NsDC-T cell as well as preparation method and application thereof
By performing gene sequencing and bioinformatics analysis on tumor tissue, tumor neoantigen-specific DC-T cells were prepared, which solved the problem of limited target selection in CAR-T therapy, realized multi-target combined intervention, and improved the efficacy of tumor treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-28
- Publication Date
- 2026-03-17
AI Technical Summary
Current CAR-T cell therapies have limited target selection, making it difficult to achieve multi-target combined intervention and thus unable to effectively control various malignant tumors.
By performing gene sequencing on tumor tissue and using bioinformatics analysis to predict neoantigen peptides, synthesizing polypeptide antigens, and preparing tumor neoantigen-specific DC-T cells, multi-site recognition and all-round strangulation can be achieved.
A standardized method for preparing NsDC-T cells is provided, which can screen suitable NsDC-T cells based on different tumor-specific neoantigens, thereby improving the efficacy of cell immunotherapy.
Smart Images
Figure CN121674337A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell culture technology, and in particular to an NsDC-T cell, its preparation method, and its application. Background Technology
[0002] The main methods of cancer treatment include surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, endocrine therapy, and interventional therapy, among which immunotherapy, a novel treatment approach, has developed rapidly due to the emergence of CAR-T therapy. Currently, CAR-T therapy targets known proto-oncogenes or tumor suppressor genes, and the selected targets are usually very limited. For malignant tumors, intervention with a single target or a few targets is insufficient; instead, it is necessary to find a wider range of precise targets and conduct combined interventions.
[0003] Tumor neoantigen-specific DC-T cells, also known as "NsDC-T cell therapy," involve sequencing the genes of tumor tissue, adjacent tissue, and normal human tissue. Using bioinformatics, all abnormal genes causing the tumor are identified, peptides are synthesized in vitro, and tumor neoantigen-specific DC-T cells are cultured. These cells then recognize multiple sites and kill tumors from all directions, ultimately achieving the goal of controlling or eradicating the tumor. Summary of the Invention
[0004] To address the technical problems existing in the prior art, this invention provides a method for the efficient expansion of gamma-delta T cells. This invention is the first to provide a standardized method for preparing NsDC-T cells, and allows for the screening of NsDC-T cells suitable for different tumor treatments based on different neoantigens, thus facilitating cell immunotherapy.
[0005] Specifically, the present invention first provides a method for preparing NsDC-T cells, which includes the following steps: 1) Identification and preparation of new antigens 1.1) Targets for the synthesis of WT1-DC vaccines: WT1-1: WAPPLDFAPPGASAYGSL; WT1-2: CYTWNQMNL; 1.2) Whole genome sequencing was performed on the patient's tumor tissue, and new antigenic peptides that could induce an immune response were predicted and screened through bioinformatics analysis; 1.3) Combine the target of the WT1-DC vaccine in 1.1) with the neoantigen peptide in 1.2) as a polypeptide antigen for uptake by immature DC cells; 2) Isolation and culture of T cells and DC cells 2.1) Collect peripheral blood from cancer patients and isolate PBMC cells; 2.2) Inoculate PBMCs into culture flasks, add DC basal medium, and incubate in a CO2 incubator for 1-2 hours; 2.3) Collect the non-adherent cells from step 2.2), wash with PBS, resuspend in T cell culture medium, and transfer to another culture flask. Add T cell culture medium, incubate in a CO2 incubator for 24-48 hours, add T cell activating factor, and continue culturing in a CO2 incubator until day 5-7. Add T cell proliferation factor, expand the T cell culture flask to two culture flasks, add T cell culture medium and adjust the cell density, and continue culturing in a CO2 incubator. 2.4) Add DC cell culture medium containing DC initiation factor to the original culture medium from which non-adherent cells were removed in step 2.3) and continue culturing until day 3-5. Replace half of the DC cell culture medium and culture until day 6-9. Add the polypeptide antigen synthesized in step 1) to allow immature DC cells to take up the antigen. Culture until day 7-10. Add maturation factor to DC cells to promote DC maturation. 3) Activation and expansion of NsDC-T cells 3.1) After continuous culture for 8-10 days, collect the T cells from step 2.3) and the DC cells from step 2.4), co-culture them at a certain ratio, transfer them into a culture bag, add T cell culture medium, and continue culturing; During 3.2), the culture medium was continuously supplemented until the cell count reached 5 × 10⁻⁶. 9 -5×10 13 More than one; 4) Screening and identification of NsDC-T cells 4.1) The viability of NsDC-T cells was detected using a cell counter and trypan blue staining. 4.2) Flow cytometry was used to detect the expression of CD3, CD8, and CD56 molecules on the cell surface; 4.3) Before harvesting cells, take a small amount of culture for bacterial and fungal culture, and test for mycoplasma, chlamydia, and endotoxin.
[0006] Preferably, the neoantigen peptide is selected from the following long peptides: RP1: MPPKTPRKNGRHRRRCRRGTP; B3GALNT2: NFRLNWAVDQTGKWQELEYP; TP53 long peptide 1: DSDGLAPPQRLIRVEGNLRV; TP53 long peptide 2: ACPGRDRRTVEENLRKKGEP; Prediction 1: WRQEGPLAL; Prediction 2: LAMAAQLPM; Prediction 3: YSYNLMmLK; Prediction 4: FSISGSYVL; Prediction 5: VTAAEFRIYK; Prediction 6: TLYRAYLLK; Prediction 7: GRIINGFAL; Prediction 8: VRVTAADAL; Prediction 9: SR The combination of ALVAQCL; or, the neoantigen peptide selected from the combination of PIK3CA long peptide: YFMKQMNDARHGGWTTKMDW; PRRT2 long peptide: SPPANGAPPECCSSWLRRIE; TP53 long peptide: SQHMTEVVRHCPHHERCSDS; YC1: KLLDLICHI; YC2: FLPLNYFIEI; YC3: SMINKSSFSV; YC4: WMCLGVLPTV; YC5: YCLDDWVLRL; YC6: KETGFHSGL; YC7: TAATWISQF; YC8: TQYAAGEPQL; YC9: CEAGALRCI.
[0007] Preferably, the DC initiation factors in step 2) include 10-15 ng / mL IL-4 and 10-15 ng / mL GM-CSF.
[0008] Preferably, in step 2), the T cell activating factors include 10-15 μg / mL anti-CD3 monoclonal antibody and 10-20 ng / mL IL-2; Preferably, in step 2), the T cell proliferation factors include 10-20 ng / mL IL-2, 10-20 ng / mL IL-7 and 10-20 ng / mL IL-15.
[0009] Preferably, the DC cell culture medium in step 2) is X-IVIVO. 15 Basic culture medium.
[0010] Preferably, the T cell culture medium in step 2) is X-IVIVO. 15 Add 10-15 ng / mL IL-2 to the basal culture medium.
[0011] Preferably, the concentration of the polypeptide antigen used in step 2) is 1-3 µg / mL.
[0012] Preferably, in step 2), the DC maturation-promoting factors are polyIC-HMV and R848, and the concentration used is 5-20 µg / mL.
[0013] Preferably, the screening criteria in step 4.1) are: the percentage of live cells should be above 90%, and the number should be 5.0 × 10⁻⁶. 9 More than one.
[0014] Preferably, the screening criteria in step 4.2) are: the proportion of CD3+CD8+ cells should be above 50%, and the proportion of CD3+CD56+ cells should be above 20%.
[0015] Preferably, the screening criteria in step 4.3) are: negative pathogen detection and endotoxin <0.5 Eu.
[0016] Another object of the present invention is to provide NsDC-T cells prepared by the above method.
[0017] Another object of the present invention is to provide the use of the above-mentioned NsDC-T cells in the preparation of antitumor drugs.
[0018] Preferably, the tumor is not limited to lung cancer or breast cancer, but may also include other common tumors.
[0019] Compared with the prior art, the above-mentioned technical solution of the present invention has the following advantages: The present invention provides a standardized method for preparing NsDC-T cells for the first time, and can screen NsDC-T cells suitable for different tumor treatments according to different neoantigens, thus providing convenience for cell immunotherapy. Attached Figure Description
[0020] Figure 1 .T cell morphology diagram.
[0021] Figure 2 DC cell morphology diagram.
[0022] Figure 3 The specific killing effect of NsDC-T cells on tumor cells in vitro.
[0023] Figure 4 In vivo anti-lung cancer tumor activity of NsDC-T cells.
[0024] Figure 5 In vivo anti-breast cancer tumor activity of NsDC-T cells. Detailed Implementation
[0025] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.
[0026] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.
[0027] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.
[0028] Example 1 A method for preparing NsDC-T cells, comprising the following steps: 1. Identification and preparation of new antigens 1.1) Targets for synthesizing WT1-DC vaccines (WT1-1: WAPPLDFAPPGASAYGSL; WT1-2: CYTWNQMNL); 1.2) Whole genome sequencing was performed on the patient's tumor tissue, and new antigenic peptides that could elicit a strong immune response were predicted and screened through bioinformatics analysis; 1.3) Combine the target of the WT1-DC vaccine in 1.1) with the neoantigen peptide in 1.2) as a peptide antigen for subsequent DC cell loading.
[0029] 2. Isolation and culture of T cells and DC cells 2.1) Collect peripheral blood from cancer patients and isolate PBMC cells; 2.2) Inoculate PBMCs into T75 bottles, add DC basal medium, and incubate in a CO2 incubator for 1 to 2 hours; 2.3) Collect the non-adherent cells from step 2.2), wash with PBS, resuspend in T cell culture medium, and transfer to another culture flask. Add T cell culture medium, incubate in a CO2 incubator for 24-48 hours, add T cell activating factor, and continue culturing in a CO2 incubator until day 5-7. Add T cell proliferation factor, expand the T cell culture to two culture flasks, add T cell culture medium and adjust the cell density, and continue culturing in a CO2 incubator. 2.4) Add DC cell culture medium containing DC initiation factor to the original T75 flask from which non-adherent cells were removed in step 2.3) and continue culturing until the third day. Replace half of the DC cell culture medium and culture until the sixth day. Add the polypeptide antigen synthesized in step 1) to allow immature DC cells to take up the antigen. Culture until the seventh day. Add maturation factor to DC cells to promote DC maturation.
[0030] Among them, DC initiation factors include IL-4 and GM-CSF; T cell activating factors include anti-CD3 monoclonal antibodies and IL-2; T cell proliferation factors include IL-2, IL-7, and IL-15.
[0031] DC cell culture medium was x-vivo. 15 Basic culture medium; T cell culture medium was x-vivo. 15 IL-2 added to basal culture medium; The concentration of the polypeptide antigen used is 1-3 µg / mL; DC maturation-promoting factors are polyIC-HMV and R848, and the concentration used is 5-20 µg / mL; 3. Activation and expansion of NsDC-T cells 3.1) After continuous culture until day 8, collect the T cells from step 2.3) and the DC cells from step 2.4), co-culture them at a certain ratio, transfer them into a culture bag, add T cell culture medium, and continue culturing; During 3.2), the culture medium was continuously supplemented until the cell count reached 5 × 10⁻⁶. 9 More than one.
[0032] 4. Screening and identification of NsDC-T cells 4.1) NsDC-T cell viability was detected using a cell counter and trypan blue staining. The screening criteria were: viable cells should be above 90% and the number should be 5.0 × 10⁻⁶. 9 More than one; 4.2) Flow cytometry was used to detect the expression of CD3, CD8, and CD56 molecules on the cell surface; the screening criteria were: the proportion of CD3+CD8+ cells should be above 50%, and the proportion of CD3+CD56+ cells should be above 20%.
[0033] 4.3) Before harvesting cells, take a small amount of culture for bacterial and fungal culture, and test for mycoplasma, chlamydia, and endotoxin; screening criteria: negative pathogen test, endotoxin <0.5 Eu.
[0034] Example 2 A method for preparing NsDC-T cells, taking lung cancer patients as an example, includes the following steps: 1. Identification and preparation of new antigens 1.1) Targets for synthesizing WT1-DC vaccines (WT1-1: WAPPLDFAPPGASAYGSL; WT1-2: CYTWNQMNL); 1.2) Whole genome sequencing was performed on the patient's tumor tissue, and new antigenic peptides that could elicit a strong immune response were predicted and screened through bioinformatics analysis; The sequence of the neoantigen peptide is shown in Table 1 below.
[0035] Table 1. Sequences of neoantigen peptides: 1.3) The target of the WT1-DC vaccine in 1.1) is mixed with the neoantigen peptide in 1.2) in equal mass as a polypeptide antigen for subsequent DC cell loading.
[0036] 2. Isolation and culture of T cells and DC cells 2.1) Collect 50 mL of peripheral blood from tumor patients and separate approximately 5 × 10⁶ PBMCs from the blood using density gradient centrifugation. 7 indivual; 2.2) After resuspending the PBMCs in 10 mL of DC basal medium, transfer them to a T75 bottle, add another 10 mL of DC basal medium, and incubate in a carbon dioxide incubator for 1 hour. 2.3) Collect the non-adherent cells from step 2.2), wash twice with PBS, resuspend in 10 mL of T cell culture medium, and transfer to another T75 flask. Add T cell culture medium to adjust the cell concentration to 1 × 10⁻⁶ cells / mL. 6 Cells were cultured at a density of 1 × 10⁶ / mL in a CO₂ incubator for 24 hours, then T cell activating factor was added. Culture continued until day 5, when T cell proliferation factor was added again. The T cell culture was expanded to two T175 flasks, and the cell concentration was adjusted to 1 × 10⁶ / mL. 6 Add T cell culture medium to the cells at a concentration of 1 × 10⁶ cells / mL and adjust the cell count. Incubate in a CO₂ incubator and continue culturing until day 7. Expand the T cell culture and adjust the cell concentration to 1 × 10⁶ cells / mL. 6 Add T cell culture medium to cells at a density of 1 / mL and adjust the cells, then incubate in a carbon dioxide incubator. 2.4) Add 20 mL of DC cell culture medium and DC initiation factor to the original T75 flask from which non-adherent cells were removed in step 2.3) and continue culturing until the third day. Replace half of the DC cell culture medium and culture until the 6th day. Add the polypeptide antigen synthesized in step 1) to allow immature DC cells to take up the antigen. Culture until the 7th day. Add maturation factor to DC cells to promote DC maturation.
[0037] Among them, the DC initiation factors include 10 ng / mL IL-4 and 10 ng / mL GM-CSF; T cell activating factors include 10 μg / mL anti-CD3 monoclonal antibody and 10 ng / mL IL-2; T cell proliferation factors include 10 ng / mL IL-2, 15 ng / mL IL-7, and 15 ng / mL IL-15.
[0038] DC cell culture medium was x-vivo. 15 Basic culture medium; T cell culture medium was x-vivo. 15 Add 10 ng / mL IL-2 to the basal culture medium; The concentration of the polypeptide antigen used is 3 µg / mL; DC maturation-promoting factors were 5 µg / mL polyIC-HMV and 10 µg / mL R848; 3. Activation and expansion of NsDC-T cells 3.1) After continuous culture until day 8, collect T cells from step 2.3). Figure 1 In steps 2.4 and 2.5, the DC cells ( Figure 2 They were co-cultured at a ratio of 1:10, and after 24 hours of co-culture, they were transferred to a culture bag, and 400 mL of T cell culture medium was added for further culture. 3.2) On day 11, add T cell culture medium to 1500 mL and continue culturing; on day 13, add T cell culture medium to 2000 mL and continue culturing; on day 15, collect cell count.
[0039] 4. Screening and identification of NsDC-T cells 4.1) NsDC-T cell viability was detected using a cell counter and trypan blue staining. The screening criteria were: viable cells should be above 95% and the number should be 5.0 × 10⁻⁶. 9 More than one; 4.2) Flow cytometry was used to detect the expression of CD3, CD8, and CD56 molecules on the cell surface; the screening criteria were: 72.1% of CD3+CD8+ cells and 28.8% of CD3+CD56+ cells.
[0040] 4.3) Before harvesting cells, take a small amount of culture for bacterial and fungal culture, and test for mycoplasma, chlamydia, and endotoxin; screening criteria: negative pathogen test, endotoxin <0.5 Eu.
[0041] Example 3 A method for preparing NsDC-T cells, taking breast cancer patients as an example, includes the following steps: 1. Identification and preparation of new antigens 1.1) Targets for synthesizing WT1-DC vaccines (WT1-1: WAPPLDFAPPGASAYGSL; WT1-2: CYTWNQMNL); 1.2) Whole genome sequencing was performed on the patient's tumor tissue, and new antigenic peptides that could elicit a strong immune response were predicted and screened through bioinformatics analysis; The sequence of the neoantigen peptide is shown in Table 2 below.
[0042] Table 2 Sequences of neoantigen peptides: 1.3) The target of the WT1-DC vaccine in 1.1) is mixed with the neoantigen peptide in 1.2) in equal mass as a polypeptide antigen for subsequent DC cell loading.
[0043] 2. Isolation and culture of T cells and DC cells 2.1) Collect 50 mL of peripheral blood from tumor patients and separate approximately 1 × 10⁻⁶ PBMCs from the blood using density gradient centrifugation. 8 indivual; 2.2) After resuspending the PBMCs in 10 mL of DC basal medium, transfer them to a T75 bottle, add another 10 mL of DC basal medium, and incubate in a CO2 incubator for 2 hours. 2.3) Collect the non-adherent cells from step 2.2), wash twice with PBS, resuspend in 10 mL of T cell culture medium, and transfer to another T75 flask. Add T cell culture medium to adjust the cell concentration to 2 × 10⁻⁶ cells / mL. 6 Cells were cultured at a density of 10 cells / mL in a CO2 incubator for 24 hours. T cell activating factor was then added, and the cells were cultured for another 5 days. T cell proliferation factor was then added, and the T cell count was expanded to two T175 flasks, adjusting the cell concentration to 2 × 10⁶ cells / mL. 6 Add T cell culture medium to the cells at a concentration of 2 × 10⁶ cells / mL and adjust the cell count. Incubate in a CO₂ incubator and continue culturing until day 7. Expand the T cell culture and adjust the cell concentration to 2 × 10⁶ cells / mL. 6 Add T cell culture medium to cells at a density of 1 / mL and adjust the cells, then incubate in a carbon dioxide incubator. 2.4) Add 20 mL of DC cell culture medium and DC initiation factor to the original T75 flask from which non-adherent cells were removed in step 2.3) and continue culturing until the third day. Replace half of the DC cell culture medium and culture until the 6th day. Add the polypeptide antigen synthesized in step 1) to allow immature DC cells to take up the antigen. Culture until the 7th day. Add maturation factor to DC cells to promote DC maturation.
[0044] Among them, the DC initiation factors include 15 ng / mL IL-4 and 15 ng / mL GM-CSF; T cell activating factors include 15 μg / mL anti-CD3 monoclonal antibody and 20 ng / mL IL-2; T cell proliferation factors include 20 ng / mL IL-2, 20 ng / mL IL-7, and 20 ng / mL IL-15.
[0045] DC cell culture medium was x-vivo. 15 Basic culture medium; T cell culture medium was x-vivo. 15 Add 10 ng / mL IL-2 to the basal culture medium; The concentration of the polypeptide antigen used is 5 µg / mL; DC maturation-promoting factors were 20 µg / mL polyIC-HMV and 15 µg / mL R848; 3. Activation and expansion of NsDC-T cells 3.1) After continuous culture until day 8, collect the T cells from step 2.3) and the DC cells from step 2.4) respectively, and co-culture them at a ratio of 1:20. After co-culturing for 24 hours, transfer them to a culture bag, add 400 mL of T cell culture medium, and continue culturing. 3.2) On day 10, add T cell culture medium to 1500 mL and continue culturing; on day 12, add T cell culture medium to 2000 mL and continue culturing; on day 15, collect cell count.
[0046] 4. Screening and identification of NsDC-T cells 4.1) NsDC-T cell viability was detected using a cell counter and trypan blue staining; the selection criteria were: cell viability 95% and cell number ≥ 5.0 × 10⁻⁶. 9 More than one; 4.2) Flow cytometry was used to detect the expression of CD3, CD8, and CD56 molecules on the cell surface; the screening criteria were: 69.5% of CD3+CD8+ cells and 32.6% of CD3+CD56+ cells.
[0047] 4.3) Before harvesting cells, take a small amount of culture for bacterial and fungal culture, and test for mycoplasma, chlamydia, and endotoxin; screening criteria: negative pathogen test, endotoxin <0.5 Eu.
[0048] Example 4 4.1 In vitro specific killing effect of NsDC-T cells on tumor cells 2×10 6 Lung cancer cells (A549) and breast cancer cells (MCF-7) were added to cell culture dishes, and after they adhered, 1×10⁻⁶ cells were added to each dish. 3 NsDC-T cells prepared in Example 2 and Example 3 were used, with the blank group receiving an equal volume of culture medium. After 48 hours of culture, the cell killing rate was analyzed using the LDH detection kit instructions. The results are as follows: Figure 3As shown, NsDC-T cells have a significant killing effect on lung cancer cells a549 and breast cancer cells mcf-7.
[0049] 4.2 In vivo antitumor activity of NsDC-T cells Lung cancer mouse models and breast cancer mouse models were established by subcutaneously inoculating C57BL / 6J mice with lung cancer cells (a549) and breast cancer cells (mcf-7), respectively. The tumors on the skin surface were allowed to grow to 2 cm². 3 1×10 mmol / L was administered via the tail vein. 4 NsDC-T cells prepared in Example 2 and NsDC-T cells prepared in Example 3 (5 mice per group). After NsDC-T cell infusion, tumor measurements were performed weekly and tumor volume was calculated. Tumor growth curves were plotted based on the tumor volume. Figure 4-5 It was found that NsDC-T cells could significantly inhibit tumor growth and reduce tumor volume significantly. This indicates that the constructed NsDC-T cells have good anti-tumor activity in vivo.
[0050] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method of making NsDC-T cells comprising the steps of: 1) Identification and preparation of neoantigens 1.1) Synthesis of WT1-DC vaccine target, WT1-1: WAPVLDFAPPGASAYGSL; WT1-2: CYTWNQMNL; 1.2) Whole gene sequencing from patient tumor tissue, and prediction and screening of neoantigen peptides capable of causing immune response through bioinformatics analysis; 1.3) Combination of WT1-DC vaccine target in 1.1) and neoantigen peptides in 1.2) as polypeptide antigens for immature DC cell uptake; 2) Separation and culture of T cells and DC cells 2.1) Collection of peripheral blood from tumor patients, and separation of PBMC cells; 2.2) Inoculation of PBMC into culture bottles, addition of DC basic culture medium, and placement in a carbon dioxide incubator for 1-2 hours; 2.3) Collection of non-adherent cells in step 2.2), washing with PBS, resuspension in T cell culture medium, and transfer to another culture bottle, addition of T cell culture medium, placement in a carbon dioxide incubator for 24-48 hours, addition of T cell activation factors, and continued placement in a carbon dioxide incubator for 5-7 days, addition of T cell proliferation factors, and expansion of T cells to two culture bottles, addition of T cell culture medium and adjustment of cell density, and continued placement in a carbon dioxide incubator for culture; 2.4) Addition of DC cell culture medium containing DC initiation factors to the original culture of step 2.3) from which non-adherent cells have been removed, continued culture for 3-5 days, half-volume medium exchange of DC cell culture medium, culture for 6-9 days, addition of polypeptide antigens synthesized in step 1) for uptake of antigens by immature DC cells, culture for 7-10 days, addition of maturation promoting factors to DC cells to promote DC maturation; 3) Activation and expansion of NsDC-T cells; 4) Screening and identification of NsDC-T cells.
2. The method of claim 1, wherein the neoantigen peptides are selected from the group consisting of RP1 long peptide: MPPKTPRKNGRHRRRCRRGTP; B3GALNT2 long peptide: NFRLNWAVDQTGKWQELEYP; TP53 long peptide 1: DSDGLAPPQRLIRVEGNLRV; TP53 long peptide 2: ACPGRDRRTVEENLRKKGEP; prediction 1: WRQEGPLAL; prediction 2: LAMAAQLPM; prediction 3: YSYNLMmLK; prediction 4: FSISGSYVL; prediction 5: VTAAEFRIYK; prediction 6: TLYRAYLLK; prediction 7: GRIINGFAL; prediction 8: VRVTAADAL; prediction 9: SRALVAQCL; Alternatively, the neoantigen peptides are selected from the group consisting of PIK3CA long peptide: YFMKQMNDARHGGWTTKMDW; PRRT2 long peptide: SPPANGAPPECCSSWLRRIE; TP53 long peptide: SQHMTEVVRHCPHHERCSDS; YC1: KLLDLICHI; YC2: FLPLNYFIEI; YC3: SMINKSSFSV; YC4: WMCLGVLPTV; YC5: YCLDDWVLRL; YC6: KETGFHSGL; YC7: TAATWISQF; YC8: TQYAAGEPQL; YC9: CEAGALRCI.
3. The method of claim 1, wherein, 3) Activation and expansion of NsDC-T cells, including: 3.1) continuous culture to day 8-10, respectively collect T cells in step 2.3) and DC cells in step 2.4), co-culture according to a certain number ratio, and transfer them into a culture bag, supplement T culture medium, and continue to culture; 3.2) during the period, continuously supplement the culture medium until the number of cells reaches 5×10 9 -5×10 13 or more.
4. The method of claim 1, wherein, 3) activation and expansion of NsDC-T cells, 4) screening and identification of NsDC-T cells, including: 4.1) cell viability of NsDC-T cells was detected by cell counter and trypan blue staining method; 4.2) expression of CD3, CD8 and CD56 molecules on the surface of cells was detected by flow cytometry; 4.3) before harvesting the cells, a small amount of culture was taken for bacterial and fungal culture, and mycoplasma, chlamydia and endotoxin were detected.
5. The method of claim 1, wherein, DC priming factors in step 2) include 10-15 ng / mL IL-4 and 10-15 ng / mL GM-CSF; and / or, T cell activation factors in step 2) include 10-15 μg / mL anti-CD3 monoclonal antibody, 10-20 ng / mL IL-2; T cell proliferation factors include 10-20 ng / mL IL-2, 10-20 ng / mL IL-7 and 10-20 ng / mL IL-15; and / or, in step 2) the DC cell culture medium is X-IVIVO 15 base medium; And / or, the T cell culture medium in step 2) is X-IVIVO 15 Basal medium plus 10-15 ng / mL IL-2.
6. The method of claim 1, wherein, The concentration of the polypeptide antigen used in step 2) is 1-3 µg / mL.
7. The method of claim 1, wherein, DC maturation factors in step 2) are polyIC-HMV and R848, and the concentration used is 5-20 µg / mL.
8. The method of claim 1, wherein, In step 4.1), the viable cells should be more than 90%, and the number should be more than 5.0 x 10 9 In step 4.2), the ratio of CD3+CD8+ cells should be more than 50%, and the ratio of CD3+CD56+ cells should be more than 20%; in step 4.3), the pathogen detection should be negative, and the endotoxin should be less than 0.5 Eu.
9. NsDC-T cells prepared by the method of any one of claims 1-8.
10. Use of the NsDC-T cells of claim 9 in the preparation of an anti-tumor drug.