CN110093376B

CN110093376B - Construction method of LRFFT1 cells

Info

Publication number: CN110093376B
Application number: CN201910439145.4A
Authority: CN
Inventors: 焦顺昌; 张嵘; 张天赋; 周子珊; 宋玉洁; 陈小彬; 吴子明
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: CN109136280A; CN110093376A

Abstract

The invention uses human peripheral blood to sequence ctDNA or tumor tissue to sequence all exons, screens out mutation sites to predict antigen epitope, connects and synthesizes mutation polypeptide expression gene sequence; meanwhile, constructing a lentiviral vector, packaging lentivirus, transfecting APC cells, completing the transformation of specific LV cells, co-culturing with PBMC (peripheral blood mononuclear cell) separated from peripheral blood in vitro, screening effective polypeptide, and transforming common T cells into RFF cells with more accurate killing capacity through second impact stimulated by the accurate effective polypeptide; the TCR-T technical principle is utilized for modification; the modified T cells are knocked out of immunosuppressive targets by using a gene editing technology, so that specific killer T cells are accurately protected from in vivo inhibition, the lethality of the T cells to tumor cells is improved, and the LRFFT1 cells with both attack and defense are obtained.

Description

Construction method of LRFFT1 cells

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method of LRFFT1 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 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-protection technology of T cells, so that a small number of specific T cells directly face a strong tumor immune microenvironment. The general lack of accurate and effective analysis of patient antigens is also lacking.

Disclosure of Invention

The invention uses the peripheral blood of a patient to carry out ctDNA sequencing or tumor tissue to carry out whole exon sequencing, screens out mutation sites to carry out antigen epitope prediction, and connects and synthesizes a mutant polypeptide expression gene sequence; meanwhile, constructing a lentiviral vector, packaging lentivirus, transfecting APC cells, completing the transformation of specific LV cells, co-culturing with PBMC (peripheral blood mononuclear cell) separated from peripheral blood in vitro, screening effective polypeptide, and transforming common T cells into RFF cells with more accurate killing capacity through second impact stimulated by the accurate effective polypeptide; the TCR-T technical principle is utilized for modification; the modified T cells are knocked out of immunosuppressive targets by using a gene editing technology, so that specific killer T cells are accurately protected from in vivo inhibition, the lethality of the T cells to tumor cells is improved, and the LRFFT1 cells with both attack and defense are obtained.

The LRFFT1 cell provided by the invention can be widely applied to individualized and accurate treatment of solid tumors.

For the interpretation of terminology:

l: lentiviral transfection technique

R: accurate polypeptide secondary impact technology

FF: polypeptide mixing technology

T: TCR-T technology

1: target spot knockout protection technology

For example: LRFFT1 cells, i.e. cells that are finally obtained by engineering or by technical means of the above L, R, FF, T, 1 protocols.

The LRFFT1 cell engineering protocol is summarized as follows:

1. epitope prediction

1) Using human peripheral blood to perform ctDNA sequencing or commercially available engineering cell lines (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, etc.) to perform MHC type detection and whole exon sequencing to detect RNA mutation;

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;

3) 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. synthesis of Gene sequences encoding 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. lentiviral packaging

Constructing a lentivirus expression plasmid for expressing the epitope peptide by the gene sequence synthesized in the step, and then packaging the lentivirus;

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

1) Antigen presenting cells are transfected with lentiviruses expressing 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;

4) secondly impacting the T cells by the screened 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. knocking out cell surface immunosuppressive signal molecule to obtain LRFFT1 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 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 LRFFT1 scheme is more than 50 percent;

the LRFFT1 cell knocks out immunosuppressive targets such as PD1, CTLA4, TIM3 and LAG3, so that the killing capacity to tumors is not limited, and the killing efficiency is higher.

Drawings

FIG. 1: detecting the efficiency of lentivirus transfection APC; wherein, 1A: control, 1B: and (4) a transfection group.

FIG. 2: and (3) carrying out LFF 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: the LRFF scheme.

FIG. 5: the TCR distribution frequency.

FIG. 6: knock-out of inhibitory targets; wherein, 6A: before knockout, 6B: and (4) knocking out.

FIG. 7: detecting the knockout efficiency of the original TCR; wherein, 7A: before knockout, 7B: and (4) knocking out.

FIG. 8: the efficiency of expression of the specific TCR; wherein, 8A: prior to transfection, 8B: after 7 days of transfection.

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.

The technical scheme is detailed as follows:

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. Synthesis of Gene sequences encoding 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. Lentiviral packaging

1) Cloning the synthesized epitope peptide gene sequence into pCDH-MSCV-MCS-EF1-copGFP plasmid to construct slow virus expression plasmid expressing epitope peptide;

2) and (3) slow virus packaging:

a. recovering 293T cells, and packaging the cells for lentiviruses after two generations;

b. cell transfection: (T175 culture flask)

Taking a 15mL centrifuge tube (marked as A), gently adding 400 mu L of Lipofectamine 2000 into 4mL DMEM, gently mixing uniformly, and standing and culturing at room temperature for 5 min;

taking two 15mL centrifuge tubes (marked as B and C, B is a control group, and C is an experimental group), adding the following reagents, gently mixing, and standing and culturing at room temperature for 5 min;

transferring the liquid in tube A into tube B and tube C, mixing, and standing at room temperature for 20 min.

Pouring out old culture medium in T175, washing cells once by using PBS, changing the cells into new 25mL DMEM (without antibiotics and serum), gently adding A, B or A, C mixed solution, gently shaking, and culturing in a 5% CO2 incubator at 37 ℃;

after 6h of transfection, the medium containing the transfection complex was aspirated off and replaced with fresh medium preheated at 37 ℃; culturing for 48h, and collecting;

3) lentivirus concentration and titer determination:

after the slow virus is successfully packaged, collecting the supernatant of the slow virus, and centrifuging for 10min at the temperature of 4 ℃ at 4000 g; filtering the supernatant with a 0.45 μm filter to remove cell debris; following viral supernatant: mixing concentrated reagents according to the proportion of 5:1, and standing for 2h or overnight at 4 ℃; centrifuging the incubated mixed solution at 4 ℃ for 30min at 4000g, and then, observing that an off-white precipitate is formed at the bottom of the tube; carefully remove the supernatant (do not touch the pellet); adding a proper volume of DMEM or PBS, and gently blowing and beating the heavy suspension precipitate; subpackaging the viruses according to the requirements, preserving at-80 ℃ (injecting the lentiviruses instead of repeated freeze thawing, and reducing the titer of the lentiviruses by 10-20% every freeze thawing time);

the day before the measurement, 293H cells with good growth status were digested and counted, and then diluted to 5X 10⁴PermL, added to 96-well plates, 100. mu.L/well, and 8-10 wells prepared for each virus. Culturing in a 37 deg.C 5% CO2 incubator

Taking a certain amount of virus liquid to infectCell: a 10-fold gradient dilution was made in EP tubes. The dilution method is as follows: for each virus, 10 1.5mL EP tubes were prepared, 90. mu.L of culture medium was added to each tube, and 10. mu.L of virus stock solution, designated 10, was added to the first tube⁰(ii) a After mixing, add 10 μ L of the mixture into a second tube and mix it as 10^-1(ii) a By analogy with that (10)⁰-10^-8) Adding 10 mu L of diluted virus solution into corresponding cell holes, marking, culturing for 48-72h, and observing the result;

and (3) calculating the titer: for lentiviruses with fluorescent labels, titer can be determined using fluorescence techniques; the results were observed under a fluorescent microscope, and the number of the last two fluorescent cell clones were counted, and assuming X and Y, the titer (TU/mL) was (X + Y × 10) × 1000/2/well of the virus content (μ L).

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

1) Preparing a whole cell culture medium containing 300U/mL rIL-2, 10% FBS by using RPMI-1640, and recording the whole cell culture medium as RPMI-10-IL-2; APC concentration was adjusted to 1X 10 by using RPMI-10-IL-2⁶Per mL; infecting APCs (including but not limited to peripheral blood mononuclear cells, dendritic cells, neutrophils, B lymphocytes, macrophages) at MOI 5-20 using lentiviruses expressing epitope peptides; culturing at 37 deg.C for 72 hr;

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 culture flask; and (3) placing the mixture into a cell culture box at the temperature of 30-37 ℃ for 14 days to obtain the LFF 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 cells of the LFF protocol, 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 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: t cells +1640+10% FBS +200U/mL IL 2;

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) when the experimental group is obviously larger than the negative control, the polypeptide is effective and accurate.

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 the T cells obtained by secondary impact of the precise polypeptide, namely LRFF cells;

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

1) Directly stimulating the LRFF cells obtained in the step 7 by using the screened accurate polypeptide as an antigen 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 an immunosuppressive Signal molecule knockout CRISPR vector

1) Cell surface inhibitory signaling 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;

11. 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) completing the construction of a TCR knockout vector and virus packaging according to the steps 3) to 5) in the step 10;

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

1) infecting the CD8+ T cells obtained in the step 8 with the viruses obtained in the steps 10 and 11, and simultaneously knocking out the original TCR and the immunosuppressive signal molecule;

2) after knockout, 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;

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

4) 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;

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

6) when the culture is carried out for 14-21 days, the TCR-T cells of which the immunosuppressive signal molecules are knocked out, namely the LRFFT1 cells can be harvested.

13. 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) 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) In the tumor growth to100-120mm³The 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 LRFFT1 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 prediction results of the mutation sites and the epitopes detected by sequencing.

TABLE 1 epitope prediction

2. Lentivirus transfection APC efficiency assay

2) the proportion of GFP positivity in the APC was determined using flow cytometry (as shown in FIG. 1).

3. LFF cell typing assay

After the LFF protocol cell culture was completed, CD4+ and CD8+ cells were typed and the results are shown in fig. 2: 89.1% of CD8+ T cells and 8.11% of CD4+ T cells.

4. Screening of precision Polypeptides by LFF 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 polypeptides No. 3 and No. 7 is greater than that of the negative control, and belongs to effective accurate polypeptides.

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

LFF protocol cells were stimulated with the screened polypeptides No. 3 and No. 7 to flow-detect the proportion of T cells specific for the precise polypeptide, and the results are shown in fig. 4, with FL1+ being specific T cells: the LRFF protocol cells, polypeptides No. 3 and No. 7, caused significantly higher proportion of IFN- γ releasing cells than non-stimulated cells (control), indicating that the LRFF protocol can yield T cells specific for the precise polypeptide; meanwhile, the sorting of CD8+ IFN-gamma + cells (FL1+) was performed by a flow cytometer.

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 20 of high-frequency distribution), wherein the distribution frequency of TCR6 is higher, which indicates that the TCR is closely related to the mutant antigen, and the TCR is amplified according to the TCR6 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 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. 6: the knockout efficiency of sgRNA1 is highest, and the expression of inhibitory signal molecules PD-1 can be effectively blocked; the sgRNA is preferably sgRNA1 and sgRNA 2; 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.; can effectively block the expression of inhibitory signal molecules.

Table 3 inhibitory target sgRNA sequences

8. 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 7: the expression of the original TCR can be effectively reduced, at which point transfection of the lentivirus expressing the specific TCR can be performed.

9. 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 8: the constructed TCR was normally expressed with a cellular proportion of TCR + of 67.4%.

10. Killing effect of LRFFT1 cell on target cell

The killing efficiency of the target cells from the mutant epitope was detected by using the control cells and the LRFFT1 cells, respectively, and the effective-to-target ratio was set to 40:1 by using the untreated cells as the control (Mock), and the result is shown in fig. 9, in which the LRFFT1 cells had a stronger killing effect on the target cells than the control.

11. Detection of LRFFT1 cell-released cytokine

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 LRFFT1 cells and tumor cells are co-cultured in a ratio of 1:1, the released IFN-gamma is detected, and the result shows that: LRFFT1 cells produced a greater amount of IFN- γ than was produced by effector cells themselves (T cells only) after co-culture with tumor cells, consistent with killing results indicating: the T cells expressing specific TCR, combined with the knockout of inhibitory targets, can more effectively improve the anti-tumor capacity.

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

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) the LRFFT1 cell has a significant influence on the survival improvement of tumor-bearing mice.

13. Clinical cases:

a woman: age 58

And (3) disease diagnosis: ovarian cancer complicated by breast cancer;

the first course of treatment: LRFFT1 cells at 1X 10 cell counts once a month ⁹2 times for each cell;

the second course of treatment: LRFFT1 cells at 1X 10 cell counts once a half year ⁹2 times for each cell;

after the administration, the patient survives for 22 months without progress;

other cases are as follows:

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

Claims

1. A method for constructing LRFFT1 cells, which comprises 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 lentivirus vector for expressing the mutant polypeptide, and packaging the lentivirus;

4) transfecting antigen presenting cells with the lentivirus and co-culturing with PBMC to obtain LFF cells;

5) the mutant polypeptide is used as an antigen to stimulate the LFF cell, 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 LFF 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 knocking out cell surface immunosuppressive signal molecules by using a CRISPR technology to obtain the LRFFT1 cell.

2. The method of claim 1, wherein the sequencing in step 1 is ctDNA sequencing using human peripheral blood from tumor patients or commercially available engineered cell lines, or whole exon sequencing using tumor tissues from 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 claim 1, wherein the epitope-linked linker in step 2 has a higher IC50 than the IC50 of the flanking epitopes.

5. The method of claim 1, wherein in step 4 the antigen presenting cells and PBMCs are mixed in a ratio of 1: 5-20 of the above-mentioned materials.

6. The method of claim 1, wherein in step 6, the cells are sorted by flow cytometry and selected from CD8+ CD137+ or CD8+ IFN- γ + cells.

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

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