CN110157745B

CN110157745B - Construction method of HAFFT1 cell

Info

Publication number: CN110157745B
Application number: CN201910438772.6A
Authority: CN
Inventors: 焦顺昌; 张嵘; 张天赋; 周子珊; 陈小彬; 李营营; 彭刚
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: 2021-06-18
Anticipated expiration: 2039-05-24
Also published as: CN109136282A; CN110157745A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a construction method of HAFFT1 cells. The method modifies the cells stimulated by the small molecule modified polypeptide by the TCR-T technology, and then knocks out the immunosuppressive target of the modified T cells, thereby accurately protecting specific killer T cells from in vivo inhibition and improving the lethality of the T cells to tumor cells. The proportion of specific T cells (TCR +) for recognizing tumor antigens of the cells modified by the HAFFT1 scheme is more than 80 percent.

Description

Construction method of HAFFT1 cell

The technical field is as follows:

the invention belongs to the technical field of biology, and particularly relates to a construction method of HAFFT1 cells.

Background art:

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 use lentiviruses as vectors have been attempted for transfection delivery, they are not as safe and convenient as polypeptides. While direct stimulation by simply mixing polypeptides is simple and convenient, it is inefficient. The secondary stimulation of specific precision polypeptides 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-defense technology of T cells, so that a small number of specific T cells directly face a strong tumor microenvironment.

The invention content is as follows:

in order to solve the technical problems, the invention provides a construction method of HAFFT1 cells, which comprises the steps of modifying cells stimulated by small molecule modified polypeptide by a TCR-T technology, knocking out an immunosuppressive target of the modified T cells, accurately protecting specific killer T cells from in vivo inhibition, and improving the killing power of the T cells to tumor cells.

The construction method of the HAFFT1 cell comprises the following main steps: 1) extracting peripheral blood of a patient, sequencing ctDNA exons, or sequencing all exons by tumor tissues, screening out mutation sites, predicting antigen epitope, synthesizing mutant polypeptide and modifying small molecules; 2) preparing immortalized DCs by using peripheral blood, loading the mutant polypeptides modified by the small molecules, and incubating with PBMC to obtain HAFF cells; 3) stimulating the HAFF cells by using the mutant polypeptide modified by the small molecules as an antigen, and screening to obtain an accurate polypeptide; 4) carrying out co-incubation on the immortalized DC cells and PBMC (peripheral blood mononuclear cell) by using the accurate polypeptide to prepare HAFF' cells; 5) stimulating HAFF' cells by taking the accurate polypeptide as an antigen, screening to obtain specific T cells capable of identifying the accurate polypeptide, obtaining high-frequency TCR of the specific cells through sequencing, knocking out the original TCR, expressing the high-frequency TCR, and constructing TCR-T cells; 6) and knocking out inhibitory signal molecules of the TCR-T cells to prepare HAFFT1 cells.

The specific preparation steps of HAFFT1 cells are as follows:

1. whole exon sequencing

Using human peripheral blood to carry out ctDNA sequencing or using tumor tissues to carry out whole exon sequencing, comparing a sequencing result with a genome of a normal cell, and screening out a mutation site;

the peripheral blood can also be a commercial engineering cell line, such as 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 and the like, and the whole exon sequencing is carried out;

2. epitope prediction

(1) 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;

(2) analyzing the IC50 of the potential antigen epitope by using prediction software, and determining the potential antigen epitope with IC50 < 1000nM as the antigen epitope;

3. synthesis of polypeptides and modification

(1) Synthesizing the 'epitope' into mutant polypeptide;

(2) the small molecule (H4) and the polypeptide antigen (P) are modified in the following linkage mode (amino terminal to carboxyl terminal: N-C): H-P, P-H, H-P-H;

4. immortalized DC-loaded mutant polypeptides

(1) Infecting dendritic cells in peripheral blood by TAX-GFP lentivirus, and selecting an ideal clone as an immortalized DC;

(2) loading the immortal DC with small molecule modified antigen polypeptide;

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

Co-incubating the DC loaded with the mutant polypeptide with PBMC to obtain HAFF cells;

6. screening for accurate Polypeptides

Collecting T cells in HAFF cells, stimulating the T cells by using the small molecule modified mutant polypeptide obtained in the step 3, and screening the precise polypeptide by checking the secretion of IFN-gamma;

7. production of HAFF' cells from screened precision polypeptide

Repeating

steps

3, 4 and 5 by replacing the mutant polypeptide with the precise polypeptide to prepare the precise polypeptide HAFF' cell;

8. determination of specific cell high-frequency TCR and construction of expression vector

(1) Stimulating HAFF' cells by using the precise polypeptide modified by small molecules, staining the stimulated cells by CD8, CD137 and IFN-gamma, and selecting CD8+ CD137+, or CD8+ IFN-gamma + T cells; extracting a genome, sequencing and analyzing the TCR, and determining a high-frequency TCR sequence according to TCR distribution frequency;

(2) designing a primer according to the sequence of the high-frequency TCR, and amplifying to obtain a TCR gene; constructing a TCR gene expression vector and packaging viruses;

9. constructing a CRISPR vector for knocking out an immunosuppressive signal molecule, and packaging viruses;

10. constructing a CRISPR vector for knocking out TCR and packaging virus;

11. TCR engineering

Infecting the CD8+ T cells screened in the step 8 with the virus obtained in the

steps

9 and 10, and knocking out the original TCR and the immunosuppressive signal molecule; then transferring into the TCR expression vector constructed in the step 8; preparing HAFFT1 cells;

the inhibitory signal molecule may be PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160 or 2B4(CD244), TIGIT.

Has the advantages that:

1. the HAFFT1 cell provided by the invention takes a tumor antigen as 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 specific cells obtained by the invention 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 HAFFT1 scheme is more than 80 percent;

3. the HAFFT1 cell obtained by the invention knocks out immunosuppressive targets such as PD1, CTLA4, TIM3, LAG3 and the like, so that the killing capability to tumors is not limited, and the killing efficiency is higher.

Description of the drawings:

FIG. 1 microscopic examination of DC morphology;

FIG. 2 efficiency of DC loading of polypeptides;

FIG. 3 AFF cell typing assay;

FIG. 4 screening for a precision polypeptide;

FIG. 5 flow assay specific T cell ratios;

FIG. 6 TCR distribution frequency;

FIG. 7 knock-out of inhibitory targets;

FIG. 8 detection of knockout efficiency of an original TCR;

figure 9 expression efficiency of specific TCRs;

figure 10 LDH release detection killing efficiency;

FIG. 11 ELISA detection of cytokine IFN-. gamma.release;

FIG. 12 improvement in survival of tumor-bearing mice by HAFFT1 cells.

The specific implementation mode is as follows:

the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

For the interpretation of the terminology HAFFT1, where H: small molecule modified polypeptide antigen technology; a: immortalized DC technology; FF: mixed polypeptide stimulation techniques; t: TCR-T technology; 1: target spot knockout protection technology. HAFFT1 cells represent cells prepared by a combination of the above techniques.

Example 1

In this embodiment, a lung cancer patient is taken as an example, and a targeted HAFFT1 cell and a preparation method thereof are provided:

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

1) 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;

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

Entrusted technology service company synthesizes the 'antigen epitope' into mutant polypeptide;

4. small molecule modified polypeptide

1) The small molecule (H4) is linked to the polypeptide antigen (P) in the following manner (amino-terminus to carboxy-terminus: N-C): H-P, P-H, H-P-H;

2) culturing the T cells by a mixed polypeptide stimulation mode through the small molecule modified polypeptide H-P or P-H or H-P-H, wherein the proportion of the T cells specific to the antigen polypeptide in the obtained T cells is higher than that of the T cells cultured by the polypeptide antigen (P) through single impact;

5. immortalized DC

1) Extracting 100ml of peripheral blood of a patient;

2) performing Ficol density gradient centrifugation to separate PBMC;

3) separating dendritic cells by adopting a Meitian whirlwind dendritic cell separation kit, and suspending the dendritic cells in a culture medium;

4) infecting the separated dendritic cells with TAX-GFP lentivirus, performing static culture in an incubator at 37 ℃, and observing;

5) after the cells grow in a cloning way, selecting and cloning the cells in a 96-well plate and culturing the cells respectively;

6) monoclonal phenotypic analysis;

7) selecting an ideal clone as an immortalized DC;

6. the immortalized DC loads a mutant polypeptide modified by a small molecule;

1) preparing a polypeptide solution: preparing the mutant polypeptides modified in the step 4, 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;

7. 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 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 ℃₂Culturing, and recording as day 0;

4) the co-cultured cells were observed at day 5 according to the cell densityThe co-cultured cells were transferred to a large flask supplemented with fresh medium OKM-100+ 12% FBS, 20mL in 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 75cm²Transfer to 175cm in the bottle²In 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) Culturing for 14-21 days to obtain HAFF cell.

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

1) collecting the obtained HAFF cells by centrifugation, collecting T cells by centrifugation at 1500rpm for 5min, 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 plated on 96-well flat-bottom plates using a line gun at 200. mu.L/well and 2X 10 cells/well⁵cells; respectively adding 10 mu L of 1mg/mL of the small molecular modification mutation polypeptide in the step 4, wherein the final concentration is 50 mu g/mL, and each polypeptide is provided with 3 multiple holes;

3) setting a positive control: t cells +100ng/mL OKT 3; negative control: 1640+ 10% FBS +200U/mL IL2

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 deg.C);

9. the accurate polypeptide evaluation standard is as follows:

1) if the positive control and the negative control are normal, the data is credible, otherwise, the test is required to be carried out again;

2) the experimental group is obviously higher than the negative control group, and the effective accurate polypeptide is obtained;

10. production of HAFF' cells from screened precision polypeptide

1) Preparing a precise polypeptide HAFF' cell by steps 4, 5, 6 and 7;

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

1) directly stimulating the HAFF' cells obtained in the step 10 by using the screened accurate polypeptides as antigens, and stimulating 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 + T cells;

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

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 cDNA, designing a primer according to a sequence of the high-frequency TCR, and carrying out amplification to obtain a TCR gene;

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

13. 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(CD 244);

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;

14. 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 13 to complete the construction of the TCR knockout vector and the virus packaging;

15. 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 11 with the virus obtained in methods 13 and 14, and simultaneously performing the knockout of the original TCR and the knockout of the immunosuppressive signaling molecule;

3) after infection, CD8+ T cells are cultured in the culture medium for 0-5 days, preferably 3 days, and then transferred into the TCR expression vector constructed in the step 12;

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) 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;

7) culturing for 14-21 days to obtain TCR-T with immunosuppressive signal molecule knocked out to obtain HAFFT1 cell;

16. 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 lentiviral vector into lentiviral particles, infecting tumor cells with appropriate HLA match, stably over-expressing the specific antigen, and detecting the expression level and the expression intensity in a flow mode;

3) inoculating the tumor cell line stably over-expressing the specific antigen peptide to an NGS mouse to make an ectopic tumor-bearing animal model; will be 5X 10⁵Tumor cells expressing specific antigens were suspended in 100. mu.l of physiological saline, and subcutaneously injected into the right flank of 30 NSG mice, respectively, while numbering the mice;

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 HAFFT1 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 experimental results are as follows:

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. Morphological Observation of immortalized DCs

After induction of DC maturation, the morphology was observed microscopically and obvious dendritic cells could be observed (FIG. 1);

3. flow detection of DC antigen load efficiency

The predicted mutant antigen is synthesized according to the table 1, FITC labeling is carried out, and the efficiency result of detecting DC-presented polypeptide antigen by using a flow cytometer after the antigen is loaded with DC shows that: the load efficiency of DC was 99.6% (as in fig. 2);

HAFF protocol cell typing assay

After the culture of the cells in the HAFF protocol was completed, the typing of CD4+ and CD8+ cells was examined, and the results are shown in FIG. 3: 68% of CD8+ T cells and 9.45% of CD4+ T cells;

5. screening of precise Polypeptides by HAFF cells

The cultured T cells are stimulated by 10 polypeptides respectively, effective polypeptides are detected by detecting the secretion of IFN-gamma, and the result is shown in figure 4 that the polypeptide No. 5 stimulates the T cells to secrete the IFN-gamma which is obviously higher than the IFN-gamma secreted and released by the contrast component, so that the polypeptide is determined to be the effective polypeptide.

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

The HAFF' protocol cells were stimulated with the selected polypeptide No. 5 to flow-detect the proportion of T cells specific for the precise polypeptide, the results are shown in FIG. 5, with specific T cells in the box: the proportion of cells releasing IFN- γ by the HAFF 'protocol cells, polypeptide No. 5, was significantly higher than that of non-stimulated cells (control), indicating that the HAFF' protocol, allows to obtain T cells specific for the effective polypeptide; sorting of CD8+ IFN- γ + T cells (boxed) simultaneously with 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 10 of high-frequency distribution), and the distribution frequency of TCR2 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 that needs to be replaced)

TCR-. beta.after replacement:

MRIRLLCCVA FSLLWAGPVI AGITQAPTSQ ILAAGRRMTL RCTQDMRHNA MYWYRQDLGL

GLRLIHYSNT AGTTGKGEVP DGYSVSRANT DDFPLTLASA VPSQTSVYF CASSQGSSGRAKNIQYF

GQGTRLTVV (replacement CDR3 sequence on the horizontal line)

9. Detection of inhibitory target knockout efficiency

An inhibitory target PD-1 on a CD8+ T cell is knocked out by using a CRISPR technology, sgRNA sequences are shown in a table 3, and the knocking-out efficiency result of the inhibitory target is shown in a figure 7: the knockout efficiency of sgRNA1 is highest, and the expression of inhibitory signal molecules PD-1 can be effectively blocked; 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

10. Detection of the original TCR knockdown efficiency

The original TCR was knocked out using CRISPR technology, 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

CD8+ T cells were transfected with lentivirus packaging specific TCRs, and the efficiency of TCR expression was assayed by flow at day 7, with the results shown in figure 9: the constructed TCR was normally expressed with a cellular proportion of TCR + of 88.4%.

Killing of target cells by HAFFT cells

The killing efficiency of HAFF ' cells, HAFFT cells and HAFFT1 cells on target cells from mutant epitopes is detected, the effective target ratio is set to be 40:1, untreated cells are used as a control (Mock), and the result is shown in FIG. 10, compared with the control group, the HAFF ' cells, the HAFFT cells and the HAFFT1 cells all have certain killing effect on the target cells, and the killing efficiency on tumors is HAFFT1> HAFFT cells > HAFF ' cells; the T cells expressing the specific TCR are shown, and the killing efficiency of the T cells on tumor cells can be effectively improved by knocking out the inhibitory target;

detection of cytokine release by HAFFT1 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) and effector cells (MOCK) without any modification, HAFF' scheme cells, HAFFT scheme cells and HAFFT1 cells can produce a large amount of IFN-gamma after being co-cultured with tumor cells, particularly HAFFT and HAFFT1 cells, and because specific TCR (HAFFT) is expressed and inhibitory signals are knocked out (HAFFT1), the effector cells can release more IFN-gamma, and the result is consistent with the result of killing experiments and indicates that: the T cells expressing specific TCR can effectively improve the anti-tumor capacity by combining the blocking 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) HAFFT1 cells have 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: HAFFT1 cells at 1X 10 counts once a month ⁹2 times for each cell;

the second course of treatment: HAFFT1 cells at a number of 1X 10 cells once a 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	Intrahepatic bile duct cancer	CASSSNDNIPYNEQFF	2016.8-Up to now
2	Ovarian cancer	CASSTERPEQYF	2016.7-Up to now
				3	Ovarian cancer	CSAPSGLAGALFYEQYF	2016.9-Up to now
4	Gastric adenocarcinoma liver metastasis	CASTSLDYEQYF	2017.1-Up to now
				5	Stomach cancer	CASSFGRERGFYNEQFF	2017.5-Up to now
6	Lung cancer	CASSPRDRGLTNYGYTF	2017.5-Up to now
				7	Esophageal cancer	CASSQEILGGGTDTQYF	2017.9-Up to now
8	Adenocarcinoma of lung	CASSSGKWGTEAFF	2018.1-Up to now
				9	Adenocarcinoma of lung	CASSDWPLNEQFF	2018.3-Up to now
10	Stomach cancer	CASSPGTSRDNEQFF	2018.4-Up to now

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

Claims

1. A method for constructing HAFFT1 cells, which is characterized by comprising the following steps: loading immortalized DC cells by using the accurate polypeptide, and co-incubating the immortalized DC cells with PBMC to prepare HAFF' cells; stimulating HAFF' cells by taking the accurate polypeptide as an antigen, screening to obtain specific T cells capable of identifying the accurate polypeptide, obtaining high-frequency TCR of the specific cells through sequencing, knocking out the original TCR and expressing the high-frequency TCR to construct TCR-T cells; knocking out inhibitory signal molecules of the TCR-T cells to prepare HAFFT1 cells;

the preparation method of the precise polypeptide comprises the following steps:

(1) performing ctDNA exon sequencing on peripheral blood or performing whole exon sequencing on tumor tissues, screening out mutation sites, performing epitope prediction, synthesizing mutant polypeptide and performing small molecule modification;

the small molecule (H) and the polypeptide antigen (P) are modified in a linking mode (from an amino terminal to a carboxyl terminal: N-C): H-P, P-H, H-P-H;

(2) 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 modified by the small molecules, and incubating with PBMC (peripheral blood mononuclear cell) to obtain HAFF cells;

(3) stimulating the HAFF cells by using the mutant polypeptide modified by the small molecules as an antigen, and obtaining an accurate polypeptide by checking IFN-gamma secretion screening;

the determination method of the high-frequency TCR is as follows: stimulating HAFF' cells by using the precise polypeptide modified by small molecules, staining the stimulated cells by CD8, CD137 and IFN-gamma, and selecting CD8+ CD137+, or CD8+ IFN-gamma + T cells; and extracting a genome, sequencing and analyzing the TCR, and determining a high-frequency TCR sequence according to the TCR distribution frequency.

2. The method of claim 1 for constructing HAFFT1 cells, wherein the patient peripheral blood may be 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 cancer cells, CRL-6323B16F1, CRL-25394T 1, U14 mouse cervical cancer cells, BV-2 mouse glioma cells, or G422 mouse glioma cells.

4. The method of claim 1 for constructing HAFFT1 cells, wherein the prediction of epitopes is performed by extending 8 amino acids to both sides of a mutated amino acid site as a potential epitope; potential epitopes were analyzed for IC50 and potential epitopes with IC50 < 1000nM were identified as epitopes.

5. The method of claim 1 for constructing HAFFT1 cells, wherein the method of knocking out native TCR genes is CRISPR technology.

6. The method of constructing a HAFFT1 cell of claim 1, wherein the immunosuppressive signal molecule comprises: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4(CD244), TIGIT.