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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PCT/CN2021/096585
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English (en)
French (fr)
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金华君
何周
马星明
李甜甜
黄晨
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上海君赛生物科技有限公司
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Priority to US18/000,122 priority Critical patent/US20230226111A1/en
Priority to JP2022573182A priority patent/JP2023527531A/ja
Priority to CN202180038540.7A priority patent/CN115698267A/zh
Priority to KR1020227046117A priority patent/KR20230019460A/ko
Priority to EP21811798.4A priority patent/EP4159843A1/en
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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Abstract

本申请涉及一种肿瘤浸润淋巴细胞的种子细胞培养基及其应用，所述培养基包括以下的组分：细胞培养成分、细胞因子和免疫检查点抗体或其抗原结合片段，其中所述细胞因子包括IL-2，所述免疫检查点包括：PD-1、LAG-3、TIGIT和/或CTLA-4，所述细胞培养成分为血清培养基或无血清培养基。本申请所述的种子细胞培养基可以加快培养肿瘤浸润淋巴细胞的种子细胞，和/或缩短肿瘤浸润淋巴细胞所需的扩增时间。

Description

肿瘤浸润淋巴细胞的种子细胞培养基及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种肿瘤浸润淋巴细胞的种子细胞培养基及其应用。
背景技术
1998年美国国立癌症研究所(NCI)外科分部(Surgery Branch)报道了首例肿瘤浸润淋巴细胞(TIL-)用于治疗病人的案例。针对TIL-的广泛研究表明从黑色素瘤的病人的肿瘤组织内能够分离获得具有抗肿瘤活性的细胞。近些年发现，对过继性转移治疗(ACT)来说体积大、难以治愈的肿瘤尤其是实体肿瘤而言，TIL-具有非常显著地疗效。
TIL-用于肿瘤免疫治疗需要较大量的细胞，这对于TIL-细胞扩增在技术上和工艺的控制上提出了较大的挑战。过去的通常做法是采用基于IL-2的初步扩增(pre-Rapid Expansion Procedure，pre-REP)后随后进行“快速扩增过程”(REP)。该过程需要大量过量的经过辐射的来源于多个供体的同种异体PBMC(也称为单个核细胞，MNC)作为饲养细胞(feeder cell)以及抗CD3抗体(OKT3)和高剂量IL-2。多供体来源的同种异体辐射的PBMC细胞存在着潜在的污染风险，而较高浓度的IL-2在后续的回输中可能存在细胞耗竭的风险。
因此亟需提供有利于快速获得肿瘤浸润淋巴细胞的种子细胞的培养基。
发明内容
本申请提供了一种肿瘤浸润淋巴细胞的种子细胞培养基及其应用。本申请所述的种子细胞培养基及其制备方法具备至少一个选自下组的有益效果：1)大幅下调IL-2的用量；2)减少甚至无需在后续回输中使用IL-2；3)减少较高浓度的IL-2在后续回输所带来的细胞耗竭的风险；4)后续快速扩增过程(REP)无需使用外源同种异体PBMC或其他细胞作为饲养细胞，减少外源污染；5)促使后续快速扩增过程(REP)达到10 10-10 11细胞的产量；6)显著地提高获得肿瘤浸润淋巴细胞的种子细胞，乃至扩增肿瘤浸润淋巴细胞的培养效率；缩短培养时间。
一方面，本申请提供了一种肿瘤浸润淋巴细胞的种子细胞培养基，其包括以下的组分： 细胞培养成分、细胞因子和免疫检查点抗体或其抗原结合片段，其中所述细胞因子包括IL-2，所述免疫检查点包括：PD-1、LAG-3、TIGIT和/或CTLA-4，所述细胞培养成分为血清培养基或无血清培养基。
在某些实施方式中，所述IL-2的浓度为约3000IU/mL以下。
在某些实施方式中，所述IL-2的浓度为约2000IU/mL-约3000IU/mL。
在某些实施方式中，所述细胞因子包括IL-7。
在某些实施方式中，所述IL-7的浓度为约200-约1000U/mL。
在某些实施方式中，所述细胞因子包括IL-15。
在某些实施方式中，所述IL-15的浓度为约200-约500U/mL。
在某些实施方式中，所述细胞因子包括肿瘤坏死因子TNF。
在某些实施方式中，所述细胞因子包括TNFα。
在某些实施方式中，所述TNFα的浓度为约10-约100pg/mL。
在某些实施方式中，所述细胞因子包括集落刺激因子。
在某些实施方式中，所述细胞因子包括GM-CSF、G-CSF和/或M-CSF。
在某些实施方式中，所述GM-CSF的浓度为约200-约5000U/mL。
在某些实施方式中，所述G-CSF的浓度为约300U/mL-约1000U/mL。
在某些实施方式中，所述M-CSF的浓度为约300U/mL-约800U/mL。
在某些实施方式中，所述细胞因子包括干扰素。
在某些实施方式中，所述细胞因子包括IFN-γ、IFN-α和/或IFN-β。
在某些实施方式中，所述IFN-γ的浓度为约10-约1000ng/mL。
在某些实施方式中，所述IFN-γ的浓度为约300-约1000U/mL。
在某些实施方式中，所述IFN-α的浓度为约500U/mL-约1000U/mL。
在某些实施方式中，所述IFN-β的浓度为约200U/mL-约500U/mL。
在某些实施方式中，所述细胞因子还包括：IL-4、IL-1α、IL-1β、IL-6、IL-9、IL-18、IL-12、IL-21和/或IL-10。
在某些实施方式中，所述IL-4的浓度为约200-约500U/mL。
在某些实施方式中，所述IL-1α的浓度为约200-约500U/mL。
在某些实施方式中，所述IL-1β的浓度为约200-约500U/mL。
在某些实施方式中，所述IL-6的浓度为约200-约500U/mL。
在某些实施方式中，所述IL-9的浓度为约200-约500U/mL。
在某些实施方式中，所述IL-18的浓度为约200-约500U/mL。
在某些实施方式中，所述IL-12的浓度为约200-约1000U/mL。
在某些实施方式中，所述IL-21的浓度为约200-约500U/mL。
在某些实施方式中，所述IL-10的浓度为约200-约500U/mL。
在某些实施方式中，所述免疫检查点抗体或其抗原结合片段为人PD-1抗体或其抗原结合片段。
在某些实施方式中，所述免疫检查点抗体或其抗原结合片段为所述PD-1抗体或其抗原结合片段的，所述PD-1抗体或其抗原结合片段的浓度为约1-约100μg/mL。
在某些实施方式中，所述免疫检查点抗体或其抗原结合片段为人LAG-3抗体或其抗原结合片段。
在某些实施方式中，所述免疫检查点抗体或其抗原结合片段为LAG-3抗体或其抗原结合片段，所述LAG-3抗体或其抗原结合片段的浓度为约3-约10μg/mL。
在某些实施方式中，所述TIGIT抗体或其抗原结合片段为人TIGIT抗体或其抗原结合片段。
在某些实施方式中，所述免疫检查点抗体或其抗原结合片段为TIGIT抗体或其抗原结合片段，所述TIGIT抗体或其抗原结合片段的浓度为约1-约25μg/mL。
在某些实施方式中，所述免疫检查点抗体或其抗原结合片段为人CTLA-4抗体或其抗原结合片段。
在某些实施方式中，所述免疫检查点抗体或其抗原结合片段为CTLA-4抗体或其抗原结合片段，所述CTLA-4抗体或其抗原结合片段的浓度为约1-约100μg/mL。
在某些实施方式中，所述的种子细胞培养基还包括共刺激受体抗体或其抗原结合片段，其中所述共刺激受体或其抗原结合片段包括：CD40抗体或其抗原结合片段、OX-40抗体或其抗原结合片段、CD137抗体或其抗原结合片段和/或CD28抗体或其抗原结合片段。
在某些实施方式中，所述免疫检查点抗体或其抗原结合片段包括CD137抗体或其抗原结合片段。
在某些实施方式中，所述CD137抗体或其抗原结合片段为人CD137抗体或其抗原结合片段。
在某些实施方式中，所述CD137抗体或其抗原结合片段的浓度为约1-约100μg/mL。
在某些实施方式中，所述免疫检查点抗体或其抗原结合片段包括CD28抗体或其抗原结合片段。
在某些实施方式中，所述CD28抗体或其抗原结合片段为人CD28抗体或其抗原结合片段。
在某些实施方式中，所述CD28抗体或其抗原结合片段的浓度为约1-约10μg/mL。
在某些实施方式中，所述CD40抗体或其抗原结合片段为人CD40抗体或其抗原结合片段。
在某些实施方式中，所述CD40抗体或其抗原结合片段的浓度为约5-约10μg/mL。
在某些实施方式中，所述OX-40抗体或其抗原结合片段为人OX-40抗体或其抗原结合片段。
在某些实施方式中，所述OX-40抗体或其抗原结合片段的浓度为约3-约10μg/mL。
在某些实施方式中，所述血清培养基包括血清。
在某些实施方式中，所述血清包括人AB血清。
在某些实施方式中，所述血清的浓度为约1-约10％(v/v)。
在某些实施方式中，所述血清培养基包括AIM-V培养基。
在某些实施方式中，所述无血清培养基包括X-VIVO培养基。
在某些实施方式中，所述细胞培养成分包括抗生素。
在某些实施方式中，所述细胞培养成分包括青霉素-链霉素混合液PS。
在某些实施方式中，所述青霉素-链霉素混合液PS的浓度为约1-约200U/mL。
在某些实施方式中，所述的种子细胞培养基还包括M2型巨噬细胞抑制剂，所述M2型巨噬细胞抑制剂包括RRx001和/或CNI-1493。
在某些实施方式中，所述M2型巨噬细胞抑制剂的浓度为约0.1-约100μM。
在某些实施方式中，所述的种子细胞培养基还包括调控T细胞(Treg)抑制剂，所述调控T细胞抑制剂包括CAL-101、达沙替尼(dasatinib)、伊马替尼(imatinib)和/或帕比司他(Panobinostat)。
在某些实施方式中，所述调控T细胞抑制剂的浓度为约0.1-约100μM。
在某些实施方式中，所述的种子细胞培养基还包括骨髓来源的抑制性细胞(MDSC)抑制剂，所述骨髓来源的抑制性细胞抑制剂包括AG490、地西他滨(decitabine)、舒尼替尼和/或BBI608。
在某些实施方式中，所述骨髓来源的抑制性细胞抑制剂的浓度为约0.1-约100μg/mL。
在某些实施方式中，所述的种子细胞培养基还包括T细胞激活剂，所述T细胞激活剂包括LYC-55716、GNE-1858和/或亚甲基蓝。
在某些实施方式中，所述T细胞激活剂的浓度为约1-约10μM。
在某些实施方式中，所述的种子细胞培养基还包括T细胞分化抑制剂，所述T细胞分化抑制剂包括TWS119。
在某些实施方式中，所述T细胞分化抑制剂的浓度为约1-约10μM。
另一方面，本申请提供了本申请所述的种子细胞培养基培养获得的肿瘤浸润淋巴细胞的种子细胞或其细胞群。
另一方面，本申请提供了药物组合物，其包括本申请所述的肿瘤浸润淋巴细胞的种子细胞或其细胞群和药学上可接受的载体。
另一方面，本申请提供了所述的肿瘤浸润淋巴细胞的种子细胞或其细胞群和本申请所述的药物组合物在制备治疗癌症的药物中的应用。
在某些实施方式中，所述癌症选自下组：黑色素瘤、胶质瘤、胃癌、肺癌、胃肠道间质瘤、肠癌、肝癌、宫颈癌、卵巢癌、乳腺癌、子宫内膜间质肉瘤、盆腔低分化腺癌和胆管癌。
另一方面，本申请提供了一种所述的种子细胞培养基在扩增肿瘤浸润淋巴细胞中的应用。
另一方面，本申请提供了一种培养肿瘤浸润淋巴细胞的种子细胞的方法，其包括以下的步骤：在本申请所述的种子细胞培养基中培养分离的肿瘤浸润淋巴细胞。
另一方面，本申请提供了一种扩增肿瘤浸润淋巴细胞的方法，其包括以下的步骤，在本申请所述的种子细胞培养基中培养分离的肿瘤浸润淋巴细胞，获得所述肿瘤浸润淋巴细胞的种子细胞。
在某些实施方式中，所述分离的肿瘤浸润淋巴细胞来源于选自下组的样本：有需要的受试者的腹水、手术切除原发灶样本、同时性和异时性手术切除转移灶样本、穿刺样本和体液。
在某些实施方式中，所述体液包括血液、组织液、淋巴液和/或体腔积液。
在某些实施方式中，所述分离的肿瘤浸润淋巴细胞来源于选自下组的肿瘤：黑色素瘤、胶质瘤、胃癌、肺癌、胃肠道间质瘤、肠癌、肝癌、宫颈癌、卵巢癌、乳腺癌、子宫内膜间质肉瘤、盆腔低分化腺癌和胆管癌。
在某些实施方式中，所述分离的肿瘤浸润淋巴细胞来自肿瘤组织被切割后的组织块，所述肿瘤组织被切割后的组织块的直径为约1mm-约10mm。
在某些实施方式中，所述培养的时间为约3-20天。
在某些实施方式中，所述培养的温度为约30-42℃。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本 申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1A-图1B～图9A-图9B显示的是使用本申请所述的种子细胞培养基从实体肿瘤组织中培养所获得的TIL种子细胞的流式细胞检测结果。
图10A-图10B～图12A-图12B显示的是使用本申请所述的种子细胞培养基从肿瘤患者的体内积液中培养所获得的TIL种子细胞的流式细胞检测结果。
图13A-图13B～图15A-图15B显示的是使用现有技术中的种子细胞培养基从肿瘤患者的体内积液中培养所获得的TIL种子细胞的流式细胞检测结果。
图16A-图16B、图17-图20显示的是针对使用本申请所述的种子细胞培养基制备的TIL回输产品临床回输后，TIL回输产品和患者在不同时间点外周血的TCR组库进行高通量测序分析的检测结果。
图21显示的是使用本申请所述的种子细胞培养基制备的TIL回输产品进行临床回输后患者外周血白细胞、中性粒细胞和淋巴细胞增殖状况的结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“干扰素”通常是指抑制病毒复制和细胞增殖并调节免疫应答的具有高度种特异性的蛋白家族。典型的合适的干扰素包括但不限于：重组干扰素α-2b，重组干扰素α-2a，重组干扰素α-2C，干扰素α-n1，共有α干扰素，或干扰素α-n3。
在本申请中，术语“集落刺激因子”通常是指一类分泌型的糖蛋白，其与造血干细胞表面上的受体蛋白结合，从而激活细胞内信号传导途径，其可导致细胞增殖并分化成特定类型的 血细胞(通常是白细胞，对于红细胞形成，参见促红细胞生成素)。它们可以人工合成并外源性施用。
在本申请中，术语“抗原结合片段”通常是指抗体分子的一部分，其包含负责抗体与抗原之间的特异性结合的氨基酸。抗原中由抗体特异性地识别和结合的部分是称作如上文所述的“表位”。如上文所述，抗原结合结构域可典型地包含抗体轻链可变区(VL)和抗体重链可变区(VH)；然而，其并非必须包含两者。Fd片段例如具有两个VH区并且通常保留完整抗原结合结构域的一些抗原结合功能。抗体的抗原结合片段的实例包括(1)Fab片段，具有VL、VH、恒定轻链(CL)和CH1结构域的单价片段；(2)F(ab’)2片段，具有由铰链区的二硫桥连接的两个Fab片段的二价片段；(3)具有两个VH和CH1结构域的Fd片段；(4)具有抗体单臂的VL和VH结构域的Fv片段，(5)dAb片段(Ward等人,“Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,”Nature 341:544-546(1989)，其以引用的方式整体并入本文)，其具有VH结构域；(6)分离的互补决定区(CDR)，和(7)单链Fv(scFv)，例如源于scFV-文库。尽管Fv片段的两个结构域VL和VH是由独立基因编码，但其可通过合成连接子使用重组方法接合，合成连接子使得其被制备为其中VL和VH区配对以形成单价分子的单一蛋白链(称为单链Fv(scFv))(可参见例如Huston等人,“Protein Engineering of Antibody Binding Sites:Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,”Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))。这些抗体片段使用所属领域的技术人员已知的常规技术获得，且以与完整抗体相同的方式评估所述片段的功能。本申请中，术语“抗原结合片段”还包括与抗原，特别是细胞表面受体分子抗原特异性结合的配体。配体通常是可以以高亲和力及特异性方式与受体蛋白(例如所述抗原)结合以引发功能反应的多肽或化合物。例如，所述配体可以包括CD40的配体CD40L，CD137的配体CD137L，CD28的配体CD80和/或OX-40的配体OX-40L。
在本申请中，术语“免疫检查点”通常是指CD4 +和CD8 +T细胞的细胞表面上的一组分子。这些分子可以有效地用作下调调节或抑制抗肿瘤免疫应答的“制动器”。可以通过抑制DNA、RNA或蛋白质水平来抑制这些抑制性分子。
在本申请中，术语“共刺激受体”通常是指在免疫细胞，如T细胞表面表达的激活免疫应答反应所依赖的一类受体。共刺激受体在抗原呈递细胞存在且已提供第一信号的前提下，通过与其相应的激活型抗体、配体或抗原结合片段相结合后打开共刺激信号，进而完全激活免 疫效应细胞。共刺激受体介导的信号通路的激活通常对于免疫应答的有效发生起着关键作用。常见的共刺激受体包括但不限于CD28、CD40、CD137和OX-40。
在本申请中，术语“肿瘤坏死因子TNF”通常是指TNF蛋白，并且不是TNF配体的超家族。人TNFα的Genbank登录号为NP_000585.2。术语“TNFα”涵盖TNFα的前体形式、前TNFα(pro-TNFα)、全长TNFα、以及由细胞内过程产生的任何形式的TNFα。该术语还涵盖TNFα的天然存在的和非天然存在的变体，例如剪接变体、等位基因变体、和同种型。TNFα能结合两个受体，TNFR1(1型TNF受体；CD120a；p55/60)和TNFR2(2型TNF受体；CD120b；p75/80)。TNFα起促炎性细胞因子的作用，例如在神经炎症中起作用。
在本申请中，术语“肿瘤浸润淋巴细胞”通常是指从肿瘤组织中分离出的浸润淋巴细胞。免疫组织化学染色显示，肿瘤浸润淋巴细胞可以包括CD3 +T细胞、CD20 +、CD79α+B细胞、浆细胞和CD56 +NK细胞；也可以包括T细胞、少量的B细胞、NK细胞、巨噬细胞和树突状细胞。所述肿瘤浸润淋巴细胞可以通过直接的细胞毒性T淋巴细胞和分泌细胞因子产生高效、特异性强的抗肿瘤杀伤效应。在本申请中，所述肿瘤浸润淋巴细胞可以包括单核白血球细胞，所述单核白血球细胞离开血流并迁移至一肿瘤。所述肿瘤浸润淋巴细胞可以为选自于由T细胞、B细胞、自然杀手细胞、自然杀手T细胞所组成的群组。
在本申请中，术语“种子细胞”通常是指使用包括本申请的培养基在内的任何合适的培养基从肿瘤组织中培养出来的肿瘤浸润淋巴细胞。这些细胞即可以作为后续进一步扩大培养的起始细胞群，也可以作为终端用途的细胞群，比如用于对受试者个体进行回输治疗的细胞群。所述种子细胞可以从肿瘤组织中经过任何不同的培养时长获得，所述种子细胞的培养时长可以根据所述种子细胞的后续用途来确定，而不局限于特定的培养时长。
在本申请中，术语“IL-15”通常是指充当T细胞(特别是肠上皮内淋巴细胞(IEL))和自然杀伤(NK)细胞的有效生长、存活和活化因子的促炎细胞因子，也可称为“IL-15抗原”和“白细胞介素15”。已在多种炎性疾患，包括CD、类风湿性关节炎(RA)和牛皮癣中证实了IL-15的表达增加(Malamut等人，2010)。IL-15被认为是CD免疫病理的中心调控因子和RCD中淋巴瘤形成的非冗余驱动因子。
在本申请中，术语“IL-2”通常是指从正常外周血淋巴细胞产生并以低浓度存在于体内的淋巴因子衍生出的蛋白质。最初，Morgan等(1976)Science193：1007-1008描述了IL-2，起初称为T细胞生长因子，因为它能够诱导刺激的T淋巴细胞增殖。该术语也包括IL-2的表达后修饰的蛋白质，例如糖基化、乙酰化、磷酸化等。
在本申请中，术语“IL-7”通常是指一种由IL-7基因编码的蛋白质。IL-7是骨髓和胸腺中 的基质细胞分泌的造血生长因子。它可以由角质形成细胞，树突状细胞，肝细胞，神经元和上皮细胞产生。IL-7可以涉及对T细胞和B细胞的作用。
在本申请中，术语“PD-1”通常是指属于CD28家族的免疫抑制性受体。PD-1主要在之前活化的T细胞上体内表达，并结合两种配体，PD-1和PD-2。完整hPD-1序列可见于GenBank登录号U64863。
在本申请中，术语“LYC-55716”通常是指一种RORγ激活剂。其CAS编号为2055536-64-4。
在本申请中，术语“GNE-1858”通常是指一种HPK1抑制剂，其CAS编号为T11438。
在本申请中，术语“TWS119”通常是指一种GSK-3抑制剂，其CAS编号为601514-19-6。
在本申请中，术语“RRX-001”通常是指一种具有放射增敏活性的表观遗传调节剂。所述RRX-001可下调CD47/SIRPα轴并使肿瘤微环境中的TAM和其他免疫抑制细胞重新极化为免疫刺激表型。其CAS编号为925206-65-1。
在本申请中，术语“CNI-1493”通常是指一种可以阻止炎性细胞因子(包括TNF)产生的化合物，其CAS编号为164301-51-3。
发明详述
一方面，本申请提供了一种肿瘤浸润淋巴细胞的种子细胞培养基，其包括以下的组分：细胞培养成分、细胞因子和免疫检查点抗体或其抗原结合片段，其中所述细胞因子包括IL-2，所述免疫检查点包括：PD-1、LAG-3、TIGIT和/或CTLA-4，所述细胞培养成分为血清培养基或无血清培养基。
在本申请中，所述细胞因子可以为人源的细胞因子。
在本申请中，所述免疫检查点抗体或其抗原结合片段可以为人源化和/或全人源的抗体或其抗原结合片段。
例如，所述IL-2的浓度可以为约3000IU/mL以下(例如，可以为约2900IU/mL以下、约2800IU/mL以下、约2700IU/mL以下、约2600IU/mL以下、约2500IU/mL以下、约2400IU/mL以下、约2300IU/mL以下、约2200IU/mL以下、约2100IU/mL以下、约2000IU/mL以下、约1900IU/mL以下、约1800IU/mL以下、约1700IU/mL以下、约1600IU/mL以下、约1500IU/mL以下、约1400IU/mL以下、约1300IU/mL以下、约1100IU/mL以下、约1100IU/mL以下、约1000IU/mL以下、约900IU/mL以下、约800IU/mL以下、约700IU/mL以下、约600IU/mL以下、约500IU/mL以下、约400IU/mL以下、约300IU/mL以下或更低)。例如，所述IL-2 的浓度可以为约2000IU/mL以下、可以为约2500IU/mL以下或者，可以为约3000IU/mL以下。
例如，所述细胞因子可以包括IL-7。
例如，所述IL-7的浓度可以为约5-约100ng/mL(例如，可以为约5-约95ng/mL、约5-约90ng/mL、约5-约85ng/mL、约5-约80ng/mL、约5-约75ng/mL、约5-约70ng/mL、约5-约65ng/mL、约5-约60ng/mL、约5-约55ng/mL、约5-约50ng/mL、约5-约45ng/mL、约5-约40ng/mL、约5-约35ng/mL、约5-约30ng/mL、约5-约25ng/mL、约5-约20ng/mL或约5-约10ng/mL)。例如，所述IL-7的浓度可以为约200-约1000U/mL(例如，可以为至少约200U/mL、至少约500U/mL、或者至少约1000U/mL)。在本申请中，所述IL-7的浓度可以为约200-1000IU/mL。例如可以为至少约200IU/mL、至少约500IU/mL或者至少约1000IU/mL。
例如，所述细胞因子可以包括IL-15。
例如，所述IL-15的浓度可以为约5-约100ng/mL(例如，可以为约5-约95ng/mL、约5-约90ng/mL、约5-约85ng/mL、约5-约80ng/mL、约5-约75ng/mL、约5-约70ng/mL、约5-约65ng/mL、约5-约60ng/mL、约5-约55ng/mL、约5-约50ng/mL、约5-约45ng/mL、约5-约40ng/mL、约5-约35ng/mL、约5-约30ng/mL、约5-约25ng/mL、约5-约20ng/mL或约5-约10ng/mL)。例如，所述IL-15的浓度可以为约200-约500U/mL(例如，可以为至少约200U/mL、至少约250U/mL、至少约300U/mL、或者至少约500U/mL)。在本申请中，所述IL-15的浓度可以为200-500IU/mL。例如可以为至少约200IU/mL、至少约250IU/mL、至少约300IU/mL或者至少约500IU/mL。
例如，所述细胞因子可以包括肿瘤坏死因子TNF。
例如，所述细胞因子可以包括TNFα。
例如，所述TNFα的浓度可以为约10-约100pg/mL(例如，可以为约10-约95pg/mL、约10-约90pg/mL、约10-约80pg/mL、约10-约70pg/mL、约10-约60pg/mL、约10-约50pg/mL、约10-约40pg/mL、约15-约40pg/mL、约10-约25pg/mL、约15-约25pg/mL、约10-约30pg/mL、约10-约20pg/mL或者约10-约15pg/mL)。在本申请中，所述TNF-α的浓度可以为约10-100pg/mL。例如，可以为约10-100pg/mL、约15-100pg/mL、约20-100pg/mL或约25-100pg/mL。例如，可以为至少约10pg/mL、至少约15pg/mL、至少约20pg/mL或者至少约25pg/mL。
例如，所述细胞因子可以包括集落刺激因子。
例如，所述细胞因子可以包括GM-CSF、G-CSF和/或M-CSF。
例如，所述GM-CSF的浓度可以为约200-约5000U/mL(例如，可以为约200-约800U/mL、 约200-约500U/mL、约300-约5000U/mL、约500-约5000U/mL、约1000-约5000U/mL、约1500-约5000U/mL、约2000-约5000U/mL、约2500-约5000U/mL、约3000-约5000U/mL、约3500-约5000U/mL、约4000-约5000U/mL或约45000-约5000U/mL)。例如，可以为约200-约1000U/mL、可以为约200-约800U/mL、可以为约200-约500U/mL。例如，可以为至少约200U/mL、至少约500U/mL或者至少约800U/mL。
例如，所述G-CSF的浓度可以为约300U/mL-约1000U/mL。例如，所述G-CSF的浓度可以为约500-1000U/mL。例如，可以为至少约500U/mL或者至少约1000U/mL。
例如，所述M-CSF的浓度可以为约300U/mL-约800U/mL。例如，所述M-CSF的浓度可以为约300U/mL-约500U/mL。例如，可以为至少约300U/mL、至少约500U/mL或者至少约800U/mL。
例如，所述细胞因子可以包括干扰素。
例如，所述细胞因子可以包括IFN-γ、IFN-α和/或IFN-β。
例如，所述IFN-γ的浓度可以为约10-约1000ng/mL(例如，可以为约15-约1000ng/mL、约20-约1000ng/mL、约30-约1000ng/mL、约40-约1000ng/mL、约50-约1000ng/mL、约60-约1000ng/mL、约70-约1000ng/mL、约80-约1000ng/mL、约90-约1000ng/mL、约100-约1000ng/mL、约200-约1000ng/mL、约300-约1000ng/mL、约400-约1000ng/mL、约500-约1000ng/mL、约600-约1000ng/mL、约700-约1000ng/mL、约800-约1000ng/mL或约900-约1000ng/mL)。例如，所述IFN-γ的浓度可以为约10-约1000ng/mL。例如，所述IFN-γ的浓度可以为约300-约1000ng/mL、约300-约800ng/mL、或约300-约500ng/mL。例如，可以为至少约300ng/mL、至少约500ng/mL、至少约800ng/mL或者至少约1000ng/mL。
例如，所述IFN-α的浓度可以为约500U/mL-约1000U/mL。例如，可以为至少约500ng/mL。
例如，所述IFN-β的浓度可以为约200U/mL-约500U/mL。例如，所述IFN-β的浓度可以为约250-约500ng/mL。例如，可以为至少约200ng/mL、至少约250ng/mL或者至少约500ng/mL。
例如，所述细胞因子还可以包括IL-4、IL-1α、IL-1β、IL-6、IL-9、IL-18、IL-12、IL-21和/或IL-10。
例如，所述IL-4的浓度可以为约200-约500U/mL。例如可以为至少约200IU/mL或者至少约500IU/mL。
例如，所述IL-1α的浓度可以为约200-约500U/mL。例如可以为至少约200IU/mL或者至少约500IU/mL。
例如，所述IL-1β的浓度可以为约200-约500U/mL。例如可以为至少约200IU/mL或者至少约500IU/mL。
例如，所述IL-6的浓度可以为约200-约500U/mL。例如可以为至少约200IU/mL或者至少约500IU/mL。
例如，所述IL-9的浓度可以为约200-约500U/mL。例如可以为至少约200IU/mL或者至少约500IU/mL。
例如，所述IL-18的浓度可以为约200-约500U/mL。例如可以为至少约200IU/mL或者至少约500IU/mL。
例如，所述IL-12的浓度可以为约200-约1000U/mL。例如，所述IL-12的浓度可以为约200-约1000U/mL、约200-约500U/mL或约300-约1000U/mL。例如可以为至少约200IU/mL、至少约250IU/mL、至少约300IU/mL或者至少约500IU/mL。
例如，所述IL-21的浓度可以为约200-约500U/mL。例如，所述IL-21的浓度可以为约250-约1000U/mL。例如可以为至少约200IU/mL、至少约250IU/mL或者至少约500IU/mL。
例如，所述IL-10的浓度可以为约200-约500U/mL。例如可以为至少约200IU/mL、至少约250IU/mL、至少约300IU/mL或者至少约500IU/mL。
例如，所述免疫检查点抗体或其抗原结合片段可以为PD-1抗体或其抗原结合片段。
例如，所述PD-1抗体或其抗原结合片段可以为人PD-1抗体或其抗原结合片段。
例如，所述PD-1抗体或其抗原结合片段的浓度可以为约1-约100μg/mL(例如，可以为约3-约100μg/mL、可以为约5-约100μg/mL、可以为约10-约100μg/mL、可以为约15-约100μg/mL、约16-约100μg/mL、约17-约100μg/mL、约18-约100μg/mL、约19-约100μg/mL、约20-约100μg/mL、约25-约100μg/mL、约30-约100μg/mL、约36-约100μg/mL、约40-约100μg/mL、约45-约100μg/mL、约50-约100μg/mL、约55-约100μg/mL、约60-约100μg/mL、约65-约100μg/mL、约70-约100μg/mL、约80-约100μg/mL或约90-约100μg/mL)。在本申请中，所述PD-1mAb的浓度可以为约3-100μg/mL。例如，可以为至少约3μg/mL、至少约5μg/mL、至少约10μg/mL、至少约15μg/mL、至少约20μg/mL、至少约25μg/mL、至少约36μg/mL、至少约50μg/mL或者至少约100μg/mL。
例如，所述免疫检查点抗体或其抗原结合片段可以为人LAG-3抗体或其抗原结合片段。
例如，所述免疫检查点抗体或其抗原结合片段可以为LAG-3抗体或其抗原结合片段，所述LAG-3抗体或其抗原结合片段的浓度为约3-约10μg/mL。例如，可以为约5-约10μg/mL。例如，可以为至少约3μg/mL、至少约5μg/mL或者至少约10μg/mL。
例如，所述TIGIT抗体或其抗原结合片段可以为人TIGIT抗体或其抗原结合片段。
例如，所述免疫检查点抗体或其抗原结合片段可以为TIGIT抗体或其抗原结合片段，所述TIGIT抗体或其抗原结合片段的浓度为约1-约25μg/mL。在本申请中，所述TIGIT mAb的浓度可以为约2.5-20μg/mL。例如，可以为约5-20μg/mL。例如，可以为至少约2.5μg/mL、至少约5μg/mL或者至少约20μg/mL。
例如，所述免疫检查点抗体或其抗原结合片段可以为人CTLA-4抗体或其抗原结合片段。
例如，所述免疫检查点抗体或其抗原结合片段可以为CTLA-4抗体或其抗原结合片段，所述CTLA-4抗体或其抗原结合片段的浓度为约1-约100μg/mL。在本申请中，所述CTLA-4mAb的浓度可以为约3-50μg/mL、约5-50μg/mL或者约10-50μg/mL。例如，可以为至少约3μg/mL、至少约5μg/mL、至少约10μg/mL或者至少约50μg/mL。
在本申请中，所述的种子细胞培养基还可以包括共刺激受体抗体或其抗原结合片段，其中所述共刺激受体免疫检查点抗体或其抗原结合片段可以包括：CD40抗体或其抗原结合片段、OX-40抗体或其抗原结合片段、CD137抗体或其抗原结合片段、和/或CD28抗体或其抗原结合片段。
例如，所述共刺激抗体或其抗原结合片段可以包括CD137抗体或其抗原结合片段。
例如，所述CD137抗体或其抗原结合片段可以为人CD137抗体或其抗原结合片段。
例如，所述CD137抗体或其抗原结合片段的浓度可以为约1-约100μg/mL(例如，可以为约2-约100μg/mL、约3-约100μg/mL、约3-约20μg/mL、约4-约100μg/mL、约5-约100μg/mL、约6-约100μg/mL、约7-约100μg/mL、约8-约100μg/mL、约9-约100μg/mL、约10-约100μg/mL、约12-约100μg/mL、约20-约100μg/mL、约10-约90μg/mL、约10-约80μg/mL、约10-约70μg/mL、约10-约50μg/mL、约10-约20μg/mL或者约3-约12μg/mL)。在本申请中，所述CD137mAb的浓度可以为约3-20μg/mL。例如，可以为至少约3μg/mL、至少约5μg/mL、至少约10μg/mL、至少约12μg/mL或者至少约20μg/mL。
例如，所述共刺激抗体或其抗原结合片段可以包括OX40抗体或其抗原结合片段。
例如，所述OX-40抗体或其抗原结合片段可以为人OX-40抗体或其抗原结合片段。
例如，所述OX-40抗体或其抗原结合片段的浓度可以为约3-约10μg/mL。在本申请中，所述OX-40mAb的浓度可以为约5-10μg/mL。例如，可以为至少约3μg/mL、至少约5μg/mL或者至少约10μg/mL。
例如，所述共刺激抗体或其抗原结合片段可以包括CD28抗体或其抗原结合片段。
例如，所述免疫检查点抗体或其抗原结合片段可以包括CD28抗体或其抗原结合片段。
例如，所述CD28抗体或其抗原结合片段可以为人CD28抗体或其抗原结合片段。
例如，所述CD28抗体或其抗原结合片段的浓度可以为约1-约10μg/mL。(例如，可以为约2-约10μg/mL、约3-约10μg/mL、约3-约5μg/mL、约4-约10μg/mL、约5-约10μg/mL、约6-约10μg/mL、约7-约10μg/mL或约8-约10μg/mL)。在本申请中，所述CD28mAb的浓度可以为约3-10μg/mL。例如，可以为至少约3μg/mL、至少约5μg/mL或者至少约10μg/mL。
例如，所述共刺激抗体或其抗原结合片段可以包括CD40抗体或其抗原结合片段。
例如，所述CD40抗体或其抗原结合片段可以为人CD40抗体或其抗原结合片段。
例如，所述CD40抗体或其抗原结合片段的浓度可以为约5-约10μg/mL。在本申请中，所述CD40mAb的浓度可以为约6-10μg/mL或约5-8μg/mL。例如，可以为至少约5μg/mL。
例如，所述血清培养基可以包括血清。
例如，所述血清可以包括人AB血清。
例如，所述血清的浓度可以为约1-约10％(v/v)(例如，可以为约1.5-约10％、约2-约10％、约3-约10％、约4-约10％、约5-约10％、约1-约9％、约1-约8％、约1-约7％或约1-约6％)。
例如，所述血清培养基可以包括AIM-V培养基。
例如，所述无血清培养基可以包括X-VIVO培养基或者CTS TMOpTmizer TM培养基。
例如，所述细胞培养成分可以包括抗生素。
例如，所述细胞培养成分可以包括青霉素-链霉素混合液PS。
例如，所述青霉素-链霉素混合液PS的浓度可以为约1-约200U/mL(例如，可以为约1-约5U/mL、约1-约10U/mL、约1-约20U/mL、约1-约30U/mL、约1-约40U/mL、约1-约50U/mL、约1-约60U/mL、约1-约70U/mL、约1-约80U/mL、约1-约90U/mL、约1-约100U/mL、约1-约120U/mL、约1-约140U/mL、约1-约160U/mL、约1-约180U/mL或者约1-约200U/mL)。例如，所述青霉素-链霉素混合液PS的浓度可以为约100U/mL。
例如，所述的种子细胞培养基还可以包括M2型巨噬细胞抑制剂，所述M2型巨噬细胞抑制剂可以包括RRx001和/或CNI-1493。
例如，所述M2型巨噬细胞抑制剂的浓度可以为约0.1-约100μM(例如，可以为约0.5-约100μM、约1-约100μM、约2-约100μM、约2.5-约100μM、约3-约100μM、约2-约3μM、约5-约100μM、约10-约100μM、约20-约100μM、约0.1-约90μM、约0.5-约90μM、约1-约90μM、约5-约90μM、约0.1-约80μM)。
例如，所述的种子细胞培养基还可以包括调控T细胞(Treg)抑制剂，所述调控T细胞 抑制剂可以包括CAL-101、达沙替尼(dasatinib)、伊马替尼(imatinib)和/或帕比司他(Panobinostat)。
例如，所述调控T细胞抑制剂的浓度可以为约0.1-约100μM(例如，可以为约0.5-约100μM、约1-约100μM、约5-约100μM、约10-约100μM、约20-约100μM、约0.1-约90μM、约0.5-约90μM、约1-约90μM、约5-约90μM、约0.1-约80μM)。例如，所述调控T细胞抑制剂的浓度可以为约1-约5μM、约1-约3μM、可以为约1.5-约5μM、可以为约2-约5μM、可以为约2.5-约5μM或者可以为约3-约5μM。
例如，所述的种子细胞培养基还可以包括骨髓来源的抑制性细胞抑制剂，所述骨髓来源的抑制性细胞抑制剂可以包括AG490、地西他滨(decitabine)、舒尼替尼和/或BBI608。
例如，所述骨髓来源的抑制性细胞抑制剂的浓度可以为约0.1-约100μg/mL(例如，可以为约0.5-约100μg/mL、约1-约100μg/mL、约2-约100μg/mL、约5-约100μg/mL、约6-约100μg/mL、约7-约100μg/mL、约8-约100μg/mL、约9-约100μg/mL、约10-约100μg/mL、约20-约100μg/mL、约10-约90μg/mL、约10-约80μg/mL、约10-约70μg/mL或约10-约50μg/mL)。例如，所述骨髓来源的抑制性细胞抑制剂的浓度可以为约1-约3μg/mL，或者可以为约1.5-约3μg/mL。
在本申请中，所述的种子培养基可以包括所述调控T细胞(Treg)抑制剂、所述骨髓来源的抑制性细胞抑制剂和所述M2型巨噬细胞抑制剂中的至少一种、至少两种和至少三种。
在本申请中，所述的种子细胞培养基还可以包括T细胞激活剂，所述T细胞激活剂可以包括LYC-55716、GNE-1858和/或亚甲基蓝。
例如，所述T细胞激活剂的浓度可以为约1-约10μM。例如，可以为约1-约9μM、约1-约8μM、约1-约7μM、约1-约6μM、约2-约10μM、约3-约10μM、约4-约10μM或约5-约10μM。
在本申请中，所述的种子细胞培养基还可以包括T细胞分化抑制剂，所述T细胞分化抑制剂可以包括TWS119。
例如，所述T细胞分化抑制剂的浓度可以为约1-约10μM。例如，可以为约1-约9μM、约1-约8μM、约1-约7μM、约1-约6μM、约2-约10μM、约3-约10μM、约4-约10μM或约5-约10μM。
在本申请中，所述RRX-001的浓度可以为约0.1-约100μM。例如，可以为至少约3μM。
在本申请中，所述Sunitinib的浓度可以为约1.5-约3μM。例如，可以为至少约1.5μM或者至少约3μM。
在本申请中，所述CNI-1493的浓度可以为约2-约3μM。例如，可以为至少约2μM、至少约2.5μM或者至少约3μM。
在本申请中，所述Imatinib的浓度可以为约2-约5μM。例如，可以为至少约2μM或者至少约5μM。
在本申请中，所述CAL-101的浓度可以为约1.5-约5μM。例如，可以为至少约1.5μM或者至少约5μM。
在本申请中，所述Dasatinib的浓度可以为约2-约3μM。例如，可以为至少约2μM、至少约2.5μM或者至少约3μM。
在本申请中，所述LYC-55716的浓度可以为约1-约10μM。例如，可以为至少约1μM或者至少约10μM。
在本申请中，所述GNE-1858的浓度可以为约4-约5nM。例如，可以为至少约4nM、至少约4.5nM或者至少约5nM。
在本申请中，所述亚甲基蓝的浓度可以为约1-约2μM。例如，可以为至少约1μM、至少约1.5μM或者至少约2μM。
在本申请中，所述TWS119的浓度可以为约1-约10μM。例如，可以为至少约1μM、至少约5μM或者至少约10μM。
在本申请中，所述的种子培养基可以包括IL-2、IL-7、PD-1抗体或其抗原结合片段、TNFα、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约1000U/mL IL-7、至少约100μg/mL PD-1抗体或其抗原结合片段、至少约10pg/mL TNFα、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-9、IL-15、PD-1抗体或其抗原结合片段、CTLA-4抗体或其抗原结合片段、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约2000U/mL IL-2、至少约500U/mL IL-9、至少约500U/mL IL-15、至少约50μg/mL CTLA-4抗体或其抗原结合片段、至少约50μg/mL PD-1抗体或其抗原结合片段、至少约3％人血清、无血清培养基AIM-V和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-7、G-CSF、PD-1抗体或其抗原结合片段、TNFα、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约2000U/mL IL-2、至少约500U/mL IL-7、至少约1000U/mLG-CSF、至少约36μg/mL PD-1抗体或其抗原结合片段、至少约20pg/mL TNFα、至少约3％人血清、无血清培养基AIM-V和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-12、IL-21、GM-CSF、LAG3抗体或其抗原结合片段、CTLA-4抗体或其抗原结合片段、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约200U/mL IL-12、至少约500U/mL IL-21、至少约800U/mL GM-CSF、至少约10μg/mL LAG-3抗体或其抗原结合片段、至少约10μg/mL CTLA-4抗体或其抗原结合片段、至少约5％人血清、无血清培养基CTS TMOpTmizer TM和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-1β、IL-2、IL-6、IL-21、IFN-γ、TIGIT抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、TNFα、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约500U/mL IL-1β、至少约2500U/mL IL-2、至少约200U/mL IL-6、至少约200U/mL IL-21、至少约1000U/mL IFN-γ、至少约20μg/mL TIGIT抗体或其抗原结合片段、至少约50μg/mL PD-1抗体或其抗原结合片段、至少约25pg/mL TNFα、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-1α、IL-2、IL-9、IL-18、IFN-α、IFN-β、LAG-3抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、TNFα、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约500U/mL IL-1α、至少约2000U/mL IL-2、至少约500U/mL IL-9、至少约200U/mL IL-18、至少约500U/mL IFN-α、至少约500U/mL IFN-β、至少约5μg/mL LAG-3抗体或其抗原结合片段、至少约25μg/mL PD-1抗体或其抗原结合片段、至少约25pg/mL TNFα、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-4、IL-10、IL-21、CD137抗体或其抗原结合片段、LAG-3抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、TNFα、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约200U/mL IL-4、至少约200U/mL IL-10、至少约250U/mL IL-21、至少约10μg/mL CD137抗体或其抗原结合片段、 至少约3μg/mL LAG-3抗体或其抗原结合片段、至少约20μg/mL PD-1抗体或其抗原结合片段、至少约10pg/mL TNFα、至少约5％人血清、无血清培养基X-VIVO 15和1 00U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-6、IL-12、IL-21、CD137抗体或其抗原结合片段、CD28抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、TNFα、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约500U/mL IL-6、至少约500U/mL IL-12、至少约200U/mL IL-21、至少约20μg/mL CD137抗体或其抗原结合片段、至少约10μg/mL CD28抗体或其抗原结合片段、至少约10μg/mL PD-1抗体或其抗原结合片段、至少约10pg/mL TNFα、至少约3％人血清、无血清培养基CTS TMOpTmizer TM和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-1β、IL-2、IL-7、IL-21、G-CSF、GM-CSF、IFN-γ、LAG-3抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、TNFα、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约200U/mL IL-1β、至少约3000U/mL IL-2、至少约200U/mL IL-7、至少约200U/mL IL-21、至少约500U/mL G-CSF、至少约500U/mL GM-CSF、至少约1000U/mL IFN-γ、至少约5μg/mL LAG-3抗体或其抗原结合片段、至少约3μg/mL PD-1抗体或其抗原结合片段、至少约15pg/mL TNFα、至少约5％人血清、无血清培养基CTS TMOpTmizer TM和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-4、IL-12、GM-CSF、M-CSF、IFN-β、IFN-γ、TIGIT抗体或其抗原结合片段、CTLA-4抗体或其抗原结合片段、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约2500U/mL IL-2、至少约500U/mL IL-4、至少约500U/mL IL-12、至少约500U/mL GM-CSF、至少约300U/mL M-CSF、至少约200U/mL IFN-β、至少约800U/mL IFN-γ、至少约5μg/mL TIGIT抗体或其抗原结合片段、至少约10μg/mL CTLA-4抗体或其抗原结合片段、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-7、IL-15、IL-2、IL-7、IL-15、GM-CSF、CD137抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、TNFα人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约200U/mL IL-7、至少约200U/mL IL-15、至少约800U/mL GM-CSF、至少约10μg/mL CD137抗体或其抗原结合片段、至少约20μg/mL PD-1抗体或其抗原结合片段、至少约20pg/mL TNFα、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-4、IL-10、IL-10、G-CSF、M-CSF、CD137抗体或其抗原结合片段、CD28抗体或其抗原结合片段、OX-40抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约2500U/mL IL-2、至少约500U/mL IL-4、至少约500U/mL IL-10、至少约250U/mL IL-10、至少约500U/mL G-CSF、至少约300U/mLM-CSF、至少约5μg/mL CD137抗体或其抗原结合片段、至少约3μg/mL CD28抗体或其抗原结合片段、至少约10μg/mL OX-40抗体或其抗原结合片段、至少约10μg/mL PD-1抗体或其抗原结合片段、至少约5％人血清、无血清培养基AIM-V和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-7、IL-21、IFN-α、IFN-γ、CD137抗体或其抗原结合片段、CD28抗体或其抗原结合片段、OX-40抗体或其抗原结合片段、至少约LAG-3抗体或其抗原结合片段、CTLA-4抗体或其抗原结合片段、TNFα、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约200U/mL IL-7、至少约500U/mL IL-21、至少约500U/mL IFN-α、至少约800U/mL IFN-γ、至少约5μg/mL CD137抗体或其抗原结合片段、至少约3μg/mL CD28抗体或其抗原结合片段、至少约5μg/mL OX-40抗体或其抗原结合片段、至少约3μg/mL LAG-3抗体或其抗原结合片段、至少约5μg/mL CTLA-4抗体或其抗原结合片段、至少约10pg/mL TNFα、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-7、IL-15、IFN-β、IFN-γ、CD137抗体或其抗原结合片段、CD40抗体或其抗原结合片段、OX-40抗体或其抗原结合片段、TIGIT抗体或其抗原结合片段、PD-1抗体或其抗原结合片段人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约500U/mL IL-7、至少约500U/mL IL-15、至少约250U/mL IFN-β、至少约1000U/mL IFN-γ、至少约5μg/mL CD137 抗体或其抗原结合片段、至少约5μg/mL CD40抗体或其抗原结合片段、至少约5μg/mL OX-40抗体或其抗原结合片段、至少约5μg/mL TIGIT抗体或其抗原结合片段、至少约15μg/mL PD-1抗体或其抗原结合片段、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-7、IL-15、GM-CSF、IFN-γ、CD137抗体或其抗原结合片段、CD28抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、TNFα、至少约5％人血清、无血清培养基CTS TMOpTmizer TM和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约200U/mL IL-7、至少约200U/mL IL-15、至少约500U/mL GM-CSF、至少约1000U/mL IFN-γ、至少约3μg/mL CD137抗体或其抗原结合片段、至少约3μg/mL CD28抗体或其抗原结合片段、至少约3μg/mL PD-1抗体或其抗原结合片段、至少约10pg/mL TNFα、至少约5％人血清、无血清培养基CTS TMOpTmizer TM和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-7、IL-12、G-CSF、GM-CSF、IFN-α、IFN-γ、CD28抗体或其抗原结合片段、CD40抗体或其抗原结合片段、TIGIT抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、TNFα、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约200U/mL IL-7、至少约300U/mL IL-12、至少约300U/mL G-CSF、至少约200U/mL GM-CSF、至少约500U/mL IFN-α、至少约500U/mL IFN-γ、至少约3μg/mL CD28抗体或其抗原结合片段、至少约5μg/mL CD40抗体或其抗原结合片段、至少约2.5μg/mL TIGIT抗体或其抗原结合片段、至少约3μg/mL PD-1抗体或其抗原结合片段、至少约10pg/mL TNFα、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-7、PD-1抗体或其抗原结合片段、RRx-001、CAL-101、人血清、无血清培养基CTS TMOpTmizer TM和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约1000U/mL IL-7、至少约50μg/mL PD-1抗体或其抗原结合片段、至少约3μM RRx-001、至少约1.5μM CAL-101、至少约5％人血清、无血清培养基CTS TMOpTmizer TM和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-10、IL-18、LAG-3抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、Sunitinib、Imatinib、人血清、无血清培养基X-VIVO 15 和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约2500U/mL IL-2、至少约200U/mL IL-10、至少约200U/mLIL-18、至少约10μg/mL LAG-3抗体或其抗原结合片段、至少约5μg/mL PD-1抗体或其抗原结合片段、至少约3μM Sunitinib、至少约5μM Imatinib、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-1β、IL-6、GM-CSF、M-CSF、CNI-1493、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约200U/mL IL-1β、至少约500U/mL IL-6、至少约500U/mL GM-CSF、至少约500U/mL M-CSF、至少约5μM CNI-1493、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-9、IL-10、IL-21、GM-CSF、OX40抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、Sunitinib、人血清、无血清培养基X-VIVO15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约2000U/mL IL-2、至少约200U/mL IL-9、至少约200U/mL IL-10、至少约500U/mL IL-21、至少约500U/mL GM-CSF、至少约5μg/mL OX40抗体或其抗原结合片段、至少约36μg/mL PD-1抗体或其抗原结合片段、至少约1.5μM Sunitinib、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-6、IL-12、CD28抗体或其抗原结合片段、OX-40抗体或其抗原结合片段、CTLA-4抗体或其抗原结合片段、TNFα、Imatinib、人血清、无血清培养基CTS TMOpTmizer TM和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约2500U/mL IL-2、至少约500U/mL IL-6、至少约1000U/mL IL-12、至少约10μg/mL CD28抗体或其抗原结合片段、至少约3μg/mL OX-40抗体或其抗原结合片段、至少约5μg/mL CTLA-4抗体或其抗原结合片段、至少约15pg/mL TNFα、至少约5μM Imatinib、至少约5％人血清、无血清培养基CTS TMOpTmizer TM和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-7、IL-15、CD137抗体或其抗原结合片段、CD28抗体或其抗原结合片段、LAG-3抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、Dasatinib、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约200U/mL IL-7、至少 约300U/mL IL-15、至少约12μg/mL CD137抗体或其抗原结合片段、至少约5μg/mL CD28抗体或其抗原结合片段、至少约5μg/mL LAG-3抗体或其抗原结合片段、至少约3μg/mL PD-1抗体或其抗原结合片段、至少约3μM Dasatinib、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-6、IL-12、G-CSF、M-CSF、IFN-β、IFN-γ、CTLA-4抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、Dasatinib、LYC-55716、GNE-1858、人血清、无血清培养基X-VIVO 15和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约3000U/mL IL-2、至少约500U/mL IL-6、至少约500U/mL IL-12、至少约1000U/mL G-CSF、至少约500U/mL M-CSF、至少约500U/mL IFN-β、至少约800U/mL IFN-γ、至少约3μg/mL CTLA-4抗体或其抗原结合片段、至少约5μg/mL PD-1抗体或其抗原结合片段、至少约2.5μM Dasatinib、至少约10μM LYC-55716、至少约4.5nM GNE-1858、至少约5％人血清、无血清培养基X-VIVO 15和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-1α、IL-9、GM-CSF、CD137抗体或其抗原结合片段、CD28抗体或其抗原结合片段、LAG-3抗体或其抗原结合片段、TIGIT抗体或其抗原结合片段、CNI-1493、亚甲基蓝、TWS119、人血清、无血清培养基AIM-V和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括至少约2500U/mL IL-2、至少约500U/mL IL-1α、至少约500U/mL IL-9、至少约800U/mL GM-CSF、至少约3μg/mL CD137抗体或其抗原结合片段、至少约3μg/mL CD28抗体或其抗原结合片段、至少约5μg/mL LAG-3抗体或其抗原结合片段、至少约2.5μg/mL TIGIT抗体或其抗原结合片段、至少约2.5μM CNI-1493、至少约1μM亚甲基蓝、至少约5μM TWS119、至少约5％人血清、无血清培养基AIM-V和100U/mL青霉素-链霉素混合液PS双抗。
在本申请中，所述的种子培养基可以包括IL-2、IL-1α、IL-12、IL-21、G-CSF、M-CSF、IFN-α、IFN-β、IFN-γ、CD28抗体或其抗原结合片段、CD40抗体或其抗原结合片段、LAG-3抗体或其抗原结合片段、PD-1抗体或其抗原结合片段、CNI-1493、Dasatinib、GNE-1858、亚甲基蓝、人血清、无血清培养基AIM-V和青霉素-链霉素混合液PS双抗。
例如，所述的种子培养基可以包括约3000U/mL IL-2、约200U/mL IL-1α、约500U/mL IL-12、约500U/mL IL-21、约300U/mL G-CSF、约800U/mL M-CSF、约500U/mL IFN-α、约300U/mL IFN-β、约300U/mL IFN-γ、约3μg/mL CD28抗体或其抗原结合片段、约5μg/mL  CD40抗体或其抗原结合片段、约5μg/mL LAG-3抗体或其抗原结合片段、约3μg/mL PD-1抗体或其抗原结合片段、约3μM CNI-1493、约2μM Dasatinib、约5nM GNE-1858、约1.5μM亚甲基蓝、约5％人血清、无血清培养基AIM-V和100U/mL青霉素-链霉素混合液PS双抗。
另一方面，本申请提供了本申请所述的种子细胞培养基培养获得的肿瘤浸润淋巴细胞的种子细胞或其细胞群。
另一方面，本申请提供了药物组合物，其包括本申请所述的肿瘤浸润淋巴细胞的种子细胞或其细胞群和药学上可接受的载体。
另一方面，本申请提供了所述的肿瘤浸润淋巴细胞的种子细胞或其细胞群和本申请所述的药物组合物在制备治疗癌症的药物中的应用。
本申请所述种子培养基可以适用于扩增源自不同种类的癌症的肿瘤浸润淋巴细胞。本申请所述的种子培养基对于癌症种类具有广谱性和普适性。在某些情况下，所述种子培养基可以适用于培养源自不同种类的癌症的肿瘤浸润淋巴细胞，可以适用于获得源自不同种类的癌症的肿瘤浸润淋巴细胞的种子细胞或其细胞群。在某些情况下，本申请所述的种子培养基和/或本申请所述的药物组合物可以用于制备治疗癌症的药物。在本申请中，所述癌症可以包括：宫颈癌(例如，可以包括复发的宫颈癌)、卵巢癌、盆腔低分化腺癌、肠癌(例如，可以包括发生了转移，例如肝转移的肠癌)、胃癌、黑色素瘤、乳腺癌和/或子宫内膜间质肉瘤。例如，所述癌症可以选自下组：黑色素瘤、胶质瘤、胃癌、肺癌、胃肠道间质瘤、肠癌、肝癌、宫颈癌、卵巢癌、乳腺癌、子宫内膜间质肉瘤、盆腔低分化腺和胆管癌。
另一方面，本申请提供了一种所述的种子细胞培养基在扩增肿瘤浸润淋巴细胞中的应用。
另一方面，本申请提供了一种培养肿瘤浸润淋巴细胞的种子细胞的方法，其包括以下的步骤：在本申请所述的种子细胞培养基中培养分离的肿瘤浸润淋巴细胞。
另一方面，本申请提供了一种扩增肿瘤浸润淋巴细胞的方法，其包括以下的步骤，在本申请所述的种子细胞培养基中培养分离的肿瘤浸润淋巴细胞，获得所述肿瘤浸润淋巴细胞的种子细胞。
例如，所述分离的肿瘤浸润淋巴细胞可以来源于选自下组的样本：有需要的受试者的腹水、手术切除原发灶样本、同时性和异时性手术切除转移灶样本、穿刺样本和体液。
例如，所述体液可以包括血液、组织液、淋巴液和/或体腔积液。例如，所述体液可以包括腹腔积液(例如可以为胃癌患者的腹腔积液、或者乳腺癌患者的腹腔积液)和/或胸腔积液(例如可以为子宫内膜间质肉瘤患者的胸腔积液)。
例如，所述分离的肿瘤浸润淋巴细胞可以来源于选自下组的肿瘤：黑色素瘤、胶质瘤、 胃癌、肺癌、胃肠道间质瘤、肠癌、肝癌、宫颈癌、卵巢癌、乳腺癌、子宫内膜间质肉瘤、盆腔低分化腺癌和胆管癌例如，所述分离的肿瘤浸润淋巴细胞可以来源于选自下组的癌症的肿瘤组织：宫颈癌(例如，可以包括复发的宫颈癌)、卵巢癌、盆腔低分化腺癌、肠癌(例如，可以包括发生了转移，例如肝转移的肠癌)、胃癌、黑色素瘤、乳腺癌和/或子宫内膜间质肉瘤。
例如，所述分离的肿瘤浸润淋巴细胞可以来自肿瘤组织被切割后的组织块，所述肿瘤组织被切割后的组织块的直径为约1mm-约10mm，例如，可以是约2mm-约10mm、约3mm-约10mm、约4mm-约10mm、约5mm-约10mm、约6mm-约10mm、约7mm-约10mm或者约8mm-约10mm。
例如，所述培养的时间可以为约3-20天。例如，所述培养时间可以为约3-15天，例如，可以为约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天或者约20天。
例如，所述培养的温度可以为约30-42℃。例如，可以为至少约30℃、至少约31℃、至少约32℃、至少约33℃、至少约34℃、至少约35℃、至少约36℃、至少约37℃、至少约38℃、至少约39℃、至少约40℃、至少约40℃或者至少约42℃。
例如，所述培养的CO 2浓度为约1％-10％，例如，可以为至少约1％、至少约2％、至少约3％、至少约4％、至少约5％、至少约6％、至少约7％、至少约8％、至少约9％或者至少约10％。
在本申请中，使用本申请所述的种子培养基，可以显著提高肿瘤浸润淋巴细胞的种子细胞的细胞活性(例如，可以显著提高肿瘤浸润淋巴细胞的种子细胞的细胞总数，例如，可以使肿瘤浸润淋巴细胞的种子细胞的数量达到至少约10 8数量级；例如，可以显著提高肿瘤浸润淋巴细胞的种子细胞的细胞活率，例如，可以使肿瘤浸润淋巴细胞的种子细胞的细胞活率达到至少约85％；例如，可以显著提高肿瘤浸润淋巴细胞的种子细胞分泌细胞因子(例如IFN-γ)的数量)。在本申请中，使用本申请所述的种子培养基，可以显著提高肿瘤浸润淋巴细胞的种子细胞中CD3 +细胞的比例。
在本申请中，使用本申请所述的种子培养基，可以显著提高肿瘤浸润淋巴细胞的种子细胞的在回输至供体受试者(例如肿瘤患者)后部分回输产品TIL的TCR克隆的频率在供体受试者外周血中有显著的大幅提升，并且供体受试者外周血的TCR组库与TIL回输产品的TCR组库的相似度也显著升高，并能长时间保持不变。此外，供体受试者在进行了经本申请所述的种子培养基培养的TIL细胞的自体回输后，其体内的淋巴细胞能够产生较为活跃的增殖。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和 用途等，而不用于限制本申请发明的范围。
实施例
实施例1 TIL种子细胞培养基
使用如下的组分(货号)制备TIL种子细胞培养基。
Figure PCTCN2021096585-appb-000001
按表1-表3配制不同TIL种子细胞培养基。基础培养基按中按比例加入血清和双抗后可以放置于4℃储存不超过1个月。使用前培养基先从4℃取出，放置于37℃水浴预热，然后在使用前加入其他组分(各类白细胞介素、集落刺激因子、干扰素、TNF-α、各类抗体及其他分子)至工作浓度后用于TIL细胞培养。另外，作为对比，按CN110099998A说明书实施例5所记载的配方和方法配制含有6000IU/mL的IL-2、Glutamax和抗生素的CM1培养基(以下称为CM1)用于Pre-REP培养来源于肿瘤样本的TIL细胞(对应于本文的种子细胞)。
表1：TIL种子细胞培养基1-9
Figure PCTCN2021096585-appb-000002
Figure PCTCN2021096585-appb-000003
Figure PCTCN2021096585-appb-000004
表2：TIL种子细胞培养基10-17
Figure PCTCN2021096585-appb-000005
Figure PCTCN2021096585-appb-000006
表3：TIL种子细胞培养基18-25
Figure PCTCN2021096585-appb-000007
Figure PCTCN2021096585-appb-000008
实施例2 实体肿瘤样本(手术切除组织)处理与TIL种子细胞培养
1)配制含有终浓度100U/mL青霉素、100μg/mL链霉素和50μg/mL庆大霉素的生理盐水待用；
2)在二级生物安全柜中无菌环境下将获得的新鲜分离的肿瘤组织样本置于已加入30mL步骤1)配制的生理盐水的10cm培养皿中洗涤，再转移至新的加入30mL步骤1)配制的生理盐水的10cm皿中洗涤，共重复洗3次；
3)用无菌手术刀片剔除脂肪组织与坏死组织，将肿瘤组织切割成为直径3×3×3mm 3的小块，每个G-REX10培养罐(购自Wilsonwolf)中放置12块随机选取的肿瘤组织块，每个G-REX10培养罐对应一种实施例1所制备的TIL种子培养基；多余的肿瘤组织块用CryoStor10(购自BioLifeSolutions)冻存液通过程序降温仪液氮冻存；
4)用实施例1所制备的各组TIL种子细胞培养基分别加入到不同G-REX10培养罐中，每罐40mL，对肿瘤组织块37℃5％CO 2进行培养，第12天收获TIL种子细胞后统计细胞总数与活率，流式细胞仪检测细胞的表型，并用HTRF IFN-γ检测试剂盒(Cisbio Human IFN  gamma kit货号：62HIFNGPET)根据说明书记载的方法检测IFN-γ分泌水平。
实施例3 肿瘤患者体内积液(腹腔积液、胸腔积液)处理与TIL种子细胞培养
将来源于肿瘤患者的体内积液(腹腔积液T012、T013和胸腔积液T014)300g离心8分钟，去上清后，细胞沉淀用D-PBS洗涤3次，每次均300g离心8分钟，最终将细胞用TIL种子培养基重悬后37℃5％CO 2进行培养，第12天收获TIL种子细胞后统计细胞总数与活率，流式细胞仪检测细胞的表型，并用HTRF IFN-γ检测试剂盒(Cisbio Human IFN gamma kit货号：62HIFNGPET)根据说明书记载的方法检测IFN-γ分泌水平。
以下每个组别中的腹腔积液或胸腔积液TIL种子细胞均来源于6mL体内积液。
实施例3所处理的各肿瘤组织(体内积液)信息如表4所示：
表4
样本编号 肿瘤类型
T001 宫颈癌
T002 卵巢癌
T003 盆腔低分化腺癌
T004 宫颈癌(复发)
T005 肠癌肝转移
T006 胃癌
T007 胃癌
T008 胃癌
T009 肠癌(穿刺)
T010 宫颈癌(穿刺)
T011 黑色素瘤(穿刺)
T012 胃癌(腹腔积液)
T013 乳腺癌(腹腔积液)
T014 子宫内膜间质肉瘤(胸腔积液)
T015 宫颈癌(复发)
T016 宫颈癌
将实施例2和实施例4中处理的各肿瘤组织和体内积液按表5安排的各组培养基进行培 养，“+”表示使用了此编号培养基，“-”表示未使用该培养基；肿瘤组织T003、T005和T007设置CM1培养基作为对照
表5
Figure PCTCN2021096585-appb-000009
培养结果如表6-表17及图1-图15所示。其中，表6-表17分别记载了表5中T001、T002、T003、T004、T005、T006、T007、T008、T015、T012、T013和T014的TIL种子细胞培养结果；图1-图12分别为T001、T002、T003、T004、T005、T006、T007、T008、T015、T012、T013和T014使用本申请的种子细胞培养基进行TIL种子细胞培养后的代表性流式细胞分析结果，图13、14和15分别为T002、T003和T007用CM1培养基培养后的流式细胞分析结果。
表6：T001 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000010
表7：T002 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000011
表8：T003 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000012
表9：T004 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000013
表10：T005 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000014
表11：T006 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000015
Figure PCTCN2021096585-appb-000016
表12：T007 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000017
表13：T008 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000018
表14：T015 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000019
Figure PCTCN2021096585-appb-000020
表15：T012 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000021
表16：T013 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000022
表17：T014 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000023
T016使用15号培养基培养，细胞总数为1.13×10 8，细胞活率为95.01％，上清中分泌的IFN-γ浓度为8446.32pg/mL，CD3 +细胞比例为92.64％。
表6-表14和T016的结果显示，使用本申请各组种子细胞培养基进行培养的实体肿瘤组织在12天收获时均能收获达到至少10 8左右的细胞总数，部分肿瘤样本更能获得总数高达接近6×10 8和超过7×10 8的细胞，如T003、T005和T006。而使用CM1进行平行培养的T003、T005和T007三个样本所收获的细胞总数虽也超过1×10 8或2×10 8，但与其各自相应的其他组培养基培养的细胞相比细胞总数均相对较少。使用本申请各组种子培养基培养所获得的TIL种子细胞的活率均较高，均在85％以上且大部分超过90％。而使用CM1进行平行培养的T003、T005和T007三个样本所收获的种子细胞的活率与其各自对应的其他组培养基培养的细胞的活率相比均明显更低，其中T005和T007两个样本的CM1种子细胞活率均低于80％。此外，使用本申请各组种子培养基培养所获得的TIL种子细胞的IFN-γ分泌量均处于较高水平。使用CM1进行平行培养的T003、T005和T007三个样本所收获的TIL种子细胞的IFN-γ分泌量与其各自对应的其他相比也都明显更低。
图1-9显示，使用本申请各组种子培养基从实体肿瘤组织中培养所获得的TIL种子细胞的流式表型正常，绝大部分样本的CD3 +细胞占比超过90％，部分样本的CD3 +细胞占比在99％左右，如T002和T006。表6-表14中的CD3 +细胞比例数据也显示了同样的结果。大部分样本的CD4 +和CD8 +比例接近1:1，部分样本中CD8 +细胞占绝大部分比例，如T006和T012。其中，图1A-图9A分别显示CD3 +细胞的占比，图1B-图9B分别显示CD4 +细胞的占比。其中，图1-图9分别依次表示T001用1号培养基、T002用3号培养基、T003用2号培养基、T004用10号培养基、T005用7号培养基、T006用8号培养基、T007用16号培养基、T008用11号培养基、T015用15号培养基培养后的细胞表型流式图。
表15-表17的结果显示，使用本申请的各组种子细胞培养基也能成功地从肿瘤患者的体内积液，如胸腔积液和腹腔积液中成功培养获得TIL种子细胞。部分样本甚至能够从低至10mL以下的体内积液中培养获得超过2×10 8的TIL种子细胞。并且各组种子培养基从体内积液中培养获得TIL种子细胞的活率均较高，全部高于85％，绝大部分超过90％，同时所有组别TIL种子细胞的IFN-γ分泌量也都处于较高的水平。
图10-12显示，使用本申请各组种子培养基从肿瘤患者体内积液中培养所获得的TIL种子细胞的流式表型正常，CD3 +细胞占比均在80％以上，T012和T014的种子细胞的CD3 +细胞占比超过90％。表15-表17中的CD3 +细胞比例数据也显示了同样的结果。其中，图10-图12依次表示T012用25号培养基、T013用9号培养基、T014用12号培养基培养后的细胞表型流式图。
图13-15依次显示，使用CM1培养基培养T003、T005和T007三个实体肿瘤组织样本 获得的TIL种子细胞的流式表型正常，CD3 +细胞占比均较高，其中T003和T007的CD3 +细胞占比在99％左右，而两者的CD4 +细胞占比与其各自对应的其他组培养基培养后收获的TIL种子细胞相比明显更高。
实施例4 实体肿瘤样本(穿刺组织)处理与TIL种子细胞培养
1)配制含有终浓度100U/mL青霉素、100μg/mL链霉素和50μg/mL庆大霉素的生理盐水待用；
2)在二级生物安全柜中无菌环境下将细针(直径1mm)穿刺获得的肿瘤穿刺组织样本置于已加入30mL步骤1)配制的生理盐水的10cm培养皿中洗涤，再转移至新的加入30mL步骤1)配制的生理盐水的10cm皿中洗涤，共重复洗3次；
3)用无菌手术刀片将肿瘤穿刺组织切割成为直径1×1×3mm 3的小块，每个G-REX24培养板(购自Wilsonwolf)的一个孔中放置3块随机选取的肿瘤组织块，每个G-REX24培养板的一个孔对应一种本申请实施例1的特定配方的TIL种子培养基，CM1培养基作为对照，每孔中加入培养基40mL；
4)对上述肿瘤穿刺组织37℃5％CO 2进行培养，第12天收获TIL种子细胞后统计细胞总数与活率，流式细胞仪检测细胞的表型，并用HTRF IFN-γ检测试剂盒(Cisbio Human IFN gamma kit货号：62HIFNGPET)根据说明书记载的方法检测IFN-γ分泌水平。
肿瘤穿刺组织样本的组别和培养结果具体信息见表18-表20：
表18：T009 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000024
表19：T010 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000025
表20：T011 TIL种子细胞培养结果
Figure PCTCN2021096585-appb-000026
表18-表20的结果显示，三个肿瘤穿刺组织样本T009、T010和T011分别使用实施例1中列举的2号、15号和12号TIL种子细胞培养基培养12天后均能获得0.3×10 8左右的TIL种子细胞。使用CM1培养基对上述组织进行平行培养后，仅恶性黑色素瘤穿刺组织样本T011能够获得数量大致相当的TIL细胞，另外2个穿刺组织样本12天后显微镜观察均未见明显的TIL细胞。12号培养基培养获得T011肿瘤穿刺组织来源的TIL种子细胞与CM1TIL相比活率和IFN-γ分泌量均更高，CD3 +细胞比例两者大致相当。
此外，用2号、15号和12号TIL种子细胞培养基培养的上述3个肿瘤穿刺组织来源的TIL细胞的细胞活率也均超过86％，最高达95％以上，IFN-γ分泌水平和CD3 +细胞比例也都在较高水平。
实施例5 TIL临床回输与体内TCR表型变化
为了验证本申请的培养方法所获得的TIL在临床上的安全性与有效性，开展了针对实体肿瘤的TIL回输治疗临床试验。本项临床试验已获得上海市第十人民医院伦理委员会批准通过，具体参见http://www.chictr.org.cn/，注册号：ChiCTR2100044705；或参见www.clinicaltrials.gov，注册号：NCT04766320。
研究方案
1)低强度清淋预处理：在TIL回输前第6天-回输前第4天连续三天按6mg/kg/天剂量对患者施用环磷酰胺，在回输前第3天-回输前1天连续三天按5mg/m 2/天对患者施用氟达拉滨；
2)在施用氟达拉滨后次日，对患者经静脉回输10 10-10 11自体TIL细胞；
3)观测并记录回输后患者的状况，不同时间点抽取外周血检测PBMC的组成及变化，回输后1-3个月影像学进行效果评估。
回输TIL细胞制备
1)按照前述实施例2记载的的方法制备来源于患者手术切除肿瘤组织的TIL种子细胞；
2)扩大培养基的配制：按中国申请CN110099998A实施例5所记载的配方和方法，在使用当天向AIM-V培养基中补充IL-2，终浓度从3000IU/mL降低为1000IU/mL，按CN110099998A实施例6记载的浓度，向1000IU/mL IL-2的AIM-V培养基中补加CD3抗体 (OKT3)至终浓度30ng/mL，作为TIL细胞扩大培养使用的扩大培养基；
3)制备回输产品(Infusion Product，IP)：选取由前述手术切除肿瘤组织培养获得的种子细胞1×10 8，在G-REX100培养罐中用步骤2)中配制的1000mL扩大培养基37℃5％CO 2条件下扩大培养12-14天，收获细胞后离心，D-PBS洗涤，重悬于生理盐水，经过质控检测(CD3 +比例、CD4 +及CD8 +比例、IFN-γ分泌水平、无菌、内毒素、渗透压等)各项指标符合《中华人民共和国药典》要求或国内外已上市免疫细胞治疗产品的相对应一般标准后放行，获得回输产品。
上述涉及TIL回输产品的制备全过程均在GMP级生产车间中按照相关法规要求进行。
受试者T015(宫颈癌复发)回输：选取实施例2中获得的T015TIL种子细胞按上述方法进行扩大培养后，最终获得4×10 10回输产品细胞，重悬于200mL生理盐水，经静脉对经过低强度清淋预处理的患者进行了自体回输，回输后不注射药用IL-2。分别在回输前、回输后第19天(D19)、第30天(D30)和第47天(D47)抽取患者外周血后检测其PBMC组成及改变，同时分离上述不同时间点PBMC，与回输产品(IP)一起针对TCR组库(TCR repertoire)进行高通量测序分析。
结果如图16-20所示。图16显示回输产品中CD3 +细胞占比超过90％，CD3 +细胞中CD8 +细胞与CD4 +细胞比例超过2:1。图17显示，患者外周血中回输产品TIL的TCR克隆(即IP克隆)所占的频率在回输后开始上升，至D30到达峰值，D47较D30下降。图18显示患者外周血中的IP克隆种类的频率在回输后开始上升，至D30到达峰值，D47的频率较D30未见明显降低。图19显示，回输产品中频率排名前10的TCR克隆(分别命名为IP-1-IP-10)频率在回输后绝大部分均有显著上升，大部分在D30到达峰值，排名第2与第9的TCR克隆在D30后频率继续上升。图20显示回输前、D19、D30和D47的外周血T细胞的TCR组库分别与IP的TCR组库的相似度指数MOI(Morisita Index)值。随着回输后时间的推移，外周血T细胞的TCR组库与回输产品的TCR组库的相似度指数逐渐升高，到D30天到达峰值，D47天未见明显下降。
上述结果表明，使用本申请TIL种子培养基培养获得的TIL种子细胞能够经过扩大培养后达到10 10-10 11数量级细胞，并在自体回输低强度清淋预处理的肿瘤患者后在不施用IL-2药物的条件下能够在体内长时间显著增殖。
实施例6 TIL临床回输与体内细胞增殖
研究方案
1)低强度清淋预处理：在TIL回输前第3天-回输前1天连续三天按6mg/kg/天剂量对患者施用环磷酰胺，不施用氟达拉滨；
2)在施用环磷酰胺后次日，对患者经静脉回输10 10-10 11自体TIL细胞；
3)观测并记录回输后患者的状况，不同时间点抽取外周血检测PBMC的组成及变化，回输后1-3个月影像学进行效果评估。
回输TIL细胞制备同实施例5。
受试者T016(宫颈癌患者)回输：选取实施例2中获得的T016TIL种子细胞按实施例5方法进行扩大培养后，最终获得5.2×10 10回输产品细胞，重悬于250mL生理盐水，经静脉对经过低强度清淋预处理的患者进行了自体回输，回输后不注射药用IL-2。分别在回输前第4天、回输前第3天(施用环磷酰胺)、回输前第2天(施用环磷酰胺)、回输前1天(施用环磷酰胺)、回输后第1天、第6天、第8天、第13天和第17天采集受试者外周血，进行白细胞、中性粒细胞和淋巴细胞计数。
结果如图21所示。图21显示，在环磷酰胺预处理后10天左右(回输后第6天-第8天)，受试者外周血中的白细胞和中性粒细胞数量均降至最低水平，与文献报道一致(M E Gershwin,E J Goetzl,A D Steinberg Cyclophosphamide:use in practice Ann Intern Med.1974Apr；80(4):531-40)。外周血中淋巴细胞水平在环磷酰胺预处理后到回输后第1天降至最低水平，但从回输后第1天起开始持续上升，即使环磷酰胺药效仍然在持续作用下中性粒细胞和白细胞总体水平处于最低水平时仍然可见淋巴细胞数量的明显增加，到回输后第13天淋巴细胞已基本回复到环磷酰胺预处理前的水平。上述结果表明，使用本申请TIL种子培养基培养获得的TIL种子细胞能够经过扩大培养后达到10 10-10 11数量级细胞，并且患者在进行自体回输后体内表现出淋巴细胞活跃的持续增殖。
以上详细描述了本申请的实施方式，但是，本申请并不限于上述实施方式中的具体细节，在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，这些简单变型均属于本申请的保护范围。另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本申请对各种可能的组合方式不再另行说明。此外，本申请的各种不同的实施方式之间也可以进行任意组合，只要其不违背本申请的思想，其同样应当视为本申请所公开的内容。

Claims (78)

  1. 一种肿瘤浸润淋巴细胞的种子细胞培养基，其包括以下的组分：细胞培养成分、细胞因子和免疫检查点抗体或其抗原结合片段，其中所述细胞因子包括IL-2，所述免疫检查点包括：PD-1、LAG-3、TIGIT和/或CTLA-4，所述细胞培养成分为血清培养基或无血清培养基。
  2. 根据权利要求1所述的种子细胞培养基，其中所述IL-2的浓度为约3000IU/mL以下。
  3. 根据权利要求1-2中任一项所述的种子细胞培养基，其中所述IL-2的浓度为约2000IU/mL-约3000IU/mL。
  4. 根据权利要求1-3中任一项所述的种子细胞培养基，其中所述细胞因子包括IL-7。
  5. 根据权利要求4所述的种子细胞培养基，其中所述IL-7的浓度为约200-约1000U/mL。
  6. 根据权利要求1-5中任一项所述的种子细胞培养基，其中所述细胞因子包括IL-15。
  7. 根据权利要求6所述的种子细胞培养基，其中所述IL-15的浓度为约200-约500U/mL。
  8. 根据权利要求1-7中任一项所述的种子细胞培养基，其中所述细胞因子包括肿瘤坏死因子TNF。
  9. 根据权利要求1-8中任一项所述的种子细胞培养基，其中所述细胞因子包括TNFα。
  10. 根据权利要求9所述的种子细胞培养基，其中所述TNFα的浓度为约10-约100pg/mL。
  11. 根据权利要求1-10中任一项所述的种子细胞培养基，其中所述细胞因子包括集落刺激因子。
  12. 根据权利要求1-11中任一项所述的种子细胞培养基，其中所述细胞因子包括GM-CSF、G-CSF和/或M-CSF。
  13. 根据权利要求12所述的种子细胞培养基，其中所述GM-CSF的浓度为约200-约5000U/mL。
  14. 根据权利要求12-13中任一项所述的种子细胞培养基，其中所述G-CSF的浓度为约300-约1000U/mL。
  15. 根据权利要求12-14中任一项所述的种子细胞培养基，其中所述M-CSF的浓度为约300-约800U/mL。
  16. 根据权利要求1-15中任一项所述的种子细胞培养基，其中所述细胞因子包括干扰素。
  17. 根据权利要求1-16中任一项所述的种子细胞培养基，其中所述细胞因子包括IFN-γ、IFN-α和/或IFN-β。
  18. 根据权利要求17所述的种子细胞培养基，其中所述IFN-γ的浓度为约10-约1000U/mL。
  19. 根据权利要求17-18中任一项所述的种子细胞培养基，其中所述IFN-α的浓度为约500-约1000U/mL。
  20. 根据权利要求17-19中任一项所述的种子细胞培养基，其中所述IFN-β的浓度为约200- 约500U/mL。
  21. 根据权利要求1-20中任一项所述的种子细胞培养基，其中所述细胞因子还包括：IL-4、IL-1α、IL-1β、IL-6、IL-9、IL-18、IL-12、IL-21和/或IL-10。
  22. 根据权利要求21所述的种子细胞培养基，其中所述IL-4的浓度为约200-约500U/mL。
  23. 根据权利要求21-22中任一项所述的种子细胞培养基，其中所述IL-1α的浓度为约200-约500U/mL。
  24. 根据权利要求21-23中任一项所述的种子细胞培养基，其中所述IL-1β的浓度为约200-约500U/mL。
  25. 根据权利要求21-24中任一项所述的种子细胞培养基，其中所述IL-6的浓度为约200-约500U/mL。
  26. 根据权利要求21-25中任一项所述的种子细胞培养基，其中所述IL-9的浓度为约200-约500U/mL。
  27. 根据权利要求21-26中任一项所述的种子细胞培养基，其中所述IL-18的浓度为约200-约500U/mL。
  28. 根据权利要求21-27中任一项所述的种子细胞培养基，其中所述IL-12的浓度为约200-约500U/mL。
  29. 根据权利要求21-28中任一项所述的种子细胞培养基，其中所述IL-21的浓度为约200-约500U/mL。
  30. 根据权利要求21-29中任一项所述的种子细胞培养基，其中所述IL-10的浓度为约200-约500U/mL。
  31. 根据权利要求1-30中任一项所述的种子细胞培养基，其中所述免疫检查点抗体或其抗原结合片段为人PD-1抗体或其抗原结合片段。
  32. 根据权利要求1-31中任一项所述的种子细胞培养基，其中所述免疫检查点抗体或其抗原结合片段为PD-1抗体或其抗原结合片段，所述PD-1抗体或其抗原结合片段的浓度为约1-约100μg/mL。
  33. 根据权利要求1-32中任一项所述的种子细胞培养基，其中所述免疫检查点抗体或其抗原结合片段为人LAG-3抗体或其抗原结合片段。
  34. 根据权利要求1-33中任一项所述的种子细胞培养基，其中所述免疫检查点抗体或其抗原结合片段为LAG-3抗体或其抗原结合片段，所述LAG-3抗体或其抗原结合片段的浓度为约3-约10μg/mL。
  35. 根据权利要求1-34中任一项所述的种子细胞培养基，其中所述TIGIT抗体或其抗原结合 片段为人TIGIT抗体或其抗原结合片段。
  36. 根据权利要求1-35中任一项所述的种子细胞培养基，其中所述免疫检查点抗体或其抗原结合片段为TIGIT抗体或其抗原结合片段，所述TIGIT抗体或其抗原结合片段的浓度为约1-约25μg/mL。
  37. 根据权利要求1-36中任一项所述的种子细胞培养基，其中所述免疫检查点抗体或其抗原结合片段为人CTLA-4抗体或其抗原结合片段。
  38. 根据权利要求1-37中任一项所述的种子细胞培养基，其中所述免疫检查点抗体或其抗原结合片段为CTLA-4抗体或其抗原结合片段，所述CTLA-4抗体或其抗原结合片段的浓度为约1-约100μg/mL。
  39. 根据权利要求1-38中任一项所述的种子细胞培养基，其还包括共刺激受体抗体或其抗原结合片段，其中所述共刺激受体抗体或其抗原结合片段包括：CD40抗体或其抗原结合片段、OX-40抗体或其抗原结合片段、CD137抗体或其抗原结合片段和/或CD28抗体或其抗原结合片段。
  40. 根据权利要求39所述的种子细胞培养基，其中所述CD137抗体或其抗原结合片段为人CD137抗体或其抗原结合片段。
  41. 根据权利要求39-40中任一项所述的种子细胞培养基，其中所述CD137抗体或其抗原结合片段的浓度为约1-约100μg/mL。
  42. 根据权利要求39-41中任一项所述的种子细胞培养基，其中所述CD28抗体或其抗原结合片段为人CD28抗体或其抗原结合片段。
  43. 根据权利要求39-42中任一项所述的种子细胞培养基，其中所述CD28抗体或其抗原结合片段的浓度为约1-约10μg/mL。
  44. 根据权利要求39-43中任一项所述的种子细胞培养基，其中所述CD40抗体或其抗原结合片段为人CD40抗体或其抗原结合片段。
  45. 根据权利要求39-44中任一项所述的种子细胞培养基，其中所述CD40抗体或其抗原结合片段的浓度为约5-约10μg/mL。
  46. 根据权利要求39-45中任一项所述的种子细胞培养基，其中所述OX-40抗体或其抗原结合片段为人OX-40抗体或其抗原结合片段。
  47. 根据权利要求39-46中任一项所述的种子细胞培养基，其中所述OX-40抗体或其抗原结合片段的浓度为约3-约10μg/mL。
  48. 根据权利要求1-47中任一项所述的种子细胞培养基，其中所述血清培养基包括血清。
  49. 根据权利要求48所述的种子细胞培养基，其中所述血清包括人AB血清。
  50. 根据权利要求48-49中任一项所述的种子细胞培养基，其中所述血清的浓度为约1-约10％(v/v)。
  51. 根据权利要求1-50中任一项所述的种子细胞培养基，其中所述血清培养基包括AIM-V培养基。
  52. 根据权利要求1-51中任一项所述的种子细胞培养基，其中所述无血清培养基包括X-VIVO培养基。
  53. 根据权利要求1-52中任一项所述的种子细胞培养基，其中所述细胞培养成分包括抗生素。
  54. 根据权利要求1-53中任一项所述的种子细胞培养基，其中所述细胞培养成分包括青霉素-链霉素混合液PS。
  55. 根据权利要求54所述的种子细胞培养基，其中所述青霉素-链霉素混合液PS的浓度为约1-约200U/mL。
  56. 根据权利要求1-55中任一项所述的种子细胞培养基，其还包括M2型巨噬细胞抑制剂，所述M2型巨噬细胞抑制剂包括RRx001和/或CNI-1493。
  57. 根据权利要求56所述的种子细胞培养基，其中所述M2型巨噬细胞抑制剂的浓度为约0.1-约100μM。
  58. 根据权利要求1-57中任一项所述的种子细胞培养基，其还包括调控T细胞(Treg)抑制剂，所述调控T细胞抑制剂包括CAL-101、达沙替尼(dasatinib)、伊马替尼(imatinib)和/或帕比司他(Panobinostat)。
  59. 根据权利要求58所述的种子细胞培养基，其中所述调控T细胞抑制剂的浓度为约0.1-约100μM。
  60. 根据权利要求1-59中任一项所述的种子细胞培养基，其还包括骨髓来源的抑制性细胞抑制剂，所述骨髓来源的抑制性细胞抑制剂包括AG490、地西他滨(decitabine)、舒尼替尼和/或BBI608。
  61. 根据权利要求60所述的种子细胞培养基，其中所述骨髓来源的抑制性细胞抑制剂的浓度为约0.1-约100μg/mL。
  62. 根据权利要求1-61中任一项所述的种子细胞培养基，其还包括T细胞激活剂，所述T细胞激活剂包括LYC-55716、GNE-1858和/或亚甲基蓝。
  63. 根据权利要求62所述的种子细胞培养基，其中所述T细胞激活剂的浓度为约1-约10μM。
  64. 权利要求1-63中任一项所述的种子细胞培养基，其还包括T细胞分化抑制剂，所述T细胞分化抑制剂包括TWS119。
  65. 根据权利要求64所述的种子细胞培养基，其中所述T细胞分化抑制剂的浓度为约1-约 10μM。
  66. 利用权利要求1-65中任一项所述的种子细胞培养基培养获得的肿瘤浸润淋巴细胞的种子细胞或其细胞群。
  67. 药物组合物，其包括权利要求66所述的肿瘤浸润淋巴细胞的种子细胞或其细胞群和药学上可接受的载体。
  68. 权利要求66所述的肿瘤浸润淋巴细胞的种子细胞或其细胞群和权利要求67所述的药物组合物在制备治疗癌症的药物中的应用。
  69. 根据权利要求68所述的应用，其中所述癌症选自下组：黑色素瘤、胶质瘤、胃癌、肺癌、胃肠道间质瘤、肠癌、肝癌、宫颈癌、卵巢癌、乳腺癌、子宫内膜间质肉瘤、盆腔低分化腺癌和胆管癌。
  70. 权利要求1-65中任一项所述的种子细胞培养基在扩增肿瘤浸润淋巴细胞中的应用。
  71. 一种培养肿瘤浸润淋巴细胞的种子细胞的方法，其包括以下的步骤：在权利要求1-65中任一项所述的种子细胞培养基中培养分离的肿瘤浸润淋巴细胞。
  72. 一种扩增肿瘤浸润淋巴细胞的方法，其包括以下的步骤，在权利要求1-65中任一项所述的种子细胞培养基中培养分离的肿瘤浸润淋巴细胞，获得所述肿瘤浸润淋巴细胞的种子细胞。
  73. 根据权利要求71-72中任一项所述的方法，其中所述分离的肿瘤浸润淋巴细胞来源于选自下组的样本：有需要的受试者的腹水、手术切除原发灶样本、同时性和异时性手术切除转移灶样本、穿刺样本和体液。
  74. 根据权利要求73所述的方法，其中所述体液包括血液、组织液、淋巴液和/或体腔积液。
  75. 根据权利要求71-74中任一项所述的方法，其中所述分离的肿瘤浸润淋巴细胞来源于选自下组的肿瘤：黑色素瘤、胶质瘤、胃癌、肺癌、胃肠道间质瘤、肠癌、肝癌、宫颈癌、卵巢癌、乳腺癌、子宫内膜间质肉瘤、盆腔低分化腺癌和胆管癌。
  76. 根据权利要求71-75中任一项所述的方法，其中所述分离的肿瘤浸润淋巴细胞来自肿瘤组织被切割后的组织块，所述肿瘤组织被切割后的组织块的直径为约1mm-约10mm。
  77. 根据权利要求71-76中任一项所述的方法，其中所述培养的时间为约3-20天。
  78. 根据权利要求71-77中任一项所述的方法，其中所述培养的温度为约30-42℃。
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