CN110408657B

CN110408657B - Construction method of AFFT1 cells

Info

Publication number: CN110408657B
Application number: CN201910438763.7A
Authority: CN
Inventors: 焦顺昌; 张嵘; 周子珊; 解佳森; 陈小彬; 陈红利
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: 2021-03-16
Anticipated expiration: 2039-05-24
Also published as: CN110408657A

Abstract

The application relates to a preparation method of AFFT1 cells, wherein RFF cells are modified by using a TCR-T technical principle, and the modified T cells are subjected to immunosuppression target knockout by using a gene editing technology, so that specific killer T cells are accurately protected from in vivo inhibition, and the lethality of the T cells to tumor cells is improved.

Description

Construction method of AFFT1 cells

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an AFFT1 cell and a preparation method thereof.

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 of them have not performed in vitro co-culture and in vitro amplification despite the in vitro antigen-impact, and have difficulty in achieving desired effects because a thin specific cell directly faces a complicated tumor microenvironment. 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-protective technology of T cells, so that a small number of specific T cells directly face a powerful immune microenvironment.

Disclosure of Invention

The RFF cells are modified by the TCR-T technical principle, and the modified T cells are knocked out of immunosuppressive targets by the genetic editing technology, so that the specific killer T cells are accurately protected from in vivo inhibition, and the lethality of the T cells to tumor cells is improved.

For the interpretation of terminology:

a: immortalized DC technology

FF: polypeptide mixing technology

R: accurate polypeptide secondary impact technology

T: TCR-T technology

1: target spot knockout protection technology

AFFT1 cell engineering protocol

1. Whole exon sequencing

1) Sequencing of whole exons using human peripheral blood for ctDNA detection 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.);

2) sequencing information was analyzed using software: on one hand, obtaining MHC type information; on the other hand, comparing the sequencing result of the whole exon with the genome of a normal cell, and screening out a mutation site;

2. epitope prediction

1) Taking the mutated amino acid site as the center, extending 10 amino acids to both sides, and taking the 21 amino acid polypeptide as the potential antigen epitope;

2) the IC50 of potential epitopes was analyzed using prediction software (recommendation software: NetMHCpan 3.0, PickPocket, and Artificial Neural Networks (ANN), and if IC50 is less than 1000nM, the potential epitope is considered as the "epitope";

3. synthesis of mutant Polypeptides

The polypeptide is synthesized by a technical service company;

1)

4. immortalized DC

1) Draw 100ml of peripheral blood

2) Ficol density gradient centrifugation PBMC

3) The kit for separating the dendritic cells of the Meitian whirlwind separates the dendritic cells and suspends the dendritic cells in a culture medium.

4) Infecting the separated dendritic cells with TAX-GFP slow virus, culturing at 37 deg.C in incubator, and observing

5) After the cells are cloned and grown, selecting and cloning in a 96-well plate to be respectively cultured

6) Monoclonal phenotypic analysis

7) Selecting an ideal clone as an immortalized DC;

5. immortalized DC-loaded mutant polypeptides

1) Preparing a polypeptide solution: preparing the mutant polypeptides synthesized in the step 3, wherein the final concentration of each polypeptide is 10-100 mug/mL, preferably 50 mug/mL, for later use;

2) centrifugally collecting the obtained immortalized DC, resuspending the immortalized DC by using a prepared polypeptide solution, and placing the immortalized DC in a cell culture plate for carrying out polypeptide loading;

3)37℃5％CO₂impacting for 1-4 h, preferably 4h, for standby;

6. co-incubation of mutant polypeptide-loaded DCs with PBMCs

1) Stimulation factor OKM-25 pre-plated, 40. mu.L of OKM-25+4mL PBS, 2 mL/dish (4.5 cm)²) Room temperature 4h, 4 ℃ for standby;

2) the mutant polypeptide-loaded DCs were compared to previously extracted PBMCs at a 1: 1-1: 50, preferably 1: 10, and transferring to a cell culture plate or a culture bottle pre-paved with OMK 25;

3) shaking evenly, 5% CO at 37 ℃₂Culturing, and recording as day 0;

4) observing the condition of the co-cultured cells, on day 5, according to the cell density, the co-cultured cells were transferred to a large flask supplemented with fresh medium OKM-100+ 12% FBS, 20mL at 75cm²In a culture bottle;

5) on the 7 th day of co-culture, 20mL of fresh OKM-100+ 12% FBS was added;

6) co-culture on day 10 in OKM-200+ 5% FBS medium, the co-cultured cells were cultured from 75cm²The flask was transferred to 175cm²In a big bottle; the specific transfer method comprises the following steps: beating 25mL of culture solution OKM-200+ 5% FBS, transferring into a large bottle, and repeating for 2 times; make up to 200mL with medium OKM-200+ 5% FBS.

7) After culturing for 14-21 days, AFF scheme cells can be obtained.

7. The polypeptide is used as an antigen to directly stimulate T cells to screen accurate polypeptide:

1) centrifuging to collect the obtained AFF program 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 counting to 1 × 10⁶cells/mL；

2) The T-cells were dispensed into 96-well flat-bottom plates with a line gun at 200. mu.L/well and a cell count of 2X 10⁵cells; then respectively adding 10 μ L of 1mg/mL mutant polypeptide synthesized in step 3 to a final concentration of 50 μ g/mL, each polypeptideSetting 3 multiple holes;

3) setting a positive control: t-cells +100ng/mL OKT 3; negative control: 1640+10% FBS +200U/mL IL 2; two T-cells controls are used as background release detection, namely the first T-cell addition and the last T-cell addition; 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 ℃);

8. 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 is used as an antigen, and T-cells is used as a baseline;

3) each group of experiments comprises two baselines, a high baseline and a low baseline, the difference of the two baselines is a system error, and when data are analyzed, detection values > low baseline, > high baseline and > high baseline + system error are respectively marked; the detection value is higher than the high baseline and the system error is the effective accurate polypeptide;

9. preparation of AFF cells from screened precision polypeptides

1) Preparing the precision polypeptide AFF cells by methods 4, 5 and 6;

10. culturing and separating the mutant antigen specific killer T cells:

1) stimulating the AFF cells obtained in the step 9 by directly taking the polypeptide as an antigen for stimulation, and standing by after stimulating for 12-72 hours;

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

CD8+ T cell TCR frequency detection and cloning of high frequency TCRs:

1) sorting to obtain cells, and immediately extracting a genome;

2) carrying out TCR sequencing analysis on the genome, and determining a high-frequency TCR sequence according to TCR distribution frequency;

3) extracting mRNA of PBMC, carrying out 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;

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

12. construction of an immunosuppressive Signal molecule knockout CRISPR vector

1) PBMC surface immunosuppressive signal molecules include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4(CD244)

2) Analyzing exons of inhibitory signal molecules, finding out a CDS (coding sequence) region of mRNA (messenger ribonucleic acid) of the gene on pubmed, and predicting knockout targets of each exon respectively;

3) designing a forward primer and a reverse primer required for synthesizing the sgRNA, and carrying out amplification reaction on the forward primer and the reverse primer 1: 1, treating at 95 ℃ for 5-60 min after mixing, and then slowly cooling to form a DNA sequence of the sgRNA;

4) performing double enzyme digestion on a CRISPR lentiviral expression vector, connecting the CRISPR lentiviral expression vector with double-stranded DNA corresponding to sgRNA, transferring the double-stranded DNA into a clone competent cell, and after 12h, selecting a single clone for sequencing, and reserving the clone with correct sequencing;

5) extracting CRISPR lentiviral vector plasmids carrying DNA sequences corresponding to the sgRNAs, and carrying out virus packaging;

13. construction of TCR knockout CRISPR vectors

1) Finding out CDS region of mRNA of TCR gene on pubmed, analyzing conservation region of TCR, and predicting knockout target of conservation region;

2) repeating the steps 3) -5) in the method 12 to complete the construction of the TCR knockout vector and the virus packaging;

14. constructing TCR-T of knockout immunosuppressive signal molecule:

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

2) infecting the CD8+ T cells screened in step 10 with the virus obtained in methods 12 and 13, and simultaneously knocking out the original TCR and the immunosuppressive signaling molecule;

3) after infection, CD8+ T cells are cultured in a culture medium for 0-5 days, preferably 3 days, and then transferred into the constructed TCR expression vector;

4) infected CD8+ T cells were resuspended in OKM100+ 12% FBS and plated on pre-plating with stimulation factor OKM-25, recorded as day 0;

5) the cells were observed for cell status and cell density, and on day 5, the co-cultured cells were transferred to 75cm²In a bottle, fresh culture solution OKM-100+ 12% FBS is supplemented;

6) cells were removed from 75cm²Transfer to 175cm in the bottle²A large bottle, wherein the culture solution is OKM-200+ 5% FBS;

7) and when the cells are cultured for 14-21 days, the TCR-T cells of which the immunosuppressive signal molecules are knocked out, namely AFFT1 cells, can be harvested.

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

1) Constructing a lentivirus 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) The tumor cell line stably over-expressing the specific antigen peptide is inoculated to NGS mice to be used as an ectopic tumor-bearing animal model. 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 to AFFT1 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.

The invention has the advantages of

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 PBMCs 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 AFFT1 scheme is more than 70 percent;

the AFFT1 cell knocks out immunosuppressive targets such as PD1, CTLA4, TIM3 and LAG3, so that the killing capacity on tumors is not limited, and the killing efficiency is higher.

Drawings

FIG. 1 microscopic examination of DC morphology

FIG. 2 efficiency of DC Loading of Polypeptides

FIG. 3AFF cell typing assay

FIG. 4 screening for precision Polypeptides

FIG. 5 flow assay of specific T cell ratios

FIG. 6TCR distribution frequency

FIG. 7 knock-out of inhibitory targets

FIG. 8 detection of knockout efficiency of the original TCR

FIG. 9 expression efficiency of specific TCR

FIG. 10 flow assay killing efficiency

FIG. 11ELISA detection of cytokine IFN-. gamma.Release

FIG. 12 animal tumor-bearing model survival curves

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope thereof

1. Whole exon sequencing

1) Taking peripheral blood of a lung cancer patient, and carrying out ctDNA sequencing and HLA typing detection;

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;

2. epitope prediction

2) the IC50 of potential epitopes was analyzed using prediction software (recommendation software: NetMHCpan 3.0, PickPocket, and Artificial Neural Networks (ANN)), and if IC50 is less than 1000nM, the potential epitope is considered as an "epitope";

3. synthetic polypeptides

The epitope peptide synthesis method adopts a polypeptide solid phase synthesis method (synthesized by technical service company)

4. Immortalized DC

1) Draw 100ml of peripheral blood

2) Ficol density gradient centrifugation PBMC

6) Monoclonal phenotypic analysis

7) Selecting an ideal clone as an immortalized DC;

5. immortalized DC-loaded mutant polypeptides

2) centrifugally collecting the obtained immortalized DC, resuspending the immortalized DC by using a prepared polypeptide solution, and placing the immortalized DC into a cell culture plate for loading the polypeptide;

3)37℃5％CO₂impacting for 1-4 h, preferably 4h, for standby;

6. co-incubation of mutant polypeptide-loaded DCs with PBMCs

2) the mutant polypeptide-loaded DCs were incubated with PBMCs at a 1: 50-1: 500, preferably 1: 100, and transferring the cells into a cell culture plate or a culture bottle which is pre-paved with OMK 25;

3) shaking evenly, 5% CO at 37 ℃₂Cultivation, mark asDay 0;

5) on the 7 th day of co-culture, 20mL of fresh OKM-100+ 12% FBS was added;

6) co-culture on day 10 in OKM-200+ 5% FBS medium, the co-cultured cells were cultured from 75cm²The one in the flask was transferred to 175cm²In a big bottle; the transfer method comprises the following steps: beating 25mL of culture solution OKM-200+ 5% FBS, transferring into a large bottle, and repeating for 2 times; make up to 200mL with medium OKM-200+ 5% FBS.

8) After culturing for 14-21 days, AFF scheme cells can be obtained.

2) The T-cells were dispensed into 96-well flat-bottom plates with a line gun at 200. mu.L/well and a cell count of 2X 10⁵cells; respectively adding 10 mu L of 1mg/mL mutant polypeptide synthesized in the step 3, wherein the final concentration is 50 mu g/mL, and each polypeptide is provided with 3 multiple holes;

8. the accurate polypeptide evaluation standard is as follows:

2) the polypeptide is used as an antigen, and T-cells is used as a baseline;

9. preparation of AFF cells from screened precision polypeptides

1) Preparing the precision polypeptide AFF cells by methods 4, 5 and 6;

10. culturing and separating the mutant antigen specific killer T cells:

1) directly taking the screened precise polypeptide as an antigen for stimulation, stimulating the AFF cell obtained in the step 9 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;

CD8+ T cell TCR frequency detection and cloning of high frequency TCRs:

1) sorting to obtain cells, and immediately extracting a genome;

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

12. construction of an immunosuppressive Signal molecule knockout CRISPR vector

13. construction of TCR knockout CRISPR vectors

14. constructing TCR-T of knockout immunosuppressive signal molecule:

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

2) infecting the CD8+ T cells obtained in step 10 with the virus obtained in methods 12 and 13, and simultaneously performing the knockout of the original TCR and the knockout of the immunosuppressive signaling molecule;

5) observing cell condition and cell density, and on day 5, transferring the co-cultured cells to a large culture flask, and supplementing fresh culture solution OKM-100+ 12% FBS;

6) cells were removed from 75cm²Transfer to 175cm in the bottle²The post-large bottle culture solution is OKM-200+ 5% FBS;

4) The cells were returned in groups when tumors grew to around 100-120mm3, 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 without any genetic manipulation (control group) according to the tumor volume⁷One group was given to AFFT1 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.

Results of the experiment

1. Mutation site and epitope prediction

Table 1 shows the sequencing-detected mutation sites and epitope prediction results, with the mutated amino acids underlined;

TABLE 1 epitope prediction

2. Morphological Observation of immortalized DCs

After induction of DC maturation, morphology was observed microscopically and visible as distinct dendritic cells (fig. 1);

detection of DC antigen Loading efficiency

Synthesizing a predicted mutant antigen according to the table 1, labeling biotin, and detecting the distribution condition of the biotin on the cell surface by using PE-labeled affinity streptomycin after the antigen is loaded with DC so as to detect the efficiency of extracting polypeptide antigen by the DC; the results are shown in FIG. 2: the dark color is the detection result without loading the labeled polypeptide, the light color is the detection result with loading the biotin polypeptide, and the result shows that: the loading efficiency of DC is 99.4%;

AFF protocol cell typing assay

After the AFF protocol cell culture was completed, typing of CD4+, CD8+, NK and NKT cells was examined, and the results are shown in FIG. 3: 83.2% for CD8+ T cells, 19.16% for CD4+ T cells, 14.2% for NKT cells, 2.46% for NKT cells;

5. screening of precise polypeptides by AFF cells

The results of stimulating cultured T cells with 10 polypeptides, respectively, and detecting effective polypeptides by detecting IFN- γ secretion are shown in FIG. 4: the release amount of IFN-gamma caused by the polypeptide No. 6 is greater than a high baseline and a system error, and the polypeptide belongs to effective and accurate polypeptide;

6. identification and sorting of T cells specific for precision polypeptides

The AFF protocol cells were stimulated with the selected polypeptide No. 6, and the ratio of T cells specific to the precise polypeptide was detected by flow assay, the result is shown in FIG. 5, in black frame (P5) as specific T cells: the AFF protocol cells, the ratio of cells releasing IFN- γ caused by polypeptide # 6, was significantly higher than the cells without stimulation (control), indicating that specific T cells to the precise polypeptide could be obtained with the AFF protocol; simultaneously sorting CD8+ IFN-gamma + cells (in black box) by flow cytometry;

7. identification and cloning of high frequency TCR

Extracting genome of the cells obtained by sorting, sequencing TCR, wherein the distribution condition of TCR is shown in figure 6 (the first 20 of high-frequency distribution), and the distribution frequency of TCR3 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:

the base sequence:

known TCR-. beta.s:

amino acids:

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

TCR-. beta.after replacement:

the horizontal line is the replaced CDR3 sequence

8. Detection of inhibitory target knockout efficiency

The inhibitory target PD-1 on the PBMC is knocked out by using the CRISPR technology, the sgRNA sequence is shown in Table 3, and the knocking-out efficiency result of the inhibitory target is shown in FIG. 7: the knockout efficiency of sgRNA1 is highest, and the expression of inhibitory signal molecules PD-1 can be effectively blocked; sgRNA preferably sgRNA1, sgRNA2, sgRNA3 and sgRNA 4; the method can also be used for knocking out inhibitory signal molecules such as Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4(CD244), etc.;

table 3 inhibitory target sgRNA sequences

9. Detection of inhibitory target knockout efficiency

The inhibitory targets on PBMCs were knocked out using CRISPR technique, and the results are shown in figure 7: can effectively block the expression of inhibitory signal molecules;

10. 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 8: the expression of the original TCR can be effectively reduced, and at the moment, the transfection of expression specificity TCR slow virus can be carried out;

11. 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 9: the constructed TCR can be normally expressed, and the cell proportion of the TCR + is 76.5 percent

Killing of target cells by AFFT1 cells

The detection of the killing efficiency of the target cells derived from the mutant epitope is performed by using the AFF cells, the AFFT cells and the AFFT1 cells respectively, and untreated cells are used as a control (Mock), and the results are shown in FIG. 10, wherein compared with the control group, the AFF cells, the AFFT cells and the AFFT1 cells all have a certain killing effect on the target cells, and the killing efficiency (in a red frame) on tumors is AFFT1> AFFT cells > AFF cells; the T cells expressing the specific TCR are shown, and the killing efficiency of the tumor cells can be effectively improved by the closure of the inhibitory target;

detection of cytokine release by AFFT1 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. 11 shows that the detection of IFN-gamma released when the cells are co-cultured with the tumor cells in different culture modes shows that: compared with IFN-gamma produced by effector cells (T cells only), after the cells are co-cultured with tumor cells, AFF scheme cells, AFFT scheme cells and AFFT1 cells can produce a large amount of IFN-gamma, particularly AFFT and AFFT1 cells, because specific TCR (AFFT) is expressed, and inhibitory signals are knocked out (AFFT1), the effector cells can release more IFN-gamma, and the result is consistent with the result of killing experiments and shows that: the T cells expressing specific TCR can effectively improve the anti-tumor capacity by combining with the knockout of inhibitory targets;

14. construction of specific antigen expression target cell and tumor model survival experiment

Successfully constructs a specific antigen expression tumor target cell line and establishes a tumor-bearing animal model, and the result shows that (figure 12) the AFFT1 cell has obvious influence on the survival improvement of tumor-bearing mice.

15. Clinical cases:

for a male: age 61

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

The first course of treatment: AFFT1 cells at 1X 10 cell counts per month ⁹2 times for each cell;

the second course of treatment: AFFT1 cells at 1X 10 cell counts per half year ⁹2 times for each cell;

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

other cases are as follows:

patient numbering	Disease diagnosis	CDR3 region sequences for high frequency TCR	Progression free survival time
				1	Liver inner containerCancer of the canal	CATSRGTVSYEQYF	2016.4-Up to now
2	Ovarian cancer	CASSQEGAFYGYTF	2016.5-Up to now
				3	Ovarian cancer	CASSIDHVSSSYNSPLHF	2017.4-Up to now
4	Gastric adenocarcinoma liver metastasis	CASSEGTESSYEQYF	2017.5-Up to now
				5	Stomach cancer	CASSIDGTATYEQYF	2017.11-Up to now
6	Lung cancer	CASSYLSETYEQYF	2017.8-Up to now
				7	Esophageal cancer	CASSSRLAGGTDTQYF	2018.1-Up to now
8	Adenocarcinoma of lung	CATSRDWLSNGNTEAFF	2018.3-Up to now
				9	Adenocarcinoma of lung	CATSIYSGETQYF	2018.3-Up to now
10	Stomach cancer	CASSITEGSPLHF	2018.4-Up to now

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

Claims

1. A preparation method of AFFT1 cells is characterized in that the preparation method of the AFFT1 cells comprises the following steps:

1) screening mutation sites: carrying out ctDNA exon sequencing on peripheral blood of a patient or carrying out whole exon sequencing on tumor tissues of the patient to screen out mutation sites;

2) synthesis of mutant polypeptides: performing epitope prediction according to the mutation site, and synthesizing mutant polypeptide;

the prediction of the antigen epitope takes a mutated amino acid site as a center, and extends 10 amino acids to two sides as a potential antigen epitope; analyzing the potential epitope IC50, and determining the potential epitope IC50 < 1000nM as the epitope;

3) obtaining AFF cells: infecting dendritic cells in peripheral blood by using TAX-GFP (T-activating fluorescent protein) lentivirus, selecting an ideal clone as an immortal DC, loading the mutant polypeptide, and incubating with PBMC (peripheral blood mononuclear cell) to obtain AFF (endothelial cell factor) cells;

4) obtaining the accurate polypeptide: stimulating the AFF cells by using the mutant polypeptide as an antigen, and screening to obtain an accurate polypeptide; the evaluation criteria of the precise polypeptide: 3) setting a positive control: t-cells +100ng/mL OKT 3; negative control: 1640+10% FBS +200U/mL IL 2; if the positive control and the negative control are normal, the data is credible; the polypeptide is used as an antigen, and T-cells is used as a baseline; each group of experiments comprises two baselines, namely a high baseline and a low baseline, and the difference between the two baselines is a system error; the detection value is higher than the high baseline and the system error is the effective accurate polypeptide;

5) obtaining AFF' cells: preparing AFF' cells by loading the DC cells with the precise polypeptide;

6) stimulating the AFF' cells by taking the precise polypeptide as an antigen, screening to obtain specific T cells capable of identifying the precise polypeptide, obtaining a TCR beta chain CDR3 region sequence of the specific T cells through sequencing, and replacing a known pair of TCR beta chain CDR3 regions with the CDR3 sequence;

7) obtaining AFFT1 cells: knocking out the original TCR gene and the surface immunosuppressive signal molecule PD-1 in the peripheral blood T cells of the patient, transferring the TCR gene which is obtained in the step 6) and can be specifically combined with the precise polypeptide, and preparing the AFFT1 cells.

2. The method of claim 1, wherein the patient's peripheral blood is replaced with a commercially available engineered cell line.

3. The method of claim 2, wherein the engineered cell lines are H1299, H226, H358, H1563, H2228, A549, Renca, LLC mouse Lewis lung carcinoma cells, CRL-6323B16F1, CRL-25394T 1, U14 mouse cervical carcinoma cells, BV-2 mouse glioma cells, or G422 mouse glioma cells.

4. The method of claim 1, wherein the method for knocking out the TCR gene and the surface immunosuppressive signal molecule of the peripheral blood cells of the patient is CRISPR technology.

5. The method of claim 1, wherein the surface immunosuppressive signaling molecule further comprises: tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4(CD244), TIGIT.