Method for in vitro expansion of tumor-infiltrating lymphocytes

Abstract

Disclosed is a method for in vitro expansion of tumor-infiltrating lymphocytes. Developed are a new method for isolating and enriching TIL cells in tumor tissues and an optimized culture method for TIL cells independent of feeder cells. 1010 grade or more of TIL cells having an anti-tumor function can be obtained. By means of cell phenotype analysis, compared with TILs expanded by the traditional two-step method, the cultured TIL cells have a higher proportion of memory T cells and a lower proportion of exhausted T cells and regulatory T cells.

Description

A method for expanding tumor-infiltrating lymphocytes in vitro TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a method for expanding tumor-infiltrating lymphocytes in vitro. BACKGROUND

[0002] In recent years, chimeric antigen receptor T cells (CAR-T) and T cell receptor-engineered T cells (TCR-T) targeting B cell maturation antigen (BCMA) and CD19 have achieved remarkable efficacy in hematological tumors. However, the treatment of solid tumors with CAR-T / TCR-T faces some bottlenecks and challenges. The main reasons are as follows: 1) it is difficult to screen specific targets for solid tumors; 2) T cells are difficult to infiltrate into tumor sites; 3) the tumor microenvironment is in an immunosuppressive state, thereby inhibiting the anti-tumor function of T cells. Since the solid tumor microenvironment greatly affects the infiltration of T cells and inhibits the anti-tumor immune response of tumor-infiltrating T cells. Therefore, the treatment of solid tumors with CAR-T / TCR-T still faces challenges. In contrast, due to the tissue specificity of tumor-infiltrating lymphocytes (TIL), cell immunotherapy based on TIL cell adoptive transfer can better overcome the bottlenecks of CAR-T / TCR-T treatment of solid tumors. TIL cell immunotherapy refers to the isolation of lymphocytes from the tumor tissue of a patient, mainly composed of T cells, B cells and natural killer (NK) cells, stimulated by adding cytokines in vitro to culture and expand a large number of lymphocytes (mainly CD3 +The present application relates to a method for treating solid tumors by using tumor infiltrating lymphocytes (TILs) to achieve cellular immunotherapy. TILs are derived from tumor tissues, and thus contain T cells that can specifically recognize tumor-associated antigens or neoantigens. TILs can selectively migrate to tumor sites. However, the anti-tumor function of TILs is significantly weakened due to the tumor immunosuppressive microenvironment, such as the presence of high expression of PD-L1 molecules, regulatory T cells (Tregs), tumor-associated macrophages, and myeloid-derived suppressor cells (MDSCs).

[0003] SUMMARY

[0004] To overcome the deficiencies of the prior art, the present application provides a method for expanding tumor infiltrating lymphocytes (TILs) in vitro.

[0005] To achieve the above-mentioned object, the present application adopts the following technical solutions:

[0006] In one aspect, the present application provides a method for expanding tumor infiltrating lymphocytes (TILs) in vitro, which comprises culturing TILs using an expansion medium, wherein the expansion medium comprises a culture medium and interleukin.

[0007] Further, the culture medium is selected from a serum-free culture medium.

[0008] Further, the serum-free culture medium comprises OptiVitro T cell serum-free medium, X-vivo 15 medium, TexMACS medium, culture medium, AIM V medium, ImmunoCult-XF T cell expansion medium, and RPMI-1640 medium.

[0009] Further, the serum-free culture medium is selected from OptiVitro T medium.

[0010] Further, the serum-free culture medium further comprises a serum substitute.

[0011] Further, the serum substitute comprises one or more of CTS Immune Cell Serum Replacement, Ultrose G Serum Substitute, and OptiVitro T cell serum-free additive components.

[0012] Further, the serum replacement is selected from CTS Immune Cell Serum Replacement and / or OptiVitro T Cell Serum-Free Supplement.

[0013] Further, the serum replacement is added at a ratio of 0%-10% CTS Immune Cell Serum Replacement, 0%-10% OptiVitro T Cell Serum-Free Supplement.

[0014] Further, the serum-free medium comprises X-vivo 15 medium and CTS Immune Cell Serum Replacement.

[0015] Further, the serum-free medium comprises OptiVitro T medium and OptiVitro T Cell Serum-Free Supplement.

[0016] Further, the interleukin comprises one or more of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23.

[0017] Further, the interleukin is selected from IL-2, IL-7, and IL-15.

[0018] Further, the IL-2 is at a concentration of 300-9000 IU / mL.

[0019] Further, the IL-2 is at a concentration of 6000 IU / mL.

[0020] Further, the IL-7 is at a concentration of 0-10 ng / mL.

[0021] Further, the IL-7 is at a concentration of 5 ng / mL.

[0022] Further, the IL-15 is at a concentration of 0-30 ng / mL.

[0023] Further, the IL-15 is at a concentration of 15 ng / mL.

[0024] Further, the expansion culture time is 7-28 days.

[0025] Further, the expansion culture time is 14-21 days.

[0026] Further, the method further comprises activating the tumor infiltrating lymphocytes prior to culturing the tumor infiltrating lymphocytes using the expansion medium.

[0027] Further, the tumor infiltrating lymphocytes are activated using an activator.

[0028] Further, the activator comprises one or several of antibodies, ligands, receptors of CD3, CD28, CD137, ICOS, CD40L, OX40.

[0029] Further, the activator is selected from CD3 / CD28 antibodies.

[0030] Further, the concentration of the CD3 antibodies is 0-5 μg / mL.

[0031] Further, the concentration of the CD28 antibodies is 0-5 μg / mL.

[0032] Further, the total concentration of the CD3 / CD28 antibodies is 0-10 μg / mL.

[0033] Further, the total concentration of the CD3 / CD28 antibodies is 1-2 μg / mL.

[0034] Further, the total concentration of the CD3 / CD28 antibodies is 2 μg / mL.

[0035] Further, the concentration ratio of anti-CD3 antibodies to anti-CD28 antibodies is 10:1-1:10.

[0036] Further, the concentration ratio of anti-CD3 antibodies to anti-CD28 antibodies is 1:1.

[0037] Further, the CD3 / CD28 antibodies are used in soluble or coated form.

[0038] Further, the CD3 / CD28 antibodies are used in coated form.

[0039] Further, the tumor infiltrating lymphocytes are obtained by digesting tumor tissue.

[0040] Further, the tumor tissue is digested using enzymatic digestion.

[0041] Further, the enzymes comprise collagenase and / or nucleases.

[0042] Further, the collagenase comprises collagenase I and / or collagenase II.

[0043] Further, serum albumin is added during the digestion of the tumor tissue.

[0044] Further, the tumor tissue is digested for 0-120 min.

[0045] Further, the tumor tissue is digested for 15-60 min.

[0046] Further, the tumor tissue digestion time is 30-60 min.

[0047] Further, the method further comprises the step of isolating tumor infiltrating lymphocytes after digestion of tumor tissue.

[0048] Further, the tumor infiltrating lymphocytes are isolated by density gradient centrifugation and / or magnetic bead method.

[0049] Further, the magnetic bead method is CD3 magnetic bead separation.

[0050] Further, the density gradient centrifugation includes Ficoll, Percoll density gradient centrifugation.

[0051] Further, the density gradient centrifugation is Ficoll density gradient centrifugation.

[0052] Further, the speed of Ficoll density gradient centrifugation is 300-500 g.

[0053] Further, the speed of Ficoll density gradient centrifugation is 400 g.

[0054] Further, the temperature of Ficoll density gradient centrifugation is 15-25℃.

[0055] Further, the temperature of Ficoll density gradient centrifugation is 20℃.

[0056] Further, the time of Ficoll density gradient centrifugation is 10-60 min.

[0057] Further, the time of Ficoll density gradient centrifugation is 25 min.

[0058] Further, the tumor tissue includes liver cancer, bile duct cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, bladder cancer, epithelial cancer, head and neck cancer, gastric cancer, esophageal cancer, nasopharyngeal carcinoma.

[0059] Further, the tumor tissue is liver cancer tissue.

[0060] Further, the tumor tissue includes fresh tissue or preserved tissue.

[0061] Further, the preserved tissue is preserved using a tissue preservative.

[0062] Further, the tumor tissue includes fresh tissue or is preserved using a preservative.

[0063] Further, the inoculation density of the tumor infiltrating lymphocytes is 1.00×10 5 / mL-2.00×10 6 / mL.

[0064] Further, the tumor infiltrating lymphocytes are seeded at a density of 5.00 x 10 5 / mL-1.00 x 10 6 / mL.

[0065] Further, the method further comprises a freezing step.

[0066] Further, the freezing is performed using a freezing solution.

[0067] Further, the freezing solution comprises: 1) CryoStor CS10 and HSA (human serum albumin), and normal saline, or CryoStor CS10 and HSA, and Compound Electrolyte Injection; or 2) DMSO, HSA, Compound Electrolyte Injection, Glucose Sodium Chloride, and Dextran-40 Glucose; or 3) and HSA, and Compound Electrolyte Injection, or and HSA, and NaCl.

[0068] In another aspect, the present application provides a tumor infiltrating lymphocyte expansion product, the product comprising a culture medium, interleukins, the interleukins comprising one or several of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23.

[0069] Further, the interleukins are selected from IL-2, IL-7, and IL-15.

[0070] Further, the concentration of IL-2 is 300-9000 IU / mL.

[0071] Further, the concentration of IL-2 is 6000 IU / mL.

[0072] Further, the concentration of IL-7 is 0-10 ng / mL.

[0073] Further, the concentration of IL-7 is 5 ng / mL.

[0074] Further, the concentration of IL-15 is 0-30 ng / mL.

[0075] Further, the concentration of IL-15 is 15 ng / mL.

[0076] Further, the culture medium is selected from a serum-free culture medium.

[0077] Further, the serum-free culture medium comprises OptiVitro T Cell Serum-Free Medium, X-vivo 15 Medium, TexMACS Medium, media, AIM V media, ImmunoCult-XF T Cell Expansion Medium, RPMI-1640 media.

[0078] Further, the serum-free media is selected from OptiVitro T media.

[0079] Further, the serum-free media further comprises a serum replacement.

[0080] Further, the serum replacement comprises one or more of CTS Immune Cell Serum Replacement, Ultrose G Serum Substitute, OptiVitro T Cell Serum-Free Additive Component.

[0081] Further, the serum replacement is selected from CTS Immune Cell Serum Replacement and / or OptiVitro T Cell Serum-Free Additive Component.

[0082] Further, the serum replacement is added at a ratio of 0%-10% CTS Immune Cell Serum Replacement, 0%-10% OptiVitro T Cell Serum-Free Additive Component.

[0083] Further, the serum-free media comprises X-vivo 15 media and CTS Immune Cell Serum Replacement.

[0084] Further, the serum-free media comprises OptiVitro T media and OptiVitro T Cell Serum-Free Additive Component.

[0085] In another aspect, the present application provides a tumor tissue preservation solution, the tumor tissue preservation solution comprising a serum-free media, interleukin.

[0086] Further, the serum-free media comprises OptiVitro T Cell Serum-Free Media, X-vivo 15 media, TexMACS media, media, AIM V media, ImmunoCult-XF T Cell Expansion Medium, RPMI-1640 media.

[0087] Further, the serum-free media is selected from OptiVitro T media.

[0088] Further, the interleukins include one or more of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, and IL-23.

[0089] Further, the interleukins are selected from IL-7 and IL-15.

[0090] Further, the concentration of IL-7 is 0-10 ng / mL.

[0091] Further, the concentration of IL-7 is 5 ng / mL.

[0092] Further, the concentration of IL-15 is 0-30 ng / mL.

[0093] Further, the concentration of IL-15 is 15 ng / mL.

[0094] Further, the preservation temperature of the tumor tissue is 0-25℃.

[0095] Further, the preservation temperature of the tumor tissue is 2-8℃.

[0096] Further, the preservation time of the tumor tissue is 0-72 h.

[0097] Further, the preservation time of the tumor tissue is 0-30 h.

[0098] Further, the tumor tissue includes liver cancer, cholangiocarcinoma, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, bladder cancer, epithelial cancer, head and neck cancer, gastric cancer, esophageal cancer, and nasopharyngeal carcinoma.

[0099] Further, the tumor tissue is liver cancer tissue.

[0100] In another aspect, the present application provides a tumor infiltrating lymphocyte population obtained by the method described above.

[0101] In another aspect, the present application provides a pharmaceutical composition comprising the tumor infiltrating lymphocyte population described above.

[0102] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0103] In another aspect, the present application provides the use of the tumor infiltrating lymphocyte population described above in the preparation of a product for preventing and / or treating tumors.

[0104] Further, the tumor includes a hematological tumor and a solid tumor.

[0105] Further, the tumor is selected from a solid tumor.

[0106] Further, the solid tumor includes liver cancer, bile duct cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, bladder cancer, epithelial cancer, head and neck cancer, gastric cancer, esophageal cancer, nasopharyngeal cancer.

[0107] In another aspect, the present application provides a tumor-infiltrating lymphocyte freezing solution, the freezing solution comprising: 1) CryoStor CS10 and HSA, and normal saline, or CryoStor CS10 and HSA, and compound electrolyte injection; or 2) DMSO, HSA, compound electrolyte injection, glucose sodium chloride, and dextran glucose; or 3) and HSA, and compound electrolyte injection, or and HSA, and NaCl.

[0108] Further, the compound electrolyte injection is PLA (Plasma-Lyte A).

[0109] Further, the volume ratio of CryoStor CS10 in 1) is 25%-75%.

[0110] Further, the volume ratio of CryoStor CS10 in 1) is 50%.

[0111] Further, the volume ratio of HSA in 1) is 0%-25%.

[0112] Further, the volume ratio of HSA in 1) is 12.5%.

[0113] Further, the volume ratio of normal saline in 1) is 25%-50%.

[0114] Further, the volume ratio of normal saline in 1) is 37.5%.

[0115] Further, the volume ratio of PLA in 1) is 25%-50%.

[0116] Further, the volume ratio of PLA in 1) is 37.5%.

[0117] Further, the volume ratio of DMSO in 2) is 0%-15%.

[0118] Further, the volume ratio of DMSO in 2) is 7.5%.

[0119] Further, the volume ratio of HSA in 2) is 0%-25%.

[0120] Further, the volume ratio of HSA in 2) is 20%.

[0121] Further, the volume ratio of PLA in 2) is 25%-50%.

[0122] Further, the volume ratio of PLA in 2) is 31.25%.

[0123] Further, the volume ratio of glucose sodium chloride in 2) is 25%-50%.

[0124] Further, the volume ratio of glucose sodium chloride in 2) is 31.25%.

[0125] Further, the volume ratio of dextran glucose in 2) is 0%-25%.

[0126] Further, the volume ratio of dextran glucose in 2) is 10%.

[0127] Further, the volume ratio of in 3) is 0%-75%.

[0128] Further, the volume ratio of in 3) is 40%-60%.

[0129] Further, the volume ratio of in 3) is 49% or 50%.

[0130] Further, the volume ratio of HSA in 3) is 0%-25%.

[0131] Further, the volume ratio of HSA in 3) is 2% or 12.5%.

[0132] Further, the volume ratio of PLA in 3) is 25%-60%.

[0133] Further, the volume ratio of PLA in 3) is 25%-50%.

[0134] Further, the volume ratio of PLA in 3) is 49% or 37.5%.

[0135] Further, the volume ratio of NaCl in 3) is 25%-50%.

[0136] Further, the volume ratio of NaCl in 3) is 37.5%.

[0137] Further, the cryopreservation solution comprises

[0138] 50% CryoStor CS10, 12.5% HSA, and 37.5% physiological saline; or

[0139] 50% CryoStor CS10, 12.5% HSA, and 37.5% PLA; or

[0140] 7.5% DMSO, 20% HSA, 31.25% PLA, 31.25% glucose sodium chloride and 10% dextran glucose; or

[0141] 49% 2% HSA and 49% PLA; or

[0142] 50% 12.5% HSA and 37.5% PLA; or

[0143] 50% 12.5% HSA and 37.5% NaCl.

[0144] Further, the density of the cryopreserved cells in the cryopreservation solution is 5.00 x 10 6 / mL-8.00 x 10 7 / mL.

[0145] In another aspect, the present application provides a method for cryopreservation of tumor infiltrating lymphocytes, comprising using the cryopreservation solution as described above.

[0146] Further, the method comprises resuspending the tumor infiltrating lymphocytes using the cryopreservation solution for cryopreservation.

[0147] Further, the cell density of the resuspended tumor infiltrating lymphocytes is 1.00 x 10 7 / mL-1.00 x 10 8 / mL.

[0148] Further, the cell density of the resuspended tumor infiltrating lymphocytes is 5.00 x 10 7 / mL.

[0149] Further, the method further comprises cryopreservation of the resuspended tumor infiltrating lymphocytes after cooling.

[0150] Further, the method of cooling comprises programmed cooling or stepwise cooling.

[0151] Further, the temperature of the cooling is -70°C to -196°C.

[0152] Further, the temperature of the cooling is -135°C.

[0153] In another aspect, the present application provides a method for treating tumors, comprising using the tumor infiltrating lymphocyte population as described above or the pharmaceutical composition as described above.

[0154] Further, the tumor comprises hematological tumors, solid tumors.

[0155] Further, the tumor is selected from solid tumors.

[0156] Further, the solid tumor includes liver cancer, bile duct cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, bladder cancer, epithelial cancer, head and neck cancer, gastric cancer, esophageal cancer, nasopharyngeal cancer.

[0157] Advantages and beneficial effects of the present application:

[0158] The present application develops a new method for separating and enriching TIL cells in tumor tissues, and a TIL cell optimized culture method independent of feeder cells, which can obtain 10 10 or more TIL cells with anti-tumor function in 3-4 weeks. Through cell phenotype analysis, compared with TILs expanded by traditional two-step method, TIL cells cultured by the technology of the present application have higher proportion of memory T cells, lower proportion of exhausted T cells and regulatory T cells. BRIEF DESCRIPTION OF DRAWINGS

[0159] Figure 1 is a comparison result graph of liver cancer tumor tissue storage time; wherein, 1A is the proportion of DRAQ7 - CD3 + T cells, CD4 + T cells and CD8 + T cells in CD3 + T cells after the tissue is stored for 24h, 30h and 48h; 1B is the proportion of dead cells in CD45 + cells (DRAQ7 + ), the proportion of dead cells in CD3 + cells (DRAQ7 + ), the number of CD45 + live cells obtained per gram of tissue and the number of CD3 + live cells obtained per gram of tissue in TIL freshly isolated at different storage time points; 1C is a graph of live cell count, cell viability and average cell diameter analysis results when cultured to the 7th day; 1D is a graph of CD3 + CD45 + T cell proportion analysis of TIL at different storage time points when cultured to the 7th day;

[0160] Figure 2 is a comparison result graph of liver cancer tumor tissue enzyme digestion time; wherein, 2A is the proportion of DRAQ7 - CD3 + T cells, CD4 + T cells and CD8 + T cells in CD3 + T cells after enzyme digestion for 15 minutes, 30 minutes and 60 minutes; 2B is the proportion of CD45 +The proportion of dead cells in cells (DRAQ7) + CD3 + The proportion of dead cells in cells (DRAQ7) + CD45 was obtained per gram of tissue. + The number of live cells and the CD3 obtained per gram of tissue isolation + 2C shows the results of live cell count (analysis results of 3 replicates); 2D shows the analysis results of live cell count, cell viability, and average cell diameter at day 7 of culture; 2D shows the CD3 count of TILs at different storage time points at day 7 of culture. + CD45 + T cell ratio analysis chart;

[0161] Figure 3 shows the comparison results of CD3 / CD28 antibody concentrations activating TILs; 3A is a time-dynamic analysis of the number of TIL cells amplified under different CD3 / CD28 antibody concentrations; 3B is a time-dynamic analysis of the fold increase of TILs under different CD3 / CD28 antibody concentrations; 3C is a time-dynamic analysis of the CD45 cell amplification rate under different CD3 / CD28 antibody concentrations. + CD3 + The proportion of T cells is shown in diagram 3D; CD4 cells are represented at different CD3 / CD28 antibody concentrations. + T cells account for CD3 + The proportion of T cells; 3E represents CD8 cells at different CD3 / CD28 antibody concentrations. + T cells account for CD3 + The proportion of T cells; 3F shows the CD4 cell ratio at different CD3 / CD28 antibody concentrations. - CD8 - Cells account for CD3 + The proportion of T cells; 3G represents the depleted phenotype (PD-1TIM3) at different CD3 / CD28 antibody concentrations. + LAG3 + ) cells account for CD8 + A graph showing the proportion of T cells;

[0162] Figure 4 shows the results of comparing the effects of different culture media and additives on TIL proliferation and phenotype; 4A is the dynamic cell number and fold increase analysis of TIL under different culture conditions; 4B is the TIL cell viability and average cell diameter under different culture conditions; 4C is the CD45 concentration under different culture conditions. + CD3 + The proportion of T cells; 4D shows the CD8+ ratio under different culture conditions. + T cells account for CD3 + The proportion of T cells; 4E represents CD4 under different culture conditions. + T cells account for CD3+ T cell ratio plot; 4F is the PD-1 TIM3 + LAG3 + CD8 + T cell ratio plot;

[0163] Figure 5 is a graph of the results of detection of the effect of IL-7 and IL-15 on the proliferation of TIL and memory phenotype T cells; wherein 5A is a graph of the dynamic cell number of TIL expansion under different culture conditions; 5B is a graph of the TIL cell expansion fold under different culture conditions; 5C is a graph of the CD8 + T cells (Tc) and CD4 + T cells (Th) accounted for CD3 + T cell ratio plot (statistical graph is the analysis result of 3 repeated experiments); 5D is a graph of the CD45RA + CCR7 + Terminally differentiated T cells (TD) accounted for CD3 + T cell ratio plot, and CD45RA + CCR7 + TCM cells accounted for CD8 + T cell ratio plot (statistical graph is the analysis result of 3 repeated experiments);

[0164] Figure 6 is a graph of the results of detection of the effect of different freezing solutions on TIL; wherein 6A is a graph of the survival rate of cells after freezing and thawing treated with different freezing solutions; 6B is a graph of the IFN-γ level in the co-culture supernatant detected by ELISA after the cells were co-cultured with target cells SK-Hep-1 overnight (effector to target ratio was 1:1 and 3:1, respectively) after 1.5 and 3h of recovery; 6C is a graph of the effect of the optimized formula of freezing solution 5. DETAILED DESCRIPTION

[0165] The following provides definitions of some terms used in the specification. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0166] The present application provides a method for expanding tumor infiltrating lymphocytes in vitro, which comprises culturing tumor infiltrating lymphocytes using an expansion medium, wherein the expansion medium comprises a culture medium and interleukin.

[0167] In some embodiments of the application, expansion generally refers to a change in the number of cells produced by culturing, and expanded cells can also produce a change in the number and / or ratio of cells, a change in secretion capacity, a change in killing capacity, or a change in expression capacity, or any combination thereof. In some embodiments, the number of cells can be increased by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold); in some embodiments, the number of cells can be increased by at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold); in some embodiments, the number of cells can be increased by at least about 100-fold (or 200, 300, 400, 500, 600, 700, 800, or 900-fold); in some embodiments, the number of cells can be increased by at least about 1000-fold (or 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000-fold); in some embodiments, the number of cells can be increased by at least about 10,000-fold (or 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, or 90,000-fold); in some embodiments, the number of cells can be increased by at least about 100,000-fold (or 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, or 900,000-fold); in some embodiments, the number of cells can be increased by at least about 1,000,000-fold (or 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, or 9,000,000-fold); in some embodiments, the number of cells can be increased by at least about 10,000,000-fold (or 20,000,000, 30,000,000, 40,000,000, 50,000,000, 60,000,000, 70,000,000, 80,000,000, or 90,000,000-fold); in some embodiments, the number of cells can be increased by at least about 100,000,000-fold (or 200,000,000, 300,000,000, 400,000,000, 500,000,000, 600,000,000, 700,000,000, 800,000,000, or 900,000,000-fold); in some embodiments, the number of cells can be increased by at least about 1,000,000,000-fold (or 2,000,000,000, 3,000,000,000, 4,000,000,000, 5,000,000,000, 6,000,000,000, 7,000,000,000, 8,000,000,000, or 9,000,000,000-fold); in some embodiments, the number of cells can be increased by at least about 10,000,000,000-fold (or 20,000,000,000, 30,000,000,000, 40,000,000,000, 50,000,000,000, 60,000,000,000, 70,000,000,000, 80,000,000,000, or 90,000,000,000-fold); in some embodiments, the number of cells can be increased by at least about 100,000,000,000-fold (or 200,000,000,000, 300,000,000,000, 400,000,000,000, 500,000,000,000, 600,000,000,000, 700,000,000,000, 800,000,000,000, or 900,000,000,000-fold); in some embodiments, the number of cells can be increased by at least about 1,000,000,000,000-fold (or 2,000,000,000,000, 3,000,000,000,000, 4,000,000,000,000, 5,000,000,000,000, 6,000,000,000,000, 7,000,000,000,000, 8,000,000,000,000, or 9,000,000,000,000-fold). 3 4 5 6 7 8 9 10

[0168] In some embodiments of the application, comprising or including is inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials.

[0169] In some embodiments of the application, the culture medium comprises a serum-containing medium or a serum-free medium.

[0170] In alternative embodiments of the application, the culture medium is selected from a serum-free medium. A serum-free medium refers to a liquid nutrient medium that does not contain serum, plasma, platelet plasma (PRP), or other blood-derived substances or blood, and contains a variety of well-defined biologically active substances, inorganic salts, water, and the like.

[0171] In some embodiments of the application, the serum-free medium comprises OptiVitro T Cell Serum-Free Medium, X-vivo 15 Medium, TexMACS Medium, T Medium, AIM V Medium, ImmunoCult-XF T Cell Expansion Medium, RPMI-1640 Medium.

[0172] ​​​​​​​​In embodiments of the application, the serum-free medium is selected from OptiVitro T medium.

[0173] The serum-free medium further comprises a serum substitute.

[0174] In embodiments of the application, the serum substitute comprises, but is not limited to, one or more of CTS Immune Cell Serum Replacement, Ultrose G Serum Substitute, OptiVitro T Cell Serum-Free Additive Component.

[0175] In embodiments of the application, the serum substitute is selected from CTS Immune Cell Serum Replacement and / or OptiVitro T Cell Serum-Free Additive Component.

[0176] In embodiments of the application, the serum-free medium comprises X-vivo 15 medium and CTS Immune Cell Serum Replacement.

[0177] In embodiments of the application, the serum-free medium comprises OptiVitro T medium and OptiVitro T Cell Serum-Free Additive Component.

[0178] In embodiments of the application, the interleukin comprises, but is not limited to, one or more of IL-2 (interleukin 2), IL-7 (interleukin 7), IL-10 (interleukin 10), IL-12 (interleukin 12), IL-15 (interleukin 15), IL-21 (interleukin 21), IL-23 (interleukin 23), including wild-type thereof or a mutant / modified thereof.

[0179] In embodiments of the application, the interleukin is selected from IL-2 (interleukin 2), IL-7 (interleukin 7), and IL-15 (interleukin 15).

[0180] In some embodiments of the application, IL-2, IL-7, or IL-15 can comprise IL-2, IL-7, or IL-15 protein or a variant thereof. Variants of IL-2, IL-7, or IL-15 protein can include a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or substitution of an amino acid, but retains at least one biological activity of IL-2, IL-7, or IL-15. Variants of IL-2, IL-7, or IL-15 protein can also include a fusion protein thereof linked to other proteins. The amino acid sequence variants can be naturally occurring variants, but other amino acid sequence variants can be post-translation variants, e.g., glycosylation variants. Any variant of IL-2, IL-7, or IL-15 can be used as long as it retains an appropriate level of IL-2, IL-7, or IL-15 activity. The form of IL-2, IL-7, or IL-15 can vary. For example, one form can include wild-type human IL-2, IL-7, or IL-15.

[0181] In specific embodiments of the application, the method of expanding TILs comprises culturing using X-vivo 15 medium and CTS Immune Cell Serum Replacement, IL-2, IL-7, and IL-15.

[0182] In specific embodiments of the application, the method of expanding TILs comprises culturing using OptiVitro T medium and OptiVitro T Cell Serum-Free Additive Component, IL-2, IL-7, and IL-15.

[0183] In some embodiments of the application, the expansion method further comprises activating tumor infiltrating lymphocytes prior to culturing the tumor infiltrating lymphocytes using the expansion medium; activating the tumor infiltrating lymphocytes using an activator; the activator comprises one or several of antibodies, ligands, receptors of CD3, CD28, CD137, ICOS, CD40L, OX40.

[0184] In specific embodiments of the application, the activator is selected from CD3 / CD28 antibodies.

[0185] In some embodiments of the application, the concentration of the CD3 / CD28 antibodies can be any concentration that activates T cells.

[0186] In some embodiments of the application, the total concentration of the CD3 / CD28 antibody is 0-10 μg / mL. In the context of the present application, 0-10 μg / mL is greater than 0 μg / mL and less than or equal to 10 μg / mL. For example, it can be 0.1 μg / mL, it can be 0.3 μg / mL, it can be 0.5 μg / mL, it can be 0.7 μg / mL, it can be 1 μg / mL, it can be 1.1 μg / mL, it can be 1.2 μg / mL, it can be 1.3 μg / mL, it can be 1.4 μg / mL, it can be 1.5 μg / mL, it can be 1.7 μg / mL, it can be 1.8 μg / mL, it can be 1.9 μg / mL, it can be 2 μg / mL, it can be 3 μg / mL, it can be 4 μg / mL, it can be 5 μg / mL, it can be 6 μg / mL, it can be 7 μg / mL, it can be 8 μg / mL, it can be 9 μg / mL, it can be 10 μg / mL, and concentrations between any two of the foregoing.

[0187] In some embodiments of the application, the total concentration of the CD3 / CD28 antibody is 1-2 μg / mL. For example, it can be 1 μg / mL, it can be 1.1 μg / mL, it can be 1.2 μg / mL, it can be 1.3 μg / mL, it can be 1.4 μg / mL, it can be 1.5 μg / mL, it can be 1.7 μg / mL, it can be 1.8 μg / mL, it can be 1.9 μg / mL, it can be 2 μg / mL, and concentrations between any two of the foregoing.

[0188] In some embodiments of the application, the total concentration of the CD3 / CD28 antibody is 2 μg / mL.

[0189] In some embodiments of the application, the CD3 / CD28 antibody is used in soluble or coated form, preferably in coated form.

[0190] In some embodiments of the application, coating or antibody coating means that the specific antibody is immobilized on a solid support.

[0191] Soluble means that the antibody is added to the culture system together with other reagents.

[0192] In some embodiments of the application, the activated TIL cells comprise activated TIL cells activated using CD3 / CD28 antibody coating.

[0193] The tumor infiltrating lymphocytes are obtained by digesting tumor tissue.

[0194] In some embodiments of the present application, the method for expanding tumor infiltrating lymphocytes in vitro comprises digesting the tumor tissue to obtain a single cell suspension of the tumor tissue. Further, the tumor infiltrating lymphocytes are isolated for subsequent TIL preparation.

[0195] In some embodiments of the present application, the enzyme digestion method is preferably used to digest the tissue.

[0196] In some embodiments of the present application, the enzyme comprises collagenase and / or nuclease, and the collagenase comprises collagenase I and / or collagenase II.

[0197] In some embodiments of the present application, the time for digesting the tumor tissue can be any time that can digest the tumor tissue.

[0198] In alternative embodiments of the present application, the time for digesting the tumor tissue is 0-120 min. In the present application, 0-120 min is greater than 0 min and less than or equal to 120 min. For example, it can be 1 min, it can be 3 min, it can be 6 min, it can be 8 min, it can be 10 min, it can be 13 min, it can be 15 min, it can be 17 min, it can be 19 min, it can be 20 min, it can be 21 min, it can be 23 min, it can be 25 min, it can be 27 min, it can be 28 min, it can be 30 min, it can be 33 min, it can be 35 min, it can be 36 min, it can be 38 min, it can be 39 min, it can be 40 min, it can be 42 min, it can be 44 min, it can be 45 min, it can be 47 min, it can be 49 min, it can be 50 min, it can be 52 min, it can be 53 min, it can be 55 min, it can be 57 min, it can be 59 min, it can be 60 min, it can be 65 min, it can be 70 min, it can be 75 min, it can be 80 min, it can be 85 min, it can be 90 min, it can be 95 min, it can be 100 min, it can be 110 min, it can be 120 min, and any time between any two of the above.

[0199] In optional embodiments of the application, the tumor tissue is digested for 15-60 min. For example, it can be 15 min, it can be 17 min, it can be 19 min, it can be 20 min, it can be 21 min, it can be 23 min, it can be 25 min, it can be 27 min, it can be 28 min, it can be 30 min, it can be 33 min, it can be 35 min, it can be 36 min, it can be 38 min, it can be 39 min, it can be 40 min, it can be 42 min, it can be 44 min, it can be 45 min, it can be 47 min, it can be 49 min, it can be 50 min, it can be 52 min, it can be 53 min, it can be 55 min, it can be 57 min, it can be 59 min, it can be 60 min, and any time between any two of the aforementioned times.

[0200] In optional embodiments of the application, the tumor tissue is digested for 30-60 min. For example, it can be 33 min, it can be 35 min, it can be 36 min, it can be 38 min, it can be 39 min, it can be 40 min, it can be 42 min, it can be 44 min, it can be 45 min, it can be 47 min, it can be 49 min, it can be 50 min, it can be 52 min, it can be 53 min, it can be 55 min, it can be 57 min, it can be 59 min, it can be 60 min, and any time between any two of the aforementioned times.

[0201] In some embodiments of the application, the preservation temperature of the tumor tissue can be any temperature that is capable of obtaining T cells with activity from the tumor tissue.

[0202] In optional embodiments of the application, the preservation temperature of the tumor tissue is 0-25°C. In the present application, 0-25°C is greater than 0°C and less than or equal to 25°C. For example, it can be greater than 0°C, it can be 2°C, it can be 3°C, it can be 4°C, it can be 5°C, it can be 6°C, it can be 7°C, it can be 8°C, it can be 10°C, it can be 12°C, it can be 14°C, it can be 16°C, it can be 18°C, it can be 20°C, it can be 22°C, it can be 25°C, and any temperature between any two of the aforementioned temperatures.

[0203] In optional embodiments of the application, the preservation temperature of the tumor tissue is 2-8°C. For example, it can be 2°C, it can be 3°C, it can be 4°C, it can be 5°C, it can be 6°C, it can be 7°C, it can be 8°C, and any temperature between any two of the aforementioned temperatures.

[0204] In some embodiments of the application, the preservation time of the tumor tissue can be any time that can obtain T cells with activity from the tumor tissue.

[0205] In alternative embodiments of the application, the preservation time of the tumor tissue is 0-72h. In the present application, 0-72h is greater than 0h and less than or equal to 72h. For example, it can be 0.1h, it can be 2h, it can be 4h, it can be 6h, it can be 8h, it can be 10h, it can be 12h, it can be 14h, it can be 16h, it can be 18h, it can be 20h, it can be 22h, it can be 24h, it can be 26h, it can be 28h, it can be 30h, it can be 32h, it can be 34h, it can be 36h, it can be 38h, it can be 40h, it can be 42h, it can be 44h, it can be 46h, it can be 48h, it can be 50h, it can be 52h, it can be 54h, it can be 56h, it can be 58h, it can be 60h, it can be 62h, it can be 64h, it can be 66h, it can be 68h, it can be 70h, it can be 72h, and a time between any two of the above.

[0206] In alternative embodiments of the application, the preservation time of the tumor tissue is 0-30h. In the present application, 0-30h is greater than 0h and less than or equal to 30h. For example, it can be 0.1h, it can be 2h, it can be 4h, it can be 6h, it can be 8h, it can be 10h, it can be 12h, it can be 14h, it can be 16h, it can be 18h, it can be 20h, it can be 22h, it can be 24h, it can be 26h, it can be 28h, it can be 30h, and a time between any two of the above.

[0207] In some embodiments of the application, the inoculation density of the tumor infiltrating lymphocytes can be any density that the cells can grow after inoculation.

[0208] In alternative embodiments of the application, the inoculation density of the tumor infiltrating lymphocytes is 1.00x10 5 / mL-2.00x10 6 / mL. For example, it can be 1.00x10 5 / mL, it can be 1.50x10 5 / mL, it can be 2.00x10 5 / mL, it can be 2.50x10 5 / mL, it can be 3.00x10 5 / mL, it can be 3.50x10 5 / mL, it can be 4.00x10 5 / mL, it can be 4.50x105 / mL, can be 5.00 x 10 5 / mL, can be 5.50 x 10 5 / mL, can be 6.00 x 10 5 / mL, can be 6.50 x 10 5 / mL, can be 7.00 x 10 5 / mL, can be 7.50 x 10 5 / mL, can be 8.00 x 10 5 / mL, can be 8.50 x 10 5 / mL, can be 9.00 x 10 5 / mL, can be 9.50 x 10 5 / mL, can be 1.00 x 10 6 / mL, can be 1.50 x 10 6 / mL, can be 2.00 x 10 6 / mL, and any two points therebetween.

[0209] In optional embodiments of the application, the tumor infiltrating lymphocytes are seeded at a density of 5.00 x 10 5 / mL to 1.00 x 10 6 / mL. For example, can be 5.00 x 10 5 / mL, can be 5.50 x 10 5 / mL, can be 6.00 x 10 5 / mL, can be 6.50 x 10 5 / mL, can be 7.00 x 10 5 / mL, can be 7.50 x 10 5 / mL, can be 8.00 x 10 5 / mL, can be 8.50 x 10 5 / mL, can be 9.00 x 10 5 / mL, can be 9.50 x 10 5 / mL, can be 1.00 x 10 6 / mL, can be 1.50 x 10 6 / mL, can be 2.00 x 10 6 / mL, and any two points therebetween.

[0210] In some embodiments of the application, the tumor tissue digestion further comprises the addition of serum albumin.

[0211] In specific embodiments of the present application, the method for expanding tumor infiltrating lymphocytes in vitro comprises digesting tumor tissue using collagenase and nuclease and serum albumin to obtain a single cell suspension of tumor tissue.

[0212] The present application provides a pharmaceutical composition comprising the above-mentioned population of tumor infiltrating lymphocytes.

[0213] The pharmaceutical composition described in the present application can be formulated in any manner suitable for delivery. For example, it can be administered as nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, or combinations thereof.

[0214] In some embodiments of the present application, administration refers to a method of giving a dose of a pharmaceutical composition to a subject (e.g., a patient). Administration can be by any suitable means, including intravenous, intramuscular, intradermal, subcutaneous, transdermal, intratumoral, and / or mucosal.

[0215] In some embodiments of the present application, the dose of the pharmaceutical composition can be determined according to the severity of the disease, the response of the disease, any treatment-related toxicity, the age and health status of the patient. In some embodiments, the number of tumor infiltrating lymphocytes of the pharmaceutical composition described in the present application can be about 1.00 x 10 8 , about 2.00 x 10 8 , about 3.00 x 10 8 , about 4.00 x 10 8 , about 5.00 x 10 8 , about 6.00 x 10 8 , about 7.00 x 10 8 , about 8.00 x 10 8 , about 9.00 x 10 8 , about 1.00 x 10 9 , about 2.00 x 10 9 , about 3.00 x 10 9 , about 4.00 x 10 9 , about 5.00 x 10 9 , about 6.00 x 10 9 , about 7.00 x 10 9 , about 8.00 x 10 9 , about 9.00 x 10 9 , about 1.00 x 10 10 , about 2.00 x 10 10 , about 3.00 x 10 10 , about 4.00 x 10 10about 5.00 x 105 10 about 6.00 x 105 10 about 7.00 x 105 10 about 8.00 x 105 10 about 9.00 x 105 10 about 1.00 x 105 11 about 2.00 x 105 11 about 3.00 x 105 11 about 4.00 x 105 11 about 5.00 x 105 11 about 6.00 x 105 11 about 7.00 x 105 11 about 8.00 x 105 11 about 9.00 x 105 11 about 1.00 x 105 12 about 2.00 x 105 12 about 3.00 x 105 12 about 4.00 x 105 12 about 5.00 x 105 12 about 6.00 x 105 12 about 7.00 x 105 12 about 8.00 x 105 12 about 9.00 x 105 12 about 1.00 x 105 13 about 2.00 x 105 13 about 3.00 x 105 13 about 4.00 x 105 13 about 5.00 x 105 13 about 6.00 x 105 13 about 7.00 x 105 13 about 8.00 x 105 13 about 9.00 x 105 13 and ranges of amounts between any of the above recited amounts.

[0216] In some embodiments of the present application, the number of tumor infiltrating lymphocytes provided in the pharmaceutical composition can range from about 1.00 x 105 8 to 5.00 x 105 8 to 1.00 x 105 8 to 5.00 x 105 9 to 1.00 x 105 9 to 5.00 x 105 9 to 1.00 x 105 9 to 5.00 x 105 10 to 1.00 x 105 10 to 5.00 x 105 10about 5.00 x 10 10 about 5.00 x 10 11 about 5.00 x 10 11 about 5.00 x 10 12 about 5.00 x 10 12 about 5.00 x 10 12 about 5.00 x 10 12 about 5.00 x 10 13 .

[0217] In some embodiments of the application, the concentration of tumor infiltrating lymphocytes provided in the pharmaceutical composition is about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%, about 0.009%, about 0.008%, about 0.007%, about 0.006%, about 0.005%, about 0.004%, about 0.003%, about 0.002%, about 0.001%, about 0.0009%, about 0.0008%, about 0.0007%, about 0.0006%, about 0.0005%, about 0.0004%, about 0.0003%, about 0.0002%, or about 0.0001% w / w, w / v, or v / v, and concentrations between any of the above recited concentrations.

[0218] In some embodiments of the application, the concentration of tumor infiltrating lymphocytes provided in the pharmaceutical composition is about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w / w, w / v, or v / v.

[0219] The pharmaceutical composition also includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0220] In some embodiments of the application, pharmaceutically acceptable means it is used in the manufacture of a pharmaceutical composition that is generally safe, non-toxic and does not biologically or otherwise undesirable.

[0221] In some embodiments of the application, a pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers in active pharmaceutical ingredient is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the application is contemplated. Other active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0222] The pharmaceutical composition can also include a pharmaceutically acceptable salt.

[0223] In some embodiments of the application, pharmaceutically acceptable salts include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, p-toluenesulfonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and the like.

[0224] The application provides the use of the above-mentioned tumor infiltrating lymphocyte population in the preparation of a product for treating tumors.

[0225] In some embodiments of the application, treatment refers to obtaining a desired pharmacological and / or physiologic effect. This effect can be prophylactic with respect to fully or partially preventing a disease or symptom thereof and / or therapeutic with respect to fully or partially curing a disease and / or adverse effect caused by the disease. As used herein, treatment includes any action that promotes well-being of a mammal, particularly a human, with respect to a disease, including: (a) preventing the disease from occurring in an individual that can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relief from one or more symptoms of the disease. Treatment also means to include the delivery of a pharmaceutical agent to provide a pharmacological effect, even in the absence of a disease or condition. For example, treatment includes the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease, such as in the case of a vaccine.

[0226] In some embodiments of the application, tumor tissue or tumor refers to an abnormal mass of tissue resulting from excessive cell division. Tumor tissue or tumor comprises tumor cells, which are neoplastic cells that have abnormal growth properties and lack useful body functions. Tumors, tumor tissue, and tumor cells can be benign, pre-malignant, or malignant, or can represent lesions that do not have any cancerous potential. Tumor tissue or tumor can also comprise tumor-associated non-tumor cells, such as vascular cells that form blood vessels to supply the tumor or tumor tissue. Non-tumor cells can be induced to replicate and develop by tumor cells, such as the induction of angiogenesis in a tumor or tumor tissue.

[0227] Patient tumor samples can be obtained using methods known in the art, generally including surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Typically, the tumor sample can be from any solid tumor, and can also be a blood tumor, such as a tumor obtained from a hematological malignancy.

[0228] In some embodiments of the application, the tumor cells are selected from a solid tumor.

[0229] In some embodiments of the application, a solid tumor refers to an abnormal mass of tissue that generally does not contain cysts or fluid areas. Solid tumors can be benign (not cancer) or malignant (cancer). Different types of solid tumors are named for the type of cell that forms them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Sarcomas are cancers that arise from connective or supportive tissue, such as bone or muscle. Carcinomas are cancers that arise from glandular and epithelial cells that line the tissues of the body. Lymphomas are cancers of the lymphoid organs, such as lymph nodes, spleen, and thymus. Because these cells are present in most tissues of the body, lymphomas can occur in many organs. Exemplary solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung cancer, bladder carcinoma, epithelial carcinoma, head and neck cancer, stomach cancer, esophageal cancer, nasopharyngeal cancer, glioblastoma multiforme, astrocytomas, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, cutaneous T-cell lymphoma (CTCL), melanoma, neuroblastoma, and retinoblastoma.

[0230] This application is further illustrated by the following specific examples. It is to be understood that the particularity of the examples described herein is presented by way of example and not as a limitation to the application. The main features of the application can be used in various embodiments without departing from the scope of the application.

[0231] Example 1 Development of a method for ex vivo preservation of tumor tissue

[0232] 1. Experimental Materials

[0233] Tumor tissue preservation solution, formula: OptiVitro T cell serum-free medium (ExCell, TE000-N072) + 5 ng / mL IL-7 (Yiqiao, GMP-11821-HNAE) + 15 ng / mL IL-15 (Yiqiao, GMP-10360-HNAE), used for the preservation of liver cancer tissue samples removed by surgery. The main components of OptiVitro T cell serum-free medium are: human albumin, glucose, amino acids, vitamins, inorganic salts, trace metal elements, lipid components, buffer substances, recombinant human insulin, recombinant human transferrin, etc.

[0234] 2 Experimental method

[0235] The stability of tumor tissue in the preservation solution (2-8°C) was explored by experiment, and 24 hours, 30 hours and 48 hours after surgery were selected as the preservation time. The tumor tissues at different preservation time points were digested to obtain tumor tissue single cell suspension, and TIL was obtained by Ficoll density gradient centrifugation. Then, the TIL was inoculated into the culture plate coated with CD3 / CD28 antibody at the same number, and cultured with OptiVitro medium, and 6000 IU / mL IL-2, 5 ng / mL IL-7 and 5 ng / mL IL-15 were added. The expansion cell number, cell viability, cell diameter and cell phenotype of TIL were detected on the 7th day of culture. The results were compared to select the best experimental conditions.

[0236] Statistical analysis: single factor analysis of variance, Tukey algorithm correction; ***P<0.001.

[0237] 3 Experimental results

[0238] Figures 1A and 1B (TIL cell batch number PO0101) show that compared with 24 hours, after the tumor tissue was stored for 48 hours, the proportion of CD45 + or CD3 + dead cells (DRAQ7 + ) in the isolated single cells was significantly increased (24 hours: DRAQ7 + CD45 + , 12.76%; DRAQ7 + CD3 + , 14.50%; 48 hours: DRAQ7 + CD45 + , 21.50%; DRAQ7 + CD3 + , 25.78%). In contrast, when the tumor tissue was stored for 30 hours, the proportion of CD45 +or CD3 + Dead cells (DRAQ7 + ) ratio did not change significantly (30 hours: DRAQ7 + CD45 + , 14.36%; DRAQ7 + CD3 + , 17.21%). Moreover, with the extension of the storage time of the tissue, the number of DRAQ7 - CD45 + or DRAQ7 - CD3 + cells isolated per gram of tissue showed a downward trend (24 hours: DRAQ7 - CD45 + , 4.72 x 10 5 ; DRAQ7 - CD3 + , 2.89 x 10 5 ; 30 hours: DRAQ7 - CD45 + , 3.72 x 10 5 ; DRAQ7 - CD3 + , 2.14 x 10 5 ; 48 hours: DRAQ7 - CD45 + , 1.64 x 10 5 ; DRAQ7 - CD3 + , 9.84 x 10 4 ); importantly, compared with the 30-hour group, the number of DRAQ7 - CD45 + (3.72 x 10 5 vs 1.64 x 10 5 ) or DRAQ7 - CD3 + (2.14 x 10 5 vs 9.84 x 10 4 ) cells isolated showed a more significant downward trend.

[0239] Further, when the TILs were cultured to the 7th day, from the results of FIG. 1C, the number of live cells, cell viability, and average cell diameter of the three groups showed no significant difference (24 hours: live cell number, 9.26 x 10 4 ; cell viability, 73.67%; average cell diameter, 12.43 mm; 30 hours: live cell number, 1.16 x 10 5; cell viability, 70.93%; average cell diameter, 12.5 mm; 48 hours: viable cell number, 8.93 x 10 4 ; cell viability, 71.57%; average cell diameter, 13.3 mm). However, compared to the 24- and 30-hour groups, the CD45 + CD3 + cell proportion was significantly down-regulated (Fig. ID; 24 hours, 90%; 30 hours, 95.1%; 48 hours, 51.4%). The above results show that the tissue preservation solution used in the present application can preserve the surgical resection of liver cancer tumor tissue for at least 30 hours to ensure that the TIL activity and in vitro culture proliferation activity obtained by isolation are not affected.

[0240] Example 2 Development of enzymatic digestion method for tumor tissue

[0241] 1 Experimental method

[0242] In the processing of fresh tumor tissue samples, the tissue was digested by enzymatic digestion to obtain a tumor tissue single cell suspension. Further, tumor infiltrating lymphocytes were isolated by Ficoll density gradient centrifugation (centrifugation parameters: 400g, 20°C, 25min, acceleration 7, deceleration 6) for subsequent TIL preparation. First, a collagenase + nuclease system (specific formula: 0.24 IU / mL collagenase I / II + 10 IU / mL universal nuclease + 15% human serum albumin, buffer OptiVitro T cell serum-free medium) was used to digest the tumor tissue (specific parameters: 37°C, 15-60 minutes). On this basis, the optimal digestion time was explored, and the activity of tumor tissue single cell suspension digested for 15 minutes, 30 minutes and 60 minutes was compared.

[0243] The TIL obtained by density gradient centrifugation were inoculated into the same number of culture plates coated with CD3 / CD28 antibodies, using OptiVitro medium, and adding 6000 IU / mL IL-2, 5 ng / mL IL-7 and 5 ng / mL IL-15 for culture. The expansion of TIL cell number, cell viability, cell diameter, and cell phenotype were detected on the 7th day of culture. The results were compared to select the best experimental conditions.

[0244] Statistical analysis: one-way ANOVA, Tukey's algorithm correction; *, P < 0.05; ns, no significant difference.

[0245] 2 Experimental results

[0246] The results of Figures 2A and 2B (TIL cell batch number PO0101) show that the CD45+ Live cells and CD3 + There was no significant difference in the proportion of live cells in T cells, and CD3 + CD4 in T cells + T cells and CD8 + The proportion of T cells did not change significantly. However, as the digestion time was prolonged, the DRAQ7 - CD45 + and DRAQ7 - CD3 + There was a trend of increase in the number of cells (15-minute group: CD45 + DRAQ7 - , 3.53 x 10 5 ; CD3 + DRAQ7 - , 2.45 x 10 5 ; 30-minute group: CD45 + DRAQ7 - , 3.87 x 10 5 ; CD3 + DRAQ7 - , 2.96 x 10 5 ; 60-minute group: CD45 + DRAQ7 - , 4.74 x 10 5 ; CD3 + DRAQ7 - , 3.87 x 10 5 ).

[0247] Further, when the TILs were cultured to day 7, it was found from the results of Figure 2C that there was a trend of increase in the number of live cells (15-minute group, 4.83 x 10 5 ; 30-minute group, 5.63 x 10 5 ; 60-minute group, 6.47 x 10 5 ) as the digestion time was prolonged, while there was no significant difference in the live cell rate and the average cell diameter. Moreover, when the TILs were cultured to day 7, there was a slight downward trend in the proportion of CD45 + CD3 + cells in the TILs of the 60-minute group compared to the 15-minute and 30-minute groups (Figure 2D). In summary, when the digestion time was 30 minutes, a relatively high TIL isolation yield was obtained, and after culture and expansion, the purity of CD45 + CD3 + cells was high.

[0248] Example 3 Establishment of a TIL cell in vitro expansion method not dependent on feeder cells (TIL cell activation parameters)

[0249] 1. Experimental methods

[0250] Freshly isolated TILs were activated by anti-CD3 / CD28 antibodies by coating the bottom surface of the culture flask with anti-human CD3 / CD28 antibodies at 37°C for 3 hours or at 4°C for 12-16 hours. The coating of anti-CD3 / CD28 antibodies was performed by using different concentrations, including 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, to select the optimal concentration.

[0251] Briefly, the TILs obtained by density gradient centrifugation were inoculated into culture plates coated with different concentrations of CD3 / CD28 antibodies at the same number, and cultured in OptiVitro medium supplemented with 6000 IU / mL IL-2, 5 ng / mL IL-7, and 5 ng / mL IL-15. On days 7, 14, and 21 of culture, the number of expanded TILs, cell viability, cell diameter, and cell phenotype were detected. The results were compared to select the optimal experimental conditions.

[0252] Statistical analysis: Two-way ANOVA with Tukey's correction; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0253] 2. Experimental results

[0254] First, the number and fold of expansion of TIL cells (TIL cell batch numbers PO0301 and PO0302) cultured for 21 days were analyzed. Compared with the 0.1 μg / mL and 0.5 μg / mL CD3 / CD28 antibody groups, the number of cells in the 1 μg / mL and 2 μg / mL CD3 / CD28 antibody groups was significantly increased (Figure 3A). Correspondingly, the cell expansion fold was also significantly increased (Figure 3B). These results suggest that the TILs in the 1 μg / mL and 2 μg / mL groups have a relatively high expansion fold.

[0255] Further, the phenotype of TILs cultured for 7, 14, and 21 days was analyzed. As shown in Figure 3C, after 21 days of culture, the percentage of CD45 + CD3 + T cells was significantly increased in the other three groups compared with the 0.1 μg / mL group, suggesting that long-term culture in the 0.1 μg / mL group is not conducive to the expansion of CD45 + CD3 + T cells. The percentage of CD4 + T cells (Figure 3D), CD8 + T cells (Figure 3E), CD4 - CD8 -Cells (Figure 3F) accounted for CD3 + The proportion of T cells in each group showed a gradual downward trend (Figure 3D), and the proportion of CD8 + The proportion of T cells in each group showed a gradual downward trend (Figure 3D), and the proportion of CD8 + The proportion of T cells in each group showed a gradual downward trend (Figure 3D), and the proportion of CD8 - The proportion of T cells in each group showed a gradual downward trend (Figure 3D), and the proportion of CD8 - The proportion of T cells in each group showed a gradual downward trend (Figure 3D), and the proportion of CD8 + The proportion of T cells in each group showed a gradual downward trend (Figure 3D), and the proportion of CD8

[0256] Based on the above data, while ensuring that the TIL has a high expansion fold and the proportion of CD8 + The optimal coating concentration of anti-human CD3 / CD28 antibody for TIL is 2 μg / mL.

[0257] Example 4 Establishment of TIL cell in vitro expansion method independent of feeder cells (TIL cell culture medium screening)

[0258] 1. Experimental method

[0259] In order to optimize the culture medium and additives for TIL, four groups of culture conditions were compared: OptiVitro T cell serum-free medium + OptiVitro T cell serum-free additive component (hereinafter referred to as OptiVitro), X-vivo 15 (hereinafter referred to as X-vivo), X-vivo 15 + 5% SR, and AIM V + 5% SR. The most suitable culture medium for TIL expansion was selected. On day 0, the same number of cells were inoculated and cultured in the above four culture media.

[0260] The additives are serum substitutes, and the final concentration of CTS Immune Cell Serum Replacement (SR) from Thermo is 5%, and the final concentration of OptiVitro T cell serum-free additive component is 5%.

[0261] 2. Experimental results

[0262] Figure 4A shows that, comparing the number of TIL cells and the fold increase under the four culture conditions, using OptiVitro as the culture medium for TILs resulted in a relatively high number of cells and a fold increase. Simultaneously, analysis of TIL cell viability and mean cell diameter during culture revealed that OptiVitro, X-vivo, and X-vivo + 5% SR maintained a TIL viability of over 70%, and by day 21, the cell viability of these three groups remained above 90%. Correspondingly, the mean cell diameter of TILs cultured in these three groups ranged from 10 μm to 15 μm; however, the TIL cell viability and mean cell diameter obtained from the AIM V + 5% SR group were significantly reduced (Figure 4B).

[0263] Furthermore, the phenotypes of TILs obtained under four culture conditions were analyzed. In the OptiVitro, X-vivo, and X-vivo+5% SR groups, CD45... + CD3 + The proportion of T cells remained above 80% in all groups, while the CD45 concentration in the AIM V+5% SR group was significantly higher. + CD3 + T cells failed to expand specifically, and their proportion gradually decreased. Notably, with prolonged culture time, CD8+ cells in the OptiVitro group and the X-vivo+5% SR group showed a decrease. + T cells account for CD3 + The proportion of T cells gradually increased, and was even higher in the OptiVitro group; conversely, the proportion of CD8 cells was lower in the X-vivo and AIM V+5% SR groups. + T cells account for CD3 + The proportion of T cells gradually decreased, indicating that OptiVitro is more suitable for CD8 cells. + T cell expansion (Figure 4D). Correspondingly, except for the AIM V+5% SR group, CD4+ was increased in all groups. + T cells account for CD3 + The proportion of T cells remained stable (Figure 4E). Further analysis of the exhausted phenotype CD8 + T cells showed that, with prolonged culture time, PD-1TIM3 levels increased in TILs obtained from OptiVitro, X-vivo, and X-vivo + 5% SR groups. + LAG3 + CD8 + The proportion of T cells gradually decreased (Figure 4F).

[0264] Based on the above data, amplifying TILs using OptiVitro medium yields relatively high amplification folds and exhibits relatively high CD8 counts. +T cell ratio, lower PD-1 TIM3 + LAG3 + CD8 + T cell (exhausted CTLs) ratio.

[0265] Example 5 Establishment of TIL cell expansion method in vitro without feeder cells (cytokine usage regimen)

[0266] 1. Experimental method

[0267] This experiment compared the necessity of IL-7 / IL-15 in the TIL culture system by adding IL-7 or IL-15 to the complete medium with IL-2. The following experimental groups were designed: A, 600 IU / mL IL-2; B, 6000 IU / mL IL-2 + 5 ng / mL IL-7; C, 6000 IU / mL IL-2 + 15 ng / mL IL-15; D, 6000 IU / mL IL-2 + 5 ng / mL IL-7 + 15 ng / mL IL-15.

[0268] Briefly, TIL obtained by density gradient centrifugation were inoculated into culture plates coated with different concentrations of CD3 / CD28 antibodies at the same number, and cultured with OptiVitro medium, different concentrations of IL-2, IL-7 and IL-15 according to the above experimental grouping. On the 7th, 14th and 21st days of culture, the number of expanded TIL cells, cell viability, cell diameter and cell phenotype were detected. The results were compared to select the best experimental conditions.

[0269] 2. Experimental results

[0270] Cell counting results showed that there was no significant difference in the number of TIL cells and the expansion ratio between groups A, B, C and D on the 7th day of culture. However, compared with group A, the number of viable cells and the expansion ratio of group B with additional IL-7 and group C with additional IL-15 were significantly decreased on the 14th day of culture. On the contrary, it was unexpectedly found that the number of viable cells and the expansion ratio of group D with simultaneous addition of IL-7 and IL-15 were significantly increased, indicating that the simultaneous addition of IL-7 and IL-15 can synergistically enhance the expansion of TIL (Figures 5A and 5B).

[0271] Further, the phenotype of TIL expanded to 14 days was analyzed. Compared with group A, the proportion of CD8 + T cells accounted for CD3 + The proportion of T cells was significantly up-regulated, and the simultaneous addition of IL-7 and IL-15 in group D can further up-regulate the proportion of CD8 +The proportion of T cells (Figure 5C). More importantly, Group D can simultaneously significantly down-regulate CCR7 - CD45RA + The proportion of terminally differentiated T cells (TD) among CD8 + T cells and significantly up-regulate CCR7 + CD45RA - The proportion of central memory T cells (TCM) among CD8 + T cells (Figure 5D).

[0272] The above results show that the addition of IL-7 alone can down-regulate the proportion of terminally differentiated cells among CD8 + T cells, but cannot improve the proliferation ability of the cells; the addition of IL-15 alone can up-regulate the proportion of central memory cells among CD8 + T cells, but cannot improve the proliferation ability of the cells. Only the simultaneous addition of IL-7 and IL-15 can synergistically increase the expansion number of TILs and the proportion of CD8 + T cells, while down-regulating the proportion of terminally differentiated cells and up-regulating the proportion of central memory cells.

[0273] Example 6 Establishment of a TIL cell in-vitro expansion method not dependent on feeder cells (TIL cell initial culture density)

[0274] 1 Experimental method

[0275] The initial inoculation density of TIL culture was optimized. The experimental groups were: A, 1.00 x 10 5 / mL; B, 5.00 x 10 5 / mL; C, 7.50 x 10 5 / mL; D, 1.00 x 10 6 / mL; E, 2.00 x 10 6 / mL.

[0276] Briefly, the TIL obtained by density gradient centrifugation was inoculated into a culture plate coated with CD3 / CD28 antibodies according to the above experimental groups, with different cell densities, using OptiVitro medium, and adding 6000 IU / mL IL-2, 5 ng / mL IL-7 and 5 ng / mL IL-15 for culture. The expansion cell number, cell viability, cell diameter and cell phenotype of TIL were detected on the 7th day and the 14th day of culture. The results obtained were compared to select the best experimental conditions.

[0277] 2 Experimental results

[0278] The results are shown in Table 1. When cultured to day 7, the cell expansion multiples of group A (3.66±0.19 times) and group E (10.3±0.27 times) were relatively low; in comparison, the cell expansion multiples of groups B, C and D were significantly increased (group B: 21.7±2.13 times; group C: 20.6±0.11 times; group D: 17.1±1.12 times). The expansion multiples of TILs cultured for 14 days also showed the same trend (group A: 80.5±24.9 times; group B: 332.0±33.6 times; group C: 372.2±41.6 times; group D: 293.7±17.7 times; group E: 90.6±10.7 times). The above results show that the inoculation density between 5.00×10 5 / mL and 1.00×10 6 / mL is conducive to the expansion of TIL cells.

[0279] Table 1 TIL culture status and expansion multiples at different inoculation densities

[0280] Analysis of the phenotype of TILs cultured for 7 days and 14 days (Table 2) shows that in group A, the proportion of CD45-negative cells was always above 50%, indicating that the inoculation density of group A was not suitable and immune cells were not specifically expanded. In groups B-D, with the increase of inoculation density, the proportion of CD45-negative cells and myeloid cells gradually decreased, and the proportion of CD3 + T cells gradually increased, indicating that high-density inoculation is conducive to the expansion of CD3 + T cells. In summary, the inoculation density between 5.00×10 5 / mL and 1.00×10 6 / mL is the highest in terms of expansion multiples and purity of TIL cells.

[0281] Table 2 Statistics of cell phenotype changes of TILs after culture at different inoculation densities

[0282] Example 7 Establishment and optimization of TIL final product cryopreservation system

[0283] 1. Experimental method

[0284] The cryopreservation conditions and parameters of TILs were optimized, and the cryopreservation density of TILs was 5.00×10 7 / mL. A total of 8 cell cryopreservation solutions were evaluated. Cryopreservation solutions 1 and 5 served as controls, while solutions 2, 3, 4, 6, 7, and 8 were optimized formulations. Cryopreservation solution 1: CryoStor CS10 (StemCell technology, Cat#100-1061); Cryopreservation solution 2: 50% CryoStor CS10 + 12.5% ​​HSA + 37.5% physiological saline; Cryopreservation solution 3: 50% CryoStor CS10 + 12.5% ​​HSA + 37.5% Plasma-Lyte A injection (PLA); Cryopreservation solution 4: 7.5% DMSO + 20% HSA + 31.25% PLA + 31.25% glucose sodium chloride + 10% dextran glucose; Cryopreservation solution 5: (Sartorius, Cat#05-713-1B, main components: DMSO, methylcellulose, inorganic salts, amino acids, vitamins); cryopreservation solution 6:49% + 2% HSA + 49% PLA; cryopreservation solution 7:50% +12.5% ​​HSA + 37.5% PLA; cryopreservation solution 8:50% +12.5% ​​HSA + 37.5% NaCl. The specific cryopreservation method for the final product is as follows: Harvest the final product cells into 250mL centrifuge tubes, centrifuge to remove the supernatant (centrifugation parameters: 500g, 10min, 20℃, acceleration 7, deceleration 6), wash the cells once with washing buffer (physiological saline containing 1% HSA), centrifuge to remove the supernatant (centrifugation parameters: 500g, 10min, 20℃, acceleration 7, deceleration 6). Finally, resuspend the cells in the above cryopreservation solution to 5.00×10⁻⁶. 7 Cells were collected at a density of / mL, filled into 5mL cryovials / 250mL cryopreservation bags, placed in a programmed cooling apparatus, cooled to -135℃, and then stored in a gas phase liquid nitrogen tank.

[0285] Statistical analysis: One-way ANOVA was used; *, P<0.1; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0286] 2 Experimental Results

[0287] The cell viability of TIL cells before and after freezing with the 5 freezing solutions is shown in Figure 6A. Except for freezing solution 1, the cell viability of the cells after freezing and thawing in the other groups was more than 90%. After 1.5 and 3 hours of cell recovery, the cells were co-cultured with target cells SK-Hep-1 overnight, and the effector-to-target ratio was 1:1 and 3:1, respectively. The IFN-γ concentration in the co-culture supernatant was detected (Figure 6B). When the effector-to-target ratio was 3:1, the IFN-γ release level of the cells treated with freezing solutions 2, 3, and 5 was higher than that before freezing, and was significantly better than that of the cells treated with freezing solutions 1 and 4. Among them, the effect of freezing solution 2 was the best. When the effector-to-target ratio was 1:1, the IFN-γ release level of the cells treated with freezing solutions 2 and 5 was the smallest in the decrease amplitude compared with that before freezing, but was significantly better than that of the cells treated with freezing solutions 1, 3, and 4.

[0288] In addition, as shown in Figure 6C (freezing solution 6, freezing solution 7, and freezing solution 8), the cell recovery activity, recovery yield, and cell activation level of the cells treated with freezing solutions 6, 7, and 8 were similar to those of the cells treated with freezing solution 5, and freezing solutions 6, 7, and 8 significantly reduced the cost of freezing reagents.

[0289] In summary, the viability and activation ability of the TIL cells treated with freezing solution 1 after freezing and thawing have a large room for improvement. The effects of freezing solution 2 and freezing solution 3, which are optimized based on freezing solution 1, are significantly better than that of freezing solution 1. At the same time, it is accidentally found that the effect of freezing solution 2 is also better than that of freezing solution 5 and its optimized formula.

[0290] The above description of the embodiments is only used to understand the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A method for in vitro expansion of tumor-infiltrating lymphocytes, characterized in that, The method includes culturing tumor-infiltrating lymphocytes using an amplification medium, the amplification medium comprising culture medium and interleukin.

2. The method according to claim 1, characterized in that, The culture medium is selected from serum-free culture medium.

3. The method according to claim 2, characterized in that, The serum-free culture media include OptiVitro T cell serum-free medium, X-vivo 15 medium, and TexMACS medium. Culture media: AIM V medium, ImmunoCult-XF T cell expansion medium, RPMI-1640 medium.

4. The method according to claim 3, characterized in that, The serum-free culture medium was selected from OptiVitro T medium.

5. The method according to claim 4, characterized in that, The serum-free culture medium also includes serum substitutes.

6. The method according to claim 5, characterized in that, The serum substitutes include one or more of CTS Immune Cell Serum Replacement, Ultrose G Serum Substitute, and OptiVitro T cell serum-free additives.

7. The method according to claim 6, characterized in that, The serum substitute is selected from CTS Immune Cell Serum Replacement and / or OptiVitro T cell serum-free additives.

8. The method according to claim 7, characterized in that, The serum substitute is added at a ratio of 0%-10% CTS Immune Cell Serum Replacement and 0%-10% OptiVitro T cell serum-free additives.

9. The method according to any one of claims 1-8, characterized in that, The serum-free culture media include X-vivo 15 medium and CTS Immune Cell Serum Replacement.

10. The method according to any one of claims 1-8, characterized in that, The serum-free culture medium includes OptiVitro T medium and OptiVitro T cell serum-free additive components.

11. The method according to claim 1, characterized in that, The interleukins include one or more of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, and IL-23.

12. The method according to claim 11, characterized in that, The interleukins are selected from IL-2, IL-7 and IL-15.

13. The method according to claim 12, characterized in that, The concentration of IL-2 is 300-9000 IU / mL.

14. The method according to claim 13, characterized in that, The concentration of IL-2 was 6000 IU / mL.

15. The method according to claim 12, characterized in that, The concentration of IL-7 is 0-10 ng / mL.

16. The method according to claim 15, characterized in that, The concentration of IL-7 was 5 ng / mL.

17. The method according to claim 12, characterized in that, The concentration of IL-15 is 0-30 ng / mL.

18. The method according to claim 17, characterized in that, The concentration of IL-15 was 15 ng / mL.

19. The method according to claim 1, characterized in that, The amplification culture time is 7-28 days.

20. The method according to claim 19, characterized in that, The amplification culture time is 14-21 days.

21. The method according to claim 1, characterized in that, The method also includes activating tumor-infiltrating lymphocytes before culturing them in an amplification medium.

22. The method according to claim 21, characterized in that, Use activators to activate tumor-infiltrating lymphocytes.

23. The method according to claim 22, characterized in that, The activator includes one or more of the following: antibodies, ligands, and receptors of CD3, CD28, CD137, ICOS, CD40L, and OX40.

24. The method according to claim 23, characterized in that, The activator is selected from CD3 / CD28 antibodies.

25. The method according to claim 24, characterized in that, The concentration of the CD3 antibody is 0-5 μg / mL.

26. The method according to claim 24, characterized in that, The concentration of the CD28 antibody is 0-5 μg / mL.

27. The method according to claim 24, characterized in that, The total concentration of the CD3 / CD28 antibody is 0-10 μg / mL.

28. The method according to claim 27, characterized in that, The total concentration of the CD3 / CD28 antibody is 1-2 μg / mL.

29. The method according to claim 28, characterized in that, The total concentration of the CD3 / CD28 antibody was 2 μg / mL.

30. The method according to claim 29, characterized in that, The concentration ratio of anti-CD3 antibody to anti-CD28 antibody is 10:1 to 1:

10.

31. The method according to claim 30, characterized in that, The concentration ratio of anti-CD3 antibody to anti-CD28 antibody was 1:

1.

32. The method according to claim 24, characterized in that, The CD3 / CD28 antibody is used in soluble or coated form.

33. The method according to claim 32, characterized in that, The CD3 / CD28 antibody is used in a coated form.

34. The method according to claim 1, characterized in that, The tumor-infiltrating lymphocytes were obtained by digesting tumor tissue.

35. The method according to claim 34, characterized in that, Tumor tissue was digested using enzymatic digestion.

36. The method according to claim 35, characterized in that, The enzymes include collagenase and / or nuclease.

37. The method according to claim 36, characterized in that, The collagenases include collagenase I and / or collagenase II.

38. The method according to claim 35, characterized in that, Serum albumin is also added during the digestion of tumor tissue.

39. The method according to claim 38, characterized in that, The digestion time of tumor tissue is 0-120 minutes.

40. The method according to claim 39, characterized in that, The digestion time for tumor tissue is 15-60 minutes.

41. The method according to claim 40, characterized in that, The digestion time for tumor tissue is 30-60 minutes.

42. The method according to claim 38, characterized in that, The method also includes the step of separating tumor-infiltrating lymphocytes after digesting tumor tissue.

43. The method according to claim 42, characterized in that, Tumor-infiltrating lymphocytes were isolated by density gradient centrifugation and / or magnetic bead method.

44. The method according to claim 43, characterized in that, The magnetic bead method is CD3 magnetic bead separation.

45. The method according to claim 1, characterized in that, The density gradient centrifugation includes Ficoll and Percoll density gradient centrifugation.

46. ​​The method according to claim 45, characterized in that, The density gradient centrifugation is Ficoll density gradient centrifugation.

47. The method according to claim 46, characterized in that, The speed of Ficoll density gradient centrifugation is 300-500g.

48. The method according to claim 47, characterized in that, The Ficoll density gradient centrifugation speed was 400g.

49. The method according to claim 46, characterized in that, The temperature for Ficoll density gradient centrifugation is 15-25℃.

50. The method according to claim 49, characterized in that, The temperature for Ficoll density gradient centrifugation was 20℃.

51. The method according to claim 46, characterized in that, The Ficoll density gradient centrifugation time is 10-60 min.

52. The method according to claim 51, characterized in that, The Ficoll density gradient centrifugation time was 25 min.

53. The method according to claim 34, characterized in that, The tumor tissues include liver cancer, bile duct cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, bladder cancer, epithelial cancer, head and neck cancer, stomach cancer, esophageal cancer, and nasopharyngeal cancer.

54. The method according to claim 53, characterized in that, The tumor tissue is liver cancer tissue.

55. The method according to claim 54, characterized in that, The tumor tissue includes fresh or preserved tissue.

56. The method according to claim 55, characterized in that, The preserved tissue was preserved using a tissue preservation solution.

57. The method according to claim 1, characterized in that, The seeding density of the tumor-infiltrating lymphocytes was 1.00 × 10⁻⁶. 5 / mL-2.00×10 6 / mL.

58. The method according to claim 57, characterized in that, The seeding density of the tumor-infiltrating lymphocytes was 5.00 × 10⁻⁶. 5 / mL-1.00×10 6 / mL.

59. The method according to claim 1, characterized in that, The method also includes a cryopreservation step.

60. The method according to claim 59, characterized in that, Use cryopreservation solution for freezing.

61. The method according to claim 60, characterized in that, The cryopreservation solution includes: 1) CryoStor CS10 and HSA, and physiological saline, or CryoStor CS10 and HSA, and compound electrolyte injection; or 2) DMSO, HSA, compound electrolyte injection, glucose sodium chloride and dextran glucose; or 3) And HSA, and compound electrolyte injection, or And HSA, and NaCl.

62. A product for expanding tumor-infiltrating lymphocytes, characterized in that, The product includes culture medium and interleukins, wherein the interleukins include one or more of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, and IL-23.

63. The product according to claim 62, characterized in that, The interleukins are selected from IL-2, IL-7 and IL-15.

64. The product according to claim 63, characterized in that, The concentration of IL-2 is 300-9000 IU / mL.

65. The product according to claim 64, characterized in that, The concentration of IL-2 was 6000 IU / mL.

66. The product according to claim 63, characterized in that, The concentration of IL-7 is 0-10 ng / mL.

67. The product according to claim 66, characterized in that, The concentration of IL-7 was 5 ng / mL.

68. The product according to claim 63, characterized in that, The concentration of IL-15 is 0-30 ng / mL.

69. The product according to claim 68, characterized in that, The concentration of IL-15 was 15 ng / mL.

70. The product according to claim 62, characterized in that, The culture medium is selected from serum-free culture medium.

71. The product according to claim 70, characterized in that, The serum-free culture media include OptiVitro T cell serum-free medium, X-vivo 15 medium, and TexMACS medium. Culture media: AIM V medium, ImmunoCult-XF T cell expansion medium, RPMI-1640 medium.

72. The product according to claim 71, characterized in that, The serum-free culture medium was selected from OptiVitro T medium.

73. The product according to claim 72, characterized in that, The serum-free culture medium also includes serum substitutes.

74. The product according to claim 73, characterized in that, The serum substitutes include one or more of CTS Immune Cell Serum Replacement, Ultrose G Serum Substitute, and OptiVitro T cell serum-free additives.

75. The product according to claim 74, characterized in that, The serum substitute is selected from CTS Immune Cell Serum Replacement and / or OptiVitro T cell serum-free additives.

76. The product according to claim 75, characterized in that, The serum substitute is added at a ratio of 0%-10% CTS Immune Cell Serum Replacement and 0%-10% OptiVitro T cell serum-free additives.

77. The product according to any one of claims 62-76, characterized in that, The serum-free culture media include X-vivo 15 medium and CTS Immune Cell Serum Replacement.

78. The product according to any one of claims 62-76, characterized in that, The serum-free culture medium includes OptiVitro T medium and OptiVitro T cell serum-free additive components.

79. A tumor tissue preservation solution, characterized in that, The tumor tissue preservation solution includes serum-free culture medium and interleukin.

80. The tumor tissue preservation solution according to claim 79, characterized in that, The serum-free culture media include OptiVitro T cell serum-free medium, X-vivo 15 medium, and TexMACS medium. Culture media: AIM V medium, ImmunoCult-XF T cell expansion medium, RPMI-1640 medium.

81. The tumor tissue preservation solution according to claim 80, characterized in that, The serum-free culture medium was selected from OptiVitro T medium.

82. The tumor tissue preservation solution according to claim 79, characterized in that, The interleukins include one or more of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, and IL-23.

83. The tumor tissue preservation solution according to claim 82, characterized in that, The interleukin is selected from IL-7 and IL-15.

84. The tumor tissue preservation solution according to claim 83, characterized in that, The concentration of IL-7 is 0-10 ng / mL.

85. The tumor tissue preservation solution according to claim 84, characterized in that, The concentration of IL-7 was 5 ng / mL.

86. The tumor tissue preservation solution according to claim 83, characterized in that, The concentration of IL-15 is 0-30 ng / mL.

87. The tumor tissue preservation solution according to claim 86, characterized in that, The concentration of IL-15 was 15 ng / mL.

88. The tumor tissue preservation solution according to claim 79, characterized in that, The tumor tissue was stored at a temperature of 0-25℃.

89. The tumor tissue preservation solution according to claim 88, characterized in that, The tumor tissue was stored at a temperature of 2-8℃.

90. The tumor tissue preservation solution according to claim 79, characterized in that, The tumor tissue was preserved for 0-72 hours.

91. The tumor tissue preservation solution according to claim 90, characterized in that, The tumor tissue was preserved for 0-30 hours.

92. The tumor tissue preservation solution according to claim 79, characterized in that, The tumor tissues include liver cancer, bile duct cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, bladder cancer, epithelial cancer, head and neck cancer, stomach cancer, esophageal cancer, and nasopharyngeal cancer.

93. The tumor tissue preservation solution according to claim 92, characterized in that, The tumor tissue is liver cancer tissue.

94. Tumor-infiltrating lymphocyte population, characterized in that, The tumor-infiltrating lymphocyte population is obtained by the method according to any one of claims 1-61.

95. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the tumor-infiltrating lymphocyte population of claim 94.

96. The pharmaceutical composition according to claim 95, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier, diluent, or excipient.

97. The use of the tumor-infiltrating lymphocyte population of claim 94 in the preparation of products for the prevention and / or treatment of tumors.

98. The application according to claim 97, characterized in that, The tumors include hematologic malignancies and solid tumors.

99. The application according to claim 98, characterized in that, The tumor was selected from solid tumors.

100. The application according to claim 99, characterized in that, The solid tumors include liver cancer, bile duct cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, bladder cancer, epithelial cancer, head and neck cancer, stomach cancer, esophageal cancer, and nasopharyngeal cancer.

101. A cryopreservation solution for tumor-infiltrating lymphocytes, characterized in that, The cryopreservation solution includes: 1) CryoStor CS10 and HSA, and physiological saline, or CryoStor CS10 and HSA, and compound electrolyte injection; or 2) DMSO, HSA, compound electrolyte injection, glucose sodium chloride and dextran glucose; or 3) And HSA, and compound electrolyte injection, or And HSA, and NaCl.

102. The cryopreservation solution according to claim 101, characterized in that, The compound electrolyte injection solution is PLA.

103. The cryopreservation solution according to claim 101, characterized in that, 1) The volume ratio of CryoStor CS10 is 25%-75%.

104. The cryopreservation solution according to claim 103, characterized in that, 1) The volume ratio of CryoStor CS10 is 50%.

105. The cryopreservation solution according to claim 101, characterized in that, 1) The volume ratio of HSA in the solution is 0%-25%.

106. The cryopreservation solution according to claim 105, characterized in that, 1) The volume ratio of HSA in the mixture is 12.5%.

107. The cryopreservation solution according to claim 101, characterized in that, 1) The volume ratio of physiological saline in the solution is 25%-50%.

108. The cryopreservation solution according to claim 107, characterized in that, 1) The volume ratio of physiological saline in the solution is 37.5%.

109. The cryopreservation solution according to claim 101, characterized in that, 1) The volume ratio of PLA is 25%-50%.

110. The cryopreservation solution according to claim 109, characterized in that, 1) The volume ratio of PLA is 37.5%.

111. The cryopreservation solution according to claim 101, characterized in that, 2) The volume ratio of DMSO is 0%-15%.

112. The cryopreservation solution according to claim 111, characterized in that, 2) The volume ratio of DMSO is 7.5%.

113. The cryopreservation solution according to claim 101, characterized in that, 2) The volume ratio of HSA in the solution is 0%-25%.

114. The cryopreservation solution according to claim 113, characterized in that, 2) The volume ratio of HSA in the mixture is 20%.

115. The cryopreservation solution according to claim 101, characterized in that, 2) The volume ratio of PLA is 25%-50%.

116. The cryopreservation solution according to claim 115, characterized in that, 2) The volume ratio of PLA is 31.25%.

117. The cryopreservation solution according to claim 101, characterized in that, 2) The volume ratio of glucose to sodium chloride is 25%-50%.

118. The cryopreservation solution according to claim 117, characterized in that, 2) The volume ratio of glucose to sodium chloride is 31.25%.

119. The cryopreservation solution according to claim 101, characterized in that, 2) The volume ratio of dextran to glucose is 0%-25%.

120. The cryopreservation solution according to claim 119, characterized in that, 2) The volume ratio of dextran to glucose is 10%.

121. The cryopreservation solution according to claim 101, characterized in that, 3) The volume ratio is 0%-75%.

122. The cryopreservation solution according to claim 121, characterized in that, 3) The volume ratio is 40%-60%.

123. The cryopreservation solution according to claim 122, characterized in that, 3) The volume ratio is 49% or 50%.

124. The cryopreservation solution according to claim 101, characterized in that, 3) The volume ratio of HSA is 0%-25%.

125. The cryopreservation solution according to claim 124, characterized in that, 3) The volume ratio of HSA is 2% or 12.5%.

126. The cryopreservation solution according to claim 101, characterized in that, 3) The volume ratio of PLA is 25%-60%.

127. The cryopreservation solution according to claim 126, characterized in that, 3) The volume ratio of PLA is 25%-50%.

128. The cryopreservation solution according to claim 127, characterized in that, 3) The volume ratio of PLA is 49% or 37.5%.

129. The cryopreservation solution according to claim 101, characterized in that, 3) The volume ratio of NaCl is 25%-50%.

130. The cryopreservation solution according to claim 129, characterized in that, 3) The volume ratio of NaCl is 37.5%.

131. The cryopreservation solution according to any one of claims 101-130, characterized in that, The cryopreservation solution comprises: 50% CryoStor CS10, 12.5% ​​HSA, and 37.5% physiological saline; or 50% CryoStor CS10, 12.5% ​​HSA, and 37.5% PLA; or 7.5% DMSO, 20% HSA, 31.25% PLA, 31.25% glucose sodium chloride, and 10% dextran glucose; or 49%... 2% HSA and 49% PLA; or 50% 12.5% ​​HSA and 37.5% PLA; or 50% 12.5% ​​HSA and 37.5% NaCl.

132. The cryopreservation solution according to claim 131, characterized in that, The density of the cells frozen in the cryopreservation solution was 5.00 × 10⁻⁶. 6 / mL-8.00×10 7 / mL.

133. A method for cryopreserving tumor-infiltrating lymphocytes, characterized in that, The method includes using the cryopreservation solution according to any one of claims 101-132.

134. The cryopreservation method according to claim 133, characterized in that, The method includes cryopreservation of tumor-infiltrating lymphocytes by resuspending them in cryopreservation solution.

135. The cryopreservation method according to claim 134, characterized in that, The cell density of the resuspended tumor-infiltrating lymphocytes was 1.00 × 10⁻⁶. 7 / mL-1.00×10 8 / mL.

136. The cryopreservation method according to claim 135, characterized in that, The cell density of the resuspended tumor-infiltrating lymphocytes was 5.00 × 10⁻⁶. 7 / mL.

137. The cryopreservation method according to claim 133, characterized in that, The method also includes freezing the resuspended tumor-infiltrating lymphocytes after cooling.

138. The cryopreservation method according to claim 137, characterized in that, The cooling methods include programmed cooling or gradual cooling.

139. The cryopreservation method according to claim 138, characterized in that, The cooling temperature is between -70°C and -196°C.

140. The cryopreservation method according to claim 139, characterized in that, The temperature at which the temperature was lowered was -135℃.

141. A method for treating tumors, characterized in that, The method includes using the tumor-infiltrating lymphocyte population of claim 94 or the pharmaceutical composition of any one of claims 95-96.

142. The method according to claim 141, characterized in that, The tumors include hematologic malignancies and solid tumors.

143. The method according to claim 142, characterized in that, The tumor was selected from solid tumors.

144. The method according to claim 143, characterized in that, The solid tumors include liver cancer, bile duct cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, bladder cancer, epithelial cancer, head and neck cancer, stomach cancer, esophageal cancer, and nasopharyngeal cancer.

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