CN110093375B

CN110093375B - Construction method of MRFFT2 cell

Info

Publication number: CN110093375B
Application number: CN201910439135.0A
Authority: CN
Inventors: 焦顺昌; 张嵘; 宋玉洁; 周子珊; 解佳森; 袁翰; 李营营
Original assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Current assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Priority date: 2018-09-30
Filing date: 2019-05-24
Publication date: 2021-01-22
Anticipated expiration: 2039-05-24
Also published as: CN110093375A; CN109136279A

Abstract

The invention relates to a construction method of MRFFT2 cells, which screens out mutant polypeptides through epitope prediction, connects and synthesizes a mutant polypeptide expression gene sequence; simultaneously, an MVA virus vector is constructed, MVA virus is packaged, APC cells are transfected, transformation of specific MV cells is completed, co-culture is carried out on the specific MV cells in vitro and PBMC separated from peripheral blood, effective polypeptide is screened out, common T cells are transformed into RFF cells with more accurate killing capacity through second impact stimulated by accurate effective polypeptide, transformation is carried out by utilizing the TCR-T technical principle, the transformed T cells are sealed by antibody medicines for immune inhibitory signals in vitro, the specific killer T cells are accurately protected from in vivo inhibition, and the killing power of the T cells to tumor cells is improved.

Description

Construction method of MRFFT2 cell

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method of MRFFT2 cells.

Background

Currently, the existing LAK, DC, CIK, DC-CIK cells and methods have proven largely ineffective in the specific immunotherapy of tumors, while NK, CAR-NK, TIL, etc. cell technologies remain to be mature and CAR-T cells have drawbacks in safety and solid tumor therapy. The prior art generally produces specific killing by DC presenting T cells by engineering the DC cells. Some laboratories have attempted to transfect presenting T cells using viruses as vectors to induce specific killing of T cells. We have also used mutant mixed polypeptides to directly stimulate PBMCs and induce T cells. There are also laboratories that use TCR-T technology for targeted presentation of MAGE a3 antigen.

The above treatment methods are not mature, and especially, the technologies for inducing DC cells in vitro and loading tumor antigens on DC cells are more researched theoretically, but there are many problems in the specific implementation process, and clear molecules related to signal transduction pathways critical for the development of tumor cells are not used as the induced antigens, so that the realization of specific cell-targeted immunotherapy is difficult to implement smoothly because the tumor antigens are not clear and the immune suppression of tumor microenvironment is hindered. In addition, some antigen-specific cells are not co-cultured and expanded in vitro, and thus a thin specific cell is directly exposed to a complex tumor immune microenvironment, even though the antigen is subjected to in vitro impact, and thus it is difficult to achieve a desired effect. Although some can also be presented and co-cultured in vitro, the target is single (MAGE-3), and only acts on individual cancer species such as non-small cell lung cancer. Although methods that are slow-toxic to vectors have been attempted for transfection presentation, they are not as safe and convenient as polypeptides. While direct stimulation by simply mixing polypeptides is simple and convenient, it is inefficient. Secondary stimulation of the anisotropically precise polypeptide is not as direct as tumor-specific antigens transduced by T cell receptors. Existing TCR-ts lack an accurate TCR covering more tumor species in solutions for treating hematological and solid tumors.

None of the above schemes consider the self-protection technology of T cells, so that a small number of specific T cells directly face a strong tumor immune microenvironment.

Disclosure of Invention

The invention carries out ctDNA sequencing by using peripheral blood of a patient or carries out whole exon sequencing by using tumor tissues, screens out mutation sites for antigen epitope prediction, and connects and synthesizes a mutant polypeptide expression gene sequence; simultaneously, an MVA virus vector is constructed, MVA virus is packaged, APC cells are transfected, transformation of specific MV cells is completed, co-culture is carried out on the modified MVA cells and PBMC separated from peripheral blood in vitro, effective polypeptide is screened out, common T cells are transformed into RFF cells with more accurate killing capacity through second impact stimulated by accurate effective polypeptide, transformation is carried out by utilizing the TCR-T technical principle, the transformed T cells are sealed by antibody medicines for immune inhibitory signals in vitro, the specific killer T cells are accurately protected from in vivo inhibition, and the killing power of the T cells to tumor cells is improved.

For the interpretation of terminology:

m: MVA virus transfection technique

R: accurate polypeptide secondary impact technology

FF: polypeptide mixing technology

T: TCR-T technology

2: antibody medicine in-vitro sealing protection technology

For example: the MRFFT2 cell is a cell obtained by modifying the technical scheme or the technical means of the M, R, FF, T and 2.

MRFFT2 cell engineering protocol:

1. epitope prediction

1) Using human peripheral blood for ctDNA sequencing or commercially available engineered cell lines (e.g., H1299, H226, H358, H1563, H2228, A549, Renca, LLC mouse Lewis lung cancer cells, CRL-6323B16F1, CRL-25394T 1, U14 mouse cervical cancer cells, BV-2 mouse glioma cells, G422 mouse glioma cells, etc.) for MHC class detection and whole exon sequencing;

2) prediction of antigenic epitopes using MHC class and gene mutation information: taking the mutated amino acid site as the center, extending 8 amino acids to both sides, and taking the polypeptide of 17 amino acids as a potential antigen epitope;

4) analyzing the IC50 of the potential epitope by using prediction software, and if the IC50 is less than 1000nM, the potential epitope is considered as the epitope;

2. polypeptide attachment

1) The software is used for analyzing the IC50 at the joint after any epitope is connected pairwise, and the IC50 is considered as weak immunogenicity when the IC50 is more than or equal to 1000nM and can be connected; IC50 < 1000nM is considered strong immunogenicity and unable to link;

2) according to the results, the epitopes with weak immunogenicity are connected together, and the IC50 at the joint is higher than the IC50 of the epitopes on both sides (namely, the joint avoids generating strong binding antigen as much as possible);

3. synthetic polypeptides

1) Reducing the connected polypeptide into a nucleic acid sequence, and performing codon optimization;

2) synthesizing the gene sequence of the epitope peptide by using a solid phase synthesis method; or synthesized by a technical service company;

4. construction of an antigenic epitope peptide-expressing MVA Virus

Constructing an MVA virus expression plasmid for expressing the epitope peptide of the antigen by using the gene sequence synthesized in the step, and packaging the virus;

5. transfection of Antigen Presenting Cells (APC) and Co-culture with PBMC

1) Antigen presenting cells were transfected with mva viruses expressing antigenic epitope peptides (including but not limited to: peripheral blood mononuclear cells, dendritic cells, neutrophils, B lymphocytes, macrophages);

2) APC completed by the treatment are collected, and APC: PBMC ═ 1: 5-20 to obtain effector cells;

6. screening for effective and accurate polypeptides and re-stimulating T cells using the accurate polypeptides

1) And (3) centrifugally collecting the T cells obtained by the scheme, and using the polypeptide as an antigen to directly stimulate the T cells to screen accurate polypeptides:

2) setting a positive control: t cells +100ng/mL OKT 3; negative control: t cells +1640+10% FBS +200U/mL IL 2;

3) the accurate polypeptide evaluation standard is as follows:

a. if the positive control and the negative control are normal, the data is credible;

b. the experimental group is obviously larger than the negative control group and is effective accurate polypeptide;

7. construction of TCR-T cells

1) Staining the stimulated T cells with CD8, CD137 and IFN-gamma, and sorting by flow cytometry;

2) sorting out specific cells capable of identifying the accurate polypeptide, sequencing to determine a high-frequency TCR sequence and amplifying;

3) constructing a TCR gene expression vector and packaging viruses;

4) knocking out the original TCR gene in the peripheral blood T cell, transferring the TCR gene constructed in the previous step, and culturing to obtain the TCR-T cell;

8. the immunosuppressive signal is sealed in vitro by antibody drug to obtain MRFFT2 cell

Cell surface inhibitory signaling molecules include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4(CD 244);

9. constructing specific antigen expression target cells and tumor model survival experiments.

The invention has the beneficial effects that:

1. the tumor antigen is a mutant antigen, is different from other tissues, has strong target specificity, is not easy to generate off-target effect and has high safety;

2. the obtained specific cells have high proportion, can usually identify the specific cells of the tumor antigen, the distribution of PBMC is less than 0.5 percent, and the proportion of the specific T cells (TCR +) for identifying the tumor antigen of the cells modified by the MRFFT2 scheme is more than 50 percent;

the MRFFT2 cell has unlimited killing capacity and higher killing efficiency on tumors due to the blocking operation of immunosuppressive signals such as PD1, CTLA4, TIM3, LAG3 and the like.

Drawings

FIG. 1: carrying out MVA virus transfection APC efficiency detection; wherein, 1A: control, 1B: and (4) a transfection group.

FIG. 2: and (4) carrying out MRFF cell typing detection.

FIG. 3: and (4) screening of the precise polypeptide.

FIG. 4: detecting the proportion of specific T cells by flow; wherein, 4A: control, 4B: MRFF scheme.

FIG. 5: the TCR distribution frequency.

FIG. 6: detecting the knockout efficiency of the original TCR; wherein, 6A: after knock-out, 6B: before knockout.

FIG. 7: the efficiency of expression of the specific TCR; wherein, 7A: 7 days after transfection, 7B: before transfection.

FIG. 8: blocking effects of immunosuppressive signals; wherein, 8A: before blocking, 8B: and (5) sealing.

FIG. 9: LDH release detection killing efficiency.

FIG. 10: ELISA was used to detect the release of the cytokine IFN-. gamma..

FIG. 11: animal tumor-bearing model survival curves.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

1. Epitope prediction

1) Performing ctDNA sequencing and HLA typing detection on peripheral blood of a lung cancer patient;

2) sequencing information was analyzed using software: comparing the sequence result of the ctDNA with the genome of a normal cell, and screening out a mutation site;

3) taking the mutated amino acid site as the center, extending 8 amino acids to both sides, and taking the polypeptide of 17 amino acids as a potential antigen epitope;

4) the IC50 of potential epitopes was analyzed using prediction software (recommendation software: NetMHCpan 3.0, PickPocket, and Artificial Neural Networks (ANN)), and if IC50 < 1000nM, the potential epitope is considered to be an epitope.

2. Polypeptide attachment

1) The software is used for analyzing the IC50 at the joint after any epitope is connected pairwise, and the IC50 is considered as weak immunogenicity when the IC50 is more than or equal to 1000nM and can be connected; the vaccine is considered to be strong immunogenicity when the IC50 is less than 1000nM and can not be connected (the calculation result of IC50 of 3 prediction software is considered here, the vaccine can be considered to be weak immunogenicity when the IC50 calculated by more than 2 software is more than or equal to 1000nM, and can be considered to be strong immunogenicity when the IC50 calculated by more than 2 software is less than 1000 nM);

2) according to the results, the epitopes are connected together, and the IC50 at the joint is higher than the IC50 of the epitopes on both sides (namely, the joint avoids generating strong binding antigen as much as possible); if necessary, the weak immunogenic peptide is used as a connecting peptide to separate the strong immunogenic peptide; or adding patient's own amino acids to the linker to reduce the likelihood of producing a strong antigen.

3. Synthetic polypeptides

if the nucleic acid sequence after the completion of the ligation is short (< 100bp), the amino acid sequence can be properly repeated, but when the sequence is reduced to a gene sequence, the occurrence of inverted repeat, direct repeat and mirror repeat sequences in the gene sequence should be avoided as much as possible

2) The gene sequence of the epitope peptide was synthesized by a solid phase synthesis method (synthesized by a technical service company).

4. Construction of an antigenic epitope peptide-expressing MVA Virus

1) Constructing a shuttle plasmid: cloning the synthesized epitope peptide gene sequence into pIIdHR-P7.5 plasmid;

2) construction of recombinant MVA viruses: CEF cells (chick embryo fibroblasts) or BHK-21 cells (hamster kidney fibroblasts) grow in a single layer to 70% -90% coverage rate, MVA is infected and shuttle plasmids are transfected in sequence, and the single layer cells are obtained by culturing; freezing and thawing the monolayer cells, and crushing to obtain an rMVA solution; inoculating the rMVA solution to RK-13 cells (rabbit kidney cells) for culture to obtain rMVA infected RK-13 aggregation sites, and selecting the aggregation sites to screen and purify to obtain the required rMVA; removing wtMVA in the rMVA, culturing the rMVA in a CEF or BHK-21 cell monolayer, screening the rMVA without a selection gene K1L, purifying to obtain rMVA-epitope peptide, and performing amplification separation to obtain the rMVA-epitope peptide;

5. transfection of Antigen Presenting Cells (APC) and Co-culture with PBMC

2) antigen Presenting Cells (APC) at 1-10X 10⁶Laying the solution in a 6-24 pore plate;

3) infecting APC with rMVA virus expressing antigen polypeptide at MOI of 0.1-2, after infecting for 2-24h, adding 0.5-2mL of 1-20% AIM V culture medium containing 100-2000IU/mL IL-2, 10-100ng/mL hTNF-alpha, 1000-5000IU/mL IL-6 and 10-100ng/mL IL-1 beta into each well, and continuing culturing for 2-48 h;

4) APC completed by the treatment are collected, and APC: PBMC ═ 1: 5-20, PBMC of about 5X 10⁷Adding 50mL of OKM100 culture medium into a T75 cell culture flask, and placing the flask into a cell culture box at 30-37 ℃ for 14 days to obtain MFF scheme effector cells.

6. Screening for effective accurate polypeptides

The polypeptide is used as an antigen to directly stimulate effector cells to screen accurate polypeptide:

1) centrifuging to collect the above MFF protocol cells, centrifuging at 1500rpm for 5min to collect T cells, adding 10mL PBS to resuspend the cells and counting, centrifuging at 1500rpm for 5min, collecting T cells, resuspending with 1640+10% FBS +200U/mL IL2, and adjusting the count to 1 × 10⁶cells/mL；

2) The T cells were plated on 96-well flat-bottom plates using a line gun at 200. mu.L/well and 2X 10 cells/well⁵cells; then respectively adding 10 mu L of 1mg/mL mutant polypeptide with the final concentration of 50 mu g/mL, and arranging 3 compound holes on each polypeptide;

3) setting a positive control: t cells +100ng/mL OKT 3; negative control: 1640+10% FBS +200U/mL IL 2; two T cell controls were used as background release detection, first T cell addition, and last T cell addition, respectively; taking the difference of two background releases as a system error;

4)37℃、5％CO₂after 24h of stimulation, centrifugation is carried out at 1500rpm for 10min, and 140 microliter of supernatant is transferred to a new 96-well plate;

5) centrifuging the 96-well plate at 1500rpm for 10min, and taking a sample for ELISA detection (or storing the sample at-80 ℃);

ELISA System for detection of IFN-. gamma.:

1) currently, the ELISA kits tested for detecting IFN-. gamma.are Biolegend: LEGEND MAX Human IFN-. gamma.ELISA Kit with Pre-coated Plates (cat # 430107) and daceae: the Human IFN-gamma ELISA Kit (cat # DKW12-1000-096) is strictly operated according to the manufacturer's instructions;

2) ELISA manual plate packing system (15 plates): human IFN-gamma DuoSet 15plate (cat # DY 285B). times.1, DuoSet ELISA Ancillary Reagent Kit 2 (cat # DY 008). times.3;

the accurate polypeptide evaluation standard is as follows:

1) if the positive control and the negative control are normal, the data is credible;

2) the polypeptide of the experimental group which is obviously higher than that of the negative control group is the effective accurate polypeptide.

7. Using the screened precise polypeptide to secondarily impact T cells

1) Culturing PBMC in step 5 to day 2-14, taking 2 × 10⁷Adding the accurate polypeptide with the final concentration of 10-100 mug/mL into the effector cells, and impacting for 1-4 h;

2) after 4h of impact, the plates were transferred to a 6-well plate of OKM25 prepacked plates or T25cm²Culture flask supplemented with OKM100+ 12% FBS, 5% CO at 37 deg.C₂Culturing, transferring to T75 flask according to cell growth condition, and maintaining cell density at 1 × 10⁶cells/mL；

3) When the cells enter a T175 culture bottle, the culture medium is OKM200+ 5% FBS, and the cells are cultured for 10-14 days to obtain T cells obtained by secondary impact of accurate polypeptide, namely MRFF cells;

8. culture and isolation of mutant antigen-specific killer T cells

1) Directly taking the screened precise polypeptide as an antigen for stimulation, stimulating the MRFF cells obtained in the step 7 for 12-72 hours for later use;

2) staining the stimulated T cells with CD8, CD137 and IFN-gamma, sorting the cells by a flow cytometer, and selecting CD8+ CD137+ or CD8+ IFN-gamma + cells;

9. TCR frequency detection and high-frequency TCR cloning of CD8+ T cells

1) Extracting the genome of the sorted CD8+ CD137+ or CD8+ IFN-gamma + cells, detecting TCR frequency, and determining a high-frequency TCR sequence;

2) extracting mRNA of the sorted cells, performing reverse transcription to obtain secondary DNA, designing a primer according to a sequence of the high-frequency TCR, and amplifying to obtain a TCR gene;

3) constructing a TCR gene expression vector and packaging viruses;

10. construction of TCR-T cells

1) Recovering PBMCs, and sorting CD8+ cells by magnetic beads for later use;

2) carrying out gene knockout on the original TCR of the CD8+ T cell by using a CRISPR technology, and transferring the original TCR into a constructed TCR expression vector when detecting that the TCR is not expressed;

3) and carrying out amplification culture on the CD8+ T cells with the transferred TCR gene until 10-21 days, and then harvesting the TCR-T cells.

11. Blocking of immunosuppressive signals

1) Immunosuppressive signaling molecules include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4(CD 244);

2) centrifuging at 1000rpm for 5min, and collecting cultured TCR-T cells;

3) washed once with PBS, centrifuged at 1000rpm for 5min, resuspended TCR-T in OKM-200+ 5% FBS and adjusted to 1X 10⁷/mL；

4) Adding single drug (such as PD1/PDL1) of inhibitory signal molecules to the mixture to obtain the MRFFT2 cell, wherein the final concentration is 50-200 mug/mL, preferably 150 mug/mL, and the mixture is sealed at 0-37 ℃ for 1-4h, preferably 1h at 37 ℃.

12. Construction of specific antigen expression target cell and tumor model survival experiment

1) Constructing a lentiviral vector capable of expressing the screened precise polypeptide (specific antigen).

2) Packaging the specific antigen expression lentivirus vector into lentivirus particles, infecting tumor cells with appropriate HLA match, stably over-expressing the specific antigen, and detecting the expression level and the expression intensity by flow.

3) Stable overexpressionThe tumor cell line of the specific antigen peptide is inoculated to NGS mice to make ectopic tumor-bearing animal models. Will be 5X 10⁵Tumor cells expressing specific antigens were suspended in 100. mu.l of physiological saline and injected subcutaneously into the right flank of 30 NSG mice, respectively, and the mice were numbered.

4) Growth of tumor to 100-120mm³The cells were returned from the left and right groups, and the animal model was randomly divided into three groups of 5-6 mice each, one group given placebo saline, and one group given 1X 10T cells (control group) without any genetic manipulation according to the tumor volume⁷One group was given MRFFT2 cells at 1X 10⁷And 7 days after the first injection of the cells, performing the second injection, 7 days after the third injection of the cells, continuously observing for 60 days, counting survival data, and drawing a survival curve.

And (3) test results:

1. mutation site and epitope prediction

Table 1 shows the results of prediction of mutation sites and epitopes detected by sequencing;

TABLE 1 epitope prediction

2. MVA virus transfection APC efficiency assay

4) detection of GFP Positive proportion in APC Using flow cytometry

3. MFF cell typing assay

After the MFF protocol cell culture was completed, CD4+ and CD8+ cells were typed and the results are shown in fig. 2: CD8+ T cells were 68% and CD4+ T cells were 9.45%.

4. Screening for precision Polypeptides by MFF cells

The cultured T cells were stimulated with 12 polypeptides, respectively, and effective polypeptides were detected by detecting IFN-. gamma.secretion, as shown in FIG. 3: the release amount of IFN-gamma caused by the polypeptide No. 3 is greater than that of the negative control group, and the polypeptide belongs to effective and accurate polypeptide.

5. Identification and sorting of T cells specific for precision polypeptides

The MFF protocol cells were stimulated with the selected polypeptide No. 3, and the proportion of T cells specific for the precise polypeptide was detected by flow assay, with the results shown in fig. 4, in black boxes as specific T cells: the proportion of cells releasing IFN- γ by MRFF protocol cells, polypeptide No. 3, was significantly higher than cells without stimulation (control), indicating that MRFF protocol, can yield T cells specific for the precise polypeptide; simultaneously sorting CD8+ IFN-gamma + cells (in black box) by flow cytometry;

6. identification and cloning of high frequency TCR

Extracting genome of the cells obtained by sorting, sequencing TCR, and displaying the distribution condition of TCR as shown in figure 5 (the first 22 of high-frequency distribution), wherein the distribution frequency of TCR1 and TCR17 is higher, which shows that the TCR is closely related to the mutant antigen, and the TCR is amplified according to the TCR sequence to construct a lentivirus expression vector.

TABLE 2 sequence case of CDR3 of TCR beta chain

Known TCR- α:

amino acid sequence:

MMKSLRVLLV ILWLQLSWVW SQQKEVEQNS GPLSVPEGAI ASLNCTYSDR

GSQSFFWYRQ YSGKSPELIM FIYSNGDKED GRFTAQLNKA SQYVSLLIRD SQPSDSATYL CAVNFGGGKL IFGQGTELSV KPN

the base sequence:

known TCR-. beta.s:

amino acids:

MRIRLLCCVA FSLLWAGPVI AGITQAPTSQ ILAAGRRMTL RCTQDMRHNA

MYWYRQDLGL GLRLIHYSNT AGTTGKGEVP DGYSVSRANT DDFPLTLASA

VPSQTSVYFC ASSLSFGTEA FFGQGTRLTV V

the horizontal line is the CDR3 sequence, the sequence to be replaced

TCR-. beta.after replacement:

the horizontal line is the replaced CDR3 sequence

7. Detection of the original TCR knockdown efficiency

The original TCR on PBMC was knocked out using CRISPR technique, and the results are shown in figure 6: the expression of the original TCR can be effectively reduced, at which point transfection of the lentivirus expressing the specific TCR can be performed.

8. Detection of specific TCR expression

PBMCs were transfected with lentiviruses packaging specific TCRs, and the efficiency of TCR expression was measured by flow at day 7, with the results shown in figure 7: the constructed TCR can be normally expressed, and the cell proportion of the TCR + is 25.1 percent

9. Blockade of immunosuppressive signals

When 150. mu.g/mL of fluorescently labeled monoclonal antibody Keytruda was added to the PBS buffer system, the binding was as shown in FIG. 8, and 90% of the cells were effectively blocked.

10. Killing effect of MRFFT2 cell on target cell

The control cells and the MRFFT2 cells were used to detect the killing efficiency of the target cells from the mutant epitope, the effective-to-target ratio was set to 40:1, and the untreated cells were used as the control (Mock), and the results are shown in fig. 9, in which the MRFFT2 cells had stronger killing effect on the target cells than the control group.

11. Detection of cytokine release by MRFFT2 cells

When tumor cells and effector cells are co-cultured, because the effector cells can recognize mutant antigens on the tumor cells, a series of cytokines can be generated, IFN-gamma is one of the most main cytokines in the anti-tumor effect, FIG. 10 shows that when MRFFT2 cells and tumor cells are co-cultured, the cell ratio is 1:1, the released IFN-gamma is detected, and the result shows that: MRFFT2 cells produced a larger amount of IFN-. gamma.after co-culture with tumor cells than IFN-. gamma.produced by effector cells themselves (T cells only), consistent with the results of the killing experiments, suggesting that: the T cells expressing specific TCR, combined with the knockout of inhibitory targets, can more effectively improve the anti-tumor capacity.

The specific antigen expression tumor target cell line is successfully constructed, a tumor-bearing animal model is established, and the result shows that (figure 11) MRFFT2 cells have a significant influence on the survival improvement of tumor-bearing mice.

13. Clinical cases:

for a male: 60 years old

And (3) disease diagnosis: poorly differentiated adenocarcinoma of both lungs;

the first course of treatment: MRFFT2 cells, 1X 10 in number, once a month ⁹2 times for each cell;

the second course of treatment: MRFFT2 cells were administered once a half year at a number of 1X 10⁹2 times for each cell;

after the administration, the medicine survives for 20 months without progress;

other cases are as follows:

patient numbering	Disease diagnosis	Progression free survival time
			1	Intrahepatic bile duct cancer	2016.5-Up to now
2	Ovarian cancer	2016.5-Up to now
			3	Ovarian cancer	2016.7-Up to now
4	Gastric adenocarcinoma liver metastasis	2016.8-Up to now
			5	Stomach cancer	2016.8-Up to now

Note: the term "to date" means "the day before the application date".

Claims

1. A method for constructing MRFFT2 cells, which is characterized by comprising the following steps:

1) screening out a mutation site by sequencing a tumor cell, taking the mutated amino acid site as a center, extending 8 amino acids to two sides respectively, taking the 17-amino acid polypeptide as a potential epitope, analyzing the IC50 of the potential epitope by using prediction software, and considering the potential epitope as the epitope if the IC50 is less than 1000 nM;

2) analyzing IC50 at joints after any epitope is connected pairwise by using the software, considering weak immunogenicity when IC50 is more than or equal to 1000nM, connecting the epitopes together by using the joints with the weak immunogenicity, and synthesizing the gene sequence of the connected mutant polypeptide;

3) constructing a MVA virus vector expressing the mutant polypeptide, and packaging the MVA virus;

4) transfecting antigen presenting cells with the MVA virus and co-culturing with PBMC to obtain MFF cells;

5) the mutant polypeptide is used as an antigen to stimulate the MFF cells, and effective and accurate polypeptide is screened out by detecting the secretion of IFN-gamma; the precision polypeptide evaluation standard is as follows: setting a positive control: t cells +100ng/mL OKT 3; negative control: t cells +1640+10% FBS +200U/mL IL 2; if the positive control and the negative control are normal, the data is credible; the release amount of IFN-gamma in the experimental group is obviously larger than that of IFN-gamma in the negative control group, and the IFN-gamma is effective accurate polypeptide;

6) stimulating the MFF cells by using the accurate polypeptide as an antigen, dyeing CD8, CD137 and IFN-gamma of the stimulated cells, sorting the cells by using a flow cytometer, screening specific cells capable of identifying the accurate polypeptide, sequencing and obtaining a high-frequency TCR gene of the specific cells;

7) knocking out the original TCR gene in the peripheral blood T cell by using a CRISPR technology, and transferring the high-frequency TCR gene to obtain a TCR-T cell;

8) and (3) blocking the cell surface immunosuppressive signal molecules in vitro by an antibody medicament to obtain the MRFFT2 cell.

2. The method for constructing MRFFT2 cells according to claim 1, wherein the sequencing in step 1 is ctDNA sequencing using human peripheral blood of tumor patients or commercially available engineered cell lines, or whole exon sequencing using tumor tissues of patients.

3. The method of claim 2, wherein the commercially available engineered cell line comprises: h1299, H226, H358, H1563, H2228, A549, Renca, LLC mouse Lewis lung cancer cell, CRL-6323B16F1, CRL-25394T 1, U14 mouse cervical cancer cell, BV-2 mouse glioma cell, G422 mouse glioma cell.

4. The method of constructing MRFFT2 cell according to claim 1, wherein the IC50 of the linker connecting the epitopes in step 2 is higher than the IC50 of the flanking epitopes.

5. The method of constructing MRFFT2 cells according to claim 1, wherein the ratio of antigen presenting cells to PBMCs in step 4 is 1: 5-20 of the above-mentioned materials.

6. The method of claim 1, wherein the MRFFT2 cells are selected from CD8+ CD137+ or CD8+ IFN- γ + cells by sorting with a flow cytometer in step 6.

7. The method of constructing the MRFFT2 cell of claim 1, wherein the antigen presenting cell comprises: peripheral blood mononuclear cells, dendritic cells, neutrophils, B lymphocytes, macrophages.

8. The method of claim 1, wherein the cell surface immunosuppressive signaling molecule comprises: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4(CD 244).