Methods for Producing Immune Cell Compositions

Abstract

The present application relates to methods for generating immune cell compositions comprising tumor-infiltrating lymphocytes, and uses of the immune cell compositions thereby generated.

Description

[Technical field]

[0001] The present application relates to methods for generating immune cell compositions, and in particular, to methods for generating immune cell compositions that include tumor infiltrating lymphocytes (TILs). [Background technology]

[0002] Cancer immunotherapy is one of the methods for treating cancer, which activates the immune system of the human body to treat cancer. One type of immunotherapy used in cancer immunotherapy is immune cell therapy, which is a cell therapy in which immune cells in the body are collected, enhanced or modified using genetic engineering technology, and then returned to the body.

[0003] One such cell therapy is a method using tumor infiltrating lymphocytes (TILs), where TILs refer to lymphocytes that gather around cancer cells. The treatment using TILs involves harvesting naturally occurring T cells that infiltrate a patient's tumor, followed by a process of activating and expanding the T cells. The large number of activated T cells that are activated and expanded through the above process are then infused back into the patient, resulting in the destruction of the tumor.

[0004] In other words, the process of culturing and expanding TIL is essential for cancer treatment using tumor-infiltrating lymphocytes.However, in this process, the isolation, culture and expansion of TIL from tumor tissue is complicated, and it is difficult to expand TIL with a higher yield.Therefore, a more efficient process for TIL culture and expansion is required, and the present application relates to a method for producing immune cell composition with a high yield. Summary of the Invention [Problem to be solved by the invention]

[0005] The present application is directed to providing methods for efficiently producing immune cell compositions at high yields, as compared to conventional low yield production methods.

[0006] In one embodiment, the present application provides a method for efficiently culturing and expanding immune cells.

[0007] In one particular embodiment, the present application provides a method for efficiently culturing and expanding tumor infiltrating lymphocytes (TILs). [Means for solving the problem]

[0008] One aspect of the present application provides a method for producing an immune cell composition.

[0009] Another aspect of the present application provides an immune cell composition comprising TILs.

[0010] Yet another aspect of the present application provides the use of an immune cell composition comprising TILs. Effect of the Invention

[0011] The method for producing an immune cell composition according to the present application has higher efficiency than that of a conventional method for producing an immune cell composition, specifically, a lower concentration of IL-2 is used in the method according to the present application, and immune cells can be obtained in a higher yield, compared to the conventional method for producing an immune cell composition. [Brief description of the drawings]

[0012] 1-4 show an example of a process for producing an immune cell composition.

[0013] [Figure 1] FIG. 1 shows a process for producing a semi-product (intermediate product) (a first immune cell population produced and obtained by isolating and culturing an immune cell population from a sample).

[0014] [Diagram 2]1 illustrates a process for producing a final product (an immune cell composition produced by obtaining a third immune cell population expanded from a second immune cell population) from a cryopreserved half-product.

[0015] [Diagram 3] A process for producing a final product from a tumor sample is shown, which is an example process that includes a cryopreservation step.

[0016] [Figure 4] A process for producing a final product from a tumor sample is shown, which is an example that does not include a cryopreservation step.

[0017] [Diagram 5] 1 shows experimental results obtained by measuring tumor infiltrating lymphocyte (TIL) proliferation yield.

[0018] [Figure 6] 1 shows the results of experiments to optimize IL-2 concentrations in TIL generation. 2 shows the results of experiments to optimize IL-2 concentrations in TIL culture.

[0019] [Figure 7] 1 shows the results of an experiment to optimize IL-2 concentrations in the generation of TILs. 2 shows the results of an experiment to optimize IL-2 concentrations in TIL expansion.

[0020] [Figure 8] 1 shows the results of experiments on the cell proliferation rate of TILs depending on the volume of composition used in culture.

[0021] [Figure 9] 1 shows experimental results obtained by measuring the number of cultured cells per tumor tissue in TIL cultures.

[0022] [Figure 10]The results of determining the percentage of T cells in cultured and expanded TILs are shown.

[0023] [Figure 11] A representative example of the isolation / culture of TILs from tumor samples and processing for mass culture (expansion) of TILs is shown.

[0024] [Figure 12] 1 shows the results of determining the percentage of CD8+ T cells and CD4+ T cells in TILs (sample no. BC16208) expanded from cryopreserved and thawed half-products.

[0025] [Figure 13] The results of confirming the types of T cells among the CD4+ T cells and CD8+ T cells identified in FIG. 12 are shown.

[0026] [Figure 14] The results of checking the percentages of CD8+ T cells and CD4+ T cells in TILs (sample no. BC17058) expanded from cryopreserved and thawed half-products are shown.

[0027] [Figure 15] The results of confirming the types of T cells among the CD4+ T cells and CD8+ T cells identified in FIG. 14 are shown.

[0028] [Figure 16] 1 shows the results of determining the percentage of CD8+ T cells and CD4+ T cells in TILs (sample no. BC18050) expanded from cryopreserved and thawed half-products.

[0029] [Figure 17] The results of confirming the types of T cells among the CD4+ T cells and CD8+ T cells identified in FIG. 16 are shown.

[0030] [Figure 18]1 shows the results of determining the percentage of immune cells present in TILs (sample no. BC16028) expanded from cryopreserved and thawed semi-products. [Figure 19] 1 shows the results of determining the percentage of immune cells present in TILs (sample no. BC16028) expanded from cryopreserved and thawed semi-products.

[0031] [Figure 20] 1 shows the results of determining the percentage of immune cells present in TILs (sample no. BC17058) expanded from cryopreserved and thawed semi-products. [Figure 21] 1 shows the results of determining the percentage of immune cells present in TILs (sample no. BC17058) expanded from cryopreserved and thawed semi-products.

[0032] [Figure 22] 1 shows the results of determining the percentage of immune cells present in TILs (sample no. BC18050) expanded from cryopreserved and thawed semi-products. [Figure 23] 1 shows the results of determining the percentage of immune cells present in TILs (sample no. BC18050) expanded from cryopreserved and thawed semi-products.

[0033] [Figure 24] The graph shows the results of confirming changes in mouse body weight in each group after TIL administration.

[0034] [Diagram 25] The graph shows the results of confirming changes in mouse body weight in each group after simultaneous administration of TIL and IL-2.

[0035] [Figure 26] The graph shows the results of confirming changes in tumor volume in mice in each group after administration of TILs. [Figure 27]The graph shows the results of confirming changes in tumor volume in mice in each group after administration of TILs. [Figure 28] The graph shows the results of confirming changes in tumor volume in mice in each group after administration of TILs. [Figure 29] The graph shows the results of confirming changes in tumor volume in mice in each group after administration of TILs.

[0036] [Diagram 30] The mice were euthanized 38 days after TIL administration, and tumors were excised from the mice to confirm tumor size in each group. [Diagram 31] The mice were euthanized 38 days after TIL administration, and tumors were excised from the mice to confirm tumor size in each group.

[0037] [Diagram 32] The graph shows the results of confirming changes in tumor size in mice in each group after simultaneous administration of TIL and IL-2. [Diagram 33] The graph shows the results of confirming changes in tumor size in mice in each group after simultaneous administration of TIL and IL-2. [Diagram 34] The graph shows the results of confirming changes in tumor size in mice in each group after simultaneous administration of TIL and IL-2. [Diagram 35] The graph shows the results of confirming changes in tumor size in mice in each group after simultaneous administration of TIL and IL-2.

[0038] [Diagram 36] The mice were euthanized 38 days after TIL administration, and tumors were excised from the mice to confirm tumor size in each group. [Figure 37] The mice were euthanized 38 days after TIL administration, and tumors were excised from the mice to confirm tumor size in each group.

[0039] [Figure 38] The graph shows the results of confirming changes in mouse body weight in each group after simultaneous administration of TIL and IL-2.

[0040] [Figure 39] The graph shows the results of confirming changes in tumor volume in mice in each group after simultaneous administration of TIL and IL-2. [Diagram 40] The graph shows the results of confirming changes in tumor volume in mice in each group after simultaneous administration of TIL and IL-2. [Diagram 41] The graph shows the results of confirming changes in tumor volume in mice in each group after simultaneous administration of TIL and IL-2. [Diagram 42] The graph shows the results of confirming changes in tumor volume in mice in each group after simultaneous administration of TIL and IL-2.

[0041] [Diagram 43] The mice were euthanized 70 days after the simultaneous administration of TIL and IL-2, and the tumors were excised from the mice to confirm the tumor volume in each group.

[0042] In one embodiment of the present application, a method for producing an immune cell composition is provided.

[0043] In one embodiment of the present application, an immune cell composition produced by a method according to an embodiment of the present application is provided.

[0044] In one embodiment of the present application, there is provided a use of an immune cell composition for treating cancer in a subject.

[0045] In one embodiment of the present application, a method for treating cancer in a subject using an immune cell composition.

[0046] The embodiments of the present application are described in detail below.

[0047] In one embodiment of the present application, a method is provided for generating an immune cell composition comprising: (a) culturing a tumor sample in a first container; wherein the tumor sample is cultured in a first composition in a first container, the first composition comprising a medium, serum, and 500-1500 IU / mL of IL-2; wherein the first container is a gas-permeable container or a container having a gas supply system; Here, the culture is carried out for 13 to 15 days. wherein a first additive composition is added to the first container one or two or more times during the culture, the first additive composition comprising medium, serum, and IL-2; (b) obtaining a first population of immune cells from the cultured product obtained from (a); (c) seeding a second composition and a second population of immune cells into a second container; wherein the second immune cell population is derived from the first immune cell population; Here, the second composition contains a medium, serum, 1000 to 3000 IU / mL of IL-2, an anti-CD3 antibody, and feeder cells; wherein the second container is a gas-permeable container or a container having a gas supply system; (d) expanding the second immune cell population to generate a third immune cell population; wherein a second additive composition is added to the second container one or two or more times during growth, the second additive composition comprising medium, serum, and IL-2; wherein the propagation is carried out for 13 to 15 days; and (e) obtaining a third population of immune cells from the expansion product of (d) to produce an immune cell composition; Here, the third immune cell population has at least 2500 times more cells compared to the second immune cell population.

[0048] In one embodiment, the method may further comprise the step of preparing said tumor sample, said step of preparing said tumor sample may be carried out prior to (a).

[0049] In one embodiment, in (a), the tumor sample may be obtained from a tumor fragment obtained from the subject, the tumor sample being approximately 1 mm 2 ~4mm 2 The cross-sectional area may be

[0050] In one embodiment, in (a), 12 to 24 tumor samples may be cultured.

[0051] In one embodiment, the serum concentration of the first composition may be 5% by volume or less.

[0052] In one embodiment, the concentration of serum in the first composition may be about 3% by volume or less.

[0053] In one embodiment, the medium of the first composition may be CTS-AIM-V medium.

[0054] In one embodiment, the concentration of IL-2 in the first composition may be about 1000 IU / mL.

[0055] In one embodiment, the volume of the first composition may be 30% or less of the maximum allowable capacity of the first container.

[0056] In one embodiment, the first container may be a T75 flask and the volume of the first composition may be 6 ml.

[0057] In one embodiment, the first additive composition may have the same compositional properties and compositional ratios as the first composition.

[0058] In one embodiment, the IL-2 concentration of the first additive composition may be about 1000 IU / mL, the serum concentration of the first additive composition may be about 3% by volume, and the medium of the first additive composition may be CTS-AIM-V medium.

[0059] In one embodiment, the first additive composition may be added to the first container two or more times during the culture.

[0060] Here, in one embodiment, during the culture, the first additive composition may be added to the first container on days 7, 10, and 12 from the start of the culture, and the amount of the first additive composition added may be the same at each addition point.

[0061] In one embodiment, the second immune cell population may be obtained from the first immune cell population.

[0062] In one embodiment, the second immune cell population may be obtained by a method comprising washing the first immune cell population.

[0063] In one embodiment, the medium of the second composition may be CTS-AIM-V.

[0064] In one embodiment, the serum concentration of the second composition may be 5% by volume or less.

[0065] In one embodiment, the serum concentration of the second composition may be about 3% by volume.

[0066] In one embodiment, the concentration of IL-2 in the second composition may be about 2000 IU / mL.

[0067] In one embodiment, the anti-CD3 antibody may be OKT3.

[0068] In one embodiment, the anti-CD3 antibody concentration of the second composition may be from 10 ng / mL to 100 ng / mL.

[0069] In one embodiment, the anti-CD3 antibody concentration of the second composition may be about 30 ng / mL.

[0070] In one embodiment, the feeder cells may be inactivated allogeneic peripheral blood mononuclear cells.

[0071] In one embodiment, the feeder cells may be included in the second composition at a ratio of approximately 1:200 (number of cells of the second immune cell population:number of feeder cells) based on the number of cells in the second immune cell population.

[0072] In one embodiment, in the seeding step, the number of cells of the second immune cell population seeded into the second container may be 1E5 to 10E5.

[0073] In one embodiment, in the seeding step, the number of cells of the second immune cell population seeded into the second container may be about 4E5.

[0074] In one embodiment, the volume of the second composition may be 30% or less of the maximum allowable capacity of the second container.

[0075] In one embodiment, the second container may be GREX100M and the volume of the second composition may be about 20 ml.

[0076] In one embodiment, during expansion, the immune cells do not have to be removed from the second container or added exogenously.

[0077] In one embodiment, the second additive composition may be a composition having the same compositional properties and compositional ratio as the second composition, except that the anti-CD3 antibody and feeder cells are excluded.

[0078] In one embodiment, the IL-2 concentration of the second additive composition may be about 2000 IU / mL, the serum concentration of the second additive composition may be about 3% by volume, and the medium of the second additive composition may be CTS-AIM-V.

[0079] In one embodiment, during growth, the second additive composition may be added to the second container two or more times.

[0080] Here, in one embodiment, the amount of the second additive composition added may be different at each addition point.

[0081] Here, in one embodiment, during growth, the second additive composition may be added to the second container on days 4, 6, 8, and 10 from the start of growth, and the amount of the second additive composition added may be gradually increased at each addition point.

[0082] Here, in one embodiment, 20 ml of the second additive composition may be added to the second container on day 4 from the start of growth, 60 ml on day 6 from the start of growth, 180 ml on day 8 from the start of growth, and 540 ml on day 10 from the start of growth.

[0083] In one embodiment, the cell number of the third immune cell population may be at least 4000 times greater than the cell number of the second immune cell population.

[0084] In one embodiment, the immune cell composition may comprise a third immune cell population.

[0085] In one embodiment, the immune cell composition may include a fourth immune cell population obtained from the third immune cell population.

[0086] In one embodiment, the tumor sample may be from a solid tumor from a subject with breast cancer, colon cancer, lung cancer, gastric cancer, renal cancer, ovarian cancer, or skin cancer, including malignant melanoma.

[0087] In one embodiment, the tumor sample may be derived from a malignant pleural effusion of a subject with lung cancer.

[0088] In one embodiment, the method may further comprise the steps of cryopreserving the first immune cell population; and thawing the cryopreserved first immune cell population, wherein the steps of cryopreserving the first immune cell population and thawing the cryopreserved first immune cell population may occur after (b).

[0089] In one embodiment of the present application, an immune cell composition comprising tumor-infiltrating lymphocytes is provided.

[0090] In one embodiment, the immune cell composition may be produced by a method for producing an immune cell composition according to an embodiment of the present application.

[0091] In one embodiment of the present application, a method for treating cancer in a subject is provided, comprising administering an immune cell composition comprising tumor infiltrating lymphocytes, wherein the immune cell composition may be produced by a method for producing an immune cell composition according to an embodiment of the present application.

[0092] In one embodiment, the subject may be the subject from which the tumor-infiltrating lymphocytes are derived.

[0093] In one embodiment of the present application, there is provided a use of an immune cell composition comprising tumor infiltrating lymphocytes for treating cancer in a subject, wherein the immune cell composition may be produced by a method for producing an immune cell composition according to an embodiment of the present application.

[0094] In one embodiment, the subject may be the subject from which the tumor-infiltrating lymphocytes are derived. [Disclosure of the Invention]

[0095] The present invention will be described in more detail below through specific embodiments and examples with reference to the accompanying drawings. Please note that the accompanying drawings include some, but not all, embodiments. The contents of the present invention disclosed in this application may be implemented in various ways and are not limited to the specific embodiments described in this application. Those skilled in the art to which the present invention belongs can imagine various modifications and other embodiments of the contents of the present invention disclosed in this application. Therefore, it should be understood that the contents of the present invention disclosed in this application are not limited to the specific embodiments described in this application, and the modifications and other embodiments are included within the scope of the claims. [Terminology definition]

[0096] Immune (cell) composition The term "immune cell composition" or "immune composition" as used herein refers to a composition comprising a population of immune cells. In the present application, immune cells include tumor infiltrating lymphocytes (TILs). Tumor infiltrating lymphocytes (TILs) refer to immune cells that migrate to and live around tumors. TILs can recognize and kill cancer cells. TILs may include various immune cells, including T cells, B cells, and natural killer cells. T cells are known to make up the majority of TILs.

[0097] Semi-product (half-product) The method for producing an immune cell composition disclosed in the present application may include a step of producing a half-product. Here, the half-product refers to a first immune cell population produced and obtained through isolating and culturing an immune cell population from a sample. According to conventional terminology used in the field of immune cell therapy technology, the half-product refers to an immune cell population produced by a pre-Rapid Expansion Protocol (pre-REP). That is, according to conventional terminology in the field of immune cell therapy technology, the half-product may be referred to as a pre-REP product.

[0098] End product The method for producing an immune cell composition disclosed herein may include a step of producing a final product (finished product). Here, the final product refers to an immune cell composition or immune composition produced by obtaining a third immune cell population expanded from a second immune cell population. According to conventional terminology used in the field of immune cell therapy technology, the final product refers to an immune cell population produced by a Rapid Expansion Protocol (REP). That is, according to conventional terminology used in the field of immune cell therapy technology, the final product may be referred to as a REP product. It may also be referred to as a drug product.

[0099] Subject As used herein, a subject refers to an individual having a tumor, including a human. The tumor may be a solid tumor or a liquid tumor. Solid tumors include tumors in the lung, breast, bladder, ovary, etc., and liquid tumors include, but are not limited to, hematological tumors such as leukemia, myeloma, lymphoma, etc.

[0100] container As used herein, a container refers to a container used to culture tumor tissue or tumor sample or grow immune cell populations. In this application, the container is used to culture or grow tumor tissue or immune cell populations with a composition comprising medium, serum, plasma, IL-2, OKT3, and / or feeder cells. The container may be a gas-permeable container through which gas passes or a container with a gas supply system that provides gas. That is, the container may be a container manufactured to allow gas to pass through the container, or a container that is not manufactured to pass gas but has a separate gas supply system that allows gas to pass through. The container may be, for example, a 24-well plate, a T25, T75, or T125 flask, a flask such as GREX10, GREX10M, GREX10M-CS, GREX100, GREX100M, GREX100M-CS, GREX500, GREX500M, or GREX500M-CS flask, or a culture bag, but the application is not limited thereto.

[0101] Culture medium As used herein, culture medium refers to a nutrient source for culturing cells and / or tissues. Culture medium may vary depending on the type of object to be cultured. Culture medium may be AlyS505N, AIM-V medium, or RPMI medium, but the present application is not limited thereto. According to one embodiment of the present application, the culture medium is supplemented with serum, plasma, IL-2, OKT3, and / or feeder cells to be used for culturing and / or expanding tissues and / or immune cells.

[0102] serum As used herein, the term "serum" or "blood serum" refers to an additive added to a basal culture medium, and the serum may contain high molecular weight proteins, low molecular weight nutrients, and components necessary for cell growth. The serum may be a human serum, such as commercially available human AB serum, but the present application is not limited thereto. Here, the concentration of serum in the medium may be approximately 10 vol% or less, approximately 9 vol% or less, approximately 8 vol% or less, approximately 7 vol% or less, approximately 6 vol% or less, approximately 5 vol% or less, approximately 4 vol% or less, or approximately 3 vol% or less.

[0103] plasma As used herein, the term "plasma" or "blood plasma" refers to an additive added to a basal culture medium, and plasma refers to fibrinogen-free serum. Plasma contains components necessary for cell growth. Plasma as used herein includes, but is not limited to, plasma isolated from a patient's blood. In the present application, plasma contained in the medium may be used instead of serum. Here, the concentration of plasma in the medium may be approximately 10 vol% or less, approximately 9 vol% or less, approximately 8 vol% or less, approximately 7 vol% or less, approximately 6 vol% or less, approximately 5 vol% or less, approximately 4 vol% or less, or approximately 3 vol% or less. IL-2

[0104] The term "IL-2" as used herein is an abbreviation for interleukin-2, which refers to a factor that promotes T cell proliferation. IL-2 includes various types of IL-2, such as human recombinant IL-2. In the present application, IL-2 may be included in the first composition, and the concentration of IL-2 may be approximately 10,000 IU / mL, approximately 9,000 IU / mL, approximately 8,000 IU / mL, approximately 7,000 IU / mL, approximately 6,000 IU / mL, approximately 5,000 IU / mL, approximately 4,000 IU / mL, approximately 3,000 IU / mL, approximately 2,000 IU / mL, approximately 1,000 IU / mL, or approximately 500 IU / mL. Further, in the present application, IL-2 may be included in a second composition along with the OKT3 and feeder cells, wherein the concentration of IL-2 may be approximately 10,000 IU / mL, approximately 9,000 IU / mL, approximately 8,000 IU / mL, approximately 7,000 IU / mL, approximately 6,000 IU / mL, approximately 5,000 IU / mL, approximately 4,000 IU / mL, approximately 3,000 IU / mL, approximately 2,000 IU / mL, approximately 1,000 IU / mL, or approximately 500 IU / mL.

[0105] Immune Cell Populations As used herein, the term "immune cell population" refers to a group or population essentially consisting of a plurality of immune cells, including TILs, wherein the first immune cell population refers to an immune cell population obtained by initially culturing a tumor tissue of a subject, the second immune cell population refers to an immune cell population derived from the first immune cell population and used by splitting the first immune cell population as needed, and the third immune cell population refers to an immune cell population obtained by expanding the second immune cell population.

[0106] frozen storage As used herein, the term "cryopreservation" refers to the storage of immune cell populations at ultra-low temperatures to suppress damage to immune cells and preserve immune cell populations for long periods of time. For example, cryopreservation may be used herein to refer to the process of storing cells or immune cell populations in a liquid nitrogen tank. In one embodiment, the cryopreservation period may be at most approximately 1 year, approximately 2 years, approximately 3 years, approximately 4 years, approximately 5 years, approximately 6 years, approximately 7 years, approximately 8 years, approximately 9 years, approximately 10 years, approximately 11 years, approximately 12 years, approximately 13 years, approximately 14 years, approximately 15 years, approximately 16 years, approximately 17 years, approximately 18 years, approximately 19 years, or approximately 20 years. A cryopreservation medium is used to cryopreserve the immune cell population. The cryopreservation medium may include approximately 10% DMSO, and the medium includes, but is not limited to, Cryostor CS10 medium. Cryopreservation may optionally be included between each step of the method for producing the immune cell composition.

[0107] sowing As used herein, the term "seeding" refers to dispensing cells into a container, and in this application, seeding includes dispensing a second immune cell population into a second container. For example, seeding of a second immune cell population may be performed by suspending approximately 2E4 cells / mL in the second composition and dispensing it in different amounts depending on the type of container. In one example, 20mL of cell suspension may be dispensed into GREX100M, and the second immune cell population may be seeded at a cell number of approximately 4E5 cells.

[0108] Anti-CD3 antibody As used herein, the term "anti-CD3 antibody" refers to an antibody that targets a membrane protein on the surface of T cells, i.e., the CD3 receptor. Anti-CD3 antibodies include OKT3 and also include all types of monoclonal and polyclonal anti-CD3 antibodies, including human, humanized and murine antibodies. OKT3 as used herein includes all monoclonal antibodies, such as human, humanized, chimeric or murine antibodies, biosimilars and variants thereof. Other examples of anti-CD3 antibodies include otelixizumab, teplizumab and visilizumab.

[0109] Feeder cells As used herein, the term "feeder cells" refers to cells that are included in a composition for culturing or growing immune cells to activate and grow immune cells. For example, in the present application, feeder cells may be included in a second composition to activate and grow immune cells of the second composition. Feeder cells include peripheral blood mononuclear cells (PBMCs), which refer to peripheral blood cells with round cell nuclei. As used herein, PBMCs include irradiated allogeneic peripheral blood mononuclear cells.

[0110] Proliferation As used herein, the term "expansion" refers to the increase in a cell or immune cell population by a predetermined fold or more over a predetermined period of time.

[0111] approximately As used herein, the term "approximately" or "about" means within a statistically significant range. Unless otherwise stated, as used herein, the term "approximately" or "about" means having a value within a range of 20% or 10% of a given value.

[0112] Includes / Has As used herein, the term "comprising" is meant not to exclude optional additional materials or steps. [Immune cell therapy] overview

[0113] Cancer is a group of diseases in which cells continue to divide due to uncontrollable cell cycles, including lung cancer, breast cancer, and colorectal cancer. In recent years, cancer immunotherapy has been studied as one of the methods for treating cancer, which refers to a method of treating cancer by activating the immune system of the human body. As a cancer immunotherapy used for cancer immunotherapy, immune cell therapy is a cell therapy that collects immune cells from the body and strengthens or genetically modifies them before returning them to the body.

[0114] This method is called adoptive cell transfer (ACT) and is classified into treatment using TILs and treatment using T cell receptor (TCR)-expressing T cells or chimeric antigen receptor (CAR)-expressing T cells that are modified through genetic engineering. In one embodiment of the present application, a treatment using TILs is disclosed.

[0115] Tumor infiltrating lymphocytes (TIL) and treatment using same Tumor-infiltrating lymphocytes (TILs) are lymphocytes that congregate around cancer cells. Treatment with TILs involves harvesting naturally occurring T cells that have infiltrated a patient's tumor, activating and expanding these T cells, and then reintroducing large numbers of activated T cells back into the patient to suppress or destroy the tumor.

[0116] Conventional methods for generating immune cell compositions overview

[0117] The process for generating immune cell compositions, ie, immune cell compositions that contain TILs, generally involves the following steps. 1. Sample processing Generally, a sample processing step is first performed to obtain an immune cell composition containing TILs, where sample means a tumor sample obtained from a patient, and the tumor sample is cut into small pieces during the sample processing step. 2. culture Generally, after tumor sample is processed, the small tumor fragments obtained are cultured in the medium for culturing tumor.Generally, the culture is carried out in the medium containing high concentration of IL-2.Through the culture step, a first immune cell population is obtained.

[0118] 3. Proliferation After the culture step, the first immune cell population proceeds to an expansion step. Generally, the expansion step can be referred to as a rapid expansion protocol (REP). The expansion step is generally performed in a medium containing high concentrations of IL-2, anti-CD3 antibodies, and feeder cells.

[0119] 4. Obtaining and formulating The immune cell population that has undergone the expansion step is obtained in a contaminating manner and formulated by transferring it into a container suitable for administration to a patient. [Limitations of the Prior Art]

[0120] In order to use TILs to treat cancer, TILs must first be isolated, cultured, and expanded from cancer tissue. The series of processes for isolating, culturing, and then expanding TILs from cancer tissue is quite complicated, and such a series of processes may affect the yield of TILs. Thus, conventional methods have a problem of low yield because each process is not optimized or standardized or has low efficiency. To overcome this, it is necessary to optimize and standardize each process to create a method with higher efficiency and higher yield than conventional methods. Therefore, the method for generating immune cell compositions provided in the present application has been developed to obtain a high yield of TILs by optimizing and standardizing a series of processes for TIL culture and expansion. Methods for Producing Immune Cell Compositions and Uses of Immune Cell Compositions Disclosed in the Present Application

[0121] The present application discloses methods for producing immune cell compositions (immune compositions).

[0122] In one embodiment, the method for producing an immune cell composition of the present application comprises the steps of: Production of a half-product; cryopreservation; thawing of the cryopreserved first immune cell population; and production of a final product. The present invention may include any one or more of the above.

[0123] In one embodiment, a method for generating an immune cell composition may comprise culturing a tumor sample; obtaining a first immune cell population; seeding a second immune cell population; expanding the second immune cell population; and generating the immune cell composition.

[0124] In one embodiment, a method for generating an immune cell composition may comprise culturing a tumor sample; obtaining a first immune cell population; cryopreserving the first immune cell population; thawing the cryopreserved first immune cell population; seeding a second immune cell population; expanding the second immune cell population; and generating the immune cell composition.

[0125] Each step that may be included in the method for producing the immune cell composition of the present application is disclosed in detail below.

[0126] I. Formation of Half-Products The method for producing an immune cell composition disclosed herein may include a process for producing a half-product and / or a process for producing a final product, where half-product refers to a first immune cell population obtained from the cultured composition after culturing a tumor sample. The process for producing a half-product may include the contents described below. This stage may correspond to a pre-rapid proliferation protocol (pre-REP) according to conventional terminology in the field of immune cell therapy.

[0127] In one embodiment, the production of the half-products comprises: Culturing the tumor sample; and Obtaining a first population of immune cells may include:

[0128] In one embodiment, the production of the half-product may further comprise a step of preparing a tumor sample, wherein the step of preparing the tumor sample is performed before the step of culturing the tumor sample.

[0129] Each process involved in the production of the semi-products is described in detail below.

[0130] 1. Tumor Sample Preparation Overview To obtain the first immune cell population, a tumor sample must first be prepared, where the tumor sample is derived from the subject's tumor tissue and contains tumor-infiltrating lymphocytes (TILs) that have the characteristics of residing around cancer cells.

[0131] In one embodiment, preparation of the tumor sample may include processing the tumor tissue obtained from the subject. In another embodiment, the tumor tissue obtained from the subject may be used as the tumor sample without further processing.

[0132] The process for preparing the tumor samples is described in detail below.

[0133] Obtaining tumor tissue The tumor tissue used to prepare the tumor sample is obtained from a subject having a tumor, which may include a human having a tumor. The tumor tissue may be obtained directly by the practitioner of the present invention, or the tumor tissue may be obtained by obtaining the tumor tissue from a hospital.

[0134] In one embodiment, the tumor tissue may be obtained from a human having a solid tumor or a liquid tumor. A human having a solid tumor may be, for example, a patient having breast cancer, colorectal cancer, lung cancer, gastric cancer, renal cancer, ovarian cancer, or skin cancer, including malignant melanoma, but the present application is not limited thereto. A human having a liquid tumor may be, for example, a patient having a blood cancer, including leukemia or lymphoma, but the present application is not limited thereto.

[0135] Here, the tumor tissue contains tumor infiltrating lymphocytes (TILs).

[0136] Tumor tissue is taken from a subject through various routes. For example, tumor tissue can be obtained by taking the tumor of a subject (e.g., a cancer patient). As another example, tumor tissue can be obtained by taking malignant pleural effusion observed in a cancer patient. As a specific example, in the case of a lung cancer patient, tumor tissue obtained from a subject can be obtained by taking pleural effusion of the patient.

[0137] Tumor tissue may be obtained by methods known in the art. In one embodiment, tumor tissue may be obtained by biopsy. Biopsies include, but are not limited to, excision biopsy and aspiration biopsy using a needle of a syringe to remove a portion of a tumor. Tumor tissue may be obtained by biopsy and other methods known in the art. In one embodiment, tumor tissue may be obtained via excision biopsy to remove a portion of a tumor in a patient with breast and / or lung cancer.

[0138] In one embodiment, the tumor tissue may be breast and / or lung cancer tissue obtained from a breast and / or lung cancer patient.

[0139] Tumor tissue processing In one embodiment, the tumor tissue obtained from the subject may be prepared as a tumor sample after undergoing further processing, or the tumor tissue obtained from the subject may be prepared as a tumor sample without any processing.

[0140] Tumor tissue processing is described in detail below.

[0141] In one embodiment, the processing of the tumor tissue may include, but is not limited to, washing, removing fatty tissue, and / or forming small fragments.

[0142] 1) Cleaning The treatment of the tumor tissue may include washing the tumor tissue. In one embodiment, the washing may include washing the tumor tissue with a medium. In one embodiment, the washing may include removing antibiotics from the tumor tissue that has been transplanted into a transport medium containing antibiotics.

[0143] In one embodiment, the tumor tissue may be washed 1, 2, 3, 4, 5 or more times with medium. In a particular embodiment, the tumor sample may be washed 2 or 3 times with medium.

[0144] In one embodiment, the tumor tissue may be washed using antibiotic-free medium. In a specific embodiment, the tumor tissue may be washed with antibiotic-free RPMI1640 medium. In a specific embodiment, the tumor tissue may be washed three times with antibiotic-free RPMI1640 medium.

[0145] 2) Removal of lipid tissue The processing of the tumor tissue may include removal of lipid tissue, which may be performed or omitted depending on whether the tumor sample contains a large amount of fat. The removal of lipid tissue includes separating lipid tissue from the tumor tissue. In one embodiment, the removal of lipid tissue may be performed using tweezers and scissors.

[0146] 3) Formation of small fragments of tumor tissue Processing the tumor tissue may include forming the tumor tissue into small pieces. The tumor tissue may be formed into small pieces using a cutter (e.g., scissors or a knife) or a solution containing digestive enzymes, but the present application is not limited thereto.

[0147] In one embodiment, the tumor tissue may be cut to have a side width of approximately 0.1 to 10 mm, approximately 0.4 to 5 mm, approximately 0.6 to 4 mm, approximately 0.8 to 3 mm, or approximately 1 to 2 mm. In a specific embodiment, the tumor tissue may be cut to have a side width of approximately 1 to 2 mm.

[0148] In one embodiment, the tumor tissue has a cross-sectional area of ​​approximately 0.01 to 100 mm 2 , approximately 0.1 to 50 mm 2 , approximately 0.2 to 10 mm 2 , approximately 0.5 to 5 mm 2 , approximately 0.7 to 4 mm 2 , or approximately 1 to 2 mm 2 In certain embodiments, the tumor tissue may be cut into pieces of approximately 1 to 2 mm 2 may be cut to have a cross-sectional area of

[0149] In another embodiment, the tumor tissue may be formed into small pieces by a digestive solution containing digestive enzymes, where the tumor tissue may be cut into small pieces by a digestive solution containing enzymes such as collagenase.

[0150] 2. Culturing Tumor Samples Overview At this stage, the tumor sample is cultured.

[0151] In one embodiment, the first immune cell population may be generated by culturing a tumor sample. The process of generating the first immune cell population by culturing a tumor sample is described in detail below.

[0152] Culturing the Tumor Sample The tumor sample is cultured in a first composition in a first container. The first immune cell population may be generated by culturing the tumor sample.

[0153] Here, the tumor sample is cultured in an environment suitable for culture. In one embodiment, the tumor sample may be cultured in a 37° C., 5% CO2 environment.

[0154] 1) The number of tumor sample fragments contained in the first container A predetermined number of cut tumor sample fragments are placed in a first composition in a first container and cultured, where the number of tumor sample fragments placed in the first container can vary depending on the cell culture area or volume of the first container.

[0155] In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 tumor sample fragments may be placed in and cultured in the first container. In a particular embodiment, 12, 16, 20, 24, 28, or 32 tumor sample fragments may be placed in and cultured in the first container.

[0156] In one embodiment, when the first container is a T75 flask, 12, 16, 20, 24, 28, or 32 tumor sample fragments may be placed and cultured in the first container. In a particular embodiment, when the first container is a T75 flask, 24 tumor sample fragments may be placed and cultured in the first container.

[0157] 2) First container The tumor sample is cultured in a first composition in a first container, where the first container refers to a container used in culturing the tumor sample. The first container in this application is used to culture the tumor sample together with a composition comprising medium, serum, and IL-2.

[0158] The container may be a gas permeable container through which gas passes or a container having a gas supply system that provides gas, i.e., the container may be a container that is manufactured to allow gas to pass through the container, or a container that is not manufactured to allow gas to pass through but has a separate gas supply system that allows the gas to pass through.

[0159] In one embodiment, the first container is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350, 400, 450, 500, 550, 600, 700, The first container may be a container having a maximum allowable volume of 800, 900, 1000, 1100, 1200, 1300, 1400, 1600, 1800, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, or 10000 mL, or a container having a maximum allowable volume within a range set by two values ​​selected from the aforementioned values. In certain embodiments, the first container may be a container having a maximum allowable volume of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 mL, or a container having a maximum allowable volume within a range set by two values ​​selected from the aforementioned values.

[0160] In one embodiment, the container may be, for example, a 24-well plate, a T25, T75, or T125 flask, a flask such as a GREX10, GREX10M, GREX10M-CS, GREX100, GREX100M, GREX100M-CS, GREX500, GREX500M, or GREX500M-CS flask, or a culture bag, but the application is not limited thereto.

[0161] In one embodiment, the tumor sample may be cultured in a T75 flask. In a particular embodiment, 24 tumor sample fragments may be cultured in a T75 flask.

[0162] 3) First Composition In the first container, the tumor sample is cultured in a first composition, where the first composition comprises any one or more of medium, serum, plasma, and an antibiotic, IL-2, and an antibody.

[0163] In one embodiment, the first composition may comprise medium, serum, and IL-2. In another embodiment, the first composition may comprise medium, plasma, and IL-2.

[0164] In one embodiment, the first composition may comprise culture medium; any one of serum and plasma; and IL-2, and may further comprise any one or more of an antibody and an antibiotic.

[0165] In a specific embodiment, the first composition comprises medium, serum, and IL-2. In another specific embodiment, the first composition comprises medium, serum, IL-2, and an antibiotic. In yet another specific embodiment, the first composition comprises medium, serum, IL-2, and an antibody. In yet another specific embodiment, the first composition comprises medium, serum, IL-2, an antibiotic, and an antibody.

[0166] Generally, in tissue or cell culture, the volume of composition used in a vessel (e.g., flask) may vary depending on the type of vessel, for example, 2 mL of composition is generally used in a 24-well plate, and 20 mL of composition is generally used in a T75 flask.

[0167] However, the amount of the first composition initially added to the first container for culturing the tumor sample of the present application may differ from the amount generally used above.

[0168] In one embodiment, the amount of the first composition used to culture the tumor sample (i.e., added to the first container) may be approximately 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5% or less of the maximum allowable volume of the first container. In a particular embodiment, the amount of the first composition added to the first container for culturing the tumor sample may be approximately 30% or less of the maximum allowable volume of the first container. Here, the maximum allowable volume refers to the maximum volume that can be added to the first container.

[0169] In one embodiment, when the first container is a 24-well plate, the amount of the first composition initially added may be 1.5 mL or less, or 1 mL or less. In a particular embodiment, when the first container is a 24-well plate, the amount of the first composition initially added may be approximately 1 mL.

[0170] In another embodiment, when the first vessel is a T75 flask, the amount of the first composition used may be 15 mL, 12 mL, 10 mL, 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, or 3 mL, or less. In a particular embodiment, when the first vessel is a T75 flask, the amount of the first composition used may be approximately 6 mL.

[0171] The amount of the initial first composition of the present application can affect the culture environment of tumor sample, thereby helping to optimize the generation of the first immune cell population.Specifically, when the amount of the initial composition is less than that of the maximum allowable amount of the composition, the substances required for culture can be provided by simply adding the composition during culture without replacing it, thereby reducing the stress that the cultured cells undergo.In addition, the control of the amount of the composition can have a positive effect on the proliferation of cultured cells by affecting the interaction between cells.

[0172] Media, serum, plasma, antibiotics and / or IL-2 that may be included in the first composition are described below.

[0173] i) Culture medium a) Type of basal medium The term "basal medium" as used herein refers to a minimum nutrient source for culturing cells and / or tissues. As the basal medium, any medium suitable for culturing immune cells including TILs may be used.

[0174] In one embodiment, the medium may be AlyS505N medium, AIM-V medium, or RPMI1640 medium, but the present application is not limited thereto. In a specific embodiment, the tumor sample may be cultured using CTS-AIM-V medium.

[0175] In one embodiment, the medium contained in the first composition may be AlyS505N medium, AIM-V medium, or RPMI 1640 medium. In a specific embodiment, the medium contained in the first composition may be CTS-AIM-V medium.

[0176] ii) Additives a) Serum In one embodiment, serum may be added to the basic culture medium (e.g., the above-mentioned AlyS505N, AIM-V, or RPMI1640 medium). In one embodiment, the first composition of the present application may include serum. Serum contains high molecular weight proteins and low molecular weight nutrients and may contain components necessary for cell growth. Serum includes human serum, but the present application is not limited thereto. In one embodiment, serum may be human AB serum.

[0177] In one embodiment, approximately 10 vol% or less human serum may be added to the medium. In another embodiment, approximately 9 vol% or less, approximately 8 vol% or less, approximately 7 vol% or less, approximately 6 vol% or less, approximately 5 vol% or less, approximately 4 vol% or less, or approximately 3 vol% or less human serum may be added to the medium. In a particular embodiment, approximately 3 vol% human serum may be added to the CTS-AIM-V medium and used to culture tumor fragments.

[0178] In one embodiment, when the first composition contains serum, the concentration of serum contained in the first composition may be approximately 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 vol% or less. In certain embodiments, the concentration of serum contained in the first composition may be approximately 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 vol% or less. In certain embodiments, the concentration of serum contained in the first composition may be approximately 5, 4, 3, 2, or 1 vol% or less.

[0179] In one embodiment, the concentration of human serum contained in the first composition may be approximately 5, 4, 3, 2, or 1 vol%. In a particular embodiment, the concentration of human serum contained in the first composition may be approximately 3 vol%.

[0180] b) Plasma In one embodiment, plasma may be added to the basal culture medium. In one embodiment, the first composition of the present application may include plasma. Plasma refers to serum that is free of fibrinogen. Plasma may contain components necessary for cell growth.

[0181] The plasma used in this application may be human plasma or plasma isolated from a patient's blood, but the application is not limited thereto.

[0182] In one embodiment, plasma may be used in place of serum in addition to the medium.

[0183] In one embodiment, approximately 10 vol% or less of plasma may be added to the medium and used. In one embodiment, approximately 9 vol% or less, approximately 8 vol% or less, approximately 7 vol% or less, approximately 6 vol% or less, approximately 5 vol% or less, approximately 4 vol% or less, or approximately 3 vol% or less of plasma may be added to the medium and used. In a particular embodiment, approximately 3 vol% of plasma may be added to the CTS-AIM-V medium and used to culture tumor fragments.

[0184] In one embodiment, when the first composition contains plasma, the concentration of plasma contained in the first composition may be approximately 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 vol% or less. In certain embodiments, the concentration of plasma contained in the first composition may be approximately 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 vol% or less. In certain embodiments, the concentration of plasma contained in the first composition may be approximately 5, 4, 3, 2, or 1 vol% or less.

[0185] In one embodiment, the concentration of plasma contained in the first composition may be approximately 5, 4, 3, 2, or 1 vol.%. In a particular embodiment, the concentration of plasma contained in the first composition may be approximately 3 vol.%.

[0186] c) IL-2 As used herein, IL-2 (interleukin-2) refers to a factor that promotes T cell proliferation. IL-2 includes various types of IL-2, including human recombinant IL-2 type.

[0187] In one embodiment, the first composition may comprise IL-2.

[0188] In one embodiment, the concentration of IL-2 added to the medium may be approximately 10,000 IU / mL or less. In one embodiment, approximately 9,000 IU / mL, approximately 8,000 IU / mL, approximately 7,000 IU / mL, approximately 6,000 IU / mL, approximately 5,000 IU / mL, approximately 4,000 IU / mL, approximately 3,000 IU / mL, approximately 2,000 IU / mL, approximately 1,000 IU / mL, or approximately 500 IU / mL of IL-2 may be added to the medium. In a particular embodiment, approximately 1,000 IU / mL of IL-2 may be added to the CTS-AIM-V medium and used to culture the tumor fragments.

[0189] In one embodiment, the first composition is about 10,000 IU / mL, about 9,500 IU / mL, about 9,000 IU / mL, about 8,500 IU / mL, about 8,000 IU / mL, about 7,500 IU / mL, about 7,000 IU / mL, about 6,500 IU / mL, about 6,000 IU / mL, about 5,500 IU / mL, about 5,000 IU / mL, about 4,500 IU / mL, about 4,000 IU / mL, about 3,500 IU / mL, about 3,500 IU / mL, about 4,000 IU / mL, about 5,500 IU / mL, about 5,000 IU / mL, about 4,500 IU / mL, about 4,000 IU / mL, about 5,000 IU / mL, about 6,000 IU / mL, about 7,500 IU / mL, about 7,000 IU / mL, about 8,500 IU / mL, about 8,000 IU / mL, about 9,000 IU / mL, about 9,000 IU / mL, about 10,000 IU / mL, about 11,000 IU / mL, about 12,000 IU / mL, about 13,000 IU / mL, about 14,000 IU / mL, about 15,000 IU / mL, about 16,000 IU / mL, about 17,000 IU / mL, about 18,000 IU / mL, about 19,000 IU / mL, about 20,000 IU / mL, about 21,000 IU / mL, about 22,000 IU The formulation may contain approximately 1,000 IU / mL, approximately 2,500 IU / mL, approximately 2,000 IU / mL, approximately 1,500 IU / mL, approximately 1,000 IU / mL, approximately 900 IU / mL, approximately 800 IU / mL, approximately 700 IU / mL, approximately 600 IU / mL, approximately 500 IU / mL, approximately 400 IU / mL, approximately 300 IU / mL, approximately 200 IU / mL, or approximately 100 IU / mL of IL-2, or a concentration within a range defined by two values ​​selected from the aforesaid values. In certain embodiments, the first composition may comprise approximately 2,000 IU / mL, approximately 1,500 IU / mL, approximately 1,000 IU / mL, approximately 900 IU / mL, approximately 800 IU / mL, approximately 700 IU / mL, approximately 600 IU / mL, or approximately 500 IU / mL of IL-2, or a concentration within a range set by two values ​​selected from the foregoing values. In certain embodiments, the first composition may comprise approximately 1,000 IU / mL of IL-2.

[0190] d) Antibiotics In one embodiment, the first composition may further comprise an antibiotic.

[0191] Antibiotics may be used to prevent bacterial or fungal contamination during cell culture and / or growth.

[0192] Whether or not to use antibiotics in the production of the half-product may be determined by appropriately considering the type of tumor of the tumor tissue obtained from the subject, the condition (state) of the cultured tumor sample, and the condition of the cultured and / or expanded cells. For example, if the tumor sample is derived from breast cancer of the subject, the first composition added initially may not contain antibiotics. In another example, if the tumor sample is derived from breast cancer of the subject, a first composition containing antibiotics may be used depending on the condition of the tumor sample. In yet another example, if the tumor sample is derived from head and neck cancer of the subject, a first composition containing antibiotics may be used.

[0193] When the first composition contains an antibiotic, the antibiotic may be contained at a concentration that does not affect cell culture and / or growth, where the concentration of the antibiotic that shows cytotoxicity may be taken into consideration. The appropriate concentration of the antibiotic contained in the first composition may vary depending on the type of antibiotic.

[0194] Examples of antibiotics contained in the first composition include ampicillin, gentamicin, kanamycin, neomycin, penicillin, and streptomycin, but the present application is not limited thereto. That is, any antibiotic known in the art may be used as an antibiotic to be added to a medium for cell culture and / or growth, and may be used after being appropriately selected by a person skilled in the art as necessary. In a specific embodiment, the first composition may contain gentamicin.

[0195] e) Antibodies As described above, in one embodiment of the present application, the first composition may further comprise an antibody. When the first composition comprises an antibody, the antibody comprised in the first composition may be any one or more selected from an antibody targeting CD137 (4-1BB) and an antibody blocking the binding of PD-1 and PD-L1. In one embodiment, the antibody comprised in the first composition may be any one or more selected from an anti-CD137 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody.

[0196] The anti-CD137 antibody is an antibody that targets CD137 (4-1BB). In one embodiment, the anti-CD137 antibody includes any type of monoclonal or polyclonal anti-CD137 antibody, such as a human, humanized or murine antibody. In one embodiment, when the first composition includes an anti-CD137 antibody, the anti-CD137 antibody may be any one or more selected from urelumab, utomirumab, and variants thereof, but the present application is not limited thereto.

[0197] An example of an antibody that inhibits the binding of PD-1 and PD-L1 may include an anti-PD-1 antibody or an anti-PD-L1 antibody. In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody includes any type of monoclonal or polyclonal anti-PD-1 antibody or PD-L1 antibody, including human, humanized, or murine antibodies. In one embodiment, when the first composition includes an anti-PD-1 antibody, the anti-PD-1 antibody may be any one or more selected from nivolumab, pembrolizumab, cemiplimab, dostarlimab, and variants thereof, but the application is not limited thereto. In one embodiment, when the first composition includes an anti-PD-L1 antibody, the anti-PD-L1 antibody may be any one or more selected from atezolizumab, avelumab, durvalumab, and variants thereof, but the application is not limited thereto.

[0198] In one embodiment, when the first composition comprises an antibody, the concentration of the antibody contained in the first composition may be approximately 1 ng / mL, approximately 10 ng / mL, approximately 100 ng / mL, approximately 1 μg / mL, approximately 2 μg / mL, approximately 3 μg / mL, approximately 4 μg / mL, approximately 5 μg / mL, approximately 10 μg / mL, approximately 15 μg / mL, approximately 20 μg / mL, approximately 25 μg / mL, approximately 30 μg / mL, approximately 35 μg / mL, approximately 40 μg / mL, approximately 45 μg / mL, approximately 50 μg / mL, approximately 60 μg / mL, approximately 70 μg / mL, approximately 80 μg / mL, approximately 90 μg / mL, or approximately 100 μg / mL, or a value included in a range set by two values ​​selected from the aforementioned values. In certain embodiments, the concentration of the antibody contained in the first composition may be approximately 10 μg / mL.

[0199] As described above, the first composition of the present application comprises a medium and an additive.

[0200] In one embodiment, the first composition may include medium, serum, and IL-2. In another embodiment, the first composition may include medium, plasma, and IL-2. In yet another embodiment, the first composition may include medium, serum, IL-2, and an antibiotic. In yet another embodiment, the first composition may include medium, plasma, IL-2, and an antibiotic. However, the present application is not limited thereto.

[0201] In a specific embodiment, the first composition may include a medium, serum, and IL-2, the medium may be a CTS-AIM-V medium, the serum may be approximately 3 vol% human AB serum, and approximately 1,000 IU / mL IL-2 may be included in the first composition. In a specific embodiment, the first composition may include a medium, serum, IL-2, and an antibiotic. Here, in the first composition, the medium may be a CTS-AIM-V medium, the antibiotic may be gentamicin, the serum may be approximately 3 vol% human AB serum, and approximately 1,000 IU / mL IL-2 may be included.

[0202] 4)Culture period The tumor sample is cultured in the first container for a predetermined period of time, hi one embodiment, the tumor sample is cultured for approximately one day or more. In one embodiment, the tumor sample may be cultured for approximately 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, 7 or more days, 8 or more days, 9 or more days, 10 or more days, 11 or more days, 12 or more days, 13 or more days, 14 or more days, 15 or more days, 16 or more days, 17 or more days, 18 or more days, 19 or more days, 20 or more days, 21 or more days, 22 or more days, 23 or more days, or 24 or more days. In certain embodiments, the tumor sample may be cultured in the first container for approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In certain embodiments, the tumor sample may be cultured in the first container for approximately 14 days.

[0203] 5) Addition of the first additive composition While the tumor sample is cultured in the first container, a first additive composition may be added to the first container, where the first additive composition may include any one or more of medium, serum, plasma, antibiotics, and IL-2. The composition of the first additive composition is disclosed in detail in the relevant paragraph.

[0204] In one embodiment, the first additive composition may be added 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times, hi a particular embodiment, the first additive composition may be added 3 times.

[0205] Here, in one embodiment, the first additive composition is added on any one or more days selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 days after the start of the culture, and may be added 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times during the culture period. In a specific embodiment, the first additive composition may be added three times on the 7th, 10th, and 12th days.

[0206] In the process of adding the first additive composition, the first additive composition may be added by a method to minimize the stress of the environment in which the tumor sample is cultured.

[0207] In the process of adding the first additive composition, the amount of the first additive composition added can vary depending on the type of container and / or the addition time.

[0208] In one embodiment, the first additive composition may be added in the same amount at each addition time. In another embodiment, the first additive composition may be added in a different amount at each addition time. In another embodiment, the first additive composition may be added by increasing its amount at each addition time.

[0209] In one embodiment, the first additive composition may be added in the same or different amounts of approximately 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 12 mL, 14 mL, 16 mL, 18 mL, 20 mL, 24 mL, 28 mL, 32 mL, 36 mL, 40 mL, 45 mL, or 50 mL, or may be added in an amount greater than or equal to the aforementioned amount at each addition time, but the present application is not limited thereto. The amount of the first additive composition to be added may be determined as an appropriate value taking into consideration various conditions (e.g., the volume of the container, the condition of the cultured cells, and / or the addition time). In a specific embodiment, the first additive composition may be added in the same amount of 3 mL at each addition time. In a specific embodiment, when the first container is a T75 flask, the first additive composition may be added in the same amount of 3 mL at each addition time.

[0210] In certain embodiments, the first additive composition may be added to the first container on days 7, 10, and 12 from the initiation of the culture, and 3 mL of the first additive composition may be added at each addition time.

[0211] In one embodiment, the first additive composition added to the first container may comprise any one or more selected from culture medium, serum, plasma, antibiotics, IL-2, and antibodies.

[0212] In one embodiment, the first additive composition may comprise medium, serum, and IL-2. In another embodiment, the first additive composition may comprise medium, plasma, and IL-2.

[0213] In one embodiment, the first additive composition may comprise culture medium; any one of serum and plasma; and IL-2, and further comprises any one or more of an antibody and an antibiotic.

[0214] In certain embodiments, the first additive composition added comprises medium, serum, and IL-2. In certain embodiments, the first additive composition comprises medium, serum, IL-2, and an antibiotic. In another specific embodiment, the first additive composition comprises medium, serum, IL-2, and an antibody. In certain embodiments, the first additive composition comprises medium, serum, IL-2, an antibiotic, and an antibody.

[0215] The embodiments regarding the components contained in the first additive composition are the same as those in the description of the first composition.

[0216] a) Culture medium The embodiment of the medium that can be included in the first additive composition is as described above in the first composition. For example, in a specific embodiment, the medium included in the first additive composition can be AIM-V medium, but the present application is not limited thereto. In a specific embodiment, the medium included in the first additive composition can be CTS-AIM-V medium.

[0217] b) Serum When the first additive composition contains serum, the embodiment of serum is as described above in the first composition.For example, in certain embodiments, serum can be human serum, but the application is not limited thereto.For example, in certain embodiments, the concentration of serum contained in the first additive composition can be approximately 3vol%, but the application is not limited thereto.

[0218] c) Plasma When the first additive composition contains plasma, the embodiment related to the plasma is as described above in the first composition.For example, in a specific embodiment, the plasma may be human plasma, but the present application is not limited thereto.For example, in a specific embodiment, the concentration of the plasma contained in the first additive composition may be approximately 3 vol%, but the present application is not limited thereto.

[0219] d) Antibiotics When the first additive composition contains an antibody, the embodiment of the antibiotic is as described above in the first composition. As described above, whether or not to use an antibiotic may be determined by appropriately considering the type of tumor obtained from the subject, the condition of the cultured tumor sample, and the condition of the cultured and / or expanded cells. For example, in certain embodiments, the antibiotic contained in the first additive composition may be gentamicin.

[0220] e) IL-2 When the first additive composition includes IL-2, the embodiments related to IL-2 are as described above for the first composition. For example, in certain embodiments, the concentration of IL-2 included in the first additive composition may be 1,000 IU / mL.

[0221] f) Antibodies When the first additive composition includes an antibody, the embodiments related to the antibody are as described above for the first additive composition. For example, the antibody included in the first additive composition may be any one or more selected from an anti-CD137 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody.

[0222] In one embodiment, the composition of the first additive composition added to the first container may be different for each addition time, while in another embodiment, the composition of the first additive composition added to the first container may be the same for all addition times.

[0223] In one embodiment, the makeup of the first additive composition added to the first container can be the same as that of the first composition, hi another embodiment, the makeup of the first additive composition added to the first container can be different from that of the first composition.

[0224] In certain embodiments, the first additive composition added to the first container may include medium, serum, and IL-2. In certain embodiments, the first additive composition added to the first container may include CTS-AIM-V medium, approximately 3 vol% human serum, and approximately 1,000 IU / mL IL-2.

[0225] In one embodiment, immune cells are not removed from the first container or added from outside during the culture, meaning that immune cells are not transplanted into a container other than the first container or new immune cells are not added from outside to the first container during the culture.

[0226] Specific examples regarding the generation of the first immune cell population may be described below, but the present application is not limited thereto. - 1~4mm 2 24 pieces of tumor specimens with a cross-sectional area of ​​100 μg / ml are cultured in a T75 flask, and 6 mL of the first composition is used at the start of the culture, where the first composition includes CTS-AIM-V medium, 3 vol% human serum, and 1,000 IU / mL IL-2. The tumor specimens are cultured in the first container at 37° C. in a 5% CO2 environment for 14 days. - on days 7, 10, and 12 from the start of the culture, a first additive composition having the same composition as the first composition is added to the first container, where 3 mL of the first additive composition is added on days 7, 10, and 12, respectively.

[0227] 3. Obtaining the First Immune Cell Population Overview The first immune cell population is obtained from a culture product obtained through culturing the tumor sample described above.

[0228] Obtaining the first immune cell population The immune cell population refers to a group that substantially includes a plurality of immune cells, and the immune cells include TILs. Here, the first immune cell population refers to an immune cell population obtained by culturing a tumor sample.

[0229] The method of obtaining the first immune cell population includes, but is not limited to, various known methods for obtaining cells generated in the process of culturing cells using tissue. In one embodiment, the first immune cell population may be obtained through filtering tumor tissue, washing, processing, isolating cells, and / or separating cells from a composition cultured by the process of culturing tumor samples. The first immune cell population may be obtained by methods known in the art.

[0230] Disclosed herein are exemplary processes that may be included in obtaining the first immune cell population.

[0231] 1) Tumor tissue filtering (straining) The acquisition of the first immune cell population may include filtering the tumor tissue fragment. In one embodiment, the filtering of the tumor tissue fragment may be performed using a cell strainer to obtain the first immune cell population from the cultured composition. Here, the size of the mesh pore of the cell strainer may vary depending on the type of immune cell obtained. In a particular embodiment, the tumor tissue fragment may be filtered using a cell strainer with a mesh pore of 40 μm.

[0232] 2) Cell collection Obtaining the first immune cell population may include obtaining or harvesting cells. Methods known in the art may be used to obtain or harvest cells from a cultured composition or filtered cell solution. In one embodiment, centrifugation may be used to obtain or harvest the first immune cell population. In one example, centrifugation may be performed under conditions of approximately 100g, 150g, 200g, 250g, 300g, 400g, 450g, 500g, 600g, 700g, 800g, 900g, or 1,000g, or a g-value greater than or equal to the aforementioned g-values, but the present application is not limited thereto. In another example, centrifugation may be performed under conditions of approximately 100 RPM, 200 RPM, 300 RPM, 400 RPM, 500 RPM, 600 RPM, 700 RPM, 800 RPM, 900 RPM, 1000 RPM, 1100 RPM, 1200 RPM, 1300 RPM, 1400 RPM, 1500 RPM, 1600 RPM, 1700 RPM, 1800 RPM, 1900 RPM, 2000 RPM, 2200 RPM, 2500 RPM, 3000 RPM, 3500 RPM, 4000 RPM, or 5000 RPM, or RPM values ​​greater than or equal to the aforementioned RPM values, but the present application is not limited thereto.

[0233] 3) Cell sorting Obtaining the first immune cell population may include sorting of cells contained in the cultured composition.

[0234] Cell sorting refers to the method used to "select desired cells" from the above-mentioned isolated cell population. For cell sorting, any method conventionally used in the art may be used. For example, methods such as fluorescence-activated cell sorting (FACS), centrifugation, or isolation by cell adhesion to a container may be used for cell sorting.

[0235] 4) Cell washing Obtaining the first immune cell population may include washing the immune cell population contained in the cultured composition. The cell washing process described in this paragraph may be performed any number of times during the stage of obtaining the first immune cell population. For example, cell washing may be performed during the process included in obtaining the first immune cell population. In another example, cell washing may be performed on a cell population that has been filtered, harvested, and / or sorted as described above. In yet another example, cell washing may be performed simultaneously with cell harvesting.

[0236] In one embodiment, the cultured immune cell population may be washed with a buffer solution including PBS or DPBS. The cultured immune cell population may be washed 1, 2, 3, 4 or 5 times or more. Here, for example, centrifugation may be used. In a particular embodiment, the cultured immune cell population may be washed 3 times with DPBS to obtain a first immune cell population.

[0237] In one embodiment, obtaining the first immune cell population may comprise cell harvesting, wherein obtaining the first immune cell population may further comprise cell washing, which may be performed any suitable number of times at any suitable time.

[0238] In one embodiment, obtaining the first immune cell population may include filtering the tumor tissue; and harvesting the cells, wherein obtaining the first immune cell population may further include washing the cells, and the washing may be performed any suitable number of times at any suitable time.

[0239] In another embodiment, obtaining the first immune cell population may comprise filtering the tumor tissue; harvesting cells; and sorting the cells, wherein obtaining the first immune cell population may further comprise washing the cells, which may be performed any suitable number of times at any suitable time.

[0240] Number of cells in the first immune cell population obtained Through obtaining the first immune cell population, a predetermined number or more of cells are obtained. In one embodiment, for each tumor sample cultured to generate the first immune cell population, approximately 1E4, approximately 2E4, approximately 3E4, approximately 4E4, approximately 5E4, approximately 6E4, approximately 7E4, approximately 8E4, approximately 9E4, approximately 1E5, approximately 2E5, approximately 3E5, approximately 4E5, approximately 5E5, approximately 6E5, approximately 7E5, approximately 8E5, approximately 9E5, approximately 1E6, approximately 2E6, approximately 3E6, approximately 4E6, or approximately 5E6 or more cells may be obtained. In certain embodiments, for one tumor sample cultured to generate the first immune cell population, approximately 1E5, approximately 2E5, approximately 3E5, approximately 4E5, approximately 5E5, approximately 6E5, approximately 7E5, approximately 8E5, approximately 9E5, or approximately 1E6 cells may be obtained. In one example, a tumor sample cultured to generate the first immune cell population may be approximately 1 to 4 mm 2 For tumor samples having a cross-sectional area of ​​approximately 8E5 or more cells may be obtained for each tumor sample. The number of cells obtained through culture to generate the first immune cell population may vary depending on the type of tumor and the size of the tumor sample, and is not limited to the above embodiment.

[0241] II. Cryopreservation Overview The methods for producing immune cell compositions disclosed herein may optionally include cryopreservation of the results of each treatment between each treatment step included in the method.

[0242] In one example, after obtaining the first immune cell population, the method may optionally include cryopreserving the first immune cell population or the half-product. In cryopreserving the first immune cell population or the half-product, the first immune cell population or the half-product is included in a cryopreservation composition and cryopreserved. The cryopreservation of the first immune cell population is described in detail below.

[0243] Media for cryopreservation As described above, the first immune cell population is contained in the cryopreservation composition and cryopreserved.The cryopreservation composition has a composition suitable for cryopreservation.The medium suitable for cryopreservation can be contained therein.The medium suitable for cryopreservation includes all types of medium for cryopreservation known in the art.

[0244] In one embodiment, the cryopreservation composition may contain DMSO at a concentration of approximately 0.1%, approximately 0.5%, approximately 1%, approximately 3%, approximately 5%, approximately 8%, approximately 10%, approximately 13%, or approximately 15%, or at a concentration higher than the aforementioned percentage values, but the present application is not limited thereto. In a specific embodiment, the cryopreservation composition may contain DMSO at a concentration of 10%. For example, CryoStor® CS10 containing 10% DMSO may be used.

[0245] Cell concentration and number of cryopreserved cells The first immune cell population may be included in a cryopreservation composition at a particular concentration and cryopreserved.

[0246] In one embodiment, the first immune cell population may be included in the cryopreservation composition at a concentration of approximately 1E5 cells / mL, approximately 1E6 cells / mL, approximately 2E6 cells / mL, approximately 3E6 cells / mL, approximately 4E6 cells / mL, approximately 5E6 cells / mL, approximately 6E6 cells / mL, approximately 7E6 cells / mL, approximately 8E6 cells / mL, approximately 9E6 cells / mL, or approximately 1E7 cells / mL, but this application is not limited thereto. The first immune cell population may be included in the cryopreservation composition at a concentration suitable for cryopreservation. In a specific embodiment, the first immune cell population may be included in the cryopreservation composition at a concentration of 2E6 cells / mL.

[0247] In one embodiment, approximately 0.1 mL, approximately 0.2 mL, approximately 0.3 mL, approximately 0.4 mL, approximately 0.5 mL, approximately 0.7 mL, approximately 1 mL, approximately 2 mL, approximately 3 mL, approximately 4 mL, approximately 5 mL, approximately 7 mL, or approximately 10 mL, or an amount greater than or equal to the aforementioned mL amounts, of the cryopreservation composition may be placed in a cryovial and cryopreserved. In a particular embodiment, approximately 1 mL of the cryopreservation composition may be placed in a cryovial and cryopreserved.

[0248] For example, a first immune cell population may be suspended in the cryopreservation composition at a concentration of approximately 2E6 cells / mL and approximately 1 mL of the cryopreservation composition may be placed into a cryovial and frozen.

[0249] Frozen storage period The first immune cell population may be cryopreserved for a period of time and then used through expansion to generate an immune cell composition. The cryopreservation period is not limited. In one embodiment, the first immune cell population may be cryopreserved for a period of at most about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, about 18 years, about 19 years, or about 20 years, or a period longer than or equal to the aforementioned periods. For example, the first immune cell population may be cryopreserved for 1 hour and thawed before being used to generate an immune cell composition. In another example, the first immune cell population may be cryopreserved for 1 day and then thawed before being used to generate an immune cell composition. In yet another example, after being cryopreserved for 10 years, the first immune cell population may be thawed and used to generate an immune cell composition.

[0250] Frozen storage temperature The first immune cell population is frozen at a temperature suitable for long-term storage. In one embodiment, the first immune cell population may be frozen at approximately -40°C or lower, -60°C or lower, -80°C or lower, approximately -100°C or lower, approximately -120°C or lower, approximately -140°C or lower, approximately -160°C or lower, approximately -180°C or lower, or approximately -190°C or lower. In a particular embodiment, the first immune cell population may be frozen at approximately -190°C or lower. For example, the first immune cell population may be frozen at approximately -190°C or lower in liquid nitrogen (LN2) or gaseous liquid nitrogen (LN2).

[0251] III. Thawing of cryopreserved first immune cell population Overview In the methods for producing immune cell compositions disclosed herein, a step of cryopreserving the results of each process may optionally be included between each process included in the method, and if such a cryopreservation process is performed, a step of thawing the cryopreserved results of each process may further be included after the cryopreservation step.

[0252] In one example, a cryopreserved first immune cell population may be thawed and used to generate an immune cell composition.

[0253] How to unzip Thawing may be performed through methods known in the art for thawing cryopreserved cells. In one embodiment, the cryopreserved first immune cell population may be thawed using a thermostat (water bath). In another embodiment, the cryopreserved first immune cell population may be thawed using a thawing device. Here, the thawing device may be a device for thawing cells by a dry thawing method. The dry thawing method may prevent contamination of cells. Here, the dry thawing method includes both anhydrous methods and methods in which water is present in a form blocked from the thawed subject (e.g., in the form of a water-containing pack), but the present application is not limited thereto. For example, the cryopreserved first immune cell population may be thawed in a thermostat at approximately 37° C. for approximately 2 minutes. In another example, the cryopreserved first immune cell population may be thawed using a thawing device including a water-containing pack for approximately 7 minutes at approximately 37° C.

[0254] As described above, the method for producing an immune (cell) composition disclosed in the present application comprises the steps of producing a final product by using a half-product comprising a first immune cell population immediately after producing the half-product comprising a first immune cell population; or optionally storing the half-product frozen for a predetermined period of time and thawing it to produce the final product.

[0255] IV. PRODUCTION OF AN END PRODUCT The methods for producing immune cell compositions disclosed herein may include producing a semi-product and / or producing an end product, where end product refers to an immune cell composition produced by obtaining a third immune cell population expanded from a second immune cell population, the second immune cell population being derived from a first immune cell population or from the first immune cell population that has been thawed following cryopreservation.

[0256] In one embodiment, the production of the immune cell composition or the production of the end product comprises: seeding a second immune cell population; Expanding the second immune cell population; and Producing an immune cell composition may include:

[0257] Below, each step involved in producing the final product is disclosed in detail.

[0258] 1. Seeding of the Second Immune Cell Population Overview A second immune cell population derived from the first immune cell population or the first immune cell population that has been thawed following cryopreservation is suspended in a second composition and seeded into a second container.

[0259] Seeding refers to dispensing cells into a container, and means dispensing a second immune cell population into a second container. Here, an appropriate number of cells for proliferation can be distributed according to the cell culture area or volume of the container. In one embodiment, the second immune cell population is suspended in a second composition and seeded into the second container in an appropriate amount.

[0260] In one embodiment, the second immune cell population may be suspended in the second composition and the cells may be seeded into the second container at approximately 1E3-1E4, approximately 1E4-1E5, approximately 1E5-1E6, or approximately 1E6-1E7 cell numbers. In a particular embodiment, the second immune cell population may be suspended in the second composition and the cells may be seeded into the second container at approximately 1E5, 2E5, 3E5, 4E5, 5E5, 6E5, 7E5, 8E5, 9E5, 1E6, 1.1E6, 1.2E6, 1.3E6, 1.4E6, or 1.5E6 cell numbers, or at a cell number within a range set by two values ​​selected from the aforementioned values. In certain embodiments, the second immune cell population may be suspended in a second composition and the cells may be seeded into a second container at a cell number of 4E5.

[0261] Second Immune Cell Population At this stage, the second immune cell population seeded in the second container is derived from the first immune cell population described above or the first immune cell population thawed after cryopreservation, i.e., the second immune cell population may be obtained from the first immune cell population described above or the first immune cell population thawed after cryopreservation.

[0262] In one embodiment, the method of obtaining a second immune cell population from the first immune cell population or a first immune cell population that has been thawed after cryopreservation may include various known methods for obtaining cells in a cell culture process, but the present application is not limited thereto.

[0263] For example, the method of obtaining a second immune cell population from the first immune cell population or a first immune cell population that has been thawed after cryopreservation may comprise any one or more of the following steps: Obtaining the second immune cell population may further include, but is not limited to, checking cell viability if necessary.

[0264] 1) Cell washing Obtaining the second immune cell population may include washing the cell population. In one embodiment, the second immune cell population may be obtained by washing the first immune cell population or the thawed first immune cell population. For example, centrifugation may be used for cell washing. In one embodiment, the first immune cell population or the thawed first immune cell population may be washed with a buffer such as DPBS or PBS, or washed with a medium.

[0265] 2) Centrifugation In one embodiment, the method of centrifuging the first immune cell population or the thawed first immune cell population may be used to obtain the second immune cell population. In one embodiment, the centrifugation may be performed on the washed immune cell population. One embodiment of the centrifugation method is the same as described in the relevant paragraph.

[0266] 3) Cell counting, division, or binding In one embodiment, cell counting can be performed on the first immune cell population to obtain the second immune cell population.For example, the second immune cell population can be obtained by counting the cell number of the first immune cell population after cell washing, and then dividing the immune cell population to have an appropriate cell number for seeding as described below.Furthermore, the second immune cell population can be obtained by combining the immune cell populations cultured in different first containers.

[0267] In one embodiment, obtaining the second immune cell population may include cell washing.

[0268] In one embodiment, obtaining the second immune cell population may include cell washing and cell counting, where the immune cell population that has undergone cell counting may be split to have an appropriate cell number for seeding.

[0269] Second container The second immune cell population may be seeded in a second container, where the second container refers to a container used to grow the second immune cell population. The second container is used to grow the second immune cell population together with a second composition comprising medium, serum, plasma, IL-2, anti-CD-3 antibody, feeder cells, and / or antibiotics. The second container is a gas-permeable container through which gas can pass, or a container having a gas supply system to provide gas. That is, the container may be a container manufactured to allow gas to pass through the container, or a container that is not manufactured to allow gas to pass through but has a separate gas supply system that allows gas to pass through.

[0270] In one embodiment, the second container is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350, 400, 450, 500, 550, 600, 700, The second container may be a container having a maximum allowable volume of 800, 900, 1000, 1100, 1200, 1300, 1400, 1600, 1800, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, or 10000 mL, or a container having a maximum allowable volume within a range set by two values ​​selected from the aforementioned values. In certain embodiments, the second container may be a container having a maximum allowable volume of approximately 500, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1600, 1800, or 2000 mL, or a container having a maximum allowable volume within a range set by two values ​​selected from the aforementioned values.

[0271] In one embodiment, the second container may be, for example, a 24-well plate, a T25, T75, or T125 flask, a flask such as a GREX10, GREX10M, GREX10M-CS, GREX100, GREX100M, GREX100M-CS, GREX500, GREX500M, or GREX500M-CS flask, or a culture bag, but the present application is not limited thereto. In a particular embodiment, the second container may be a GREX100M.

[0272] Second Composition A second immune cell population is suspended in a second composition and seeded into a second container, where the second composition comprises any one or more selected from medium, serum, plasma, IL-2, anti-CD3 antibody, feeder cells, and antibiotics.

[0273] In one embodiment, the second composition may comprise culture medium; serum; and IL-2. In another embodiment, the second composition may comprise culture medium; plasma; and IL-2. In one embodiment, the second composition may further comprise, in addition to the above-mentioned substances, an antibody (e.g., any one or more selected from an anti-CD137 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody) listed in the content table for the first composition of the present application, and the antibody embodiments are the same as those described in the relevant paragraphs.

[0274] In a specific embodiment, the second composition comprises medium, serum, IL-2, an anti-CD3 antibody, and feeder cells. In another specific embodiment, the second composition comprises medium, plasma, IL-2, an anti-CD3 antibody, and feeder cells. In yet another specific embodiment, the second composition comprises medium, one of serum and plasma, IL-2, an anti-CD3 antibody, an antibiotic, and feeder cells.

[0275] Generally, in the culture and / or growth of tissue or cells, the volume of the composition used in the container (e.g., flask) may vary depending on the type of container. For example, generally, for GREX10, 40 mL of the composition is used, for GREX100, 400 mL of the composition is used, and for GREX100M, 1,000 mL of the composition is used.

[0276] However, the amount of the composition initially added to the second container to expand the second immune cell population of the present application may differ from the amounts commonly used described above.

[0277] In one embodiment, the amount of the second composition initially added to the second container may be approximately 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5% or less of the maximum allowable capacity of the second container. In certain embodiments, the amount of the second composition initially added to the second container may be approximately 30% or less of the maximum allowable capacity of the second container. In certain embodiments, the amount of the second composition initially added to the second container may be approximately 10% or less of the maximum allowable capacity of the second container. Here, the maximum allowable capacity refers to the maximum amount that can be added to the second container.

[0278] In one embodiment, when the second container is GREX100M, the amount of the second composition added initially may be 200mL, 150mL, 100mL, 80mL, 60mL, 40mL, 20mL, or 10mL, or less. In a particular embodiment, when the second container is GREX100M, the amount of the second composition added initially may be approximately 20mL.

[0279] 1) Culture medium The medium used in this application means a nutrient source for culturing cells and / or tissues. As the medium, any medium suitable for culturing immune cells including TILs may be used.

[0280] In one embodiment, the medium may be AlyS505N medium, AIM-V medium, or RPMI medium, but the present application is not limited thereto. In a specific embodiment, CTS-AIM-V medium may be used.

[0281] In one embodiment, the medium contained in the second composition may be AlyS505N medium, AIM-V medium, or RPMI medium. In a specific embodiment, the medium contained in the second composition may be CTS-AIM-V medium.

[0282] Serum, plasma, IL-2, anti-CD-3 antibody, feeder cells, and / or antibiotics may be added to the medium and used in the proliferation of immune cells, i.e., the second composition may further comprise serum, plasma, IL-2, anti-CD-3 antibody, feeder cells, and / or antibiotics in addition to the medium.

[0283] 2) Additives a) Serum In one embodiment, serum may be added to a basic culture medium (e.g., the above-mentioned AlyS505N medium, AIM-V medium, or RPMI1640 medium). In one embodiment, the second composition of the present application may include serum. Serum contains high molecular weight proteins and low molecular weight nutrients, and may contain components necessary for cell growth. The serum may include human serum, but the present application is not limited thereto.

[0284] In one embodiment, human serum may be used by adding to the medium at a concentration of approximately 10 vol% or less. In another embodiment, human serum may be used by adding to the medium at a concentration of approximately 9 vol% or less, approximately 8 vol% or less, approximately 7 vol% or less, approximately 6 vol% or less, approximately 5 vol% or less, approximately 4 vol% or less, or approximately 3 vol% or less. In a particular embodiment, human serum may be used by adding to the CTS-AIM-V medium at approximately 3 vol%.

[0285] In one embodiment, when serum is included in the second composition, the serum concentration in the second composition may be approximately 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 vol.%, or less than or equal to the aforementioned vol.%. In certain embodiments, the serum concentration in the second composition may be approximately 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 vol.%, or less than or equal to the aforementioned vol.%. In certain embodiments, the serum concentration in the second composition may be approximately 5, 4, 3, 2, or 1 vol.%, or less than or equal to the aforementioned vol.%.

[0286] In one embodiment, the concentration of human serum contained in the second composition may be approximately 5, 4, 3, 2 or 1 vol%. In a particular embodiment, the concentration of human serum contained in the second composition may be approximately 3 vol%.

[0287] b) Plasma In one embodiment, plasma may be added to the basal medium. In one embodiment, the second composition of the present application may include plasma. Plasma refers to fibrinogen-free serum. Plasma contains the components necessary for cell growth.

[0288] The plasma used in this application may be human plasma or plasma isolated from a patient's blood, but the application is not limited thereto.

[0289] In one embodiment, plasma may be used in place of serum in addition to the medium.

[0290] In one embodiment, plasma may be used by adding to the medium at a concentration of approximately 10 vol% or less. In another embodiment, plasma may be used by adding to the medium at a concentration of approximately 9 vol% or less, approximately 8 vol% or less, approximately 7 vol% or less, approximately 6 vol% or less, approximately 5 vol% or less, approximately 4 vol% or less, or approximately 3 vol% or less. In a particular embodiment, plasma may be added to the CTS-AIM-V medium at approximately 3 vol% and used.

[0291] In one embodiment, when the second composition contains plasma, the concentration of plasma contained in the second composition may be approximately 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 vol.%, or less than or equal to the aforementioned vol.%. In certain embodiments, the concentration of plasma contained in the second composition may be approximately 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 vol.%, or less than or equal to the aforementioned vol.%. In certain embodiments, the concentration of plasma contained in the second composition may be approximately 5, 4, 3, 2, or 1 vol.%, or less than or equal to the aforementioned vol.%.

[0292] In one embodiment, the concentration of plasma contained in the second composition may be approximately 5, 4, 3, 2 or 1 vol.%. In a particular embodiment, the concentration of plasma contained in the second composition may be approximately 3 vol.%.

[0293] c) IL-2 As used herein, IL-2 is an abbreviation for interleukin-2, which refers to a factor that promotes T cell proliferation. IL-2 includes various types of IL-2, including human recombinant IL-2 types.

[0294] In one embodiment, the second composition may comprise IL-2.

[0295] In one embodiment, IL-2 may be added to the medium at a concentration of approximately 10,000 IU / mL. In one embodiment, the concentration of IL-2 added to the medium may be approximately 9000 IU / mL, approximately 8,000 IU / mL, approximately 7,000 IU / mL, approximately 6,000 IU / mL, approximately 5,000 IU / mL, approximately 4,000 IU / mL, approximately 3,000 IU / mL, approximately 2,000 IU / mL, approximately 1,000 IU / mL, or approximately 500 IU / mL. In a particular embodiment, IL-2 may be added to the CTS-AIM-V medium at a concentration of approximately 2,000 IU / mL.

[0296] In one embodiment, the second composition is about 10,000 IU / mL, about 9,500 IU / mL, about 9,000 IU / mL, about 8,500 IU / mL, about 8,000 IU / mL, about 7,500 IU / mL, about 7,000 IU / mL, about 6,500 IU / mL, about 6,000 IU / mL, about 5,500 IU / mL, about 5,000 IU / mL, about 4,500 IU / mL, about 4,000 IU / mL, about 3,500 IU / mL, about 3,500 IU / mL, about 4,000 IU / mL, about 5,000 IU / mL, about 6,500 IU / mL, about 6,000 IU / mL, about 5,500 IU / mL, about 5,000 IU / mL, about 4,500 IU / mL, about 4,000 IU / mL, about 3,500 IU / mL, about 3,500 IU / mL, about 4,000 IU / mL, about 4,500 IU / mL, about 5,000 IU / mL, about 5,000 IU / mL, about 6,000 IU / mL, about 7,500 IU / mL, about 7,000 IU / mL, about 8,500 IU / mL, about 8,000 IU / mL, about 9,000 IU / mL, about 9,000 IU / mL, about 10 ... The formulation may contain IL-2 at a concentration of approximately 1,000 IU / mL, approximately 2,500 IU / mL, approximately 2,000 IU / mL, approximately 1,500 IU / mL, approximately 1,000 IU / mL, approximately 900 IU / mL, approximately 800 IU / mL, approximately 700 IU / mL, approximately 600 IU / mL, approximately 500 IU / mL, approximately 400 IU / mL, approximately 300 IU / mL, approximately 200 IU / mL, or approximately 100 IU / mL, or at a concentration within a range set by two values ​​selected from the aforementioned values. In certain embodiments, the second composition may comprise IL-2 at a concentration of approximately 3,000 IU / mL, approximately 2,500 IU / mL, approximately 2,000 IU / mL, approximately 1,500 IU / mL, or approximately 1,000 IU / mL, or a concentration within a range set by any two values ​​selected from the foregoing values. In certain embodiments, the second composition may comprise IL-2 at a concentration of approximately 2,000 IU / mL.

[0297] d) Anti-CD3 antibody Anti-CD3 antibody refers to an antibody that targets a membrane protein on the surface of T cells, namely the CD3 receptor.

[0298] In one embodiment, the second composition may comprise an anti-CD3 antibody. In one embodiment, the anti-CD3 antibody may be any one selected from OKT3, otelixizumab, teplizumab, and visilizumab, or a combination of two or more thereof. In a particular embodiment, the anti-CD3 antibody included in the second composition may be OKT3.

[0299] In one embodiment, the anti-CD3 antibody may be used while added to the culture medium at a concentration of approximately 1 ng / mL, approximately 2 ng / mL, approximately 3 ng / mL, approximately 4 ng / mL, approximately 5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 45 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, approximately 100 ng / mL, approximately 200 ng / mL, approximately 300 ng / mL, approximately 400 ng / mL, approximately 500 ng / mL, or approximately 1,000 ng / mL, or a concentration within a range set by two values ​​selected from the aforementioned values.

[0300] In one embodiment, when an anti-CD3 antibody is included in the second composition, the concentration of the anti-CD3 antibody included in the second composition may be approximately 1 ng / mL, approximately 2 ng / mL, approximately 3 ng / mL, approximately 4 ng / mL, approximately 5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 45 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, approximately 100 ng / mL, approximately 200 ng / mL, approximately 300 ng / mL, approximately 400 ng / mL, approximately 500 ng / mL, or approximately 1000 ng / mL, or a concentration within a range set by two values ​​selected from the aforesaid values. In certain embodiments, the concentration of anti-CD3 antibody in the second composition may be approximately 30 ng / mL.

[0301] e) Feeder cells i) Feeder cell percentage The feeder cells used in the present application are cells that are included in the second composition and help activate and expand TILs when expanding the second immune cell population. In one embodiment, the feeder cells include peripheral blood mononuclear cells (PBMCs), where PBMCs refer to peripheral blood cells with round cell nuclei.

[0302] In one embodiment, the feeder cells may be peripheral blood mononuclear cells (PBMCs). In one embodiment, the feeder cells may be inactivated PBMCs. In one embodiment, the feeder cells may be allogeneic PBMCs. In one embodiment, the feeder cells may be inactivated allogeneic PBMCs. Here, the PBMCs may be inactivated by irradiation, and the method of inactivating the PBMCs is not limited thereto. For example, the PBMCs may be inactivated through irradiation of approximately 60 Gy.

[0303] The feeder cells may be added to the second composition at a predetermined ratio to the second immune cell population (TIL). In one embodiment, after the second immune cell population is suspended in the second composition, the feeder cells may be included in the second composition to have the following ratios (cells included in the second immune cell population:feeder cells) with the cells in the second immune cell population: approximately 1:1000, approximately 1:900, approximately 1:800, approximately 1:700, approximately 1:600, approximately 1:500, approximately 1: 450, approximately 1:400, approximately 1:350, approximately 1:300, approximately 1:280, approximately 1:260, approximately 1:240, approximately 1:220, approximately 1:200, approximately 1:180, approximately 1:160, approximately 1:140, approximately 1:120, approximately 1:100, approximately 1:90, approximately 1:80, approximately 1:70, approximately 1:60, or approximately 1:50.

[0304] In certain embodiments, the feeder cells may be included in the second composition such that the ratio to the immune cell population (cells included in the second immune cell population:feeder cells) is 1:200 (±100).

[0305] In certain embodiments, the feeder cells may be included in the second composition such that the ratio to the immune cell population (cells included in the second immune cell population:feeder cells) is approximately 1:200.

[0306] ii) Isolation of PBMCs In one embodiment, the feeder cell can be PBMC. Wherein, the PBMC can be isolated from the blood of a healthy person. When the feeder cell is an inactivated allogeneic PBMC, the inactivated allogeneic PBMC is prepared by inactivating (e.g., inactivating by irradiation) peripheral blood cells isolated from the blood of a healthy patient.

[0307] Isolation of PBMCs from healthy individuals may be carried out through known methods. For example, the method of isolating PBMCs may include the common isolation method using Ficoll solution.

[0308] f) Antibiotics In one embodiment, the second composition may further comprise an antibiotic.

[0309] Antibiotics may be used to prevent bacterial or fungal contamination during cell culture and / or growth.

[0310] The use or non-use of antibiotics in the production of the final product may be determined by appropriate consideration of the condition of the first or second immune cell population used and the conditions of the cultured and / or expanded cells.

[0311] When the second composition contains an antibiotic, the antibiotic may be included at a concentration that does not affect cell culture and / or growth, where the concentration of the antibiotic that shows cytotoxicity may be taken into consideration. The concentration of the antibiotic included in the second composition may vary depending on the type of antibiotic.

[0312] Examples of antibiotics contained in the second composition include, but are not limited to, ampicillin, gentamicin, kanamycin, neomycin, penicillin, and streptomycin. That is, any antibiotic known in the art may be used as an antibiotic to be added to a medium for cell culture and / or growth. In certain embodiments, the second composition may include gentamicin.

[0313] As described above, the second composition in the present application includes a medium and an additive. In one embodiment, the second composition may include a medium, serum, IL-2, an anti-CD3 antibody, and a feeder cell. In another embodiment, the second composition may include a medium, plasma, IL-2, an anti-CD3 antibody, and a feeder cell. In yet another embodiment, the second composition may include a medium, serum, IL-2, an anti-CD3 antibody, a feeder cell, and an antibiotic. In yet another embodiment, the second composition may include a medium, plasma, IL-2, an anti-CD3 antibody, a feeder cell, and an antibiotic. However, the present application is not limited thereto.

[0314] In a specific embodiment, the second composition comprises a medium, serum, IL-2, OKT3, and feeder cells, the medium is CTS-AIM-V medium, the serum is human AB serum, the second composition comprises human AB serum at a concentration of approximately 3 vol%, the second composition comprises IL-2 at a concentration of approximately 2,000 IU / mL, the second composition comprises OKT3 at a concentration of approximately 30 ng / mL, and the feeder cells are inactivated allogeneic PBMCs, where the inactivated allogeneic PBMCs may be included in the second composition at a ratio of 1:200 (cells included in the second immune cell population:feeder cells) relative to the cells included in the immune cell population.

[0315] In a specific embodiment, the second composition comprises a medium, serum, IL-2, OKT3, an antibiotic, and feeder cells, the medium is CTS-AIM-V medium, the serum is human AB serum, the second composition comprises human AB serum at a concentration of approximately 3 vol%, the second composition comprises IL-2 at a concentration of approximately 2,000 IU / mL, the second composition comprises OKT3 at a concentration of 30 ng / mL, and the feeder cells are inactivated allogeneic PBMCs, where the inactivated allogeneic PBMCs may be included in the second composition at a ratio of 1:200 (cells included in the second immune cell population:feeder cells) relative to the cells included in the immune cell population.

[0316] Specific examples of seeding the second immune cell population are described below, but the application is not limited thereto.

[0317] A second immune cell population is obtained from the first immune cell population or the first immune cell population thawed after cryopreservation. The second immune cell population is suspended in a second composition and seeded into a second container GREX100M, where the cells are seeded at approximately 4E5 cell number per container. Here, the second composition includes CTS-AIM-V medium, 3 vol% human serum, 2,000 IU / mL IL-2, 30 ng / mL OKT3, and inactivated allogeneic PBMCs. Here, based on the time point after the second immune cell population is suspended in the second composition, the second composition includes inactivated allogeneic PBMCs at a ratio of approximately 1:200 (cells included in the second immune cell population: inactivated allogeneic PBMCs) to the cells included in the second immune cell population. That is, the second immune cell population is suspended in the second composition, and 20 mL of the second composition containing the second immune cell population is added per container.

[0318] 2. Expansion of a second immune cell population Overview The method for producing an immune cell composition of the present application comprises expanding a second immune cell population. In one embodiment, the method for producing an immune cell composition of the present application comprises expanding a second immune cell population to produce a third immune cell population.

[0319] The seeded second immune cell population is expanded in the second composition in the second container during an expansion period, through which a third immune cell population is generated. The third immune cell population refers to an immune cell population obtained by expanding the second immune cell population.

[0320] Expanding the second immune cell population can include adding a second additive composition.

[0321] The expansion of the second immune cell population is described in detail below.

[0322] Adding the second additive composition to the second container During expansion of the second immune cell population, a second additive composition may be added to the second container, where the second additive composition may comprise any one or more selected from medium, serum, plasma, antibiotics, IL-2, anti-CD3 antibodies, and feeder cells. In certain embodiments, the second additive composition may comprise medium, serum, and IL-2.

[0323] The process for adding the second additive composition is described in detail below.

[0324] 1) Number of additions A second additive composition is added (supplemented) to the second container while the second immune cell population is cultured in the second composition in the second container.

[0325] In one embodiment, during growth, the second additive composition may be added to the second container 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or more than the foregoing, In a particular embodiment, the second additive composition may be added 4 times during the growth period.

[0326] When the second additive composition is added two or more times during growth, the second additive composition may be added in the same amount at every addition time, or in different amounts at every addition time. The amount of the second additive composition added is described in detail in the relevant paragraph.

[0327] 2) Addition time Here, in one embodiment, the second additive composition may be added on any one or more days selected from the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st days from the start of growth, and may be added 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times during the growth period. In a specific embodiment, the second additive composition is added 4 times, on the 4th, 6th, 8th, and 10th days.

[0328] 3) Addition amount The amount of the second additive composition added can vary depending on the type of container and / or the time of addition. In one embodiment, the second additive composition may be added in the same amount at each addition time. In another embodiment, the second additive composition may be added in a different amount at each addition time. In yet another embodiment, the second additive composition may be added by increasing its amount at each addition time.

[0329] In one embodiment, the second additive composition may be present in an amount of approximately 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 32 mL, 35 mL, 37 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 80 mL, 90 mL, 100 mL, 120 mL, 140 mL, 145 ... The second additive composition may be added in the same or different amounts, such as 1 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, 500 mL, 550 mL, 600 mL, 650 mL, 700 mL, 750 mL, 800 mL, 850 mL, 900 mL, 950 mL, 1000 mL, 1500 mL, 2 L, 3 L, 4 L, or 5 L, or an amount greater than or equal to the aforementioned value at each addition time. However, the present application is not limited thereto, and the amount of the second additive composition to be added may be determined as an appropriate value taking into account various conditions (e.g., the volume of the container, the condition of the cultured cells, and / or the time of addition). In certain embodiments, the second additive composition may be added by increasing the amount added at each addition time, such that 20 mL, 60 mL, 180 mL, and 540 mL of the second additive composition may be added to the second container on days 4, 6, 8, and 10, respectively.

[0330] 4) Configuration of the second additive composition The second additive composition added to the second container may comprise any one or more selected from medium, serum, plasma, IL-2, anti-CD3 antibody, and feeder cells.

[0331] In one embodiment, the second additive composition may comprise culture medium; serum; and IL-2. In another embodiment, the second additive composition may comprise culture medium; plasma; and IL-2.

[0332] In certain embodiments, the second additive composition may comprise medium; serum; and IL-2. In another specific embodiment, the second additive composition may further comprise, in addition to medium, serum, and IL-2, any one or more of an anti-CD3 antibody and feeder cells.

[0333] Embodiments of each component that can be included in the second additive composition are described above in the second composition.

[0334] a) Culture medium The embodiment of the medium that can be included in the second additive composition is described above in the second composition. For example, in a specific embodiment, the medium included in the second additive composition can be CTS-AIM-V medium, but the present application is not limited thereto.

[0335] b) Serum When the second additive composition contains serum, the embodiment of serum is the same as that described above in the second composition.For example, in a specific embodiment, serum can be human serum, but the application is not limited thereto.For example, in a specific embodiment, the concentration of serum contained in the second additive composition can be approximately 3 vol%, but the application is not limited thereto.

[0336] c) Plasma When the second additive composition contains plasma, the embodiment related to the plasma is the same as that described above in the second composition. For example, in a specific embodiment, the plasma may be human plasma, but the present application is not limited thereto. For example, in a specific embodiment, the concentration of plasma contained in the second additive composition may be approximately 3 vol%, but the present application is not limited thereto.

[0337] d) IL-2 When the second additive composition includes IL-2, the embodiment regarding IL-2 is the same as that described above in the second composition. For example, in a specific embodiment, the concentration of IL-2 included in the second additive composition may be approximately 2,000 IU / mL, but the present application is not limited thereto.

[0338] e) Anti-CD3 antibody When the second additive composition contains an anti-CD3 antibody, the embodiment regarding the anti-CD3 antibody is the same as that described above in the second composition. For example, in a specific embodiment, the anti-CD3 antibody may be OKT3, but the present application is not limited thereto. For example, in a specific embodiment, the concentration of the anti-CD3 antibody contained in the second additive composition may be approximately 30ng / mL, but the present application is not limited thereto.

[0339] f) Feeder cells When the second additive composition contains feeder cells, the embodiment regarding the feeder cells is the same as that described above in the second composition.For example, in certain embodiments, the feeder cells may be inactivated allogeneic PBMCs, but the present application is not limited thereto.For example, in certain embodiments, the feeder cells may be contained in the second additive composition at a ratio of 1:200 (immune cells or cells contained in the second immune cell population:feeder cells) with respect to the immune cells or cells contained in the second immune cell population contained in the second container that are initially seeded in the second container.

[0340] g) Antibiotics When the second additive composition includes an antibiotic, the embodiments regarding the antibiotic are the same as those described above for the second composition.

[0341] In one embodiment, the composition of the second additive composition added to the second container can vary at each addition time, hi another embodiment, the composition of the second additive composition added to the second container can be the same at each addition time.

[0342] In one embodiment, the composition of the second additive composition added to the second container may be the same as that of the second composition. In another embodiment, the composition of the second additive composition added to the second container may be different from that of the second composition. In a specific embodiment, the second additive composition may be a composition having the same compositional components and compositional ratios as the second composition, except for the anti-CD3 antibody and the feeder cells.

[0343] In one embodiment, the second additive composition added to the second container may include medium, serum, and IL-2. In a particular embodiment, the second additive composition added to the second container may include CTS-AIM-V medium, approximately 3 vol% human serum, and approximately 2,000 IU / mL IL-2.

[0344] Whether or not to add new immune cells during proliferation During expansion of the second immune cell population in the second container, no additional immune cells may be added exogenously to the second immune cell population.

[0345] Furthermore, in such expansion processes, container changes may be optionally performed, but it is advantageous to provide convenience in this process by not requiring the cell population to be transferred from the second container to another container during expansion of the second immune cell population.

[0346] This means that in the methods of the present application, there is no need to transplant the second immune cell population into a container other than the second container during expansion, or to add additional new immune cells from outside to the second container.

[0347] Growth Period The second immune cell population may be expanded in the second container for a predetermined period of time, hi one embodiment, the second immune cell population is expanded for approximately one day or more. In one embodiment, the second immune cell population may be expanded for approximately 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, 7 or more days, 8 or more days, 9 or more days, 10 or more days, 11 or more days, 12 or more days, 13 or more days, 14 or more days, 15 or more days, 16 or more days, 17 or more days, 18 or more days, 19 or more days, 20 or more days, 21 or more days, 22 or more days, 23 or more days, or 24 or more days. In certain embodiments, the second immune cell population may be expanded in the second container for approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In certain embodiments, the second immune cell population may be expanded in the second container for approximately 14 days.

[0348] Growth efficiency The second immune cell population seeded in the second container may be expanded a predetermined number or more times through expansion of the second immune cell population, i.e., the seeded second immune cell population is expanded a predetermined number or more times in the second container.

[0349] In one embodiment, the seeded second immune cell population can be expanded in a second container, thus generating a third immune cell population.Compared with the second immune cell population, the third immune cell population has a cell number that is greater than or equal to a certain multiple.In one embodiment, the third immune cell population has at least approximately 100 times more cells than the second immune cell population. In one embodiment, the third immune cell population is at least about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1,000 times, about 1,100 times, about 1,200 times, about 1,300 times, about 1,400 times, about 1,500 times, about 1,600 times, about 1,700 times, about 1,800 times, about 1,900 times, about 2,000 times, about 2,200 times, about 3,300 times, about 4,400 times, about 5,500 times, about 6,000 times, about 7,000 times, about 8,000 times, about 9,000 times, about 10,000 times, about 11,000 times, about 12,000 times, about 13,000 times, about 14,000 times, about 15,000 times, about 16,000 times, about 17,000 times, about 18,000 times, about 19,000 times, about 20,000 times, about 21,000 times, about 22,000 times, about 23,000 times, about 24,000 times, about 25,000 times, about 26,000 times, about 27,000 times, about 28,000 times, about 29,000 times, about 30,000 times, about 31,000 times, about 32,000 times, about 33,000 times, about 34,000 times, about 35,000 times, about 36,000 times, about In certain embodiments, the third immune cell population may have at least approximately 2,500 times more cells than the second immune cell population. In certain embodiments, the third immune cell population may have at least approximately 9,400 times more cells than the second immune cell population, and in certain embodiments, the third immune cell population may have approximately 2,500-9,400 times more cells than the second immune cell population.

[0350] A specific example of expanding the second immune cell population may be described as follows, but the present application is not limited thereto. - the second immune cell population suspended in the second composition and then seeded is expanded for a proliferation period of 14 days. - A second additive composition is added to the second container on days 4, 6, 8, and 10 from the start of expansion, where the added second additive composition comprises CTS-AIM-V medium, 3 vol% human serum, and 2,000 IU / mL IL-2. More specifically, the second additive composition may be added to the second container at 20 mL on day 4, 60 mL on day 6, 180 mL on day 8, and 540 mL on day 10 from the start of expansion. A tertiary immune cell population is generated from the second immune cell population through a 14 day expansion process.

[0351] 3. Generation of immune cell compositions (immune compositions) Overview The immune cell composition or method for producing the immune composition of the present application may comprise the step of preparing an immune (cell) composition comprising immune cells as an active ingredient, obtained through the process described above.

[0352] After expansion of the second immune cell population is completed, the immune (cell) composition is prepared by obtaining a third immune cell population from the expansion product.

[0353] In one embodiment, the immune (cell) composition may comprise a third immune cell population as an active ingredient. In another embodiment, the immune (cell) composition may comprise a fourth immune cell population derived from the third immune cell population as an active ingredient.

[0354] An immune cell composition or immune composition refers to a composition that contains an "immune cell population" as an active ingredient. Here, the immune cell population may contain TILs, and the immune cell composition (immune composition) of the present application may contain TILs as an active ingredient.

[0355] In one embodiment, generating the immune cell composition may include obtaining a third immune cell population from the expansion product.

[0356] In one embodiment, the production of the immune cell composition may further comprise obtaining a fourth immune cell population from the third immune cell population, in addition to obtaining a third immune cell population from the expanded product.

[0357] The generation of immune cell compositions is described in detail below.

[0358] Obtaining the third immune cell population The third immune cell population is obtained from the expanded product. The method of obtaining the third immune cell population may include various known methods for obtaining cells in the process of culturing or expanding cells, but the present application is not limited thereto. For example, the obtaining of the third immune cell population may further include the process conventionally performed in the art, including cell counting and / or checking cell viability, in addition to the process described below, but the present application is not limited thereto.

[0359] In one embodiment, obtaining the immune cell population may include the following process: harvesting the immune cell population generated by the expansion process from the second container (cell harvesting): the third immune cell population may be obtained by harvesting the immune cell population from the expansion product.

[0360] In one embodiment, obtaining the third immune cell population may further comprise the following process: washing the harvested immune cell population (cell washing).

[0361] In one embodiment, obtaining the third immune cell population includes collecting the immune cell population generated through proliferation from the second container. Here, as a method for collecting the immune cell population, a method known in the art may be used, but the present application is not limited thereto. In one embodiment, centrifugation may be used to collect the immune cell population from the second container. Centrifugation may be performed under conditions such as, for example, 200 g, 7 minutes, room temperature, and Acc9 / Dcc9, but the present application is not limited thereto.

[0362] In one embodiment, the acquisition of the third immune cell population may further include, in addition to collecting the immune cell population generated through proliferation from the second container, washing the collected immune cell population. In washing the cells, a method known in the art may be used. For example, centrifugation may be used to wash the cells, but the application is not limited thereto. Here, washing the immune cell population may be performed with a buffer solution. Here, as the buffer solution, for example, PBS or DPBS may be used, but the application is not limited thereto. In one embodiment, the collected immune cell population may be washed 1, 2, 3, 4, or 5 times or more with DPBS. In a specific embodiment, the immune cell population may be washed 3 times with DPBS.

[0363] Third Immune Cell Population In one embodiment, the third immune cell population is at least approximately 100 times, approximately 200 times, approximately 300 times, approximately 400 times, approximately 500 times, approximately 600 times, approximately 700 times, approximately 800 times, approximately 900 times, approximately 1,000 times, approximately 1,100 times, approximately 1,200 times, approximately 1,300 times, approximately 1,400 times, approximately 1,500 times, approximately 1,600 times, approximately 1,700 times, approximately 1,800 times, approximately 1,900 times, approximately 2,000 times, approximately 2,200 times, approximately 3,000 times, approximately 4,000 times, approximately 5,000 times, approximately 6,000 times, approximately 7,000 times, approximately 8,000 times, approximately 9,0 ...0,000 times, approximately 11,000 times, approximately 12,000 times, approximately 13,000 times, approximately 14,000 times, approximately 15,000 times, approximately 16,000 times, approximately 17,000 times, approximately 18,00 fold, approximately 2,300 times, approximately 2,400 times, approximately 2,500 times, approximately 2,600 times, approximately 2,700 times, approximately 2,800 times, approximately 3,000 times, approximately 3,500 times, approximately 4,000 times, approximately 4,500 times, approximately 5,000 times, approximately 5,500 times, approximately 6,000 times, approximately 6,500 times, approximately 7,000 times, approximately 7,500 times, approximately 8,000 times, approximately 8,500 times, approximately 9,000 times, approximately 9,500 times, approximately 10,000 times, or approximately 20,000 times more cells.

[0364] In certain embodiments, the third immune cell population may have at least approximately 2,500 times more cells than the second immune cell population. In certain embodiments, the third immune cell population may have at least approximately 9,400 times more cells than the second immune cell population. In certain embodiments, the third immune cell population may have approximately 2,500 to 9,400 times more cells than the second immune cell population.

[0365] In one embodiment, the third immune cell population comprises immune cells. The third immune cell population may be obtained by culturing and / or expanding TILs present in the vicinity of tumor tissue, whereby the immune cells are TILs. Here, the percentage of immune cells among the cells contained in the third immune cell population may be approximately 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or higher. In a particular embodiment, the percentage of TILs among the cells contained in the third immune cell population may be approximately 97%, 98%, 99%, 99.5%, or 99.9% or higher. Here, the cells contained in the third immune cell population may refer to viable cells.

[0366] In one embodiment, the third immune cell population comprises T cells. Here, the third immune cell population comprises TILs, and the TILs comprise T cells. That is, the T cells contained in the third immune cell population may be a type of TIL. In one embodiment, the percentage of T cells among the cells contained in the third immune cell population may be approximately 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or higher. In certain embodiments, the percentage of T cells among the cells contained in the third immune cell population may be approximately 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or higher. In certain embodiments, the percentage of T cells among the cells contained in the third immune cell population may be approximately 99% or higher. In certain embodiments, the percentage of T cells among the cells contained in the third immune cell population may be approximately 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or higher. Here, the cells contained in the third immune cell population may refer to viable cells.

[0367] In one embodiment, the percentage of natural killer cells (NK cells) among the cells contained in the third immune cell population may be 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% or less. In a particular embodiment, the percentage of NK cells among the cells contained in the third immune cell population may be 10% or less. In a particular embodiment, the percentage of NK cells may be 1% or less. In a particular embodiment, the percentage of NK cells may be 0.2% or less. Here, the cells contained in the third immune cell population may refer to viable cells.

[0368] In one embodiment, the percentage of B cells among the cells contained in the third immune cell population may be 5, 4, 3, 2, or 1% or less. In a specific embodiment, the percentage of B cells may be 1% or less. In a specific embodiment, the percentage of B cells may be 0.2% or less. Here, the cells contained in the third immune cell population may refer to viable cells.

[0369] Optionally, the process includes obtaining a fourth immune cell population from the third immune cell population. In one embodiment, generating the immune cell composition, in addition to obtaining the third immune cell population, may further comprise obtaining a fourth immune cell population from the third immune cell population.

[0370] Here, the fourth immune cell population may be obtained from the third immune cell population through cell washing, processing, isolation, and / or cell sorting, including but not limited to, methods known in the art, for example, the fourth immune cell population may be a processed third immune cell population that has been processed to be suitable for administration to a subject.

[0371] Cell sorting Obtaining the fourth immune cell population from the third immune cell population may include cell sorting. Cell sorting refers to a method used to select only desired cells from a composition containing a cell population. For cell sorting, a method conventionally used in the art may be used. For example, a method such as fluorescence-activated cell sorting (FACS), centrifugation, or isolation by cell adhesion to a container may be used for cell sorting.

[0372] A population of immune cells processed to a state suitable for administration to a subject The third immune cell population, or the third immune cell population obtained via cell washing, may be processed into a state suitable for administration to a subject (eg, a patient).

[0373] In one example, the obtained third immune cell population or the washed third immune cell population; or the immune cell population obtained by separating it may be treated with a pharma- ceutically acceptable substance. For example, the immune cell population may be suspended in a pharma-ceutically acceptable substance such as a diluent and / or excipient, and may be treated to a state suitable for administration to a patient. For example, the pharma-ceutically acceptable substance may be isotonic saline, Ringer's solution, phosphate buffer, or any non-toxic pharma-ceutically acceptable substance known in the art.

[0374] Immune cell composition or immune composition The immune (cell) composition prepared by the method of the present application comprises a third or fourth immune cell population as an active ingredient.

[0375] In one embodiment, the immune cell composition is a composition comprising a third immune cell population or a fourth immune cell population, and may be processed into a state suitable for administration to a subject. That is, in one embodiment, the immune cell composition may be a composition in a state suitable for administration to a subject.

[0376] The characteristics of the immune cell composition of the present application are described in detail below.

[0377] Characterization of immune cell composition As described above, the immune cell composition may include a third immune cell population or a fourth immune cell population obtained from a third immune cell population.

[0378] In one embodiment, the immune cell composition comprises immune cells. Here, the immune cells may be TILs. Here, the percentage of immune cells in the cells contained in the immune cell composition may be approximately 60, 65, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or higher. In a specific embodiment, the percentage of TILs in the cells contained in the immune cell composition may be approximately 97%, 98%, 99%, 99.5%, or 99.9% or higher. Here, the cells contained in the immune cell composition may refer to viable cells.

[0379] In one embodiment, the immune cell composition comprises T cells. Here, the immune cell composition comprises TILs, and the TILs comprise T cells. That is, the T cells contained in the immune cell composition may be a type of TIL. In one embodiment, the percentage of T cells among the cells contained in the immune cell composition may be approximately 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or higher. In a particular embodiment, the percentage of T cells among the cells contained in the immune cell composition may be approximately 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or higher. In certain embodiments, the percentage of T cells among the cells contained in the immune cell composition may be approximately 99% or higher. In certain embodiments, the percentage of T cells among the cells contained in the immune cell composition may be approximately 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or higher. Here, cells contained in the immune cell composition may refer to viable cells.

[0380] In one embodiment, the percentage of NK cells among the cells contained in the immune cell composition may be 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% or less. In a specific embodiment, the percentage of NK cells among the cells contained in the immune cell composition may be 10% or less. In a specific embodiment, the percentage of NK cells may be 1% or less. In a specific embodiment, the percentage of NK cells may be 0.2% or less. Here, the cells contained in the immune cell composition may refer to viable cells.

[0381] In one embodiment, the percentage of B cells among the cells contained in the immune cell composition may be 5, 4, 3, 2, or 1% or less. In a specific embodiment, the percentage of B cells may be 1% or less. In a specific embodiment, the percentage of B cells may be 0.2% or less. Here, the cells contained in the immune cell composition may refer to viable cells.

[0382] Substances that may be further included in the immune (cell) composition As described in the combination administration section below, an immune cell composition comprising a third immune cell population or a fourth immune cell population derived from the third immune cell population may be administered together with an agent that can increase the therapeutic effect.

[0383] Here, the substance to be co-administered may be, for example, an interleukin such as IL-2 and / or an immune checkpoint inhibitor, but the present application is not limited thereto.

[0384] In one embodiment, the immune checkpoint inhibitor may be a substance that blocks the binding of PD-L1 and PD-1, such as an anti-PD-L1 agent or an anti-PD-1 agent. In one embodiment, the immune checkpoint inhibitor may be any one or more selected from nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, durvalumab, and variants thereof. In a particular embodiment, the immune checkpoint inhibitor may be nivolumab.

[0385] The above-mentioned substance that can be co-administered may further be included in the immune cell composition. In this case, the substance that can be co-administered in the immune cell composition is administered to the subject at the same time as the administration of the immune cell composition. Here, when the immune cell composition contains a co-administered substance, the concentration of the co-administered substance may be determined in consideration of the amount suitable for administration to the subject.

[0386] In one embodiment, the immune cell composition may further comprise any one or more selected from IL-2 and an immune checkpoint inhibitor. In a specific embodiment, the immune cell composition may further comprise IL-2. In a specific embodiment, the immune cell composition may further comprise nivolumab.

[0387] Uses of the generated immune (cell) compositions Overview The generated immune cell composition may be used for administration to a subject in need thereof. In one embodiment, the generated immune cell composition may be cryopreserved for storage. The cryopreserved immune cell composition may be thawed and used for administration to a subject in need thereof. Here, the period for cryopreserving the immune cell composition is not limited thereto. Furthermore, here, the method for cryopreserving the immune cell composition may be, but is not limited to, a method known in the art for cryopreserving an immune cell composition. The administration of the immune cell composition may have the effect of delaying the symptoms or progression of a particular medical condition, compared to a subject to which the immune cell composition is not administered. In one embodiment, the immune cell composition may be filled into a container and administered to a subject.

[0388] In one embodiment, a method of treating a tumor in a subject is provided comprising administering to the subject an immune cell composition or immune composition generated by the method for generating an immune cell composition of the present application.

[0389] In one embodiment, there is provided a use of an immune cell composition or immune composition produced by the method for producing an immune cell composition of the present application for treating a tumor in a subject.

[0390] Filling the container with immune cell composition An immune cell composition comprising the third immune cell population or a fourth immune cell population from the third immune cell population may be filled into a container. Here, the container may be an injection container for injecting tumor infiltrating lymphocytes (TILs) expanded by the method of the present application into a subject. Here, the immune cell composition may be filled into the container in consideration of an amount suitable for administration to a subject (e.g., a patient). Here, the condition of the patient and the condition of the immune cells contained in the immune cell composition may be taken into consideration.

[0391] In one embodiment, the immune cells are administered in a culture medium having a concentration of immune cells of approximately 1E8 / 200mL, 2E8 / 200mL, 3E8 / 200mL, 4E8 / 200mL, 5E8 / 200mL, 6E8 / 200mL, 7E8 / 200mL, 8E8 / 200mL, 9E8 / 200mL, 1E9 / 200mL, 2E9 / 200mL, 3E9 / 200mL, 4E9 / 200mL, 5E9 / 200mL, 6E9 / 200mL, 7E9 / 200mL, 8E9 / 200mL, 9E8 / 200mL, The container may be filled with E9 / 200mL, 1E10 / 200mL, 2E10 / 200mL, 3E10 / 200mL, 4E10 / 200mL, 5E10 / 200mL, 6E10 / 200mL, 7E10 / 200mL, 8E10 / 200mL, 9E10 / 200mL, 1E11 / 200mL, 2E11 / 200mL, 3E11 / 200mL, 4E11 / 200mL, or 5E11 / 200mL or more, but the present application is not limited thereto. In certain embodiments, the immune cells may be filled into the container such that the concentration of the immune cells is approximately 1E9 / 200mL or more. In certain embodiments, the immune cells may be filled into the container such that the concentration of the immune cells is approximately 1E10 / 200mL or more. In certain embodiments, the immune cells may be loaded into the container such that the concentration of the immune cells is between 1E10 / 200 mL and 1.3E11 / 200 mL.

[0392] Subjects to be administered The immune cell composition may be administered to a subject in need thereof. The subject includes a human having a tumor, where the tumor includes a solid tumor or a liquid tumor, where the solid tumor includes, but is not limited to, breast cancer, lung cancer, gastric cancer, ovarian cancer, renal cancer, or skin cancer, including malignant melanoma. Liquid tumors include, but are not limited to, blood cancer, including leukemia or lymphoma, or pleural effusion in patients with lung cancer. In certain embodiments, the immune cell composition may be administered to a human having breast cancer. In certain embodiments, the immune cell composition may be administered to a human having lung cancer.

[0393] In one embodiment, the subject in need of the immune cell composition may be the subject who provided the tumor tissue in I. Production of the semi-product. For example, the immune cell composition produced using this method may be administered back to the same subject via a tumor sample derived from the subject (obtained from tumor tissue obtained from the subject).

[0394] Method of administration The method of administering the immune cell composition to a subject in need thereof may be one of those known in the art. In one embodiment, the immune cell composition may be administered intra-arterially, intravenously, intradermally, or intraperitoneally. In a particular embodiment, the immune cell composition may be administered intra-arterially or intravenously.

[0395] In one embodiment, the immune cell composition may be administered with an additional substance. Here, co-administration may include administration of the immune cell composition before or after administration of the additional substance, or administration simultaneously with the additional substance. The additional substance includes any substance known in the art as a substance that enhances a therapeutic effect for a subject. In one embodiment, the additional substance may include IL-2, cyclophosphamide, and / or fludarabine. Co-administration with an additional substance is disclosed in detail below.

[0396] dose The dose of immune cell composition administered to a subject in need thereof may be an effective amount suitable for treatment, where the dose may vary depending on parameters including tumor volume, body weight, and age of the subject. In one embodiment, the dose is approximately 1E6, approximately 1.5E6, approximately 2E6, approximately 2.5E6, approximately 3E6, approximately 3.5E6, approximately 4E6, approximately 4.5E6, approximately 5E6, approximately 5.5E6, approximately 6E6, approximately 6.5E6, approximately 7E6, approximately 7.5E6, approximately 8E6, approximately 8.5E6, approximately 9E6, approximately 9.5E6, approximately 1E7, approximately 1.5E7, approximately 2E7, approximately 2.5E7, approximately 3 ....5E6, approximately 5.5E6, approximately 5.5E6, approximately 6E6, approximately 6.5E6, approximately 7 .5E7, approximately 4E7, approximately 4.5E7, approximately 5E7, approximately 5.5E7, approximately 6E7, approximately 6.5E7, approximately 7E7, approximately 7.5E7, approximately 8E7, approximately 8.5E7, approximately 9E7, approximately 9.5E7, approximately 1E8, approximately 1.5E8, approximately 2E8, approximately 2.5E8, approximately 3E8, approximately 3.5E8, approximately 4E8, approximately 4.5E8, approximately 5E8, approximately 5.5E8, approximately 6E8, approximately 6.5E8, approximately 7E8, approximately 7.5E8, approximately 8E8, approximately 8.5E8, approximately 9E8, approximately 9.5E8, approximately 1E9, approximately 1.5E9, approximately 2E9, approximately 2.5E9, approximately 3E9, approximately 3.5E9, approximately 4E9, approximately 4.5E9, approximately 5E9, approximately 5.5E9, approximately 6E9, approximately 6.5E9, approximately 7E9, approximately 7.5E9, approximately 8E9, approximately 8.5E9, approximately 9E9, approximately 9.5E9, approximately 1E10, oh Approximately 1.5E10, approximately 2E10, approximately 2.5E10, approximately 3E10, approximately 3.5E10, approximately 4E10, approximately 4.5E10, approximately 5E10, approximately 5.5E10, approximately 6E10, approximately 6.5E10, approximately 7E10, approximately 7.5E10, approximately 8E10, approximately 8.5E10, approximately 9E10, approximately 9.5E10, approximately 1E11, approximately 1.5E11, approximately 2E11, approximately 2.5E11, approximately 3E11, approximately 3.An immune cell composition comprising about 5E11, about 4E11, about 4.5E11, about 5E11, about 5.5E11, about 6E11, about 6.5E11, about 7E11, about 7.5E11, about 8E11, about 8.5E11, about 9E11, about 9.5E11, or about 1E12 or more immune cells may be administered once or multiple times to a subject in need thereof, but the application is not limited thereto.

[0397] Combination Administration As described above, the immune cell composition may be administered with an additional substance. Here, the co-administered additional substance may be administered before or after administration of the immune cell composition, or may be administered simultaneously with the immune cell composition. For example, the additional substance may be administered on the day the immune cell composition is administered. In another example, the additional substance may be administered to the subject on the day before the day the immune cell composition is administered. In yet another example, the additional substance may be administered to the subject on the day after the day the immune cell composition is administered. In yet another example, the additional substance may be administered multiple times, where the additional substance may be administered once on the same day as the day the immune cell composition is administered and multiple times on the day after the day the immune cell composition is administered. Furthermore, the co-administered additional substance may be administered via the same route as that of the immune cell composition, or via a different route.

[0398] The additional substance includes any substance known in the art as a substance that enhances the therapeutic effect for the subject. In one embodiment, the additional substance may include IL-2, an immune checkpoint inhibitor (e.g., a PD-1 or PD-L1 blocker such as nivolumab, pembrolizumab, cemiplimab, dostallimab, atezolizumab, avelumab, or durvalumab), cyclophosphamide, and / or fludarabine.

[0399] In one embodiment, the immune cell composition may be administered in conjunction with an immune checkpoint inhibitor and / or IL-2.

[0400] In certain embodiments, the immune cell composition may be administered with IL-2, where the amount of IL-2 administered to the subject may be an effective amount suitable for treatment, where the amount of IL-2 administered to the subject may vary depending on parameters including, but not limited to, the tumor volume, weight, and age of the subject.

[0401] In one embodiment, IL-2 is administered to a subject in need thereof at approximately 1E3 IU, approximately 5E3 IU, approximately 1E4 IU, approximately 5E4 IU, approximately 1E5 IU, approximately 5E5 IU, approximately 1E6 IU, approximately 5E6 IU, approximately 1E7 IU, approximately 5E7 IU, approximately 1E8 IU, approximately 5E8 IU, approximately 1E9 IU, approximately It may be administered in one or more doses of approximately 5E9 IU, approximately 1E10 IU, approximately 5E10 IU, approximately 1E11 IU, approximately 5E11 IU, approximately 1E12 IU, approximately 5E12 IU, approximately 1E13 IU, approximately 5E13 IU, approximately 1E14 IU, approximately 5E14 IU, approximately 1E15 IU, or approximately 5E15 IU or more.

[0402] In certain embodiments, IL-2 may be administered to a subject in need thereof in one or more doses at 720,000 IU / kg or less per dose.

[0403] In certain embodiments, IL-2 may be administered to a subject in need thereof in one or more doses at 18E6 IU / animal or less per dose.

[0404] For example, when IL-2 is administered intravenously, it may be administered once or repeatedly multiple times according to the following administration method. - Administer 18E6IU / day for 5 days - Take a break for 2 to 6 days (interruption of administration) - Administer 18E6IU / day for 5 days - Take a 3-week break (interruption of treatment).

[0405] In another example, when IL-2 is administered subcutaneously, administration according to the following method of administration may be done once or repeated multiple times. - Administer 18E6IU / day for 5 days - Take a 2-day break (interruption of administration) - Administered for 3 weeks: in the first week (Week 1), 18E6IU is administered on days 1 and 2; 9E6IU is administered on days 3, 4, and 5; and no drug is administered on days 6 and 7. The administration in the second (Week 2) and third (Week 3) of the three weeks is also the same as that in the first week.

[0406] In a particular embodiment, IL-2 may be administered at the following doses: 2E6 IU / head per day for 14 days.

[0407] Illustrative examples of the methods disclosed herein are presented below. The following illustrative examples are illustrative methods for easy understanding of the methods disclosed herein, and the scope of the methods disclosed herein is not limited by the following examples. 1. Illustrative Example of Producing a Semi-Product (First Immune Cell Population Generated and Obtained from a Tumor Sample) 1) Preparation of tumor samples Tumor tissues from patients with breast or lung cancer are obtained from medical institutions. The tumor tissues are washed three times using RPMI1640 medium without antibiotics. Lipid tissue is removed from the washed tumor tissues. The tumor tissues from which the lipid tissue has been removed are cut into pieces of approximately 1 mm 2 ~Approximately 2mm 2 The tumor sample is then cut to have a cross-sectional area of ​​100 mm, thereby preparing a tumor sample. 2) Generation of the first immune cell population 24 pieces of tumor sample are placed in a T75 flask together with CTS-AIM-V medium containing approximately 3 vol% human AB serum and approximately 1,000 IU / mL IL-2 (first composition, approximately 6 mL in total), and cultured for 14 days in a 5% CO2 environment at 37°C. During the culture, CTS-AIM-V medium containing approximately 3 vol% human AB serum and approximately 1,000 IU / mL IL-2 (first additive composition to be added, approximately 3 mL in total) is added to the T75 flask on days 7, 10, and 12 from the start of the culture. 3) Obtaining the first immune cell population Using a cell strainer with a mesh pore of 40 μm, tumor tissue fragments are filtered from the cultured composition. The cultured immune cell population is washed three times with DPBS to obtain a first immune cell population. 4) Cryopreservation of the first immune cell population The first immune cell population is suspended at a concentration of approximately 2E6 cells / mL using CryoStor® CS10 and added to a cryovial in 1 mL, which is then stored in gaseous liquid nitrogen (LN2) at approximately -190°C or lower. 2. Illustrative Example of Producing a Final Product (Immune Cell Composition Generated by Obtaining a Third Immune Cell Population) from a Cryopreserved Semi-Product 1) Thawing of the cryopreserved first immune cell population The cryopreserved first immune cell population is thawed in a water bath at approximately 37° C. for approximately 2 minutes. 2) Seeding of the second immune cell population The second immune cell population divided from the first immune cell population and CTS-AIM-V medium containing approximately 3 vol% human AB serum, approximately 2,000 IU / mL IL-2, approximately 30 ng / mL OKT3, and irradiated PBMCs with a ratio of approximately 1:200 (TIL:PBMC) to the second immune cell population (TIL) (second composition, approximately 20 mL in total) are seeded into GREX-100M. Here, seeding is performed to contain approximately 4E5 immune cells in GREX-100M. 3) Expansion of a second immune cell population During the expansion, CTS-AIM-V medium (added second additive composition) containing approximately 3 vol% human AB serum and approximately 2,000 IU / mL IL-2 is added to GREX-100M at 20 mL on day 4, 60 mL on day 6, 180 mL on day 8, and 540 mL on day 10 from the start of expansion. Here, expansion is performed for 14 days, during which the second immune cell population is not transplanted into a new container or new immune cells are not added from the outside. During the expansion period, the second immune cells are expanded 2,500-fold or more. 4) Generation of immune cell compositions The composition containing the immune cell population expanded through the above processes is centrifuged to obtain a third immune cell population, thereby generating an immune cell composition. 3. Illustrative Examples of Producing a Final Product (Immune Cell Composition Generated by Obtaining a Third Immune Cell Population) Through Cryopreservation 1) Obtaining a sample for generating an immune cell composition Tumor tissues from patients with breast or lung cancer are obtained from medical institutions. The tumor tissues are washed three times using RPMI1640 medium without antibiotics. Lipid tissue is removed from the washed tumor tissues. The tumor tissues from which the lipid tissue has been removed are cut into pieces of approximately 1 mm 2 ~Approximately 2mm 2 The tumor sample is then cut to have a cross-sectional area of ​​100 mm, thereby preparing a tumor sample. 2) Generation of the first immune cell population 24 pieces of tumor sample are placed in a T75 flask together with CTS-AIM-V medium containing approximately 3 vol% human AB serum and approximately 1,000 IU / mL IL-2 (first composition, approximately 6 mL in total), and cultured for 14 days in a 5% CO2 environment at 37°C. During the culture, CTS-AIM-V medium containing approximately 3 vol% human AB serum and approximately 1,000 IU / mL IL-2 (first additive composition to be added, approximately 3 mL in total) is added to the T75 flask on days 7, 10, and 12 from the start of the culture. 3) Obtaining the first immune cell population Using a cell strainer with a mesh pore of 40 μm, the tumor tissue fragments are filtered from the culture composition. The cultured immune cell population is washed three times with DPBS to obtain a first immune cell population. 4) Cryopreservation of the first immune cell population The first immune cell population is suspended at a concentration of approximately 2E6 cells / mL using CryoStor® CS10 and added to a cryovial in 1 mL, which is then stored in gaseous liquid nitrogen (LN2) at approximately -190°C or lower. 5) Thawing of the cryopreserved first immune cell population The cryopreserved first immune cell population is thawed in a water bath at approximately 37° C. for approximately 2 minutes. 6) Seeding of the second immune cell population The second immune cell population divided from the first immune cell population and CTS-AIM-V medium (second composition, total approximately 20 mL) containing approximately 3 vol% human AB serum, approximately 2,000 IU / mL IL-2, approximately 30 ng / mL OKT3, and irradiated PBMCs with a ratio of approximately 1:200 (TIL:PBMC) to the second immune cell population (TIL) are seeded into GREX-100M. Here, seeding is performed to contain approximately 4E5 immune cells in GREX-100M. 7) Expansion of a second immune cell population During the expansion, CTS-AIM-V medium (added second additive composition) containing approximately 3 vol% human AB serum and approximately 2,000 IU / mL IL-2 is added to GREX-100M at 20 mL on day 4, 60 mL on day 6, 180 mL on day 8, and 540 mL on day 10 from the start of expansion. Here, expansion is performed for 14 days, during which the second immune cell population is not transplanted into a new container or new immune cells are not added from the outside. During the expansion period, the second immune cells are expanded 2,500-fold or more. 8) Generation of immune cell compositions The composition containing the immune cell population expanded through the above processes is centrifuged to obtain a third immune cell population, thereby generating an immune cell composition. 4. Illustrative Examples of Producing a Final Product (Immune Cell Composition Generated by Obtaining a Third Immune Cell Population) Without Cryopreservation 1) Obtaining a sample for generating an immune cell composition Tumor tissues from patients with breast or lung cancer are obtained from medical institutions. The tumor tissues are washed three times using RPMI1640 medium without antibiotics. Lipid tissue is removed from the washed tumor tissues. The tumor tissues from which the lipid tissue has been removed are cut into pieces of approximately 1 mm 2 ~Approximately 2mm 2 The tumor sample is then cut to have a cross-sectional area of ​​100 mm, thereby preparing a tumor sample. 2) Generation of the first immune cell population 24 pieces of tumor sample are placed in a T75 flask together with CTS-AIM-V medium containing approximately 3 vol% human AB serum and approximately 1,000 IU / mL IL-2 (first composition, approximately 6 mL in total), and cultured for 14 days in a 5% CO2 environment at 37°C. During the culture, CTS-AIM-V medium containing approximately 3 vol% human AB serum and approximately 1,000 IU / mL IL-2 (first additive composition to be added, approximately 3 mL in total) is added to the T75 flask on days 7, 10, and 12 from the start of the culture. 3) Obtaining the first immune cell population Using a cell strainer with a mesh pore of 40 μm, tumor tissue fragments are filtered from the culture composition. The first immune cell population is obtained by washing the cultured immune cell population three times with DPBS. 4) Seeding of the second immune cell population The second immune cell population divided from the first immune cell population and CTS-AIM-V medium (second composition, total approximately 20 mL) containing approximately 3 vol% human AB serum, approximately 2,000 IU / mL IL-2, approximately 30 ng / mL OKT3, and irradiated PBMCs with a ratio of approximately 1:200 (TIL:PBMC) to the second immune cell population (TIL) are seeded into GREX-100M. Here, seeding is performed to contain approximately 4E5 immune cells in GREX-100M. 5) Expansion of a second immune cell population During the expansion, CTS-AIM-V medium (added second additive composition) containing approximately 3 vol% human AB serum and approximately 2,000 IU / mL IL-2 is added to GREX-100M at 20 mL on day 4, 60 mL on day 6, 180 mL on day 8, and 540 mL on day 10 from the start of expansion. Here, expansion is performed for 14 days, during which the second immune cell population is not transplanted into a new container or new immune cells are not added from the outside. During the expansion period, the second immune cells are expanded 2,500-fold or more. 6) Generation of immune cell compositions The composition containing the immune cell population expanded through the above processes is centrifuged to obtain a third immune cell population, thereby generating an immune cell composition.

[0408] Exemplary embodiments of the method provided by the present application are disclosed below. The following exemplary embodiments are provided for easy understanding of the method disclosed in the present application, and the scope of the present invention disclosed in the present application is not limited to the following examples. PR01.

[0409] The method for generating a first immune cell population comprises: (a) culturing a tumor sample in a first container; wherein the tumor sample is cultured in a first composition in a first container, the first composition comprising medium, serum, and IL-2; the first container is a gas-permeable container or a container having a gas supply system; The culture was carried out for 7 to 21 days. A first additive composition is added to the first container one or two or more times during the culture; and (b) obtaining a first population of immune cells from the composition cultured in (a). PR02.

[0410] The method according to PR01, wherein the method further comprises the step of preparing a tumor sample. PR03.

[0411] The tumor sample is obtained from a tumor fragment obtained from the subject, the tumor sample being approximately 1 mm 2 ~4mm 2 The method according to any one of PR01 and PR02, wherein the cross-sectional area is PR04

[0412] The method according to any one of PR01 to PR04, wherein in (a) 12 to 24 tumor samples are cultured. PR05.

[0413] The method according to any one of PR01 to PR04, wherein the serum is human serum. PR06.

[0414] The method according to any one of PR01 to PR04, wherein the concentration of serum in the first composition is 5 vol% or lower. PR07.

[0415] The method according to any one of PR01 to PR04, wherein the concentration of serum in the first composition is approximately 3 vol%. PR08.

[0416] The method according to any one of PR01 to PR07, wherein the medium in the first composition is AIM-V medium. PR09.

[0417] The method according to any one of PR01 to PR07, wherein the medium in the first composition is a CTS-AIM-V medium. PR10.

[0418] The method according to any one of PR01 to PR09, wherein the concentration of IL-2 in the first composition is 500 IU / mL to 3,000 IU / mL. PR11.

[0419] The method according to any one of PR01 to PR09, wherein the concentration of IL-2 in the first composition is approximately 1,000 IU / mL. PR12.

[0420] The method according to any one of PR01 to PR11, wherein the first vessel is a T75 flask. PR13.

[0421] The method according to any one of PR01 to PR12, wherein the volume of the first composition is 30% or less of the maximum allowable volume of the first container. PR14.

[0422] The method according to any one of PR01 to PR12, wherein when the first vessel is a T75 flask, the volume of the first composition is 6 mL. PR15.

[0423] The method according to any one of PR01 to PR14, wherein the first additive composition comprises medium, serum, and IL-2. PR16.

[0424] The method according to any one of PR01 to PR14, wherein the first additive composition comprises CTS-AIM-V medium, 3 vol% serum, and 1,000 IU / mL IL-2. PR17.

[0425] The method according to any one of PR01 to PR14, wherein the first additive composition has the same compositional properties and composition ratio as the first composition. PR18.

[0426] The method according to any one of PR01 to PR17, wherein during the culture, the first additive composition is added to the first container two or more times. PR19.

[0427] The method according to any one of PR01 to PR17, wherein during the culture, the first additive composition is added to the first container on days 7, 10, and 12 from the start date of the culture, and the amount of the first additive composition added is the same at each addition time. PR20.

[0428] During the culture, the first additive composition may be added to the first container at 3 mL on day 7, 3 mL on day 10, and 3 mL on day 12 from the start of the culture. The method according to any one of PR01 to PR17. PR21.

[0429] Cultivation is carried out for 13 to 15 days using one of the methods PR01 to PR20. PR22.

[0430] The culture is carried out for 14 days according to one of the methods PR01 to PR21. F01.

[0431] Cryopreserving the first immune cell population generated by any one of PR01 to PR22. 2. A method for cryopreserving a first population of immune cells, comprising: T01.

[0432] A method of thawing a cryopreserved first immune cell population, comprising thawing a cryopreserved first immune cell population according to F01. R01.

[0433] A method for generating an immune cell composition, comprising: (a) seeding a second composition and a second population of immune cells into a second container; wherein the second immune cell population is derived from the first immune cell population; the second composition comprises a medium, serum, 1,000 IU / mL to 3,000 IU / mL of IL-2, an anti-CD3 antibody, and feeder cells; The second container is a gas permeable container or a container having a gas supply system; (b) expanding the second immune cell population to generate a third immune cell population; wherein during growth, the second additive composition is added to the second container one or two or more times; Growth is carried out for 7 to 21 days; and (c) obtaining a third population of immune cells from the expansion product obtained in (b) and producing an immune cell composition; Here, the third immune cell population has at least 2500 times more cells than the second immune cell population. R02.

[0434] The method according to R01, wherein the first immune cell population is an immune cell population generated by any one of PR01 to PR22, or an immune cell population thawed by T01. R03.

[0435] The method according to any one of R01 and R02, wherein the medium in the second composition is AIM-V medium. R04.

[0436] The method according to any one of R01 to R03, wherein the serum in the second composition is human serum. R05.

[0437] The method according to any one of R01 to R03, wherein the concentration of serum in the second composition is 5 vol% or lower. R06.

[0438] The method according to any one of R01 to R03, wherein the concentration of serum in the second composition is approximately 3 vol%. R07.

[0439] The method according to any one of R01 to R06, wherein the concentration of IL-2 in the second composition is approximately 2,000 IU / mL. R08.

[0440] The method according to any one of R01 to R07, wherein the anti-CD3 antibody is OKT3. R09.

[0441] The method according to any one of R01 to R08, wherein the concentration of the anti-CD3 antibody in the second composition is 10 ng / mL to 100 ng / mL. R10.

[0442] The method according to any one of R01 to R08, wherein the concentration of the anti-CD3 antibody in the second composition is approximately 30 ng / mL. R11.

[0443] The method according to any one of R01 to R10, wherein the feeder cells are allogeneic PBMCs. R12.

[0444] The method according to any one of R01 to R10, wherein the feeder cells are inactivated allogeneic PBMC. R13.

[0445] The method according to any one of R01 to R12, wherein the feeder cells comprise the second composition such that the ratio of the number of feeder cells is approximately 1:200 (number of cells of the second immune cell population:feeder cells) based on the number of cells of the second immune cell population. R14.

[0446] The method according to any one of R01 to R13, wherein during seeding, the number of cells of the second immune cell population seeded in the second container is 1E5 to 10E5. R15.

[0447] The method according to any one of R01 to R13, wherein during seeding, the number of cells of the second immune cell population seeded in the second container is approximately 4E5. R16.

[0448] The method according to any one of R01 to R15, wherein the second container is GREX100M. R17.

[0449] The method according to any one of R01 to R16, wherein the volume of the second composition is 30% or less of the maximum allowable volume of the second container. R18.

[0450] The method according to any one of R01 to R16, wherein when the second container is GREX100M, the volume of the second composition is approximately 20 mL. R19.

[0451] The method according to any one of R01 to R18, wherein during expansion, immune cells are not removed from the second container or added exogenously. R20.

[0452] Growth is performed for 13-15 days according to any one of the methods R01-R19. R21.

[0453] Growth is performed for 14 days according to one of the methods R01 to R19. R22.

[0454] The method according to any one of R01 to R21, wherein the second additive composition comprises a medium, serum, and IL-2. R23.

[0455] The method according to any one of R01 to R21, wherein the second additive composition comprises CTS-AIM-V medium, 3 vol% serum, and 2,000 IU / mL IL-2. R24.

[0456] The method according to any one of R01 to R21, wherein the second additive composition has the same compositional properties and composition ratio as the second composition, except for the anti-CD3 antibody and the feeder cells. R25.

[0457] The method according to any one of R01 to R24, wherein the second additive composition is added to the second container two or more times. R26.

[0458] The method according to any one of R01 to R24, wherein the second additive composition is added to the second container two or more times, and the amount of the second additive composition added is different each addition time. R27.

[0459] The method according to any one of R01 to R24, wherein the second additive composition is added to the second container on days 4, 6, 8, and 10 from the start date of growth, and the amount of the second additive composition added is different at each addition time. R28.

[0460] The method according to any one of R01 to R24, wherein the second additive composition is added to the second container at 20 mL on day 4, 60 mL on day 6, 180 mL on day 8, and 540 mL on day 10 from the start of growth. R29.

[0461] The method according to any one of R01 to R28, wherein the third immune cell population has at least 4000 times more cells than the second immune cell population. R30.

[0462] The method according to any one of R01 to R29, wherein the immune cell composition comprises a third immune cell population. R31.

[0463] The method according to any one of R01 to R29, wherein the immune cell composition comprises a fourth immune cell population obtained from the third immune cell population. R32.

[0464] The method according to any one of R01 to R29, further comprising a step of genetically manipulating immune cells. R33.

[0465] The method according to R32, wherein the step of genetically manipulating the immune cells is performed during or prior to culturing the tumor sample in the first container. R34.

[0466] The method according to R32, wherein the step of genetically manipulating the immune cells is performed during or prior to seeding the second composition and the second immune cell population into the second container. R35.

[0467] The method according to R32, wherein the step of genetically manipulating the immune cell comprises introducing a transgene into the immune cell. R36.

[0468] The method according to R32, wherein the step of genetically manipulating the immune cell comprises the step of introducing mRNA into the immune cell. R37.

[0469] The method according to R32, wherein the step of manipulating the genes of the immune cells includes a step of manipulating the genes of the immune cells using a CRISPR-Cas system. R38.

[0470] The method according to R32, wherein the step of genetically manipulating the immune cell comprises genetically manipulating the immune cell with a transposon. R39.

[0471] The method according to R32, wherein the step of manipulating the gene of the immune cell comprises reducing expression of the gene by siRNA or antisense oligonucleotide. C01.

[0472] An immune cell composition containing TILs, generated by R01 to R39. C02.

[0473] The immune cell composition according to C01, further comprising IL-2. C03.

[0474] The immune cell composition according to any one of C01 and C02, wherein the immune cell composition further comprises an immune checkpoint inhibitor. C04.

[0475] The immune cell composition according to any one of C01 and C02, wherein the immune cell composition further comprises nivolumab. U01.

[0476] Use of an immune cell composition comprising TILs, generated in accordance with R01 to R39, for treating a tumor in a subject. U02.

[0477] The use according to U01, wherein the subject is the subject from which the tumor-infiltrating lymphocytes are derived. TM01.

[0478] A method for treating a tumor in a subject, comprising administering to the subject an immune cell composition comprising TILs generated in accordance with R01 to R39. TM02.

[0479] The method according to TM01, wherein the subject is a TIL-derived subject. TM03.

[0480] The method according to any one of TM01 and TM02, wherein the method further comprises administering IL-2. TM04.

[0481] The method according to any one of TM01 to TM03, further comprising administering an immune checkpoint inhibitor. TM05.

[0482] The method according to any one of TM01 to TM03, further comprising administering nivolumab.

[0483] Characteristics of the methods for generating immune cell compositions disclosed herein IL-2 concentration In general, in the production of immune cell compositions containing TIL, a high concentration (e.g., 6,000 IU / mL) of IL-2 is added to the medium for TIL culture. However, the method disclosed herein uses a first composition containing a lower concentration of IL-2 in the culture. The inventors have demonstrated through experiments that the use of a composition containing a lower concentration of IL-2 is sufficient for the culture of immune cell populations containing TIL (see Experimental Example 3).

[0484] Furthermore, in addition to the composition for culture, generally, a high concentration (e.g., 3,000 IU / mL) of IL-2 is added to the composition for proliferation. However, the second composition for proliferation disclosed herein may contain a lower concentration of IL-2. The inventors of the present application have demonstrated through experiments that the use of a composition supplemented with a low concentration of IL-2 is sufficient for the proliferation of immune cell populations including TIL (see Experimental Example 3).

[0485] In conclusion, the above is significant in that it establishes an effective treatment that can adequately culture and expand immune cell populations, including TILs, using low concentrations of IL-2.

[0486] Culture and / or Growth Compositions 1) Amount of composition for initial culture and / or composition for growth Generally, in the culture and / or expansion of cells, including TILs, the amount of the composition for culture and / or expansion varies depending on the container in which the culture and / or expansion occurs. Moreover, here, the determined amount of the composition for culture and / or expansion is generally determined by the maximum allowable volume of the container. However, in the method disclosed herein, the amount of the composition for culture (e.g., the first composition) and / or the composition for expansion (e.g., the second composition) is less than the maximum allowable volume of the container at the start of cell culture and / or expansion. That is, the amount of the composition for culture and / or expansion is set to be less than the maximum allowable volume of the container, and thus the cell concentration is adjusted to be optimal for culture and / or expansion, and subsequently, the culture and / or expansion is started. The optimal concentration of cells in the composition may have a positive effect on the interaction between cells, and thus plays a role in enhancing the efficiency of cell culture and / or expansion. With reference to Experimental Example 4 and FIG. 8, it can be confirmed that when cells were cultured by varying the amount of the composition using a 24-well plate, the cell number was higher when 1 mL was added. According to this, it means that through optimization of the concentration of cells in the composition, the efficiency of cell culture and / or proliferation can be further increased.

[0487] 2) Addition of the first additive composition and / or the second additive composition Generally, in the production of immune cell compositions containing TILs, the culture and / or expansion compositions are replaced during the culture and expansion of TILs. However, according to the method disclosed in the present application, in the process of culturing and expanding immune cell populations containing TILs, the culture or expansion compositions are not replaced, but are only added by adding them during the culture and expansion. This has two advantages.

[0488] First, it can provide the materials necessary for TIL culture or expansion while minimizing the stress that may be imposed on the TILs during complex TIL culture or expansion procedures, and further, it can prevent the loss of immune cell populations that may occur upon replacement of culture and / or expansion compositions.

[0489] Secondly, through the addition of the composition, the concentration of cells cultured and / or grown in the composition may be continuously maintained as an effective optimal concentration for culture and / or growth, meaning that the cell culture and / or growth environment is continuously maintained as an optimized environment for culture and / or growth, through which immune cell compositions can be produced in higher yields.

[0490] In conclusion, by adjusting the amount of the composition during culture and / or expansion, the concentration of cells in the composition is optimized, and further, by adding the culture and / or expansion composition during culture and / or expansion, the optimal concentration of cells in the composition is maintained. Furthermore, because the composition is added rather than replaced during this process, immune cell compositions including TILs can be produced more efficiently and with higher yields.

[0491] Growth efficiency Generally, during the proliferation of immune cells including TILs, TILs are proliferated by approximately 100 to 1,000 times. However, in the method disclosed in the present application, during proliferation, TILs are proliferated by approximately 2,500 times or more on average. In the present application, the composition for proliferation has a lower IL-2 concentration and also has a high proliferation rate of approximately 2,500 times or more, compared to other common treatments. This indicates that the inventors have established an efficient immune cell production method with a higher proliferation rate using a smaller amount of additives than other treatments, which is shown in Experimental Example 2 and FIG. 5.

[0492] Frozen storage period According to the method of producing an immune cell composition disclosed herein, a first immune cell population may be obtained and then cryopreserved to be stored for a certain period or longer, and the cryopreserved first immune cell population may be expanded after thawing when needed and used to produce an immune cell composition. Here, as an example, the first immune cell population may be cryopreserved and stored for approximately 1, 2, 3, 4, or 5 years or longer, and then expanded and used to produce an immune cell composition. As another example, the first immune cell population may be cryopreserved for a short period, thawed, and then used to produce an immune cell composition. Therefore, it is very important that the first immune cells obtained by the method disclosed herein can be used for expansion after short-term or long-term cryopreservation depending on the situation. For example, if the production of the semi-product and the production of the final product are carried out at different locations or by different companies, the first immune cell population may be transported to another location after cryopreservation. Additionally, for example, if a patient's cancer recurs or metastasizes, the previously cryopreserved first population of immune cells may be thawed, expanded, and used to treat the patient. [Experimental Example]

[0493] The present invention provided in this application will be described in more detail with reference to the following experimental examples and examples. These examples are provided for the sole purpose of illustrating the contents disclosed in this application, and it is obvious to those skilled in the art that the scope of the contents disclosed in this application should not be construed as being limited by these examples.

[0494] Table 1. Reagent information [Table 1] Experimental example 1. TIL generation Experimental Example 1-1. TIL isolation / culture treatment 1. TIL Isolation / Culture Treatment - Day 0 The tumor tissue removed from the operating room of the hospital was washed three times with RPMI1640 medium and transferred to a 100 mm Petri dish. The lipid tissue in the tumor tissue was removed using sterilized surgical scissors and tweezers, and the tumor tissue was cut into 1-2 mm pieces using sterilized surgical scissors and tweezers. 24 pieces of tumor tissue cut into 1-2 mm pieces were placed in a T75 flask, 6 mL of the prepared culture composition was added, and the mixture was then cultured at 37°C for 7 days in a 5% CO2 incubator.

[0495] The culture composition consisted of the following: CTS-AIM-V medium, 3 vol% human serum, and 1,000 IU / mL IL2. - Day 7 3 mL of the prepared culture composition was added to each flask and cultured at 37° C. in a 5% CO 2 incubator for 3 days. - Day 10 3 mL of the prepared culture composition was added to each flask and cultured at 37° C. in a 5% CO 2 incubator for 2 days. - Day 12 3 mL of the prepared culture composition was added to each flask and cultured at 37° C. in a 5% CO 2 incubator for 2 days. - Day 14 To collect the cells, a 40 μm cell strainer was placed on top of a 50 mL tube. The tumor tissue was filtered by passing the cultured cells through the cell strainer using a 10 mL pipette, and only the cultured cells were collected in a 50 mL conical tube, which was then centrifuged at 450 g for 5 minutes at room temperature. 5 mL of DPBS was added to the precipitated cells, which were then suspended by pipetting. The suspended cells were counted, and cell viability was determined using a hemocytometer (C-chip). After centrifugation at 450 g for 5 minutes, the cells were suspended in Cryo-stor CS10 at 2E6 cells / mL, filled into a cryovial at 1 mL, and stored in a liquid nitrogen tank (LN2) or a gaseous liquid nitrogen tank (LN2).

[0496] Experimental Example 1-2. TIL proliferation treatment 1. Isolation of allo-feeder PBMCs - Leukapheresis and irradiation in healthy volunteers Blood bags from healthy volunteers were leukoreduced using a leukoreducer in the blood collection room of the hospital, and the leukoreduced blood bags were inactivated by exposure to X-rays. Isolation of PBMCs from Irradiated Blood Bags Leukoreduced irradiated blood was mixed with DPBS at a ratio of 1:2. After 15 mL of Ficoll solution was added to a 50 mL tube, 30 mL of blood mixture was layered on the Ficoll layer without mixing with Ficoll and centrifuged at 800 g for 20 min at room temperature under Acc9 / Dcc0 conditions. The separated PBMC layer was collected and then transferred to a new 50 mL tube. For washing, DPBS was added to the 50 mL tube to which the PBMC layer was transferred, and then centrifugation was performed at 800 g for 5 min at room temperature. After centrifugation, the supernatant was completely removed and the precipitated PBMCs were suspended in 50 mL of medium for culturing the final product. The suspended cells were counted using a hemocytometer set to determine the cell viability. After cell counting, 3.2E9 PBMCs were prepared. 2. End product formation - Day 0 (cell seeding) Eleven vials of the half-product stored in a liquid nitrogen tank (LN2) were thawed in a water bath at 37°C for 2 minutes. The thawed cells were washed with CTS-AIM-V medium and centrifuged at 1,500 rpm for 5 minutes at room temperature. After discarding the supernatant, the resulting pellet was suspended in CTS-AIM-V growth composition, and the cells were counted using a hemocytometer set to determine cell viability. After cell counting, 1.6E7 cells were prepared. 4E5 half-product TILs and 8E7 irradiated PBMCs were placed in each of 40 GREX100M (1L) culture vessels, and 20 mL of the final product growth composition was added. The resulting cell mixture was cultured at 37°C in a 5% CO2 incubator.

[0497] The final product growth composition consisted of the following: CTS-AIM-V medium, 3% human serum, 2,000 IU / mL IL-2, and 30 ng / mL OKT3. - Day 4 IL-2 and fractionated human serum were added to CTS-AIM-V medium to 2,000 IU / mL and 3%, respectively, to prepare the additive composition in the required amount. 20 mL of the prepared additive composition was further dispensed into each GREX flask and cultured at 37°C in a 5% CO2 incubator. - Day 6 IL-2 and fractionated human serum were added to CTS-AIM-V medium to 2,000 IU / mL and 3%, respectively, to prepare the additive composition in the required amount. 60 mL of the prepared additive composition was further dispensed into each GREX flask and cultured at 37°C in a 5% CO2 incubator. - Day 8 IL-2 and fractionated human serum were added to CTS-AIM-V medium to 2,000 IU / mL and 3%, respectively, to prepare the additive composition in the required amount. 180 mL of the prepared additive composition was further dispensed into each GREX flask and cultured at 37°C in a 5% CO2 incubator. - Day 10 IL-2 and fractionated human serum were added to CTS-AIM-V medium to 2,000 IU / mL and 3%, respectively, to prepare the additive composition in the required amount. 540 mL of the prepared additive composition was further dispensed into each GREX flask and cultured at 37°C in a 5% CO2 incubator. - Day 14 The cultured cells were carefully aspirated while leaving 200 mL of medium, and the remaining 200 mL of medium was gently shaken to suspend and collect in a 500 mL Corning bottle. The collected cells were centrifuged at 2000 g for 7 minutes at room temperature. After centrifugation, the supernatant was completely removed, and the precipitated cells were suspended in 260 mL of excipient. The suspended cells were counted using a hemocytometer set to determine the cell viability. After cell counting, 200 mL of cells were loaded into an infusion bag.

[0498] Experimental example 2. Experiments on TIL proliferation yield According to the experimental protocol for TIL generation, an experiment was carried out to measure the proliferation yield of TIL (Example 1).

[0499] After obtaining TILs generated according to the experimental protocol for TIL generation (Experimental Example 1), cells were counted using a hemocytometer set to determine cell viability.

[0500] Referring to FIG. 5, the results of the TIL expansion yield experiment confirmed that TILs expanded at least 2,500-fold and up to 9,400-fold during the TIL expansion treatment.

[0501] Experimental example 3. IL-2 optimization experiments Experimental Example 3-1. Experiments on optimizing IL-2 during TIL isolation / culture 1. TIL isolation / culture treatment for IL-2 optimization - Day 0 The tumor tissue removed from the operating room of the hospital was washed three times with RPMI1640 medium and transferred to a 100 mm Petri dish. The lipid tissue in the tumor tissue was removed using sterilized surgical scissors and tweezers, and the tumor tissue was cut into 1-2 mm pieces using sterilized surgical scissors and tweezers. Two pieces of tumor tissue cut into 1-2 mm pieces were placed in a 24-well plate, and 1 mL of the prepared culture composition according to the IL-2 concentration was added, followed by culturing at 37°C for 7 days in a 5% CO2 incubator. - Day 7 0.5 mL of the culture composition prepared according to the IL-2 concentration was added to each well, and the cells were cultured at 37° C. in a 5% CO 2 incubator for 3 days. - Day 10 0.5 mL of the prepared culture composition according to the IL-2 concentration was added to each well, and the cells were cultured at 37° C. in a 5% CO 2 incubator for 2 days. - Day 12 0.5 mL of the prepared culture composition according to the IL-2 concentration was added to each well, and the cells were cultured at 37° C. in a 5% CO 2 incubator for 2 days. - Day 14 To collect the cells, a 40 μm cell strainer was placed on top of a 50 mL tube. The tumor tissue was filtered by passing the cultured cells through the cell strainer using a 10 mL pipette, and only the cultured cells were collected in a 50 mL conical tube, which was then centrifuged at 450 g for 5 minutes at room temperature. 1 mL of DPBS was added to the precipitated cells, which were then suspended by pipetting. The suspended cells were counted using a hemocytometer (C-chip) to determine the cell viability, and thereby the cell proliferation rate for each concentration of IL-2.

[0502] Referring to FIG. 6, in the presence of human AB serum, when the concentration of IL-2 was 1,000 IU / mL or higher, the cell proliferation rate did not show a significant difference.

[0503] Experimental Example 3-2. Experiments on optimizing IL-2 concentration during TIL proliferation 1. Isolation of Allofeder PBMCs PBMC isolation was performed in the same manner as in Experimental Example 1-2. TIL expansion treatment 2. TIL expansion treatment for IL-2 optimization - Day 0 (cell seeding) The half-product vials stored in a liquid nitrogen tank (LN2) were thawed in a water bath at 37°C for 2 minutes. The thawed cells were washed with CTS-AIM-V medium and centrifuged at 1,500 rpm for 5 minutes at room temperature. After discarding the supernatant, the resulting pellet was suspended in CTS-AIM-V growth composition, and the cells were counted using a hemocytometer set to determine cell viability. 5E4 half-product TIL cells and 1E7 irradiated PBMCs were placed in each T75 flask culture vessel, and 3 mL of growth composition was added. The resulting cell mixture was further cultured at 37°C in a 5% CO2 incubator. - Day 4 IL-2 was placed in CTS-AIM-V medium containing 3 vol% human serum at 1000, 2000, 4000, and 6,000 IU / mL, respectively, to prepare the additive composition in the required amount. 6 mL of the prepared additive composition was further dispensed into each T75 flask and cultured at 37°C in a 5% CO2 incubator. - Day 6 IL-2 was placed in CTS-AIM-V medium containing 3 vol% human serum at 1000, 2000, 4000, and 6,000 IU / mL, respectively, to prepare the additive composition in the required amount. 18 mL of the prepared additive composition was further dispensed into each T75 flask and cultured at 37°C in a 5% CO2 incubator. - Day 8 IL-2 was placed in CTS-AIM-V medium containing 3 vol% human serum at 1000, 2000, 4000, and 6,000 IU / mL, respectively, to prepare the additive composition in the required amount. 54 mL of the prepared additive composition was further dispensed into each T75 flask and cultured at 37°C in a 5% CO2 incubator. - Day 10 IL-2 was placed in CTS-AIM-V medium containing 3 vol% human serum at 1000, 2000, 4000, and 6,000 IU / mL, respectively, to prepare the additive composition in the required amount. 162 mL of the prepared additive composition was further dispensed into each T75 flask and cultured at 37°C in a 5% CO2 incubator. - Day 14 cultured cells were carefully aspirated while leaving 150 mL of medium, and the remaining 150 mL of medium was gently shaken to suspend the cells, and collected in a 50 mL tube. The collected cells were centrifuged at 2000 g for 7 minutes at room temperature. After centrifugation, the supernatant was completely removed, and the precipitated cells were suspended in 30 mL of DPBS. The suspended cells were counted using a hemocytometer set to determine the cell viability.

[0504] 7, it can be seen that the composition supplemented with 2,000 IU / mL of IL-2 is sufficient for expanding TILs, and furthermore, when considering both cell expansion yield and cell viability, it can be seen that the composition supplemented with 2,000 IU / mL of IL-2 is the most efficient for TIL expansion.

[0505] Experimental example 4. Experiments on optimization of culture composition amounts 1. TIL isolation / culture process for optimization of culture composition volumes - Day 0 The tumor tissue removed from the operating room of the hospital was washed three times with RPMI1640 medium and transferred to a 100 mm Petri dish. The lipid tissue in the tumor tissue was removed using sterilized surgical scissors and tweezers, and the tumor tissue was cut into pieces of 1-2 mm in size using sterilized surgical scissors and tweezers. Two pieces of tumor tissue cut into pieces of 1-2 mm in size were placed in a 24-well plate, and 1 mL or 2 mL of the prepared culture composition was added, followed by culturing at 37°C for 7 days in a 5% CO2 incubator. - Day 7 To the wells to which 1 mL of the composition was added on day 0, 0.5 mL of the prepared culture composition was added, and from the wells to which 2 mL of the composition was added on day 0, 1 mL of the composition was carefully removed and then 1 mL of the composition was added thereto, followed by culturing at 37° C. for 3 days in a 5% CO2 incubator. - Day 10 To wells to which 1 mL of the composition was added on day 0, 0.5 mL of the prepared culture composition was added, and from wells to which 2 mL of the composition was added on day 0, 1 mL of the composition was carefully removed and then 1 mL of the composition was added thereto, followed by culturing at 37° C. for 3 days in a 5% CO2 incubator. - Day 12 To wells to which 1 mL of the composition was added on day 0, 0.5 mL of the prepared culture composition was added, and from wells to which 2 mL of the composition was added on day 0, 1 mL of the composition was carefully removed and then 1 mL of the composition was added thereto, followed by culturing at 37° C. for 3 days in a 5% CO2 incubator. - Day 14 To collect cells, a 40 μm cell strainer was placed on top of a 50 mL tube. The tumor tissue was filtered by passing the cultured cells through the cell strainer using a 10 mL pipette, and only the cultured cells were collected in a 50 mL conical tube, which was then centrifuged at 450 g for 5 minutes at room temperature. 1 mL of DPBS was added to the precipitated cells, which were then suspended by pipetting. The suspended cells were counted using a hemocytometer (C-chip) to determine the cell viability, thereby determining the cell proliferation rate according to the amount of culture composition added on day 0.

[0506] Referring to FIG. 8, as a result of an experiment using a 24-well plate, it was confirmed that the cell proliferation rate was higher when 1 mL of the composition was used than when 2 mL of the composition was used.

[0507] Experimental Example 5. Experiments for counting cells obtained per tumor fragment In the same manner as in Experimental Example 1-1, TIL isolation / culture processing and experiments were performed using lung cancer (LC) tumor tissue, head and neck cancer (HNC) tumor tissue, and breast cancer (BC) tumor tissue. As a result, the average number of cells obtained per tumor fragment was confirmed. Referring to FIG. 9, in the case of LC tumor tissue, approximately 4E5 cells were obtained on average per tumor fragment. In the case of HNC tumor tissue, approximately 6E5 cells were obtained on average per tumor fragment. In the case of BC tumor tissue, approximately 6E5 cells were obtained on average per tumor fragment.

[0508] Experimental Example 6. Experiments to confirm the percentage of T cells in cultured and expanded TILs 1. Preparation of antibodies to confirm T cell percentages As antibodies to confirm the T cell percentage, antibodies against CD3 and CD45 were prepared, and an isotype control antibody was also prepared.

[0509] Table 2. Information about antibodies to check T cell percentages [Table 2] 2. Pre-processing for FACS Analysis Cultured and expanded TILs were placed in 15 mL or 50 mL conical tubes, washed twice with FACS buffer (2% FBS in DPBS) and suspended in FACS buffer. 5 The suspended cells were placed in a 1.7 mL microcentrifuge tube and centrifuged at 1,500 rpm for 5 minutes at 4°C, and all but 200 μL of the supernatant was discarded. The remaining supernatant was vortexed to resuspend the cells, and 5 μL of each antibody was placed in each tube as below.

[0510] Table 3. Antibodies added to the tubes [Table 3] After the antibody was added, the cells were incubated in the dark at 4°C for 30 minutes. After the reaction, the cells were washed three times with FACS buffer and centrifuged at 1,500 rpm at 4°C for 5 minutes, and the supernatant was discarded except for approximately 200 μL. The remaining supernatant was vortexed to suspend the cells, and 300 μL of FACS buffer was added to perform FACS analysis. 3. FACS Data Analysis After loading the data from the FACS machine into a FSC / SSC dot plot, the lymphocyte region was gated. After loading the gated lymphocyte population into the dot plot, CD3-APC-cy7 was set on the x-axis, CD45-Amcyan was set on the y-axis, and four quadrants were set. The percentage of CD3 positive and CD45 positive T cells in the four quadrants was confirmed.

[0511] 10, the percentage of T cells in the TILs generated according to Experimental Example 1 was measured, and it was found that 99.7%, 99.4%, or 98.4% of the generated TILs were T cells. In other words, it was confirmed that a high proportion of T cells were present in the TILs generated according to the generation process disclosed in the present application.

[0512] Experimental Example 7. Expansion using the half-products stored frozen for a long period of time (production of the final product) and confirmation of the percentage of T cells in the expanded TILs Propagation of frozen stored semi-products 1. Isolation of Allofeder PBMCs Blood obtained from normal donors was prepared through leukapheresis and irradiated with 60 Gy of X-rays. 15 mL of Ficoll solution was placed at the bottom of a 50 mL conical tube without air bubbles. After dividing the collected blood into multiple 50 mL conical tubes, the amount of blood was measured. The blood divided into multiple 50 mL conical tubes was diluted in a ratio of 1:2 (blood:DPBS). 30 mL of diluted blood was carefully added to a 50 mL conical tube filled with 15 mL of Ficoll solution, then centrifuged at 800 g for 20 minutes at room temperature under Acc9 / Dcc0 conditions. Only the PBMC layer was collected and transferred to a new 50 mL conical tube, then centrifuged at 800 g for 5 minutes at room temperature under Acc9 / Dcc9 conditions. After centrifugation, the supernatant was completely removed, DPBS was filled up to 50mL, washed, and then centrifuged at 800g for 5 minutes at room temperature under Acc9 / Dcc9 conditions. After centrifugation, the supernatant was completely removed, and the precipitated cells were suspended in DPBS. The suspended cells were counted using a hemocytometer set to determine the cell viability. After cell counting, the required number of cells was prepared according to the ratio of 1:200 (TIL:PBMC cultured for 2 weeks). The prepared cells were centrifuged at 800g for 5 minutes at room temperature under Acc9 / Dcc9 conditions. After centrifugation, the supernatant was completely removed, and the precipitated cells were suspended in the final product culture complete medium and used in the manufacture of the final product. 2. End product formation - Day 0 (Thawing and cell seeding) A vial of half-product that had been stored in a liquid nitrogen tank (LN2) for a long period of one year or longer was thawed. The TIL half-product was transferred from the thawed vial to a 50 mL conical tube. The TIL half-product was washed with AIM-V medium (volume ratio of 9:1 (medium:TIL half-product)). The TIL half-product was centrifuged at 1,500 rpm for 5 minutes at room temperature under Acc9 / Dcc9 conditions, the supernatant was discarded, and the precipitated cells were suspended in CTS-AIM-V medium, followed by cell counting and confirmation of cell viability. The half-product TIL cells of 4E5 and the irradiated PBMCs (alloPBMCs) of 8E7 were placed in a GREX100M (1 L) culture vessel, respectively, and then the final product expansion composition was added to a final volume of 20 mL. Incubation was performed at 37° C. in a 5% CO2 incubator.

[0513] The final product growth composition consisted of the following: CTS-AIM-V medium, 3 vol% human serum, 2,000 IU / mL IL-2, and 30 ng / mL OKT3. - Day 4 The required amount of growth composition was prepared by supplementing CTS-AIM-V medium with IL-2 and fractionated human serum to 2,000 IU / mL and 3%, respectively. 20 mL of the prepared growth composition was further added to each GREX flask and incubated at 37° C. in a 5% CO2 incubator. - Day 6 The required amount of growth composition was prepared by supplementing CTS-AIM-V medium with IL-2 and fractionated human serum to 2,000 IU / mL and 3%, respectively. 60 mL of the prepared growth composition was further added to each GREX flask and incubated at 37° C. in a 5% CO incubator. - Day 8 The required amount of growth composition was prepared by supplementing CTS-AIM-V medium with IL-2 and fractionated human serum to 2,000 IU / mL and 3%, respectively. 180 mL of the prepared growth composition was further added to each GREX flask and incubated at 37° C. in a 5% CO2 incubator. - Day 10 The required amount of growth composition was prepared by supplementing CTS-AIM-V medium with IL-2 and fractionated human serum to 2,000 IU / mL and 3%, respectively. 540 mL of the prepared growth composition was further added to each GREX flask and incubated at 37° C. in a 5% CO2 incubator. - Day 14 cultured cells were carefully aspirated while leaving 200 mL of medium, and the remaining 200 mL of medium was gently shaken to suspend and collect in a 500 mL Corning bottle. 150 mL of DPBS was placed in the flask, where cell harvesting was performed to collect the cells in the vessel (repeated twice). The collected cells were centrifuged at 2,000 g for 7 minutes at room temperature. After centrifugation, the supernatant was completely removed, and DPBS was added up to 500 mL for washing (2,000 g, 7 minutes, room temperature, Acc9 / Dcc9, repeated three times). After centrifugation, the supernatant was completely removed, and the precipitated cells were suspended in DPBS. The suspended cells were counted using a hemocytometer set to determine cell viability. Then, a portion of the cells was used for the following confirmation test, and the remaining cells were centrifuged at 3 × 10 in CS10. 7 The cells were suspended at 100 cells / mL, aliquoted into cryogenic vials, and frozen (-80°C) in a cryocontainer using a deep freezer.

[0514] Confirmation of cell viability upon thawing of frozen preservatives method The frozen half-products were thawed, and cell viability was confirmed after thawing. One vial each of TIL half-products from three different cases (BC16208, BC17058, and BC18050) that had been stored in liquid nitrogen for different periods of time was thawed. After thawing, cell viability was confirmed.

[0515] result As a result of checking the cell viability after thawing, the cell viability after thawing was 91.25% in the case of BC16208, 95.88% in the case of BC17058, and 69.37% in the case of BC18050.

[0516] Table 4. Confirmation of cell viability upon thawing of semi-finished products [Table 4] Confirmation of yield of final product method Thawed TIL half-products from three cases (BC16208, BC17058, BC18050) were used to prepare final products, and the final harvested cell numbers, cell viability, and cell proliferation rates were measured.

[0517] result In the case of BC16208, it is 1.31x10 9 In the case of BC17058, the number of cells was 1.71x10 9 In the case of BC18050, the number of cells is 1.42x10 9 It was confirmed that the cells in the above cases proliferated approximately 3,270, 4,275, and 3,540 times, respectively. It was confirmed that all of the above cases showed high cell viability of 90% or higher.

[0518] Table 5. Conditions for seeding semi-product cells to prepare the final product [Table 5] Table 6. Product yield in the production of the final product [Table 6] Confirmation of cell percentage in final product FACS analysis The cell percentage in the final product was confirmed by FACS analysis. The cells used for FACS analysis were suspended in FACS buffer (2% FBS in DPBS) and centrifuged (1,500 rpm, 4°C, 5 min) (repeated twice). After centrifugation, the cells were suspended in FACS buffer. 1–5 × 10 of suspended cells were 5The cells were dispensed into 1.7 mL microtubes. For surface marker staining, each tube was treated with 2 μL of antibody. Incubation was performed in the dark at 4° C. for 30 minutes. After incubation, 800 μL of FACS buffer was added, followed by centrifugation (1,500 rpm, 4° C., 5 minutes). After discarding the supernatant, the cells were suspended by vortexing, 800 μL of FACS buffer was added thereto, followed by centrifugation (1,500 rpm, 4° C., 5 minutes) (repeated three times). After suspension using 200 μL of FACS buffer, the cells were transferred to a 5 mL FACS tube and analyzed using a BD FACS CantoTM II flow cytometer.

[0519] result FACS analysis of the final product produced confirmed the expression of T cell markers CD3 and CD45. To perform the analysis on viable cells only, cells were first gated on SSC-A and FSC-A (top left graphs in Figures 12, 14, and 16). The gated viable cells are now displayed as lymphocytes in the top left graphs in Figures 12, 14, and 16. As a result of the experiment, the percentage of T lymphocytes expressing both CD3 and CD45 (CD3+ / CD45+) was 99.5% in the case of BC16208, 99.4% in the case of BC17058, and 99.1% in the case of BC18050. Furthermore, in the case of BC16208, the percentage of CD4+ T cells was 83.6%, and the percentage of CD8+ T cells was 13.2%, in the case of BC17058, the percentage of CD4+ T cells was 93.2%, and the percentage of CD8+ T cells was 4.71%, and in the case of BC18050, the percentage of CD4+ T cells was 78.4%, and the percentage of CD8+ T cells was 14.0% (see Figures 12, 14, and 16). For reference, in the table below, the percentage of each type of T cells among CD3+ / CD45+ lymphocytes is a percentage converted based on the FACS results with CD3+ / CD45+ lymphocytes as 100%. The analysis results are summarized in Table 7 below.

[0520] Table 7 Checking cell percentages per case [Table 7] Furthermore, the percentage of memory type CD4+ T cells in the CD4+ T cells confirmed above was confirmed through CD45RA and CCR7 expression. As a result, the percentage of effector memory T cells (Tem) was confirmed to be 81.35%, 83%, 84%, and 73.11% in each case of BC16208, BC17058, and BC18050 (see Figure 13, Figure 15, and Figure 17). Tscm indicates stem cell memory T cells, Tcm indicates central memory T cells, Tem indicates effector memory T cells, and Teff indicates effector T cells. The analysis results are summarized in Table 8 below.

[0521] Table 8. Confirmation of CD4+ T cell type in each case [Table 8] Furthermore, the percentage of memory type CD8+ T cells among the CD8+ T cells identified above was confirmed through CD45RA and CCR7 expression. As a result, the percentage of effector memory T cells (Tem) was confirmed to be 11.40%, 2.20%, and 10.90% in each case of BC16208, BC17058, and BC18050. The analysis results are summarized in Table 9 below.

[0522] Table 9 Confirmation of CD8+ T cell type in each case [Table 9] Verify the purity of the final product Analysis by FACS Cells to be used in FACS analysis were suspended in FACS buffer (2% FBS in DPBS) and centrifuged (1,500 rpm, 4°C, 5 min) (repeated twice). After centrifugation, the cells were suspended in FACS buffer. 1-5 × 10 of suspended cells were 5 The cells were dispensed into 1.7 mL microtubes. For surface marker staining, each tube was treated with 2 μL of antibody. Incubation was performed in the dark at 4° C. for 30 min. After incubation, 800 μL of FACS buffer was added, followed by centrifugation (1,500 rpm, 4° C., 5 min). After discarding the supernatant, the cells were suspended by vortexing, 800 μL of FACS buffer was added thereto, followed by centrifugation (1,500 rpm, 4° C., 5 min) (repeated three times). After suspension using 200 μL of FACS buffer, the cells were transferred to 5 mL FACS tubes and analyzed using a BD FACS CantoTM II flow cytometer.

[0523] Results To confirm the purity of the final product, the expression of CD19, a B cell marker, and CD56, a NK cell marker in non-T cells, was confirmed by FACS analysis. To perform an analysis on viable cells only, cells were first gated on SSC-A and FSC-A (left graphs in Figures 18, 20, and 22). Here, the gated viable cells are displayed as lymphocytes in the left graphs in Figures 18, 20, and 22. In the cases of BC16208, BC17058, and BC18050, the CD19 expression rates were confirmed to be 0.03%, 0.01%, and 0.01%, respectively. Furthermore, in the cases of BC16208, BC17058, and BC18050, the CD56 expression rates were confirmed to be 0.02%, 0.00%, and 0.03%, respectively (see Figures 18 to 23). The analysis results are summarized in Table 10 below.

[0524] Table 10. Verify the purity of the final product [Table 10] Experimental Example 8. Confirmation of the effect of immune cell composition 1 containing TIL - Confirmation of tumor-inhibiting efficacy of TIL derived from breast cancer patients The in vivo tumor-inhibitory efficacy of TILs cultured from tumor tissue of breast cancer patients was confirmed in patient-derived xenografts (PDX) from the same patients.

[0525] Experimental Example 8-1. Confirmation of the tumor-inhibiting effect of TIL and confirmation of the tumor-inhibiting effect of simultaneous administration of TIL and IL-2 method 1. Preparation of patient-derived xenografts (PDX) PDX were prepared by the following method.

[0526] Acclimated 5-week-old NOD-SCID mice (NOD.CB17-Prkdc scid The mice were restrained in a 10-mm x 10-mm tether (Koatech). Mice were anesthetized with 20 mg / mL tribromoethanol (1x Avertin) administered intraperitoneally at 250 mg / kg. Subjects were identified by attaching ear tags to the anesthetized mice's ears using an ear punch. Hair was removed from the surgical site using an epilator. An incision was made in the abdominal subcutaneous tissue approximately 1.5 cm long. Blunt separation between the subcutaneous tissue and the peritoneum was performed using straight forceps (iris forceps). A 1-2 mm cut was made. 2 A patient-derived tumor tissue measuring the size of 100 mm was implanted into the right fourth mammary fat pad of the animal. After implantation was completed, the incised subcutaneous tissue was sutured using autoclips and disinfected with povidone. The animal was placed in a cage and covered with leaf litter to maintain body temperature. The animal information was recorded on the cage name tag, and the animal was confined to an individually ventilated cage (IVC) rack, after which it was confirmed that the animal had woken up from anesthesia. 2. Preparation of TILs Frozen TILs (i.e., cryopreserved final product, BC20082-TIL sample, produced according to the method of Experimental Example 1 or Experimental Example 7) cryopreserved in liquid nitrogen after expansion were thawed in a water bath at 37°C. To remove DMSO present in the cryopreservation solution, the volumes of warm CTS-AIM-V medium and the thawed final product were mixed in a ratio of 9:1. Centrifugation was performed at 1,500 rpm for 5 minutes at room temperature. After discarding the supernatant, the precipitated cells were suspended in CTS-AIM-V medium and counted. After centrifugation at 1,500 rpm for 5 minutes at room temperature, the supernatant was removed and the cell pellet was suspended in DPBS, and the number of TILs suitable for administration to mice was titrated as shown in the table below. The doses of TILs are listed in the table below. G1 is the control. G2 is 1 x 10 7 G3 was administered 5 × 10 TILs and is referred to as the low-dose TIL administration group. 7 G4 is the IL-2 administration group, receiving 1.5 × 10 of IL-2 daily from day 0 to day 5. 5 G4 is referred to as IL-2 administration control. G5 is a group administered TIL and IL-2 simultaneously, which was administered 1 × 10 IU daily from day 0 to day 5 after TIL administration. 7 TILs and 1.5 × 10 5 IU of IL-2 every day from day 0 to day 5 after TIL administration. G5 is referred to as the low-dose TIL+IL-2 co-administration group. G6 is the TIL+IL-2 co-administration group, which is administrated with 5×10 7 TILs of 1.5x10 5 IU of IL-2 was administered to each mouse. G6 is referred to as the high-dose TIL+IL-2 co-administration group.

[0527] Table 11. Dosage information for TIL and / or IL-2 [Table 11] 3. TIL Injection and Mouse Observation Approximately 13 to 63 mm 3Mice with tumor volumes of 1.5×10 were grouped. The mice were restrained using a restrainer, and then the blood vessels were relaxed by placing the tail in warm water. TIL and IL-2 were administered intravenously at 1 min / head using a 30G insulin syringe. TIL was administered once on day 0. In the simultaneous administration group, IL-2 was administered at 1.5×10 on days 0, 1, 2, 3, 4, and 5 after TIL administration. 5 IU / animal. That is, IL-2 was administered at 1.5 × 10 5 After administration, tumor volumes were measured twice a week and body weights were measured once a week. Tumor volumes were recorded when the tumor volume reached 2,000 mm 3 Observe until it reaches 2,000 mm 3 When the tumor volume exceeded 100 μg / kg, the mice were euthanized using CO2. After euthanasia of the mice, the tumors were removed and the tumor sizes were measured.

[0528] result 1. Changes in mouse weight Confirmation of changes in mouse body weight due to TIL administration The changes in body weight after administration of TILs to mice were observed in G1, G2 (low-dose TIL administration group), and G3 (high-dose TIL administration group). The results are shown in Figure 24. As a result of the observation, it was confirmed that there was no change in body weight due to cell administration.

[0529] Confirmation of changes in body weight due to simultaneous administration of TIL and IL-2 Furthermore, changes in body weight after administration of TIL to mice were observed in G4 (IL-2 administration control), G5 (low-dose TIL+IL-2 co-administration group), and G6 (high-dose TIL+IL-2 co-administration group). The results are shown in Figure 25. As a result of the observation, it was confirmed that there was no change in body weight due to cell administration. 2. Measurement of changes in tumor volume Confirmation of changes in tumor volume due to TIL administration In G1, G2 (low-dose TIL administration group), and G3 (high-dose TIL administration group), changes in tumor volume after administration of TIL to mice were observed. The results are shown in Figures 26 and 27. Figures 28 and 29 are images of the tumor implantation site of the mouse taken 28 days after TIL administration. Furthermore, the mouse was euthanized 38 days after TIL administration, the tumor was excised, and the tumor volume was measured. The results are shown in Figure 30, and an image of the excised tumor is shown in Figure 31. As a result of the measurement, it was confirmed that tumor growth was suppressed in G2 (low-dose TIL administration group) and G3 (high-dose TIL administration group), and the tumor volume was smaller than that of the control.

[0530] Confirmation of changes in tumor volume by simultaneous administration of TIL and IL-2 In G4 (IL-2 administration control), G5 (low-dose TIL+IL-2 co-administration group), and G6 (high-dose TIL+IL-2 co-administration group), changes in tumor volume were observed after administration of TIL to mice. The results are shown in Figures 32 and 33. Figures 34 and 35 are images of the tumor implantation site of the mouse 28 days after TIL administration. Furthermore, on the 38th day after TIL administration, the mouse was euthanized, the tumor was excised, and the tumor volume was measured. The results are shown in Figure 36, and an image of the excised tumor is shown in Figure 37. As a result of the measurement, it was confirmed that tumor growth was suppressed in G5 (low-dose TIL+IL-2 co-administration group) and G6 (high-dose TIL+IL-2 co-administration group), and the tumor volume was smaller than that of the control.

[0531] Experimental Example 9. Confirmation of efficacy of immune cell composition containing TIL - Confirmation of tumor-inhibiting efficacy of TIL derived from lung cancer patients The in vivo tumor-inhibitory efficacy of TILs cultured from tumor tissues of lung cancer patients was confirmed in patient-derived xenografts (PDXs) derived from the same patients.

[0532] method 1.PDX preparation PDX were prepared by the following method.

[0533] Acclimated 5-week-old NOD-SCID mice (NOD.CB17-Prkdc scid The mice were restrained in a suction cup (Koatech). Mice were anesthetized with 20 mg / mL tribromoethanol (1x Avertin) administered intraperitoneally at 250 mg / kg. Subjects were identified by attaching ear tags to the anesthetized mice's ears using an ear punch. Hair was removed from the surgical site using an epilator. An incision was made in the subcutaneous tissue above the thigh muscle, approximately 1 cm. Blunt separation between the subcutaneous tissue and fascia was performed using straight forceps (Iris Forceps). A 1-2 mm cut was made. 2 A patient-derived tumor tissue with a size of 100 mm was implanted subcutaneously in the flank of the animal at the same position on the right side. After implantation was completed, the incised subcutaneous tissue was sutured using autoclips and disinfected with povidone. The animal was placed in a cage and covered with leaf litter to maintain body temperature. The animal information was recorded on the cage name tag, and the animal was confined to an individually ventilated cage (IVC) rack, after which it was confirmed that the animal had woken up from anesthesia. 2. Preparation of TILs Frozen TILs (i.e., the cryopreserved final product, LC20001-TIL sample, produced according to the method of Experimental Example 1 or Experimental Example 7) cryopreserved in liquid nitrogen after expansion were thawed in a water bath at 37°C. To remove DMSO present in the cryopreservation solution, the warm CTS-AIM-V medium and the final product were mixed in a volume ratio of 9:1. After centrifugation at 1,500 rpm for 5 minutes at room temperature and removal of the supernatant, the precipitated cells were suspended in CTS-AIM-V medium and counted. After centrifugation at 1,500 rpm for 5 minutes at room temperature, the supernatant was removed and the cell pellet was suspended in DPBS, and the number of TILs was counted at 5 x 10 per 200 μL. 7 The dose of TIL is shown in the table below. The control was a group that received only IL-2, where 1.5×10 5 IU of IL-2 was administered daily from day 0 to day 5. The control is called IL-2. The cell administration group was a group in which TIL and IL-2 were administered simultaneously, where 5×10 7 of TILs and 1.5 × 10 5IU of IL-2 was administered. The cell-administered group is referred to as TIL+IL-2.

[0534] Table 12. Dosage information for TIL and / or IL-2 [Table 12] 3. Simultaneous administration of TIL and IL-2 Approximately 13 to 63 mm 3 Mice with tumor volumes of 1.5×10 were grouped. The mice were restrained using a restrainer, and then the blood vessels were relaxed by placing the tail in warm water. TIL and IL-2 were administered intravenously at 1 min / head using a 30G insulin syringe. TIL was administered once on day 0. In the simultaneous administration group, IL-2 was administered at 1.5×10 on days 0, 1, 2, 3, 4, and 5 after TIL administration. 5 IU / animal. That is, IL-2 was administered at 1.5 × 10 5 IU / head. After administration, tumor volumes were measured twice a week and body weights were measured once a week. Tumor volumes were recorded when the tumor volume reached 2,000 mm 3 Observe until it reaches 2,000 mm 3 When the tumor volume exceeded 100 μg / kg, the mice were euthanized using CO2. After euthanasia of the mice, the tumors were removed and the tumor sizes were measured.

[0535] result 1. Changes in mouse weight After intravenous administration of the control (IL-2) and the cell administration group (TIL+IL-2), changes in body weight were observed. The results are shown in Figure 38. As a result of the observation, it was confirmed that there was no change in body weight due to cell administration. 2. Measurement of changes in tumor volume The tumor volumes were measured twice one week after intravenous administration in the control group administered only IL-2 and the cell administration group administered simultaneously with TIL and IL-2. The results are shown in Figures 39 to 41. Figure 42 is an image of the tumor implantation site in the mouse 64 days after TIL administration. Furthermore, on the 70th day after TIL administration, the mouse was euthanized, the tumor was excised, and the tumor volume was measured. The results are shown in Figure 43. As a result of the measurements, it can be confirmed that tumor growth was suppressed in the cell administration group, and the tumor volume was smaller than that of the control.

Claims

1. 1. A method for producing an immune cell composition, comprising: (a) culturing a tumor sample in a first container; wherein the tumor specimen is cultured in a first composition in the first container, the first composition comprising medium, serum, and 500-1500 IU / mL IL-2; wherein the first container is a gas-permeable container or a container having a gas supply system; wherein the culturing is carried out for 13 to 15 days; wherein a first additive composition is added to the first container one or two or more times during the culturing, the first additive composition comprising medium, serum, and IL-2; (b) obtaining a first population of immune cells from the cultured product obtained from (a); (c) seeding a second composition and a second population of immune cells into a second container; wherein the second immune cell population is derived from the first immune cell population; wherein the second composition comprises a medium, serum, 1000 to 3000 IU / mL IL-2, an anti-CD3 antibody, and a feeder cell; wherein the second container is a gas permeable container or a container having a gas supply system; (d) expanding the second immune cell population to generate a third immune cell population; wherein a second additive composition is added to the second container one or two or more times during said expansion, said second additive composition comprising medium, serum, and IL-2; wherein the propagation is for 13 to 15 days; and (e) obtaining the third immune cell population from the expansion product obtained from (d) and producing the immune cell composition; A method for providing the above.

2. The method further comprises preparing the tumor sample, The step of preparing the tumor sample is performed prior to (a). The method of claim 1.

3. In (a), the tumor sample is obtained from a tumor fragment obtained from the subject; The tumor sample was approximately 1 mm ２ ~4mm ２ having a cross-sectional area of The method according to claim 1 or 2.

4. In (a), 12 to 24 of the tumor samples are cultured.

4. The method according to any one of claims 1 to 3.

5. The concentration of serum in the first composition is 5 vol% or less.

5. The method according to any one of claims 1 to 4.

6. The serum concentration of the first composition is about 3 vol%.

6. The method according to any one of claims 1 to 5.

7. The medium of the first composition is CTS-AIM-V medium.

7. The method according to any one of claims 1 to 6.

8. The concentration of IL-2 in the first composition is about 1000 IU / mL.

8. The method according to any one of claims 1 to 7.

9. The volume of the first composition is 30% or less of the maximum allowable capacity of the first container.

9. The method according to any one of claims 1 to 8.

10. The first additive composition is a composition having the same compositional properties and ratios as the first composition.

10. The method according to any one of claims 1 to 9.

11. the concentration of the IL-2 in the first additive composition is 1000 IU / mL; The concentration of the serum in the first additive composition is 3 vol %; The medium of the first additive composition is CTS-AIM-V medium.

11. The method according to any one of claims 1 to 10.

12. During the incubation, the first additive composition is added to the first vessel two or more times.

12. The method according to any one of claims 1 to 11.

13. During the culture, the first additive composition is added to the first container on days 7, 10, and 12 from the start of the culture; The amount of the first additive composition added at each addition point is the same. The method of claim 12.

14. The first container is a 24-well plate, a T25 flask, a T75 flask, a T125 flask, GREX10, GREX10M, GREX10M-CS, GREX100, GREX100M, GREX100M-CS, GREX500, GREX500M, GREX500M-CS, or a culture bag.

14. The method according to any one of claims 1 to 13.

15. The first container is a T75 flask and the volume of the first composition is 6 ml.

15. The method according to any one of claims 1 to 14.

16. The second immune cell population is obtained from the first immune cell population.

16. The method according to any one of claims 1 to 15.

17. The second immune cell population is obtained by a method comprising washing the first immune cell population.

17. The method of claim 16.

18. 18. The method of any one of claims 1 to 17, wherein the medium of the second composition is CTS-AIM-V.

19. The concentration of the serum in the second composition is 5 vol% or less.

19. The method according to any one of claims 1 to 18.

20. The concentration of the serum in the second composition is 3 vol% or less.

20. The method of any one of claims 1 to 19.

21. The concentration of the IL-2 in the second composition is about 2000 IU / mL.

21. The method according to any one of claims 1 to 20.

22. The anti-CD3 antibody is OKT3.

22. The method of any one of claims 1 to 21.

23. The concentration of the anti-CD3 antibody in the second composition is between 10 ng / mL and 100 ng / mL.

23. The method of any one of claims 1 to 22.

24. The concentration of the anti-CD3 antibody in the second composition is about 30 ng / mL.

24. The method of any one of claims 1 to 23.

25. The feeder cells are inactivated allogeneic peripheral blood mononuclear cells 25. The method of any one of claims 1 to 24.

26. The feeder cells are included in the second composition at a ratio of approximately 1:200 (number of cells in the second immune cell population: number of feeder cells) based on the number of cells in the second immune cell population.

26. The method of any one of claims 1 to 25.

27. In the seeding step, the number of cells of the second immune cell population seeded in the second container is 1E5 to 10E5.

27. The method of any one of claims 1 to 26.

28. In the seeding step, the number of cells of the second immune cell population seeded into the second container is about 4E5.

28. The method of any one of claims 1 to 27.

29. The volume of the second composition is 30% or less of the maximum allowable volume of the second container.

29. The method of any one of claims 1 to 28.

30. During the expansion, immune cells are not removed from the second container or are added exogenously.

30. The method of any one of claims 1 to 29.

31. The second additive composition has the same compositional properties and ratios as the second composition, except for the anti-CD3 antibody and the feeder cells.

31. The method of any one of claims 1 to 30.

32. the concentration of the IL-2 in the second additive composition is about 2000 IU / mL; the concentration of the serum in the second additive composition is about 3% by volume; The medium of the second additive composition is CTS-AIM-V.

32. The method of any one of claims 1 to 31.

33. During the growth, the second additive composition is added to the second vessel two or more times.

33. The method of any one of claims 1 to 32.

34. The amount of the second additive composition added varies at each addition point.

34. The method of claim 33.

35. During the growth, the second additive composition is added to the second container on days 4, 6, 8, and 10 from the start of the growth, with the amount of the second additive composition added gradually increasing at each addition point.

35. The method of claim 33 or 34.

36. The second container is a 24-well plate, a T25 flask, a T75 flask, a T125 flask, GREX10, GREX10M, GREX10M-CS, GREX100, GREX100M, GREX100M-CS, GREX500, GREX500M, GREX500M-CS, or a culture bag.

36. The method of any one of claims 1 to 35.

37. The second container is GREX 100M and the volume of the second composition is about 20 ml.

37. The method of any one of claims 1 to 36.

38. 20 ml of the second additive composition is added to the second container on day 4 from the start of the growth, 60 ml on day 6 from the start of the growth, 180 ml on day 8 from the start of the growth, and 540 ml on day 10 from the start of the growth.

38. The method of claim 37.

39. The immune cell composition comprises the third immune cell population.

39. The method of any one of claims 1 to 38.

40. The immune cell composition includes a fourth immune cell population obtained from the third immune cell population.

40. The method of any one of claims 1 to 39.

41. The tumor sample is derived from a solid tumor from a subject with breast cancer, colon cancer, lung cancer, gastric cancer, renal cancer, ovarian cancer, or skin cancer, including malignant melanoma.

41. The method of any one of claims 1 to 40.

42. The tumor sample is derived from a malignant pleural effusion of a subject with lung cancer.

42. The method of any one of claims 1 to 41.

43. The method comprises: Cryopreserving the first population of immune cells; and thawing the cryopreserved first population of immune cells. Further equipped with wherein the steps of cryopreserving the first immune cell population and thawing the cryopreserved first immune cell population are performed after step (b) of obtaining a first immune cell population from the culture product obtained from (a).

43. The method of any one of claims 1 to 42.

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