CN109136276 A CN 109136276A CN 201811153181 A CN201811153181 A CN 201811153181A CN 109136276 A CN109136276 A CN 109136276A
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cell
polypeptide
rfft2
culture
tcr
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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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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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Priority to CN201910438775.XA priority patent/CN110129371B/en
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Abstract

The present invention provides a kind of construction methods of RFFT2 cell, belong to field of biotechnology.This method is that a kind of attack-defense integrated, accuracy be high, lethal high T cell for constructing a kind of RFFT2 cell for cellular immunotherapy.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 polypeptide 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 is 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 RFFT2 cell.

Description

A kind of construction method of RFFT2 cell
Technical field
The present invention relates to field of biotechnology more particularly to a kind of construction methods of RFFT2 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, to excite and enhance the autoimmune function of body 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 RFFT2 cell, constructs the new TCR-T cell of one kind and (is named as RFFT2 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 RFFT2 cell, the construction method the following steps are included:
S1) PBMC cell loading causes the polypeptide of Tumor mutations, then carries out to the PBMC cell after load polypeptide primary more Peptide 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) cultivate PBMC cell, knock out cell in original tcr gene, and be transferred to obtained in step S7 can be with essence The tcr gene that quasi- polypeptid specificity combines, is prepared RFFT cell；
S9 inhibitive ability of immunity signaling molecule closing) is carried out to RFFT cell obtained by step S8 with monoclonal antibody medicine, to obtain RFFT2 Cell, the inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT、2B4(CD244)。
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.
Further, the polypeptide for causing Tumor mutations is synthesized to obtain by following methods:
1) exon is sequenced
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；
2) Epitope prediction
Centered on the amino acid sites of mutation, extends 10 amino acid to two sides, obtain the more of one section of 21 amino acid Peptide, in this, as potentially antigenic epitope；
IC50 < 1000nM then thinks that this potentially antigenic epitope is epitope；
3) synthesis polypeptide
Using polypeptide solid-state reaction method synthetic antigen epitope peptide.
Further, the tumour cell derive from engineering cell system, including H1299, H226, H358, H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL-6323 B16F1, CRL-2539 4T1, U14 Mouse Uterus Neck cancer cell, the small glioma cell of BV-2 mouse, G422 mouse glioma cell.
Further, before step S8, building knock out tcr gene CRISPR slow virus carrier, with its infection by PBMC cell passes through the CD8+T cell of magnetic bead sorting, is used to prepare RFFT cell.
Further, step S9 includes that RFFT cell obtained by step S8 is carried out weight with culture solution OKM-200+5%FBS It is outstanding, the monoclonal antibody medicine of the inhibition signaling molecule of final concentration of 50~200 μ g/mL is then added, 37 DEG C of closing 1h obtain RFFT2 Cell.
The invention has the following beneficial effects:
1) the present invention provides a kind of new cell construction methods, to construct a kind of TCR-T for cellular immunotherapy Cell (is named as RFFT2 cell), is a kind of super T cell having conditions in both attack and defence.It is effective by filtering out in building process It is simultaneously carried out secondary polypeptide impact to PBMC cell by accurate polypeptide.It should be noted that being adopted in secondary accurate polypeptide impact Stimulation is impacted with the polypeptide of dosage.Then on the basis of stimulating twice, joint TCR-T technology carries out third time stimulation, will be general Logical T cell transform the T cell for more having specific cytotoxicity as, and killing-efficiency is high, accuracy is high.
2) in the present invention, while association's outer closure, in vitro culture and amplification in vitro realize the accuracy of killing, spy Anisotropic and safety covers more polyoma kind, and improving has the T cell of high accuracy lethal effect to complicated tumor microenvironment Adaptive faculty.
3) present invention is by the stimulation of joint polypeptide and the lethal effect of TCR-T technological guide T cell, and directly uses slow virus The lethal effect of transfection inducing T cell is compared, 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 cell 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 inhibition target spot；
Fig. 7 shows RFFT2 cell described in the embodiment of the present invention to the lethal effect of target cell；
Fig. 8 shows the detection of RFFT2 cell cytokine release described in the embodiment of the present invention；
Fig. 9 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 a kind of construction method of RFFT2 cell, constructs a kind of for cellular immunotherapy TCR-T cell (is named as RFFT2 cell) herein.The construction method may comprise steps of:
S1) PBMC cell loading causes the polypeptide of Tumor mutations, then carries out to the PBMC cell after load polypeptide primary more Peptide 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) knock out original tcr gene in the peripheral blood in patients T cell, and be transferred to obtained in step S7 can be with The tcr gene that accurate polypeptid specificity combines, culture obtain RFFT cell；
S9 inhibitive ability of immunity signaling molecule closing) is carried out to RFFT cell obtained by step S8 with monoclonal antibody medicine, to obtain RFFT2 Cell, the inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT、2B4(CD244)。
Herein, it is as follows to define the meaning that " RFFT2 " cell is related to:
The accurate polypeptide secondary pulse technology of R-；
FF-mixed polypeptide technology；
T-TCR-T technology；
2-close guard technology in vitro.
That is, " RFFT2 " cell is by combining mixed polypeptide technology, accurate polypeptide secondary pulse technology, TCR-T herein (T cell receptor (TCR) mosaic type T cell) technology, external one for closing RFFT2 scheme composed by guard technology and being prepared 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 RFFT2 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 in conjunction in vitro culture and external closing guard technology, keep improved T thin Born of the same parents improve the adaptive faculty to internal complicated immune environment.
Before step S1, 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 using tumour cell, then sequencing information is analyzed using software, on the one hand To MHC type information；On the other hand, full exon sequencing result is filtered out into mutation compared with the genome of normal cell Amino acid sites.
In exon sequencing, tumour cell may come from engineering cell system, can be from peripheral blood in patients.
Engineering cell system includes H1299, H226, H358, H1563, H2228, A549, Renca, LLC Mice Bearing Lewis Lung Cancer Cell, 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.Engineering cell system refers to using technique for gene engineering or cell-fusion techniques to host cell Inhereditary material carry out modification transformation or recombination, obtain 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.
In step sl, with causing the polypeptide of Tumor mutations to carry out polypeptide impact to the PBMC cell after load polypeptide, directly PBMC cell after stimulation load polypeptide, carries out first time stimulation.
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 every 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) as being impacted in tissue culture plate；
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.
It can specifically include:
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, the original tcr gene of cell can be knocked out with CRISPR technology.
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, the CRISPR slow virus carrier for knocking out original tcr gene can be constructed respectively, and can express step The tcr gene expression vector of tcr gene determined by rapid S7.It is thin by the CD8+T of magnetic bead sorting by PBMC cell with its infection Born of the same parents.
Specifically, magnetic bead point is first passed through by PBMC cell with the CRISPR slow virus carrier infection for knocking out original tcr gene The CD8+T cell of choosing, knocks out original TCR；Then it is transferred to the tcr gene expression load that can express tcr gene determined by step S7 Body is transferred to tcr gene determined by step S7 with its infection cell.Then with OKM100+12%FBS by metainfective CD8+T Cell is resuspended, and is placed on the pre- bed board of stimulating factor OKM-25 and continues to cultivate, to obtain RFFT cell.
The building process for knocking out the CRISPR slow virus carrier of original tcr gene is 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 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 repeats no more herein.
In step S9, inhibitive ability of immunity signaling molecule closing is carried out to RFFT cell obtained by step S8 with monoclonal antibody medicine, with To RFFT2 cell, the inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160,2B4(CD244).Guard technology is closed in vitro by antibody drug, T cell is protected, and improves it to multiple in vivo The adaptive faculty of miscellaneous immune microenvironment.
In one embodiment, RFFT cell obtained by step S8 is resuspended with culture solution OKM-200+5%FBS, then The monoclonal antibody medicine of the inhibition signaling molecule of final concentration of 150 μ g/mL is added, 37 DEG C of closing 1h obtain RFFT2 cell.
For example, the detailed operation of step S9 is specifically as follows:
1) 1000rpm is centrifuged 5min, collects cultured RFFT cell；
2) it being washed once with PBS, 1000rpm is centrifuged 5min, TCR-T is resuspended with OKM-200+5%FBS, and adjust to 1 × 107/mL；
3) the monoclonal antibody medicine (such as PD-1 antibody) of addition inhibition signaling molecule, final concentration of 50~200 μ g/mL, preferably 150 μ g/mL, 0~37 DEG C of 1~4h of closing, preferably 37 DEG C of 1h.
The embodiment of the invention provides a kind of new T cells (RFFT2 cell) for immunotherapy of tumors, pass through joint Mixed polypeptide technology, TCR-T technology, closes guard technology at accurate polypeptide secondary pulse in vitro, transform T cell as accuracy Good, lethal height can cover a variety of tumor kinds, the TCR-T cell strong to complicated immune microenvironment adaptive faculty.
Hereafter further the construction method of RFFT2 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 T-cells
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.
(11) building knocks out the CRISPR slow virus 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) 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.
(12) TCR-T
1) recovery PBMC, it is spare with magnetic bead sorting CD8+ cell；
2) the CRISPR slow virus carrier to obtain in step 11 infects CD8+T cell, while carrying out original TCR's It knocks out；
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 removes infection cell, to be transferred to tcr gene constructed by step 10；
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. RFFT is thin Born of the same parents.
The external closing of (13) inhibitive ability of immunity signaling molecule
1) 1000rpm is centrifuged 5min, collects cultured RFFT cell；
2) it being washed once with PBS, 1000rpm is centrifuged 5min, TCR-T is resuspended with OKM-200+5%FBS, and adjust to 1 × 107/mL；
3) the monoclonal antibody medicine of inhibition signaling molecule, such as PD-1 antibody, final concentration of 150 μ g/mL, 37 DEG C of closings are added 1h。
(14) tumor-bearing mice survival assay
1) it takes tumor cell line 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 RFFT2 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 60 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 (FL2-H subset 54.4% indicates that PI stain contaminates to the load efficiency of PBMC by 54.4% To cell account for 54.4%).
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: it is 2 good, No. 4, No. 6, burst size > high baseline+system of IFN-γ caused by No. 10 polypeptides misses Difference belongs to effective accurate polypeptide.
The detection of 4.RFF cell-specific cell proportion
It with No. 6 polypeptides of screening, is repeatedly stimulated on FF cell base, after culture, with flow cytometer detection to precisely more The T cell ratio of peptide specific, as a result as shown in figure 3, being specific T-cells in box: compared with non-treated control group, RFF cell, after repeat impact stimulates, the cell proportion of release IFN-γ caused by No. 6 polypeptides, hence it is evident that be higher than without multiple The cell (FF cell) for impacting stimulation, illustrates, accurate polypeptide repeatedly stimulates, and specific T-cells ratio can be improved；Simultaneously with stream Formula cell instrument carries out the sorting of CD8+IFN- γ+(in box) cell.
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 is (high as shown in Figure 4 20) TCR9 distribution frequency is higher for frequency division cloth preceding, illustrates, 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.
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. inhibition target spot closes Efficiency testing in vitro
Inhibition signaling molecule antibody is marked with fluorescence, then with the closing feelings of flow cytometer detection PD-1 antibody on cell Condition.As a result as shown in fig. 6, the cell proportion that can detecte fluorescence signal is 96.9%.
8.RFFT2 cell is to the lethal effect of target cell
Killing effect is carried out with the target cell of RFF cell, RFFT cell and RFFT2 cell to mutant antigen epitope source respectively The detection of rate, using non-treated cell as control (Mock), as a result as shown in fig. 7, compared with the control group, RFF cell, RFFT Cell and RFFT2 cell have certain fragmentation effect to target cell, and in 20:1 and 40:1 (effector cell: target cell), with Mock group difference is obvious.Wherein, the killing-efficiency > RFFT cell > RFF cell of RFFT2 cells against tumor.
The detection of 9.RFFT2 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. 8 is difference The detection for the IFN-γ that training method cell and tumour cell discharge when co-culturing.The result shows that: it is generated with effector cell itself IFN-γ compare (only effector cell), with tumour cell co-culture after, RFF Protocols Cell, RFFT Protocols Cell and RFFT2 are thin Born of the same parents can produce a large amount of IFN-γ, especially RFFT and RFFT2 cell, releasable more IFN-γ, this result and killing Experimental result unanimously illustrates: the T cell of expression specificity TCR, in conjunction with inhibition target spot closing can more effectively improve it is anti- Tumour ability.
10. tumor-bearing mice survival assay
As a result as shown in figure 9, feeding back the existence improvement of the RFFT2 cells against tumor tumor-bearing mice of the embodiment of the present invention has Significantly affect effect.P value shows less than 0.01 (p value=0.0017) with statistical significance.
11. clinical case
Administration process:
First course for the treatment of: monthly RFFT2 cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year RFFT2 cell, quantity 1 × 109A cell, totally 2 times.
Table 3
Number Gender Age Medical diagnosis on disease The Progression free survival time after administration
1 Female 76 Oophoroma 2017.4- so far
2 Female 71 Cervical carcinoma 2017.4- so far
3 Male 71 Gastric cancer 2017.3- so far
4 Male 77 Lung cancer 2017.9- so far
5 Male 65 Lung cancer 2017.1- so far
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 (9)

1. a kind of construction method of RFFT2 cell, which is characterized in that the construction method the following steps are included:
S1) PBMC cell loading causes the polypeptide of Tumor mutations, then carries out a polypeptide punching to the PBMC cell after load polypeptide It hits；
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 PBMC cell) is cultivated, during the cultivation process, precisely polypeptide carries out multiple polypeptide impact obtained by step S3；
S5 continue to cultivate after) impacting, obtain RFF cell；
S6) the gained RFF cell using accurate polypeptide as antigenic stimulus, screening can identify that the specificity of the accurate polypeptide is thin Born of the same parents；
S7 the TCR β chain CDR3 region sequence of the 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 in cell is knocked out, and being transferred in step S7 gained can be with accurate polypeptide The tcr gene of specific binding, culture obtain RFFT cell；
S9 inhibitive ability of immunity signaling molecule closing) is carried out to RFFT cell obtained by step S8 with monoclonal antibody medicine, it is thin that culture obtains RFFT2 Born of the same parents, the inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4(CD244)。
2. the construction method of RFFT2 cell according to claim 1, which is characterized in that in step s 2, after the impact Expand culture, obtaining FF cell composition includes:
PBMC cell after polypeptide is impacted is cultivated 5 days in the cell culture apparatus for overlaying cell stimulation factor OKM-25；
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.
3. the RFFT2 born of the same parents according to claim 1 for cellular immunotherapy, which is characterized in that in step S4, repeat more Peptide impacts 3~4 times.
4. the construction method of RFFT2 cell according to claim 3, which is characterized in that in step s 4, every 3~4 days Carry out a polypeptide impact.
5. the RFFT2 born of the same parents according to claim 1 for cellular immunotherapy, which is characterized in that in polypeptide impact, use is dense The polypeptide solution that degree is 10 μ of μ g/mL~100 g/mL carries out polypeptide impact.
6. the construction method of RFFT2 cell according to claim 1, which is characterized in that the polypeptide for causing Tumor mutations It synthesizes to obtain by following methods:
1) exon is sequenced
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；
2) Epitope prediction
Centered on the amino acid sites of mutation, to two sides extend 10 amino acid, using the polypeptide of one section of 21 amino acid as Potentially antigenic epitope；
IC50 < 1000nM then thinks that this potentially antigenic epitope is epitope；
3) synthesis polypeptide
Using polypeptide solid-state reaction method synthetic antigen epitope peptide.
7. the construction method of RFFT2 cell according to claim 6, which is characterized in that the tumour cell derives from work Journey cell line, including H1299, H226, H358, H1563, H2228, A549, Renca, LLC Lewis lung cancer cells, CRL- 6323B16F1, CRL-2539 4T1, U14 mouse carcinoma of uterine cervix cell, the small glioma cell of BV-2 mouse, G422 mouse Nerve Glioma cell.
8. the construction method of RFFT2 cell according to claim 1, which is characterized in that original with knocking out in step S8 The CRISPR slow virus carrier of TCR and the CRISPR slow virus carrier for knocking out surface inhibitive ability of immunity signaling molecule go infection of PBMCs Cell, while carrying out the knockout of original TCR and the removal of inhibitive ability of immunity signaling molecule.
9. the construction method of RFFT2 cell according to claim 1, which is characterized in that described to use monoclonal antibody medicine to step S8 Gained RFFT cell carries out the closing of inhibitive ability of immunity signaling molecule to obtain RFFT2 cell
RFFT cell obtained by step S8 is resuspended with culture solution OKM-200+5%FBS, then it is added final concentration of 50~ The monoclonal antibody medicine of the inhibition signaling molecule of 200 μ g/mL, 37 DEG C of closing 1h, obtains RFFT2 cell.
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