A method and culture medium for culturing tumor infiltrating lymphocytes

Abstract

The present invention relates to a method for culturing tumor-infiltrating lymphocytes and a culture medium. The present invention relates to the field of cell culture. Specifically, the present invention provides a culture medium containing an NKG2D antibody for culturing TIL cells. NKG2D mAb can significantly improve the viability of TIL cells, promote the proliferation of TIL, increase the activation level of TIL and the proportion of CD8+ cells, and enhance the killing ability of TIL against various tumor cells. Among them, the number of TIL cells cultured in the culture medium supplemented with NKG2D mAb is more than twice that of the control culture medium without NKG2D mAb, and the INF-γ secretion amount reaches more than 1.3 times. NKG2D mAb can be added to various basal culture media for TIL cells to culture TIL cells derived from multiple cancer types and prepare high-quality TIL cell products.

Description

Technical Field

[0001] The invention relates to the field of cell culture, and in particular to a culture method and culture medium for tumor infiltrating lymphocytes. Background Art

[0002] Tumor infiltrating lymphocytes, or TIL cells, are specific types of white blood cells derived from the patient's own tumor tissue, mainly T cells, which can recognize and attack cancer cells. These cells are usually extracted from the patient's tumor, then amplified in large quantities in the laboratory, and then infused back into the patient after activation and proliferation to provide a more powerful immune response to fight cancer.

[0003] TIL cells have the advantages of high specificity, strong lethality and few side effects, and have become an important part of personalized precision medicine.

[0004] However, the proliferation process of TIL cells is complex and challenging, involving multiple technical and biological difficulties. TIL cell proliferation faces multiple difficulties, including the heterogeneity of tumor tissues, low cell proliferation efficiency, cell senescence and apoptosis, the influence of the immunosuppressive microenvironment, and individual differences and high costs in clinical applications.

[0005] Therefore, there is an urgent need to optimize culture techniques and improve culture conditions in order to develop new TIL culture methods. Summary of the invention

[0006] The purpose of the present invention is to provide an optimized TIL cell culture method. Specifically, the purpose of the present invention is to provide a TIL cell culture method in which NKG2D antibodies are added to the culture medium.

[0007] In a first aspect of the present invention, a method for culturing tumor infiltrating lymphocytes (TIL) cells is provided, comprising the steps of:

[0008] (a) providing a TIL culture medium, wherein the TIL culture medium comprises: (i) a TIL cell culture component; and (ii) an NKG2D antibody.

[0009] (b) TIL cells were cultured in the TIL medium described in (a).

[0010] In another preferred embodiment, the NKG2D antibody includes an active fragment of a NKG2D antibody or a derivative thereof.

[0011] In another preferred embodiment, the NKG2D antibody is selected from the following group: monoclonal antibody, polyclonal antibody, single chain antibody, single domain antibody, or a combination thereof.

[0012] In another preferred embodiment, the NKG2D antibody is a monoclonal antibody.

[0013] In another preferred embodiment, the NKG2D antibody is a recombinant monoclonal antibody.

[0014] In another preferred embodiment, the concentration of the NKG2D antibody is 0.1 μg / ml to 10 μg / ml.

[0015] In another preferred embodiment, the concentration of the NKG2D antibody is 0.5 μg / ml to 5 μg / ml.

[0016] In another preferred embodiment, the concentration of the NKG2D antibody is 1 μg / ml to 3 μg / ml.

[0017] In another preferred embodiment, the concentration of the NKG2D antibody is 2 μg / ml.

[0018] In another preferred embodiment, the TIL cell culture components include: TIL cell basal culture medium, serum or serum replacement, and cytokine combination.

[0019] In another preferred embodiment, the TIL cell basal culture medium is selected from the following group: RPMI 1640, X-VIVO, DMEM, AIM-V, OpTmizer TM .

[0020] In another preferred embodiment, the cytokine combination includes: IL-2, CD antibody, and 4-1BBL antibody.

[0021] In another preferred embodiment, the cytokine combination includes: 100-500 U / ml IL-2, 10-30 ng / ml CD3 monoclonal antibody, and 10-30 ng / ml 4-1BBL monoclonal antibody.

[0022] In another preferred embodiment, the cytokine combination includes: 300U / ml IL-2, 15ng / ml CD3 monoclonal antibody, and 15ng / ml 4-1BBL monoclonal antibody.

[0023] In another preferred embodiment, the concentration of the serum replacement in the culture medium is 1% to 10%.

[0024] In another preferred embodiment, the concentration of the serum replacement in the culture medium is 2% to 5%.

[0025] In another preferred embodiment, the TIL cells are derived from tumor tissue.

[0026] In another preferred embodiment, the tumor includes a solid tumor, a hematological tumor, or a combination thereof.

[0027] In another preferred embodiment, the tumor is a solid tumor.

[0028] In another preferred embodiment, the tumor is selected from the following group: cervical cancer, lung cancer, melanoma, colorectal cancer, liver cancer, and esophageal cancer.

[0029] In another preferred embodiment, the tumor tissue originates from humans or other mammals.

[0030] In another preferred embodiment, the TIL cells are genetically engineered TIL cells.

[0031] In another preferred embodiment, the genetic engineering modification includes: introducing exogenous genes into the cell genome, delivering exogenous vectors into cells, expressing exogenous proteins (including introducing mRNA or plasmids to express exogenous proteins), and knocking out predetermined endogenous genes in the cell genome.

[0032] In another preferred embodiment, the genetically engineered TIL cells express an exogenous activating protein, the activating protein comprises a DAP10 element and an activating element, and the sequence of the activating element is shown in SEQ ID NO: 1.

[0033] In another preferred embodiment, the amino acid sequence of the activated protein is shown in SEQ ID NO: 2.

[0034] In another preferred embodiment, the method further comprises the step of: (c) harvesting the cultured TIL cells and detecting the cultured TIL cells.

[0035] In another preferred embodiment, the number of cells cultured by the method is Q1, and the number of cells cultured under the same conditions without adding the NKG2D antibody is Q0, Q1 / Q0≥1.5; preferably, Q1 / Q0≥2; more preferably, Q1 / Q0≥2.5.

[0036] In another preferred embodiment, about 1×10 6 ~3×10 7 TIL cells, preferably about 2×10 6 ~1×10 7 TIL cells, preferably about 3×10 6 ~8×10 6 TIL cells.

[0037] In another preferred embodiment, the cell viability of the cultured TIL cells is ≥80%; preferably ≥85%; more preferably ≥90%.

[0038] In another preferred embodiment, the cultured TIL cells have one or more characteristics selected from the following group:

[0039] (1) CD8 + T cells and CD4+ The proportion of T cells increases.

[0040] (2) CD25 + CD69 + The proportion of T cells increases, or the proportion of activated cells increases;

[0041] (3) CD45 + CD3 + The proportion of TIL cells is increased, or the cell purity is improved.

[0042] In another preferred embodiment, the cultured TIL cells have enhanced tumor cell killing ability or cytotoxicity.

[0043] In another preferred embodiment, the secretion amount of INF-γ after co-culture of the cultured TIL cells and tumor cells is ≥600 pg / mL; preferably ≥700 pg / mL; more preferably ≥800 pg / mL.

[0044] In a second aspect of the present invention, a tumor infiltrating lymphoid (TIL) cell culture medium is provided, the culture medium comprising: (i) TIL cell culture components; and (ii) NKG2D antibody.

[0045] In another preferred embodiment, the NKG2D antibody includes an active fragment of a NKG2D antibody or a derivative thereof.

[0046] In another preferred embodiment, the NKG2D antibody is selected from the following group: monoclonal antibody, polyclonal antibody, single chain antibody, single domain antibody, or a combination thereof.

[0047] In another preferred embodiment, the NKG2D antibody is a monoclonal antibody.

[0048] In another preferred embodiment, the NKG2D antibody is a recombinant monoclonal antibody.

[0049] In another preferred embodiment, the concentration of the NKG2D antibody is 0.1 μg / ml to 10 μg / ml.

[0050] In another preferred embodiment, the concentration of the NKG2D antibody is 0.5 μg / ml to 5 μg / ml.

[0051] In another preferred embodiment, the concentration of the NKG2D antibody is 1 μg / ml to 3 μg / ml.

[0052] In another preferred embodiment, the concentration of the NKG2D antibody is 2 μg / ml.

[0053] In another preferred embodiment, the cell culture components include: TIL cell basal culture medium, serum or serum replacement, and cytokine combination.

[0054] In another preferred embodiment, the TIL cell basal culture medium is selected from the following group: RPMI 1640, X-VIVO, DMEM, AIM-V, OpTmizer TM .

[0055] In another preferred embodiment, the cytokine combination includes: IL-2, CD3 antibody, and 4-1BBL antibody.

[0056] In another preferred embodiment, the cytokine combination includes: 100-500 U / ml IL-2, 10-30 ng / ml CD3 monoclonal antibody, and 10-30 ng / ml 4-1BBL monoclonal antibody.

[0057] In another preferred embodiment, the cytokine combination includes: 300U / ml IL-2, 15ng / ml CD3 monoclonal antibody, and 15ng / ml 4-1BBL monoclonal antibody.

[0058] In the third aspect of the present invention, a use of an NKG2D antibody is provided for preparing a TIL cell culture medium as described in the second aspect of the present invention.

[0059] In another preferred embodiment, the NKG2D antibody includes an active fragment of a NKG2D antibody or a derivative thereof.

[0060] In another preferred embodiment, the NKG2D antibody is selected from the following group: monoclonal antibody, polyclonal antibody, single chain antibody, single domain antibody, or a combination thereof.

[0061] In another preferred embodiment, the NKG2D antibody is a monoclonal antibody.

[0062] In another preferred embodiment, the NKG2D antibody is a recombinant monoclonal antibody.

[0063] In another preferred embodiment, the concentration of the NKG2D antibody in the culture medium is 0.1 μg / ml to 10 μg / ml.

[0064] In another preferred embodiment, the concentration of the NKG2D antibody in the culture medium is 0.5 μg / ml to 5 μg / ml.

[0065] In another preferred embodiment, the concentration of the NKG2D antibody in the culture medium is 1 μg / ml to 3 μg / ml.

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The number of TILs after culture from different cancer types is shown.

[0068] Figure 2 The viability of TIL cultured from different cancer types is shown.

[0069] Figure 3 The results show that CD8 + / CD4 + T cell ratio.

[0070] Figure 4 The CD25 expression of TILs from different cancer types was shown + CD69 + T cell percentage.

[0071] Figure 5 The CD39 expression of TILs from different cancer types was shown - CD69 - T cell percentage.

[0072] Figure 6 The CD45 expression of TILs from different cancer types was shown + CD3 + T cell percentage.

[0073] Figure 7 The figure shows the survival rate of tumor cells after co-culture of TILs from different cancer types with corresponding tumor cells.

[0074] Figure 8 The cytotoxicity of TILs of different cancer types on corresponding tumor cells is shown.

[0075] Fig. 9 The figure shows the level of IFN-γ secreted by TIL after co-culture of TILs from different cancer types with corresponding tumor cells.

[0076] Fig.10 A schematic diagram of the construct of genetically engineered TIL cells is shown.

[0077] Fig.11 The number of engineered TILs from different cancer types after culture is shown.

[0078] Fig.12 The figure shows the viability of engineered TILs derived from different cancer types after culture. DETAILED DESCRIPTION

[0079] After extensive and in-depth research, the inventors unexpectedly found that adding NKG2D monoclonal antibody (NKG2D mAb) to the TIL culture process can obtain high-quality TIL cells. Specifically, NKG2D mAb can significantly increase TIL cell viability, promote TIL proliferation, and increase TIL activation levels and CD8 + The cell ratio is increased to enhance the killing ability of TIL against various tumor cells. Among them, the number of TIL cells cultured in the culture medium with NKG2D mAb added is more than twice that of the control culture medium without NKG2D mAb, and the INF-γ secretion is more than 1.3 times. NKG2D mAb can be added to various TIL cell basal culture media for the culture of TIL cells derived from various cancer types. The present invention was completed on this basis.

[0080] Culture of TIL cells

[0081] The culture and proliferation of TIL cells is complex and challenging. First, tumor tissue is usually composed of heterogeneous cells, including tumor cells, fibroblasts, vascular cells, and immune cells. This heterogeneity makes it difficult to separate and purify high-quality TIL cells from tumor tissue. In some tumor types or specific areas of the tumor, the infiltration rate of TIL cells is low, making it difficult to obtain a sufficient number of TIL cells for initial expansion.

[0082] Secondly, some TIL cells may show low proliferation efficiency during in vitro expansion, which may be due to the intrinsic characteristics of the cells or insufficient external culture conditions. For example, some TIL cells may be insensitive to proliferation factors (such as IL-2), resulting in low expansion efficiency. Even after successful expansion, different TIL cell subsets may show different anti-tumor activities. Some TIL cells may lose their killing ability or the ability to secrete cytokines, affecting the therapeutic effect. Furthermore, during the expansion process, TIL cells may undergo cell senescence and apoptosis, resulting in a decrease in cell number and decreased function. Therefore, the culture method of TIL cells needs to be optimized and improved to cultivate higher quality TIL cell products.

[0083] NKG2D

[0084] NKG2D is an activating immune receptor that regulates innate and adaptive immune responses. In humans, NKG2D is usually expressed on all NK cells, CD8 T cells, subsets of γδ T cells, and some autoreactive CD4 T cells. The NKG2D receptor recognizes a wide range of structurally diverse ligands. The expression of NKG2D ligands on the surface of tumor cells can activate NK cells and co-stimulate effector T cells by binding to NKG2D, thereby making tumor cells more sensitive to immune cell-mediated killing. Different research groups have shown that NKG2D-CAR can effectively control the growth of multiple tumor types such as multiple myeloma, ovarian cancer, and osteosarcoma in preclinical mouse models. NKG2D antibodies can also bind to NKG2D efficiently and mediate changes in cell signaling pathways. Currently, NKG2D antibodies have been used to treat diseases such as Crohn's disease.

[0085] NKG2D Antibody

[0086] As used herein, the terms "NKG2D antibody," "anti-NKG2D antibody," and "anti-human NKG2D antibody" are used interchangeably.

[0087] As used herein, the term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

[0088] The term "heavy chain" refers to a full-length heavy chain or a fragment thereof, including a variable region VH, which comprises an amino acid sequence sufficient to provide antigen specificity, and three constant regions CH1, CH2 and CH3, and a hinge. The term "light chain" refers to a full-length light chain or a fragment thereof, including a variable region VL, which comprises an amino acid sequence sufficient to provide antigen specificity, and a constant region CL.

[0089] The term "complementarity determining region (CDR)" refers to an amino acid sequence found in the hypervariable region of a heavy chain or light chain of an immunoglobulin. The heavy chain and light chain may include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3), respectively. CDRs may provide residues that play an important role in the binding of an antibody to an antigen or epitope. The terms "specific binding" or "specific recognition" are well known to those of ordinary skill in the art and indicate that an antibody and an antigen specifically interact to produce an immunological activity.

[0090] In the present disclosure, antibodies may include, but are not limited to, polyclonal or monoclonal; and / or humanized, animal (e.g., mouse, rabbit, etc.) derived antibodies, or chimeric antibodies (e.g., mouse-human chimeric antibodies). In addition, antibodies of the present disclosure may include monospecific antibodies and multispecific antibodies, such as bispecific antibodies and trispecific antibodies, but are not limited thereto.

[0091] As used herein, the term "antigen binding fragment" refers to a fragment derived from a complete immunoglobulin structure, which fragment includes a portion capable of binding to an antigen (such as a CDR). For example, an antigen binding fragment may be or may include scFv, (scFv)2, Fab, Fab', F(ab')2, or any combination thereof, but is not limited thereto. In the context of the present disclosure, the antigen binding fragment may be a fragment derived from an antibody, comprising at least one complementary determining region, for example, selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2.

[0092] For example, the antibody or antigen-binding fragment may be chemically or recombinantly synthesized (not naturally occurring). In a preferred embodiment, the NKG2D antibody of the present invention is a recombinant monoclonal antibody.

[0093] NKG2D antibody for TIL cell culture

[0094] In the present invention, NKG2D antibody is used for the culture of TIL cells, which can significantly improve the viability of TIL cells, promote TIL proliferation, and increase the activation level of TIL and CD8 + cell ratio, and enhance the killing ability of TIL against various tumor cells.

[0095] Adding inhibitory antibodies against NKG2D to TIL can effectively prevent TIL cells from being overactivated and entering the exhaustion stage, thereby effectively improving TIL cell viability.

[0096] The present invention provides a culture medium for TIL cells, comprising a TIL cell basal culture medium and an NKG2D antibody. In a preferred embodiment, the concentration of the NKG2D antibody in the culture medium is 0.1 μg / ml to 10 μg / ml, preferably 0.5 μg / ml to 5 μg / ml, and more preferably 1 μg / ml to 3 μg / ml.

[0097] In the present invention, the type of TIL cell basal culture medium is not limited, and any culture medium known in the art that can be used for TIL cell culture can be used for the TIL cell culture of the present invention.

[0098] In the present invention, the source of TIL cells is tumor tissue, preferably solid tumor tissue, including but not limited to: cervical cancer, lung cancer, melanoma, colorectal cancer, liver cancer, esophageal cancer, etc.

[0099] In a preferred embodiment, the TIL cells are genetically engineered TIL cells, for example, engineered TIL cells that introduce exogenous genes into the cell genome, deliver exogenous vectors into the cells, and express exogenous proteins.

[0100] TIL cell culture method of the present invention

[0101] The present invention also provides a method for culturing TIL cells, comprising culturing TIL cells using a culture medium to which NKG2D antibodies are added according to the present invention, comprising the steps of: (a) culturing the TIL cells in a culture medium to which NKG2D antibodies are added; and (b) harvesting the cultured TIL cells; and optionally (c) detecting the cultured TIL cells.

[0102] In a preferred embodiment, the tumor tissue containing TIL cells is treated and then placed in the culture medium of the present invention for culture. During the culture process, the medium is replaced at intervals, for example, the medium is replaced every day or every two days. In a preferred embodiment, the culture conditions are 37° C., 5% CO2.

[0103] At a time point after the TIL cells are cultured for 5 to 15 days, the cultured TIL cells are detected. In a preferred embodiment, cell counting and cell viability detection are performed on the cultured TIL cells. After culturing in a medium containing NKG2D antibodies, the number of TIL cells is more than twice the number of cells cultured in a control medium without NKG2D antibodies.

[0104] In a preferred embodiment, flow cytometry is used to detect the phenotype of the cultured TIL cells. For example, the CD8 + Cells and CD4 + The proportion of cells with direct anti-tumor cytotoxic effector function; CD25 + CD69 + The cell ratio represents the proportion of activated TIL cells; CD39 - CD69 - Characterize the proportion of stem cells; use CD45 + CD3 + Characterize the purity of TIL cells, etc.

[0105] In the present invention, compared with the control culture medium without NKG2D antibody, under the same conditions, the TIL cells harvested by culturing in the culture medium containing NKG2D antibody have a higher proportion of CD8 + cells, a higher proportion of activated cells and a higher purity.

[0106] In a preferred embodiment, TIL cells harvested by culturing in a culture medium containing NKG2D antibodies have stronger cytotoxicity and INF-γ secretion against tumor cells, and exhibit better tumor cell killing ability.

[0107] The main advantages of the present invention include:

[0108] (1) In the present invention, adding NKG2D antibody to TIL cell culture medium can significantly improve the culture quality of TIL cells and can be applied to a variety of TIL cell culture media.

[0109] (2) The TIL cells cultured in the culture medium containing NKG2D antibodies of the present invention have a cell proliferation number that is increased by more than 2 times compared to cells cultured in the culture medium without NKG2D antibodies under the same conditions.

[0110] (3) The TIL cells cultured in the culture medium containing NKG2D antibodies of the present invention have stronger cytotoxicity and INF-γ secretion than the cells cultured in the culture medium without NKG2D antibodies under the same conditions.

[0111] (4) The TIL cells cultured in the culture medium containing NKG2D antibodies of the present invention have higher cell viability, cell purity and activation level compared with cells cultured in the culture medium without NKG2D antibodies under the same conditions.

[0112] (5) The culture medium and culture method of the present invention can be used to culture TIL cells derived from various cancer types, including non-engineered TIL and engineered TIL, and are universal.

[0113] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0114] Example 1: Preparation and culture of TIL cells

[0115] 1. Tumor tissue processing

[0116] Place the tumor blocks of different cancer types in a 10 cm culture dish and wash once with PBS; remove the necrotic area and connective tissue on the tumor block with sterile ophthalmic scissors or a scalpel, and then cut into 1-3 mm 3 Small pieces.

[0117] 2. Culture of TIL cells

[0118] The culture medium used was Gibco™ Advanced RPMI 1640 medium (Cat. No.: 12633020), supplemented with 2.5% serum replacement, 300U / ml IL-2, 15ng / ml CD3 monoclonal antibody, and 15ng / ml 4-1BBL monoclonal antibody.

[0119] The tumor pieces were evenly distributed into the culture medium, and 2 mL of culture medium and 1-5 mg of tumor tissue were added to each well; one group of culture medium contained NKG2D mAb (GMP-A075) with a final concentration of 2 μg / ml, and the other group of normal culture was used as the control group (Ctr).

[0120] If there are no obvious adherent cells when observing the cells under a microscope, change the medium every other day. If the number of lymphocytes does not gradually increase or the number of cancer cells does not decrease, continue to change the medium. If the density of lymphocytes increases significantly, such as the appearance of cell clusters, use a 40μm filter to filter out the tumor tissue. After the expansion operation is completed, place the cells in a 37℃, 5% CO2 incubator for continued culture.

[0121] Example 2: Isolation and purification of TIL cells

[0122] On the 9th day of TIL initial culture, the viability and cell count of each group of TIL were tested. The steps included:

[0123] On the 9th day of culture, cells were collected. The collected cell suspension was filtered through a 40µm filter to remove tumor masses; centrifuged at 1000 rpm for 5 min, and an appropriate amount of culture medium was added to resuspend the TIL precipitate, and cell viability was detected and cell counted.

[0124] The results of cell proliferation test are as follows Figure 1 The results showed that after adding NKG2D mAb to culture, the number of TILs in different cancer types increased significantly, with an average increase of 1.6×10 6 The number of cells increased by 2.7 times on average.

[0125] The results of cell viability test after culture are as follows Figure 2 In different cancer types, the addition of NKG2D mAb can increase the viability of TIL cells, with an average increase of 7%.

[0126] The above results indicate that TIL cells cultured with NKG2D mAb added to the culture medium have better proliferation rate and cell viability.

[0127] Example 3: Phenotypic detection of TIL cells

[0128] On the 9th day of initial culture of TIL, cell phenotype detection was performed on each group of TILs. The steps are as follows:

[0129] 1. Take 5×10 5 The cells were centrifuged at 400 g for 5 minutes, and 1 ml of 2% FBS in PBS was added to resuspend the cells. The cells were centrifuged at 400 g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 50 µl of 2% FBS in PBS. Then, Fc block was added to block Fc receptors (1×10 6 Cells were incubated with 5 µl Fc block at room temperature for 10 minutes.

[0130] 2. Transfer half of the cells in the control group as Blank, and add 50 µl of diluted antibodies (Anti-Human CD25, Biolegend, 302604; Anti-human CD69, Biolegend, 310910; Anti-Human CD8, Biolegend, 344721; Anti-human CD4, Biolegend, 317429; Anti-Human CD3, Biolegend, 317332; Anti-human CD45; Anti-Human CD39, Biolegend, 328208) to the remaining cells. 6 Cells were incubated with 5 µl of antibody for 8 minutes at room temperature.

[0131] 3. Add 50 µl of 7-AAD dye (7-AAD Viability Staining Solution, Biolegend, 420404) prepared with 2% FBS in PBS to each 1×10 cells in the antibody group. 6 Use 5 µl of cells and incubate at room temperature for 7 minutes. Add 1 ml of 2% FBS in PBS and centrifuge at 400g for 5 minutes. Discard the supernatant and add 200 µl of 2% FBS in PBS to resuspend the cells and test on the instrument.

[0132] CD8 + TIL has direct anti-tumor cytotoxic effector function. Figure 3 The results showed that CD8 + The results showed that in the group with NKG2D mAb, CD8 + The number of TILs was greater than that in the Ctr group.

[0133] CD25 + CD69 + TIL cells represent activated TIL cells. Figure 4 CD25 + CD69 + The results showed that CD25+ CD69 + The percentage of T cells was higher than that of Ctr group.

[0134] CD39 - CD69 - Represents stem cells, Figure 5 CD39 - CD69 - The results showed that there was no significant difference in the proportion of stem cells between the NKG2DmAb group and the Ctr group, indicating that the addition of NKG2D mAb did not affect the proportion of stem cells in TIL.

[0135] Figure 6 CD45 + CD3 + The phenotype indicates the purity of TIL. The results showed that the purity of TIL cells in the NKG2D mAb culture group was higher.

[0136] The above results show that adding NKG2D mAb to the culture medium can obtain CD8 + TIL cells with higher proportion, higher activation and higher purity.

[0137] Example 4: TIL cytotoxicity assay

[0138] After co-culture of TILs from different tumor tissues with corresponding tumor cell lines at an effector-target ratio of 5:1 for 24 hours, the CCK-8 kit was used to detect tumor cell survival rate and TIL cytotoxicity. The steps are as follows:

[0139] Day1 takes 1×10 4 Tumor cells HELA, NCIH520, HCC827, A375, SW480, HepG2, Eca-109 (ATCC) were seeded in 24-well plates. On Day 2, TIL cells from different tumor tissues were diluted to a density of 2.5×10 5 / ml. Aspirate the culture medium in the original wells of the 24-well plate. Add 200μl TIL cell solution to each well. After 24 hours, aspirate the culture medium in the wells, centrifuge and use the cell supernatant for ELISA detection. Wash the cells in the wells with PBS, and detect the activity of tumor cells with CCK8 (Mei Lun Bio, MA0218-5).

[0140] The results are as follows Figure 7 As shown in Table 1. In the NKG2D mAb group, the survival rate of tumor cells in tumor cell lines of different cancer types was significantly lower than that in the normal culture group (Ctr). At the same time, the cytotoxicity of TIL cultured with NKG2D mAb to tumor cells was significantly higher than that of TIL cultured with normal culture (Ctr) ( Figure 8), cytotoxicity increased by an average of 12%.

[0141] Table 1 Tumor cell survival rate (%)

[0142]

[0143] Example 5: Detection of INF-γ secretion by TIL

[0144] After TILs from different tumor tissues were co-cultured with the corresponding tumor cell lines at an effector-target ratio of 5:1 for 24 hours, the cell supernatant was collected and the ELISA kit Human IFN-γ Precoated ELISA Kit (Dakoway, 2307-3) was used to detect the amount of IFN-γ secretion in the cell supernatant. All reagents and consumables were provided by the kit, and the operation was carried out according to the kit instructions.

[0145] like Fig. 9 As shown in Table 2, when NKG2D mAb was added during TIL culture and co-cultured with tumor cells, the secretion of TILINF-γ increased significantly, with an average increase of 1.3 times.

[0146] Table 2 IFN-γ secretion level (pg / ml)

[0147]

[0148] Example 6: Cultivation of engineered TIL cells

[0149] In this embodiment, the culture effect of the culture method of the present invention on genetically engineered TIL cells was verified.

[0150] Specifically, exogenous membrane-bound IL-15 protein, DAP10 activating protein and hEGFRt molecular switch element were expressed on TIL cells, and the following Fig.10 The construct shown in the figure, the molecular sequence of the construct was synthesized by GenScript, and the synthesized gene sequence was cloned into the basic vector pLV-EF1a-c-MYC-IRES-EGFP (Wuhan Miaoling) to obtain the target gene plasmid vector. The sequence of each element is shown in Table 3.

[0151] Table 3 Nucleotide sequence or amino acid sequence of each element

[0152]

[0153] The target gene plasmid vector, psPAX2 vector and pMD2.G vector three-plasmid system is used to package the lentivirus and infect TIL cells, and the engineered TIL cells of the present invention are harvested.

[0154] After engineering transformation, TIL cells derived from different cancer types were cultured in a culture medium with or without NKG2D mAb, and the cell number and cell viability were detected. The experimental method was the same as that of Example 1 and Example 2. The results are shown in FIG. Fig.11 and Fig.12 shown.

[0155] The results of the engineered TIL cell proliferation and viability test are as follows Fig.10 and Fig.11 The results showed that after adding NKG2D mAb to culture, the number of TILs of different cancer types increased significantly, and the cell viability also increased significantly.

[0156] The above results show that adding NKG2D mAb to the culture medium can not only better culture ordinary TIL cells, but also better culture engineered TIL cells, indicating that the TIL cell culture method of the present invention is universal.

[0157] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for culturing tumor infiltrating lymphocytes (TIL) cells, characterized in that: Includes steps: (a) providing a TIL culture medium, the TIL culture medium comprising: (i) a TIL cell culture component, the TIL cell culture component comprising a cytokine combination, the cytokine combination comprising: 100-500 U / ml IL-2, 10-15 ng / ml CD3 monoclonal antibody and 10-30 ng / ml 4-1BBL monoclonal antibody; and (ii) an NKG2D antibody, the concentration of the NKG2D antibody being 0.5 μg / ml-5 μg / ml; (b) culturing TIL cells in the TIL culture medium described in (a) to obtain cultured TIL cells; The number of cells cultured by the method is Q1, and the number of cells cultured without adding the NKG2D antibody under the same conditions is Q0, and Q1 / Q0≥1.5; Furthermore, the cell viability of the cultured TIL cells is ≥85%; The secretion amount of INF-γ after the cultured TIL cells are co-cultured with tumor cells is ≥600pg / mL.

2. The method according to claim 1, characterized in that The concentration of the NKG2D antibody was 2 μg / ml.

3. The method according to claim 1, characterized in that The TIL cell culture components also include: TIL cell basal culture medium, serum or serum substitute.

4. The method according to claim 1, wherein: The cytokine combination includes: 300U / ml IL-2, 15ng / ml CD3 monoclonal antibody, and 15ng / ml 4-1BBL monoclonal antibody.

5. The method according to claim 1, characterized in that The TIL cells are derived from tumor tissue; the tumor includes solid tumors and hematological tumors.

6. The method according to claim 1, characterized in that The TIL cells are genetically engineered TIL cells.

7. The method according to claim 6, characterized in that The genetically engineered TIL cells express an exogenous activating protein, wherein the activating protein comprises a DAP10 element and an activating element, and the sequence of the activating element is shown in SEQ ID NO:

1.

8. The method according to claim 1, characterized in that Compared with cells cultured under the same conditions without adding the NKG2D antibody, the cultured TIL cells also have one or more characteristics selected from the following group: (1) CD8 + T cells and CD4 + The proportion of T cells increased; (2) CD25 + CD69 + The proportion of T cells increases, or the proportion of activated cells increases; (3) CD45 + CD3 + The proportion of TIL cells is increased, or the cell purity is improved.

9. A culture medium for tumor infiltrating lymphocytes (TIL) cells, characterized in that: The culture medium includes: (i) TIL cell culture components, which include a cytokine combination, wherein the cytokine combination is: 100~500U / ml IL-2, 10~15ng / ml CD3 monoclonal antibody and 10~30ng / ml 4-1BBL monoclonal antibody; and (ii) NKG2D antibody, wherein the concentration of the NKG2D antibody is 0.5μg / ml~5μg / ml.

10. The culture medium according to claim 9, characterized in that The concentration of the NKG2D antibody was 2 μg / ml.

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