Preparation method of infiltrating lymphocytes with high proportion of neoantigen reactive T cells

Abstract

The invention discloses a preparation method of infiltrating lymphocytes with high proportion of neoantigen reactive T cells. The proportion of the reactive T cells with tumor neoantigen specificity can be increased without using a gene editing technology. In the TIL cell culture process, DC presents tumor neoantigen and TIL is sorted and cultured, the proportion of T cells with neoantigen reactivity in TIL is increased, the anti-tumor activity of the TIL cell preparation is enhanced, then the prepared infiltrated lymphocytes are rapidly and massively amplified, and the TIL cell preparation has the advantages that the tumor neoantigen reactivity is improved. And batch preparation of the neoantigen reactive TIL cell preparation with high reactivity to tumors is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor cell preparation, and in particular to a method for preparing infiltrating lymphocytes with a high proportion of neoantigen-reactive T cells. Background Art

[0002] Immunotherapy is an emerging approach to cancer therapy in which T cell-mediated responses to tumor antigens act to eliminate tumor cells. Tumor-infiltrating lymphocytes (TIL) are T cells that infiltrate tumor tissues and can effectively recognize tumor cell neoantigens. They have the ability to efficiently kill tumor cells, including killing tumor cells that have metastasized from the lesion to other sites. Neoantigens are peptides presented by the major histocompatibility complex (MHC) that have new sequences formed by somatic mutations and represent a particularly promising class of immunotherapy targets because they only appear on tumors and are specific, thus causing minimal off-target effects. Therefore, the development of neoantigen-specific tumor-infiltrating lymphocytes that are highly reactive to tumors is meaningful for the treatment of tumors, but due to the heterogeneous nature of TIL cells, when the existing technology can achieve large-scale expansion of natural tumor-infiltrating lymphocytes (TIL), the proportion of reactive T cells with tumor antigen specificity in the total cells obtained is usually not high, affecting the therapeutic effect of clinical preparations. Although TCR-T cells can be prepared using gene editing technology, the entire process is time-consuming and costly. Summary of the invention

[0003] Therefore, based on the above background, the present invention provides a method for preparing infiltrating lymphocytes with a high proportion of neoantigen-reactive T cells. The prepared infiltrating lymphocytes have a high proportion of neoantigen-reactive T cells, which is conducive to the preparation of highly effective preparations.

[0004] The technical solution of the present invention is:

[0005] A method for preparing infiltrating lymphocytes with a high proportion of neoantigen reactive T cells comprises the following steps:

[0006] S1: extracting TIL cells from the patient's tumor tissue to obtain the second group of TIL cells;

[0007] S2: Obtain the genomic data of the patient's tumor tissue and normal tissue, identify and screen the mutant genes, and synthesize new antigen peptides based on the screened genes;

[0008] S3: extracting PBMC cells from the patient's own fresh whole blood, resuspending the obtained PBMC cells, and adding liquid DC cell culture medium to expand and culture DC cells;

[0009] S4: replacing the liquid DC cell culture medium with the DC cells obtained in step S3, adding the solution of the new antigen polypeptide obtained in step S2, culturing, and loading the new antigen polypeptide;

[0010] S5: sorting the second group of TIL cells obtained in step S1 using a CD39+TIL cell sorting kit, enriching and culturing them to obtain a third group of TIL cells;

[0011] and co-culturing the DC cells loaded with the neoantigen polypeptide obtained in step S4 with the third group of TIL cells to obtain a fourth group of TIL cells;

[0012] S6: Cryopreserving or expanding the fourth group of TIL cells.

[0013] Furthermore, the DC cell culture medium in steps S3 and S4 is RPMI 1640 culture medium containing 5wt% human AB serum, 1% double antibody, 1:100 glutamine, 800IU / ml GM-CSF, and 200U / ml IL-4.

[0014] Furthermore, the culture medium used for culturing the sorted cells in step S5 is RPMI 1640 culture medium containing 10wt% human AB serum, 10mg / ml gentamicin, 1:100GlutaMAX, 25mM HEPES, 6000IU / ml IL-2, 30ng / mg IL-7, 15ng / mg IL-21, and 5ng / ml TGF-β.

[0015] Furthermore, when the DC cells are co-cultured with the third group of TIL cells in step S5, the ratio of the number of the DC cells to the third group of TIL cells is (15-40):1;

[0016] The co-culture medium is AIM-V CTS medium containing 5wt% human AB serum, 1wt% double antibody, 10mg / ml gentamicin, 1:10GlutaMAX, 25mM HEPES, 3000IU / ml IL-2, 30ng / ml OKT-3, 30ng / ml IL-7, 15ng / mg IL-21, and 5ng / ml TGF-β.

[0017] Furthermore, the amplification culture of the fifth group of TIL cells in step S6 includes the following steps:

[0018] (1) Day 0 to 2

[0019] ① If the total number of cells is ≥2E9, continue to culture in the original medium;

[0020] ② If the total number of cells is less than 2E9, add complete culture medium according to the counting results and adjust the cell density to 1×10 6 Pieces / ml;

[0021] The amplification culture conditions are: CO 2 The concentration is 3-8%, O 2 The concentration is 18-25%, the temperature is 35-40℃, the rocking speed is 0, and the rocking angle is 0.

[0022] (2) Day 3-4

[0023] Perfusion expansion culture, the perfusion rate is 1L / day,

[0024] The amplification culture conditions are: CO 2 The concentration is 3-8%, O 2 The concentration is 18-25%, the temperature is 35-40℃, the swing rate is 1-2, and the swing angle is 1-2;

[0025] (3) Days 5 to 7

[0026] The perfusion rate was adjusted to 2 L / day;

[0027] The amplification culture conditions are: CO 2 The concentration is 3-8%, O 2 The concentration is 18-25%, the temperature is 35-40℃, the rocking speed is 3-6, and the rocking angle is 3-6.

[0028] Furthermore, the specific operation of extracting TIL cells from the patient's tumor tissue in step S1 includes the following steps:

[0029] S1.1: Cleaning

[0030] Tumor tissue was obtained under sterile conditions, rinsed with tumor cleaning solution, and necrotic tissue and yellow fat tissue were cut off.

[0031] S1.2: Digestion

[0032] Cut the tumor tissue into tissue fragments, add the mixed digestion solution to the tissue fragments, add it in a water bath, observe the digestion state, continue to mechanically cut, repeat the water bath heating and mechanical cutting 3-4 times to obtain a cell suspension;

[0033] S1.3 Termination of digestion

[0034] RPMI1640 containing human AB serum was added to the cell suspension to terminate the digestion;

[0035] S1.4 Collect the filtrate

[0036] The cell suspension digested in step S1.3 is sieved through a cell sieve to obtain the filtrate;

[0037] S1.5 Centrifugation

[0038] After centrifuging the filtrate in step S1.4, discard the supernatant, add complete medium for resuspending to obtain a single cell suspension. The cells obtained at this time are called the first group of cells;

[0039] S1.6 Expansion culture

[0040] The single cell suspension obtained in step S1.5 was incubated at 35-40°C and 2-8% CO 2 to obtain a second group of TIL cells.

[0041] Furthermore, in step S2, whole exome sequencing is performed on the patient's tumor tissue and normal tissue using NGS sequencing technology to obtain whole exome genome data of the tumor tissue and normal tissue, and compare and identify mutant genes;

[0042] Then, HLA typing test was performed on peripheral blood to obtain HLA-A typing data. The affinity with MHC-I allele molecules was predicted by Net-MHC4.0 software, and high-affinity mutant genes were screened out. New antigen peptides were synthesized based on the screened genes.

[0043] Based on the same inventive concept, the present invention also provides tumor infiltrating lymphocytes prepared by the above-mentioned method for preparing infiltrating lymphocytes with a high proportion of new antigen-reactive T cells.

[0044] Based on the same inventive concept, the present invention also applies the tumor infiltrating lymphocytes prepared by the above-mentioned method for preparing infiltrating lymphocytes with a high proportion of new antigen-reactive T cells to the preparation of tumor treatment preparations.

[0045] The beneficial effects achieved by the technical solution of the present invention are:

[0046] The present invention can achieve an increase in the proportion of reactive T cells specific to tumor neoantigens without the use of gene editing technology. In the process of TIL cell culture, DC presents tumor neoantigens and TIL is sorted and cultured to increase the proportion of T cells reactive with neoantigens in TIL, enhance the anti-tumor activity of TIL cell preparations, and then rapidly and massively amplify the prepared infiltrating lymphocytes, which is conducive to the batch preparation of neoantigen reactive TIL cell preparations with high reactivity to tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Attached Figure 1 It is a schematic diagram of the preparation process of the present invention. DETAILED DESCRIPTION

[0050] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0053] Example 1: A method for preparing infiltrating lymphocytes with a high proportion of neoantigen-reactive T cells, comprising the following steps:

[0054] S1: extracting TIL cells from the patient's tumor tissue to obtain the second group of TIL cells;

[0055] Extraction of TILs from tumor tissue

[0056] S1.1 Cleaning: Obtain tumor tissue under sterile conditions, rinse with tumor cleaning solution (50 μg / ml gentamicin Hanks balanced salt solution), and cut off necrotic tissue and yellow fat tissue.

[0057] S1.2 Digestion: Cut the tumor tissue into 1-3 mm pieces. 3 Add 10 ml of mixed digestion solution (10 ml of 2 wt% IV collagenase working solution + 10 ml of 0.25 wt% pancreatin + 20 ml of normal saline) to the tissue fragments and place them in a 37°C water bath for 30 min. Observe the digestion status, continue mechanically chopping, and place them in a 37°C water bath for 30 min. Repeat 3-4 times to obtain a cell suspension.

[0058] S1.3 Termination of digestion: Add 3 ml of RPMI1640 containing 10 wt% human AB serum to each tube to terminate the digestion.

[0059] S1.4 Collect the filtrate: Sieve through a 70um cell sieve and collect the filtrate.

[0060] Centrifugation: 2100rpm, turn off the brake, centrifuge for 10min; discard the supernatant after centrifugation, resuspend with complete culture medium (RPMI1640 containing 10% human AB serum, 1% double antibody, 10mg / ml gentamicin, 1:100GlutaMAX, 25mM HEPES, 6000IU / ml IL-2), and the cells obtained at this time are called the first group of cells.

[0061] Counting and spreading: Mix the single cell suspension by pipetting, count the cells, and adjust the cell density to 4×10 with complete culture medium (RPMI 1640 containing 10% human AB serum, 1% double antibody, 10 mg / ml gentamicin, 1:100 GlutaMAX, 25 mM HEPES, and 6000 IU / ml IL-2). 6 Each bottle was filled with T25, 5 ml per bottle, and placed in a 37°C, 5% CO 2 After culturing in a carbon dioxide incubator for 7 days, the obtained cells are called the second group of TIL cells.

[0062] S2: Obtain the genomic data of the patient's tumor tissue and normal tissue, identify and screen the mutant genes, and synthesize new antigen peptides based on the screened genes;

[0063] Then, HLA typing test was performed on peripheral blood to obtain HLA-A typing data. The affinity with MHC-I allele molecules was predicted by Net-MHC4.0 software, and high-affinity mutant genes were screened out. New antigen peptides were synthesized based on the screened genes.

[0064] S3: extracting PBMC cells from the patient's own fresh whole blood, resuspending the obtained PBMC cells, and adding liquid DC cell culture medium to expand and culture DC cells;

[0065] S3.1 Draw fresh anticoagulated whole blood from the patient, dilute and mix the anticoagulated whole blood: normal saline volume = 1:1, calculate the amount of lymphocyte separation solution used in the ratio of diluted peripheral blood: lymphocyte separation solution = 1:1, take the corresponding volume of lymphocyte separation solution and put it into a 50ml centrifuge tube, slowly add the diluted anticoagulated blood into the 50ml centrifuge tube (do not mix with the separation solution, the two should be clearly stratified), centrifuge at 700g, 20min, 20℃, speed up 6, speed down 0.

[0066] S3.2 After centrifugation, the liquid levels in the tube from top to bottom are the diluted plasma layer, PBMC layer, separation liquid layer and red blood cell layer. Aspirate the plasma layer, carefully aspirate the PBMC layer (i.e., the buffy coat layer) and transfer it to a 15mL centrifuge tube. Add physiological saline to the PBMC layer cells at a volume ratio of 1:1, mix and wash, centrifuge at 2100rpm, 10min, 20℃, repeat this step 1 to 2 times to obtain PBMC.

[0067] S3.3 Mix the PBMC suspension by pipetting, count the cells, and adjust the cell density to 1×10 using culture medium AIM-V. 6 10 ml / ml was spread on 10 cm cell culture dishes, 10 ml per dish, and placed in 37°C, 5% CO 2After culturing in a carbon dioxide incubator for 90 minutes, the supernatant and non-adherent cells were discarded, the adherent cells were washed with RPMI 1640 medium, and then further cultured with AIM-V medium for 60 minutes, the supernatant and non-adherent cells were discarded, and DC medium (RPMI 1640 medium + 5wt% human serum + 1wt% double antibody, 1:100 glutamine, 800IU / ml GM-CSF, 200U / ml IL-4) was added and cultured for four days to obtain DC cell fluid.

[0068] S4: replacing the DC cell solution obtained in step S3 with a liquid DC cell culture medium, adding the solution of the new antigen polypeptide obtained in step S2, culturing, and loading the new antigen polypeptide;

[0069] The DC cell fluid was replaced, and the new antigen polypeptide synthesized in step S2 was dissolved in PBS and added to the DC cell culture dish. The final concentration of the new antigen polypeptide was 10 ug / ml, and cultured overnight.

[0070] S5: sorting the second group of TIL cells obtained in step S1 using a CD39+TIL cell sorting kit, enriching and culturing them to obtain a third group of TIL cells;

[0071] The second group of TIL cells was sorted for CD39+ using a BD flow cytometer, T cells were enriched, and the cells were counted. The cell density was adjusted to 1×10 with complete culture medium (RPMI 1640 containing 10 wt% human AB serum, 10 mg / ml gentamicin, 1:100 GlutaMAX, 25 mM HEPES, 6000 IU / ml IL-2, 30 ng / mg IL-7, 15 ng / mg IL-21, and 5 ng / ml TGF-β). 6 / ml, spread the bottle; replenish the liquid every other day to keep the density at 1×10 6 / m, cultured for 7 days, the cells obtained are called the third group of TIL cells.

[0072] and co-culturing the DC cells loaded with the neoantigen polypeptide obtained in step S4 with the third group of TIL cells to obtain a fourth group of TIL cells;

[0073] Co-culture of DC cells loaded with neoantigens with the third group of TILs to activate TILs: Count the DC cells loaded with neoantigens and the third group of TIL cells, and co-culture them with a 1:1 mixed culture medium (AIM-V CTS culture medium with 5% human AB serum, 1% double antibody, 10 mg / ml gentamicin, 1:10 GlutaMAX, 25 mM HEPES, 3000 IU / ml IL-2, 30 ng / ml OKT-3, 30 ng / ml IL-7, 15 ng / mg IL-21, 5 ng / ml TGF-β, 100 ng / ml CD185) at a ratio of TIL: DC = 20:1, with a total density of 1×10 6 On the third day, half of the total cell culture suspension was taken and centrifuged at 1800 rpm / min for 5 minutes. The supernatant was discarded and an equal volume of mixed culture medium was added to continue the culture. The liquid was replenished every other day to keep the density at 1×10 6 / ml; when the total cell volume is greater than 100ml, the cells are transferred to a cell culture bag for culture for 7 days. The obtained cells are called the fourth group of TIL cells.

[0074] S6: Cryopreserving or expanding the fourth group of TIL cells.

[0075] In this step, the operation of freezing the fourth group of TIL cells is as follows:

[0076] ① Count and centrifuge the fourth group of TIL cells to be frozen, replace the supernatant with CS10 freezing solution containing 0.5% HSA, and adjust the cell density in the freezing solution to 1×10 8 Aliquot into cryotubes and store in a 4℃ refrigerator.

[0077] ②After cell packaging is completed, take out all cryopreservation tubes from the 4℃ temporary storage refrigerator, insert them into the cryopreservation rack, and transfer them to a programmed cooling device whose chamber temperature has dropped to 4℃ for freezing.

[0078] ③The parameters of the program cooling instrument are as follows:

[0079] ①Profile Steps:

[0080] ②1.Wait at 4.0℃

[0081] ③2.Ramp 1.0℃ / min until Sample=-10.0℃

[0082] ④3.Ramp 15.0℃ / min until Chamber=-35.0℃

[0083] ⑤4.Ramp 10.0℃ / min until Chamber=-22.0℃

[0084] ⑥5.Ramp 1.0℃ / min until Sample=-40.0℃

[0085] ⑦6.Ramp 10.0℃ / min until Chamber=-90.0℃

[0086] ⑧7.End

[0087] After the programmed cooling ends, open the door, take out the cell cryopreservation tube, and transfer it to the gas phase liquid nitrogen tank for long-term cryopreservation.

[0088] In this step, Xuri-W25 can be used to expand and culture the fourth group of TIL cells. The expansion and culture of the fourth group of TIL cells using Xuri-W25 includes the following steps:

[0089] (1) Day 0 to 2

[0090] The fourth group of TIL cells was transferred to the Xuri-W25 cell expansion system for culture.

[0091] ① If the total number of cells is ≥2E9, transfer all the cell suspension in the bag to the Xuri-W25 cell expansion system for culture, with a final volume of 2L, and calculate the cell density at this time;

[0092] ② If the total number of cells is less than 2E9, transfer all the cell suspension in the bag to the Xuri-W25 cell expansion system for culture. According to the counting results, add an appropriate amount of complete culture medium to adjust the cell density to 1×10 6 / ml. Recorded as day 0;

[0093] The complete culture medium in this step is AIM-V CTS culture medium containing 5wt% human AB serum, 1wt% double antibody, 3000IU / ml IL-2, 30ng / ml OKT, 15ng / mg IL-21, 5ng / ml TGF-β, and 100ng / ml CD185;

[0094] Xuri-W25 cell expansion system parameters are set as: C0 2 The concentration is 5%, O 2 The concentration is 21%, the temperature is 37°C, the swing speed is 0, and the angle is 0.

[0095] (2) Day 3-4

[0096] When the TIL cells are cultured in the Xuri-W25 cell expansion system to the third day, ① if the total volume is less than 2L, add complete culture medium (AIM-V CTS culture medium containing 5wt% human AB serum, 1wt% double antibody, 3000IU / ml IL-2, 30ng / ml OKT, 15ng / mg IL-21, 5ng / ml TGF-β, 100ng / mlCD185) to replenish to 2L; ② if the total volume is equal to 2L, no replenishment is required;

[0097] Turn on perfusion at a rate of 1 L / day.

[0098] Xuri-W25 cell expansion system parameters are set as: C0 2 The concentration is 5%, O 2 The concentration is 21%, the temperature is 37°C, the swing speed is 2, and the angle is 2.

[0099] (3) Days 5 to 7:

[0100] The perfusion rate was adjusted to 2 L / day.

[0101] Xuri-W25 cell expansion system parameters are set as: C0 2 The concentration is 5%, O 2 The concentration is 21%, the temperature is 37°C, the swing speed is 4, and the angle is 4.

[0102] The TIL cells expanded and cultured in this step can be directly made into injections for direct clinical use according to needs, or can be cryopreserved for subsequent use.

[0103] I: If the injection is prepared directly, the preparation steps are as follows:

[0104] 1) Continuous flow centrifugation was performed using the Gentle Flex Pro high-throughput fully enclosed automated cell processing system. The supernatant was replaced with saline containing 0.5% HSA and the cell density was adjusted to 5×10 7 ~8×10 7 Pieces / ml.

[0105] 2) Packaging: Use Gentle P-Pac high-precision automatic packaging system to pack into cell return bags with specifications of 5-8×10 9 / 100ml / bag, move to 4℃ transport box for clinical use.

[0106] Ⅱ: If cryopreservation is required, the procedure is as follows:

[0107] 1) Continuous flow centrifugation was performed using the Gentle Flex Pro high-throughput fully enclosed automated cell processing system, and the supernatant was replaced with CS10 cryopreservation solution containing 0.5% HSA, and the cell density was adjusted to 5×10 7 Pieces / ml.

[0108] 2) After the cell packaging is completed, take out all the cryopreservation bags from the 4°C temporary storage refrigerator, put them into the cryopreservation clips, insert them into the cryopreservation rack, and transfer them to the programmed cooling device whose chamber temperature has dropped to 4°C for freezing.

[0109] 3) The parameters of the program cooling instrument are as follows:

[0110] ①Profile Steps:

[0111] ②1.Wait at 4.0℃

[0112] ③2.Ramp 1.0℃ / min until Sample=-10.0℃

[0113] ④3.Ramp 15.0℃ / min until Chamber=-35.0℃

[0114] ⑤4.Ramp 10.0℃ / min until Chamber=-20.0℃

[0115] ⑥5.Ramp 1.0℃ / min until Sample=-40.0℃

[0116] ⑦6.Ramp 10.0℃ / min until Chamber=-90.0℃

[0117] ⑧7.End

[0118] After the programmed cooling ends, open the door, take out the cell freezing bag, and transfer it to the gas phase liquid nitrogen tank for long-term freezing.

[0119] In this embodiment, unless otherwise specified, the tumor tissue, normal tissue or whole blood of a patient are all from the same patient.

[0120] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing infiltrating lymphocytes with a high proportion of neoantigen reactive T cells, characterized in that: The steps include: S1: extracting TIL cells from the patient's tumor tissue to obtain the second group of TIL cells; S2: Obtain the genomic data of the patient's tumor tissue and normal tissue, identify and screen the mutant genes, and synthesize new antigen peptides based on the screened genes; S3: extracting PBMC cells from the patient's own fresh whole blood, resuspending the obtained PBMC cells, and adding liquid DC cell culture medium to expand and culture DC cells; S4: replacing the liquid DC cell culture medium with the DC cells obtained in step S3, adding the solution of the new antigen polypeptide obtained in step S2, culturing, and loading the new antigen polypeptide; S5: sorting the second group of TIL cells obtained in step S1 using a CD39+TIL cell sorting kit, enriching and culturing them to obtain a third group of TIL cells; and co-culturing the DC cells loaded with the neoantigen polypeptide obtained in step S4 with the third group of TIL cells to obtain a fourth group of TIL cells; S6: Cryopreserving or expanding the fourth group of TIL cells.

2. The method for preparing infiltrating lymphocytes with a high proportion of neoantigen reactive T cells according to claim 1, characterized in that: The DC cell culture medium in steps S3 and S4 is RPMI 1640 culture medium containing 5 wt % human AB serum, 1 % double antibody, 1:100 glutamine, 800 IU / ml GM-CSF, and 200 U / ml IL-4.

3. The method for preparing infiltrating lymphocytes with a high proportion of neoantigen reactive T cells according to claim 1, characterized in that: The culture medium used for culturing the sorted cells in step S5 is RPMI 1640 culture medium containing 10wt% human AB serum, 10mg / ml gentamicin, 1:100GlutaMAX, 25mM HEPES, 6000IU / ml IL-2, 30ng / mg IL-7, 15ng / mg IL-21, and 5ng / ml TGF-β.

4. The method for preparing infiltrating lymphocytes with a high proportion of neoantigen reactive T cells according to claim 1, characterized in that: When the DC cells are co-cultured with the third group of TIL cells in step S5, the ratio of the number of the DC cells to the number of the third group of TIL cells is (15-40):1; The co-culture medium is AIM-V CTS medium containing 5wt% human AB serum, 1wt% double antibody, 10mg / ml gentamicin, 1:10GlutaMAX, 25mM HEPES, 3000IU / ml IL-2, 30ng / ml OKT-3, 30ng / ml IL-7, 15ng / mg IL-21, and 5ng / ml TGF-β.

5. The method for preparing infiltrating lymphocytes with a high proportion of neoantigen reactive T cells according to claim 1, characterized in that: The amplification and culture of the fifth and fourth groups of TIL cells in step S6 includes the following steps: (1) Day 0 to 2 ① If the total number of cells is ≥2E9, continue to culture in the original medium; ② If the total number of cells is less than 2E9, add complete culture medium according to the counting results and adjust the cell density to 1×10 6 Pieces / ml; The amplification culture conditions are: CO2 concentration of 3-8%, O2 concentration of 18-25%, temperature of 35-40°C, rocking speed of 0, and rocking angle of 0. (2) Day 3-4 Perfusion expansion culture, the perfusion rate is 1L / day, The amplification culture conditions are: CO2 concentration of 3-8%, O2 concentration of 18-25%, temperature of 35-40°C, rocking speed of 1-2, and rocking angle of 1-2; (3) Days 5 to 7 The perfusion rate was adjusted to 2 L / day; The amplification culture conditions are: CO2 concentration of 3-8%, O2 concentration of 18-25%, temperature of 35-40°C, rocking speed of 3-6, and rocking angle of 3-6.

6. The method for preparing infiltrating lymphocytes with a high proportion of neoantigen reactive T cells according to claim 1, characterized in that: The specific operation of extracting TIL cells from the patient's tumor tissue in step S1 includes the following steps: S1.1: Cleaning Tumor tissue was obtained under sterile conditions, rinsed with tumor cleaning solution, and necrotic tissue and yellow fat tissue were cut off. S1.2: Digestion Cut the tumor tissue into tissue fragments, add the mixed digestion solution to the tissue fragments, add it in a water bath, observe the digestion state, continue to mechanically cut, repeat the water bath heating and mechanical cutting 3-4 times to obtain a cell suspension; S1.3 Termination of digestion RPMI1640 containing human AB serum was added to the cell suspension to terminate the digestion; S1.4 Collect the filtrate The cell suspension digested in step S1.3 is sieved through a cell sieve to obtain the filtrate; S1.5 Centrifugation After centrifuging the filtrate in step S1.4, discard the supernatant, add complete medium for resuspending to obtain a single cell suspension. The cells obtained at this time are called the first group of cells; S1.6 Expansion culture The single cell suspension obtained in step S1.5 is cultured at 35-40° C. and 2-8% CO 2 to obtain a second group of TIL cells.

7. The method for preparing infiltrating lymphocytes with a high proportion of neoantigen reactive T cells according to claim 1, characterized in that: In step S2, NGS sequencing technology is used to perform whole exome sequencing on the patient's tumor tissue and normal tissue to obtain whole exome genome data of the tumor tissue and normal tissue, and compare and identify mutant genes; Then, HLA typing test was performed on peripheral blood to obtain HLA-A typing data. The affinity with MHC-I allele molecules was predicted by Net-MHC4.0 software, and high-affinity mutant genes were screened out. New antigen peptides were synthesized based on the screened genes.

8. Tumor infiltrating lymphocytes prepared by the method for preparing infiltrating lymphocytes with a high proportion of neoantigen-reactive T cells as described in any one of claims 1 to 7.

9. Use of tumor infiltrating lymphocytes prepared by the method for preparing infiltrating lymphocytes with a high proportion of neoantigen-reactive T cells as described in any one of claims 1 to 7 in preparing tumor therapeutic preparations.

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