Seed cell culture medium for tumor-infiltrating lymphocytes and uses thereof

Abstract

The present application relates to a seed cell medium for tumor-infiltrating lymphocytes and uses thereof, the medium comprising cell culture components, cellular factors, and an immune checkpoint antibody or an antigen-binding fragment thereof, the cellular factors including IL-2, the immune checkpoints including PD-1, LAG-3, TIGIT, and / or CTLA-4, and the cell culture components being a serum medium or a serum-free medium. The seed cell medium described herein can accelerate the culture of seed cells for tumor-infiltrating lymphocytes and / or shorten the expansion time required for tumor-infiltrating lymphocytes.

Description

[Technical Field]

[0001] The present application relates to the field of biomedicine, and more particularly to a seed cell culture medium for tumor-infiltrating lymphocytes and uses thereof. [Background technology]

[0002] In 1998, the Surgery Branch of the National Cancer Institute (NCI) reported the first case of tumor-infiltrating lymphocytes (TILs) being used to treat a patient. Extensive research on TILs has shown that cells with anti-tumor activity can be isolated from tumor tissue in melanoma patients. In recent years, adoptive cell transfer (ACT) has been found to have a significant therapeutic effect on large, difficult-to-treat tumors, especially solid tumors.

[0003] The use of TILs in tumor immunotherapy requires a large number of cells, posing significant challenges in terms of technical and process control of TIL cell expansion. Conventionally, an IL-2-based initial rapid expansion procedure (pre-Rapid Expansion Procedure, pre-REP) is typically followed by a rapid expansion procedure (REP). This method requires a large number of over-irradiated allogeneic PBMCs (mononuclear cells, also known as MNCs) collected from multiple donors as feeder cells, anti-CD3 antibody (OKT3), and a large amount of IL-2. Irradiated allogeneic PBMCs from multiple donors carry the potential risk of contamination, while high concentrations of IL-2 may lead to cell depletion during subsequent reinfusion.

[0004] Therefore, there is an urgent need to provide a culture medium that can rapidly obtain seed cells from tumor-infiltrating lymphocytes. Summary of the Invention [Problem to be solved by the invention]

[0005] The present application provides a seed cell culture medium for tumor-infiltrating lymphocytes and uses thereof. The seed cell culture medium and the method for preparing the same described in the present application have at least one beneficial effect selected from the following: 1) substantial downregulation of the amount of IL-2 used, 2) reduced or no need to use IL-2 in subsequent reinfusions, 3) reduced risk of cell depletion associated with high concentrations of IL-2 in subsequent reinfusions, 4) no need to use exogenous allogeneic PBMCs or other cells as feeder cells in the subsequent rapid expansion process (REP), thereby reducing exogenous contamination, and 5) increased cell proliferation rate of 10% or more in the subsequent rapid expansion process (REP). 10 ~10 11 6) The efficiency of obtaining seed cells for tumor-infiltrating lymphocytes and the proliferation efficiency of tumor-infiltrating lymphocytes are significantly improved, thereby shortening the culture time. [Means for solving the problem]

[0006] Meanwhile, the present application provides a seed cell culture medium for tumor-infiltrating lymphocytes, comprising cell culture components, cytokines, and immune checkpoint antibodies or antigen-binding fragments thereof, wherein the cytokines include IL-2, and the immune checkpoints include PD-1, LAG-3, TIGIT, and / or CTLA-4, and the cell culture components are a serum medium or a serum-free medium.

[0007] In some embodiments, the concentration of IL-2 is about 3000 IU / mL or less.

[0008] In some embodiments, the concentration of the IL-2 is about 2000 IU / mL to about 3000 IU / mL.

[0009] In some embodiments, the cytokine comprises IL-7.

[0010] In some embodiments, the concentration of IL-7 is about 200 U / mL to about 1000 U / mL.

[0011] In some embodiments, the cytokine comprises IL-15.

[0012] In some embodiments, the concentration of IL-15 is about 200 U / mL to about 500 U / mL.

[0013] In some embodiments, the cytokine comprises tumor necrosis factor, TNF.

[0014] In some embodiments, the cytokine comprises TNFα.

[0015] In some embodiments, the concentration of TNFα is from about 10 pg / mL to about 100 pg / mL.

[0016] In some embodiments, the cytokine comprises a colony-stimulating factor.

[0017] In some embodiments, the cytokines include GM-CSF, G-CSF, and / or M-CSF.

[0018] In some embodiments, the GM-CSF has a concentration of about 200 U / mL to about 5000 U / mL.

[0019] In some embodiments, the concentration of the G-CSF is about 300 U / mL to about 1000 U / mL.

[0020] In some embodiments, the concentration of the M-CSF is about 300 U / mL to about 800 U / mL.

[0021] In some embodiments, the cytokine comprises an interferon.

[0022] In some embodiments, the cytokines include IFN-γ, IFN-α, and / or IFN-β.

[0023] In some embodiments, the concentration of the IFN-γ is from about 10 ng / mL to about 1000 ng / mL.

[0024] In some embodiments, the concentration of the IFN-γ is about 300 U / mL to about 1000 U / mL.

[0025] In some embodiments, the concentration of the IFN-α is about 500 U / mL to about 1000 U / mL.

[0026] In some embodiments, the concentration of the IFN-β is about 200 U / mL to about 500 U / mL.

[0027] In some embodiments, the cytokines further comprise IL-4, IL-1α, IL-1β, IL-6, IL-9, IL-18, IL-12, IL-21, and / or IL-10.

[0028] In some embodiments, the concentration of IL-4 is about 200 U / mL to about 500 U / mL.

[0029] In some embodiments, the concentration of IL-1α is about 200 U / mL to about 500 U / mL.

[0030] In some embodiments, the concentration of IL-1β is about 200 U / mL to about 500 U / mL.

[0031] In some embodiments, the concentration of IL-6 is about 200 U / mL to about 500 U / mL.

[0032] In some embodiments, the concentration of IL-9 is about 200 U / mL to about 500 U / mL.

[0033] In some embodiments, the concentration of IL-18 is about 200 U / mL to about 500 U / mL.

[0034] In some embodiments, the concentration of IL-12 is from about 200 U / mL to about 1000 U / mL.

[0035] In some embodiments, the concentration of IL-21 is about 200 U / mL to about 500 U / mL.

[0036] In some embodiments, the concentration of IL-10 is about 200 U / mL to about 500 U / mL.

[0037] In some embodiments, the immune checkpoint antibody or antigen-binding fragment thereof is a human PD-1 antibody or antigen-binding fragment thereof.

[0038] In some embodiments, the immune checkpoint antibody or antigen-binding fragment thereof is the PD-1 antibody or antigen-binding fragment thereof, and the concentration of the PD-1 antibody or antigen-binding fragment thereof is about 1 μg / mL to about 100 μg / mL.

[0039] In some embodiments, the immune checkpoint antibody or antigen-binding fragment thereof is a human LAG-3 antibody or antigen-binding fragment thereof.

[0040] In some embodiments, the immune checkpoint antibody or antigen-binding fragment thereof is a LAG-3 antibody or antigen-binding fragment thereof, and the concentration of the LAG-3 antibody or antigen-binding fragment thereof is about 3 μg / mL to about 10 μg / mL.

[0041] In some embodiments, the TIGIT antibody or antigen-binding fragment thereof is a human TIGIT antibody or antigen-binding fragment thereof.

[0042] In some embodiments, the immune checkpoint antibody or antigen-binding fragment thereof is a TIGIT antibody or antigen-binding fragment thereof, and the concentration of the TIGIT antibody or antigen-binding fragment thereof is about 1 μg / mL to about 25 μg / mL.

[0043] In some embodiments, the immune checkpoint antibody or antigen-binding fragment thereof is a human CTLA-4 antibody or antigen-binding fragment thereof.

[0044] In some embodiments, the immune checkpoint antibody or antigen-binding fragment thereof is a CTLA-4 antibody or antigen-binding fragment thereof, and the concentration of the CTLA-4 antibody or antigen-binding fragment thereof is about 1 μg / mL to about 100 μg / mL.

[0045] In some embodiments, the seed cell culture medium further comprises a costimulatory receptor antibody or antigen-binding fragment thereof, wherein the costimulatory receptor or antigen-binding fragment thereof comprises a CD40 antibody or antigen-binding fragment thereof, an OX-40 antibody or antigen-binding fragment thereof, a CD137 antibody or antigen-binding fragment thereof, and / or a CD28 antibody or antigen-binding fragment thereof.

[0046] In some embodiments, the immune checkpoint antibody or antigen-binding fragment thereof comprises a CD137 antibody or antigen-binding fragment thereof.

[0047] In some embodiments, the CD137 antibody or antigen-binding fragment thereof is a human CD137 antibody or antigen-binding fragment thereof.

[0048] In some embodiments, the concentration of the CD137 antibody or antigen-binding fragment thereof is from about 1 μg / mL to about 100 μg / mL.

[0049] In some embodiments, the immune checkpoint antibody or antigen-binding fragment thereof comprises a CD28 antibody or antigen-binding fragment thereof.

[0050] In some embodiments, the CD28 antibody or antigen-binding fragment thereof is a human CD28 antibody or antigen-binding fragment thereof.

[0051] In some embodiments, the concentration of the CD28 antibody or antigen-binding fragment thereof is about 1 μg / mL to about 10 μg / mL.

[0052] In some embodiments, the CD40 antibody or antigen-binding fragment thereof is a human CD40 antibody or antigen-binding fragment thereof.

[0053] In some embodiments, the concentration of the CD40 antibody or antigen-binding fragment thereof is about 5 μg / mL to about 10 μg / mL.

[0054] In some embodiments, the OX-40 antibody or antigen-binding fragment thereof is a human OX-40 antibody or antigen-binding fragment thereof.

[0055] In some embodiments, the concentration of the OX-40 antibody or antigen-binding fragment thereof is about 3 μg / mL to about 10 μg / mL.

[0056] In some embodiments, the serum medium comprises serum.

[0057] In some embodiments, the serum comprises human AB serum.

[0058] In a specific embodiment, the serum has a concentration of about 1% to about 10% (v / v).

[0059] In some embodiments, the serum medium comprises AIM-V medium.

[0060] In some embodiments, the serum-free medium comprises X-VIVO medium.

[0061] In some embodiments, the cell culture components include antibiotics.

[0062] In some embodiments, the cell culture components comprise a penicillin-streptomycin mixture, PS.

[0063] In some embodiments, the concentration of the penicillin-streptomycin mixture PS is from about 1 U / mL to about 200 U / mL.

[0064] In some embodiments, the seed cell medium further comprises an M2 macrophage inhibitor, wherein the M2 macrophage inhibitor comprises RRx001 and / or CNI-1493.

[0065] In some embodiments, the concentration of the M2 macrophage inhibitor is about 0.1 μM to about 100 μM.

[0066] In some embodiments, the seed cell medium further comprises a regulatory T cell (Treg) inhibitor, wherein the regulatory T cell inhibitor comprises CAL-101, dasatinib, imatinib, and / or panobinostat.

[0067] In some embodiments, the concentration of the regulatory T cell inhibitor is from about 0.1 μM to about 100 μM.

[0068] In some embodiments, the seed cell medium further comprises a myeloid-derived suppressor cell (MDSC) inhibitor, wherein the myeloid-derived suppressor cell inhibitor comprises AG490, decitabine, sunitinib, and / or BBI608.

[0069] In some embodiments, the concentration of the myeloid-derived suppressor cell inhibitor is from about 0.1 μg / mL to about 100 μg / mL.

[0070] In some embodiments, the seed cell medium further comprises a T cell activator, wherein the T cell activator comprises LYC-55716, GNE-1858, and / or methylene blue.

[0071] In some embodiments, the concentration of the T cell activator is from about 1 μM to about 10 μM.

[0072] In some embodiments, the seed cell medium further comprises a T cell differentiation inhibitor, wherein the T cell differentiation inhibitor comprises TWS119.

[0073] In some embodiments, the concentration of the T cell differentiation inhibitor is about 1 μM to about 10 μM.

[0074] In another aspect, the present application provides a tumor-infiltrating lymphocyte seed cell or a cell population thereof obtained by culturing in the seed cell medium described herein.

[0075] In another aspect, the present application provides a pharmaceutical composition comprising a tumor-infiltrating lymphocyte seed cell or cells thereof described herein and a pharmaceutically acceptable carrier.

[0076] In another aspect, the present application provides the use of a tumor-infiltrating lymphocyte seed cell or cell populations described herein and a pharmaceutical composition described herein in the preparation of a cancer treatment drug.

[0077] In some embodiments, the cancer is selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic small cell adenocarcinoma, and cholangiocarcinoma.

[0078] In another aspect, the present application provides the use of the seed cell culture medium in expanding tumor-infiltrating lymphocytes.

[0079] In another aspect, the present application provides a method for culturing seed cells of tumor-infiltrating lymphocytes, comprising culturing isolated tumor-infiltrating lymphocytes in a seed cell medium described herein.

[0080] In another aspect, the present application provides a method for expanding tumor-infiltrating lymphocytes, comprising the step of culturing isolated tumor-infiltrating lymphocytes in a seed cell medium described herein to obtain seed cells of the tumor-infiltrating lymphocytes.

[0081] In some embodiments, the isolated tumor-infiltrating lymphocytes are derived from a sample selected from the group consisting of ascites, surgical resection samples of primary tumors, surgical resection samples of synchronous and metachronous metastases, aspirate samples, and body fluids of a subject in need thereof.

[0082] In some embodiments, the bodily fluid comprises blood, interstitial fluid, lymphatic fluid, and / or body cavity fluid.

[0083] In some embodiments, the isolated tumor-infiltrating lymphocytes are derived from a tumor selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, and cholangiocarcinoma.

[0084] In some embodiments, the isolated tumor-infiltrating lymphocytes are derived from a tissue mass obtained by excising tumor tissue, and the diameter of the tissue mass obtained by excising the tumor tissue is about 1 mm to about 10 mm.

[0085] In some embodiments, the culture period is about 3 to 20 days.

[0086] In some embodiments, the temperature of the culture is about 30-42°C. [Effects of the Invention]

[0087] Those skilled in the art can readily ascertain other aspects and advantages of the present application from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present application. Those skilled in the art will recognize that the present application will enable those skilled in the art to make modifications to the particular embodiments disclosed without departing from the spirit and scope of the invention of the present application. Accordingly, the accompanying drawings and description of the present application are illustrative only and not restrictive. [Brief explanation of the drawings]

[0088] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments described in detail herein and the accompanying drawings, the brief description of which is as follows:

[0089] [Fig. 1A-1B-9A-9B] 1 shows the flow cytometry results of TIL seed cells obtained from solid tumor tissue cultured in the seed cell medium described herein.

[0090] [Fig. 10A-10B-12A-12B] 1 shows the flow cytometry results of TIL seed cells obtained from the body cavity fluid of a tumor patient cultured in the seed cell medium described in this application.

[0091] [Fig. 13A-13B-15A-15B] 1 shows the flow cytometry results of TIL seed cells obtained by culturing from body cavity fluid of a tumor patient in a prior art seed cell medium.

[0092] [Fig. 16A-16B-17-20] 1 shows the results of high-throughput sequence analysis assays of TCR repertoires in TIL reinfusions prepared using the seed cell culture medium described herein and in the peripheral blood of patients at various time points after clinical reinfusion.

[0093] [Figure 21] 1 shows the results of the proliferation status of peripheral blood leukocytes, neutrophils, and lymphocytes in patients after clinical reinfusion of TIL reinfusion prepared using the seeding cell medium described herein. DETAILED DESCRIPTION OF THE INVENTION

[0094] In the following, the embodiments of the present invention will be described using specific examples, but those skilled in the art will easily understand other advantages and effects of the present invention from the disclosure in this application.

[0095] Definition of Terms

[0096] As used herein, the term "interferon" generally refers to a highly species-specific family of proteins that inhibit viral replication and cellular proliferation and regulate immune responses. Exemplary suitable interferons include, but are not limited to, recombinant interferon alpha-2b, recombinant interferon alpha-2a, recombinant interferon alpha-2c, interferon alpha-n1, covalent alpha interferon, or interferon alpha-n3.

[0097] As used herein, the term "colony-stimulating factor" generally refers to a secreted glycoprotein that binds to a receptor protein on the surface of hematopoietic stem cells, thereby activating an intracellular signaling pathway that leads to cell proliferation and differentiation into specific types of blood cells (usually white blood cells; for red blood cells, see erythropoietin). They can be artificially synthesized and administered exogenously.

[0098] As used herein, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that contains amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that an antibody specifically recognizes and binds to is referred to as an "epitope," as described above. As described above, an antigen-binding domain typically contains an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but does not necessarily contain both. For example, an Fd fragment contains two VH regions and typically retains some of the antigen-binding function of the complete antigen-binding domain. Examples of antibody antigen-binding fragments include, but are not limited to, (1) Fab fragments, which are monovalent fragments containing VL, VH, a constant light chain (CL), and a CH1 domain; (2) F(ab')2 fragments, which are bivalent fragments in which two Fab fragments are linked by a disulfide bond in the hinge region; (3) Fd fragments containing VH and two CH1 domains; (4) Fv fragments containing the VL and VH domains of a single antibody arm; (5) dAb fragments containing a VH domain (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which is incorporated herein by reference in its entirety); (6) isolated complementarity-determining regions (CDRs); and (7) single-chain Fvs (scFvs), for example, obtained from an scFV library. The two domains of the Fv fragment, VL and VH, are encoded by separate genes but can be joined by recombinant methods using a synthetic linker, which is prepared as a single protein chain (called single-chain Fv (scFv)) in which the VL and VH domains pair to form a monovalent molecule (see, e.g., Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli / Troc. Natl. Acad. Sci. USA 85:5879-5883 (1988)).These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the functionality of the fragments can be evaluated in the same manner as for intact antibodies. As used herein, the term "antigen-binding fragment" also includes ligands that specifically bind to antigens, particularly cell surface receptor molecules. Ligands are typically polypeptides or compounds that can bind to receptor proteins (e.g., the antigen) with high affinity and specificity and induce a functional response. For example, the ligands may include CD40L, a ligand for CD40; CD137L, a ligand for CD137; CD80, a ligand for CD28; and / or OX-40L, a ligand for OX-40.

[0099] In this application, the term "immune checkpoint" generally refers to CD4 + and CD8 + This refers to a group of molecules on the cell surface of T cells. These molecules are effectively used as "brakes" to downregulate or inhibit anti-tumor immune responses. These inhibitory molecules are inhibited by suppressing DNA, RNA, and protein levels.

[0100] As used herein, the term "costimulatory receptor" generally refers to a receptor expressed on the surface of immune cells, such as T cells, and dependent on them for activation of an immune response. When an antigen-presenting cell is present and a first signal is given, the costimulatory receptor binds to the corresponding activating antibody, ligand, or antigen-binding fragment to activate the costimulatory signal, thereby fully activating the immune effector cell. Activation of signal transduction pathways via costimulatory receptors is often critical for the effective generation of an immune response. Common costimulatory receptors include, but are not limited to, CD28, CD40, CD137, and OX-40.

[0101] As used herein, the term "tumor necrosis factor (TNF)" generally refers to the TNF protein and does not refer to the TNF ligand superfamily. The GenBank accession number for human TNFα is NP_000585.2. The term "TNFα" encompasses the precursor of TNFα, pro-TNFα (pro-TNFa), full-length TNFα, and any form of TNFα produced by intracellular processes. The term also encompasses naturally occurring and non-naturally occurring variants of TNFα, including splice variants, allelic variants, and isoforms. TNFα binds to two receptors: TNFR1 (TNF receptor type 1; CD120a; p55 / 60) and TNFR2 (TNF receptor type 2; CD120b; p75 / 80). TNFα acts as a proinflammatory cytokine, including in neuroinflammation.

[0102] In this application, the term "tumor-infiltrating lymphocytes" generally refers to infiltrating lymphocytes isolated from tumor tissue. Immunohistochemical staining has shown that tumor-infiltrating lymphocytes express CD3 + T cells, CD20+, CD79α+ B cells, plasma cells, CD56 + The tumor-infiltrating lymphocytes may include NK cells, as well as T cells, a small number of B cells, NK cells, macrophages, and dendritic cells. The tumor-infiltrating lymphocytes can exert an efficient and specific anti-tumor killing effect directly through cytotoxic T lymphocytes and secreted cytokines. In the present application, the tumor-infiltrating lymphocytes may include mononuclear leukocytes, which leave the bloodstream and migrate to tumors. The tumor-infiltrating lymphocytes may be selected from the group consisting of T cells, B cells, natural killer cells, and natural killer T cells.

[0103] As used herein, the term "seed cells" generally refers to tumor-infiltrating lymphocytes cultured from tumor tissue using any appropriate medium, including the medium of the present application. These cells can be used as a starting cell population for subsequent further expansion culture, or as a cell population for end use, such as reinfusion therapy into an individual subject. The seed cells may be obtained from tumor tissue after any of various culture periods, and the culture period for the seed cells may be determined depending on the subsequent use of the seed cells and is not limited to a specific culture period.

[0104] In this application, the term "IL-15" generally refers to a proinflammatory cytokine that acts as an effective proliferation, survival, and activation factor for T cells (especially intestinal intraepithelial lymphocytes (IELs)) and natural killer (NK) cells, and is also referred to as "IL-15 antigen" or "interleukin-15." Increased expression of IL-15 has been demonstrated in various inflammatory diseases, including CD, rheumatoid arthritis (RA), and psoriasis (Malamut et al., 2010). IL-15 is thought to be a central regulator of the immunopathology of CD and a nonredundant driver of lymphomagenesis in RCD.

[0105] As used herein, the term "IL-2" generally refers to a protein derived from lymphokines produced by normal peripheral blood lymphocytes and present at low concentrations in the body. IL-2 was originally described by Morgan et al. (1976, Science 193: 1007-1008) and was initially called T-cell growth factor due to its ability to induce proliferation of stimulated T lymphocytes. IL-2 also includes proteins that have undergone modifications, such as glycosylation, acetylation, and phosphorylation, after expression.

[0106] As used herein, the term "IL-7" generally refers to the protein encoded by the IL-7 gene. IL-7 is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus. It can be produced by keratinocytes, dendritic cells, hepatocytes, neurons, and epithelial cells. IL-7 may be involved in effects on T cells and B cells.

[0107] As used herein, the term "PD-1" generally refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed in vivo primarily on preactivated T cells and binds to two ligands, PD-1 and PD-2. The complete nucleotide sequence of hPD-1 is available in GenBank accession number U64863.

[0108] In this application, the term "LYC-55716" generally refers to the RORy activator. The CAS number is 2055536-64-4.

[0109] In this application, the term "GNE-1858" generally refers to the HPK1 inhibitor with CAS number T11438.

[0110] In this application, the term "TWS119" generally refers to the GSK-3 inhibitor with CAS number 601514-19-6.

[0111] As used herein, the term "RRX-001" generally refers to an epigenetic modulator with radiosensitizing activity. RRX-001 downregulates the CD47 / SIRPα axis, repolarizing tumor amniotic membrane receptors (TAMs) and other immunosuppressive cells in the tumor microenvironment toward an immunostimulatory phenotype. Its CAS number is 925206-65-1.

[0112] As used herein, the term "CNI-1493" generally refers to a compound capable of inhibiting the production of inflammatory cytokines (including TNF) with CAS number 164301-51-3.

[0113] Detailed Description of the Invention

[0114] On the other hand, the present application provides a seed cell medium for tumor-infiltrating lymphocytes, comprising as ingredients cell culture components, cytokines, and immune checkpoint antibodies or antigen-binding fragments thereof, wherein the cytokines include IL-2, and the immune checkpoints include PD-1, LAG-3, TIGIT, and / or CTLA-4, and the cell culture components are a serum medium or a serum-free medium.

[0115] In the present application, the cytokine may be a cytokine of human origin.

[0116] In the present application, the immune checkpoint antibody or antigen-binding fragment thereof may be a humanized and / or fully human antibody or antigen-binding fragment thereof.

[0117] For example, the concentration of IL-2 may be about 3000 IU / mL or less (e.g., about 2900 IU / mL or less, about 2800 IU / mL or less, about 2700 IU / mL or less, about 2600 IU / mL or less, about 2500 IU / mL or less, about 2400 IU / mL or less, about 2300 IU / mL or less, about 2200 IU / mL or less, about 2100 IU / mL or less, about 2000 IU / mL or less, about 1900 IU / mL or less, about 1800 IU / mL or less, (e.g., about 1700 IU / mL or less, about 1600 IU / mL or less, about 1500 IU / mL or less, about 1400 IU / mL or less, about 1300 IU / mL or less, about 1100 IU / mL or less, about 1100 IU / mL or less, about 1000 IU / mL or less, about 900 IU / mL or less, about 800 IU / mL or less, about 700 IU / mL or less, about 600 IU / mL or less, about 500 IU / mL or less, about 400 IU / mL or less, about 300 IU / mL or less, or less.) For example, the IL-2 concentration may be about 2000 IU / mL or less, about 2500 IU / mL or less, or about 3000 IU / mL or less.

[0118] For example, the cytokine may include IL-7.

[0119] For example, the concentration of IL-7 may be about 5 to about 100 ng / mL (e.g., about 5 ng / mL to about 95 ng / mL, about 5 ng / mL to about 90 ng / mL, about 5 ng / mL to about 85 ng / mL, about 5 ng / mL to about 80 ng / mL, about 5 ng / mL to about 75 ng / mL, about 5 ng / mL to about 70 ng / mL, about 5 ng / mL to about 65 ng / mL, about 5 ng / mL to about 60 ng / mL, about 5 ng / mL to 55 ng / mL, about 5 ng / mL to 50 ng / mL, about 5 ng / mL to 45 ng / mL, about 5 ng / mL to 40 ng / mL, about 5 ng / mL to 35 ng / mL, about 5 ng / mL to 30 ng / mL, about 5 ng / mL to 25 ng / mL, about 5 ng / mL to 20 ng / mL, or about 5 ng / mL to 10 ng / mL). For example, the concentration of IL-7 may be about 200 U / mL to about 1000 U / mL (e.g., at least about 200 U / mL, at least about 500 U / mL, or at least about 1000 U / mL). In the present application, the concentration of IL-7 may be about 200 to 1000 IU / mL. For example, it may be at least about 200 IU / mL, at least about 500 IU / mL, or at least about 1000 IU / mL.

[0120] For example, the cytokine may include IL-15.

[0121] For example, the concentration of IL-15 may be about 5 ng / mL to about 100 ng / mL (e.g., about 5 ng / mL to about 95 ng / mL, about 5 ng / mL to about 90 ng / mL, about 5 ng / mL to about 85 ng / mL, about 5 ng / mL to about 80 ng / mL, about 5 ng / mL to about 75 ng / mL, about 5 ng / mL to about 70 ng / mL, about 5 ng / mL to about 65 ng / mL, about 5 ng / mL to about 60 ng / mL, about 5 ng / mL to 55 ng / mL, about 5 ng / mL to 50 ng / mL, about 5 ng / mL to 45 ng / mL, about 5 ng / mL to 40 ng / mL, about 5 ng / mL to 35 ng / mL, about 5 ng / mL to 30 ng / mL, about 5 ng / mL to 25 ng / mL, about 5 ng / mL to 20 ng / mL, or about 5 ng / mL to 10 ng / mL). For example, the concentration of IL-15 may be about 200 to about 500 U / mL (e.g., at least about 200 U / mL, at least about 250 U / mL, at least about 300 U / mL, or at least about 500 U / mL). In the present application, the concentration of IL-15 may be about 200 to 500 IU / mL. For example, it may be at least about 200 IU / mL, at least about 250 IU / mL, at least about 300 IU / mL, or at least about 500 IU / mL.

[0122] For example, the cytokine may include tumor necrosis factor TNF.

[0123] For example, the cytokine may include TNFα.

[0124] For example, the concentration of the TNFα may be about 10 μM to about 100 pg / mL (e.g., about 10 pg / mL to about 95 pg / mL, about 10 pg / mL to about 90 pg / mL, about 10 pg / mL to about 80 pg / mL, about 10 pg / mL to about 70 pg / mL, about 10 pg / mL to about 60 pg / mL, about 10 pg / mL to about 50 pg / mL, about 10 pg / mL to about 40 pg / mL, about 15 pg / mL to about 40 pg / mL, about 10 pg / mL to about 25 pg / mL, about 15 pg / mL to about 25 pg / mL, about 10 pg / mL to about 30 pg / mL, about 10 pg / mL to about 20 pg / mL, or about 10 pg / mL to about 15 pg / mL). In the present application, the concentration of TNF-α may be about 10 to 100 pg / mL, for example, about 10 to 100 pg / mL, about 15 to 100 pg / mL, about 20 to 100 pg / mL, or about 25 to 100 pg / mL, for example, at least about 10 pg / mL, at least about 15 pg / mL, at least about 20 pg / mL, or at least about 25 pg / mL.

[0125] For example, the cytokine may include a colony stimulating factor.

[0126] For example, the cytokines may include GM-CSF, G-CSF, and / or M-CSF.

[0127] For example, the concentration of the GM-CSF may be about 200 U / mL to about 5000 U / mL (e.g., about 200 U / mL to about 800 U / mL, about 200 U / mL to about 500 U / mL, about 300 U / mL to about 5000 U / mL, about 500 U / mL to about 5000 U / mL, about 1000 U / mL to about 5000 U / mL, about 1500 U / mL to about 5000 U / mL, about 2000 U / mL to about 5000 U / mL, about 2500 U / mL to about 5000 U / mL, about 3000 U / mL to about 5000 U / mL, about 3500 U / mL to about 5000 U / mL, about 4000 U / mL to about 5000 U / mL, about 4000 U / mL to about 5000 U / mL, U / mL, or about 45,000 U / mL to about 5,000 U / mL). For example, it may be about 200 to about 1,000 U / mL, about 200 to about 800 U / mL, or about 200 to about 500 U / mL. For example, it may be at least about 200 U / mL, at least about 500 U / mL, or at least about 800 U / mL.

[0128] For example, the concentration of the G-CSF may be about 300 U / mL to about 1000 U / mL. For example, the concentration of the G-CSF may be about 500 to 1000 U / mL. For example, the concentration may be at least about 500 U / mL or at least about 1000 U / mL.

[0129] For example, the concentration of the M-CSF may be about 300 U / mL to about 800 U / mL. For example, the concentration of the M-CSF may be about 300 U / mL to about 500 U / mL. For example, the concentration may be at least about 300 U / mL, at least about 500 U / mL, or at least about 800 U / mL.

[0130] For example, the cytokine may include an interferon.

[0131] For example, the cytokines may include IFN-γ, IFN-α, and / or IFN-β.

[0132] For example, the concentration of the IFN-γ may be about 10 ng / mL to about 1000 ng / mL (e.g., about 15 ng / mL to about 1000 ng / mL, about 20 ng / mL to about 1000 ng / mL, about 30 ng / mL to about 1000 ng / mL, about 40 ng / mL to about 1000 ng / mL, about 50 ng / mL to about 1000 ng / mL, about 60 ng / mL to about 1000 ng / mL, about 70 ng / mL to about 1000 ng / mL, about 80 ng / mL to about 1000 ng / mL, about 9 The concentration of IFN-γ may be about 10 ng / mL to about 1000 ng / mL, about 100 ng / mL to about 1000 ng / mL, about 200 ng / mL to about 1000 ng / mL, about 300 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 1000 ng / mL, about 500 ng / mL to about 1000 ng / mL, about 600 ng / mL to about 1000 ng / mL, about 700 ng / mL to about 1000 ng / mL, about 800 ng / mL to about 1000 ng / mL, or about 900 ng / mL to about 1000 ng / mL). For example, the concentration of the IFN-γ may be about 10 ng / mL to about 1000 ng / mL. For example, the concentration of IFN-γ may be about 300 ng / mL to about 1000 ng / mL, about 300 ng / mL to about 800 ng / mL, or about 300 ng / mL to about 500 ng / mL, e.g., at least about 300 ng / mL, at least about 500 ng / mL, at least about 800 ng / mL, or at least about 1000 ng / mL.

[0133] For example, the concentration of the IFN-α may be about 500 U / mL to about 1000 U / mL, for example, at least about 500 ng / mL.

[0134] For example, the concentration of the IFN-β may be about 200 U / mL to about 500 U / mL. For example, the concentration of the IFN-β may be about 250 U / mL to about 500 U / mL. For example, the concentration of the IFN-β may be at least about 200 ng / mL, at least about 250 ng / mL, or at least about 500 ng / mL.

[0135] For example, the cytokines may include IL-4, IL-1α, IL-1β, IL-6, IL-9, IL-18, IL-12, IL-21, and / or IL-10.

[0136] For example, the concentration of IL-4 may be about 200 U / mL to about 500 U / mL, for example, at least about 200 IU / mL or at least about 500 IU / mL.

[0137] For example, the concentration of IL-1α may be about 200 U / mL to about 500 U / mL, for example, at least about 200 IU / mL or at least about 500 IU / mL.

[0138] For example, the concentration of IL-1β may be about 200 U / mL to about 500 U / mL, for example, at least about 200 IU / mL or at least about 500 IU / mL.

[0139] For example, the concentration of IL-6 may be about 200 U / mL to about 500 U / mL, for example, at least about 200 IU / mL or at least about 500 IU / mL.

[0140] For example, the concentration of IL-9 may be about 200 U / mL to about 500 U / mL, for example, at least about 200 IU / mL or at least about 500 IU / mL.

[0141] For example, the concentration of IL-18 may be about 200 U / mL to about 500 U / mL, for example, at least about 200 IU / mL or at least about 500 IU / mL.

[0142] For example, the concentration of IL-12 may be about 200 U / mL to about 1000 U / mL. For example, the concentration of IL-12 may be about 200 U / mL to about 1000 U / mL, about 200 U / mL to about 500 U / mL, or about 300 U / mL to about 1000 U / mL. For example, the concentration of IL-12 may be at least about 200 IU / mL, at least about 250 IU / mL, at least about 300 IU / mL, or at least about 500 IU / mL.

[0143] For example, the concentration of IL-21 may be about 200 U / mL to about 500 U / mL. For example, the concentration of IL-21 may be about 250 U / mL to about 1000 U / mL. For example, the concentration may be at least about 200 IU / mL, at least about 250 IU / mL, or at least about 500 IU / mL.

[0144] For example, the concentration of IL-10 may be about 200 U / mL to about 500 U / mL, e.g., at least about 200 IU / mL, at least about 250 IU / mL, at least about 300 IU / mL, or at least about 500 IU / mL.

[0145] For example, the immune checkpoint antibody or antigen-binding fragment thereof may be a PD-1 antibody or antigen-binding fragment thereof.

[0146] For example, the PD-1 antibody or antigen-binding fragment thereof may be a human PD-1 antibody or antigen-binding fragment thereof.

[0147] For example, the concentration of the PD-1 antibody or antigen-binding fragment thereof may be about 1 μg / mL to about 100 μg / mL (e.g., about 3 μg / mL to about 100 μg / mL, about 5 μg / mL to about 100 μg / mL, about 10 μg / mL to about 100 μg / mL, about 15 μg / mL to about 100 μg / mL, about 16 μg / mL to about 100 μg / mL, about 17 μg / mL to about 100 μg / mL, about 18 μg / mL to about 100 μg / mL, about 19 μg / mL to about 100 μg / mL, about 20 μg / mL to about 100 μg / mL, about 2 (5 μg / mL to about 100 μg / mL, about 30 μg / mL to about 100 μg / mL, about 36 μg / mL to about 100 μg / mL, about 40 μg / mL to about 100 μg / mL, about 45 μg / mL to about 100 μg / mL, about 50 μg / mL to about 100 μg / mL, about 55 μg / mL to about 100 μg / mL, about 60 μg / mL to about 100 μg / mL, about 65 μg / mL to about 100 μg / mL, about 70 μg / mL to about 100 μg / mL, about 80 μg / mL to about 100 μg / mL, or about 90 μg / mL to about 100 μg / mL). In the present application, the concentration of the PD-1 mAb may be about 3 to 100 μg / mL. For example, it may be at least about 3 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 15 μg / mL, at least about 20 μg / mL, at least about 25 μg / mL, at least about 36 μg / mL, at least about 50 μg / mL, or at least about 100 μg / mL.

[0148] For example, the immune checkpoint antibody or antigen-binding fragment thereof may be a human LAG-3 antibody or antigen-binding fragment thereof.

[0149] For example, the immune checkpoint antibody or antigen-binding fragment thereof may be a LAG-3 antibody or antigen-binding fragment thereof, and the concentration of the LAG-3 antibody or antigen-binding fragment thereof may be about 3 μg / mL to about 10 μg / mL, for example, about 5 μg / mL to about 10 μg / mL, or at least about 3 μg / mL, at least about 5 μg / mL, or at least about 10 μg / mL.

[0150] For example, the TIGIT antibody or antigen-binding fragment thereof may be a human TIGIT antibody or antigen-binding fragment thereof.

[0151] For example, the immune checkpoint antibody or antigen-binding fragment thereof may be a TIGIT antibody or antigen-binding fragment thereof, and the concentration of the TIGIT antibody or antigen-binding fragment thereof is about 1 μg / mL to about 25 μg / mL. In the present application, the concentration of the TIGIT mAb may be about 2.5 μg / mL to 20 μg / mL, for example, 5 to 20 μg / mL, or for example, at least about 2.5 μg / mL, at least about 5 μg / mL, or at least about 20 μg / mL.

[0152] For example, the immune checkpoint antibody or antigen-binding fragment thereof may be a human CTLA-4 antibody or antigen-binding fragment thereof.

[0153] For example, the immune checkpoint antibody or antigen-binding fragment thereof may be a CTLA-4 antibody or antigen-binding fragment thereof, and the concentration of the CTLA-4 antibody or antigen-binding fragment thereof is about 1 μg / mL to about 100 μg / mL. In the present application, the concentration of the CTLA-4 mAb may be about 3 to 50 μg / mL, about 5 to 50 μg / mL, or about 10 to 50 μg / mL. For example, it may be at least about 3 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, or at least about 50 μg / mL.

[0154] In the present application, the seed cell culture medium may further comprise a costimulatory receptor antibody or antigen-binding fragment thereof, and the costimulatory receptor immune checkpoint antibody or antigen-binding fragment thereof may comprise a CD40 antibody or antigen-binding fragment thereof, an OX-40 antibody or antigen-binding fragment thereof, a CD137 antibody or antigen-binding fragment thereof, and / or a CD28 antibody or antigen-binding fragment thereof.

[0155] For example, the costimulatory antibody or antigen-binding fragment thereof may comprise a CD137 antibody or antigen-binding fragment thereof.

[0156] For example, the CD137 antibody or antigen-binding fragment thereof may be a human CD137 antibody or antigen-binding fragment thereof.

[0157] For example, the concentration of the CD137 antibody or antigen-binding fragment thereof may be about 1 μg / mL to about 100 μg / mL (e.g., about 2 μg / mL to about 100 μg / mL, about 3 μg / mL to about 100 μg / mL, about 3 μg / mL to about 20 μg / mL, about 4 μg / mL to about 100 μg / mL, about 5 μg / mL to about 100 μg / mL, about 6 μg / mL to about 100 μg / mL, about 7 μg / mL to about 100 μg / mL, about 8 μg / mL to about 1 (The concentration of the CD137 mAb may be about 3 to about 12 μg / mL, about 9 μg / mL to about 100 μg / mL, about 10 μg / mL to about 100 μg / mL, about 12 μg / mL to about 100 μg / mL, about 20 μg / mL to about 100 μg / mL, about 10 μg / mL to about 90 μg / mL, about 10 μg / mL to about 80 μg / mL, about 10 μg / mL to about 70 μg / mL, about 10 μg / mL to about 50 μg / mL, about 10 μg / mL to about 20 μg / mL, or about 3 μg / mL to about 12 μg / mL). In the present application, the concentration of the CD137 mAb may be about 3 to 20 μg / mL. For example, it may be at least about 3 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 12 μg / mL, or at least about 20 μg / mL.

[0158] For example, the costimulatory antibody or antigen-binding fragment thereof can comprise an OX40 antibody or antigen-binding fragment thereof.

[0159] For example, the OX-40 antibody or antigen-binding fragment thereof may be a human OX-40 antibody or antigen-binding fragment thereof.

[0160] For example, the concentration of the OX-40 antibody or antigen-binding fragment thereof may be about 3 μg / mL to about 10 μg / mL. In the present application, the concentration of the OX-40 mAb may be about 5 μg / mL to 10 μg / mL. For example, it may be at least about 3 μg / mL, at least about 5 μg / mL, or at least about 10 μg / mL.

[0161] For example, the costimulatory antibody or antigen-binding fragment thereof may comprise a CD28 antibody or antigen-binding fragment thereof.

[0162] For example, the immune checkpoint antibody or antigen-binding fragment thereof may comprise a CD28 antibody or antigen-binding fragment thereof.

[0163] For example, the CD28 antibody or antigen-binding fragment thereof may be a human CD28 antibody or antigen-binding fragment thereof.

[0164] For example, the concentration of the CD28 antibody or antigen-binding fragment thereof may be about 1 μg / mL to about 10 μg / mL (e.g., about 2 μg / mL to about 10 μg / mL, about 3 μg / mL to about 10 μg / mL, about 3 μg / mL to about 5 μg / mL, about 4 μg / mL to about 10 μg / mL, about 5 μg / mL to about 10 μg / mL, about 6 μg / mL to about 10 μg / mL, about 7 μg / mL to about 10 μg / mL, or about 8 μg / mL to about 10 μg / mL). In the present application, the concentration of the CD28 mAb may be about 3 to 10 μg / mL. For example, it may be at least about 3 μg / mL, at least about 5 μg / mL, or at least about 10 μg / mL.

[0165] For example, the costimulatory antibody or antigen-binding fragment thereof may comprise a CD40 antibody or antigen-binding fragment thereof.

[0166] For example, the CD40 antibody or antigen-binding fragment thereof may be a human CD40 antibody or antigen-binding fragment thereof.

[0167] For example, the concentration of the CD40 antibody or antigen-binding fragment thereof may be about 5 μg / mL to about 10 μg / mL. In the present application, the concentration of the CD40 mAb may be about 6 to 10 μg / mL, or about 5 to 8 μg / mL. For example, it may be at least about 5 μg / mL.

[0168] For example, the serum medium may contain serum.

[0169] For example, the serum may comprise human AB serum.

[0170] For example, the concentration of the serum may be about 1% to about 10% (v / v) (e.g., about 1.5% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, or about 1% to about 6%).

[0171] For example, the serum medium may contain AIM-V medium.

[0172] For example, the serum-free medium is X-VIVO medium or CTS medium. TM OpTmizer TM It may contain a culture medium.

[0173] For example, the cell culture components may include antibiotics.

[0174] For example, the cell culture components may include a penicillin-streptomycin mixture, PS.

[0175] For example, the concentration of the penicillin-streptomycin mixture PS may be about 1 U / mL to about 200 U / mL (e.g., about 1 U / mL to about 5 U / mL, about 1 U / mL to about 10 U / mL, about 1 U / mL to about 20 U / mL, about 1 U / mL to about 30 U / mL, about 1 U / mL to about 40 U / mL, about 1 U / mL to about 5 ...20 U / mL, about 1 U / mL to about 20 U / mL, about 1 U / mL to about 20 U / mL, about 1 U / mL to about 20 U / mL, about 1 U / mL to about 20 U / mL, about 1 U / mL to about 20 U / mL, about 1 U / mL to about 20 U / mL, about 1 U / (For example, the concentration of the penicillin-streptomycin mixture PS may be about 100 U / mL.)

[0176] For example, the seed cell culture medium may further contain an M2-type macrophage inhibitor, and the M2-type macrophage inhibitor may contain RRx001 and / or CNI-1493.

[0177] For example, the concentration of the M2 macrophage inhibitor may be about 0.1 μM to about 100 μM (e.g., about 0.5 μM to about 100 μM, about 1 μM to about 100 μM, about 2 μM to about 100 μM, about 2.5 μM to about 100 μM, about 3 μM to about 100 μM, about 2 μM to about 3 μM, about 5 μM to about 100 μM, about 10 μM to about 100 μM, about 20 μM to about 100 μM, about 0.1 μM to about 90 μM, about 0.5 μM to about 90 μM, about 1 μM to about 90 μM, about 5 μM to about 90 μM, or about 0.1 μM to about 80 μM).

[0178] For example, the seed cell culture medium may further comprise a regulatory T cell (Treg) inhibitor, and the regulatory T cell inhibitor may comprise CAL-101, dasatinib, imatinib, and / or panobinostat.

[0179] For example, the concentration of the regulatory T cell inhibitor may be about 0.1 μM to about 100 μM (e.g., about 0.5 μM to about 100 μM, about 1 μM to about 100 μM, about 5 μM to about 100 μM, about 10 μM to about 100 μM, about 20 μM to about 100 μM, about 0.1 μM to about 90 μM, about 0.5 μM to about 90 μM, about 1 μM to about 90 μM, about 5 μM to about 90 μM, or about 0.1 μM to about 80 μM). For example, the concentration of the regulatory T cell inhibitor may be about 1 μM to about 5 μM, about 1 μM to about 3 μM, about 1.5 μM to about 5 μM, about 2 μM to about 5 μM, about 2.5 μM to about 5 μM, or about 3 μM to about 5 μM.

[0180] For example, the seed cell medium may further comprise a myeloid-derived suppressor cell inhibitor, and the myeloid-derived suppressor cell inhibitor may comprise AG490, decitabine, sunitinib, and / or BBI608.

[0181] For example, the concentration of the myeloid-derived suppressor cell inhibitor may be about 0.1 μg / mL to about 100 μg / mL (e.g., about 0.5 μg / mL to about 100 μg / mL, about 1 μg / mL to about 100 μg / mL, about 2 μg / mL to about 100 μg / mL, about 5 μg / mL to about 100 μg / mL, about 6 μg / mL to about 100 μg / mL, about 7 μg / mL to about 1 (The concentration of the myeloid-derived suppressor cell inhibitor may be about 1 μg / mL to about 3 μg / mL, or about 1.5 μg / mL to about 3 μg / mL.)

[0182] In the present application, the seed medium may contain at least one, at least two, or at least three of the regulatory T cell (Treg) inhibitor, the myeloid-derived suppressor cell inhibitor, and the M2 macrophage inhibitor.

[0183] In the present application, the seed cell culture medium may further contain a T cell activator, and the T cell activator may include LYC-55716, GNE-1858, and / or methylene blue.

[0184] For example, the concentration of the T cell activator may be about 1 μM to about 10 μM, e.g., about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 2 μM to about 10 μM, about 3 μM to about 10 μM, about 4 μM to about 10 μM, or about 5 μM to about 10 μM.

[0185] In the present application, the seed cell culture medium may further contain a T cell differentiation inhibitor, and the T cell differentiation inhibitor may include TWS119.

[0186] For example, the concentration of the T cell differentiation inhibitor may be about 1 μM to about 10 μM, e.g., about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 2 μM to about 10 μM, about 3 μM to about 10 μM, about 4 μM to about 10 μM, or about 5 μM to about 10 μM.

[0187] In the present application, the concentration of RRX-001 may be about 0.1 μM to about 100 μM, for example, at least about 3 μM.

[0188] In the present application, the concentration of sunitinib may be about 1.5 μM to about 3 μM, for example, at least about 1.5 μM or at least about 3 μM.

[0189] In the present application, the concentration of CNI-1493 may be about 2 μM to about 3 μM, for example, at least about 2 μM, at least about 2.5 μM, or at least about 3 μM.

[0190] In the present application, the concentration of imatinib may be about 2 μM to about 5 μM, for example, at least about 2 μM or at least about 5 μM.

[0191] In the present application, the concentration of CAL-101 may be about 1.5 μM to about 5 μM, for example, at least about 1.5 μM or at least about 5 μM.

[0192] In the present application, the concentration of dasatinib may be about 2 μM to about 3 μM, for example, at least about 2 μM, at least about 2.5 μM, or at least about 3 μM.

[0193] In the present application, the concentration of LYC-55716 may be about 1 μM to about 10 μM, for example, at least about 1 μM or at least about 10 μM.

[0194] In the present application, the concentration of GNE-1858 may be about 4 to about 5 nM, for example, at least about 4 nM, at least about 4.5 nM, or at least about 5 nM.

[0195] In the present application, the concentration of methylene blue may be about 1 μM to about 2 μM, for example, at least about 1 μM, at least about 1.5 μM, or at least about 2 μM.

[0196] In the present application, the concentration of TWS119 may be about 1 μM to about 10 μM, for example, at least about 1 μM, at least about 5 μM, or at least about 10 μM.

[0197] In the present application, the seed medium may contain IL-2, IL-7, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0198] For example, the seed medium may contain at least about 3000 U / mL IL-2, at least about 1000 U / mL IL-7, at least about 100 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 10 pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0199] In the present application, the seed medium may contain IL-2, IL-9, IL-15, PD-1 antibody or its antigen-binding fragment, CTLA-4 antibody or its antigen-binding fragment, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0200] For example, the seed medium may contain at least about 2000 U / mL IL-2, at least about 500 U / mL IL-9, at least about 500 U / mL IL-15, at least about 50 μg / mL CTLA-4 antibody or antigen-binding fragment thereof, at least about 50 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 3% human serum, serum-free medium AIM-V, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0201] In the present application, the seed medium may contain IL-2, IL-7, G-CSF, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0202] For example, the seed medium may contain at least about 2000 U / mL IL-2, at least about 500 U / mL IL-7, at least about 1000 U / mL G-CSF, at least about 36 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 20 pg / mL TNFα, at least about 3% human serum, serum-free medium AIM-V, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0203] In the present application, the seed medium may contain IL-2, IL-12, IL-21, GM-CSF, LAG3 antibody or its antigen-binding fragment, CTLA-4 antibody or its antigen-binding fragment, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0204] For example, the seed medium may contain at least about 3000 U / mL of IL-2, at least about 200 U / mL of IL-12, at least about 500 U / mL of IL-21, at least about 800 U / mL of GM-CSF, at least about 10 μg / mL of a LAG-3 antibody or an antigen-binding fragment thereof, at least about 10 μg / mL of a CTLA-4 antibody or an antigen-binding fragment thereof, at least about 5% human serum, and a serum-free medium CTS. TM OpTmizer TMand 100 U / mL penicillin-streptomycin mixture PS double antibody may be included.

[0205] In the present application, the seed medium may contain IL-1β, IL-2, IL-6, IL-21, IFN-γ, TIGIT antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0206] For example, the seed medium may contain at least about 500 U / mL IL-1β, at least about 2500 U / mL IL-2, at least about 200 U / mL IL-6, at least about 200 U / mL IL-21, at least about 1000 U / mL IFN-γ, at least about 20 μg / mL TIGIT antibody or antigen-binding fragment thereof, at least about 50 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 25 pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15, 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0207] In the present application, the seed medium may contain IL-1α, IL-2, IL-9, IL-18, IFN-α, IFN-β, LAG-3 antibody or an antigen-binding fragment thereof, PD-1 antibody or an antigen-binding fragment thereof, TNFα, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0208] For example, the seed medium may contain at least about 500 U / mL IL-1α, at least about 2000 U / mL IL-2, at least about 500 U / mL IL-9, at least about 200 U / mL IL-18, at least about 500 U / mL IFN-α, at least about 5 μg / mL LAG-3 antibody or antigen-binding fragment thereof, at least about 25 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 25 pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0209] In the present application, the seed medium may contain IL-2, IL-4, IL-10, IL-21, CD137 antibody or its antigen-binding fragment, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0210] For example, the seed medium may contain at least about 3000 U / mL IL-2, at least about 200 U / mL IL-4, at least about 200 U / mL IL-10, at least about 250 U / mL IL-21, at least about 10 μg / mL CD137 antibody or antigen-binding fragment thereof, at least about 3 μg / mL LAG-3 antibody or antigen-binding fragment thereof, at least about 20 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 10 pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0211] In the present application, the seed medium may contain IL-2, IL-6, IL-12, IL-21, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0212] For example, the seed medium may contain at least about 3000 U / mL of IL-2, at least about 500 U / mL of IL-6, at least about 500 U / mL of IL-12, at least about 200 U / mL of IL-21, at least about 20 μg / mL of a CD137 antibody or an antigen-binding fragment thereof, at least about 10 μg / mL of a CD28 antibody or an antigen-binding fragment thereof, at least about 10 μg / mL of a PD-1 antibody or an antigen-binding fragment thereof, at least about 10 pg / mL of TNFα, at least about 3% human serum, and a serum-free medium CTS. TM OpTmizer TM and 100 U / mL penicillin-streptomycin mixture PS double antibody may be included.

[0213] In the present application, the seed medium may contain IL-1β, IL-2, IL-7, IL-21, G-CSF, GM-CSF, IFN-γ, LAG-3 antibody or an antigen-binding fragment thereof, PD-1 antibody or an antigen-binding fragment thereof, TNFα, human serum, serum-free medium X-VIVO 15, penicillin-streptomycin mixture PS double antibody.

[0214] For example, the seed medium may contain at least about 200 U / mL of IL-1β, at least about 3000 U / mL of IL-2, at least about 200 U / mL of IL-7, at least about 200 U / mL of IL-21, at least about 500 U / mL of G-CSF, at least about 500 U / mL of GM-CSF, at least about 1000 U / mL of IFN-γ, at least about 5 μg / mL of a LAG-3 antibody or an antigen-binding fragment thereof, at least about 3 μg / mL of a PD-1 antibody or an antigen-binding fragment thereof, at least about 15 pg / mL of TNFα, at least about 5% human serum, and a serum-free medium CTS. TM OpTmizer TM and 100 U / mL penicillin-streptomycin mixture PS double antibody may be included.

[0215] In the present application, the seed medium may contain IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody or an antigen-binding fragment thereof, CTLA-4 antibody or an antigen-binding fragment thereof, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0216] For example, the seed medium may contain at least about 2500 U / mL IL-2, at least about 500 U / mL IL-4, at least about 500 U / mL IL-12, at least about 500 U / mL GM-CSF, at least about 300 U / mL M-CSF, at least about 200 U / mL IFN-β, at least about 800 U / mL IFN-γ, at least about 5 μg / mL TIGIT antibody or antigen-binding fragment thereof, at least about 10 μg / mL CTLA-4 antibody or antigen-binding fragment thereof, at least about 5% human serum, serum-free medium X-VIVO 15, 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0217] In the present application, the seed medium may contain IL-2, IL-7, IL-15, GM-CSF, CD137 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0218] For example, the seed medium may contain at least about 3000 U / mL IL-2, at least about 200 U / mL IL-7, at least about 200 U / mL IL-15, at least about 800 U / mL GM-CSF, at least about 10 μg / mL CD137 antibody or antigen-binding fragment thereof, at least about 20 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 20 pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0219] In the present application, the seed medium may contain IL-2, IL-4, IL-10, IL-10, G-CSF, M-CSF, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, OX-40 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0220] For example, the seed medium may contain at least about 2500 U / mL IL-2, at least about 500 U / mL IL-4, at least about 500 U / mL IL-10, at least about 250 U / mL IL-10, at least about 500 U / mL G-CSF, at least about 300 U / mL L-CSF, at least about 5 μg / mL CD137 antibody or antigen-binding fragment, at least about 3 μg / mL CD28 antibody or antigen-binding fragment thereof, at least about 10 μg / mL OX-40 antibody or antigen-binding fragment thereof, at least about 10 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 5% human serum, serum-free medium AIM-V, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0221] In the present application, the seed medium may contain IL-2, IL-7, IL-21, IFN-α, IFN-γ, a CD137 antibody or an antigen-binding fragment thereof, a CD28 antibody or an antigen-binding fragment thereof, an OX-40 antibody or an antigen-binding fragment thereof, at least about a LAG-3 antibody or an antigen-binding fragment thereof, a CTLA-4 antibody or an antigen-binding fragment thereof, TNFα, human serum, serum-free medium X-VIVO 15, and a penicillin-streptomycin mixture PS double antibody.

[0222] For example, the seed medium may contain at least about 3000 U / mL IL-2, at least about 200 U / mL IL-7, at least about 500 U / mL IL-21, at least about 500 U / mL IFN-α, at least about 800 U / mL IFN-γ, at least about 5 μg / mL CD137 antibody or antigen-binding fragment thereof, at least about 3 μg / mL CD28 antibody or antigen-binding fragment thereof, at least about 5 μg / mL OX-40 antibody or antigen-binding fragment thereof, at least about 3 μg / mL LAG-3 antibody or antigen-binding fragment thereof, at least about 5 μg / mL CTLA-4 antibody or antigen-binding fragment thereof, at least about 10 pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0223] In the present application, the seed medium may contain IL-2, IL-7, IL-15, IFN-β, IFN-γ, a CD137 antibody or an antigen-binding fragment thereof, a CD40 antibody or an antigen-binding fragment thereof, an OX-40 antibody or an antigen-binding fragment thereof, a TIGIT antibody or an antigen-binding fragment thereof, a PD-1 antibody or an antigen-binding fragment thereof, human serum, serum-free medium X-VIVO 15, and a penicillin-streptomycin mixture PS double antibody.

[0224] For example, the seed medium may contain at least about 3000 U / mL IL-2, at least about 500 U / mL IL-7, at least about 500 U / mL IL-15, at least about 250 U / mL IFN-β, at least about 1000 U / mL IFN-γ, at least about 5 μg / mL of a CD137 antibody or antigen-binding fragment thereof, at least about 5 μg / mL of a CD40 antibody or antigen-binding fragment thereof, at least 5 μg / mL of an OX-40 antibody or antigen-binding fragment thereof, at least 5 μg / mL of a TIGIT antibody or antigen-binding fragment thereof, at least 15 μg / mL of a PD-1 antibody or antigen-binding fragment thereof, at least 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL of a penicillin-streptomycin mixture PS double antibody.

[0225] In the present application, the seed medium may contain IL-2, IL-7, IL-15, GM-CSF, IFN-γ, a CD137 antibody or an antigen-binding fragment thereof, a CD28 antibody or an antigen-binding fragment thereof, a PD-1 antibody or an antigen-binding fragment thereof, TNFα, at least about 5% human serum, and a serum-free medium CTS. TM OpTmizer TM and penicillin-streptomycin mixture PS double antibody may be included.

[0226] For example, the seed medium may contain at least about 3000 U / mL of IL-2, at least about 200 U / mL of IL-7, at least about 200 U / mL of IL-15, at least about 500 U / mL of GM-CSF, at least about 1000 U / mL of IFN-γ, at least about 3 μg / mL of a CD137 antibody or an antigen-binding fragment thereof, at least about 3 μg / mL of a CD28 antibody or an antigen-binding fragment thereof, at least about 3 μg / mL of a PD-1 antibody or an antigen-binding fragment thereof, at least about 10 pg / mL of TNFα, at least about 5% human serum, and a serum-free medium CTS. TM OpTmizer TM and 100 U / mL penicillin-streptomycin mixture PS double antibody may be included.

[0227] In the present application, the seed medium may contain IL-2, IL-7, IL-12, G-CSF, GM-CSF, IFN-α, IFN-γ, CD28 antibody or its antigen-binding fragment, CD40 antibody or its antigen-binding fragment, TIGIT antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0228] For example, the seed medium may contain at least about 3000 U / mL IL-2, at least about 200 U / mL IL-7, at least about 300 U / mL IL-12, at least about 300 U / mL G-CSF, at least about 200 U / mL GM-CSF, at least about 500 U / mL IFN-α, at least about 3 μg / mL CD28 antibody or antigen-binding fragment thereof, at least 5 μg / mL CD40 antibody or antigen-binding fragment thereof, at least 2.5 μg / mL TIGIT antibody or antigen-binding fragment thereof, at least 3 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least 10 pg / mL TNFα, at least 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0229] In the present application, the seed medium contains IL-2, IL-7, PD-1 antibody or its antigen-binding fragment, RRx-001, CAL-101, human serum, serum-free medium CTS TM OpTmizer TM and penicillin-streptomycin mixture PS double antibody may be included.

[0230] For example, the seed medium may contain at least about 3000 U / mL of IL-2, at least about 1000 U / mL of IL-7, at least about 50 μg / mL of a PD-1 antibody or an antigen-binding fragment thereof, at least about 3 μM of RRx-001, at least about 1.5 μM of CAL-101, at least about 5% human serum, and the serum-free medium CTS. TM OpTmizer TM and 100 U / mL penicillin-streptomycin mixture PS double antibody may be included.

[0231] In the present application, the seed medium may contain IL-2, IL-10, IL-18, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, sunitinib, imatinib, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0232] For example, the seed medium may contain at least about 2500 U / mL IL-2, at least about 200 U / mL IL-10, at least about 200 U / mL IL-18, at least about 10 μg / mL LAG-3 antibody or antigen-binding fragment thereof, at least about 5 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 3 μM sunitinib, at least about 5 μM imatinib, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0233] In the present application, the seed medium may contain IL-2, IL-1β, IL-6, GM-CSF, M-CSF, CNI-1493, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0234] For example, the seed medium may contain at least about 3000 U / mL IL-2, at least about 200 U / mL IL-1β, at least about 500 U / mL IL-6, at least about 500 U / mL GM-CSF, at least about 500 U / mL M-CSF, at least about 5 μM CNI-1493, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0235] In the present application, the seed medium may contain IL-2, IL-9, IL-10, IL-21, GM-CSF, OX40 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, sunitinib, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0236] For example, the seed medium may contain at least about 2000 U / mL IL-2, at least about 200 U / mL IL-9, at least about 200 U / mL IL-10, at least about 500 U / mL IL-21, at least about 500 U / mL GM-CSF, at least about 5 μg / mL OX40 antibody or antigen-binding fragment thereof, at least about 36 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 1.5 μM sunitinib, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0237] In the present application, the seed medium may contain IL-2, IL-6, IL-12, CD28 antibody or an antigen-binding fragment thereof, OX-40 antibody or an antigen-binding fragment thereof, CTLA-4 antibody or an antigen-binding fragment thereof, TNFα, imatinib, human serum, serum-free medium CTS TM OpTmizer TM and penicillin-streptomycin mixture PS double antibody may be included.

[0238] For example, the seed medium may contain at least about 2500 U / mL of IL-2, at least about 500 U / mL of IL-6, at least about 1000 U / mL of IL-12, at least about 10 μg / mL of a CD28 antibody or an antigen-binding fragment thereof, at least about 3 μg / mL of an OX-40 antibody or an antigen-binding fragment thereof, at least about 5 μg / mL of a CTLA-4 antibody or an antigen-binding fragment thereof, at least about 15 pg / mL of TNFα, at least about 5 μM of imatinib, at least about 5% human serum, and a serum-free medium CTS. TM OpTmizer TM and 100 U / mL penicillin-streptomycin mixture PS double antibody may be included.

[0239] In the present application, the seed medium may contain IL-2, IL-7, IL-15, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, dasatinib, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0240] For example, the seed medium may contain at least about 3000 U / mL IL-2, at least about 200 U / mL IL-7, at least about 300 U / mL IL-15, at least about 12 μg / mL CD137 antibody or antigen-binding fragment thereof, at least about 5 μg / mL CD28 antibody or antigen-binding fragment thereof, at least about 5 μg / mL LAG-3 antibody or antigen-binding fragment thereof, at least about 3 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 3 μM dasatinib, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0241] In the present application, the seed medium may contain IL-2, IL-6, IL-12, G-CSF, M-CSF, IFN-β, IFN-γ, CTLA-4 antibody or an antigen-binding fragment thereof, PD-1 antibody or an antigen-binding fragment thereof, dasatinib, LYC-55716, GNE-1858, human serum, serum-free medium X-VIVO 15, and penicillin-streptomycin mixture PS double antibody.

[0242] For example, the seed medium may contain at least about 3000 U / mL IL-2, at least about 500 U / mL IL-6, at least about 500 U / mL IL-12, at least about 1000 U / mL G-CSF, at least about 500 U / mL M-CSF, at least about 500 U / mL IFN-β, at least about 800 U / mL IFN-γ, at least about 3 μg / mL CTLA-4 antibody or antigen-binding fragment thereof, at least about 5 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 2.5 μM dasatinib, at least about 10 μM LYC-55716, at least about 4.5 nM GNE-1858, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0243] In the present application, the seed medium may contain IL-2, IL-1α, IL-9, GM-CSF, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, LAG-3 antibody or its antigen-binding fragment, TIGIT antibody or its antigen-binding fragment, CNI-1493, methylene blue, TWS119, human serum, No. 1 serum medium AIM-V and penicillin-streptomycin mixture PS double antibody.

[0244] For example, the seed medium may contain at least about 2500 U / mL IL-2, at least about 500 U / mL IL-1α, at least about 500 U / mL IL-9, at least about 800 U / mL GM-CSF, at least about 3 μg / mL CD137 antibody or antigen-binding fragment thereof, at least about 3 μg / mL CD28 antibody or antigen-binding fragment thereof, at least about 5 μg / mL LAG-3 antibody or antigen-binding fragment thereof, at least about 2.5 μg / mL TIGIT antibody or antigen-binding fragment thereof, at least about 2.5 μM CNI-1493, at least about 1 μM methylene blue, at least about 5 μM TWS119, at least about 5% human serum, serum-free medium AIM-V, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0245] In the present application, the seed medium may contain IL-2, IL-1α, IL-12, IL-21, G-CSF, M-CSF, IFN-α, IFN-β, IFN-γ, a CD28 antibody or an antigen-binding fragment thereof, a CD40 antibody or an antigen-binding fragment thereof, a LAG-3 antibody or an antigen-binding fragment thereof, a PD-1 antibody or an antigen-binding fragment thereof, CNI-1493, dasatinib, GNE-1858, methylene blue, human serum, serum-free medium AIM-V, and a penicillin-streptomycin mixture PS double antibody.

[0246] For example, the seed medium may contain about 3000 U / mL of IL-2, about 200 U / mL of IL-1α, about 500 U / mL of IL-12, about 500 U / mL of IL-21, about 300 U / mL of G-CSF, about 800 U / mL of M-CSF, about 500 U / mL of IFN-α, about 300 U / mL of IFN-β, about 300 U / mL of IFN-γ, about 3 μg / mL of a CD28 antibody or an antigen-binding fragment thereof, about 5 μg / mL of a CD40 antibody or an antigen-binding fragment thereof, about 5 μg / mL of a LAG-3 antibody or an antigen-binding fragment thereof, about 3 μg / mL of a PD-1 antibody or an antigen-binding fragment thereof, about 3 μM of CNI-1493, about 2 μM of dasatinib, about 5 nM of GNE-1858, about 1.5 μM of IFN-γ, about 1 ... The PS double antibody may be added in the serum-free medium AIM-V, 0.1 μM methylene blue, 5% human serum, and 100 U / mL penicillin-streptomycin mixture.

[0247] In another aspect, the present application provides a tumor-infiltrating lymphocyte seed cell or a cell population thereof obtained by culturing in the seed cell medium described herein.

[0248] In another aspect, the present application provides a pharmaceutical composition comprising a tumor-infiltrating lymphocyte seed cell or cells thereof described herein and a pharmaceutically acceptable carrier.

[0249] In another aspect, the present application provides the use of the tumor-infiltrating lymphocyte seed cells or cell populations thereof and the pharmaceutical composition of the present application in the preparation of a cancer treatment drug.

[0250] The seed medium described herein can be applied to the expansion of tumor-infiltrating lymphocytes derived from different types of cancer. The seed medium described herein has a broad spectrum and versatility, regardless of the type of cancer. In some aspects, the seed medium may be suitable for culturing tumor-infiltrating lymphocytes derived from different types of cancer, or for obtaining seed cells or cell populations derived from tumor-infiltrating lymphocytes derived from different types of cancer. In certain embodiments, the seed medium described herein and / or the pharmaceutical composition described herein can be used to prepare a medicament for the treatment of cancer. In the present application, the cancer may include cervical cancer (which may include, for example, recurrent cervical cancer), ovarian cancer, pelvic poorly differentiated adenocarcinoma, intestinal cancer (which may include, for example, metastatic intestinal cancer, e.g., liver metastasis), gastric cancer, melanoma, breast cancer, and / or endometrial stromal sarcoma. For example, the cancer may be selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, and bile duct carcinoma.

[0251] In another aspect, the present application provides the use of the seed cell culture medium in expanding tumor-infiltrating lymphocytes.

[0252] In another aspect, the present application provides a method for culturing seed cells of tumor-infiltrating lymphocytes, comprising culturing isolated tumor-infiltrating lymphocytes in a seed cell medium described herein.

[0253] In another aspect, the present application provides a method for expanding tumor-infiltrating lymphocytes, comprising the step of culturing isolated tumor-infiltrating lymphocytes in a seed cell medium described herein to obtain seed cells of the tumor-infiltrating lymphocytes.

[0254] For example, the isolated tumor-infiltrating lymphocytes may be derived from a sample selected from the group consisting of ascites, a surgical resection sample of a primary lesion, a surgical resection sample of a synchronous or metachronous metastasis, a puncture sample, and a body fluid of a subject in need thereof.

[0255] For example, the bodily fluid may include blood, tissue fluid, lymphatic fluid, and / or body cavity fluid. For example, the bodily fluid may include peritoneal fluid (e.g., peritoneal fluid from a patient with gastric cancer, peritoneal fluid from a patient with breast cancer), and / or pleural fluid (e.g., pleural fluid from a patient with endometrial stromal sarcoma).

[0256] For example, the isolated tumor-infiltrating lymphocytes may be derived from a tumor tissue selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, poorly differentiated pelvic adenocarcinoma, and bile duct cancer. For example, the isolated tumor-infiltrating lymphocytes may be derived from a tumor tissue selected from the group consisting of cervical cancer (which may include, for example, recurrent cervical cancer), ovarian cancer, poorly differentiated pelvic adenocarcinoma, intestinal cancer (which may include, for example, metastatic intestinal cancer, e.g., liver metastasis), gastric cancer, melanoma, breast cancer, and / or endometrial stromal sarcoma.

[0257] For example, the separated tumor-infiltrating lymphocytes may be derived from a tissue mass obtained by cutting tumor tissue, and the diameter of the tissue mass obtained by cutting tumor tissue may be about 1 mm to about 10 mm, for example, about 2 mm to about 10 mm, about 3 mm to about 10 mm, about 4 mm to about 10 mm, about 5 mm to about 10 mm, about 6 mm to about 10 mm, about 7 mm to about 10 mm, or about 8 mm to about 10 mm.

[0258] For example, the culture period may be about 3 to 20 days. For example, the culture period may be about 3 to 15 days, e.g., about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days.

[0259] For example, the culture temperature may be about 30 to 42° C. For example, it may be at least about 30° C., at least about 31° C., at least about 32° C., at least about 33° C., at least about 34° C., at least about 35° C., at least about 36° C., at least about 37° C., at least about 38° C., at least about 39° C., at least about 40° C., or at least about 42° C.

[0260] For example, the CO concentration for culture may be about 1 to 10%, for example, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

[0261] In the present application, the use of the seed medium described in the present application can significantly increase the cell activity of tumor-infiltrating lymphocyte seed cells (e.g., can significantly increase the total cell number of tumor-infiltrating lymphocyte seed cells, e.g., can increase the number of tumor-infiltrating lymphocyte seed cells by at least about 10 8 For example, the viability of tumor-infiltrating lymphocyte seed cells may be increased to at least about 85%, and the amount of cytokines (e.g., IFN-γ) secreted by tumor-infiltrating lymphocyte seed cells may be significantly increased. + The proportion of cells can be significantly increased.

[0262] In the present application, the use of the seed medium described herein significantly increases the frequency of TCR clones in the peripheral blood of a portion of the reinfused TILs after reinfusion of tumor-infiltrating lymphocyte seed cells into a donor subject (e.g., a tumor patient). Furthermore, the similarity between the TCR repertoire in the donor's peripheral blood and that of the reinfused TILs is significantly higher and maintained over time. Furthermore, autologous reinfusion of TIL cells cultured in the seed medium described herein results in more vigorous proliferation of lymphocytes in the donor subject's body.

[0263] The following examples are not intended to be limited by theory and are used only to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the present application.

[0264] Example

[0265] Example 1 TIL seed cell culture medium

[0266] TIL seed cell medium was prepared using the following components (product numbers): TIFF0007785699000001.tif224170

[0267] Various TIL seed cell culture media were prepared according to Tables 1 to 3. Serum and double antibodies were added to the basal medium at the specified ratios and stored at 4°C for up to one month. Before use, the medium was removed from 4°C and preheated in a 37°C water bath. Other components (various interleukins, colony-stimulating factors, interferons, TNF-α, various antibodies, and other molecules) were added to working concentrations prior to use for TIL cell culture. For comparison, CM1 medium (hereinafter referred to as CM1) containing 6000 IU / mL of IL-2, Glutamax, and antibiotics was prepared according to the formulation and method described in Example 5 of the CN110099998A specification and used for Pre-REP culture of tumor sample-derived TIL cells (corresponding to the seed cells in this application). [Table 1] TIFF0007785699000003.tif239170TIFF0007785699000004.tif248170TIFF0007785699000005.tif248170TIFF0007785699000006.tif62170 [Table 2] TIFF0007785699000008.tif241170TIFF0007785699000009.tif240170TIFF0007785699000010.tif229170TIFF0007785699000011.tif31170 [Table 3] TIFF0007785699000013.tif240170TIFF0007785699000014.tif240170TIFF0007785699000015.tif248170

[0268] Example 2 Processing of solid tumor samples (surgically removed tissue) and culturing of TIL seed cells

[0269] 1) A saline solution containing penicillin at a final concentration of 100 U / mL, streptomycin at 100 μg / mL, and gentamicin at 50 μg / mL was prepared. 2) In a sterile environment in a Class II safety cabinet, the obtained freshly isolated tumor tissue sample was added to a 10 cm dish containing 30 mL of the saline prepared in step 1) and washed, then transferred to a new 10 cm dish containing 30 mL of the saline prepared in step 1) and washed, for a total of three times. 3) Remove adipose tissue and necrotic tissue using a sterile surgical blade, and extract tumor tissue measuring 3 × 3 × 3 mm. 3 The tumor tissue was cut into small pieces, and 12 randomly selected pieces were placed in G-REX10 culture flasks (purchased from Wilsonwolf), each containing one of the TIL seed cell culture media prepared in Example 1. The remaining tumor tissue pieces were cryopreserved in liquid nitrogen using a programmed freezer with CryoStor10 (purchased from BioLifeSolutions) freezing medium. 4) 40 mL of the TIL seed cell medium prepared in Example 1 for each group was added to each G-REX10 culture flask, and the tumor tissue mass was cultured at 37°C in the presence of 5% CO2. After harvesting the TIL seed cells on day 12, the total cell count and viability were counted, and the cell phenotype was detected using a flow cytometer. IFN-γ secretion levels were measured using an HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, product number: 62HIFNGPET) according to the method described in the instruction manual.

[0270] Example 3 Treatment of body cavity fluid (ascites, pleural effusion) from tumor patients and culture of TIL seed cells

[0271] Tumor patient-derived body cavity fluids (ascites T012, T013, and pleural effusion T014) were centrifuged at 300 g for 8 minutes. The supernatant was removed, and the cell pellet was washed three times with D-PBS, centrifuged at 300 g for 8 minutes each time. Finally, the cells were resuspended in TIL seed medium and cultured at 37°C in 5% CO2. On day 12, TIL seed cells were harvested, and the total cell count and viability were measured. The cell phenotype was measured using a flow cytometer, and IFN-γ secretion levels were measured using an HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, product number: 62 HIFNGPET) according to the instructions in the manufacturer's manual.

[0272] The TIL seed cells in the ascites or pleural effusion of each of the following groups were obtained from 6 mL of body cavity fluid.

[0273] Table 4 shows information on each tumor tissue (body cavity fluid) treated in Example 3. [Table 4]

[0274] Each tumor tissue and body cavity fluid treated in Examples 2 and 4 was cultured in the medium for each group listed in Table 5, with "+" indicating that the corresponding medium was used and "-" indicating that the corresponding medium was not used. Tumor tissues T003, T005, and T007 were cultured in CM1 medium as a control. [Table 5] TIFF0007785699000018.tif66170

[0275] The culture results are shown in Tables 6 to 17 and Figures 1 to 15. Tables 6 to 17 show the results of TIL seed cell culture for T001, T002, T003, T004, T005, T006, T007, T008, T015, T012, T013, and T014 in Table 5, respectively, Figures 1 to 12 show representative flow cytometry analysis results for T001, T002, T003, T004, T005, T006, T007, T008, T015, T012, T013, and T014, respectively, after TIL seed cell culture using the seed cell medium of the present application, and Figures 13, 14, and 15 show the results of flow cytometry analysis for T002, T003, and T007, respectively, after culturing them in CM1 medium. [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17]

[0276] T016 was cultured using medium 15, and the total cell number was 1.13 × 10 8 , cell viability was 95.01%, the secreted IFN-γ concentration in the supernatant was 8446.32 pg / mL, and CD3 + The percentage of cells was 92.64%.

[0277] From the results of Tables 6 to 14 and T016, all of the solid tumor tissues cultured using the seed cell medium of each group of the present invention had at least 10 8 The total cell numbers achieved were approximately 6 × 10 for tumor samples such as T003, T005, and T006. 8 Near, 7x10 8 On the other hand, in the three samples T003, T005, and T007 cultured in parallel using CM1, the total number of cells obtained was 1 × 10 8 or 2 x 10 8 Although the total cell number exceeded 100%, the total cell number was relatively low compared to cells cultured in the corresponding medium of each group. The viability of TIL seed cells cultured using the seed medium of each group of the present application was high, all above 85%, and most above 90%. In three samples, T003, T005, and T007, cultured in parallel using CM1, the viability of the resulting seed cells was significantly reduced compared to the viability of cells cultured in the corresponding medium of each group. Among these, the viability of CM1 seed cells in two samples, T005 and T007, was lower than 80%. Furthermore, the IFN-γ secretion levels of TIL seed cells cultured using the seed medium of each group of the present application were all higher. In three samples, T003, T005, and T007, cultured in parallel using CM1, the IFN-γ secretion levels of the resulting TIL seed cells were also significantly reduced compared to the corresponding samples.

[0278] As shown in Figures 1 to 9, the flow phenotype of TIL seed cells obtained from solid tumor tissues cultured using the seed cell medium of each group of the present application was normal, and in most samples, CD3 + The percentage of cells was over 90%, and in some samples, such as T002 and T006, CD3 + The percentage of CD3 cells was found to be approximately 99%. + The cell ratio data show the same results. In most samples, CD4 + and CD8 + The ratio was close to 1:1, and in some samples, such as T006 and T012, the CD8 + Here, Figures 1A to 9A show the CD3 + Figures 1B to 9B show the percentage of CD4 + 1 to 9 are flow diagrams of cell phenotypes after culturing T001 in medium #1, T002 in medium #3, T003 in medium #2, T004 in medium #10, T005 in medium #7, T006 in medium #8, T007 in medium #16, T008 in medium #11, and T015 in medium #15, respectively.

[0279] The results in Tables 15 to 17 demonstrate that TIL seed cells can be successfully cultured from body cavity fluids such as pleural effusion and ascites of tumor patients using the seed cell culture medium of each group of the present invention. 8 In some samples, more than 100 TIL seed cells were obtained. Furthermore, the survival rates of TIL seed cells obtained from the coelomic fluid cultured in each group were high, exceeding 85% in all cases and exceeding 90% in most cases. Furthermore, the TIL seed cells in all groups secreted high levels of IFN-γ.

[0280] As shown in Figures 10 to 12, the flow phenotype of TIL seed cells obtained from the body cavity fluid of tumor patients cultured using the seed medium of each group of the present application was normal, and CD3 + The percentage of cells was over 80%, and CD3 in seed cells of T012 and T014 + The percentage of CD3 cells in Tables 15 to 17 exceeded 90%. +The data on cell ratios also show the same results. Figures 10 to 12 show the flow diagrams of cell phenotypes after culturing T012 in medium #25, T013 in medium #9, and T014 in medium #12, respectively.

[0281] As shown in Figures 13 to 15, the flow phenotype of TIL seed cells obtained by culturing three solid tumor tissue samples, T003, T005, and T007, in CM1 medium was normal, and CD3 + The proportion of CD3 cells was high in both T003 and T007. + The percentage of CD3 cells was about 99%, while the percentage of CD3 + The percentage of cells was significantly higher than that of TIL seed cells after culture in the corresponding media of the other groups.

[0282] Example 4 Processing of solid tumor samples (aspirated tissue) and culturing of TIL seed cells

[0283] 1) A physiological saline solution containing penicillin at a final concentration of 100 U / mL, streptomycin at 100 μg / mL, and gentamicin at 50 μg / mL was prepared.

[0284] 2) In a sterile environment in a Class I safety cabinet, tumor puncture tissue samples obtained by puncture with a fine needle (1 mm diameter) were added to a 10 cm dish containing 30 mL of the saline solution prepared in step 1) and washed, then transferred to a new 10 cm dish containing 30 mL of the saline solution prepared in step 1), and washed three times in total.

[0285] 3) Using a sterile surgical blade, puncture the tumor tissue to a diameter of 1 x 1 x 3 mm. 3 The tumor tissue was cut into small chunks of 1000 μg each, and three randomly selected chunks were placed in one well of each G-REX24 culture plate (purchased from Wilsonwolf). One well of each G-REX24 culture plate corresponded to one of the TIL seed cell media with a specific formulation in Example 1 of the present application, and 40 mL of CM1 medium was added per well as a control.

[0286] 4) The tumor aspirate tissue was cultured at 37°C in 5% CO2. On day 12, TIL seed cells were harvested, after which the total cell count and viability were measured. The cell phenotype was measured using a flow cytometer, and the IFN-γ secretion level was measured using an HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, product number: 62HIFNGPET) according to the method described in the instruction manual.

[0287] Tables 18 to 20 show specific information on the groups of tumor puncture tissue samples and the culture results. [Table 18] [Table 19] [Table 20]

[0288] From the results of Tables 18 to 20, the three tumor puncture tissue samples T009, T010, and T011 were cultured for 12 days using TIL seed cell culture media #2, #15, and #12 described in Example 1, respectively, and each produced approximately 0.3 × 10 8 It was confirmed that TIL seed cells were obtained. When the above tissues were cultured in parallel using CM1 medium, only the malignant melanoma aspirate tissue sample T011 yielded approximately the same number of TIL cells, while the other two aspirate tissue samples did not show any obvious TIL cells under microscopic observation after 12 days. In T011, which was cultured in medium #12, the tumor aspirate-derived TIL seed cells had higher survival rates and IFN-γ secretion levels, and CD3 TILs compared to CM1 TILs. + The proportion of cells was also almost the same in both cases.

[0289] In addition, the TIL cells derived from the above three tumor aspirates cultured in TIL seed media #2, #15, and #12 also showed cell viability of over 86%, reaching a maximum of 95%, and increased IFN-γ secretion levels and CD3 + The proportion of cells was also confirmed to be at a high level.

[0290] Example 5 Clinical reinfusion of TILs and in vivo changes in TCR phenotype

[0291] To verify the clinical safety and efficacy of TILs obtained by this culture method, a clinical trial of TIL reinfusion therapy for solid tumors was conducted. This clinical trial was approved by the Ethics Committee of Shanghai Tenth People's Hospital. For details, please see http: / / www.chictr.org.cn / , registration number: ChiCTR2100044705; or www.clinicaltrials.gov, registration number: NCT04766320.

[0292] Research Plan

[0293] 1) Low-intensity lymphatic clearance pretreatment: Patients received cyclophosphamide at a dose of 6 mg / kg / day for 3 consecutive days from day 6 to day 4 before TIL reinfusion, and fludarabine at a dose of 5 mg / m for 3 consecutive days from day 3 to day 1 before TIL reinfusion. 2 The dose was administered at 100 mg / day. 2) On the day after fludarabine administration, 10 ~10 11 Autologous TIL cells were reinfused intravenously. 3) The patient's condition after reinfusion was observed and recorded, peripheral blood was collected at different time points to detect the composition and changes of PBMCs, and the effects were evaluated by imaging 1 to 3 months after reinfusion.

[0294] Preparation of TIL cell reinfusion solution

[0295] 1) TIL seed cells were prepared from surgically resected tumor tissue of a patient by the method described in Example 2 above. 2) Preparation of expansion medium: According to the formulation and method described in Example 5 of Chinese patent application CN110099998A, AIM-V medium was supplemented with IL-2 on the day, and the final concentration was reduced from 3000 IU / mL to 1000 IU / mL. According to the concentration described in Example 6 of CN110099998A, CD3 antibody (OKT3) was added to AIM-V medium containing 1000 IU / mL of IL-2 to a final concentration of 30 ng / mL, which was used as the expansion medium for TIL cell expansion. 3) Preparation of infusion product (IP): 1 × 10 cells obtained by culturing the surgically resected tumor tissue. 8 Select seed cells and expand them in a G-REX100 culture flask using 1000 mL of the expansion medium prepared in step 2) at 37°C, 5% CO2 for 12-14 days. The cells are then harvested, centrifuged, washed with D-PBS, and resuspended in saline. If the cells are found to be CD3+ / CD4+ / CD5+ / CD6+ / CD8+ / CD9+ / CD10+ / CD11+ / CD12+ / CD13+ / CD14+ / CD15+ / CD16+ / CD17+ / CD18+ / CD19 ... + Percentage, CD4 + and CD8 + All the indicators of the immunotherapy product (including the ratio of IFN-γ, IFN-γ secretion level, sterility, endotoxin, osmolality, etc.) met the requirements of the Pharmacopoeia of the People's Republic of China or the corresponding general specifications for immuno-cell therapy products already on the market at home and abroad, and thus the reinfusion product was obtained.

[0296] The entire manufacturing process of the TIL reinjection was carried out in a GMP-grade manufacturing facility in accordance with relevant regulatory requirements.

[0297] Reinfusion of subject T015 (recurrent cervical cancer): T015 TIL seed cells obtained in Example 2 were selected and expanded according to the method described above, and 4 × 10 10 The reinfusion cells were obtained, resuspended in 200 mL of saline, and autologous reinfusion was performed intravenously in patients who underwent low-intensity lymphatic clearance pretreatment (without the administration of IL-2). Peripheral blood samples were collected from the patients before reinfusion and on days 19 (D19), 30 (D30), and 47 (D47) after reinfusion to measure the PBMC composition and changes. PBMCs were isolated at various time points and subjected to high-throughput sequencing of the TCR repertoire along with the reinfusion (IP).

[0298] The results are shown in Figures 16 to 20. As shown in Figure 16, CD3 + The percentage of cells is over 90% and CD3 + CD8 in cells + Cells and CD4 + The cell ratio exceeded 2:1. As shown in Figure 17, the frequency of TCR clones (i.e., IP clones) in the patient's peripheral blood began to increase after reinfusion, peaked at D30, and decreased at D47 compared to D30. As shown in Figure 18, the frequency of IP clone types in the patient's peripheral blood began to increase after reinfusion, peaked at D30, and did not decrease significantly at D47 compared to D30. As shown in Figure 19, the frequencies of the top 10 TCR clones (named IP-1 to IP-10, respectively) in the reinfusion significantly increased after reinfusion, with most peaking at D30. The second and ninth ranked TCR clones continued to increase in frequency even after D30. Figure 20 shows the Morishita Index (MOI) values ​​for the TCR repertoires of peripheral blood T cells before reinfusion, at D19, D30, and D47, compared to the TCR repertoires of IP. The Morishita index of the TCR repertoire of peripheral blood T cells and that of the reinfused specimens gradually increased over time after reinfusion, peaked on day D30, and showed no significant decline on day D47.

[0299] From the above results, it was found that TIL seed cells cultured using the TIL seed medium of the present invention could be expanded to 10 10 ~10 11 These cells reached a cell number of 1000 and were shown to be capable of significant long-term proliferation in vivo in the absence of IL-2 administration after autologous reinfusion from tumor patients who underwent low-intensity lymphatic clearance pretreatment.

[0300] Example 6 Clinical reinfusion of TILs and cell proliferation in vivo

[0301] Research Plan

[0302] 1) Low-intensity lymphatic clearance pretreatment: Patients received cyclophosphamide at a dose of 6 mg / kg / day without fludarabine for 3 consecutive days, starting from 3 days before TIL reinfusion to 1 day before TIL reinfusion. 2) 10 days after cyclophosphamide administration 10 ~10 11 Autologous TIL cells were reinfused intravenously into the patient. 3) The patient's condition after reinfusion was observed and recorded, peripheral blood was collected at different time points to detect the composition and changes of PBMCs, and the effects were evaluated by imaging 1 to 3 months after reinfusion.

[0303] The reinjected TIL cells were prepared in the same manner as in Example 5.

[0304] Reinfusion of subject T016 (cervical cancer patient): T016 TIL seed cells obtained in Example 2 were selected and expanded according to the method of Example 5 to obtain 5.2 × 10 10 The reinfusion cells were obtained, resuspended in 250 mL of saline, and autologous reinfusion was performed intravenously in patients who had undergone low-intensity lymphatic clearance pretreatment, without the injection of pharmaceutical IL-2 after reinfusion. Peripheral blood samples were collected from the subjects on days 4, 3, 2, and 1 before reinfusion (cyclophosphamide administration), and 1, 6, 8, 13, and 17 after reinfusion, and the white blood cell, neutrophil, and lymphocyte counts were counted.

[0305] The results are shown in Figure 21. As shown in Figure 21, the white blood cell and neutrophil counts in the subjects' peripheral blood decreased to their lowest levels approximately 10 days after cyclophosphamide pretreatment (6-8 days after reinfusion), which is consistent with literature reports (ME Gershwin, EJ Goetzl, AD Steinberg, Cyclophosphamide: use in practice, Ann Intern Med. 1974 Apr;80(4):531-40). Peripheral blood lymphocyte counts decreased to their lowest levels after cyclophosphamide pretreatment until 1 day after reinfusion, but continued to increase from 1 day after reinfusion. Even though the therapeutic effect of cyclophosphamide persisted and total white blood cell and neutrophil levels were at their lowest levels, a significant increase in lymphocyte counts was observed. By 13 days after reinfusion, lymphocyte counts had almost returned to their pre-cyclophosphamide pretreatment levels. From the above results, it was found that TIL seed cells cultured using the TIL seed medium of the present invention could be expanded to 10 10 ~10 11 The number of lymphocytes reached 1000, and vigorous and sustained lymphocyte proliferation was confirmed in vivo after autologous reinfusion.

[0306] Although the embodiments of the present application have been described in detail above, the present application is not limited to the details of the above embodiments. Various simple modifications of the technical solutions of the present application may be made within the scope of the technical idea of ​​the present application, all of which fall within the patent scope of the present application. It should be noted that the various specific technical features described in the above specific embodiments may be combined in any appropriate manner as long as they are not contradictory, and to avoid unnecessary repetition, the present application will not separately describe various possible combinations. Furthermore, the various embodiments of the present application may also be combined with each other, and such combinations shall be deemed to be disclosed by the present application unless they are contrary to the spirit of the present application.

Claims

1. A seed cell culture medium for tumor-infiltrating lymphocytes, comprising as ingredients a cell culture component, a cytokine, and an immune checkpoint antibody or an antigen-binding fragment thereof, wherein the cytokine includes IL-2, and the immune checkpoint includes PD-1, LAG-3, TIGIT, and / or CTLA-4, the cell culture component is a serum medium or a serum-free medium, and the concentration of the IL-2 is 2000 IU / mL to 3000 IU / mL.

2. 2. The seed cell culture medium according to claim 1, wherein the cytokines comprise IL-7, IL-15, tumor necrosis factor (TNF), colony-stimulating factor, interferon, IL-4, IL-1α, IL-1β, IL-6, IL-9, IL-18, IL-12, IL-21, and / or IL-10.

3. The concentration of the IL-7 is 200 U / mL to 1000 U / mL, the concentration of the IL-15 is 200 U / mL to 500 U / mL, the concentration of the IL-4 is 200 U / mL to 500 U / mL, the concentration of the IL-1α is 200 U / mL to 500 U / mL, the concentration of the IL-1β is 200 U / mL to 500 U / mL, and the concentration of the IL-6 is 200 U / mL to 500 U / mL.

3. The seed cell culture medium according to claim 2, wherein the IL-9 has a concentration of 200 U / mL to 500 U / mL, the IL-18 has a concentration of 200 U / mL to 500 U / mL, the IL-12 has a concentration of 200 U / mL to 500 U / mL, the IL-21 has a concentration of 200 U / mL to 500 U / mL, and / or the IL-10 has a concentration of 200 U / mL to 500 U / mL.

4. 2. The seed cell culture medium of claim 1, wherein the cytokine comprises TNFα, and the concentration of the TNFα is 10 pg / mL to 100 pg / mL.

5. 2. The seed cell culture medium according to claim 1, wherein the cytokines comprise GM-CSF, G-CSF, and / or M-CSF, the GM-CSF having a concentration of 200 U / mL to 5000 U / mL, the G-CSF having a concentration of 300 U / mL to 1000 U / mL, and the M-CSF having a concentration of 300 U / mL to 800 U / mL.

6. The seed cell culture medium according to claim 1, wherein the cytokines comprise IFN-γ, IFN-α, and / or IFN-β, the concentration of the IFN-γ is 10 U / mL to 1000 U / mL, the concentration of the IFN-α is 500 U / mL to 1000 U / mL, and the concentration of the IFN-β is 200 U / mL to 500 U / mL.

7. The seed cell culture medium according to claim 1, wherein the immune checkpoint antibody or antigen-binding fragment thereof is a PD-1 antibody or antigen-binding fragment thereof, a LAG-3 antibody or antigen-binding fragment thereof, a TIGIT antibody or antigen-binding fragment thereof, and / or a CTLA-4 antibody or antigen-binding fragment thereof.

8. The seed cell culture medium according to claim 1, wherein the concentration of the PD-1 antibody or antigen-binding fragment thereof is 1 μg / mL to 100 μg / mL, the concentration of the LAG-3 antibody or antigen-binding fragment thereof is 3 μg / mL to 10 μg / mL, the concentration of the TIGIT antibody or antigen-binding fragment thereof is 1 μg / mL to 25 μg / mL, and / or the concentration of the CTLA-4 antibody or antigen-binding fragment thereof is 1 μg / mL to 100 μg / mL.

9. The seed cell culture medium of claim 1, further comprising a costimulatory receptor antibody or an antigen-binding fragment thereof, wherein the costimulatory receptor antibody or an antigen-binding fragment thereof comprises a CD40 antibody or an antigen-binding fragment thereof, an OX-40 antibody or an antigen-binding fragment thereof, a CD137 antibody or an antigen-binding fragment thereof, and / or a CD28 antibody or an antigen-binding fragment thereof.

10. The seed cell culture medium according to claim 9, wherein the concentration of the CD137 antibody or antigen-binding fragment thereof is 1 μg / mL to 100 μg / mL, the concentration of the CD28 antibody or antigen-binding fragment thereof is 1 μg / mL to 10 μg / mL, the concentration of the CD40 antibody or antigen-binding fragment thereof is 5 μg / mL to 10 μg / mL, and / or the concentration of the OX-40 antibody or antigen-binding fragment thereof is 3 μg / mL to 10 μg / mL.

11. The seed cell culture medium of claim 1 , wherein the serum medium contains serum.

12. The seed cell culture medium according to claim 11, wherein the serum has a concentration of 1% to 10% (v / v).

13. 10. The seed cell medium of claim 1, wherein the cell culture components include an antibiotic.

14. 2. The seed cell medium of claim 1, wherein the cell culture components comprise a penicillin-streptomycin mixture (PS).

15. The seed cell culture medium according to claim 14, wherein the concentration of the penicillin-streptomycin mixture PS is 1 U / mL to 200 U / mL.

16. The seed cell culture medium according to claim 1, further comprising an M2-type macrophage inhibitor, a regulatory T cell (Treg) inhibitor, a myeloid-derived suppressor cell inhibitor, a T cell activator, and / or a T cell differentiation inhibitor, wherein the M2-type macrophage inhibitor comprises RRx001 and / or CNI-1493, the regulatory T cell inhibitor comprises CAL-101, dasatinib, imatinib, and / or panobinostat, the myeloid-derived suppressor cell inhibitor comprises AG490, decitabine, sunitinib, and / or BBI608, the T cell activator comprises LYC-55716, GNE-1858, and / or methylene blue, and the T cell differentiation inhibitor comprises TWS119.

17. A tumor-infiltrating lymphocyte seed cell or cell population thereof obtained by culturing in the seed cell medium according to claim 1.

18. A pharmaceutical composition comprising the tumor-infiltrating lymphocyte seed cells or cell populations thereof according to claim 17 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition for use as a cancer therapeutic agent, comprising the tumor-infiltrating lymphocyte seed cells or cell populations thereof according to claim 17.

20. 20. The pharmaceutical composition of claim 19, wherein the cancer is selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, and bile duct carcinoma.

21. 1. A method for culturing seed cells of tumor-infiltrating lymphocytes, comprising: A method comprising culturing isolated tumor-infiltrating lymphocytes in the seed cell medium of claim 1.

22. The method of claim 21, wherein the isolated tumor-infiltrating lymphocytes are derived from a sample selected from the group consisting of ascites, a surgically resected sample of a primary lesion, a surgically resected sample of a synchronous or metachronous metastatic lesion, a puncture sample, and a body fluid from a subject in need thereof.

23. The method of claim 22, wherein the bodily fluid comprises blood, tissue fluid, lymphatic fluid and / or body cavity fluid.

24. The method described in claim 21, wherein the isolated tumor-infiltrating lymphocytes are derived from a tumor selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, and bile duct carcinoma.

25. 1. A method for expanding tumor-infiltrating lymphocytes, comprising: A method comprising the step of culturing isolated tumor-infiltrating lymphocytes in the seed cell medium of claim 1 to obtain seed cells of said tumor-infiltrating lymphocytes.

26. 26. The method of claim 25, wherein the isolated tumor-infiltrating lymphocytes are derived from a sample selected from the group consisting of ascites, a surgical resection sample of a primary tumor, a surgical resection sample of a synchronous or metachronous metastasis, an aspirate sample, and a body fluid from a subject in need thereof.

27. 27. The method of claim 26, wherein the body fluid comprises blood, interstitial fluid, lymphatic fluid, and / or body cavity fluid.

28. 26. The method of claim 25, wherein the isolated tumor-infiltrating lymphocytes are derived from a tumor selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, and cholangiocarcinoma.

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