CN110129372B

CN110129372B - Construction method of RFFT1 cells

Info

Publication number: CN110129372B
Application number: CN201910438798.0A
Authority: CN
Inventors: 焦顺昌; 张嵘; 周子珊; 解佳森; 王海燕; 李营营
Original assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Current assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Priority date: 2018-09-30
Filing date: 2019-05-24
Publication date: 2021-06-18
Anticipated expiration: 2039-05-24
Also published as: CN109182383A; CN110129372A

Abstract

The invention provides a construction method of RFFT1 cells, and belongs to the technical field of biology. The invention constructs the T cell with integrated attack and defense, high accuracy and high killing property. The construction method comprises the following steps: the PBMC cells are loaded with polypeptide causing tumor mutation, and then polypeptide impact is carried out on the PBMC cells loaded with the polypeptide; performing expanded culture after impact to obtain FF cells; so that the tumor-mutated polypeptide is used as an antigen to directly stimulate FF cells to screen precise polypeptide; culturing PBMC cells, and performing multiple impacts by using the accurate polypeptide in the culture process; continuing culturing after impacting to obtain RFF cells; screening to obtain specific cells capable of identifying the precision polypeptide; obtaining a TCR gene from a specific cell; culturing PBMC (peripheral blood mononuclear cell), knocking out the original TCR gene and the surface immunosuppressive signal molecule, and transferring the obtained TCR gene to prepare an RFFT cell; and (3) carrying out immunosuppressive signal molecule blocking on the obtained RFFT cells by using a monoclonal antibody medicine to obtain the RFFT1 cells.

Description

Construction method of RFFT1 cells

Technical Field

The invention relates to the technical field of biology, in particular to a construction method of RFFT1 cells.

Background

Tumor cell immunotherapy is an emerging tumor therapy model, which collects immune cells from a patient, cultures and expands in vitro, and then transfuses them back into the patient to stimulate and enhance the body's autoimmune function to treat tumors. Tumor cell immunotherapy is the fourth method of tumor treatment following surgery, radiation therapy and chemotherapy.

When the normal or bioengineered human body cells are transplanted or input into the body of a patient, the newly input cells can replace damaged cells or have stronger immune killing function, thereby achieving the purpose of treating diseases.

The bioengineered cells are modified by special methods during in vitro culture process, and can effectively kill tumor cells in patients. For example, chinese patent application CN201210194280.5 provides a human cytokine-induced killer cell. Chinese patent application CN201510034781.0 provides a tumor cell specific polyclonal T cell. The chinese patent application CN201510013987.5 provides an anti-tumor T cell and a preparation method thereof. Chinese patent application CN201711060030.1 provides a CAR-T cell for treating AIDS-associated lymphoma, and a preparation method and application thereof. CN201610824893.0 provides a double-antigen specific T cell regulated by an antibody, a preparation method and an application thereof.

In the prior art, T cells are generally presented through DC cells to generate specific killing T cells, or viruses are used as vectors to induce the specific killing of the T cells through a lentivirus transfection technology. But the effect is not good because of unclear tumor antigen and the obstacle of tumor microenvironment immunosuppression; or the effect is poor because of the simple and thin specific cells directly facing the complex tumor microenvironment, or the target is single and only effective for individual tumors.

Disclosure of Invention

The invention aims to provide a construction method of RFFT1 cells, so as to construct a novel TCR-T cell (named as RFFT1 cell) for cellular immunotherapy, and realize the precision, specificity and safety of killing.

The invention provides a construction method of RFFT1 cells, which comprises the following steps:

s1) sequencing the whole exon of the tumor cell; comparing the sequencing result of the whole exon with the genome of a normal cell, and screening out a mutant amino acid site; predicting the epitope by taking the mutated amino acid site as a center; synthesizing mutant polypeptide by polypeptide solid phase synthesis method; the PBMC cells are loaded with the polypeptide causing tumor mutation, and then the PBMC cells loaded with the polypeptide are subjected to polypeptide impact for one time;

s2) expanding culture after impact to obtain FF cells;

s3) so that the tumor mutated polypeptide acts as an antigen to directly stimulate the FF cells to screen for the precise polypeptide;

s4), culturing PBMC cells, and performing multiple polypeptide impacts by using the accurate polypeptide in the culture process;

s5) continuing culturing after impact to obtain RFF cells;

s6) stimulating the obtained RFF cells by taking the precision polypeptide as an antigen, and screening to obtain specific cells capable of identifying the precision polypeptide;

s7) obtaining a TCR beta chain CDR3 region sequence of a specific cell through sequencing, and obtaining a TCR gene through amplifying the TCR beta chain CDR3 region sequence;

s8), knocking out the original TCR gene and cell surface immunosuppressive signal molecule in the cell, and transferring the TCR gene which is obtained in the step S7 and can be specifically combined with the precise polypeptide to prepare the RFFT1 cell, wherein the immunosuppressive signal molecule comprises: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4(CD 244).

Further, the tumor cells are derived from commercially available engineered cell lines, including 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.

Furthermore, the prediction of the antigen epitope is that the mutated amino acid site is used as the center, 10 amino acids are extended towards two sides, and a section of polypeptide with 21 amino acids is used as a potential antigen epitope.

Further, in step S2, the expanding culturing after the impacting to obtain the FF cell composition comprises:

culturing the PBMC cells after the polypeptide impact in a cell culture device pre-paved with a cell stimulating factor OKM-25 for 5 days;

transferring to a cell culture device containing a culture solution OKM-100+ 12% FBS for continuous culture till the 10 th day;

transferring the cells to a cell culture device containing a culture solution OKM-200+ 5% FBS, and continuing culturing for 14-21 days.

Further, in step S4, the polypeptide impact is repeated 3-4 times.

Furthermore, in the polypeptide impact, polypeptide impact is carried out by using polypeptide solution with the concentration of 10 to 100 mu g/mL.

Further, the impact time of the polypeptide impact is 1-4 h.

In step S8, the cellular immunosuppressive signal molecules and the original TCR genes of the cells are sequentially knocked out by CRISPR technique.

In step S8, infecting PBMC cells with CRISPR lentiviral vectors that have the original TCR knocked out and CRISPR lentiviral vectors that have surface immunosuppressive signal molecules knocked out, and simultaneously knocking out the original TCR and removing the immunosuppressive signal molecules; then, the vector is transferred to a TCR gene expression vector for expressing the TCR gene obtained in step S7, so that the TCR gene obtained in step S7 is transferred.

The invention has the following beneficial effects:

1) the invention provides a novel cell construction method, which is used for constructing a novel TCR-T cell (named as RFFT1 cell) which is a super T cell with both attack and defense. In the method, T cells are modified by combining a polypeptide mixing technology, an accurate polypeptide secondary impact technology, a TCR-T technology and a target spot knockout protection technology. Firstly, mixed polypeptide impact stimulation is carried out, then effective accurate polypeptide is screened out to carry out polypeptide impact on PBMC again, and additional polypeptide impact stimulation is carried out. On the basis of two times of stimulation, the TCR-T technology is combined for third stimulation, so that common T cells are transformed into T cells with specific killing effect, and the killing efficiency and the precision are high.

2) In the invention, the precision, specificity and safety of searching and killing are realized by combining a target spot knockout protection technology, in-vitro culture and in-vitro amplification, more tumor species are covered, and the adaptability of the T cells with high-precision killing effect to a complex tumor microenvironment is improved.

3) Compared with the killing effect of directly transfecting and inducing T cells by using lentiviruses, the method is simple and convenient and has high safety.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

Figure 1 shows antigen loading efficiency detection;

FIG. 2 shows the results of a precision polypeptide screen;

FIG. 3 shows the flow assay specific T cell ratios;

FIG. 4 shows TCR distribution of specific cells;

FIG. 5 shows the flow assay of the knockout of an existing TCR on a cell;

FIG. 6 shows an in vitro blocking of an immunosuppressive signaling molecule;

FIG. 7 shows the expression profile for constructing a TCR;

FIG. 8 shows the killing of target cells by RFFT1 cells according to an embodiment of the invention;

FIG. 9 shows the detection of cytokine release by RFFT1 cells according to an embodiment of the invention;

figure 10 shows tumor-bearing mouse survival curves.

Detailed Description

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.

Tumor cell immunotherapy is an emerging mode of tumor therapy. In the aspect of tumor cell immunotherapy, the existing LAK, DC, CIK, DC-CIK cells have been proved to be basically ineffective. The present invention provides a novel T cell useful for cellular immunotherapy. The invention modifies T cells, provides the TCR-T cells, has strong lethality and high accuracy, and covers various tumors.

The embodiment of the invention provides a construction method of RFFT1 cells, and a TCR-T cell (named as RFFT1 cell) for cellular immunotherapy is constructed. The construction method may include the steps of:

s1) sequencing the whole exon of the tumor cell; comparing the sequencing result of the whole exon with the genome of a normal cell, and screening out a mutant amino acid site; predicting the epitope by taking the mutated amino acid site as a center; synthesizing mutant polypeptide by polypeptide solid phase synthesis method; the PBMC cells are loaded with polypeptide causing tumor mutation, and then polypeptide impact is carried out on the PBMC cells loaded with the polypeptide;

s2) expanding culture after impact to obtain FF cells;

s4), culturing the PBMC cells, and performing polypeptide impact by using the accurate polypeptide in the culture process;

s5) continuing culturing after impact to obtain RFF cells;

s8) knocking out the original TCR gene and the surface immunosuppressive signal molecule in the T cell, and transferring the TCR gene which is obtained in the step S7 and can be specifically combined with the precise polypeptide to prepare the RFFT1 cell, wherein the immunosuppressive signal molecule comprises: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4(CD 244).

Herein, the definition of "RFFT 1" cells refers to the following meanings:

r-precise polypeptide secondary impact technology;

FF-mixed polypeptide technology;

T-TCR-T technology;

1-target knockout protection technology.

That is, the "RFFT 1" cell herein is a novel T cell prepared by an RFFT1 scheme composed of a combination of a mixed polypeptide technology, an accurate polypeptide secondary impact technology, a TCR-T (T cell receptor (TCR) chimeric T cell) technology, and a target knockout protection technology, and can be used for tumor immunotherapy. As the name implies, "FF" cells are T cells derived from the FF protocol. "RFF" cells are T cells derived from the RFF protocol. "RFFT" cells are T cells derived from the RFFT protocol.

The embodiments of the present invention provide a TCR-T cell for use in cellular immunotherapy. In the RFFT1 scheme of the embodiment of the invention, firstly, the mixed polypeptide is used for directly impacting PBMC cells loaded with the polypeptide to perform first stimulation; secondly, screening the accurate polypeptide, taking the screened accurate polypeptide as an antigen to directly stimulate FF cells, and performing secondary stimulation; followed by a third stimulation by TCR-T technique. T cells are transformed through three times of stimulation, and the transformed T cells achieve the precision, specificity and safety of searching and killing. Meanwhile, the adaptation of the modified T cell to the complex immune environment in vivo is improved by combining the in vitro culture and target spot knockout protection technology.

In step S1, the tumorigenic polypeptide may be synthesized by: 1) sequencing exons; 2) predicting the epitope; 3) and (3) synthesizing the polypeptide.

1) Exon sequencing

Sequencing the tumor cells by using the whole exons, and then analyzing sequencing information by using software to obtain MHC type information on one hand; on the other hand, the whole exon sequencing result is compared with the genome of normal cells, and the mutated amino acid site is selected.

In exon sequencing, tumor cells may be derived from engineered cell lines or from the patient's peripheral blood.

The engineered cell lines may include 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. The engineering cell line is a cell line which is obtained by modifying or recombining genetic materials of host cells by adopting a genetic engineering technology or a cell fusion technology and has unique characteristics of stable heredity.

2) Epitope prediction

In the prediction of epitope, 10 amino acids are extended from the site of mutated amino acid as the center to both sides, and the 21 amino acid polypeptide is used as "potential epitope". The IC50 of potential epitopes was analyzed using prediction software (recommended software: NetMHCpan3.0, PickPocket, Artificial Neural Networks (ANN)). A potential epitope is considered to be an "epitope" if IC50 < 1000 nM.

3) Synthetic polypeptides

The epitope peptide is synthesized by a polypeptide solid phase synthesis method.

Specifically, the loading of the PBMC cells with the polypeptide causing tumor mutation and then performing polypeptide impact on the PBMC cells loaded with the polypeptide can specifically be as follows:

1) preparing a polypeptide solution: dissolving polypeptide with RPMI 1640+ 10% FBS (fetal bovine serum) or OKM100+ 12% FBS, wherein the final concentration of the polypeptide is 10-100 mug/mL, preferably 50 mug/mL, for later use;

2) resuscitating PBMC 1 day ahead, blowing cells, sucking 15mL, counting and centrifuging;

3) resuspending PBMCs with the formulated polypeptide solution;

4) placing the cell culture plate in a cell culture plate for impacting;

5)37℃5％CO₂and impacting for 1-4h, preferably 4h to obtain impacted PBMC cells for later use.

In step S2, the expanding culture after the impact to obtain FF cells may specifically be:

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

2) transferring the impacted PBMC 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 75cm²Transfer to 175cm in the bottle²In a big bottle;

7) 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) FF cell composition can be obtained after culturing for 14-21 days. And extracting the T cells from the FF cell composition by centrifugation to obtain FF cells.

In step S3, T cells are isolated and extracted from the FF cell composition such that the tumor mutated polypeptide acts as an antigen to directly stimulate the T cells to screen for a precise polypeptide.

The screening criteria for the precise polypeptide were:

polypeptide as antigen with FF cells as baseline; the two are independently repeated, the high detection value is a high base line, and the low detection value is a low base line;

the difference between the two baselines is a system error, and when data are analyzed, the detection values > low baseline, > high baseline and > high baseline + system error are respectively marked; and (5) the detection value is greater than the high baseline and the system error is the effective accurate polypeptide.

In step S3, the step of directly stimulating T cells with the polypeptide as an antigen to screen precise polypeptide may specifically be:

1) centrifuging the obtained FF cell composition at 1500rpm for 5min to collect T cells, adding 10mL PBS to resuspend the cells and counting, centrifuging at 1500rpm for 5min, collecting T cells, resuspending with 1640+ 10% FBS +200U/mL IL2, and adjusting the count to 1 × 10⁶Per 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/well⁵A plurality of; 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 OKT3(CD3 monoclonal antibody); negative control: RPMI 1640+ 10% FBS +200U/mL IL2 (Interleukin 2); two T cell controls were used as background release detection, first T cell addition, and last T cell addition, respectively; taking the difference of two background releases as a system error;

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

5) centrifuging 96-well plate at 1500rpm for 10min, and collecting sample for ELISA (enzyme-linked immunosorbent assay) (or storing at-80 deg.C);

6) screening of the precise polypeptide:

based on the accurate polypeptide screening standard, the detection value is greater than the high baseline and the system error is the effective accurate polypeptide.

In step S4, PBMC cells are cultured and a second stimulation is performed by polypeptide shock with the precision polypeptide during the culturing process.

Compared to step S1, an additional amount of precision polypeptide impact is used in step S4. That is, the PBMC cells are subjected to multiple polypeptide shock stimuli in this step. Specifically, the polypeptide impact can be repeated for 3-4 times.

During the culture process, the culture protocol is similar to the expanded culture of the PBMC cells loaded with the polypeptide in the step S2. Firstly culturing for a period of time in a device pre-paved with cell stimulating factors OKM-25, and then sequentially transferring to an OKM-100+ 12% FBS culture medium and an OKM-200+ 5% FBS culture medium for continuous culture.

Step S4 may specifically include the following steps:

culturing the PBMC cells in a cell culture device pre-paved with a cell stimulating factor OKM-25, and performing polypeptide impact on the accurate polypeptide obtained in the step S3 after culturing for a period of time;

and then transferring the cells to a cell culture device (culture plate or culture bottle) containing a culture solution OKM-100+ 12% FBS for continuous culture, and performing polypeptide impact on the precise polypeptides obtained in the step S3 every 3-4 days.

The impact time per polypeptide impact may be 1-4 h. Ordinary T cells are transformed into RFF cells with more accurate killing capacity through the impact stimulation of the added accurate polypeptide.

In step S5, the culture is continued after the impact, and RFF cells are obtained. A suitable medium may be OKM200+ 5% FBS. Transferring the polypeptide after impacting to a device containing culture solution OKM200+ 5% FBS for continuous culture at 37 ℃ and 5% CO₂And culturing to obtain the T cell, namely the RFF cell, obtained by secondary impact of the accurate polypeptide.

In step S6, the RFF cells obtained by stimulating the precise polypeptide as an antigen, staining CD8, CD137 and IFN-gamma of the stimulated cells, sorting the cells by a flow cytometer, and screening to obtain specific cells capable of identifying the precise polypeptide.

In step S7, the sequence of the CDR3 region of the TCR β chain of the specific cell is obtained by sequencing. The TCR gene is obtained by amplifying the CDR3 region sequence of the TCR beta chain.

1) Extracting a genome from the cell sorted in step S6;

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

3) extracting mRNA of PBMC, reverse transcribing to obtain DNA, designing primer according to the sequence of high frequency TCR, and amplifying to obtain TCR gene.

In step S8, the original TCR gene of the cell can be knocked out by CRISPR technique (Clustered regulated interstitial short palindromic repeats).

In step S8, the original TCR gene and surface immunosuppressive signal molecule in the peripheral blood T cell are knocked out, and the TCR gene obtained in step S7 and capable of specifically binding to the precision polypeptide is transferred to obtain RFFT1 cell. T cells were engineered by a third stimulation using TCR-T technology.

In step S8, the original TCR gene and the surface immunosuppressive signal molecule can be knocked out by CRISPR lentivirus transfection, and the TCR gene obtained in step S7 can be transferred to be capable of specifically binding to the precise polypeptide.

Specifically, 3 lentiviral vectors were constructed separately: constructing a TCR gene expression vector based on the TCR gene obtained in step S7; constructing a CRISPR lentiviral vector for knocking out the original TCR; and (3) constructing a CRISPR lentiviral vector for knocking out the surface immunosuppressive signal molecule. The constructed lentiviral vectors were then used to infect cells: infecting PMBC cells with the obtained CRISPR lentiviral vector for knocking out the original TCR and the CRISPR lentiviral vector for knocking out the surface immunosuppressive signal molecule, knocking out the original TCR and knocking out the immunosuppressive signal molecule; then, the cells are infected with the TCR gene expression vector, and the TCR gene obtained in step S7 is transferred. After infection, co-culture was continued to obtain RFFT1 cells.

The construction process of the CRISPR lentiviral vector for knocking out the TCR gene can be as follows:

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) 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;

3) 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;

4) and (3) extracting CRISPR lentiviral vector plasmids carrying DNA sequences corresponding to the sgRNAs, and packaging the viruses.

The construction of the CRISPR lentiviral vector for knocking out the immunosuppressive signal molecule can refer to the construction of the CRISPR lentiviral vector for knocking out TCR genes.

The construction process of the TCR gene expression vector capable of expressing the TCR gene determined in step S7 is as follows: and (4) performing virus packaging based on the determined TCR gene.

The embodiment of the invention provides a novel cell construction method, which is used for constructing a novel T cell (RFFT1 cell) for tumor immunotherapy, and the T cell is transformed into the TCR-T cell which has good precision, high killing property, capability of covering various tumor species and strong adaptability to complex immune microenvironment by combining a mixed polypeptide technology, an accurate polypeptide secondary impact technology, a TCR-T technology and a target spot knockout protection technology.

The construction method of RFFT1 cells according to the present invention is further described in detail below.

(one) sequencing of all exons

1) Performing whole exon sequencing using the engineered cell line;

2) sequencing information was analyzed using software: on one hand, obtaining MHC type information; on the other hand, the whole exon sequencing result is compared with the genome of normal cells, and the mutation site is selected.

(II) 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: NetMHCpan3.0, PickPocket, and Artificial Neural Networks (ANN)), and if IC50 < 1000nM, the potential epitope is considered to be an "epitope".

(III) Synthesis of Polypeptides

The epitope peptide synthesis method adopts a polypeptide solid phase synthesis method

1) Anchoring: anchoring the first amino acid to the solid phase resin;

2) deprotection: protected amino acid the protecting group of the amino group is removed using an alkaline solvent;

3) and (3) activation: activating the amino acid carboxyl group to be linked using an activating agent;

4) and (3) bonding: the activated carboxyl group reacts with the naked amino group of the previous amino acid to form peptide;

5) and (5) repeating the steps 2-4 to completely synthesize the whole epitope peptide chain.

(IV) PBMC Loading mutant Polypeptides

1) Preparing a polypeptide solution: dissolving polypeptides by 1640+ 10% FBS or OKM100+ 12% FBS, wherein the final concentration of each polypeptide is 50 mug/mL for later use;

3) resuspending the PBMC in the prepared polypeptide solution and placing into a cell culture plate for impact;

4)37℃5％CO₂impacting for 4h for standby.

(V) expanded culture of PBMC after polypeptide impact

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) transferring the impacted PBMC to a culture flask pre-paved with OMK 25;

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

4) co-cultured cells were observed, and on day 5, the cells were transferred to a large flask, supplemented with fresh medium OKM-100+ 12% FBS, 20mL at 75cm, depending on cell density²In 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 one in the flask was transferred to 175cm²In a big bottle;

8) Culturing for 14-21 days to obtain FF cell composition.

(VI) the polypeptide is used as an antigen to directly stimulate T cells to screen precise polypeptides:

1) the T cells in the FF cell composition are the T cells obtained by the FF protocol (FF cells). Centrifuging to collect the obtained FF cell composition, centrifuging at 1500rpm for 5min to collect FF 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) FF cells were plated on 96-well flat-bottom plates with a row 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: FF cells +100ng/mL OKT 3; negative control: 1640+ 10% FBS +200U/mL IL 2; two FF cell controls are used as background release detection, namely a first FF cell addition and a last FF cell addition; taking the difference of two background releases as a system error;

5) the 96-well plate is centrifuged again at 1500rpm for 10min, and samples are taken for ELISA detection (or the samples are stored at-80 ℃).

(VII) accurate polypeptide evaluation standard:

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; and (5) the detection value is greater than the high baseline and the system error is the effective accurate polypeptide.

(VIII) preparation of RFF cells from the selected precision polypeptide

1) PBMC were cultured in FF cell composition culture protocol (stimulating factor OKM)25 Pre-plating, 40. mu.L of OKM-25+4mL PBS, 2 mL/dish (4.5 cm)²) Room temperature 4h, 4 ℃ for standby; the culture conditions were 37 ℃ and 5% CO₂) By day 3, re-impacting the precision polypeptide;

2) take 2X 10⁷Adding polypeptide with the final concentration of 50 mu g/mL into the T cells, and impacting for 4 hours;

3) after impacting for 4h, the height is changed to 25cm²Culture flask supplemented with OKM100+ 12% FBS, 5% CO at 37 deg.C₂Culturing, transferring to 75cm according to cell growth²The cell density in the culture flask was kept as high as 1X 10⁶Per mL;

4) repeating the precision polypeptide shock, i.e., repeating steps 2) and 3), at days 7, 10, and 14 of culture;

5) cells entered 175cm²When the cells are cultured in a culture bottle, the culture medium is OKM200+ 5% FBS, and the cells are cultured for 10-21 days to obtain the T cells, namely the RFF cells, obtained by secondary impact of the precise polypeptides.

(nine) culture and isolation of mutant antigen-specific killer T cells

1) Stimulating RFF cells by directly taking the precise polypeptide as an antigen for 12-72 hours for later use;

2) the stimulated cells were stained with CD8, CD137, IFN-. gamma.and sorted by flow cytometry, selecting CD8+ CD137+, or CD8+ IFN-. gamma. + cells.

(Ten) TCR frequency detection of CD8+ T cells and cloning of high frequency TCR

1) Extracting the genome from the cells obtained by sorting in the step nine;

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 TCR gene expression vector and packaging virus.

(eleven) construction of CRISPR lentiviral vector for knocking out immunosuppressive signal molecule

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

2) finding out a CDS region of mRNA of the gene on pubmed, and predicting a knockout target point 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) and (3) extracting CRISPR lentiviral vector plasmids carrying DNA sequences corresponding to the sgRNAs, and packaging the viruses.

(twelfth) construction of CRISPR Lentiviral vector for knocking out original TCR

(thirteen) TCR-T

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

2) infecting CD8+ T cells by the CRISPR lentiviral vector for knocking out the immunosuppressive signal molecules obtained in the step eleven and the CRISPR lentiviral vector for knocking out the original TCR obtained in the step twelfth, and knocking out the immunosuppressive signal molecules and the original TCR simultaneously;

3) after infection, culturing CD8+ T cells in a culture medium for 3 days, transferring into the TCR gene expression vector constructed in the step ten, and transferring into the TCR gene obtained in the step ten;

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 75cm²The one in the flask was transferred to 175cm²The post-large bottle culture solution is OKM-200+ 5% FBS;

7) after being cultured for 14-21 days, the TCR-T cells, namely the RFFT1 cells, of which the immunosuppressive signal molecules are knocked out can be harvested.

(fourteen) tumor-bearing mouse survival experiments

1) And (4) inoculating the engineering cell line to an NSG 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 injected subcutaneously into the right flank of 30 NSG mice, respectively, and the mice were numbered.

2) Growth of tumor to 100-120mm³The cells were returned to the left and right groups, and the animal model was randomly divided into three groups of 5 mice each, one group given placebo physiological saline, and one group given 1X 10T cells (control group) without any genetic manipulation according to the tumor volume⁷One group was given to RFFT1 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 50 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 the epitope prediction results, and the underlined are the mutated amino acids.

TABLE 1 epitope prediction

2. Antigen load efficiency detection

Synthesizing a predicted mutant antigen according to the table 1, labeling biotin, and detecting the distribution of the biotin on the cell surface by using PE-labeled affinity streptomycin after the antigen is loaded on PBMC so as to detect the efficiency of extracting polypeptide antigen by the PBMC; the results are shown in figure 1-antigen loading efficiency assay: a is a detection result without loading the marker polypeptide, b is a detection result with loading the biotin polypeptide, and the result shows that: the loading efficiency of PBMC was 42.7% (FL4-H subset 42.7% indicates that PE-labeled cells account for 42.7%).

3. Screening for accurate Polypeptides

As shown in FIG. 2, 12 polypeptides were used to stimulate cultured FF cells and effective polypeptides were detected by detecting IFN-. gamma.secretion, and the results are shown in FIG. 2: 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.

RFF cell-specific cell proportion detection

The screened polypeptide No. 6 was subjected to multiple stimulations on the basis of FF cells, and after culturing, the ratio of T cells specific to the precise polypeptide was measured by flow assay (FACSCaliburTM (BD biosciences)), and the results are shown in FIG. 3, in boxes with specific T cells: the proportion of cells that released IFN-. gamma.after multiple shock stimuli (RFF cells) was significantly higher than that of cells without multiple shock stimuli (FF cells) compared to the untreated control group. Therefore, the specific T cell proportion can be improved by multiple times of stimulation of the precise polypeptide; sorting of CD8+ IFN- γ + cells (in boxes) was also performed by flow cytometry.

5. Identification and cloning of high frequency TCR

The cells obtained by sorting were subjected to genome extraction and TCR sequencing, the distribution of TCRs is shown in fig. 4 (top 20 of high frequency distribution), and the distribution frequency of TCR16 is high, which indicates that this TCR is closely related to the mutant antigen, and the TCR was amplified according to the TCR sequence.

TABLE 2 sequence case of CDR3 of TCR beta chain

6. Knockout of original TCR

And (3) knocking out the original TCR on the PBMC by using a CRISPR technology, and detecting the knocking-out condition in a flow mode. The results are shown in FIG. 5: the original TCR can be effectively knocked out.

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

7. Knockout of immunosuppressive signaling molecules

And (3) knocking out the inhibitory signal molecules on the PBMC by using a CRISPR technology, and detecting the knocking-out condition of the knocked-out signal molecules in a flow mode. The results are shown in FIG. 6: there may be blocking of the expression of inhibitory signal molecules.

8. Specific TCR expression profiles

The expression of TCR was detected by flow assay, and the results are shown in fig. 7: the constructed TCR can be normally expressed, transfection is carried out for 3 days, and the cell proportion of TCR + is 48.3%; the proportion of cells transfected for 7 days was 48.7% TCR +.

9. Killing effect of RFFT1 cells on target cells

As shown in fig. 8, RFF cells, RFFT cells, and RFFT1 cells all had a certain killing effect on target cells compared to the control group, and the killing efficiency of RFF cells, RFFT cells, and RFFT1 cells on target cells derived from mutant epitopes was measured, and the results were shown in 20: 1 (effector cells: target cells), the difference with the Mock group is obvious; the overall trend is that the killing efficiency: RFFT1 cells > RFFT cells > RFF cells; the T cells expressing specific TCR can effectively improve the killing efficiency of tumor cells through the knockout of inhibitory targets.

10. Detection of cytokine release by RFFT1 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. 9 is a graph of the detection of released IFN-gamma when cells and tumor cells of different culture schemes are co-cultured, and the results show that: after co-culture with tumor cells, RFF protocol cells, RFFT protocol cells and RFFT1 cells all produced significant amounts of IFN- γ compared to IFN- γ produced by the effector cells themselves (only effector cells), especially RFFT and RFFT1 cells, which released more IFN- γ due to the expression of specific tcr (RFFT) while inhibitory signals were knocked out (RFFT1), consistent with the results of killing experiments, indicating that: the T cells expressing specific TCR can effectively improve the anti-tumor capability through the knockout of inhibitory targets.

11. Experiment on survival of tumor-bearing mice

The results are shown in fig. 10, where reinfusion of RFFT1 cells of the present example provided a significant improvement in mouse survival. A P value less than 0.01(P value 0.0008) indicates statistical significance.

12. Clinical cases

The administration process comprises the following steps:

the first course of treatment: RFFT1 cells were administered once a month, 1X 10 in number ⁹2 times for each cell;

the second course of treatment: RFFT1 cells at a rate of 1X 10 cells/half year ⁹2 cells in total.

TABLE 3

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method of constructing RFFT1 cells, the method comprising: s1) sequencing the whole exon of the tumor cell; comparing the sequencing result of the whole exon with the genome of a normal cell, and screening out a mutant amino acid site; predicting the epitope by taking the mutated amino acid site as a center; synthesizing mutant polypeptide by polypeptide solid phase synthesis method; the PBMC cells are loaded with the mutant polypeptide, and then the PBMC cells loaded with the polypeptide are subjected to polypeptide impact for one time; s2) expanding culture after impact to obtain FF cells; s3) so that the tumor-mutated polypeptide acts as an antigen to directly stimulate the FF cells to screen for the precise polypeptide, wherein the precise polypeptide criteria are: setting two independent repeats by taking FF cells as a baseline, wherein a high detection value is a high baseline, and a low detection value is a low baseline; the difference between the two baselines is the systematic error; the experimental group with the detection value of more than the high baseline and the system error is the effective accurate polypeptide; s4), culturing PBMC cells, and performing multiple polypeptide impacts by using the accurate polypeptide in the culture process; s5) continuing culturing after impact to obtain RFF cells; s6) stimulating the obtained RFF cells by taking the precise polypeptide as an antigen, and screening specific cells capable of identifying the precise polypeptide; s7) obtaining a TCR beta chain CDR3 region sequence of the specific cell through sequencing, and obtaining a TCR gene through amplifying the TCR beta chain CDR3 region sequence; s8), knocking out the original TCR gene and surface immunosuppressive signal molecule in the cell, and transferring the TCR gene which is obtained in the step S7 and can be specifically combined with the precise polypeptide to prepare the RFFT1 cell, wherein the immunosuppressive signal molecule comprises: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4(CD 244).

2. The method of constructing RFFT1 cells of claim 1,

the tumor cells are derived from commercial engineering cell lines and comprise 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 and G422 mouse glioma cells.

3. The method of constructing RFFT1 cells of claim 1,

the prediction of the antigen epitope is that a mutated amino acid site is used as a center, 10 amino acids are extended towards two sides, and a section of polypeptide with 21 amino acids is used as a potential antigen epitope.

4. The method for constructing RFFT1 cells according to claim 1, wherein the polypeptide impact is repeated 3 to 4 times in step S4.

5. The method for constructing RFFT1 cells of claim 1, wherein the polypeptide impact is performed with a polypeptide solution having a concentration of 10 μ g/mL to 100 μ g/mL.