Method for improving adaptability of T cells to tumor microenvironment

Abstract

The invention relates to the technical field of medicine and health, in particular to a method for improving the adaptability of T cells to a tumor microenvironment, and the key point of the technical scheme is that the method comprises the following steps: S1, preparing tumor neoantigen mRNA; s2, collecting and separating PBMCs (peripheral blood mononuclear cells) to obtain initial T cells; s3, inducing by using the initial T cells to obtain DC cells; s4, modifying the DC cells by using the tumor neoantigen mRNA; s5, carrying out electric transfer knockout on the PD-1 of the initial T cell through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9); and S6, adding the modified DC cells into the T cells of which the PD-1 is knocked out, and carrying out co-incubation amplification, so as to obtain the treated T cells. The tumor neoantigen mRNA is used for modifying the DC cell, the CRISPR-Cas9 is combined for modifying the T cell, then the DC cell and the T cell are incubated and amplified together, and the method has the advantages that the safety is improved, and the adaptability of the T cell to the tumor microenvironment is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine and health, and more specifically, to a method for improving the adaptability of T cells to tumor microenvironment. Background Art

[0002] Neoantigens are new antigens encoded by mutated genes in tumor cells. They are new abnormal proteins that are different from those expressed by normal cells, mainly produced by gene point mutations, deletion mutations, gene fusions, etc. The polypeptide fragments formed by these proteins after enzymatic hydrolysis are presented to T cells through DC cells as antigens, which can promote T cells to become mature activated T cells that specifically recognize neoantigens and proliferate the number of these activated T cells. By utilizing the immune activity of neoantigens, synthetic neoantigens are designed according to the mutation of tumor cells, and immune cells such as T cells, NK cells and macrophages are genetically modified in vitro to produce neoantigen immune cells, and patients are injected with immune to achieve the effect of treatment.

[0003] PD-L1 is programmed death ligand 1, which is the ligand of programmed death receptor 1 (PD-1). Under normal circumstances, T cells have the function of killing cancer cells. However, PD-L1 is expressed on the surface of cancer cells, which binds to PD-1 on the surface of T cells, reducing the activity of T cells, inducing T cell apoptosis and disintegration, and inhibiting T cell proliferation. Cancer cells can save themselves and thus achieve immune escape.

[0004] Based on this principle, there are currently inhibitors targeting the PD-1 immune checkpoint on the market. This drug blocks the immune escape mechanism caused by the tumor, thereby activating the body's own immune cells and killing tumor cells to achieve the purpose of treating the tumor. Although PD-1 inhibitors are effective immunotherapy drugs, they may cause a variety of side effects during use, ranging from mild skin reactions to severe immune-related inflammatory reactions. At the same time, traditional neoantigen immunotherapy is subject to the dual restrictions of HLA (human leukocyte antigen) and antigen expression, which limits the patients suitable for neoantigen immunotherapy. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for improving the adaptability of T cells to the tumor microenvironment, which has the advantages of improving safety and adaptability to the tumor microenvironment.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: a method for improving the adaptability of T cells to the tumor microenvironment, comprising the following steps:

[0007] S1. Preparation of tumor neoantigen mRNA;

[0008] S2, collect and separate PBMC to obtain initial T cells;

[0009] S3, using the initial T cells to induce DC cells;

[0010] S4, using the tumor neoantigen mRNA to modify the DC cells;

[0011] S5, knocking out PD-1 of the initial T cells by CRISPR-Cas9 electroporation;

[0012] S6. Add the modified DC cells to the T cells in which PD-1 has been knocked out, incubate and expand them together to obtain treated T cells.

[0013] In one embodiment, the S1 comprises the following steps:

[0014] S11. Design and construct an expression vector based on the tumor neoantigen gene sequence, and obtain a recombinant plasmid after codon optimization;

[0015] S12, linearizing the recombinant plasmid using an endonuclease, and then purifying and recovering the plasmid to obtain a linearized plasmid;

[0016] S13. Using an in vitro transcription reagent, perform in vitro transcription on the linearized plasmid, and then purify and recover it to obtain the tumor neoantigen mRNA.

[0017] In one embodiment, S2 comprises the following steps:

[0018] S21, collect peripheral blood, add lymph separation fluid, centrifuge, aspirate buffy coat cells, add physiological saline to resuspend, centrifuge and discard supernatant, then add culture medium to resuspend, and count cells;

[0019] S22, adding culture medium again for incubation, collecting the upper suspended cells, centrifuging and discarding the supernatant, then adding culture medium for resuspending, counting the cells, and obtaining the initial T cells.

[0020] In one embodiment, the cell ratio of the initial T cells in S3 to the initial T cells in S5 is 1:(3-4.5).

[0021] In one embodiment, the S3 comprises the following steps:

[0022] S31, adding culture medium to the initial T cells, and adding IL-4 and GM-CSF for culturing;

[0023] S32, replace the culture medium, add IL-4 and GM-CSF and continue culturing;

[0024] S33, then add TMF-α and continue culturing to obtain the DC cells.

[0025] In one embodiment, the final concentrations of cytokines in S3 are: IL-4 is 40-60 ng / ml, GM-CSF is 50-150 ng / ml, and TMF-α is 200-300 ng / ml.

[0026] In one embodiment, the S4 comprises the following steps: counting the DC cells, and every 1×10 7 cells, adding 25-30ug of the tumor-specific antigen mRNA, electroporating, and then transferring to a culture medium containing recombinant human IL-2 protein for culture, to obtain the modified DC cells;

[0027] The electroporation transduction modification operation of the DC cells occurs on the 7th to 8th day of their culture.

[0028] In one embodiment, the S5 comprises the following steps:

[0029] S51, mixing Cas9 protein and sgRNA to obtain a Cas9 protein mixture;

[0030] S52, taking the initial T cells, each 1×10 7 The cells are added with 18-25ug of the Cas9 protein mixture, electroporated, and then inoculated into a culture bottle, and anti-human CD3 monoclonal antibody is added for culture to obtain the PD-1 knocked-out T cells.

[0031] In one embodiment, the voltage of the electric shock transduction in S4 and S5 is 360V, and the transient time is 1ms.

[0032] In one embodiment, the S6 includes the following steps: adding the modified DC cells to the T cells from which PD-1 has been knocked out, mixed culturing for 8-10 days, sampling and testing, and rehydration culture to obtain the treated T cells.

[0033] The above method for improving the adaptability of T cells to the tumor microenvironment has the following beneficial effects:

[0034] First, using tumor neoantigen mRNA to modify DC cells and using CRISPR-Cas9 to modify T cells, while improving the efficiency of DC cell transfection and PD-1 knockout, the modified DC cells and T cells are co-incubated and amplified, which can enhance process versatility, reduce production costs and improve safety;

[0035] Second, the cells obtained through culture have strong adaptability to the tumor microenvironment;

[0036] Third, the T cells treated by the present invention have specific immunogenicity for each patient, which can maximize the inclusion of tumor patients and stimulate the autologous immune system to attack tumor cells, allowing more patients to benefit from personalized treatment in the long term. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a flow cytometry result diagram of DC cells in Example 1;

[0038] Figure 2 This is a graph showing the detection result of the T cell PD-1 knockout efficiency in Example 1;

[0039] Figure 3 This is a comparison chart of the determination of γ-interferon content in the cytotoxic experiment of Example 1, Comparative Example 1, Comparative Example 2 and common T cells. DETAILED DESCRIPTION

[0040] The present invention is described in further detail below in conjunction with the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. When there is a conflict, the definitions in this specification shall prevail.

[0041] As used herein, the term "prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0042] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0043] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0044] A method for improving the adaptability of T cells to a tumor microenvironment, comprising the following steps:

[0045] S1. Preparation of tumor neoantigen mRNA;

[0046] S2, collect and separate PBMC to obtain initial T cells;

[0047] S3, using initial T cells to induce DC cells;

[0048] S4. Modify DC cells using tumor neoantigen mRNA;

[0049] S5, knocking out PD-1 in naive T cells by CRISPR-Cas9 electroporation;

[0050] S6. Add the modified DC cells to the T cells in which PD-1 has been knocked out, incubate and expand them together to obtain treated T cells.

[0051] The present invention uses tumor neoantigen mRNA to modify DC cells and CRISPR-Cas9 to modify T cells. While improving the DC cell transfection efficiency and PD-1 knockout efficiency, the modified DC cells and T cells are co-incubated and amplified, which can enhance the versatility of the process, reduce production costs and improve safety. The cultured cells have strong adaptability to the tumor microenvironment.

[0052] In addition, the T cells treated by the present invention have specific immunogenicity for each patient, which can maximize the inclusion of tumor patients and stimulate the autologous immune system to attack tumor cells, allowing more patients to benefit in the long term through personalized treatment.

[0053] Specifically, S1 includes the following steps:

[0054] S11. Design and construct an expression vector based on the tumor neoantigen gene sequence, and obtain a recombinant plasmid after codon optimization;

[0055] In actual production and preparation, the tumor neoantigen gene sequence is obtained based on genetic testing of the target tumor patient.

[0056] S12, linearizing the recombinant plasmid using an endonuclease, and then purifying and recovering the plasmid to obtain a linearized plasmid;

[0057] Furthermore, the endonucleases include the endonuclease EcoRI and the endonuclease BamHI. At room temperature, a PCR tube is added with the recombinant plasmid and the endonuclease, and the plasmid is linearized in a water bath, and then purified and recovered using a PCR purification kit to obtain a linearized plasmid.

[0058] S13, using an in vitro transcription reagent to perform in vitro transcription on the linearized plasmid, and then purify and recover it to obtain tumor neoantigen mRNA;

[0059] In actual production, the linearized plasmid is mixed with the reagents required for in vitro transcription, and then in vitro transcription is performed at room temperature. The mRNA purification kit is then used to purify and recover the mRNA to obtain tumor neoantigen mRNA.

[0060] Specifically, S2 includes the following steps:

[0061] S21, collect peripheral blood, add lymph separation fluid, centrifuge, aspirate buffy coat cells, add physiological saline to resuspend, centrifuge and discard supernatant, then add culture medium to resuspend, and count cells;

[0062] Furthermore, the peripheral blood collection volume is 80-100 ml, and the mixing ratio of peripheral blood and lymph separation fluid is 2:1.

[0063] S22, adding culture medium again for incubation, collecting the upper suspended cells, centrifuging and discarding the supernatant, then adding culture medium for resuspending, counting the cells, and obtaining initial T cells;

[0064] In actual production and preparation, according to 1×10 6 The cells were added into 581 culture medium at a density of cells / ml, and then transferred into T175 culture flasks and incubated in a 37°C, 5% CO2 incubator. The initial T cells obtained included adherent cells and suspension cells.

[0065] Specifically, the cell ratio of the initial T cells in S3 to the initial T cells in S5 is 1: (3-4.5). Under this ratio, the harvested T cells and DC cells have a better antigen presentation effect.

[0066] Specifically, S3 includes the following steps:

[0067] S31, adding the culture medium to the initial T cells, and adding IL-4 and GM-CSF for culture;

[0068] In actual production and preparation, some of the collected T cells were divided into 1×10 6 The cells were cultured in 581 medium at a density of 1:1 cells / ml, and IL-4 (40-60 ng / ml) and GM-CSF (50-150 ng / ml) were added at the final concentrations. The cells were cultured in a 37°C, 5% CO2 incubator.

[0069] S32, replace the culture medium, add IL-4 and GM-CSF and continue culturing;

[0070] The specific operation of replacing the culture medium is as follows: on the third day of culture, discard the original culture medium, re-add an equal volume of 581 culture medium, and then add IL-4 (40-60ng / ml) and GM-CSF (50-150ng / ml) at the final concentration, and culture in a 37°C, 5% CO2 incubator.

[0071] S33, then add TMF-α and continue culturing to obtain DC cells;

[0072] In actual production preparation, TMF-α (200-300 ng / ml) is added at a final concentration on the 5th to 6th day of culture and cultured in a 37°C, 5% CO2 incubator.

[0073] By setting the final concentrations of IL-4 (40-60 ng / ml), GM-CSF (50-150 ng / ml) and TMF-α (200-300 ng / ml), it is easier to induce high-purity DC cells.

[0074] Specifically, S4 includes the following steps: counting DC cells, and every 1×10 7 Add 25-30ug of tumor-specific antigen mRNA to cells, perform electroporation transduction, and then transfer to a culture medium containing recombinant human IL-2 protein to obtain modified DC cells;

[0075] The electroporation transduction modification of DC cells occurred on the 7th to 8th day of their culture.

[0076] Specifically, S5 includes the following steps:

[0077] S51, mixing Cas9 protein and sgRNA to obtain a Cas9 protein mixture;

[0078] It should be further explained that the mixing mass ratio of Cas9 protein and sgRNA is 1:1.

[0079] S52, take the initial T cells, each 1×10 7The cells were added with 18-25ug of Cas9 protein mixture, electroporated, and then inoculated into culture flasks, and anti-human CD3 monoclonal antibodies were added for culture to obtain T cells with PD-1 knockout.

[0080] In actual production and preparation, the final concentration of anti-human CD3 monoclonal antibody culture is 50 ng / ml.

[0081] Specifically, the voltage of the electric shock transduction in S4 and S5 was 360 V and the transient time was 1 ms.

[0082] Specifically, S6 includes the following steps: adding the modified DC cells to the T cells in which PD-1 has been knocked out, co-culturing for 8-10 days, sampling and testing, and rehydration culture to obtain the treated T cells;

[0083] The specific operation of mixed culture is: culture in a 37°C, 5% CO2 incubator for 8-10 days, then take samples to detect the DC cell ratio and T cell PD-1 knockout efficiency, and replenish fluid depending on the cell production situation, and finally obtain processed T cells.

[0084] The following is a specific example, in which the plasmid was purchased from Guangzhou Aigi Biotechnology, and the Cas9 protein and sgRNA were purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.

[0085] Example 1

[0086] Example 1 provides a method for improving the adaptability of T cells to the tumor microenvironment, comprising the following steps:

[0087] S1. Preparation of tumor neoantigen mRNA:

[0088] According to the tumor neoantigen gene sequence, an expression vector (WT1 non-small cell lung cancer neoantigen, polypeptide sequence: CMTWNQMNL) was designed and constructed, and the plasmid was codon optimized to obtain a recombinant plasmid;

[0089] At room temperature, take a PCR tube and add the recombinant plasmid, endonuclease EcoRI and endonuclease BamHI, and linearize the plasmid in a water bath at 37°C for 1 h, and then purify and recover it using a PCR purification kit (Quanshijin, EP101-01) to obtain a linearized plasmid;

[0090] The linearized plasmid was transcribed in vitro using an in vitro transcription reagent at 37°C in a water bath for 1 h, and then purified and recovered using an mRNA purification kit (Quanshijin, ER701-01) to obtain tumor neoantigen mRNA.

[0091] S2. Collect and separate PBMC to obtain initial T cells:

[0092] Collect 80-100 ml of peripheral blood, add lymph separation solution according to volume (peripheral blood: lymph separation solution = 2:1), centrifuge at 1100 g for 10 min, aspirate the buffy coat cells, add physiological saline to resuspend, centrifuge at 500 g / min for 5 min, remove the supernatant, add 1 ml of 581 culture medium to resuspend, and count the cells;

[0093] Then according to 1×10 6 Cells / ml were added into 581 medium and transferred into a T175 culture flask. The cells were incubated at 37°C with 5% CO2 for 2 h. The upper suspended cells were collected and centrifuged at 500 g for 5 min. The supernatant was discarded and 1 ml of 581 medium was added for resuspending. The cells were counted to obtain initial T cells, which included adherent cells and suspended cells.

[0094] S3. Use initial T cells to induce DC cells:

[0095] The adherent cells obtained in S2 were cultured at a volume of 1 × 10 6 The cells were added to 581 medium at a density of cells / ml, and IL-4 (40-60 ng / ml) and GM-CSF (50-150 ng / ml) were added at the final concentrations, and cultured in a 37°C, 5% CO2 incubator;

[0096] On the third day, the culture medium was discarded, and an equal volume of 581 culture medium was added, and IL-4 (40-60 ng / ml) and GM-CSF (50-150 ng / ml) were added at the final concentrations, and the cells were placed in a 37°C, 5% CO2 incubator for continued culture;

[0097] On day 5-6, TMF-α was added at a final concentration of 200-300 ng / ml and cultured in a 37°C, 5% CO2 incubator to obtain DC cells.

[0098] S4. Modification of DC cells using tumor neoantigen mRNA:

[0099] On days 7-8, DC cells were collected and counted at 1×10 7 25-30ug of tumor-specific antigen mRNA was added to the cells, and electroporation was performed at 360V for 1ms in an electroporator. The cells were then transferred to a T175 culture flask containing 20ml of 1000IU recombinant human IL-2 protein and 581 culture medium, and cultured in a 37°C, 5% CO2 incubator for 4h to obtain modified DC cells.

[0100] S5. Knockout of PD-1 in naive T cells by CRISPR-Cas9 electroporation:

[0101] Mix Cas9 protein and sgRNA at a mass ratio of 1:1 to obtain a Cas9 protein mixture;

[0102] Take the upper layer of suspended cells obtained in S2 (3-4.5 times the number of adherent cells used in S3) and add 1×10 7 Add 18-25ug of Cas9 protein mixture to cells, perform electroporation at 360V, 1ms in an electroporator, and then press 1×10 6 The cells were inoculated at a density of cells / ml into a T175 culture flask with 581 culture medium, and anti-human CD3 monoclonal antibody (50ng / ml) was added at a final concentration. The cells were cultured in a 37°C, 5% CO2 incubator. The cells were replenished with fluid depending on their growth. When the culture medium exceeded 500ml, the cells were transferred to a cell culture bag for culture to obtain PD-1 knocked-out T cells.

[0103] S6. Add the modified DC cells to the T cells in which PD-1 has been knocked out, incubate and amplify them together to obtain treated T cells:

[0104] On the 7th and 8th days, the modified DC cells were added to the T cells in which PD-1 had been knocked out, mixed, and cultured in a 37°C, 5% CO2 incubator for 8-10 days. Samples were taken to detect the DC cell ratio and the knockout efficiency of T cell PD-1, and the fluid was replenished depending on the cell production situation. When the culture medium exceeded 500 ml, it was transferred to a cell culture bag for culture, and the co-incubation and amplification were completed to obtain the treated T cells.

[0105] Comparative Example 1

[0106] Comparative Example 1 provides a method for improving the adaptability of T cells to the tumor microenvironment based on tumor neoantigen mRNA, comprising the following steps:

[0107] S1. Preparation of tumor neoantigen mRNA:

[0108] According to the tumor neoantigen gene sequence, an expression vector (WT1 non-small cell lung cancer neoantigen, polypeptide sequence: CMTWNQMNL) was designed and constructed, and the plasmid was codon optimized to obtain a recombinant plasmid;

[0109] At room temperature, take a PCR tube and add the recombinant plasmid, endonuclease EcoRI and endonuclease BamHI, and linearize the plasmid in a water bath at 37°C for 1 h, and then purify and recover it using a PCR purification kit (Quanshijin, EP101-01) to obtain a linearized plasmid;

[0110] The linearized plasmid was transcribed in vitro using an in vitro transcription reagent at 37°C in a water bath for 1 h, and then purified and recovered using an mRNA purification kit (Quanshijin, ER701-01) to obtain tumor neoantigen mRNA.

[0111] S2. Collect and separate PBMC to obtain initial T cells:

[0112] Collect 80-100 ml of peripheral blood, add lymph separation solution according to volume (peripheral blood: lymph separation solution = 2:1), centrifuge at 1100 g for 10 min, aspirate the buffy coat cells, add physiological saline to resuspend, centrifuge at 500 g / min for 5 min, remove the supernatant, add 1 ml of 581 culture medium to resuspend, and count the cells;

[0113] Then according to 1×10 6 Cells / ml were added into 581 medium and transferred into a T175 culture flask. The cells were incubated at 37°C with 5% CO2 for 2 h. The upper suspended cells were collected and centrifuged at 500 g for 5 min. The supernatant was discarded and 1 ml of 581 medium was added for resuspending. The cells were counted to obtain initial T cells, which included adherent cells and suspended cells.

[0114] S3. Use initial T cells to induce DC cells:

[0115] The adherent cells obtained in S2 were cultured at a volume of 1 × 10 6 The cells were added to 581 medium at a density of cells / ml, and IL-4 (40-60 ng / ml) and GM-CSF (50-150 ng / ml) were added at the final concentrations, and cultured in a 37°C, 5% CO2 incubator;

[0116] On the third day, the culture medium was discarded, and an equal volume of 581 culture medium was added, and IL-4 (40-60 ng / ml) and GM-CSF (50-150 ng / ml) were added at the final concentrations, and the cells were placed in a 37°C, 5% CO2 incubator for continued culture;

[0117] On day 5-6, TMF-α was added at a final concentration of 200-300 ng / ml and cultured in a 37°C, 5% CO2 incubator to obtain DC cells.

[0118] S4. Modification of DC cells using tumor neoantigen mRNA:

[0119] On days 7-8, DC cells were collected and counted at 1×10 7 25-30ug of tumor-specific antigen mRNA was added to the cells, and electroporation was performed at 360V for 1ms in an electroporator. The cells were then transferred to a T175 culture flask containing 20ml of 1000IU recombinant human IL-2 protein and 581 culture medium, and cultured in a 37°C, 5% CO2 incubator for 4h to obtain modified DC cells.

[0120] S5. T cell culture:

[0121] Take the upper layer of suspended cells obtained in S2 (3-4.5 times the number of adherent cells used in S3) and add 1×10 6 cells / ml were inoculated into a T175 culture flask with 581 culture medium, and anti-human CD3 monoclonal antibody (50ng / ml) was added at a final concentration. The cells were cultured in a 37°C, 5% CO2 incubator. The cells were replenished with fluid depending on the cell growth. When the culture medium exceeded 500ml, the cells were transferred to a cell culture bag for culture to obtain cultured T cells.

[0122] S6. Add the modified DC cells to the cultured T cells, incubate and amplify them together to obtain treated T cells:

[0123] On the 7th and 8th days, the modified DC cells were added to the cultured T cells, mixed, and cultured in a 37°C, 5% CO2 incubator for 8-10 days. Samples were taken to detect the DC cell ratio and T cell PD-1 knockout efficiency, and fluid was replenished depending on the cell production situation. When the culture medium exceeded 500 ml, it was transferred to a cell culture bag for culture, and co-incubation and amplification were completed to obtain treated T cells.

[0124] Comparative Example 2

[0125] Comparative Example 2 provides a method for improving the adaptability of T cells to the tumor microenvironment based on CRISPR-Cas9 technology, comprising the following steps:

[0126] S1. Collect and separate PBMC to obtain initial T cells:

[0127] Collect 80-100 ml of peripheral blood, add lymph separation solution according to volume (peripheral blood: lymph separation solution = 2:1), centrifuge at 1100 g for 10 min, aspirate the buffy coat cells, add physiological saline to resuspend, centrifuge at 500 g / min for 5 min, remove the supernatant, add 1 ml of 581 culture medium to resuspend, and count the cells;

[0128] Then according to 1×10 6 Cells / ml were added into 581 medium and transferred into a T175 culture flask. The cells were incubated at 37°C with 5% CO2 for 2 h. The upper suspended cells were collected and centrifuged at 500 g for 5 min. The supernatant was discarded and 1 ml of 581 medium was added for resuspending. The cells were counted to obtain initial T cells, which included adherent cells and suspended cells.

[0129] S2. Use initial T cells to induce DC cells:

[0130] The adherent cells obtained in S1 were cultured at a volume of 1 × 10 6The cells were added to 581 medium at a density of cells / ml, and IL-4 (40-60 ng / ml) and GM-CSF (50-150 ng / ml) were added at the final concentrations, and cultured in a 37°C, 5% CO2 incubator;

[0131] On the third day, the culture medium was discarded, and an equal volume of 581 culture medium was added, and IL-4 (40-60 ng / ml) and GM-CSF (50-150 ng / ml) were added at the final concentrations, and the cells were placed in a 37°C, 5% CO2 incubator for continued culture;

[0132] On day 5-6, TMF-α was added at a final concentration of 200-300 ng / ml and cultured in a 37°C, 5% CO2 incubator to obtain DC cells.

[0133] S3. Knockout of PD-1 in naive T cells by CRISPR-Cas9 electroporation:

[0134] Mix Cas9 protein and sgRNA at a mass ratio of 1:1 to obtain a Cas9 protein mixture;

[0135] Take the upper layer of suspended cells obtained in S1 (3-4.5 times the number of adherent cells used in S2) and add 1×10 7 Add 18-25ug of Cas9 protein mixture to cells, perform electroporation at 360V, 1ms in an electroporator, and then press 1×10 6 The cells were inoculated at a density of cells / ml into a T175 culture flask with 581 culture medium, and anti-human CD3 monoclonal antibody (50ng / ml) was added at a final concentration. The cells were cultured in a 37°C, 5% CO2 incubator. The cells were replenished with fluid depending on their growth. When the culture medium exceeded 500ml, the cells were transferred to a cell culture bag for culture to obtain PD-1 knocked-out T cells.

[0136] S4. Add DC cells to T cells in which PD-1 has been knocked out, and incubate and expand together:

[0137] On the 7th to 8th day, DC cells were added to T cells in which PD-1 had been knocked out, mixed, and cultured in a 37°C, 5% CO2 incubator for 8-10 days. Samples were taken to detect the DC cell ratio and T cell PD-1 knockout efficiency, and fluid was replenished depending on the cell production situation. When the culture medium exceeded 500 ml, it was transferred to a cell culture bag for culture to complete the co-incubation and amplification.

[0138] Effect test:

[0139] (1) Flow cytometry was used to detect the DC cell ratio of S6 in Example 1. The results were as follows: Figure 1As shown, the CD80 positive rate is greater than 80%, and the CD86 positive rate is greater than 80%. The results show that under the culture process parameters set in Example 1, high-purity DC cells can be easily obtained.

[0140] (2) Figure 2 For the knockout efficiency test of T cell PD-1 in Example 1, the gene sequencing results showed that there was a staggered peak in the target sequence, indicating that the target gene had been effectively knocked out.

[0141] (3) The unmodified ordinary T cells, the cells prepared in Example 1, Comparative Example 1 and Comparative Example 2 were subjected to cytotoxicity experiments with A549 non-small cell lung cancer cell line, with an effector-target ratio of 1:25. After 48 hours of co-culture, the supernatant was collected and the γ-interferon content in the supernatant was detected by ELISA kit. The results are shown in Table 1:

[0142] Mean concentration (pg / ml) Example 1 6978.5 Comparative Example 1 4652.1 Comparative Example 2 3154.7 Normal T cells 587.2

[0143] Table 1

[0144] From Table 1 and Figure 3 It can be seen that co-culture of DC cells and T cells can improve the killing ability of T cells against tumor cells. From the comparison of the data in Example 1 and Comparative Example 1, the PD-1 of T cells was not knocked out, and PD-L1 was expressed on the surface of cancer cells, which combined with PD-1 on the surface of T cells, reduced the activity of T cells, induced T cell apoptosis and disintegration, inhibited T cell proliferation, knocked out T cell PD-1, and improved the adaptability of T cells to the tumor microenvironment. From the comparison of the data in Example 1 and Comparative Example 2, the electroporation modification of tumor neoantigen mRNA can stimulate the autologous immune system to attack tumor cells to a greater extent.

[0145] As can be seen from the above examples, the present invention discloses a preparation method for improving the adaptability of T cells to the tumor microenvironment based on electroporation of tumor neoantigen mRNA and CRISPR-Cas9 technology. The process has strong versatility, low production cost, high safety, and the cells obtained by culture have strong adaptability to the tumor microenvironment, which can stimulate the autologous immune system to attack tumor cells, allowing more patients to benefit from individualized treatment in the long term.

[0146] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for improving the adaptability of T cells to the tumor microenvironment, characterized in that: The following steps are involved: S1. Preparation of tumor neoantigen mRNA; S2, collect and separate PBMC to obtain initial T cells; S3, using the initial T cells to induce DC cells; S4, using the tumor neoantigen mRNA to modify the DC cells; S5, knocking out PD-1 of the initial T cells by CRISPR-Cas9 electroporation; S6. Add the modified DC cells to the T cells in which PD-1 has been knocked out, incubate and expand them together to obtain treated T cells.

2. A method for improving the adaptability of T cells to the tumor microenvironment according to claim 1, characterized in that: The S1 comprises the following steps: S11. Design and construct an expression vector based on the tumor neoantigen gene sequence, and obtain a recombinant plasmid after codon optimization; S12, linearizing the recombinant plasmid using an endonuclease, and then purifying and recovering the plasmid to obtain a linearized plasmid; S13. Using an in vitro transcription reagent, perform in vitro transcription on the linearized plasmid, and then purify and recover it to obtain the tumor neoantigen mRNA.

3. A method for improving the adaptability of T cells to the tumor microenvironment according to claim 1, characterized in that: S2 includes the following steps: S21, collect peripheral blood, add lymph separation fluid, centrifuge, aspirate buffy coat cells, add physiological saline to resuspend, centrifuge and discard supernatant, then add culture medium to resuspend, and count cells; S22, adding culture medium again for incubation, collecting the upper suspended cells, centrifuging and discarding the supernatant, then adding culture medium for resuspending, counting the cells, and obtaining the initial T cells.

4. The method for improving the adaptability of T cells to the tumor microenvironment according to claim 1, characterized in that: The cell ratio of the naive T cells in S3 to the naive T cells in S5 was 1:(3-4.5).

5. The method for improving the adaptability of T cells to the tumor microenvironment according to claim 1, characterized in that: The S3 comprises the following steps: S31, adding culture medium to the initial T cells, and adding IL-4 and GM-CSF for culturing; S32, replace the culture medium, add IL-4 and GM-CSF and continue culturing; S33, then add TMF-α and continue culturing to obtain the DC cells.

6. A method for improving the adaptability of T cells to the tumor microenvironment according to claim 5, characterized in that: The final concentrations of cytokines in the S3 are: IL-4 is 40-60 ng / ml, GM-CSF is 50-150 ng / ml, and TMF-α is 200-300 ng / ml.

7. The method for improving the adaptability of T cells to the tumor microenvironment according to claim 1, characterized in that: The S4 comprises the following steps: counting the DC cells, and every 1×10 7 cells, adding 25-30ug of the tumor-specific antigen mRNA, electroporating, and then transferring to a culture medium containing recombinant human IL-2 protein for culture, to obtain the modified DC cells; The electroporation transduction modification operation of the DC cells occurs on the 7th to 8th day of their culture.

8. The method for improving the adaptability of T cells to the tumor microenvironment according to claim 7, characterized in that: The S5 comprises the following steps: S51, mixing Cas9 protein and sgRNA to obtain a Cas9 protein mixture; S52, taking the initial T cells, each 1×10 7 The cells are added with 18-25ug of the Cas9 protein mixture, electroporated, and then inoculated into a culture bottle, and anti-human CD3 monoclonal antibody is added for culture to obtain the PD-1 knocked-out T cells.

9. A method for improving the adaptability of T cells to the tumor microenvironment according to claim 8, characterized in that: The voltage of the electric shock transduction in the S4 and S5 was 360 V, and the transient time was 1 ms.

10. The method for improving the adaptability of T cells to the tumor microenvironment according to claim 1, characterized in that: The S6 comprises the following steps: adding the modified DC cells to the PD-1 knocked-out T cells, co-culturing for 8-10 days, sampling and testing, and rehydration culture to obtain the treated T cells.

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