CN109294998A - A kind of RFF1 cell - Google Patents

A kind of RFF1 cell Download PDF

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Publication number
CN109294998A
CN109294998A CN201811153225.5A CN201811153225A CN109294998A CN 109294998 A CN109294998 A CN 109294998A CN 201811153225 A CN201811153225 A CN 201811153225A CN 109294998 A CN109294998 A CN 109294998A
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cell
polypeptide
pbmc
rff1
impact
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CN109294998B (en
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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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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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    • C12N2510/00—Genetically modified cells
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    • C12N2800/00—Nucleic acids vectors
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Abstract

The present disclosure provides a kind of RFF1 cells, belong to field of biotechnology.The present invention, which discloses, provides a kind of RFF1 cell for cellular immunotherapy, is a kind of super T cell that attacking and defending takes into account, and accuracy is high, killing rate is high.The cell is prepared by following methods: PBMC cell loading causes the polypeptide of Tumor mutations, then carries out polypeptide impact to the PBMC cell after load polypeptide；Expand culture after impact, obtains FF cell；So that the polypeptide of Tumor mutations directly stimulates the FF cell to screen accurate polypeptide as antigen；Knock out the inhibitive ability of immunity signaling molecule on PBMC cell；With the accurate polypeptide load PBMC and with knock out inhibitive ability of immunity signaling molecule PBMC mixing with cells after train altogether, during total training, carry out repeat impact with accurate polypeptide, obtain RFF1 cell.The cell can be used for cellular immunotherapy technology.

Description

A kind of RFF1 cell
Technical field
The present invention relates to field of biotechnology more particularly to a kind of for the RFF1 cell of cellular immunotherapy and its preparation Method.
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 human body cell specific process that bioengineering was transformed is transformed in incubation in vitro, can effectively kill and remove Patient's body tumour cell.Such as, Chinese patent application CN201210194280.5 provides a kind of killing for human cell factor induction Hurt cell.Chinese patent application CN201510034781.0 provides a kind of polyclonal T cell of tumor cell specific.Chinese patent Application CN201510013987.5 provide a kind of antitumor T cell and preparation method thereof.Chinese patent application CN201711060030.1 provides a kind of CAR-T cell and its preparation method and application treated AIDS and merge lymthoma. CN201610824893.0 provides a kind of double T cells with antigenic specificity and its preparation method and application of antibody regulation.
Summary of the invention
The present invention is intended to provide a kind of RFF1 cell for cellular immunotherapy, the impact of joint polypeptide and external training altogether, General T cell is transform to the super T cell having conditions in both attack and defence as, solving immunocyte, attacking and defending cannot be simultaneous in cellular immunotherapy The problem of Gu, accuracy is good, killing rate is high.
The present invention provides a kind of RFF1 cell for cellular immunotherapy, wherein the preparation method packet of the RFF1 cell Include following steps:
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, to obtain FF cell；
S3) directly stimulate the FF cell to screen accurate polypeptide so that the polypeptide of Tumor mutations is as antigen；
S4) knock out PBMC cell on inhibitive ability of immunity signaling molecule, the signaling molecule include: PD-1, Tim-3, LAG3,CTLA-4,BTLA,VISTA,CD160,TIGIT,2B4(CD244)；
S5 PBMC cell, then the PBMC cell with knockout inhibitive ability of immunity signaling molecule) are loaded with the accurate polypeptide of screening Mixing co-cultures；
S6) during total training, multiple polypeptide impact is carried out with accurate polypeptide；
S7 continue to cultivate after) impacting, obtain RFF1 cell.
Further, step S2 includes:
PBMC cell after polypeptide is impacted is cultivated in the cell culture apparatus for overlaying cell stimulation factor OKM-25；
After cultivating a period of time, it is transferred to and continues to train in the cell culture apparatus containing culture solution OKM-100+12%FBS It supports；
After cultivating a period of time, transfers in the cell culture apparatus containing culture solution OKM-200+5%FBS and continue to train It supports.
Further, in step S5, the PBMC of PBMC cell and knockout inhibitive ability of immunity signaling molecule after loading polypeptide Cell is mixed with the ratio of 1:1~1:20.
Further, it in step S6, during total training, repeats polypeptide and impacts 3~4 times.
Further, in step s 6, a polypeptide impact was carried out every 3~4 days.
Further, in step S6, carrying out polypeptide impact with accurate polypeptide during total training includes: that load sudden change is more The PBMC of peptide is trained after mixing with the PBMC for knocking out inhibitive ability of immunity signaling molecule in the cell for overlaying cell stimulation factor OKM-25 It supports and is cultivated in device, the accurate polypeptide obtained by step S3 carries out polypeptide impact after cultivating a period of time；
It is transferred to and continues to cultivate in the cell culture apparatus containing culture solution OKM100+12%FBS, respectively every 3~4 days Precisely polypeptide carries out polypeptide impact obtained by step S3.
Further, in step S7, continuing culture after the impact to obtain RFF1 cell includes: to contain after polypeptide impacts Continue to cultivate in the cell culture apparatus for having culture solution OKM200+5%FBS, to obtain RFF1 cell.
Further, the inhibitive ability of immunity signaling molecule on PBMC cell is knocked out using CRISPR technology.
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, the engineering cell system include 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 glioma cell.
The invention has the following beneficial effects:
The present invention provides a kind of RFF1 cell for cellular immunotherapy, is a kind of super T cell having conditions in both attack and defence.It is logical It crosses and filters out effective precisely polypeptide and it is subjected to secondary polypeptide impact to PBMC cell, more there is specific cytotoxicity T cell, killing-efficiency are higher.
In the prior art, it is in T cell generally by DC cell delivery, generates the T cell of specific killing；Or made with virus For carrier, pass through the specific killing of slow-virus transfection technological guide T cell.In this application, skill is impacted by secondary polypeptide The specific killing of art inducing T cell.First time polypeptide impacts first, directly stimulates PBMC cell with mixed polypeptide；Then with Accurate polypeptide directly stimulates PBMC, and during co-cultivation, repeatedly with the stimulation of accurate polypeptide.By repeatedly stimulating general T Cell is transformed into the super T cell for more accurately killing ability.It is simple and convenient, highly-safe compared to slow-virus transfection. In addition, combining target spot in the application knocks out guard technology and external training and amplification in vitro altogether, it is micro- to complicated tumour to improve T cell The adaptive faculty of environment covers a variety of tumor kinds.
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 the flow diagram of the preparation method of RFF1 cell described in the embodiment of the present invention；
Fig. 2 shows antigen load Efficiency testings；
Fig. 3 shows accurate polypeptide the selection result；
Fig. 4 shows flow cytometer detection specific T-cells ratio；
Fig. 5 shows the knockout situation of flow cytometer detection inhibitive ability of immunity signaling molecule；
Fig. 6 shows the lethal effect of RFF1 cells against tumor；
Fig. 7 shows the release of RFF1 cell by cell factor IFN-γ；
Fig. 8 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 cell, DC cell, CIK cell, DC-CIK cell be proved to invalid substantially.The embodiment of the present invention provides a kind of new T Cell is used for cellular immunotherapy.
The embodiment of the present invention provides a kind of RFF1 cell for cellular immunotherapy, wherein the preparation of the RFF1 cell 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, to obtain FF cell；
S3) directly stimulate the FF cell to screen accurate polypeptide so that the polypeptide of Tumor mutations is as antigen；
S4) knock out PBMC cell on inhibitive ability of immunity signaling molecule, the signaling molecule include: PD-1, Tim-3, LAG3,CTLA-4,BTLA,VISTA,CD160,TIGIT,2B4(CD244)；
S5 PBMC cell, then the PBMC cell with knockout inhibitive ability of immunity signaling molecule) are loaded with the accurate polypeptide of screening Mixing co-cultures；
S6) during total training, repeat impact is carried out with accurate polypeptide；
S7 continue to cultivate after) impacting, obtain RFF1 cell.
It is as follows to define the meaning that " RFF1 " cell is related to:
The accurate polypeptide secondary pulse technology of R-；
FF-mixed polypeptide technology；
1-target spot knocks out guard technology.
Herein, " RFF1 " cell is by combining accurate polypeptide secondary pulse technology, mixed polypeptide technology and target spot Knock out the T cell that guard technology is transformed.Wherein, " FF " cell is not pass through accurate polypeptide two by mixed polypeptide technology Obtained T cell is transformed in Secondary Shocks (FF scheme)." RFF " cell is to be transformed by accurate polypeptide secondary pulse (RFF scheme) The T cell arrived.
The embodiment of the present invention provides a kind of RFF1 cell for cellular immunotherapy, has filtered out really effective accurate Polypeptide, and secondary polypeptide impact is carried out to PBMC cell with it, " the super T cell " being had conditions in both attack and defence, specific cytotoxicity It is stronger.Joint target spot knocks out guard technology on this basis, improves T cell to the adaptive faculty of intracorporal immunosupress microenvironment, Enhance the self-protection of T cell.
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-6323B16F1, 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 the something lost using technique for gene engineering or cell-fusion techniques to host cell It passes substance and carries 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, polypeptide impact is carried out to the PBMC cell after load polypeptide with the mixed polypeptide of cause Tumor mutations, PBMC cell directly 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:
Mixed polypeptide is dissolved with RPMI 1640+10%FBS (fetal calf serum) or OKM100+12%FBS, the end of polypeptide is dense Degree is 10~100 μ g/mL, and preferably 50 μ g/mL carry out polypeptide impact with this.
Further, the time of polypeptide impact can be 1~4h, such as preferably can be able to be 4h with 1h, 3h or 4h.
In step s 2, expand culture after impact to obtain FF cell.PBMC cell and cell after polypeptide is impacted pierce Swash factor OKM-25 to train altogether, to obtain FF cell.
Step S2 is specifically as follows:
PBMC cell after polypeptide is impacted is cultivated in the cell culture apparatus for overlaying cell stimulation factor OKM-25；
After cultivating a period of time, it is transferred to and continues to train in the cell culture apparatus containing culture solution OKM-100+12%FBS It supports；
It is transferred in the cell culture apparatus containing culture solution OKM-200+5%FBS after culture a period of time and continues to train It supports.
Specifically, FF cell composition can be obtained after expanding culture.It can be from the FF cell composition by centrifugation Separation and Extraction T cell (i.e. FF cell).
In step S3, directly stimulate T cell to screen accurate polypeptide using polypeptide as antigen.
The screening criteria of accurate polypeptide are as follows:
Using FF cell as baseline, two independent to be repeated, and detected value is high for high baseline, low for 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.
Step S3 can specifically include:
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:
Polypeptide is as antigen using FF cell as baseline；
Every group of experiment is two baselines, high baseline and low baseline (detected value is high for high baseline, low for low baseline), two The difference of a baseline is systematic error, when data are analyzed, 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.
In step S4, the inhibitive ability of immunity signaling molecule on PBMC cell is knocked out using CRISPR technology.The signal point Son includes: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGHT, 2B4 (CD244).
Specifically, building knocks out the CRISPR slow virus carrier of inhibitive ability of immunity signaling molecule, to infection of PBMCs cell, Obtain knocking out the PBMC cell of inhibitive ability of immunity signaling molecule.
Operation can specifically include following steps in detail:
1) exon for analyzing inhibition signaling molecule, finds the area CDS of the mRNA of gene on pubmed database, point Each exon knock out 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；
5) with RPMI1640+10%FBS recovery PBMC, at the 1st day to the 4th day, CRISPR slow-virus infection is carried out；
6) after infecting, it is to knock out inhibitive ability of immunity signaling molecule that 0-3 days are cultivated in RPMI 1640+10%FBS PBMC。
In step S5, the accurate polypeptide screened with step S3 loads PBMC cell, and with knock out inhibitive ability of immunity signal The PBMC cell of molecule is mixed with the ratio of 1:1~1:20, can preferably be mixed with the ratio of 1:10.
In step S6, polypeptide impact is carried out again with accurate polypeptide.Attack time can be 1-4h.
Compared to step S1, impacted in step s 6 using the accurate polypeptide of dosage.That is, accurate more with what is screened Peptide carries out multiple polypeptide impact stimulation to the cell after total training.During total training, repeats polypeptide and impact 3~4 times.
Further, in step s 6, a polypeptide impact can be carried out every 3~4 days, it every time can be with 1~4h.
For example, being overlay carefully after the PBMC for loading accurate polypeptide is mixed with the PBMC for knocking out inhibitive ability of immunity signaling molecule It is cultivated in the cell culture apparatus of born of the same parents' stimulating factor OKM-25, the accurate polypeptide obtained by step S3 carries out more after culture a period of time Peptide impact；
It is transferred to after polypeptide impact and continues to cultivate in the cell culture apparatus containing culture solution OKM100+12%FBS, every Precisely polypeptide carries out polypeptide impact obtained by step S3 respectively within 3~4 days.
Continue culture in step S7, after impact to obtain RFF1 cell.It can specifically include: containing training after polypeptide impact Continue to cultivate in the cell culture apparatus of nutrient solution OKM200+5%FBS, to obtain RFF1 cell.
The present invention provides a kind of RFF1 cell for cellular immunotherapy by a kind of cells in vitro preparation method, is A kind of super T cell having conditions in both attack and defence.During the preparation process, really effective precisely polypeptide is filtered out and with it to PBMC cell Carry out secondary pulse.Polypeptide impact is carried out with mixed polypeptide for the first time, carries out multiple polypeptide with the accurate polypeptide of screening for the second time Impact, to more be there is the RFF cell of specific cytotoxicity, accuracy is high, lethality is high.During the preparation process, pass through target Point knocks out guard technology and external training and amplification in vitro altogether, enhances gained RFF1 cell to the complicated tumour of patient's body The adaptability of microenvironment.
Hereafter the RFF1 cell further described in the embodiment of the present invention for cellular immunotherapy is described in detail.
(1) full exon sequencing
1) full exon sequencing is carried out using LLC Lewis lung cancer cells；
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: NetMHCpan 3.0, 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), make whole epitope peptide chain complete synthesis.
(4) PBMC load sudden change polypeptide and to load polypeptide after PBMC cell carry out polypeptide impact
1) prepare polypeptide solution: with RPMI 1640+10%FBS dissolve polypeptide, the final concentration of 50 μ g/mL of every polypeptide, 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, 5%CO2, 4h is impacted, the PBMC cell after being impacted 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 tissue culture plate or 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, 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, 20mL are mended in T75 culture bottle；
5) it co-cultures the 7th day, adds the fresh OKM-100+12%FBS of 20mL；
6) co-culture the 10th day, culture solution OKM-200+5%FBS, by co-cultured cell from T25 bottles that be transferred to In the big bottle of T75；
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, acquisition FF cell composition.
(6) polypeptide directly stimulates the accurate polypeptide of FF cell screening as antigen
1) T cell, i.e. FF cell are extracted from FF cell composition by centrifuge separation.1500rpm is centrifuged 5min and collects T Cell is added 10mL PBS and cell is resuspended and counts, and 1500rpm is centrifuged 5min, collects T cell with 1640+10%FBS+200U/ ML IL2 is resuspended, and counting is adjusted 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 the mutant polypeptide of 10 μ L 1mg/mL, final concentration of 50 μ g/mL, 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, sample is taken to carry out ELISA detection or by sample as -80 DEG C save.
(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 FF cell 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) the inhibitive ability of immunity signaling molecule on PBMC is knocked out using CRISPR technology
1) surface PBMC inhibitive ability of immunity signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160,TIGHT,2B4(CD244)；
2) exon for analyzing inhibition signaling molecule, finds the area CDS of the mRNA of gene on pubmed database, point Each exon knock out the prediction of target spot；
3) forward primer and reverse primer needed for design synthesis sgRNA, forward primer and reverse primer 1:1 are mixed Afterwards, 95 DEG C of 5~60min of processing, then slow cooling is carried out, form the DNA sequence dna of sgRNA；
4) CRISPR Lentiviral is subjected to double digestion, and double-stranded DNA corresponding with sgRNA is attached, turned Enter and clone competent cell, after 12h, picking monoclonal is sequenced, and retains sequencing correctly clone；
5) the CRISPR slow virus carrier plasmid for carrying sgRNA corresponding DNA sequence is extracted, viral packaging is carried out；
6) with 1640+10%FBS recovery PBMC, at the 1st day to the 4th day, CRISPR slow-virus infection is carried out；
7) after infecting, it is the PBMC for knocking out inhibitive ability of immunity signaling molecule that 0-3 days are cultivated in 1640+10%FBS.
(9) preparation of RFF1 cell
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 of accurate polypeptide will be loaded and knock out the PBMC of inhibitive ability of immunity signaling molecule, carried out with the ratio of 1:10 Mixing, and be 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) when culture was to the 3rd day, impacting again for accurate polypeptide is carried out；
5) 2 × 10 are taken7PBMC cell, the polypeptide of final concentration of 50 μ g/mL is added, impacts 4h；
6) after impacting 4h, it is transferred to T25 culture bottle, mends OKM100+12%FBS, 37 DEG C of 5%CO2Culture, grows according to cell Situation is transferred in T75 culture bottle, and holding cell density as far as possible is 1 × 106A/mL；
7) when cultivating the 7th day, 10 days and 14 days, step 5) and 6) is repeated；
8) when cell is entered in T75 culture bottle, culture medium OKM200+5%FBS, culture can be obtained for 10~21 days RFF1 cell.
(10) 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 RFF1 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. 2 --- 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 55.4% indicates the cell that PI stain contaminates to the load efficiency of PBMC by 55.4% It accounts for 55.4%).
3. screening accurate polypeptide
As shown in figure 3, stimulating the FF cell of culture respectively with 13 polypeptides, by detecting the secretion of IFN-γ, detection has The polypeptide of effect, as a result as shown in Figure 2: burst size > high baseline+systematic error of IFN-γ caused by No. 6 and No. 9 polypeptides belongs to Effective accurate polypeptide.
4. specific T-cells ratio detects
It with No. 9 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 4, being specific T-cells in box: compared with non-treated control group, RFF1 cell, after repeat impact stimulates, the cell proportion of release IFN-γ caused by No. 9 polypeptides, hence it is evident that be higher than without more The cell (FF cell) of Secondary Shocks stimulation.The sorting of CD8+IFN- γ+cell (in box) is carried out with flow cytometer.
5. the knockout of inhibitive ability of immunity signaling molecule
Using CRISPR technology, the inhibition signaling molecule on PBMC is knocked out, as a result as shown in Figure 5.
Lethal effect of the 6.RFF1 cell to target cell
The detection of killing-efficiency is carried out to the target cell in mutant antigen epitope source with RFF1 of embodiment of the present invention cell, with Non-treated cell is as control (Mock), as a result as shown in fig. 6, compared with the control group, RFF1 cell pair of the embodiment of the present invention Target cell has certain fragmentation effect, in 20:1 and 40:1 (effector cell: target cell), with the obvious (RFF1- of Mock group difference 1: the 3rd, 10 day in step 9 has carried out accurate polypeptide and has impacted again；RFF1-2: it the 3rd in step 9, has carried out within 7,10,14 days precisely Polypeptide impacts again).
The detection of 7.RFF1 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. 7 is difference Training method cell, when being co-cultured with tumour cell, the detection of the IFN-γ of release, the results showed that produced with effector cell itself Raw IFN-γ compares (only effector cell), and after co-culturing with tumour cell, RFF1 cell of the embodiment of the present invention be can produce largely IFN-γ, illustrate: the knockout of inhibition target spot can more effectively improve anti-tumor capacity (RFF1-1: the 3rd in step 9, Accurate polypeptide is carried out within 10 days to impact again；RFF1-2: it the 3rd in step 9, has carried out accurate polypeptide and has impacted again within 7,10,14 days).
8. tumor-bearing mice survival assay
As a result as shown in figure 8, feeding back the existence improvement of the RFF1 cells against tumor tumor-bearing mice of the embodiment of the present invention has Significantly affect effect.
9. clinical case
Administration process:
First course for the treatment of: monthly RFF1 cell, quantity 1 × 109A cell, totally 2 times；
Second course for the treatment of: every half a year RFF1 cell, quantity 1 × 109A cell, totally 2 times.
Table 2
Number Gender Age Medical diagnosis on disease The Progression free survival time after administration
1 Female 67 Oophoroma 2016.4- so far
2 Female 61 Gastric cancer 2017.11- so far
3 Male 63 Gastric cancer 2017.11- 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 RFF1 cell for cellular immunotherapy, which is characterized in that the preparation method of the RFF1 cell include with Lower step:
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) knock out PBMC cell on inhibitive ability of immunity signaling molecule, the signaling molecule include: PD-1, Tim-3, LAG3, CTLA-4,BTLA,VISTA,CD160,TIGIT,2B4(CD244)；
S5 PBMC, then the PBMC mixing with cells with knockout inhibitive ability of immunity signaling molecule) are loaded with the accurate polypeptide of screening, trained altogether It supports；
S6) during total training, multiple polypeptide impact is carried out with accurate polypeptide；
S7 continue to cultivate after) impacting, to obtain RFF1 cell.
2. the RFF1 cell according to claim 1 for cellular immunotherapy, which is characterized in that in step s 2, institute Expand culture after stating impact, includes: to obtain FF cell
PBMC cell after polypeptide is impacted is cultivated in the cell culture apparatus for overlaying cell stimulation factor OKM-25；
After cultivating a period of time, it is transferred to and continues to cultivate in the cell culture apparatus containing culture solution OKM-100+12%FBS；
After cultivating a period of time, transfers in the cell culture apparatus containing culture solution OKM-200+5%FBS and continue to cultivate.
3. the RFF1 cell according to claim 1 for cellular immunotherapy, which is characterized in that in step S5, load PBMC cell after accurate polypeptide is mixed with the PBMC cell for knocking out inhibitive ability of immunity signaling molecule with the ratio of 1:1~1:20.
4. the RFF1 cell according to claim 1 for cellular immunotherapy, which is characterized in that in step S6, altogether During training, repeats polypeptide and impact 3~4 times.
5. the RFF1 cell according to claim 4 for cellular immunotherapy, which is characterized in that in step s 6, often A polypeptide impact was carried out every 3~4 days.
6. the RFF1 cell according to claim 1 for cellular immunotherapy, which is characterized in that in step S6, altogether Carrying out polypeptide impact with accurate polypeptide during training includes:
By the PBMC for loading accurate polypeptide with knock out inhibitive ability of immunity signaling molecule PBMC mix after overlay cytositimulation because It is cultivated in the cell culture apparatus of sub- OKM-25, the accurate polypeptide obtained by step S3 carries out polypeptide impact after cultivating a period of time；
It is transferred to and continues to cultivate in the cell culture apparatus containing culture solution OKM100+12%FBS, every 3~4 days respectively with step Precisely polypeptide carries out polypeptide impact obtained by rapid S3.
7. the RFF1 cell according to claim 1 for cellular immunotherapy, which is characterized in that utilize CRISPR technology Knock out the inhibitive ability of immunity signaling molecule on PBMC cell.
8. the RFF1 cell according to claim 1 for cellular immunotherapy, which is characterized in that the cause Tumor mutations Polypeptide synthesize 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.
9. the RFF1 cell according to claim 8 for cellular immunotherapy, which is characterized in that the tumour cell comes Derived from engineering cell system, the engineering cell system includes H1299, H226, H358, H1563, H2228, A549, Renca, LLC small Mouse Lewis lung carcinoma cell, CRL-6323B16F1, CRL-2539 4T1, U14 mouse carcinoma of uterine cervix cell, the small colloid of BV-2 mouse Oncocyte, G422 mouse glioma cell.
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