WO2022135525A1 - 肿瘤浸润淋巴细胞的制备方法及其用途 - Google Patents

肿瘤浸润淋巴细胞的制备方法及其用途 Download PDF

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WO2022135525A1
WO2022135525A1 PCT/CN2021/140841 CN2021140841W WO2022135525A1 WO 2022135525 A1 WO2022135525 A1 WO 2022135525A1 CN 2021140841 W CN2021140841 W CN 2021140841W WO 2022135525 A1 WO2022135525 A1 WO 2022135525A1
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til
present application
cell
cells
agonist
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PCT/CN2021/140841
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English (en)
French (fr)
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刘雅容
赵佩佩
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苏州沙砾生物科技有限公司
上海沙砾生物科技有限公司
珠海拓域生物科技有限公司
珠海沙砾生物科技有限公司
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Priority to EP21909520.5A priority Critical patent/EP4269568A1/en
Priority to CN202180015572.5A priority patent/CN115175988B/zh
Priority to CN202410639644.9A priority patent/CN118480505A/zh
Priority to US18/269,407 priority patent/US20240043801A1/en
Publication of WO2022135525A1 publication Critical patent/WO2022135525A1/zh

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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present application relates to the field of biomedicine, in particular to a method for preparing tumor-infiltrating lymphocytes and uses thereof.
  • Tumor therapy using adoptively autologously transferred tumor-infiltrating lymphocytes is an effective approach to treat patients with poor prognosis.
  • adoptive autologous transfer of tumor-infiltrating lymphocytes to treat tumors requires a large number of tumor-infiltrating lymphocytes. Therefore, how to provide a robust and reliable method for culturing tumor-infiltrating lymphocytes is an urgent problem to be solved.
  • the present application provides a method for culturing tumor-infiltrating lymphocytes.
  • the tumor-infiltrating lymphocytes obtained by the culture method of the present application have one or more effects selected from the group consisting of: increased number of TIL cells, increased cytokine secretion capacity, increased tumor cell killing capacity, improved proportion of T cell subsets, increased proportion of central memory T cells, decreased proportion of regulatory T cells, increased proportion of activated T cells, increased proportion of tumor-specific T cells, Increased proportion of stem-like T cells, and improved gene editing effects.
  • the present application provides a method of culturing tumor-infiltrating lymphocytes (TILs), comprising: subjecting TILs derived from tumor tissue and not expanded in vitro through at least one stage of in vitro expansion, wherein in at least one stage In the in vitro expansion, the TIL is contacted with a CD28 agonist.
  • TILs tumor-infiltrating lymphocytes
  • the TIL derived from tumor tissue and not expanded in vitro is subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage of in vitro expansion, the The TILs expanded in vitro from the first stage are contacted with the CD28 agonist.
  • the CD28 agonist comprises an anti-CD28 antibody and/or antigen-binding fragment thereof.
  • the second stage in vitro expansion is performed for up to about 13 days.
  • the second stage in vitro expansion is performed for about 3 days to about 13 days.
  • the culturing method of the present application further comprises: co-culturing the TIL with feeder cells in at least one stage of the in vitro expansion.
  • the TIL is contacted with the CD28 agonist and co-cultured with the feeder cells in a single stage of the in vitro expansion.
  • the TIL is contacted with the CD28 agonist for a period of time before being co-cultured with the feeder cells in a single stage of the in vitro expansion.
  • the certain period of time is at least about 2 hours.
  • the certain period of time is from about 6 hours to about 72 hours.
  • the certain period of time is from about 12 hours to about 48 hours.
  • the certain period of time is about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours.
  • the feeder cells comprise antigen presenting cells.
  • the feeder cells comprise one or more selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.
  • the feeder cells are peripheral mononuclear cells.
  • the feeder cells are irradiated feeder cells.
  • co-culturing the TIL with the feeder cell comprises contacting the surface of the feeder cell with the surface of the TIL.
  • the co-cultivation of the TIL with the feeder cells comprises adding the feeder cells to the cell culture medium of the TIL.
  • the feeder cells are added to the cell culture medium of the TIL at a ratio of the feeder cells to the TIL from about 40:1 to about 400:1.
  • the culturing method of the present application further comprises: in at least one stage of the in vitro expansion, contacting the TIL with one or more T cell growth factors.
  • the TIL is contacted with the CD28 agonist and with the one or more T cell growth factors in a single stage of the in vitro expansion.
  • the TIL is contacted with the CD28 agonist and the one or more T cell growth factors substantially simultaneously in a single stage of the in vitro expansion.
  • the T cell growth factor is selected from one or more of the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21, gamma interferon, and their functionally active fragments.
  • the T cell growth factor comprises IL-2 and/or a functionally active fragment thereof.
  • contacting the TIL with the one or more T cell growth factors comprises adding the T cell growth factors to the cell culture medium of the TIL.
  • the initial concentration of the T cell growth factor in the cell culture medium of the TIL is at least about 300 IU/mL.
  • said TIL that has been contacted with said CD28 agonist during at least one in vitro expansion phase exhibits an improvement compared to a corresponding TIL that has not been contacted with said CD28 agonist during the in vitro expansion phase the amplification effect.
  • the improved expansion effect comprises one or more selected from the group consisting of an increased number of TIL cells, an improved proportion of T cell subsets, an increased cytokine secretion capacity, and an increased tumor cell killing ability.
  • the improved proportion of T cell subsets comprises one or more selected from the group consisting of: increased proportion of central memory T cells, decreased proportion of regulatory T cells, increased activated T cells ratio, increased proportion of tumor-specific T cells, and increased proportion of stem-like T cells.
  • said TIL that has been contacted with said CD28 agonist during at least one in vitro expansion phase exhibits an improvement compared to a corresponding TIL that has not been contacted with said CD28 agonist during the in vitro expansion phase effect of gene editing.
  • the improved gene editing effect comprises increased gene knockout efficiency.
  • the culturing method of the present application further comprises: in at least one stage of the in vitro expansion, combining the TIL with one or more other T cell activators other than the CD28 agonist touch.
  • the TIL is contacted with the CD28 agonist and with the one or more other T cell activators in a single stage of the in vitro expansion.
  • the TIL is contacted with the CD28 agonist and the one or more other T cell activators at substantially the same time in a single stage of the in vitro expansion.
  • the other T cell activator comprises one or more selected from the group consisting of CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, and their functionally active fragments.
  • the other T cell activator comprises an agonist of one or more targets selected from the group consisting of CD3, HVEM, CD40L, OX40 and 4-1BB.
  • the other T cell activator comprises a CD3 agonist.
  • the other T cell activator comprises an anti-CD3 antibody and/or antigen-binding fragment thereof.
  • the contacting the TIL with the CD28 agonist and with the one or more other T cell activators comprises one or more means selected from the group consisting of: (1) bringing The CD28 agonist and the other T cell activator are added to the cell culture medium of the TIL; (2) the engineered cells expressing the CD28 agonist and the other T cell activator are added to the TIL In the cell culture medium of TIL; (3) adding the solid-phase medium containing the CD28 agonist and the other T cell activator to the cell culture medium of the TIL.
  • the initial concentration of the other T cell activator in the cell culture medium of the TIL is at least about 30 ng/mL.
  • the initial concentration of the other T cell activator in the cell culture medium of the TIL is from about 30 ng/mL to about 300 ng/mL.
  • the solid phase medium has a diameter of about 500 nanometers to about 10 micrometers.
  • the solid phase medium has a diameter of from about 1 nanometer to about 500 nanometers.
  • the diameter of the solid medium is measured by transmission electron microscopy.
  • the solid phase medium comprises a polymer
  • the solid phase medium comprises at least about 25 ⁇ g of the CD28 agonist and the other T cell activator per mg of the solid medium.
  • the solid phase medium comprising the CD28 agonist and the other T cell activator is added in a ratio of the solid phase medium to the TIL of about 2:1 to about 1:2 into the cell culture medium of the TIL.
  • the solid phase medium comprising the CD28 agonist and the other T cell activator is added at a ratio of the solid phase medium to the TIL of about 1:100 to about 1:2000 into the cell culture medium of the TIL.
  • the TIL derived from tumor tissue and not expanded in vitro is TIL derived from fragments of the tumor tissue.
  • the fragments have a volume of about 1 cubic millimeter to about 27 cubic millimeters.
  • the application provides a method for culturing tumor-infiltrating lymphocytes (TILs), comprising:
  • step (A) contacting the first TIL population derived from tumor tissue and not expanded in vitro with one or more T cell growth factors; wherein the second TIL cell population is obtained through the step (A);
  • the CD28 agonist comprises an anti-CD28 antibody and/or antigen-binding fragment thereof.
  • step (B) is performed for up to about 13 days.
  • the step (B) is performed for about 3 days to about 13 days.
  • the culture method of the present application further comprises: in the step (A) and/or the step (B), co-culturing the TIL with the feeder cells.
  • the second TIL population is co-cultured with the feeder cells.
  • the second TIL population is co-cultured with the feeder cells after contacting the second TIL population with the CD28 agonist for a certain period of time.
  • the certain period of time is at least about 2 hours.
  • the certain period of time is from about 6 hours to about 72 hours.
  • the certain period of time is from about 12 hours to about 48 hours.
  • the certain period of time is about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours.
  • the feeder cells comprise antigen presenting cells.
  • the feeder cells comprise one or more selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.
  • the feeder cells are peripheral mononuclear cells.
  • the feeder cells are irradiated feeder cells.
  • co-culturing the TIL with the feeder cell comprises contacting the surface of the feeder cell with the surface of the TIL.
  • the co-cultivation of the TIL with the feeder cells comprises adding the feeder cells to the cell culture medium of the TIL.
  • the feeder cells are added to the cell culture medium of the TIL at a ratio of the feeder cells to the TIL from about 40:1 to about 400:1.
  • the culture method of the present application further comprises: in the step (A) and/or the step (B), contacting the TIL with one or more T cell growth factors.
  • step (B) the second TIL population is contacted with the one or more T cell growth factors.
  • step (B) wherein, in step (B), the second TIL population is contacted with the CD28 agonist and the one or more T cell growth factors at substantially the same time.
  • the T cell growth factor is selected from one or more of the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21, gamma interferon, and their functionally active fragments.
  • the T cell growth factor comprises IL-2 and/or a functionally active fragment thereof.
  • contacting the TIL with the one or more T cell growth factors comprises adding the T cell growth factors to the cell culture medium of the TIL.
  • the initial concentration of the T cell growth factor in the cell culture medium of the TIL is at least about 300 IU/mL.
  • said TIL contacted with said CD28 agonist in said step (B) is compared to a corresponding TIL that has not been contacted with said CD28 agonist in said step (B) Shows improved amplification.
  • the improved expansion effect comprises one or more selected from the group consisting of an increased number of TIL cells, an improved proportion of T cell subsets, an increased cytokine secretion capacity, and an increased tumor cell killing ability.
  • the improved proportion of T cell subsets comprises one or more selected from the group consisting of: increased proportion of central memory T cells, decreased proportion of regulatory T cells, increased activated T cells ratio, increased proportion of tumor-specific T cells, and increased proportion of stem-like T cells.
  • said TIL contacted with said CD28 agonist in said step (B) is compared to a corresponding TIL that has not been contacted with said CD28 agonist in said step (B) Shows improved gene editing effects.
  • the improved gene editing effect comprises increased gene knockout efficiency.
  • the culture method of the present application further comprises: in the step (A) and/or the step (B), making the TIL and one or more other than the CD28 agonist exposure to other T cell activators.
  • step (B) the second population of TILs is contacted with the CD28 agonist and with the one or more other T cell activators.
  • step (B) wherein, in step (B), the second TIL population is contacted with the CD28 agonist and the one or more other T cell activators at substantially the same time.
  • the other T cell activator comprises one or more selected from the group consisting of CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, and their functionally active fragments.
  • the other T cell activator comprises an agonist of one or more targets selected from the group consisting of CD3, HVEM, CD40L, OX40 and 4-1BB.
  • the other T cell activator comprises a CD3 agonist.
  • the other T cell activator comprises an anti-CD3 antibody and/or antigen-binding fragment thereof.
  • the contacting the TIL with the CD28 agonist and with the one or more other T cell activators comprises one or more means selected from the group consisting of: (1) bringing The CD28 agonist and the other T cell activator are added to the cell culture medium of the TIL; (2) the engineered cells expressing the CD28 agonist and the other T cell activator are added to the TIL In the cell culture medium of TIL; (3) adding the solid-phase medium containing the CD28 agonist and the other T cell activator to the cell culture medium of the TIL.
  • the initial concentration of the other T cell activator in the cell culture medium of the TIL is at least about 30 ng/mL.
  • the initial concentration of the other T cell activator in the cell culture medium of the TIL is from about 30 ng/mL to about 300 ng/mL.
  • the solid phase medium has a diameter of about 500 nanometers to about 10 micrometers.
  • the solid phase medium has a diameter of from about 1 nanometer to about 500 nanometers.
  • the diameter of the solid medium is measured by transmission electron microscopy.
  • the solid phase medium comprises a polymer
  • the solid phase medium comprises at least about 25 ⁇ g of the CD28 agonist and the other T cell activator per mg of the solid medium.
  • the solid phase medium comprising the CD28 agonist and the other T cell activator is added in a ratio of the solid phase medium to the TIL of about 2:1 to about 1:2 into the cell culture medium of the TIL.
  • the solid phase medium comprising the CD28 agonist and the other T cell activator is added at a ratio of the solid phase medium to the TIL of about 1:100 to about 1:2000 into the cell culture medium of the TIL.
  • the TIL derived from tumor tissue and not expanded in vitro is TIL derived from fragments of the tumor tissue.
  • the fragments have a volume of about 1 cubic millimeter to about 27 cubic millimeters.
  • the present application provides a tumor-infiltrating lymphocyte (TIL) obtained by the method of the present application.
  • TIL tumor-infiltrating lymphocyte
  • the application provides a composition comprising the TIL of the application.
  • the application provides a pharmaceutical composition comprising a TIL of the application and/or a composition of the application, and optionally a pharmaceutically acceptable carrier.
  • the application provides a method of affecting tumor cell growth comprising administering to a subject a TIL of the application and/or a pharmaceutical composition of the application.
  • the present application provides a use of the TIL of the present application and/or the pharmaceutical composition of the present application in the preparation of a medicament for preventing and/or treating tumors.
  • the tumor is a solid tumor.
  • the tumor is one or more selected from the group consisting of melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, stomach cancer, colorectal cancer cancer, and kidney cancer.
  • Figure 1 shows the results of the proliferative ability of TILs obtained by the second-stage in vitro expansion of the mixed antibody group with CD28 antibody added and the control group without CD28 antibody added for donor A.
  • FIG. 2 shows the results of the T cell subset ratio of TIL cells obtained by the second-stage in vitro expansion of the mixed antibody group with CD28 antibody added and the control group without CD28 antibody added for donor B-1.
  • FIG 3 shows the results of the T cell subset ratio of TIL cells obtained by the second stage in vitro expansion of the mixed antibody group with CD28 antibody and the control group without CD28 antibody for donor B-2.
  • Figure 4 shows the results of the T cell subset ratio of TIL cells obtained by the second-stage in vitro expansion of the magnetic bead group with CD28 antibody and the control group without CD28 antibody for donor C-1.
  • FIG. 5 shows the results of the T cell subset ratio of TIL cells obtained by the second-stage in vitro expansion of the magnetic beads group with CD28 antibody added and the control group without CD28 antibody added for donor C-2.
  • Figure 6 shows the results of the T-cell subset ratio of TIL cells obtained by the second-stage in vitro expansion of the nanomatrix group with CD28 antibody and the control group without CD28 antibody.
  • Figure 7 shows the results of the cell killing ability of TIL cells obtained by the second-stage in vitro expansion of the nanomatrix group with CD28 antibody added and the control group without CD28 antibody added for donor E.
  • Figure 8 shows the results of intracellular factor expression detection of TIL cells cultured in the second-stage in vitro expansion of the mixed antibody group supplemented with CD28 antibody and the control group without CD28 antibody for donor F.
  • Figure 9 shows the results of intracellular factor expression detection of TIL cells cultured in the second-stage in vitro expansion of the magnetic beads group with CD28 antibody and the control group without CD28 antibody for donor G-1.
  • FIG 10 shows the results of intracellular factor expression detection of TIL cells cultured in the second-stage in vitro expansion of the magnetic beads group with CD28 antibody added and the control group without CD28 antibody added for donor G-2.
  • FIG 11 shows the results of intracellular factor expression detection of TIL cells cultured in the second-stage in vitro expansion of the magnetic beads group with CD28 antibody added and the control group without CD28 antibody for donor G-3.
  • Figure 12 shows the results of intracellular factor expression detection of TIL cells cultured in the second-stage in vitro expansion of the magnetic beads group with CD28 antibody added and the control group without CD28 antibody added for donor G-4.
  • Figure 13 shows the results of intracellular factor expression detection of TIL cells cultured in the second-stage in vitro expansion of the CD28 antibody-added nanomatrix group and the non-CD28 antibody-added control group for Donor H.
  • Figure 14 shows the cytokine secretion detection results of TIL cells cultured in the second-stage in vitro expansion of the CD28 antibody-added nanomatrix group and the non-CD28 antibody-added control group for donor I-1.
  • Figure 15 shows the results of cytokine secretion detection after co-incubation of TIL cells and tumor cells in the second-stage in vitro expansion of the CD28 antibody-added nanomatrix group and the non-CD28 antibody-added control group for donor I-2 .
  • Figure 16 shows the results of gene knockout efficiency after co-incubation of TIL cells and tumor cells in the second-stage in vitro expansion of the CD28 antibody-added nanomatrix group and the non-CD28 antibody-added control group for donor J-1 .
  • Figure 17 shows the results of gene knockout efficiency after co-incubation of TIL cells and tumor cells in the second-stage in vitro expansion of the CD28 antibody-added nanomatrix group and the non-CD28 antibody-added control group for donor J-2 .
  • Figure 18 shows the results of the proliferative capacity of TILs for donors K-1, K-2 and K-3 for the third stage in vitro expansion with the addition of a CD28 agonist compared to the third stage expansion without the addition of the CD28 agonist.
  • Figure 19A shows the TIL proliferative capacity of experimental groups to which different forms of CD28 agonists were added.
  • Figure 19B shows the proportion of tumor-specific T cells (CD103 + CD39 + ) in TILs of experimental groups of different forms of nano-sized CD28 agonists.
  • Figures 19C-19F show the proportions of depleted T cells (CD39 + , PD1 + , LAG3 + , TIM3 + ) in TILs of experimental groups with different forms of nano-sized CD28 agonists.
  • Figure 19G shows the proportion of regulatory T cells (Treg) in TILs of experimental groups with different forms of nano-sized CD28 agonists.
  • FIG. 19H-19J show the proportion of activated T cells (CD25 + , 41BB + , CD28 + ) in TILs of experimental groups with different forms of nano-sized CD28 agonists.
  • FIG. 19K shows the proportion of stem-like T cells (CD69 ⁇ CD39 ⁇ ) in TILs of the experimental groups for different forms of nano-sized CD28 agonists.
  • Figure 19L shows the proportion of central memory T cells (Tcm, eg CD45RO + CD62L + ) in TILs of experimental groups with different forms of nano-sized CD28 agonists.
  • Figure 19M shows the expression ratio of intracellular factor (CD107a) in TILs of experimental groups with different forms of nano-sized CD28 agonists.
  • Figure 19N shows the expression ratio of intracellular factor (CD107a) in TILs of experimental groups with different forms of nano-sized CD28 agonists. TIL cells of each group were incubated with CD3 antibody overnight before flow cytometry.
  • Figure 190 shows the cytokine secretion capacity in TILs of experimental groups of different forms of nano-sized CD28 agonists, wherein TIL cells in the CD3 antibody stimulated group were incubated with CD3 antibody overnight before cytokine detection.
  • the term "expression” generally refers to the process of transcription and/or translation that occurs within a cell of a gene encoding a polypeptide of interest.
  • the level of transcription of the gene encoding the polypeptide of interest in the host cell can be determined by measuring the amount of the corresponding mRNA present in the cell. For example, quantitative measurement of mRNA transcribed from a gene encoding a polypeptide of interest can be performed by PCR or by RNA hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
  • the level of translation of a gene encoding a polypeptide of interest can be measured by a variety of methods, such as by ELISA, by polypeptide biological activity assays, or by Western blotting or radioimmunoassays (see Sambrook et al., supra).
  • stage in the terms “one stage in vitro expansion”, “single stage in vitro expansion”, or “first stage in vitro expansion” etc. generally refers to a period of expansion that TILs undergo in vitro process.
  • each stage can be divided by the change in the number of TIL cells, in one embodiment, when the number of TIL cells increases by at least about 1-fold, it can be considered that TIL cells have entered the next stage.
  • TIL cells when the number of TIL cells increases 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 At least about 50-fold, TIL cells can be considered to have entered the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the conditions of TIL cell culture.
  • TIL cells can be considered to enter the next stage of in vitro expansion when T cell activators and/or T cell growth factors are added or supplemented to the cell culture medium. In one embodiment, after the TIL cells are centrifuged and/or cell washed, the TIL cells can be considered to have entered the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the number of days in which the TIL cells are cultured.
  • 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 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, the TIL cells can be considered to have entered the next stage of in vitro expansion.
  • first stage in vitro expansion generally refers to the stage of expansion using T cell growth factors after primary TILs are obtained from tissue.
  • the tissue of the present application may be selected from the group consisting of tumor tissue and pleural effusion, and the pleural effusion of the present application may be 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 the preREP (pre-rapid expansion) stage.
  • the term "second-stage in vitro expansion” generally refers to a stage in which tissue is removed from a subject and expanded, and then expanded again.
  • the number of TIL cells expanded in vitro in the second stage of the present application is increased, eg, at least about 10-fold (or at least about 20, 30 fold), compared to the TILs expanded in vitro in the first stage. , 40, 50, 60, 70, 80 or 90-fold), or in one embodiment the number of cells can be increased by at least about 100-fold.
  • the culture conditions of the second stage in vitro expansion may be different from that of the first stage in vitro expansion, eg, the added culture substance may be different.
  • the second stage of in vitro expansion of the present application may also be referred to as the REP (rapid expansion) stage.
  • in vivo generally refers to an event that occurs in a subject.
  • in vitro generally refers to events that occur outside the body of a subject.
  • ex vivo generally refers to events 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 subject's body by surgery or treatment.
  • secretion generally refers to the transfer of an expressed polypeptide or protein by a cell to the extracellular environment.
  • secretory capacity generally refers to the ability of a cell to express a polypeptide or protein and to transfer the polypeptide or protein of the present application to the extracellular environment.
  • irradiation generally refers to the treatment of a substance by means of radiation.
  • irradiating may refer to irradiating a substance with X-rays, alpha rays, beta rays, or gamma 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.
  • the engineered cells may be genetically modified to express TILs of T cell activators and/or T cell growth factors according to the present application.
  • co-culture generally refers to the culturing of two or more different populations of cells with some degree of contact between them.
  • Contacting of two or more different populations of cells of the present application may, in one embodiment, be by direct contact, ie, in which cells of one population are in direct physical contact with cells of another population.
  • indirect contact mediated by shared culture medium may be used.
  • the shared medium of the present application may contain metabolites produced and released by at least one population of co-cultured cells and used to culture cells of another population.
  • the term "contacting" generally means that two or more substances of different types are brought into contact together in any order, in any manner, and for any length of time.
  • 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, eg, one or more feeder cells can be added.
  • the medium of cells, T cell activators and/or T cell growth factors is added to and/or replaced with the medium of TIL cells, for example, one or more feeder cells, T cell activators and/or T cell growth factors may be added to The medium of factors is used for the cultivation of TIL cells; in one embodiment, the metabolites produced and released by the feeder cells can be used for the cultivation of TIL cells by indirect contact, for example.
  • 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 “concurrently contacting”, “co-contacting”, “concurrently in contact with”, “simultaneously” and “co-contacting” generally refer to the administration of two or more substances to a subject and/or cell such that the substances both in the subject and/or in the context 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.
  • “simultaneously contacting” as used herein 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 can be increased by at least about 100-fold.
  • the term “expanded” generally means that the cells of the application have been expanded by one or more of the above.
  • polymer generally refers to a molecule consisting of separate chemical moieties linked together, which may be the same or different in this application.
  • the term “polymer” may refer to individual chemical moieties linked tail to tail 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, polysaccharides, dextran, hydrogels, polyethylene glycol, or poloxamers.
  • Poloxamers are nonionic triblock copolymers with a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) and two pendant hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).
  • the materials encompassed by this application can be formulated with, or administered with, any of the polymers described herein or known in the art.
  • antibody generally refers to an immunoglobulin reactive against a specified protein or peptide or fragment thereof.
  • Such antibodies include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, monoclonal antibodies, monospecific antibodies, polyclonal antibodies, multispecific antibodies, nonspecific antibodies, bispecific antibodies, multispecific antibodies
  • Antibodies can be from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, and from any subclass (eg, IgGl, IgG2, IgG3, and IgG4).
  • the antibody may have a heavy chain constant region selected from, eg, IgGl, IgG2, IgG3, or IgG4.
  • the antibody may also have a light chain selected from, for example, kappa ( ⁇ ) or lambda ( ⁇ ).
  • the antibodies of the present application can be derived from any species, including but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, chicken, and bovine.
  • the constant region of the antibody can be altered, eg, mutated, to modify the properties of the antibody (eg, to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine residues, effect organ cell function, or complement function).
  • the antibody specifically binds to a predetermined antigen, eg, an antigen associated with a disorder, eg, an inflammatory, immune, autoimmune, neurodegenerative, metabolic, and/or malignant disorder.
  • 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 alleviate the immune response induced by the murine antibody.
  • a hybridoma that secretes a mouse-specific monoclonal antibody can be established, and then the variable region gene can be cloned from the mouse hybridoma cell, and the constant region gene of the human antibody can be cloned according to the needs.
  • the human constant region gene is connected into a chimeric gene and inserted into an expression vector, and the chimeric antibody molecule can be expressed in a eukaryotic system or a prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody
  • CDR-grafted antibody generally refers to the grafting of murine CDR sequences into the framework of human antibody variable regions, i.e. different Types of human germline antibody framework sequences produced in antibodies.
  • the heterologous reaction induced by chimeric antibodies can be overcome because they carry a large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database.
  • monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the antibodies or ligands described herein may be fully human monoclonal antibodies.
  • Related technologies for the preparation of fully human antibodies include: human hybridoma technology, EBV transformation of B lymphocytes, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.
  • CDR generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • 6 CDRs are provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242), Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins, "J. Mol. Biol. 196:901 (1987); and MacCallum et al., “Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography,” J. Mol. Biol. 262:732 (1996)).
  • CDR L1, CDR L2, CDR L3 or L1, L2, L3 the Kabat definition of CDRs can be applied to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domains, as well as the heavy chain variable domains of CDR1, CDR2 and CDR3 (CDR H1, CDR H2, CDR H3 or H1, H2, H3).
  • anti-CD3 antibody generally refers to an antibody or variant thereof that targets CD3, such as a monoclonal antibody, including human, humanized, chimeric or murine antibodies, directed against T cells of mature T cells CD3 receptor among antigen receptors.
  • Anti-CD3 antibodies can include OKT-3.
  • Anti-CD3 antibodies can include SP34.
  • Anti-CD3 antibodies can also include other anti-CD3 antibodies including, for example, in one embodiment otelixizumab, teplizumab, and visilizumab.
  • IL-2 or "IL2” generally refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2, which in one embodiment may include humans and mammals forms, conservative amino acid substitutions, glycoform modifications or variants, or active fragments thereof.
  • the GeneID encoding the IL-2 gene may be 3558.
  • the term "antigen-presenting cell”, “antigen-presenting cell”, or “APC” generally refers to an immunizing agent that displays on its surface a foreign antigen complexed with the major histocompatibility complex (MHC).
  • Systemic cells such as helper cells (eg, B cells, dendritic cells, etc.). T cells can recognize these complexes using their T cell receptors (TCRs).
  • TCRs T cell receptors
  • APCs can process and present antigens to T cells.
  • the antigen presenting cells may comprise selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.
  • expansion effect generally refers to the effect that occurs after a cell has been expanded. Changes in the effect of expansion can include changes in the number and/or ratio of cells, changes in secretion capacity, changes in killing capacity, or changes in expression capacity, or any combination thereof. Variations of the present application may be increased or decreased.
  • nanoparticle generally refers to at least one microscopic particle having a size of less than 100 nm.
  • nanoparticles typically have diameters 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 delivered to the desired site.
  • the nanoparticles of the present application may comprise a CD28 antibody or antigen-binding fragment thereof.
  • the nanoparticles of the present application may comprise a CD28 antibody or antigen-binding fragment thereof and a CD3 antibody or antigen-binding fragment thereof.
  • Anti-CD28 antibodies can include 15E8.
  • an artificial antigen-presenting cell generally refers to an artificially constructed immune cell for presenting exogenous antigens.
  • the complex of the histocompatibility complex MHC.
  • an isolated artificial antigen presenting cell may be included, which may comprise expression of HLA-A/B/C (the gene encoding the GeneID of which may be 3105, 3106 or 3107), CD64 (the gene encoding it GeneID can be 2209), CD80 (GeneID encoding it can be 941), ICOS-L (GeneID encoding it can be 23308) and CD58 (GeneID encoding it can be 965) cells, and can be modified To express more than one T cell activator, the above of the present application may include 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 (ie, different from the first polypeptide or protein or of 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 fragment, analog or derivative thereof) polypeptide or protein.
  • a fusion protein may comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide.
  • the heterologous proteins, polypeptides or peptides of the present application may or may not be different types of prophylactic or therapeutic drugs.
  • fusion protein may retain or increase the activity of the heterologous protein, polypeptide, or the original polypeptide or protein prior to protein fusion.
  • 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 refers to killing target cells by contacting the cells of the present application with an effective amount of a substance.
  • the agents of the present application may be TIL cells. Killing of the present application may include killing cells by themselves or by promoting CDC, apoptosis, ADCC, and/or phagocytosis of other cells or substances, or by a combination of two or more of these mechanisms.
  • administration generally refers to the delivery of 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 this application.
  • TIL cells of the present application are included.
  • the term "subject” generally refers to cells or animals, which may be mammals such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, 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 of skill in the art.
  • the term "feeder” generally refers to a cultured cell that grows in vitro and secretes at least one factor into the culture medium and can be used to support the growth of another cell of interest.
  • the feeder cells may comprise antigen presenting cells.
  • the term "specifically binds” generally refers to an antibody that recognizes a specific antigen, but does not substantially recognize or bind to other molecules in a sample.
  • an antibody can specifically bind a specific antigen of the present application from one species, the antibody of the present application can also specifically bind an antigen of the present application or a cognate antigen from another species or species. Such interspecies reactivity may not by itself alter the classification of the antibody as specific.
  • antibodies that specifically bind to an antigen can also bind to different allelic forms of the antigen.
  • complete culture process generally refers to the complete process of starting cells from isolated tumor tissue from a patient, going through one or more expansions, and finally obtaining cells that can be administered to a subject. .
  • 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.
  • the cell culture medium includes buffers, salts, carbohydrates, amino acids, vitamins and necessary trace elements.
  • the cell culture medium 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 formulation” generally refers to a preparation which may allow the biological activity of the active ingredient to be effective and which may be free of receptors for which the formulation will be administered. Additional components that are unacceptably toxic to the subject. Such formulations are sterile. "Pharmaceutically acceptable” excipients (carriers, additives) are those that can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient used.
  • TIL tumor infiltrating lymphocytes
  • TILs can include, but are not limited to, CD8 + cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + 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 subject tissue sample, and "secondary TILs” can be any TIL cell population that has been expanded or expanded in the present application.
  • the tumor-infiltrating lymphocytes of the present application may not be isolated and purified, or may be mutually infiltrated with tumor cells.
  • TIL of the present application may refer to a population of TIL cells.
  • central memory T cells generally refers to T cells that have long-term memory and are able to receive antigenic restimulation.
  • Central memory T cells can have the phenotype of CD45RA - CCR7 + and/or CD45RO + CD62L + , for example, central memory T cells can be identified by CD45RA- and CCR7 + .
  • central memory T cells can be identified by CD45RO + and CD62L + .
  • Central memory T cells can have stronger anti-tumor growth ability than ordinary T cells.
  • regulatory T cells generally refers to a subset of T cells that control autoimmune reactivity in the body. Regulatory T cells can have the phenotype of CD4 + CD25 + Foxp3 + , eg, regulatory T cells can be identified by CD4 + , CD25 + , and Foxp3 + . Regulatory T cells may have the ability to suppress the antitumor 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 may have the phenotype of PD1 + , LAG3 + or CD28 + , eg, activated T cells may be identified by PD1 + , LAG3 + or CD28 + .
  • Activated T cells may have the ability to resist tumor growth.
  • tumor-specific T cells generally refers to T cells that can specifically fight tumor growth.
  • Tumor-specific T cells can have a CD103 + CD39 + phenotype, eg, tumor-specific T cells can be identified by CD103 + and CD39 + .
  • Tumor-specific T cells may have a more specific anti-tumor growth ability than ordinary T cells.
  • stem-like T cells generally refers to a class of T cells that can have the potential to self-proliferate and/or differentiate.
  • Stem-like T cells can have a TCF1 + phenotype, for example, stem-like T cells can be identified by TCF1 + .
  • Tumor-specific T cells may have stronger and/or longer-term anti-tumor growth ability than normal T cells.
  • tumor fragments generally refers to tumor fragments that can be formed by mechanical disruption, enzymatic hydrolysis, and/or other disruption methods after tumor tissue is removed from a 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 carriers, stabilizers , a mixture of diluents, dispersing agents, suspending agents, thickening agents and/or 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 administration to humans.
  • 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), and the like.
  • flavoring agents such as peppermint, peppermint, peppermint, trehalose, trehalose, trehalose, trehalose, glycerin, trehalose, glycerin, glycerin, sorbitol, sorbitol, and the like.
  • antimicrobial agents such as sweeteners, antioxidants, antistatic agents, lipids , protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), and the like.
  • salt-forming counterions such as sodium
  • 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.
  • a functionally active fragment of the growth factor IL-2 may retain or partially retain the biologically active function of full-length IL-2 that causes cell proliferation.
  • T cell activator generally refers to a substance that binds to the corresponding binding receptor on T cells and mediates T cell co-stimulatory responses.
  • T cell activators can be substances other than antigen receptors that are required by T cells to generate an effective immune response.
  • T cell activators may refer to T cell costimulatory molecules.
  • 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 activation receptors, BTLA (the gene encoding it can be 151888), Toll ligand receptor, OX40 (the gene encoding it can be 7293), CD2 (the gene encoding it can be 914), CD7 (the gene encoding it can be GeneID 914) is 924), CD27 (the gene encoding it can be 939), CD28 (the gene encoding it can be 940), CD30 (the gene encoding it can be 943), CD40 (the gene encoding it can be 958) ), CDS, ICAM-1 (the gene encoding it can be GeneID 3383), LFA-1 (CD11a/CD18) (the gene encoding it can be 3689), 4-1BB (CD137) (the gene encoding it can be Gene
  • the term "T cell growth factor” generally refers to a biologically active polypeptide or small molecule compound that causes cell proliferation.
  • the T cell growth factor may be selected from one or more of the following group: IL-2 (the gene encoding it may be GeneID 3558), IL-4 (the gene encoding it may be 3565) , IL-7 (the gene GeneID encoding it can be 3574), IL-10 (the gene encoding it can be 3586), IL-12 (the gene encoding it can be 3592 or 3593), IL-15 (the gene encoding it can be 3592 or 3593) Its gene GeneID may be 3600), and gamma interferon (the gene encoding it may be 3458).
  • IL-2 the gene encoding it may be GeneID 3558
  • IL-4 the gene encoding it may be 3565
  • IL-7 the gene GeneID encoding it can be 3574
  • IL-10 the gene encoding it can be 3586
  • IL-12 the gene en
  • substantially simultaneously generally means that the TIL can be in contact with two or more substances simultaneously during a period of time during the contacting process, but may not be limited to the fact that the TIL is always simultaneously with two or more substances during the entire contacting process touch. In one embodiment, substantially simultaneously can mean that TIL can interact with 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 at the same time.
  • solid phase medium generally refers to a solid phase material that has a binding function.
  • the solid-phase medium in the present 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 binding and/or non-covalent binding.
  • the solid phase medium of the present application can bind one or more T cell activators.
  • the solid-phase medium of the present application may refer to the binding of CD28 antibody or its antigen-binding fragment and CD3 antibody or its antigen-binding fragment in and/or on the surface of the medium through covalent binding and/or non-covalent binding.
  • the solid phase medium of the present application can be microspheres of about 500 nanometers to about 10 micrometers in diameter comprising the OKT3 antibody and the 15E8 antibody.
  • the solid phase medium of the present application may be a polymeric material.
  • the solid phase medium 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 having a diameter of about 1 nanometer to about 500 nanometers comprising the OKT3 antibody and the 15E8 antibody.
  • the term "nanomatrix" generally refers to a material having a diameter of from about 1 nanometer to about 500 nanometers.
  • the nanomatrix may have a binding function, for example, the nanomatrix of the present application may bind 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.
  • gene editing generally refers to a type of genetic engineering that utilizes one or more nucleases and/or nickases to insert, replace or remove DNA from a target DNA (eg, the TCR ⁇ genome of a cell).
  • a target DNA eg, the TCR ⁇ genome of a cell
  • gene knockout generally refers to genetic engineering means that silence a gene and/or disable the expression of the protein it encodes.
  • gene knockout can refer to the targeted disruption of a gene in a cell or in vivo, resulting in a complete loss of its function.
  • the gene knockout of the present application can use site-specific nucleases.
  • the gene knockout of the present application can use zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and/or CRISPR/Cas-based systems.
  • ZFNs zinc finger nucleases
  • TALENs TAL effector nucleases
  • CRISPR/Cas-based systems for example, the gene knockout of the present application can use the CRISPR/Cas9 system.
  • dendritic cells generally refers to antigen-presenting cells that are present in vivo, in vitro, ex vivo or in a host or subject or which can 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 morphologies such as lamellae (lamellipodia) extending in multiple directions of the dendritic cell body. In general, 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 are able to elicit primary T cell responses in vitro and in vivo.
  • MHC co-stimulatory
  • in vitro expansion generally refers to culturing to produce changes in the number of cells, and the expanded cells can also produce changes in the number and/or ratio of cells, changes in secretion capacity, changes in killing capacity or expression Changes in abilities, or any combination of them. Variations of the present application may be increased or decreased.
  • in vitro expansion can 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), which may not belong to the in vitro expansion of the present application.
  • peripheral mononuclear cells or “peripheral blood mononuclear cells” generally refers to cells in peripheral blood that have 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 one cell population that acts as an intercellular regulator of another cell.
  • Cytokines of the present application may be lymphokines, monokines and polypeptide hormones.
  • 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-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 the substance of the application.
  • the term “diameter” generally refers to the largest diameter and/or the mean diameter of the largest cross-section of the substance of the present application.
  • the method of determining the diameter of the substance may be a method commonly used in the art, such as transmission electron microscopy.
  • tumor generally refers to any new pathological tissue proliferation.
  • the tumors of the present application may be benign or malignant.
  • the tumor of the present 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, stomach cancer, colorectal cancer, and kidney cancer .
  • tumor tissue generally refers to a sample from a tumor in a subject, including any solid tumor and/or any tissue that is not a 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 can be a small molecule preparation that binds CD28.
  • a 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 TIL cells or TIL cell populations according to different T cell subsets.
  • different T cell subsets of the present application have different immune activity and/or differentiation capacity.
  • T cell subsets of the present application can be differentiated based on T cell surface markers.
  • central memory T cells can have the phenotype of CD45RA - CCR7 + and/or CD45RO + CD62L + .
  • regulatory T cells can have a CD4 + CD25 + Foxp3 + phenotype.
  • activated T cells can have the phenotype of CD25 + , CD28 + , PD1 + , 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 through the first stage.
  • the number of TIL cells can refer to the number of cells of the third TIL population expanded in vitro through the second stage.
  • the number of TIL cells can refer to the cells of TIL finally obtained by any one of the culturing methods of the present application.
  • the number of TIL cells can be measured by methods commonly used in the art, such as, but not limited to, manual cell counting with a cytometer and/or automated cell counter counting.
  • the terms “about” and “approximately” generally refer to within a statistically significant range of values. Such a range 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 variation encompassed by the term “about” or “approximately” may depend on the particular system under study, and can be readily understood by one of ordinary skill in the art.
  • the terms “above,” “below,” “at most,” and “at least” may include this number.
  • the present application provides a method of culturing tumor-infiltrating lymphocytes (TILs), which may comprise: subjecting TILs derived from tumor tissue and not expanded in vitro through at least one stage of in vitro expansion, wherein in at least one In the staged in vitro expansion of the present application, the TIL of the present application may be brought into contact with a CD28 agonist.
  • TILs tumor-infiltrating lymphocytes
  • the present application provides a method of culturing tumor-infiltrating lymphocytes (TILs). It may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with one or more T cell growth factors; wherein the step (A) obtains a second TIL cell population ; (B) contacting a second population of TILs of the present application with a CD28 agonist.
  • TILs tumor-infiltrating lymphocytes
  • the present application provides a method of culturing tumor-infiltrating lymphocytes (TILs). It may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with one or more T cell growth factors; wherein the step (A) obtains a second TIL cell population (B) contacting a second TIL population of the present application with a CD28 agonist and co-culturing the second TIL population with feeder cells.
  • TILs tumor-infiltrating lymphocytes
  • the present application provides a method of culturing tumor-infiltrating lymphocytes (TILs). It may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with one or more T cell growth factors; wherein the step (A) obtains a second TIL cell population ; (B) contacting a second TIL population of the present application with a CD28 agonist and one or more T cell growth factors.
  • TILs tumor-infiltrating lymphocytes
  • the present application provides a method of culturing tumor-infiltrating lymphocytes (TILs). It may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with one or more T cell growth factors; wherein the step (A) obtains a second TIL cell population (B) After contacting the second TIL population of the present application with a CD28 agonist and one or more T cell growth factors for a certain period of time, it is then co-cultured with the feeder cells of the present application.
  • TILs tumor-infiltrating lymphocytes
  • the first stage in vitro amplification of the present application can be used in any substitution with step (A) in the method of the above aspect.
  • the second stage in vitro amplification of the present application can be used arbitrarily alternatively to step (B) in the method of the above aspect.
  • the TILs expanded in vitro in the first stage of the present application can be arbitrarily replaced with the second population of TILs obtained through step (A) of the method of the above aspect.
  • the TILs expanded in vitro in the second stage of the present application can be arbitrarily replaced with the third population of TILs obtained through step (B) of the method of the above aspect.
  • the third-stage in vitro amplification of the present application can be optionally used in place of any additional step (C) in the method of the above aspect, if necessary.
  • the TILs of the present application expanded in vitro in the third stage can be optionally replaced with the fourth TIL population obtained through any additional step (C) in the method of the above aspect. use.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and expanded in vitro in the second stage of the present application
  • the TILs expanded in vitro in the first stage of the present application can be contacted with the CD28 agonists of the present application.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and expanded in vitro in the first stage of the present application
  • tumor tissue-derived TILs of the present application that have not been expanded in vitro can be contacted with the CD28 agonists of the present application.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and expanded in vitro in the first stage of the present application
  • the tumor tissue-derived TIL that has not been expanded in vitro can be contacted with the CD28 agonist of the present application, and in the second-stage in vitro expansion of the present application, the first-phase in vitro expansion of the present application can be made.
  • Staged in vitro expanded TILs are contacted with the CD28 agonists of the present application.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first-stage in vitro expansion, a second-stage in vitro expansion, and a third-stage in vitro expansion, and the present application
  • the TIL derived from the tumor tissue of the present application and not expanded in vitro can be contacted with the CD28 agonist of the present application.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first-stage in vitro expansion, a second-stage in vitro expansion, and a third-stage in vitro expansion, and the present application
  • the TILs expanded in vitro by the first phase of the present application can be contacted with the CD28 agonist of the present application.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first-stage in vitro expansion, a second-stage in vitro expansion, and a third-stage in vitro expansion, and the present application
  • the TILs expanded in the second-phase in vitro expansion of the present application may be contacted with the CD28 agonist of the present application.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first-stage in vitro expansion, a second-stage in vitro expansion, and a third-stage in vitro expansion, and the present application
  • the TIL derived from the tumor tissue of the present application and not expanded in vitro can be contacted with the CD28 agonist of the present application
  • the second stage of in vitro expansion of the present application it can be The TILs expanded in vitro from the first stage of the present application are contacted with the CD28 agonists of the present application.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first-stage in vitro expansion, a second-stage in vitro expansion, and a third-stage in vitro expansion, and the present application
  • the TIL derived from the tumor tissue of the present application and not expanded in vitro can be contacted with the CD28 agonist of the present application
  • the third stage of in vitro expansion of the present application it can be The in vitro expanded TILs of the second stage of the present application are contacted with the CD28 agonists of the present application.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first-stage in vitro expansion, a second-stage in vitro expansion, and a third-stage in vitro expansion, and the present application
  • the TILs expanded in vitro in the first stage of the present application can be contacted with the CD28 agonist of the present application
  • the TILs expanded in vitro by the present application can be made to contact
  • the second stage of in vitro expanded TIL is contacted with the CD28 agonist of the present application.
  • the tumor tissue-derived TILs of the present application that have not been expanded in vitro can be subjected to a first-stage in vitro expansion, a second-stage in vitro expansion, and a third-stage in vitro expansion, and the present application
  • the TIL derived from the tumor tissue of the present application and not expanded in vitro can be contacted with the CD28 agonist of the present application
  • the second stage of in vitro expansion of the present application it can be
  • the TILs expanded in vitro in the first stage of the present application are contacted with the CD28 agonist of the present application
  • the TILs expanded in vitro in the second stage of the present application can be brought into contact with the CD28 agonist of the present application.
  • each stage of in vitro expansion can be divided by the change in the number of TIL cells, in one embodiment, TIL cells can be considered when the number of TIL cells increases by at least about 1-fold Entered the next stage of in vitro expansion. In some embodiments, when the number of TIL cells increases 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 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 have entered the next stage of in vitro expansion.
  • each stage of in vitro expansion may also be demarcated by changes in the conditions of TIL cell culture.
  • TIL cells can be considered to enter the next stage of in vitro expansion when T cell activators and/or T cell growth factors are added or supplemented to the cell culture medium.
  • TIL cells can be considered to have entered the next stage of in vitro expansion.
  • TIL cells can be considered to 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 have entered the next stage of in vitro expansion.
  • each stage can also be divided by the number of days in which the TIL cells are cultured.
  • 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 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, the TIL cells can be considered to have entered the next stage of in vitro expansion.
  • the in vitro expansion in the present application may only comprise a complete culture process.
  • the in vitro expansion of the present application may refer to culturing to produce changes in the number of cells, and the expanded cells may also produce changes in the number and/or ratio of cells, changes in secretion capacity, and changes in killing capacity or changes in expressive power, or any combination of them.
  • the change therein can be an increase or decrease.
  • the in vitro expansion of the present application can be for expansion purposes; in order to detect the function of TIL cells, such as detecting the ability of TIL cells to release cytokines, the manipulation steps performed on TIL cells (such as adding TIL cells to TIL cells) One or more substances are added to the culture medium to detect the ability of TIL cells to release cytokines), which may not belong to the in vitro expansion of this application.
  • the manipulation steps performed on TIL cells such as adding TIL cells to TIL cells
  • One or more substances are added to the culture medium to detect the ability of TIL cells to release cytokines
  • only contacting the TIL of the present application with the CD28 agonist, but not contacting the TIL of the present application with the TIL growth factor may not belong to the in vitro expansion of the present application.
  • the CD28 agonists of the present application may comprise anti-CD28 antibodies and/or antigen-binding fragments thereof.
  • the CD28 agonist of the present application may comprise an anti-CD28 antibody selected from the group consisting of murine antibodies, humanized antibodies, fully human antibodies, and chimeric antibodies.
  • the CD28 agonist of the present application may comprise an antigen-binding fragment of an anti-CD28 antibody selected from the group consisting of Fab, Fab', F(ab) 2 and Fv fragments.
  • the second stage in vitro expansion of the present application can be performed for up to about 13 days.
  • the number of days during which the second-stage in vitro expansion of the present application is performed can be calculated from the start time 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 carried out for about 0 days. For example, about 24 hours after the start of the second-stage in vitro expansion, it can be considered that the second-stage in vitro expansion has been carried out for about one day. For example, on the day when the second-stage in vitro expansion starts, it can be considered that the second-stage in vitro expansion has been carried out for about 0 days.
  • the number of days for the second-stage in vitro expansion of the present application may be calculated by the number of days for the second-stage in vitro expansion.
  • the second-stage in vitro expansion of the present application can be performed for up to about 13 days, up to about 12 days, up to about 11 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, or up to about 1 day.
  • the second stage in vitro expansion of the present application can be performed for about 3 days to about 13 days. In one embodiment, the second stage in vitro expansion of the present application can be performed for about 1 day to about 13 days. For example, the second stage in vitro expansion of the present application can be performed for about 2 days to about 13 days, about 3 days to about 13 days, about 4 days to about 13 days, about 5 days to about 13 days, about 6 days to about 6 days 13 days, about 7 days to about 13 days, about 8 days to about 13 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, or about 12 days to about 13 days sky.
  • the second stage in vitro expansion of the present application can be performed for about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 2 days 7 days, about 2 days to about 8 days, about 2 days to about 9 days, about 2 days to about 10 days, about 2 days to about 11 days, about 2 days to about 12 days, or about 2 days to about 13 days sky.
  • the second stage in vitro expansion of the present application can be performed for about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 3 days 8 days, about 3 days to about 9 days, about 3 days to about 10 days, about 3 days to about 11 days, about 3 days to about 12 days, or about 3 days to about 13 days.
  • the second stage in vitro expansion of the present application can be performed for about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day.
  • the second stage of in vitro expansion of 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 third stage in vitro expansion of the present application can be performed for up to about 13 days.
  • the number of days for the third-stage in vitro expansion of the present application can be calculated from the start of the third-stage in vitro expansion. For example, when the third-stage in vitro expansion starts, it can be considered that the third-stage in vitro expansion has been carried out for about 0 days. For example, it can be considered that the third-stage in vitro expansion has been carried out for about 24 hours after the start of the third-stage in vitro expansion for about 1 day. For example, on the day when the third-stage in vitro expansion starts, it can be considered that the third-stage in vitro expansion has been carried out for about 0 days.
  • the third-stage in vitro expansion of the present application can be performed for up to about 13 days, up to about 12 days, up to about 11 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, or up to about 1 day.
  • the third stage in vitro expansion of the present application can be performed for about 3 days to about 13 days.
  • the third stage in vitro expansion of the present application can be performed for about 1 day to about 13 days.
  • the third stage in vitro expansion of the present application can be carried out for about 2 days to about 13 days, about 3 days to about 13 days, about 4 days to about 13 days, about 5 days to about 13 days, about 6 days to about 6 days 13 days, about 7 days to about 13 days, about 8 days to about 13 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, or about 12 days to about 13 days sky.
  • the third stage in vitro expansion of the present application can be performed for about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 2 days 7 days, about 2 days to about 8 days, about 2 days to about 9 days, about 2 days to about 10 days, about 2 days to about 11 days, about 2 days to about 12 days, or about 2 days to about 13 days sky.
  • the third stage in vitro expansion of the present application can be carried out for about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 3 days 8 days, about 3 days to about 9 days, about 3 days to about 10 days, about 3 days to about 11 days, about 3 days to about 12 days, or about 3 days to about 13 days.
  • the third stage in vitro expansion of the present application can be performed for about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day.
  • the second stage of in vitro expansion of the present application can be considered as the REP (rapid expansion protocol) stage.
  • the third stage of in vitro expansion of the present application can be considered the reREP stage.
  • the method of the present application may further comprise: in at least one stage of the in vitro expansion of the present application, the TIL of the present application may be co-cultured with feeder cells.
  • the TIL of the present application in a single stage of the in vitro expansion of the present application, can be contacted with the CD28 agonist of the present application and co-cultured with the feeder cells of the present application, in one embodiment, the single stage of the present application
  • the in vitro expansion of the application may refer to the in vitro expansion of the application at the same stage, for example, the same in the first stage of the in vitro amplification of the The third stage of in vitro expansion, etc.
  • the TIL of the present application in the first stage in vitro expansion of the present application, can be contacted with the CD28 agonist of the present application and co-cultured with the feeder cells of the present application. In one embodiment, in the second stage in vitro expansion of the present application, the TIL of the present application can be contacted with the CD28 agonist of the present application and co-cultured with the feeder cells of the present application. In one embodiment, in the third stage in vitro expansion of the present application, the TIL of the present application can be contacted with the CD28 agonist of the present application and co-cultured with the feeder cells of the present application.
  • the TIL of the present application in a single stage of the in vitro expansion of the present application, may be contacted with the CD28 agonist of the present application for a certain period of time, and then co-cultured with the feeder cells of the present application.
  • the TIL of the present application in the first stage of in vitro expansion of the present application, may be contacted with the CD28 agonist of the present application for a certain period of time, and then 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, may be contacted with the CD28 agonist of the present application for a certain period of time, and then co-cultured with the feeder cells of the present application.
  • the TIL of the present application in the third stage of the in vitro expansion of the present application, may be contacted with the CD28 agonist of the present application for a certain period of time, and then co-cultured with the feeder cells of the present application.
  • the certain time of the present application may be at least about 2 hours. In one embodiment, the certain 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 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 About 72 hours.
  • the certain period of time of the present application may be from about 6 hours to about 72 hours. In one embodiment, the certain time of the present application may 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 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 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, 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 time of the present application may 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 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 may 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. In one embodiment, 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. In one embodiment, the feeder cells of the present application may be peripheral mononuclear cells. In one embodiment, the feeder cells of the present application may be irradiated feeder cells.
  • the feeder cells of the present application may be isolated artificial antigen-presenting cells (aAPCs), and the artificial antigen-presenting cells of the present application may 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.
  • co-culturing the TIL of the present application with 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, co-culturing the TILs of the present application with the feeder cells of the present application comprises adding the feeder cells of the present application to the cell culture medium of the TILs of the present application.
  • the feeder cells of the present application may be added to the cell culture medium of the TILs of the present application at a ratio of the feeder cells of the present application to the TIL of the present application at a ratio of about 40:1 to about 400:1.
  • the present application may be at about 40:1 to about 400:1, at about 40:1 to about 300:1, at about 40:1 to about 200:1, at 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, at about 90:1 to about 400:1, at about 100:1 to about 400:1, at about 200:1 to about 400:1, or at about 300:1 to about 400:1 of the present application
  • the methods of the present application may further comprise: in at least one stage of the in vitro expansion of the present application, contacting the TIL of the present application with one or more T cell growth factors.
  • a TIL of the present application can be contacted with a CD28 agonist 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 the in vitro expansion of the present application, can be contacted with the CD28 agonist of the present application and with one or more T cell growth factors of the present application.
  • the TIL of the present application in the second stage of the in vitro expansion of the present application, can be contacted with the CD28 agonist of the present application and with one or more T cell growth factors of the present application.
  • the TIL of the present application in the third stage in vitro expansion of the present application, can be contacted with the CD28 agonist of the present application and with one or more T cell growth factors of the present application.
  • the TIL of the present application is contacted with the CD28 agonist of the present application and the one or more T cell growth factors of the present application at substantially the same time in a single stage of the in vitro expansion of the present application.
  • the TIL of the application in the first stage of the in vitro expansion of the application, is contacted with the CD28 agonist of the application and one or more T cell growth factors of the application at substantially the same time.
  • the TIL of the application is contacted with the CD28 agonist of the application and the one or more T cell growth factors of the application at substantially the same time.
  • the TIL of the application is contacted with the CD28 agonist of the application and one or more T cell growth factors of the application at substantially the same time.
  • the T cell growth factor of the present application may be selected from one or more of the following group: IL-2, IL-7, IL-12, IL-15, IL-21, gamma interferon, and their functionally active fragments.
  • the T cell growth factors of the present application may comprise IL-2 and/or functionally active fragments thereof.
  • functionally active fragments of IL-2 can comprise fragments of IL-2 known in the art that can bind to the IL-2 receptor of T cells.
  • contacting a TIL of the present application with one or more T cell growth factors of the present application may comprise adding the T cell growth factors 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 may be at least about 300 IU/mL.
  • the initial concentration of the IL-2 of the present application in the cell culture medium of the TIL of the present application may be at least about 350 IU/mL, at least about 400 IU/mL, at least about 500 IU/mL, at least about 600 IU/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 1500IU/mL, at least about 2000IU/mL, at least about 2500IU/mL, at least about 2600IU/mL, at least about 2700IU/mL, at least about 2800IU/mL, at least about 2900IU/mL, at least about 3000IU/mL, at least about 3100IU/mL mL, at least about 3200 IU/
  • a TIL of the present application that has been contacted with a CD28 agonist of the present application during at least one phase of in vitro expansion may exhibit a Improved amplification.
  • the corresponding TIL that has not been contacted with the CD28 agonist of the present application may refer to TIL cells derived from the same donor and that have not been contacted with the CD28 agonist of the present application.
  • the corresponding TIL that has not been contacted with the CD28 agonist of the present application may refer to TIL cells derived from the same donor that have been isolated in the same manner and have not been contacted with the CD28 agonist of the present application.
  • the corresponding TILs that have not been contacted with the CD28 agonists of the present application may refer to TIL cells derived from the same tumor source from the same donor and have not been contacted with the CD28 agonists of the present application. In one embodiment, the corresponding TILs that have not been contacted with the CD28 agonists of the present application may refer to TIL cells from the same tumor source from the same donor that have been isolated in the same manner and have not been contacted with the CD28 agonists of the present application.
  • the corresponding TILs that have not been in contact with the CD28 agonists of the present application may refer to dividing TIL cells derived from the same donor into two groups, wherein one group of TIL cells that have not been in contact with the CD28 agonists of the present application may be are the corresponding TILs that have not been in contact with the CD28 agonists of the present application. In one embodiment, the corresponding TIL that has not been in contact with the CD28 agonist of the present application may refer to dividing TIL cells from the same donor and isolated in the same way into two groups, one of which has not been contacted with the CD28 agonist of the present application.
  • the contacted TIL cells can be corresponding TILs that have not been contacted with the CD28 agonist of the present application.
  • the corresponding TILs that have not been in contact with the CD28 agonist of the present application may refer to dividing TIL cells derived from the same tumor from the same donor into two groups, one of which has not been in contact with the CD28 agonist of the present application.
  • the TIL cells may be corresponding TILs that have not been contacted with the CD28 agonist of the present application.
  • the corresponding TILs that have not been in contact with the CD28 agonist of the present application may refer to dividing TIL cells from the same tumor source from the same donor and isolated in the same manner into two groups, one of which has not been contacted with the present application.
  • the TIL cells to which the CD28 agonist of the present application is contacted may be the corresponding TIL that has not been contacted with the CD28 agonist of the present application.
  • the improved expansion effect of the present application may comprise one or more selected from the group consisting of increased number of TIL cells, improved proportion of T cell subsets, increased cytokine secretion capacity, and Enhanced tumor cell killing ability.
  • the increased number of TIL cells of the present application may refer to the increase in the number of TIL cells of the present application in at least one in vitro expansion phase compared to the corresponding TIL of the present application that has not been contacted with the CD28 agonist of the present application during the in vitro expansion phase.
  • the number of cells of the TIL of the present application that the agonist has contacted 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 number of TIL cells of the present application may refer to the increase in the number of TIL cells of the present application in at least one in vitro expansion phase compared to the corresponding TIL of the present application that has not been contacted with the CD28 agonist of the present application during the in vitro expansion phase.
  • the number of cells of the TIL of the present application to which the agonist has been exposed 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 cytokine secretion capacity of the present application may refer to increased cytokine secretion capacity of TIL cells selected from the group consisting of CD107a, GZMB, IL-2, TNF and IFN ⁇ . In one embodiment, the increased cytokine secretion capacity of the present application may refer to the difference between the present application and the The cytokine secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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-fold , 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 capacity of the present application may refer to the difference between the present application and the The cytokine secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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 cytokine secretion capacity of the present application may refer to the difference between the present application and the The CD107a secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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-fold, 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 capacity of the present application may refer to the difference between the present application and the The CD107a secretory capacity of the TILs of the present application to which the CD28 agonist has been exposed 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 cytokine secretion capacity of the present application may refer to the difference between the present application and the The GZMB secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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-fold, 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 capacity of the present application may refer to the difference between the present application and the The GZMB secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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 cytokine secretion capacity of the present application may refer to the difference between the present application and the The IL-2 secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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-fold 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 times, at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion capacity of the present application may refer to the difference between the present application and the The IL-2 secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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 cytokine secretion capacity of the present application may refer to the difference between the present application and the The TNF secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed can be increased 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 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 capacity of the present application may refer to the difference between the present application and the The TNF secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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 cytokine secretion capacity of the present application may refer to the difference between the present application and the The IFN ⁇ secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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-fold, 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 capacity of the present application may refer to the difference between the present application and the The IFN ⁇ secretion capacity of the TILs of the present application to which the CD28 agonist has been exposed 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 determination of the cytokine secretion capacity of the TILs of the present application may be by measuring the cytokine expression capacity of TIL cells. In one embodiment, the cytokine secretion capacity of the TILs of the present application is determined by measuring the cytokine release capacity of TIL cells. In one embodiment, the cytokine secretion capacity of the TIL of the present application is determined by the CBA method (Cytometric Bead Array).
  • the increased tumor cell killing ability of the present application may refer to the difference between the present application and the The tumor cell killing rate of the TILs of the present application to which the CD28 agonist has been exposed 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-fold , 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 tumor cell killing ability of the present application may refer to the difference between the present application and the
  • the tumor cell killing rate of a CD28 agonist-exposed TIL of the present application 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 tumor cell killing rate of the TILs of the present application can be measured by CFSE and DAPI staining.
  • tumor cell killing of TILs of the present application may refer to the ability of TILs to kill solid tumor cells.
  • the tumor cell killing of TILs of the present application may refer to the ability of TILs to kill cervical cancer cells.
  • the tumor cell killing of TILs of the present application may refer to the ability of TILs to kill HeLa cells.
  • the improved proportion of T cell subsets of the present application may comprise one or more selected from the group consisting of: increased proportion of central memory T cells, decreased proportion of regulatory T cells, increased activation T-cell ratio, increased tumor-specific T-cell ratio, and increased stem-like T-cell ratio.
  • the increased proportion of central memory T cells of the present application may be an increase in the proportion of CD45RA - CCR7 + and/or CD45RO + CD62L + cells in TIL cells.
  • the proportion of central memory T cells in TIL 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.
  • the reduced proportion of regulatory T cells of the present application may be an increase in the proportion of CD4 + CD25 + Foxp3 + cells in TIL cells.
  • the proportion of regulatory T cells in TIL 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 increased proportion of activated T cells of the present application may be an increase in the proportion of CD25 + , CD28 + , PD1 + or 41BB + cells in TIL cells.
  • the proportion of activated T cells in TIL 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
  • the proportion of CD25 + cells in TIL 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 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 times, at least about 6 times, or can
  • the proportion of CD28 + cells in TIL 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 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 times, at least about 6 times, or can
  • the proportion of PD1 + cells in TIL 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 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 times, at least about 6 times, at
  • the proportion of 41BB + cells in TIL 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 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 times, at least about 6 times, or can
  • the TIL of the present application that has been contacted with the CD28 agonist of the present application during at least one in vitro expansion phase is compared to the corresponding TIL of the present application that has not been contacted with the CD28 agonist of the present application during the in vitro expansion phase Can exhibit improved gene editing effects.
  • the improved gene editing effects of the present application may comprise increased gene knockout efficiency.
  • the TIL of the present application that has been contacted with the CD28 agonist of the present application during at least one in vitro expansion phase is compared to the corresponding TIL of the present application that has not been contacted with the CD28 agonist of the present application during the in vitro expansion phase
  • the gene knockout efficiency 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%
  • the method of the present application may further comprise: in at least one stage of the in vitro expansion of the present application, combining the TIL of the present application with one or more other T cell activators other than the CD28 agonist of the present application touch.
  • other T cell activators may comprise agonists of one or more targets selected from the group consisting of CD3, HVEM, CD40L, OX40 and 4-1BB.
  • the TIL of the present application is contacted with the CD28 agonist of the present application and with one or more other T cell activators of the present application.
  • the TILs of the present application can be contacted with the CD28 agonists of the present application and with one or more other T cell activators of the present application in the first stage of in vitro expansion of the present application.
  • the TILs of the present application can be contacted with the CD28 agonists of the present application and with one or more other T cell activators of the present application in the second stage of in vitro expansion of the present application.
  • the TIL of the present application can be contacted with the CD28 agonist of the present application and contacted with one or more other T cell activators of the present application in the third stage of in vitro expansion of the present application.
  • the TIL of the present application can be contacted with the CD28 agonist of the present application and one or more other T cell activators of the present application in a single stage of the present application's in vitro expansion at substantially the same time .
  • the TILs of the present application can be contacted with the CD28 agonists of the present application and one or more other T cell activators of the present application at substantially the same time in the first stage of the in vitro expansion of the present application.
  • the TIL of the present application can be contacted with the CD28 agonist of the present application and one or more other T cell activators of the present application at substantially the same time in the second stage of the in vitro expansion of the present application.
  • the TIL of the present application can be contacted with the CD28 agonist of the present application and one or more other T cell activators of the present application at substantially the same time in the third stage of the in vitro expansion of the present application.
  • other T cell activators of the present application may comprise one or more selected from the group consisting of CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, and their functionally active fragments.
  • other T cell activators of the present application may comprise agonists of one or more targets selected from the group consisting of CD3, HVEM, CD40L, OX40 and 4-1BB.
  • other T cell activators of the present application may comprise antibodies selected from the group consisting of CD3, HVEM, CD40L, OX40, and 4-1BB, and antigen-binding fragments thereof.
  • other T cell activators of the present application may comprise CD3 agonists.
  • other T cell activators of the present application may comprise anti-CD3 antibodies and/or antigen-binding fragments thereof, such as may be OKT3 from Miltenyi Biotech.
  • contacting a TIL of the present application with a CD28 agonist of the present application and with one or more other T cell activators of the present application may comprise one or more means selected from the group consisting of: (1) adding the CD28 agonist of the present application and other T cell activators of the present application to the cell culture medium of the TIL of the present application; (2) activating the expression of the CD28 agonist of the present application and other T cell activators of the present application
  • the engineered cells of the present application are added to the cell culture medium of the TIL of the present application; (3) the solid phase medium comprising the CD28 agonist of the present application and other T cell activators of the present application is added to the cell culture of the TIL of the present application. base.
  • contacting a TIL of the present application with a CD28 agonist of the present application and contacting one or more other T cell activators of the present application may comprise contacting a CD28 agonist of the present application with a CD28 agonist of the present application Solid phase media of other T cell activators are added to the cell culture medium of the TILs of the present application.
  • contacting a TIL of the present application with a CD28 agonist of the present application and contacting one or more other T cell activators of the present application may comprise contacting a solid comprising a CD28 antibody of the present application and a CD3 antibody of the present application Phase medium is added to the cell culture medium of the TILs of this application.
  • the initial concentration of the other T cell activator in the cell culture medium of the TIL 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 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 the TIL of the present application can be at least about 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 30 ng/mL to about 300 ng/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 30 ng/mL to about 300 ng/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 medium of the present application may have a diameter of about 500 nanometers to about 10 micrometers.
  • the diameter of the solid phase medium of the present application can be measured by transmission electron microscopy.
  • the solid phase medium of the present application may have a diameter of about 1 nanometer to about 500 nanometers.
  • the solid phase medium of the present application may have a diameter of about 100 nanometers to about 500 nanometers.
  • the solid phase medium of the present application may have a diameter of about 200 nanometers to about 500 nanometers.
  • the diameter of the solid phase medium of the present application can be measured by transmission electron microscopy.
  • 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.
  • the solid phase medium of the present application comprises at least about 25 ⁇ g of the CD28 agonist of the present application and other T cell activators of the present application per mg.
  • the solid phase medium comprising the CD28 agonist of the present application and other T cell activators of the present application is added at a ratio of the solid phase medium of the present application to the TIL of the present application at a ratio of about 1:100 to about 1:2000. into the cell culture medium of the TIL of the present application.
  • the solid phase medium comprising the CD28 agonist of the present application and other T cell activators of the present application is added in a ratio of about 2:1 to about 1:2 of the present application solid phase medium to the present application TIL into the cell culture medium of the TIL of the present application.
  • the CD28 of the present application may be contained in a ratio of the solid phase medium of the present application to the TIL of the present application in a ratio of about 2:1 to about 1:2.
  • Solid phase media of agonists and other T cell activators of the present application are added to the cell culture medium of the TILs 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 may be about 2:1 to about 1:2, about 2:1 to about 1:1, or about 1:1 - a ratio of about 1:2 of the solid phase medium of the application to the TIL of the application, the solid phase medium comprising the CD28 agonist of the application and other T-cell activators of the application, such as CD3 agonists, is added to the cells of the TIL of the application in the culture medium.
  • the CD28 of the present application may be contained in the ratio of the solid phase medium of the present application to the TIL of the present application in a ratio of about 1:100 to about 1:2000.
  • Solid phase media of agonists and other T cell activators of the present application are added to the cell culture medium of the TILs 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, at about 1:1600-about 1:2000, or at a ratio of about 1:1800-about 1:2000 of the application's solid phase medium to the application's TIL, the application's CD28 agonist and the application's other TILs will be included
  • a solid-phase medium of a cell activator such as a CD3 agonist, is added to the cell culture medium of the TIL of the present application.
  • the CD3 agonist of the present application may be a CD3 antibody or an antigen binding protein thereof.
  • the antibody or antigen-binding protein thereof of the present application comprises at least one CDR in the VH of the variable region of the antibody heavy chain.
  • the application CDRs may be defined according to IMGT nomenclature, the application CDRs may be defined according to Chothia, or the application CDRs 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 NOs: 1 and 11; the CDRs 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 proteins 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 NOs: 2 and 12; the CDRs 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 proteins 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 NOs: 3 and 13; the CDRs 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 proteins of the present application may have CD3 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 any one of SEQ ID NOs: 1 and 11, and the HCDR2 of the present application may comprise SEQ ID NO: 2
  • the amino acid sequence shown in any one of and 12, and the application HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 3 and 13;
  • the application CDRs may be defined according to the IMGT nomenclature;
  • the application CDRs Can be as defined by Kabat; for example, an antigen binding protein of the present application can have CD3 binding capacity.
  • 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: 1, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 2
  • the amino acid sequence of HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 3; the CDRs 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: 11, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 12
  • the amino acid sequence of HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 13; 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 or antigen-binding protein thereof of the present application comprises at least one CDR in the variable region VL of the antibody light chain.
  • the present CDRs may be defined according to IMGT nomenclature, or the present CDRs may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1, and LCDR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 4 and 14;
  • the CDRs 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 proteins of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR2, and LCDR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 5 and 15;
  • the present application CDRs 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 proteins of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise LCDR3, and LCDR3 of the present application can comprise the amino acid sequence shown in any one of SEQ ID NOs: 6 and 16;
  • the CDRs of the present application 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 proteins 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 NOs: 4 and 14, and the LCDR2 of the present application may comprise SEQ ID NO: 5
  • the amino acid sequence shown in any one of and 15, and the application LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 6 and 16;
  • the application CDRs may be defined according to the IMGT nomenclature;
  • the application CDRs Can be as defined by Kabat; for example, an antigen binding protein of the present application can have CD3 binding capacity.
  • 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: 4, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 5
  • the amino acid sequence of , and LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 6; the CDRs 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 LCDR1-3 as SP34, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 14, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 15
  • the amino acid sequence of , and LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 16; 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 HCDR1-3 and LCDR1-3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1 and 11, and the HCDR2 of the present application may comprise SEQ ID NO: 1 and 11.
  • the HCDR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 3 and 13
  • the LCDR1 of the present application may comprise the amino acid sequence of SEQ ID NO: 4 and 14
  • the amino acid sequence shown in any one, LCDR2 of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 5 and 15, and LCDR3 of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 6 and 16.
  • the amino acid sequence shown; the CDRs 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 proteins 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 and LCDR1-3 as OKT3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 1, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: : the amino acid sequence shown in 2, the application HCDR3 can include the amino acid sequence shown in SEQ ID NO: 3, the application LCDR1 can include the amino acid sequence shown in SEQ ID NO: 4, the application LCDR2 can include SEQ ID NO: 5
  • the amino acid sequence shown, and LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 6; the CDRs 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: 11, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 12
  • the amino acid sequence of the present application, HCDR3 of the present application can include the amino acid sequence shown in SEQ ID NO: 13, LCDR1 of the present application can include the amino acid sequence shown in SEQ ID NO: 14, and LCDR2 of the present application can include the amino acid sequence shown in SEQ ID NO: 15 Sequence, and LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 16;
  • 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 or antigen-binding protein thereof of the present application 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 NOs: 7 and 17; 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: 7; 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: 17; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody or antigen-binding protein thereof of the present application 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 NOs: 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 VL as OKT3, and the VL 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 or antigen-binding protein of the present application 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: 18; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody or antigen-binding protein thereof of the present application may comprise a heavy chain variable region VH and a light chain variable region VL, and the present application VH may comprise any one of SEQ ID NOs: 7 and 17.
  • the amino acid sequence shown, the VL of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 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 and VL as OKT3, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 7, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 8
  • the amino acid sequence of ; 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: 17, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 18
  • the amino acid sequence of for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody or antigen-binding protein thereof of the present application may comprise a heavy chain, and the heavy chain of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 9 and 19;
  • the antigen binding protein may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same heavy chain as OKT3, and the heavy chain 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 heavy chain as SP34, and the heavy chain 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 or antigen-binding protein thereof of the present application may comprise a light chain, and the light chain of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 10 and 20;
  • the antigen binding protein may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same light chain as OKT3, and the light chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 10; 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 light chain as SP34, and the light chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 20; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody or antigen-binding protein thereof of the present application 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 NOs: 9 and 19, the present application
  • the light chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 10 and 20; 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 heavy chain and light chain as OKT3, and the heavy chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 9, and the light chain of the present application may comprise SEQ ID NO: : the amino acid sequence shown in 10; 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 heavy chain and light chain as SP34, and the heavy chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 19, and the light chain of the present application may comprise SEQ ID NO: : the amino acid sequence shown in 20; 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 or antigen-binding protein thereof of the present application comprises at least one CDR in the VH of the variable region of the antibody heavy chain.
  • the present CDRs may be defined according to IMGT nomenclature, or the present CDRs 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: 21; the CDRs of the present application may be defined according to the IMGT nomenclature; the CDRs of the present application may be defined according to As defined by Kabat; for example, an antigen binding protein of the present application may have CD28 binding capacity.
  • 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: 22; the CDRs of the present application may be defined according to the IMGT nomenclature; the CDRs of the present application may be defined according to As defined by Kabat; for example, an antigen binding protein of the present application may have CD28 binding capacity.
  • 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: 23; the CDRs of the present application may be defined according to the IMGT nomenclature; the CDRs of the present application may be defined according to As defined by Kabat; for example, an antigen binding protein of the present application may have CD28 binding capacity.
  • 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: 21, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22, And the HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 23;
  • the CDRs 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 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: 21, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22
  • the amino acid sequence of HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 23; the CDRs 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 or antigen-binding protein thereof of the present application comprises at least one CDR in the variable region VL of the antibody light chain.
  • the present CDRs may be defined according to IMGT nomenclature, or the present CDRs may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1, and LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 24; the CDRs of the present application may be defined according to the IMGT nomenclature; the CDRs of the present application may be defined according to As defined by Kabat; for example, an antigen binding protein of the present application may have CD28 binding capacity.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR2, and LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 25; the CDRs of the present application may be defined according to the IMGT nomenclature; the CDRs of the present application may be defined according to As defined by Kabat; for example, an antigen binding protein of the present application may have CD28 binding capacity.
  • 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: 26; the CDRs of the present application may be defined according to the IMGT nomenclature; the CDRs of the present application may be defined according to As defined by Kabat; for example, an 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 LCDR1-3, wherein LCDR1 of the present application can comprise the amino acid sequence shown in SEQ ID NO: 24, and LCDR2 of the present application can comprise the amino acid sequence shown in SEQ ID NO: 25, And LCDR3 of the present application can comprise the amino acid sequence shown in SEQ ID NO: 26; CDRs of the present application can be defined according to IMGT nomenclature; CDRs of the present application can be defined according to Kabat; 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 the same LCDR1-3 as 15E8, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 24, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 25
  • the amino acid sequence of the present application, and LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 26; the CDRs 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 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: 21, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22
  • the amino acid sequence of HCDR3 of the present application can include the amino acid sequence shown in SEQ ID NO: 23
  • LCDR1 of the present application can include the amino acid sequence shown in SEQ ID NO: 24
  • LCDR2 of the present application can include the amino acid sequence shown in SEQ ID NO: 25 Sequence
  • LCDR3 of the present application can comprise the amino acid sequence shown in SEQ ID NO: 26
  • the CDRs of the present application can be defined according to the IMGT nomenclature
  • the CDRs of the present application can be defined according to Kabat
  • the antigen-binding proteins of the present application can be Has CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same HCDR1-3 and LCDR1-3 as 15E8, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 21, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: : the amino acid sequence shown in 22, the application HCDR3 can include the amino acid sequence shown in SEQ ID NO: 23, the application LCDR1 can include the amino acid sequence shown in SEQ ID NO: 24, the application LCDR2 can include SEQ ID NO: 25
  • the amino acid sequence shown, and LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 26; the CDRs 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 or antigen-binding protein thereof of the present application 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 NOs: 27 and 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 may comprise the same VH as a 15E8, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 27; 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 as another 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 or antigen-binding protein of the present application 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: 29; for example, the antigen-binding protein of the present application Can have CD28 binding ability.
  • the antibody of the present application or the antigen-binding protein thereof 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: 29; for example, the antigen-binding protein of the present application may have CD28 binding ability.
  • the antibody or antigen-binding protein thereof of the present application may comprise a heavy chain variable region VH and a light chain variable region VL, and the application VH may comprise any one of SEQ ID NOs: 27 and 28.
  • the amino acid sequence shown, the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 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 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: 27, and the VL of the present application may comprise SEQ ID NO: 29
  • 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: 28, and the VL of the present application may comprise SEQ ID NO: 29; for example, the antigen-binding protein of the present application may have CD28-binding ability.
  • the antibody or antigen-binding protein thereof of the present application may comprise a heavy chain, and the heavy chain of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 30 and 31;
  • the antigen binding protein may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same heavy chain as a 15E8, and the heavy chain 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 capacity.
  • the antibody of the present application or the antigen-binding protein thereof may comprise the same heavy chain as another 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 Has CD28 binding ability.
  • the antibody or antigen-binding protein of the present application may comprise a light chain, and the light chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 32; 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 light chain as 15E8, and the light chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 32; for example, the antigen-binding protein of the present application may have CD28 binding ability.
  • the antibody or antigen-binding protein thereof of the present application 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 NOs: 30 and 31, the present application
  • the light chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 32 and 20; 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: 30, and the light chain of the present application may comprise SEQ ID NO: 30
  • the amino acid sequence shown in ID NO: 32; 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: 31, and the light chain of the present application may comprise The amino acid sequence shown in SEQ ID NO: 32; for example, the antigen-binding protein of the present application may have CD28 binding ability.
  • the TILs of the present application may be TILs derived from fragments of tumor tissue of the present application. In one embodiment, the TILs of the present application can be obtained by processing tumor tissue into tumor fragments. In one embodiment, the tumor fragments of the present application are about 1-27 cubic millimeters in volume.
  • 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 mm3, about 9 mm3, about 10 mm3, about 11 mm3, about 12 mm3, about 13 mm3, about 15 mm3, about 17 mm3, about 19 mm3, about 20 mm3 , about 21 mm3, about 23 mm3, about 24 mm3, about 25 mm3, about 26 mm3 or 27 mm3.
  • the application provides a method for culturing tumor-infiltrating lymphocytes (TILs), which may comprise: (A) contacting a first population of TILs derived from tumor tissue and not expanded in vitro with IL-2; wherein, Obtaining the second TIL cell population through the steps (A); (B) contacting the second TIL population of the present application with the CD28 agonist and IL-2 at substantially the same time.
  • TILs tumor-infiltrating lymphocytes
  • the application provides a method for culturing tumor-infiltrating lymphocytes (TILs), which may comprise: (A) contacting a first population of TILs derived from tumor tissue and not expanded in vitro with IL-2; wherein, The second TIL cell population is obtained through the step (A); (B) the second TIL population of the present application is contacted with the CD28 agonist and IL-2 at the same time, and after a certain period of time, it is then co-cultured with the feeder cells.
  • TILs tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TILs), which may comprise: (A) subjecting a first TIL population derived from tumor tissue and not expanded in vitro to a concentration of 300-9000 IU/mL wherein, the second TIL cell population is obtained through the step (A); (B) the second TIL population of the present application is substantially at the same time with CD28 agonist, CD3 antibody and a concentration of 300-9000IU/ mL of IL-2, and after a certain period of time, co-culture with PBMC cells at a ratio of feeder cells to the TIL of about 40:1 to about 400:1.
  • TILs tumor-infiltrating lymphocytes
  • the present application provides a method of culturing tumor-infiltrating lymphocytes (TILs).
  • TILs tumor-infiltrating lymphocytes
  • the method for obtaining TIL cells from the subject's tissue sample can be that the patient obtains an in situ tumor sample or a metastatic tumor sample through surgery, and the weight can be at least about 1 g, or multiple pieces of tissue can be combined.
  • Tumor tissue is transported in a sample transport solution, such as a commercially commonly used tumor tissue transport solution, tumor tissue preservation solution or tumor tissue transport solution, at about 2-8 degrees, and processed within 48 hours.
  • Tissue pieces can be mechanically disrupted to a size of approximately 1-27 mm3 each, transferred into a gas permeable culture bag or Grex, and added with T cell serum-free medium at a concentration of 300-9000 IU/mL (for example, it can be 1000-9000 IU/mL, For example, 6000 IU/mL) of IL-2 can be cultured for about 3-14 days.
  • the cells in the culture medium can be collected and transferred together with the tissue pieces into a gas permeable culture bag, or Grex, or Xuri equipment.
  • the T cell serum-free medium can be supplemented with the CD28 antibody, CD3 antibody and CD28 antibody of the application, including CD3 antibody and CD28 antibody.
  • Magnetic beads e.g.
  • Dynabeads and/or nanomatrices comprising CD3 antibody and CD28 antibody and IL at a concentration of 300-9000 IU/mL (e.g. can be 1000-9000 IU/mL, e.g. can be 6000 IU/mL) -2, after activating the application for a certain period of time, add irradiated PBMC (TIL and PBMC in a ratio of about 1:40 to about 1:400), and expand and culture for about 3-14 days. After filtering the tissue block, the cells in the culture medium can be collected, washed, frozen, and tested using a cell processing system.
  • irradiated PBMC TIL and PBMC in a ratio of about 1:40 to about 1:400
  • the CD3 ratio of the final product can be greater than 80%, the cell viability can be greater than 70%, and the T cells greater than 80% can be memory effector T cells and effector T cells. IFNy may be secreted upon stimulation, and/or may 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 culture method of the present application.
  • TIL tumor-infiltrating lymphocyte
  • the TIL provided by the present application may comprise one or one batch of the culturing method of the present application to obtain TIL.
  • the TILs provided by the present application may comprise multiple or multiple batches of TILs cultured by the culturing 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 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 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.
  • the tumor is a melanoma
  • from about 2.3 x 109 to about 13.7 x 1010 TILs can be administered.
  • about 1 ⁇ 10 9 to about 12 ⁇ 10 10 TILs can be administered.
  • about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 TILs can be administered.
  • about 3 ⁇ 10 10 to about 12 ⁇ 10 10 TILs can be administered.
  • about 4 ⁇ 10 10 to about 10 ⁇ 10 10 TILs can be administered.
  • about 5 ⁇ 10 10 to about 8 ⁇ 10 10 TILs can be administered.
  • about 6 ⁇ 10 10 to about 8 ⁇ 10 10 TILs can be administered. In some embodiments, about 7 ⁇ 10 10 to about 8 ⁇ 10 10 TILs can be administered. In some embodiments, the therapeutically effective dose may be from about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 . In some embodiments, a therapeutically effective dose may be from about 1 x 109 to about 12 x 1010 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, a therapeutically effective dose may be from about 4 ⁇ 10 10 to about 10 ⁇ 10 10 TILs.
  • the therapeutically effective dose may be from about 5 ⁇ 10 10 to about 8 ⁇ 10 10 TILs. In some embodiments, a therapeutically effective dose can 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 compositions 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 , approx. 7 ⁇ 10 6 , approx. 8 ⁇ 10 6 , approx. 9 ⁇ 10 6 , approx. 1 ⁇ 10 7 , approx. 2 ⁇ 10 7 , approx. 3 ⁇ 10 7 , approx. 4 ⁇ 10 7 , approx. 5 ⁇ 10 7 , approx. 6 ⁇ 10 7 , approx. 7 ⁇ 10 7 , approx. 8 ⁇ 10 7 , approx. 9 ⁇ 10 7 , approx. 1 ⁇ 10 8 , approx. 2 ⁇ 10 8 , approx.
  • 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 , approx. 5 ⁇ 10 7 to 1 ⁇ 10 8 , approx. 1 ⁇ 10 8 to 5 ⁇ 10 8 , approx. 5 ⁇ 10 8 to 1 ⁇ 10 9 , approx.
  • the concentration of TIL provided in the compositions of the present application may be less than, eg, 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%,
  • 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 TILs provided in the compositions of the present application may be provided in concentrations ranging 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 TILs provided in the compositions of the present application may be provided in concentrations ranging 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 from 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 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.
  • the amount of TIL provided in the compositions of the present application can 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,
  • the TIL can be administered in a single dose. Such administration can be by injection, eg, intravenous injection. In some embodiments, the TIL can be administered in multiple doses. The dosage can be one, two, three, four, five, six or more than six times per year. Dosing can be once a month, once every two weeks, once a week or once every 2 days. In some embodiments, the administration of TIL can be continuous.
  • the application provides a pharmaceutical composition.
  • it may comprise a TIL of the present application and/or a composition of the present application, together with a pharmaceutically acceptable carrier.
  • the present application provides a kit, which can include T cell activators, T cell growth factors and/or feeder cells used in the method for culturing tumor-infiltrating lymphocytes (TILs) of the present application and a method for culturing tumors described in the present application. Instructions for the steps of the infiltrating lymphocyte (TIL) method.
  • the present application provides a kit, and the kit of the present application may comprise the TIL of the present application and/or the pharmaceutical composition of the present application.
  • the present application provides a method of affecting tumor cell growth, which can include administering to a subject a TIL of the present application and/or a pharmaceutical composition of the present application.
  • affecting tumor growth can comprise a reduction in 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 the volume before administration %, 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 % or about 0.1%.
  • 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 a medicament, and the medicament of the present application can be used for the prevention and/or treatment of tumors.
  • the tumors of the present application are selected from solid tumors.
  • the tumor of the present application can 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, Rectal cancer, and kidney cancer.
  • the present application provides a method of 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 tumors of the present application are selected from solid tumors.
  • the tumor of the present application can 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, Rectal cancer, and kidney cancer.
  • the present application provides a TIL of the present application and/or a pharmaceutical composition of the present application, which can be used for preventing and/or treating tumors.
  • the tumors of the present application are selected from solid tumors.
  • the tumor of the present application can 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, Rectal cancer, and kidney cancer.
  • TIL tumor infiltrating lymphocyte
  • PBMC peripheral blood mononuclear cells
  • erythrocytes can be split after centrifugation. Add erythrocyte lysate at 1:2 to 1:3 of the cell sediment volume and erythrocyte lysate, mix well, lyse at room temperature for 10 minutes, and gently mix the centrifuge tube for 2- 3 times to ensure the lysis effect. After the lysis is completed, add PBS or normal saline to wash the cells. After splitting, the cells were washed twice, centrifuged at 400g for 6 minutes, and the samples were counted before the last centrifugation.
  • a pre-concentration step can be performed to concentrate the blood volume to within 120mL.
  • the neatcell program can be used to separate and wash PBMC.
  • the washing solution is normal saline, and the intermediate volume is 20 mL; the resuspended solution is the basal medium, and 80 mL/batch is added.
  • each donor PBMC is a 100mL bag. In the flat state, the liquid level can be no more than 1 cm, and the X-ray irradiation is 50Gy.
  • the PBMC cells in the blood samples were isolated and cryopreserved according to the above procedures for manual isolation and cryopreservation of PBMC.
  • a culture flask and bag with a gas permeable surface such as a G-Rex100 culture flask (Wilson Wolf Manufacturing)
  • 300 mL of rewarmed complete medium the complete medium can optionally be X-vivo 15 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 add a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/mL) mL, for example can be 6000 IU/mL) of IL-2.
  • preREP stage The first stage of in vitro expansion and harvest
  • the medium is replenished or half-changed every 3-7 days to ensure cell nutrition.
  • Use complete medium X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium, can be optionally added to the complete medium.
  • samples can be counted on the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th or 14th day, if the number of cells is 5 ⁇ 10 5 When it reaches 5 ⁇ 10 8 , the following first-stage in vitro amplification harvesting step is entered.
  • X-vivo 15 medium or other commercial T cell medium such as Stem
  • T cell medium such as Stem
  • Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added to adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 cells/mL, suspended in a 24-well culture plate , 1mL/well, add IL-2 at a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/mL, for example, it can be 6000IU/mL). While adding IL-2 to each experimental group, the corresponding CD28 agonist was added according to the following formula:
  • Control group CD3 antibody, such as OKT3 about 30ng/mL, was added to the medium at the same time.
  • Mixed antibody group add CD3 antibody and CD28 antibody to the medium at the same time, for example, about 30ng/mL of OKT3 and about 30ng/mL of CD28 antibody.
  • Magnetic bead group Add magnetic beads (about 1 to 10 ⁇ m in diameter, Dynabeads, Thermo Fisher) containing CD3 antibody and CD28 antibody to the medium at the same time, for example, at a ratio of magnetic beads to TIL of about 1:2-2:1. beads.
  • Nanomatrix group add transACT (about 100 to 500 nm in diameter, Miltenyi) containing CD3 antibody and CD28 antibody in the culture medium at the same time, for example, add transACT at a ratio of transACT to TIL of about 1:100-1:2000.
  • Tn can be 0 hours to 14 days, such as 24 hours or 48 hours
  • Tn can be 0 hours to 14 days, such as 24 hours or 48 hours
  • Resuscitate the feeder cells mixed by 1-5 donors transfer the activated TIL cells, tissue blocks and feeder cells into G-Rex100 culture flasks or gas-permeable bags, supplement complete medium, take samples every 1-3 days and count them according to The cell state was supplemented with liquid or half volume of medium was exchanged until the total number of cells was greater than 1 ⁇ 10 9 or the second stage of in vitro expansion culture for 13 days, and then the culture was terminated.
  • TIL third TIL population expanded in vitro in the second stage.
  • Samples were counted during the three washings. According to the counting results, the supernatant was discarded after the last centrifugation, and 3 ⁇ 10 6 cells were taken for quality control testing; all the remaining cells were added to the freezing solution, and the cell density was adjusted to 1-3 ⁇ 10 8 cells/ mL cryopreserved.
  • Resuscitated therapeutic tumor-infiltrating lymphocytes can be administered to the subject by intravenous infusion.
  • FIG. 1 The proliferation ability analysis of the experimental group and the control group added with different forms of CD28 agonists is shown in FIG. 1 .
  • the numerical value of the ordinate in Figure 1 indicates that the number of TIL cells in the third TIL population obtained by the second stage of in vitro expansion in each test group was compared to the second TIL population before the start of the second stage of in vitro expansion. multiple.
  • the results showed that for Donor A, the proliferation of TILs obtained by adding CD28 antibody in the second stage of in vitro expansion was stronger than that of TILs cultured in the control group (without adding CD28 antibody).
  • 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-through antibodies in this example were purchased from BD or Biolegend. Add 1 x 10 5 to 5 x 10 5 cell samples per group into a flow tube or V-bottom 96-well plate. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, flow tube 1mL/tube, 96-well plate 250 ⁇ L/well, discard supernatant. Add the prepared antibody working solution for cell surface staining. The concentration of the antibody (BD or Biolegend) is 1:100 to 1:200, and the activity detection dye is 1:10000. Flow tube 100 ⁇ L/tube, 50 ⁇ L/well of 96-well plate for staining, and incubate at 2-8°C for 30 minutes in the dark.
  • BD Transcription Factor Buffer Set
  • BD Transcription Factor Buffer Set
  • BD Fixation/Permeabilization
  • Working Solution A use double distilled water to dilute 5 ⁇ permeation washing solution (BD, Perm/Wash Buffer) is 1 ⁇ working solution B, pre-cooled at four degrees for use.
  • an appropriate amount of PBS was added to wash the cells twice (250 ⁇ L/time for a 96-well plate, 1 mL/time for a flow tube), centrifuged at 600 g for 3 minutes, and the supernatant was discarded after centrifugation.
  • Cell fixation and membrane rupture Sufficiently resuspend the cells, add an appropriate amount (100 ⁇ L/well of 96-well plate, 1 mL/tube of flow 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 the membrane was fixed and ruptured, 1 ⁇ working solution B was added to wash the cells (250 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuged at 2-8°C, 350g for 6 minutes, and washed 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 at 2-8 °C for 30 minutes in the dark. After staining, 1 ⁇ working solution B was added to wash the cells (250 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuged at 2-8°C for 6 minutes at 350 g, and washed twice.
  • cells were washed once with PBS (250 ⁇ L/time for 96-well plate, 1 mL/time for flow tube), centrifuged at 600 g for 3 minutes at room temperature, and the supernatant was discarded after centrifugation. Resuspend the cells in 100-500 ⁇ L PBS for flow cytometry.
  • Fig. 2 shows the ratio of T cell subsets of TIL cells cultured in the mixed antibody group and the control group for donor B-1.
  • the results showed that the addition of CD28 antibody in the second stage of in vitro expansion resulted in an improved proportion of T cell subsets in TILs compared to the control group (without the addition of CD28 antibody).
  • higher proportion of activated T cells CD28 + or 41BB +
  • lower proportion of regulatory T cells Treg, eg CD4 + CD25 + Foxp3 +
  • TCF1 + stem-like T cells
  • Tcm central memory T cells
  • Fig. 3 shows the ratio of T cell subsets of TIL cells cultured in the mixed antibody group and the control group for donor B-2.
  • the results showed that the addition of CD28 antibody in the second stage of in vitro expansion resulted in an improved proportion of T cell subsets in TILs compared to the control group (without the addition of CD28 antibody).
  • a higher proportion of tumor-specific T cells CD103 + CD39 +
  • a higher proportion of activated T cells CD25 +
  • Reg regulatory T cells
  • Fig. 4 shows the ratio of T cell subsets of TIL cells cultured in the magnetic bead group and the control group for donor C-1.
  • the results showed that the addition of CD28 antibody in the second stage of in vitro expansion (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had an improved proportion of T cell subsets.
  • a higher proportion of activated T cells CD28 + , PD1 + or 41BB +
  • TCF1 + stem-like T cells
  • Tcm central memory T cells
  • Fig. 5 shows the ratio of T cell subsets of TIL cells cultured in the magnetic bead group and the control group for donor C-2.
  • the results showed that the addition of CD28 antibody in the second stage of in vitro expansion (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had an improved proportion of T cell subsets.
  • TCF1 + stem-like T cells
  • 41BB + activated T cells
  • Tcm central memory T cells
  • Treg eg CD4 + CD25 + Foxp3 +
  • tumor specific T cells CD103 + CD39 +
  • FIG. 6 shows the proportion of T cell subsets of TIL cells cultured in the nanomatrix group and the control group for Donor D.
  • FIG. 6 The results showed that the addition of CD28 antibody in the second stage of in vitro expansion (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without the addition of CD28 antibody), the obtained TIL had an improved proportion of T cell subsets.
  • a higher proportion of tumor-specific T cells CD103 + CD39 +
  • a higher proportion of activated T cells CD25 + or PD1 +
  • Tcm central memory T cells
  • the cell killing ability was detected for the third TIL population obtained by the second-stage in vitro expansion culture of each test group in Example 1.
  • TIL obtained for each experimental group for assay and target cells (eg, Hela tumor cells) for co-culture.
  • target cells eg, 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 PBS resuspension to a final concentration of CFSE of 0.5 ⁇ mol/L. After incubation at 37°C for 6 minutes, add medium containing 10% FBS to wash, centrifuge at 600g for 5 minutes, and use X-vivo 15 medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi, etc.
  • Killing rate % Dapi + CFSE + cell number/total CFSE + ⁇ 100%.
  • FIG. 7 shows the cell killing ability of TIL cells cultured in the nanomatrix group and the control group for Donor E.
  • FIG. 7 shows the cell killing ability of TIL cells cultured in the nanomatrix group and the control group for Donor E.
  • FIG. 7 shows the cell killing ability of TIL cells cultured in the nanomatrix group and the control group for Donor E.
  • FIG. 7 shows the cell killing ability of TIL cells cultured in the nanomatrix group and the control group for Donor E.
  • FIG. 7 shows the cell killing ability of TIL cells cultured in the nanomatrix group and the control group for Donor E.
  • FIG. 8 shows the results of the detection of intracellular factor expression in TIL cells cultured in the mixed antibody group and the control group for donor F.
  • FIG. The results showed that the addition of CD28 antibody in the second stage of in vitro expansion, compared with the control group (without the addition of CD28 antibody), the obtained TIL had higher intracellular factor expression ability. For example, higher CD107a expression capacity.
  • FIG. 9 shows the results of the detection of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group for donor G-1.
  • the results showed that the addition of CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody) in the second stage of in vitro expansion, compared with the control group (without adding CD28 antibody), the obtained TIL had a higher ability to express intracellular factors. For example, higher CD107a expression capacity.
  • Fig. 10 shows the results of the detection of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group for donor G-2.
  • the results showed that the addition of CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody) in the second stage of in vitro expansion, compared with the control group (without adding CD28 antibody), the obtained TIL had a higher ability to express intracellular factors. For example, higher CD107a expression capacity.
  • Fig. 11 shows the results of the detection of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group for donor G-3.
  • the results showed that the addition of CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody) in the second stage of in vitro expansion, compared with the control group (without adding CD28 antibody), the obtained TIL had a higher ability to express intracellular factors. For example, higher CD107a expression capacity.
  • FIG. 12 shows the results of the detection of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group for donor G-4.
  • the results showed that the addition of CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody) in the second stage of in vitro expansion, compared with the control group (without adding CD28 antibody), the obtained TIL had a higher ability to express intracellular factors. For example, higher CD107a expression capacity.
  • FIG. 13 shows the results of the detection of intracellular factor expression in TIL cells cultured in the nanomatrix group and the control group for Donor H.
  • FIG. The results showed that the addition of CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody) in the second stage of in vitro expansion, compared with the control group (without adding CD28 antibody), the obtained TIL had higher intracellular factor expression ability. For example, higher CD107a expression ability, higher IFN ⁇ expression ability, or higher GZMB expression ability.
  • Cytokine secretion detection was performed on the third TIL population obtained by the second-stage in vitro expansion culture of each test group in Example 1.
  • BD Human Th1/Th2/Th17 cytokine standard lyophilized powder
  • 2mL Assay Diluent Diluent (BD) the concentration of each cytokine in the standard solution was 5000pg/mL
  • 1:2, 1 :4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 serial dilution then mixed with Capture Beads (BD) and detected by PE Detection Reagent Reagents (BD) were mixed and transferred to a 15mL conical bottom centrifuge tube, labeled "Standard Tube". Take 1 tube containing Assay Diluent Diluent only as a negative control.
  • Use Assay Diluent to dilute the third TIL population obtained in the second-stage in vitro expansion culture of each test group add 10 ⁇ L/well to the plate, and then mix with each Capture Beads (BD) at 2 ⁇ L/Beads/well, and 10 ⁇ L/well The wells were mixed with PE Detection Reagent (BD) and incubated for 3 hours at room temperature in the dark.
  • BD Capture Beads
  • FIG. 14 shows the results of cytokine secretion detection of TIL cells cultured in the nanomatrix group and the control group for donor I-1.
  • the results showed that the addition of CD28 antibody in the second stage of in vitro expansion (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.
  • TIL obtained from each test group was incubated with tumor cells overnight according to the method in Example 4 (TIL cell killing ability detection).
  • FIG. 15 shows the results of cytokine secretion detection after co-incubation with tumor cells of TIL cells cultured in the nanomatrix group and the control group for donor I-2.
  • the results showed that the addition of CD28 antibody in the second stage of in vitro expansion (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.
  • TIL cells of each test group in Example 1 after 48 hours of in vitro expansion and culture in the second stage were used to detect the gene knockout efficiency.
  • Nuclease-free water (commercial source: Shanghai Youfan Biotechnology Co., Ltd.; RT121-02) was used to prepare sgRNA (sequence shown in SEQ ID NO: 33, GGAGAATGACGAGTGGACCC), and the concentration was adjusted to 50 ⁇ mol/L.
  • sgRNA sequence shown in SEQ ID NO: 33, GGAGAATGACGAGTGGACCC
  • concentration was adjusted to 50 ⁇ mol/L.
  • sgRNA:P3Buffer:Cas9 nuclease 2:2:1
  • P3Buffer commercial source: Lonza; V4XP-3032
  • 61.7 ⁇ mol/L Cas9 nuclease commercial source: Suzhou
  • T cell culture medium at 1 mL/well and place in a CO 2 incubator to preheat. Take the TIL cells of each test group in Example 1 after 48 hours of in vitro expansion and culture in the second stage, and count them after mixing. Take 5 ⁇ 10 5 cells from each test group and add P3Buffer (20 ⁇ L), and mix the cells evenly. ; The cells were added to a new PCR tube and mixed with 5 ⁇ L of RNP; the mixture of cells and RNP was added to the electroporation plate and electroporated (human T cell stimulated (E0115)) in an electroporator (Lonza).
  • the TIL cells of each test group after culture were taken for cell counting, 2 ⁇ 10 5 cells were taken from each test group, centrifuged at 500 g for 3 minutes, and the supernatant was discarded after centrifugation.
  • FIG. 16 shows the results of gene knockout efficiency after co-incubation of TIL cells cultured with the nanomatrix group and the control group with tumor cells for donor J-1.
  • the results show that the addition of CD28 antibody in the second stage of in vitro expansion (eg, adding transACT containing CD3 antibody and CD28 antibody), the resulting TIL has improved gene knockout efficiency compared to the control group (without CD28 antibody). For example, increased TCR ⁇ gene knockout efficiency.
  • FIG. 17 shows the results of gene knockout efficiency after co-incubation of TIL cells cultured with the nanomatrix group and the control group with tumor cells for donor J-2.
  • the results show that the addition of CD28 antibody in the second stage of in vitro expansion (eg, adding transACT containing CD3 antibody and CD28 antibody), the resulting TIL has improved gene knockout efficiency compared to the control group (without CD28 antibody). For example, increased TCR ⁇ gene knockout efficiency.
  • the first TIL population was subjected to the first stage in vitro expansion (preREP) in the same manner and the second stage in vitro expansion in the same manner ( REP) to obtain a third TIL population.
  • the third TIL group was randomly divided into 3 groups.
  • IL-2 was added 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 did not add CD3 antibody about 30ng/mL.
  • the CD28 agonist group was added with a CD3 agonist and a CD28 agonist, for example, transACT was added at a ratio of transACT to TIL of about 1:100-1:2000.
  • the TILs (fourth cell population) obtained by culturing for 3 days were tested for the proliferation ability of TIL cells by the cell viability detection method using the CellTiter-Glo kit (commercial source: Promega).
  • FIG. 18 The fluorescence value of the ordinate in FIG. 18 reflects the proliferation ability of TIL cells expanded in vitro in the third stage in different ways in each test group.
  • the results showed that for donors K-1, K-2 and K-3, the third-stage in vitro expansion with the addition of a CD28 agonist had similar TIL proliferative capacity compared with the third-stage expansion without the addition of a CD28 agonist.
  • a nano-sized CD28 agonist can be an agonist comprising a CD3 antibody and a CD28 antibody about 100 nm in diameter;
  • Nanomatrix A can be transACT (about 100 to 500 nm in diameter, Miltenyi) and Nanomatrix B can be CytoSinct ( About 100 nm in diameter, GENSCRIPT, eg, added at a ratio of transACT to TIL of about 1:100 to 1:1000).
  • FIG. 19A Analysis of the proliferative capacity of the experimental groups to which different forms of CD28 agonists were added is shown in Figure 19A.
  • the value of the ordinate in the figure represents the expansion fold of the number of TIL cells in the third TIL population obtained by the second stage of in vitro expansion in each experimental group compared with the second TIL population before the second stage of in vitro expansion. .
  • the results show that different forms of nano-sized CD28 agonists can have the effect of enhancing the proliferation ability of TILs.
  • Figure 19B shows that different forms of nano-sized CD28 agonists have the ability to increase the proportion of tumor-specific T cells (CD103 + CD39 + ).
  • Figures 19C-19F show that different forms of nano-sized CD28 agonists can have the ability to reduce the proportion of exhausted T cells (CD39 + , PD1 + , LAG3 + , TIM3 + ).
  • Figure 19G shows that different forms of nano-sized CD28 agonists have the ability to reduce the proportion of regulatory T cells (Treg).
  • Figures 19H-19J show that different forms of nano-sized CD28 agonists have the ability to increase the proportion of activated T cells (CD25 + , 41BB + , CD28 + ).
  • Figure 19K shows that different forms of nano-sized CD28 agonists have the ability to increase the proportion of stem-like T cells (CD69 - CD39- ).
  • Figure 19L shows that different forms of nano-sized CD28 agonists can have the ability to increase the ratio of central memory T cells (Tcm, eg CD45RO + CD62L + ).
  • Figure 19M shows that different forms of nano-sized CD28 agonists were added to the second-stage in vitro expansion culture, and the expression ratio of intracellular factor (CD107a) was detected by flow cytometry. The results show that different forms of nano-sized CD28 agonists can increase the expression of intracellular factors in TIL cells.
  • Figure 19N shows that different forms of nano-sized CD28 agonists were added to the second-stage in vitro expansion culture, and the expression ratio of intracellular factor (CD107a) was detected by flow cytometry. Antibodies were incubated overnight. The results show that different forms of nano-sized CD28 agonists can increase the expression of intracellular factors in TIL cells.
  • Figure 19O shows the detection results of cytokine secretion in the test groups added with different forms of CD28 agonists.
  • Figure 190 shows that different forms of nano-sized CD28 agonists were added to the second-stage in vitro expansion culture, and the cytokine secretion ability of TIL cells was verified by cytokine secretion detection, wherein the TIL cells in the CD3 antibody stimulation group were in cells Incubate with CD3 antibody overnight before factor detection. The results show that different forms of nano-sized CD28 agonists can enhance the cytokine secretion ability of TIL cells.

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Abstract

提供了肿瘤浸润淋巴细胞的制备方法及其用途，具体涉及一种培养肿瘤浸润淋巴细胞的方法，其包含：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，在至少一个阶段的所述体外扩增中，使所述TIL与CD28激动剂接触。还提供了一种肿瘤浸润淋巴细胞预防和/或治疗肿瘤的方法。

Description

肿瘤浸润淋巴细胞的制备方法及其用途 技术领域
本申请涉及生物医药领域，具体涉及肿瘤浸润淋巴细胞的制备方法及其用途。
背景技术
使用过继性自体转移肿瘤浸润淋巴细胞治疗肿瘤是一种治疗预后不良患者的有效方法。但是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。因此如何提供一种稳健可靠的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
本申请提供了一种肿瘤浸润淋巴细胞的培养方法，本申请的培养方法获得的肿瘤浸润淋巴细胞具有选自以下组的一种或多种的效果：增加的TIL细胞数量，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，改善的T细胞亚群比例，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，增加的干细胞样T细胞比例，和改善的基因编辑效果。
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，在至少一个阶段的所述体外扩增中，使所述TIL与CD28激动剂接触。
在一种实施方式中，使所述源自肿瘤组织且未经体外扩增的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与所述CD28激动剂接触。
在一种实施方式中，所述CD28激动剂包含抗CD28的抗体和/或其抗原结合片段。
在一种实施方式中，所述第二阶段体外扩增进行至多约13天。
在一种实施方式中，所述第二阶段体外扩增进行约3天至约13天。
在一种实施方式中，本申请的培养方法还包含：在至少一个阶段的所述体外扩增中，使所述TIL与饲养细胞共培养。
在一种实施方式中，其中，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激 动剂接触且与所述饲养细胞共培养。
在一种实施方式中，其中，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂接触一定时间之后，再与所述饲养细胞共培养。
在一种实施方式中，所述一定时间为至少约2小时。
在一种实施方式中，所述一定时间为约6小时至约72小时。
在一种实施方式中，所述一定时间为约12小时至约48小时。
在一种实施方式中，所述一定时间为约6小时、约12小时、约24小时、约48小时、或约72小时。
在一种实施方式中，所述饲养细胞包含抗原呈递细胞。
在一种实施方式中，所述饲养细胞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，所述饲养细胞为外周单个核细胞。
在一种实施方式中，所述饲养细胞为经过辐照的饲养细胞。
在一种实施方式中，所述TIL与所述饲养细胞共培养包含使所述饲养细胞的表面与所述TIL的表面相接触。
在一种实施方式中，所述TIL与所述饲养细胞共培养包含将所述饲养细胞添加至所述TIL的细胞培养基中。
在一种实施方式中，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。
在一种实施方式中，本申请的培养方法还包含：在至少一个阶段的所述体外扩增中，使所述TIL与一种或多种T细胞生长因子接触。
在一种实施方式中，其中，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂接触且与所述一种或多种T细胞生长因子接触。
在一种实施方式中，其中，在单个阶段的所述体外扩增中，使所述TIL基本上同时与所述CD28激动剂以及所述一种或多种T细胞生长因子接触。
在一种实施方式中，所述T细胞生长因子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，所述T细胞生长因子包含IL-2和/或其功能活性片段。
在一种实施方式中，所述TIL与所述一种或多种T细胞生长因子接触包含将所述T细胞生长因子添加至所述TIL的细胞培养基中。
在一种实施方式中，所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度为至少约300IU/mL。
在一种实施方式中，与在体外扩增阶段未曾与所述CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与所述CD28激动剂接触过的所述TIL显示出改善的扩增效果。
在一种实施方式中，所述改善的扩增效果包含选自以下组的一种或多种：增加的TIL细胞数量，改善的T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。
在一种实施方式中，所述改善的T细胞亚群比例包含选自以下组的一种或多种：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，与在体外扩增阶段未曾与所述CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与所述CD28激动剂接触过的所述TIL显示出改善的基因编辑效果。
在一种实施方式中，所述改善的基因编辑效果包含提高的基因敲除效率。
在一种实施方式中，本申请的培养方法还包含：在至少一个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂之外的一种或多种其他T细胞激活剂接触。
在一种实施方式中，其中，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂接触且与所述一种或多种其它T细胞激活剂接触。
在一种实施方式中，其中，在单个阶段的所述体外扩增中，使所述TIL基本上同时与所述CD28激动剂以及所述一种或多种其它T细胞激活剂接触。
在一种实施方式中，所述其它T细胞激活剂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，所述其它T细胞激活剂包含选自以下组的一种或多种靶点的激动剂：CD3、HVEM、CD40L、OX40和4-1BB。
在一种实施方式中，所述其它T细胞激活剂包含CD3激动剂。
在一种实施方式中，所述其它T细胞激活剂包含抗CD3的抗体和/或其抗原结合片段。
在一种实施方式中，所述使TIL与所述CD28激动剂接触且与所述一种或多种其它T细胞激活剂接触包含选自以下组的一种或多种方式：(1)将所述CD28激动剂与所述其它T细胞激活剂添加至所述TIL的细胞培养基中；(2)将表达所述CD28激动剂与所述其它T细胞激活剂的工程化细胞添加至所述TIL的细胞培养基中；(3)将包含所述CD28激动剂与所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
在一种实施方式中，所述其它T细胞激活剂在所述TIL的细胞培养基中的初始浓度为至少约30ng/mL。
在一种实施方式中，所述其它T细胞激活剂在所述TIL的细胞培养基中的初始浓度为约30ng/mL-约300ng/mL。
在一种实施方式中，所述固相介质的直径为约500纳米至约10微米。
在一种实施方式中，所述固相介质的直径为约1纳米至约500纳米。
在一种实施方式中，所述固相介质的直径通过透射电子显微镜测量。
在一种实施方式中，所述固相介质包含聚合物。
在一种实施方式中，每mg所述固相介质包含至少约25μg的所述CD28激动剂和所述其它T细胞激活剂。
在一种实施方式中，以约2:1-约1:2的所述固相介质与所述TIL的比例，将包含所述CD28激动剂和所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
在一种实施方式中，以约1:100-约1:2000的所述固相介质与所述TIL的比例，将包含所述CD28激动剂和所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
在一种实施方式中，所述源自肿瘤组织且未经体外扩增的TIL为源自所述肿瘤组织的碎片的TIL。
在一种实施方式中，所述碎片的体积为约1立方毫米至约27立方毫米。
一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
(A)使源自肿瘤组织且未经体外扩增的第一TIL群与一种或多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL细胞群；
(B)使所述第二TIL群与CD28激动剂接触。
在一种实施方式中，所述CD28激动剂包含抗CD28的抗体和/或其抗原结合片段。
在一种实施方式中，所述步骤(B)进行至多约13天。
在一种实施方式中，所述步骤(B)进行约3天至约13天。
在一种实施方式中，本申请的培养方法还包含：所述步骤(A)和/或所述步骤(B)中，使所述TIL与饲养细胞共培养。
在一种实施方式中，其中，在所述步骤(B)中，使所述第二TIL群与所述饲养细胞共培养。
在一种实施方式中，其中，在所述步骤(B)中，使所述第二TIL群与所述CD28激动剂接触一定时间之后，再与所述饲养细胞共培养。
在一种实施方式中，所述一定时间为至少约2小时。
在一种实施方式中，所述一定时间为约6小时至约72小时。
在一种实施方式中，所述一定时间为约12小时至约48小时。
在一种实施方式中，所述一定时间为约6小时、约12小时、约24小时、约48小时、或约72小时。
在一种实施方式中，所述饲养细胞包含抗原呈递细胞。
在一种实施方式中，所述饲养细胞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，所述饲养细胞为外周单个核细胞。
在一种实施方式中，所述饲养细胞为经过辐照的饲养细胞。
在一种实施方式中，所述TIL与所述饲养细胞共培养包含使所述饲养细胞的表面与所述TIL的表面相接触。
在一种实施方式中，所述TIL与所述饲养细胞共培养包含将所述饲养细胞添加至所述TIL的细胞培养基中。
在一种实施方式中，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。
在一种实施方式中，本申请的培养方法还包含：在所述步骤(A)和/或所述步骤(B)中，使所述TIL与一种或多种T细胞生长因子接触。
在一种实施方式中，其中，在所述步骤(B)中，使所述第二TIL群与所述一种或多种T细胞生长因子接触。
在一种实施方式中，其中，在所述步骤(B)中，使所述第二TIL群基本上同时与所述CD28激动剂以及所述一种或多种T细胞生长因子接触。
在一种实施方式中，所述T细胞生长因子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，所述T细胞生长因子包含IL-2和/或其功能活性片段。
在一种实施方式中，所述TIL与所述一种或多种T细胞生长因子接触包含将所述T细胞生长因子添加至所述TIL的细胞培养基中。
在一种实施方式中，所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度为至少约300IU/mL。
在一种实施方式中，与在所述步骤(B)中未曾与所述CD28激动剂接触的相应TIL相 比，在所述步骤(B)中与所述CD28激动剂接触过的所述TIL显示出改善的扩增效果。
在一种实施方式中，所述改善的扩增效果包含选自以下组的一种或多种：增加的TIL细胞数量，改善的T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。
在一种实施方式中，所述改善的T细胞亚群比例包含选自以下组的一种或多种：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，与在所述步骤(B)中未曾与所述CD28激动剂接触的相应TIL相比，在所述步骤(B)中与所述CD28激动剂接触过的所述TIL显示出改善的基因编辑效果。
在一种实施方式中，所述改善的基因编辑效果包含提高的基因敲除效率。
在一种实施方式中，本申请的培养方法还包含：在所述步骤(A)和/或所述步骤(B)中，使所述TIL与所述CD28激动剂之外的一种或多种其他T细胞激活剂接触。
在一种实施方式中，其中，在所述步骤(B)中，使所述第二TIL群与所述CD28激动剂接触且与所述一种或多种其它T细胞激活剂接触。
在一种实施方式中，其中，在所述步骤(B)中，使所述第二TIL群基本上同时与所述CD28激动剂以及所述一种或多种其它T细胞激活剂接触。
在一种实施方式中，所述其它T细胞激活剂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，所述其它T细胞激活剂包含选自以下组的一种或多种靶点的激动剂：CD3、HVEM、CD40L、OX40和4-1BB。
在一种实施方式中，所述其它T细胞激活剂包含CD3激动剂。
在一种实施方式中，所述其它T细胞激活剂包含抗CD3的抗体和/或其抗原结合片段。
在一种实施方式中，所述使TIL与所述CD28激动剂接触且与所述一种或多种其它T细胞激活剂接触包含选自以下组的一种或多种方式：(1)将所述CD28激动剂与所述其它T细胞激活剂添加至所述TIL的细胞培养基中；(2)将表达所述CD28激动剂与所述其它T细胞激活剂的工程化细胞添加至所述TIL的细胞培养基中；(3)将包含所述CD28激动剂与所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
在一种实施方式中，所述其它T细胞激活剂在所述TIL的细胞培养基中的初始浓度为至少约30ng/mL。
在一种实施方式中，所述其它T细胞激活剂在所述TIL的细胞培养基中的初始浓度为约30ng/mL-约300ng/mL。
在一种实施方式中，所述固相介质的直径为约500纳米至约10微米。
在一种实施方式中，所述固相介质的直径为约1纳米至约500纳米。
在一种实施方式中，所述固相介质的直径通过透射电子显微镜测量。
在一种实施方式中，所述固相介质包含聚合物。
在一种实施方式中，每mg所述固相介质包含至少约25μg的所述CD28激动剂和所述其它T细胞激活剂。
在一种实施方式中，以约2:1-约1:2的所述固相介质与所述TIL的比例，将包含所述CD28激动剂和所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
在一种实施方式中，以约1:100-约1:2000的所述固相介质与所述TIL的比例，将包含所述CD28激动剂和所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
在一种实施方式中，所述源自肿瘤组织且未经体外扩增的TIL为源自所述肿瘤组织的碎片的TIL。
在一种实施方式中，所述碎片的体积为约1立方毫米至约27立方毫米。
一方面，本申请提供一种肿瘤浸润淋巴细胞(TIL)，所述TIL经过本申请的方法获得。
一方面，本申请提供一种组合物，其包含本申请的TIL。
一方面，本申请提供一种药物组合物，其包含本申请的TIL和/或本申请的组合物，以及任选地药学上可接受的载体。
一方面，本申请提供一种影响肿瘤细胞生长的方法，包含向受试者施用本申请的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，所述药物用于预防和/或治疗肿瘤。
在一种实施方式中，所述肿瘤为实体瘤。
在一种实施方式中，所述肿瘤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。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是对于供者A，第二阶段体外扩增添加CD28抗体的混合抗体组与不添加CD28抗体的对照组，获得的TIL的增殖能力的结果。
图2显示的是对于供者B-1，第二阶段体外扩增添加CD28抗体的混合抗体组与不添加CD28抗体的对照组，获得的TIL细胞的T细胞亚群比例的结果。
图3显示的是对于供者B-2，第二阶段体外扩增添加CD28抗体的混合抗体组与不添加CD28抗体的对照组，获得的TIL细胞的T细胞亚群比例的结果。
图4显示的是对于供者C-1，第二阶段体外扩增添加CD28抗体的磁珠组与不添加CD28抗体的对照组，获得的TIL细胞的T细胞亚群比例的结果。
图5显示的是对于供者C-2，第二阶段体外扩增添加CD28抗体的磁珠组与对不添加CD28抗体的照组，获得的TIL细胞的T细胞亚群比例的结果。
图6显示的是对于供者D，第二阶段体外扩增添加CD28抗体的纳米基质组与不添加CD28抗体的对照组，获得的TIL细胞的T细胞亚群比例的结果。
图7显示的是对于供者E，第二阶段体外扩增添加CD28抗体的纳米基质组与不添加CD28抗体的对照组，获得的TIL细胞的细胞杀伤能力的结果。
图8显示的是对于供者F，第二阶段体外扩增添加CD28抗体的混合抗体组与不添加CD28抗体的对照组培养所得的TIL细胞的胞内因子表达检测结果。
图9显示的是对于供者G-1，第二阶段体外扩增添加CD28抗体的磁珠组与不添加CD28抗体的对照组培养所得的TIL细胞的胞内因子表达检测结果。
图10显示的是对于供者G-2，第二阶段体外扩增添加CD28抗体的磁珠组与不添加CD28抗体的对照组培养所得的TIL细胞的胞内因子表达检测结果。
图11显示的是对于供者G-3，第二阶段体外扩增添加CD28抗体的磁珠组与不添加CD28抗体的对照组培养所得的TIL细胞的胞内因子表达检测结果。
图12显示的是对于供者G-4，第二阶段体外扩增添加CD28抗体的磁珠组与不添加CD28抗体的对照组培养所得的TIL细胞的胞内因子表达检测结果。
图13显示的是对于供者H，第二阶段体外扩增添加CD28抗体的纳米基质组与不添加CD28抗体的对照组培养所得的TIL细胞的胞内因子表达检测结果。
图14显示的是对于供者I-1，第二阶段体外扩增添加CD28抗体的纳米基质组与不添加CD28抗体的对照组培养所得的TIL细胞的细胞因子分泌检测结果。
图15显示的是对于供者I-2，第二阶段体外扩增添加CD28抗体的纳米基质组与不添加CD28抗体的对照组培养所得的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16显示的是对于供者J-1，第二阶段体外扩增添加CD28抗体的纳米基质组与不添加CD28抗体的对照组培养所得的TIL细胞与肿瘤细胞共同孵育后的基因敲除效率结果。
图17显示的是对于供者J-2，第二阶段体外扩增添加CD28抗体的纳米基质组与不添加CD28抗体的对照组培养所得的TIL细胞与肿瘤细胞共同孵育后的基因敲除效率结果。
图18显示的是对于供者K-1、K-2和K-3，第三阶段体外扩增添加CD28激动剂与第三阶段扩增不添加CD28激动剂相比的TIL增殖能力结果。
图19A显示的是添加不同形式的CD28激动剂的试验组的TIL增殖能力。图19B显示的是，不同形式的纳米尺寸的CD28激动剂的试验组的TIL中肿瘤特异性T细胞(CD103 +CD39 +)比例。图19C-19F显示的是，不同形式的纳米尺寸的CD28激动剂的试验组的TIL中耗竭T细胞(CD39 +、PD1 +、LAG3 +、TIM3 +)比例。图19G显示的是，不同形式的纳米尺寸的CD28激动剂的试验组的TIL中调节性T细胞(Treg)比例。图19H-19J显示的是，不同形式的纳米尺寸的CD28激动剂的试验组的TIL中活化T细胞(CD25 +、41BB +、CD28 +)比例。图19K显示的是，不同形式的纳米尺寸的CD28激动剂都的试验组的TIL中干细胞样T细胞(CD69 -CD39 -)比例。图19L显示的是，不同形式的纳米尺寸的CD28激动剂的试验组的TIL中的中心记忆T细胞(Tcm，例如CD45RO +CD62L +)比例。图19M显示的是，不同形式的纳米尺寸的CD28激动剂的试验组的TIL中胞内因子(CD107a)表达比例。图19N显示的是，不同形式的纳米尺寸的CD28激动剂的试验组的TIL中胞内因子(CD107a)表达比例，流式检测之前各组的TIL细胞与CD3抗体孵育过夜。图19O显示的是，不同形式的纳米尺寸的CD28激动剂的试验组的TIL中细胞因子分泌能力，其中CD3抗体刺激组的TIL细胞在细胞因子检测前与CD3抗体孵育过夜。
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“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(参见Sambrook等，分子克隆：实验手册，Cold Spring Harbor Laboratory Press(1989))。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(参见Sambrook等，同上)。
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。
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。本申请的第二阶段体外扩增也可以称为REP(快速扩增)阶段。
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”通常是指细胞将表达的多肽或蛋白转移到细胞外环境。
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根据本申请的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“抗体”通常是指对指定蛋白质或肽或其片段有反应性的免疫球蛋白。此类抗体包括但不限于人抗体、灵长类化抗体、嵌合抗体、单克隆抗体、单特异性抗体、多克隆抗体、多特异性抗体、非特异性抗体、双特异性抗体、多特异性抗体、人源化抗体、合成抗体、重组抗体、杂合抗体、突变型抗体、嫁接偶联抗体(即偶联或融合至其它蛋白质、放射性标记物、细胞毒素的抗体)、和体外生成的抗体。抗体可来自任何类的抗体，包括但不限于IgG、IgA、IgM、IgD、和IgE，及来自任何亚类(例如IgG1、IgG2、IgG3、和IgG4)的抗体。抗体可具有选自例如IgG1、IgG2、IgG3、或IgG4的重链恒定区。抗体还可具有选自例如卡帕(κ)或拉姆达(λ)的轻链。本申请的抗体可衍生自任何物种，包括但不限于小鼠、人、骆驼、美洲驼、鱼、鲨鱼、山羊、家兔、鸡、和牛。抗体的恒定区可进行改变，例如突变，以修饰抗体的特性(例如以提高或降低下述一项或多项：Fc受体结合、抗体糖基化、半胱氨酸残基的数 目、效应器细胞功能、或补体功能)。通常，抗体特异性结合预定抗原，例如与病症有关的抗原，病症例如炎性的、免疫性的、自身免疫性的、神经变性性的、代谢的、和/或恶性的病症。
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由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication 91-3242)提供，Chothia等人,“Canonical Structures For the Hypervariable Regions of Immunoglobulins,”J.Mol.Biol.196:901(1987)；和MacCallum等人,“Antibody-Antigen Interactions:Contact Analysis and Binding Site Topography,”J.Mol.Biol.262:732(1996))。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OKT-3。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“扩增效果”通常是指细胞经过扩增后出现的效果。扩增效果的变化可以包括，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，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。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)”通常是指体外生长和分泌至少一种因子至培养基并且可以用于支持培养另一种所关注的细胞生长的培养细胞。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抗原，但是基本不识别或结合样品中其它分子的抗体。例如，如果一种抗体可以特异性结合来自一个物种的本申请的特异性抗原，则本申请的抗体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抗原或同源抗原。这种种间反应性本身可以不会改变抗体作为特异性的分类。在某些情形中，特异性结合至抗原的抗体还可以结合至抗原的不同等位形式。
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细胞群。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细胞群。
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 +和/或CD45RO +CD62L +的表型，例如可以是通过CD45RA -和CCR7 +来鉴定中心记忆T细胞。例如可以是通过CD45RO +和CD62L +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PD1 +、LAG3 +或CD28 +的表型，例如可以是通过PD1 +、LAG3 +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。
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。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。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-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)、和γ干扰素(编码其的基因GeneID可以为3458)。
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“基因编辑”通常是指利用一个或多个核酸酶和/或切口酶，将靶DNA(例如细胞的TCRβ基因组)插入、替换或去除DNA的一种基因工程。
在本申请中，术语“基因敲除”通常是指使基因沉默和/或无法表达出其编码的蛋白质的基因工程手段。例如，基因敲除可以是指细胞内或体内基因的定向破坏，导致其功能完全丧失。例如，本申请的基因敲除可以使用位点特异性核酸酶。例如，本申请的基因敲除可以使用锌指核酸酶(ZFN)、TAL效应物核酸酶(TALEN)和/或基于CRISPR/Cas的系统。例如，本申请的基因敲除可以使用CRISPR/Cas9的系统。
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-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细胞群中的比例。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 +和/或CD45RO +CD62L +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 +、PD1 +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肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：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的体外扩增中，可以使本申请的TIL与CD28激动剂接触。
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群与一种或多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL细胞群；(B)使本申请的第二TIL群与CD28激动剂接触。
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群与一种或多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL细胞群；(B)使本申请的第二TIL群与CD28激动剂接触且使第二TIL群与饲养细胞共培养。
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群与一种或多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL细胞群；(B)使本申请的第二TIL群与CD28激动剂以及一种或多种T细胞生长因子接触。
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群与一种或多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL细胞群；(B)使本申请的第二TIL群与CD28激动剂以及一种或多种T细胞生长因子接触一定时间之后，再与本申请的饲养细胞共培养。
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)得到的的第四TIL群任意替换使用。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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，可以使经本申请的第一阶段体外扩增的TIL与本申请的CD28激动剂接触。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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，可以使经本申请的源自肿瘤组织且未经体外扩增的TIL与本申请的CD28激动剂接触。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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，可以使经本申请的源自肿瘤组织且未经体外扩增的TIL与本申请的CD28激动剂接触，且在本申请的第二阶段体外扩增中，可以使经本申请的第一阶段体外扩增的TIL与本申请的CD28激动剂接触。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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，可以使经本申请的源自肿瘤组织且未经体外扩增的TIL与本申请的CD28激动剂接触。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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，可以使经本申请的第一阶段体外扩增的TIL与本申请的CD28激动剂接触。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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，可以使经本申请的第二阶段体外扩增的TIL与本申请的CD28激动剂接触。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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，可以使经本申请的源自肿瘤组织且未经体外扩增的TIL与本申请的CD28激动剂接触，且在本申请的第二阶段体外扩增中，可以使经本申请的第一阶段体外扩增的TIL与本申请的CD28激动剂接触。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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，可以使经本申请的源自肿瘤组织且未经体外扩增的TIL与本申请的CD28激动剂接触，且在本申请的第三阶段体外扩增中，可以使经本申请的第二阶段体外扩增的TIL与本申请的CD28激动剂接触。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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 以使经本申请的第一阶段体外扩增的TIL与本申请的CD28激动剂接触，且在本申请的第三阶段体外扩增中，可以使经本申请的第二阶段体外扩增的TIL与本申请的CD28激动剂接触。
在一种实施方式中，可以使本申请的源自肿瘤组织且未经体外扩增的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，可以使经本申请的源自肿瘤组织且未经体外扩增的TIL与本申请的CD28激动剂接触，且在本申请的第二阶段体外扩增中，可以使经本申请的第一阶段体外扩增的TIL与本申请的CD28激动剂接触，且在本申请的第三阶段体外扩增中，可以使经本申请的第二阶段体外扩增的TIL与本申请的CD28激动剂接触。
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，当细胞培养基中添加了或补充添加了CD28激动剂后，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。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。
在一种实施方式中，本申请中的体外扩增可以只包含完整的培养过程。在一种实施方式中，本申请的体外扩增可以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。其中的变化可以是提高或者降低。在一种实施方式中，在本申请的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细胞释放细胞因子的能力)，可以不属于本申请的体外扩增。例如，在单个阶段的本申请的体外扩增中，仅使本申请的TIL与CD28激动剂接触，而不使本申请的TIL与TIL生长因子接触，可以不属于本申请的体外扩增。例如，在单个阶段的本申请的体外扩增中，仅使本申请的TIL与CD28激动剂接触，而不使本申请的TIL与IL-2接触，可以不属于本申请的体外扩增。
在一种实施方式中，本申请的CD28激动剂可以包含抗CD28的抗体和/或其抗原结合片段。
在一种实施方式中，本申请的CD28激动剂可以包含抗CD28抗体的选自以下组：鼠源抗体、人源化抗体、全人源抗体和嵌合抗体。
在一种实施方式中，本申请的CD28激动剂可以包含抗CD28抗体的抗原结合片段的选自以下组：Fab，Fab’，F(ab) 2和Fv片段。
在一种实施方式中，本申请的第二阶段体外扩增可以进行至多约13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。例如，本申请的第二阶段体外扩增可以进行至多约13天、至多约12天、至多约11天、至多约10天、至多约9天、至多约8天、至多约7天、至多约6天、至多约5天、至多约4天、至多约3天、至多约2天或至多约1天。在一种实施方式中，本申请的第二阶段体外扩增可以进行约3天至约13天。在一种实施方式中，本申请的第二阶段体外扩增可以进行约1天至约13天。例如，本申请的第二阶段体外扩增可以进行约2天至约13天、约3天至约13天、约4天至约13天、约5天至约13天、约6天至约13天、约7天至约13天、约8天至约13天、约9天至约13天、约10天至约13天、约11天至约13天、或约12天至约13天。例如，本申请的第二阶段体外扩增可以进行约2天至约3天、约2天至约4天、约2天至约5天、约2天至约6天、约2天至约7天、约2天至约8天、约2天至约9天、约2天至约10天、约2天至约11天、约2天至约12天、或约2天至约13天。例如，本申请的第二阶 段体外扩增可以进行约3天至约4天、约3天至约5天、约3天至约6天、约3天至约7天、约3天至约8天、约3天至约9天、约3天至约10天、约3天至约11天、约3天至约12天、或约3天至约13天。例如，本申请的第二阶段体外扩增可以进行约13天、约12天、约11天、约10天、约9天、约8天、约7天、约6天、约5天、约4天、约3天、约2天或约1天。在一种实施方式中，本申请的第二阶段体外扩增可以认为是REP(rapid expansion protocol)阶段。在一种实施方式中，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，本申请的第三阶段体外扩增可以进行至多约13天。在一种实施方式中，本申请第三阶段体外扩增进行的天数可以是从第三阶段体外扩增开始进行计算。例如，第三阶段体外扩增开始的当时，可以认为是第三阶段体外扩增进行了约0天。例如，第三阶段体外扩增开始后进行了约24小时，可以认为是第三阶段体外扩增进行了约1天。例如，第三阶段体外扩增开始的当天，可以认为是第三阶段体外扩增进行了约0天。例如，第三阶段体外扩增开始后的第二天，可以认为是第三阶段体外扩增进行了约1天。例如，本申请的第三阶段体外扩增可以进行至多约13天、至多约12天、至多约11天、至多约10天、至多约9天、至多约8天、至多约7天、至多约6天、至多约5天、至多约4天、至多约3天、至多约2天或至多约1天。在一种实施方式中，本申请的第三阶段体外扩增可以进行约3天至约13天。在一种实施方式中，本申请的第三阶段体外扩增可以进行约1天至约13天。例如，本申请的第三阶段体外扩增可以进行约2天至约13天、约3天至约13天、约4天至约13天、约5天至约13天、约6天至约13天、约7天至约13天、约8天至约13天、约9天至约13天、约10天至约13天、约11天至约13天、或约12天至约13天。例如，本申请的第三阶段体外扩增可以进行约2天至约3天、约2天至约4天、约2天至约5天、约2天至约6天、约2天至约7天、约2天至约8天、约2天至约9天、约2天至约10天、约2天至约11天、约2天至约12天、或约2天至约13天。例如，本申请的第三阶段体外扩增可以进行约3天至约4天、约3天至约5天、约3天至约6天、约3天至约7天、约3天至约8天、约3天至约9天、约3天至约10天、约3天至约11天、约3天至约12天、或约3天至约13天。例如，本申请的第三阶段体外扩增可以进行约13天、约12天、约11天、约10天、约9天、约8天、约7天、约6天、约5天、约4天、约3天、约2天或约1天。在一种实施方式中，本申请的第二阶段体外扩增可以认为是REP(rapid expansion protocol)阶段。在一种实施方式中，本申请的第三阶段体外扩增可以认为是reREP阶段。
在一种实施方式中，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，可以使本申请TIL与本申请CD28激动剂接触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，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，本申请的在本申请第一阶段体外扩增中，可以使本申请TIL与本申请CD28激动剂接触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，可以使本申请TIL与本申请CD28激动剂接触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，可以使本申请TIL与本申请CD28激动剂接触且与本申请饲养细胞共培养。
在一种实施方式中，在单个阶段的本申请体外扩增中，可以使本申请TIL与本申请CD28激动剂接触一定时间之后，再与本申请饲养细胞共培养。在一种实施方式中，在本申请第一阶段体外扩增中，可以使本申请TIL与本申请CD28激动剂接触一定时间之后，再与本申请饲养细胞共培养。在一种实施方式中，在本申请第二阶段体外扩增中，可以使本申请TIL与本申请CD28激动剂接触一定时间之后，再与本申请饲养细胞共培养。在一种实施方式中，在本申请第三阶段体外扩增中，可以使本申请TIL与本申请CD28激动剂接触一定时间之后，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：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 CD28激动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CD28激动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CD28激动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CD28激动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，使本申请TIL基本上同时与本申请CD28激动剂以及本申请一种或多种T细胞生长因子接触。例如，在本申请第一阶段体外扩增中，使本申请TIL基本上同时与本申请CD28激动剂以及本申请一种或多种T细胞生长因子接触。例如，在本申请第二阶段体外扩增中，使本申请TIL基本上同时与本申请CD28激动剂以及本申请一种或多种T细胞生长因子接触。例如，在本申请第三阶段体外扩增中，使本申请TIL基本上同时与本申请CD28激动剂以及本申请一种或多种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，与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL可以显示出改善的扩 增效果。在一种实施方式中，未曾与本申请CD28激动剂接触的相应TIL可以是指源自同一供体的且未曾与本申请CD28激动剂接触的TIL细胞。在一种实施方式中，未曾与本申请CD28激动剂接触的相应TIL可以是指源自同一供体的经过同样方式分离的且未曾与本申请CD28激动剂接触的TIL细胞。在一种实施方式中，未曾与本申请CD28激动剂接触的相应TIL可以是指源自同一供体的同一肿瘤来源的且未曾与本申请CD28激动剂接触的TIL细胞。在一种实施方式中，未曾与本申请CD28激动剂接触的相应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与本申请CD28激动剂接触的TIL细胞。在一种实施方式中，未曾与本申请CD28激动剂接触的相应TIL可以是指将源自同一供体的TIL细胞分为两组，其中一组未曾与本申请CD28激动剂接触的TIL细胞可以为未曾与本申请CD28激动剂接触的相应TIL。在一种实施方式中，未曾与本申请CD28激动剂接触的相应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，其中一组未曾与本申请CD28激动剂接触的TIL细胞可以为未曾与本申请CD28激动剂接触的相应TIL。在一种实施方式中，未曾与本申请CD28激动剂接触的相应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，其中一组未曾与本申请CD28激动剂接触的TIL细胞可以为未曾与本申请CD28激动剂接触的相应TIL。在一种实施方式中，未曾与本申请CD28激动剂接触的相应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，其中一组未曾与本申请CD28激动剂接触的TIL细胞可以为未曾与本申请CD28激动剂接触的相应TIL。
在一种实施方式中，本申请的改善的扩增效果可以包含选自以下组的一种或多种：增加的TIL细胞数量，改善的T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。
在一种实施方式中，本申请的增加的TIL细胞数量可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL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。在一种实施方式中，本申请的增加的TIL细胞数量可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的细胞数量可以增加至少约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％。
在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指TIL细胞的选自以下组的细胞因子分泌能力提高：CD107a、GZMB、IL-2、TNF和IFNγ。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的细胞因子分泌能力可以增加至少约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。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的细胞因子分泌能力可以增加至少约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％。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的CD107a分泌能力可以增加至少约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。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的CD107a分泌能力可以增加至少约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％。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的GZMB分泌能力可以增加至少约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。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的GZMB分泌能力可以增加至少约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％。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的IL-2分泌能力可以增加至少约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。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的IL-2分泌能力可以增加至少约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％。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28 激动剂接触过的本申请TIL的TNF分泌能力可以增加至少约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。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的TNF分泌能力可以增加至少约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％。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的IFNγ分泌能力可以增加至少约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。在一种实施方式中，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL的IFNγ分泌能力可以增加至少约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％。在一种实施方式中，本申请的TIL的细胞因子分泌能力的测定可以是通过测量TIL细胞的细胞因子表达能力。在一种实施方式中，本申请的TIL的细胞因子分泌能力通过测量TIL细胞的细胞因子释放能力以测定。在一种实施方式中，本申请的TIL的细胞因子分泌能力是通过CBA法(Cytometric Bead Array)测定。
在一种实施方式中，本申请的提高的肿瘤细胞杀伤能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激 动剂接触过的本申请TIL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。在一种实施方式中，本申请的提高的肿瘤细胞杀伤能力可以是指与在体外扩增阶段未曾与本申请CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请CD28激动剂接触过的本申请TIL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％。在一种实施方式中，本申请的TIL的肿瘤细胞杀伤率可以通过CFSE和DAPI染色法测量。在一种实施方式中，本申请的TIL的肿瘤细胞杀伤可以是指TIL杀伤实体瘤细胞的能力。在一种实施方式中，本申请的TIL的肿瘤细胞杀伤可以是指TIL杀伤宫颈癌细胞的能力。在一种实施方式中，本申请的TIL的肿瘤细胞杀伤可以是指TIL杀伤Hela细胞的能力。
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 +和/或CD45RO +CD62L +细胞的比例的增加。例如，在TIL细胞中中心记忆T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％。
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 +细胞的比例的增加。例如，在TIL细胞中调节性T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％。
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 +、PD1 +或41BB +细胞的比例的增加。例如，在TIL细胞中活化T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TIL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TIL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TIL细胞中PD1 +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TIL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。
在一种实施方式中，与在体外扩增阶段未曾与本申请的CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请的CD28激动剂接触过的本申请的TIL可以显示出改善的基因编辑效果。在一种实施方式中，本申请的改善的基因编辑效果可以包含提高的基因敲除效率。在一种实施方式中，与在体外扩增阶段未曾与本申请的CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与本申请的CD28激动剂接触过的本申请的TIL的基因敲除效率可以增加至少约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％，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。
在一种实施方式中，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TIL与本申请CD28激动剂之外的一种或多种其他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，其它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、HVEM、CD40L、OX40和4-1BB。使本申请的TIL与本申请的CD28激动剂接触且与本申请的一种或多种其它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，可以使本申请的TIL与本申请的CD28激动剂接触且与本申请的一种或多种其它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，可以使本申请的TIL与本申请的CD28激动剂接触且与本申请的一种或多种其它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，可以使本申请的TIL与本申请的CD28激动剂接触且与本申请的一种或多种其它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基本上同时与本申请的CD28激动剂以及本申请的一种或多种其它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，可以使本申请的TIL基本上同时与本申请的CD28激动剂以及本申请的一种或多种其它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，可以使本申请的TIL基本上同时与本申请的CD28激动剂以及本申请的一种或多种其它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，可以使本申请的TIL基本上同时与本申请的CD28激动剂以及本申请的一种或多种其它T细胞激活剂接触。
在一种实施方式中，本申请的其它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，本申请的其它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、HVEM、CD40L、OX40和4-1BB。在一种实施方式中，本申请的其它T细胞激活剂可以包含选自以下组：CD3、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，本申请的其它T细胞激活剂可以包含CD3激动剂。在一种实施方式中，本申请的其它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。
在一种实施方式中，使本申请的TIL与本申请的CD28激动剂接触且与本申请的一种或多种其它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CD28 激动剂与本申请的其它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CD28激动剂与本申请的其它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CD28激动剂与本申请的其它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CD28激动剂接触且与本申请的一种或多种其它T细胞激活剂接触可以包含将包含本申请的CD28激动剂与本申请的其它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CD28激动剂接触且与本申请的一种或多种其它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，所述其它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CD28激动剂和本申请的其它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CD28激动剂和本申请其它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CD28激动剂和本申请其它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CD28激动剂和本申请其它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CD28激动剂和本申请其它T细胞激活剂，例如CD3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CD28激动剂和本申请其它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，将包含本申请CD28激动剂和本申请其它T细胞激活剂，例如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：1和11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：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HCDR3，且本申请HCDR3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HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：1和11中任一项所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：2和12中任一项所示的氨基酸序列，且本申请HCDR3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与OKT3相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：1所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：2所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：3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：11所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：12所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：13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：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LCDR2，且本申请LCDR2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LCDR3，且本申请LCDR3可以包含SEQ  ID NO：6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LCDR1-3，其中本申请LCDR1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LCDR2可以包含SEQ ID NO：5和15中任一项所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：6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与OKT3相同的LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：4所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：5所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：6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：14所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：15所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：16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：1和11中任一项所示的氨基酸序列，本申请HCDR2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HCDR3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LCDR1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LCDR2可以包含SEQ ID NO：5和15中任一项所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：6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与OKT3相同的HCDR1-3和LCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：1所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：2所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：3所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：4所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：5所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：6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：11所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：12所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：13所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：14所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：15所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：16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：7和17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：7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：17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：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VL，且本申请VL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VL，且本申请VL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重链可变区VH和轻链可变区VL，且本申请VH可包含SEQ ID NO：7和17中任一项所示的氨基酸序列，本申请VL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和VL，且本申请 VH可包含SEQ ID NO：7所示的氨基酸序列，本申请VL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和VL，且本申请VH可包含SEQ ID NO：17所示的氨基酸序列，本申请VL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重链，且本申请重链可包含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相同的重链，且本申请重链可包含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相同的重链，且本申请重链可包含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轻链，且本申请轻链可包含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相同的轻链，且本申请轻链可包含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相同的轻链，且本申请轻链可包含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重链和轻链，且本申请重链可包含SEQ ID NO：9和19中任一项所示的氨基酸序列，本申请轻链可包含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相同的重链和轻链，且本申请重链可包含SEQ ID NO：9所示的氨基酸序列，本申请轻链可包含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相同的重链和轻链，且本申请重 链可包含SEQ ID NO：19所示的氨基酸序列，本申请轻链可包含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：21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：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HCDR3，且本申请HCDR3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HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：21所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：22所示的氨基酸序列，且本申请HCDR3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与15E8相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：21所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：22所示的氨基酸序列，且本申请HCDR3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；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：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LCDR2，且本申请LCDR2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LCDR3，且本申请LCDR3可以包含SEQ ID NO：26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：24所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：25所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：26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：24所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：25所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：26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：21所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：23所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：24所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：25所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：26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：21所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：23所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：24所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：25所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：26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：27和28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：27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：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。
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：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。
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：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重链可变区VH和轻链可变区VL，且本申请VH可包含SEQ ID NO：27和28中任一项所示的氨基酸序列，本申请VL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。
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：27所示的氨基酸序列，本申请VL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。
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：28所示的氨基酸序列，本申请VL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重链，且本申请重链可包含SEQ ID NO：30和31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：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。
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：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。
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：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重链和轻链，且本申请重链可包含SEQ ID NO：30和31中任一项所示的氨基酸序列，本申请轻链可包含SEQ ID NO：32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：30所示的氨基酸序列，本申请轻链可包含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。
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：31所示的氨基酸序列，本申请轻链可包含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27立方毫米。
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IL-2接触；其中，经所述步骤(A)得到第二TIL细胞群；(B)使本申请的第二TIL群基本上同时与CD28激动剂以及IL-2接触。
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IL-2接触；其中，经所述步骤(A)得到第二TIL细胞群；(B)使本申请的第二TIL群基本上同时与CD28激动剂以及IL-2接触，一定时间之后，再与饲养细胞共培养。
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群与浓度为300-9000IU/mL的IL-2接触；其中，经所述步骤(A)得到第二TIL细胞群；(B)使本申请的第二TIL群基本上同时与CD28激动剂、CD3抗体以及浓度为300-9000IU/mL的IL-2接触，一定时间之后，再以约40:1-约400:1的饲养细胞与所述TIL的比例与PBMC细胞共培养。
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。收集培养基中细胞，可以与组织块共同转移入透气培养袋、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)以及浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2，活化本申请的一定时间后，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。过滤组织块，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70％，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IFNγ，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融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1 肿瘤浸润淋巴细胞(TIL)细胞的培养方法
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，将3-5名供者PBMC悬液按照0.5:1至1:2的比例混合，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和袋子，例如G-Rex100培养瓶(Wilson Wolf Manufacturing)，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-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。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第一阶段体外扩增及收获(preREP阶段)
1.3.1第一阶段体外扩增
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，例如6000IU/mL的IL-2。第一阶段体外扩增的3-14天，例如可以第3、4、5、6、7、8、9、10、11、12、13或14天时取样计数，若细胞数目处于5×10 5至5×10 8之间时进入下述第一阶段体外扩增的收获步骤。
1.3.2第一阶段体外扩增的收获
收集第一阶段体外扩增结束细胞，离心，弃去培养基，使用PBS或生理盐水洗涤细胞一次，获得经第一阶段体外扩增的TIL(第二TIL群)，并取样计数留取约5×10 5至2×10 8个细胞进入下述第一阶段体外扩增步骤；取约5×10 5个细胞可以进行质量控制检测；其余细胞加入等量2倍冻存液冻存。
1.4第二阶段体外扩增(REP阶段)
1.4.1第二阶段体外扩增的TIL活化
取5×10 5至2×10 8的第一阶段体外扩增的细胞量，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)的IL-2。各个试验组加入IL-2的同时，按照以下配方添加对应的CD28激动剂：
对照组：同时在培养基中添加CD3抗体，例如OKT3约30ng/mL。
混合抗体组：同时在培养基中添加CD3抗体和CD28抗体，例如OKT3约30ng/mL、CD28抗体约30ng/mL。
磁珠组：同时在培养基中添加包含CD3抗体和CD28抗体的磁珠(直径约1至10μm Dynabeads，Thermo Fisher)，例如以约1:2-2:1的磁珠与TIL的比例加入磁珠。
纳米基质组：同时在培养基中添加包含CD3抗体和CD28抗体的transACT(直径约100至500nm，Miltenyi)，例如以约1:100-1:2000的transACT与TIL的比例加入transACT。
1.4.2第二阶段体外扩增的扩大培养
以上各个试验组，在第二阶段体外扩增加入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，例如24小时或48小时)，复苏1-5名供者混合的饲养细胞；将活化的TIL细胞，组织块及饲养细胞转入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或第二阶段体外扩增培养达13天，终止培养。
1.4.3肿瘤浸润淋巴细胞的收获
取第二阶段体外扩增的细胞，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，获得经第二阶段体外扩增的TIL(第三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。
1.5肿瘤浸润淋巴细胞的应用
可以将复苏后的治疗性肿瘤浸润淋巴细胞给予受试者静脉滴注。
实施例2 TIL增殖能力检测
对于实施例1中各个试验组第二阶段体外扩增培养获得的第三TIL群进行细胞计数。
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如图1所示。图1中纵坐标的数值表示，各个试验组第二阶段体外扩增获得的第三TIL群相比于第二阶段体外扩 增开始前的第二TIL群，TIL细胞数量扩增至的扩增倍数。结果显示，对于供者A，第二阶段体外扩增添加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3 TIL流式检测
对于实施例1中各个试验组第二阶段体外扩增培养获得的第三TIL群进行流式检测。
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250μL/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250μL/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250μL/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250μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。表面染色结束后，PBS清洗细胞一次(96孔板250μL/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。
不同刺激物培养的TIL的流式结果分析如图2到图6所示。
图2表示对于供者B-1，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，第二阶段体外扩增添加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3表示对于供者B-2，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，第二阶段体外扩增添加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4表示对于供者C-1，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，第二阶段体外扩增添加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 +、PD1 +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5表示对于供者C-2，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，第二阶段体外扩增添加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6表示对于供者D，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，第二阶段体外扩增添加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PD1 +)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4 TIL细胞杀伤能力检测
对于实施例1中各个试验组第二阶段体外扩增培养获得的第三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1×10 6个细胞/mL。对各个试验组第二阶段体外扩增培养获得的第三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。
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7表示对于供者E，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，第二阶段体外扩增添加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5 TIL胞内因子表达检测
对于实施例1中各个试验组第二阶段体外扩增培养获得的第三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1:1000加入Golgistop(BD)，按照体积比1:200添加CD107a抗体(BD)。
检测步骤
取各个试验组第二阶段体外扩增培养获得的第三TIL群离心后，使用600μL上述胞内因子表达检测所需培养基重悬为5×10 5个细胞/mL，加入96孔板内，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1:100，50μL/组染色，2-8℃避光孵育30分钟。染色结束后清洗细胞，加入100μL/孔Fixation/Permeabilization Buffer(BD)进行固定破膜，4℃避光孵育20分钟。固定破膜结束，加入1×BD Perm/Wash buffer(BD)清洗。可以继续使用1×BD Perm/Wash buffer(BD)配制IFNγ抗体(BD)和GZMB抗体(BD)，抗体浓度为1:100，50μL/组染色，4℃避光染色30分钟；染色结束后，加入1×BD Perm/Wash buffer(BD)清洗，使用PBS重悬，进行流式上机检测。
图8表示对于供者F，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，第二阶段体外扩增添加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9表示对于供者G-1，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，第二阶段体外扩增添加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10表示对于供者G-2，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，第二阶段体外扩增添加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11表示对于供者G-3，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，第二阶段体外扩增添加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12表示对于供者G-4，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，第二阶段体外扩增添加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13表示对于供者H，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，第二阶段体外扩增添加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IFNγ表达能力或更高的GZMB表达能力。
实施例6 TIL细胞因子分泌检测
对于实施例1中各个试验组第二阶段体外扩增培养获得的第三TIL群进行细胞因子分泌检测。
标准品准备
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，随后与Capture Beads(BD)混合及PE Detection  Reagent检测试剂(BD)混合，转移至15mL锥底离心管，标记为“标准品管”。取1管仅含有Assay Diluent稀释液作为阴性对照。
检测步骤
使用Assay Diluent稀释各个试验组第二阶段体外扩增培养获得的第三TIL群，按照10μL/孔加入孔板，随后按照2μL/Beads/孔与每种Capture Beads(BD)混合，以及按照10μL/孔与PE Detection Reagent检测试剂(BD)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14表示对于供者I-1，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，第二阶段体外扩增添加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IFNγ分泌能力。
取各个试验组获得的TIL参照实施例4(TIL细胞杀伤能力检测)的方法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15表示对于供者I-2，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，第二阶段体外扩增添加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IFNγ分泌能力。
实施例7 TIL基因敲除效率检测
取实施例1中各个试验组在第二阶段体外扩增培养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：33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中各个试验组在第二阶段体外扩增培养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BB515 Mouse 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16表示对于供者J-1，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基因敲除效率结果。结果显示，第二阶段体外扩增添加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17表示对于供者J-2，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基因敲除效率结果。结果显示，第二阶段体外扩增添加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8 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群经过相同方式的第一阶段体外扩增(preREP)和相同方式的第二阶段体外扩增(REP)得到第三TIL群。将第三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(第四细胞群)，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。
对于REP阶段结束后，以不同方式进行体外扩增的试验组的增殖能力分析如图18所示。图18的纵坐标荧光值反映了各个试验组以不同方式进行第三阶段体外扩增的TIL细胞的增殖能力。结果显示，对于供者K-1、K-2和K-3，第三阶段体外扩增添加CD28激动剂，与第三阶段扩增不添加CD28激动剂相比，具有类似的TIL增殖能力。
实施例9 不同CD28激动剂形式的激活效果
对于实施例1中各个试验组第二阶段体外扩增培养7天、10天或大于10天中，检测使用不同形式的CD28激动剂的激活效果。例如，纳米尺寸的CD28激动剂可以是包含CD3抗体和CD28抗体的直径约为100nm的激动剂；例如，纳米基质A可以是transACT(直径约100至500nm，Miltenyi)，纳米基质B可以是CytoSinct(直径约为100nm，GENSCRIPT，例如以约1:100至1:1000的transACT与TIL的比例加入)。
添加不同形式的CD28激动剂的试验组的增殖能力分析如图19A所示。图中纵坐标的数值表示，各个试验组第二阶段体外扩增获得的第三TIL群相比于第二阶段体外扩增开始前的第二TIL群，TIL细胞数量扩增至的扩增倍数。结果显示，不同形式的纳米尺寸的CD28激动剂都可以具有提高TIL增殖能力的效果。
添加不同形式的CD28激动剂的试验组的流式结果分析如图19B到图19L所示。
图19B显示的是，不同形式的纳米尺寸的CD28激动剂都可以具有提高肿瘤特异性T细胞(CD103 +CD39 +)比例的能力。
图19C-19F显示的是，不同形式的纳米尺寸的CD28激动剂都可以具有降低耗竭T细胞(CD39 +、PD1 +、LAG3 +、TIM3 +)比例的能力。
图19G显示的是，不同形式的纳米尺寸的CD28激动剂都可以具有降低调节性T细胞(Treg)比例的能力。
图19H-19J显示的是，不同形式的纳米尺寸的CD28激动剂都可以具有提高活化T细胞(CD25 +、41BB +、CD28 +)比例的能力。
图19K显示的是，不同形式的纳米尺寸的CD28激动剂都可以具有提高干细胞样T细胞(CD69 -CD39 -)比例的能力。
图19L显示的是，不同形式的纳米尺寸的CD28激动剂都可以具有提高中心记忆T细胞(Tcm，例如CD45RO +CD62L +)比例的能力。
添加不同形式的CD28激动剂的试验组的胞内因子表达检测结果如图19M到19N所示。
图19M显示的是，不同形式的纳米尺寸的CD28激动剂加入到第二阶段体外扩增培养中，通过流式检测胞内因子(CD107a)表达比例。结果显示，不同形式的纳米尺寸的CD28激动剂都可以具有提高TIL细胞的胞内因子表达。
图19N显示的是，不同形式的纳米尺寸的CD28激动剂加入到第二阶段体外扩增培养中，通过流式检测胞内因子(CD107a)表达比例，流式检测之前各组的TIL细胞与CD3抗体孵育过夜。结果显示，不同形式的纳米尺寸的CD28激动剂都可以具有提高TIL细胞的胞内因子表达。
添加不同形式的CD28激动剂的试验组的细胞因子分泌检测结果如图19O所示。
图19O显示的是，不同形式的纳米尺寸的CD28激动剂加入到第二阶段体外扩增培养中，通过细胞因子分泌检测验证TIL细胞的细胞因子分泌能力，其中CD3抗体刺激组的TIL细胞在细胞因子检测前与CD3抗体孵育过夜。结果显示，不同形式的纳米尺寸的CD28激动剂都可以具有提高TIL细胞的细胞因子分泌能力。
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 (106)

  1. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：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，在至少一个阶段的所述体外扩增中，使所述TIL与CD28激动剂接触。
  2. 根据权利要求1所述的方法，其中，使所述源自肿瘤组织且未经体外扩增的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与所述CD28激动剂接触。
  3. 根据权利要求1-2中任一项所述的方法，所述CD28激动剂包含抗CD28的抗体和/或其抗原结合片段。
  4. 根据权利要求2-3中任一项所述的方法，所述第二阶段体外扩增进行至多约13天。
  5. 根据权利要求2-4中任一项所述的方法，所述第二阶段体外扩增进行约3天至约13天。
  6. 根据权利要求1-5中任一项所述的方法，其还包含：在至少一个阶段的所述体外扩增中，使所述TIL与饲养细胞共培养。
  7. 根据权利要求6所述的方法，其中，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂接触且与所述饲养细胞共培养。
  8. 根据权利要求6-7中任一项所述的方法，其中，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂接触一定时间之后，再与所述饲养细胞共培养。
  9. 根据权利要求8所述的方法，所述一定时间为至少约2小时。
  10. 根据权利要求8-9中任一项所述的方法，所述一定时间为约6小时至约72小时。
  11. 根据权利要求8-10中任一项所述的方法，所述一定时间为约12小时至约48小时。
  12. 根据权利要求8-10中任一项所述的方法，所述一定时间为约6小时、约12小时、约24小时、约48小时、或约72小时。
  13. 根据权利要求6-12中任一项所述的方法，所述饲养细胞包含抗原呈递细胞。
  14. 根据权利要求6-13中任一项所述的方法，所述饲养细胞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。
  15. 根据权利要求6-14中任一项所述的方法，所述饲养细胞为外周单个核细胞。
  16. 根据权利要求6-15中任一项所述的方法，所述饲养细胞为经过辐照的饲养细胞。
  17. 根据权利要求6-16中任一项所述的方法，所述TIL与所述饲养细胞共培养包含使所述饲养细胞的表面与所述TIL的表面相接触。
  18. 根据权利要求6-17中任一项所述的方法，所述TIL与所述饲养细胞共培养包含将所述饲养细胞添加至所述TIL的细胞培养基中。
  19. 根据权利要求6-18中任一项所述的方法，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。
  20. 根据权利要求1-19中任一项所述的方法，其还包含：在至少一个阶段的所述体外扩增中，使所述TIL与一种或多种T细胞生长因子接触。
  21. 根据权利要求20所述的方法，其中，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂接触且与一种或多种所述T细胞生长因子接触。
  22. 根据权利要求20-21中任一项所述的方法，其中，在单个阶段的所述体外扩增中，使所述TIL基本上同时与所述CD28激动剂以及一种或多种所述T细胞生长因子接触。
  23. 根据权利要求20-22中任一项所述的方法，所述T细胞生长因子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。
  24. 根据权利要求20-23中任一项所述的方法，所述T细胞生长因子包含IL-2和/或其功能活性片段。
  25. 根据权利要求20-24中任一项所述的方法，所述TIL与一种或多种所述T细胞生长因子接触包含将所述T细胞生长因子添加至所述TIL的细胞培养基中。
  26. 根据权利要求20-25中任一项所述的方法，所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度为至少约300IU/mL。
  27. 根据权利要求1-26中任一项所述的方法，与在体外扩增阶段未曾与所述CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与所述CD28激动剂接触过的所述TIL显示出改善的扩增效果。
  28. 根据权利要求27所述的方法，所述改善的扩增效果包含选自以下组的一种或多种：增加的TIL细胞数量，改善的T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。
  29. 根据权利要求28所述的方法，所述改善的T细胞亚群比例包含选自以下组的一种或多种：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。
  30. 根据权利要求1-29中任一项所述的方法，与在体外扩增阶段未曾与所述CD28激动剂接触的相应TIL相比，在至少一个体外扩增阶段中与所述CD28激动剂接触过的所述TIL显示出改善的基因编辑效果。
  31. 根据权利要求30所述的方法，所述改善的基因编辑效果包含提高的基因敲除效率。
  32. 根据权利要求1-31中任一项所述的方法，其还包含：在至少一个阶段的所述体外扩增中， 使所述TIL与所述CD28激动剂之外的一种或多种其他T细胞激活剂接触。
  33. 根据权利要求32所述的方法，其中，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂接触且与一种或多种所述其它T细胞激活剂接触。
  34. 根据权利要求32-33中任一项所述的方法，其中，在单个阶段的所述体外扩增中，使所述TIL基本上同时与所述CD28激动剂以及一种或多种所述其它T细胞激活剂接触。
  35. 根据权利要求32-34中任一项所述的方法，所述其它T细胞激活剂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。
  36. 根据权利要求32-35中任一项所述的方法，所述其它T细胞激活剂包含选自以下组的一种或多种靶点的激动剂：CD3、HVEM、CD40L、OX40和4-1BB。
  37. 根据权利要求32-36中任一项所述的方法，所述其它T细胞激活剂包含CD3激动剂。
  38. 根据权利要求32-37中任一项所述的方法，所述其它T细胞激活剂包含抗CD3的抗体和/或其抗原结合片段。
  39. 根据权利要求32-38中任一项所述的方法，所述使TIL与所述CD28激动剂接触且与一种或多种所述其它T细胞激活剂接触包含选自以下组的一种或多种方式：(1)将所述CD28激动剂与所述其它T细胞激活剂添加至所述TIL的细胞培养基中；(2)将表达所述CD28激动剂与所述其它T细胞激活剂的工程化细胞添加至所述TIL的细胞培养基中；(3)将包含所述CD28激动剂与所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  40. 根据权利要求32-39中任一项所述的方法，所述其它T细胞激活剂在所述TIL的细胞培养基中的初始浓度为至少约30ng/mL。
  41. 根据权利要求32-40中任一项所述的方法，所述其它T细胞激活剂在所述TIL的细胞培养基中的初始浓度为约30ng/mL-约300ng/mL。
  42. 根据权利要求39-41中任一项所述的方法，所述固相介质的直径为约500纳米至约10微米。
  43. 根据权利要求39-41中任一项所述的方法，所述固相介质的直径为约1纳米至约500纳米。
  44. 根据权利要求42-43中任一项所述的方法，所述固相介质的直径通过透射电子显微镜测量。
  45. 根据权利要求39-44中任一项所述的方法，所述固相介质包含聚合物。
  46. 根据权利要求39-45中任一项所述的方法，每mg所述固相介质包含至少约25μg的所述 CD28激动剂和所述其它T细胞激活剂。
  47. 根据权利要求39-46中任一项所述的方法，以约2:1-约1:2的所述固相介质与所述TIL的比例，将包含所述CD28激动剂和所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  48. 根据权利要求39-46中任一项所述的方法，以约1:100-约1:2000的所述固相介质与所述TIL的比例，将包含所述CD28激动剂和所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  49. 根据权利要求1-48中任一项所述的方法，所述源自肿瘤组织且未经体外扩增的TIL为源自所述肿瘤组织的碎片的TIL。
  50. 根据权利要求49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。
  51. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
    (A)使源自肿瘤组织且未经体外扩增的第一TIL群与一种或多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL细胞群；
    (B)使所述第二TIL群与CD28激动剂接触。
  52. 根据权利要求51所述的方法，所述CD28激动剂包含抗CD28的抗体和/或其抗原结合片段。
  53. 根据权利要求51-52中任一项所述的方法，所述步骤(B)进行至多约13天。
  54. 根据权利要求51-53中任一项所述的方法，所述步骤(B)进行约3天至约13天。
  55. 根据权利要求51-54中任一项所述的方法，其还包含：所述步骤(A)和/或所述步骤(B)中，使所述TIL与饲养细胞共培养。
  56. 根据权利要求55所述的方法，其中，在所述步骤(B)中，使所述第二TIL群与所述饲养细胞共培养。
  57. 根据权利要求55-56中任一项所述的方法，其中，在所述步骤(B)中，使所述第二TIL群与所述CD28激动剂接触一定时间之后，再与所述饲养细胞共培养。
  58. 根据权利要求57所述的方法，所述一定时间为至少约2小时。
  59. 根据权利要求57-58中任一项所述的方法，所述一定时间为约6小时至约72小时。
  60. 根据权利要求57-59中任一项所述的方法，所述一定时间为约12小时至约48小时。
  61. 根据权利要求57-59中任一项所述的方法，所述一定时间为约6小时、约12小时、约24小时、约48小时、或约72小时。
  62. 根据权利要求55-61中任一项所述的方法，所述饲养细胞包含抗原呈递细胞。
  63. 根据权利要求55-62中任一项所述的方法，所述饲养细胞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。
  64. 根据权利要求55-63中任一项所述的方法，所述饲养细胞为外周单个核细胞。
  65. 根据权利要求55-64中任一项所述的方法，所述饲养细胞为经过辐照的饲养细胞。
  66. 根据权利要求55-65中任一项所述的方法，所述TIL与所述饲养细胞共培养包含使所述饲养细胞的表面与所述TIL的表面相接触。
  67. 根据权利要求55-66中任一项所述的方法，所述TIL与所述饲养细胞共培养包含将所述饲养细胞添加至所述TIL的细胞培养基中。
  68. 根据权利要求55-67中任一项所述的方法，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。
  69. 根据权利要求51-68中任一项所述的方法，其还包含：在所述步骤(A)和/或所述步骤(B)中，使所述TIL与一种或多种T细胞生长因子接触。
  70. 根据权利要求69所述的方法，其中，在所述步骤(B)中，使所述第二TIL群与一种或多种所述T细胞生长因子接触。
  71. 根据权利要求69-70中任一项所述的方法，其中，在所述步骤(B)中，使所述第二TIL群基本上同时与所述CD28激动剂以及一种或多种所述T细胞生长因子接触。
  72. 根据权利要求69-71中任一项所述的方法，所述T细胞生长因子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。
  73. 根据权利要求69-72中任一项所述的方法，所述T细胞生长因子包含IL-2和/或其功能活性片段。
  74. 根据权利要求69-73中任一项所述的方法，所述TIL与一种或多种所述T细胞生长因子接触包含将所述T细胞生长因子添加至所述TIL的细胞培养基中。
  75. 根据权利要求69-74中任一项所述的方法，所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度为至少约300IU/mL。
  76. 根据权利要求51-75中任一项所述的方法，与在所述步骤(B)中未曾与所述CD28激动剂接触的相应TIL相比，在所述步骤(B)中与所述CD28激动剂接触过的所述TIL显示出改善的扩增效果。
  77. 根据权利要求76所述的方法，所述改善的扩增效果包含选自以下组的一种或多种：增加的TIL细胞数量，改善的T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。
  78. 根据权利要求77所述的方法，所述改善的T细胞亚群比例包含选自以下组的一种或多种：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。
  79. 根据权利要求51-78中任一项所述的方法，与在所述步骤(B)中未曾与所述CD28激动剂接触的相应TIL相比，在所述步骤(B)中与所述CD28激动剂接触过的所述TIL显示出改善的基因编辑效果。
  80. 根据权利要求79所述的方法，所述改善的基因编辑效果包含提高的基因敲除效率。
  81. 根据权利要求51-80中任一项所述的方法，其还包含：在所述步骤(A)和/或所述步骤(B)中，使所述TIL与所述CD28激动剂之外的一种或多种其他T细胞激活剂接触。
  82. 根据权利要求81所述的方法，其中，在所述步骤(B)中，使所述第二TIL群与所述CD28激动剂接触且与一种或多种所述其它T细胞激活剂接触。
  83. 根据权利要求81-82中任一项所述的方法，其中，在所述步骤(B)中，使所述第二TIL群基本上同时与所述CD28激动剂以及一种或多种所述其它T细胞激活剂接触。
  84. 根据权利要求81-83中任一项所述的方法，所述其它T细胞激活剂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。
  85. 根据权利要求81-84中任一项所述的方法，所述其它T细胞激活剂包含选自以下组的一种或多种靶点的激动剂：CD3、HVEM、CD40L、OX40和4-1BB。
  86. 根据权利要求81-85中任一项所述的方法，所述其它T细胞激活剂包含CD3激动剂。
  87. 根据权利要求81-86中任一项所述的方法，所述其它T细胞激活剂包含抗CD3的抗体和/或其抗原结合片段。
  88. 根据权利要求81-87中任一项所述的方法，所述使TIL与所述CD28激动剂接触且与一种或多种所述其它T细胞激活剂接触包含选自以下组的一种或多种方式：(1)将所述CD28激动剂与所述其它T细胞激活剂添加至所述TIL的细胞培养基中；(2)将表达所述CD28激动剂与所述其它T细胞激活剂的工程化细胞添加至所述TIL的细胞培养基中；(3)将包含所述CD28激动剂与所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  89. 根据权利要求81-88中任一项所述的方法，所述其它T细胞激活剂在所述TIL的细胞培养基中的初始浓度为至少约30ng/mL。
  90. 根据权利要求81-89中任一项所述的方法，所述其它T细胞激活剂在所述TIL的细胞培养 基中的初始浓度为约30ng/mL-约300ng/mL。
  91. 根据权利要求88-90中任一项所述的方法，所述固相介质的直径为约500纳米至约10微米。
  92. 根据权利要求88-90中任一项所述的方法，所述固相介质的直径为约1纳米至约500纳米。
  93. 根据权利要求91-92中任一项所述的方法，所述固相介质的直径通过透射电子显微镜测量。
  94. 根据权利要求88-93中任一项所述的方法，所述固相介质包含聚合物。
  95. 根据权利要求88-94中任一项所述的方法，每mg所述固相介质包含至少约25μg的所述CD28激动剂和所述其它T细胞激活剂。
  96. 根据权利要求88-95中任一项所述的方法，以约2:1-约1:2的所述固相介质与所述TIL的比例，将包含所述CD28激动剂和所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  97. 根据权利要求88-95中任一项所述的方法，以约1:100-约1:2000的所述固相介质与所述TIL的比例，将包含所述CD28激动剂和所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  98. 根据权利要求51-97中任一项所述的方法，所述源自肿瘤组织且未经体外扩增的TIL为源自所述肿瘤组织的碎片的TIL。
  99. 根据权利要求98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。
  100. 一种肿瘤浸润淋巴细胞(TIL)，所述TIL经过权利要求1-99中任一项所述的方法获得。
  101. 一种组合物，其包含权利要求100所述的TIL。
  102. 一种药物组合物，其包含权利要求100所述的TIL和/或权利要求101中任一项所述的组合物，以及任选地药学上可接受的载体。
  103. 一种影响肿瘤细胞生长的方法，包含向受试者施用权利要求100所述的TIL和/或权利要求102所述的药物组合物。
  104. 权利要求100所述的TIL和/或权利要求102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/或治疗肿瘤。
  105. 根据权利要求104所述的应用，其中，所述肿瘤为实体瘤。
  106. 根据权利要求104-105中任一项所述的应用，其中，所述肿瘤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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