WO2021239083A1 - 肿瘤浸润淋巴细胞的种子细胞培养基及其应用 - Google Patents

肿瘤浸润淋巴细胞的种子细胞培养基及其应用 Download PDF

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WO2021239083A1
WO2021239083A1 PCT/CN2021/096585 CN2021096585W WO2021239083A1 WO 2021239083 A1 WO2021239083 A1 WO 2021239083A1 CN 2021096585 W CN2021096585 W CN 2021096585W WO 2021239083 A1 WO2021239083 A1 WO 2021239083A1
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culture medium
cell culture
antigen
antibody
binding fragment
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English (en)
French (fr)
Chinese (zh)
Inventor
金华君
何周
马星明
李甜甜
黄晨
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Shanghai Gencells Therapeutics Co Ltd
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Shanghai Gencells Therapeutics Co Ltd
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Priority to EP21811798.4A priority Critical patent/EP4159843A4/en
Priority to KR1020227046117A priority patent/KR20230019460A/ko
Priority to CN202180038540.7A priority patent/CN115698267A/zh
Priority to US18/000,122 priority patent/US20230226111A1/en
Priority to JP2022573182A priority patent/JP7785699B2/ja
Publication of WO2021239083A1 publication Critical patent/WO2021239083A1/zh
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Definitions

  • This application relates to the field of biomedicine, in particular to a seed cell culture medium for tumor infiltrating lymphocytes and its application.
  • TIL- tumor infiltrating lymphocytes
  • TIL-used in tumor immunotherapy requires a larger amount of cells, which poses a greater challenge in terms of technology and process control for the expansion of TIL-cells.
  • pre-REP pre-Rapid Expansion Procedure
  • REP rapid expansion procedure
  • This process requires a large amount of irradiated allogeneic PBMC (also known as mononuclear cells, MNC) from multiple donors as feeder cells, anti-CD3 antibodies (OKT3) and high-dose IL-2 .
  • Allogeneic irradiated PBMC cells from multi-donor sources have a potential risk of contamination, and higher concentrations of IL-2 may have the risk of cell depletion in the subsequent reinfusion.
  • This application provides a seed cell culture medium for tumor infiltrating lymphocytes and its application.
  • the seed cell culture medium and the preparation method thereof described in the present application have at least one beneficial effect selected from the following group: 1) significantly reduce the amount of IL-2; 2) reduce or even eliminate the need to use IL-2 in subsequent reinfusion; 3 ) Reduce the risk of cell exhaustion caused by higher concentrations of IL-2 in the subsequent reinfusion; 4)
  • the subsequent rapid expansion process (REP) does not require the use of exogenous allogeneic PBMC or other cells as feeder cells, reducing exogenous Contamination; 5) Promote the subsequent rapid expansion process (REP) to achieve the yield of 10 10 -10 11 cells; 6) Significantly increase the seed cells for obtaining tumor infiltrating lymphocytes, and even expand the culture efficiency of tumor infiltrating lymphocytes; shorten the culture time.
  • the present application provides a seed cell culture medium for tumor infiltrating lymphocytes, which includes the following components: cell culture components, cytokines, and immune checkpoint antibodies or antigen-binding fragments thereof, wherein the cytokines include IL -2, the immune checkpoint includes: PD-1, LAG-3, TIGIT and/or CTLA-4, and the cell culture component is a serum medium or a serum-free medium.
  • the concentration of IL-2 is about 3000 IU/mL or less.
  • the concentration of IL-2 is about 2000 IU/mL to about 3000 IU/mL.
  • the cytokine includes IL-7.
  • the concentration of IL-7 is about 200 to about 1000 U/mL.
  • the cytokine includes IL-15.
  • the concentration of IL-15 is about 200 to about 500 U/mL.
  • the cytokine includes tumor necrosis factor TNF.
  • the cytokine includes TNF ⁇ .
  • the concentration of TNF ⁇ is about 10 to about 100 pg/mL.
  • the cytokine includes colony stimulating factor.
  • the cytokine includes GM-CSF, G-CSF and/or M-CSF.
  • the concentration of the GM-CSF is about 200 to about 5000 U/mL.
  • the concentration of the G-CSF is about 300 U/mL to about 1000 U/mL.
  • the concentration of the M-CSF is about 300 U/mL to about 800 U/mL.
  • the cytokine includes interferon.
  • the cytokine includes IFN- ⁇ , IFN- ⁇ , and/or IFN- ⁇ .
  • the concentration of IFN- ⁇ is about 10 to about 1000 ng/mL.
  • the concentration of the IFN- ⁇ is about 300 to about 1000 U/mL.
  • the concentration of the IFN- ⁇ is about 500 U/mL to about 1000 U/mL.
  • the concentration of the IFN- ⁇ is about 200 U/mL to about 500 U/mL.
  • the cytokine further includes: IL-4, IL-1 ⁇ , IL-1 ⁇ , IL-6, IL-9, IL-18, IL-12, IL-21 and/or IL- 10.
  • the concentration of IL-4 is about 200 to about 500 U/mL.
  • the concentration of IL-1 ⁇ is about 200 to about 500 U/mL.
  • the concentration of IL-1 ⁇ is about 200 to about 500 U/mL.
  • the concentration of IL-6 is about 200 to about 500 U/mL.
  • the concentration of IL-9 is about 200 to about 500 U/mL.
  • the concentration of IL-18 is about 200 to about 500 U/mL.
  • the concentration of IL-12 is about 200 to about 1000 U/mL.
  • the concentration of IL-21 is about 200 to about 500 U/mL.
  • the concentration of IL-10 is about 200 to about 500 U/mL.
  • the immune checkpoint antibody or antigen-binding fragment thereof is a human PD-1 antibody or antigen-binding fragment thereof.
  • the immune checkpoint antibody or antigen-binding fragment thereof is of 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 to about 100 ⁇ g/mL.
  • the immune checkpoint antibody or antigen-binding fragment thereof is a human LAG-3 antibody or antigen-binding fragment thereof.
  • 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 to about 10 ⁇ g/mL .
  • the TIGIT antibody or antigen-binding fragment thereof is a human TIGIT antibody or antigen-binding fragment thereof.
  • 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 to about 25 ⁇ g/mL.
  • the immune checkpoint antibody or antigen-binding fragment thereof is a human CTLA-4 antibody or antigen-binding fragment thereof.
  • 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 to about 100 ⁇ g/mL .
  • the seed cell culture medium further includes a costimulatory receptor antibody or antigen-binding fragment thereof, wherein the costimulatory receptor or antigen-binding fragment thereof includes: CD40 antibody or antigen-binding fragment thereof, OX -40 antibody or its antigen-binding fragment, CD137 antibody or its antigen-binding fragment and/or CD28 antibody or its antigen-binding fragment.
  • the immune checkpoint antibody or antigen-binding fragment thereof includes a CD137 antibody or antigen-binding fragment thereof.
  • the CD137 antibody or antigen-binding fragment thereof is a human CD137 antibody or antigen-binding fragment thereof.
  • the concentration of the CD137 antibody or antigen-binding fragment thereof is about 1 to about 100 ⁇ g/mL.
  • the immune checkpoint antibody or antigen-binding fragment thereof includes a CD28 antibody or antigen-binding fragment thereof.
  • the CD28 antibody or antigen-binding fragment thereof is a human CD28 antibody or antigen-binding fragment thereof.
  • the concentration of the CD28 antibody or antigen-binding fragment thereof is about 1 to about 10 ⁇ g/mL.
  • the CD40 antibody or antigen-binding fragment thereof is a human CD40 antibody or antigen-binding fragment thereof.
  • the concentration of the CD40 antibody or antigen-binding fragment thereof is about 5 to about 10 ⁇ g/mL.
  • the OX-40 antibody or antigen-binding fragment thereof is a human OX-40 antibody or antigen-binding fragment thereof.
  • the concentration of the OX-40 antibody or antigen-binding fragment thereof is about 3 to about 10 ⁇ g/mL.
  • the serum culture medium includes serum.
  • the serum includes human AB serum.
  • the concentration of the serum is about 1 to about 10% (v/v).
  • the serum medium includes AIM-V medium.
  • the serum-free medium includes X-VIVO medium.
  • the cell culture components include antibiotics.
  • the cell culture component includes a penicillin-streptomycin mixture PS.
  • the concentration of the penicillin-streptomycin mixture PS is about 1 to about 200 U/mL.
  • the seed cell culture medium further includes an M2 type macrophage inhibitor, and the M2 type macrophage inhibitor includes RRx001 and/or CNI-1493.
  • the concentration of the M2 type macrophage inhibitor is about 0.1 to about 100 ⁇ M.
  • the seed cell culture medium further includes regulatory T cell (Treg) inhibitors, and the regulatory T cell inhibitors include CAL-101, dasatinib, and imatinib ( imatinib) and/or Panobinostat (Panobinostat).
  • Treg regulatory T cell
  • the regulatory T cell inhibitors include CAL-101, dasatinib, and imatinib ( imatinib) and/or Panobinostat (Panobinostat).
  • the concentration of the regulatory T cell inhibitor is about 0.1 to about 100 ⁇ M.
  • the seed cell culture medium further includes a bone marrow-derived suppressor cell (MDSC) inhibitor, and the bone marrow-derived suppressor cell inhibitor includes AG490, decitabine, and Nitinib and/or BBI608.
  • MDSC bone marrow-derived suppressor cell
  • the concentration of the bone marrow-derived suppressor cytostatic agent is about 0.1 to about 100 ⁇ g/mL.
  • the seed cell culture medium further includes a T cell activator, and the T cell activator includes LYC-55716, GNE-1858 and/or methylene blue.
  • the concentration of the T cell activator is about 1 to about 10 ⁇ M.
  • the seed cell culture medium further includes a T cell differentiation inhibitor, and the T cell differentiation inhibitor includes TWS119.
  • the concentration of the T cell differentiation inhibitor is about 1 to about 10 ⁇ M.
  • this application provides the seed cells of tumor infiltrating lymphocytes obtained by culturing the seed cell culture medium described in this application or their cell populations.
  • the present application provides a pharmaceutical composition, which includes the seed cell of the tumor-infiltrating lymphocyte or its cell population and a pharmaceutically acceptable carrier as described in the present application.
  • the present application provides the application of the seed cells of tumor infiltrating lymphocytes or cell populations thereof and the pharmaceutical composition of the present application in the preparation of drugs for the treatment of cancer.
  • the cancer is selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, bowel cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial cancer Stromal sarcoma, poorly differentiated pelvic adenocarcinoma, and cholangiocarcinoma.
  • this application provides an application of the seed cell culture medium in the expansion of tumor infiltrating lymphocytes.
  • this application provides a method for culturing seed cells of tumor-infiltrating lymphocytes, which includes the following steps: culturing isolated tumor-infiltrating lymphocytes in the seed cell culture medium described in this application.
  • this application provides a method for amplifying tumor infiltrating lymphocytes, which includes the following steps: culturing isolated tumor infiltrating lymphocytes in the seed cell culture medium described in this application to obtain the tumor infiltrating lymphocytes The seed cell of the cell.
  • the isolated tumor-infiltrating lymphocytes are derived from a sample selected from the group consisting of ascites from a subject in need, a sample of a primary tumor after surgical resection, and simultaneous and metachronous surgical resection of metastases Samples, puncture samples and body fluids.
  • the body fluid includes blood, tissue fluid, lymph fluid, and/or body cavity fluid.
  • the isolated tumor-infiltrating lymphocytes are derived from tumors selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, colon cancer, liver cancer, cervical cancer, Ovarian cancer, breast cancer, endometrial stromal sarcoma, poorly differentiated pelvic adenocarcinoma, and cholangiocarcinoma.
  • the isolated tumor-infiltrating lymphocytes are derived from a tissue mass after tumor tissue is cut, and the diameter of the tissue mass after the tumor tissue is cut is about 1 mm to about 10 mm.
  • the culture time is about 3-20 days.
  • the temperature of the culture is about 30-42°C.
  • Figures 1A- Figure 1B- Figure 9A- Figure 9B show the flow cytometric detection results of TIL seed cells obtained by culturing from solid tumor tissues using the seed cell culture medium described in this application.
  • Figures 10A- Figure 10B- Figure 12A- Figure 12B show the flow cytometric detection results of TIL seed cells obtained by culturing from the body fluid of tumor patients using the seed cell culture medium described in this application.
  • Figures 13A-13B-15A-15B show the flow cytometric detection results of TIL seed cells obtained by culturing from the body fluid of tumor patients using the seed cell culture medium in the prior art.
  • FIGs 16A-16B, Figure 17-20 show the TIL reinfusion products prepared using the seed cell culture medium described in this application after clinical reinfusion, the TIL reinfusion products and the TCR of the patient's peripheral blood at different time points The detection results of high-throughput sequencing analysis of the repertoire.
  • Figure 21 shows the results of the proliferation status of patients' peripheral blood leukocytes, neutrophils and lymphocytes after clinical reinfusion using the TIL reinfusion product prepared from the seed cell culture medium described in this application.
  • interferon generally refers to a family of proteins with high species specificity that inhibit virus replication and cell proliferation and regulate immune responses.
  • suitable interferons include but are not limited to: recombinant interferon alpha-2b, recombinant interferon alpha-2a, recombinant interferon alpha-2C, interferon alpha-n1, consensus alpha interferon, or interferon alpha-n3.
  • colony stimulating factor generally refers to a type of secreted glycoprotein that binds to receptor proteins on the surface of hematopoietic stem cells, thereby activating intracellular signaling pathways, which can lead to cell proliferation and differentiation into A specific type of blood cells (usually white blood cells, for red blood cell formation, see erythropoietin). They can be artificially synthesized and applied exogenously.
  • the term "antigen-binding fragment” generally refers to a part of an antibody molecule that contains amino acids responsible for the specific binding between an antibody and an antigen.
  • the part of the antigen that is specifically recognized and bound by the antibody is called the "epitope" as described above.
  • the antigen-binding domain may typically include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily include both.
  • Fd fragments have, for example, two VH regions and generally retain some of the antigen binding functions of the complete antigen binding domain.
  • antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments with VL, VH, constant light chain (CL) and CH1 domains; (2) F(ab')2 fragments, with two hinge regions A bivalent fragment of two Fab fragments connected by a sulfur bridge; (3) Fd fragment with two VH and CH1 domains; (4) Fv fragment with VL and VH domains of one antibody arm, (5) dAb fragment (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), which has a VH domain; ( 6) Isolated complementarity determining regions (CDR), and (7) single-chain Fv (scFv), for example derived from scFV-library.
  • CDR complementarity determining regions
  • scFv single-chain Fv
  • the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined by a synthetic linker using recombinant methods.
  • the synthetic linker allows it to be prepared as a single protein in which the VL and VH regions are paired to form a monovalent molecule Chain (called single-chain Fv (scFv)) (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichiacoli," Proc.Natl .Acad.Sci.USA 85:5879-5883(1988)).
  • scFv single-chain Fv
  • the term "antigen-binding fragment” also includes ligands that specifically bind to antigens, especially cell surface receptor molecules.
  • Ligands are generally polypeptides or compounds that can bind to receptor proteins (such as the antigen) with high affinity and specificity to trigger a functional response.
  • the ligand may include CD40L, a ligand of CD40, CD137L, a ligand of CD137, CD80, a ligand of CD28, and/or OX-40L, a ligand of OX-40.
  • immune checkpoint generally refers to a group of molecules on the cell surface of CD4 + and CD8 + T cells. These molecules can be effectively used as a "brake” to down-regulate or suppress the anti-tumor immune response. These inhibitory molecules can be inhibited by inhibiting DNA, RNA or protein levels.
  • the term "co-stimulatory receptor” generally refers to a type of receptor expressed on the surface of immune cells, such as T cells, on which an immune response is activated.
  • the costimulatory receptor turns on the costimulatory signal by combining with its corresponding activated antibody, ligand or antigen-binding fragment, thereby completely activating the immune effector cell.
  • the activation of costimulatory receptor-mediated signaling pathways usually plays a key role in the effective occurrence of immune responses.
  • Common costimulatory receptors include but are not limited to CD28, CD40, CD137 and OX-40.
  • TNF tumor necrosis factor TNF
  • TNF ⁇ tumor necrosis factor
  • the term “TNF ⁇ ” encompasses precursor forms of TNF ⁇ , pro-TNF ⁇ (pro-TNF ⁇ ), full-length TNF ⁇ , and any form of TNF ⁇ produced by intracellular processes.
  • the term also encompasses naturally-occurring and non-naturally-occurring variants of TNF ⁇ , such as splice variants, allelic variants, and isoforms.
  • TNF ⁇ can bind to two receptors, TNFR1 (type 1 TNF receptor; CD120a; p55/60) and TNFR2 (type 2 TNF receptor; CD120b; p75/80). TNF ⁇ acts as a pro-inflammatory cytokine, for example in neuroinflammation.
  • tumor infiltrating lymphocytes generally refers to infiltrating lymphocytes isolated from tumor tissues. Immunohistochemical staining showed that tumor-infiltrating lymphocytes can include CD3 + T cells, CD20 + , CD79 ⁇ + B cells, plasma cells and CD56 + NK cells; they can also include T cells, a small amount of B cells, NK cells, and macrophages. And dendritic cells. The tumor-infiltrating lymphocytes can produce high-efficiency and strong specific anti-tumor killing effects through direct cytotoxic T lymphocytes and secretion of cytokines.
  • the tumor-infiltrating lymphocytes may include mononuclear leukocytes, which leave the bloodstream and migrate to a tumor.
  • the tumor-infiltrating lymphocytes may be selected from the group consisting of T cells, B cells, natural killer cells, and natural killer T cells.
  • seed cell generally refers to tumor-infiltrating lymphocytes cultured from tumor tissue using any suitable medium including the medium of this application. These cells can be used as a starting cell population for further expansion in the subsequent cultivation, or as a cell population for end-use purposes, such as a cell population for reinfusion therapy to individual subjects.
  • the seed cells can be obtained from tumor tissues for any different culture duration, and the culture duration of the seed cells can be determined according to the subsequent use of the seed cells, and is not limited to a specific culture duration.
  • IL-15 generally refers to pro-inflammatory cytokines that act as effective growth, survival and activation factors for T cells (especially intestinal intraepithelial lymphocytes (IEL)) and natural killer (NK) cells, It can also be referred to as "IL-15 antigen” and "Interleukin 15". Increased expression of IL-15 has been demonstrated in a variety of inflammatory diseases, including CD, rheumatoid arthritis (RA) and psoriasis (Malamut et al., 2010). IL-15 is considered to be a central regulator of CD immunopathology and a non-redundant driver of lymphoma formation in RCD.
  • IL-2 generally refers to a protein derived from lymphokines that are produced from normal peripheral blood lymphocytes and exist in the body at a low concentration. Originally, Morgan et al. (1976) Science 193: 1007-1008 described IL-2, originally called T cell growth factor, because it can induce proliferation of stimulated T lymphocytes. The term also includes proteins modified after the expression of IL-2, such as glycosylation, acetylation, phosphorylation and the like.
  • IL-7 generally refers to a protein encoded by the IL-7 gene.
  • IL-7 is a hematopoietic growth factor secreted by stromal cells in bone marrow and thymus. It can be produced by keratinocytes, dendritic cells, hepatocytes, neurons and epithelial cells. IL-7 can be involved in the effects on T cells and B cells.
  • PD-1 generally refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed in vivo on previously activated T cells and binds to two ligands, PD-1 and PD-2. The complete hPD-1 sequence can be found in GenBank accession number U64863.
  • LYC-55716 generally refers to a ROR ⁇ activator. Its CAS number is 2055536-64-4.
  • GNE-1858 generally refers to an HPK1 inhibitor with a CAS number of T11438.
  • TWS119 generally refers to a GSK-3 inhibitor with a CAS number of 601514-19-6.
  • RRX-001 generally refers to an epigenetic modulator with radiosensitizing activity.
  • the RRX-001 can down-regulate the CD47/SIRP ⁇ axis and repolarize TAM and other immunosuppressive cells in the tumor microenvironment into an immunostimulatory phenotype. Its CAS number is 925206-65-1.
  • CNI-1493 generally refers to a compound that can prevent the production of inflammatory cytokines (including TNF), and its CAS number is 164301-51-3.
  • the present application provides a seed cell culture medium for tumor infiltrating lymphocytes, which includes the following components: cell culture components, cytokines, and immune checkpoint antibodies or antigen-binding fragments thereof, wherein the cytokines include IL -2, the immune checkpoint includes: PD-1, LAG-3, TIGIT and/or CTLA-4, and the cell culture component is a serum medium or a serum-free medium.
  • the cytokine may be a human-derived cytokine.
  • the immune checkpoint antibody or antigen-binding fragment thereof may be a humanized and/or fully human antibody or antigen-binding fragment thereof.
  • the concentration of IL-2 can be about 3000IU/mL or less (for example, it can be about 2900IU/mL or less, about 2800IU/mL or less, about 2700IU/mL or less, about 2600IU/mL or less, about 2500IU/mL or less , About 2400IU/mL or less, about 2300IU/mL or less, about 2200IU/mL or less, about 2100IU/mL or less, about 2000IU/mL or less, about 1900IU/mL or less, about 1800IU/mL or less, about 1700IU/mL or less, about 1600IU/mL or less, about 1500IU/mL or less, about 1400IU/mL or less, about 1300IU/mL or less, about 1100IU/mL or less, about 1100IU/mL or less, about 1000IU/mL or less, about 900IU/mL or less, about 800IU/mL or less mL or less, about 700 IU/mL or less (for
  • the cytokine may include IL-7.
  • the concentration of IL-7 may be about 5 to about 100 ng/mL (for example, it may be about 5 to about 95 ng/mL, about 5 to about 90 ng/mL, about 5 to about 85 ng/mL, about 5 About 80ng/mL, about 5 to about 75ng/mL, about 5 to about 70ng/mL, about 5 to about 65ng/mL, about 5 to about 60ng/mL, about 5 to about 55ng/mL, about 5 to about 50ng /mL, about 5 to about 45ng/mL, about 5 to about 40ng/mL, about 5 to about 35ng/mL, about 5 to about 30ng/mL, about 5 to about 25ng/mL, about 5 to about 20ng/mL Or about 5 to about 10 ng/mL).
  • the concentration of IL-7 may be about 200 to about 1000 U/mL (for example, it may be at least about 200 U/mL, at least about 500 U/mL, or at least about 1000 U/mL).
  • the concentration of IL-7 may be about 200-1000 IU/mL.
  • it can be at least about 200 IU/mL, at least about 500 IU/mL, or at least about 1000 IU/mL.
  • the cytokine may include IL-15.
  • the concentration of IL-15 may be about 5 to about 100 ng/mL (for example, it may be about 5 to about 95 ng/mL, about 5 to about 90 ng/mL, about 5 to about 85 ng/mL, about 5 About 80ng/mL, about 5 to about 75ng/mL, about 5 to about 70ng/mL, about 5 to about 65ng/mL, about 5 to about 60ng/mL, about 5 to about 55ng/mL, about 5 to about 50ng /mL, about 5 to about 45ng/mL, about 5 to about 40ng/mL, about 5 to about 35ng/mL, about 5 to about 30ng/mL, about 5 to about 25ng/mL, about 5 to about 20ng/mL Or about 5 to about 10 ng/mL).
  • the concentration of IL-15 may be about 200 to about 500 U/mL (for example, it may be 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).
  • the concentration of IL-15 may be 200-500 IU/mL.
  • it can 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.
  • the cytokine may include tumor necrosis factor TNF.
  • the cytokine may include TNF ⁇ .
  • the concentration of the TNF ⁇ may be about 10 to about 100 pg/mL (for example, it may be about 10 to about 95 pg/mL, about 10 to about 90 pg/mL, about 10 to about 80 pg/mL, about 10 to about 70 pg/mL. /mL, about 10-about 60pg/mL, about 10-about 50pg/mL, about 10-about 40pg/mL, about 15-about 40pg/mL, about 10-about 25pg/mL, about 15-about 25pg/mL , About 10 to about 30 pg/mL, about 10 to about 20 pg/mL, or about 10 to about 15 pg/mL).
  • the concentration of the TNF- ⁇ may be about 10-100 pg/mL.
  • it can be about 10-100 pg/mL, about 15-100 pg/mL, about 20-100 pg/mL, or about 25-100 pg/mL.
  • it can be 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.
  • the cytokine may include colony stimulating factor.
  • the cytokine may include GM-CSF, G-CSF and/or M-CSF.
  • the concentration of the GM-CSF may be about 200 to about 5000 U/mL (for example, it may be about 200 to about 800 U/mL, about 200 to about 500 U/mL, about 300 to about 5000 U/mL, about 500 to about 500 U/mL.
  • it may be about 200 to about 1000 U/mL, may be about 200 to about 800 U/mL, or about 200 to about 500 U/mL.
  • it can be at least about 200 U/mL, at least about 500 U/mL, or at least about 800 U/mL.
  • the concentration of the G-CSF may be about 300 U/mL to about 1000 U/mL.
  • the concentration of the G-CSF may be about 500-1000 U/mL.
  • it can be at least about 500 U/mL or at least about 1000 U/mL.
  • the concentration of the M-CSF may be about 300 U/mL to about 800 U/mL.
  • the concentration of the M-CSF may be about 300 U/mL to about 500 U/mL.
  • it can be at least about 300 U/mL, at least about 500 U/mL, or at least about 800 U/mL.
  • the cytokine may include interferon.
  • the cytokine may include IFN- ⁇ , IFN- ⁇ , and/or IFN- ⁇ .
  • the concentration of the IFN- ⁇ may be about 10 to about 1000 ng/mL (for example, it may be about 15 to about 1000 ng/mL, about 20 to about 1000 ng/mL, about 30 to about 1000 ng/mL, about 40 to about 1000 ng/mL).
  • the concentration of the IFN- ⁇ may be about 10 to about 1000 ng/mL.
  • the concentration of the IFN- ⁇ may be about 300 to about 1000 ng/mL, about 300 to about 800 ng/mL, or about 300 to about 500 ng/mL.
  • it can be 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.
  • the concentration of the IFN- ⁇ may be about 500 U/mL to about 1000 U/mL.
  • it can be at least about 500 ng/mL.
  • the concentration of the IFN- ⁇ may be about 200 U/mL to about 500 U/mL.
  • the concentration of the IFN- ⁇ may be about 250 to about 500 ng/mL.
  • it can be at least about 200 ng/mL, at least about 250 ng/mL, or at least about 500 ng/mL.
  • the cytokine may also include IL-4, IL-1 ⁇ , IL-1 ⁇ , IL-6, IL-9, IL-18, IL-12, IL-21 and/or IL-10.
  • the concentration of IL-4 may be about 200 to about 500 U/mL.
  • it can be at least about 200 IU/mL or at least about 500 IU/mL.
  • the concentration of the IL-1 ⁇ may be about 200 to about 500 U/mL.
  • it can be at least about 200 IU/mL or at least about 500 IU/mL.
  • the concentration of IL-1 ⁇ may be about 200 to about 500 U/mL.
  • it can be at least about 200 IU/mL or at least about 500 IU/mL.
  • the concentration of IL-6 may be about 200 to about 500 U/mL.
  • it can be at least about 200 IU/mL or at least about 500 IU/mL.
  • the concentration of IL-9 may be about 200 to about 500 U/mL.
  • it can be at least about 200 IU/mL or at least about 500 IU/mL.
  • the concentration of IL-18 may be about 200 to about 500 U/mL.
  • it can be at least about 200 IU/mL or at least about 500 IU/mL.
  • the concentration of IL-12 may be about 200 to about 1000 U/mL.
  • the concentration of the IL-12 may be about 200 to about 1000 U/mL, about 200 to about 500 U/mL, or about 300 to about 1000 U/mL.
  • it can 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.
  • the concentration of IL-21 may be about 200 to about 500 U/mL.
  • the concentration of IL-21 may be about 250 to about 1000 U/mL.
  • it can be at least about 200 IU/mL, at least about 250 IU/mL, or at least about 500 IU/mL.
  • the concentration of IL-10 may be about 200 to about 500 U/mL.
  • it can 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.
  • the immune checkpoint antibody or antigen-binding fragment thereof may be a PD-1 antibody or antigen-binding fragment thereof.
  • the PD-1 antibody or antigen-binding fragment thereof may be a human PD-1 antibody or antigen-binding fragment thereof.
  • the concentration of the PD-1 antibody or antigen-binding fragment thereof may be about 1 to about 100 ⁇ g/mL (for example, about 3 to about 100 ⁇ g/mL, about 5 to about 100 ⁇ g/mL, or about 10 to about 100 ⁇ g/mL, about 15 to about 100 ⁇ g/mL, about 16 to about 100 ⁇ g/mL, about 17 to about 100 ⁇ g/mL, about 18 to about 100 ⁇ g/mL, about 19 to about 100 ⁇ g/mL, about 20-about 100 ⁇ g/mL, about 25-about 100 ⁇ g/mL, about 30-about 100 ⁇ g/mL, about 36-about 100 ⁇ g/mL, about 40-about 100 ⁇ g/mL, about 45-about 100 ⁇ g/mL, about 50- About 100 ⁇ g/mL, about 55 to about 100 ⁇ g/mL, about 60 to about 100 ⁇ g/mL, about 65 to about 100 ⁇ g/mL, about 70 to about 100 ⁇ g/
  • the concentration of the PD-1 mAb may be about 3-100 ⁇ g/mL.
  • it can be at least about 3 ⁇ g/mL, at least about 5 ⁇ g/mL, at least about 10 ⁇ g/mL, at least about 15 ⁇ g/mL, at least about 20 ⁇ g/mL, at least about 25 ⁇ g/mL, at least about 36 ⁇ g/mL, at least about 50 ⁇ g/mL.
  • the immune checkpoint antibody or antigen-binding fragment thereof may be a human LAG-3 antibody or antigen-binding fragment thereof.
  • 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 is about 3 to about 10 ⁇ g/mL.
  • the concentration of the LAG-3 antibody or antigen-binding fragment thereof is about 3 to about 10 ⁇ g/mL.
  • it can be about 5 to about 10 ⁇ g/mL.
  • it can be at least about 3 ⁇ g/mL, at least about 5 ⁇ g/mL, or at least about 10 ⁇ g/mL.
  • the TIGIT antibody or antigen-binding fragment thereof may be a human TIGIT antibody or antigen-binding fragment thereof.
  • 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 to about 25 ⁇ g/mL.
  • the concentration of the TIGIT mAb may be about 2.5-20 ⁇ g/mL.
  • it can be about 5-20 ⁇ g/mL.
  • it can be at least about 2.5 ⁇ g/mL, at least about 5 ⁇ g/mL, or at least about 20 ⁇ g/mL.
  • the immune checkpoint antibody or antigen-binding fragment thereof may be a human CTLA-4 antibody or antigen-binding fragment thereof.
  • 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 to about 100 ⁇ g/mL.
  • the concentration of the CTLA-4mAb may be about 3-50 ⁇ g/mL, about 5-50 ⁇ g/mL, or about 10-50 ⁇ g/mL.
  • it can be at least about 3 ⁇ g/mL, at least about 5 ⁇ g/mL, at least about 10 ⁇ g/mL, or at least about 50 ⁇ g/mL.
  • the seed cell culture medium may also include a costimulatory receptor antibody or antigen-binding fragment thereof, wherein the costimulatory receptor immune checkpoint antibody or antigen-binding fragment thereof may include: CD40 antibody or antigen-binding fragment thereof Binding fragment, OX-40 antibody or antigen-binding fragment thereof, CD137 antibody or antigen-binding fragment thereof, and/or CD28 antibody or antigen-binding fragment thereof.
  • the costimulatory antibody or antigen-binding fragment thereof may include a CD137 antibody or antigen-binding fragment thereof.
  • the CD137 antibody or antigen-binding fragment thereof may be a human CD137 antibody or antigen-binding fragment thereof.
  • the concentration of the CD137 antibody or antigen-binding fragment thereof may be about 1 to about 100 ⁇ g/mL (for example, it may be about 2 to about 100 ⁇ g/mL, about 3 to about 100 ⁇ g/mL, about 3 to about 20 ⁇ g/mL.
  • the concentration of the CD137mAb may be about 3-20 ⁇ g/mL.
  • it can be at least about 3 ⁇ g/mL, at least about 5 ⁇ g/mL, at least about 10 ⁇ g/mL, at least about 12 ⁇ g/mL, or at least about 20 ⁇ g/mL.
  • the costimulatory antibody or antigen-binding fragment thereof may include an OX40 antibody or antigen-binding fragment thereof.
  • the OX-40 antibody or antigen-binding fragment thereof may be a human OX-40 antibody or antigen-binding fragment thereof.
  • the concentration of the OX-40 antibody or antigen-binding fragment thereof may be about 3 to about 10 ⁇ g/mL.
  • the concentration of the OX-40mAb may be about 5-10 ⁇ g/mL.
  • it can be at least about 3 ⁇ g/mL, at least about 5 ⁇ g/mL, or at least about 10 ⁇ g/mL.
  • the costimulatory antibody or antigen-binding fragment thereof may include a CD28 antibody or antigen-binding fragment thereof.
  • the immune checkpoint antibody or antigen-binding fragment thereof may include a CD28 antibody or antigen-binding fragment thereof.
  • the CD28 antibody or antigen-binding fragment thereof may be a human CD28 antibody or antigen-binding fragment thereof.
  • the concentration of the CD28 antibody or antigen-binding fragment thereof may be about 1 to about 10 ⁇ g/mL.
  • it can be about 2 to about 10 ⁇ g/mL, about 3 to about 10 ⁇ g/mL, about 3 to about 5 ⁇ g/mL, about 4 to about 10 ⁇ g/mL, about 5 to about 10 ⁇ g/mL, about 6 to about 10 ⁇ g /mL, about 7 to about 10 ⁇ g/mL, or about 8 to about 10 ⁇ g/mL).
  • the concentration of the CD28mAb may be about 3-10 ⁇ g/mL.
  • it can be at least about 3 ⁇ g/mL, at least about 5 ⁇ g/mL, or at least about 10 ⁇ g/mL.
  • the costimulatory antibody or antigen-binding fragment thereof may include a CD40 antibody or antigen-binding fragment thereof.
  • the CD40 antibody or antigen-binding fragment thereof may be a human CD40 antibody or antigen-binding fragment thereof.
  • the concentration of the CD40 antibody or antigen-binding fragment thereof may be about 5 to about 10 ⁇ g/mL.
  • the concentration of the CD40mAb may be about 6-10 ⁇ g/mL or about 5-8 ⁇ g/mL. For example, it can be at least about 5 ⁇ g/mL.
  • the serum culture medium may include serum.
  • the serum may include human AB serum.
  • the concentration of the serum may be about 1 to about 10% (v/v) (for example, it may be about 1.5 to about 10%, about 2 to about 10%, about 3 to about 10%, about 4 to about 10%). 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%).
  • the serum medium may include AIM-V medium.
  • the serum-free medium may include X-VIVO medium or CTS TM OpTmizer TM medium.
  • the cell culture components may include antibiotics.
  • the cell culture component may include a penicillin-streptomycin mixture PS.
  • the concentration of the penicillin-streptomycin mixture PS may be about 1 to about 200 U/mL (for example, it may be about 1 to about 5 U/mL, about 1 to about 10 U/mL, about 1 to about 20 U/mL.
  • the concentration of the penicillin-streptomycin mixture PS may be about 100 U/mL.
  • the seed cell culture medium may also include an M2 type macrophage inhibitor, and the M2 type macrophage inhibitor may include RRx001 and/or CNI-1493.
  • the concentration of the M2 type macrophage inhibitor may be about 0.1 to about 100 ⁇ M (for example, about 0.5 to about 100 ⁇ M, about 1 to about 100 ⁇ M, about 2 to about 100 ⁇ M, about 2.5 to about 100 ⁇ M, about 3- about 100 ⁇ M, about 2 to about 3 ⁇ M, about 5 to about 100 ⁇ M, about 10 to about 100 ⁇ M, about 20 to about 100 ⁇ M, about 0.1 to about 90 ⁇ M, about 0.5 to about 90 ⁇ M, about 1 to about 90 ⁇ M, about 5 About 90 ⁇ M, about 0.1 to about 80 ⁇ M).
  • the seed cell culture medium may also include regulatory T cell (Treg) inhibitors, and the regulatory T cell inhibitors may include CAL-101, dasatinib, imatinib, and / Or Panobinostat (Panobinostat).
  • Treg regulatory T cell
  • CAL-101 dasatinib
  • imatinib imatinib
  • Panobinostat Panobinostat
  • the concentration of the regulatory T cell inhibitor may be about 0.1 to about 100 ⁇ M (for example, it may be about 0.5 to about 100 ⁇ M, about 1 to about 100 ⁇ M, about 5 to about 100 ⁇ M, about 10 to about 100 ⁇ M, about 20 to about 100 ⁇ M.
  • the concentration of the regulatory T cell inhibitor may be about 1 to about 5 ⁇ M, about 1 to about 3 ⁇ M, about 1.5 to about 5 ⁇ M, about 2 to about 5 ⁇ M, about 2.5 to about 5 ⁇ M, or It is about 3 to about 5 ⁇ M.
  • the seed cell culture medium may also include a bone marrow-derived suppressor cytostatic agent
  • the bone marrow-derived suppressor cytostatic agent may include AG490, decitabine, sunitinib and/or BBI608.
  • the concentration of the bone marrow-derived suppressor cytostatic agent may be about 0.1 to about 100 ⁇ g/mL (for example, it may be about 0.5 to about 100 ⁇ g/mL, about 1 to about 100 ⁇ g/mL, about 2 to about 100 ⁇ g/mL.
  • the concentration of the bone marrow-derived suppressor cytostatic agent may be about 1 to about 3 ⁇ g/mL, or may be about 1.5 to about 3 ⁇ g/mL.
  • the seed culture medium may include at least one of the regulatory T cell (Treg) inhibitor, the bone marrow-derived suppressor cell inhibitor, and the M2 macrophage inhibitor, At least two and at least three.
  • Treg regulatory T cell
  • the bone marrow-derived suppressor cell inhibitor the bone marrow-derived suppressor cell inhibitor
  • the M2 macrophage inhibitor At least two and at least three.
  • the seed cell culture medium may also include a T cell activator, and the T cell activator may include LYC-55716, GNE-1858 and/or methylene blue.
  • the concentration of the T cell activator may be about 1 to about 10 ⁇ M.
  • it can be about 1 to about 9 ⁇ M, about 1 to about 8 ⁇ M, about 1 to about 7 ⁇ M, about 1 to about 6 ⁇ M, about 2 to about 10 ⁇ M, about 3 to about 10 ⁇ M, about 4 to about 10 ⁇ M, or about 5 to about 10 ⁇ M.
  • the seed cell culture medium may also include a T cell differentiation inhibitor, and the T cell differentiation inhibitor may include TWS119.
  • the concentration of the T cell differentiation inhibitor may be about 1 to about 10 ⁇ M.
  • it can be about 1 to about 9 ⁇ M, about 1 to about 8 ⁇ M, about 1 to about 7 ⁇ M, about 1 to about 6 ⁇ M, about 2 to about 10 ⁇ M, about 3 to about 10 ⁇ M, about 4 to about 10 ⁇ M, or about 5 to about 10 ⁇ M.
  • the concentration of the RRX-001 may be about 0.1 to about 100 ⁇ M.
  • it can be at least about 3 ⁇ M.
  • the concentration of the sunitinib may be about 1.5 to about 3 ⁇ M.
  • it can be at least about 1.5 ⁇ M or at least about 3 ⁇ M.
  • the concentration of CNI-1493 can be about 2 to about 3 ⁇ M.
  • it can be at least about 2 ⁇ M, at least about 2.5 ⁇ M, or at least about 3 ⁇ M.
  • the concentration of the Imatinib may be about 2 to about 5 ⁇ M.
  • it can be at least about 2 ⁇ M or at least about 5 ⁇ M.
  • the concentration of CAL-101 may be about 1.5 to about 5 ⁇ M.
  • it may be at least about 1.5 ⁇ M or at least about 5 ⁇ M.
  • the concentration of Dasatinib may be about 2 to about 3 ⁇ M.
  • it can be at least about 2 ⁇ M, at least about 2.5 ⁇ M, or at least about 3 ⁇ M.
  • the concentration of LYC-55716 may be about 1 to about 10 ⁇ M.
  • it can be at least about 1 ⁇ M or at least about 10 ⁇ M.
  • the concentration of GNE-1858 may be about 4 to about 5 nM.
  • it can be at least about 4 nM, at least about 4.5 nM, or at least about 5 nM.
  • the concentration of the methylene blue may be about 1 to about 2 ⁇ M.
  • it can be at least about 1 ⁇ M, at least about 1.5 ⁇ M, or at least about 2 ⁇ M.
  • the concentration of the TWS119 may be about 1 to about 10 ⁇ M.
  • it can be at least about 1 ⁇ M, at least about 5 ⁇ M, or at least about 10 ⁇ M.
  • the seed culture medium may include IL-2, IL-7, PD-1 antibody or antigen-binding fragments thereof, TNF ⁇ , human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin Mixed liquid PS double antibody.
  • the seed culture medium may include at least about 3000 U/mL IL-2, at least about 1000 U/mL IL-7, at least about 100 ⁇ g/mL PD-1 antibody or antigen-binding fragment thereof, at least about 10 pg/mL TNF ⁇ , At least about 5% human serum, serum-free medium X-VIVO 15 and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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.
  • the seed culture medium may include 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 ⁇ g/mL CTLA-4 antibody or its antigen binding Fragment, at least about 50 ⁇ g/mL PD-1 antibody or its antigen binding fragment, at least about 3% human serum, serum-free medium AIM-V and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-7, G-CSF, PD-1 antibody or its antigen-binding fragment, TNF ⁇ , human serum, serum-free medium X-VIVO 15 and penicillin -Streptomycin mixture PS double antibody.
  • the seed culture medium may include 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 ⁇ g/mL PD-1 antibody or antigen-binding fragment thereof , At least about 20pg/mL TNF ⁇ , at least about 3% human serum, serum-free medium AIM-V and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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.
  • the seed culture medium may include at least about 3000 U/mL IL-2, at least about 200 U/mL IL-12, at least about 500 U/mL IL-21, at least about 800 U/mL GM-CSF, at least about 10 ⁇ g/mL.
  • mL LAG-3 antibody or antigen-binding fragment thereof at least about 10 ⁇ g/mL CTLA-4 antibody or antigen-binding fragment thereof, at least about 5% human serum, serum-free medium CTS TM OpTmizer TM and 100 U/mL penicillin-streptomycin Mixed liquid PS double antibody.
  • the seed culture medium may include IL-1 ⁇ , IL-2, IL-6, IL-21, IFN- ⁇ , TIGIT antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment , TNF ⁇ , human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include at least about 500 U/mL IL-1 ⁇ , 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 mL IFN- ⁇ , at least about 20 ⁇ g/mL TIGIT antibody or its antigen-binding fragment, at least about 50 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, at least about 25pg/mL TNF ⁇ , at least about 5% human serum, serum-free culture Base X-VIVO 15 and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-1 ⁇ , IL-2, IL-9, IL-18, IFN- ⁇ , IFN- ⁇ , LAG-3 antibodies or antigen-binding fragments thereof, PD-1 Antibody or its antigen-binding fragment, TNF ⁇ , human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include at least about 500 U/mL IL-1 ⁇ , 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 mL IFN- ⁇ , at least about 500U/mL IFN- ⁇ , at least about 5 ⁇ g/mL LAG-3 antibody or its antigen-binding fragment, at least about 25 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, at least about 25 pg/mL TNF ⁇ , At least about 5% human serum, serum-free medium X-VIVO 15 and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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, TNF ⁇ , human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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 ⁇ g/mL mL CD137 antibody or its antigen-binding fragment, at least about 3 ⁇ g/mL LAG-3 antibody or its antigen-binding fragment, at least about 20 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, at least about 10pg/mL TNF ⁇ , at least about 5% Human serum, serum-free medium X-VIVO 15 and 1 00U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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, TNF ⁇ , human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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 200 U/mL IL-21, at least about 20 ⁇ g/mL.
  • mL CD137 antibody or antigen-binding fragment thereof at least about 10 ⁇ g/mL CD28 antibody or antigen-binding fragment thereof, at least about 10 ⁇ g/mL PD-1 antibody or antigen-binding fragment thereof, at least about 10 pg/mL TNF ⁇ , at least about 3% human serum , Serum-free medium CTS TM OpTmizer TM and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-1 ⁇ , IL-2, IL-7, IL-21, G-CSF, GM-CSF, IFN- ⁇ , LAG-3 antibodies or antigen-binding fragments thereof , PD-1 antibody or its antigen-binding fragment, TNF ⁇ , human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include at least about 200 U/mL IL-1 ⁇ , at least about 3000 U/mL IL-2, at least about 200 U/mL IL-7, at least about 200 U/mL IL-21, at least about 500 U/mL mL G-CSF, at least about 500 U/mL GM-CSF, at least about 1000 U/mL IFN- ⁇ , at least about 5 ⁇ g/mL LAG-3 antibody or antigen-binding fragment thereof, at least about 3 ⁇ g/mL PD-1 antibody or antigen Bound fragment, at least about 15pg/mL TNF ⁇ , at least about 5% human serum, serum-free medium CTS TM OpTmizer TM and 100 U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN- ⁇ , IFN- ⁇ , TIGIT antibody or antigen-binding fragment thereof, CTLA -4 antibody or its antigen-binding fragment, human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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 mL M-CSF, at least about 200 U/mL IFN- ⁇ , at least about 800 U/mL IFN- ⁇ , at least about 5 ⁇ g/mL TIGIT antibody or its antigen-binding fragment, at least about 10 ⁇ g/mL CTLA-4 antibody or its antigen-binding fragment , At least about 5% human serum, serum-free medium X-VIVO 15 and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-7, IL-15, IL-2, IL-7, IL-15, GM-CSF, CD137 antibody or its antigen binding fragment, PD -1 antibody or its antigen-binding fragment, TNF ⁇ human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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 ⁇ g/mL mL CD137 antibody or its antigen-binding fragment, at least about 20 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, at least about 20 pg/mL TNF ⁇ , at least about 5% human serum, serum-free medium X-VIVO 15 and 100 U/mL Penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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.
  • the seed culture medium may include 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, and at least about 500 U/mL IL-10.
  • mL G-CSF at least about 300 U/mLM-CSF, at least about 5 ⁇ g/mL CD137 antibody or antigen-binding fragment thereof, at least about 3 ⁇ g/mL CD28 antibody or antigen-binding fragment thereof, at least about 10 ⁇ g/mL OX-40 antibody or Antigen-binding fragments, at least about 10 ⁇ g/mL PD-1 antibody or antigen-binding fragments thereof, at least about 5% human serum, serum-free medium AIM-V and 100 U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-7, IL-21, IFN- ⁇ , IFN- ⁇ , CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, OX -40 antibody or its antigen-binding fragment, at least about LAG-3 antibody or its antigen-binding fragment, CTLA-4 antibody or its antigen-binding fragment, TNF ⁇ , human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin Mixed liquid PS double antibody.
  • the seed culture medium may include 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- ⁇ , at least about 800 U/mL mL IFN- ⁇ , at least about 5 ⁇ g/mL CD137 antibody or its antigen-binding fragment, at least about 3 ⁇ g/mL CD28 antibody or its antigen-binding fragment, at least about 5 ⁇ g/mL OX-40 antibody or its antigen-binding fragment, at least about 3 ⁇ g/ mL LAG-3 antibody or its antigen-binding fragment, at least about 5 ⁇ g/mL CTLA-4 antibody or its antigen-binding fragment, at least about 10pg/mL TNF ⁇ , at least about 5% human serum, serum-free medium X-VIVO 15 and 100U /mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-7, IL-15, IFN- ⁇ , IFN- ⁇ , CD137 antibody or its antigen-binding fragment, CD40 antibody or its antigen-binding fragment, OX -40 antibody or its antigen-binding fragment, TIGIT 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.
  • the seed culture medium may include 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- ⁇ , at least about 1000 U/mL mL IFN- ⁇ , at least about 5 ⁇ g/mL CD137 antibody or its antigen-binding fragment, at least about 5 ⁇ g/mL CD40 antibody or its antigen-binding fragment, at least about 5 ⁇ g/mL OX-40 antibody or its antigen-binding fragment, at least about 5 ⁇ g/ mL TIGIT antibody or its antigen-binding fragment, at least about 15 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, at least about 5% human serum, serum-free medium X-VIVO 15 and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-7, IL-15, GM-CSF, IFN- ⁇ , CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, PD -1 antibody or its antigen-binding fragment, TNF ⁇ , at least about 5% human serum, serum-free medium CTS TM OpTmizer TM and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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 500 U/mL GM-CSF, at least about 1000 U/mL mL IFN- ⁇ , at least about 3 ⁇ g/mL CD137 antibody or antigen-binding fragment thereof, at least about 3 ⁇ g/mL CD28 antibody or antigen-binding fragment thereof, at least about 3 ⁇ g/mL PD-1 antibody or antigen-binding fragment thereof, at least about 10 pg/mL mL TNF ⁇ , at least about 5% human serum, serum-free medium CTS TM OpTmizer TM and 100 U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-7, IL-12, G-CSF, GM-CSF, IFN- ⁇ , IFN- ⁇ , 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, TNF ⁇ , human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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 mL GM-CSF, at least about 500 U/mL IFN- ⁇ , at least about 500 U/mL IFN- ⁇ , at least about 3 ⁇ g/mL CD28 antibody or antigen-binding fragment thereof, at least about 5 ⁇ g/mL CD40 antibody or antigen-binding fragment thereof, at least About 2.5 ⁇ g/mL TIGIT antibody or its antigen-binding fragment, at least about 3 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, at least about 10 pg/mL TNF ⁇ , at least about 5% human serum, serum-free medium X-VIVO 15 And 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-7, PD-1 antibody or antigen-binding fragments thereof, RRx-001, CAL-101, human serum, serum-free medium CTS TM OpTmizer TM And penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include at least about 3000 U/mL IL-2, at least about 1000 U/mL IL-7, at least about 50 ⁇ g/mL PD-1 antibody or antigen-binding fragment thereof, at least about 3 ⁇ M RRx-001, At least about 1.5 ⁇ M CAL-101, at least about 5% human serum, serum-free medium CTS TM OpTmizer TM and 100 U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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.
  • the seed culture medium may include 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 ⁇ g/mL LAG-3 antibody or antigen-binding fragment thereof , At least about 5 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, at least about 3 ⁇ M Sunitinib, at least about 5 ⁇ M Imatinib, at least about 5% human serum, serum-free medium X-VIVO 15 and 100U/mL penicillin-streptomycin Mixed liquid PS double antibody.
  • the seed culture medium may include IL-2, IL-1 ⁇ , IL-6, GM-CSF, M-CSF, CNI-1493, human serum, serum-free medium X-VIVO 15 and penicillin -Streptomycin mixture PS double antibody.
  • the seed culture medium may include at least about 3000 U/mL IL-2, at least about 200 U/mL IL-1 ⁇ , at least about 500 U/mL IL-6, at least about 500 U/mL GM-CSF, at least about 500 U/mL mL M-CSF, at least about 5 ⁇ M CNI-1493, at least about 5% human serum, serum-free medium X-VIVO 15 and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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-VIVO15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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 mL GM-CSF, at least about 5 ⁇ g/mL OX40 antibody or its antigen-binding fragment, at least about 36 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, at least about 1.5 ⁇ M Sunitinib, at least about 5% human serum, serum-free medium X-VIVO 15 and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-6, IL-12, CD28 antibody or its antigen-binding fragment, OX-40 antibody or its antigen-binding fragment, CTLA-4 antibody or its antigen Binding fragment, TNF ⁇ , Imatinib, human serum, serum-free medium CTS TM OpTmizer TM and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include at least about 2500 U/mL IL-2, at least about 500 U/mL IL-6, at least about 1000 U/mL IL-12, at least about 10 ⁇ g/mL CD28 antibody or antigen-binding fragment thereof, At least about 3 ⁇ g/mL OX-40 antibody or antigen-binding fragment thereof, at least about 5 ⁇ g/mL CTLA-4 antibody or antigen-binding fragment thereof, at least about 15 pg/mL TNF ⁇ , at least about 5 ⁇ M Imatinib, at least about 5% human serum, no Serum medium CTS TM OpTmizer TM and 100 U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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.
  • the seed culture medium may include 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 ⁇ g/mL CD137 antibody or antigen-binding fragment thereof, At least about 5 ⁇ g/mL CD28 antibody or its antigen-binding fragment, at least about 5 ⁇ g/mL LAG-3 antibody or its antigen-binding fragment, at least about 3 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, at least about 3 ⁇ M Dasatinib, at least about 5% human serum, serum-free medium X-VIVO 15 and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-6, IL-12, G-CSF, M-CSF, IFN- ⁇ , IFN- ⁇ , CTLA-4 antibodies or antigen-binding fragments thereof , PD-1 antibody or its antigen-binding fragment, Dasatinib, LYC-55716, GNE-1858, human serum, serum-free medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include 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 mL M-CSF, at least about 500 U/mL IFN- ⁇ , at least about 800 U/mL IFN- ⁇ , at least about 3 ⁇ g/mL CTLA-4 antibody or antigen-binding fragment thereof, at least about 5 ⁇ g/mL PD-1 antibody or antigen Bound fragment, at least about 2.5 ⁇ M Dasatinib, at least about 10 ⁇ M LYC-55716, at least about 4.5nM GNE-1858, at least about 5% human serum, serum-free medium X-VIVO 15 and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-1 ⁇ , 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, serum-free medium AIM-V and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include at least about 2500 U/mL IL-2, at least about 500 U/mL IL-1 ⁇ , at least about 500 U/mL IL-9, at least about 800 U/mL GM-CSF, at least about 3 ⁇ g/mL mL CD137 antibody or its antigen-binding fragment, at least about 3 ⁇ g/mL CD28 antibody or its antigen-binding fragment, at least about 5 ⁇ g/mL LAG-3 antibody or its antigen-binding fragment, at least about 2.5 ⁇ g/mL TIGIT antibody or its antigen-binding fragment , At least about 2.5 ⁇ M CNI-1493, at least about 1 ⁇ M methylene blue, at least about 5 ⁇ M TWS119, at least about 5% human serum, serum-free medium AIM-V and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include IL-2, IL-1 ⁇ , IL-12, IL-21, G-CSF, M-CSF, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , CD28 Antibody or its antigen-binding fragment, CD40 antibody or its antigen-binding fragment, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, CNI-1493, Dasatinib, GNE-1858, methylene blue, human serum, Serum-free medium AIM-V and penicillin-streptomycin mixture PS double antibody.
  • the seed culture medium may include about 3000 U/mL IL-2, about 200 U/mL IL-1 ⁇ , about 500 U/mL IL-12, about 500 U/mL IL-21, about 300 U/mL G-CSF, About 800U/mL M-CSF, about 500U/mL IFN- ⁇ , about 300U/mL IFN- ⁇ , about 300U/mL IFN- ⁇ , about 3 ⁇ g/mL CD28 antibody or its antigen-binding fragment, about 5 ⁇ g/mL CD40 antibody Or its antigen-binding fragment, about 5 ⁇ g/mL LAG-3 antibody or its antigen-binding fragment, about 3 ⁇ g/mL PD-1 antibody or its antigen-binding fragment, about 3 ⁇ M CNI-1493, about 2 ⁇ M Dasatinib, about 5nM GNE-1858, About 1.5 ⁇ M methylene blue, about 5% human serum, serum-free medium AIM-V and 100U/mL penicillin-streptomycin mixture PS double antibody.
  • this application provides the seed cells of tumor infiltrating lymphocytes obtained by culturing the seed cell culture medium described in this application or their cell populations.
  • the present application provides a pharmaceutical composition, which includes the seed cell of the tumor-infiltrating lymphocyte or its cell population and a pharmaceutically acceptable carrier as described in the present application.
  • the present application provides the application of the seed cells of tumor infiltrating lymphocytes or cell populations thereof and the pharmaceutical composition of the present application in the preparation of drugs for the treatment of cancer.
  • the seed culture medium described in this application can be suitable for amplifying tumor-infiltrating lymphocytes derived from different types of cancers.
  • the seed culture medium described in this application has a broad spectrum and universal applicability to cancer types.
  • the seed culture medium may be suitable for culturing tumor infiltrating lymphocytes derived from different types of cancers, and may be suitable for obtaining seed cells or cell populations of tumor infiltrating lymphocytes derived from different types of cancers.
  • the seed culture medium described in this application and/or the pharmaceutical composition described in this application can be used to prepare drugs for the treatment of cancer.
  • the cancer may include: cervical cancer (for example, may include recurrent cervical cancer), ovarian cancer, pelvic poorly differentiated adenocarcinoma, intestinal cancer (for example, may include intestinal cancer that has metastasized, such as liver metastasis) ), gastric cancer, melanoma, breast cancer and/or endometrial stromal sarcoma.
  • the cancer may be selected from the following group: melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, bowel cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, Pelvic poorly differentiated gland and cholangiocarcinoma.
  • this application provides an application of the seed cell culture medium in the expansion of tumor infiltrating lymphocytes.
  • this application provides a method for culturing seed cells of tumor-infiltrating lymphocytes, which includes the following steps: culturing isolated tumor-infiltrating lymphocytes in the seed cell culture medium described in this application.
  • this application provides a method for amplifying tumor infiltrating lymphocytes, which includes the following steps: culturing isolated tumor infiltrating lymphocytes in the seed cell culture medium described in this application to obtain the tumor infiltrating lymphocytes The seed cell of the cell.
  • the isolated tumor-infiltrating lymphocytes may be derived from samples selected from the following group: ascites from subjects in need, samples from surgical resection of primary tumors, samples from simultaneous and metachronous surgical resection of metastases, and puncture samples And body fluids.
  • the body fluid may include blood, tissue fluid, lymph fluid, and/or body cavity fluid.
  • the body fluid may include ascites fluid (for example, it may be the abdominal effusion of a patient with gastric cancer, or the abdominal effusion of a breast cancer patient) and/or pleural fluid (for example, it may be the pleural effusion of a patient with endometrial stromal sarcoma). liquid).
  • the isolated tumor infiltrating lymphocytes may be derived from tumors 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 Cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, and cholangiocarcinoma.
  • the isolated tumor infiltrating lymphocytes may be derived from tumor tissues selected from the group of cancers: cervical cancer (for example, may include recurrent cervical cancer).
  • ovarian cancer ovarian cancer, pelvic poorly differentiated adenocarcinoma, bowel cancer (for example, can include bowel cancer that has metastasized, such as liver metastasis), gastric cancer, melanoma, breast cancer, and/or endometrial stromal sarcoma.
  • bowel cancer for example, can include bowel cancer that has metastasized, such as liver metastasis
  • gastric cancer melanoma
  • breast cancer and/or endometrial stromal sarcoma.
  • the isolated tumor-infiltrating lymphocytes may be derived from a tissue mass after the tumor tissue is cut, and the diameter of the tissue mass after the tumor tissue is cut is about 1 mm to about 10 mm, for example, may be 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.
  • the culture time may be about 3-20 days.
  • the culture time may be about 3-15 days, for example, 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.
  • the temperature of the culture may be about 30-42°C.
  • it can 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, at least about 40°C, or at least about 42°C.
  • the CO 2 concentration of the culture is about 1%-10%, for example, it can be 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%.
  • the use of the seed culture medium described in this application can significantly increase the cell activity of seed cells of tumor infiltrating lymphocytes (for example, it can significantly increase the total number of seed cells of tumor infiltrating lymphocytes, for example, can make tumor
  • the number of seed cells of infiltrating lymphocytes reaches at least about 10 8 orders of magnitude; for example, the cell viability of seed cells of tumor infiltrating lymphocytes can be significantly increased, for example, the cell viability of seed cells of tumor infiltrating lymphocytes can reach at least about 10 85%; for example, it can significantly increase the amount of cytokines (such as IFN- ⁇ ) secreted by the seed cells of tumor-infiltrating lymphocytes).
  • using the seed culture medium described in this application can significantly increase the proportion of CD3 + cells in the seed cells of tumor infiltrating lymphocytes.
  • the use of the seed culture medium described in this application can significantly improve the TCR clones of the TIL of the product TIL after being reinfused into the donor subject (for example, tumor patients).
  • the frequency has increased significantly in the peripheral blood of donor subjects, and the similarity between the TCR repertoire of donor subjects’ peripheral blood and the TCR repertoire of TIL reinfusion products is also significantly increased, and can be maintained for a long time. constant.
  • the lymphocytes in the donor subject can proliferate more actively.
  • TIL seed cell culture medium The following components (product numbers) were used to prepare TIL seed cell culture medium.
  • TIL seed cell culture media prepare different TIL seed cell culture media.
  • the basal medium can be stored at 4°C for no more than 1 month after adding serum and double antibodies in proportion to the medium.
  • the culture medium should be taken out from 4°C, placed in a 37°C water bath to preheat, and then other components (various interleukins, colony stimulating factors, interferons, TNF- ⁇ , various antibodies and other molecules) are added before use. ) To the working concentration for TIL cell culture.
  • CM1 medium (hereinafter referred to as CM1) containing 6000IU/mL IL-2, Glutamax and antibiotics was prepared according to the formula and method described in Example 5 of the CN110099998A specification for Pre-REP culture derived from tumor samples TIL cells (corresponding to the seed cells herein).
  • step 2 Place the freshly isolated tumor tissue sample in a sterile environment in a secondary biological safety cabinet in a 10 cm petri dish that has been added with 30 mL of physiological saline prepared in step 1), and then transfer to a new 30 mL step 1 ) Wash in a 10 cm dish of the prepared physiological saline, and repeat the washing 3 times in total;
  • each G-REX10 culture tank corresponds to a kind of TIL seed medium prepared in Example 1; excess tumor tissue pieces are cryopreserved with CryoStor10 (purchased from BioLifeSolutions) cryopreservation solution through a program cooling device with liquid nitrogen;
  • TIL seed cell culture medium prepared in Example 1 was added to different G-REX10 culture tanks, 40 mL per tank, and tumor tissue masses were cultured at 37°C with 5% CO 2 and TIL seeds were harvested on the 12th day After the cells were counted, the total number and viability of the cells were counted, the phenotype of the cells was detected by flow cytometry, and the secretion level of IFN- ⁇ was detected with the HTRF IFN- ⁇ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instructions.
  • HTRF IFN- ⁇ detection kit Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET
  • TIL seed cells in the ascites or pleural effusion in each of the following groups were derived from 6 mL of in vivo effusion.
  • Tumor tissues T003, T005 and T007 are set with CM1 medium as a control
  • Table 6 to Table 17 respectively record the TIL seed cell culture results of T001, T002, T003, T004, T005, T006, T007, T008, T015, T012, T013 and T014 in Table 5;
  • Figures 1 to 12 are respectively Representative flow cytometric analysis results of T001, T002, T003, T004, T005, T006, T007, T008, T015, T012, T013, and T014 after using the seed cell culture medium of the present application for TIL seed cell culture,
  • Figure 13, 14 And 15 are the flow cytometric analysis results of T002, T003 and T007 cultured in CM1 medium, respectively.
  • T016 was cultured with No. 15 medium, the total number of cells was 1.13 ⁇ 10 8 , the cell viability was 95.01%, the concentration of IFN- ⁇ secreted in the supernatant was 8446.32 pg/mL, and the proportion of CD3 + cells was 92.64%.
  • Table 6-Table 14 and T016 show that the solid tumor tissues cultured using the seed cell culture medium of each group of this application can harvest at least about 10 8 total cells when harvested on 12 days, and some tumor samples can even obtain the total number Up to close to 6 ⁇ 10 8 and more than 7 ⁇ 10 8 cells, such as T003, T005 and T006. Although the total number of cells harvested from the three samples of T003, T005 and T007 that were cultured in parallel using CM1 exceeded 1 ⁇ 10 8 or 2 ⁇ 10 8 , the total number of cells was all compared with the cells cultured in other groups of media. Relatively small.
  • the viability of the TIL seed cells obtained by culturing the seed culture medium of each group of the application is relatively high, which is above 85% and most of them exceed 90%.
  • the viability of the seed cells harvested from the three samples T003, T005 and T007 that were cultured in parallel with CM1 was significantly lower than the viability of the cells cultured in their respective other groups of media.
  • T005 and T007 were two of them.
  • the viability of CM1 seed cells of the samples were all lower than 80%.
  • the IFN- ⁇ secretion of the TIL seed cells obtained by culturing the seed media of each group of the application is at a relatively high level.
  • the IFN- ⁇ secretion of TIL seed cells harvested from the three samples T003, T005 and T007 that were cultured in parallel using CM1 was also significantly lower than their respective counterparts.
  • Figures 1-9 show that the flow phenotype of TIL seed cells cultured from solid tumor tissues using each set of seed media in this application is normal, and most of the samples contain more than 90% of CD3 + cells. Some samples The proportion of CD3 + cells is around 99%, such as T002 and T006. The CD3+ cell ratio data in Table 6 to Table 14 also show the same result. The ratio of CD4 + to CD8 + in most samples is close to 1:1, and CD8 + cells in some samples account for the majority of the ratio, such as T006 and T012. Among them, Figure 1A- Figure 9A respectively show the proportion of CD3 + cells, and Figure 1B- Figure 9B respectively show the proportion of CD4 + cells. Among them, Fig. 1 to Fig. 9 respectively show medium No.
  • Table 15-17 show that the use of the seed cell culture medium of each group of the application can also successfully cultivate TIL seed cells from the body fluid of tumor patients, such as pleural effusion and ascites effusion. Some samples can even be cultured to obtain more than 2 ⁇ 10 8 TIL seed cells from as little as 10 mL of body fluid.
  • the viability of TIL seed cells obtained by culturing the seed culture medium from the body fluid in each group was higher, all higher than 85%, and most of them exceeded 90%.
  • the IFN- ⁇ secretion of TIL seed cells in all groups was also All are at a high level.
  • Figures 10-12 show that the flow phenotypes of TIL seed cells obtained from the effusion of tumor patients using the seed media of each group of the application are normal, and the proportion of CD3 + cells is more than 80%, and T012 and T014 are CD3 + cells of seed cells accounted for more than 90%.
  • the CD3 + cell ratio data in Table 15-17 also show the same result.
  • Figures 10-12 show the flow cytometry diagrams of the cell phenotypes after culturing with medium No. 25 for T012, medium No. 9 for T013, and medium No. 12 for T014.
  • FIGS 13-15 sequentially show that the flow phenotype of TIL seed cells obtained by culturing T003, T005 and T007 solid tumor tissue samples with CM1 medium is normal, and the proportion of CD3 + cells is relatively high.
  • T003 and T007 have CD3 +
  • the proportion of cells is about 99%, and the proportion of CD4 + cells of the two is significantly higher than the TIL seed cells harvested after culture in other groups of their respective counterparts.
  • each G-REX24 culture plate Cut the tumor puncture tissue into small pieces with a diameter of 1 ⁇ 1 ⁇ 3mm 3 with a sterile surgical blade, and place 3 randomly selected tumor tissue pieces in one hole of each G-REX24 culture plate (purchased from Wilsonwolf).
  • One hole of each G-REX24 culture plate corresponds to a TIL seed medium with a specific formula of Example 1 of this application, CM1 medium is used as a control, and 40 mL of medium is added to each well;
  • the above-mentioned tumor puncture tissue was cultured at 37°C with 5% CO 2 and the total number and viability of TIL seed cells were harvested on the 12th day.
  • the phenotype of the cells was detected by flow cytometry, and the HTRF IFN- ⁇ detection kit was used ( Cisbio Human IFN gamma kit (Cat. No.: 62HIFNGPET) was used to detect the secretion level of IFN- ⁇ according to the method described in the instructions.
  • TIL seed cells derived from T011 tumor puncture tissue cultured on No. 12 medium have higher viability and IFN- ⁇ secretion, and the ratio of CD3 + cells is roughly the same.
  • the cell viability rates of the above three tumor puncture tissue-derived TIL cells cultured with No. 2, No. 15 and No. 12 TIL seed cell culture media also exceeded 86%, up to 95%, and the level of IFN- ⁇ secretion was stable.
  • the proportion of CD3 + cells is also at a higher level.
  • infusion Product select 1 ⁇ 10 8 seed cells obtained from the aforementioned surgical resection of tumor tissue culture, and use the 1000 mL expansion medium prepared in step 2) in a G-REX100 culture tank at 37°C Expanded culture for 12-14 days under 5% CO 2 conditions, harvested cells, centrifuged, washed with D-PBS, resuspended in saline, and passed quality control tests (CD3 + ratio, CD4 + and CD8 + ratio, IFN- ⁇ secretion level , Sterility, endotoxin, osmotic pressure, etc.) After the indicators meet the requirements of the Pharmacopoeia of the People's Republic of China or the corresponding general standards of immune cell therapy products already on the market at home and abroad, they will be released and the products will be reinfused.
  • T015 (cervical cancer recurrence) reinfusion The T015TIL seed cells obtained in Example 2 were selected for expansion and culture according to the above method, and finally 4 ⁇ 10 10 reinfusion product cells were obtained, which were resuspended in 200 mL of normal saline and intravenously Autologous reinfusion was performed on patients who had undergone low-intensity cleansing pretreatment, and no medicinal IL-2 was injected after reinfusion.
  • the PBMC composition and changes were detected before the reinfusion, 19 days (D19), 30 days (D30) and 47 days (D47) after the reinfusion, and the PBMC composition and changes were detected at the same time.
  • the return product (IP) is used for high-throughput sequencing analysis of the TCR repertoire.
  • Figure 16 shows that the proportion of CD3 + cells in the reinfusion product exceeds 90%, and the ratio of CD8 + cells to CD4 + cells in the CD3 + cells exceeds 2:1.
  • Figure 17 shows that the frequency of TCR clones (ie IP clones) of the product TIL in the patient's peripheral blood began to increase after the reinfusion, reaching the peak at D30, and D47 decreased compared with D30.
  • Figure 18 shows that the frequency of IP clone species in the patient's peripheral blood began to increase after reinfusion and reached a peak on D30. The frequency of D47 was not significantly lower than that of D30.
  • Figure 19 shows that the frequency of the top 10 TCR clones (named IP-1-IP-10, respectively) among the retransmission products has a significant increase in frequency after retransmission, and most of them reach the peak at D30, ranking second. The frequency of the 9th TCR clone continued to rise after D30.
  • Figure 20 shows the similarity index MOI (Morisita Index) values of the TCR repertoire of peripheral blood T cells of D19, D30 and D47 and the TCR repertoire of IP before reinfusion, respectively. With the passage of time after reinfusion, the similarity index between the TCR repertoire of peripheral blood T cells and the TCR repertoire of reinfusion products gradually increased, reaching a peak on D30, and there was no significant decrease on D47.
  • MOI Mosita Index
  • the TIL seed cells obtained by culturing with the TIL seed medium of the present application can reach the order of 10 10 -10 11 cells after being expanded and cultured, and after autologous infusion of low-intensity cleansing pretreated tumor patients without the administration of IL -2 Under the condition of drugs, it can proliferate significantly in the body for a long time.
  • the preparation of the reinfused TIL cells was the same as in Example 5.
  • Subject T016 (cervical cancer patient) reinfusion the T016TIL seed cells obtained in Example 2 were selected and expanded and cultured according to the method in Example 5, and finally 5.2 ⁇ 10 10 reinfusion product cells were obtained, and resuspended in 250 mL of normal saline. Patients who had undergone low-intensity cleansing pretreatment were autologously injected intravenously, and no medicinal IL-2 was injected after the infusion.
  • Figure 21 shows that about 10 days after cyclophosphamide pretreatment (day 6 to day 8 after reinfusion), the number of leukocytes and neutrophils in the peripheral blood of the subjects decreased to the lowest level, which is consistent with the literature reports. Consistent (ME Gershwin, EJ Goetzl, AD Steinberg Cyclophosphamide: use in practice Ann Intern Med. 1974 Apr; 80(4): 531-40).
  • the level of lymphocytes in the peripheral blood drops to the lowest level after cyclophosphamide pretreatment to the first day after reinfusion, but continues to rise from the first day after reinfusion, even though the efficacy of cyclophosphamide is still under continuous action
  • the overall level of neutrophils and white blood cells was at the lowest level, there was still a significant increase in the number of lymphocytes.
  • the lymphocytes had basically returned to the level before cyclophosphamide pretreatment.

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SHANSHAN JIANG, YAN TANG, KE PAN, DESHENG WENG, JINGJING ZHAO, JIA HE, JIANCHUAN XIA: "In Vitro Culturing and Characteristics of Tumor-infi ltrating Lymphocyte of Hepatocellular Carcinoma", CHINESE JOURNAL OF CELL BIOLOGY, vol. 36, no. 7, 1 July 2014 (2014-07-01), CN , pages 970 - 975, XP055874789, ISSN: 1674-7666, DOI: 10.11844/cjcb.2014.07.0367 *
WARD ET AL.: "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli", NATURE, vol. 341, 1989, pages 544 - 546

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4308175A4 (en) * 2021-03-16 2025-02-26 H. Lee Moffitt Cancer Center & Research Institute, Inc. Til enhancement via ex vivo stimulation with cd40 agonists
WO2026000621A1 (zh) * 2024-06-26 2026-01-02 广州百吉生物制药有限公司 一种肿瘤浸润淋巴细胞体外扩增方法
CN119410579A (zh) * 2024-12-02 2025-02-11 中国医学科学院输血研究所 一种培养nk细胞的方法及应用

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US20230226111A1 (en) 2023-07-20
JP7785699B2 (ja) 2025-12-15
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