CN109294998B

CN109294998B - RFF1 cell

Info

Publication number: CN109294998B
Application number: CN201811153225.5A
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: 2018-09-30
Publication date: 2020-11-06
Anticipated expiration: 2038-09-30
Also published as: CN109294998A

Abstract

The invention discloses and provides an RFF1 cell, and belongs to the technical field of biology. The invention discloses and provides an RFF1 cell for cellular immunotherapy, which is a super T cell with both attack and defense, and has high accuracy and high killing rate. The cell is prepared by the following 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; performing expanded culture after impact to obtain FF cells; so that the tumor mutated polypeptide is used as antigen to directly stimulate the FF cell to screen precise polypeptide; knocking out immunosuppressive signaling molecules on PBMC cells; and loading PBMC with the accurate polypeptide, mixing the PBMC with the immune inhibitory signal molecule knocked out, co-culturing, and performing multiple impacts with the accurate polypeptide in the co-culturing process to obtain the RFF1 cell. The cells can be used in cellular immunotherapy technology.

Description

RFF1 cell

Technical Field

The invention relates to the technical field of biology, in particular to an RFF1 cell for cellular immunotherapy and a preparation method thereof.

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 human body cells are reformed by a special method in the in vitro culture process, and can effectively kill the tumor cells in the body of a patient. 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.

Disclosure of Invention

The invention aims to provide an RFF1 cell for cellular immunotherapy, which is combined with polypeptide impact and in-vitro co-culture to transform a common T cell into a super T cell with both attack and defense, so that the problem that the attack and defense of immune cells in the cellular immunotherapy cannot be considered is solved, and the RFF1 cell has high accuracy and high killing rate.

The invention provides an RFF1 cell for cellular immunotherapy, wherein the preparation method of the RFF1 cell comprises the following steps:

s1) loading the polypeptide causing tumor mutation to the PBMC cells, and then carrying out one-time polypeptide impact on the PBMC cells loaded with the polypeptide;

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) knocking out immunosuppressive signaling molecules on PBMC cells, said signaling molecules comprising: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGIT, 2B4(CD 244);

s5) loading PBMC cells with the screened precise polypeptide, mixing with the PBMC cells with the immunosuppressive signal molecules knocked out, and co-culturing;

s6), performing multiple polypeptide impacts by using the precise polypeptide in the co-culture process;

s7) followed by the impact, RFF1 cells were obtained.

Further, step S2 includes:

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

after culturing for a period of time, transferring the cells to a cell culture device containing a culture solution OKM-100+ 12% FBS for continuous culture;

after culturing for a certain period of time, the cells were transferred to a cell culture apparatus containing the culture medium OKM-200+ 5% FBS for further culturing.

Further, in step S5, the PBMC cells loaded with the polypeptide and the PBMC cells knocked out of the immunosuppressive signal molecule are mixed in a ratio of 1: 1-1: 20.

Further, in step S6, the polypeptide impact is repeated 3-4 times during the co-cultivation process.

Further, in step S6, polypeptide impact is performed every 3 to 4 days.

Further, in step S6, the polypeptide impacting with the precision polypeptide during co-cultivation comprises: mixing the PBMC loaded with the mutant polypeptide and the PBMC of which the immunosuppressive signal molecules are knocked out, culturing in a cell culture device paved with cell stimulating factors OKM-25 in advance, and performing polypeptide impact on the polypeptide by using the accurate polypeptide obtained in the step S3 after culturing for a period of time;

and (4) transferring the cells to a cell culture device containing a culture solution OKM100+ 12% FBS for continuous culture, and performing polypeptide impact on the polypeptides obtained in the step S3 every 3-4 days.

Further, in step S7, the culturing after the impact to obtain RFF1 cells comprises: the polypeptide was cultured continuously in a cell culture apparatus containing a culture solution of OKM200+ 5% FBS after the impact to obtain RFF1 cells.

Further, immunosuppressive signal molecules on PBMC cells are knocked out using CRISPR technology.

Further, the tumor-causing mutant polypeptide is synthesized by the following method:

1) exon sequencing

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;

2) epitope prediction

Taking the mutated amino acid site as the center, extending 10 amino acids to two sides to obtain a section of polypeptide with 21 amino acids as a potential antigen epitope;

the potential epitope is considered as the epitope when IC50 is less than 1000 nM;

3) synthetic polypeptides

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

Further, the tumor cells are derived from engineering cell lines, and the engineering cell lines 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.

The invention has the following beneficial effects:

the invention provides an RFF1 cell for cellular immunotherapy, which is a super T cell with both attack and defense. By screening effective accurate polypeptide and performing secondary polypeptide impact on PBMC cells, T cells with specific killing effect are obtained, and the killing efficiency is higher.

In the prior art, T cells are generally presented through DC cells to generate specific killed T cells; or using virus as carrier, inducing T cell specific killing by slow virus transfection technology. In the present application, specific killing of T cells is induced by a secondary polypeptide impact technique. Firstly, carrying out first polypeptide impact, and directly stimulating PBMC (peripheral blood mononuclear cell) by using mixed polypeptide; PBMC are then stimulated directly with the precision polypeptide and, during co-culture, multiple times with the precision polypeptide. Ordinary T cells are transformed into super T cells with more accurate killing capacity through multiple stimulations. Compared with lentivirus transfection, the method is simple and convenient and has high safety. In addition, the target knockout protection technology, in-vitro co-culture and in-vitro amplification are combined, the adaptability of the T cells to a complex tumor microenvironment is improved, and multiple tumor species are covered.

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.

FIG. 1 shows a schematic flow chart of a method for preparing RFF1 cells according to an embodiment of the invention;

figure 2 shows antigen loading efficiency detection;

FIG. 3 shows the results of a precision polypeptide screen;

FIG. 4 shows the flow assay specific T cell ratios;

FIG. 5 shows flow-based detection of knockdown of an immunosuppressive signaling molecule;

fig. 6 shows the killing effect of RFF1 cells on tumors;

FIG. 7 shows the release of the cytokine IFN-. gamma.by RFF1 cells;

figure 8 shows the tumor-bearing mouse survival curve.

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 cells, DC cells, CIK cells and DC-CIK cells have been proved to be ineffective basically. The embodiment of the invention provides a novel T cell which is used for cellular immunotherapy.

The embodiment of the invention provides an RFF1 cell for cellular immunotherapy, wherein the preparation method of the RFF1 cell comprises the following steps:

s2) expanding culture after impact to obtain FF cells;

s6), in the co-culture process, carrying out multiple impacts by using the accurate polypeptide;

s7) followed by the impact, RFF1 cells were obtained.

Definitions the meaning involved in "RFF 1" cells is as follows:

r-precise polypeptide secondary impact technology;

FF-mixed polypeptide technology;

1-target knockout protection technology.

Herein, the "RFF 1" cell is a T cell obtained by modifying through combining an accurate polypeptide secondary impact technology, a mixed polypeptide technology and a target knockout protection technology. Wherein, the 'FF' cell is a T cell obtained by a mixed polypeptide technology without the transformation of precise polypeptide secondary impact (FF scheme). "RFF" cells are T cells engineered by precision polypeptide secondary impact (RFF protocol).

The embodiment of the invention provides RFF1 cells for cellular immunotherapy, which screens out real effective accurate polypeptide and uses the polypeptide to carry out secondary polypeptide impact on PBMC cells to obtain 'super T cells' with both attack and defense, and the specific killing effect is stronger. On the basis, the target spot knockout protection technology is combined, the adaptability of the T cell to the in vivo immunosuppressive microenvironment is improved, and the self protection of the T cell is enhanced.

Prior to 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

Carrying out whole exon sequencing by using tumor cells, 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 engineering cell lines 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. 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: NetMHCpan 3.0, PickPocket, Artificial Neural Networks (ANN)). A potential epitope is considered to be an "epitope" if IC50 < 1000 nM.

3) Synthetic polypeptides

In step S1, the PBMC cells loaded with the polypeptide are directly stimulated by polypeptide-impacting with the mixed polypeptide causing tumor mutation to perform the first stimulation.

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:

the polypeptide impact is carried out by dissolving the mixed polypeptide in RPMI1640+ 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.

Furthermore, the time for polypeptide impact may be 1 to 4 hours, for example, 1 hour, 3 hours, or 4 hours, preferably 4 hours.

In step S2, the culture is expanded after the impact to obtain FF cells. The PBMC cells after the polypeptide impact are co-cultured with a cell stimulating factor OKM-25 to obtain FF cells.

Step S2 may specifically be:

after culturing for a while, the cells were transferred to a cell culture apparatus containing the culture medium OKM-200+ 5% FBS for further culturing.

Specifically, FF cell compositions can be obtained after extensive culture. T cells (i.e., FF cells) can be isolated from the FF cell composition by centrifugation.

In step S3, the T cells are directly stimulated with the polypeptide as an antigen to screen for the precise polypeptide.

The screening criteria for the precise polypeptide were:

taking FF cells as a baseline, and repeating the FF cells for two times independently, wherein the high detection value is a high baseline, and the low detection value is a low baseline;

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.

Step S3 may specifically include:

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: RPMI1640+ 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:

polypeptide as antigen with FF cells as baseline;

each group of experiments comprises two baselines, namely a high baseline and a low baseline (the high detected value is the high baseline, and the low detected value is the low baseline), the difference of the two baselines is a system error, and the detected values > the low baseline, > the high baseline and > the high baseline + the system error are respectively marked during data analysis; and (5) the detection value is greater than the high baseline and the system error is the effective accurate polypeptide.

In step S4, immunosuppressive signal molecules on PBMC cells are knocked out using CRISPR technology. The signal molecule comprises: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGHT, 2B4(CD 244).

Specifically, a CRISPR lentiviral vector for knocking out the immunosuppressive signal molecule is constructed so as to infect the PBMC cell, and the PBMC cell for knocking out the immunosuppressive signal molecule is obtained.

The detailed operation may specifically include the following steps:

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

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

5) resuscitating PBMC with RPMI1640+ 10% FBS, and performing CRISPR lentivirus infection from day 1 to day 4;

6) after infection, PBMC with knockout of immunosuppressive signaling molecules are obtained by culturing in RPMI1640+ 10% FBS for 0-3 days.

In step S5, the PBMC cells are loaded with the precision polypeptide selected in step S3 and contacted with the PBMC cells knocked out for immunosuppressive signaling molecules at a ratio of 1: 1-1: the mixing ratio is 20, and preferably 1: 10.

In step S6, polypeptide impact is performed again with the precision polypeptide. The impact time may be 1-4 hours.

Compared to step S1, an additional amount of precision polypeptide impact is used in step S6. That is, the co-cultured cells are subjected to multiple polypeptide shock stimulations with the selected precision polypeptide. In the co-culture process, the polypeptide impact can be repeated for 3-4 times.

Further, in step S6, the polypeptide impact may be performed every 3 to 4 days for 1 to 4 hours each time.

For example, PBMC loaded with the precision polypeptide and PBMC knocked out of immunosuppressive signal molecules are mixed and cultured in a cell culture device pre-paved with cell stimulating factor OKM-25, and after a period of time, polypeptide impact is performed with the precision polypeptide obtained in step S3;

and (4) transferring the polypeptide after the polypeptide is impacted to a cell culture device containing a culture solution OKM100+ 12% FBS for continuous culture, and respectively impacting the polypeptide by using the accurate polypeptide obtained in the step S3 every 3-4 days.

In step S7, the culture was continued after the impact to obtain RFF1 cells. The method specifically comprises the following steps: the polypeptide was cultured continuously in a cell culture apparatus containing a culture solution of OKM200+ 5% FBS after the impact to obtain RFF1 cells.

The invention provides an RFF1 cell for cellular immunotherapy through a cell in-vitro preparation method, and the RFF1 cell is a super T cell with both attack and defense. In the preparation process, the truly effective and accurate polypeptide is screened out and is used for carrying out secondary impact on PBMC cells. The mixed polypeptide is used for polypeptide impact for the first time, and the screened accurate polypeptide is used for polypeptide impact for multiple times for the second time, so that the RFF cell with specific killing effect is obtained, and the RFF cell is high in accuracy and killing power. In the preparation process, the adaptability of the obtained RFF1 cell to the complex tumor microenvironment in the body of a patient is enhanced through a target knockout protection technology, in-vitro co-culture and in-vitro amplification.

The following is a further detailed description of the RFF1 cells for cellular immunotherapy according to the embodiments of the present invention.

(one) sequencing of all exons

1) Performing whole exon sequencing by using LLC mouse Lewis lung cancer cells;

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: NetMHCpan 3.0, 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) loading the PBMC with the mutant polypeptide and subjecting the PBMC loaded with the polypeptide to polypeptide impact

1) Preparing a polypeptide solution: dissolving the polypeptides by RPMI1640+ 10% FBS, wherein the final concentration of each polypeptide is 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) impact into a cell culture plate;

5)37℃、5％CO₂and impacting for 4h to obtain impacted PBMC cells for later use.

(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 cell culture plate or a culture flask 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, transferring the co-cultured cells to a large culture flask according to the cell density on the 5 th day, supplementing fresh culture solution OKM-100+ 12% FBS, and placing 20mL of the co-cultured cells in a T75 culture flask;

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

6) on day 10 of co-culture, the medium was OKM-200+ 5% FBS, and the co-cultured cells were transferred from one of the T25 flasks to a T75 large flask;

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

(VI) polypeptide as antigen for directly stimulating FF cell to screen precise polypeptide

1) Centrifugation of FF cell compositionsT cells, namely FF cells were extracted. Centrifuging at 1500rpm for 5min to collect T cells, adding 10mL PBS to resuspend the cells and counting, centrifuging at 1500rpm for 5min, collecting T cells, resuspending with 1640+ 10% FBS +200U/mL IL2, and adjusting the count to 1 × 10⁶Per mL;

3) setting a positive control: FF cells +100ng/mL OKT 3; negative control: 1640+ 10% FBS +200U/mLIL 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) and centrifuging the 96-well plate at 1500rpm for 10min, and taking a sample for ELISA detection or storing the sample at-80 ℃.

(VII) accurate polypeptide evaluation standard:

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

2) polypeptide as antigen with FF cells as 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) knocking out immunosuppressive signal molecules on PBMC by using CRISPR technology

1) PBMC surface immunosuppressive signal molecules include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, TIGHT, 2B4(CD 244);

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

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;

6) resuscitating PBMC with 1640+ 10% FBS, and performing CRISPR lentivirus infection from day 1 to day 4;

7) after infection, the PBMC with the knockout immunosuppressive signal molecule is obtained by culturing in 1640+ 10% FBS for 0-3 days.

Preparation of (nine) RFF1 cells

2) PBMCs loaded with precision polypeptides and PBMCs knock-out of immunosuppressive signal molecules were mixed at a ratio of 1:10, and transferring the mixture to a culture bottle pre-paved with OMK 25;

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

4) culturing until 3 days, and performing re-impacting of the precise polypeptide;

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

6) after 4h of impact, the cells were transferred to a T25 flask supplemented with OKM100+ 12% FBS and 5% CO at 37 ℃₂Culturing, transferring to T75 flask according to cell growth condition, and maintaining cell density at 1 × 10⁶Per mL;

7) repeating steps 5) and 6) at 7, 10 and 14 days of culture);

8) when the cells enter a T75 culture flask, the culture medium is OKM200+ 5% FBS, and the RFF1 cells can be obtained after culturing for 10-21 days.

(Ten) tumor-bearing mouse survival experiments

1) And (4) inoculating the tumor 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 RFF1 cells 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 2-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 55.4% (FL 2-H55.4% means 55.4% of the cells stained with PI stain).

3. Screening for accurate Polypeptides

As shown in FIG. 3, 13 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 polypeptides No. 6 and No. 9 is greater than high baseline and systematic error, and the polypeptide belongs to effective and accurate polypeptides.

4. Specific T cell ratio detection

The screened polypeptide No. 9 is stimulated for multiple times on the basis of FF cells, and after culture, the proportion of T cells specific to the precise polypeptide is detected in a flow mode, and the result is shown in figure 4, and specific T cells are shown in a box: compared with the untreated control group, the RFF1 cells have the IFN-gamma releasing cell rate caused by the polypeptide No. 9 after multiple impact stimulation, which is obviously higher than that of the cells without multiple impact stimulation (FF cells). Sorting of CD8+ IFN- γ + cells (in boxes) was performed by flow cytometry.

5. Knockout of immunosuppressive signaling molecules

Inhibitory signal molecules on PBMCs were knocked out using CRISPR technique, and the results are shown in figure 5.

Killing of target cells by RFF1 cells

The killing efficiency of the RFF1 cells of the embodiment of the present invention on the target cells derived from the mutant epitope was tested, and the untreated cells were used as a control (Mock), and the results are shown in fig. 6, where the RFF1 cells of the embodiment of the present invention all had a certain killing effect on the target cells, in the range of 20: 1 and 40: 1 (effector cells: target cells), the difference from the Mock group was significant (RFF 1-1: precise polypeptide re-strike on

days

3 and 10 in the ninth step; RFF 1-2: precise polypeptide re-strike on

days

3, 7, 10 and 14 in the ninth step).

Detection of cytokine Release by RFF1 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. 7 shows that the detection of IFN-gamma released when the cells are co-cultured with the tumor cells in different culture modes shows that: the RFF1 cells of the example of the invention produced significant amounts of IFN- γ after co-culture with tumor cells, compared to IFN- γ produced by the effector cells themselves (effector cells only), demonstrating that: the knockout of the inhibitory target can more effectively improve the antitumor ability (RFF 1-1: precise polypeptide re-strike on

days

3 and 10 in the ninth step; RFF 1-2: precise polypeptide re-strike on

days

3, 7, 10 and 14 in the ninth step).

8. Experiment on survival of tumor-bearing mice

The results are shown in fig. 8, and the reinfusion of RFF1 cells of the present example had a significant effect on the improvement in survival of tumor-bearing mice.

9. Clinical cases

The administration process comprises the following steps:

the first course of treatment: RFF1 cells at 1X 10 times per month ⁹2 times for each cell;

the second course of treatment: RFF1 cells at a rate of 1X 10/min ⁹2 cells in total.

TABLE 2

Numbering	Sex	Age (age)	Disease diagnosis	Progression-free survival time after completion of administration
					1	Woman	67	Ovarian cancer	2016.4-Up to now
2	Woman	61	Stomach cancer	2017.11-Up to now
					3	For male	63	Stomach cancer	2017.11-Up to now

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. An RFF1 cell for cellular immunotherapy, wherein the RFF1 cell is prepared by the following steps:

s1) Synthesis of a tumor-mutagenizing polypeptide

1) Exon sequencing

Sequencing the whole exon of the tumor cell;

2) epitope prediction

Taking a mutated amino acid site as a center, extending 10 amino acids to two sides, and taking a section of polypeptide with 21 amino acids as a potential antigen epitope;

3) synthetic polypeptides

Synthesizing antigen epitope peptide by using a polypeptide solid phase synthesis method, and marking the antigen epitope peptide as a polypeptide causing tumor mutation;

the PBMC cells are loaded with the polypeptide causing the 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; wherein the accurate polypeptide evaluation standard is as follows: 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;

s5) loading PBMC with the screened precise polypeptide, mixing with PBMC cells with the immunosuppressive signal molecules knocked out, and co-culturing;

s6) in the co-culture process, performing multiple polypeptide impacts by using the accurate polypeptide, and repeating the polypeptide impacts for 3-4 times;

s7) to obtain RFF1 cells.

2. The RFF1 cell for cellular immunotherapy, according to claim 1, wherein the expanding culture after impact to obtain FF cells in step S2 comprises:

3. The RFF1 cell for cellular immunotherapy, according to claim 1, wherein the PBMC cells loaded with the precision polypeptide and the PBMC cells knocked out with immunosuppressive signal molecule are mixed at a ratio of 1: 1-1: 20 in step S5.

4. The RFF1 cell for cellular immunotherapy, according to claim 1, wherein in step S6, the polypeptide impact is performed every 3-4 days.

5. The RFF1 cell for cellular immunotherapy, according to claim 1, wherein the polypeptide impacting with the precise polypeptide during the co-culture process in the step S6 comprises:

mixing PBMC loaded with the accurate polypeptide and PBMC of which immunosuppressive signal molecules are knocked out, culturing in a cell culture device paved with cell stimulating factors OKM-25 in advance, and performing polypeptide impact on the accurate polypeptide obtained in the step S3 after culturing for a period of time;

6. The RFF1 cell for cellular immunotherapy, according to claim 1, wherein an immunosuppressive signal molecule on PBMC cells is knocked out using CRISPR technology.

7. The RFF1 cell for cellular immunotherapy, wherein the tumor cell is derived from an engineered cell line comprising 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.