WO2023011434A1 - 一种修饰的免疫细胞及其用途 - Google Patents

一种修饰的免疫细胞及其用途 Download PDF

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WO2023011434A1
WO2023011434A1 PCT/CN2022/109580 CN2022109580W WO2023011434A1 WO 2023011434 A1 WO2023011434 A1 WO 2023011434A1 CN 2022109580 W CN2022109580 W CN 2022109580W WO 2023011434 A1 WO2023011434 A1 WO 2023011434A1
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cells
present application
cell
til
gene
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PCT/CN2022/109580
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English (en)
French (fr)
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刘雅容
孙静玮
盛耀
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苏州沙砾生物科技有限公司
珠海拓域生物科技有限公司
上海沙砾生物科技有限公司
珠海沙砾生物科技有限公司
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Priority to CN202280046106.8A priority Critical patent/CN117580944A/zh
Publication of WO2023011434A1 publication Critical patent/WO2023011434A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • This application relates to the field of biomedicine, in particular to a modified immune cell and its use.
  • Immunotherapy as well as the use of adoptive autologous transfer of tumor-infiltrating lymphocytes to treat tumors, is an effective approach in the treatment of patients with poor prognosis.
  • the immune cells used in immunotherapy have the problem of low cell activity or weak proliferation ability.
  • adoptive autologous transfer of tumor-infiltrating lymphocytes to treat tumors requires a large number of tumor-infiltrating lymphocytes, and currently the tumor-infiltrating lymphocytes from the patient's tumor have weak survival and expansion capabilities in the body, and the ability to kill target cells is not strong. Functionally limited by multiple inhibitions from the tumor microenvironment.
  • the present application provides a method for culturing immune cells, the method comprising: reducing the expression and/or weakening the activity of members of the YEATS domain protein family of the immune cells.
  • the present application provides a method for culturing immune cells, the method comprising: reducing the expression and/or weakening the activity of members of the YEATS domain protein family of the immune cells.
  • the present application provides an immune cell obtained through the method of the present application.
  • the present application provides a composition comprising the immune cells of the present application.
  • the present application provides a pharmaceutical composition, which comprises the immune cells of the present application and/or the composition of the present application, and optionally a pharmaceutically acceptable carrier.
  • the present application provides a method for affecting cell growth, comprising administering the immune cells of the present application, the composition of the present application and/or the pharmaceutical composition of the present application.
  • the present application provides the application of the immune cells of the present application, the composition of the present application and/or the pharmaceutical composition of the present application in the preparation of medicines, and the medicines are used to prevent and/or treat diseases and/or symptoms .
  • Figure 1 shows the analysis results of the proliferation ability of TIL cultured with feeder cells at different addition times.
  • Figure 2 and Figure 3 show the proportion of CD45RA - CCR7 + central memory T cells (Tcm) in TIL cells cultured after adding OKT3 and IL-2 0 hours, 24 hours or 48 hours after adding feeder cells. .
  • Figure 4 shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells obtained by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • Figures 5 and 6 show the proportion of activated T cells in TIL cells cultured by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • Figure 7 shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells cultured by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • Figure 8 shows the ratio of TCF1 + stem cell-like T cells in TIL cells obtained by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • Figure 9 shows the analysis results of the proliferation ability of the test group and the control group added with different forms of CD28 agonists.
  • Figure 10 and Figure 11 respectively show the ratio of T cell subsets of TIL cells cultured in the mixed antibody group and the control group for TIL from different donors.
  • Figure 12 and Figure 13 respectively show the ratio of T cell subsets of TIL cells cultured in the magnetic bead group and the control group for TIL from different donors.
  • Figure 14 shows the ratio of T cell subsets of TIL cells cultured in the nano-matrix group and the control group.
  • Figure 15 shows the cell killing ability of TIL cells cultured in the nano-matrix group and the control group.
  • Figure 16 shows the detection results of intracellular factor expression in TIL cells cultured in the mixed antibody group and the control group.
  • Figure 17, Figure 18, Figure 19 and Figure 20 respectively show the detection results of intracellular factor expression of TIL cells cultured from the magnetic bead group and the control group for TIL from different donors.
  • Figure 21 shows the detection results of intracellular factor expression in TIL cells cultured in the nano-matrix group and the control group.
  • Figure 22 shows the results of cytokine secretion detection of TIL cells cultured in the nano-matrix group and the control group.
  • Figure 23 shows the cytokine secretion detection results after the co-incubation of TIL cells and tumor cells obtained from the nano-matrix group and the control group.
  • Figure 24 and Figure 25 respectively show the gene knockout efficiency results of TIL cells cultured in the nano-matrix group and the control group for TILs from different donors.
  • Fig. 26, Fig. 27 and Fig. 28 respectively show the proliferative ability analysis results of the test groups subjected to in vitro expansion in different ways in the terminal stimulation stage for TILs from different donors.
  • Figure 29A shows the results of cell proliferation ability of TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 29B shows the results of CD45RA - CCR7 + central memory T cell (Tcm) ratio of TIL cells obtained by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 29C shows the ratio of TCF1 + stem cell-like T cells in TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 29D shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in the cultured TIL cells obtained by adding feeder cells to TIL after 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2.
  • Figure 29E shows the ratio of activated T cells (PD-1 + ) in TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 29F shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours after adding feeder cells.
  • Figure 29G shows the ratio of activated T cells (CD28 + ) in TIL cells obtained by adding feeder cells after 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2.
  • Figure 29H shows the proportion of activated T cells (41BB + ) in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours after adding feeder cells.
  • Figure 29I shows the proportion of activated T cells (CD25 + ) in TIL cells cultured after adding feeder cells for 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2.
  • Figure 29J shows the TILs cultured with feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours, and the detection results of intracellular factor expression of TIL cells obtained from culture.
  • Figure 29K shows the cytokine secretion test results of TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 29L shows the TILs cultured with feeder cells after adding OKT3 and IL-2 for 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days, and the results of the cell proliferation ability of the TIL cells obtained by culturing picture.
  • Figure 29M shows TILs cultured with feeder cells after adding OKT3 and IL-2 at 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days, and CD8 + T cells of the resulting TIL cells Proportion.
  • Figure 29N shows the CD45RO + CD62L + CD45RO + CD62L + of TIL cells cultured after adding OKT3 and IL-2 at 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells. T cell ratio.
  • Figure 29O shows the addition of feeder cells to cultured TIL after adding OKT3 and IL-2 for 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days, and the proportion of NK T cells in the cultured TIL cells .
  • Figure 29P shows the CD4 + CD25 + CD4 + CD25 + of TIL cells cultured after adding OKT3 and IL-2 at 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells. Foxp3 + regulatory T cell (Treg) ratio.
  • Figure 29Q shows the results of the cell killing ability of TIL cells cultured by adding feeder cells 48 hours after adding OKT3 and IL-2.
  • Figures 30A-30B show, for TIL cells derived from donors 713 and 812, the amount of fluorescence after expansion of TIL cells in each group.
  • Figure 31 shows the test results of the killing ability of TIL cells derived from donor 812 co-cultured with tumor cells at an effect-to-target ratio of 1:1.
  • 32A-32B show the detection results of apoptosis in TIL cells derived from donor 812.
  • Figures 33A-33C show, for TIL cells derived from donors 713 and 812, the proportion of central memory T cells (CD45RO + CD62L + ) among TIL cells in each group.
  • 34A-34D show, for TIL cells derived from donors 713 and 812, the proportion of naive T cells (CD45RO ⁇ CD62L + ) in TIL cells of each group.
  • Figures 35A-35E show, for TIL cells derived from donor 713, the proportions of CD38-positive, LAG-3-positive and TIM-3-positive cells among TIL cells in each group.
  • Figures 36A-36E show, for TIL cells derived from donor 812, the proportions of CD38-positive, LAG-3-positive and TIM-3-positive cells among TIL cells in each group.
  • Figures 37A-37D show the ratio of GZMB, TNF- ⁇ and IFN- ⁇ expressing cells in TIL cells of each group for TIL cells derived from donor 713 under the condition of no activator stimulation.
  • Figures 38A-38E show the percentages of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells derived from donor 812 under the condition of no activator stimulation.
  • Figures 39A-39B show the ratio of TNF- ⁇ expressing cells among TIL cells in each group of TIL cells derived from donor 713 under the condition of transACT stimulation.
  • Figures 40A-40F show the percentages of cells expressing GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells derived from donor 812 under the condition of CD3 antibody stimulation.
  • Figure 41, Figure 42, Figure 43 and Figure 44 show, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group without additional stimulation.
  • Figure 45 and Figure 46 show, for TIL cells derived from different donors, the amount of fluorescence after expansion of TIL cells in each group stimulated by anti-CD3 antibody (Miltenyi Biotech, OKT3).
  • Figure 47 shows the test results of the killing ability of TIL cells derived from donor B co-cultured with tumor cells at an effect-to-target ratio of 1:1.
  • Figure 48 shows the test results of the killing ability of TIL cells derived from donor C co-cultured with tumor cells at an effect-to-target ratio of 1:3.
  • Figure 49 and Figure 50 show the test results of the killing ability of TIL cells derived from donor D co-cultured with tumor cells at an effect-to-target ratio of 1:1 and an effect-to-target ratio of 1:3.
  • Figure 51, Figure 52 and Figure 53 show, for TIL cells from different donors, the multiplication factor of long-term culture of TIL cells in each group after withdrawal of IL-2.
  • Figure 54 and Figure 55 show, for TIL cells from different donors, the long-term culture viability of TIL cells in each group after IL-2 was withdrawn.
  • Figure 56 and Figure 57 show, for TIL cells derived from donor C, the ratio of TIM-3 positive and CD101 positive cells in TIL cells of each group.
  • Figure 58 shows, for TIL cells derived from donors, the ratio of CD45RA-negative CCR7-positive memory T cells (Tcm) among TIL cells in each group.
  • Figure 59, Figure 60, Figure 61 and Figure 62 show the expression of CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells from different donors under the condition of no stimulation cell ratio.
  • Figure 63, Figure 64, Figure 65 and Figure 66 show that, under the condition of overnight stimulation with CD3 antibody containing about 30ng/mL, for TIL cells from different donors, CD107a, GZMB, TNF - Proportion of ⁇ and IFN- ⁇ expressing cells.
  • Fig. 67, Fig. 68 and Fig. 69 show is, under the condition containing phorbol-myristate-acetate (PMA, 25ng/ml) and ionomycin (Ionomycin, 1 ⁇ g/ml) to stimulate overnight,
  • PMA phorbol-myristate-acetate
  • Ionomycin Ionomycin 1 ⁇ g/ml
  • Figure 70 and Figure 71 show the Shannon diversity index of TCR V ⁇ clones of CD4 + T cells and CD8 + T cells on day 8 and day 18 after gene editing of TIL cells.
  • Figure 72 shows, for TIL cells derived from donor C, the secretion detection results of cytokines IFN- ⁇ and GZMB of TIL cells cultured in each group after IL-2 was withdrawn.
  • Figures 73A-73B show, for TIL cells derived from donors 619 and 003, the amount of fluorescence after expansion of TIL cells in each group.
  • Figure 73C shows, for the TIL cells derived from donor 410, the amount of fluorescence after TGF ⁇ -1 stimulated the expansion of TIL cells in each group.
  • FIG. 73D shows, for the TIL cells derived from donor 410, the amount of fluorescence after the expansion of OKT3-induced stimulated TIL cells in each group.
  • FIG. 73E shows, for the TIL cells derived from donor 410, the TGF ⁇ -1 and OKT3 induced by each group stimulated the amplified fluorescence of the TIL cells.
  • Figures 74A-74C show the test results of the killing ability of TIL cells derived from donors 619 and 921 co-cultured with tumor cell A375 at an effect-to-target ratio of 1:1 and 1:3.
  • Figure 74D shows the test results of the killing ability of TIL cells derived from donor 921 co-cultured with tumor cells at an effect-to-target ratio of 1:1.
  • Figure 74E shows that, for TIL cells derived from donor 921, they were co-cultured with tumor cells at an effect-to-target ratio of 1:1
  • Figures 74F-74G show the results of the killing ability detection of TIL cells derived from donor 811 co-cultured with tumor cells at an effect-to-target ratio of 1:1 and 1:3.
  • 75A-75B show the detection results of apoptosis of TIL cells derived from donor 811.
  • Figures 75C-75D show the detection results of apoptosis in TIL cells derived from donor 921.
  • Figures 75E-75F show the results of apoptosis detection of TIL cells derived from donor 614.
  • Figures 76A-76B show the proportions of PD-1 positive, LAG-3 positive and CD38 positive cells among TIL cells in each group for TIL cells derived from donor 811.
  • Figures 76C-76E show, for TIL cells derived from donors 614 and 921, the proportion of Fas-L positive cells in TIL cells in each group.
  • Figures 76F-76G show, for TIL cells derived from donors 410 and 811, the ratio of regulatory T cell Treg (CD4 + CD25 + FoxP3 + ) in TIL cells of each group.
  • FIG. 76H shows, for TIL cells derived from donor 811, the ratio of regulatory T cell Treg (CD4 + CD25 + FoxP3 + ) in TIL cells of each group.
  • Figure 76I shows the ratio of regulatory T cells Treg (CD4 + CD25 + FoxP3 + ) in TIL cells of each group for TIL cells derived from donor 811 .
  • Figure 77 shows the test results of the killing ability of TIL cells derived from donor 313 co-cultured with tumor cells at an effect-to-target ratio of 0.7:1.
  • 78A-78B show the detection results of apoptosis in TIL cells from donor 105.
  • Figure 79A-79F shows, for TIL cells derived from donors 105, 222, 313 and 812, the proportion of LAG-3 positive, PD-1 positive, CD38 positive, and TIM-3 positive cells in TIL cells of each group .
  • Figure 80 shows, for TIL cells derived from donor 313, the ratio of CD45RO-positive CD62L-positive central memory T cells (Tcm) among TIL cells in each group.
  • Figures 81A-81B show that, under the condition that CD3 antibody (Miltenyi Biotech, OKT3), CD3 antibody was pretreated with 30ng/mL 96-well plate) stimulated overnight, for the TIL cells derived from donor 105, the TIL cells of each group The proportion of cells expressing GZMB and TNF- ⁇ .
  • CD3 antibody Miltenyi Biotech, OKT3
  • Figures 82A-82C show the ratio of TNF- ⁇ , IFN- ⁇ and CD107a expressing cells in TIL cells from donors 713 and 812 under the condition of no activator stimulation.
  • Figures 83A-83D show the proportion of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells derived from donors 713 and 812 under the condition of overnight transACT stimulation.
  • 84A-84C show, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group.
  • Figure 85 shows, for TIL cells derived from donor 222, the amount of fluorescence after expansion of TIL cells in each group.
  • Figures 86A-86B show the test results of the killing ability of TIL cells derived from donor 222 co-cultured with tumor cells at an effect-to-target ratio of 1:3 and 0.7:1.
  • Figures 86C-86H show the test results of the killing ability of TIL cells derived from donors 313, 222, 812 and 709 co-cultured with tumor cells at an effect-to-target ratio of 1:3, 0.7:1 and 1:1.
  • 87A-87B show the detection results of apoptosis in TIL cells derived from donor 812.
  • Figures 88A-88F show, for TIL cells derived from donors 313, 222, 812 and 709, the proportions of PD-1 positive, LAG-3 positive, CD38 positive, and TIM-3 positive cells in TIL cells of each group .
  • 89A-89B show, for TIL cells derived from donors 313 and 709, the ratio of CD45RO-positive CD62L-positive central memory T cells (Tcm) among TIL cells in each group.
  • Figures 90A-90D show the percentages of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells from donors 713 and 812 under the condition of no activator stimulation .
  • Figures 91A-91B show the percentages of CD107a, GZMB, TNF- ⁇ and IFN- ⁇ expressing cells in TIL cells from donor 713 under transACT stimulation overnight.
  • 92A-92C show, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group. The results show that the gene-edited TIL cells of the present application can have significant expansion ability.
  • Figures 93A-93C show, for TIL cells from donors 105, 313 and 222, the amount of fluorescence after expansion of TIL cells in each group.
  • Figures 94A-94C show the test results of the killing ability of TIL cells derived from donors 313 and 222 co-cultured with tumor cells at an effect-to-target ratio of 1:3 and 0.7:1.
  • 95A-95B show the detection results of apoptosis in TIL cells from donor 105.
  • Figures 96A-96E show, for TIL cells derived from donors 105, 313, and 222, the proportions of PD-1 positive, LAG-3 positive, CD38 positive, and TIM-3 positive cells among TIL cells in each group.
  • Figure 97 shows, for TIL cells derived from donor 313, the ratio of CD45RO-positive CD62L-positive central memory T cells (Tcm) among TIL cells in each group.
  • Figures 98A-98B show that, under the condition that CD3 antibody (Miltenyi Biotech, OKT3), CD3 antibody was pretreated with 30ng/mL 96-well plate) stimulated overnight, for the TIL cells derived from donor 105, each group of TIL cells Proportions of cells expressing GZMB, TNF- ⁇ and IFN- ⁇ .
  • CD3 antibody Miltenyi Biotech, OKT3
  • Figures 99A-99B show, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group.
  • Figure 100 shows, for TIL cells derived from donor 713, the amount of fluorescence after expansion of TIL cells in each group.
  • Figure 101 shows the test results of the killing ability of TIL cells derived from donor 709 co-cultured with tumor cells at an effect-to-target ratio of 1:1.
  • 102A-102B show the detection results of apoptosis in TIL cells from donor 812.
  • Figures 103A-103D show, for TIL cells derived from donors 812 and 709, the proportions of LAG-3 positive and CD38 positive cells in TIL cells of each group.
  • Figure 104 shows, for TIL cells derived from donor 709, the ratio of CD45RO-positive CD62L-positive central memory T cells (Tcm) among TIL cells in each group.
  • Fig. 105A shows the ratio of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells derived from donor 713 under the condition of no activator stimulation.
  • Fig. 105B shows the ratio of CD107a expressing cells in TIL cells of each group for TIL cells derived from donor 713 under the condition of overnight transACT stimulation.
  • Figure 106 shows, for the TIL cells derived from donor 713, the amount of fluorescence after expansion of TIL cells in each group.
  • Figure 107 shows, for the TIL cells derived from donor 713, the amount of fluorescence after expansion of TIL cells in each group.
  • Figures 108A-108B show the test results of the killing ability of TIL cells derived from donor 713 co-cultured with tumor cells at an effect-to-target ratio of 1:1 and 1:3.
  • Figures 109A-109D show the detection results of apoptosis of TIL cells derived from donors 713 and 811.
  • Figures 110A-110F show, for TIL cells derived from donors 713, 811, and 812, the proportions of CD101-positive, LAG-3-positive and CD38-positive cells in TIL cells of each group.
  • Figure 111A shows the ratio of GZMB-expressing cells in TIL cells of each group for TIL cells derived from donor 713 under the condition of no activator stimulation.
  • Figures 111B-111C show the ratio of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells derived from donor 713 under the condition of transACT stimulation overnight.
  • YEATS domain protein family generally refers to a family member protein having a YEATS domain or a functionally active fragment thereof.
  • members of the YEATS domain protein family can recognize modified proteins.
  • the YEATS domain can recognize histone modifications, such as histone acetylation.
  • members of the protein family comprising a YEATS domain may comprise ENL (UniProt No Q03111), AF9 (UniProt No P42568), and YEATS2 (UniProt No Q9ULM3) and GAS41 (UniProt No O95619).
  • YEATS inhibitor generally refers to a substance capable of affecting the activity and/or function of YEATS protein.
  • Suitable inhibitor molecules may include antagonist antibodies or antibody fragments, fragments or derivatives of small molecules, peptides, antisense oligonucleotides, small organic molecules, and the like.
  • a method of identifying an inhibitor of the present application comprises contacting a cell expressing a molecule inhibited by the present application with a candidate inhibitor molecule, detecting a detectable change in one or more biological activities associated with the molecule inhibited by the present application, YEATS inhibition
  • the agent may comprise a substance capable of reducing the expression and/or activity of a nucleic acid molecule encoding a protein comprising a YEATS domain.
  • a YEATS inhibitor can inhibit the function of YEATS by binding to the histone-binding domain of the YEATS protein.
  • the YEATS inhibitor may include SGC-iMLLT (CAS NO.: 2255338-25-9) and any known YEATS inhibitor, as well as derivatives of the above compounds.
  • a YEATS inhibitor can be specific, having higher binding activity for one or more proteins that comprise members of the YEATS domain family.
  • JAK-STAT pathway inhibitory molecule generally refers to an inhibitory molecule of a signaling pathway.
  • the JAK-STAT pathway inhibitory molecule may comprise a substance that inhibits activation of the JAK-STAT pathway or inhibits the activity of a JAK-STAT pathway transducer.
  • JAK Java kinase
  • Janus kinase can act as a factor that transmits JAK-STAT pathway signals.
  • the UniProt accession number for JAK could be P23458.
  • cytokine signaling inhibitory molecule generally refers to a substance that inhibits cytokine signaling.
  • an exemplary cytokine signaling inhibitory molecule may be SOCS, or a variant thereof, or a substance comprising a functionally active fragment as described above.
  • SOCS1 Sytokine Signaling 1
  • SOCS1 can inhibit JAK-STAT pathway transduction.
  • UniProt accession number for SOCS1 could be O15524.
  • SOCS1 may encompass unprocessed SOCS1, any form of processed SOCS1, variants of SOCS1 or substances comprising functionally active fragments of SOCS1.
  • TGF ⁇ R2 generally refers to a cytokine receptor.
  • TGF ⁇ R2 can bind to TGF ⁇ and mediate downstream signals, playing a key role in the growth, development and differentiation of cells and tissues.
  • NCBI Gene accession number for TGF ⁇ R2 can be 7048.
  • TGF ⁇ R2 may encompass unprocessed TGF ⁇ R2, any form of processed TGF ⁇ R2, variants of TGF ⁇ R2 or substances comprising functionally active fragments of TGF ⁇ R2.
  • FLI1 generally refers to a gene encoding a protein or the protein.
  • FLI1 may encode a protein comprising the DNA binding domain of ETS (erythroid transformation specificity).
  • ETS erythroid transformation specificity
  • FLI1 can mediate downstream signals and play a key role in the growth, development, and differentiation of cells and tissues.
  • NCBI Gene accession number for FLI1 can be 2313.
  • FLI1 may encompass unprocessed FLI1, any form of processed FLI1, variants of FLI1 or substances comprising functionally active fragments of FLI1.
  • CISH Cytokine Inducible SH2 Containing Protein
  • CISH can encode a protein comprising an SH2 domain and a SOCS box domain.
  • CISH can mediate downstream signals and play key roles in the growth, development, and differentiation of cells and tissues.
  • NCBI Gene accession number for CISH could be 1154.
  • CISH may encompass unprocessed CISH, any form of processed CISH, variants of CISH or substances comprising functionally active fragments of CISH.
  • CBLB generally refers to a gene encoding a protein or the protein.
  • CBLB can encode a protein comprising an E3 ubiquitin ligase.
  • NCBI Gene accession number for CBLB could be 868.
  • CBLB can mediate downstream signaling and play a key role in the growth, development, and differentiation of cells and tissues.
  • CBLB may encompass unprocessed CBLB, any form of processed CBLB, variants of CBLB or substances comprising functionally active fragments of CBLB.
  • ANKRD11 generally refers to a gene encoding a protein or the protein.
  • ANKRD11 may encode an ankyrin repeat domain-containing protein.
  • ANKRD11 can mediate downstream signals and play a key role in the growth, development, and differentiation of cells and tissues.
  • NCBI Gene accession number for ANKRD11 can be 29123.
  • ANKRD11 may encompass unprocessed ANKRD11, any form of processed ANKRD11, variants of ANKRD11 or substances comprising functionally active fragments of ANKRD11.
  • BATF generally refers to a gene encoding a protein or the protein.
  • BATF can encode a protein that contains a leucine zipper.
  • BATF can mediate downstream signals and play a key role in the growth, development, and differentiation of cells and tissues.
  • NCBI Gene accession number for BATF could be 10538.
  • BATF may encompass unprocessed BATF, any form of processed BATF, variants of BATF or substances comprising functionally active fragments of BATF.
  • a gene regulatory system generally refers to a system that regulates the expression or activity of a target gene.
  • a gene regulatory system can comprise a gene regulatory molecule.
  • the gene regulatory system can regulate the expression or activity of a gene, such as making the gene in an inactivated or activated state, increasing or decreasing the number of the gene, making the gene in a state of increasing or decreasing the amount of transcription, and/or making the gene
  • the transcription product of the gene is in an inactivated or activated state; for example, the gene regulatory system can regulate the expression or activity of the gene, such as increasing or decreasing the amount of the expression product of the gene in a single cell, and/or making the expression of the gene
  • the number of cells expressing the product is increased or decreased.
  • guide nucleic acid molecule generally refers to a nucleic acid molecule that can be used for gene editing.
  • a guide nucleic acid molecule can provide information on insertion or deletion of nucleotides to guide the editing process.
  • the guide nucleic acid molecule can be a guide RNA or guide RNA (guide RNA, gRNA).
  • guide RNA guide RNA
  • gRNA may refer to an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific location within the target DNA.
  • perfect complementarity may not be required, e.g., as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex.
  • an enzyme protein generally refers to a protein with enzymatic activity.
  • an enzyme protein may refer to a Cas protein.
  • a Cas protein can contain at least one RNA recognition or binding domain that can interact with a gRNA.
  • Cas proteins can also comprise nuclease domains (e.g., DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and/or other structures area.
  • a nuclease domain may have catalytic activity for nucleic acid cleavage. Cleavage can include the breaking of covalent bonds in nucleic acid molecules.
  • the Cas protein can be a wild-type protein (ie, a naturally occurring protein), a modified Cas protein (ie, a Cas protein variant), or a fragment of a wild-type or modified Cas protein.
  • the Cas protein can also be an active variant or fragment of a wild-type or modified Cas protein.
  • Cas protein may cover unprocessed Cas protein, any form of processed Cas protein, variants of Cas protein or substances comprising functionally active fragments of Cas protein.
  • ribonucleoprotein complex generally refers to a complex formed by a protein and nucleic acid.
  • a protein in a ribonucleoprotein complex can have nuclease activity.
  • ribonucleoprotein complexes can cut target sequences under the guidance of nucleic acids therein.
  • the ribonucleoprotein complex can be a complex formed by Cas protein and gRNA.
  • exon generally refers to the portion of a gene that can be expressed as a protein.
  • an exon may refer to the ability to be expressed as a protein during protein biosynthesis.
  • splicing the exon sequence of a target gene can reduce the activity or function of the target gene.
  • the term "protospacer adjacent motif (PAM)” generally refers to the short sequence following the target sequence.
  • PAM sequence can be used to determine the position of cleavage.
  • those skilled in the art can easily determine the position of a suitable target sequence, and can easily design a gRNA sequence for cutting the target sequence.
  • the term "decreased expression” generally refers to a decrease in the expression level of a product or its gene and/or a decrease in the proportion of cells capable of expressing the product. For example, there may be a decrease in the amount of cells expressing the product of the gene or a decrease in the proportion of cells that contain the product of the gene expression, or a decrease in the proportion of cells that secrete the product of the gene expression.
  • the decrease in the expression of the gene can be indirectly indicated by detecting the knockout amount of the gene in the genome of the cell. For example, by detecting the proportion of cells in which the gene is knocked out in a cell population, it can be indirectly indicated that the expression of the gene is reduced.
  • the term "activity" generally refers to the biological function of a substance.
  • the activity of a gene can refer to the transcriptional and/or translational state of the gene.
  • the weakening of the activity of a gene may mean that the transcriptional function of the gene is weakened, the gene cannot be normally transcribed, or the function of the transcription product of the gene is inhibited.
  • CD80 generally refers to a cell stimulating molecule.
  • CD80 can be a ligand for CD28.
  • CD80 can be found at GenBank Accession No. P33681.
  • the CD80 protein of the present application may also cover its functionally active fragments, not limited to substances containing functionally active fragments of CD80 produced after processing and/or modification in cells.
  • CD80 of the present application may comprise functionally active fragments of CD80 and other arbitrary structural domains.
  • CD86 generally refers to a cell stimulating molecule.
  • CD86 can be a ligand for CD28.
  • CD86 can be found at GenBank Accession No. P42081.
  • the CD86 protein of the present application may also cover its functionally active fragments, not limited to substances containing functionally active fragments of CD86 produced after processing and/or modification in cells.
  • CD86 of the present application may comprise a functionally active fragment of CD86 and other arbitrary structural domains.
  • secretion generally means that a substance can be localized extracellularly of a cell.
  • secreted substances can be transported to the extracellular space of the cell after intracellular synthesis.
  • whether a substance is secreted can be tested by an enzyme-linked immunosorbent assay or other detection method.
  • T cell receptor generally refers to a complex of membrane proteins involved in the activation of T cells in response to the presentation of an antigen.
  • TCRs may be responsible for recognizing antigens bound to major histocompatibility complex molecules.
  • TCRs can consist of heterodimers of alpha ( ⁇ ) and beta ( ⁇ ) chains, or of gamma and delta ( ⁇ / ⁇ ) chains.
  • TCRs can exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions.
  • a TCR can be a TCR that is modified on any cell that expresses a TCR.
  • the type of TCR can be analyzed by a TCR subtype analysis reagent.
  • the term "clonal diversity” generally refers to a substance having multiple clonotypes.
  • the clonal diversity of TCRs may mean that TCRs may have different sequence structures and/or antigen recognition abilities.
  • the diversity of TCR is often distinguished by ⁇ chain subtypes, which can include V ⁇ 23, V ⁇ 7.2, V ⁇ 5.2, V ⁇ 11, V ⁇ 16, V ⁇ 3, etc. When a T cell population has more ⁇ chain subtypes, it can be This T cell population is thought to have a higher clonal diversity.
  • CD4 + cells generally refers to CD4 positive cells, such as T cells.
  • the terms “CD4 + cells”, “CD4 positive cells” may be used synonymously.
  • These cells can be identified by methods known in the art, such as by staining the cells with a fluorescently labeled antibody directed against CD4 and using fluorescence activated cell sorting.
  • existing data can prove that an increase in the proportion of CD4 + cells can increase the ability of the cell population to secrete IFN- ⁇ and/or TNF, and can improve the effect of T cell populations in promoting tumor suppression.
  • Tay, RE, Richardson, EK et al. 2020. Cancer Gene Therapy, 1-13.
  • this application can provide a method to affect CD4 + cells proportional method.
  • CD8 + cells generally refer to CD8 positive cells, such as T cells.
  • CD8 + cells CD8 positive cells
  • CD8 positive cells may be used synonymously. These cells can be identified by methods known in the art, such as by staining the cells with a fluorescently labeled antibody directed against CD8 and using fluorescence activated cell sorting.
  • IC50 value or “IC50 value” generally refers to the concentration of a target substance required to obtain 50% inhibition of a biological process. IC50 values can be converted to absolute inhibition constants (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol. (1973) 22:3099).
  • KD value or “KD value” generally refers to the dissociation constant, which can be determined by surface plasmon resonance.
  • surface plasmon resonance analysis uses a BIAcore system (Pharmacia Biosensor, Piscataway, NJ) to measure ligands (substances immobilized on a biosensor substrate) and analytes (substances in solution) by surface plasmon resonance (SPR). ) real-time binding interactions.
  • SPR surface plasmon resonance
  • SPR surface plasmon resonance
  • Surface plasmon analysis can also be performed by immobilizing the analyte (substance on the biosensor substrate) and presenting the ligand.
  • the term "encoding” generally refers to the ability to directly or indirectly deduce the structure or composition information of another type of molecule related to it according to basically definite rules.
  • the nucleotide sequence can be deduced from the amino acid sequence, for example, based on the characteristics of deoxyribonucleic acid transcription complementary nucleic acid, including nucleic acid that can be translated into a polypeptide.
  • deoxyribonucleic acid can encode RNA transcribed from deoxyribonucleic acid.
  • a deoxyribonucleic acid may similarly encode a polypeptide translated from RNA transcribed from the deoxyribonucleic acid.
  • the term "immune cell” generally refers to cells involved in carrying out innate and adaptive immune responses.
  • lymphocytes such as T cells (including thymocytes) and B cells
  • natural killer (NK) cells such as T cells (including thymocytes) and B cells
  • NK natural killer
  • NKT cells NKT cells
  • macrophages monocytes, eosinophils, basophils cells, neutrophils, dendritic cells, and mast cells.
  • the modified immune effector cells are T cells, such as CD4+ T cells, CD8+ T cells (also known as cytotoxic T cells or CTLs), regulatory T cells (Treg), Th1 cells, Th2 cells , Th17 cells ⁇ T cells and/or ⁇ T cells.
  • the immune cells of the present application also include immune cells derived from stem cell differentiation.
  • the immune cells of the present application also include immune cells differentiated from pluripotent stem cells.
  • the stem cells of the present application can be obtained by induction.
  • the aforementioned stem cells of the present application may include induced pluripotent stem cells (iPSCs).
  • a CAR generally refers to an engineered antigen receptor.
  • a CAR may comprise an extracellular antigen-binding domain fused via a hinge and transmembrane domain to a cytoplasmic domain comprising a signaling domain.
  • the CAR extracellular domain can bind to an antigen expressed by a target cell in an MHC-independent manner, resulting in activation and proliferation of the cell.
  • the extracellular domain of the CAR can recognize a tag fused to an antibody or antigen-binding fragment thereof.
  • a single CAR construct can be made to target multiple different antigens by substituting one antibody for another.
  • the extracellular domain of the CAR may comprise an antigen-binding fragment derived from an antibody.
  • Antigen binding domains useful in the present disclosure may include, for example, scFvs, antibodies, antigen binding regions of antibodies, variable regions of heavy/light chains, and/or single chain antibodies.
  • T cell receptor generally refers to an engineered antigen receptor.
  • a TCR may comprise a TCR alpha and/or TCR beta chain that has been isolated and cloned from a population of T cells that recognize a particular target antigen.
  • the TCR ⁇ and/or TCR ⁇ genes i.e., TRAC and TRBC
  • TRAC and TRBC can be derived from T cell populations isolated from individuals with specific malignancies or from T cells that have been isolated from humanized mice immunized with specific tumor antigens or tumor cells cloned from the population.
  • Engineered TCRs can recognize antigens by the same mechanism as their endogenous counterparts (e.g., by recognizing their cognate antigens presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of target cells), thereby It can lead to the activation and proliferation of TCR engineered cells.
  • MHC major histocompatibility complex
  • small molecule compound generally refers to peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, molecular weight less than about 10,000 gram/mole of organic or inorganic substances (i.e. including heterogeneous organic and organometallic compounds), organic or inorganic substances of molecular weight less than about 5,000 g/mole, organic or inorganic substances of molecular weight less than about 1,000 g/mole, molecular weight of less than about 500 Gram/mole organic or inorganic substances, as well as salts, esters and other pharmaceutically acceptable forms of such drugs.
  • organic or inorganic substances i.e. including heterogeneous organic and organometallic compounds
  • NK cell is also called “natural killer cell”, and generally refers to a cell with large granules in the cytoplasm.
  • NK cells are developed from bone marrow lymphoid stem cells and can differentiate and develop depending on the bone marrow or thymus microenvironment.
  • the proportion of NK cells in TIL cells can be changed by the method of this application.
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies.
  • the term “antibody” also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specifically bind CD3). Typically, such fragments will include the antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 that can be combined with the J chain to form a multivalent A basic 4-chain unit for combinations.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site.
  • immunoglobulins can be assigned to different classes, or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
  • the term "antigen-binding fragment” generally refers to one or more polypeptide fragments that have the ability to specifically bind an antigen (eg, CD3).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • solid phase medium generally refers to a solid phase material that incorporates a function.
  • a solid phase medium in this application may refer to a material that binds one or more substances in the medium and/or on the surface of the medium through covalent bonding and/or non-covalent bonding.
  • the solid-phase medium of the present application may refer to a medium in which CD28 antibody or its antigen-binding fragment and CD3 antibody or its antigen-binding fragment are bound in the medium and/or on the surface of the medium through covalent binding and/or non-covalent binding.
  • the solid phase medium of the present application may be a polymeric material.
  • the term "expression” generally refers to the process of transcription and/or translation of a gene encoding a target polypeptide within a cell.
  • the level of transcription of a gene encoding a polypeptide of interest in a host cell can be determined by measuring the amount of corresponding mRNA present in the cell. For example, mRNA transcribed from a gene encoding a polypeptide of interest can be quantitatively measured by PCR or by RNA hybridization.
  • the level of translation of a gene encoding a polypeptide of interest can be measured by various methods, such as by ELISA, by a polypeptide bioactivity assay, or by Western blot or radioimmunoassay.
  • expression may generally also refer to the transcription and/or translation process by which the product occurs.
  • expression of a cytokine can be the process by which a cell transcribes and/or translates the cytokine.
  • expression of a cytokine can be determined by detecting the amount of the corresponding mRNA present in the cell or by detecting the amount of the cytokine produced by the cell, or both.
  • stage in the term “one-stage in vitro expansion”, “single-stage in vitro expansion”, or “first-stage in vitro expansion” generally refers to a period of expansion that TIL undergoes in vitro process.
  • each stage can be divided by changes in the number of TIL cells.
  • the number of TIL cells increases by at least about 1-fold, the TIL cells can be considered to have entered the next stage.
  • each stage can also be divided by the conditions of TIL cell culture.
  • T cell activators and/or T cell growth factors when added or supplemented to the cell culture medium, it can be considered that the TIL cells enter the next stage of in vitro expansion. In one embodiment, after the TIL cells are centrifuged and/or washed, the TIL cells can be considered to enter the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the days of TIL cell culture.
  • TIL cells when the TIL cells are cultured in vitro for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days 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, about 20 days, about 30 days, about 40 days, After about 50 days or about 100 days, TIL cells can be considered to enter the next stage of in vitro expansion.
  • the term "the first stage of in vitro expansion” generally refers to the stage of expansion using T cell growth factors after primary TILs are obtained from tissues.
  • the tissue of the present application can be selected from the following group: tumor tissue and pleural effusion, and the pleural effusion of the present application can be the pleural effusion of a patient with metastatic cancer.
  • the amplification of the present application may be autologous or allogeneic in vivo amplification, or may be in vitro amplification.
  • the first stage of in vitro expansion of the present application may also be referred to as preREP (Pre-Rapid Expansion) stage.
  • TILs derived from tumor tissue and not expanded in vitro can be referred to as the first population of TILs.
  • the TILs obtained through the first stage of in vitro expansion in the culture method of the present application divided by the two-step method can be called the second TIL population.
  • the term "second-stage in vitro expansion” generally refers to the stage of expanding again after the tissue taken from the subject is expanded.
  • the number of TIL cells expanded in vitro by the second stage of the present application is increased, for example, can be increased by at least about 10 times (or at least about 20, 30, 40, 50, 60, 70, 80 or 90 fold), or in one embodiment the number of cells may be increased by at least about 100 fold.
  • the culture conditions of the second-stage in vitro expansion may be different from those of the first-stage in vitro expansion, for example, the culture substances added may be different.
  • the second stage of in vitro expansion in the culture method of the present application divided by the two-step method can also be called the REP (Rapid Expansion) stage.
  • the TILs obtained through the second stage of in vitro expansion in the culture method of the present application divided by the two-step method can be called the third TIL population.
  • in vivo generally refers to an event that occurs in the body of a subject.
  • in vitro generally refers to events that occur outside the body of a subject.
  • ex vivo generally refers to an event involving treatment or surgery on cells, tissues and/or organs that have been removed from a subject.
  • the cells, tissues and/or organs can be returned to the body of the subject by surgery or therapy.
  • secretion capacity generally refers to the ability of a cell to express a polypeptide or protein and transfer the polypeptide or protein of the present application to the extracellular environment.
  • irradiation generally refers to the treatment of matter by radiation.
  • irradiating may refer to irradiating a substance with X-rays, ⁇ -rays, ⁇ -rays or ⁇ -rays.
  • engineered cell generally refers to a cell that has been genetically modified by adding additional genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • engineered cells can be genetically modified to express TILs of T cell activators and/or T cell growth factors of the present application.
  • co-culture generally refers to the cultivation of two or more different populations of cells with some degree of contact between them.
  • the "contact" of two or more different populations of cells of the present application may, in one embodiment, be through direct contact, ie, wherein cells of one population are in direct physical contact with cells of another population. Or in one embodiment it may be through indirect contact mediated by a common culture medium.
  • the common culture medium of the present application may contain metabolites produced and released by at least one population of co-cultured cells and be used to culture another population of cells.
  • one or more feeder cells, T cell activators and/or T cell growth factors can be added to the medium of TIL cells by direct contact, for example, one or more feeder cells can be added Culture medium for cells, T cell activators and/or T cell growth factors is added to and/or replaces the medium for TIL cells, e.g., one or more feeder cells, T cell activators and/or T cell growth factors may be added to The culture medium of factors is used for culturing TIL cells; in one embodiment, it can be used for culturing TIL cells through indirect contact, for example, metabolites produced and released by feeder cells.
  • the term "mixture” generally refers to a combination of two or more different substances.
  • the CD28 antibody or antigen-binding fragment thereof of the present application and the CD3 antibody or antigen-binding fragment thereof can be added to the cell culture medium as a mixture after mixing.
  • the terms “concurrent contact”, “co-contact”, “simultaneously with”, “simultaneously” and “commonly” generally refer to the administration of two or more substances to a subject and/or cell such that the substances Also present in the subject and/or in the environment of the cell culture.
  • Simultaneous contacting can include simultaneous administration in different compositions, administration in different compositions at different times, or administration in a composition in which two or more active pharmaceutical ingredients are present.
  • “simultaneous contacting” in this application may generally refer to substantially simultaneous contacting.
  • the term “expansion” generally refers to a several-fold increase in the number of cells over a period of time.
  • the number of cells can be increased by at least about 3-fold (or 4, 5, 6, 7, 8 or 9-fold), in one embodiment the number of cells can be increased by at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80 or 90 fold), or in one embodiment the number of cells may be increased by at least about 100 fold.
  • the term “expanded” generally means that the cells of the present application have undergone one or more expansions as described above.
  • polymer generally refers to a molecule consisting of individual chemical moieties linked together, the polymer moieties herein being the same or different.
  • the term “polymer” may refer to individual chemical moieties joined end to end to form a linear molecule, as well as individual chemical moieties linked together in branched (eg, "multi-armed” or "star") structures .
  • the polymer may include, for example, a polysaccharide, dextran, hydrogel, polyethylene glycol, or poloxamer.
  • Poloxamers are nonionic triblock copolymers having a polyoxypropylene (poly(propylene oxide)) central hydrophobic chain flanked by two polyoxyethylene (poly(ethylene oxide)) hydrophilic chains.
  • the materials encompassed herein may be formulated with, or administered with, any polymer described herein or known in the art.
  • chimeric antibody generally refers to an antibody in which the variable region of a murine antibody is fused with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • a chimeric antibody you can establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and clone the constant region gene of the human antibody as needed, and combine the mouse variable region gene with the Human constant region genes are connected into chimeric genes and then inserted into expression vectors to express chimeric antibody molecules in eukaryotic or prokaryotic systems.
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) usually refers to the antibody variable region framework grafted to the human antibody CDR sequence, that is, different Types of antibodies produced in the framework sequences of human germline antibodies. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database.
  • the term "fully human antibody”, “fully human antibody” or “fully human antibody”, also known as “fully human monoclonal antibody”, the variable region and constant region of the antibody can be human Source, remove immunogenicity and toxic side effects.
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the antibody or ligand described in this application may be a fully human monoclonal antibody.
  • the relevant technologies for the preparation of fully human antibodies can be: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.
  • CDR generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • One of the most commonly used definitions of the 6 CDRs can be given by Kabat E.A. et al., Chothia et al. and MacCallum et al.
  • the Kabat definition of CDR can be applied to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and the heavy chain variable domain CDR1, CDR2 and CDR3 (CDR H1, CDR H2, CDR H3 or H1, H2, H3).
  • anti-CD3 antibody generally refers to CD3-targeting antibodies or variants thereof, such as monoclonal antibodies, including human, humanized, chimeric or murine antibodies, which are directed against mature T-cell T cells CD3 receptor among antigen receptors.
  • Anti-CD3 antibodies can include OKT3.
  • Anti-CD3 antibodies can include SP34.
  • Anti-CD3 antibodies can also include other anti-CD3 antibodies including, for example, otelixizumab, teplizumab, and visilizumab in one embodiment.
  • IL-2 or "IL2” generally refers to the T-cell growth factor known as interleukin 2, and includes all forms of IL-2, which can include in one embodiment humans and mammals forms, conservative amino acid substitutions, glycoform modifications or variants, or active fragments thereof.
  • the GeneID of the gene encoding IL-2 may be 3558.
  • the term "antigen presenting cell”, “antigen presenting cell”, or “APC” generally refers to an immune cell displaying on its surface exogenous antigen complexed with major histocompatibility complex (MHC).
  • Systemic cells such as accessory cells (eg, B cells, dendritic cells, etc.).
  • T cells can recognize these complexes using their T cell receptor (TCR).
  • APCs can process antigens and present them to T cells.
  • the antigen presenting cells may comprise a group selected from peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.
  • TIL characteristics generally refers to the characteristics of TIL cells obtained through the cultivation method of this application. Changes in TIL properties can include: increased number of TIL cells, increased proportion of viable cells, increased viability, improved proportion of T cell subsets, increased cytokine secretion, increased tumor cell killing capacity, increased T cell Receptor (TCR) clonal diversity and increased number of TIL cells in tissue, or any combination thereof. The changes in this application can be increased or decreased.
  • an increase in the viability of TIL cells can refer to an increase in the time that TIL cells exist in vivo.
  • increased viability can refer to an increase in the time a cell exists in a tissue of a subject, such as a tumor, spleen, bone marrow, lung tissue, and blood.
  • increased viability can be an increase in the viability of TIL cells after IL-2 is withdrawn from the culture medium.
  • nanoparticle generally refers to microscopic particles having at least one dimension smaller than 100 nm.
  • nanoparticles have a diameter in the range of 50 nm to 500 nm (ie, 0.05 ⁇ m to 0.5 ⁇ m); are structurally stable in physiological environments; and can accommodate smaller molecules (such as drugs or other bioactive agents), which can then be Deliver to desired site.
  • a nanoparticle of the present application may comprise a CD28 antibody or an antigen-binding fragment thereof.
  • a nanoparticle of the present application may comprise a CD28 antibody or an antigen-binding fragment thereof and a CD3 antibody or an antigen-binding fragment thereof.
  • an anti-CD3 antibody can include OKT3.
  • an anti-CD28 antibody can include 15E8.
  • artificial antigen-presenting cell generally refers to the artificially constructed immune cells used to present foreign antigens, for example, the way of presenting foreign antigens can be that the surface of artificial antigen-presenting cells contains foreign Histocompatibility complex (MHC) complex.
  • MHC foreign Histocompatibility complex
  • isolated artificial antigen-presenting cells may be included, which may contain genes expressing HLA-A/B/C (the gene GeneID encoding it may be 3105, 3106, or 3107), CD64 (the gene encoding it GeneID can be 2209), CD80 (the gene GeneID encoding it can be 941), ICOS-L (the gene GeneID encoding it can be 23308) and CD58 (the gene GeneID encoding it can be 965), and can be modified To express more than one T cell activator, the above of the present application may contain this number.
  • fusion protein generally refers to an amino acid sequence comprising a first polypeptide or protein or a fragment, analog or derivative thereof and a heterologous polypeptide or protein (i.e., different from the first polypeptide or protein or the amino acid sequence of a second polypeptide or protein, or a fragment, analog or derivative thereof, or generally not part of the first polypeptide or protein, or a fragment, analog or derivative thereof) of a fragment, analog or derivative thereof peptide or protein.
  • a fusion protein may comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide.
  • the heterologous protein, polypeptide or peptide of the present application may or may not be different types of preventive or therapeutic drugs.
  • two different proteins, polypeptides or peptides with immunomodulatory activity can be fused together to form a fusion protein.
  • the fusion protein may retain or increase activity compared to the activity of the original polypeptide or protein prior to fusion of the heterologous protein, polypeptide or protein.
  • the fusion protein of the present application may be a fusion protein fused with a CD28 antibody or an antigen-binding fragment thereof and a CD3 antibody or an antigen-binding fragment thereof.
  • the term "killing ability" generally means that the target cells are killed by contacting the cells of the present application with an effective amount of substances.
  • the substance of the present application may be TIL cells. Killing in the present application may include killing cells by itself or promoting CDC, apoptosis, ADCC, and/or phagocytosis of other cells or substances, or by a combination of two or more of these mechanisms.
  • administering generally refers to delivering a substance to a subject in need thereof by any route known in the art.
  • Pharmaceutically acceptable carriers and formulations or compositions are also well known in the art. Routes of administration may include: intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral and/or mucosal.
  • kit generally refers to two or more components packaged together in a container, receptacle or other container, one of which corresponds to the substance of the present application.
  • TIL cells of the present application are included.
  • the term "subject” generally refers to a cell or an animal, which can be a mammal such as a human, a non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), a domestic animal (dog and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs) and laboratory animals (mice, rats, rabbits, guinea pigs).
  • Human subjects include fetal, neonatal, infant, adolescent and adult subjects.
  • Subjects include animal disease models, such as tumor animal models, and other animal models known to those skilled in the art.
  • feeder generally refers to a cultured cell that can be used to support the growth of another cell of interest in culture. For example, by growing in vitro and secreting at least one factor into the culture medium.
  • feeder cells may include antigen presenting cells.
  • the term "specific binding” generally refers to a binding substance that recognizes a specific target substance, but does not substantially recognize or bind to other molecules in a sample.
  • a binding substance can specifically bind the specific target substance of the application from one species
  • the binding substance of the present application can also specifically bind the target substance of the application from one or more other species or homologous target substances. This cross-species reactivity by itself may not alter the classification of the binding substance as specific.
  • a binding substance that specifically binds to a target substance may also bind to a different allelic form of the target substance.
  • complete culture process generally refers to the complete process of separating cells from tumor tissues isolated from patients, expanding them once or more, and finally obtaining cells that can be administered to subjects .
  • cell culture medium generally refers to a nutrient solution in which cells, such as mammalian cells, are grown.
  • the formulation of cell culture media is well known in the art.
  • cell culture media include buffers, salts, carbohydrates, amino acids, vitamins and necessary trace elements.
  • Cell culture media may or may not contain serum, peptone, and/or protein.
  • Cell culture media can be supplemented with additional components or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, etc., depending on the cells to be cultured requirements and/or desired cell culture parameters.
  • the term "pharmaceutical composition” or “pharmaceutical preparation” generally refers to a preparation, which may allow the biological activity of the active ingredient to be effective, and may not contain any substances that are harmful to the subject to which the preparation will be administered.
  • the tester was unacceptably toxic for additional components.
  • Such preparations are sterile.
  • “Pharmaceutically acceptable” excipients carriers, additives are those which can reasonably be administered to a subject mammal to provide an effective dosage of the active ingredient employed.
  • TIL tumor infiltrating lymphocytes
  • TILs may include, but are not limited to, CD8 + cytotoxic T cells (lymphocytes), Th1 and Th17CD4 + T cells, natural killer cells, dendritic cells, and M1 macrophages.
  • TILs can include primary TILs and secondary TILs.
  • Primary TILs can be those TIL cells obtained from a tissue sample of a subject, and "secondary TILs" can be any population of TILs that have been or have been expanded in this application.
  • the tumor-infiltrating lymphocytes of the present application may not be isolated and purified, or may infiltrate with tumor cells.
  • TIL in the present application may refer to a group of TILs.
  • central memory T cells generally refers to T cells that have long-term memory and are capable of receiving antigen restimulation.
  • Central memory T cells may have a phenotype of CD45RA ⁇ CCR7 + , for example, central memory T cells may be identified by CD45RA ⁇ and CCR7 + .
  • Central memory T cells can have a stronger ability to resist tumor growth than ordinary T cells.
  • regulatory T cells generally refers to a subset of T cells that control autoimmune reactivity in vivo. Regulatory T cells may have a phenotype of CD4 + CD25 + Foxp3 + , for example, regulatory T cells may be identified by CD4 + , CD25 + and Foxp3 + . Regulatory T cells may have the ability to suppress the anti-tumor growth of T cells.
  • activated T cells generally refers to T cells that have been activated to have the ability to resist tumor growth.
  • Activated T cells can have a phenotype of PD-1 + (PD1 + ), LAG-3 + (LAG3 + ) or CD28 + , for example, activated T cells can be identified by PD-1 + , LAG-3 + or CD28 + .
  • Activated T cells may have the ability to fight tumor growth.
  • tumor-specific T cells generally refers to T cells that can specifically fight tumor growth.
  • Tumor-specific T cells may have a phenotype of CD103 + CD39 + , for example, tumor-specific T cells may be identified by CD103 + and CD39 + .
  • Tumor-specific T cells can have a more specific ability to resist tumor growth than ordinary T cells.
  • stem cell-like T cell generally refers to a type of T cell that may have the potential of self-proliferation and/or differentiation.
  • Stem cell-like T cells may have a TCF1 + phenotype, for example, stem cell-like T cells may be identified by TCF1 + .
  • Tumor-specific T cells may have stronger and/or longer-term anti-tumor growth capabilities than ordinary T cells.
  • tumor fragments generally refers to tumor fragments that can be formed by mechanical disruption, enzymatic hydrolysis and/or other disruption methods after the tumor tissue is removed from the subject.
  • composition or “pharmaceutical composition” generally refers to at least one cell and at least one and optionally more than one other pharmaceutically acceptable chemical components such as carrier, stabilizer , diluents, dispersants, suspending agents, thickeners and/or mixtures of excipients.
  • the term "pharmaceutically acceptable carrier” generally refers to one or more non-toxic materials that do not interfere with the active ingredient.
  • a pharmaceutically acceptable carrier may not interfere with the biological activity of the active ingredient; for example, a pharmaceutically acceptable carrier may not interfere with the effectiveness of the biological activity possessed by the active ingredient.
  • Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for human administration.
  • contemplated carriers, excipients, and/or additives may include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids , protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), etc.
  • pharmaceutically acceptable carrier carrier
  • carrier can be understood as a carrier (vector) that does not include the nucleic acid form used in genetic engineering.
  • the term "functionally active fragment” generally refers to a fragment that has a partial region of a full-length protein or nucleic acid, but retains or partially retains the biological activity or function of the full-length protein or nucleic acid.
  • a functionally active fragment may retain or partially retain the ability of the full-length protein to bind another molecule.
  • the functionally active fragment of the growth factor IL-2 may retain or partially retain the biologically active function of the full-length IL-2 to cause cell proliferation.
  • T cell activator generally refers to a substance that binds to the corresponding binding receptor on T cells and mediates costimulatory responses of T cells.
  • T cell activators may be substances other than antigen receptors that are required for T cells to mount an effective immune response.
  • a T cell activator may refer to a T cell co-stimulatory molecule.
  • the T cell activator of the present application may comprise its variant, homologue or any substance comprising its functionally active fragment.
  • T cell activators may include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), NK cell activating receptors, BTLA (the gene GeneID encoding it can be 151888), Toll ligand receptor, OX40 (the gene GeneID encoding it can be 7293), CD2 (the gene GeneID encoding it can be 914), CD7 (the gene GeneID encoding it can be 914), CD7 (the gene GeneID encoding it can be 924), CD27 (the gene GeneID encoding it can be 939), CD28 (the gene GeneID encoding it can be 940), CD30 (the gene GeneID encoding it can be 943), CD40 (the gene GeneID encoding it can be 958 ), CDS, ICAM-1 (the gene GeneID encoding it can be 3383), LFA-1 (CD11a/CD18) (the gene
  • T cell growth factor generally refers to a biologically active polypeptide or small molecule compound that causes cell proliferation.
  • the T cell growth factor of the present application may comprise its variant, homologue or any substance comprising its functionally active fragment.
  • the T cell growth factor can be selected from one or more of the following groups: IL-2 (the gene GeneID encoding it can be 3558), IL-4 (the gene GeneID encoding it can be 3565) , IL-6 (the gene GeneID encoding it can be 3569), IL-7 (the gene GeneID encoding it can be 3574), IL-10 (the gene GeneID encoding it can be 3586), IL-12 (the gene GeneID encoding it can be 3586), IL-12 (the gene GeneID encoding it can be 3586), Gene GeneID can be 3592 or 3593), IL-15 (the gene GeneID encoding it can be 3600), IL-21 (the gene GeneID encoding it can be 59067), TNF- ⁇ (the gene GeneID encoding it can be 100137091) , gamma interferon (the gene GeneID encoding it can be 3458) and the like.
  • IL-2 the gene GeneID encoding it can be 3558
  • IL-4 the gene
  • substantially simultaneously usually means that TIL can be contacted with two or more substances at the same time during a period of time in the contact process, but it is not limited to that TIL is always in contact with two or more substances at the same time during the entire contact process. touch.
  • substantially simultaneously can mean that TIL can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% of each of the two or more substances are in contact simultaneously.
  • solid phase medium generally refers to a solid phase material having a binding function.
  • a solid phase medium in this application may refer to a material that binds one or more substances in the medium and/or on the surface of the medium through covalent bonding and/or non-covalent bonding.
  • the solid phase media of the present application can be combined with one or more T cell activators.
  • the solid-phase medium of the present application may refer to a medium in which CD28 antibody or its antigen-binding fragment and CD3 antibody or its antigen-binding fragment are bound in the medium and/or on the surface of the medium through covalent binding and/or non-covalent binding. Material.
  • the solid phase medium of the present application may be microspheres comprising the OKT3 antibody and the 15E8 antibody with a diameter of about 500 nanometers to about 10 micrometers.
  • the solid phase medium of the present application may be a polymeric material.
  • the solid phase media of the present application can be microspheres having a diameter of at least about 500 nanometers.
  • the solid phase medium of the present application may be a nanomatrix.
  • the solid phase medium of the present application may be a nanomatrix comprising the OKT3 antibody and the 15E8 antibody with a diameter of about 1 nanometer to about 500 nanometers.
  • the term "nanomatrix" generally refers to a material with a diameter ranging from about 1 nanometer to about 500 nanometers.
  • the nanomatrix can have a binding function, for example, the nanomatrix of the present application can be combined with one or more T cell activators.
  • the nanomatrix may comprise a polymer, for example, the nanomatrix of the present application may comprise a degradable polymer.
  • the nanomatrix may comprise polysaccharides, and/or dextran.
  • dendritic cell generally refers to an antigen-presenting cell present in vivo, in vitro, ex vivo or within a host or subject, or which may be derived from hematopoietic stem cells or monocytes.
  • Dendritic cells and their precursors can be isolated from various lymphoid organs such as spleen, lymph nodes as well as bone marrow and peripheral blood.
  • the dendritic cells of the present application may have characteristic morphology such as thin layers (lamellipodia) extending in multiple directions from the dendritic cell body.
  • dendritic cells can express high levels of MHC and co-stimulatory (eg, B7-1 and B7-2) molecules.
  • Dendritic cells can induce antigen-specific differentiation of T cells in vitro and can elicit primary T cell responses in vitro and in vivo.
  • in vitro expansion generally refers to cultured to produce changes in the number of cells, expanded cells may also produce changes in the number and/or ratio of cells, changes in secretion capacity, changes in killing capacity or expression of Changes in capabilities, or any combination of them.
  • the changes in this application can be increased or decreased.
  • in vitro expansion may be for the purpose of expansion; in order to detect the function of TIL cells, such as detecting the ability of TIL cells to release cytokines, the operation steps performed on TIL cells (such as adding a One or more substances to detect the ability of TIL cells to release cytokines), may not belong to the in vitro expansion of this application.
  • peripheral mononuclear cells or “peripheral blood mononuclear cells” generally refer to cells in peripheral blood having a single nucleus.
  • the peripheral blood mononuclear cells of the present application may include lymphocytes, monocytes and/or dendritic cells.
  • cytokine generally refers to a protein released by a population of cells that acts as an intercellular regulator of another cell.
  • the cytokines of the present application may be lymphokines, monokines and polypeptide hormones.
  • the cytokines of the present application may include interleukins (ILs) such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-15, IL-21, and/or IL-12.
  • ILs interleukins
  • the term cytokine may include proteins from natural sources or from recombinant cell culture, biologically active equivalents of native sequence cytokines, and functionally active fragments thereof.
  • the term “diameter” generally refers to the diameter of a cross-section of a substance of the present application.
  • the term “diameter” generally refers to the maximum diameter and/or average diameter of the largest cross-section of the material of the present application.
  • the method for determining the diameter of a substance may be a method commonly used in the art, such as transmission electron microscopy.
  • the term "neoplastic” generally refers to any new pathological growth of tissue.
  • the tumors of this application may be benign or malignant.
  • the tumors of this application may be solid or hematological.
  • the term “tumor” may be selected from one or more of the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer .
  • tumor tissue generally refers to a sample of any tissue from a tumor in a subject, including any solid tumor and/or non-solid tumor in a subject.
  • CD28 agonist generally refers to a compound that binds to the cell surface CD28 protein and elicits a response in the cell.
  • a CD28 agonist of the present application may be a small molecule agent that binds CD28.
  • the CD28 agonist of the present application can be an antibody or antigen-binding fragment thereof that binds CD28.
  • the term "proportion of T cell subsets" generally refers to the proportion of different T cell subsets in TIL cells or TIL populations.
  • different T cell subsets of the present application have different immunological activity and/or differentiation ability.
  • T cell subsets of the present application can be differentiated based on T cell surface markers.
  • central memory T cells can have a CD45RA - CCR7 + phenotype.
  • regulatory T cells can have a phenotype of CD4 + CD25 + Foxp3 + .
  • activated T cells can have a phenotype of CD25 + , CD28 + , PD-1 + , or 41BB + .
  • tumor-specific T cells can have a CD103 + CD39 + phenotype.
  • stem-like T cells can have a TCF1 + phenotype.
  • the term "number of TIL cells” generally refers to the number of cells in the TIL cells of the present application.
  • the number of TIL cells may refer to the number of cells in the TIL population obtained at any stage of the present application.
  • the number of TIL cells can refer to the number of cells of the first TIL population derived from tumor tissue and not expanded in vitro.
  • the number of TIL cells can refer to the number of cells of the second TIL population expanded in vitro by the first stage.
  • the number of TIL cells can refer to the number of cells of the third TIL population expanded in vitro by the second stage.
  • the number of TIL cells may refer to the TIL cells finally obtained by any of the culturing methods in the present application.
  • the number of TIL cells can be measured by methods commonly used in the art, for example, including but not limited to manual cell counting on a cell counting board and/or automatic cell counter counting.
  • the terms “about” and “approximately” generally mean within a statistically meaningful range of values. Such ranges may be within an order of magnitude of a given value or range, may be within 50%, may be within 20%, may be within 10%, may be within 5%.
  • the permissible variations encompassed by the term “about” or “approximately” may depend on the particular system under study and are readily understood by those of ordinary skill in the art.
  • the terms “above”, “below”, “at most” and “at least” may be inclusive of numbers.
  • the present application provides a method for culturing immune cells, which reduces the expression and/or activity of YEATS domain protein family members and/or functionally active fragments thereof of the immune cells.
  • the member of the YEATS domain protein family can comprise a YEATS domain.
  • the YEATS domain protein family member may comprise AF9 and/or ENL.
  • the target gene of the present application may be a gene encoding a member of the YEATS domain protein family and/or a functionally active fragment thereof.
  • an immune cell obtained by reducing the expression and/or attenuating the activity of at least one target gene of the immune cell may exhibit improved immune cell properties compared to an immune cell whose expression and/or activity of the target gene is not altered .
  • the immune cells whose expression and/or activity of the target gene have not been changed may refer to immune cells derived from the same donor and which have not reduced the expression and/or activity of at least one target gene of the immune cells immune cells.
  • the immune cells whose expression and/or activity of the target gene have not been changed can refer to the immune cells derived from the same donor and which have not made other genes other than the target gene of the immune cells (for example, knocking out the other gene). , have substantially no effect on cell function) immune cells with reduced expression and/or weakened activity.
  • the corresponding immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may refer to those isolated from the same donor in the same way and that have not reduced the expression of the at least one target gene of the immune cells. Immune cells with reduced expression and/or attenuated activity of at least one target gene. In one embodiment, the corresponding immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may refer to the same tumor origin from the same donor and have not reduced the immune cells. Immune cells with reduced expression and/or reduced activity of at least one gene of interest.
  • the corresponding immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may refer to the same tumor source from the same donor and isolated in the same way.
  • the immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may refer to dividing immune cells derived from the same donor into two groups, one of which is
  • the immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may be corresponding immune cells that have not reduced the expression and/or weakened the activity of the at least one target gene of the immune cells .
  • the corresponding immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may refer to dividing immune cells from the same donor and separated in the same way into Two groups, wherein a group of immune cells that have not reduced the expression and/or activity of at least one target gene of the immune cells may be those that have not reduced the expression and/or activity of at least one target gene of the immune cells Weakened corresponding immune cells.
  • the corresponding immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may refer to dividing the immune cells of the same tumor origin from the same donor into two A group, wherein a group of immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may be those that have not reduced the expression and/or weakened the activity of the at least one target gene of the immune cells corresponding immune cells.
  • the corresponding immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may refer to those isolated from the same tumor source from the same donor in the same way.
  • the immune cells are divided into two groups, wherein a group of immune cells that have not reduced the expression and/or weakened the activity of at least one target gene of the immune cells may be those that have not reduced the expression of at least one target gene of the immune cells and/or corresponding immune cells with reduced activity.
  • the reduced expression and/or weakened activity of at least one target gene may mean that the target gene of the natural cell is in a certain degree of expression state, and after the treatment of the present application, the expression level of the target gene of the cell can be reduced. Decreasing, that is, reducing the expression level of the target gene can be to make the natural cell change from expressing the target gene to substantially not expressing the target gene or expressing a decrease in the amount of the target gene.
  • the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and/or basophils.
  • the immune cells comprise monocytes, macrophages and/or dendritic cells.
  • the immune cells of the present application also include immune cells derived from stem cell differentiation.
  • the immune cells of the present application also include immune cells differentiated from pluripotent stem cells.
  • the stem cells of the present application can be obtained by induction.
  • the aforementioned stem cells of the present application may include induced pluripotent stem cells (iPSCs).
  • the immune cells comprise B cells, T cells, natural killer cells and/or natural killer-like T cells (NKT).
  • an "unmodified immune cell” or “unmodified immune cell” can refer to one in which the genome has not been modified and contains no gene regulation system or contains a control gene regulation system (e.g., empty vector control, non-targeting gRNA, plus scrambling siRNA, etc.) cells or cell populations.
  • the immune cells comprise ⁇ T cells and/or ⁇ T cells.
  • the immune cells comprise tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • the TILs are TILs derived from fragments of tumor tissue, pleural effusion and/or peritoneal effusion and/or TILs derived from thawed after cryopreservation.
  • the TILs of the present application may be TILs derived from fragments of tumor tissue, pleural effusion and/or peritoneal effusion and/or TILs derived from resuscitation after cryopreservation.
  • TILs of the present application can be obtained by processing tumor tissue into tumor fragments.
  • the tumor fragments of the present application have a volume of about 1-27 cubic millimeters.
  • the tumor fragments of the present application have a volume of about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters, about 8 cubic millimeters , about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 15 cubic millimeters, about 17 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters, about 21 cubic millimeters , about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters, or about 27 cubic millimeters.
  • the immune cells comprise engineered immune receptors displayed on the cell surface.
  • the engineered immune receptor specifically binds to an antigen expressed on a target cell.
  • the immune cells comprise chimeric antigen receptors and/or T cell receptors.
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: reducing the expression and/or activity of members of the YEATS domain-containing protein family and/or functionally active fragments thereof in the TIL.
  • TIL tumor-infiltrating lymphocytes
  • TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, Decrease the expression and/or activity of the members of the protein family comprising the YEATS domain and/or their functionally active fragments in the TIL.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage in vitro expansion and the second stage in vitro expansion, and in the first stage of the present application
  • the expression and/or activity of the YEATS domain protein family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage in vitro expansion and the second stage in vitro expansion, and in the first stage of the present application
  • the expression and/or activity of the YEATS domain protein family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage in vitro expansion and the second stage in vitro expansion, and in the first stage of the present application
  • the expression and/or activity of the YEATS domain protein family members and/or their functionally active fragments in the TIL can be reduced, and in the second-stage in vitro expansion of the present application, all The expression and/or activity of members of the YEATS domain protein family and/or functionally active fragments thereof in the TILs are reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the first stage of in vitro amplification of the present application, the expression and/or activity of the YEATS domain protein family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the second stage of in vitro amplification of the present application, the expression and/or activity of the YEATS domain protein family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the third stage of in vitro amplification of the present application, the expression and/or activity of the YEATS domain protein family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the first stage of in vitro amplification of the application, the expression and/or activity of the YEATS domain protein family members and/or functionally active fragments thereof in the TIL can be reduced, and in the second stage of the application in vitro During the amplification, the expression and/or activity of the YEATS domain protein family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the first stage of in vitro amplification of the present application, the expression and/or activity of the YEATS domain protein family members and/or functionally active fragments thereof in the TIL can be reduced, and in the third stage of the present application in vitro During the amplification, the expression and/or activity of the YEATS domain protein family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the second stage of in vitro amplification of the present application, the expression and/or activity of members of the YEATS domain protein family and/or functionally active fragments thereof in the TIL can be reduced, and in the third stage of the present application in vitro During the amplification, the expression and/or activity of the YEATS domain protein family members and/or their functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the first stage of in vitro amplification of the application, the expression and/or activity of the YEATS domain protein family members and/or functionally active fragments thereof in the TIL can be reduced, and in the second stage of the application in vitro During the expansion, the expression and/or activity of the YEATS domain protein family member and/or its functionally active fragment in the TIL can be reduced, and in the third stage of in vitro expansion of the present application, the expression and/or activity of the TIL in the TIL can be reduced. Reduced expression and/or activity of a YEATS domain protein and/or a functionally active fragment thereof.
  • each stage of in vitro expansion can be divided by the change in the number of TIL cells.
  • the number of TIL cells increases by at least about 1-fold, it can be considered that the TIL cells have entered the next stage of in vitro expansion.
  • the number of TIL cells is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times, or at least about 1000 times, TIL cells can be considered to enter the next stage of in vitro expansion.
  • each stage of in vitro expansion can also be divided by changing the conditions of TIL cell culture.
  • TIL cells when cell activators and/or cell growth factors are added or supplemented to the cell culture medium, TIL cells can be considered to enter the next stage of in vitro expansion.
  • TIL cells can be considered to enter the next stage of in vitro expansion when IL-2 is added or supplemented to the cell culture medium.
  • IL-2 is added or supplemented to the cell culture medium.
  • it can be considered that TIL cells enter the next stage of in vitro expansion.
  • feeder cells are added or supplemented to the cell culture medium
  • TIL cells can be considered to enter the next stage of in vitro expansion.
  • TIL cells when TIL cells have undergone centrifugation and/or cell washing operations, it can be considered that TIL cells have entered the next stage of in vitro expansion.
  • each stage can also be divided by the days of TIL cell culture.
  • TIL cells when TIL cells are cultured in vitro for about 1 day, about 2 days, 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, about 20 days, about 30 days, about 40 days, about 50 days or about After 100 days, TIL cells can be considered to enter the next stage of in vitro expansion.
  • an immune cell obtained by reducing the expression and/or attenuating the activity of a member of the YEATS domain protein family exhibits improved cellular properties compared to an immune cell in which the expression and/or activity of the member of the YEATS domain protein family is not altered .
  • the improved cell properties comprise one or more selected from the group consisting of improved cell proliferation, increased proportion of viable cells, improved proportion of cell subsets, increased cytokine secretion and increased tumor cell Lethality.
  • the improved proportion of cell subpopulations comprises one or more selected from the group consisting of increased proportion of activated cells, decreased proportion of regulatory cells, decreased proportion of exhausted cells, increased central memory cells and/or Proportion of naive cells, increased proportion of naive cells, decreased proportion of apoptotic cells and increased proportion of stem-like cells.
  • the number of immune cells improved in the present application refers to the immune cells whose expression and/or activity of the YEATS domain protein family members are not changed.
  • the number of immune cells of the present application with reduced expression and/or reduced activity can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • an increased proportion of viable cells can be manifested as an increase in immune cell viability.
  • the proportion of living cells of the present application may be increased compared with immune cells whose expression and/or activity of members of the YEATS domain protein family have not changed, and the YEATS domain protein family has been reduced in at least one in vitro expansion stage.
  • the proportion of viable cells of the immune cells of the present application with reduced expression and/or attenuated activity of the member may be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, At least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the increased cytokine secretion ability of the present application may refer to the increase of cytokine secretion ability of immune cells selected from the following group: IL-2, IL-6, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ .
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of cytokine-secreting cells in immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least About 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of cytokine-secreting cells in the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least About 3%, at least about 2%, at least about 1%
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of cells secreting CD107a in the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of cells secreting CD107a in the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of IL-2-secreting cells in the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, At least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, At least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of IL-2-secreting cells in the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, At least about 3%, at least about 2%, at least about 1%,
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of IL-6-secreting cells in the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, At least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, At least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of IL-6-secreting cells in the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, At least about 3%, at least about 2%, at least about 1%,
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of cells that secrete IFN (for example, the IFN of the present application can be IFN- ⁇ ) in the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold , at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times , at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of IFN-secreting cells in the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of cells that secrete TNF (for example, the TNF of the present application can be TNF- ⁇ ) in the immune cells of the present application with reduced expression and/or activity weakening of family members can be increased by at least about 1 times, at least about 2 times, at least about 3 times , at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times , at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of TNF-secreting cells in the immune cells of the present application with reduced expression and/or attenuated activity of family members can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of cells secreting GZMB in immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one stage of in vitro expansion
  • the proportion of cells secreting GZMB in the immune cells of the present application with reduced expression and/or attenuated activity of family members can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about about
  • the determination of the cytokine secreting ability of the immune cells of the present application may be by measuring the cytokine expressing ability of the immune cells.
  • the cytokine secreting ability of the immune cells of the present application can be determined by the method of cell flow cytometry.
  • the cytokine secreting ability of the immune cells of the present application is determined by measuring the cytokine releasing ability of the immune cells.
  • the cytokine secreting ability of the immune cells of the present application is determined by flow cytometry or CBA (Cytometric Bead Array).
  • the improved tumor cell killing ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one in vitro expansion stage
  • the tumor cell killing rate of the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the improved tumor cell killing ability of the present application may refer to the immune cells whose expression and/or activity of the members of the YEATS domain protein family are not changed, making the YEATS domain protein in at least one in vitro expansion stage
  • the tumor cell killing rate of the immune cells of the present application with reduced expression and/or reduced activity of family members can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50% %, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13% %, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3 %, at least about 2%, at least about 1%, at least about 0.5%
  • the tumor cell killing rate of immune cells of the present application can be measured by IncuCyte system or CFSE and DAPI staining.
  • the tumor cell killing of immune cells in the present application may refer to the ability of immune cells to kill solid tumor cells.
  • the improved cell subpopulation ratio of the present application may comprise one or more selected from the following group: increased CD8 + cell ratio, increased central memory cell and/or naive cell ratio, decreased regulatory cell ratio , increased proportion of activated cells, increased proportion of tumor-specific cells, and increased proportion of stem-like cells.
  • the increased proportion of CD8 + cells of the present application may be an increase in the proportion of CD8 positive cells among immune cells.
  • the proportion of CD8 + cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the increased proportion of activated cells of the present application may be an increase in the proportion of CD28 + , CD25 + , and/or 41BB + cells among immune cells.
  • the proportion of activated cells in immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% , at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% , at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1% , at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or may be increased
  • the proportion of CD28 + cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the proportion of CD25 + cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the proportion of 41BB + cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the reduced proportion of exhausted cells of the present application can be an increase in the proportion of PD-1 + , LAG-3 + , TIM-3 + , and/or CD39 + cells among immune cells.
  • the proportion of exhausted cells in immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% , at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% , at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1% , at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.
  • the proportion of PD-1 + cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about
  • the proportion of LAG-3 + cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about
  • the proportion of TIM-3 + cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about
  • the proportion of CD39 + cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the reduced proportion of regulatory cells of the present application may be a reduction in the proportion of CD4 + CD25 + Foxp3 + cells among immune cells.
  • the proportion of regulatory cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the reduced proportion of apoptotic cells of the present application may be a decrease in the proportion of CD95 + caspass3 + cells and/or CD95 + DR5 + cells in immune cells.
  • the proportion of apoptotic cells in immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%,
  • the increased proportion of stem cells of the present application may be an increase in the proportion of CD69 ⁇ CD39 ⁇ cells and/or TCF1 + cells among immune cells.
  • the proportion of cells with stemness among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least About 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the increased proportion of central memory cells of the present application may be an increase in the proportion of CD45RA ⁇ CCR7 + or CD45RO + CD62L + cells among immune cells.
  • the proportion of central memory cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • an increased proportion of naive T cells of the present application may be an increase in the proportion of CD45RO ⁇ CD62L + cells among immune cells.
  • the proportion of naive cells in immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% , at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% , at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1% , at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the culturing method of the present application may include a gene editing step for cells.
  • a gene editing step for cells comprises: subjecting the immune cells to at least one stage of in vitro expansion, wherein, in the at least one stage of the in vitro expansion, a gene regulation system can be introduced into the immune cells.
  • the gene regulation system can disrupt the target gene at the DNA level.
  • the gene regulation system can disrupt the region of the gene of interest or a fragment thereof in the genome of the immune cell.
  • the DNA region or its fragment where the target gene is located in the immune cell is cleaved and the expression ability of the target gene is reduced or the activity of the target gene is inhibited.
  • the editing effect of the gene regulation system on the target gene can be long-term and sustained.
  • the gene regulation system may comprise a guide nucleic acid molecule and an enzyme protein.
  • the enzyme protein may have nucleic acid scissor activity, and the guide nucleic acid molecule may guide the enzyme protein to specifically cut the region where the target gene is located or a fragment thereof.
  • the guide nucleic acid molecule and the enzyme protein may exist in the form of ribonucleoprotein complexes (RNP), or each independently.
  • the enzyme protein may comprise a Cas protein.
  • reducing the expression and/or weakening the activity of at least one target gene of an immune cell in the present application may comprise: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the immune cell middle.
  • RNP ribonucleoprotein complex
  • the enzyme protein may comprise a Cas protein, a Cas protein homologue, or a functionally active fragment thereof.
  • the guide nucleic acid molecule can comprise a guide RNA (gRNA).
  • the gRNA can be used to bind to the sequence of the target gene.
  • the combination of the gRNA and the sequence of the target gene may be fully complementary, partially complementary, or hybridized to the sequence of the target gene under moderate stringency or stringent conditions.
  • the combination of the gRNA and the sequence of the target gene can allow the CRISPR system of the gRNA to specifically cut the target gene.
  • the guide nucleic acid molecules described in the application can be combined with regions or fragments thereof selected from the following groups: SEQ ID NO: 64-69.
  • SEQ ID NO: 64-69 there may be a protospacer adjacent motif (PAM) downstream of the region targeted by the guide nucleic acid molecule of the present application, and the protospacer adjacent motif (PAM) may be GGG, TGG or AGG.
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of a protospacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, GGG, and CGG .
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule may comprise the first about 10 to about 30 PAM regions shown by AGG, TGG, GGG and/or CGG in the DNA where the gene encoding the YEATS domain protein family member and/or its functionally active fragment is located
  • the target sequence consists of nucleotides.
  • the guide nucleic acid molecule may comprise the first about 15 to about 25 PAM regions shown in AGG, TGG, GGG and/or CGG in the DNA where the gene encoding the YEATS domain protein family member and/or its functionally active fragment is located , about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, About 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about A target sequence consisting of 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by TGG in the DNA of the gene encoding the YEATS domain protein family member and/or its functionally active fragment.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, 17, 25, About 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17
  • the target sequence consists of about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr19:6270571-6270590.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by TGG in the DNA of the gene encoding the YEATS domain protein family member and/or its functionally active fragment.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, 17, 25, About 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17
  • the target sequence consists of about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr19:6270679-6270698.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by TGG in the DNA of the gene encoding the YEATS domain protein family member and/or its functionally active fragment.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, 17, 25, About 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17
  • the target sequence consists of about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr9:20620738-20620757.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by AGG in the DNA of the gene encoding the YEATS domain protein family member and/or its functionally active fragment.
  • the guide nucleic acid molecule may comprise the first about 15 to about 25, about 17 to about 25, about 17 to about 25, about 17 to about 25 of the PAM region indicated by AGG in the DNA where the gene encoding the YEATS domain protein family member and/or its functionally active fragment is located.
  • the target sequence consists of about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr9:20448206-20448225.
  • the guide nucleic acid molecule can comprise a sequence as shown in any one of SEQ ID NO:70-75.
  • the immune cells obtained by reducing the expression and/or weakening the activity of at least one target gene of the immune cells express the product of the target gene
  • the proportion of cells can be reduced and/or the expression level of the target gene in a single cell can be reduced.
  • the immune cells obtained by reducing the expression and/or weakening the activity of at least one target gene of the immune cells express the The proportion of cells that are products of the gene of interest is reduced by at least about 5%.
  • the proportion of cells expressing the product of a gene encoding a member of the YEATS domain protein family and/or a functionally active fragment thereof is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60% %, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14% %, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the proportion of cells expressing the product of a gene encoding a member of the YEATS domain protein family and/or a functionally active fragment thereof can range from an observable proportion of cells to 0%.
  • the proportion of cells expressing the product of a gene encoding a member of the YEATS domain protein family and/or a functionally active fragment thereof can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or At least about 1%.
  • the gene encoding the YEATS domain protein family member and/or its functionally active fragment is expressed
  • the cell fraction of the product can be up to about 95%.
  • the proportion of cells expressing the product of a gene encoding a member of the YEATS domain protein family and/or a functionally active fragment thereof may be at most about 95%, at most about 90%, at most about 80%, at most about 70% , up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15% , up to about 14%, up to about 13%, up to about 12%, up to about 11%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, or up to about 5% %.
  • the proportion of cells expressing the product of the gene encoding the member of the YEATS domain protein family and/or its functionally active fragment can be detected by flow cytometry.
  • a single cell in the immune cells obtained by reducing the expression and/or weakening of at least one target gene of the immune cells can be decreased by at least about 5%.
  • the expression of the gene of interest in a single cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least About 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the expression level of the target gene in a single cell can range from an observable amount to 0%.
  • the expression level of the target gene in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40% , at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12% , at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the expression of the target gene in a single cell of the immune cells obtained by reducing the expression and/or weakening of the activity of at least one target gene of the immune cell can be the expression and/or activity of the target gene Or up to about 95% of immune cells with unchanged activity.
  • the expression level of the gene encoding a member of the YEATS domain protein family and/or its functionally active fragment (for example, the gene encoding AF9 and/or ENL) in a single cell of an immune cell can be the At most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most of the immune cells whose expression and/or activity of functionally active fragments thereof are unchanged About 40%, up to about 30%, up to about 20%, up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15%, up to about 14%, up to about 13%, up to About 12%, up to about 11%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, or up to about 5%.
  • it comprises: subjecting the immune cells to at least one stage of in vitro expansion, wherein, during at least one stage of the in vitro expansion, the expression of members of the YEATS domain protein family of the immune cells is reduced and/or or reduced activity.
  • the TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro are subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage In the stage of in vitro amplification, the expression and/or activity of the members of the YEATS domain protein family of the TILs amplified in the first stage in vitro are reduced.
  • the first stage of in vitro expansion is performed for at least about 7 days.
  • the second stage of in vitro expansion is performed for at least about 7 days.
  • the immune cells may be contacted with the one or more cell activators and the YEATS domain-containing protein and/or its function in the immune cells may be in a single stage of the in vitro expansion of the present application Expression and/or activity of the active fragment is reduced.
  • the cell activator may comprise an agonist for one or more targets selected from the group consisting of: CD3, CD28, HVEM, CD40L, OX40, and 4-1BB.
  • the immune cells of the present application are reduced in expression and/or activity of members of the YEATS domain protein family and contacted with one or more cell activating agents of the present application.
  • the expression and/or activity of the TIL of the present application and the members of the YEATS domain protein family of the present application can be reduced and/or the activity can be weakened and combined with one or more cell activators of the present application. touch.
  • the expression and/or activity of the members of the YEATS domain protein family of the TIL of the present application can be reduced and contacted with one or more cell activators of the present application.
  • the expression and/or activity of the members of the YEATS domain protein family of the TIL of the present application can be reduced and contacted with one or more cell activators of the present application.
  • the immune cells of the present application can substantially simultaneously reduce the expression and/or activity of members of the YEATS domain protein family and activate one or more cells of the present application. agent contact.
  • the immune cells of the present application can first reduce the expression and/or weaken the activity of the members of the YEATS domain protein family, for example, 2 hours in advance, 4 hours in advance, or 2 hours in advance. 8 hours, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more cell activators of the present application.
  • the immune cells of the present application can be contacted with one or more cell activators of the present application first, for example, 2 hours in advance, 4 hours in advance, and 8 hours in advance , 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then reduce the expression and/or activity of the members of the YEATS domain protein family.
  • the TILs of the present application can be made to substantially simultaneously reduce the expression and/or activity of members of the YEATS domain protein family and contact one or more cell activators of the present application.
  • TILs of the present application can be made to substantially simultaneously reduce the expression and/or activity of members of the YEATS domain protein family and contact one or more cell activators of the present application.
  • the TILs of the present application can substantially simultaneously reduce the expression and/or activity of members of the YEATS domain protein family and contact one or more cell activators of the present application. .
  • the second stage in vitro expansion of the present application is performed for at least about 9 days.
  • the second stage in vitro expansion of the present application can be performed for at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the second stage of in vitro expansion of the present application can be carried out for about 9 days to about 14 days
  • the second stage of in vitro expansion of the present application can be carried out for about 9 days to about 14 days, about 10 days to about 14 days, About 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days days to about 13 days, about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the second stage of in vitro expansion in the present application can be considered as the REP (rapid expansion protocol) stage.
  • the first stage of in vitro expansion of the present application can be considered the preREP stage.
  • the number of days for the second stage of in vitro expansion in the present application can be calculated from the start of the second stage of in vitro expansion. For example, when the second-stage in vitro expansion starts, it can be considered that the second-stage in vitro expansion has been performed for about 0 days. For example, if the second-stage in vitro expansion proceeds for about 24 hours, it can be considered that the second-stage in vitro expansion has been performed for about one day. For example, the day when the second-stage in vitro expansion starts can be considered as about 0 days after the second-stage in vitro expansion.
  • the number of days for the second stage of in vitro expansion in the present application can be calculated based on the number of days for the second stage of in vitro expansion. For example, on the second day after the start of the second-stage in vitro expansion, it can be considered that the second-stage in vitro expansion has been performed for about one day.
  • the cell activator of the present application may comprise one or more selected from the following group: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, and their functional activities fragment.
  • the cell activator of the present application may comprise an agonist of one or more targets selected from the following group: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the cell activator of the present application may comprise antibodies and antigen-binding fragments thereof selected from the group consisting of CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • a cell activator of the present application may comprise a CD3 agonist.
  • the cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, such as OKT3 from Miltenyi Biotech, or SP34 from BD.
  • a cell activator of the present application may comprise a CD28 agonist.
  • the cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, such as Merck's 15E8.
  • the cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain VL and heavy chain VH of OKT3 of Miltenyi Biotech, may comprise the light chain VL and heavy chain of SP34 of BD VH.
  • a cell activator of the present application may comprise a CD28 agonist.
  • the cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain VL and heavy chain VH of Merck's 15E8.
  • the cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of Miltenyi Biotech's OKT3, and may comprise the light chain of BD's SP34 LCDR1-3 and heavy chain HCDR1-3, the anti-CD3 antibody and/or antigen-binding fragment thereof of the present application may have CD3-binding ability.
  • a cell activator of the present application may comprise a CD28 agonist.
  • the cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of Merck's 15E8, the anti-CD28 antibody of the present application and/or Or an antigen-binding fragment thereof may have CD28-binding ability.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VH of the heavy chain of the antibody.
  • the CDRs of this application may be defined according to IMGT nomenclature, the CDRs of this application may be defined according to Chothia, or the CDRs of this application may be defined according to Kabat.
  • contacting the immune cells of the present application with one or more cell activators of the present application may include one or more methods selected from the following groups: (1) adding the cell activators of the present application to the cell activators of the present application In the cell culture medium of immune cells; (2) Add the engineered cells expressing the cell activator of the present application to the cell culture medium of the immune cells of the present application; (3) Add the solid phase containing the cell activator of the present application The medium is added to the cell culture medium of the immune cells of the application.
  • contacting the immune cells of the present application with one or more cell activators of the present application may comprise adding a solid phase medium comprising the cell activators of the present application to the cell culture medium of the immune cells of the present application.
  • contacting the immune cells of the present application with one or more cell activators of the present application may comprise adding the solid phase medium comprising the CD28 antibody and the CD3 antibody of the present application to the cell culture medium of the immune cells of the present application.
  • the initial concentration of the cell activator in the cell culture medium of the immune cells of the present application may be at least about 30 ng/mL.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the immune cells of the present application can be at least about 30 ng/mL;
  • the initial concentration of the CD3 antibody of the present application in the cell culture medium of the immune cells of the present application can be At least about 30 ng/mL.
  • the selection of the initial concentration of the CD28 antibody of the present application can be independent of the selection of the initial concentration of the CD3 antibody of the present application; for example, the initial concentration of the CD28 antibody of the present application and the CD3 antibody of the present application in the cell culture medium of the immune cells of the present application Concentrations can be combined arbitrarily.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the immune cells of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL.
  • the initial concentration of the CD3 antibody of the present application in the cell culture medium of the immune cells of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the immune cells of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL, and the CD3 antibody of the present application in the cell culture medium of the immune cells of the present application
  • the initial concentration of can be arbitrarily selected from about 30ng/mL to about 300ng/mL, and the selection of the initial concentration of the CD28 antibody of the present application can be independent of the selection of the initial concentration of the CD3 antibody of the present application.
  • the diameter of the solid phase media of the present application can be from about 500 nanometers to about 10 micrometers.
  • the diameter of the solid phase medium of the present application can be measured by a transmission electron microscope.
  • the diameter of the solid phase media of the present application can be from about 1 nanometer to about 500 nanometers.
  • the diameter of the solid phase media of the present application can be from about 100 nanometers to about 500 nanometers.
  • the diameter of the solid phase media of the present application can be from about 200 nanometers to about 500 nanometers.
  • the diameter of the solid phase medium of the present application can be measured by a transmission electron microscope.
  • the solid phase medium of the present application may comprise a polymer.
  • the solid phase media of the present application may comprise dextran.
  • the solid phase medium of the present application contains at least about 25 ⁇ g of the cell activator of the present application per mg.
  • the solid phase medium containing the cell activator of the present application is added to the cell culture medium of the immune cells of the present application at a ratio of the solid phase medium of the present application to the immune cells of the present application of about 1:100-about 1:2000.
  • the solid phase medium containing the cell activator of the present application is added to the cell culture medium of the immune cells of the present application at a ratio of about 2:1 to about 1:2 of the solid phase medium of the present application to the immune cells of the present application.
  • the ratio of the solid phase medium of the present application to the immune cells of the present application can be about 2:1 to about 1:2.
  • the solid phase medium of the cell activator is added to the cell culture medium of the immune cells of the present application.
  • the diameter of the solid phase medium of the present application when the diameter of the solid phase medium of the present application is about 100 nanometers to about 500 nanometers, it can be about 2:1-about 1:2, about 2:1-about 1:1, or about 1:1 -
  • the ratio of the solid medium of the present application to the immune cells of the present application is about 1:2
  • the solid phase medium comprising the cell activator of the present application such as CD3 agonist and/or CD28 agonist, is added to the cell culture of the immune cells of the present application Base.
  • the ratio of the solid phase medium of the present application to the immune cells of the present application can be about 1:100-about 1:2000.
  • the solid phase medium of the cell activator is added to the cell culture medium of the immune cells of the present application.
  • the diameter of the solid phase medium of the present application when the diameter of the solid phase medium of the present application is about 100 nanometers to about 500 nanometers, it can be about 1:100-about 1:2000, about 1:200-about 1:2000, about 1:300- About 1:2000, about 1:400-about 1:2000, about 1:500-about 1:2000, about 1:600-about 1:2000, about 1:700-about 1:2000, about About 1:800-about 1:2000, about 1:900-about 1:2000, about 1:1000-about 1:2000, about 1:1200-about 1:2000, about 1:1400-about 1 :2000, about 1:1600-about 1:2000, or about 1:1800-about 1:2000 of the ratio of the solid phase medium of the application to the immune cells of the application, for example, the CD28 agonist of the application and the CD3
  • the solid phase medium of the agonist is added to the cell culture medium of the immune cells of the present application.
  • the method of the present application may further comprise: contacting the immune cells of the present application with one or more cell growth factors during at least one stage of the in vitro expansion of the present application.
  • immune cells of the present application can be contacted with a cell activator of the present application and contacted with one or more cellular growth factors of the present application.
  • the TIL of the present application in the first stage of in vitro expansion of the present application, can be contacted with the cell activator of the present application and contacted with one or more cell growth factors of the present application.
  • the TIL of the present application in the second stage of in vitro expansion of the present application, can be contacted with the cell activator of the present application and contacted with one or more cell growth factors of the present application.
  • the TIL of the present application in the third stage of in vitro expansion of the present application, can be contacted with the cell activator of the present application and contacted with one or more cell growth factors of the present application.
  • the immune cells of the present application are contacted with the cell activating agent of the present application and one or more cell growth factors of the present application substantially simultaneously.
  • immune cells of the present application can be contacted with one or more cellular growth factors of the present application and one or more cellular activators of the present application substantially simultaneously.
  • the immune cells of the present application can be first contacted with one or more cell growth factors of the present application, for example, 2 hours in advance, 4 hours in advance, or 8 hours in advance , 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more cell activators of the present application.
  • one or more cell growth factors of the present application for example, 2 hours in advance, 4 hours in advance, or 8 hours in advance , 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc.
  • the immune cells of the present application can be contacted with one or more cell activators of the present application first, for example, 2 hours in advance, 4 hours in advance, and 8 hours in advance , 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more cell growth factors of the present application.
  • the immune cells of the present application can be contacted with the cell activator of the present application and one or more cell growth factors of the present application substantially simultaneously.
  • the TILs of the present application can be contacted with the cell activator of the present application and one or more cell growth factors of the present application substantially simultaneously.
  • the TILs of the present application can be contacted with the cell activator of the present application and one or more cell growth factors of the present application substantially simultaneously.
  • the cell growth factor of the present application can be selected from one or more of the following groups: IL-2, IL-7, IL-12, IL-15, IL-21, gamma interferon, and their functionally active fragments .
  • the cell growth factors of the present application may comprise IL-2 and/or functionally active fragments thereof.
  • a functionally active fragment of IL-2 can comprise a fragment of IL-2 known in the art that can bind to a cellular IL-2 receptor.
  • contacting the immune cells of the present application with one or more cell growth factors of the present application may comprise adding the cell growth factors of the present application to the cell culture medium of the immune cells of the present application.
  • the initial concentration of the cell growth factors of the present application in the cell culture medium of the immune cells of the present application can be at least about 300 IU/mL.
  • the initial concentration of IL-2 of the present application in the cell culture medium of the immune cells of the present application can be at least about 350IU/mL, at least about 400IU/mL, at least about 500IU/mL, at least about 600IU/mL, at least about 700IU/mL mL, at least about 800 IU/mL, at least about 900 IU/mL, at least about 1000 IU/mL, at least about 1100 IU/mL, at least about 1200 IU/mL, at least about 1300 IU/mL, at least about 1400 IU/mL, at least about 1500 IU/mL, At least about 2000 IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL, at least about 2700 IU/mL, at least about 2800 IU/mL, at least about 2900 IU/mL, at least about 3000 IU/mL, at least about 3100 IU/mL, at least about 3200
  • the method of the present application may further comprise: in at least one stage of the in vitro expansion of the present application, the immune cells of the present application may be co-cultured with the feeder cells.
  • the immune cells of the present application can be contacted with one or more cell activators and/or one or more cell growth factors and co-cultured with the feeder cells of the present application, e.g.
  • the in vitro amplification of the present application at a single stage may refer to the in vitro amplification of the present application at the same stage, for example, the same in the first stage of in vitro amplification of the present application, the same in the second stage of in vitro amplification of the present application, Or the third stage of in vitro amplification in the present application.
  • the TILs of the present application can be contacted with one or more cell activators and/or one or more cell growth factors and co-cultured with the feeder cells of the present application.
  • the TIL of the present application in the second stage of in vitro expansion of the present application, can be contacted with one or more cell activators and/or one or more cell growth factors of the present application and with the feeder cells of the present application. Co-culture.
  • the TIL of the present application in the third stage of in vitro expansion of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application and with the feeder cells of the present application. Co-culture.
  • the immune cells of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then contacted with the present application.
  • Feeder cell co-culture For example, in the first stage of in vitro expansion of the present application, the TILs of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then fed with the present application. Cell co-culture.
  • the TILs of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then fed with the present application.
  • Cell co-culture For example, in the third stage of in vitro expansion of the present application, TILs of the present application can be brought into contact with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then fed with the present application. Cell co-culture.
  • the immune cells of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then contacted with the present application.
  • Feeder cell co-culture For example, the certain period of time for this application may be at least about 2 hours.
  • the certain period of time of the present application can be at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least About 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, or at least about 72 hours.
  • a certain period of time for the present application may be from about 2 hours to about 72 hours.
  • the certain period of time of the present application can be about 6 hours to about 7 hours, about 6 hours to about 8 hours, about 6 hours to about 9 hours, about 6 hours to about 10 hours, about 6 hours to about 11 hours, about 6 hours to about 12 hours, about 6 hours to about 13 hours, about 6 hours to about 14 hours, about 6 hours to about 15 hours, about 6 hours to about 16 hours, about 6 hours to about 17 hours, about 6 hours to about 18 hours, about 6 hours to about 19 hours, about 6 hours to about 20 hours, about 6 hours to about 21 hours, about 6 hours to about 22 hours, about 6 hours to about 23 hours, about 6 hours to about 24 hours, about 6 hours to about 36 hours, about 6 hours to about 48 hours, about 6 hours to about 60 hours, or about 6 hours to about 72 hours.
  • the certain period of time of the present application can be about 12 hours to about 13 hours, about 12 hours to about 14 hours, about 12 hours to about 15 hours, about 12 hours to about 16 hours, about 12 hours to about 17 hours, about 12 hours to about 18 hours, about 12 hours to about 19 hours, about 12 hours to about 20 hours, about 12 hours to about 21 hours, about 12 hours to about 22 hours, about 12 hours to about 23 hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, or about 12 hours to about 72 hours.
  • the certain period of time in the present application can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours , about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours.
  • feeder cells of the present application may comprise antigen presenting cells.
  • the feeder cells of the present application may comprise one or more selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen-presenting cells.
  • the feeder cells of the present application may be peripheral mononuclear cells.
  • the feeder cells of the present application can be irradiated feeder cells.
  • the feeder cells of the present application can be isolated artificial antigen-presenting cells (aAPC), and the artificial antigen-presenting cells of the present application can comprise cells expressing HLA-A/B/C, CD64, CD80, ICOS-L and/or CD58 , and can be modified to express more than one cell activator of the present application.
  • the feeder cells of the present application can be irradiated, eg, can be gamma irradiated, or can be X-ray irradiated.
  • the co-cultivation of the immune cells of the present application and the feeder cells of the present application may include contacting the surface of the feeder cells of the present application with the surface of the immune cells of the present application.
  • the co-culture of the immune cells of the present application and the feeder cells of the present application comprises adding the feeder cells of the present application to the cell culture medium of the immune cells of the present application.
  • the present application can add the feeder cells of the present application to the cell culture medium of the immune cells of the present application at a ratio of about 40:1 to about 400:1 of the feeder cells of the present application to the immune cells of the present application.
  • the present application can be about 40:1 to about 400:1, about 40:1 to about 300:1, about 40:1 to about 200:1, about 40:1 to about 100:1, and About 40:1-about 90:1, about 40:1-about 80:1, about 40:1-about 70:1, about 40:1-about 60:1, about 40:1-about 50:1, about 50:1-about 400:1, about 60:1-about 400:1, about 70:1-about 400:1, about 80:1-about 400:1, about 90:1 to about 400:1, about 100:1 to about 400:1, about 200:1 to about 400:1, or about 300:1 to about 400:1 of the feeder cells of the present application and the present application
  • the feeder cells of the present application are added to the cell culture medium
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) making tumor-infiltrating lymphocytes derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion The first TIL group is contacted with one or more cell growth factors; wherein, the second TIL group is obtained through the step (A); (B) reducing the expression of the second TIL group YEATS domain protein family member and /or the activity is weakened; wherein, the third TIL group is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: (A) making tumor-infiltrating lymphocytes derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion The increased first TIL population is contacted with one or more cell growth factors; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population is contacted with SGC-iMLLT, and SGC-
  • the initial concentration of iMLLT in the cell culture medium of the TIL may be at least about 1 ⁇ M to about 200 ⁇ M such that the TIL is contacted with one or more cell activators and with SGC-iMLLT substantially simultaneously; wherein, via the The above step (B) obtains the third TIL group.
  • the first-stage in vitro expansion of the present application can be optionally used interchangeably with step (A) in the method of the above aspect.
  • the second-stage in vitro expansion of the present application can be used in any substitution with step (B) in the method of the above aspects.
  • the TILs expanded in vitro in the first stage of the present application can be used in any substitution with the second population of TILs obtained through step (A) of the method of the above aspects.
  • the second-stage in vitro expanded TILs of the present application can be used in any substitution with the third TIL population obtained through step (B) of the method of the above aspects.
  • the third-stage in vitro amplification of the present application can be used in any replacement with any added step (C) in the method of the above aspects.
  • the third-stage in vitro expanded TILs of the present application can be used in any replacement with the fourth TIL population obtained through any additional step (C) in the method of the above aspects .
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion The first TIL population is contacted with various cell growth factors; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with various cell growth factors, and with various The cell activator is contacted to reduce the expression and/or activity of the members of the YEATS domain protein family, and the TIL is co-cultured with the feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion The first TIL population is contacted with a cell growth factor; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a cell growth factor, a cell activator, or The expression and/or activity of the YEATS domain protein family member is reduced and the TIL is co-cultured with the feeder cells, and the YEATS domain protein family member may comprise AF9 and/or ENL; wherein, the second step (B) is obtained Three TIL groups.
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • the method of obtaining TIL cells from a tissue sample of a subject can be obtained by surgically obtaining an orthotopic tumor sample or a metastatic tumor sample, the weight of which can be at least about 1 g, or multiple pieces of tissue can be combined.
  • Tumor tissue, pleural effusion and/or peritoneal effusion should be transported at about 2-8 degrees in sample transport fluid, such as commonly used commercial tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid, and processed within 48 hours .
  • the tissue pieces can be mechanically broken to about 1-27 cubic millimeters in size, transferred into a gas-permeable culture bag or Grex, and added with a cell serum-free medium and a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/mL, such as Can be 6000IU/mL) IL-2 culture for about 3-14 days.
  • YEATS domain protein family member can comprise AF9 and/or ENL, for example can be formed by carrying the ribonucleoprotein complex (RNP) that comprises the gRNA of the present application and Cas protein Transduction is carried out so that the cell ratio of the gene encoding the YEATS domain protein family member in the TIL is about 95% or less), after activating the
  • Cells in the culture medium can be collected using a cell processing system, washed and frozen, and detected.
  • the CD3 ratio of the final product can be greater than 80%, the cell viability can be greater than 50%, and the cells greater than 80% can be memory effector cells and effector cells.
  • IFN- ⁇ can be secreted after stimulation, and/or can be characterized by an up-regulation of the proportion of activated cells.
  • the present application provides an immune cell, and the immune cell of the present application can be cultured according to the cultivation method of the present application.
  • the immune cells provided in the present application may comprise one or a batch of immune cells cultured by the cultivation method of the present application.
  • the immune cells provided in the present application may comprise multiple or multiple batches of immune cells cultured by the cultivation method of the present application and combined in any proportion.
  • immune cells expanded using the methods of the present application can be administered to a patient as a pharmaceutical composition.
  • the pharmaceutical composition may be a suspension of immune cells in a sterile buffer.
  • Immune cells expanded using the PBMCs of the present application can be administered by any suitable route known in the art.
  • the cells can be administered as a single intra-arterial or intravenous infusion, which can last for about 30 to 60 minutes.
  • Other suitable routes of administration may include intraperitoneal, intrathecal and intralymphatic administration.
  • any suitable dose of immune cells can be administered.
  • about 2.3 x 10 9 to about 13.7 x 10 10 immune cells may be administered.
  • about 1 x 109 to about 12 x 1010 immune cells may be administered.
  • about 1.2 x 1010 to about 4.3 x 1010 immune cells may be administered.
  • about 3 x 1010 to about 12 x 1010 immune cells may be administered.
  • about 4 x 1010 to about 10 x 1010 immune cells may be administered.
  • about 5 x 1010 to about 8 x 1010 immune cells may be administered.
  • the therapeutically effective dose may be from about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 . In some embodiments, the therapeutically effective dose may be from about 1 ⁇ 10 9 to about 12 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 3 ⁇ 10 10 to about 12 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 4 ⁇ 10 10 to about 10 ⁇ 10 10 immune cells.
  • the therapeutically effective dose may be from about 5 ⁇ 10 10 to about 8 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 6 ⁇ 10 10 to about 8 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 7 ⁇ 10 10 to about 8 ⁇ 10 10 immune cells.
  • the number of immune cells provided in the composition of the present application may be about 1 ⁇ 10 6 , about 2 ⁇ 10 6 , about 3 ⁇ 10 6 , about 4 ⁇ 10 6 , about 5 ⁇ 10 6 , About 6 ⁇ 10 6 , About 7 ⁇ 10 6 , About 8 ⁇ 10 6 , About 9 ⁇ 10 6 , About 1 ⁇ 10 7 , About 2 ⁇ 10 7 , About 3 ⁇ 10 7 , About 4 ⁇ 10 7 , About 5 ⁇ 10 7 , about 6 ⁇ 10 7 , about 7 ⁇ 10 7 , about 8 ⁇ 10 7 , about 9 ⁇ 10 7 , about 1 ⁇ 10 8 , about 2 ⁇ 10 8 , about 3 ⁇ 10 8 , about 4 ⁇ 10 8 , about 5 ⁇ 10 8 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 , about 8 ⁇ 10 8 , about 9 ⁇ 10 8 , about 1 ⁇ 10 9 , about 2 ⁇ 10 9 , about 3 ⁇ 10 9 , about 4 ⁇ 10 9 , about 5 ⁇ 10 8 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8
  • the number of immune cells provided in the composition of the present application may range from about 1 ⁇ 10 6 to 5 ⁇ 10 6 , about 5 ⁇ 10 6 to 1 ⁇ 10 7 , about 1 ⁇ 10 7 to 5 ⁇ 10 7 , about 5 ⁇ 10 7 to 1 ⁇ 10 8 , about 1 ⁇ 10 8 to 5 ⁇ 10 8 , about 5 ⁇ 10 8 to 1 ⁇ 10 9 , about 1 ⁇ 10 9 to 5 ⁇ 10 9 , about 5 ⁇ 10 9 to 1 ⁇ 10 10 , about 1 ⁇ 10 10 to 5 ⁇ 10 10 , about 5 ⁇ 10 10 to 1 ⁇ 10 11 , about 5 ⁇ 10 11 to 1 ⁇ 10 12 , about 1 ⁇ 10 12 to 5 ⁇ 10 12 , or about 5 ⁇ 10 12 to 1 ⁇ 10 13 .
  • the concentration of immune cells provided in the compositions of the present application may be less than, for example, about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40% of the composition. %, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, About 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2 %, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%, about 0.009%, about 0.008%, About 0.007%, about 0.006%, about 0.005%, about 0.004%, about 0.003%, about 0.002%, about 0.001%, about 0.0009%, about 0.0009%, about
  • the concentration of immune cells provided in the composition of the present application can be greater than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30% of the composition , about 20%, about 19.75%, about 19.50%, about 19.25%, about 19%, about 18.75%, about 18.50%, about 18.25%, about 18%, about 17.75%, about 17.50%, about 17.25%, about 17%, about 16.75%, about 16.50%, about 16.25%, about 16%, about 15.75%, about 15.50%, about 15.25%, about 15%, about 14.75%, about 14.50%, about 14.25%, about 14% , about 13.75%, about 13.50%, about 13.25%, about 13%, about 12.75%, about 12.50%, about 12.25%, about 12%, about 11.75%, about 11.50%, about 11.25%, about 11%, about 10.75%, about 10.50%, about 10.25%, about 10%, about 9.75%, about 9.50%, about 9.25%,
  • the concentration of immune cells provided in the composition of the present application can range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% of the composition. % to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to About 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17% %, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v/v.
  • the immune cells provided in the composition of the present application may be present in a concentration range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% of the composition % to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to About 1%, or about 0.1% to about 0.9% w/w, w/v or v/v.
  • the amount of immune cells provided in the composition of the present application can be equal to or less than about 10 g, about 9.5 g, about 9.0 g, about 8.5 g, about 8.0 g, about 7.5 g, about 7.0 g, about 6.5g, about 6.0g, about 5.5g, about 5.0g, about 4.5g, about 4.0g, about 3.5g, about 3.0g, about 2.5g, about 2.0g, about 1.5g, about 1.0g, about 0.95g , about 0.9g, about 0.85g, about 0.8g, about 0.75g, about 0.7g, about 0.65g, about 0.6g, about 0.55g, about 0.5g, about 0.45g, about 0.4g, about 0.35g, about 0.3g, about 0.25g, about 0.2g, about 0.15g, about 0.1g, about 0.09g, about 0.08g, about 0.07g, about 0.06g, about 0.05g, about 0.04g, about 0.03g, about 0.02g ,
  • the amount of immune cells provided in the compositions of the present application may be greater than about 0.0001 g, about 0.0002 g, about 0.0003 g, about 0.0004 g, about 0.0005 g, about 0.0006 g, about 0.0007 g, about 0.0008 g, about 0.0009g, about 0.001g, about 0.0015g, about 0.002g, about 0.0025g, about 0.003g, about 0.0035g, about 0.004g, about 0.0045g, about 0.005g, about 0.0055g, about 0.006g, About 0.0065g, about 0.007g, about 0.0075g, about 0.008g, about 0.0085g, about 0.009g, about 0.0095g, about 0.01g, about 0.015g, about 0.02g, about 0.025g, about 0.03g, about 0.035 g, about 0.04g, about 0.045g, about 0.05g, about 0.055g, about 0.06g, about
  • the immune cells can be administered in a single dose. Such administration may be by injection, for example intravenously. In some embodiments, immune cells can be administered in multiple doses. Doses may be once, twice, three, four, five, six or more than six times per year. Dosage can be monthly, biweekly, weekly, or every 2 days. In some embodiments, the administration of immune cells can be administered sequentially.
  • the present application provides a pharmaceutical composition.
  • it may comprise the immune cells of the present application and/or the composition of the present application, and a pharmaceutically acceptable carrier.
  • the present application provides a kit.
  • the kit of the present application may include the cell activator, cell growth factor and/or feeder cells of the method for culturing immune cells of the present application, and instructions describing the steps of the method of culturing immune cells of the present application.
  • the present application provides a kit, which may include the immune cells of the present application and/or the pharmaceutical composition of the present application.
  • the present application provides a method for affecting the growth of cells, such as tumor cells, which may include administering the immune cells of the present application and/or the pharmaceutical composition of the present application to a subject.
  • affecting tumor growth can comprise reducing the volume of the tumor to, for example, about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40% of its pre-administration volume.
  • the present application provides the application of the immune cells of the present application and/or the pharmaceutical composition of the present application in the preparation of medicines, and the medicines of the present application can be used to prevent and/or treat diseases and/or symptoms.
  • the diseases and/or symptoms of the present application may include tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • the present application provides a method for preventing and/or treating diseases and/or symptoms, which may include administering the immune cells of the present application and/or the pharmaceutical composition of the present application to a subject.
  • the diseases and/or symptoms of the present application may include tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • the present application provides a TIL of the present application and/or the pharmaceutical composition of the present application, which can be used to prevent and/or treat diseases and/or symptoms.
  • the diseases and/or symptoms of the present application may include tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), the method may include: reducing the expression and/or weakening the activity of at least one target gene of the TIL, and making the TIL in Co-cultivation with feeder cells followed by exposure to T cell activators and/or T cell growth factors for a certain period of time.
  • TIL tumor-infiltrating lymphocytes
  • the method may comprise: reducing the expression and/or attenuating the activity of at least one target gene of the TIL after co-cultivating the TIL with the feeder cells.
  • the method may comprise: co-cultivating the TIL with the feeder cells after reducing the expression and/or attenuating the activity of at least one target gene of the TIL.
  • the method can comprise: after contacting the TIL with the T cell activator and/or the T cell growth factor and before the TIL is co-cultivated with the feeder cells, at least one of the TILs The expression and/or activity of a target gene is reduced.
  • the method may comprise reducing expression and/or attenuating the activity of at least one target gene of the TIL substantially simultaneously with contacting the TIL with the T cell activator and/or the T cell growth factor .
  • the method may comprise: reducing the expression and/or attenuating the activity of at least one target gene of the TIL substantially simultaneously with the co-cultivation of the TIL with the feeder cells.
  • the present application provides a method for culturing tumor infiltrating lymphocytes (TIL), the method may include: reducing the expression and/or attenuating the activity of at least one target gene of the TIL.
  • the present application also provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: reducing the expression and/or weakening the activity of at least one target gene of the TIL, wherein the TIL Contains TIL obtained from co-culture with feeder cells after exposure to T cell activators and/or T cell growth factors for a certain period of time.
  • TIL tumor-infiltrating lymphocytes
  • the present application also provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: contacting the TIL with a T cell activator and/or a T cell growth factor for a certain period of time and then contacting with a feeder Cell co-cultivation, wherein the TIL comprises TIL obtained by reducing the expression and/or weakening the activity of at least one target gene of the TIL.
  • TIL tumor-infiltrating lymphocytes
  • the present application also provides a method for culturing tumor-infiltrating lymphocytes (TIL), the method may include: reducing the expression and/or weakening the activity of at least one target gene of the TIL, and making the TILs were exposed to CD28 agonists.
  • TIL tumor-infiltrating lymphocytes
  • the present application also provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: reducing the expression and/or weakening the activity of at least one target gene of the TIL, wherein the TIL Contains TILs obtained by exposure to a CD28 agonist.
  • TIL tumor-infiltrating lymphocytes
  • the present application also provides a method for culturing tumor infiltrating lymphocytes (TIL), the method may comprise: contacting the TIL with a CD28 agonist, wherein the TIL comprises at least one of the TIL Reduced expression and/or attenuated activity of the target gene in TILs obtained.
  • TIL tumor infiltrating lymphocytes
  • the CD28 agonist comprises an anti-CD28 antibody and/or its antigen-binding fragment, CD80 and/or its functionally active fragment and/or CD86 and/or its functionally active fragment, and recombinant proteins of the above substances.
  • TILs obtained by reducing the expression and/or activity of at least one target gene of said TIL may exhibit improved TIL properties compared to TILs with unchanged expression and/or activity of the target gene.
  • the TIL whose expression and/or activity of the target gene is unchanged may refer to a TIL derived from the same donor and which has not reduced the expression and/or activity of at least one target gene of said TIL cell.
  • the TIL whose expression and/or activity of the target gene has not been changed may refer to genes other than the target gene of the TIL derived from the same donor (for example, knocking out the other gene, for TIL cells with reduced expression and/or attenuated activity that have essentially no effect on cell function.
  • the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL may refer to a TIL that has been isolated in the same way from the same donor and that has not made the TIL TIL cells with reduced expression and/or reduced activity of at least one gene of interest.
  • the corresponding TIL that has not reduced the expression and/or attenuated the activity of at least one target gene of said TIL may refer to the same tumor origin from the same donor and has not reduced at least one target gene of said TIL.
  • a TIL cell with reduced expression and/or reduced activity of a gene of interest may refer to a TIL that has been isolated in the same way from the same donor and that has not made the TIL TIL cells with reduced expression and/or reduced activity of at least one gene of interest.
  • the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL may refer to the same tumor source from the same donor that has been isolated in the same way and that has not been subjected to the same method. TIL cells with reduced expression and/or reduced activity of at least one target gene of TIL.
  • the corresponding TIL that has not reduced the expression and/or attenuated the activity of at least one target gene of said TIL may refer to dividing TIL cells derived from the same donor into two groups, one of which has not
  • the TIL cells that reduce the expression and/or attenuate the activity of the at least one target gene of the TIL may be corresponding TILs that have not reduced the expression and/or attenuation of the at least one target gene of the TIL.
  • the corresponding TIL that has not reduced the expression and/or attenuated the activity of at least one target gene of said TIL may refer to dividing TIL cells derived from the same donor in the same way into two groups , wherein a group of TIL cells that have not reduced the expression and/or weakened activity of at least one target gene of said TIL may be corresponding TILs that have not reduced the expression and/or weakened activity of at least one target gene of said TIL .
  • the corresponding TIL that has not reduced the expression and/or attenuated the activity of at least one target gene of said TIL may refer to dividing the TIL cells of the same tumor origin from the same donor into two groups, One group of TIL cells that have not reduced the expression and/or weakened the activity of the at least one target gene of the TILs may be the corresponding TILs that have not reduced the expression and/or weakened the activity of the at least one target gene of the TILs.
  • the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL may refer to the TIL cells isolated in the same manner from the same tumor source of the same donor Divided into two groups, wherein a group of TIL cells that have not reduced the expression and/or activity of the at least one target gene of the TIL may be those that have not reduced the expression and/or activity of the at least one target gene of the TIL Corresponding TIL attenuated.
  • the reduced expression and/or weakened activity of at least one target gene may mean that the target gene of the natural cell is in a certain degree of expression state, and after the treatment of the present application, the expression level of the target gene of the cell can be reduced. Decreasing, that is, reducing the expression level of the target gene can be to make the natural cell change from expressing the target gene to substantially not expressing the target gene or expressing a decrease in the amount of the target gene.
  • the improved TIL properties of the present application comprise one or more selected from the following group: increased TIL cell number and expansion capacity, increased live cell ratio, increased viability, improved TIL Proportions of cell subpopulations, increased cytokine secretion, increased tumor cell killing, increased anti-apoptosis, and increased T cell receptor (TCR) clonal diversity.
  • the improved T cell subset ratio of the present application may comprise one or more selected from the following group: increased CD4 + cell ratio, decreased CD8 + cell ratio, increased central memory T cell proportion, decreased proportion of regulatory T cells, increased proportion of activated T cells, increased proportion of tumor-specific T cells, and increased proportion of stem-like T cells.
  • the increased proportion of central memory T cells of the present application may be an increase in the proportion of CD45RA ⁇ CCR7 + cells among TIL cells.
  • the reduced ratio of regulatory T cells of the present application may be a reduction in the ratio of CD4 + CD25 + Foxp3 + cells among TIL cells.
  • the increased proportion of activated T cells of the present application may be an increase in the proportion of CD25 + , CD28 + , PD-1 + or 41BB + cells in TIL cells.
  • reducing the expression and/or weakening the activity of at least one target gene of TIL in the method of the present application may comprise introducing a gene regulation system into the TIL cells.
  • the gene regulation system can disrupt the target gene at the DNA level.
  • the gene regulation system can disrupt the region of the target gene or a fragment thereof in the genome of the TIL cell.
  • the DNA region where the target gene is located or its fragment in TIL cells is cleaved and the expression ability of the target gene is reduced or the activity of the target gene is inhibited.
  • the editing effect of the gene regulation system on the target gene can be long-term and sustained.
  • the gene regulation system may comprise a guide nucleic acid molecule and an enzyme protein.
  • the enzyme protein may have nucleic acid scissor activity, and the guide nucleic acid molecule may guide the enzyme protein to specifically cut the region where the target gene is located or a fragment thereof.
  • the guide nucleic acid molecule and the enzyme protein may exist in the form of ribonucleoprotein complexes (RNP), or each independently.
  • the enzyme protein may comprise a Cas protein.
  • reducing the expression and/or weakening the activity of at least one target gene of TIL in the present application may comprise: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the TIL.
  • RNP ribonucleoprotein complex
  • the enzyme protein may comprise a Cas protein, a Cas protein homologue, or a functionally active fragment thereof.
  • the guide nucleic acid molecule can comprise a guide RNA (gRNA).
  • the gRNA can be used to bind to the sequence of the target gene.
  • the combination of the gRNA and the sequence of the target gene may be fully complementary, partially complementary, or hybridized to the sequence of the target gene under moderate stringency or stringent conditions.
  • the combination of the gRNA and the sequence of the target gene can allow the CRISPR system of the gRNA to specifically cut the target gene.
  • the target gene of the present application may include a gene encoding a cytokine signaling inhibitory molecule.
  • the cytokine signal inhibitory molecule encoded by the target gene of the present application may comprise a Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway inhibitory molecule.
  • the JAK/STAT pathway inhibitory molecule of the present application can bind Janus kinase (JAK).
  • the cytokine signaling inhibitory molecule may comprise a suppressor of cytokine signaling (SOCS), such as suppressor of cytokine signaling 1 (SOCS1).
  • SOCS suppressor of cytokine signaling 1
  • the guide nucleic acid molecule described in the present application can be combined with the region where exon 2 of the SOCS1 gene is located or a fragment thereof.
  • the guide nucleic acid molecules described in the present application can be combined with regions or fragments thereof selected from the following groups: SEQ ID NO: 34 and SEQ ID NO: 35.
  • SEQ ID NO: 34 SEQ ID NO: 34
  • SEQ ID NO: 35 SEQ ID NO: 35
  • PAM protospacer adjacent motif downstream of the region targeted by the guide nucleic acid molecule of the present application
  • PAM protospacer adjacent motif
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine the target sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the PAM of the target gene, and at the same time design suitable gRNA for the target sequence.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 15 to about 25 nucleotides of the PAM region indicated by CGG in the DNA where the SOCS1 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr16:11255292-11255311.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by AGG in the DNA where the SOCS1 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr16:11255230-11255249.
  • the guide nucleic acid molecule can comprise a sequence as shown in any one of SEQ ID NO: 36 and SEQ ID NO: 37.
  • the target gene of the present application may include a gene for a protein capable of binding TGF ⁇ .
  • TGF ⁇ R2 a gene for a protein capable of binding TGF ⁇ .
  • the guide nucleic acid molecules described in the present application can be combined with regions or fragments thereof selected from the following groups: SEQ ID NO: 38 and SEQ ID NO: 39.
  • SEQ ID NO: 38 and SEQ ID NO: 39 there may be a protospacer adjacent motif (PAM) downstream of the region targeted by the guide nucleic acid molecule of the present application, and the protospacer adjacent motif (PAM) may be GGG or CGG.
  • PAM protospacer adjacent motif
  • GGG protospacer adjacent motif
  • those skilled in the art can easily determine the target sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the PAM of the target gene, and at the same time design suitable gRNA for the target sequence.
  • the guide nucleic acid molecule can bind to a region defined by any one set of genomic coordinates shown in Table 7 or a fragment thereof.
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of a protospacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, GGG, and CGG .
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by GGG in the DNA where the TGF ⁇ R2 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr3:30672268-30672287.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by CGG in the DNA where the TGF ⁇ R2 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to A target sequence of about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr3:30672178-30672197.
  • the guide nucleic acid molecule may comprise a sequence as shown in any one of SEQ ID NO:40 and SEQ ID NO:41.
  • the target gene of the present application may include a gene encoding a protein including a DNA-binding domain of ETS (erythrocyte transformation specificity).
  • ETS erythrocyte transformation specificity
  • FLI1 erythrocyte transformation specificity
  • the guide nucleic acid molecule is capable of binding to a region defined by any one set of genomic coordinates shown in Table 8 or a fragment thereof.
  • the guide nucleic acid molecule described in the present application can be combined with a region or fragment thereof selected from the following: SEQ ID NO:42.
  • a protospacer adjacent motif (PAM) downstream of the region targeted by the guide nucleic acid molecule of the present application may be TGG.
  • PAM protospacer adjacent motif
  • TGG protospacer adjacent motif
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of a protospacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, GGG, and CGG .
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by TGG in the DNA where the FLI1 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr11:128758312-128758331.
  • the guide nucleic acid molecule can comprise the sequence shown in SEQ ID NO: 43.
  • the target gene of the present application may include a gene encoding a protein comprising an SH2 domain and a SOCS box domain.
  • a gene encoding a protein comprising an SH2 domain and a SOCS box domain For example CISH.
  • the guide nucleic acid molecule is capable of binding to a region defined by any one set of genome coordinates shown in Table 9 or a fragment thereof.
  • the guide nucleic acid molecules described in the application can be combined with regions or fragments thereof selected from the following groups: SEQ ID NO: 44, 45, 46, and 47.
  • there may be a protospacer adjacent motif (PAM) downstream of the region targeted by the guide nucleic acid molecule of the present application and the protospacer adjacent motif (PAM) may be TGG or AGG.
  • PAM protospacer adjacent motif
  • TGG TGG or AGG.
  • those skilled in the art can easily determine the target sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the PAM of the target gene, and at the same time design suitable gRNA for the target sequence.
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of a protospacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, CGG, and GGG .
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by TGG in the DNA where the CISH gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr3:50608013-50608032.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by AGG in the DNA where the CISH gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to A target sequence of about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr3:50608368-50608387.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by TGG in the DNA where the CISH gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to A target sequence of about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr3:50608580-50608599.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by AGG in the DNA where the CISH gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to A target sequence of about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr3:50608564-50608583.
  • the guide nucleic acid molecule can comprise a sequence as shown in any one of SEQ ID NO: 48-51.
  • the target gene of the present application may comprise a gene encoding a protein comprising E3 ubiquitin ligase.
  • a protein comprising E3 ubiquitin ligase for example CBLB.
  • the guide nucleic acid molecule is capable of binding to a region defined by any one set of genomic coordinates shown in Table 10 or a fragment thereof.
  • the guide nucleic acid molecules described in the present application can be combined with regions or fragments thereof selected from the following groups: SEQ ID NO: 52 and SEQ ID NO: 53.
  • SEQ ID NO: 52 and SEQ ID NO: 53 there may be a protospacer adjacent motif (PAM) downstream of the region targeted by the guide nucleic acid molecule of the present application, and the protospacer adjacent motif (PAM) may be TGG or AGG.
  • PAM protospacer adjacent motif
  • TGG protospacer adjacent motif
  • those skilled in the art can easily determine the target sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the PAM of the target gene, and at the same time design suitable gRNA for the target sequence.
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of a protospacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, GGG, and CGG .
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by TGG in the DNA where the CBLB gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr3: 105746016-105746035.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by AGG in the DNA where the CBLB gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr3:105751606-105751625.
  • the guide nucleic acid molecule can comprise a sequence as shown in any one of SEQ ID NO:54 and SEQ ID NO:55.
  • the target gene of the present application may comprise a gene encoding a protein comprising an ankyrin repeat domain.
  • ANKRD11 ankyrin repeat domain
  • the guide nucleic acid molecule is capable of binding to a region defined by any one set of genome coordinates shown in Table 11 or a fragment thereof.
  • the guide nucleic acid molecules described in the present application can be combined with regions or fragments thereof selected from the following groups: SEQ ID NO: 56 and SEQ ID NO: 57.
  • SEQ ID NO: 56 and SEQ ID NO: 57 there may be a protospacer adjacent motif (PAM) downstream of the region targeted by the guide nucleic acid molecule of the present application, and the protospacer adjacent motif (PAM) may be TGG or AGG.
  • PAM protospacer adjacent motif
  • TGG protospacer adjacent motif
  • those skilled in the art can easily determine the target sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the PAM of the target gene, and at the same time design suitable gRNA for the target sequence.
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of a protospacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, GGG, and CGG .
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by TGG in the DNA where the ANKRD11 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr16:89305232-89305251.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by AGG in the DNA where the ANKRD11 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to A target sequence of about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr16:89305208-89305227.
  • the guide nucleic acid molecule can comprise a sequence as shown in any one of SEQ ID NO:58 and SEQ ID NO:59.
  • the target gene of the present application may include a gene encoding a protein comprising a leucine zipper.
  • a protein comprising a leucine zipper such as BATF.
  • the guide nucleic acid molecule is capable of binding to a region defined by any one set of genomic coordinates shown in Table 12 or a fragment thereof.
  • the guide nucleic acid molecules described in the present application can be combined with regions or fragments thereof selected from the following groups: SEQ ID NO: 60 and SEQ ID NO: 61.
  • SEQ ID NO: 60 and SEQ ID NO: 61 there may be a protospacer adjacent motif (PAM) downstream of the region targeted by the guide nucleic acid molecule of the present application, and the protospacer adjacent motif (PAM) may be CGG or GGG.
  • PAM protospacer adjacent motif
  • those skilled in the art can easily determine the target sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the PAM of the target gene, and at the same time design suitable gRNA for the target sequence.
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of a protospacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, GGG, and CGG .
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by CGG in the DNA where the BATF gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr14:75546524-75546543.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by GGG in the DNA where the BATF gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to A target sequence of about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the target sequence may be derived from human chr14:75546605-75546624.
  • the guide nucleic acid molecule can comprise a sequence as shown in any one of SEQ ID NO:62 and SEQ ID NO:63.
  • the proportion of cells expressing the product of the target gene in the TIL obtained by reducing the expression and/or weakening the activity of at least one target gene of the TIL compared with the TIL in which the expression and/or activity of the target gene is not changed The expression level of said gene of interest in individual cells may be reduced and/or may be reduced.
  • the target gene is expressed in the TIL obtained by reducing the expression and/or weakening the activity of at least one target gene of the TIL compared with the TIL whose expression and/or activity of the target gene are not changed
  • the cell fraction of the product is reduced by at least about 5%.
  • the proportion of cells expressing the product of the gene of interest is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the proportion of cells expressing the product of the gene of interest can range from an observable proportion of cells to 0%.
  • the proportion of cells expressing the product of the gene of interest can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, At least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the proportion of cells expressing the product of the target gene can be detected by flow cytometry.
  • the proportion of cells expressing the product of the target gene in the TIL obtained by reducing the expression and/or weakening the activity of at least one target gene of the TIL in the method of the present application may be at most about 95%.
  • the proportion of cells expressing the product of the gene of interest may be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40% %, up to about 30%, up to about 20%, up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15%, up to about 14%, up to about 13%, up to about 12% %, up to about 11%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, or up to about 5%.
  • the proportion of cells expressing the product of the target gene can be detected by flow cytometry.
  • the TIL obtained by reducing the expression and/or activity of at least one target gene of the TIL is described in a single cell.
  • the expression level of the gene of interest may be reduced by at least about 5%.
  • the expression of the gene of interest in a single cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least About 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the expression level of the target gene in a single cell can range from an observable amount to 0%.
  • the expression level of the target gene in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40% , at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12% , at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the expression level of the target gene in a single cell in the TIL obtained by reducing the expression and/or activity of at least one target gene of the TIL in the method of the present application can be the expression and/or activity of the target gene Up to about 95% of TIL is unchanged.
  • the amount of expression of the target in a single cell in the TIL can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most About 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15%, up to About 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%.
  • the method of the present application may further comprise: subjecting TILs derived from tumor tissue and not expanded in vitro to undergo at least one stage of in vitro expansion, wherein, in at least one stage of in vitro expansion of the present application , TILs of the present application can be co-cultured with feeder cells.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and/or the activity can be weakened and co-cultured with the feeder cells of the present application
  • the in vitro amplification of the present application in a single stage may refer to the in vitro amplification of the present application in the same stage, for example, the same in the first stage in vitro expansion of the present application, and the same in the second stage in vitro expansion of the present application. increase, or in vitro expansion in the third stage of this application, etc.
  • the expression and/or activity of at least one target gene of the TIL in the first stage of in vitro expansion of the present application, can be reduced and co-cultured with the feeder cells of the present application. In one embodiment, in the second stage of in vitro expansion of the present application, the expression and/or activity of at least one target gene of the TIL can be reduced and co-cultured with the feeder cells of the present application. In one embodiment, in the third stage of in vitro expansion of the present application, the expression and/or activity of at least one target gene of the TIL can be reduced and co-cultured with the feeder cells of the present application.
  • each stage of in vitro expansion can be divided by the change in the number of TIL cells.
  • TIL cells when the number of TIL cells increases by at least about 1-fold, TIL cells can be considered Into the next stage of in vitro expansion.
  • the number of TIL cells when the number of TIL cells is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times, or at least about 1000 times, TIL cells can be considered to enter the next stage of in vitro expansion.
  • each stage of in vitro expansion can also be divided by changing the conditions of TIL cell culture.
  • T cell activators and/or T cell growth factors are added or supplemented to the cell culture medium, it can be considered that the TIL cells enter the next stage of in vitro expansion.
  • TIL cells can be considered to enter the next stage of in vitro expansion when IL-2 is added or supplemented to the cell culture medium.
  • IL-2 is added or supplemented to the cell culture medium.
  • TIL cells can be considered to enter the next stage of in vitro expansion when feeder cells are added or supplemented to the cell culture medium.
  • each stage can also be divided by the days of TIL cell culture.
  • TIL cells when the TIL cells are cultured in vitro for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days 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, about 20 days, about 30 days, about 40 days, After about 50 days or about 100 days, TIL cells can be considered to enter the next stage of in vitro expansion.
  • the second stage of in vitro expansion can be performed for at least about 7 days.
  • the second stage of in vitro expansion can be performed for at least about 9 days.
  • the second stage of in vitro expansion can be performed for up to about 14 days.
  • the second stage of in vitro expansion can be performed for up to about 13 days.
  • the second stage of in vitro expansion can be performed for about 7 days to about 14 days, about 9 days to about 14 days, about 7 days to about 13 days, or about 9 days to about 13 days.
  • the second stage in vitro expansion of the present application can be performed for at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the second stage of in vitro expansion of the present application can be carried out for about 9 days to about 14 days
  • the second stage of in vitro expansion of the present application can be carried out for about 9 days to about 14 days, about 10 days to about 14 days, About 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days days to about 13 days, about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the second stage of in vitro expansion in the present application can be considered as the REP (rapid expansion protocol) stage.
  • the first stage of in vitro expansion can be performed for at least about 7 days.
  • the first stage of in vitro expansion can be performed for about 7 days to about 14 days.
  • the first stage of in vitro expansion of the present application can be performed for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least About 14 days.
  • the first stage of in vitro expansion of the present application can be performed for about 7 days to about 14 days, about 8 days to about 14 days, about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days to about 13 days , about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the first stage of in vitro expansion of the present application can be considered the preREP stage.
  • the number of days for the second-stage in vitro expansion of the present application can be calculated from the start of the second-stage in vitro expansion. For example, when the second-stage in vitro expansion starts, it can be considered that the second-stage in vitro expansion has been performed for about 0 days. For example, if the second-stage in vitro expansion proceeds for about 24 hours, it can be considered that the second-stage in vitro expansion has been performed for about one day. For example, the day when the second-stage in vitro expansion starts can be considered as about 0 days after the second-stage in vitro expansion.
  • the number of days for the second stage of in vitro expansion in the present application can be calculated based on the number of days for the second stage of in vitro expansion. For example, on the second day after the start of the second-stage in vitro expansion, it can be considered that the second-stage in vitro expansion has been performed for about one day.
  • the cultivation method of the present application can be divided into two steps.
  • the first TIL population derived from tumor tissue and not expanded in vitro can be contacted with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • the TIL population can be made The expression of at least one target gene of the second TIL population is reduced and/or the activity is weakened, and the second TIL population is co-cultured with feeder cells after being contacted with a T cell activator and/or a T cell growth factor for a certain period of time , wherein, the third TIL group is obtained through the step (B).
  • step (A) may be performed for about 7 days to about 14 days.
  • step (B) may be performed for about 7 days to about 14 days.
  • the cultivation method of the present application can be divided into three steps.
  • (A) the first TIL population derived from tumor tissue and not expanded in vitro can be contacted with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • (B) the TIL population can be made The expression of at least one target gene of the second TIL population is reduced and/or the activity is weakened, and the second TIL population can be contacted with a T cell activator and/or a T cell growth factor, wherein, after the step ( B) Obtaining the third TIL population;
  • (C) The third TIL population can be co-cultured with feeder cells, wherein the fourth TIL population is obtained through the step (C).
  • step (A) may be performed for about 7 days to about 14 days.
  • step (B) can be performed for about 0 days to about 8 days.
  • step (C) may be performed for about 5 days to about 14 days.
  • the cultivation method of the present application can be divided into four steps.
  • the first TIL population derived from tumor tissue and not expanded in vitro can be contacted with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • the TIL population can be made The second TIL population is contacted with a T cell activator and/or a T cell growth factor, wherein a third TIL population is obtained through the step (B);
  • at least one target of the third TIL population can be made The expression of the gene is reduced and/or the activity is weakened, wherein, the fourth TIL group is obtained through the step (C);
  • the fourth TIL group can be co-cultured with feeder cells, wherein, after the step (D) ) to obtain the fifth TIL group.
  • step (A) may be performed for about 7 days to about 14 days.
  • step (B) can be performed for about 0 days to about 4 days.
  • step (C) can be performed for about 0 days to about 4 days.
  • step (D) may be performed for about 5 days to about 14 days.
  • step (A) of the culturing method of the present application is to obtain a second TIL population from the recovered and/or continued culture of the in vitro TIL population.
  • the TIL population in vitro may comprise a TIL population obtained by in vitro expansion of a first TIL population derived from tumor tissue and not expanded in vitro.
  • the in vitro TIL population can comprise a TIL population obtained by contacting the first TIL population with a T cell growth factor.
  • said in vitro TIL population can comprise a TIL population obtained by cryopreservation of said first TIL population.
  • the in vitro TIL population may comprise a TIL population obtained by contacting the first TIL population with a T cell growth factor and cryopreserving.
  • the step (A) of the present application is to obtain the second TIL population from the recovered and/or continued culture of the in vitro TIL population, the step (A) can be carried out for about 2 hours to about 4 days.
  • the TILs of the present application in a single stage of the in vitro expansion of the present application, can be contacted with one or more T cell activators and/or one or more T cell growth factors of the present application for a certain period of time Afterwards, co-cultivate with the feeder cells of the present application.
  • the certain period of time of the application may be at least about 2 hours.
  • the certain period of time of the present application can be at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours or at least About 72 hours.
  • the certain period of time for this application may be from about 6 hours to about 72 hours.
  • the certain period of time of the present application can be about 6 hours to about 7 hours, about 6 hours to about 8 hours, about 6 hours to about 9 hours, about 6 hours to about 10 hours, about 6 hours to about About 11 hours, about 6 hours to about 12 hours, about 6 hours to about 13 hours, about 6 hours to about 14 hours, about 6 hours to about 15 hours, about 6 hours to about 16 hours, about 6 hours to about 17 hours hours hours, about 6 hours to about 18 hours, about 6 hours to about 19 hours, about 6 hours to about 20 hours, about 6 hours to about 21 hours, about 6 hours to about 22 hours, about 6 hours to about 23 hours, From about 6 hours to about 24 hours, from about 6 hours to about 36 hours, from about 6 hours to about 48 hours, from about 6 hours to about 60 hours, or from about 6 hours to about 72 hours.
  • the certain period of time of the present application can be about 12 hours to about 13 hours, about 12 hours to about 14 hours, about 12 hours to about 15 hours, about 12 hours to about 16 hours, about 12 hours to About 17 hours, about 12 hours to about 18 hours, about 12 hours to about 19 hours, about 12 hours to about 20 hours, about 12 hours to about 21 hours, about 12 hours to about 22 hours, about 12 hours to about 23 hours hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, or about 12 hours to about 72 hours.
  • the certain time of the present application can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, About 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours hours, about 23 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours.
  • the feeder cells of the present application may comprise antigen presenting cells.
  • the feeder cells of the present application may comprise one or more selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen-presenting cells.
  • the feeder cells of the present application may be peripheral mononuclear cells.
  • the feeder cells of the present application may be irradiated feeder cells.
  • the feeder cells of the present application can be isolated artificial antigen-presenting cells (aAPC), and the artificial antigen-presenting cells of the present application can comprise cells expressing HLA-A/B/C, CD64, CD80, ICOS-L and/or CD58 , and can be modified to express more than one T cell activator of the present application.
  • the feeder cells of the present application can be irradiated, for example, can be gamma irradiated, or can be X-ray irradiated.
  • the co-cultivation of the TIL of the present application and the feeder cell of the present application may comprise contacting the surface of the feeder cell of the present application with the surface of the TIL of the present application. In one embodiment, the co-cultivation of the TIL of the present application and the feeder cell of the present application comprises adding the feeder cell of the present application to the cell culture medium of the TIL of the present application.
  • the present application can add the feeder cells of the present application to the cell culture medium of the TIL of the present application at a ratio of about 40:1 to about 400:1 of the feeder cells of the present application to the TIL of the present application.
  • the present application can be about 40:1 to about 400:1, about 40:1 to about 300:1, about 40:1 to about 200:1, about 40:1 to about 100:1, about 40:1-about 90:1, about 40:1-about 80:1, about 40:1-about 70:1, about 40:1-about 60:1, about 40:1-about 50:1, about 50:1-about 400:1, about 60:1-about 400:1, about 70:1-about 400:1, about 80:1-about 400 :1, about 90:1-about 400:1, about 100:1-about 400:1, about 200:1-about 400:1, or about 300:1-about 400:1 of the present application
  • the ratio of the feeder cells to the TIL of the present application, the feeder cells of the present application are added to the cell
  • the method of the present application may further comprise: subjecting TILs derived from tumor tissue and not expanded in vitro to undergo at least one stage of in vitro expansion, wherein, in at least one stage of in vitro expansion of the present application , contacting TILs of the present application with one or more T cell activators.
  • said TILs are contacted with said one or more T cell activators during a single stage of in vitro expansion of the application.
  • the T cell activator may comprise an agonist for one or more targets selected from the group consisting of: CD3, CD28, HVEM, CD40L, OX40, and 4-1BB.
  • the expression and/or activity of at least one target gene of the TIL is reduced and combined with one or more T cell activators of the present application touch.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and can be combined with one or more T cell activation of the application. agent contact.
  • the expression and/or activity of at least one target gene of the TIL in the second stage of in vitro expansion of the application, can be reduced and can be combined with one or more T cell activation of the application. agent contact.
  • the expression and/or activity of at least one target gene of the TIL in the third stage of in vitro expansion of the present application, can be reduced and can be combined with the activation of one or more T cells of the present application. agent contact.
  • the expression and/or activity of at least one target gene of the TIL in a single stage of the in vitro expansion of the present application, can be reduced and/or the activity of the TIL can be substantially simultaneously reduced and the TIL can be combined with one of the present application. or multiple T cell activators.
  • the expression and/or activity of at least one target gene of the TIL in a single stage of the in vitro amplification of the present application, can be reduced first, for example, 2 hours in advance, 4 hours in advance , 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more T cell activators of the present application.
  • the TILs of the present application in the in vitro expansion of the present application in a single stage, can be contacted with one or more T cell activators of the present application first, for example, 2 hours in advance, 4 hours in advance. hours, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then reduce the expression and/or activity of at least one target gene of the TIL.
  • the expression and/or activity of at least one target gene of the TIL in the first stage of in vitro expansion of the present application, can be reduced and/or the activity of the TIL can be substantially simultaneously reduced and the TIL can be contacted with one or more of the present application. Exposure to multiple T cell activators. In one embodiment, in the second stage of in vitro expansion of the present application, the expression and/or activity of at least one target gene of the TIL can be reduced and/or the activity of the TIL can be substantially simultaneously reduced and the TIL can be contacted with one or more of the present application. Exposure to multiple T cell activators.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and/or the activity of at least one target gene of the TIL can be substantially simultaneously reduced and the TIL can be contacted with one or more of the present application. Exposure to multiple T cell activators.
  • the T cell activator of the present application may comprise one or more selected from the following group: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT , and their functionally active fragments.
  • the T cell activator of the present application may comprise an agonist of one or more targets selected from the following group: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the T cell activator of the present application may comprise antibodies selected from the group consisting of CD3, CD28, HVEM, CD40L, OX40 and 4-1BB and their antigen-binding fragments.
  • the T cell activator of the present application may comprise a CD3 agonist.
  • the T cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, it may be OKT3 from Miltenyi Biotech, or it may be SP34 from BD.
  • the T cell activator of the present application may comprise a CD28 agonist.
  • the T cell activator of the present application may comprise an anti-CD28 antibody and/or its antigen-binding fragment, such as Merck's 15E8, and the T cell activator of the present application may comprise CD80 and/or its function Active fragments and/or CD86 and/or functionally active fragments thereof, as well as recombinant proteins of the above substances.
  • the T cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain VL and heavy chain VH of Miltenyi Biotech's OKT3, may comprise the SP34 of BD Light chain VL and heavy chain VH.
  • the T cell activator of the present application may comprise a CD28 agonist.
  • the T cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, for example may comprise the light chain VL and heavy chain VH of Merck's 15E8.
  • the T cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of Miltenyi Biotech's OKT3, may comprise The light chain LCDR1-3 and heavy chain HCDR1-3 of SP34 of BD, the anti-CD3 antibody and/or antigen-binding fragment thereof of the present application may have CD3-binding ability.
  • the T cell activator of the present application may comprise a CD28 agonist.
  • the T cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, for example may comprise Merck's 15E8 light chain LCDR1-3 and heavy chain HCDR1-3, the present application's Anti-CD28 antibodies and/or antigen-binding fragments thereof may have CD28-binding ability.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VH of the heavy chain of the antibody.
  • the CDRs of this application may be defined according to IMGT nomenclature, the CDRs of this application may be defined according to Chothia, or the CDRs of this application may be defined according to Kabat.
  • the CD3 agonist of the present application may be a CD3 antibody or an antigen-binding protein thereof.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VH of the heavy chain of the antibody.
  • the CDRs of this application may be defined according to IMGT nomenclature, the CDRs of this application may be defined according to Chothia, or the CDRs of this application may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1, and the HCDR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 2 and 12;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR2, and the HCDR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 3 and 13;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR3, and the HCDR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 4 and 14;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise HCDR1-3, wherein the HCDR1 of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 2 and 12, and the HCDR2 of the present application can comprise SEQ ID NO: 3 and the amino acid sequence shown in any one of 13, and the HCDR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 4 and 14;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDR of the present application It may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same HCDR1-3 as OKT3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 3 amino acid sequence, and the HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 4; the CDR of the present application may be defined according to the IMGT nomenclature; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same HCDR1-3 as SP34, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 13
  • the amino acid sequence of the present application, and the HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 14; the CDR of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VL of the light chain of the antibody.
  • the CDRs of the present application may be defined according to the IMGT nomenclature, or the CDRs of the present application may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1, and the LCDR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 5 and 15;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR2, and the LCDR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 6 (DTS) and 16;
  • the CDR of the present application may be according to the IMGT nomenclature Defined;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR3, and the LCDR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 7 and 17;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1-3, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 5 and 15, and the LCDR2 of the present application may comprise SEQ ID NO: 6 (DTS) and the amino acid sequence shown in any one of 16, and the application LCDR3 can comprise the amino acid sequence shown in any one of SEQ ID NO:7 and 17;
  • the application CDR can be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same LCDR1-3 as OKT3, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 5, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 6 (DTS ), and the application LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 7; the CDR of the application may be defined according to the IMGT nomenclature; for example, the antigen binding protein of the application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same LCDR1-3 as SP34, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 15, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 16
  • the amino acid sequence of the present application, and the LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 17; the CDR of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise HCDR1-3 and LCDR1-3, wherein the HCDR1 of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 2 and 12, and the HCDR2 of the present application can comprise SEQ ID NO:
  • the amino acid sequence shown in any one of ID NO: 3 and 13 can comprise the amino acid sequence shown in any one of SEQ ID NO: 4 and 14, and the LCDR1 of this application can comprise SEQ ID NO: 5 and 15
  • the amino acid sequence shown in any one, the application LCDR2 can comprise the amino acid sequence shown in any one of SEQ ID NO: 6 (DTS) and 16, and the application LCDR3 can comprise SEQ ID NO: 7 and 17 in any One of the amino acid sequences shown;
  • the CDRs of this application can be defined according to IMGT nomenclature; the CDRs of this application can be defined according to Kabat; for example, the antigen binding protein of this application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1-3 and LCDR1-3 identical to OKT3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR2 of the present application may comprise SEQ ID NO :
  • the amino acid sequence shown in 3 the HCDR3 of the present application may contain the amino acid sequence shown in SEQ ID NO: 4
  • the LCDR1 of the present application may contain the amino acid sequence shown in SEQ ID NO: 5
  • the LCDR2 of the present application may contain the amino acid sequence shown in SEQ ID NO: 6
  • the amino acid sequence shown in (DTS), and the application LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 7;
  • the application CDR can be defined according to IMGT nomenclature;
  • the antigen binding protein of the application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same HCDR1-3 as SP34, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 13 HCDR3 of this application may include the amino acid sequence shown in SEQ ID NO: 14, LCDR1 of this application may include the amino acid sequence shown in SEQ ID NO: 15, and LCDR2 of this application may include the amino acid sequence shown in SEQ ID NO: 16 Sequence, and the LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 17; the CDR of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain variable region VH, and the VH of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 8 and 18; for example, The antigen-binding protein of the present application may have CD3-binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH as OKT3, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 8; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH as SP34, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 18; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a light chain variable region VL, and the VL of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 9 and 19; for example, The antigen-binding protein of the present application may have CD3-binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VL as OKT3, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 9; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VL as SP34, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 19; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, and the VH of the present application may comprise any one of SEQ ID NO: 8 and 18.
  • the amino acid sequence shown, the VL of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 9 and 19; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH and VL as OKT3, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 8, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 9 Amino acid sequence; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH and VL as SP34, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 18, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 19 Amino acid sequence; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise a heavy chain, and the heavy chain of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 10 and 20;
  • the present application The antigen binding protein may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain as OKT3, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 10;
  • the antigen-binding protein of the present application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain as SP34, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 20;
  • the antigen-binding protein of the present application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise a light chain, and the light chain of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 11 and 21;
  • the present application The antigen binding protein may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same light chain as OKT3, and the light chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 11;
  • the antigen-binding protein of the present application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same light chain as SP34, and the light chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 21;
  • the antigen-binding protein of the present application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain and a light chain
  • the heavy chain of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 10 and 20
  • the present application The light chain may comprise the amino acid sequence shown in any one of SEQ ID NO: 11 and 21; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain and light chain as OKT3, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 10, and the light chain of the present application can comprise SEQ ID NO : the amino acid sequence shown in 11; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain and light chain as SP34, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 20, and the light chain of the present application can comprise SEQ ID NO : the amino acid sequence shown in 21; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the CD28 agonist of the present application may be a CD28 antibody or an antigen-binding protein thereof.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VH of the heavy chain of the antibody.
  • the CDRs of the present application may be defined according to the IMGT nomenclature, or the CDRs of the present application may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1, and the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR2, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 23; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR3, and the HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 24; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1-3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 23, And the HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 24;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDR of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same HCDR1-3 as 15E8, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 23
  • the amino acid sequence of the HCDR3 of the present application may include the amino acid sequence shown in SEQ ID NO: 24; the CDR of the present application may be defined according to the IMGT nomenclature; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VL of the light chain of the antibody.
  • the CDRs of the present application may be defined according to the IMGT nomenclature, or the CDRs of the present application may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1, and the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 25; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR2, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 26 (AAS); the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application It may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • AAS amino acid sequence shown in SEQ ID NO: 26
  • the CDR of the present application may be defined according to the IMGT nomenclature
  • the CDR of the present application It may be defined according to Kabat
  • the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR3, and the LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 27; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1-3, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 26 (AAS).
  • Amino acid sequence, and the application LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27; the application CDR may be defined according to the IMGT nomenclature; the application CDR may be defined according to Kabat; for example, the antigen binding protein of the application May have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same LCDR1-3 as 15E8, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 26 (AAS ), and the application LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27; the CDR of the application may be defined according to the IMGT nomenclature; for example, the antigen binding protein of the application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1-3 and LCDR1-3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 23 HCDR3 of this application may include the amino acid sequence shown in SEQ ID NO: 24, LCDR1 of this application may include the amino acid sequence shown in SEQ ID NO: 25, and LCDR2 of this application may include the amino acid sequence shown in SEQ ID NO: 30 sequence, and the application LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26 (AAS); the application CDR may be defined according to the IMGT nomenclature; the application CDR may be defined according to Kabat; for example, the antigen of the application A binding protein may have CD28 binding ability.
  • the application CDR may be defined according to the IMGT nomenclature
  • the application CDR may be defined according to Kabat
  • the antigen of the application A binding protein
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1-3 and LCDR1-3 identical to 15E8, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 of the present application may comprise SEQ ID NO : the amino acid sequence shown in 23, the HCDR3 of the present application may contain the amino acid sequence shown in SEQ ID NO: 24, the LCDR1 of the present application may contain the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 of the present application may contain the amino acid sequence shown in SEQ ID NO: 26
  • the amino acid sequence shown in (AAS), and the application LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 27;
  • the application CDR can be defined according to IMGT nomenclature;
  • the antigen binding protein of the application can have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain variable region VH, and the VH of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 28 and 29; for example, The antigen-binding protein of the present application may have CD28-binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a VH identical to a 15E8, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 28; for example, the antigen-binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same VH as another 15E8, and the VH of the present application can comprise the amino acid sequence shown in SEQ ID NO: 29; for example, the antigen-binding protein of the present application can have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a light chain variable region VL, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 30; for example, the antigen-binding protein of the present application May have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VL as 15E8, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 30; for example, the antigen-binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, and the VH of the present application may comprise any one of SEQ ID NO:28 and 29.
  • the amino acid sequence shown in the present application VL may comprise the amino acid sequence shown in SEQ ID NO: 30; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH and VL as a 15E8, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 28, and the VL of the present application may comprise SEQ ID NO: 30
  • the amino acid sequence shown; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH and VL as another 15E8, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 29, and the VL of the present application may comprise SEQ ID NO: The amino acid sequence shown in 30; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise a heavy chain, and the heavy chain of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 31 and 32;
  • the present application The antigen binding protein may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain identical to a 15E8, and the heavy chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 31; for example, the antigen-binding protein of the present application may have CD28 binding capacity.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain as another 15E8, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 32; for example, the antigen-binding protein of the present application can be Has CD28 binding ability.
  • the antibody of the present application or its antigen binding protein can comprise a light chain, and the light chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 33;
  • the antigen binding protein of the present application can have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same light chain as 15E8, and the light chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 33;
  • the antigen-binding protein of the present application can have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain and a light chain
  • the heavy chain of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 31 and 32
  • the present application The light chain may comprise the amino acid sequence shown in SEQ ID NO: 33; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same heavy chain and light chain as a 15E8, and the heavy chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 31, and the light chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 31.
  • the amino acid sequence shown in ID NO: 33; for example, the antigen-binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same heavy chain and light chain as another 15E8, and the heavy chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 32, and the light chain of the present application may comprise The amino acid sequence shown in SEQ ID NO: 33; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • contacting the TIL of the present application with one or more T cell activators of the present application may comprise one or more methods selected from the following group: (1) activating the T cell of the present application (2) adding engineered cells expressing the T cell activator of the present application to the cell culture medium of TIL of the present application; (3) adding the T cell culture medium containing the TIL of the present application The solid phase medium of the cell activator was added to the cell culture medium of the TIL of the present application.
  • contacting the TILs of the present application with one or more T cell activators of the present application may comprise adding a solid phase medium comprising the T cell activators of the present application to the cell culture of the TILs of the present application. Base.
  • contacting the TILs of the present application with one or more T cell activators of the present application may comprise adding a solid phase medium comprising the CD28 antibody and the CD3 antibody of the present application to the cells of the TILs of the present application medium.
  • the initial concentration of the T cell activator in the TIL cell culture medium of the present application may be at least about 30 ng/mL.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of TIL of the present application can be at least about 30 ng/mL;
  • the initial concentration of the CD3 antibody of the present application in the cell culture medium of TIL of the present application can be at least about 30 ng/mL; 30ng/mL.
  • the selection of the initial concentration of the CD28 antibody of the present application can be independent of the selection of the initial concentration of the CD3 antibody of the present application; for example, the initial concentration of the CD28 antibody of the present application and the CD3 antibody of the present application in the cell culture medium of the TIL of the present application Any combination is possible.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the TIL of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL.
  • the initial concentration of the CD3 antibody of the present application in the cell culture medium of the TIL of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the TIL of the present application can be arbitrarily selected from about 30ng/mL-about 300ng/mL, and the initial concentration of the CD3 antibody of the present application in the cell culture medium of the TIL of the present application
  • the concentration can be arbitrarily selected from about 30ng/mL to about 300ng/mL, and the selection of the initial concentration of the CD28 antibody of the present application can be independent of the selection of the initial concentration of the CD3 antibody of the present application.
  • the solid phase media of the present application may have a diameter of about 500 nanometers to about 10 microns. In one embodiment, the diameter of the solid phase medium of the present application can be measured by a transmission electron microscope.
  • the diameter of the solid phase medium of the present application may be from about 1 nanometer to about 500 nanometers. In one embodiment, the diameter of the solid phase medium of the present application may be from about 100 nanometers to about 500 nanometers. In one embodiment, the diameter of the solid phase medium of the present application may be from about 200 nanometers to about 500 nanometers. In one embodiment, the diameter of the solid phase medium of the present application can be measured by a transmission electron microscope.
  • the solid phase medium of the present application may comprise a polymer. In one embodiment, the solid phase medium of the present application may comprise dextran.
  • each mg of the solid medium of the present application comprises at least about 25 ⁇ g of the T cell activator of the present application.
  • the solid phase medium comprising the T cell activator of the present application is added to the cell culture of the TIL of the present application at a ratio of the solid phase medium of the present application to the TIL of the present application of about 1:100-about 1:2000 Base. In one embodiment, the solid phase medium comprising the T cell activator of the present application is added to the cell culture of the TIL of the present application at a ratio of the solid phase medium of the present application to the TIL of the present application of about 2:1 to about 1:2 Base.
  • the ratio of the solid phase medium of the present application to the TIL of the present application can be about 2:1 to about 1:2.
  • the solid phase medium of cell activator was added to the cell culture medium of TIL in this application.
  • the diameter of the solid phase medium of the present application when the diameter of the solid phase medium of the present application is about 100 nanometers to about 500 nanometers, it can be about 2:1-about 1:2, about 2:1-about 1:1, or about 1:1 -The ratio of the solid phase medium of the present application to the TIL of the present application is about 1:2, the solid phase medium comprising the T cell activator of the present application, such as CD3 agonist and/or CD28 agonist is added to the cell culture medium of the TIL of the present application middle.
  • the solid phase medium comprising the T cell activator of the present application such as CD3 agonist and/or CD28 agonist
  • the ratio of the solid phase medium of the present application to the TIL of the present application can be about 1:100-about 1:2000.
  • the solid phase medium of cell activator was added to the cell culture medium of TIL in this application.
  • the diameter of the solid phase medium of the present application when the diameter of the solid phase medium of the present application is about 100 nanometers to about 500 nanometers, it can be about 1:100-about 1:2000, about 1:200-about 1:2000, about 1:300- About 1:2000, about 1:400-about 1:2000, about 1:500-about 1:2000, about 1:600-about 1:2000, about 1:700-about 1:2000, about About 1:800-about 1:2000, about 1:900-about 1:2000, about 1:1000-about 1:2000, about 1:1200-about 1:2000, about 1:1400-about 1 :2000, about 1:1600-about 1:2000, or about 1:1800-about 1:2000 of the ratio of the solid phase medium of the present application to the TIL of the present application, for example, the CD28 agonist and CD3 agonist of the present application can be included
  • the solid medium of the agent was added to the cell culture medium of TIL in this application.
  • the method of the present application may further comprise: subjecting TILs derived from tumor tissue and not expanded in vitro to undergo at least one stage of in vitro expansion, wherein, in at least one stage of in vitro expansion of the present application , contacting TILs of the present application with one or more T cell growth factors.
  • TILs of the present application may be contacted with a T cell activator of the present application and contacted with one or more T cell growth factors of the present application in a single stage of in vitro expansion of the present application.
  • the TIL of the present application in the first stage of in vitro expansion of the present application, can be contacted with the T cell activator of the present application and contacted with one or more T cell growth factors of the present application.
  • the TIL of the present application in the second stage of in vitro expansion of the present application, can be contacted with the T cell activator of the present application and contacted with one or more T cell growth factors of the present application.
  • the TILs of the present application in the third stage of in vitro expansion of the present application, can be contacted with the T cell activator of the present application and contacted with one or more T cell growth factors of the present application.
  • the expression and/or activity of at least one target gene of the TIL is reduced and the TIL is contacted with a T cell growth factor substantially simultaneously. In one embodiment, in a single stage of the in vitro expansion of the present application, the expression and/or activity of at least one target gene of said TIL is reduced and the TIL is contacted with a T cell growth factor substantially simultaneously.
  • the expression and/or activity of at least one target gene of the TIL can be reduced first, for example, 2 hours in advance, 4 hours in advance , 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact the TIL of the present application with one or more T cell growth factors of the present application.
  • the TIL of the present application in the in vitro expansion of the present application in a single stage, can be contacted with one or more T cell growth factors of the present application first, for example, 2 hours in advance, 4 hours in advance hours, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then reduce the expression and/or activity of at least one target gene of the TIL.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and the TIL can be contacted with T cell growth factors substantially at the same time.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and the TIL can be contacted with T cell growth factors substantially simultaneously.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and the TIL can be contacted with T cell growth factors substantially at the same time.
  • the T cell growth factor of the present application can be selected from one or more of the following groups: IL-2, IL-7, IL-12, IL-15, IL-21, interferon gamma, and their functionally active fragments.
  • the T cell growth factor of the present application may comprise IL-2 and/or a functionally active fragment thereof.
  • a functionally active fragment of IL-2 may comprise a fragment of IL-2 known in the art that binds to the IL-2 receptor of a T cell.
  • contacting the TIL of the present application with one or more T cell growth factors of the present application may comprise adding the T cell growth factor of the present application to the cell culture medium of the TIL of the present application.
  • the initial concentration of the T cell growth factor of the present application in the cell culture medium of the TIL of the present application can be at least about 300 IU/mL.
  • the initial concentration of IL-2 of the present application in the cell culture medium of TIL of the present application can be at least about 350IU/mL, at least about 400IU/mL, at least about 500IU/mL, at least about 600IU/mL, At least about 700 IU/mL, at least about 800 IU/mL, at least about 900 IU/mL, at least about 1000 IU/mL, at least about 1100 IU/mL, at least about 1200 IU/mL, at least about 1300 IU/mL, at least about 1400 IU/mL, at least about 1500 IU/mL, at least about 2000 IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL, at least about 2700 IU/mL, at least about 2800 IU/mL, at least about 2900 IU/mL, at least about 3000 IU/mL, at least about 3100 IU/mL mL, at least about 3200
  • the TIL of the present application may be the TIL derived from the fragments of the tumor tissue of the present application. In one embodiment, the TIL of the present application can be obtained by processing tumor tissue into tumor fragments. In one embodiment, the tumor fragments of the present application have a volume of about 1-27 cubic millimeters.
  • the tumor fragments of the present application have a volume of about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters , about 8 cubic millimeters, about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 15 cubic millimeters, about 17 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters , about 21 cubic millimeters, about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters, or about 27 cubic millimeters.
  • the TILs may be selected from the group consisting of TILs derived from fragments of tumor tissue, TILs derived from fragments of lymphatic metastases, TILs derived from pleural effusions, TILs derived from peritoneal effusions, and TILs derived from cryopreserved After resuscitating the TIL.
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor , wherein, the second TIL population is obtained through the step (A); (B) reducing the expression and/or activity of at least one target gene of the TIL, and making the second TIL population interact with T cells The activator and/or the T cell growth factor are contacted for a certain period of time and then co-cultured with the feeder cells, wherein the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: (A) resuscitating and/or continuing to cultivate an in vitro TIL population to obtain a second TIL population, wherein the in vitro TIL population comprising a TIL population obtained by in vitro expansion of a first TIL population, the first TIL population is a TIL population derived from tumor tissue and not amplified in vitro; (B) expressing at least one target gene of the TIL reduced and/or weakened in activity, and the second TIL population is co-cultured with feeder cells after being in contact with T cell activators and/or T cell growth factors for a certain period of time, wherein the third TIL population is obtained through the step (B) TIL group.
  • TIL tumor-infiltrating lymphocytes
  • TIL populations derived from tumor tissue and not expanded in vitro can be contacted with T cell growth factors at a certain time and/or at a certain location to obtain TIL populations in vitro.
  • the TIL populations in vitro can be continuously cultivated , to carry out step (B), on the other hand, the in vitro TIL population can be cryopreserved first, and the in vitro TIL population can be revived when necessary, and then step (B) can be carried out.
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor , wherein, the second TIL population is obtained through the step (A); (B) the expression and/or activity of the target gene of the TIL is reduced and/or the activity is weakened, and the second TIL population is combined with a T cell activator and /or co-cultivate with feeder cells after being exposed to T cell growth factors for a certain period of time, wherein the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor , wherein, the second TIL population is obtained through the step (A); (B) the proportion of cells expressing the target gene in the TIL is about 95% or less, and the second TIL population is compared with TIL The cell activator and/or the T cell growth factor are contacted for a certain period of time and then co-cultured with the feeder cells, wherein the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the first-stage in vitro expansion of the present application can be optionally used interchangeably with step (A) in the method of the above aspects.
  • the second-stage in vitro expansion of the present application can be used in any substitution with step (B) in the method of the above aspect.
  • the TILs expanded in vitro in the first stage of the present application can be used in any substitution with the second population of TILs obtained through step (A) of the method of the above aspects.
  • the second-stage in vitro expanded TILs of the present application can be used in any substitution with the third TIL population obtained through step (B) of the method of the above aspect.
  • the third-stage in vitro amplification of the present application can be used in any replacement with any added step (C) in the method of the above aspect.
  • the third-stage in vitro expanded TILs of the present application can be used in any replacement with the fourth TIL population obtained through any additional step (C) in the method of the above aspects .
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The expression and/or activity of at least one target gene of the TILs is reduced and/or the activity is weakened, and the TILs are co-cultured with the feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The expression and/or activity of the target gene of the TIL is reduced, and the TIL is co-cultured with the feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The proportion of cells expressing the target gene in the TIL is about 95% or less, and the TIL is co-cultured with feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The proportion of cells expressing the target gene in the TIL is about 95% or less, and the TIL is co-cultured with the feeder cells after at least 2 hours; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The proportion of cells expressing the target gene in the TIL is about 95% or less, and after at least 2 hours, the TIL is co-cultured with feeder cells, which may comprise peripheral mononuclear cells, and the feeder cells are added to the cell culture medium of the TIL; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The proportion of cells expressing the target gene in the TIL is about 95% or less, and after at least 2 hours, the TIL is co-cultured with feeder cells, which may include peripheral mononuclear cells, and may be at about 40: The ratio of the feeder cells to the TILs is about 1-400:1, the feeder cells are added to the cell culture medium of the TILs; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 ; Wherein, the second TIL group is obtained through the step (A); (B) the second TIL group can be contacted with IL-2, contacted with various T cell activators, and expressed in the TIL
  • the ratio of cells with the gene of interest is about 95% or less, and after at least 2 hours, the TILs are co-cultured with feeder cells, which may comprise peripheral mononuclear cells, and may be in a ratio of about 40:1 to about 400:1
  • the ratio of the feeder cells to the TIL, the feeder cells are added to the TIL cell culture medium; wherein, the third TIL population is obtained through the step (B).
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 , the initial concentration of the IL-2 in the cell culture medium of the TIL can be at least about 300IU/mL; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be made
  • the TIL population is contacted with IL-2, which may have an initial concentration of at least about 300 IU/mL in the cell culture medium of the TILs, with a CD3 antibody, the CD3 antibody in the cell culture medium of the TILs an initial concentration of at least about 30 ng/mL such that the proportion of cells in the TIL expressing the gene of interest is about 95% or less, and after at least 2 hours the TIL is co-cultured with feeder cells, which may comprise Peripheral mononuclear cells, the feeder cells can be added to the cell culture
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 ; Wherein, the second TIL group is obtained through the step (A); (B) the second TIL group can be contacted with IL-2, contacted with a nano-matrix comprising CD3 antibody and CD28 antibody, and made in the TIL The proportion of cells expressing the target gene is about 95% or less, and the TILs are co-cultured with feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 ; Wherein, the second TIL group is obtained through the step (A); (B) the second TIL group can be contacted with IL-2, with the nano-matrix comprising CD3 antibody and CD28 antibody, and the nano-matrix of the nano-matrix The diameter can be about 1 nanometer to about 500 nanometers, and each mg of the nanomatrix can contain about 25 ⁇ g of CD3 antibody and CD28 antibody, so that the proportion of cells expressing the target gene in the TIL is about 95% or less, And the TILs are co-cultured with the feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 ; Wherein, the second TIL group is obtained through the step (A); (B) the second TIL group can be contacted with IL-2, with the nano-matrix comprising CD3 antibody and CD28 antibody, and the nano-matrix of the nano-matrix
  • the diameter can be about 1 nanometer to about 500 nanometers, and each mg of the nanomatrix can contain about 25 ⁇ g of the CD3 antibody and the CD28 antibody, and the nanomatrix and the TIL can be in a ratio of about 1:100 to about 1:2000
  • the proportion of adding to the cell culture medium of the TIL, so that the proportion of cells expressing the target gene in the TIL is about 95% or less, and co-cultivating the TIL and the feeder cells; wherein, through the step (B) Obtaining
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 , the initial concentration of the IL-2 in the cell culture medium of the TIL can be at least about 300IU/mL; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be made
  • the population of TILs is contacted with IL-2, which may have an initial concentration of at least about 300 IU/mL in a cell culture medium of the TILs, with a nanomatrix comprising an antibody to CD3 and an antibody to CD28, the nanomatrix having The diameter can be about 1 nanometer to about 500 nanometers, and each mg of the nanomatrix can contain about 25 ⁇ g of the CD3 antibody and the CD28 antibody, and the nanomatrix and the TIL can be in a ratio of about 1:100 to about 1:2000
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • the method of obtaining TIL cells from a tissue sample of a subject can be obtained by surgically obtaining an orthotopic tumor sample or a metastatic tumor sample, the weight of which can be at least about 1 g, or multiple pieces of tissue can be combined.
  • the tumor tissue is transported at about 2-8 degrees in the sample transport solution, for example, a commonly used commercial tumor tissue transport solution, tumor tissue preservation solution or tumor tissue transfer solution, and processed within 48 hours.
  • the tissue pieces can be mechanically broken to a size of about 1-27 cubic millimeters per piece, transferred into a gas-permeable culture bag or Grex, and T cell serum-free medium is added with a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/mL, For example, 6000 IU/mL) of IL-2 can be cultured for about 3-14 days.
  • T cell serum-free medium is added with a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/mL, For example, 6000 IU/mL) of IL-2 can be cultured for about 3-14 days.
  • the harvested TIL cells can be frozen and resuscitated, or the cells in the culture medium can be directly collected and transferred into a gas-permeable culture bag, or Grex, or Xuri equipment, and the CD28 antibody and CD3 antibody of this application can be added to the T cell serum-free medium And CD28 antibody, magnetic beads comprising CD3 antibody and CD28 antibody (such as Dynabeads) and/or nano-matrix (such as transACT) comprising CD3 antibody and CD28 antibody, the concentration is 300-9000IU/mL (for example, it can be 1000-9000IU/mL , for example, can be 6000IU/mL) of IL-2, and with carrying SEQ ID NO: 36-37,40-41,43,48-51,54-55,58-59,62-63 and 70-75
  • RNP ribonucleoprotein complex
  • Cells in the culture medium can be collected using a cell processing system, washed and frozen, and detected.
  • the CD3 ratio of the final product can be greater than 80%, the cell viability can be greater than 50%, and the T cells greater than 80% can be memory effector T cells and effector T cells.
  • IFN- ⁇ can be secreted after stimulation, and/or can be characterized by an up-regulated proportion of activated T cells.
  • the present application provides a tumor infiltrating lymphocyte (TIL), and the TIL of the present application can be cultured according to the cultivation method of the present application.
  • TILs provided in the present application may comprise one or a batch of TILs cultured by the cultivation method of the present application.
  • the TIL provided in the present application may comprise multiple or multiple batches of TIL cultured by the cultivation method of the present application and combined in any ratio.
  • TILs expanded using the methods of the present application can be administered to a patient as a pharmaceutical composition.
  • the pharmaceutical composition may be a suspension of TIL in a sterile buffer.
  • TILs expanded using the PBMCs of the present application can be administered by any suitable route known in the art.
  • T cells can be administered as a single intra-arterial or intravenous infusion, which can last for about 30 to 60 minutes. Other suitable routes of administration may include intraperitoneal, intrathecal and intralymphatic administration.
  • any suitable dose of TIL can be administered.
  • about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 TILs may be administered.
  • about 1 ⁇ 10 9 to about 12 ⁇ 10 10 TILs may be administered.
  • about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 TILs may be administered.
  • about 3 ⁇ 10 10 to about 12 ⁇ 10 10 TILs may be administered.
  • about 4 ⁇ 10 10 to about 10 ⁇ 10 10 TILs may be administered.
  • about 5 ⁇ 10 10 to about 8 ⁇ 10 10 TILs may be administered.
  • about 6 ⁇ 10 10 to about 8 ⁇ 10 10 TILs may be administered.
  • the therapeutically effective dose may be from about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 . In some embodiments, the therapeutically effective dose may be from about 1 ⁇ 10 9 to about 12 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 3 ⁇ 10 10 to about 12 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 4 ⁇ 10 10 to about 10 ⁇ 10 10 TILs.
  • a therapeutically effective dose may be from about 5 ⁇ 10 10 to about 8 ⁇ 10 10 TILs. In some embodiments, a therapeutically effective dose may be from about 6 ⁇ 10 10 to about 8 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 7 ⁇ 10 10 to about 8 ⁇ 10 10 TILs.
  • the amount of TIL provided in the composition of the present application can be about 1 ⁇ 10 6 , about 2 ⁇ 10 6 , about 3 ⁇ 10 6 , about 4 ⁇ 10 6 , about 5 ⁇ 10 6 , about 6 ⁇ 10 6 , about 7 ⁇ 10 6 , about 8 ⁇ 10 6 , about 9 ⁇ 10 6 , about 1 ⁇ 10 7 , about 2 ⁇ 10 7 , about 3 ⁇ 10 7 , about 4 ⁇ 10 7 , about 5 ⁇ 10 7 , about 6 ⁇ 10 7 , about 7 ⁇ 10 7 , about 8 ⁇ 10 7 , about 9 ⁇ 10 7 , about 1 ⁇ 10 8 , about 2 ⁇ 10 8 , about 3 ⁇ 10 8 , about 4 ⁇ 10 8 , about 5 ⁇ 10 8 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 , about 8 ⁇ 10 8 , about 9 ⁇ 10 8 , about 1 ⁇ 10 9 , about 2 ⁇ 10 9 , about 3 ⁇ 10 9 , about 4 ⁇ 10 9 , about 5 ⁇ 10 9 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 , about
  • the amount of TIL provided in the compositions of the present application may range from about 1 ⁇ 10 6 to 5 ⁇ 10 6 , about 5 ⁇ 10 6 to 1 ⁇ 10 7 , about 1 ⁇ 10 7 to 5 ⁇ 10 7 , about 5 ⁇ 10 7 to 1 ⁇ 10 8 , about 1 ⁇ 10 8 to 5 ⁇ 10 8 , about 5 ⁇ 10 8 to 1 ⁇ 10 9 , about 1 ⁇ 10 9 to 5 ⁇ 10 9 , about 5 ⁇ 10 9 to 1 ⁇ 10 10 , about 1 ⁇ 10 10 to 5 ⁇ 10 10 , about 5 ⁇ 10 10 to 1 ⁇ 10 11 , about 5 ⁇ 10 11 to 1 ⁇ 10 12 , about 1 ⁇ 10 12 to 5 ⁇ 10 12 , or about 5 ⁇ 10 12 to 1 ⁇ 10 13 .
  • the concentration of TIL provided in the composition of the present application can be less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40% of the composition , about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2% , about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%, about 0.009%, about 0.008%, about 0.007%, about 0.006%, about 0.005%, about 0.004%, about 0.003%, about 0.002%, about 0.001%, about 0.0009%, about 0.0008%, about 0.0007%, about
  • the concentration of TIL provided in the composition of the present application can be greater than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, About 20%, about 19.75%, about 19.50%, about 19.25%, about 19%, about 18.75%, about 18.50%, about 18.25%, about 18%, about 17.75%, about 17.50%, about 17.25%, about 17 %, about 16.75%, about 16.50%, about 16.25%, about 16%, about 15.75%, about 15.50%, about 15.25%, about 15%, about 14.75%, about 14.50%, about 14.25%, about 14%, About 13.75%, about 13.50%, about 13.25%, about 13%, about 12.75%, about 12.50%, about 12.25%, about 12%, about 11.75%, about 11.50%, about 11.25%, about 11%, about 10.75 %, about 10.50%, about 10.25%, about 10%, about 9.75%, about 9.50%, about 9.25%, about 9%
  • the concentration of TIL provided in the composition of the present application can range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% of the composition to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17% , about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v /v.
  • the concentration of TIL provided in the compositions of the present application may range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% of the composition to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w/w, w/v or v/v.
  • the amount of TIL provided in the compositions of the present application may be equal to or less than about 10 g, about 9.5 g, about 9.0 g, about 8.5 g, about 8.0 g, about 7.5 g, about 7.0 g, about 6.5 g, about 6.0g, about 5.5g, about 5.0g, about 4.5g, about 4.0g, about 3.5g, about 3.0g, about 2.5g, about 2.0g, about 1.5g, about 1.0g, about 0.95g, About 0.9g, about 0.85g, about 0.8g, about 0.75g, about 0.7g, about 0.65g, about 0.6g, about 0.55g, about 0.5g, about 0.45g, about 0.4g, about 0.35g, about 0.3 g, about 0.25g, about 0.2g, about 0.15g, about 0.1g, about 0.09g, about 0.08g, about 0.07g, about 0.06g, about 0.05g, about 0.04g, about 0.03g, about 0.02g
  • the amount of TIL provided in the compositions of the present application may be greater than about 0.0001 g, about 0.0002 g, about 0.0003 g, about 0.0004 g, about 0.0005 g, about 0.0006 g, about 0.0007 g, about 0.0008 g , about 0.0009g, about 0.001g, about 0.0015g, about 0.002g, about 0.0025g, about 0.003g, about 0.0035g, about 0.004g, about 0.0045g, about 0.005g, about 0.0055g, about 0.006g, about 0.0065g, about 0.007g, about 0.0075g, about 0.008g, about 0.0085g, about 0.009g, about 0.0095g, about 0.01g, about 0.015g, about 0.02g, about 0.025g, about 0.03g, about 0.035g , about 0.04g, about 0.045g, about 0.05g, about 0.055g, about 0.06g,
  • TILs can be administered in a single dose. Such administration may be by injection, for example intravenously. In some embodiments, TILs can be administered in multiple doses. Doses may be once, twice, three, four, five, six or more than six times per year. Dosage can be monthly, biweekly, weekly, or every 2 days. In some embodiments, the administration of TILs can be administered continuously.
  • the present application provides a pharmaceutical composition.
  • it may comprise the TIL of the present application and/or the composition of the present application, and a pharmaceutically acceptable carrier.
  • the present application provides a kit
  • the kit of the present application may comprise the T cell activator, T cell growth factor and/or feeder cells of the method for culturing tumor infiltrating lymphocytes (TIL) of the present application and the method for culturing tumors described in the present application Description of the steps of the infiltrating lymphocyte (TIL) method.
  • the present application provides a kit, which may comprise the TIL of the present application and/or the pharmaceutical composition of the present application.
  • the present application provides a method for affecting the growth of tumor cells, which may include administering the TIL of the present application and/or the pharmaceutical composition of the present application to a subject.
  • affecting tumor growth can comprise reducing the volume of the tumor to, for example, about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40% of its pre-administration volume.
  • the present application provides the application of the TIL of the present application and/or the pharmaceutical composition of the present application in the preparation of medicines, and the medicines of the present application can be used to prevent and/or treat tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • the present application provides a method for preventing and/or treating tumors, which may include administering the TIL of the present application and/or the pharmaceutical composition of the present application to a subject.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • the present application provides a TIL of the present application and/or the pharmaceutical composition of the present application, which can be used for preventing and/or treating tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • PBMC peripheral blood mononuclear cells
  • red blood cells If there are many red blood cells, you can split the red blood cells after centrifugation. Add the red blood cell lysate according to the volume of the cell pellet and the red blood cell lysate at a ratio of 1:2 to 1:3, mix well, and lyse at room temperature for 10 minutes. Mix gently in the middle of the centrifuge tube 2- 3 times to ensure the lysis effect. After the lysis is completed, add PBS or saline to wash the cells. After cleavage, the cells were washed twice, centrifuged at 400g for 6 minutes, and samples were taken and counted before the last centrifugation.
  • Discard the supernatant resuspend the cells in the basal medium, adjust the cell density to about 2-3 ⁇ 107 cells/mL, the liquid level may not exceed 1 cm, and the volume in each T225 culture bottle may be less than 200 mL; , X-ray irradiation 50Gy.
  • the supernatant was discarded by centrifugation, and the cells were frozen according to the counting results, about 1-2 ⁇ 108 cells/mL, 1-2 mL/vessel; the cells were placed in a programmed cooling box and transferred to a -80°C refrigerator for freezing.
  • cpro separation kit Aseptically connect the tubing of the blood bag to the input end of the cpro separation kit (Cytiva). If the blood volume is greater than 120 mL, a pre-concentration step is performed to concentrate the blood volume to less than 120 mL.
  • the neatcell program can be used to separate and wash PBMCs.
  • the washing liquid is physiological saline, with an intermediate volume of 20 mL; the resuspension liquid is the basal medium, and 80 mL/batch is added.
  • each donor’s PBMC is a bag of 100mL. In the flat state, the height of the liquid level can not exceed 1 cm, and the X-ray irradiation is 50Gy.
  • the washing solution is normal saline; set the intermediate volume and final volume so that every 1 ⁇ 10 9 cells are not less than 2 mL; add an equal amount to 2 times for freezing Mix well. Adjust the cell density from 1 ⁇ 10 7 cells/mL to 2 ⁇ 10 8 cells/mL with 1-times cryopreservation solution, aliquot 20 mL/bag, freeze in a programmed cooling apparatus, and store in liquid nitrogen.
  • PBMC cells in blood samples were separated and frozen according to the above PBMC manual separation and cryopreservation procedures. Take a culture bottle or culture bag with a gas-permeable surface, such as a culture bag (Origen), and add 300 mL of rewarmed complete medium.
  • a culture bag with a gas-permeable surface, such as a culture bag (Origen)
  • the complete medium can be optionally selected from X-vivo15 medium or other commercial T cell culture medium , such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and can add essential amino acids and antibiotics, and the added concentration is 300-9000IU/mL (for example, it can be 1000-9000IU/mL, for example, it can be 6000 IU/mL) of IL-2.
  • T cell culture medium such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium
  • the added concentration is 300-9000IU/mL (for example, it can be 1000-9000IU/mL, for example, it can be 6000 IU/mL) of IL-2.
  • ophthalmic scissors and ophthalmic tweezers to make preliminary cuts to remove fat tissue and necrotic tissue, and continue to cut each tissue block to a size of about 27 cubic millimeters.
  • take the non-suspended tumor tissue block use a 20mL syringe to remove the internal piston, connect it to the culture bag, and use a pipette to transfer about 1g of the tissue block into the culture bag through the syringe. Put the culture bag into the carbon dioxide incubator for cultivation.
  • the scissors and tweezers were cleaned, and after initial disinfection with 75% alcohol, ultrasonic cleaning and sterilization were performed to obtain the first TIL group.
  • the liquid should be replaced every 3-7 days or half of the liquid should be replaced to ensure the nutrition of the cells.
  • Use complete medium complete medium can choose X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium, and can add necessary Amino acids and antibiotics, and IL-2 (Double Heron) with a concentration of 300-9000IU/mL (for example, 1000-9000IU/mL, for example, 6000IU/mL) was added.
  • T cell medium such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium
  • IL-2 Double Heron
  • samples can be taken and counted on the 13th or 14th day. If the number of cells is between 5 ⁇ 10 5 and 5 ⁇ 10 8 , enter the harvesting step of step (A).
  • step (A) Collect the cells after step (A) in vitro expansion, centrifuge, discard the medium, wash the cells once with PBS or normal saline, obtain the TILs amplified in vitro (the second TIL population) in step (A), and take a sample for counting About 5 ⁇ 10 5 to 2 ⁇ 10 8 cells enter the subsequent in vitro expansion step; about 5 ⁇ 10 5 cells can be taken for quality control testing; the rest of the cells are added to the cryopreservation solution and stored as cryopreserved preREP TIL in vitro cells .
  • step (A) Continue to culture the TILs (second TIL population) amplified in step (A) in vitro, or perform cell recovery on the frozen preREP TILs in vitro to activate TILs in step (B).
  • complete medium can choose X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium, and can add necessary For amino acids and antibiotics, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 cells/mL, place in a suspended 24-well culture plate, 1 mL/well, and add a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/ mL, for example may be 6000 IU/mL or 6000 IU/mL) of IL-2.
  • T cell medium such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium
  • T cell activators such as CD3 agonists and/or CD28 agonists
  • CD3 antibody Miltenyi Biotech, OKT3
  • CD28 Antibody Merck, 15E8
  • magnetic beads diameter about 1 to 10 ⁇ m Dynabeads, Thermo Fisher
  • the ratio of transACT (approximately 100 to 500 nm in diameter, Miltenyi) to TIL was added to transACT. After culturing for about 0-4 days, the third TIL population was obtained.
  • gRNA of the present application (sequence shown in any one of SEQ ID NO: 36-37, 40-41, 43, 48-51, 54-55, 58-59, 62-63 and 70-75) And add nuclease-free water to adjust the concentration to about 100 ⁇ M.
  • Cas9 Cas9
  • the above RNP was electroporated with about 1 ⁇ 10 6 cells of the third TIL population by a Lonza electroporator.
  • the electroporation procedure can be human T cell stim (EO115).
  • the fourth TIL population was obtained by culturing for about 0-4 days after gene editing by electroporation.
  • Tn for each test group can be taken from 0 hours to 12 days, such as 24 hours or 48 hours).
  • step (D) First resuscitate the feeder cells mixed with 1-5 donors; mix the activated TIL cells and feeder cells according to the ratio of TIL cells:feeder cells about 1:200, transfer them into G-Rex100 culture flasks or air-permeable bags, and replenish completely Culture medium, sampling and counting every 1-3 days, and rehydration or half-quantity replacement according to the cell state until the total number of cells is greater than 1 ⁇ 10 9 or step (D) in vitro expansion and culture for about 5 days to about 14 days, and step (D) is terminated Culture for in vitro expansion.
  • step (D) Take the cells amplified in step (D), discard the medium supernatant after centrifugation, and wash three times with PBS or physiological saline or compound electrolyte solution to obtain TILs amplified in step (D) (the fifth TIL group), the first Sampling and counting during the three washes, according to the counting results, discard the supernatant after the last centrifugation, take 3 ⁇ 10 6 cells and send them to quality control testing; add all the remaining cells to the cryopreservation solution, adjust the cell density to 1-3 ⁇ 10 8 cells/ mL frozen.
  • Tn can range from 0 hour to 14 days
  • Tn is selected from 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 5 days, 7 days, and 9 days to obtain TIL cultured at different addition times of feeder cells, and compare the cell counts test.
  • FIG. 1 shows the analysis results of the proliferation ability of TIL cultured with feeder cells at different addition times.
  • the values on the vertical axis in each group of graphs in which TIL was cultured with feeder cells at different addition times indicate the expansion factor of the number of TIL cells after the end of in vitro expansion compared with before the start of in vitro expansion.
  • the proliferation results of TILs from 4 donors showed that the TILs cultured with feeder cells added 0 hours after the addition of OKT3 and IL-2 (that is, at the same time), the proliferation ability was weaker than that of 24 hours or 48 hours after the addition of OKT3 and IL-2 Then add feeder cell cultured TIL.
  • T n can range from 0 hour to 14 days
  • Tn is selected from 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 5 days, 7 days, and 9 days to obtain TIL cultured at different addition times of feeder cells, and perform flow detection Comparative Test.
  • Transcription Factor Buffer Set manufacturer BD, product number 562574; V-bottom 96-well plate, manufacturer Corning, product number 3894; flow tube, manufacturer Corning, product number 352052.
  • the flow cytometry antibodies in this example were purchased from BD or Biolegend. Add 1 ⁇ 10 5 to 5 ⁇ 10 5 cell samples in each group into flow tubes or V-bottom 96-well plates. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, flow tube 1mL/tube, 96-well plate 200 ⁇ L/well, discard supernatant. Add the prepared antibody working solution for cell surface staining, the antibody (BD or Biolegend) concentration is 1:100 to 1:200, and the activity detection dye is 1:10000. Flow tube 100 ⁇ L/tube, 96-well plate 50 ⁇ L/well for staining, incubate at 2-8°C for 30 minutes in the dark.
  • BD Transcription Factor Buffer Set
  • BD Fixation/Permeabilization
  • BD Fixation/Permeabilization
  • BD Fixation/Permeabilization
  • BD Perm/Wash Buffer
  • Cell fixation and membrane rupture Sufficiently resuspend the cells, add an appropriate amount (96-well plate 100 ⁇ L/well, flow tube 1mL/tube) 1 ⁇ working solution A to fix and rupture the membrane, and incubate at 2-8°C for 40-50 minutes in the dark. After fixation and membrane rupture, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B to prepare intracellular antibody, the antibody concentration is 1:100 to 1:200, 50 ⁇ L/well of 96-well plate, 100 ⁇ L/tube of flow tube, and stain for 30 minutes at 2-8°C in the dark. After staining, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice. Use 100-500 ⁇ L PBS to resuspend the cells for flow cytometry detection.
  • FIGS. 2 and Figure 3 show the proportion of CD45RA - CCR7 + central memory T cells (Tcm) in TIL cells cultured after adding OKT3 and IL-2 0 hours, 24 hours or 48 hours after adding feeder cells. .
  • the results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of central memory T cells than TILs cultured with feeders at the same time.
  • FIG. 4 shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells obtained by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a lower proportion of regulatory T cells than TILs cultured with feeder cells at the same time.
  • FIGS 5 and 6 show the proportion of activated T cells in TIL cells cultured by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TIL cultured with feeder cells after 24 hours or 48 hours had a higher proportion of activated T cells, such as PD-1 + , LAG-3 + and/or CD28, than TILs cultured with feeder cells at the same time + Higher percentage of cells.
  • Figure 7 shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells cultured by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of tumor-specific T cells than TILs cultured with feeder cells at the same time.
  • Figure 8 shows the ratio of TCF1 + stem cell-like T cells in TIL cells obtained by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2. The results showed that the TIL cultured with feeder cells after 24 hours or 48 hours had a higher proportion of stem cell-like T cells than the TILs cultured with feeder cells at the same time.
  • Figure 9 shows the analysis results of the proliferation ability of the test group and the control group added with different forms of CD28 agonists.
  • the value on the ordinate in the figure indicates the expansion factor to which the number of TIL cells is amplified to when the TIL population obtained from in vitro expansion of each test group is compared with the TIL population before in vitro expansion.
  • the results showed that the in vitro expansion of step (B) in the four-step division method added CD28 antibody, and the proliferation ability of TIL obtained was stronger than that of TIL cultured in the control group (without adding CD28 antibody).
  • FIG 10 shows the ratio of T cell subsets of TIL cells cultured in the mixed antibody group and the control group.
  • step (B) in the four-step division method was expanded in vitro and CD28 antibody was added, and compared with the control group (without adding CD28 antibody), the TIL obtained had an improved ratio of T cell subsets.
  • higher proportion of activated T cells CD28 + or 41BB +
  • lower proportion of regulatory T cells Treg, e.g. CD4 + CD25 + Foxp3 +
  • TCF1 + stem cell-like T cells
  • Tcm central memory T cells
  • FIG 11 shows the ratio of T cell subsets of TIL cells cultured in the mixed antibody group and the control group.
  • step (B) in the four-step division method was expanded in vitro and CD28 antibody was added, and compared with the control group (without adding CD28 antibody), the TIL obtained had an improved ratio of T cell subsets.
  • a higher proportion of tumor-specific T cells CD103 + CD39 +
  • a higher proportion of activated T cells CD25 +
  • Reg regulatory T cells
  • FIG 12 shows the ratio of T cell subsets of TIL cells cultured in the magnetic bead group and the control group.
  • step (B) in the four-step division method was expanded in vitro and added with CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had improved Proportions of T cell subsets.
  • CD28 antibody for example, adding magnetic beads containing CD3 antibody and CD28 antibody
  • TCF1 + stem cell-like T cells
  • Tcm central memory T cells
  • Figure 13 shows the ratio of T cell subsets of TIL cells cultured in the magnetic bead group and the control group.
  • step (B) in the four-step division method was expanded in vitro and added with CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had improved Proportions of T cell subsets.
  • TCF1 + stem cell-like T cells
  • 41BB + activated T cells
  • Tcm central memory T cells
  • Reg Proportion of regulatory T cells
  • CD103 + CD39 + tumor-specific T cells
  • FIG 14 shows the ratio of T cell subsets of TIL cells cultured in the nano-matrix group and the control group.
  • step (B) in the four-step division method was expanded in vitro and added CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had improved T Proportions of cell subpopulations.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • the obtained TIL had improved T Proportions of cell subpopulations.
  • a higher proportion of tumor-specific T cells CD103 + CD39 +
  • a higher proportion of activated T cells CD25 + or PD-1 +
  • Tcm central memory T cells
  • target cells such as Hela tumor cells
  • CFSE (6(6)-Carboxyfluorescein diacetate N-succinimidyl ester, Sigma, 21888-25MG-F): wash tumor cells with PBS, resuspend tumor cells in 500 ⁇ L of PBS; add CFSE to 500 ⁇ L of PBS , mixed with 500 ⁇ L of tumor cell resuspension in PBS, to a final concentration of CFSE of 0.5 ⁇ mol/L. After incubating at 37°C for 6 minutes, add medium containing 10% FBS to wash, centrifuge at 600g for 5 minutes, use X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi, etc.
  • TIL cells Resuspend tumor cells in branded T cell medium at a concentration of 5 x 105 cells/mL.
  • the TIL cells in each test group were centrifuged at 600g for 5 minutes, and the TIL cells were resuspended according to the effect-to-target ratio (ratio of TIL cells to tumor cells) of 3:1 (that is, the concentration of resuspended TIL cells was 1.5 ⁇ 10 6 cells/mL).
  • ratio of TIL cells to tumor cells was 1.5 ⁇ 10 6 cells/mL.
  • a group of control groups containing only tumor cells was set up, and different reagents were added according to different groups of experiments. Centrifuge the plate at 200g for 1 minute, and incubate at 37°C for 4 hours to overnight.
  • Killing rate% Dapi + CFSE + cell number/total CFSE + ⁇ 100%.
  • Figure 15 shows the cell killing ability of TIL cells cultured in the nano-matrix group and the control group.
  • step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher cell killing ability.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • Figure 16 shows the detection results of intracellular factor expression in TIL cells cultured in the mixed antibody group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added with CD28 antibody.
  • the TIL obtained had a higher ability to express intracellular factors. For example, higher CD107a expression capacity.
  • Figure 17 shows the detection results of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher expression of intracellular factors. For example, higher CD107a expression capacity.
  • Figure 18 shows the detection results of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher expression of intracellular factors. For example, higher CD107a expression capacity.
  • Figure 19 shows the detection results of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher expression of intracellular factors. For example, higher CD107a expression capacity.
  • Figure 20 shows the detection results of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher expression of intracellular factors. For example, higher CD107a expression capacity.
  • Figure 21 shows the detection results of intracellular factor expression in TIL cells cultured in the nano-matrix group and the control group.
  • the results showed that the step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher Intracellular factor expression ability.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • the control group without adding CD28 antibody
  • Figure 22 shows the results of cytokine secretion detection of TIL cells cultured in the nano-matrix group and the control group.
  • the results showed that the step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher Cytokine secretion capacity.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • control group without adding CD28 antibody
  • the TIL obtained from each test group was incubated with the tumor cells overnight, and after the incubation, the supernatant was taken to detect cytokine secretion according to the detection steps in this example.
  • Fig. 23 shows the cytokine secretion detection results after co-incubating TIL cells and tumor cells obtained from the nano-matrix group and the control group.
  • the results showed that the step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher Cytokine secretion capacity.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • TIL cells of each test group in Example 1 were expanded and cultured in vitro for 48 hours in step (B) of the four-step division method, and the gene knockout efficiency was detected.
  • Nuclease-free water (commercial source: Shanghai Youfan Biotechnology Co., Ltd.; RT121-02) was used to prepare sgRNA (sequence shown in SEQ ID NO: 1, GGAGAATGACGAGTGGACCC), and the concentration was adjusted to 50 ⁇ mol/L. Take 2 ⁇ L of gRNA and add it to a PCR tube, Nuclease-free water is used as a negative control, incubate in a PCR instrument at 95°C for 2 minutes, then cool at room temperature for 10 minutes.
  • P3Buffer commercial source: Lonza; V4XP-3032
  • 61.7 ⁇ mol/L Cas9 nuclease commercial source: Suzhou Kerui Gene Biotechnology Co., Ltd.; C01-2019-11-001
  • T cell medium at 1 mL/well and place in a CO 2 incubator to preheat.
  • an electroporation apparatus Lionza
  • feeder cells irradiated PBMC cells
  • the TIL cells of each test group after the end of the culture were taken for cell counting, and 2 ⁇ 10 5 cells were taken from each test group, centrifuged at 500g for 3 minutes, and the supernatant was discarded after centrifugation.
  • Fixable Viability Dye eFluor 780 (commercial source: eBioscience; 65-0865-18) and dilute it 10000 times in PBS; take 100 ⁇ L of diluted Fixable Viability Dye eFluor 780 in PBS solution, add 1 ⁇ L TCR- ⁇ -APC (commercial source: eBioscience; 17-9986-42), 1 ⁇ L of BB515Mouse Anti-Hu CD8 (commercial source: BD Pharmingen; 564526), 1 ⁇ L of PE-Cy7Mouse Anti-Hu CD4 (commercial source: BD Pharmingen ;557852), mix well.
  • Figure 24 shows the gene knockout efficiency results of TIL cells cultured in the nano-matrix group and the control group.
  • step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), and compared with the control group (without adding CD28 antibody), the resulting TILs had improved genes.
  • Knockout efficiency capability For example, improved TCR ⁇ gene knockout efficiency.
  • Figure 25 shows the results of gene knockout efficiency of TIL cells cultured in the nano-matrix group and the control group.
  • step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), and compared with the control group (without adding CD28 antibody), the resulting TILs had improved genes.
  • Knockout efficiency capability For example, improved TCR ⁇ gene knockout efficiency.
  • the first TIL population derived from tumor tissue and not expanded in vitro was obtained, and the first TIL population was subjected to steps (A), step (B), and step (C) in the four-step division method in the same manner and step (D) to obtain a fifth population of TILs.
  • the fifth TIL group was randomly divided into 3 groups. While adding IL-2 to the T cell culture medium of each test group, the blank group did not add any T cell activator, and the CD28 agonist group was added with CD3 antibody (Miltenyi Biotech, OKT3) about 30ng/mL, CD28 agonist group was added with CD3 agonist and CD28 agonist, for example, transACT was added at a ratio of transACT to TIL of about 1:100-1:2000.
  • the TIL (terminal stimulatory cell population) obtained after culturing for 3 days was used to detect the proliferation ability of TIL cells by using the CellTiter-Glo kit (commercial source: Promega) by the cell viability detection method.
  • Figure 26, Figure 27 and Figure 28 respectively show the proliferative ability analysis results of the test groups that were expanded in vitro in different ways at the terminal stimulation stage for TILs from different donors.
  • the fluorescence values on the ordinate in the figure reflect the proliferation ability of TIL cells subjected to terminal stimulation in different ways in each test group.
  • the results showed that the addition of a CD28 agonist to the terminal stimulation had similar proliferative capacity of TILs compared to the terminal stimulation without the addition of a CD28 agonist.
  • TIL activation of the second-stage expansion in 1.4 of Example 1 take the amount of cells expanded in the first stage, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, and place them in a suspended 24-well culture plate , 1 mL/well, add CD3 antibody, such as OKT3 about 30 ng/mL, add IL-2 at a concentration of about 1000-9000 IU/mL, such as 3000 or 6000 IU/mL IL-2.
  • feeder cells were added into the culture environment of tumor infiltrating lymphocytes.
  • TIL and feeder cells can be added at a ratio of 1:40-1:400, and all the cells are collected after about 9-14 days of expansion and culture in the second stage, and the results of TIL obtained from the culture are detected and counted.
  • TILs derived from tumors from different donors were regarded as different batches; the data of each batch of the test group in which OKT3 and IL-2 were added to feeder cells at the same time (0h group) were used as benchmark 1, and the data of other time points in the same batch were tested The data of each group were standardized, and the relative proliferative ability of each test group in the second stage of amplification relative to the 0h group was counted.
  • Fig. 29A shows the results of cell proliferation ability of TIL cells cultured by adding feeder cell-cultured TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. Compared with the TIL cultured with feeder cells added 0 hours after the addition of OKT3 and IL-2 (that is, at the same time), the proliferation ability of TILs cultured with feeder cells 24 hours or 48 hours after the addition of OKT3 and IL-2 was significantly enhanced.
  • Flow cytometric detection was performed on the TIL population obtained by culturing the above-mentioned feeder cells at different addition times.
  • TILs derived from tumors from different donors were regarded as different batches; the data of each batch of the test group in which OKT3 and IL-2 were added to feeder cells at the same time (0h group) were used as benchmark 1, and the data of other time points in the same batch were tested The data of each group were standardized, and the cell composition ratio of each experimental group expanded in the second stage relative to the 0h group was counted.
  • test procedure of flow detection refer to the content of Example 3 of the present application.
  • Figure 29B shows the results of CD45RA - CCR7 + central memory T cell (Tcm) ratio of TIL cells obtained by adding feeder cell-cultured TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Tcm central memory T cell
  • FIG. 29C shows the ratio of TCF1 + stem cell-like T cells in TIL cells obtained by adding feeder cell-cultured TILs after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. The results showed that TIL cultured with feeder cells added after 24 hours or 48 hours had a higher proportion of stem cell-like T cells in CD8 + than TILs cultured with feeder cells added at the same time.
  • Fig. 29D shows the proportion of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells obtained by adding feeder cell-cultured TILs after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a lower proportion of regulatory T cells than TILs cultured with feeder cells at the same time.
  • FIG. 29E shows the proportion of activated T cells (PD-1 + ) in TIL cells obtained by adding feeder cell-cultured TILs after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • the results showed that TIL cultured with feeder cells supplemented after 24 hours or 48 hours had a higher proportion of activated T cells, such as PD-1 in CD8 + and/or CD4 + , than TILs cultured with feeder cells at the same time + Higher percentage of cells.
  • Fig. 29F shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells obtained by adding feeder cells cultured TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of tumor-specific T cells in CD8 + and/or CD4 + than TILs cultured with feeder cells at the same time.
  • FIG. 29G shows the proportion of activated T cells (CD28 + ) in TIL cells cultured after adding feeder cells to TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of activated T cells, such as a higher proportion of CD8 + CD28 + cells, than TILs cultured with feeder cells at the same time.
  • FIG. 29H shows the proportion of activated T cells (41BB + ) in TIL cells cultured by adding feeder cells cultured TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TILs cultured with feeder cells supplemented after 24 hours or 48 hours had a higher proportion of activated T cells, such as 41BB + cells in CD8 + and/or CD4 + cells, than TILs cultured with feeder cells at the same time The proportion is higher.
  • Figure 29I shows the proportion of activated T cells (CD25 + ) in TIL cells obtained by adding feeder cells cultured TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TILs cultured with feeder cells supplemented after 24 hours or 48 hours had a higher proportion of activated T cells, such as CD25+ cells in CD8 + and/or CD4 + cells, than TILs cultured with feeder cells at the same time The proportion is higher.
  • Figure 29J shows the detection results of intracellular factor expression in TIL cells obtained by adding feeder cells cultured TILs after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • the results showed that the TIL cultured with feeder cells added after 24 hours or 48 hours had a higher ability to express intracellular factors than the TILs cultured with feeder cells added at the same time. For example, higher CD107a expression capacity in CD3 + , CD8 + and/or CD4 + .
  • the cytokine secretion detection method can refer to the instructions of the cytokine detection kit (BD), reconstitute the human Th1/Th2/Th17 cytokine standard freeze-dried powder (BD) with 2mL Assay Diluent diluent (BD) (Standard stock solution The concentration of each cytokine is 5000pg/mL) and in order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 serial dilution, marked as "standard tube”. Take 1 tube containing Assay Diluent dilution only as a reference.
  • BD cytokine detection kit
  • BD Assay Diluent diluent
  • Figure 29K shows the cytokine secretion detection results of TIL cells cultured by adding feeder cells cultured TILs after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. The results showed that TIL cultured with feeder cells added after 24 hours or 48 hours had higher cytokine secretion ability than TILs cultured with feeder cells added at the same time. For example, higher TNF- ⁇ secretion capacity, or higher IFN- ⁇ secretion capacity.
  • TIL activation of the second-stage expansion in 1.4 of Example 1 take the amount of cells expanded in the first stage, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, and place them in a suspended 24-well culture plate , 1 mL/well, add CD3 antibody, such as OKT3 about 30 ng/mL, add IL-2 at a concentration of about 1000-9000 IU/mL, such as 3000 or 6000 IU/mL IL-2.
  • feeder cells were added to the culture environment of tumor infiltrating lymphocytes.
  • TIL and feeder cells can be added at a ratio of 1:40-1:400, such as 1:200, and all cells are collected after about 9-14 days of second-stage expansion culture, and the results of cultured TIL are detected and counted.
  • Figure 29L shows that the TIL cultured by feeder cells was added after adding OKT3 and IL-2 for 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days, and the cell proliferation ability of the cultured TIL cells Result graph. Compared with the TILs cultured with feeder cells added 0 hours after the addition of OKT3 and IL-2 (i.e. at the same time), the proliferation ability of TILs cultured with feeder cells after 12 hours or more after the addition of OKT3 and IL-2 was significantly enhanced .
  • Flow cytometric detection was performed on the TIL population obtained by culturing the above-mentioned feeder cells at different addition times.
  • TILs derived from tumors from different donors were regarded as different batches; the data of each batch of the test group in which OKT3 and IL-2 were added to feeder cells at the same time (0h group) were used as benchmark 1, and the data of other time points in the same batch were tested The data of each group were standardized, and the cell composition ratio of each experimental group expanded in the second stage relative to the 0h group was counted.
  • test procedure of flow detection refer to the content of Example 3 of the present application.
  • Figure 29M shows the CD8 + T of TIL cells cultured after adding OKT3 and IL-2 at 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cell-cultured TILs. cell ratio. The results showed that TILs cultured with feeder cells added 12 hours or more after the addition of OKT3 and IL-2 had a higher proportion of CD8 + T cells than TILs cultured with feeder cells at the same time.
  • Figure 29N shows the CD45RO + CD62L of TIL cells cultured after adding OKT3 and IL-2 at 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells. + T cell ratio.
  • the results showed that TILs cultured with feeder cells added 12 hours or more after addition of OKT3 and IL-2 had higher memory T cells (Tcm, CD45RO + CD62L + ) compared with TILs cultured with feeder cells at the same time. proportion.
  • Figure 29O shows the addition of feeder cell-cultured TILs at 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after the addition of OKT3 and IL-2, and the NK T cells of the resulting TIL cells. Proportion. The results showed that TILs cultured with feeder cells added 12 hours or more after the addition of OKT3 and IL-2 had a higher proportion of NK T cells than TILs cultured with feeder cells at the same time.
  • Figure 29P shows the CD4 + CD25 CD4 + CD25 + Foxp3 + regulatory T cell (Treg) ratio. The results showed that TILs cultured with feeder cells added 12 hours or more after the addition of OKT3 and IL-2 had a lower proportion of regulatory T cells than TILs cultured with feeder cells at the same time.
  • TIL activation in the second stage of expansion in 1.4 of Example 1 take the amount of cells expanded in the first stage, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, and place them in a suspended 24-well culture plate , 1 mL/well, add CD3 antibody, such as OKT3 about 30 ng/mL, add IL-2 at a concentration of about 1000-9000 IU/mL, such as 3000 or 6000 IU/mL IL-2. 12 hours to 14 days after the addition of the above OKT3 and IL-2, for example 48 hours later, the feeder cells are added into the culture environment of tumor infiltrating lymphocytes. Among them, TIL and feeder cells can be added at a ratio of 1:40-1:400, and after about 9-14 days of expansion and culture in the second stage, all cells are collected, and the cell killing ability of TIL obtained from culture is detected and counted.
  • CD3 antibody such as OKT3 about 30 ng/mL
  • IL-2 at a concentration of about 1000
  • TILs obtained from each test group for detection and target cells for co-culture such as A375 melanoma cells and/or Hela cervical cancer cells.
  • CFSE (6(6)-Carboxyfluorescein diacetate N-succinimidyl ester, Sigma, 21888-25MG-F): wash tumor cells with PBS, resuspend tumor cells in 500 ⁇ L of PBS; add CFSE to 500 ⁇ L of PBS , mixed with 500 ⁇ L of tumor cell resuspension in PBS, to a final concentration of CFSE of 0.5 ⁇ mol/L. After incubating at 37°C for 6 minutes, add medium containing 10% FBS to wash, centrifuge at 600g for 5 minutes, use X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi, etc.
  • TIL cells Resuspend tumor cells in branded T cell medium at a concentration of 1 x 106 cells/mL.
  • the TIL population of each test group was centrifuged at 600g for 5 minutes, and the TIL cells were resuspended according to the effect-to-target ratio (the ratio of TIL cells to tumor cells) of 3:1 (that is, the concentration of resuspended TIL cells was 3 ⁇ 10 6 cells/mL).
  • TIL and tumor cells are co-cultured, substances that activate TIL cells can be omitted as the non-activation group, or transACT (Miltenyi, a nano-matrix material containing CD3 antibody and CD28 antibody) can be added as the activation group.
  • transACT Miltenyi, a nano-matrix material containing CD3 antibody and CD28 antibody
  • Killing rate% Dapi + CFSE + cell number/total CFSE + ⁇ 100%, or the killing rate can be expressed by Dapi + cell number/total tumor cell number.
  • Figure 29Q shows the results of the cell killing ability of TIL cells cultured by adding feeder cells cultured TILs 48 hours after the addition of OKT3 and IL-2. The results showed that 48 hours after the addition of OKT3 and IL-2, the TIL cultured with feeder cells had significant tumor cell killing ability, such as melanoma and/or cervical tumor.
  • DNA extraction solution QuickExtract DNA extraction solution, Lucigen, QE09050
  • nuclease-free water RNase/DNase free water, Tiangen
  • EDTA Etggong, 0.5M
  • Recombinant DNase I RNase-free, TAKARA
  • Genomic DNA extraction About 4 days after TIL cell knockout, take about 1 ⁇ 105 to about 2 ⁇ 105 cells, wash with PBS once, then resuspend TIL cells with 44 ⁇ L PBS, and add to the configuration 6 ⁇ L nuclease mixture (containing 1 ⁇ L DNase I and 5 ⁇ L 10 ⁇ DNase I Buffer), incubate at 37°C for 5 minutes. Add 2.5 ⁇ L of 0.5M EDTA to the sample and incubate at 80°C for 10 minutes. After centrifuging to discard the supernatant, add 50 ⁇ L of DNA extraction solution to the cell pellet, and run the following program after brief centrifugation: 75°C for 10 minutes; 95°C for 5 minutes; 4°C for maintenance. DNA sample concentration can be detected using a spectrophotometer (NanoDrop TM ).
  • PCR primers can be designed in a region of about 100 to about 200 nucleotides upstream and downstream of the PAM site. Design the PCR reaction system as follows:
  • Reagent volume 2 ⁇ PCR Premix Buffer 25 ⁇ L DNA template About 100-500ng Forward primer (10 ⁇ M) 1 ⁇ L Reverse primer (10 ⁇ M) 1 ⁇ L double distilled water Replenish to 50 ⁇ L DMSO 1.5 ⁇ L
  • the PCR products were subjected to Sanger sequencing analysis.
  • the donor 812 is a skin malignant melanoma patient
  • the donor 713 is an ovarian cancer patient.
  • TIL cells from each group were re-plated with the same total number of cells. Stimulated with 30ng/mL CD3 antibody (Miltenyi Biotech, OKT3), 3 days later, the expansion efficiency of TIL cells was analyzed with CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega).
  • FIGS 30A-30B show, for TIL cells derived from donors 713 and 812, the amount of fluorescence after expansion of TIL cells in each group. The results showed that compared with the control group (NT), the gene-edited TIL cells of the present application can have significant expansion ability.
  • TIL cells tumor cells, E: T
  • Tumor cells and TIL cells were 100 ⁇ L each, and three replicate wells were set up for each group, and a control group containing only tumor cells was set up at the same time.
  • the instructions of the apoptosis detection reagent (Incucyte Caspase-3/7 Green Dye for Apoptosis, Sartorius), add the apoptosis detection reagent at 0.2 ⁇ L/well, and add the medium to dilute Caspase 3/7 Green Dye at 25 ⁇ L/well .
  • the incucyte recorder (Sartorius) was used to record the activity of Caspase 3/7 to analyze the killing ability of TIL on tumor cells, and recorded every 3 hours, and the total recording time was about 5 days.
  • Figure 31 shows the test results of the killing ability of TIL cells derived from donor 812 co-cultured with tumor cells at an effect-to-target ratio of 1:1. The results showed that gene-edited TIL cells could have more significant tumor cell killing ability compared with the control group (NT).
  • Apoptosis detection was performed on the TIL populations obtained on the 8th day after gene editing in each test group in Example 1.
  • TILs from the gene knockout group or the control group were used to detect the apoptosis level of T cells with a cell apoptosis detection kit (BD 559763 Annexin V PE Apoptosis kit).
  • 32A-32B show the detection results of apoptosis in TIL cells derived from donor 812. The results showed that gene-edited TIL cells could have more significant anti-apoptotic ability.
  • TIL populations obtained on the 8th day after gene editing in each test group in Example 1 were detected by flow cytometry.
  • V-bottom 96-well plate manufacturer Corning, product number 3894; flow tube, manufacturer Corning, product number 352052.
  • the flow cytometry antibodies in this example were purchased from BD or Biolegend. Add 1 ⁇ 10 5 to 5 ⁇ 10 5 cell samples in each group into flow tubes or V-bottom 96-well plates. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, flow tube 1mL/tube, 96-well plate 200 ⁇ L/well, discard supernatant. Add the prepared antibody working solution for cell surface staining, the antibody (BD or Biolegend) concentration is 1:100 to 1:200, and the activity detection dye is 1:10000. Flow tube 100 ⁇ L/tube, 96-well plate 50 ⁇ L/well for staining, incubate at 2-8°C for 30 minutes in the dark.
  • Cell fixation and membrane rupture Sufficiently resuspend the cells, add an appropriate amount (96-well plate 100 ⁇ L/well, flow tube 1mL/tube) 1 ⁇ working solution A to fix and rupture the membrane, and incubate at 2-8°C for 40-50 minutes in the dark. After fixation and membrane rupture, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 600g for 3 minutes, and wash twice.
  • 1 ⁇ working solution B 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 600g for 3 minutes, and wash twice.
  • 1 ⁇ working solution B to prepare intracellular antibody, the antibody concentration is 1:100 to 1:200, 50 ⁇ L/well of 96-well plate, 100 ⁇ L/tube of flow tube, and stain for 30 minutes at 2-8°C in the dark. After staining, add 1 ⁇ working solution B to wash the cells (96-well plate 200 ⁇ L/time, flow tube 2 mL/time), centrifuge at 2-8°C, 600g for 3 minutes, and wash twice. Use 100-500 ⁇ L PBS to resuspend the cells for flow cytometry detection.
  • Figures 33A-33C show, for TIL cells derived from donors 713 and 812, the proportion of central memory T cells (CD45RO + CD62L + ) among TIL cells in each group. The results showed that the gene-edited TIL cells had a higher proportion of central memory T cells.
  • Figures 34A-345D show the proportion of naive T cells (CD45RO ⁇ CD62L + ) in TIL cells of each group for TIL cells derived from donors 713 and 812. The results showed that the gene-edited TIL cells had a higher proportion of naive T cells.
  • Figures 35A-35E show, for TIL cells derived from donor 713, the proportions of CD38-positive, LAG-3-positive and TIM-3-positive cells among TIL cells in each group.
  • Figures 36A-36E show, for TIL cells derived from donor 812, the proportions of CD38-positive, LAG-3-positive and TIM-3-positive cells among TIL cells in each group.
  • TIL populations obtained on the 7th or 8th day after the gene editing in each test group in Example 1 the expression of cytokines was detected by flow cytometry.
  • the CD3 antibody group was coated with 30ng/ml CD3 antibody (Miltenyi Biotech, OKT3) on a flat-bottomed 96-well plate one day in advance, and coated overnight at 4°C; the transACT group was added when T cells were inoculated.
  • Cell fixation and membrane rupture Sufficiently resuspend the cells, add an appropriate amount (96-well plate 100 ⁇ L/well, flow tube 1mL/tube) 1 ⁇ working solution A to fix and rupture the membrane, and incubate at 2-8°C for 40-50 minutes in the dark. After fixation and membrane rupture, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B to prepare intracellular antibodies (CD107a, GZMB, TNF- ⁇ and IFN- ⁇ , BD/BioLegend), the antibody concentration is 1:100 to 1:200, 50 ⁇ L/well in 96-well plate, 100 ⁇ L in flow tube /tube, 2-8°C for 30 minutes in the dark. After staining, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice. Use 100-500 ⁇ L PBS to resuspend the cells for flow cytometry detection.
  • Figures 37A-37D show the ratio of GZMB, TNF- ⁇ and IFN- ⁇ expressing cells in TIL cells of each group for TIL cells derived from donor 713 under the condition of no activator stimulation.
  • Figures 38A-38E show the percentages of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells derived from donor 812 under the condition of no activator stimulation.
  • Figures 39A-39B show the ratio of TNF- ⁇ expressing cells among TIL cells in each group of TIL cells derived from donor 713 under the condition of transACT stimulation.
  • Figures 40A-40F show the percentages of cells expressing GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells derived from donor 812 under the condition of CD3 antibody stimulation.
  • DNA extraction solution QuickExtract DNA extraction solution, Lucigen, QE09050
  • nuclease-free water RNase/DNase free water, Tiangen
  • EDTA Etggong, 0.5M
  • Genomic DNA extraction About 4 days after TIL cell knockout, take about 1 ⁇ 105 to about 2 ⁇ 105 cells, add the prepared nuclease mixture (DNase I), and incubate at 37°C for 5 minutes . Add 2.5 ⁇ L of 0.5M EDTA to the sample and incubate at 80°C for 10 minutes. After centrifugation, add 50 ⁇ L of DNA extraction solution, run the following program after brief centrifugation: 75°C-10 minutes; 95°C-5 minutes; 4°C-maintain. DNA sample concentration can be detected using a spectrophotometer (NanoDrop TM ).
  • PCR primers can be designed in a region of about 100 to about 200 nucleotides upstream and downstream of the PAM site. Design the PCR reaction system as follows:
  • Reagent volume 2 ⁇ PCR Premix Buffer 25 ⁇ L DNA template About 100-500ng Forward primer (10 ⁇ M) 1 ⁇ L Reverse primer (10 ⁇ M) 1 ⁇ L double distilled water Replenish to 50 ⁇ L DMSO 1.5 ⁇ L
  • the PCR products were subjected to Sanger sequencing analysis.
  • TILs can be derived from different tumor patients.
  • Donors A and B are patients with skin malignant melanoma
  • donors C are patients with pancreatic cancer
  • donors D and E are patients with non-small cell lung cancer.
  • the sgRNA represented by SEQ ID NO: 36 (CGGCGTGCGAACGGAATGTG), i.e. SC1
  • SC1 had a knockout efficiency of about 64.5% for donor A, about 40.6% for donor B, and about 40.6% for donor C.
  • the knockout efficiency is about 30.1%, and the knockout efficiency for donor D is about 10%;
  • the knockout efficiency of the sgRNA shown in SEQ ID NO: 37 (GGGCCCCCAGTAGAATCCGC), SC2, for donor A is about 60.5%,
  • the knockout efficiency was about 18.5% for donor B, about 42.3% for donor C, and about 26.3% for donor D.
  • Various gene editing methods of the present application can achieve a certain percentage of knockout efficiency.
  • TIL cells in each group were re-plated with the same total number of cells, and cultured in cell culture medium without IL-2. After 3 days, the expansion efficiency of TIL cells was analyzed with CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega).
  • Figure 41, Figure 42, Figure 43 and Figure 44 show, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group. The results show that the gene-edited TIL cells of the present application can still have significant expansion ability even if cultured in a cell culture medium that does not contain IL-2.
  • the 96-well plate was pretreated with 30ng/mL CD3 antibody (Miltenyi Biotech, OKT3) and incubated overnight at 4°C; Cell culture medium without IL-2. After 3 days, the expansion efficiency of TIL cells was analyzed with CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega).
  • Figure 45 and Figure 46 show, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group.
  • NT represents the negative control group, TILs that have not undergone gene editing
  • ST3 represents the irrelevant knockout group, TILs that have been knocked out in areas that do not affect cell function
  • SC1 represents the TILs that have been knocked out by the sgRNA shown in SEQ ID NO: 36. Knockout TIL
  • SC2 indicates TIL knockout by sgRNA shown in SEQ ID NO:37. * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, *** indicates p ⁇ 0.001, **** indicates p ⁇ 0.0001.
  • TIL cells:tumor cells, E:T effect-to-target ratio
  • the instructions of the apoptosis detection reagent (Incucyte Caspase-3/7 Green Dye for Apoptosis, Sartorius), add the apoptosis detection reagent at 0.2 ⁇ L/well, and add the medium to dilute Caspase 3/7 Green Dye at 25 ⁇ L/well .
  • the incucyte recorder (Sartorius) was used to record the activity of Caspase 3/7 to analyze the killing ability of TIL on tumor cells, and recorded every 3 hours, and the total recording time was about 5 days.
  • Figure 47 shows the test results of the killing ability of TIL cells derived from donor B co-cultured with tumor cells at an effect-to-target ratio of 1:1.
  • Figure 48 shows the test results of the killing ability of TIL cells derived from donor C co-cultured with tumor cells at an effect-to-target ratio of 1:3.
  • Figure 49 and Figure 50 show the test results of the killing ability of TIL cells derived from donor D co-cultured with tumor cells at an effect-to-target ratio of 1:1 and an effect-to-target ratio of 1:3. The results showed that gene-edited TIL cells can have more significant tumor cell killing ability.
  • IL-2 is an important factor that regulates the growth of T cells. This example tests whether the gene-edited TIL cells of the present application can survive and/or expand independently of IL-2.
  • TIL cells in each group were re-plated with the same total number of cells, and cultured in a cell culture medium that did not contain IL-2, that is, IL-2 was removed for culture.
  • the expansion efficiency and viability of TIL cells were analyzed with a cell counter every 2 to 4 days.
  • the amplification efficiency is expressed by the multiplication factor: the total number of cells on the day of IL-2 removal (the 0th day) is taken as 1, and the proliferation factor on the nth day is expressed as the total number of cells on the nth day/the total number of cells on the 0th day; Indicates the percentage of the number of surviving cells to the total number of cells.
  • Figure 51, Figure 52 and Figure 53 show the proliferation factor of TIL cells in each group for TIL cells derived from different donors.
  • Figure 54 and Figure 55 show the viability of TIL cells in each group for TIL cells from different donors. The results show that even if IL-2 is withdrawn, the gene-edited TIL cells of the present application can still have significant expansion ability and maintain a high cell viability.
  • TIL populations obtained on the 8th day after gene editing in each test group in Example 1 were detected by flow cytometry.
  • V-bottom 96-well plate manufacturer Corning, product number 3894; flow tube, manufacturer Corning, product number 352052.
  • the flow cytometry antibodies in this example were purchased from BD or Biolegend. Add 1 ⁇ 10 5 to 5 ⁇ 10 5 cell samples in each group into flow tubes or V-bottom 96-well plates. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, flow tube 1mL/tube, 96-well plate 200 ⁇ L/well, discard supernatant. Add the prepared antibody working solution for cell surface staining, the antibody (BD or Biolegend) concentration is 1:100 to 1:200, and the activity detection dye is 1:10000. Flow tube 100 ⁇ L/tube, 96-well plate 50 ⁇ L/well for staining, incubate at 2-8°C for 30 minutes in the dark.
  • Figure 56 and Figure 57 show the ratio of TIM-3-positive and CD101-positive cells in TIL cells in each group for TIL cells derived from donors. The results showed that the gene-edited TIL cells had a lower proportion of exhausted cells.
  • Figure 58 shows, for TIL cells derived from donors, the ratio of CD45RA-negative CCR7-positive memory T cells (Tcm) among TIL cells in each group. The results showed that gene-edited TIL cells had a higher proportion of central memory T cells.
  • TIL populations obtained on the 7th or 8th day after the gene editing in each test group in Example 1 the expression of cytokines was detected by flow cytometry.
  • CD3 antibody group 30 ng/ml CD3 antibody (Miltenyi Biotech, OKT3) was used to coat flat-bottomed 96-well plates 1 day in advance, and coated overnight at 4°C; on the day of plating in the PMA group, phorbol-myristate- Acetate (PMA, 25ng/ml) and ionomycin (Ionomycin, 1 ⁇ g/ml) are sufficient.
  • PMA phorbol-myristate- Acetate
  • Ionomycin 1 ⁇ g/ml
  • Cell fixation and membrane rupture Sufficiently resuspend the cells, add an appropriate amount (96-well plate 100 ⁇ L/well, flow tube 1mL/tube) 1 ⁇ working solution A to fix and rupture the membrane, and incubate at 2-8°C for 40-50 minutes in the dark. After fixation and membrane rupture, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B to prepare intracellular antibodies (CD107a, GZMB, TNF- ⁇ and IFN- ⁇ , BD/BioLegend), the antibody concentration is 1:100 to 1:200, 50 ⁇ L/well in 96-well plate, 100 ⁇ L in flow tube /tube, 2-8°C for 30 minutes in the dark. After staining, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice. Use 100-500 ⁇ L PBS to resuspend the cells for flow cytometry detection.
  • Figure 59, Figure 60, Figure 61 and Figure 62 show the expression of CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells from different donors under the condition of no stimulation cell ratio.
  • Figure 63, Figure 64, Figure 65 and Figure 66 show that under the condition of CD3 antibody stimulation overnight, for TIL cells from different donors, CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group The proportion of expressing cells.
  • Figure 67, Figure 68 and Figure 69 show the expression of CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells from different donors under the condition of overnight stimulation with PMA and Ionomycin cell ratio.
  • TIL cells in each group were re-plated with the same total number of cells, and cultured in a cell culture medium that did not contain IL-2, that is, IL-2 was removed for culture.
  • T cell receptor (TCR) clonal diversity in TILs was analyzed on days 8 and 18 after gene editing.
  • TCR pedigree kit Beta Mark TCR V ⁇ Repertoire Kit, Beckman Coulter
  • 0.01 ⁇ L eFluor 780-labeled dead cell dye eBioscience
  • TCR diversity of TIL cells was identified according to the instructions of the TCR repertoire kit (Beta Mark TCR V ⁇ Repertoire Kit, Beckman Coulter). Table 1 shows the correspondence between the fluorescence from tube A to tube H and the TCR V ⁇ clone. Where the TCR V ⁇ clone is other, it may mean that the T cells under this ratio do not have the TCR V ⁇ that can be identified by the TCR lineage kit. Tables 2 to 5 show the diversity of TCR V ⁇ clones of CD4 + T cells and CD8 + T cells on day 8 and day 18 after gene editing of TIL cells.
  • Table 2 shows the diversity of TCR V ⁇ clones in CD4 + T cells on day 8 after gene editing in TIL cells.
  • Table 3 shows the diversity of TCR V ⁇ clones in CD4 + T cells on day 18 after gene editing in TIL cells.
  • Table 4 shows the diversity of TCR V ⁇ clones in CD8 + T cells on day 8 after TIL cell gene editing.
  • Table 5 shows the diversity of TCR V ⁇ clones in CD8 + T cells on day 18 after TIL cell gene editing.
  • Figure 70 and Figure 71 show the Shannon diversity index of TCR V ⁇ clones of CD4 + T cells and CD8 + T cells on day 8 and day 18 after gene editing of TIL cells.
  • the Shannon diversity index reflects the diversity of TCR V ⁇ clones, and the results show that gene-edited TIL cells can maintain a higher diversity of ⁇ -chain subtypes at day 18, indicating that gene-edited TIL cells can significantly maintain TCR for a long time
  • the diversity of clones can help to achieve stronger antigen recognition and immune response to tumor cells.
  • TIL cells in each group were re-plated with the same total number of cells, and cultured in a cell culture medium that did not contain IL-2, that is, IL-2 was removed for culture. After 24 hours, the supernatant was collected for cytokine secretion detection.
  • the cytokine secretion detection method can refer to the instructions of the cytokine detection kit (BD), reconstitute the human Th1/Th2/Th17 cytokine standard freeze-dried powder (BD) with 2mL Assay Diluent diluent (BD) (Standard stock solution The concentration of each cytokine is 5000pg/mL) and in order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 serial dilution, marked as "standard tube”. Take 1 tube containing Assay Diluent dilution only as a reference.
  • BD cytokine detection kit
  • BD Assay Diluent diluent
  • Figure 72 shows, for TIL cells derived from donor C, the secretion of cytokines IFN- ⁇ and GZMB in TIL cells cultured in each group after removing IL-2. The results showed that gene-edited TIL cells could have more significant cytokine secretion assay results after cultured in cell culture medium without IL-2.
  • DNA extraction solution QuickExtract DNA extraction solution, Lucigen, QE09050
  • nuclease-free water RNase/DNase free water, Tiangen
  • EDTA Etggong, 0.5M
  • Recombinant DNase I RNase-free, TAKARA
  • Genomic DNA extraction About 4 days after TIL cell knockout, take about 1 ⁇ 105 to about 2 ⁇ 105 cells, wash with PBS once, then resuspend TIL cells with 44 ⁇ L PBS, and add to the configuration 6 ⁇ L nuclease mixture (containing 1 ⁇ L DNase I and 5 ⁇ L 10 ⁇ DNase I Buffer), incubate at 37°C for 5 minutes. Add 2.5 ⁇ L of 0.5M EDTA to the sample and incubate at 80°C for 10 minutes. After centrifuging to discard the supernatant, add 50 ⁇ L of DNA extraction solution to the cell pellet, and run the following program after brief centrifugation: 75°C for 10 minutes; 95°C for 5 minutes; 4°C for maintenance. DNA sample concentration can be detected using a spectrophotometer (NanoDrop TM ).
  • PCR primers can be designed in a region of about 100 to about 200 nucleotides upstream and downstream of the PAM site. Design the PCR reaction system as follows:
  • Reagent volume 2 ⁇ PCR Premix Buffer 25 ⁇ L DNA template About 100-500ng Forward primer (10 ⁇ M) 1 ⁇ L Reverse primer (10 ⁇ M) 1 ⁇ L double distilled water Replenish to 50 ⁇ L DMSO 1.5 ⁇ L
  • the PCR products were subjected to Sanger sequencing analysis.
  • Donor 811 is a pancreatic cancer patient
  • donors 410, 614, 619, and 003 are skin malignant melanoma patients
  • donor 921 is an ovarian cancer patient.
  • the sgRNA shown in SEQ ID NO: 41 (ACCTACAGGAGTACCTGACG), that is, G4, has knockout efficiencies of about 23.9%, 17.1%, 24.35%, 23.0%, and 50.8% for donors 614, 921, 619, 003, 410, and 811, respectively. %, 65.2%.

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Abstract

提供了一种培养免疫细胞的方法，该方法包含使该免疫细胞的YEATS结构域蛋白家族成员和/或其功能活性片段的表达降低和/或活性减弱。还提供了通过该方法获得的免疫细胞及其在制备用于预防和/或治疗肿瘤的药物中的应用。

Description

一种修饰的免疫细胞及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种修饰的免疫细胞及其用途。
背景技术
免疫治疗以及使用过继性自体转移肿瘤浸润淋巴细胞治疗肿瘤，是治疗预后不良患者的有效方法。但是免疫治疗中使用的免疫细胞存在细胞活性不强或增殖能力弱的问题。以及，过继性自体转移肿瘤浸润淋巴细胞治疗肿瘤需要大量的肿瘤浸润淋巴细胞，而且目前来自患者肿瘤的肿瘤浸润淋巴细胞在体内的存续和扩增能力弱，杀伤靶细胞的能力不强，同时受到来自肿瘤微环境的多种抑制而功能受限。
因此如何提供一种稳健可靠的免疫疗法以及肿瘤浸润淋巴细胞的培养方法是亟待解决的问题。
发明内容
本申请提供了一种培养免疫细胞的方法，所述方法包含：使所述免疫细胞的YEATS结构域蛋白家族成员的表达降低和/或活性减弱。
一方面，本申请提供了一种培养免疫细胞的方法，所述方法包含：使所述免疫细胞的YEATS结构域蛋白家族成员的表达降低和/或活性减弱。
另一方面，本申请提供了一种免疫细胞，所述免疫细胞经过本申请的方法获得。
另一方面，本申请提供了一种组合物，其包含本申请的免疫细胞。
另一方面，本申请提供了一种药物组合物，其包含本申请的免疫细胞和/或本申请的组合物，以及任选的药学上可接受的载体。
另一方面，本申请提供了一种影响细胞生长的方法，包含施用本申请的免疫细胞、本申请的组合物和/或本申请的药物组合物。
另一方面，本申请提供了本申请的免疫细胞、本申请的组合物和/或本申请的药物组合物在制备药物中的应用，所述药物用于预防和/或治疗疾病和/或症状。
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。对附图简要说明如下：
图1显示的是，饲养细胞不同添加时间培养的TIL的增殖能力分析结果。
图2和图3显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA -CCR7 +中心记忆T细胞(Tcm)比例。
图4显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。
图5和图6显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞比例。
图7显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103 +CD39 +肿瘤特异性T细胞比例。
图8显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1 +干细胞样T细胞比例。
图9显示的是，添加不同形式的CD28激动剂的试验组以及对照组的增殖能力分析结果。
图10和图11分别显示的是，对于不同供者来源的TIL，混合抗体组与对照组培养所得的TIL细胞的T细胞亚群比例。
图12和图13分别显示的是，对于不同供者来源的TIL，磁珠组与对照组培养所得的TIL细胞的T细胞亚群比例。
图14显示的是，纳米基质组与对照组培养所得的TIL细胞的T细胞亚群比例。
图15显示的是，纳米基质组与对照组培养所得的TIL细胞的细胞杀伤能力。
图16显示的是，混合抗体组与对照组培养所得的TIL细胞的胞内因子表达检测结果。
图17、图18、图19和图20分别显示的是，对于不同供者来源的TIL，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。
图21显示的是，纳米基质组与对照组培养所得的TIL细胞的胞内因子表达检测结果。
图22显示的是，纳米基质组与对照组培养所得的TIL细胞的细胞因子分泌检测结果。
图23显示的是，纳米基质组与对照组培养所得的TIL细胞与肿瘤细胞共同孵育后的细胞 因子分泌检测结果。
图24和图25分别显示的是，对于不同供者来源的TIL，纳米基质组与对照组培养所得的TIL细胞的基因敲除效率结果。
图26、图27和图28分别显示的是，对于不同供者来源的TIL，在终末刺激阶段中，以不同方式进行体外扩增的试验组的增殖能力分析结果。
图29A显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。
图29B显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA -CCR7 +中心记忆T细胞(Tcm)比例结果图。
图29C显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1 +干细胞样T细胞比例。
图29D显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。
图29E显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(PD-1 +)比例。
图29F显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103 +CD39 +肿瘤特异性T细胞比例。
图29G显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD28 +)比例。
图29H显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(41BB +)比例。
图29I显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD25 +)比例。
图29J显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的胞内因子表达检测结果。
图29K显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞因子分泌检测结果。
图29L显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。
图29M显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72 小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD8 +T细胞比例。
图29N显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RO +CD62L +T细胞比例。
图29O显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的NK T细胞比例。
图29P显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。
图29Q显示的是加入OKT3和IL-2的48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞杀伤能力结果。
图30A-30B显示的是，对于来源于供者713、812的TIL细胞，各组TIL细胞的扩增后的荧光量。
图31显示的是，对于来源于供者812的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。
图32A-32B显示的是，对于来源于供者812的TIL细胞的凋亡检测结果。
图33A-33C显示的是，对于来源于供者713、812的TIL细胞，各组TIL细胞中的中心记忆T细胞(CD45RO +CD62L +)的比例。
图34A-34D显示的是，对于来源于供者713、812的TIL细胞，各组TIL细胞中的幼稚T细胞(CD45RO -CD62L +)的比例。
图35A-35E显示的是，对于来源于供者713的TIL细胞，各组TIL细胞中CD38阳性、LAG-3阳性和TIM-3阳性细胞的比例。
图36A-36E显示的是，对于来源于供者812的TIL细胞，各组TIL细胞中CD38阳性、LAG-3阳性和TIM-3阳性细胞的比例。
图37A-37D显示的是，在无激活物刺激的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中GZMB，TNF-α和IFN-γ的表达细胞的比例。
图38A-38E显示的是，在无激活物刺激的条件下，对于来源于供者812的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图39A-39B显示的是，在transACT刺激的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中TNF-α的表达细胞的比例。
图40A-40F显示的是，在CD3抗体刺激的条件下，对于来源于供者812的TIL细胞，各组TIL细胞中GZMB，TNF-α和IFN-γ的表达细胞的比例。
图41、图42、图43和图44显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞在没有额外刺激条件下的扩增后的荧光量。
图45和图46显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞在anti-CD3抗体(Miltenyi Biotech，OKT3)刺激下的扩增后的荧光量。
图47显示的是，对于来源于供者B的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。
图48显示的是，对于来源于供者C的TIL细胞，以效靶比1:3与肿瘤细胞共培养的杀伤能力检测结果。
图49和图50显示的是，对于来源于供者D的TIL细胞，以效靶比1:1和效靶比1:3与肿瘤细胞共培养的杀伤能力检测结果。
图51、图52和图53显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞在撤去IL-2后长期培养的增殖倍数。
图54和图55显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞在撤去IL-2后长期培养的活率。
图56和图57显示的是，对于来源于供者C的TIL细胞，各组TIL细胞中TIM-3阳性和CD101阳性细胞的比例。
图58显示的是，对于来源于供者的TIL细胞，各组TIL细胞中CD45RA阴性CCR7阳性记忆T细胞(Tcm)的比例。
图59、图60、图61和图62显示的是，在没有刺激的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图63、图64、图65和图66显示的是，在含有约30ng/mL的CD3抗体刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图67、图68和图69显示的是，在含有佛波醇-十四酸酯-乙酸酯(PMA，25ng/ml)和离子霉素(Ionomycin，1μg/ml)刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图70和图71显示的是，TIL细胞基因编辑后的第8天和第18天的CD4 +T细胞和CD8 +T细胞TCR Vβ克隆的香农多样性指数。
图72显示的是，对于来源于供者C的TIL细胞，各组TIL细胞在撤去IL-2后进行培养的细胞因子IFN-γ和GZMB的分泌检测结果。
图73A-73B显示的是，对于来源于供者619、003的TIL细胞，各组TIL细胞的扩增后的荧光量。图73C显示的是，对于来源于供者410的TIL细胞，各组TGFβ-1诱导刺激TIL细胞的扩增后的荧光量。图73D显示的是，对于来源于供者410的TIL细胞，各组OKT3诱导刺激TIL细胞的扩增后的荧光量。图73E显示的是，对于来源于供者410的TIL细胞，各组TGFβ-1和OKT3诱导刺激TIL细胞的扩增后的荧光量。
图74A-74C显示的是，对于来源于供者619、921的TIL细胞，以效靶比1:1、1:3与肿瘤细胞A375共培养的杀伤能力检测结果。对于额外加入transACT(1:1000)刺激的TIL细胞，图74D显示的是，对于来源于供者921的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。对于额外加入transACT(1:1000)以及TGFβ-1(10nM)诱导刺激的TIL细胞，图74E显示的是，对于来源于供者921的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。对于A375-CD3细胞的杀伤能力检测，图74F-74G显示的是，对于来源于供者811的TIL细胞，以效靶比1:1、1:3与肿瘤细胞共培养的杀伤能力检测结果。
图75A-75B显示的是，对于来源于供者811的TIL细胞的凋亡检测结果。图75C-75D显示的是，对于来源于供者921的TIL细胞的凋亡检测结果。图75E-75F显示的是，对于来源于供者614的TIL细胞的凋亡检测结果。
图76A-76B显示的是，对于来源于供者811的TIL细胞，各组TIL细胞中PD-1阳性、LAG-3阳性和CD38阳性细胞的比例。图76C-76E显示的是，对于来源于供者614、921的TIL细胞，各组TIL细胞中Fas-L阳性细胞的比例。图76F-76G显示的是，对于来源于供者410、811的TIL细胞，各组TIL细胞中调节性T细胞Treg(CD4 +CD25 +FoxP3 +)的比例。对于用30ng/mL的OKT3刺激3天后的TIL，图76H显示的是，对于来源于供者811的TIL细胞，各组TIL细胞中调节性T细胞Treg(CD4 +CD25 +FoxP3 +)的比例。对于用TGFβ-1(10nM)刺激3天后的TIL，图76I显示的是，对于来源于供者811的TIL细胞，各组TIL细胞中调节性T细胞Treg(CD4 +CD25 +FoxP3 +)的比例。
图77显示的是，对于来源于供者313的TIL细胞，以效靶比0.7:1与肿瘤细胞共培养的杀伤能力检测结果。
图78A-78B显示的是，对于来源于供者105的TIL细胞的凋亡检测结果。
图79A-79F显示的是，对于来源于供者105、222、313和812的TIL细胞，各组TIL细胞中LAG-3阳性、PD-1阳性、CD38阳性、和TIM-3阳性细胞的比例。
图80显示的是，对于来源于供者313的TIL细胞，各组TIL细胞中CD45RO阳性CD62L阳性中心记忆T细胞(Tcm)的比例。
图81A-81B显示的是，在CD3抗体(Miltenyi Biotech，OKT3)，CD3抗体以30ng/mL预处理96孔板)刺激过夜的条件下，对于来源于供者105的TIL细胞，各组TIL细胞中GZMB和TNF-α的表达细胞的比例。
图82A-82C显示的是，在无激活物刺激的条件下，对于来源于供者713和812的TIL细胞，各组TIL细胞中TNF-α、IFN-γ和CD107a的表达细胞的比例。
图83A-83D显示的是，在transACT刺激过夜的条件下，对于来源于供者713、812的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图84A-84C显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。
图85显示的是，对于来源于供者222的TIL细胞，各组TIL细胞的扩增后的荧光量。
图86A-86B显示的是，对于来源于供者222的TIL细胞，以效靶比1:3和0.7:1与肿瘤细胞共培养的杀伤能力检测结果。图86C-86H显示的是，对于来源于供者313、222、812和709的TIL细胞，以效靶比1:3、0.7:1和1:1与肿瘤细胞共培养的杀伤能力检测结果。
图87A-87B显示的是，对于来源于供者812的TIL细胞的凋亡检测结果。
图88A-88F显示的是，对于来源于供者313、222、812和709的TIL细胞，各组TIL细胞中PD-1阳性、LAG-3阳性、CD38阳性、和TIM-3阳性细胞的比例。
图89A-89B显示的是，对于来源于供者313和709的TIL细胞，各组TIL细胞中CD45RO阳性CD62L阳性中心记忆T细胞(Tcm)的比例。
图90A-90D显示的是，在无激活物刺激的条件下，对于来源于供者713和812的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图91A-91B显示的是，在transACT刺激过夜的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图92A-92C显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以具有显著的扩增能力。
图93A-93C显示的是，对于来源于供者105、313和222的TIL细胞，各组TIL细胞的扩增后的荧光量。
图94A-94C显示的是，对于来源于供者313和222的TIL细胞，以效靶比1:3和0.7:1与肿瘤细胞共培养的杀伤能力检测结果。
图95A-95B显示的是，对于来源于供者105的TIL细胞的凋亡检测结果。
图96A-96E显示的是，对于来源于供者105、313、222的TIL细胞，各组TIL细胞中PD-1阳性、LAG-3阳性、CD38阳性、和TIM-3阳性细胞的比例。
图97显示的是，对于来源于供者313的TIL细胞，各组TIL细胞中CD45RO阳性CD62L阳性中心记忆T细胞(Tcm)的比例。
图98A-98B显示的是，在CD3抗体(Miltenyi Biotech，OKT3)，CD3抗体以30ng/mL预处理96孔板)刺激过夜的条件下，对于来源于供者105的TIL细胞，各组TIL细胞中GZMB，TNF-α和IFN-γ的表达细胞的比例。
图99A-99B显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。
图100显示的是，对于来源于供者713的TIL细胞，各组TIL细胞的扩增后的荧光量。
图101显示的是，对于来源于供者709的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。
图102A-102B显示的是，对于来源于供者812的TIL细胞的凋亡检测结果。
图103A-103D显示的是，对于来源于供者812、709的TIL细胞，各组TIL细胞中LAG-3阳性和CD38阳性细胞的比例。
图104显示的是，对于来源于供者709的TIL细胞，各组TIL细胞中CD45RO阳性CD62L阳性中心记忆T细胞(Tcm)的比例。
图105A显示的是，在无激活物刺激的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。图105B显示的是，在transACT刺激过夜的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中CD107a的表达细胞的比例。
图106显示的是，对于来源于供者713的TIL细胞，各组TIL细胞的扩增后的荧光量。
图107显示的是，对于来源于供者713的TIL细胞，各组TIL细胞的扩增后的荧光量。
图108A-108B显示的是，对于来源于供者713的TIL细胞，以效靶比1:1、1:3与肿瘤细胞共培养的杀伤能力检测结果。
图109A-109D显示的是，对于来源于供者713、811的TIL细胞的凋亡检测结果。
图110A-110F显示的是，对于来源于供者713、811、812的TIL细胞，各组TIL细胞中CD101阳性、LAG-3阳性和CD38阳性细胞的比例。
图111A显示的是，在无激活物刺激的条件下，对于来源于供者713的TIL细胞，各组 TIL细胞中GZMB表达细胞的比例。图111B-111C显示的是，在transACT刺激过夜的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
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“YEATS结构域蛋白家族”通常是指具有YEATS结构域的家族成员蛋白或其功能活性片段。例如，YEATS结构域蛋白家族成员可以识别修饰蛋白。例如，YEATS结构域可以识别组蛋白的修饰，例如组蛋白的乙酰化修饰。例如，包含YEATS结构域的蛋白家族成员可以包含ENL(UniProt No Q03111)、AF9(UniProt No P42568)、和YEATS2(UniProt No Q9ULM3)和GAS41(UniProt No O95619)。在本申请中，术语“YEATS抑制剂”通常是指能够影响YEATS蛋白的活性和/或功能的物质。合适的抑制剂分子可以包括拮抗剂抗体或抗体片段、小分子的片段或衍生物、肽、反义寡核苷酸、小的有机分子等。鉴定本申请的抑制剂的方法包括使表达本申请所抑制的分子的细胞与候选抑制剂分子接触，检测本申请所抑制的分子相关的一种或多种生物学活性可检测的变化，YEATS抑制剂可以包含能够降低编码包含YEATS结构域的蛋白的核酸分子的表达和/或活性的物质。例如，YEATS抑制剂可以通过结合YEATS蛋白的用于结合组蛋白的结构域，实现抑制YEATS功能的效果。例如，YEATS抑制剂可以包含SGC-iMLLT(CAS NO.：2255338-25-9)以及已知的任意YEATS抑制剂，以及上述化合物的衍生物。例如，YEATS抑制剂可以具有特异性，对于包含YEATS结构域的家族成员的一种或多种蛋白具有更高的结合活性。
在本申请中，术语“Janus激酶-信号转导及转录激活因子(JAK-STAT)通路抑制分子”通常是指一种信号通路的抑制分子。例如，JAK-STAT通路抑制分子可以包含抑制JAK-STAT通路激活或抑制JAK-STAT通路转导因子的活性的物质。
在本申请中，术语“Janus激酶(JAK)”通常是指一种信号通路因子。例如Janus激酶可以作为传递JAK-STAT通路信号的因子。例如，JAK的UniProt登录号可以是P23458。
在本申请中，术语“细胞因子信号抑制分子”通常是指一种抑制细胞因子信号传导的物质。例如，一种示例性细胞因子信号抑制分子可以是SOCS，或其变体，或者包含上述的功能活性片段的物质。
在本申请中，术语“细胞因子信号抑制物1(SOCS1)”通常是指一种抑制细胞因子信号传导的物质。例如，SOCS1可以抑制JAK-STAT通路转导。例如，SOCS1的UniProt登录号可以是O15524。本申请中，SOCS1可以涵盖未加工的SOCS1、任何形式加工的SOCS1、SOCS1的变体或包含SOCS1的功能活性片段的物质。
在本申请中，术语“TGFβR2”通常是指一种细胞因子的受体。例如，TGFβR2可以与TGFβ结合，并介导下游信号，在细胞和组织的生长、发育、分化中起关键作用。例如，TGFβR2的NCBI Gene登录号可以是7048。本申请中，TGFβR2可以涵盖未加工的TGFβR2、任何形式加工的TGFβR2、TGFβR2的变体或包含TGFβR2的功能活性片段的物质。
在本申请中，术语“FLI1”通常是指一种编码蛋白的基因或该蛋白。例如，FLI1可以编码包含ETS(红细胞转化特异性)的DNA结合结构域的蛋白。例如，FLI1可以介导下游信号，在细胞和组织的生长、发育、分化中起关键作用。例如，FLI1的NCBI Gene登录号可以是2313。本申请中，FLI1可以涵盖未加工的FLI1、任何形式加工的FLI1、FLI1的变体或包含FLI1的功能活性片段的物质。
在本申请中，术语“CISH(Cytokine Inducible SH2 Containing Protein)”通常是指一种编码蛋白的基因或该蛋白。例如，CISH可以编码包含SH2结构域和SOCS盒结构域的蛋白。例如，CISH可以介导下游信号，在细胞和组织的生长、发育、分化中起关键作用。例如，CISH的NCBI Gene登录号可以是1154。本申请中，CISH可以涵盖未加工的CISH、任何形式加工的CISH、CISH的变体或包含CISH的功能活性片段的物质。
在本申请中，术语“CBLB”通常是指一种编码蛋白的基因或该蛋白。例如，CBLB可以编码包含E3泛素连接酶的蛋白。例如，CBLB的NCBI Gene登录号可以是868。例如，CBLB可以介导下游信号，在细胞和组织的生长、发育、分化中起关键作用。本申请中，CBLB可以涵盖未加工的CBLB、任何形式加工的CBLB、CBLB的变体或包含CBLB的功能活性片段的物质。
在本申请中，术语“ANKRD11”通常是指一种编码蛋白的基因或该蛋白。例如，ANKRD11可以编码包含锚蛋白重复结构域的蛋白。例如，ANKRD11可以介导下游信号，在细胞和组织的生长、发育、分化中起关键作用。例如，ANKRD11的NCBI Gene登录号可以是29123。本申请中，ANKRD11可以涵盖未加工的ANKRD11、任何形式加工的ANKRD11、ANKRD11的变体或包含ANKRD11的功能活性片段的物质。
在本申请中，术语“BATF”通常是指一种编码蛋白的基因或该蛋白。例如，BATF可以编码包含亮氨酸拉链的蛋白。例如，BATF可以介导下游信号，在细胞和组织的生长、发育、分 化中起关键作用。例如，BATF的NCBI Gene登录号可以是10538。本申请中，BATF可以涵盖未加工的BATF、任何形式加工的BATF、BATF的变体或包含BATF的功能活性片段的物质。
在本申请中，术语“基因调控系统”通常是指调节目标基因表达或活性的系统。例如，基因调控系统可以包含基因调控分子。例如，基因调控系统可以调节基因的表达或活性，如使该基因处于失活或激活的状态、使该基因的数量增加或降低、使该基因处于转录量提高或降低的状态、和/或使该基因的转录产物处于失活或激活的状态；例如，基因调控系统可以调节基因的表达或活性，如使单个细胞中该基因的表达产物的量提高或降低、和/或使表达该基因的表达产物的细胞的数量提高或降低。
在本申请中，术语“指导核酸分子”通常是指一种可以用于基因编辑的核酸分子。例如，指导核酸分子可以提供核苷酸插入或删除的信息，指导编辑过程。例如，指导核酸分子可以是指导RNA或向导RNA(guide RNA，gRNA)。例如，“gRNA”可以是指结合到Cas蛋白并使Cas蛋白靶向靶DNA内特定位置的RNA分子。例如，其中gRNA与DNA靶向序列之间的杂交促进CRISPR复合物的形成，可以不必要求完全互补性，例如只要存在引起杂交并促进CRISPR复合物形成的充分互补性即可。
在本申请中，术语“酶蛋白”通常是指一种具有酶活性的蛋白。例如，酶蛋白可以是指Cas蛋白。例如，Cas蛋白可以包含至少一个RNA识别或结合结构域，该结构域可以与gRNA相互作用。Cas蛋白还可以包含核酸酶结构域(例如，DNA酶或RNA酶结构域)、DNA结合结构域、解旋酶结构域、蛋白-蛋白相互作用结构域、二聚化结构域和/或其他结构域。核酸酶结构域可以具有用于核酸切割的催化活性。切割可以包括核酸分子共价键的断裂。Cas蛋白可以为野生型蛋白(即，自然界存在的蛋白)、经修饰的Cas蛋白(即，Cas蛋白变体)或者野生型或经修饰的Cas蛋白的片段。Cas蛋白还可以是野生型或经修饰的Cas蛋白的活性变体或片段。本申请中，Cas蛋白可以涵盖未加工的Cas蛋白、任何形式加工的Cas蛋白、Cas蛋白的变体或包含Cas蛋白的功能活性片段的物质。
在本申请中，术语“核糖核蛋白复合物”通常是指一种蛋白与核酸形成的复合体。例如，核糖核蛋白复合物中的蛋白可以具有核酸酶活性。例如，核糖核蛋白复合物可以在其中核酸的指导下，对目标序列进行切割。例如，核糖核蛋白复合物可以是Cas蛋白与gRNA形成的复合物。
在本申请中，术语“外显子”通常是指基因上可以被表达为蛋白质的部分。例如，外显子可以是指在蛋白质生物合成过程中具有被表达为蛋白质的能力。例如，剪切目标基因的外 显子序列可以降低目标基因的活性或功能。
在本申请中，术语“原型间隔序列毗邻基序(PAM)”通常是指靶序列后的短序列。例如，Cas9对靶DNA的位点特异性切割时，PAM序列可以用于决定切割的位置。例如，确定了PAM的区域，本领域技术人员可以容易确定合适的靶序列位置，以及可以容易设计出用于切割目标序列的gRNA序列。
在本申请中，术语“表达降低”通常是指产物或其基因的表达量的降低和/或能够表达所述产物的细胞比例下降。例如，可以是细胞该基因表达的产物的量降低或包含该基因表达的产物的细胞比例降低，或者分泌该基因表达的产物的细胞比例降低。例如，可以通过检测细胞的基因组中该基因的敲除量，间接表示该基因的表达降低。例如，可以通过检测一个细胞群中该基因被敲除的细胞所占的比例，间接表示该基因的表达降低。
在本申请中，术语“活性”通常是指物质的生物学功能。例如，基因的活性可以是指该基因的转录和/或翻译状态。例如，基因的活性减弱可以是指该基因的转录功能减弱、该基因无法被正常转录或该基因转录产物的功能被抑制。
在本申请中，术语“CD80”通常是指一种细胞刺激分子。例如，CD80可以是CD28的配体。例如，CD80可以见于GenBank登记号P33681。本申请的CD80蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含CD80的功能活性片段的物质。例如，本申请的CD80可以包含CD80的功能活性片段以及其它任意的结构域。
在本申请中，术语“CD86”通常是指一种细胞刺激分子。例如，CD86可以是CD28的配体。例如，CD86可以见于GenBank登记号P42081。本申请的CD86蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含CD86的功能活性片段的物质。例如，本申请的CD86可以包含CD86的功能活性片段以及其它任意的结构域。
在本申请中，术语“分泌”通常是指一种物质可以定位于细胞的胞外。例如，分泌的物质可以在细胞内合成之后，被运送到细胞的胞外空间。例如，可以通过酶联免疫吸附剂测定或其它检测方法检测一种物质是否是分泌的物质。
在本申请中，术语“T细胞受体”或“TCR”通常是指响应于抗原的呈递参与T细胞的活化的膜蛋白的复合体。TCR可以负责识别结合至主要组织相容性复合体分子的抗原。TCR可以由alpha(α)和beta(β)链的异二聚体组成，或由gamma和delta(γ/δ)链构成。TCR可以以α/β和γ/δ形式存在，其是结构上相似的，但是具有独特的解剖学位置和功能。例如，TCR可以是在表达TCR的任何细胞上被修饰的TCR。例如，TCR的种类可以通过TCR亚型分析试剂进行分析。
在本申请中，术语“克隆多样性”通常是指某种物质具有多种克隆型。例如，TCR的克隆多样性可以是指TCR可以具有不同序列结构和/或抗原识别能力。例如，TCR具有的多样性常用β链亚型来区分，可以包括Vβ23、Vβ7.2、Vβ5.2、Vβ11、Vβ16、Vβ3等，当一个T细胞群具有更多的β链亚型时，可以认为该T细胞群具有更高的克隆多样性。
在本申请中，“CD4 +细胞”通常是指CD4阳性的细胞，例如可以是T细胞。术语“CD4 +细胞”，“CD4阳性细胞”可以同义使用。这些细胞可通过本领域知道的方法来鉴定，例如通过用荧光标记的针对CD4的抗体对细胞染色和使用荧光激活细胞分选。例如，已有的数据可以证明，CD4 +细胞比例的提高可以使得细胞群分泌IFN-γ和/或TNF的能力提高，并可以提高T细胞群的促进肿瘤抑制的效果。例如，请见Tay,R.E.,Richardson,E.K.等人(2020).Cancer Gene Therapy,1-13.但是，本领域缺少一种提高CD4 +细胞比例的方法，本申请可以提供一种影响CD4 +细胞比例的方法。
在本申请中，“CD8 +细胞”通常是指CD8阳性的细胞，例如可以是T细胞。术语“CD8 +细胞”，“CD8阳性细胞”可以同义使用。这些细胞可通过本领域知道的方法来鉴定，例如通过用荧光标记的针对CD8的抗体对细胞染色和使用荧光激活细胞分选。
在本申请中，术语“IC 50值”或“IC50值”通常是指目标物获得生物学过程50％抑制需要的浓度。可以使用Cheng-Prusoff方程(Biochem.Pharmacol.(1973)22:3099)将IC50值换算成绝对抑制常数(Ki)。
在本申请中，术语“K D值”或“KD值”通常是指解离常数，其可通过表面等离子体共振进行测定。通常，表面等离子体共振分析使用BIAcore系统(Pharmacia Biosensor，Piscataway，NJ)，通过表面等离子体共振(SPR)，测量配体(固定化于生物传感器基质上的物质)和分析物(溶液中的物质)之间的实时结合相互作用。也可以通过固定化分析物(生物传感器基质上的物质)和呈递配体，进行表面等离子体分析。
在本申请中，术语“编码”通常是指能够根据基本上确定的规则，由一种分子的结构或组成信息，直接或间接推断出与其相关的另一类分子的结构或组成信息。例如，可以根据氨基酸的序列推断出其核苷酸序列，例如根据脱氧核糖核酸转录互补核酸的特性，包括能翻译成多肽的核酸。例如，脱氧核糖核酸可编码从脱氧核糖核酸转录的RNA。脱氧核糖核酸可类似地编码从脱氧核糖核酸所转录的RNA翻译的多肽。
在本申请中，术语“免疫细胞”通常是指参与进行先天性和适应性免疫应答的细胞。例如，可以包括但不限于淋巴细胞(诸如T细胞(包括胸腺细胞)和B细胞)、自然杀伤(NK)细胞、NKT细胞、巨噬细胞、单核细胞、嗜酸性粒细胞、嗜碱性粒细胞、嗜中性粒细胞、树突状细胞和 肥大细胞。在一些实施方案中，修饰的免疫效应细胞是T细胞，诸如CD4+T细胞、CD8+T细胞(也称为细胞毒性T细胞或CTL)、调控性T细胞(Treg)、Th1细胞、Th2细胞、Th17细胞αβT细胞和/或γδT细胞。例如，本申请的免疫细胞还包含来源于干细胞分化的免疫细胞。例如，本申请的免疫细胞还包含来源于多能干细胞分化的免疫细胞。例如，获取本申请的干细胞可以是通过诱导产生。例如，本申请的上述干细胞可以包含诱导的多能干细胞(iPSC)。
在本申请中，术语“嵌合抗原受体”通常是指一种工程化抗原受体。例如，CAR可以包含经由铰链和跨膜结构域与包含信号传导结构域的细胞质结构域融合的细胞外抗原结合结构域。在一些实施方案中，CAR细胞外结构域可以以MHC非依赖性方式与由靶细胞表达的抗原结合，从而导致细胞的活化和增殖。在一些实施方案中，CAR的细胞外结构域可以识别与抗体或其抗原结合片段融合的标签。例如，可以使得单个CAR构建体可以通过用一种抗体取代另一种抗体来靶向多种不同的抗原。在一些实施方案中，CAR的细胞外结构域可以包含来源于抗体的抗原结合片段。可用于本公开的抗原结合结构域可以包括例如scFv、抗体、抗体的抗原结合区、重链/轻链的可变区、和/或单链抗体。
在本申请中，术语“T细胞受体”通常是指一种工程化抗原受体。例如，TCR可以包含已从识别特定靶抗原的T细胞群体中分离并克隆的TCRα和/或TCRβ链。例如，TCRα和/或TCRβ基因(即TRAC和TRBC)可以从分离自患有特定恶性肿瘤的个体的T细胞群体或已分离自用特异性肿瘤抗原或肿瘤细胞免疫的人源化小鼠的T细胞群体中克隆而来。工程化TCR可以通过与其内源对应物相同的机制识别抗原(例如，通过识别在靶细胞表面上表达的主要组织相容性复合物(MHC)蛋白的背景下呈递的其同源抗原)，从而可以导致TCR工程化细胞的活化和增殖。
在本申请中，术语“小分子化合物”通常是指肽、肽模拟物、氨基酸、氨基酸类似物、多核苷酸、多核苷酸类似物、核苷酸、核苷酸类似物、分子量小于约10,000克/摩尔的有机或无机物(即包括异源有机物和有机金属化合物)、分子量小于约5,000克/摩尔的有机或无机物、分子量小于约1,000克/摩尔的有机或无机物、分子量小于约500克/摩尔的有机或无机物，以及这类药物的盐、酯和其它药学上可接受的形式。
在本申请中，术语“NK细胞”也称为“自然杀伤细胞”，通常是指一种细胞质中具有大颗粒的细胞。NK细胞由骨髓淋巴样干细胞发育而成，可以依赖于骨髓或胸腺微环境分化、发育。在本申请中，TIL细胞中的NK细胞的比例可以通过本申请的方法加以改变。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、 单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本申请的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab')2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合CD3)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。根据重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。目前存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。
在本申请中，术语“抗原结合片段”通常指具有特异结合抗原(例如，CD3)能力的一个或多个多肽片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv和/或dAb。
在本申请中，术语“固相介质”或“介质”通常是指结合功能的固相材料。例如，本申请固相介质可以是指通过共价结合和/或非共价结合的作用，将一种或一种以上的物质结合在介质内和/或介质表面的材料。例如，本申请的固相介质可以是指通过共价结合和/或非共价结合的作用将CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段结合在介质内和/或介质表面的材料。例如，本申请的固相介质可以是聚合物材料。
在本申请中，术语“表达”通常是指编码目标多肽的基因在细胞内发生的转录和/或翻译过程。可以通过测量存在于细胞中的相应mRNA的量来确定宿主细胞中编码目标多肽的基因的转录水平。例如，可通过PCR或通过RNA杂交对编码目标多肽的基因转录的mRNA进行定量测量。可以通过多种方法测量编码目标多肽的基因的翻译水平，例如通过ELISA，通过多肽生物活性测试，或通过蛋白质印迹或放射免疫测试法。在本申请中，术语“表达”通 常也可以是指产物发生的转录和/或翻译过程。例如，细胞因子的表达可以是细胞转录和/或翻译该细胞因子的过程。例如，细胞因子的表达可以通过检测存在于细胞中的相应mRNA的量或检测通过细胞生产的该细胞因子的量，或两者进行确定。
在本申请中，术语“一个阶段的体外扩增”、“单个阶段的体外扩增”、或“第一阶段体外扩增”等中的“阶段”通常是指TIL在体外经过的一段扩增过程。在一种实施方式中，每一个阶段之间可以是通过TIL细胞数量的变化来划分的，在一种实施方式中，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的条件来划分的。在一种实施方式中，当细胞培养基中添加了或补充添加了T细胞激活剂和/或T细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，当TIL细胞进行了离心和/或细胞洗涤后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。在一种实施方式中，当TIL细胞体外培养约1天、约2天、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、约20天、约30天、约40天、约50天或约100天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
在本申请中，术语“第一阶段体外扩增”通常是指从组织中获得初级TIL后，使用T细胞生长因子进行扩增的阶段。在一种实施方式中，本申请的组织可以选自以下组：肿瘤组织和胸腔积液，本申请的胸腔积液可以是有转移癌的患者的胸腔积液。在一种实施方式中，本申请的扩增可以是自体或者异体进行的体内扩增，或者可以是体外扩增。本申请的第一阶段体外扩增也可以称为preREP(快速扩增前)阶段。例如，源自肿瘤组织且未经体外扩增的TIL可以称为第一TIL群。例如，在两步骤法划分的本申请的培养方式中经过第一阶段体外扩增获得的TIL可以称为第二TIL群。
在本申请中，术语“第二阶段体外扩增”通常是指从受试者体内取出的组织并进行扩增后，再次进行扩增的阶段。在一种实施方式中，与经第一阶段体外扩增的TIL相比，本申请的经第二阶段体外扩增的TIL细胞数量增加，例如，可以增加至少约10倍(或至少约20、30、40、50、60、70、80或90倍)，或者在一种实施方式中细胞的数量可以增加至少约100倍。在一 种实施方式中，第二阶段体外扩增可以与第一阶段体外扩增的培养条件不同，例如加入的培养物质可以不同。例如，在两步骤法划分的本申请的培养方式中第二阶段体外扩增也可以称为REP(快速扩增)阶段。例如，在两步骤法划分的本申请的培养方式中经过第二阶段体外扩增获得的TIL可以称为第三TIL群。
在本申请中，术语“体内”通常是指发生在受试者体内的事件。
在本申请中，术语“体外”通常是指在受试者体外发生的事件。
在本申请中，术语“离体”通常是指涉及对已从受试者体内移除的细胞、组织和/或器官进行治疗或进行手术的事件。在一种实施方式中，该细胞、组织和/或器官可以通过手术或治疗方法返回到受试者的身体。
在本申请中，术语“分泌能力”通常是指细胞表达多肽或蛋白并将本申请的多肽或蛋白转移到细胞外环境的能力。
在本申请中，术语“辐照”通常是指通过射线对物质进行的处理。例如，在一种实施方式中，辐照可以是指通过X射线、α射线、β射线或γ射线对物质进行辐照。
在本申请中，术语“工程化细胞”通常是指将DNA或RNA形式的额外遗传物质加入细胞的总遗传物质而被基因修饰的细胞。在一种实施方式中，工程化细胞可以经过基因修饰以表达本申请的T细胞激活剂和/或T细胞生长因子的TIL。
在本申请中，术语“共培养”通常是指将两个或更多个不同群体的细胞在它们之间有一定程度的接触的情况下培养。本申请的两个或更多个不同群体的细胞的“接触”，在一种实施方式中可以通过直接接触，即其中一个群体的细胞与另一个群体的细胞直接物理接触。或者在一种实施方式中可以通过共用培养基所介导的间接接触。本申请的共用的培养基可以含有由共培养细胞的至少一个群体所产生和释放的代谢产物，并用于培养另一个群体的细胞。
在本申请中，术语“接触”通常是指两个或更多个不同类型的物质以任何顺序、任何方式以及任何时长接触在一起。在一种实施方式中可以通过直接接触，例如可以将一种或多种饲养细胞、T细胞激活剂和/或T细胞生长因子加入TIL细胞的培养基，例如可以将包含一种或多种饲养细胞、T细胞激活剂和/或T细胞生长因子的培养基加入和/或替换TIL细胞的培养基，例如，可以将包含一种或多种饲养细胞、T细胞激活剂和/或T细胞生长因子的培养基用于TIL细胞的培养；在一种实施方式中可以通过间接接触，例如可以将饲养细胞产生和释放的代谢产物，用于培养TIL细胞。
在本申请中，术语“混合物”通常是指两个或更多个不同物质的组合。例如，本申请的CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段可以在混合后作为混合物加入细胞培养基。
在本申请中，术语“同时接触”、“共同接触”、“与...接触同时”、“同时”和“共同”通常是指向受试者和/或细胞施用两种以上物质，使得物质同时存在于受试者和/或细胞培养的环境中。同时接触可以包括以不同的组合物同时施用、以不同的组合物在不同时间施用，或以其中存在两种以上活性药物成分的组合物施用。例如，本申请中“同时接触”通常可以是指基本上同时接触。
在本申请中，术语“扩增”通常是指在一段时间内细胞的数量增加若干倍。在一种实施方式中细胞的数量可以增加至少约3倍(或4、5、6、7、8或9倍)，在一种实施方式中细胞的数量可以增加至少约10倍(或20、30、40、50、60、70、80或90倍)，或者在一种实施方式中细胞的数量可以增加至少约100倍。在本申请中，术语“经扩增”通常是指本申请的细胞经过上述一种或多种扩增。
在本申请中，术语“聚合物”通常是指由连接在一起的单独化学部分组成的分子，本申请的聚合物部分可相同或不同。在一种实施方式中，术语“聚合物”可以指尾尾相连而形成线性分子的单独化学部分，以及以分支(如“多臂”或“星型”)结构形式连接在一起的单独化学部分。在一种实施方式中聚合物可以包括例如多糖、葡聚糖、水凝胶、聚乙二醇、或泊洛沙姆。泊洛沙姆是非离子三嵌段共聚物，其具有聚氧丙烯(聚(环氧丙烷))中央疏水链，侧连两条聚氧乙烯(聚(环氧乙烷))亲水链。本申请包含的物质可以与本文所描述的或本领域已知的任何聚合物一起配制，或与它们一起给予。
在本申请中，术语“嵌合抗体(chimeric antibody)”通常是指鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，可以建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，可以根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，可以在真核系统或原核系统中表达嵌合抗体分子。
在本申请中，术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，通常是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库。
在本申请中，术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区可以都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本申请所述抗体或配体可以为全人源单克隆抗体。全人抗体制备的相关技术可以为：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
在本申请中，术语“CDR”通常是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一可以由Kabat E.A.等人，Chothia等人和MacCallum等人提供。如本申请中使用的，CDR的Kabat定义可以应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR1、CDR2和CDR3(CDR H1、CDR H2、CDR H3或H1、H2、H3)。
在本申请中，术语“抗CD3抗体”通常是指靶向CD3的抗体或其变体，例如单克隆抗体，包括人、人源化、嵌合或鼠抗体，其针对成熟T细胞的T细胞抗原受体中的CD3受体。抗CD3抗体可以包括OKT3。抗CD3抗体可以包括SP34。抗CD3抗体还可以包括其他抗CD3抗体包括例如在一种实施方式中otelixizumab、teplizumab和visilizumab。
在本申请中，术语“IL-2”或“IL2”通常是指称为白细胞介素2的T细胞生长因子，并包括所有形式的IL-2，可以包括在一种实施方式中人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体，或其活性片段。编码IL-2基因的GeneID可以为3558。
在本申请中，术语“抗原呈递细胞”、“抗原递呈细胞”、或“APC”通常是指，在其表面上展示与主要组织相容性复合物(MHC)复合的外源抗原的免疫系统细胞，如辅助细胞(例如，B细胞、树突细胞等)。T细胞可以使用其T细胞受体(TCR)识别这些复合物。APC可以加工抗原并将其递呈至T细胞。在一种实施方式中，抗原呈递细胞可以包括选自以下组：外周单个核细胞，树突状细胞，和人工抗原呈递细胞。
在本申请中，术语“TIL特性”通常是指TIL细胞经过本申请培养方法获得的特性。TIL特性的变化可以包含：增加的TIL细胞数量，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的T细胞受体(TCR)克隆多样性和提高的组织中TIL细胞数量，或它们的任何组合。本申请的变化可以是提高或者降低。
在本申请中，术语“存续”通常是指细胞在体外和/或受试者体内的存在。例如，TIL细胞存续能力的增加，可以是指TIL细胞在体内存在的时间增加。例如，存续能力增加可以是指 细胞在受试者组织内，例如肿瘤、脾脏、骨髓、肺组织及血液中存在的时间的增加。例如，存续能力增加可以是培养基中撤去IL-2之后，TIL细胞存续能力的增加。
在本申请中，术语“纳米颗粒”通常是指至少一个尺寸小于100nm的微观颗粒。通常，纳米颗粒具有50nm至500nm(即0.05μm至0.5μm)范围内的直径；在生理环境中结构稳定；且可以容纳更小的分子(如药物或其他生物活性剂)，然后可以将该分子递送至希望的部位。例如，本申请的纳米颗粒可以包含CD28抗体或其抗原结合片段。例如，本申请的纳米颗粒可以包含CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段。例如，抗CD3抗体可以包括OKT3。例如，抗CD28抗体可以包括15E8。
在本申请中，术语“人工抗原呈递细胞”通常是指人工构建的用于呈递外源抗原的免疫细胞，例如，呈递外源抗原的方式可以是人工抗原呈递细胞的表面包含外源抗原与主要组织相容性复合物(MHC)的复合物。在一个实施方案中，可以包括分离的人工抗原呈递细胞(aAPC)，其可以包含表达HLA-A/B/C(编码其的基因GeneID可以为3105、3106或3107)、CD64(编码其的基因GeneID可以为2209)、CD80(编码其的基因GeneID可以为941)、ICOS-L(编码其的基因GeneID可以为23308)和CD58(编码其的基因GeneID可以为965)的细胞，并可以被修饰以表达一种以上T细胞激活剂，本申请的以上可以包含本数。
在本申请中，术语“融合蛋白”通常是指含有第一多肽或蛋白质或其片段、类似物或衍生物的氨基酸序列和异源多肽或蛋白质(即，不同于第一多肽或蛋白质或其片段、类似物或衍生物的第二多肽或蛋白质或其片段、类似物或衍生物，或者通常不是第一多肽或蛋白质或其片段、类似物或衍生物的一部分)的氨基酸序列的多肽或蛋白质。在某些情形中，融合蛋白可包含与异源蛋白、多肽或肽融合的预防性或治疗性药物。其中，本申请的异源蛋白、多肽或肽可以是或不是不同类型的预防性或治疗性药物。例如，可将具有免疫调节活性的两种不同蛋白质、多肽或肽融合到一起形成融合蛋白。在某些情形中，与异源蛋白、多肽或蛋白质融合前的初始多肽或蛋白质的活性相比，融合蛋白可以保留或提高了活性。例如，本申请的融合蛋白可以是融合了CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段的融合蛋白。
在本申请中，术语“杀伤能力”通常是指通过使本申请的细胞接触有效量的物质从而杀伤靶细胞来实现。在一个实施方案中，本申请的物质可以是TIL细胞。本申请的杀伤可以包括通过自身或者促进其它细胞或物质的CDC、凋亡、ADCC、和/或吞噬作用，或通过两种或更多种这些机制的组合以杀伤细胞。
在本申请中，术语“施用”通常是指通过本领域已知的任意途径，将物质递送给有此需要的受试者。药用载体和制剂或组合物也是本领域众所周知的。给药途径可以包括：静脉内的、肌肉内的、真皮内的、皮下的、透皮的、粘膜的、瘤内的和/或粘膜的。
在本申请中，术语“试剂盒”通常是指一起被包装在容器、接受器或其它容器中的两种或更多种组分，其中一种对应于本申请的物质。例如，包含本申请的TIL细胞。
在本申请中，术语“受试者”通常是指细胞或动物，可以是哺乳动物，诸如人、非人灵长类动物(猿、长臂猿、大猩猩、黑猩猩、猩猩、猕猴)、家畜(狗和猫)、农场动物(家禽如鸡和鸭、马、牛、山羊、绵羊、猪)和实验动物(小鼠、大鼠、兔、豚鼠)。人受试者包括胎儿、新生儿、婴儿、青少年和成人受试者。受试者包括动物疾病模型，例如肿瘤动物模型，和本领域技术人员已知的其它动物模型。
在本申请中，术语“饲养细胞(feeder)”通常是指可以用于支持培养另一种所关注的细胞生长的培养细胞。例如，可以通过体外生长和分泌至少一种因子至培养基。在一种实施方式中，饲养细胞可以包括抗原呈递细胞。
在本申请中，术语“特异性结合”通常是指识别特异性靶点物质，但是基本不识别或结合样品中其它分子的结合物质。例如，如果一种结合物质可以特异性结合来自一个物种的本申请的特异性靶点物质，则本申请的结合物质还可以特异性结合来自其它的一个或多个物种的本申请的靶点物质或同源靶点物质。这种种间反应性本身可以不会改变结合物质作为特异性的分类。在某些情形中，特异性结合至靶点物质的结合物质还可以结合至靶点物质的不同等位形式。
在本申请中，术语“完整的培养过程”通常是指将细胞从患者体内分离的肿瘤组织中分离开始，经过一次或一次以上的扩增，最终获得可以施用于受试者的细胞的完整过程。
在本申请中，术语“细胞培养基”通常是指细胞例如哺乳动物细胞在其中生长的营养液。细胞培养基的配制在本领域中是熟知的。典型地，细胞培养基包括缓冲液、盐、碳水化合物、氨基酸、维生素以及必要的微量元素。细胞培养基可以含有或不含有血清、蛋白胨、和/或蛋白质。细胞培养基可以补充有另外的组分或浓度增加的组分，如氨基酸、盐、糖、维生素、激素、生长因子、缓冲液、抗生素、脂质、微量元素等，这取决于有待培养的细胞的要求和/或所希望的细胞培养参数。
在本申请中，术语“药物组合物”或“药物制剂”通常是指一种制备物，本申请的制备物可以允许有效成分的生物活性有效，并且可以不含有对于将会施用该制剂的受试者不可接受地有 毒的额外组分。这类制剂是无菌的。“可药用的”赋形剂(载体、添加物)是可以合理地施用至受试哺乳动物以提供有效剂量的所用有效成分的那些赋形剂。
在本申请中，术语“肿瘤浸润淋巴细胞”或“TIL”通常是指最初作为白细胞获得的细胞群，本申请的细胞已经离开受试者的血流并迁移到肿瘤中。TIL可以包括但不限于CD8 +细胞毒性T细胞(淋巴细胞)、Th1和Th17CD4 +T细胞、天然杀伤细胞、树突细胞和M1巨噬细胞。TIL可以包括初级TIL和次级TIL。“初级TIL”可以是从受试者组织样品获得的那些TIL细胞，“次级TIL”可以是本申请中已扩增或经扩增的任何TIL群。在一些实施方式中，本申请的肿瘤浸润淋巴细胞可以是未经分离纯化的，或者可以是与肿瘤细胞相互浸润的。在一种实施方式中，本申请的TIL可以是指TIL群。
在本申请中，术语“中心记忆T细胞”通常是指具有长期记忆性的，并能够接受抗原再刺激的T细胞。中心记忆T细胞可以具有CD45RA -CCR7 +的表型，例如可以是通过CD45RA -和CCR7 +来鉴定中心记忆T细胞。中心记忆T细胞可以相比普通T细胞具有更强的抗肿瘤生长的能力。
在本申请中，术语“调节性T细胞”通常是指一类控制体内自身免疫反应性的T细胞亚群。调节性T细胞可以具有CD4 +CD25 +Foxp3 +的表型，例如可以是通过CD4 +、CD25 +和Foxp3 +来鉴定调节性T细胞。调节性T细胞可以具有抑制T细胞的抗肿瘤生长的能力。
在本申请中，术语“活化T细胞”通常是指经过活化而可以具有抗肿瘤生长的能力的T细胞。活化T细胞可以具有PD-1 +(PD1 +)、LAG-3 +(LAG3 +)或CD28 +的表型，例如可以是通过PD-1 +、LAG-3 +或CD28 +来鉴定活化T细胞。活化T细胞可以具有抗肿瘤生长的能力。
在本申请中，术语“肿瘤特异性T细胞”通常是指可以特异性抗肿瘤生长的T细胞。肿瘤特异性T细胞可以具有CD103 +CD39 +的表型，例如，可以是通过CD103 +和CD39 +来鉴定肿瘤特异性T细胞。肿瘤特异性T细胞可以相比普通T细胞具有更特异性的抗肿瘤生长的能力。
在本申请中，术语“干细胞样T细胞”通常是指可以具有自我增殖和/或分化的潜能的一类T细胞。干细胞样T细胞可以具有TCF1 +的表型，例如可以是通过TCF1 +来鉴定干细胞样T细胞。肿瘤特异性T细胞可以相比普通T细胞具有更强和/或更长期的抗肿瘤生长的能力。
在本申请中，术语肿瘤“碎片”通常是指从受试者体内取出肿瘤组织后，可以通过机械破碎、酶解和/或其它破碎方法，形成的肿瘤碎片。
在本申请中，术语“组合物”或“药物组合物”通常是指至少一种细胞以及至少一种和任选多于一种的其他药学上可接受的化学组分如运载体、稳定剂、稀释剂、分散剂、助悬剂、增稠剂和/或赋形剂的混合物。
在本申请中，术语“药学上可接受的载体”通常是指不干扰活性成分的一种或多种非毒性材料。例如，药学上可接受的载体可以不干扰扰活性成分的生物活性；例如，药学上可接受的载体可以不干扰扰活性成分所具有的生物活性的有效性。这类制剂常规地可以含有盐、缓冲剂、防腐剂、相容的载体、以及任选地其他治疗剂。这类药学上可接受的制剂还可以含有适合于给予人的相容的固体或液体填料、稀释剂或包封物质。可以用于在此所描述的配制品中的其他设想的载体、赋形剂、和/或添加剂可以包括：例如，调味剂、抗微生物剂、增甜剂、抗氧化剂、抗静电剂、脂质、蛋白质赋形剂(如血清白蛋白、明胶、酪蛋白)、成盐平衡离子(如钠)等等。适合用于在此所描述的配制品中的这些和另外已知的药物载体、赋形剂和/或添加剂是本领域中已知的。本申请中，“药学上可接受的载体(carrier)”可以理解为不包含基因工程用到的核酸形式的载体(vector)。
在本申请中，术语“功能活性片段”通常是指具有全长蛋白质或核酸的部分区域，但保留或部分保留全长蛋白质或核酸的生物活性或功能的片段。例如，功能活性片段可以保留或部分保留全长蛋白质结合另一种分子的能力。例如，生长因子IL-2的功能活性片段，可以保留或部分保留全长IL-2的引起细胞增殖的生物活性功能。
在本申请中，术语“T细胞激活剂”通常是指与T细胞上的相应结合受体结合，并介导T细胞共刺激反应的物质。T细胞激活剂可以是T细胞产生有效免疫应答所需的除抗原受体之外的物质。T细胞激活剂可以是指T细胞共刺激分子。例如，本申请的T细胞激活剂可以包含其变体、同源物或包含其功能活性片段的任何物质。T细胞激活剂可以包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号淋巴细胞活化分子(SLAM蛋白)、NK细胞活化受体、BTLA(编码其的基因GeneID可以为151888)、Toll配体受体、OX40(编码其的基因GeneID可以为7293)、CD2(编码其的基因GeneID可以为914)、CD7(编码其的基因GeneID可以为924)、CD27(编码其的基因GeneID可以为939)、CD28(编码其的基因GeneID可以为940)、CD30(编码其的基因GeneID可以为943)、CD40(编码其的基因GeneID可以为958)、CDS、ICAM-1(编码其的基因GeneID可以为3383)、LFA-1(CD11a/CD18)(编码其的基因GeneID可以为3689)、4-1BB(CD137)(编码其的基因GeneID可以为3604)、B7-H3(编码其的基因GeneID可以为80381)、ICOS(CD278)(编码其的基因GeneID可以为29851)、GITR(编码其的基因GeneID可以为8784)、BAFFR(编码其的基因 GeneID可以为115650)、LIGHT(编码其的基因GeneID可以为8740)、HVEM(LIGHTR)(编码其的基因GeneID可以为8764)、KIRDS2、SLAMF7(编码其的基因GeneID可以为57823)、NKp80(KLRF1)(编码其的基因GeneID可以为51348)、NKp44(编码其的基因GeneID可以为9436)、NKp30(编码其的基因GeneID可以为259197)、NKp46(编码其的基因GeneID可以为9437)、CD19(编码其的基因GeneID可以为930)、CD4(编码其的基因GeneID可以为920)、CD8α(编码其的基因GeneID可以为925)、CD8β(编码其的基因GeneID可以为926)、IL-2Rβ、IL-2Rγ、IL7Rα(编码其的基因GeneID可以为)、ITGA4(编码其的基因GeneID可以为3676)、VLA1(编码其的基因GeneID可以为3672)、CD49a(编码其的基因GeneID可以为3672)、IA4(编码其的基因GeneID可以为3732)、CD49D(编码其的基因GeneID可以为3676)、ITGA6(编码其的基因GeneID可以为3655)、VLA-6(编码其的基因GeneID可以为3655)、CD49f(编码其的基因GeneID可以为3655)、ITGAD(编码其的基因GeneID可以为3681)、CD11d(编码其的基因GeneID可以为3681)、ITGAE(编码其的基因GeneID可以为3682)、CD103(编码其的基因GeneID可以为3682)、ITGAL(编码其的基因GeneID可以为3683)、CD11a(编码其的基因GeneID可以为3683)、LFA-1(编码其的基因GeneID可以为3683)、ITGAM(编码其的基因GeneID可以为3684)、CD11b(编码其的基因GeneID可以为3684)、ITGAX(编码其的基因GeneID可以为3687)、CD11c(编码其的基因GeneID可以为3687)、ITGB1(编码其的基因GeneID可以为3688)、CD29(编码其的基因GeneID可以为3688)、ITGB2(编码其的基因GeneID可以为3689)、CD18(编码其的基因GeneID可以为3689)、LFA-1(编码其的基因GeneID可以为3689)、ITGB7(编码其的基因GeneID可以为3695)、NKG2D(编码其的基因GeneID可以为22914)、NKG2C(编码其的基因GeneID可以为3822)、TNFR2(编码其的基因GeneID可以为7133)、TRANCE/RANKL(编码其的基因GeneID可以为8600)、DNAM1(CD226)(编码其的基因GeneID可以为10666)、SLAMF4(CD244、2B4)(编码其的基因GeneID可以为51744)、CD84(编码其的基因GeneID可以为8832)、CD96(Tactile)(编码其的基因GeneID可以为10225)、CEACAM1(编码其的基因GeneID可以为634)、CRTAM(编码其的基因GeneID可以为56253)、Ly9(CD229)(编码其的基因GeneID可以为4063)、CD160(BY55)(编码其的基因GeneID可以为11126)、PSGL1(编码其的基因GeneID可以为6404)、CD100(SEMA4D)(编码其的基因GeneID可以为10507)、CD69(编码其的基因GeneID可以为969)、SLAMF6(NTB-A、Ly108)(编码其的基因GeneID可以为114836)、SLAM(SLAMF1、CD150、IPO-3)(编码其的基因GeneID可以为6504)、BLAME(SLAMF8)(编码其的基因GeneID可以为56833)、SELPLG(CD162)(编码其的基因GeneID可以为6404)、LTBR(编码其的基因GeneID可以为4055)、LAT(编码其 的基因GeneID可以为27040)、GADS(编码其的基因GeneID可以为9402)、SLP-76(编码其的基因GeneID可以为3937)、PAG/Cbp(编码其的基因GeneID可以为55824)、CD19a、和特异性结合CD3的配体、特异性结合CD28的配体、特异性结合HVEM的配体、特异性结合CD40L的配体、特异性结合OX40的配体、和特异性结合4-1BB的配体。共刺激胞内信号传导结构域可以是指T细胞激活剂的胞内部分。胞内信号传导结构域可以包含从中衍生的分子的完整胞内部分或完整天然胞内信号传导结构域或其功能性片段。
在本申请中，术语“T细胞生长因子”通常是指引起细胞增殖的生物活性多肽或小分子化合物。例如，本申请的T细胞生长因子可以包含其变体、同源物或包含其功能活性片段的任何物质。在一种实施方式中，T细胞生长因子可以选自以下组的一种或多种：IL-2(编码其的基因GeneID可以为3558)、IL-4(编码其的基因GeneID可以为3565)、IL-6(编码其的基因GeneID可以为3569)、IL-7(编码其的基因GeneID可以为3574)、IL-10(编码其的基因GeneID可以为3586)、IL-12(编码其的基因GeneID可以为3592或3593)、IL-15(编码其的基因GeneID可以为3600)、IL-21(编码其的基因GeneID可以为59067)、TNF-α(编码其的基因GeneID可以为100137091)、γ干扰素(编码其的基因GeneID可以为3458)等等。
在本申请中，术语“基本上同时”通常是指接触过程的一段时间内TIL可以与两种以上的物质同时接触，但是可以不限于在整个接触过程中TIL总是与两种以上的物质同时接触。在一种实施方式中，基本上同时可以是指一段时间内TIL可以与至少10％、20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％的两种以上的物质的每种物质同时接触。
在本申请中，术语“固相介质”或“介质”通常是指具有结合功能的固相材料。例如，本申请固相介质可以是指通过共价结合和/或非共价结合的作用，将一种或一种以上的物质结合在介质内和/或介质表面的材料。例如，本申请的固相介质可以结合一种或一种以上的T细胞激活剂。例如，本申请的固相介质可以是指通过共价结合和/或非共价结合的作用将CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段结合在介质内和/或介质表面的材料。例如，本申请的固相介质可以是包含OKT3抗体和15E8抗体的直径为约500纳米至约10微米的微球。例如，本申请的固相介质可以是聚合物材料。例如，本申请的固相介质可以是直径至少约500纳米的微球。例如，本申请的固相介质可以是纳米基质。例如，本申请的固相介质可以是包含OKT3抗体和15E8抗体的直径为约1纳米至约500纳米的纳米基质。
在本申请中，术语“纳米基质”通常是指一种直径在约1纳米到约500纳米的材料。在本申请中，纳米基质可以具有结合功能，例如，本申请的纳米基质可以结合一种或一种以上的 T细胞激活剂。在本申请中，纳米基质可以包含聚合物，例如，本申请的纳米基质可以包含可降解聚合物。在本申请中，纳米基质可以包含多糖、和/或葡聚糖。
在本申请中，术语“树突状细胞”通常是指存在于体内、体外、离体或宿主或受试者内的或可衍生自造血干细胞或单核细胞的抗原递呈细胞。树突状细胞及其前体可以从各种淋巴器官例如脾脏、淋巴结以及骨髓和外周血分离。本申请的树突状细胞可以具有特征形态，例如在树突细胞体的多个方向上延伸的薄层(板状伪足)。通常，树突细胞可以表达高水平的MHC和共刺激(例如B7-1和B7-2)分子。树突状细胞可以在体外诱导T细胞的抗原特异性分化，并且能够在体外和体内引发原代T细胞应答。
在本申请中，术语“体外扩增”通常是指经过培养以产生细胞的数量的变化，经扩增的细胞也可以产生细胞的数量和/或比例变化，分泌能力变化，杀伤能力变化或表达能力的变化，或它们的任何组合。本申请的变化可以是提高或者降低。在本申请中，体外扩增可以是为了扩增目的；为了检测TIL细胞的功能，例如检测TIL细胞释放细胞因子能力，而对TIL细胞进行的操作步骤(例如向TIL细胞的培养基中加入一种或一种以上物质以检测TIL细胞释放细胞因子能力)，可以不属于本申请的体外扩增。
在本申请中，术语“外周单个核细胞”或“外周血单个核细胞”通常是指外周血中具有单个核的细胞。例如，在本申请中，本申请的外周血单个核细胞可以包括淋巴细胞、单核细胞和/或树突状细胞。
在本申请中，术语“细胞因子”通常是指由一个细胞群释放的对另一个细胞起细胞间调节剂作用的蛋白。本申请的细胞因子可以是淋巴细胞因子(lymphokines)、单核细胞因子(monokines)和多肽激素。本申请的细胞因子可以包括白细胞介素(ILs)如IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-15、IL-21、和/或IL-12。在本申请中，术语细胞因子可以包括来自天然来源或来自重组细胞培养物的蛋白，天然序列细胞因子的生物活性等价物，以及其功能活性片段。
在本申请中，术语“直径”通常是指本申请物质的截面的直径。例如，当本申请的物质不是球形时，则术语“直径”通常是指本申请物质的最大截面的最大直径和/或平均直径。确定物质的直径的方法可以是本领域通用的方法，例如透射电子显微镜。
在本申请中，术语“肿瘤”通常是指任何新的病理性的组织增生。本申请的肿瘤可能是良性的，也可能是恶性的。本申请的肿瘤可能是实体的，也可能是血液的。术语“肿瘤”可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
在本申请中，术语“肿瘤组织”通常是指来自对象中的肿瘤，包括对象中的任何实体肿瘤和/或非实体肿瘤的任何组织的样品。
在本申请中，术语“CD28激动剂”通常是指结合细胞表面CD28蛋白并且在细胞中引发应答的化合物。例如，本申请的CD28激动剂可以是结合CD28的小分子制剂。例如，本申请的CD28激动剂可以是结合CD28的抗体或其抗原结合片段。
在本申请中，术语“T细胞亚群比例”通常是指根据不同T细胞亚群占TIL细胞或TIL群中的比例。例如，本申请不同的T细胞亚群具有不同的免疫活性和/或分化能力。例如，本申请的T细胞亚群可以根据T细胞表面标志物进行区分。例如，中心记忆T细胞可以具有CD45RA -CCR7 +的表型。例如，调节性T细胞可以具有CD4 +CD25 +Foxp3 +的表型。例如，活化T细胞可以具有CD25 +、CD28 +、PD-1 +或41BB +的表型。例如，肿瘤特异性T细胞可以具有CD103 +CD39 +的表型。例如，干细胞样T细胞可以具有TCF1 +的表型。
在本申请中，术语“TIL细胞数量”通常是指本申请的TIL细胞中细胞数量。在本申请中，TIL细胞数量可以是指本申请任一阶段获得的TIL群中的细胞数量。例如，TIL细胞数量可以是指源自肿瘤组织且未经体外扩增的第一TIL群的细胞数量。例如，TIL细胞数量可以是指经第一阶段体外扩增的第二TIL群的细胞数量。例如，TIL细胞数量可以是指经第二阶段体外扩增的第三TIL群的细胞数量。例如，TIL细胞数量可以是指本申请任意一种培养方法最终获得的TIL的细胞。在本申请中，TIL细胞数量可以通过本领域常用的方法测量，例如可以包括但不限于细胞计数板手动细胞计数和/或自动细胞计数器计数。
在本申请中，术语“约”和“大约”通常是指在统计上有意义的数值范围内。这样的范围可以在给定值或范围的一个数量级内，可以包括在50％内，可以包括在20％内，可以包括在10％内，可以包括在5％内。术语“约”或“大约”所包含的可允许变化可以取决于所研究的特定系统，并且本领域普通技术人员可以容易地理解。术语“以上”、“以下”、“至多”和“至少”可以包括本数。
发明详述
一方面，本申请提供一种培养免疫细胞的方法，使所述免疫细胞的YEATS结构域蛋白家族成员和/或其功能活性片段的表达降低和/或活性减弱。例如，所述YEATS结构域蛋白家族成员可以包含YEATS结构域。例如，所述YEATS结构域蛋白家族成员可以包含AF9和/或ENL。
例如，本申请的目标基因可以是编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因。例如，与目标基因的表达和/或活性未改变的免疫细胞相比，使所述免疫细胞的至少 一种目标基因的表达降低和/或活性减弱获得的免疫细胞可以显示出改善的免疫细胞特性。在一种实施方式中，目标基因的表达和/或活性未改变的免疫细胞可以是指源自同一供体的且未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的免疫细胞。在一种实施方式中，目标基因的表达和/或活性未改变的免疫细胞可以是指源自同一供体的且未曾使所述免疫细胞的目标基因以外的其它基因(例如敲除该其它基因，对细胞功能基本没有影响)的表达降低和/或活性减弱的免疫细胞。
在一种实施方式中，未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞可以是指源自同一供体的经过同样方式分离的且未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的免疫细胞。在一种实施方式中，未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞可以是指源自同一供体的同一肿瘤来源的且未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的免疫细胞。在一种实施方式中，未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞可以是指源自同一供体的同一肿瘤来源的经过同样方式分离的且未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的免疫细胞。在一种实施方式中，未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞可以是指将源自同一供体的免疫细胞分为两组，其中一组未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的免疫细胞可以为未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞。在一种实施方式中，未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞可以是指将源自同一供体的经过同样方式分离的免疫细胞分为两组，其中一组未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的免疫细胞可以为未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞。在一种实施方式中，未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞可以是指将源自同一供体的同一肿瘤来源的免疫细胞分为两组，其中一组未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的免疫细胞可以为未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞。在一种实施方式中，未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞可以是指将源自同一供体的同一肿瘤来源的经过同样方式分离的免疫细胞分为两组，其中一组未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的免疫细胞可以为未曾使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱的相应免疫细胞。例如，至少一种目标基因的表达降低和/或活性减弱可以是指天然的细胞的该目 标基因的处于一定程度的表达状态，经过本申请的处理，可以使得该细胞的该目标基因的表达量降低，即该目标基因的表达量降低可以是使天然细胞从表达该目标基因转变为基本不表达该目标基因或表达该目标基因的量降低。
例如，所述免疫细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
例如，所述免疫细胞包含单核细胞、巨噬细胞和/或树突状细胞。
例如，本申请的免疫细胞还包含来源于干细胞分化的免疫细胞。例如，本申请的免疫细胞还包含来源于多能干细胞分化的免疫细胞。例如，获取本申请的干细胞可以是通过诱导产生。例如，本申请的上述干细胞可以包含诱导的多能干细胞(iPSC)。
例如，所述免疫细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。例如，“未修饰的免疫细胞”或“未改造的免疫细胞”可以是指其中基因组未被修饰并且不包含基因调控系统或包含对照基因调控系统(例如，空载体对照、非靶向gRNA、加扰siRNA等)的细胞或细胞群体。
例如，所述免疫细胞包含αβT细胞和/或γδT细胞。
例如，所述免疫细胞包含肿瘤浸润淋巴细胞(TIL)。
例如，所述TIL为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
例如，本申请的TIL可以为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。例如，可以通过将肿瘤组织处理成肿瘤碎片获得本申请的TIL。例如，本申请的肿瘤碎片的体积约为1-27立方毫米。例如，本申请的肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方毫米、约6立方毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约15立方毫米、约17立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或约27立方毫米。
例如，所述免疫细胞包含展示在细胞表面上的工程化免疫受体。
例如，所述工程化免疫受体与靶细胞上表达的抗原特异性结合。
例如，所述免疫细胞包含嵌合抗原受体和/或T细胞受体。
一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，可以包含：使所述TIL中包含YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以使所述TIL中包含YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本申请的第二阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低，且在本申请的第二阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第二阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第三阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低，且在本申请的第二阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成 员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低，且在本申请的第三阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第二阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低，且在本申请的第三阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低，且在本申请的第二阶段体外扩增中，可以使所述TIL中YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性降低，且在本申请的第三阶段体外扩增中，可以使所述TIL中YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，每一个阶段体外扩增之间可以是通过TIL细胞数量的变化来划分的，例如，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、至少约50倍、至少约100倍、至少约200倍、至少约500倍、或者至少约1000倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，每一个阶段的体外扩增之间也可以是通过TIL细胞培养的条件的变化来划分的。例如，当细胞培养基中添加了或补充添加了细胞激活剂和/或细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了IL-2后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了一种或多种基因调控系统后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了饲养细胞后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当TIL细胞进行了离心和/或 细胞洗涤的操作后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。例如，当TIL细胞体外培养约1天、约2天、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、约20天、约30天、约40天、约50天或约100天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
例如，与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱获得的免疫细胞显示出改善的细胞特性。
例如，所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力和提高的肿瘤细胞杀伤能力。
例如，所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞和/或幼稚细胞比例、增加的幼稚细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
例如，本申请的改善的免疫细胞数量是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞的细胞数量可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，增加的活细胞比例可以表现为免疫细胞活率的增加。例如，本申请的增加的活细胞比例可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞的活细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指免疫细胞的选自以下组的细胞因子分泌能力提高：IL-2、IL-6、CD107a、GZMB、TNF-α和IFN-γ。例如，本申请的提高的细胞因 子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌细胞因子的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌细胞因子的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌CD107a的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌CD107a的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白 家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌IL-2的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌IL-2的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌IL-6的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌IL-6的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌IFN(例如，本申请的IFN可以为IFN-γ)的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约 5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌IFN的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌TNF(例如，本申请的TNF可以为TNF-α)的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌TNF的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌GZMB的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、 至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞中分泌GZMB的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本申请的免疫细胞的细胞因子分泌能力的测定可以是通过测量免疫细胞的细胞因子表达能力。例如，本申请的免疫细胞的细胞因子分泌能力可以通过细胞流式检测的方法测定。例如，本申请的免疫细胞的细胞因子分泌能力通过测量免疫细胞的细胞因子释放能力测定。例如，本申请的免疫细胞的细胞因子分泌能力是通过流式细胞术或者CBA法(Cytometric Bead Array)测定。
例如，本申请的提高的肿瘤细胞杀伤能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞的肿瘤细胞杀伤率可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的肿瘤细胞杀伤能力可以是指与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，在至少一个体外扩增阶段中使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱的本申请免疫细胞的肿瘤细胞杀伤率可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本申请的免疫细胞的肿瘤细胞杀伤率可以通过IncuCyte系统或者CFSE和DAPI染色法测量。例如，本申请 的免疫细胞的肿瘤细胞杀伤可以是指免疫细胞杀伤实体瘤细胞的能力。
例如，本申请的改善的细胞亚群比例可以包含选自以下组的一种或多种：增加的CD8 +细胞比例，增加的中心记忆细胞和/或幼稚细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例，和增加的干细胞样细胞比例。
例如，本申请的增加的CD8 +细胞比例可以是免疫细胞中CD8阳性细胞的比例的增加。例如，在免疫细胞中CD8 +细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的增加的活化细胞比例可以是免疫细胞中CD28 +、CD25 +、和/或41BB +细胞的比例的增加。例如，在免疫细胞中活化细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中CD28 +细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中CD25 +细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中41BB +细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本申请的降低的耗竭细胞比例可以是免疫细胞中PD-1 +、LAG-3 +、TIM-3 +、和/或CD39 +细胞的比例的增加。例如，在免疫细胞中耗竭细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中PD-1 +细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约 19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中LAG-3 +细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中TIM-3 +细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中CD39 +细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、 至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本申请的减少的调节性细胞的比例可以是免疫细胞中CD4 +CD25 +Foxp3 +细胞的比例的减少。例如，在免疫细胞中调节性细胞比例可以减少至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的减少的凋亡细胞的比例可以是免疫细胞中CD95 +caspass3 +细胞和/或CD95 +DR5 +细胞的比例的减少。例如，在免疫细胞中凋亡细胞比例可以减少至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的增加的具有干性的细胞的比例可以是免疫细胞中CD69 -CD39 -细胞和/或TCF1 +细胞的比例的增加。例如，在免疫细胞中具有干性的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的增加的中心记忆细胞比例可以是免疫细胞中CD45RA -CCR7 +或者CD45RO +CD62L +细胞的比例的增加。例如，在免疫细胞中中心记忆细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、 至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的增加的幼稚T细胞比例可以是免疫细胞中CD45RO -CD62L +细胞的比例的增加。例如，在免疫细胞中幼稚细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的培养方法可以包含对于细胞的基因编辑步骤。例如，其包含：使所述免疫细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以将基因调控系统引入所述免疫细胞中。
例如，所述基因调控系统可以在DNA水平破坏所述目标基因。例如，所述基因调控系统可以破坏所述免疫细胞的基因组中的所述目标基因的区域或其片段。例如，使用所述基因调控系统后，免疫细胞中的所述目标基因所在的DNA区域或其片段被剪切而该目标基因的表达能力降低或该目标基因的活性被抑制。例如，所述基因调控系统对目标基因的编辑效果可以是长期的、持续的。
例如，所述基因调控系统可以包含指导核酸分子和酶蛋白。例如，所述酶蛋白可以具有核酸剪切酶活性，所述指导核酸分子可以指导所述酶蛋白特异性剪切目标基因所在的区域或其片段。例如，指导核酸分子和酶蛋白可以以核糖核蛋白复合物(RNP)形式存在、或各自独立地单独存在。例如，所述酶蛋白可以包含Cas蛋白。
例如，本申请使免疫细胞的至少一种目标基因的表达降低和/或活性减弱可以包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述免疫细胞中。例如，所述酶蛋白可以包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。例如，所述指导核酸分子可以包含指导RNA(gRNA)。
例如，所述gRNA可以用于与所述目标基因的序列结合。例如，所述gRNA与所述目标基因的序列的结合可以是完全互补、可以是部分互补、也可以是中等严紧或严紧条件杂交于所述目标基因的序列。例如，所述gRNA与所述目标基因的序列的结合可以使得gRNA的CRISPR系统特异性剪切所述目标基因。
例如，本申请所述指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO： 64-69。例如，本申请的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是GGG、TGG或AGG。例如，当确定了目标基因的PAM区域，本领域人员可以容易确定目标基因PAM的5′端上游约15至约25个核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中AGG、TGG、GGG和/或CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。
例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中TGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中TGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr19:6270571-6270590。
例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中TGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中TGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至 约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr19:6270679-6270698。
例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中TGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中TGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr9:20620738-20620757。
例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中AGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因所在DNA中AGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr9:20448206-20448225。
例如，所述指导核酸分子可以包含如SEQ ID NO：70-75中任一项所示的序列。
例如，与目标基因的表达和/或活性未改变的免疫细胞相比，使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱获得的免疫细胞中表达所述目标基因的产物的细胞比例可以降低和/或单个细胞中所述目标基因的表达量可以下降。
例如，本申请的方法中与目标基因的表达和/或活性未改变的免疫细胞相比，使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱获得的免疫细胞中表达所述目标基因的产物的细胞比例降低至少约5％。例如，表达所述编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因的产物的细胞比例降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、 至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，表达所述编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因的产物的细胞比例可以从可以观测的细胞比例到0％。例如，表达所述编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因的产物的细胞比例可以降低到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。例如，表达所述编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本申请的方法中所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱获得的免疫细胞中表达所述编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因的产物的细胞比例可以为至多约95％。例如，表达所述编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因的产物的细胞比例的细胞比例可以为至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。例如，表达所述编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本申请的方法中与目标基因的表达和/或活性未改变的免疫细胞相比，使所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱获得的免疫细胞中单个细胞中所述目标基因的表达量可以下降至少约5％。例如，单个细胞中所述目标基因的表达量可以下降至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，单个细胞中所述目标基因的表达量可以从可以观测的量到0％。例如，，单个细胞中所述目标基因的表达量可以下降到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、 至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。
例如，本申请的方法中所述免疫细胞的至少一种目标基因的表达降低和/或活性减弱获得的免疫细胞中单个细胞中所述目标基因的表达量可以为所述目标基因的表达和/或活性未改变的免疫细胞的至多约95％。例如，免疫细胞中单个细胞中所述编码YEATS结构域蛋白家族成员和/或其功能活性片段的基因(例如编码AF9和/或ENL的基因)表达量可以为所述编码YEATS结构域蛋白家族成员和/或其功能活性片段的表达和/或活性未改变的免疫细胞的至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。
例如，其包含：使所述免疫细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述免疫细胞的YEATS结构域蛋白家族成员的表达降低和/或活性减弱。
例如，其中，使所述源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使经所述第一阶段体外扩增的TIL的YEATS结构域蛋白家族成员的表达降低和/或活性减弱。
例如，所述第一阶段体外扩增进行至少约7天。
例如，所述第二阶段体外扩增进行至少约7天。
例如，可以在单个阶段的本申请的体外扩增中，使所述免疫细胞与所述一种或多种细胞激活剂接触以及使所述免疫细胞中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。例如，细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，在单个阶段的所述体外扩增中，使本申请的免疫细胞的YEATS结构域蛋白家族成员的表达降低和/或活性减弱且与本申请的一种或多种细胞激活剂接触。例如，在本申请第一阶段体外扩增中，可以使本申请的TIL与本申请的YEATS结构域蛋白家族成员的表达降低和/或活性减弱且与本申请的一种或多种细胞激活剂接触。例如，在本申请第二阶段体外扩增中，可以使本申请的TIL的YEATS结构域蛋白家族成员的表达降低和/或活性减弱且与本申请的一种或多种细胞激活剂接触。例如，在本申请第三阶段体外扩增中，可以使本申请的TIL的YEATS结构域蛋白家族成员的表达降低和/或活性减弱且与本申请的一种或多种细胞激活剂接触。
例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞基本上同时使 YEATS结构域蛋白家族成员的表达降低和/或活性减弱以及本申请的一种或多种细胞激活剂接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞先使YEATS结构域蛋白家族成员的表达降低和/或活性减弱，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本申请的一种或多种细胞激活剂接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞先与本申请的一种或多种细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使YEATS结构域蛋白家族成员的表达降低和/或活性减弱。
例如，在本申请第一阶段体外扩增中，可以使本申请的TIL基本上同时使YEATS结构域蛋白家族成员的表达降低和/或活性减弱以及本申请的一种或多种细胞激活剂接触。例如，在本申请第二阶段体外扩增中，可以使本申请的TIL基本上同时使YEATS结构域蛋白家族成员的表达降低和/或活性减弱以及本申请的一种或多种细胞激活剂接触。例如，在本申请第三阶段体外扩增中，可以使本申请的TIL基本上同时使YEATS结构域蛋白家族成员的表达降低和/或活性减弱以及本申请的一种或多种细胞激活剂接触。
例如，本申请的第二阶段体外扩增进行至少约9天。例如，本申请的第二阶段体外扩增可以进行至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本申请的第二阶段体外扩增可以进行约9天至约14天，例如，本申请的第二阶段体外扩增可以进行约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。例如，本申请的第二阶段体外扩增可以认为是REP(rapid expansion protocol)阶段。例如，本申请的第一阶段体外扩增可以认为是preREP阶段。
例如，本申请第二阶段体外扩增进行的天数可以是从第二阶段体外扩增的开始时刻进行计算。例如，第二阶段体外扩增开始的当时，可以认为是第二阶段体外扩增进行了约0天。例如，第二阶段体外扩增开始后进行了约24小时，可以认为是第二阶段体外扩增进行了约1天。例如，第二阶段体外扩增开始的当天，可以认为是第二阶段体外扩增进行了约0天。例如，本申请第二阶段体外扩增进行的天数可以是通过第二阶段体外扩增进行的天数进行计算。例如，第二阶段体外扩增开始后的第二天，可以认为是第二阶段体外扩增进行了约1天。
例如，本申请的细胞激活剂可以包含选自以下组的一种或多种：CD80、CD86、B7-H3、4-1BBL、CD27、CD30、CD134、B7h、CD40、LIGHT、以及它们的功能活性片段。例如，本 申请的细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，本申请的细胞激活剂可以包含选自以下组：CD3、CD28、HVEM、CD40L、OX40和4-1BB的抗体以及它们的抗原结合片段。例如，本申请的细胞激活剂可以包含CD3激动剂。例如，本申请的细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以是Miltenyi Biotech的OKT3，可以是BD的SP34。例如，本申请的细胞激活剂可以包含CD28激动剂。例如，本申请的细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以是Merck的15E8。
例如，本申请的细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链VL和重链VH，可以包含BD的SP34的轻链VL和重链VH。例如，本申请的细胞激活剂可以包含CD28激动剂。例如，本申请的细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链VL和重链VH。例如，本申请的细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链LCDR1-3和重链HCDR1-3，可以包含BD的SP34的轻链LCDR1-3和重链HCDR1-3，本申请的抗CD3的抗体和/或其抗原结合片段可以具有CD3结合能力。例如，本申请的细胞激活剂可以包含CD28激动剂。例如，本申请的细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链LCDR1-3和重链HCDR1-3，本申请的抗CD28的抗体和/或其抗原结合片段可以具有CD28结合能力。在本申请中，本申请抗体或其抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，本申请CDR可以是根据Chothia定义的，或本申请CDR可以是根据Kabat定义的。
例如，使本申请的免疫细胞与本申请的一种或多种细胞激活剂接触可以包含选自以下组的一种或多种方式：(1)将本申请的细胞激活剂添加至本申请的免疫细胞的细胞培养基中；(2)将表达本申请的细胞激活剂的工程化细胞添加至本申请的免疫细胞的细胞培养基中；(3)将包含本申请的细胞激活剂的固相介质添加至本申请的免疫细胞的细胞培养基中。例如，使本申请的免疫细胞与本申请的一种或多种细胞激活剂接触可以包含将包含本申请的细胞激活剂的固相介质添加至本申请的免疫细胞的细胞培养基中。例如，使本申请的免疫细胞与本申请的一种或多种细胞激活剂接触可以包含将包含本申请的CD28抗体与CD3抗体的固相介质添加至本申请的免疫细胞的细胞培养基中。
例如，所述细胞激活剂在本申请免疫细胞的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本申请的CD28抗体在本申请免疫细胞的细胞培养基中的初始浓度可以为至少 约30ng/mL；例如，本申请的CD3抗体在本申请免疫细胞的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本申请的CD28抗体初始浓度的选择可以与本申请的CD3抗体初始浓度的选择相互独立；例如，本申请的CD28抗体与本申请的CD3抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意组合。例如，本申请的CD28抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL。例如，本申请的CD3抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL。例如，本申请的CD28抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL，且本申请的CD3抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL，本申请的CD28抗体初始浓度的选择可以与本申请的CD3抗体初始浓度的选择相互独立。例如，本申请的固相介质的直径可以为约500纳米至约10微米。例如，本申请的固相介质的直径可以通过透射电子显微镜测量。例如，本申请的固相介质的直径可以为约1纳米至约500纳米。例如，本申请的固相介质的直径可以为约100纳米至约500纳米。例如，本申请的固相介质的直径可以为约200纳米至约500纳米。例如，本申请的固相介质的直径可以通过透射电子显微镜测量。
例如，本申请的固相介质可以包含聚合物。例如，本申请的固相介质可以包含葡聚糖。
例如，每mg本申请的固相介质包含至少约25μg的本申请的细胞激活剂。
例如，以约1:100-约1:2000的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂的固相介质添加至本申请免疫细胞的细胞培养基中。例如，以约2:1-约1:2的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂的固相介质添加至本申请免疫细胞的细胞培养基中。
例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约2:1-约1:2的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂的固相介质添加至本申请免疫细胞的细胞培养基中。例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约2:1-约1:2、以约2:1-约1:1、或以约1:1-约1:2的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂，例如CD3激动剂和/或CD28激动剂的固相介质添加至本申请免疫细胞的细胞培养基中。
例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂的固相介质添加至本申请免疫细胞的细胞培养基中。例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000、以约1:200-约1:2000、以约1:300-约1:2000、以约 1:400-约1:2000、以约1:500-约1:2000、以约1:600-约1:2000、以约1:700-约1:2000、以约1:800-约1:2000、以约1:900-约1:2000、以约1:1000-约1:2000、以约1:1200-约1:2000以约1:1400-约1:2000、以约1:1600-约1:2000、或以约1:1800-约1:2000的本申请固相介质与本申请免疫细胞的比例，例如可以将包含本申请CD28激动剂和CD3激动剂的固相介质添加至本申请免疫细胞的细胞培养基中。
例如，本申请的方法还可以包含：在至少一个阶段的本申请体外扩增中，使本申请免疫细胞与一种或多种细胞生长因子接触。
例如，在单个阶段的本申请体外扩增中，可以使本申请的免疫细胞与本申请的细胞激活剂接触且与本申请的一种或多种细胞生长因子接触。例如，在本申请第一阶段体外扩增中，可以使本申请的TIL与本申请的细胞激活剂接触且与本申请的一种或多种细胞生长因子接触。例如，在本申请第二阶段体外扩增中，可以使本申请的TIL与本申请的细胞激活剂接触且与本申请的一种或多种细胞生长因子接触。例如，在本申请第三阶段体外扩增中，可以使本申请的TIL与本申请的细胞激活剂接触且与本申请的一种或多种细胞生长因子接触。
例如，在单个阶段的本申请体外扩增中，使本申请免疫细胞基本上同时与本申请细胞激活剂以及本申请一种或多种细胞生长因子接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞基本上同时与本申请的一种或多种细胞生长因子以及本申请的一种或多种细胞激活剂接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞先与本申请的一种或多种细胞生长因子接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本申请的一种或多种细胞激活剂接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞先与本申请的一种或多种细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本申请的一种或多种细胞生长因子接触。
例如，在本申请第一阶段体外扩增中，可以使本申请免疫细胞基本上同时与本申请细胞激活剂以及本申请一种或多种细胞生长因子接触。例如，在本申请第二阶段体外扩增中，可以使本申请TIL基本上同时与本申请细胞激活剂以及本申请一种或多种细胞生长因子接触。例如，在本申请第三阶段体外扩增中，可以使本申请TIL基本上同时与本申请细胞激活剂以及本申请一种或多种细胞生长因子接触。
例如，本申请的细胞生长因子可以选自以下组的一种或多种：IL-2、IL-7、IL-12、IL-15、IL-21、γ干扰素、以及它们的功能活性片段。例如，本申请的细胞生长因子可以包含IL-2和/或其功能活性片段。例如，IL-2的功能活性片段可以包含本领域已知的可以与细胞的IL-2受 体结合的IL-2的片段。
例如，本申请的免疫细胞与本申请一种或多种细胞生长因子接触可以包含将本申请细胞生长因子添加至本申请免疫细胞的细胞培养基中。例如，本申请的细胞生长因子在本申请免疫细胞的细胞培养基中的初始浓度可以为至少约300IU/mL。例如，本申请IL-2在本申请免疫细胞的细胞培养基中的初始浓度可以为至少约350IU/mL、至少约400IU/mL、至少约500IU/mL、至少约600IU/mL、至少约700IU/mL、至少约800IU/mL、至少约900IU/mL、至少约1000IU/mL、至少约1100IU/mL、至少约1200IU/mL、至少约1300IU/mL、至少约1400IU/mL、至少约1500IU/mL、至少约2000IU/mL、至少约2500IU/mL、至少约2600IU/mL、至少约2700IU/mL、至少约2800IU/mL、至少约2900IU/mL、至少约3000IU/mL、至少约3100IU/mL、至少约3200IU/mL、至少约3300IU/mL、至少约3400IU/mL、至少约3500IU/mL、至少约4000IU/mL、至少约4500IU/mL、至少约5000IU/mL、至少约5500IU/mL、至少约6000IU/mL、至少约6500IU/mL、至少约7000IU/mL、至少约7500IU/mL、至少约8000IU/mL、至少约8500IU/mL、或至少约9000IU/mL。
例如，本申请的方法还可以包含：在至少一个阶段的本申请体外扩增中，可以使本申请免疫细胞与饲养细胞共培养。
例如，在单个阶段的本申请体外扩增中，可以使本申请免疫细胞与一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本申请饲养细胞共培养，例如，单个阶段的本申请体外扩增可以指在同一个阶段的本申请的体外扩增，例如，可以同在本申请的第一阶段体外扩增、同在本申请的第二阶段体外扩增、或同在本申请的第三阶段体外扩增等。
例如，本申请的第一阶段体外扩增中，可以使本申请TIL与一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本申请饲养细胞共培养。例如，本申请的在本申请第二阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本申请饲养细胞共培养。例如，本申请的在本申请第三阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本申请饲养细胞共培养。
例如，在单个阶段的本申请体外扩增中，可以使本申请免疫细胞与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。例如，在本申请第一阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。例如，在本申请第二阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细 胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。例如，在本申请第三阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。
例如，在单个阶段的本申请体外扩增中，可以使本申请免疫细胞与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。例如，本申请的一定时间可以为至少约2小时。例如，本申请的一定时间可以为至少约1小时、至少约2小时、至少约3小时、至少约4小时、至少约5小时、至少约6小时、至少约7小时、至少约8小时、至少约9小时、至少约10小时、至少约11小时、至少约12小时、至少约13小时、至少约14小时、至少约15小时、至少约16小时、至少约17小时、至少约18小时、至少约19小时、至少约20小时、至少约21小时、至少约22小时、至少约23小时、至少约24小时、至少约36小时、至少约48小时、至少约60小时或至少约72小时。例如，本申请的一定时间可以为约2小时至约72小时。例如，本申请的一定时间可以为约6小时到约7小时、约6小时到约8小时、约6小时到约9小时、约6小时到约10小时、约6小时到约11小时、约6小时到约12小时、约6小时到约13小时、约6小时到约14小时、约6小时到约15小时、约6小时到约16小时、约6小时到约17小时、约6小时到约18小时、约6小时到约19小时、约6小时到约20小时、约6小时到约21小时、约6小时到约22小时、约6小时到约23小时、约6小时到约24小时、约6小时到约36小时、约6小时到约48小时、约6小时到约60小时或约6小时到约72小时。例如，本申请的一定时间可以为约12小时到约13小时、约12小时到约14小时、约12小时到约15小时、约12小时到约16小时、约12小时到约17小时、约12小时到约18小时、约12小时到约19小时、约12小时到约20小时、约12小时到约21小时、约12小时到约22小时、约12小时到约23小时、约12小时到约24小时、约12小时到约36小时、约12小时到约48小时、约12小时到约60小时或约12小时到约72小时。例如，本申请的一定时间可以为约1小时、约2小时、约3小时、约4小时、约5小时、约6小时、约7小时、约8小时、约9小时、约10小时、约11小时、约12小时、约13小时、约14小时、约15小时、约16小时、约17小时、约18小时、约19小时、约20小时、约21小时、约22小时、约23小时、约24小时、约36小时、约48小时、约60小时或约72小时。
例如，本申请的饲养细胞可以包含抗原呈递细胞。例如，本申请的饲养细胞可以包含选自以下组的一种或多种：外周单个核细胞，树突状细胞，和人工抗原呈递细胞。例如，本申请的饲养细胞可以为外周单个核细胞。例如，本申请的饲养细胞可以为经过辐照的饲养细胞。 例如，本申请的饲养细胞可以为分离的人工抗原呈递细胞(aAPC)，本申请的人工抗原呈递细胞可以包含表达HLA-A/B/C、CD64、CD80、ICOS-L和/或CD58的细胞，并可以被修饰以表达一种以上本申请的细胞激活剂。例如，本申请的饲养细胞可以经过辐照，例如，可以经过伽马射线辐照，或可以经过X射线辐照。
例如，本申请的免疫细胞与本申请的饲养细胞共培养可以包含使本申请的饲养细胞的表面与本申请的免疫细胞的表面相接触。例如，本申请的免疫细胞与本申请的饲养细胞共培养包含将本申请的饲养细胞添加至本申请的免疫细胞的细胞培养基中。
例如，本申请可以以约40:1-约400:1的本申请饲养细胞与本申请免疫细胞的比例，将本申请饲养细胞添加至本申请免疫细胞的细胞培养基中。例如，本申请可以以约40:1-约400:1、以约40:1-约300:1、以约40:1-约200:1、以约40:1-约100:1、以约40:1-约90:1、以约40:1-约80:1、以约40:1-约70:1、以约40:1-约60:1、以约40:1-约50:1、以约50:1-约400:1、以约60:1-约400:1、以约70:1-约400:1、以约80:1-约400:1、以约90:1-约400:1、以约100:1-约400:1、以约200:1-约400:1、或以约300:1-约400:1的本申请饲养细胞与本申请免疫细胞的比例，将本申请饲养细胞添加至本申请免疫细胞的细胞培养基中。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群YEATS结构域蛋白家族成员的表达降低和/或活性减弱；其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群与SGC-iMLLT接触，SGC-iMLLT在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM，使所述TIL基本上同时与一种或多种细胞激活剂以及与SGC-iMLLT接触；其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中的术语中，本申请的第一阶段体外扩增可以与以上方面的方法中的步骤(A)任意替换使用。在一种实施方式中的术语中，本申请的第二阶段体外扩增可以与以上方面的方法中的步骤(B)任意替换使用。在一种实施方式中的术语中，本申请的经第一阶段体外扩增的TIL可以与经以上方面的方法中步骤(A)得到的第二TIL群任意替换使用。在一种实施方式中的术语中，本申请的经第二阶段体外扩增的TIL可以与经以上方面的方法中步骤(B)得到的第三TIL群任意替换使用。在一种实施方式中的术语中，如有需要，本申请 的第三阶段体外扩增可以与以上方面的方法中任意增加的步骤(C)任意替换使用。在一种实施方式中的术语中，如有需要，本申请的经第三阶段体外扩增的TIL可以与经以上方面的方法中任意增加的步骤(C)得到的第四TIL群任意替换使用。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种细胞生长因子接触、与多种细胞激活剂接触、使YEATS结构域蛋白家族成员的表达降低和/或活性减弱，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与细胞生长因子接触、与细胞激活剂接触、使YEATS结构域蛋白家族成员的表达降低和/或活性减弱且使所述TIL与饲养细胞共培养，YEATS结构域蛋白家族成员可以包含AF9和/或ENL；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织、胸腔积液和/或腹腔积液在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液，内约2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入细胞无血清培养基和浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2培养约3-14天。收集培养基中细胞，转移入透气培养袋、或Grex、或Xuri设备，细胞无血清培养基可以添加本申请的CD28抗体、CD3抗体以及CD28抗体、包含CD3抗体以及CD28抗体的磁珠(例如Dynabeads)和/或包含CD3抗体以及CD28抗体的纳米基质(例如transACT)、浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2以及使YEATS结构域蛋白家族成员的表达降低和/或活性减弱(YEATS结构域蛋白家族成员可以包含AF9和/或ENL，例如可以通过用携带包含本申请的gRNA与Cas蛋白形成的核糖核蛋白复合物(RNP)进行转导使所述TIL中编码YEATS结构域蛋白家族成员的基因的细胞比例为约95％或以下)，活化本申请的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于 50％，大于80％的细胞可以为记忆效应细胞和效应细胞。经刺激后可以分泌IFN-γ，和/或可以具有活化细胞比例上调的特征。
一方面，本申请提供一种免疫细胞，本申请的免疫细胞可以根据本申请的培养方法培养得到。在一种实施方式中，本申请提供的免疫细胞可以包含一种或一个批次的本申请的培养方法培养得到免疫细胞。在一种实施方式中，本申请提供的免疫细胞可以包含多种或多个批次的本申请的培养方法培养得到并以任意比例组合的免疫细胞。
在一些实施方式中，可以将使用本申请方法扩增的免疫细胞作为药物组合物施用于患者。在一些实施方式中，药物组合物可以是免疫细胞在无菌缓冲液中的悬液。使用本申请的PBMC扩增的免疫细胞可以通过本领域已知的任何合适途径施用。在一些实施方式中，细胞可以以单次动脉内或静脉内输注施用，输注可以持续约30至60分钟。其他合适的施用途径可以包括腹膜内、鞘内和淋巴管内施用。
在一些实施方式中，可以施用任何合适剂量的免疫细胞。在一些实施方式中，例如当肿瘤是黑色素瘤时，可以施用约2.3×10 9至约13.7×10 10个免疫细胞。在一些实施方式中，可以施用约1×10 9至约12×10 10个免疫细胞。在一些实施方式中，可以施用约1.2×10 10至约4.3×10 10个免疫细胞。在一些实施方式中，可以施用约3×10 10至约12×10 10个免疫细胞。在一些实施方式中，可以施用约4×10 10至约10×10 10个免疫细胞。在一些实施方式中，可以施用约5×10 10至约8×10 10个免疫细胞。在一些实施方式中，可以施用约6×10 10至约8×10 10个免疫细胞。在一些实施方式中，可以施用约7×10 10至约8×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约2.3×10 9至约13.7×10 10。在一些实施方式中，治疗有效剂量可以为约1×10 9至约12×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约1.2×10 10至约4.3×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约3×10 10至约12×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约4×10 10至约10×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约5×10 10至约8×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约6×10 10至约8×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约7×10 10至约8×10 10个免疫细胞。
在一些实施方式中，本申请的组合物中提供的免疫细胞的数量可以为约1×10 6、约2×10 6、约3×10 6、约4×10 6、约5×10 6、约6×10 6、约7×10 6、约8×10 6、约9×10 6、约1×10 7、约2×10 7、约3×10 7、约4×10 7、约5×10 7、约6×10 7、约7×10 7、约8×10 7、约9×10 7、约1×10 8、约2×10 8、约3×10 8、约4×10 8、约5×10 8、约6×10 8、约7×10 8、约8×10 8、约9×10 8、约1×10 9、约2×10 9、约3×10 9、约4×10 9、约5×10 9、约6×10 9、约7×10 9、约8×10 9、约9×10 9、约1×10 10、约2×10 10、 约3×10 10、约4×10 10、约5×10 10、约6×10 10、约7×10 10、约8×10 10、约9×10 10、约1×10 11、约2×10 11、约3×10 11、约4×10 11、约5×10 11、约6×10 11、约7×10 11、约8×10 11、约9×10 11、约1×10 12、约2×10 12、约3×10 12、约4×10 12、约5×10 12、约6×10 12、约7×10 12、约8×10 12、约9×10 12、约1×10 13、约2×10 13、约3×10 13、约4×10 13、约5×10 13、约6×10 13、约7×10 13、约8×10 13，或约9×10 13。在一些实施方式中，本申请的组合物中提供的免疫细胞数量的范围可以为约1×10 6至5×10 6、约5×10 6至1×10 7、约1×10 7至5×10 7、约5×10 7至1×10 8、约1×10 8至5×10 8、约5×10 8至1×10 9、约1×10 9至5×10 9、约5×10 9至1×10 10、约1×10 10至5×10 10、约5×10 10至1×10 11、约5×10 11至1×10 12、约1×10 12至5×10 12，或约5×10 12至1×10 13。
在一些实施方式中，本申请的组合物中提供的免疫细胞的浓度可以小于组合物的例如约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约0.0002％，或约0.0001％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的免疫细胞的浓度可以大于组合物的约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19.75％、约19.50％、约19.25％、约19％、约18.75％、约18.50％、约18.25％、约18％、约17.75％、约17.50％、约17.25％、约17％、约16.75％、约16.50％、约16.25％、约16％、约15.75％、约15.50％、约15.25％、约15％、约14.75％、约14.50％、约14.25％、约14％、约13.75％、约13.50％、约13.25％、约13％、约12.75％、约12.50％、约12.25％、约12％、约11.75％、约11.50％、约11.25％、约11％、约10.75％、约10.50％、约10.25％、约10％、约9.75％、约9.50％、约9.25％、约9％、约8.75％、约8.50％、约8.25％、约8％、约7.75％、约7.50％、约7.25％、约7％、约6.75％、约6.50％、约6.25％、约6％、约5.75％、约5.50％、约5.25％、约5％、约4.75％、约4.50％、约4.25％、约4％、约3.75％、约3.50％、约3.25％、约3％、约2.75％、约2.50％、约2.25％、约2％、约1.75％、约1.50％、约125％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、 约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约或0.0002％，或者约0.0001％w/w、w/v或v/v。
在一些实施方式中，本申请的组合物中提供的免疫细胞的浓度范围可以为组合物的约0.0001％至约50％、约0.001％至约40％、约0.01％至约30％、约0.02％至约29％、约0.03％至约28％、约0.04％至约27％、约0.05％至约26％、约0.06％至约25％、约0.07％至约24％、约0.08％至约23％、约0.09％至约22％、约0.1％至约21％、约0.2％至约20％、约0.3％至约19％、约0.4％至约18％、约0.5％至约17％、约0.6％至约16％、约0.7％至约15％、约0.8％至约14％、约0.9％至约12％，或约1％至约10％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的免疫细胞的浓度范围可以为组合物的约0.001％至约10％、约0.01％至约5％、约0.02％至约4.5％、约0.03％至约4％、约0.04％至约3.5％、约0.05％至约3％、约0.06％至约2.5％、约0.07％至约2％、约0.08％至约1.5％、约0.09％至约1％、或约0.1％至约0.9％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的免疫细胞的量可以等于或小于约10g、约9.5g、约9.0g、约8.5g、约8.0g、约7.5g、约7.0g、约6.5g、约6.0g、约5.5g、约5.0g、约4.5g、约4.0g、约3.5g、约3.0g、约2.5g、约2.0g、约1.5g、约1.0g、约0.95g、约0.9g、约0.85g、约0.8g、约0.75g、约0.7g、约0.65g、约0.6g、约0.55g、约0.5g、约0.45g、约0.4g、约0.35g、约0.3g、约0.25g、约0.2g、约0.15g、约0.1g、约0.09g、约0.08g、约0.07g、约0.06g、约0.05g、约0.04g、约0.03g、约0.02g、约0.01g、约0.009g、约0.008g、约0.007g、约0.006g、约0.005g、约0.004g、约0.003g、约0.002g、约0.001g、约0.0009g、约0.0008g、约0.0007g、约0.0006g、约0.0005g、约0.0004g、约0.0003g、约0.0002g，或者约0.0001g。
在一些实施方式中，本申请的组合物中提供的免疫细胞的量可以大于约0.0001g、约0.0002g、约0.0003g、约0.0004g、约0.0005g、约0.0006g、约0.0007g、约0.0008g、约0.0009g、约0.001g、约0.0015g、约0.002g、约0.0025g、约0.003g、约0.0035g、约0.004g、约0.0045g、约0.005g、约0.0055g、约0.006g、约0.0065g、约0.007g、约0.0075g、约0.008g、约0.0085g、约0.009g、约0.0095g、约0.01g、约0.015g、约0.02g、约0.025g、约0.03g、约0.035g、约0.04g、约0.045g、约0.05g、约0.055g、约0.06g、约0.065g、约0.07g、约0.075g、约0.08g、约0.085g、约0.09g、约0.095g、约0.1g、约0.15g、约0.2g、约0.25g、约0.3g、约0.35g、约0.4g、约0.45g、约0.5g、约0.55g、约0.6g、约0.65g、约0.7g、约0.75g、约0.8g、约0.85g、约0.9g、约0.95g、约1g、约1.5g、约2g、约2.5g、约3g、约3.5g、约4g、约4.5g、约5g、 约5.5g、约6g、约6.5g、约7g、约7.5g、约8g、约8.5g、约9g、约9.5g，或者约10g。
在一些实施方式中，免疫细胞可以单剂量施用。此种施用可以通过注射，例如可以静脉内注射。在一些实施方式中，免疫细胞可以多剂量施用。剂量可以是每年一次、两次、三次、四次、五次、六次或超过六次。剂量可以是每月一次、每两周一次、每周一次或每2天一次。在一些实施方式中，免疫细胞的施用可以连续施用。
一方面，本申请提供一种药物组合物。在一些实施方式中，其可以包含本申请的免疫细胞和/或本申请的组合物，与药学上可接受的载体。
一方面，本申请提供一种试剂盒，本申请的试剂盒可以包含本申请培养免疫细胞方法的细胞激活剂、细胞生长因子和/或饲养细胞与记载本申请培养免疫细胞方法的步骤的说明书。一方面，本申请提供一种试剂盒，本申请试剂盒可以包含本申请的免疫细胞和/或本申请的药物组合物。
一方面，本申请提供一种影响细胞，例如肿瘤细胞，生长的方法，可以包括向受试者施用本申请的免疫细胞和/或本申请的药物组合物。在一些实施方式中，影响肿瘤生长可以包含肿瘤的体积减少到施用前的例如约99％、约95％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％或约0.1％。
一方面，本申请提供本申请的免疫细胞和/或本申请的药物组合物在制备药物中的应用，本申请的药物可以用于预防和/或治疗疾病和/或症状。例如，本申请的疾病和/或症状可以包含肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
一方面，本申请提供一种预防和/或治疗疾病和/或症状的方法，可以包括向受试者施用本申请的免疫细胞和/或本申请的药物组合物。例如，本申请的疾病和/或症状可以包含肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
一方面，本申请提供一种本申请的TIL和/或本申请的药物组合物，其可以用于预防和/或治疗疾病和/或症状。例如，本申请的疾病和/或症状可以包含肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑 色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
肿瘤浸润淋巴细胞的培养方法
一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
例如，所述方法可以包含：使所述TIL与所述饲养细胞共培养之后，使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
例如，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱之后，使所述TIL与所述饲养细胞共培养。
例如，所述方法可以包含：在所述TIL与所述T细胞激活剂和/或所述T细胞生长因子接触之后且在所述TIL与所述饲养细胞共培养之前使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
例如，所述方法可以包含：在所述TIL与所述T细胞激活剂和/或所述T细胞生长因子接触基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
例如，所述方法可以包含：在所述TIL与所述饲养细胞共培养基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，其中所述TIL包含与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养获得的TIL。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中所述TIL包含使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL与CD28激动剂接触。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，其中所述TIL包含与CD28激动 剂接触获得的TIL。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL与CD28激动剂接触，其中所述TIL包含使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL。
例如，所述CD28激动剂包含抗CD28的抗体和/或其抗原结合片段、CD80和/或其功能活性片段和/或CD86和/或其功能活性片段，以及上述物质的重组蛋白。
例如，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL可以显示出改善的TIL特性。在一种实施方式中，目标基因的表达和/或活性未改变的TIL可以是指源自同一供体的且未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞。在一种实施方式中，目标基因的表达和/或活性未改变的TIL可以是指源自同一供体的且未曾使所述TIL的目标基因以外的其它基因(例如敲除该其它基因，对细胞功能基本没有影响)的表达降低和/或活性减弱的TIL细胞。
在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指源自同一供体的经过同样方式分离的且未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指源自同一供体的同一肿瘤来源的且未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指源自同一供体的同一肿瘤来源的经过同样方式分离的且未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指将源自同一供体的TIL细胞分为两组，其中一组未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞可以为未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指将源自同一供体的经过同样方式分离的TIL细胞分为两组，其中一组未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞可以为未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指将源自同一供体的同一肿瘤来源的TIL细胞分为两组，其中一组未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞可以为未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL。在一种实施方式中， 未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指将源自同一供体的同一肿瘤来源的经过同样方式分离的TIL细胞分为两组，其中一组未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞可以为未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL。例如，至少一种目标基因的表达降低和/或活性减弱可以是指天然的细胞的该目标基因的处于一定程度的表达状态，经过本申请的处理，可以使得该细胞的该目标基因的表达量降低，即该目标基因的表达量降低可以是使天然细胞从表达该目标基因转变为基本不表达该目标基因或表达该目标基因的量降低。
在一种实施方式中，本申请的改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的抗凋亡能力，和提高的T细胞受体(TCR)克隆多样性。在一种实施方式中，本申请的改善的T细胞亚群比例可以包含选自以下组的一种或多种：增加的CD4 +细胞比例，降低的CD8 +细胞比例，增加的中心记忆T细胞比例，降低的调节性T细胞的比例，增加的活化T细胞比例，增加的肿瘤特异性T细胞比例，和增加的干细胞样T细胞比例。在一种实施方式中，本申请的增加的中心记忆T细胞比例可以是TIL细胞中CD45RA -CCR7 +细胞的比例的增加。在一种实施方式中，本申请的减少的调节性T细胞的比例可以是TIL细胞中CD4 +CD25 +Foxp3 +细胞的比例的减少。在一种实施方式中，本申请的增加的活化T细胞比例可以是TIL细胞中CD25 +、CD28 +、PD-1 +或41BB +细胞的比例的增加。
例如，本申请的方法中使TIL的至少一种目标基因的表达降低和/或活性减弱可以包含将基因调控系统引入所述TIL细胞中。
例如，所述基因调控系统可以在DNA水平破坏所述目标基因。例如，所述基因调控系统可以破坏所述TIL细胞的基因组中的所述目标基因的区域或其片段。例如，使用所述基因调控系统后，TIL细胞中的所述目标基因所在的DNA区域或其片段被剪切而该目标基因的表达能力降低或该目标基因的活性被抑制。例如，所述基因调控系统对目标基因的编辑效果可以是长期的、持续的。
例如，所述基因调控系统可以包含指导核酸分子和酶蛋白。例如，所述酶蛋白可以具有核酸剪切酶活性，所述指导核酸分子可以指导所述酶蛋白特异性剪切目标基因所在的区域或其片段。例如，指导核酸分子和酶蛋白可以以核糖核蛋白复合物(RNP)形式存在、或各自独立地单独存在。例如，所述酶蛋白可以包含Cas蛋白。
例如，本申请使TIL的至少一种目标基因的表达降低和/或活性减弱可以包含：将包含所 述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。例如，所述酶蛋白可以包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。例如，所述指导核酸分子可以包含指导RNA(gRNA)。
例如，所述gRNA可以用于与所述目标基因的序列结合。例如，所述gRNA与所述目标基因的序列的结合可以是完全互补、可以是部分互补、也可以是中等严紧或严紧条件杂交于所述目标基因的序列。例如，所述gRNA与所述目标基因的序列的结合可以使得gRNA的CRISPR系统特异性剪切所述目标基因。
例如，本申请的目标基因可以包含编码细胞因子信号抑制分子的基因。例如，本申请的目标基因编码的细胞因子信号抑制分子可以包含Janus激酶-信号转导及转录激活因子(JAK-STAT)通路抑制分子。例如，本申请的所述JAK/STAT通路抑制分子可以结合Janus激酶(JAK)。例如，所述细胞因子信号抑制分子可以包含细胞因子信号抑制物(SOCS)，例如细胞因子信号抑制物1(SOCS1)。
例如，本申请所述指导核酸分子可以与SOCS1基因的第2外显子所在的区域或其片段结合。例如，本申请所述指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：34和SEQ ID NO：35。例如，本申请的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是CGG或AGG。例如，当确定了目标基因的PAM区域，本领域人员可以容易确定目标基因PAM的5′端上游约15至约25个核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，所述指导核酸分子可以包含能够结合SOCS1基因所在DNA中CGG所示PAM区前约15至约25个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合SOCS1基因所在DNA中CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr16:11255292-11255311。
例如，所述指导核酸分子可以包含能够结合SOCS1基因所在DNA中AGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合SOCS1基因所在DNA中AGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、 约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr16:11255230-11255249。
例如，所述指导核酸分子可以包含如SEQ ID NO：36和SEQ ID NO：37中任一项所示的序列。
例如，本申请的目标基因可以包含能够结合TGFβ的蛋白的基因。例如TGFβR2。
例如，本申请所述指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：38和SEQ ID NO：39。例如，本申请的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是GGG或CGG。例如，当确定了目标基因的PAM区域，本领域人员可以容易确定目标基因PAM的5′端上游约15至约25个核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子可以与选自表7所示的任一组基因组坐标定义的区域或其片段结合。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，所述指导核酸分子可以包含能够结合TGFβR2基因所在DNA中GGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合TGFβR2基因所在DNA中GGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr3:30672268-30672287。
例如，所述指导核酸分子可以包含能够结合TGFβR2基因所在DNA中CGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合TGFβR2基因所在DNA中CGG所示PAM区约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如， 靶序列可以是源自于人chr3:30672178-30672197。
例如，所述指导核酸分子可以包含如SEQ ID NO：40和SEQ ID NO：41中任一项所示的序列。
例如，本申请的目标基因可以包含编码包含ETS(红细胞转化特异性)的DNA结合结构域的蛋白的基因。例如FLI1。在一种实施方式中，所述指导核酸分子能够与选自表8所示的任一组基因组坐标定义的区域或其片段结合。
例如，本申请所述指导核酸分子可以与选自以下所示的区域或其片段结合：SEQ ID NO：42。例如，本申请的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是TGG。例如，当确定了目标基因的PAM区域，本领域人员可以容易确定目标基因PAM的5′端上游约15至约25个核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，所述指导核酸分子可以包含能够结合FLI1基因所在DNA中TGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合FLI1基因所在DNA中TGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr11:128758312-128758331。
例如，所述指导核酸分子可以包含如SEQ ID NO：43所示的序列。
例如，本申请的目标基因可以包含编码包含SH2结构域和SOCS盒结构域的蛋白的基因。例如CISH。在一种实施方式中，所述指导核酸分子能够与选自表9所示的任一组基因组坐标定义的区域或其片段结合。
例如，本申请所述指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：44、45、46、和47。例如，本申请的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是TGG或AGG。例如，当确定了目标基因的PAM区域，本领域人员可以容易确定目标基因PAM的5′端上游约15至约25个核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子 能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、CGG和GGG。
例如，所述指导核酸分子可以包含能够结合CISH基因所在DNA中TGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合CISH基因所在DNA中TGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr3:50608013-50608032。
例如，所述指导核酸分子可以包含能够结合CISH基因所在DNA中AGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合CISH基因所在DNA中AGG所示PAM区约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr3:50608368-50608387。
例如，所述指导核酸分子可以包含能够结合CISH基因所在DNA中TGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合CISH基因所在DNA中TGG所示PAM区约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr3:50608580-50608599。
例如，所述指导核酸分子可以包含能够结合CISH基因所在DNA中AGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合CISH基因所在DNA中AGG所示PAM区约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约 19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr3:50608564-50608583。
例如，所述指导核酸分子可以包含如SEQ ID NO：48-51中任一项所示的序列。
例如，本申请的目标基因可以包含编码包含E3泛素连接酶的蛋白的基因。例如CBLB。在一种实施方式中，所述指导核酸分子能够与选自表10所示的任一组基因组坐标定义的区域或其片段结合。
例如，本申请所述指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：52和SEQ ID NO：53。例如，本申请的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是TGG或AGG。例如，当确定了目标基因的PAM区域，本领域人员可以容易确定目标基因PAM的5′端上游约15至约25个核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，所述指导核酸分子可以包含能够结合CBLB基因所在DNA中TGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合CBLB基因所在DNA中TGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr3:105746016-105746035。
例如，所述指导核酸分子可以包含能够结合CBLB基因所在DNA中AGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合CBLB基因所在DNA中AGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列 可以是源自于人chr3:105751606-105751625。
例如，所述指导核酸分子可以包含如SEQ ID NO：54和SEQ ID NO：55中任一项所示的序列。
例如，本申请的目标基因可以包含编码包含锚蛋白重复结构域的蛋白的基因。例如，ANKRD11。在一种实施方式中，所述指导核酸分子能够与选自表11所示的任一组基因组坐标定义的区域或其片段结合。
例如，本申请所述指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：56和SEQ ID NO：57。例如，本申请的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是TGG或AGG。例如，当确定了目标基因的PAM区域，本领域人员可以容易确定目标基因PAM的5′端上游约15至约25个核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，所述指导核酸分子可以包含能够结合ANKRD11基因所在DNA中TGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合ANKRD11基因所在DNA中TGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr16:89305232-89305251。
例如，所述指导核酸分子可以包含能够结合ANKRD11基因所在DNA中AGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合ANKRD11基因所在DNA中AGG所示PAM区约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr16:89305208-89305227。
例如，所述指导核酸分子可以包含如SEQ ID NO：58和SEQ ID NO：59中任一项所示 的序列。
例如，本申请的目标基因可以包含编码包含亮氨酸拉链的蛋白的基因。例如BATF。在一种实施方式中，所述指导核酸分子能够与选自表12所示的任一组基因组坐标定义的区域或其片段结合。
例如，本申请所述指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：60和SEQ ID NO：61。例如，本申请的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是CGG或GGG。例如，当确定了目标基因的PAM区域，本领域人员可以容易确定目标基因PAM的5′端上游约15至约25个核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。例如，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、GGG和CGG。
例如，所述指导核酸分子可以包含能够结合BATF基因所在DNA中CGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合BATF基因所在DNA中CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr14:75546524-75546543。
例如，所述指导核酸分子可以包含能够结合BATF基因所在DNA中GGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合BATF基因所在DNA中GGG所示PAM区约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr14:75546605-75546624。
例如，所述指导核酸分子可以包含如SEQ ID NO：62和SEQ ID NO：63中任一项所示的序列。
例如，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因 的表达降低和/或活性减弱获得的TIL中表达所述目标基因的产物的细胞比例可以降低和/或单个细胞中所述目标基因的表达量可以下降。
例如，本申请的方法中与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目标基因的产物的细胞比例降低至少约5％。例如，表达所述目标基因的产物的细胞比例降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，表达所述目标基因的产物的细胞比例可以从可以观测的细胞比例到0％。例如，表达所述目标基因的产物的细胞比例可以降低到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。例如，表达所述目标基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本申请的方法中所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目标基因的产物的细胞比例可以为至多约95％。例如，表达所述目标基因的产物的细胞比例的细胞比例可以为至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。例如，表达所述目标基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本申请的方法中与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中单个细胞中所述目标基因的表达量可以下降至少约5％。例如，单个细胞中所述目标基因的表达量可以下降至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，单个细胞中所述目标基因的表达量可以从可以观测的量到0％。例如，，单个细胞中所述目标基因的表达量可以下降到至少约100％、至少 约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。
例如，本申请的方法中所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中单个细胞中所述目标基因的表达量可以为所述目标基因的表达和/或活性未改变的TIL的至多约95％。例如，TIL中单个细胞中所述目标的表达量可以为所述目标的表达和/或活性未改变的TIL的至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。
在一种实施方式中，本申请的方法还可以包含：使源自肿瘤组织且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的本申请体外扩增中，可以使本申请TIL与饲养细胞共培养。
在一种实施方式中，在单个阶段的本申请体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请饲养细胞共培养，在一种实施方式中，单个阶段的本申请体外扩增可以指在同一个阶段的本申请的体外扩增，例如，可以同在本申请的第一阶段体外扩增、同在本申请的第二阶段体外扩增、或同在本申请的第三阶段体外扩增等。
在一种实施方式中，本申请的第一阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请饲养细胞共培养。在一种实施方式中，本申请的在本申请第二阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请饲养细胞共培养。在一种实施方式中，本申请的在本申请第三阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请饲养细胞共培养。
在一种实施方式中，每一个阶段体外扩增之间可以是通过TIL细胞数量的变化来划分的，在一种实施方式中，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、至少约50倍、至少约100倍、至少约200倍、至少约500倍、或者至少约1000倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一 种实施方式中，每一个阶段的体外扩增之间也可以是通过TIL细胞培养的条件的变化来划分的。在一种实施方式中，当细胞培养基中添加了或补充添加了T细胞激活剂和/或T细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了IL-2后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，使所述TIL的至少一种目标基因的表达降低和/或活性减弱，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了饲养细胞后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，当TIL细胞进行了离心和/或细胞洗涤的操作后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。在一种实施方式中，当TIL细胞体外培养约1天、约2天、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、约20天、约30天、约40天、约50天或约100天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
例如，所述第二阶段体外扩增可以进行至少约7天。例如，所述第二阶段体外扩增可以进行至少约9天。例如，所述第二阶段体外扩增可以进行至多约14天。例如，所述第二阶段体外扩增可以进行至多约13天。例如，所述第二阶段体外扩增可以进行约7天至约14天、约9天至约14天、约7天至约13天或约9天至约13天。例如，本申请的第二阶段体外扩增可以进行至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本申请的第二阶段体外扩增可以进行约9天至约14天，例如，本申请的第二阶段体外扩增可以进行约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。例如，本申请的第二阶段体外扩增可以认为是REP(rapid expansion protocol)阶段。
例如，所述第一阶段体外扩增可以进行至少约7天。例如，所述第一阶段体外扩增可以进行约7天至约14天。例如，本申请的第一阶段体外扩增可以进行至少约7天、至少约8天、至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本申请的第一阶段体外扩增可以进行约7天至约14天、约8天至约14天、约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。例如，本申请的第一阶 段体外扩增可以认为是preREP阶段。
在一种实施方式中，本申请第二阶段体外扩增进行的天数可以是从第二阶段体外扩增的开始时刻进行计算。例如，第二阶段体外扩增开始的当时，可以认为是第二阶段体外扩增进行了约0天。例如，第二阶段体外扩增开始后进行了约24小时，可以认为是第二阶段体外扩增进行了约1天。例如，第二阶段体外扩增开始的当天，可以认为是第二阶段体外扩增进行了约0天。在一种实施方式中，本申请第二阶段体外扩增进行的天数可以是通过第二阶段体外扩增进行的天数进行计算。例如，第二阶段体外扩增开始后的第二天，可以认为是第二阶段体外扩增进行了约1天。
在一种实施方式中，本申请培养方法可以按照两步骤划分方式进行划分。例如，(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。例如，所述步骤(A)可以进行约7天至约14天。例如，所述步骤(B)可以进行约7天至约14天。
在一种实施方式中，本申请培养方法可以按照三步骤划分方式进行划分。例如，(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且可以使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；(C)可以使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。例如，所述步骤(A)可以进行约7天至约14天。例如，所述步骤(B)可以进行约0天至约8天。例如，所述步骤(C)可以进行约5天至约14天。
在一种实施方式中，本申请培养方法可以按照四步骤划分方式进行划分。例如，(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；(C)可以使所述第三TIL群的至少一种目标基因的表达降低和/或活性减弱，其中，经所述步骤(C)得到第四TIL群；(D)可以使所述第四TIL群与饲养细胞共培养，其中，经所述步骤(D)得到第五TIL群。例如，所述步骤(A)可以进行约7天至约14天。例如，所述步骤(B)可以进行约0天至约4天。例如，所述步骤(C)可以进行约0天至约4天。例如，所述步骤(D)可以进行约5天至约14天。
在一种实施方式中，本申请的培养方式的步骤(A)是从复苏和/或继续培养体外TIL群 得到第二TIL群。例如，所述体外TIL群可以包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群。例如，所述体外TIL群可以包含由所述第一TIL群接触T细胞生长因子获得的TIL群。例如，所述体外TIL群可以包含由所述第一TIL群冷冻保存获得的TIL群。例如，所述体外TIL群可以包含由所述第一TIL群接触T细胞生长因子且冷冻保存获得的TIL群。例如，当本申请的步骤(A)为是从复苏和/或继续培养体外TIL群得到第二TIL群时，此时步骤(A)可以进行约2小时至约4天。
在一种实施方式中，在单个阶段的本申请体外扩增中，可以使本申请TIL与本申请一种或多种T细胞激活剂和/或一种或多种T细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。在一种实施方式中，本申请的一定时间可以为至少约2小时。在一种实施方式中，本申请的一定时间可以为至少约1小时、至少约2小时、至少约3小时、至少约4小时、至少约5小时、至少约6小时、至少约7小时、至少约8小时、至少约9小时、至少约10小时、至少约11小时、至少约12小时、至少约13小时、至少约14小时、至少约15小时、至少约16小时、至少约17小时、至少约18小时、至少约19小时、至少约20小时、至少约21小时、至少约22小时、至少约23小时、至少约24小时、至少约36小时、至少约48小时、至少约60小时或至少约72小时。在一种实施方式中，本申请的一定时间可以为约6小时至约72小时。在一种实施方式中，本申请的一定时间可以为约6小时到约7小时、约6小时到约8小时、约6小时到约9小时、约6小时到约10小时、约6小时到约11小时、约6小时到约12小时、约6小时到约13小时、约6小时到约14小时、约6小时到约15小时、约6小时到约16小时、约6小时到约17小时、约6小时到约18小时、约6小时到约19小时、约6小时到约20小时、约6小时到约21小时、约6小时到约22小时、约6小时到约23小时、约6小时到约24小时、约6小时到约36小时、约6小时到约48小时、约6小时到约60小时或约6小时到约72小时。在一种实施方式中，本申请的一定时间可以为约12小时到约13小时、约12小时到约14小时、约12小时到约15小时、约12小时到约16小时、约12小时到约17小时、约12小时到约18小时、约12小时到约19小时、约12小时到约20小时、约12小时到约21小时、约12小时到约22小时、约12小时到约23小时、约12小时到约24小时、约12小时到约36小时、约12小时到约48小时、约12小时到约60小时或约12小时到约72小时。在一种实施方式中，本申请的一定时间可以为约1小时、约2小时、约3小时、约4小时、约5小时、约6小时、约7小时、约8小时、约9小时、约10小时、约11小时、约12小时、约13小时、约14小时、约15小时、约16小时、约17小时、约18小时、约19小时、约20小时、约21小时、约22小时、约23小时、约24小时、约36小时、 约48小时、约60小时或约72小时。
在一种实施方式中，本申请的饲养细胞可以包含抗原呈递细胞。在一种实施方式中，本申请的饲养细胞可以包含选自以下组的一种或多种：外周单个核细胞，树突状细胞，和人工抗原呈递细胞。在一种实施方式中，本申请的饲养细胞可以为外周单个核细胞。在一种实施方式中，本申请的饲养细胞可以为经过辐照的饲养细胞。例如，本申请的饲养细胞可以为分离的人工抗原呈递细胞(aAPC)，本申请的人工抗原呈递细胞可以包含表达HLA-A/B/C、CD64、CD80、ICOS-L和/或CD58的细胞，并可以被修饰以表达一种以上本申请的T细胞激活剂。在一种实施方式中，本申请的饲养细胞可以经过辐照，例如，可以经过伽马射线辐照，或可以经过X射线辐照。
在一种实施方式中，本申请的TIL与本申请的饲养细胞共培养可以包含使本申请的饲养细胞的表面与本申请的TIL的表面相接触。在一种实施方式中，本申请的TIL与本申请的饲养细胞共培养包含将本申请的饲养细胞添加至本申请的TIL的细胞培养基中。
在一种实施方式中，本申请可以以约40:1-约400:1的本申请饲养细胞与本申请TIL的比例，将本申请饲养细胞添加至本申请TIL的细胞培养基中。在一种实施方式中，本申请可以以约40:1-约400:1、以约40:1-约300:1、以约40:1-约200:1、以约40:1-约100:1、以约40:1-约90:1、以约40:1-约80:1、以约40:1-约70:1、以约40:1-约60:1、以约40:1-约50:1、以约50:1-约400:1、以约60:1-约400:1、以约70:1-约400:1、以约80:1-约400:1、以约90:1-约400:1、以约100:1-约400:1、以约200:1-约400:1、或以约300:1-约400:1的本申请饲养细胞与本申请TIL的比例，将本申请饲养细胞添加至本申请TIL的细胞培养基中。
在一种实施方式中，本申请的方法还可以包含：使源自肿瘤组织且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的本申请体外扩增中，使本申请TIL与一种或多种T细胞激活剂接触。
在一种实施方式中，在单个阶段的本申请的体外扩增中，使所述TIL与所述一种或多种T细胞激活剂接触。例如，T细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本申请第一阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本申请第二阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本 申请第三阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请的一种或多种T细胞激活剂接触。
在一种实施方式中，在单个阶段的本申请的体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以先使所述TIL的至少一种目标基因的表达降低和/或活性减弱，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以使本申请的TIL先与本申请的一种或多种T细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
在一种实施方式中，在本申请第一阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL接触本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本申请第二阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL接触本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本申请第三阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL接触本申请的一种或多种T细胞激活剂接触。
在一种实施方式中，本申请的T细胞激活剂可以包含选自以下组的一种或多种：CD80、CD86、B7-H3、4-1BBL、CD27、CD30、CD134、B7h、CD40、LIGHT、以及它们的功能活性片段。在一种实施方式中，本申请的T细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。在一种实施方式中，本申请的T细胞激活剂可以包含选自以下组：CD3、CD28、HVEM、CD40L、OX40和4-1BB的抗体以及它们的抗原结合片段。在一种实施方式中，本申请的T细胞激活剂可以包含CD3激动剂。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以是Miltenyi Biotech的OKT3，可以是BD的SP34。在一种实施方式中，本申请的T细胞激活剂可以包含CD28激动剂。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以是Merck的15E8，本申请的T细胞激活剂可以包含CD80和/或其功能活性片段和/或CD86和/或其功能活性片段，以及上述物质的重组蛋白。
在一种实施方式中，本申请的T细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链VL和重链VH，可以包含BD的SP34的轻链VL和重链VH。在一种实施方式中，本申请的T细胞激活剂可以包含CD28激动剂。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链VL和重链VH。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链LCDR1-3和重链HCDR1-3，可以包含BD的SP34的轻链LCDR1-3和重链HCDR1-3，本申请的抗CD3的抗体和/或其抗原结合片段可以具有CD3结合能力。在一种实施方式中，本申请的T细胞激活剂可以包含CD28激动剂。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链LCDR1-3和重链HCDR1-3，本申请的抗CD28的抗体和/或其抗原结合片段可以具有CD28结合能力。在本申请中，本申请抗体或其抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，本申请CDR可以是根据Chothia定义的，或本申请CDR可以是根据Kabat定义的。
在一种实施方式中，本申请的CD3激动剂可以为CD3抗体或其抗原结合蛋白。
在本申请中，本申请抗体或其抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，本申请CDR可以是根据Chothia定义的，或本申请CDR可以是根据Kabat定义的。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1，且本申请HCDR1可以包含SEQ ID NO：2和12中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR2，且本申请HCDR2可以包含SEQ ID NO：3和13中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR3，且本申请HCDR3可以包含SEQ ID NO：4和14中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：2和12中任一项所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：3和13中任一项所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：4和14中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：2所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：3所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：4所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：12所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：13所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：14所示的氨基酸序列；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在本申请中，本申请抗体或其抗原结合蛋白包含抗体轻链可变区VL中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，或本申请CDR可以是根据Kabat定义的。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR1，且本申请LCDR1可以包含SEQ ID NO：5和15中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR2，且本申请LCDR2可以包含SEQ ID NO：6(DTS)和16中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR3，且本申请LCDR3可以包含SEQ ID NO：7和17中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：5和15中任一项所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO： 6(DTS)和16中任一项所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：7和17中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：5所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：6(DTS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：7所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：15所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：16所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：17所示的氨基酸序列；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1-3和LCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：2和12中任一项所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：3和13中任一项所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：4和14中任一项所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：5和15中任一项所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：6(DTS)和16中任一项所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：7和17中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的HCDR1-3和LCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：2所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：3所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：4所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：5所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：6(DTS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：7所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：12所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：13所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：14所示的氨基酸序列，本申请 LCDR1可以包含SEQ ID NO：15所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：16所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：17所示的氨基酸序列；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链可变区VH，且本申请VH可包含SEQ ID NO：8和18中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的VH，且本申请VH可包含SEQ ID NO：8所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的VH，且本申请VH可包含SEQ ID NO：18所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含轻链可变区VL，且本申请VL可包含SEQ ID NO：9和19中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的VL，且本申请VL可包含SEQ ID NO：9所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的VL，且本申请VL可包含SEQ ID NO：19所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链可变区VH和轻链可变区VL，且本申请VH可包含SEQ ID NO：8和18中任一项所示的氨基酸序列，本申请VL可包含SEQ ID NO：9和19中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的VH和VL，且本申请VH可包含SEQ ID NO：8所示的氨基酸序列，本申请VL可包含SEQ ID NO：9所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的VH和VL，且本申请VH可包含SEQ ID NO：18所示的氨基酸序列，本申请VL可包含SEQ ID NO：19所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链，且本申请重链可包含SEQ ID NO：10和20中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的重链，且本申请重链可包含SEQ ID NO：10所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的重链，且本申请重链可包含SEQ ID NO：20所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含轻链，且本申请轻链可包含SEQ ID NO：11和21中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的轻链，且本申请轻链可包含SEQ ID NO：11所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的轻链，且本申请轻链可包含SEQ ID NO：21所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链和轻链，且本申请重链可包含SEQ ID NO：10和20中任一项所示的氨基酸序列，本申请轻链可包含SEQ ID NO：11和21中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的重链和轻链，且本申请重链可包含SEQ ID NO：10所示的氨基酸序列，本申请轻链可包含SEQ ID NO：11所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的重链和轻链，且本申请重链可包含SEQ ID NO：20所示的氨基酸序列，本申请轻链可包含SEQ ID NO：21所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的CD28激动剂可以为CD28抗体或其抗原结合蛋白。
在本申请中，本申请抗体或其抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，或本申请CDR可以是根据Kabat定义的。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1，且本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR2，且本申请HCDR2可以包含SEQ ID NO：23所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR3，且本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：23所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：23所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在本申请中，本申请抗体或其抗原结合蛋白包含抗体轻链可变区VL中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，或本申请CDR可以是根据Kabat定义的。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR1，且本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR2，且本申请LCDR2可以包含SEQ ID NO：26(AAS)所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR3，且本申请LCDR3可以包含SEQ ID NO：27所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：26(AAS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：27所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：26(AAS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：27所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1-3和LCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：23所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：30所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：26(AAS)所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的HCDR1-3和LCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：23所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：26(AAS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：27所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链可变区VH，且本申请VH可包含SEQ ID NO：28和29中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与一种15E8相同的VH，且本申请VH可包含SEQ ID NO：28所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与另一种15E8相同的VH，且本申请VH可包含SEQ ID NO：29所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含轻链可变区VL，且本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的VL，且本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链可变区VH和轻链可变区VL，且本申请VH可包含SEQ ID NO：28和29中任一项所示的氨基酸序列，本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与一种15E8相同的VH和VL，且本申请VH可包含SEQ ID NO：28所示的氨基酸序列，本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与另一种15E8相同的VH和VL，且本申请VH可包含SEQ ID NO：29所示的氨基酸序列，本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链，且本申请重链可包含SEQ ID NO：31和32中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与一种15E8相同的重链，且本申请重链可包含SEQ ID NO：31所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与另一种15E8相同的重链，且本申请重链可包含SEQ ID NO：32所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含轻链，且本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能 力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的轻链，且本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链和轻链，且本申请重链可包含SEQ ID NO：31和32中任一项所示的氨基酸序列，本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与一种15E8相同的重链和轻链，且本申请重链可包含SEQ ID NO：31所示的氨基酸序列，本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与另一种15E8相同的重链和轻链，且本申请重链可包含SEQ ID NO：32所示的氨基酸序列，本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，使本申请的TIL与本申请的一种或多种T细胞激活剂接触可以包含选自以下组的一种或多种方式：(1)将本申请的T细胞激活剂添加至本申请的TIL的细胞培养基中；(2)将表达本申请的T细胞激活剂的工程化细胞添加至本申请的TIL的细胞培养基中；(3)将包含本申请的T细胞激活剂的固相介质添加至本申请的TIL的细胞培养基中。在一种实施方式中，使本申请的TIL与本申请的一种或多种T细胞激活剂接触可以包含将包含本申请的T细胞激活剂的固相介质添加至本申请的TIL的细胞培养基中。在一种实施方式中，使本申请的TIL与本申请的一种或多种T细胞激活剂接触可以包含将包含本申请的CD28抗体与CD3抗体的固相介质添加至本申请的TIL的细胞培养基中。
在一种实施方式中，所述T细胞激活剂在本申请TIL的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本申请的CD28抗体在本申请TIL的细胞培养基中的初始浓度可以为至少约30ng/mL；例如，本申请的CD3抗体在本申请TIL的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本申请的CD28抗体初始浓度的选择可以与本申请的CD3抗体初始浓度的选择相互独立；例如，本申请的CD28抗体与本申请的CD3抗体在本申请TIL的细胞培养基中的初始浓度可以任意组合。例如，本申请的CD28抗体在本申请TIL的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL。例如，本申请的CD3抗体在本申请TIL的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL。例如，本申请的CD28抗体在本申请TIL的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL， 且本申请的CD3抗体在本申请TIL的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL，本申请的CD28抗体初始浓度的选择可以与本申请的CD3抗体初始浓度的选择相互独立。在一种实施方式中，本申请的固相介质的直径可以为约500纳米至约10微米。在一种实施方式中，本申请的固相介质的直径可以通过透射电子显微镜测量。在一种实施方式中，本申请的固相介质的直径可以为约1纳米至约500纳米。在一种实施方式中，本申请的固相介质的直径可以为约100纳米至约500纳米。在一种实施方式中，本申请的固相介质的直径可以为约200纳米至约500纳米。在一种实施方式中，本申请的固相介质的直径可以通过透射电子显微镜测量。
在一种实施方式中，本申请的固相介质可以包含聚合物。在一种实施方式中，本申请的固相介质可以包含葡聚糖。
在一种实施方式中，每mg本申请的固相介质包含至少约25μg的本申请的T细胞激活剂。
在一种实施方式中，以约1:100-约1:2000的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂的固相介质添加至本申请TIL的细胞培养基中。在一种实施方式中，以约2:1-约1:2的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂的固相介质添加至本申请TIL的细胞培养基中。
例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约2:1-约1:2的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂的固相介质添加至本申请TIL的细胞培养基中。例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约2:1-约1:2、以约2:1-约1:1、或以约1:1-约1:2的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂，例如CD3激动剂和/或CD28激动剂的固相介质添加至本申请TIL的细胞培养基中。
例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂的固相介质添加至本申请TIL的细胞培养基中。例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000、以约1:200-约1:2000、以约1:300-约1:2000、以约1:400-约1:2000、以约1:500-约1:2000、以约1:600-约1:2000、以约1:700-约1:2000、以约1:800-约1:2000、以约1:900-约1:2000、以约1:1000-约1:2000、以约1:1200-约1:2000以约1:1400-约1:2000、以约1:1600-约1:2000、或以约1:1800-约1:2000的本申请固相介质与本申请TIL的比例，例如可以将包含本申请CD28激动剂和CD3激动剂的固相介质添加至本申请TIL的细 胞培养基中。
在一种实施方式中，本申请的方法还可以包含：使源自肿瘤组织且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的本申请体外扩增中，使本申请TIL与一种或多种T细胞生长因子接触。
在一种实施方式中，在单个阶段的本申请体外扩增中，可以使本申请的TIL与本申请的T细胞激活剂接触且与本申请的一种或多种T细胞生长因子接触。例如，在本申请第一阶段体外扩增中，可以使本申请的TIL与本申请的T细胞激活剂接触且与本申请的一种或多种T细胞生长因子接触。例如，在本申请第二阶段体外扩增中，可以使本申请的TIL与本申请的T细胞激活剂接触且与本申请的一种或多种T细胞生长因子接触。例如，在本申请第三阶段体外扩增中，可以使本申请的TIL与本申请的T细胞激活剂接触且与本申请的一种或多种T细胞生长因子接触。
在一种实施方式中，在单个阶段的本申请体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以先使所述TIL的至少一种目标基因的表达降低和/或活性减弱，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使本申请的TIL与本申请的一种或多种T细胞生长因子接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以使本申请的TIL先与本申请的一种或多种T细胞生长因子接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
例如，在本申请第一阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。例如，在本申请第二阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。例如，在本申请第三阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。
在一种实施方式中，本申请的T细胞生长因子可以选自以下组的一种或多种：IL-2、IL-7、IL-12、IL-15、IL-21、γ干扰素、以及它们的功能活性片段。在一种实施方式中，本申请的T细胞生长因子可以包含IL-2和/或其功能活性片段。例如，IL-2的功能活性片段可以包含本领域已知的可以与T细胞的IL-2受体结合的IL-2的片段。
在一种实施方式中，本申请的TIL与本申请一种或多种T细胞生长因子接触可以包含将本申请T细胞生长因子添加至本申请TIL的细胞培养基中。在一种实施方式中，本申请的T细胞生长因子在本申请TIL的细胞培养基中的初始浓度可以为至少约300IU/mL。在一种实施方式中，本申请IL-2在本申请TIL的细胞培养基中的初始浓度可以为至少约350IU/mL、至少约400IU/mL、至少约500IU/mL、至少约600IU/mL、至少约700IU/mL、至少约800IU/mL、至少约900IU/mL、至少约1000IU/mL、至少约1100IU/mL、至少约1200IU/mL、至少约1300IU/mL、至少约1400IU/mL、至少约1500IU/mL、至少约2000IU/mL、至少约2500IU/mL、至少约2600IU/mL、至少约2700IU/mL、至少约2800IU/mL、至少约2900IU/mL、至少约3000IU/mL、至少约3100IU/mL、至少约3200IU/mL、至少约3300IU/mL、至少约3400IU/mL、至少约3500IU/mL、至少约4000IU/mL、至少约4500IU/mL、至少约5000IU/mL、至少约5500IU/mL、至少约6000IU/mL、至少约6500IU/mL、至少约7000IU/mL、至少约7500IU/mL、至少约8000IU/mL、至少约8500IU/mL、或至少约9000IU/mL。
在一种实施方式中，本申请的TIL可以为源自本申请的肿瘤组织的碎片的TIL。在一种实施方式中，可以通过将肿瘤组织处理成肿瘤碎片获得本申请的TIL。在一种实施方式中，本申请的肿瘤碎片的体积约为1-27立方毫米。在一种实施方式中，本申请的肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方毫米、约6立方毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约15立方毫米、约17立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或约27立方毫米。例如，所述TIL可以选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由第一TIL群体外扩增获得的TIL群，所述第一TIL群为源自肿瘤组织且未经体外扩增的TIL群；(B)使所 述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
例如，可以先将在一定的时间和/或一定位置的源自肿瘤组织且未经体外扩增的TIL群与T细胞生长因子接触，得到体外TIL群，一方面可以继续培养所述体外TIL群，进行步骤(B)，另一方面可以先冷冻保存所述体外TIL群，在有需要的时候复苏所述体外TIL群，进行步骤(B)。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)使所述TIL的目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中的术语中，本申请的第一阶段体外扩增可以与以上方面的方法中的步骤(A)任意替换使用。在一种实施方式中的术语中，本申请的第二阶段体外扩增可以与以上方面的方法中的步骤(B)任意替换使用。在一种实施方式中的术语中，本申请的经第一阶段体外扩增的TIL可以与经以上方面的方法中步骤(A)得到的第二TIL群任意替换使用。在一种实施方式中的术语中，本申请的经第二阶段体外扩增的TIL可以与经以上方面的方法中步骤(B)得到的第三TIL群任意替换使用。在一种实施方式中的术语中，如有需要，本申请的第三阶段体外扩增可以与以上方面的方法中任意增加的步骤(C)任意替换使用。在一种实施方式中的术语中，如有需要，本申请的经第三阶段体外扩增的TIL可以与经以上方面的方法中任意增加的步骤(C)得到的第四TIL群任意替换使用。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所 述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL的目标基因的表达降低和/或活性减弱，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，可以以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A) 可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触、与多种T细胞激活剂接触、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，可以以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为至少约300IU/mL；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为至少约300IU/mL、与CD3抗体接触，CD3抗体在所述TIL的细胞培养基中的初始浓度至少约30ng/mL、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，可以以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触、与包含CD3抗体以及CD28抗体的纳米基质接触、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触、与包含CD3抗体以及CD28抗体的纳米基质接触，所述纳米基质的直径可以为约1纳米至约500纳米，每mg所述纳米基质可以分别包含CD3抗体以及CD28抗体各为约25μg、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触、与包含CD3抗体以及CD28抗体 的纳米基质接触，所述纳米基质的直径可以为约1纳米至约500纳米，每mg所述纳米基质可以分别包含CD3抗体以及CD28抗体各为约25μg，可以以约1:100-约1:2000的所述纳米基质与所述TIL的比例添加至所述TIL的细胞培养基中、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为至少约300IU/mL；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为至少约300IU/mL、与包含CD3抗体以及CD28抗体的纳米基质接触，所述纳米基质的直径可以为约1纳米至约500纳米，每mg所述纳米基质可以分别包含CD3抗体以及CD28抗体各为约25μg，可以以约1:100-约1:2000的所述纳米基质与所述TIL的比例添加至所述TIL的细胞培养基中、使所述TIL中表达所述目标基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，可以以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液，内约2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入T细胞无血清培养基和浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2培养约3-14天。可以将收获的TIL细胞冻存后再复苏，也可以直接收集培养基中细胞，转移入透气培养袋、或Grex、或Xuri设备，T细胞无血清培养基可以添加本申请的CD28抗体、CD3抗体以及CD28抗体、包含CD3抗体以及CD28抗体的磁珠(例如Dynabeads)和/或包含CD3抗体以及CD28抗体的纳米基质(例如transACT)、浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2，以及用携带包含SEQ ID NO：36-37、40-41、43、48-51、54-55、58-59、62-63和70-75中任一项所示的gRNA与Cas蛋白形成的核糖核蛋白复合物(RNP)进行转导使所述TIL中表达所述目标基因的细胞比例为约95％或以下，活化本申请的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩 增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于50％，大于80％的T细胞可以为记忆效应T细胞和效应T细胞。经刺激后可以分泌IFN-γ，和/或可以具有活化T细胞比例上调的特征。
一方面，本申请提供一种肿瘤浸润淋巴细胞(TIL)，本申请的TIL可以根据本申请的培养方法培养得到。在一种实施方式中，本申请提供的TIL可以包含一种或一个批次的本申请的培养方法培养得到TIL。在一种实施方式中，本申请提供的TIL可以包含多种或多个批次的本申请的培养方法培养得到并以任意比例组合的TIL。
在一些实施方式中，可以将使用本申请方法扩增的TIL作为药物组合物施用于患者。在一些实施方式中，药物组合物可以是TIL在无菌缓冲液中的悬液。使用本申请的PBMC扩增的TIL可以通过本领域已知的任何合适途径施用。在一些实施方式中，T细胞可以以单次动脉内或静脉内输注施用，输注可以持续约30至60分钟。其他合适的施用途径可以包括腹膜内、鞘内和淋巴管内施用。
在一些实施方式中，可以施用任何合适剂量的TIL。在一些实施方式中，例如当肿瘤是黑色素瘤时，可以施用约2.3×10 9至约13.7×10 10个TIL。在一些实施方式中，可以施用约1×10 9至约12×10 10个TIL。在一些实施方式中，可以施用约1.2×10 10至约4.3×10 10个TIL。在一些实施方式中，可以施用约3×10 10至约12×10 10个TIL。在一些实施方式中，可以施用约4×10 10至约10×10 10个TIL。在一些实施方式中，可以施用约5×10 10至约8×10 10个TIL。在一些实施方式中，可以施用约6×10 10至约8×10 10个TIL。在一些实施方式中，可以施用约7×10 10至约8×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约2.3×10 9至约13.7×10 10。在一些实施方式中，治疗有效剂量可以为约1×10 9至约12×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约1.2×10 10至约4.3×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约3×10 10至约12×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约4×10 10至约10×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约5×10 10至约8×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约6×10 10至约8×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约7×10 10至约8×10 10个TIL。
在一些实施方式中，本申请的组合物中提供的TIL的数量可以为约1×10 6、约2×10 6、约3×10 6、约4×10 6、约5×10 6、约6×10 6、约7×10 6、约8×10 6、约9×10 6、约1×10 7、约2×10 7、约3×10 7、约4×10 7、约5×10 7、约6×10 7、约7×10 7、约8×10 7、约9×10 7、约1×10 8、约2×10 8、约3×10 8、约4×10 8、约5×10 8、约6×10 8、约7×10 8、约8×10 8、约9×10 8、约1×10 9、约2×10 9、约3×10 9、约4×10 9、约5×10 9、约6×10 9、约7×10 9、约8×10 9、约9×10 9、约1×10 10、约2×10 10、 约3×10 10、约4×10 10、约5×10 10、约6×10 10、约7×10 10、约8×10 10、约9×10 10、约1×10 11、约2×10 11、约3×10 11、约4×10 11、约5×10 11、约6×10 11、约7×10 11、约8×10 11、约9×10 11、约1×10 12、约2×10 12、约3×10 12、约4×10 12、约5×10 12、约6×10 12、约7×10 12、约8×10 12、约9×10 12、约1×10 13、约2×10 13、约3×10 13、约4×10 13、约5×10 13、约6×10 13、约7×10 13、约8×10 13，或约9×10 13。在一些实施方式中，本申请的组合物中提供的TIL数量的范围可以为约1×10 6至5×10 6、约5×10 6至1×10 7、约1×10 7至5×10 7、约5×10 7至1×10 8、约1×10 8至5×10 8、约5×10 8至1×10 9、约1×10 9至5×10 9、约5×10 9至1×10 10、约1×10 10至5×10 10、约5×10 10至1×10 11、约5×10 11至1×10 12、约1×10 12至5×10 12，或约5×10 12至1×10 13。
在一些实施方式中，本申请的组合物中提供的TIL的浓度可以小于组合物的例如约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约0.0002％，或约0.0001％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的TIL的浓度可以大于组合物的约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19.75％、约19.50％、约19.25％、约19％、约18.75％、约18.50％、约18.25％、约18％、约17.75％、约17.50％、约17.25％、约17％、约16.75％、约16.50％、约16.25％、约16％、约15.75％、约15.50％、约15.25％、约15％、约14.75％、约14.50％、约14.25％、约14％、约13.75％、约13.50％、约13.25％、约13％、约12.75％、约12.50％、约12.25％、约12％、约11.75％、约11.50％、约11.25％、约11％、约10.75％、约10.50％、约10.25％、约10％、约9.75％、约9.50％、约9.25％、约9％、约8.75％、约8.50％、约8.25％、约8％、约7.75％、约7.50％、约7.25％、约7％、约6.75％、约6.50％、约6.25％、约6％、约5.75％、约5.50％、约5.25％、约5％、约4.75％、约4.50％、约4.25％、约4％、约3.75％、约3.50％、约3.25％、约3％、约2.75％、约2.50％、约2.25％、约2％、约1.75％、约1.50％、约125％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、 约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约或0.0002％，或者约0.0001％w/w、w/v或v/v。
在一些实施方式中，本申请的组合物中提供的TIL的浓度范围可以为组合物的约0.0001％至约50％、约0.001％至约40％、约0.01％至约30％、约0.02％至约29％、约0.03％至约28％、约0.04％至约27％、约0.05％至约26％、约0.06％至约25％、约0.07％至约24％、约0.08％至约23％、约0.09％至约22％、约0.1％至约21％、约0.2％至约20％、约0.3％至约19％、约0.4％至约18％、约0.5％至约17％、约0.6％至约16％、约0.7％至约15％、约0.8％至约14％、约0.9％至约12％，或约1％至约10％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的TIL的浓度范围可以为组合物的约0.001％至约10％、约0.01％至约5％、约0.02％至约4.5％、约0.03％至约4％、约0.04％至约3.5％、约0.05％至约3％、约0.06％至约2.5％、约0.07％至约2％、约0.08％至约1.5％、约0.09％至约1％、或约0.1％至约0.9％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的TIL的量可以等于或小于约10g、约9.5g、约9.0g、约8.5g、约8.0g、约7.5g、约7.0g、约6.5g、约6.0g、约5.5g、约5.0g、约4.5g、约4.0g、约3.5g、约3.0g、约2.5g、约2.0g、约1.5g、约1.0g、约0.95g、约0.9g、约0.85g、约0.8g、约0.75g、约0.7g、约0.65g、约0.6g、约0.55g、约0.5g、约0.45g、约0.4g、约0.35g、约0.3g、约0.25g、约0.2g、约0.15g、约0.1g、约0.09g、约0.08g、约0.07g、约0.06g、约0.05g、约0.04g、约0.03g、约0.02g、约0.01g、约0.009g、约0.008g、约0.007g、约0.006g、约0.005g、约0.004g、约0.003g、约0.002g、约0.001g、约0.0009g、约0.0008g、约0.0007g、约0.0006g、约0.0005g、约0.0004g、约0.0003g、约0.0002g，或者约0.0001g。
在一些实施方式中，本申请的组合物中提供的TIL的量可以大于约0.0001g、约0.0002g、约0.0003g、约0.0004g、约0.0005g、约0.0006g、约0.0007g、约0.0008g、约0.0009g、约0.001g、约0.0015g、约0.002g、约0.0025g、约0.003g、约0.0035g、约0.004g、约0.0045g、约0.005g、约0.0055g、约0.006g、约0.0065g、约0.007g、约0.0075g、约0.008g、约0.0085g、约0.009g、约0.0095g、约0.01g、约0.015g、约0.02g、约0.025g、约0.03g、约0.035g、约0.04g、约0.045g、约0.05g、约0.055g、约0.06g、约0.065g、约0.07g、约0.075g、约0.08g、约0.085g、约0.09g、约0.095g、约0.1g、约0.15g、约0.2g、约0.25g、约0.3g、约0.35g、约0.4g、约0.45g、约0.5g、约0.55g、约0.6g、约0.65g、约0.7g、约0.75g、约0.8g、约0.85g、约0.9g、约0.95g、约1g、约1.5g、约2g、约2.5g、约3g、约3.5g、约4g、约4.5g、约5g、 约5.5g、约6g、约6.5g、约7g、约7.5g、约8g、约8.5g、约9g、约9.5g，或者约10g。
在一些实施方式中，TIL可以单剂量施用。此种施用可以通过注射，例如可以静脉内注射。在一些实施方式中，TIL可以多剂量施用。剂量可以是每年一次、两次、三次、四次、五次、六次或超过六次。剂量可以是每月一次、每两周一次、每周一次或每2天一次。在一些实施方式中，TIL的施用可以连续施用。
一方面，本申请提供一种药物组合物。在一些实施方式中，其可以包含本申请的TIL和/或本申请的组合物，与药学上可接受的载体。
一方面，本申请提供一种试剂盒，本申请的试剂盒可以包含本申请培养肿瘤浸润淋巴细胞(TIL)方法的T细胞激活剂、T细胞生长因子和/或饲养细胞与记载本申请培养肿瘤浸润淋巴细胞(TIL)方法的步骤的说明书。一方面，本申请提供一种试剂盒，本申请试剂盒可以包含本申请的TIL和/或本申请的药物组合物。
一方面，本申请提供一种影响肿瘤细胞生长的方法，可以包括向受试者施用本申请的TIL和/或本申请的药物组合物。在一些实施方式中，影响肿瘤生长可以包含肿瘤的体积减少到施用前的例如约99％、约95％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％或约0.1％。
一方面，本申请提供本申请的TIL和/或本申请的药物组合物在制备药物中的应用，本申请的药物可以用于预防和/或治疗肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
一方面，本申请提供一种预防和/或治疗肿瘤的方法，可以包括向受试者施用本申请的TIL和/或本申请的药物组合物。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
一方面，本申请提供一种本申请的TIL和/或本申请的药物组合物，其可以用于预防和/或治疗肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
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产品、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
1.1饲养细胞接收及制备
1.1.1单采血接收
记录单采血信息，批号及体积，并复温至室温。
1.1.2 PBMC(外周血单个核细胞)手动分离及冻存
使用75％酒精消毒血袋，转移至生物安全柜内。使用无菌剪刀剪开血袋后，将单采血转移至50mL离心管内，使用20mL注射器注入20mL PBS或生理盐水清洗血袋，将洗涤液一并转入50mL离心管内。每个50mL离心管内液体体积可以不超过30mL。将单采血3000g离心10分钟。离心过程中准备6-8支50mL离心管，加入已复温的淋巴细胞分离液(天津灏洋Ficoll)，20mL/支。离心结束后，弃掉上层血浆，使用PBS或生理盐水稀释细胞沉淀，将稀释后的血细胞混合液缓慢滴加上淋巴细胞分离液上层，可以不破坏界面，每管约加25mL样品，可以不超过28mL。
离心使用水平转子，500-600g离心15-30分钟，温度18-22℃，离心结束后得到的白膜层将处于生理盐水及淋巴细胞分离液Ficoll的分界面处。吸弃上层血浆及生理盐水，用移液管吸取中间白膜层至另一干净的50mL离心管内。使用PBS或生理盐水稀释收集到的白膜层，600g离心10分钟，室温。离心结束后弃上清，PBS或生理盐水清洗细胞一次，500g离心5分钟，室温。
如红细胞较多，离心结束后可以进行裂红，按照细胞沉淀体积与红细胞裂解液1:2至1:3加入红细胞裂解液，混匀，室温裂解10分钟中，中间轻柔混匀离心管2-3次，保证裂解效果，裂解完成后加入PBS或生理盐水清洗细胞。裂红后清洗细胞两次，400g离心6分钟，最后一次离心前取样计数。
弃上清，基础培养基重悬细胞，调整细胞密度约2-3×10 7个细胞/mL，液面高度可以不超过1厘米，每T225培养瓶中体积可以低于200mL；平铺状态下，X射线辐照50Gy。离心弃上清，根据计数结果冻存细胞，约1-2×10 8个细胞/mL，1-2mL/支；将细胞放入程序降温盒内转移至﹣80℃冰箱内冻存。
1.1.3 PBMC自动分离及冻存
将血袋的管路与cpro分离套件(Cytiva)输入端无菌接管。若血量大于120mL，进行预 浓缩步骤，可以将血液体积浓缩至120mL以内。可以使用neatcell程序进行PBMC分离及洗涤，洗涤液为生理盐水，中间体积20mL；重悬液为基础培养基，添加80mL/批。分离后每供者PBMC为一袋100mL，在平铺状态下，液面高度可以不超过1厘米，X射线辐照50Gy。辐照后取样计数，使用culture wash程序收集细胞并洗涤三次，洗涤液为生理盐水；设置中间体积及终体积，使得每1×10 9个细胞不少于2mL；加入等量至2倍冻存液混匀。使用1倍冻存液调整细胞密度约为1×10 7个细胞/mL至2×10 8个细胞/mL，分装20mL/袋，程序降温仪内冻存，液氮保存。
1.2肿瘤组织接收及处理
1.2.1组织接收
接收供者的肿瘤组织及血样，核对样品信息并记录，打印相应样品标签。
1.2.2组织处理及培养
使用75％酒精消毒样品管及采血管，转移至生物安全柜内。根据上述PBMC手动分离及冻存操作程序分离血样中PBMC细胞并进行冻存。取一种具有透气表面的培养瓶或培养袋，例如培养袋(Origen)，加入300mL已复温的完全培养基，完全培养基可以任意地选用X-vivo15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，并添加浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2。取数个10厘米培养皿，加入适量培养基，使用无菌眼科镊从样品管中取出肿瘤组织于10厘米培养皿中，培养基量以刚没过肿瘤组织为准，观察组织形态并记录。洗涤组织并更换培养皿。使用眼科剪及眼科镊将进行初步剪切，去除脂肪组织及坏死组织，每块组织块继续剪碎至约27立方毫米大小。取非悬浮肿瘤组织块，使用20mL注射器去除内部活塞后，与培养袋连接，使用移液管将约1g组织块通过注射器转入培养袋内。将培养袋放入二氧化碳培养箱内进行培养。清理剪刀及镊子，并用75％酒精进行初步消毒后，超声清洗后进行灭菌，得到第一TIL群。
1.3步骤(A)体外扩增及收获
1.3.1步骤(A)体外扩增
根据细胞生长状态，每3-7天补液或半量换液，保证细胞营养。使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，并添加浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2(双鹭)。步骤(A)的3-14天，例如可以第13或14天时取样计数，若细胞数目处于5×10 5至5×10 8之间 时进入步骤(A)的收获步骤。
1.3.2步骤(A)的收获
收集步骤(A)体外扩增结束细胞，离心，弃去培养基，使用PBS或生理盐水洗涤细胞一次，获得经步骤(A)体外扩增的TIL(第二TIL群)，并取样计数留取约5×10 5至2×10 8个细胞进入后续体外扩增步骤；取约5×10 5个细胞可以进行质量控制检测；其余细胞加入冻存液冻存，作为冻存的preREP TIL体外细胞。
1.4步骤(B)TIL活化
继续培养经步骤(A)体外扩增的TIL(第二TIL群)，或者对冻存的preREP TIL体外细胞进行细胞复苏，进行步骤(B)的TIL活化。
使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，调整细胞密度为5×10 5至2×10 6个细胞/mL，于悬浮24孔培养板内，1mL/孔，添加浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL或6000IU/mL)的IL-2。各TIL细胞群的培养基中同时可以添加T细胞激活剂，例如添加CD3激动剂和/或CD28激动剂，例如，约30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)、约30ng/mL的CD28抗体(Merck，15E8)、以约1:2-2:1的磁珠与TIL的比例加入磁珠(直径约1至10μm Dynabeads，Thermo Fisher)和/或以约1:100-1:2000的transACT(直径约100至500nm，Miltenyi)与TIL的比例加入transACT。培养约0-4天，获得第三TIL群。
1.5步骤(C)TIL细胞基因编辑
将本申请的gRNA(序列如SEQ ID NO：36-37、40-41、43、48-51、54-55、58-59、62-63和70-75中任一项所示)，解冻并加入无核酸酶水，配至浓度为约100μM。将约2μL的gRNA(50μM)在95℃孵育2分钟退火后加入到P3缓冲液，并加入0.3-1μL的Cas9(克睿，10mg/mL)，25℃孵育10分钟以形成核糖核蛋白复合物(RNP)。在P3缓冲液(Lonza)中，通过Lonza电转仪将上述RNP与第三TIL群的约1×10 6个细胞进行电转。例如，电转程序可以是human T cell stim(EO115)。电转的基因编辑后培养约0-4天，获得第四TIL群。
1.6步骤(D)TIL细胞基因编辑后培养
在第四TIL细胞群中加入饲养细胞进行培养。TIL与饲养细胞接触的时间需要在步骤(B)的TIL与IL-2以及T细胞激活剂接触后的若干时间T n以后(各个试验组的T n可以取0小时到12天，例如24小时或48小时)。首先复苏1-5名供者混合的饲养细胞；将活化的TIL细胞、饲养细胞按照TIL细胞：饲养细胞约为1:200的比例混合，转入G-Rex100培养瓶或者透 气袋内，补充完全培养基，每1-3天取样计数，并根据细胞状态补液或半量换液直至细胞总数大于1×10 9或步骤(D)体外扩增培养约5天至约14天，终止步骤(D)体外扩增的培养。
1.7肿瘤浸润淋巴细胞的收获
取步骤(D)扩增的细胞，离心后弃去培养基上清，并使用PBS或生理盐水或复方电解质溶液清洗三次，获得经步骤(D)扩增的TIL(第五TIL群)，第三次清洗时取样计数，根据计数结果，最后一次离心后弃上清，取3×10 6细胞送质量控制检测；其余全部细胞加入冻存液，调整细胞密度1-3×10 8个细胞/mL冻存。
实施例2饲养细胞不同添加时间培养的TIL增殖能力对比
实施例1的加入IL-2与不同形式的T细胞激活剂后的若干时间T n以后(T n可以取0小时到14天)，将饲养细胞与肿瘤浸润淋巴细胞共培养。本实施例中T n选取0小时、6小时、12小时、24小时、48小时、72小时、5天、7天、和9天获得饲养细胞不同添加时间培养的TIL，并进行细胞计数的对比试验。
图1显示的是，饲养细胞不同添加时间培养的TIL的增殖能力分析结果。饲养细胞不同添加时间培养TIL的各组图中纵坐标的数值表示，体外扩增结束后相比于体外扩增开始前，TIL细胞数量扩增至的扩增倍数。4名供者来源的TIL增殖结果显示，加入OKT3和IL-2后的0小时后(即同时)添加饲养细胞培养的TIL，增殖能力弱于加入OKT3和IL-2后的24小时或48小时后加饲养细胞培养的TIL。
实施例3饲养细胞不同添加时间培养的TIL流式检测对比
在实施例1的加入IL-2与不同形式的T细胞激活剂后的若干时间T n以后(T n可以取0小时到14天)，将饲养细胞与肿瘤浸润淋巴细胞共培养。本实施例中T n选取0小时、6小时、12小时、24小时、48小时、72小时、5天、7天、和9天获得饲养细胞不同添加时间培养的TIL，并进行流式检测的对比试验。
TIL流式检测试验材料的来源
转录因子缓冲组(Transcription Factor Buffer Set)，厂家BD，货号562574；V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色， 2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
饲养细胞不同添加时间培养的TIL的流式结果分析如图2到图8所示。
图2和图3显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA -CCR7 +中心记忆T细胞(Tcm)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高中心记忆T细胞的比例。
图4显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更少的调节性T细胞的比例。
图5和图6显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如PD-1 +、LAG-3 +和/或CD28 +细胞比例更高。
图7显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103 +CD39 +肿瘤特异性T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的肿瘤特异性T细胞的比例。
图8显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1 +干细胞样T细胞比例。结果显示，24小时或48小时后添 加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的干细胞样T细胞的比例。
实施例4加入CD28激动剂刺激的TIL增殖能力检测
对于实施例1中各个试验组培养获得的TIL群进行细胞计数。
图9显示的是，添加不同形式的CD28激动剂的试验组以及对照组的增殖能力分析结果。图中纵坐标的数值表示，各个试验组体外扩增获得的TIL群相比于体外扩增开始前的TIL群，TIL细胞数量扩增至的扩增倍数。结果显示，四步骤划分法中的步骤(B)的体外扩增添加CD28抗体，获得的TIL增殖能力强于对照组(不添加CD28抗体)培养的TIL。
实施例5加入CD28激动剂刺激的TIL流式检测
对于实施例1中各个试验组体外扩增培养获得的TIL群进行流式检测。
添加不同形式的CD28激动剂的TIL的流式结果分析如图10到图14所示。
图10显示的是，混合抗体组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如，更高的活化T细胞(CD28 +或41BB +)的比例，更低的调节性T细胞(Treg，例如CD4 +CD25 +Foxp3 +)比例，更高的干细胞样T细胞(TCF1 +)比例，和/或更高的中心记忆T细胞(Tcm，例如CD45RA -CCR7 +)的比例。
图11显示的是，混合抗体组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如，更高的肿瘤特异性T细胞(CD103 +CD39 +)比例，更高的活化T细胞(CD25 +)的比例，和/或更低的调节性T细胞(Treg，例如CD4 +CD25 +Foxp3 +)比例。
图12显示的是，磁珠组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如，更高的活化T细胞(CD28 +、PD-1 +或41BB +)的比例，更高的干细胞样T细胞(TCF1 +)比例，和/或更高的中心记忆T细胞(Tcm，例如CD45RA -CCR7 +)的比例。
图13显示的是，磁珠组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如， 更高的干细胞样T细胞(TCF1 +)比例，更高的活化T细胞(41BB +)的比例，更高的中心记忆T细胞(Tcm，例如CD45RA -CCR7 +)的比例，更低的调节性T细胞(Treg，例如CD4 +CD25 +Foxp3 +)比例，和/或更高的肿瘤特异性T细胞(CD103 +CD39 +)比例。
图14显示的是，纳米基质组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如，更高的肿瘤特异性T细胞(CD103 +CD39 +)比例，更高的活化T细胞(CD25 +或PD-1 +)的比例，和/或更高的中心记忆T细胞(Tcm，例如CD45RA -CCR7 +)的比例。
实施例6加入CD28激动剂刺激的TIL细胞杀伤能力检测
对于实施例1中各个试验组四步骤划分法中的步骤(B)体外扩增培养获得的TIL群进行细胞杀伤能力检测。
细胞准备
准备用于检测的各个试验组获得的TIL和用于共培养的靶细胞(例如Hela肿瘤细胞)。
检测步骤
用CFSE(5(6)-Carboxyfluorescein diacetate N-succinimidyl ester，Sigma，21888-25MG-F)标记肿瘤细胞：用PBS清洗肿瘤细胞，重悬肿瘤细胞于500μL的PBS中；将CFSE加入500μL的PBS中，与500μL的肿瘤细胞PBS重悬液混合，至CFSE的终浓度为0.5μmol/L。37℃孵育6分钟后，加含10％FBS的培养基清洗，600g离心5分钟，用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基重悬肿瘤细胞浓度为5×10 5个细胞/mL。对各个试验组的TIL细胞600g离心5分钟，按照效靶比(TIL细胞与肿瘤细胞的比例)3:1重悬TIL细胞(即重悬TIL细胞浓度为1.5×10 6个细胞/mL)。于U底96孔板(Corning)中加入肿瘤细胞和TIL细胞各100μL，每组设置三个复孔。同时设置一组只包含肿瘤细胞的对照组并按照实验不同分组加入不同试剂。将孔板200g离心1分钟，置于37℃孵育4小时至过夜。
孵育完成后，600g离心3分钟，弃上清，每孔加入20μL胰酶，37℃培养箱内孵育3-5分钟消化肿瘤细胞，消化完成后加入180μL含10％FBS的培养基终止消化。将Dapi(碧云天，C0060)用1:100稀释，然后每孔加入20μL稀释后的Dapi。进行流式上机检测。
杀伤率％＝Dapi +CFSE +细胞数/总CFSE +×100％。
图15显示的是，纳米基质组与对照组培养所得的TIL细胞的细胞杀伤能力。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗 体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的细胞杀伤能力。
实施例7加入CD28激动剂刺激的TIL胞内因子表达检测
对于实施例1中各个试验组四步骤划分法中的步骤(B)体外扩增培养获得的TIL群进行胞内因子表达检测。
试验准备
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比1:500添加CD107a抗体(BD)。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，100μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，50μL/组染色，2-8℃避光孵育30分钟。染色结束后清洗细胞，使用PBS重悬，进行流式上机检测。
图16显示的是，混合抗体组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图17显示的是，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图18显示的是，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图19显示的是，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图20显示的是，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28 抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图21显示的是，纳米基质组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力或更高的GZMB表达能力。
实施例8加入CD28激动剂刺激的TIL细胞因子分泌检测
对于实施例1中各个试验组四步骤划分法中的步骤(B)体外扩增培养获得的TIL群进行细胞因子分泌检测。
图22显示的是，纳米基质组与对照组培养所得的TIL细胞的细胞因子分泌检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的细胞因子分泌能力。例如，更高的IL-2分泌能力、更高的TNF分泌能力、或更高的IFN-γ分泌能力。
取各个试验组获得的TIL与肿瘤细胞过夜孵育，孵育结束后取上清液参照本实施例检测步骤进行细胞因子分泌检测。
图23显示的是，纳米基质组与对照组培养所得的TIL细胞与肿瘤细胞共同孵育后的细胞因子分泌检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的细胞因子分泌能力。例如，更高的IL-2分泌能力、更高的TNF分泌能力、或更高的IFN-γ分泌能力。
实施例9加入CD28激动剂刺激的TIL基因敲除效率检测
取实施例1中各个试验组在四步骤划分法中的步骤(B)体外扩增培养48小时后的TIL细胞，进行基因敲除效率检测。
用Nuclease-free water(商业来源：上海右凡生物科技有限公司；RT121-02)配制sgRNA(序列如SEQ ID NO:1所示，GGAGAATGACGAGTGGACCC)，调节浓度至50μmol/L。取2μL gRNA加入PCR管中，Nuclease-free water作为阴性对照，在PCR仪中95℃孵育2分钟后，室温冷却10分钟。
按照体积比sgRNA:P3Buffer:Cas9核酸酶＝2:2:1，在含有sgRNA的PCR管中依次加入P3Buffer(商业来源：Lonza；V4XP-3032)及61.7μmol/L Cas9核酸酶(商业来源：苏州克睿基因生物科技有限公司；C01-2019-11-001)，将PCR管放入PCR仪，25℃孵育10分钟以形成RNP，放入4℃备用。
按照1mL/孔加入T细胞培养基，放置于CO 2培养箱中预热。取实施例1中各个试验组在四步骤划分法中的步骤(B)体外扩增培养48小时后的TIL细胞，混匀后进行计数，每个试验组样品取5×10 5个细胞加入P3Buffer(20μL)，混匀细胞；将细胞加入新的PCR管中，与5μL的RNP混合；将细胞与RNP的混合物加入电转条板中，在电转仪(Lonza)中进行电转(human T cell stimulated(E0115))。电转程序结束后，立即加入预热的180μL的T细胞培养基，将全部体积转移到24孔悬浮板中，放置于CO 2培养箱中进行培养。24小时后进行细胞计数，按照TIL：饲养细胞＝1:200比例加入饲养细胞(辐照后的PBMC细胞)，放置于CO 2培养箱中继续培养72小时。取培养结束后的各个试验组的TIL细胞进行细胞计数，每个试验组取2×10 5个细胞，500g离心3分钟，离心后吸弃上清。
配制流式检测混合抗体：取Fixable Viability Dye eFluor 780(商业来源：eBioscience；65-0865-18)在PBS中稀释10000倍；取100μL稀释后的Fixable Viability Dye eFluor 780的PBS溶液，分别加入1μL的TCR-αβ-APC(商业来源：eBioscience；17-9986-42)、1μL的BB515Mouse Anti-Hu CD8(商业来源：BD Pharmingen；564526)、1μL的PE-Cy7Mouse Anti-Hu CD4(商业来源：BD Pharmingen；557852)，混合均匀。
每个试验组的TIL细胞样品加入100μL上述流式检测混合抗体，混合均匀后冰上孵育30分钟；孵育结束后，500g离心3分钟，离心后吸弃上清，加入200μL PBS重悬。流式细胞仪行进检测，利用Flowjo软件分析TCRβ敲除效率。
图24显示的是，纳米基质组与对照组培养所得的TIL细胞的基因敲除效率结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的基因敲除效率能力。例如，提高的TCRβ基因敲除效率。
图25显示的是，纳米基质组与对照组培养所得的TIL细胞的基因敲除效率结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的基因敲除效率能力。例如，提高的TCRβ基因敲除效率。
实施例10 REP阶段结束后添加CD28激动剂的TIL增殖能力检测
参考实施例1得到源自肿瘤组织且未经体外扩增的第一TIL群，将第一TIL群经过相同方式的四步骤划分法中的步骤(A)、步骤(B)、步骤(C)和步骤(D)得到第五TIL群。将第五TIL群随机分为3组，各个试验组的T细胞培养基加入IL-2的同时，其中空白组不添加任何T细胞激活剂，未添加CD28激动剂组添加CD3抗体(Miltenyi Biotech，OKT3)约30ng/mL，添加CD28激动剂组添加CD3激动剂和CD28激动剂，例如以约1:100-1:2000的transACT与TIL的比例加入transACT。培养3天得到的TIL(终末刺激细胞群)，通过细胞活力检测方法使用CellTiter-Glo试剂盒(商业来源：Promega)的进行TIL细胞增殖能力的检测。
图26、图27和图28分别显示的是，对于不同供者来源的TIL，终末刺激阶段以不同方式进行体外扩增的试验组的增殖能力分析结果。图中纵坐标荧光值反映了各个试验组以不同方式进行终末刺激的TIL细胞的增殖能力。结果显示，终末刺激添加CD28激动剂，与终末刺激不添加CD28激动剂相比，具有类似的TIL增殖能力。
实施例11 TIL细胞培养过程中饲养细胞不同添加时间
饲养细胞不同添加时间培养的TIL的结果统计
在实施例1的1.4的第二阶段扩增的TIL活化中，取第一阶段扩增的细胞量，调整细胞密度为5×10 5至2×10 6/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如3000或6000IU/mL的IL-2。加入上述OKT3和IL-2后的0小时、24小时、48小时以后，将饲养细胞加入肿瘤浸润淋巴细胞的培养环境中。其中，TIL与饲养细胞可以按照比率1:40-1:400加入，第二阶段扩增培养约9-14天后收集全部细胞，检测和统计培养所得TIL的结果。
增殖能力检测
对于上述饲养细胞不同添加时间培养获得的TIL进行细胞计数。
不同供者肿瘤来源的TIL作为各自不同的批次；将各个批次的加入OKT3和IL-2同时(0h组)加入饲养细胞的试验组的数据作为基准1，将同批次其它时间点试验组的数据进行标准化处理，统计各试验组在第二阶段扩增相对于0h组的相对增殖能力。
图29A显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。相比于加入OKT3和IL-2后的0小时后(即同时)添加饲养细胞培养的TIL，加入OKT3和IL-2后的24小时或48小时后加饲养 细胞培养的TIL增殖能力显著增强。
流式检测TIL细胞组成
对于上述饲养细胞不同添加时间培养获得的TIL群进行流式检测。
不同供者肿瘤来源的TIL作为各自不同的批次；将各个批次的加入OKT3和IL-2同时(0h组)加入饲养细胞的试验组的数据作为基准1，将同批次其它时间点试验组的数据进行标准化处理，统计各试验组在第二阶段扩增相对于0h组的细胞组成比例。
流式检测的试验流程可以参照本申请实施例3的内容。
图29B显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA -CCR7 +中心记忆T细胞(Tcm)比例结果图。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高CD8 +中和/或CD4 +中的中心记忆T细胞的比例。
图29C显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1 +干细胞样T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高CD8 +中的干细胞样T细胞的比例。
图29D显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更少的调节性T细胞的比例。
图29E显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(PD-1 +)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8 +中和/或CD4 +中的PD-1 +细胞比例更高。
图29F显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103 +CD39 +肿瘤特异性T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的CD8 +中和/或CD4 +中的肿瘤特异性T细胞的比例。
图29G显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD28 +)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比 例，例如CD8 +CD28 +细胞比例更高。
图29H显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(41BB +)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8 +中和/或CD4 +中的41BB +细胞比例更高。
图29I显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD25 +)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8 +中和/或CD4 +中的CD25 +细胞比例更高。
胞内因子表达检测
试验准备
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比1:500添加CD107a抗体(BD)。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，100μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，50μL/组染色，2-8℃避光孵育30分钟。染色结束后清洗细胞，使用PBS重悬，进行流式上机检测。
图29J显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的胞内因子表达检测结果。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的胞内因子表达能力。例如，更高的CD3 +中、CD8 +中和/或CD4 +中的CD107a表达能力。
细胞因子分泌检测
细胞因子分泌检测方法可以参照细胞因子检测试剂盒(BD)的说明书，将人Th1/Th2/Th17细胞因子标准品冻干粉(BD)使用2mL Assay Diluent稀释液(BD)复溶(标准品原液各细胞因子浓度均为5000pg/mL)并按顺序：1:2，1:4，1:8，1:16，1:32，1:64，1:128，1:256，1:512，1:1024梯度稀释，标记为“标准品管”。取1管仅含有Assay Diluent稀释液作为参照。按照2μL/Beads/孔加入每种Capture Beads(BD)，然后按照10μL/孔加入PE Detection Reagent检测试剂(BD)并混合配制为混合物(mix)，按照22μL/孔加入V底96孔板内，随后按照 10μL/孔加入各标准品和试验组的上清并混合，室温下避光孵育3小时。
孵育结束，每孔加入200μL Wash Buffer(BD)，500g离心3分钟。离心结束，每孔加入100μL Wash Buffer(BD)重悬，进行流式分析。
图29K显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞因子分泌检测结果。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的细胞因子分泌能力。例如，更高的TNF-α分泌能力、或更高的IFN-γ分泌能力。
饲养细胞不同添加时间培养的TIL的结果统计
在实施例1的1.4的第二阶段扩增的TIL活化中，取第一阶段扩增的细胞量，调整细胞密度为5×10 5至2×10 6/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如3000或6000IU/mL的IL-2。加入上述OKT3和IL-2后的0小时、6小时、12小时、24小时、48小时、72小时、或5天以后，将饲养细胞加入肿瘤浸润淋巴细胞的培养环境中。其中，TIL与饲养细胞可以按照比率1:40-1:400加入，例如1:200，第二阶段扩增培养约9-14天后收集全部细胞，检测和统计培养所得TIL的结果。
增殖能力检测
对于上述饲养细胞不同添加时间培养获得的TIL进行细胞计数。
图29L显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。相比于加入OKT3和IL-2后的0小时后(即同时)添加饲养细胞培养的TIL，加入OKT3和IL-2后的12小时或更多时间后加饲养细胞培养的TIL增殖能力显著增强。
流式检测TIL细胞组成
对于上述饲养细胞不同添加时间培养获得的TIL群进行流式检测。
不同供者肿瘤来源的TIL作为各自不同的批次；将各个批次的加入OKT3和IL-2同时(0h组)加入饲养细胞的试验组的数据作为基准1，将同批次其它时间点试验组的数据进行标准化处理，统计各试验组在第二阶段扩增相对于0h组的细胞组成比例。
流式检测的试验流程可以参照本申请实施例3的内容。
图29M显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD8 +T细胞比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添 加饲养细胞培养的TIL，具有更高的CD8 +T细胞的比例。
图29N显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RO +CD62L +T细胞比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的记忆T细胞(Tcm，CD45RO +CD62L +)的比例。
图29O显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的NK T细胞比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的NK T细胞的比例。
图29P显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更少的调节性T细胞的比例。
本申请培养的TIL的杀伤能力检测
对于实施例1的1.4的第二阶段扩增的TIL活化中，取第一阶段扩增的细胞量，调整细胞密度为5×10 5至2×10 6/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如3000或6000IU/mL的IL-2。加入上述OKT3和IL-2后的12小时至14天后，例如48小时以后，将饲养细胞加入肿瘤浸润淋巴细胞的培养环境中。其中，TIL与饲养细胞可以按照比率1:40-1:400加入，第二阶段扩增培养约9-14天后收集全部细胞，检测和统计培养所得TIL的细胞杀伤能力检测。
细胞准备
准备用于检测的各个试验组获得的TIL和用于共培养的靶细胞(例如A375黑色素瘤细胞和/或Hela宫颈癌细胞)。
检测步骤
用CFSE(5(6)-Carboxyfluorescein diacetate N-succinimidyl ester，Sigma，21888-25MG-F)标记肿瘤细胞：用PBS清洗肿瘤细胞，重悬肿瘤细胞于500μL的PBS中；将CFSE加入500μL的PBS中，与500μL的肿瘤细胞PBS重悬液混合，至CFSE的终浓度为0.5μmol/L。37℃孵育6分钟后，加含10％FBS的培养基清洗，600g离心5分钟，用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基重悬 肿瘤细胞浓度为1×10 6个细胞/mL。对各个试验组的TIL群600g离心5分钟，按照效靶比(TIL细胞与肿瘤细胞的比例)3:1重悬TIL细胞(即重悬TIL细胞浓度为3×10 6个细胞/mL)。于U底96孔板(Corning)中加入肿瘤细胞和TIL细胞各100μL，每组设置三个复孔。同时设置一组只包含肿瘤细胞的对照组。将孔板200g离心1分钟，置于37℃孵育4小时至过夜。其中，TIL与肿瘤细胞共培养时，可以不加激活TIL细胞的物质作为无激活组，或者加入transACT(Miltenyi，包含CD3抗体和CD28抗体的纳米基质材料)作为激活组。
孵育完成后，600g离心3分钟，弃上清，每孔加入20μL胰酶，37℃培养箱内孵育3-5分钟消化肿瘤细胞，消化完成后加入180μL含10％FBS的培养基终止消化。将Dapi(碧云天，C0060)用1：100稀释，然后每孔加入20μL稀释后的Dapi。进行流式上机检测。
杀伤率％＝Dapi +CFSE +细胞数/总CFSE +×100％，或杀伤率可以通过Dapi +细胞数/总肿瘤细胞数表示。
图29Q显示的是，加入OKT3和IL-2的48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞杀伤能力结果。结果显示，加入OKT3和IL-2后的48小时后添加饲养细胞培养的TIL均具有显著的肿瘤细胞杀伤能力，例如黑色素瘤和/或宫颈肿瘤。
结果显示，基因编辑后的TIL细胞具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力、更高的TNF-α表达能力或更高的GZMB表达能力。
实施例12 YEATS基因编辑的细胞
细胞的敲除效率检测
试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)、Recombinant DNase I(RNase-free，TAKARA)。
提取基因组DNA：在TIL细胞敲除后的约4天，取约1×10 5个至约2×10 5个细胞，用PBS清洗1次，再用44μL PBS重悬TIL细胞，用并加入配置的6μL核酸酶混合液(含1μL DNase I和5μL 10×DNase I Buffer)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃孵育10分钟。离心弃去上清后，在细胞沉淀中加入50μL DNA提取液，短暂离心后运行以下程序：75℃-10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDrop TM)检测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以下设计PCR反应体系：
试剂 体积
2×PCR Premix Buffer 25μL
DNA模板 约100-500ng
正向引物(10μM) 1μL
反向引物(10μM) 1μL
双蒸水 补充到50μL
DMSO 1.5μL
并按照以下PCR程序扩增：
Figure PCTCN2022109580-appb-000001
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本申请相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
不同的TIL可以是来源于不同的肿瘤患者，供者812为皮肤恶性黑色素瘤患者，供者713为卵巢癌患者。
结果显示，SEQ ID NO：70(CCACTCACCGCGTCTGGGCT)所示的sgRNA，即ENL-1，对于供者713、812的敲除效率分别约为4.7％和5.3％。
结果显示，SEQ ID NO：72(GTCGTGAGTGAACCCCTCCG)所示的sgRNA，即ENL-3，对于供者713、812的敲除效率分别约为51.3％和38.9％。
结果显示，SEQ ID NO：73(GTACGAACACCATCCAGTCG)所示的sgRNA，即AF9-1，对于供者713、812的敲除效率分别约为62.1％和57.9％。
结果显示，SEQ ID NO：75(GTGGATGGCCTTCAAGATGC)所示的sgRNA，即AF9- 3，对于供者713、812的敲除效率分别约为26.3％和13.9％。
本申请的各种基因编辑方式均可以实现一定比例的敲除效率。
细胞的扩增情况的检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板。用30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)刺激，3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图30A-30B显示的是，对于来源于供者713、812的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明相对于对照组(NT)，本申请的基因编辑的TIL细胞可以具有显著的扩增能力。
细胞杀伤能力检测
基因编辑后的第6天开始，将A375肿瘤细胞系铺于96孔平底板中。次日，将各组TIL细胞以效靶比(TIL细胞：肿瘤细胞，E：T)为1:1的比例与上述A375细胞共培养。肿瘤细胞和TIL细胞各100μL，每组设置三个复孔，同时设置一组只包含肿瘤细胞的对照组。
根据细胞凋亡检测试剂(Incucyte Caspase-3/7 Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7 Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
图31显示的是，对于来源于供者812的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。结果表明相对于对照组(NT)，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
细胞的凋亡检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行凋亡检测。将基因敲除组或对照组(NT-no treatment)组的TIL用细胞凋亡检测试剂盒(BD 559763 Annexin V PE Apoptosis kit)检测T细胞凋亡水平。
图32A-32B显示的是，对于来源于供者812的TIL细胞的凋亡检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的抗凋亡能力。
细胞流式检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行流式细胞仪检测。
TIL流式检测试验材料的来源
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
对于细胞内因子的流式检测，配制固定、破膜所需试剂：使用缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，4℃预冷待用。在表面染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图33A-33C显示的是，对于来源于供者713、812的TIL细胞，各组TIL细胞中的中心记忆T细胞(CD45RO +CD62L +)的比例。结果显示，基因编辑后的TIL细胞具有更高的中心记忆T细胞的比例。
图34A-345D显示的是，对于来源于供者713、812的TIL细胞，各组TIL细胞中的幼稚T细胞(CD45RO -CD62L +)的比例。结果显示，基因编辑后的TIL细胞具有更高的幼稚T细胞的比例。
图35A-35E显示的是，对于来源于供者713的TIL细胞，各组TIL细胞中CD38阳性、LAG-3阳性和TIM-3阳性细胞的比例。
图36A-36E显示的是，对于来源于供者812的TIL细胞，各组TIL细胞中CD38阳性、LAG-3阳性和TIM-3阳性细胞的比例。
结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
细胞因子表达流式检测
对于实施例1中各个试验组基因编辑后的第7天或第8天获得的TIL群进行流式细胞仪检测细胞因子表达情况。
试验准备
CD3抗体组提前1天使用30ng/ml的CD3抗体(Miltenyi Biotech，OKT3)包被平底96孔板，4℃包被过夜；transACT组在T细胞接种时添加。
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比添加：Golgistop 0.7:1000，Golgiplug 1：1000，CD107a抗体1:500即2μL/mL。不添加白介素。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，200μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体(CD107a，GZMB，TNF-α和IFN-γ，BD/BioLegend)，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图37A-37D显示的是，在无激活物刺激的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中GZMB，TNF-α和IFN-γ的表达细胞的比例。
图38A-38E显示的是，在无激活物刺激的条件下，对于来源于供者812的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图39A-39B显示的是，在transACT刺激的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中TNF-α的表达细胞的比例。
图40A-40F显示的是，在CD3抗体刺激的条件下，对于来源于供者812的TIL细胞，各组TIL细胞中GZMB，TNF-α和IFN-γ的表达细胞的比例。
结果显示，基因编辑后的TIL细胞具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力、更高的TNF-α表达能力或更高的GZMB表达能力。
表6A人AF9(MLLT3)基因组坐标
Figure PCTCN2022109580-appb-000002
Figure PCTCN2022109580-appb-000003
Figure PCTCN2022109580-appb-000004
Figure PCTCN2022109580-appb-000005
Figure PCTCN2022109580-appb-000006
Figure PCTCN2022109580-appb-000007
Figure PCTCN2022109580-appb-000008
Figure PCTCN2022109580-appb-000009
Figure PCTCN2022109580-appb-000010
Figure PCTCN2022109580-appb-000011
Figure PCTCN2022109580-appb-000012
Figure PCTCN2022109580-appb-000013
Figure PCTCN2022109580-appb-000014
Figure PCTCN2022109580-appb-000015
Figure PCTCN2022109580-appb-000016
Figure PCTCN2022109580-appb-000017
Figure PCTCN2022109580-appb-000018
Figure PCTCN2022109580-appb-000019
Figure PCTCN2022109580-appb-000020
Figure PCTCN2022109580-appb-000021
Figure PCTCN2022109580-appb-000022
表6B人ENL(MLLT1)基因组坐标
Figure PCTCN2022109580-appb-000023
Figure PCTCN2022109580-appb-000024
实施例13SOCS1基因编辑的细胞
TIL细胞的敲除效率检测
试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)。
提取基因组DNA：在TIL细胞敲除后的约4天，取约1×10 5个至约2×10 5个细胞，并加入配置的核酸酶混合液(DNase I)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃孵育10分钟。离心后，加入50μL DNA提取液，短暂离心后运行以下程序：75℃-10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDrop TM)检测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以下设计PCR反应体系：
试剂 体积
2×PCR Premix Buffer 25μL
DNA模板 约100-500ng
正向引物(10μM) 1μL
反向引物(10μM) 1μL
双蒸水 补充到50μL
DMSO 1.5μL
并按照以下PCR程序扩增：
Figure PCTCN2022109580-appb-000025
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本申请相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
不同的TIL可以是来源于不同的肿瘤患者，供者A、供者B为皮肤恶性黑色素瘤患者，供者C为胰腺癌患者，供者D、供者E为非小细胞肺癌患者。
结果显示，SEQ ID NO：36(CGGCGTGCGAACGGAATGTG)所示的sgRNA，即SC1，对于供者A的敲除效率为约64.5％，对于供者B的敲除效率为约40.6％，对于供者C的敲除效率为约30.1％，对于供者D的敲除效率为约10％；SEQ ID NO：37(GGGCCCCCAGTAGAATCCGC)所示的sgRNA，即SC2，对于供者A的敲除效率为约60.5％，对于供者B的敲除效率为约18.5％，对于供者C的敲除效率为约42.3％，对于供者D的敲除效率为约26.3％。本申请的各种基因编辑方式均可以实现一定比例的敲除效率。
TIL细胞的扩增情况的检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板，并换用不含有IL-2的细胞培养基培养。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图41、图42、图43和图44显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，即使不含有IL-2的细胞培养基培养，本申请的基因编辑的TIL细胞仍然可以具有显著的扩增能力。
基因编辑后的第6天开始，用30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)预处理96孔板，4℃过夜孵育；次日各组TIL细胞以相同的细胞总数重新铺板，并换用不含有IL-2 的细胞培养基培养。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图45和图46显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。
本申请中，NT表示阴性对照组，未经过基因编辑的TIL；ST3表示无关敲除组，在不影响细胞功能的区域进行敲除的TIL；SC1表示通过SEQ ID NO：36所示的sgRNA进行敲除的TIL；SC2表示通过SEQ ID NO：37所示的sgRNA进行敲除的TIL。*表示p<0.05，**表示p<0.01，***表示p<0.001，****表示p<0.0001。
TIL细胞杀伤能力检测
基因编辑后的第6天开始，将A375肿瘤细胞系铺于96孔板中。次日，将各组TIL细胞以效靶比(TIL细胞：肿瘤细胞，E：T)为1:1或1:3的比例与上述A375细胞共培养。
根据细胞凋亡检测试剂(Incucyte Caspase-3/7 Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7 Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
图47显示的是，对于来源于供者B的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。
图48显示的是，对于来源于供者C的TIL细胞，以效靶比1:3与肿瘤细胞共培养的杀伤能力检测结果。
图49和图50显示的是，对于来源于供者D的TIL细胞，以效靶比1:1和效靶比1:3与肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
TIL细胞的扩增效率以及活率的检测
IL-2是调节T细胞生长的重要因子，本实施例检测本申请基因编辑的TIL细胞是否可以不依赖于IL-2而依然可以存活和/或扩增。
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板，并换用不含有IL-2的细胞培养基培养，即撤去IL-2进行培养。每隔2至4天用细胞计数仪分析TIL细胞的扩增效率及活率。其中扩增效率以增殖倍数表示：将撤去IL-2当天(第0天)的细胞总数作为1，第n天的增殖倍数表示为第n天的细胞总数/第0天的细胞总数；活率表示存活细胞数量占总细胞数量的百分比。
图51、图52和图53显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的增殖倍数。
图54和图55显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的活率。结果表明，即使撤去IL-2，本申请的基因编辑的TIL细胞仍然可以具有显著的扩增能力，以及可以保持较高的细胞活率。
TIL流式检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行流式细胞仪检测。
TIL流式检测试验材料的来源
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。染色结束后，加入PBS清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图56和图57显示的是，对于来源于供者的TIL细胞，各组TIL细胞中TIM-3阳性和CD101阳性细胞的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
图58显示的是，对于来源于供者的TIL细胞，各组TIL细胞中CD45RA阴性CCR7阳性记忆T细胞(Tcm)的比例。结果显示，基因编辑后的TIL细胞具有更高中心记忆T细胞的比例。
TIL细胞因子表达流式检测
对于实施例1中各个试验组基因编辑后的第7天或第8天获得的TIL群进行流式细胞仪检测细胞因子表达情况。
试验准备
CD3抗体组提前1天使用30ng/ml的CD3抗体(Miltenyi Biotech，OKT3)包被平底96孔板，4℃包被过夜；PMA组铺板当天在培养基中添加佛波醇-十四酸酯-乙酸酯(PMA，25ng/ml)和离子霉素(Ionomycin，1μg/ml)即可。
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比添加：Golgistop 0.7:1000，Golgiplug 1：1000，CD107a抗体1:500即2μL/mL。不添加白介素。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，200μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体(CD107a，GZMB，TNF-α和IFN-γ，BD/BioLegend)，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图59、图60、图61和图62显示的是，在没有刺激的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图63、图64、图65和图66显示的是，在CD3抗体刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图67、图68和图69显示的是，在PMA和Ionomycin刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
结果显示，基因编辑后的TIL细胞具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力、更高的TNF-α表达能力或更高的GZMB表达能力。
TIL细胞的TCR克隆多样性检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板，并换用不含有IL-2的细胞培养基培养，即撤去IL-2进行培养。
在基因编辑后的第8天和第18天对TIL中T细胞受体(TCR)克隆多样性进行分析。根 据TCR谱系试剂盒(Beta Mark TCR VβRepertoire Kit，Beckman Coulter)的说明书，在A管至H管的8个管中分别加入4μL的Beta Mark TCR VβRepertoire Kit Tube A-H抗体，并在每个管中加入1μL的BV510标记的CD3抗体(BD)、1μL的PerCP-cy5.5标记的CD8抗体(BD)、1μL的PE-cy7标记的CD4抗体(BD)和0.01μL的eFluor 780标记的死活细胞染料(eBioscience)。将待测细胞分为8份，分别加入A管至H管，混合均匀，4℃孵育30分钟。染色结束，离心并弃去上清，使用PBS重悬洗涤1次，用于流式细胞仪检测。
按照TCR谱系试剂盒(Beta Mark TCR VβRepertoire Kit，Beckman Coulter)的说明书对TIL细胞的TCR多样性进行鉴定。表1显示的是，A管至H管的荧光与TCR Vβ克隆的对应关系。其中TCR Vβ克隆为其它，可以是指该比例下的T细胞没有TCR谱系试剂盒可以鉴定的TCR Vβ。表2至表5显示的是，TIL细胞基因编辑后的第8天和第18天的CD4 +T细胞和CD8 +T细胞的TCR Vβ克隆的多样性。
表1A管至H管的荧光与TCR Vβ克隆的对应关系
Figure PCTCN2022109580-appb-000026
Figure PCTCN2022109580-appb-000027
表2显示的是，TIL细胞基因编辑后的第8天的CD4 +T细胞TCR Vβ克隆的多样性。
Figure PCTCN2022109580-appb-000028
Figure PCTCN2022109580-appb-000029
表3显示的是，TIL细胞基因编辑后的第18天的CD4 +T细胞TCR Vβ克隆的多样性。
Figure PCTCN2022109580-appb-000030
Figure PCTCN2022109580-appb-000031
表4显示的是，TIL细胞基因编辑后的第8天的CD8 +T细胞TCR Vβ克隆的多样性。
Figure PCTCN2022109580-appb-000032
Figure PCTCN2022109580-appb-000033
表5显示的是，TIL细胞基因编辑后的第18天的CD8 +T细胞TCR Vβ克隆的多样性。
Figure PCTCN2022109580-appb-000034
Figure PCTCN2022109580-appb-000035
图70和图71显示的是，TIL细胞基因编辑后的第8天和第18天的CD4 +T细胞和CD8 +T细胞TCR Vβ克隆的香农多样性指数。香农多样性指数反应了TCR Vβ克隆的多样性，结果表明，基因编辑的TIL细胞在第18天时，可以保持更高的β链亚型多样性，说明基因编辑的TIL细胞可以显著地长期保持TCR克隆的多样性，从而可以有助于实现对于肿瘤细胞更强的抗原识别和免疫应答能力。
TIL细胞的细胞因子分泌检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板，并换用不含有IL-2的细胞培养基培养，即撤去IL-2进行培养。24小时后取上清进行细胞因子分泌检测。
细胞因子分泌检测方法可以参照细胞因子检测试剂盒(BD)的说明书，将人Th1/Th2/Th17细胞因子标准品冻干粉(BD)使用2mL Assay Diluent稀释液(BD)复溶(标准品原液各细胞因子浓度均为5000pg/mL)并按顺序：1:2，1:4，1:8，1:16，1:32，1:64，1:128，1:256，1:512，1:1024梯度稀释，标记为“标准品管”。取1管仅含有Assay Diluent稀释液作为参照。按照2μL/Beads/孔加入每种Capture Beads(BD)，然后按照10μL/孔加入PE Detection Reagent检测试剂(BD)并混合配制为混合物(mix)，按照22μL/孔加入V底96孔板内，随后按照10μL/孔加入各标准品和实验组的上清并混合，室温下避光孵育3小时。
孵育结束，每孔加入200μL Wash Buffer(BD)，500g离心3分钟。离心结束，每孔加入100μL Wash Buffer(BD)重悬，进行流式分析。
图72显示的是，对于来源于供者C的TIL细胞，各组TIL细胞在撤去IL-2后进行培养的细胞因子IFN-γ和GZMB的分泌情况。结果表明，基因编辑的TIL细胞在不含有IL-2的细胞培养基培养后可以具有更显著的细胞因子分泌检测结果。
实施例14 TGFBR2基因编辑的细胞
TIL细胞的敲除效率检测
试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)、Recombinant DNase I(RNase-free，TAKARA)。
提取基因组DNA：在TIL细胞敲除后的约4天，取约1×10 5个至约2×10 5个细胞，用PBS清洗1次，再用44μL PBS重悬TIL细胞，用并加入配置的6μL核酸酶混合液(含1μL DNase I和5μL 10×DNase I Buffer)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃孵育10分钟。离心弃去上清后，在细胞沉淀中加入50μL DNA提取液，短暂离心后运行以下程序：75℃-10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDrop TM)检测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以下设计PCR反应体系：
试剂 体积
2×PCR Premix Buffer 25μL
DNA模板 约100-500ng
正向引物(10μM) 1μL
反向引物(10μM) 1μL
双蒸水 补充到50μL
DMSO 1.5μL
并按照以下PCR程序扩增：
Figure PCTCN2022109580-appb-000036
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本申请相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
不同的TIL可以是来源于不同的肿瘤患者，供者811为胰腺癌患者，供者410、614、619、003为皮肤恶性黑色素瘤患者，供者921为卵巢癌患者。
结果显示，SEQ ID NO：40(ACAGTGATCACACTCCATGT)所示的sgRNA，即G1，对于供者614、921、619、003、410、811的敲除效率分别约为34.8％、22.7％、38.75％、30.65％、50.4％、54.3％。
SEQ ID NO：41(ACCTACAGGAGTACCTGACG)所示的sgRNA，即G4，对于供者614、921、619、003、410、811的敲除效率分别约为23.9％、17.1％、24.35％、23.0％、50.8％、65.2％。
本申请的各种基因编辑方式均可以实现一定比例的敲除效率。
TIL细胞的扩增情况的检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图73A-73B显示的是，对于来源于供者619、003的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以显著提高扩增能力。
对于加入TGFβ-1的TIL细胞组，基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板。额外添加10nM的TGFβ-1(Biolegend，货号781802)抑制TIL增殖，3天后用CTG试剂盒分析细胞扩增情况。
图73C显示的是，对于来源于供者410的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以显著提高扩增能力。
基因编辑后的第7天开始，用30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)预处理96孔板，4℃过夜孵育；次日各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图73D显示的是，对于来源于供者410的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以显著提高扩增能力。
对于同时用CD3抗体和TGFβ-1培养的TIL细胞，图73E显示的是，对于来源于供者410的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以显著提高扩增能力。
TIL细胞杀伤能力检测
基因编辑后的第6天开始，将A375肿瘤细胞系铺于96孔平底板中。次日，将各组TIL细胞以效靶比(TIL细胞：肿瘤细胞，E：T)为1:1或1:3的比例与上述A375细胞共培养。肿瘤细胞和TIL细胞各100μL，每组设置三个复孔，同时设置一组只包含肿瘤细胞的对照组。根据细胞凋亡检测试剂(Incucyte Caspase-3/7 Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7 Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
图74A-74C显示的是，对于来源于供者619、921的TIL细胞，以效靶比1:1、1:3与肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
对于额外加入transACT(1:1000)刺激的TIL细胞，图74D显示的是，对于来源于供者921的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。
对于额外加入transACT(1:1000)以及TGFβ-1(10nM)的TIL细胞，图74E显示的是，对于来源于供者921的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。
对于A375-CD3细胞的杀伤能力检测，图74F-74G显示的是，对于来源于供者811的TIL细胞，以效靶比1:1、1:3与肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
TIL细胞的凋亡检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行凋亡检测。将基因敲除组或对照组(NT-no treatment)组的TIL用细胞凋亡检测试剂盒(BD 559763 Annexin V PE Apoptosis kit)检测T细胞凋亡水平。
图75A-75B显示的是，对于来源于供者811的TIL细胞的凋亡检测结果。图75C-75D显示的是，对于来源于供者921的TIL细胞的凋亡检测结果。图75E-75F显示的是，对于来源于供者614的TIL细胞的凋亡检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的抗凋亡能力。
TIL流式检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行流式细胞仪检测。
TIL流式检测试验材料的来源
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
对于细胞内因子的流式检测(例如FoxP3)，配制固定、破膜所需试剂：使用缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，4℃预冷待用。在表面染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图76A-76B显示的是，对于来源于供者811的TIL细胞，各组TIL细胞中PD-1阳性、LAG-3阳性和CD38阳性细胞的比例。图76C-76E显示的是，对于来源于供者614、921的TIL细胞，各组TIL细胞中Fas-L阳性细胞的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
图76F-76G显示的是，对于来源于供者410、811的TIL细胞，各组TIL细胞中调节性T细胞Treg(CD4 +CD25 +FoxP3 +)的比例。对于用30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)刺激3天后的TIL，图76H显示的是，对于来源于供者811的TIL细胞，各组TIL细胞中调节性T细胞Treg(CD4 +CD25 +FoxP3 +)的比例。对于用TGFβ-1(10nM)刺激3天后的TIL，图76I显示的是，对于来源于供者811的TIL细胞，各组TIL细胞中调节性T细胞Treg(CD4 +CD25 +FoxP3 +)的比例。结果显示，基因编辑后的TIL细胞具有更低的调节性T细胞的比例。
表7人TGFBR2基因组坐标
Figure PCTCN2022109580-appb-000037
Figure PCTCN2022109580-appb-000038
Figure PCTCN2022109580-appb-000039
Figure PCTCN2022109580-appb-000040
Figure PCTCN2022109580-appb-000041
实施例15FLI1基因编辑的细胞
TIL细胞的敲除效率检测
试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)、Recombinant DNase I(RNase-free，TAKARA)。
提取基因组DNA：在TIL细胞敲除后的约4天，取约1×10 5个至约2×10 5个细胞，用PBS清洗1次，再用44μL PBS重悬TIL细胞，用并加入配置的6μL核酸酶混合液(含1μL DNase I和5μL 10×DNase I Buffer)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃孵育10分钟。离心弃去上清后，在细胞沉淀中加入50μL DNA提取液，短暂离心后运行以下程序：75℃-10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDrop TM)检测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以下设计PCR反应体系：
试剂 体积
2×PCR Premix Buffer 25μL
DNA模板 约100-500ng
正向引物(10μM) 1μL
反向引物(10μM) 1μL
双蒸水 补充到50μL
DMSO 1.5μL
并按照以下PCR程序扩增：
Figure PCTCN2022109580-appb-000042
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本申请相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
不同的TIL可以是来源于不同的肿瘤患者，供者105为胰腺癌患者，供者313、供者222和供者812为皮肤恶性黑色素瘤患者，供者713为卵巢癌患者。
结果显示，SEQ ID NO：43(CTTACCTGGATCCATTCATG)所示的sgRNA，即FL4，对于供者313、222、105、713和812的敲除效率分别约为47.5％、8.7％、60.0％、86.9％、和88.7％。本申请的各种基因编辑方式均可以实现一定比例的敲除效率。
TIL细胞杀伤能力检测
基因编辑后的第6天开始，将A375肿瘤细胞系铺于96孔平底板中。次日，将各组TIL细胞以效靶比(TIL细胞：肿瘤细胞，E：T)为0.7:1的比例与上述A375细胞共培养。肿瘤细胞和TIL细胞各100μL，每组设置三个复孔，同时设置一组只包含肿瘤细胞的对照组。
根据细胞凋亡检测试剂(Incucyte Caspase-3/7 Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7 Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
图77显示的是，对于来源于供者313的TIL细胞，以效靶比0.7:1与肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
TIL细胞的凋亡检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行凋亡检测。将基因敲除组或对照组(NT-no treatment)组的TIL用细胞凋亡检测试剂盒(BD 559763 Annexin V PE Apoptosis kit)检测T细胞凋亡水平。
图78A-78B显示的是，对于来源于供者105的TIL细胞的凋亡检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的抗凋亡能力。
TIL流式检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行流式细胞仪检测。
TIL流式检测试验材料的来源
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式 管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
对于细胞内因子的流式检测，配制固定、破膜所需试剂：使用缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，4℃预冷待用。在表面染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图79A-79F显示的是，对于来源于供者105、222、313和812的TIL细胞，各组TIL细胞中LAG-3阳性、PD-1阳性、CD38阳性、和TIM-3阳性细胞的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
图80显示的是，对于来源于供者313的TIL细胞，各组TIL细胞中CD45RO阳性CD62L阳性记忆T细胞(Tcm)的比例。结果显示，基因编辑后的TIL细胞具有更高中心记忆T细胞的比例。
TIL细胞因子表达流式检测
对于实施例1中各个试验组基因编辑后的第7天或第8天获得的TIL群进行流式细胞仪检测细胞因子表达情况。
试验准备
CD3抗体组提前1天使用30ng/ml的CD3抗体(Miltenyi Biotech，OKT3)包被平底96孔板，4℃包被过夜；transACT组提前1天使用transACT包被平底96孔板，4℃包被过夜。
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比添加：Golgistop 0.7:1000， Golgiplug 1：1000，CD107a抗体1:500即2μL/mL。不添加白介素。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，200μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体(CD107a，GZMB，TNF-α和IFN-γ，BD/BioLegend)，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图81A-81B显示的是，在CD3抗体(Miltenyi Biotech，OKT3，CD3抗体以30ng/mL预处理96孔板)刺激过夜的条件下，对于来源于供者105的TIL细胞，各组TIL细胞中GZMB和TNF-α的表达细胞的比例。
图82A-82C显示的是，在无激活物刺激的条件下，对于来源于供者713和812的TIL细胞，各组TIL细胞中TNF-α、IFN-γ和CD107a的表达细胞的比例。
图83A-83D显示的是，在transACT刺激过夜的条件下，对于来源于供者713、812的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
结果显示，基因编辑后的TIL细胞具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力、更高的TNF-α表达能力或更高的GZMB表达能力。
表8人FLI1基因组坐标
Figure PCTCN2022109580-appb-000043
Figure PCTCN2022109580-appb-000044
Figure PCTCN2022109580-appb-000045
Figure PCTCN2022109580-appb-000046
Figure PCTCN2022109580-appb-000047
Figure PCTCN2022109580-appb-000048
实施例16CISH基因编辑的细胞
TIL细胞的敲除效率检测
试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)、Recombinant DNase I(RNase-free，TAKARA)。
提取基因组DNA：在TIL细胞敲除后的约4天，取约1×10 5个至约2×10 5个细胞，用PBS清洗1次，再用44μL PBS重悬TIL细胞，用并加入配置的6μL核酸酶混合液(含1μL DNase I和5μL 10×DNase I Buffer)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃ 孵育10分钟。离心弃去上清后，在细胞沉淀中加入50μL DNA提取液，短暂离心后运行以下程序：75℃-10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDrop TM)检测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以下设计PCR反应体系：
试剂 体积
2×PCR Premix Buffer 25μL
DNA模板 约100-500ng
正向引物(10μM) 1μL
反向引物(10μM) 1μL
双蒸水 补充到50μL
DMSO 1.5μL
并按照以下PCR程序扩增：
Figure PCTCN2022109580-appb-000049
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本申请相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
不同的TIL可以是来源于不同的肿瘤患者，供者313、供者222和供者812为皮肤恶性黑色素瘤患者，供者709、713为卵巢癌患者。
结果显示，SEQ ID NO：48(CTGTCAGTGAAAACCACTCG)所示的sgRNA，即CI1，对于供者313的敲除效率分别约为48.6％。
SEQ ID NO：49(CTCACCAGATTCCCGAAGGT)所示的sgRNA，即CI3，对于供者313和222的敲除效率分别约为47.5％和6.3％。
SEQ ID NO：50(TTCTAGACCTCGTCCTTTGC)所示的sgRNA，即CI-6，对于供者812和713的敲除效率分别约为95％和26.8％。
SEQ ID NO：51(GTCCGCTCCACAGCCAGCAA)所示的sgRNA，即CI-8，对于供者709、812和713的敲除效率分别约为2.3％、92.3％和71.2％。
本申请的各种基因编辑方式均可以实现一定比例的敲除效率。
TIL细胞的扩增情况的检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图84A-84C显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以具有显著的扩增能力。
基因编辑后的第7天开始，用30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)预处理96孔板，4℃过夜孵育；次日各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图85显示的是，对于来源于供者222的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以具有显著的扩增能力。
TIL细胞杀伤能力检测
基因编辑后的第6天开始，将A375肿瘤细胞系铺于96孔平底板中。次日，将各组TIL细胞以效靶比(TIL细胞：肿瘤细胞，E：T)为1:1、0.7:1或1:3的比例与上述A375细胞共培养。肿瘤细胞和TIL细胞各100μL，每组设置三个复孔，同时设置一组只包含肿瘤细胞的对照组。
根据细胞凋亡检测试剂(Incucyte Caspase-3/7 Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7 Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
图86A-86B显示的是，对于来源于供者222的TIL细胞，以效靶比1:3和0.7:1与肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
对于A375-CD3细胞的杀伤效果。图86C-86H显示的是，对于来源于供者313、222、812 和709的TIL细胞，以效靶比1:1、0.7:1或1:3与肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
TIL细胞的凋亡检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行凋亡检测。将基因敲除组或对照组(NT-no treatment)组的TIL用细胞凋亡检测试剂盒(BD 559763 Annexin V PE Apoptosis kit)检测T细胞凋亡水平。
图87A-87B显示的是，对于来源于供者812的TIL细胞的凋亡检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的抗凋亡能力。
TIL流式检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行流式细胞仪检测。
TIL流式检测试验材料的来源
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
对于细胞内因子的流式检测，配制固定、破膜所需试剂：使用缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，4℃预冷待用。在表面染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图88A-88F显示的是，对于来源于供者313、222、812和709的TIL细胞，各组TIL细胞中PD-1阳性、LAG-3阳性、CD38阳性、和TIM-3阳性细胞的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
图89A-89B显示的是，对于来源于供者313和709的TIL细胞，各组TIL细胞中CD45RO阳性CD62L阳性中心记忆T细胞(Tcm)的比例。结果显示，基因编辑后的TIL细胞具有更高中心记忆T细胞的比例。
TIL细胞因子表达流式检测
对于实施例1中各个试验组基因编辑后的第7天或第8天获得的TIL群进行流式细胞仪检测细胞因子表达情况。
试验准备
CD3抗体组提前1天使用30ng/ml的CD3抗体(Miltenyi Biotech，OKT3)包被平底96孔板，4℃包被过夜；transACT组提前1天使用transACT包被平底96孔板，4℃包被过夜。
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比添加：Golgistop 0.7:1000，Golgiplug 1：1000，CD107a抗体1:500即2μL/mL。不添加白介素。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，200μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体(CD107a，GZMB，TNF-α和IFN-γ，BD/BioLegend)，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS 重悬细胞，进行流式上机检测。
图90A-90D显示的是，在无激活物刺激的条件下，对于来源于供者713和812的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图91A-91B显示的是，在transACT刺激过夜的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
结果显示，基因编辑后的TIL细胞具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力、更高的TNF-α表达能力或更高的GZMB表达能力。
表9人CISH基因组坐标
序号 染色体编号 坐标起点 坐标终点
1 chr3 50606327 50606618
2 chr3 50606627 50607183
3 chr3 50607180 50608868
4 chr3 50608873 50609778
5 chr3 50609774 50610331
6 chr3 50610328 50610669
7 chr3 50610671 50610938
8 chr3 50610940 50611907
实施例17CBLB基因编辑的细胞
TIL细胞的敲除效率检测
试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)、Recombinant DNase I(RNase-free，TAKARA)。
提取基因组DNA：在TIL细胞敲除后的约4天，取约1×10 5个至约2×10 5个细胞，用PBS清洗1次，再用44μL PBS重悬TIL细胞，用并加入配置的6μL核酸酶混合液(含1μL DNase I和5μL 10×DNase I Buffer)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃孵育10分钟。离心弃去上清后，在细胞沉淀中加入50μL DNA提取液，短暂离心后运行以下程序：75℃-10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDrop TM)检测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以 下设计PCR反应体系：
试剂 体积
2×PCR Premix Buffer 25μL
DNA模板 约100-500ng
正向引物(10μM) 1μL
反向引物(10μM) 1μL
双蒸水 补充到50μL
DMSO 1.5μL
并按照以下PCR程序扩增：
Figure PCTCN2022109580-appb-000050
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本申请相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
不同的TIL可以是来源于不同的肿瘤患者，供者313、222为皮肤恶性黑色素瘤患者，供者105为胰腺癌患者。
结果显示，SEQ ID NO：54(TTCCGCAAAATAGAGCCCCA)所示的sgRNA，即CB1，对于供者105、313和222的敲除效率分别约为70.2％、74.5％和19.3％。
SEQ ID NO：55(TGCACAGAACTATCGTACCA)所示的sgRNA，即CB3，对于供者105、313和222的敲除效率分别约为67.5％、59.1％和16.3％。
本申请的各种基因编辑方式均可以实现一定比例的敲除效率。
TIL细胞的扩增情况的检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图92A-92C显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以具有显著的扩增能力。
基因编辑后的第7天开始，用30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)预处理96孔板，4℃过夜孵育；次日各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图93A-93C显示的是，对于来源于供者105、313和222的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以具有显著的扩增能力。
TIL细胞杀伤能力检测
基因编辑后的第6天开始，将A375肿瘤细胞系铺于96孔平底板中。次日，将各组TIL细胞以效靶比(TIL细胞：肿瘤细胞，E：T)为0.7:1或1:3的比例与上述A375细胞共培养。肿瘤细胞和TIL细胞各100μL，每组设置三个复孔，同时设置一组只包含肿瘤细胞的对照组。
根据细胞凋亡检测试剂(Incucyte Caspase-3/7 Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7 Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
图94A-94C显示的是，对于来源于供者313和222的TIL细胞，以效靶比1:3和0.7:1与肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
TIL细胞的凋亡检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行凋亡检测。将基因敲除组或对照组(NT-no treatment)组的TIL用细胞凋亡检测试剂盒(BD 559763 Annexin V PE Apoptosis kit)检测T细胞凋亡水平。
图95A-95B显示的是，对于来源于供者105的TIL细胞的凋亡检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的抗凋亡能力。
TIL流式检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行流式细胞仪检测。
TIL流式检测试验材料的来源
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
对于细胞内因子的流式检测，配制固定、破膜所需试剂：使用缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，4℃预冷待用。在表面染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图96A-96E显示的是，对于来源于供者105、313、222的TIL细胞，各组TIL细胞中PD-1阳性、LAG-3阳性、CD38阳性、和TIM-3阳性细胞的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
图97显示的是，对于来源于供者313的TIL细胞，各组TIL细胞中CD45RO阳性CD62L阳性中心记忆T细胞(Tcm)的比例。结果显示，基因编辑后的TIL细胞具有更高中心记忆T细胞的比例。
TIL细胞因子表达流式检测
对于实施例1中各个试验组基因编辑后的第7天或第8天获得的TIL群进行流式细胞仪检测细胞因子表达情况。
试验准备
CD3抗体组提前1天使用30ng/ml的CD3抗体(Miltenyi Biotech，OKT3)包被平底96孔板，4℃包被过夜；transACT组提前1天使用transACT包被平底96孔板，4℃包被过夜。
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比添加：Golgistop 0.7:1000，Golgiplug 1：1000，CD107a抗体1:500即2μL/mL。不添加白介素。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，200μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体(CD107a，GZMB，TNF-α和IFN-γ，BD/BioLegend)，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图98A-98B显示的是，在CD3抗体(Miltenyi Biotech，OKT3)，CD3抗体以30ng/mL预处理96孔板)刺激过夜的条件下，对于来源于供者105的TIL细胞，各组TIL细胞中GZMB，TNF-α和IFN-γ的表达细胞的比例。
表10人CBLB基因组坐标
Figure PCTCN2022109580-appb-000051
Figure PCTCN2022109580-appb-000052
Figure PCTCN2022109580-appb-000053
Figure PCTCN2022109580-appb-000054
Figure PCTCN2022109580-appb-000055
Figure PCTCN2022109580-appb-000056
Figure PCTCN2022109580-appb-000057
Figure PCTCN2022109580-appb-000058
Figure PCTCN2022109580-appb-000059
Figure PCTCN2022109580-appb-000060
Figure PCTCN2022109580-appb-000061
Figure PCTCN2022109580-appb-000062
Figure PCTCN2022109580-appb-000063
Figure PCTCN2022109580-appb-000064
Figure PCTCN2022109580-appb-000065
Figure PCTCN2022109580-appb-000066
Figure PCTCN2022109580-appb-000067
Figure PCTCN2022109580-appb-000068
Figure PCTCN2022109580-appb-000069
实施例18 ANKRD11基因编辑的细胞
TIL细胞的敲除效率检测
试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)、Recombinant DNase I(RNase-free，TAKARA)。
提取基因组DNA：在TIL细胞敲除后的约4天，取约1×10 5个至约2×10 5个细胞，用PBS 清洗1次，再用44μL PBS重悬TIL细胞，用并加入配置的6μL核酸酶混合液(含1μL DNase I和5μL 10×DNase I Buffer)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃孵育10分钟。离心弃去上清后，在细胞沉淀中加入50μL DNA提取液，短暂离心后运行以下程序：75℃-10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDrop TM)检测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以下设计PCR反应体系：
试剂 体积
2×PCR Premix Buffer 25μL
DNA模板 约100-500ng
正向引物(10μM) 1μL
反向引物(10μM) 1μL
双蒸水 补充到50μL
DMSO 1.5μL
并按照以下PCR程序扩增：
Figure PCTCN2022109580-appb-000070
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本申请相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
不同的TIL可以是来源于不同的肿瘤患者，供者812为皮肤恶性黑色素瘤患者，供者709、713为卵巢癌患者。
结果显示，SEQ ID NO：58(CCCTTCACCGCGGGCGCCAA)所示的sgRNA，即AN-6，对于供者709、812和713的敲除效率分别约为4.4％、43.7％和45.6％。
SEQ ID NO：59(GAGCAGAAGGACTCGGACAC)所示的sgRNA，即AN-8，对于供者709、812和713的敲除效率分别约为3.5％、76.9％和83.3％。
本申请的各种基因编辑方式均可以实现一定比例的敲除效率。
TIL细胞的扩增情况的检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图99A-99B显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以显著提高扩增能力。
基因编辑后的第7天开始，用30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)预处理96孔板，4℃过夜孵育；次日各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图100显示的是，对于来源于供者713的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以显著提高扩增能力。
TIL细胞杀伤能力检测
基因编辑后的第6天开始，将A375肿瘤细胞系铺于96孔平底板中。次日，将各组TIL细胞以效靶比(TIL细胞：肿瘤细胞，E：T)为0.7:1或1:3的比例与上述A375细胞共培养。肿瘤细胞和TIL细胞各100μL，每组设置三个复孔，同时设置一组只包含肿瘤细胞的对照组。
根据细胞凋亡检测试剂(Incucyte Caspase-3/7 Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7 Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
图101显示的是，对于来源于供者709的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
TIL细胞的凋亡检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行凋亡检测。将基因敲除组或对照组(NT-no treatment)组的TIL用细胞凋亡检测试剂盒(BD 559763 Annexin V PE Apoptosis kit)检测T细胞凋亡水平。
图102A-102B显示的是，对于来源于供者812的TIL细胞的凋亡检测结果。结果表明， 基因编辑的TIL细胞可以具有更显著的抗凋亡能力。
TIL流式检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行流式细胞仪检测。
TIL流式检测试验材料的来源
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
对于细胞内因子的流式检测，配制固定、破膜所需试剂：使用缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，4℃预冷待用。在表面染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图103A-103D显示的是，对于来源于供者812、709的TIL细胞，各组TIL细胞中LAG-3阳性和CD38阳性细胞的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
图104显示的是，对于来源于供者709的TIL细胞，各组TIL细胞中CD45RO阳性CD62L阳性中心记忆T细胞(Tcm)的比例。结果显示，基因编辑后的TIL细胞具有更高中心记忆T细胞的比例。
TIL细胞因子表达流式检测
对于实施例1中各个试验组基因编辑后的第7天或第8天获得的TIL群进行流式细胞仪检测细胞因子表达情况。
试验准备
CD3抗体组提前1天使用30ng/ml的CD3抗体(Miltenyi Biotech，OKT3)包被平底96孔板，4℃包被过夜；transACT组提前1天使用transACT包被平底96孔板，4℃包被过夜。
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比添加：Golgistop 0.7:1000，Golgiplug 1：1000，CD107a抗体1:500即2μL/mL。不添加白介素。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，200μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体(CD107a，GZMB，TNF-α和IFN-γ，BD/BioLegend)，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图105A显示的是，在无激活物刺激的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图105B显示的是，在transACT刺激过夜的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中CD107a的表达细胞的比例。
结果显示，基因编辑后的TIL细胞具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力、更高的TNF-α表达能力或更高的GZMB表达能力。 表11人ANKRD11基因组坐标
Figure PCTCN2022109580-appb-000071
Figure PCTCN2022109580-appb-000072
Figure PCTCN2022109580-appb-000073
Figure PCTCN2022109580-appb-000074
Figure PCTCN2022109580-appb-000075
Figure PCTCN2022109580-appb-000076
Figure PCTCN2022109580-appb-000077
实施例19BATF基因编辑的细胞
TIL细胞的敲除效率检测
试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)、Recombinant DNase I(RNase-free，TAKARA)。
提取基因组DNA：在TIL细胞敲除后的约4天，取约1×10 5个至约2×10 5个细胞，用PBS清洗1次，再用44μL PBS重悬TIL细胞，用并加入配置的6μL核酸酶混合液(含1μL DNase I和5μL 10×DNase I Buffer)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃孵育10分钟。离心弃去上清后，在细胞沉淀中加入50μL DNA提取液，短暂离心后运行以下程序：75℃-10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDrop TM)检 测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以下设计PCR反应体系：
试剂 体积
2×PCR Premix Buffer 25μL
DNA模板 约100-500ng
正向引物(10μM) 1μL
反向引物(10μM) 1μL
双蒸水 补充到50μL
DMSO 1.5μL
并按照以下PCR程序扩增：
Figure PCTCN2022109580-appb-000078
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本申请相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
不同的TIL可以是来源于不同的肿瘤患者，供者811为胰腺癌患者，供者812为皮肤恶性黑色素瘤患者，供者713为卵巢癌患者。
结果显示，SEQ ID NO：62(GGAACTGAAGTACTTCACGT)所示的sgRNA，即BATF-2，对于供者713、811和812的敲除效率分别约为51.3％、12.6％和67.3％。
SEQ ID NO：63(GGGCGCTGTACACCACCTCG)所示的sgRNA，即BATF-3，对于供者713、811和812的敲除效率分别约为63.4％、27.8％和76.8％。
本申请的各种基因编辑方式均可以实现一定比例的敲除效率。
TIL细胞的扩增情况的检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图106显示的是，对于来源于供者713的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以具有显著提高的扩增能力。
基因编辑后的第7天开始，用30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)预处理96孔板，4℃过夜孵育；次日各组TIL细胞以相同的细胞总数重新铺板。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图107显示的是，对于来源于供者713的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以具有显著提高的扩增能力。
TIL细胞杀伤能力检测
基因编辑后的第6天开始，将A375肿瘤细胞系铺于96孔平底板中。次日，将各组TIL细胞以效靶比(TIL细胞：肿瘤细胞，E：T)为1:1或1:3的比例与上述A375细胞共培养。肿瘤细胞和TIL细胞各100μL，每组设置三个复孔，同时设置一组只包含肿瘤细胞的对照组。
根据细胞凋亡检测试剂(Incucyte Caspase-3/7 Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7 Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
图108A-108B显示的是，对于来源于供者713的TIL细胞，以效靶比1:1、1:3与肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
TIL细胞的凋亡检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行凋亡检测。将基因敲除组或对照组(NT-no treatment)组的TIL用细胞凋亡检测试剂盒(BD 559763 Annexin V PE Apoptosis kit)检测T细胞凋亡水平。
图109A-109D显示的是，对于来源于供者713、811的TIL细胞的凋亡检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的抗凋亡能力。
TIL流式检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行流式细胞仪检测。
TIL流式检测试验材料的来源
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
对于细胞内因子的流式检测，配制固定、破膜所需试剂：使用缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，4℃预冷待用。在表面染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，600g离心3分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图110A-110F显示的是，对于来源于供者713、811、812的TIL细胞，各组TIL细胞中CD101阳性、LAG-3阳性和CD38阳性细胞的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
TIL细胞因子表达流式检测
对于实施例1中各个试验组基因编辑后的第7天或第8天获得的TIL群进行流式细胞仪检测细胞因子表达情况。
试验准备
CD3抗体组提前1天使用30ng/ml的CD3抗体(Miltenyi Biotech，OKT3)包被平底96孔板，4℃包被过夜；transACT组提前1天使用transACT包被平底96孔板，4℃包被过夜。
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比添加：Golgistop 0.7:1000， Golgiplug 1：1000，CD107a抗体1:500即2μL/mL。不添加白介素。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，200μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体(CD107a，GZMB，TNF-α和IFN-γ，BD/BioLegend)，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。
图111A显示的是，在无激活物刺激的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中GZMB表达细胞的比例。
图111B-111C显示的是，在transACT刺激过夜的条件下，对于来源于供者713的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
结果显示，基因编辑后的TIL细胞具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力、更高的TNF-α表达能力或更高的GZMB表达能力。
表12人BATF基因组坐标
Figure PCTCN2022109580-appb-000079
Figure PCTCN2022109580-appb-000080
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本文所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (40)

  1. 一种培养免疫细胞的方法，所述方法包含：使所述免疫细胞的YEATS结构域蛋白家族成员和/或其功能活性片段的表达降低和/或活性减弱。
  2. 根据权利要求1所述的方法，所述免疫细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
  3. 根据权利要求1-2中任一项所述的方法，所述免疫细胞包含单核细胞、巨噬细胞和/或树突状细胞。
  4. 根据权利要求1-3中任一项所述的方法，所述免疫细胞来源于干细胞分化的免疫细胞。
  5. 根据权利要求4所述的方法，所述干细胞包含诱导的多能干细胞(iPSC)。
  6. 根据权利要求1-5中任一项所述的方法，所述免疫细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞(NKT)。
  7. 根据权利要求1-6中任一项所述的方法，所述免疫细胞包含αβT细胞和/或γδT细胞。
  8. 根据权利要求1-7中任一项所述的方法，所述免疫细胞包含肿瘤浸润淋巴细胞(TIL)。
  9. 根据权利要求8所述的方法，所述TIL为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
  10. 根据权利要求9所述的方法，所述碎片的体积为约1立方毫米至约27立方毫米。
  11. 根据权利要求1-10中任一项所述的方法，所述免疫细胞包含展示在细胞表面上的工程化免疫受体。
  12. 根据权利要求11所述的方法，所述工程化免疫受体与靶细胞上表达的抗原特异性结合。
  13. 根据权利要求1-12中任一项所述的方法，所述免疫细胞包含嵌合抗原受体和/或T细胞受体。
  14. 根据权利要求1-13中任一项所述的方法，所述使所述免疫细胞的YEATS结构域蛋白家族成员的表达降低和/或活性减弱包含抑制YEATS结构域与组蛋白的结合能力。
  15. 根据权利要求1-14中任一项所述的方法，与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱获得的免疫细胞显示出改善的细胞特性。
  16. 根据权利要求15所述的方法，所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力和提高的肿瘤细胞杀伤能力。
  17. 根据权利要求16所述的方法，所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞和/或幼稚细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
  18. 根据权利要求1-17中任一项所述的方法，所述YEATS结构域蛋白家族成员包含YEATS结构域。
  19. 根据权利要求1-18中任一项所述的方法，所述YEATS结构域蛋白家族成员包含AF9和/或ENL。
  20. 根据权利要求1-19中任一项所述的方法，其中所述使所述免疫细胞的YEATS结构域蛋白家族成员的表达降低和/或活性减弱包含将基因调控系统引入所述免疫细胞中。
  21. 根据权利要求20所述的方法，所述基因调控系统能够在DNA水平破坏所述YEATS结构域蛋白家族成员。
  22. 根据权利要求20-21中任一项所述的方法，所述基因调控系统包含指导核酸分子和酶蛋白。
  23. 根据权利要求22所述的方法，其中所述使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述免疫细胞中。
  24. 根据权利要求22-23中任一项所述的方法，所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
  25. 根据权利要求22-24中任一项所述的方法，所述指导核酸分子包含指导RNA(gRNA)。
  26. 根据权利要求22-25中任一项所述的方法，所述指导核酸分子能够与所述YEATS结构域蛋白家族成员的序列结合。
  27. 根据权利要求22-26中任一项所述的方法，所述指导核酸分子能够与选自表6A和/或表6B所示的任一组基因组坐标定义的区域或其片段结合。
  28. 根据权利要求22-27中任一项所述的方法，所述指导核酸分子能够与选自以下组所 示的区域或其片段结合：SEQ ID NO：64-69。
  29. 根据权利要求22-28中任一项所述的方法，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：AGG、TGG、CGG和GGG。
  30. 根据权利要求22-29中任一项所述的方法，所述指导核酸分子包含如SEQ ID NO：70-75中任一项所示的序列。
  31. 根据权利要求1-30中任一项所述的方法，与YEATS结构域蛋白家族成员的表达和/或活性未改变的免疫细胞相比，使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱获得的免疫细胞中表达所述目标基因的产物的细胞比例降低和/或单个细胞中所述目标基因的表达量下降。
  32. 根据权利要求1-31中任一项所述的方法，所述使所述YEATS结构域蛋白家族成员的表达降低和/或活性减弱获得的免疫细胞中，表达所述目标基因的细胞比例为约95％或以下。
  33. 一种免疫细胞，所述免疫细胞经过权利要求1-32中任一项所述的方法获得。
  34. 一种组合物，其包含权利要求33所述的免疫细胞。
  35. 一种药物组合物，其包含权利要求33所述的免疫细胞和/或权利要求34所述的组合物，以及任选的药学上可接受的载体。
  36. 一种影响细胞生长的方法，包含施用权利要求33所述的免疫细胞、权利要求34所述的组合物和/或权利要求35所述的药物组合物。
  37. 权利要求33所述的免疫细胞、权利要求34所述的组合物和/或权利要求35所述的药物组合物在制备药物中的应用，所述药物用于预防和/或治疗疾病和/或症状。
  38. 根据权利要求37所述的应用，所述疾病和/或症状包含肿瘤。
  39. 根据权利要求37-38中任一项所述的应用，所述疾病和/或症状包含实体瘤。
  40. 根据权利要求37-39中任一项所述的应用，所述疾病和/或症状包含选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
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