WO2023011433A1 - 一种修饰的肿瘤浸润淋巴细胞及其用途 - Google Patents

一种修饰的肿瘤浸润淋巴细胞及其用途 Download PDF

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WO2023011433A1
WO2023011433A1 PCT/CN2022/109577 CN2022109577W WO2023011433A1 WO 2023011433 A1 WO2023011433 A1 WO 2023011433A1 CN 2022109577 W CN2022109577 W CN 2022109577W WO 2023011433 A1 WO2023011433 A1 WO 2023011433A1
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til
cell
cells
tils
vitro
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PCT/CN2022/109577
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English (en)
French (fr)
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刘雅容
孙静玮
盛耀
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苏州沙砾生物科技有限公司
珠海拓域生物科技有限公司
上海沙砾生物科技有限公司
珠海沙砾生物科技有限公司
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Priority to CN202280046128.4A priority Critical patent/CN117616115A/zh
Publication of WO2023011433A1 publication Critical patent/WO2023011433A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • This application relates to the field of biomedicine, in particular to a modified tumor infiltrating lymphocyte and its use.
  • tumor-infiltrating lymphocytes Treatment of tumors using adoptive autologous transfer of tumor-infiltrating lymphocytes is an effective approach for patients with poor prognosis.
  • adoptive autologous transfer of tumor-infiltrating lymphocytes to treat tumors requires a large number of tumor-infiltrating lymphocytes, and the current survival and expansion of tumor-infiltrating lymphocytes from patient tumors are weak in vivo, and the ability to kill target cells is not strong. Functionally limited by multiple inhibitions of the tumor microenvironment.
  • the present application provides a method for culturing tumor infiltrating lymphocytes (TIL), the method comprising: reducing the expression and/or attenuating the activity of at least one target gene of the TIL, and making the TIL in Co-cultivation with feeder cells followed by exposure to T cell activators and/or T cell growth factors for a certain period of time.
  • TIL tumor infiltrating lymphocytes
  • the method comprises: reducing the expression and/or attenuating the activity of at least one target gene of the TIL before co-cultivating the TIL with the feeder cells.
  • the method comprises: contacting the TIL with the T cell activator and/or the T cell growth factor and before co-cultivating the TIL with the feeder The expression and/or activity of at least one target gene of the TIL is reduced.
  • a method for culturing tumor-infiltrating lymphocytes comprising: reducing the expression and/or attenuating the activity of at least one target gene of the TIL, wherein the TIL is combined with a T cell activator and/or TIL obtained by co-culture with feeder cells after T cell growth factor exposure for a certain period of time.
  • a method of culturing tumor-infiltrating lymphocytes comprising: co-cultivating the TILs with feeder cells after contacting a T cell activator and/or a T cell growth factor for a certain period of time, wherein the TILs comprise The TIL obtained by reducing the expression and/or weakening the activity of at least one target gene of the TIL.
  • the TIL obtained by reducing the expression and/or attenuating the activity of at least one target gene of said TIL exhibits improved TIL properties compared to TIL with unchanged expression and/or activity of the target gene .
  • said improved TIL properties comprise one or more selected from the group consisting of: increased TIL cell number and expansion capacity, increased live cell ratio, increased viability, improved T cell Subpopulation ratio, increased cytokine secretion capacity, increased tumor cell killing capacity, increased anti-apoptosis capacity, and increased T cell receptor (TCR) clonal diversity.
  • 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 proportion of activated T cells proportion, increased proportion of tumor-specific T cells, and increased proportion of stem cell-like T cells.
  • said reducing the expression and/or attenuating the activity of at least one target gene of said TIL comprises introducing a gene regulation system into said TIL cells.
  • the gene regulation system is capable of disrupting the target gene at the DNA level.
  • the gene regulation system comprises a guide nucleic acid molecule and an enzyme protein.
  • said reducing the expression and/or weakening the activity of at least one target gene of said TIL comprises: combining the ribonucleoprotein complex (RNP) comprising said guide nucleic acid molecule and said enzyme protein ) is introduced into the TIL.
  • RNP ribonucleoprotein complex
  • the enzyme protein comprises a Cas protein, a Cas protein homologue, or a functionally active fragment thereof.
  • the guide nucleic acid molecule comprises a guide RNA (gRNA).
  • gRNA guide RNA
  • the guide nucleic acid molecule is capable of binding to the sequence of the target gene.
  • the target gene comprises a gene encoding an NF- ⁇ B pathway inhibitory molecule.
  • the NF- ⁇ B pathway inhibitory molecule is capable of ubiquitinating a protein selected from the group consisting of receptor interacting protein 1 (RIP1), mucosa-associated lymphoid tissue lymphoma translocator protein 1 (MALT1) , receptor interacting protein 2 (RIP2) and tumor necrosis factor receptor-associated factor 6 (TRAF6).
  • RIP1 receptor interacting protein 1
  • MALT1 mucosa-associated lymphoid tissue lymphoma translocator protein 1
  • RIP2 receptor interacting protein 2
  • TNF6 tumor necrosis factor receptor-associated factor 6
  • the NF- ⁇ B pathway inhibitory molecule comprises tumor necrosis factor alpha-inducible protein 3 (TNFAIP3).
  • the guide nucleic acid molecule can bind to the region where exon 2 and/or exon 7 of the TNFAIP3 gene is located or a fragment thereof.
  • the guide nucleic acid molecule is capable of binding to a region or a fragment thereof selected from the group consisting of SEQ ID NO: 34 to 47.
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the group consisting of: GGG, TGG, CGG and AGG.
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NO: 48 to 61.
  • the expression of the target gene is expressed in the TIL obtained by reducing the expression and/or weakening of the activity of at least one target gene of the TIL compared with the TIL in which the expression and/or activity of the target gene are not changed
  • the cell ratio of the product is reduced and/or the expression level of a single cell is reduced.
  • the proportion of cells expressing the target gene is about 95% or less.
  • the method further comprises: subjecting TIL derived from tumor tissue and not expanded in vitro to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the The TILs are co-cultured with the feeder cells.
  • said TILs are co-cultured with said feeder cells in a single stage of said in vitro expansion.
  • the expression and/or activity of at least one target gene of said TIL is reduced and said TIL is co-cultured with said feeder cells.
  • 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 TILs are co-cultured with the feeder cells.
  • said first stage in vitro expansion is performed for at least about 7 days.
  • the first stage of in vitro expansion is performed for about 7 days to about 14 days.
  • said second stage in vitro expansion is performed for at least about 7 days.
  • said second stage in vitro expansion is performed for about 7 days to about 14 days.
  • said TILs are co-cultured with said feeder cells for at least about 2 hours after contacting with a T cell activator and/or a T cell growth factor.
  • said TILs are co-cultured with said feeder cells after contacting said T cell activator and/or said T cell growth factor for about 6 hours to about 72 hours.
  • said TILs are co-cultured with said feeder cells about 12 hours to about 48 hours after contacting said T cell activator and/or said T cell growth factor.
  • said TIL is contacted with said T cell activator and/or said T cell growth factor for about 6 hours, about 12 hours, about 24 hours, about 48 hours or about 72 hours with The feeder cells are co-cultured.
  • 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-cultivating said TIL with said feeder cells comprises contacting the surface of said feeder cells with the surface of said TIL.
  • the co-cultivation of the TILs with the feeder cells comprises adding the feeder cells to the cell culture medium of the TILs.
  • said feeder cells are added to the cell culture medium of said TIL at a ratio of said feeder cells to said TIL of about 40:1 to about 400:1.
  • the method further comprises: subjecting TIL derived from tumor tissue and not expanded in vitro to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the The TILs are contacted with the T cell activator.
  • said TILs are contacted with said T cell activator during a single stage of said in vitro expansion.
  • the expression and/or activity of at least one target gene of said TIL is reduced and said TIL is contacted with said T cell activator.
  • 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 TILs are contacted with the T cell activator.
  • the T cell activator comprises one or more selected from the following group: cluster of differentiation 80 (CD80), CD86, CD276, 4-1BB ligand (4-1BBL), CD27, CD30 , CD134, CD275, CD40, CD258, and functionally active fragments thereof.
  • the T cell activator comprises an agonist of one or more targets selected from the group consisting of CD3, CD28, herpes virus entry medium (HVEM), CD40L, OX40, and 4-1BB.
  • HVEM herpes virus entry medium
  • CD40L CD40L
  • OX40 OX40
  • 4-1BB 4-1BB
  • the T cell activator comprises a CD3 agonist and/or a CD28 agonist.
  • the T cell activator comprises a CD3 agonist.
  • the T cell activator comprises an anti-CD3 antibody and/or an antigen-binding fragment thereof.
  • the T cell activator comprises a CD28 agonist.
  • the T cell activator comprises an anti-CD28 antibody and/or an antigen-binding fragment thereof, CD80 and/or a functionally active fragment thereof, and/or CD86 and/or a functionally active fragment thereof.
  • said contacting TILs with said T cell activator comprises one or more means selected from the group consisting of: (1) adding said T cell activator to said TIL cell culture (2) adding the engineered cells expressing the T cell activator to the cell culture medium of the TIL; and (3) adding the solid phase medium containing the T cell activator to the TIL in the cell culture medium.
  • the initial concentration of each of said T cell activators in said TIL cell culture medium is each independently at least about 30 ng/mL.
  • the initial concentration of each of said T cell activators in said TIL cell culture medium is each independently 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 about 1 nanometer to about 500 nanometers.
  • the diameter of the solid phase medium is measured by transmission electron microscopy.
  • the solid medium comprises a polymer
  • each of said T cell activators is contained in an amount of each independently at least about 25 ⁇ g per mg of said solid medium.
  • the solid phase medium comprising the T cell activator is added to the cell culture of the TIL at a ratio of the solid phase medium to the TIL of about 2:1 to about 1:2 Base.
  • the solid phase medium comprising the T cell activator is added to the cell culture of the TIL at a ratio of the solid phase medium to the TIL of about 1:100 to about 1:2000 Base.
  • the method further comprises: subjecting TIL derived from tumor tissue and not expanded in vitro to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the The TILs are contacted with the T cell growth factors.
  • said TIL is contacted with said T cell growth factor during a single stage of said in vitro expansion.
  • said TILs are contacted with said T cell activator and said T cell growth factor in a single stage of said in vitro expansion.
  • 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 TILs are contacted with T cell growth factors.
  • said TIL is contacted with said T cell activator and said T cell growth factor substantially simultaneously.
  • the T cell growth factor is 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 factor comprises IL-2 and/or a functionally active fragment thereof.
  • contacting said TIL with said T cell growth factor comprises adding said T cell growth factor to the cell culture medium of said TIL.
  • the initial concentration of each of said T cell growth factors in said TIL cell culture medium is each independently at least about 300 IU/mL.
  • the TILs are selected from the group consisting of TILs derived from fragments of tumor tissue, TILs derived from fragments of lymphatic metastases, TILs derived from pleural effusions, TILs derived from peritoneal effusions and Derived from TILs thawed after cryopreservation.
  • the fragments have a volume from about 1 mm3 to about 27 mm3.
  • TIL tumor infiltrating lymphocytes
  • step (A) contacting the first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • step (B) reducing the expression and/or attenuating the activity of at least one target gene of the second TIL population, and after contacting the second TIL population with a T cell activator and/or a T cell growth factor for a certain period of time co-culture with feeder cells, wherein the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • step (B) reducing the expression and/or attenuating the activity of at least one target gene of the second TIL population, and after contacting the second TIL population with a T cell activator and/or a T cell growth factor for a certain period of time co-culture with feeder cells, wherein the third TIL population is obtained through the step (B).
  • said population of TILs in vitro comprises a population of TILs obtained by contacting said first population of TILs with a T cell growth factor.
  • said population of TILs in vitro comprises a population of TILs obtained by cryopreservation of said first population of TILs.
  • said step (A) is performed for about 7 days to about 14 days.
  • said step (B) is performed for about 7 days to about 14 days.
  • TIL tumor infiltrating lymphocytes
  • step (A) contacting the first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • step (B) reducing the expression and/or attenuating the activity of at least one target gene of the second TIL population, and contacting the second TIL population with a T cell activator and/or a T cell growth factor, wherein, via The step (B) obtains a third TIL population;
  • TIL tumor infiltrating lymphocytes
  • step (B) reducing the expression and/or attenuating the activity of at least one target gene of the second TIL population, and contacting the second TIL population with a T cell activator and/or a T cell growth factor, wherein, via The step (B) obtains a third TIL population;
  • said population of TILs in vitro comprises a population of TILs obtained by contacting said first population of TILs with a T cell growth factor.
  • said population of TILs in vitro comprises a population of TILs obtained by cryopreservation of said first population of TILs.
  • said step (A) is performed for about 7 days to about 14 days.
  • said step (B) is performed for about 0 days to about 8 days.
  • said step (C) is performed for about 5 days to about 14 days.
  • TIL tumor infiltrating lymphocytes
  • step (A) contacting the first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • step (B) contacting said second population of TILs with a T cell activator and/or a T cell growth factor, wherein said step (B) results in a third population of TILs;
  • TIL tumor infiltrating lymphocytes
  • step (B) contacting said second population of TILs with a T cell activator and/or a T cell growth factor, wherein said step (B) results in a third population of TILs;
  • said population of TILs in vitro comprises a population of TILs obtained by contacting said first population of TILs with a T cell growth factor.
  • said population of TILs in vitro comprises a population of TILs obtained by cryopreservation of said first population of TILs.
  • said step (A) is performed for about 7 days to about 14 days.
  • said step (B) is performed for about 0 days to about 4 days.
  • step (C) is performed for about 0 days to about 4 days.
  • said step (D) is performed for about 5 days to about 14 days.
  • the TIL obtained by reducing the expression and/or attenuating the activity of at least one target gene of said TIL exhibits improved TIL properties compared to TIL with unchanged expression and/or activity of the target gene .
  • said improved TIL properties comprise one or more selected from the group consisting of: increased TIL cell number and expansion capacity, increased live cell ratio, increased viability, improved T cell Subpopulation ratio, increased cytokine secretion capacity, increased tumor cell killing capacity, increased anti-apoptosis capacity, and increased T cell receptor (TCR) clonal diversity.
  • 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 proportion of activated T cells proportion, increased proportion of tumor-specific T cells, and increased proportion of stem cell-like T cells.
  • said reducing the expression and/or attenuating the activity of at least one target gene of said TIL comprises introducing a gene regulation system into said TIL cells.
  • the gene regulation system is capable of disrupting the target gene at the DNA level.
  • the gene regulation system comprises a guide nucleic acid molecule and an enzyme protein.
  • said reducing the expression and/or weakening the activity of at least one target gene of said TIL comprises: combining the ribonucleoprotein complex (RNP) comprising said guide nucleic acid molecule and said enzyme protein ) is introduced into the TIL.
  • RNP ribonucleoprotein complex
  • the enzyme protein comprises a Cas protein, a Cas protein homologue, or a functionally active fragment thereof.
  • the guide nucleic acid molecule comprises a guide RNA (gRNA).
  • gRNA guide RNA
  • the guide nucleic acid molecule is capable of binding to the sequence of the target gene.
  • the target gene comprises a gene encoding an NF- ⁇ B pathway inhibitory molecule.
  • the NF- ⁇ B pathway inhibitory molecule is capable of ubiquitinating a protein selected from the group consisting of receptor interacting protein 1 (RIP1), mucosa-associated lymphoid tissue lymphoma translocator protein 1 (MALT1) , receptor interacting protein 2 (RIP2) and tumor necrosis factor receptor-associated factor 6 (TRAF6).
  • RIP1 receptor interacting protein 1
  • MALT1 mucosa-associated lymphoid tissue lymphoma translocator protein 1
  • RIP2 receptor interacting protein 2
  • TNF6 tumor necrosis factor receptor-associated factor 6
  • the NF- ⁇ B pathway inhibitory molecule comprises tumor necrosis factor alpha-inducible protein 3 (TNFAIP3).
  • the guide nucleic acid molecule can bind to the region where exon 2 and/or exon 7 of the TNFAIP3 gene is located or a fragment thereof.
  • the guide nucleic acid molecule is capable of binding to a region or a fragment thereof selected from the group consisting of SEQ ID NO: 34 to 47.
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the group consisting of: GGG, TGG, CGG and AGG.
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NO: 48 to 61.
  • the expression of the target gene is expressed in the TIL obtained by reducing the expression and/or weakening of the activity of at least one target gene of the TIL compared with the TIL in which the expression and/or activity of the target gene are not changed
  • the cell ratio of the product is reduced and/or the expression level of a single cell is reduced.
  • the proportion of cells expressing the target gene is about 95% or less.
  • said TILs are co-cultured with said feeder cells for at least about 2 hours after contacting said T cell activator and/or said T cell growth factor.
  • said TILs are co-cultured with said feeder cells after contacting said T cell activator and/or said T cell growth factor for about 6 hours to about 72 hours.
  • said TILs are co-cultured with said feeder cells about 12 hours to about 48 hours after contacting said T cell activator and/or said T cell growth factor.
  • said TIL is contacted with said T cell activator and/or said T cell growth factor for about 6 hours, about 12 hours, about 24 hours, about 48 hours or about 72 hours with The feeder cells are co-cultured.
  • 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-cultivating said TIL with said feeder cells comprises contacting the surface of said feeder cells with the surface of said TIL.
  • the co-cultivation of the TILs with the feeder cells comprises adding the feeder cells to the cell culture medium of the TILs.
  • said feeder cells are added to the cell culture medium of said TIL at a ratio of said feeder cells to said TIL of about 40:1 to about 400:1.
  • the T cell activator comprises one or more selected from the following group: cluster of differentiation 80 (CD80), CD86, CD276, 4-1BB ligand (4-1BBL), CD27, CD30 , CD134, CD275, CD40, CD258, and functionally active fragments thereof.
  • the T cell activator comprises an agonist of one or more targets selected from the group consisting of CD3, CD28, herpes virus entry medium (HVEM), CD40L, OX40, and 4-1BB.
  • HVEM herpes virus entry medium
  • CD40L CD40L
  • OX40 OX40
  • 4-1BB 4-1BB
  • the T cell activator comprises a CD3 agonist and/or a CD28 agonist.
  • the T cell activator comprises a CD3 agonist.
  • the T cell activator comprises an anti-CD3 antibody and/or an antigen-binding fragment thereof.
  • the T cell activator comprises a CD28 agonist.
  • the T cell activator comprises an anti-CD28 antibody and/or an antigen-binding fragment thereof, CD80 and/or a functionally active fragment thereof, and/or CD86 and/or a functionally active fragment thereof.
  • said contacting TILs with said T cell activator comprises one or more means selected from the group consisting of: (1) adding said T cell activator to said TIL cell culture (2) adding the engineered cells expressing the T cell activator to the cell culture medium of the TIL; and (3) adding the solid phase medium containing the T cell activator to the TIL in the cell culture medium.
  • the initial concentration of each of said T cell activators in said TIL cell culture medium is each independently at least about 30 ng/mL.
  • the initial concentration of each of said T cell activators in said TIL cell culture medium is each independently 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 about 1 nanometer to about 500 nanometers.
  • the diameter of the solid phase medium is measured by transmission electron microscopy.
  • the solid medium comprises a polymer
  • each of said T cell activators is contained in an amount of each independently at least about 25 ⁇ g per mg of said solid medium.
  • the solid phase medium comprising the T cell activator is added to the cell culture of the TIL at a ratio of the solid phase medium to the TIL of about 2:1 to about 1:2 Base.
  • the solid phase medium comprising the T cell activator is added to the cell culture of the TIL at a ratio of the solid phase medium to the TIL of about 1:100 to about 1:2000 Base.
  • said TIL is contacted with said T cell activator and said T cell growth factor substantially simultaneously.
  • the T cell growth factor is 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 factor comprises IL-2 and/or a functionally active fragment thereof.
  • contacting said TIL with said T cell growth factor comprises adding said T cell growth factor to the cell culture medium of said TIL.
  • the initial concentration of each of said T cell growth factors in said TIL cell culture medium is each independently at least about 300 IU/mL.
  • the TILs are selected from the group consisting of TILs derived from fragments of tumor tissue, TILs derived from fragments of lymphatic metastases, TILs derived from pleural effusions, TILs derived from peritoneal effusions and Derived from TILs thawed after cryopreservation.
  • the fragments have a volume from about 1 mm3 to about 27 mm3.
  • the present application also provides a method for culturing tumor infiltrating lymphocytes (TIL), the method comprising: reducing the expression and/or weakening the activity of at least one target gene of the TIL, and making the TILs were exposed to CD28 agonists.
  • TIL tumor infiltrating lymphocytes
  • the method comprises: reducing the expression and/or attenuating the activity of at least one target gene of the TIL after contacting the TIL with a CD28 agonist.
  • a method for culturing tumor-infiltrating lymphocytes comprising: reducing the expression and/or attenuating the activity of at least one target gene of the TIL, wherein the TIL comprises TIL obtained by contacting with a CD28 agonist .
  • a method of culturing tumor infiltrating lymphocytes comprising: contacting the TIL with a CD28 agonist, wherein the TIL comprises reducing the expression and/or activity of at least one target gene of the TIL Weaken acquired TIL.
  • the TIL obtained by reducing the expression and/or attenuating the activity of at least one target gene of said TIL exhibits improved TIL properties compared to TIL with unchanged expression and/or activity of the target gene .
  • said improved TIL properties comprise one or more selected from the group consisting of: increased TIL cell number and expansion capacity, increased live cell ratio, increased viability, improved T cell Subpopulation proportions, increased cytokine secretion capacity, increased tumor cell killing capacity, and increased T cell receptor (TCR) clonal diversity.
  • 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 proportion of activated T cells proportion, increased proportion of tumor-specific T cells, and increased proportion of stem cell-like T cells.
  • said TILs that have been exposed to said CD28 agonist during at least one in vitro expansion phase exhibit improved compared to corresponding TILs that have not been exposed to said CD28 agonist during an in vitro expansion phase. gene editing effects.
  • the improved gene editing effect comprises increased gene knockout efficiency.
  • said reducing the expression and/or attenuating the activity of at least one target gene of said TIL comprises introducing a gene regulation system into said TIL cells.
  • the gene regulation system is capable of disrupting the target gene at the DNA level.
  • the gene regulation system comprises a guide nucleic acid molecule and an enzyme protein.
  • said reducing the expression and/or weakening the activity of at least one target gene of said TIL comprises: combining the ribonucleoprotein complex (RNP) comprising said guide nucleic acid molecule and said enzyme protein ) is introduced into the TIL.
  • RNP ribonucleoprotein complex
  • the enzyme protein comprises a Cas protein, a Cas protein homologue, or a functionally active fragment thereof.
  • the guide nucleic acid molecule comprises a guide RNA (gRNA).
  • gRNA guide RNA
  • the guide nucleic acid molecule is capable of binding to the sequence of the target gene.
  • the target gene comprises a gene encoding an NF- ⁇ B pathway inhibitory molecule.
  • the NF- ⁇ B pathway inhibitory molecule is capable of ubiquitinating a protein selected from the group consisting of receptor interacting protein 1 (RIP1), mucosa-associated lymphoid tissue lymphoma translocator protein 1 (MALT1) , receptor interacting protein 2 (RIP2) and tumor necrosis factor receptor-associated factor 6 (TRAF6).
  • RIP1 receptor interacting protein 1
  • MALT1 mucosa-associated lymphoid tissue lymphoma translocator protein 1
  • RIP2 receptor interacting protein 2
  • TNF6 tumor necrosis factor receptor-associated factor 6
  • the NF- ⁇ B pathway inhibitory molecule comprises tumor necrosis factor alpha-inducible protein 3 (TNFAIP3).
  • the guide nucleic acid molecule can bind to the region where exon 2 and/or exon 7 of the TNFAIP3 gene is located or a fragment thereof.
  • the guide nucleic acid molecule is capable of binding to a region or a fragment thereof selected from the group consisting of SEQ ID NO: 34 to 47.
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the group consisting of: GGG, TGG, CGG and AGG.
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NO: 48 to 61.
  • the expression of the target gene is expressed in the TIL obtained by reducing the expression and/or weakening of the activity of at least one target gene of the TIL compared with the TIL in which the expression and/or activity of the target gene are not changed
  • the cell ratio of the product is reduced and/or the expression level of a single cell is reduced.
  • the proportion of cells expressing the target gene is about 95% or less.
  • TIL derived from tumor tissue and not expanded in vitro undergoes at least one stage of in vitro expansion, wherein, in at least one stage of said in vitro expansion, said TIL is combined with CD28 agonist exposure.
  • the TIL derived from tumor tissue and not expanded in vitro is subjected to the first stage of in vitro expansion and the second stage of in vitro expansion, and in the second stage of in vitro expansion , contacting said first stage in vitro expanded TILs with said CD28 agonist.
  • said first stage in vitro expansion is performed for at least about 7 days.
  • the first stage of in vitro expansion is performed for about 7 days to about 14 days.
  • said second stage in vitro expansion is performed for at least about 7 days.
  • said second stage in vitro expansion is performed for about 7 days to about 14 days.
  • the CD28 agonist comprises an anti-CD28 antibody and/or an antigen-binding fragment thereof, CD80 and/or a functionally active fragment thereof, and/or CD86 and/or a functionally active fragment thereof.
  • the method further comprises: subjecting TIL derived from tumor tissue and not expanded in vitro to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the The TILs are contacted with T cell activators other than the CD28 agonist.
  • said TILs are contacted with said other T cell activator during a single stage of said in vitro expansion.
  • said TIL in a single stage of said in vitro expansion, is reduced in expression and/or attenuated in activity of at least one target gene and said TIL is contacted with said other T cell activator .
  • 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 TILs are contacted with the other T cell activators.
  • said TIL is contacted with said CD28 agonist and said other T cell activator substantially simultaneously.
  • said other T cell activators comprise agonists of one or more targets selected from the group consisting of CD3, HVEM, CD40L, OX40 and 4-1BB.
  • said other T cell activator comprises a CD3 agonist.
  • said other T cell activator comprises an anti-CD3 antibody and/or an antigen-binding fragment thereof.
  • said contacting TILs with said CD28 agonist and said other T cell activators comprises one or more methods selected from the group consisting of: (1) contacting said CD28 agonist and said other T cell activators Adding the other T cell activators mentioned above to the cell culture medium of the TIL; (2) adding the engineered cells expressing the CD28 agonist and the other T cell activators to the cell culture medium of the TIL; (3) adding the solid phase medium containing the CD28 agonist and the other T cell activators to the cell culture medium of the TIL.
  • the initial concentration of said other T cell activator in said TIL cell culture medium is at least about 30 ng/mL.
  • the initial concentration of said other T cell activator in said TIL cell culture medium 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 about 1 nanometer to about 500 nanometers.
  • the diameter of the solid phase medium is measured by transmission electron microscopy.
  • the solid medium comprises a polymer
  • said solid medium comprises at least about 25 ⁇ g of said CD28 agonist and said other T cell activator per mg of said solid medium.
  • the solid phase medium comprising the CD28 agonist and the other T cell activators is added at a ratio of the solid phase medium to the TIL of about 2:1 to about 1:2 to the cell culture medium of the TIL.
  • the solid phase medium comprising the CD28 agonist and the other T cell activators is added at a ratio of the solid phase medium to the TIL of about 1:100 to about 1:2000 to the cell culture medium of the TIL.
  • the method further comprises: subjecting TIL derived from tumor tissue and not expanded in vitro to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the The TILs are co-cultured with feeder cells following exposure to the CD28 agonist for a certain period of time.
  • said TILs are co-cultured with said feeder cells in a single stage of said in vitro expansion.
  • said TILs are contacted with a CD28 agonist and said TILs are co-cultured with said feeder cells.
  • 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 TILs are co-cultured with the feeder cells.
  • said TILs are co-cultured with said feeder cells at least about 2 hours after contacting said TILs with said CD28 agonist.
  • said TILs are co-cultured with said feeder cells after contacting said CD28 agonist for about 6 hours to about 72 hours.
  • said TILs are co-cultured with said feeder cells about 12 hours to about 48 hours after contacting said CD28 agonist.
  • said TILs are co-cultured with said feeder cells after about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours of contacting with said CD28 agonist.
  • 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-cultivating said TIL with said feeder cells comprises contacting the surface of said feeder cells with the surface of said TIL.
  • the co-cultivation of the TILs with the feeder cells comprises adding the feeder cells to the cell culture medium of the TILs.
  • said feeder cells are added to the cell culture medium of said TIL at a ratio of said feeder cells to said TIL of about 40:1 to about 400:1.
  • the method further comprises: subjecting TIL derived from tumor tissue and not expanded in vitro to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, the The TILs are contacted with T cell growth factors.
  • said TIL is contacted with said T cell growth factor during a single stage of said in vitro expansion.
  • said TILs are contacted with said CD28 agonist and said T cell growth factor in a single stage of said in vitro expansion.
  • 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 TILs are contacted with T cell growth factors.
  • said TIL is contacted with said CD28 agonist and said T cell growth factor substantially simultaneously.
  • the T cell growth factor is 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 factor comprises IL-2 and/or a functionally active fragment thereof.
  • contacting said TIL with said T cell growth factor comprises adding said T cell growth factor to the cell culture medium of said TIL.
  • the initial concentration of each of said T cell growth factors in said TIL cell culture medium is each independently at least about 300 IU/mL.
  • the TILs are selected from the group consisting of TILs derived from fragments of tumor tissue, TILs derived from fragments of lymphatic metastases, TILs derived from pleural effusions, TILs derived from peritoneal effusions and Derived from TILs thawed after cryopreservation.
  • the fragments have a volume from about 1 mm3 to about 27 mm3.
  • TIL tumor infiltrating lymphocytes
  • step (A) contacting the first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • step (B) reducing the expression and/or attenuating the activity of at least one target gene of the second TIL population, and contacting the TILs with a CD28 agonist, wherein the third TIL population is obtained through the step (B) .
  • TIL tumor infiltrating lymphocytes
  • step (B) reducing the expression and/or attenuating the activity of at least one target gene of the second TIL population, and contacting the TILs with a CD28 agonist, wherein the third TIL population is obtained through the step (B) .
  • said population of TILs in vitro comprises a population of TILs obtained by contacting said first population of TILs with a T cell growth factor.
  • said population of TILs in vitro comprises a population of TILs obtained by cryopreservation of said first population of TILs.
  • said step (A) is performed for about 7 days to about 14 days.
  • said step (B) is performed for about 7 days to about 14 days.
  • the TIL obtained by reducing the expression and/or attenuating the activity of at least one target gene of said TIL exhibits improved TIL properties compared to TIL with unchanged expression and/or activity of the target gene .
  • said improved TIL properties comprise one or more selected from the group consisting of: increased TIL cell number and expansion capacity, increased live cell ratio, increased viability, improved T cell Subpopulation proportions, increased cytokine secretion capacity, increased tumor cell killing capacity, and increased T cell receptor (TCR) clonal diversity.
  • 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 proportion of activated T cells proportion, increased proportion of tumor-specific T cells, and increased proportion of stem cell-like T cells.
  • said TILs that have been exposed to said CD28 agonist during at least one in vitro expansion phase exhibit improved compared to corresponding TILs that have not been exposed to said CD28 agonist during an in vitro expansion phase. gene editing effects.
  • the improved gene editing effect comprises increased gene knockout efficiency.
  • said reducing the expression and/or attenuating the activity of at least one target gene of said TIL comprises introducing a gene regulation system into said TIL cells.
  • the gene regulation system is capable of disrupting the target gene at the DNA level.
  • the gene regulation system comprises a guide nucleic acid molecule and an enzyme protein.
  • said reducing the expression and/or weakening the activity of at least one target gene of said TIL comprises: combining the ribonucleoprotein complex (RNP) comprising said guide nucleic acid molecule and said enzyme protein ) is introduced into the TIL.
  • RNP ribonucleoprotein complex
  • the enzyme protein comprises a Cas protein, a Cas protein homologue, or a functionally active fragment thereof.
  • the guide nucleic acid molecule comprises a guide RNA (gRNA).
  • gRNA guide RNA
  • the guide nucleic acid molecule is capable of binding to the sequence of the target gene.
  • the target gene comprises a gene encoding an NF- ⁇ B pathway inhibitory molecule.
  • the NF- ⁇ B pathway inhibitory molecule is capable of ubiquitinating a protein selected from the group consisting of receptor interacting protein 1 (RIP1), mucosa-associated lymphoid tissue lymphoma translocator protein 1 (MALT1) , receptor interacting protein 2 (RIP2) and tumor necrosis factor receptor-associated factor 6 (TRAF6).
  • RIP1 receptor interacting protein 1
  • MALT1 mucosa-associated lymphoid tissue lymphoma translocator protein 1
  • RIP2 receptor interacting protein 2
  • TNF6 tumor necrosis factor receptor-associated factor 6
  • the NF- ⁇ B pathway inhibitory molecule comprises tumor necrosis factor alpha-inducible protein 3 (TNFAIP3).
  • the guide nucleic acid molecule can bind to the region where exon 2 and/or exon 7 of the TNFAIP3 gene is located or a fragment thereof.
  • the guide nucleic acid molecule is capable of binding to a region or a fragment thereof selected from the group consisting of SEQ ID NO: 34 to 47.
  • the guide nucleic acid molecule is capable of binding to a sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the protospacer adjacent motif (PAM) selected from the group consisting of: GGG, TGG, CGG and AGG.
  • PAM protospacer adjacent motif
  • the guide nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NO: 48 to 61.
  • the expression of the target gene is expressed in the TIL obtained by reducing the expression and/or weakening of the activity of at least one target gene of the TIL compared with the TIL in which the expression and/or activity of the target gene are not changed
  • the cell ratio of the product is reduced and/or the expression level of a single cell is reduced.
  • the proportion of cells expressing the target gene is about 95% or less.
  • the CD28 agonist comprises an anti-CD28 antibody and/or an antigen-binding fragment thereof, CD80 and/or a functionally active fragment thereof, and/or CD86 and/or a functionally active fragment thereof.
  • said TIL is contacted with said CD28 agonist and said other T cell activator substantially simultaneously.
  • said other T cell activators comprise agonists of one or more targets selected from the group consisting of CD3, HVEM, CD40L, OX40 and 4-1BB.
  • said other T cell activator comprises a CD3 agonist.
  • said other T cell activator comprises an anti-CD3 antibody and/or an antigen-binding fragment thereof.
  • said contacting TILs with said CD28 agonist and said other T cell activators comprises one or more methods selected from the group consisting of: (1) contacting said CD28 agonist and said other T cell activators Adding the other T cell activators mentioned above to the cell culture medium of the TIL; (2) adding the engineered cells expressing the CD28 agonist and the other T cell activators to the cell culture medium of the TIL; (3) adding the solid phase medium containing the CD28 agonist and the other T cell activators to the cell culture medium of the TIL.
  • the initial concentration of said other T cell activator in said TIL cell culture medium is at least about 30 ng/mL.
  • the initial concentration of said other T cell activator in said TIL cell culture medium 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 about 1 nanometer to about 500 nanometers.
  • the diameter of the solid phase medium is measured by transmission electron microscopy.
  • the solid medium comprises a polymer
  • said solid medium comprises at least about 25 ⁇ g of said CD28 agonist and said other T cell activator per mg of said solid medium.
  • the solid phase medium comprising the CD28 agonist and the other T cell activators is added at a ratio of the solid phase medium to the TIL of about 2:1 to about 1:2 to the cell culture medium of the TIL.
  • the solid phase medium comprising the CD28 agonist and the other T cell activators is added at a ratio of the solid phase medium to the TIL of about 1:100 to about 1:2000 to the cell culture medium of the TIL.
  • said TILs are co-cultured with said feeder cells at least about 2 hours after contacting said TILs with said CD28 agonist.
  • said TILs are co-cultured with said feeder cells after contacting said CD28 agonist for about 6 hours to about 72 hours.
  • said TILs are co-cultured with said feeder cells about 12 hours to about 48 hours after contacting said CD28 agonist.
  • said TILs are co-cultured with said feeder cells after about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours of contacting with said CD28 agonist.
  • 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-cultivating said TIL with said feeder cells comprises contacting the surface of said feeder cells with the surface of said TIL.
  • the co-cultivation of the TILs with the feeder cells comprises adding the feeder cells to the cell culture medium of the TILs.
  • said feeder cells are added to the cell culture medium of said TIL at a ratio of said feeder cells to said TIL of about 40:1 to about 400:1.
  • said TIL is contacted with said CD28 agonist and said T cell growth factor substantially simultaneously.
  • the T cell growth factor is 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 factor comprises IL-2 and/or a functionally active fragment thereof.
  • contacting said TIL with said T cell growth factor comprises adding said T cell growth factor to the cell culture medium of said TIL.
  • the initial concentration of each of said T cell growth factors in said TIL cell culture medium is each independently at least about 300 IU/mL.
  • the TILs are selected from the group consisting of TILs derived from fragments of tumor tissue, TILs derived from fragments of lymphatic metastases, TILs derived from pleural effusions, TILs derived from peritoneal effusions and Derived from TILs thawed after cryopreservation.
  • the fragments have a volume from about 1 mm3 to about 27 mm3.
  • the present application also provides a tumor infiltrating lymphocyte (TIL), which is obtained through the method of the present application.
  • TIL tumor infiltrating lymphocyte
  • the present application also provides a composition comprising the TIL of the present application.
  • the present application also provides a pharmaceutical composition, which comprises the TIL of the present application and/or the composition of the present application, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a method for affecting the growth of tumor cells, comprising administering the TIL of the present application, the composition of the present application and/or the pharmaceutical composition of the present application to a subject.
  • the present application also provides the application of the TIL of the present application, the composition of the present application and/or the pharmaceutical composition of the present application in the preparation of medicines, and the medicines are used for preventing and/or treating tumors.
  • the tumor is a solid tumor.
  • the tumor is selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.
  • Figure 1 shows the analysis results of the proliferation ability of TIL cultured with feeder cells at different addition times.
  • Figure 2 and Figure 3 show the proportion of CD45RA - CCR7 + central memory T cells (Tcm) in TIL cells cultured after adding OKT3 and IL-2 0 hours, 24 hours or 48 hours after adding feeder cells. .
  • Figure 4 shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells obtained by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • Figures 5 and 6 show the proportion of activated T cells in TIL cells cultured by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • Figure 7 shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells cultured by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • Figure 8 shows the ratio of TCF1 + stem cell-like T cells in TIL cells obtained by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • Figure 9 shows the analysis results of the proliferation ability of the test group and the control group added with different forms of CD28 agonists.
  • Figure 10 and Figure 11 respectively show the ratio of T cell subsets of TIL cells cultured in the mixed antibody group and the control group for TIL from different donors.
  • Figure 12 and Figure 13 respectively show the ratio of T cell subsets of TIL cells cultured in the magnetic bead group and the control group for TIL from different donors.
  • Figure 14 shows the ratio of T cell subsets of TIL cells cultured in the nano-matrix group and the control group.
  • Figure 15 shows the cell killing ability of TIL cells cultured in the nano-matrix group and the control group.
  • Figure 16 shows the detection results of intracellular factor expression in TIL cells cultured in the mixed antibody group and the control group.
  • Figure 17, Figure 18, Figure 19 and Figure 20 respectively show the detection results of intracellular factor expression of TIL cells cultured from the magnetic bead group and the control group for TIL from different donors.
  • Figure 21 shows the detection results of intracellular factor expression in TIL cells cultured in the nano-matrix group and the control group.
  • Figure 22 shows the results of cytokine secretion detection of TIL cells cultured in the nano-matrix group and the control group.
  • Fig. 23 shows the cytokine secretion detection results after co-incubating TIL cells and tumor cells obtained from the nano-matrix group and the control group.
  • Figure 24 and Figure 25 respectively show the gene knockout efficiency results of TIL cells cultured in the nano-matrix group and the control group for TILs from different donors.
  • Fig. 26, Fig. 27 and Fig. 28 respectively show the proliferative ability analysis results of the test groups subjected to in vitro expansion in different ways in the terminal stimulation stage for TILs from different donors.
  • Figure 29, Figure 30, Figure 31 and Figure 32 show, for TIL cells from different donors, the amount of fluorescence of each group of TIL cells after expansion without additional stimulation.
  • Figure 33 and Figure 34 show, for TIL cells derived from different donors, the amount of fluorescence after the expansion of TIL cells in each group stimulated by anti-CD3 antibody (Miltenyi Biotech, OKT3).
  • Figure 35 shows the test results of the killing ability of TIL cells derived from donor B co-cultured with tumor cells at an effect-to-target ratio of 1:1.
  • Figure 36 and Figure 37 show the test results of the killing ability of TIL cells derived from donor C co-cultured with tumor cells at an effect-to-target ratio of 1:1 and an effect-to-target ratio of 1:3.
  • Figure 38 and Figure 39 show, for TIL cells derived from different donors, the multiplication factor of long-term culture of TIL cells in each group after withdrawal of IL-2.
  • Figure 40 and Figure 41 show, for TIL cells from different donors, the long-term culture viability of TIL cells in each group after removing IL-2.
  • Figure 42 and Figure 43 show, for TIL cells derived from donor C, the ratio of TIM-3 positive and CD101 positive cells in TIL cells of each group. The results showed that the gene-edited TIL cells had a lower proportion of exhausted cells.
  • Figure 44 shows, for TIL cells derived from donor C, the ratio of CD45RA-negative CCR7-positive memory T cells (Tcm) among TIL cells in each group.
  • Figure 45, Figure 46, Figure 47 and Figure 48 show the expression of CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group for TIL cells from different donors under the condition of no stimulation cell ratio.
  • FIG. 49, Fig. 50, Fig. 51 and Fig. 52 show is, under the condition that CD3 antibody (Miltenyi Biotech, OKT3, CD3 antibody is pretreated with 30ng/mL 96-well plates) stimulated overnight, for TIL derived from different donors Cells, the proportion of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group.
  • CD3 antibody Miltenyi Biotech, OKT3, CD3 antibody is pretreated with 30ng/mL 96-well plates
  • Fig. 53, Fig. 54, Fig. 55 and Fig. 56 show the conditions under which phorbol-myristate-acetate (PMA, 25ng/ml) and ionomycin (Ionomycin, 1 ⁇ g/ml) stimulated overnight Bottom, for TIL cells from different donors, the proportion of CD107a, GZMB, TNF- ⁇ and IFN- ⁇ expressing cells in TIL cells of each group.
  • PMA phorbol-myristate-acetate
  • Ionomycin Ionomycin
  • Figure 57 and Figure 58 show the Shannon diversity index of TCR V ⁇ clones of CD4 + T cells and CD8 + T cells on day 8 and day 18 after gene editing of TIL cells.
  • Fig. 59A shows the results of cell proliferation ability of TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 59B shows the results of CD45RA - CCR7 + central memory T cell (Tcm) ratio of TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 59C shows the ratio of TCF1 + stem cell-like T cells in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours after adding feeder cells.
  • Figure 59D shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours after adding feeder cells.
  • Figure 59E shows the ratio of activated T cells (PD-1 + ) in the cultured TIL cells obtained by adding feeder cells to culture TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 59F shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells obtained by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 59G shows the proportion of activated T cells (CD28 + ) in TIL cells cultured by adding feeder cells after 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2.
  • Figure 59H shows the proportion of activated T cells (41BB + ) in the cultured TIL cells obtained by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 59I shows the proportion of activated T cells (CD25 + ) in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours after adding feeder cells.
  • Figure 59J shows the TILs cultured with feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours, and the detection results of intracellular factor expression of TIL cells obtained from culture.
  • Figure 59K shows the cytokine secretion detection results of TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 59L shows the results of TIL cultured with feeder cells after adding OKT3 and IL-2 for 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days, and the results of the cell proliferation ability of the cultured TIL cells picture.
  • Figure 59M shows the addition of feeder cells to culture TIL after adding OKT3 and IL-2 for 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days, and culture the resulting CD8 + T cells of TIL cells Proportion.
  • Figure 59N shows the CD45RO + CD62L + CD45RO + CD62L + CD45RO + CD62L + T cell ratio.
  • Figure 59O shows the TILs cultured with feeder cells after adding OKT3 and IL-2 at 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days, and the proportion of NK T cells in the cultured TIL cells .
  • Figure 59P shows the CD4 + CD25 + CD4 + CD25 + of TIL cells cultured after adding OKT3 and IL-2 at 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells to culture TIL. Foxp3 + regulatory T cell (Treg) ratio.
  • Figure 59Q shows the results of cell killing ability of TIL cells cultured by adding feeder cells 48 hours after adding OKT3 and IL-2.
  • Figure 60A shows, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group under the non-stimulation detection condition.
  • Fig. 60B shows, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group under the stimulation detection condition of anti-CD3 antibody.
  • Figure 60C shows the results of long-term proliferation/persistence of TIL cells after withdrawal of IL-2.
  • Figure 60D shows the killing effect of the gene-edited TIL cells of the present application on the tumor cell line matched with the TIL donor tumor type.
  • Figure 60E shows the killing effect of the gene-edited TIL cells of the present application on a tumor cell line unmatched with the tumor type of the TIL donor.
  • Fig. 60F shows, for TIL cells from different donors, the percentages of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group under the condition of no stimulation test.
  • Figure 60G shows the proportion of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group under the condition of anti-CD3 antibody stimulation overnight, for TIL cells from different donors.
  • Figure 60H shows, for TIL cells derived from different donors, the ratio of expression of different exhaustion marker molecules in TIL cells of each group.
  • Figure 60I shows, for TIL cells derived from different donors, the proportion of central memory cells (CD45RO positive CD62L positive) among TIL cells in each group.
  • Figure 60J shows the results of the apoptosis detection of TIL cells derived from donor 709.
  • NF- ⁇ B pathway inhibitory molecule generally refers to an inhibitory molecule of a signaling pathway.
  • NF- ⁇ B pathway inhibitory molecules can achieve the function of inhibiting the transmission of signal pathways by ubiquitinating target proteins.
  • NF- ⁇ B pathway inhibitory molecules can ubiquitinate the following proteins: receptor interacting protein 1 (RIP1, UniProt accession number Q13546), mucosa-associated lymphoid tissue lymphoma translocator protein 1 (MALT1, UniProt accession number Q9UDY8), Receptor Interacting Protein 2 (RIP2, UniProt Accession No. O43353) and Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6, UniProt Accession No. Q9Y4K3).
  • receptor interacting protein 1 RIP1, UniProt accession number Q13546
  • MALT1, UniProt accession number Q9UDY8 mucosa-associated lymphoid tissue lymphoma translocator protein 1
  • MALT1 UniProt
  • TNFAIP3 tumor necrosis factor alpha-inducible protein 3
  • TNFAIP3 can ubiquitinate signal transduction substances of NF- ⁇ B pathway.
  • the UniProt accession number for TNFAIP3 could be P21580.
  • TNFAIP3 may encompass unprocessed TNFAIP3, any form of processed TNFAIP3, variants of TNFAIP3 or substances comprising functionally active fragments of TNFAIP3.
  • a gene regulatory system generally refers to a system that regulates the expression or activity of a target gene.
  • a gene regulatory system can comprise a gene regulatory molecule.
  • the gene regulatory system can regulate the expression or activity of a gene, such as making the gene in an inactivated or activated state, increasing or decreasing the number of the gene, making the gene in a state of increasing or decreasing the amount of transcription, and/or making the gene
  • the transcription product of the gene is in an inactivated or activated state; for example, the gene regulatory system can regulate the expression or activity of the gene, such as increasing or decreasing the amount of the expression product of the gene in a single cell, and/or making the expression of the gene
  • the number of cells expressing the product is increased or decreased.
  • guide nucleic acid molecule generally refers to a nucleic acid molecule that can be used for gene editing.
  • a guide nucleic acid molecule can provide information on insertion or deletion of nucleotides to guide the editing process.
  • the guide nucleic acid molecule can be a guide RNA or guide RNA (guide RNA, gRNA).
  • guide RNA guide RNA
  • gRNA may refer to an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific location within the target DNA.
  • perfect complementarity may not be required, e.g., as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex.
  • an enzyme protein generally refers to a protein with enzymatic activity.
  • an enzyme protein may refer to a Cas protein.
  • a Cas protein can contain at least one RNA recognition or binding domain that can interact with a gRNA.
  • Cas proteins can also comprise nuclease domains (e.g., DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and/or other structures area.
  • a nuclease domain may have catalytic activity for nucleic acid cleavage. Cleavage can include the breaking of covalent bonds in nucleic acid molecules.
  • the Cas protein can be a wild-type protein (ie, a naturally occurring protein), a modified Cas protein (ie, a Cas protein variant), or a fragment of a wild-type or modified Cas protein.
  • the Cas protein can also be an active variant or fragment of a wild-type or modified Cas protein.
  • Cas protein may cover unprocessed Cas protein, any form of processed Cas protein, variants of Cas protein or substances comprising functionally active fragments of Cas protein.
  • ribonucleoprotein complex generally refers to a complex formed by a protein and nucleic acid.
  • a protein in a ribonucleoprotein complex can have nuclease activity.
  • ribonucleoprotein complexes can cut target sequences under the guidance of nucleic acids therein.
  • the ribonucleoprotein complex can be a complex formed by Cas protein and gRNA.
  • exon generally refers to the portion of a gene that can be expressed as a protein.
  • an exon may refer to the ability to be expressed as a protein during protein biosynthesis.
  • splicing an exon sequence of a target gene can reduce the activity or function of the target gene.
  • the term "protospacer adjacent motif (PAM)” generally refers to the short sequence following the target sequence.
  • PAM sequence can be used to determine the position of cleavage.
  • those skilled in the art can easily determine the position of a suitable target sequence, and can easily design a gRNA sequence for cutting the target sequence.
  • the term "decreased expression” generally refers to a decrease in the expression level of a product or its gene and/or a decrease in the proportion of cells capable of expressing the product. For example, there may be a decrease in the amount of cells expressing the product of the gene or a decrease in the proportion of cells that contain the product of the gene expression, or a decrease in the proportion of cells that secrete the product of the gene expression.
  • the decrease in the expression of the gene can be indirectly indicated by detecting the knockout amount of the gene in the genome of the cell. For example, by detecting the proportion of cells in which the gene is knocked out in a cell population, it can be indirectly indicated that the expression of the gene is reduced.
  • the term "activity" generally refers to the biological function of a substance.
  • the activity of a gene can refer to the transcriptional and/or translational state of the gene.
  • the weakening of the activity of a gene may mean that the transcriptional function of the gene is weakened, the gene cannot be normally transcribed, or the function of the transcription product of the gene is inhibited.
  • CD80 generally refers to a cell stimulating molecule.
  • CD80 can be a ligand for CD28.
  • CD80 can be found at GenBank Accession No. P33681.
  • the CD80 protein of the present application may also cover its functionally active fragments, not limited to substances containing functionally active fragments of CD80 produced after processing and/or modification in cells.
  • CD80 of the present application may comprise functionally active fragments of CD80 and other arbitrary structural domains.
  • CD86 generally refers to a cell stimulating molecule.
  • CD86 can be a ligand for CD28.
  • CD86 can be found at GenBank Accession No. P42081.
  • the CD86 protein of the present application may also cover its functionally active fragments, not limited to substances containing functionally active fragments of CD86 produced after processing and/or modification in cells.
  • CD86 of the present application may comprise a functionally active fragment of CD86 and other arbitrary structural domains.
  • secretion generally means that a substance can be localized extracellularly of a cell.
  • secreted substances can be transported to the extracellular space of the cell after intracellular synthesis.
  • whether a substance is secreted can be tested by an enzyme-linked immunosorbent assay or other detection method.
  • T cell receptor generally refers to a complex of membrane proteins involved in the activation of T cells in response to the presentation of an antigen.
  • TCRs may be responsible for recognizing antigens bound to major histocompatibility complex molecules.
  • TCRs can consist of heterodimers of alpha ( ⁇ ) and beta ( ⁇ ) chains, or of gamma and delta ( ⁇ / ⁇ ) chains.
  • TCRs can exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions.
  • a TCR can be a TCR that is modified on any cell that expresses a TCR.
  • the type of TCR can be analyzed by a TCR subtype analysis reagent.
  • the term "clonal diversity” generally refers to a substance having multiple clonotypes.
  • the clonal diversity of TCRs may mean that TCRs may have different sequence structures and/or antigen recognition abilities.
  • the diversity of TCR is often distinguished by ⁇ chain subtypes, which can include V ⁇ 23, V ⁇ 7.2, V ⁇ 5.2, V ⁇ 11, V ⁇ 16, V ⁇ 3, etc. When a T cell population has more ⁇ chain subtypes, it can be This T cell population is thought to have a higher clonal diversity.
  • CD4 + cells generally refers to CD4 positive cells, such as T cells.
  • the terms “CD4 + cells”, “CD4 positive cells” may be used synonymously.
  • These cells can be identified by methods known in the art, such as by staining the cells with a fluorescently labeled antibody directed against CD4 and using fluorescence activated cell sorting.
  • existing data can prove that an increase in the proportion of CD4 + cells can increase the ability of the cell population to secrete IFN- ⁇ and/or TNF, and can improve the effect of T cell populations in promoting tumor suppression.
  • Tay, RE, Richardson, EK et al. 2020. Cancer Gene Therapy, 1-13.
  • this application can provide a method to affect CD4 + cells proportional method.
  • CD8 + cells generally refer to CD8 positive cells, such as T cells.
  • CD8 + cells CD8 positive cells
  • CD8 positive cells may be used synonymously. These cells can be identified by methods known in the art, such as by staining the cells with a fluorescently labeled antibody directed against CD8 and using fluorescence activated cell sorting.
  • IC50 value or “IC50 value” generally refers to the concentration of a target substance required to obtain 50% inhibition of a biological process. IC50 values can be converted to absolute inhibition constants (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol. (1973) 22:3099).
  • KD value or “KD value” generally refers to the dissociation constant, which can be determined by surface plasmon resonance.
  • surface plasmon resonance analysis uses a BIAcore system (Pharmacia Biosensor, Piscataway, NJ) to measure ligands (substances immobilized on a biosensor substrate) and analytes (substances in solution) by surface plasmon resonance (SPR). ) real-time binding interactions.
  • SPR surface plasmon resonance
  • SPR surface plasmon resonance
  • Surface plasmon analysis can also be performed by immobilizing the analyte (substance on the biosensor substrate) and presenting the ligand.
  • the term "encoding” generally refers to the ability to directly or indirectly deduce the structure or composition information of another type of molecule related to it according to basically definite rules.
  • the nucleotide sequence can be deduced from the amino acid sequence, for example, based on the characteristics of deoxyribonucleic acid transcription complementary nucleic acid, including nucleic acid that can be translated into a polypeptide.
  • deoxyribonucleic acid can encode RNA transcribed from deoxyribonucleic acid.
  • a deoxyribonucleic acid may similarly encode a polypeptide translated from RNA transcribed from the deoxyribonucleic acid.
  • small molecule compound generally refers to peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, molecular weight less than about 10,000 gram/mole of organic or inorganic substances (i.e. including heterogeneous organic and organometallic compounds), organic or inorganic substances of molecular weight less than about 5,000 g/mole, organic or inorganic substances of molecular weight less than about 1,000 g/mole, molecular weight of less than about 500 Gram/mole organic or inorganic substances, as well as salts, esters and other pharmaceutically acceptable forms of such drugs.
  • organic or inorganic substances i.e. including heterogeneous organic and organometallic compounds
  • NK cell is also called “natural killer cell”, and generally refers to a cell with large granules in the cytoplasm.
  • NK cells are developed from bone marrow lymphoid stem cells and can differentiate and develop depending on the bone marrow or thymus microenvironment.
  • the proportion of NK cells in TIL cells can be changed by the method of this application.
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies.
  • the term “antibody” also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specifically bind CD3). Typically, such fragments will include the antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 that can be combined with the J chain to form a multivalent A basic 4-chain unit for combinations.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site.
  • immunoglobulins can be assigned to different classes, or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
  • the term "antigen-binding fragment” generally refers to one or more polypeptide fragments that have the ability to specifically bind an antigen (eg, CD3).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • solid phase medium generally refers to a solid phase material that incorporates a function.
  • a solid phase medium in this application may refer to a material that binds one or more substances in the medium and/or on the surface of the medium through covalent bonding and/or non-covalent bonding.
  • the solid-phase medium of the present application may refer to a medium in which CD28 antibody or its antigen-binding fragment and CD3 antibody or its antigen-binding fragment are bound in the medium and/or on the surface of the medium through covalent binding and/or non-covalent binding.
  • the solid phase medium of the present application may be a polymeric material.
  • the term "expression” generally refers to the process of transcription and/or translation of a gene encoding a target polypeptide within a cell.
  • the level of transcription of a gene encoding a polypeptide of interest in a host cell can be determined by measuring the amount of corresponding mRNA present in the cell. For example, mRNA transcribed from a gene encoding a polypeptide of interest can be quantitatively measured by PCR or by RNA hybridization.
  • the level of translation of a gene encoding a polypeptide of interest can be measured by various methods, such as by ELISA, by a polypeptide bioactivity assay, or by Western blot or radioimmunoassay.
  • expression may generally also refer to the transcription and/or translation process by which the product occurs.
  • expression of a cytokine can be the process by which a cell transcribes and/or translates the cytokine.
  • expression of a cytokine can be determined by detecting the amount of the corresponding mRNA present in the cell or by detecting the amount of the cytokine produced by the cell, or both.
  • stage in the term “one-stage in vitro expansion”, “single-stage in vitro expansion”, or “first-stage in vitro expansion” generally refers to a period of expansion that TIL undergoes in vitro process.
  • each stage can be divided by changes in the number of TIL cells.
  • the number of TIL cells increases by at least about 1-fold, the TIL cells can be considered to have entered the next stage.
  • each stage can also be divided by the conditions of TIL cell culture.
  • T cell activators and/or T cell growth factors when added or supplemented to the cell culture medium, it can be considered that the TIL cells enter the next stage of in vitro expansion. In one embodiment, after the TIL cells are centrifuged and/or washed, the TIL cells can be considered to enter the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the days of TIL cell culture.
  • TIL cells when the TIL cells are cultured in vitro for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, After about 50 days or about 100 days, TIL cells can be considered to enter the next stage of in vitro expansion.
  • the term "the first stage of in vitro expansion” generally refers to the stage of expansion using T cell growth factors after primary TILs are obtained from tissues.
  • the tissue of the present application can be selected from the following group: tumor tissue and pleural effusion, and the pleural effusion of the present application can be the pleural effusion of a patient with metastatic cancer.
  • the amplification of the present application may be autologous or allogeneic in vivo amplification, or may be in vitro amplification.
  • the first stage of in vitro expansion of the present application may also be referred to as preREP (Pre-Rapid Expansion) stage.
  • TILs derived from tumor tissue and not expanded in vitro can be referred to as the first population of TILs.
  • the TILs obtained through the first stage of in vitro expansion in the culture method of the present application divided by the two-step method can be called the second TIL population.
  • the term "second-stage in vitro expansion” generally refers to the stage of expanding again after the tissue taken from the subject is expanded.
  • the number of TIL cells expanded in vitro by the second stage of the present application is increased, for example, can be increased by at least about 10 times (or at least about 20, 30, 40, 50, 60, 70, 80 or 90 fold), or in one embodiment the number of cells may be increased by at least about 100 fold.
  • the culture conditions of the second-stage in vitro expansion may be different from those of the first-stage in vitro expansion, for example, the culture substances added may be different.
  • the second stage of in vitro expansion in the culture method of the present application divided by the two-step method can also be called the REP (Rapid Expansion) stage.
  • the TILs obtained through the second stage of in vitro expansion in the culture method of the present application divided by the two-step method can be called the third TIL population.
  • in vivo generally refers to an event that occurs in the body of a subject.
  • in vitro generally refers to events that occur outside the body of a subject.
  • ex vivo generally refers to an event involving treatment or surgery on cells, tissues and/or organs that have been removed from a subject.
  • the cells, tissues and/or organs can be returned to the body of the subject by surgery or therapy.
  • secretion capacity generally refers to the ability of a cell to express a polypeptide or protein and transfer the polypeptide or protein of the present application to the extracellular environment.
  • irradiation generally refers to the treatment of matter by radiation.
  • irradiating may refer to irradiating a substance with X-rays, ⁇ -rays, ⁇ -rays or ⁇ -rays.
  • engineered cell generally refers to a cell that has been genetically modified by adding additional genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • engineered cells can be genetically modified to express TILs of T cell activators and/or T cell growth factors of the present application.
  • co-culture generally refers to the cultivation of two or more different populations of cells with some degree of contact between them.
  • the "contact" of two or more different populations of cells of the present application may, in one embodiment, be through direct contact, ie, wherein cells of one population are in direct physical contact with cells of another population. Or in one embodiment it may be through indirect contact mediated by a common culture medium.
  • the common culture medium of the present application may contain metabolites produced and released by at least one population of co-cultured cells and be used to culture another population of cells.
  • one or more feeder cells, T cell activators and/or T cell growth factors can be added to the medium of TIL cells by direct contact, for example, one or more feeder cells can be added Culture medium for cells, T cell activators and/or T cell growth factors is added to and/or replaces the medium for TIL cells, e.g., one or more feeder cells, T cell activators and/or T cell growth factors may be added to The culture medium of factors is used for culturing TIL cells; in one embodiment, it can be used for culturing TIL cells through indirect contact, for example, metabolites produced and released by feeder cells.
  • the term "mixture” generally refers to a combination of two or more different substances.
  • the CD28 antibody or antigen-binding fragment thereof of the present application and the CD3 antibody or antigen-binding fragment thereof can be added to the cell culture medium as a mixture after mixing.
  • the terms “concurrent contact”, “co-contact”, “simultaneously with”, “simultaneously” and “commonly” generally refer to the administration of two or more substances to a subject and/or cell such that the substances Also present in the subject and/or in the environment of the cell culture.
  • Simultaneous contacting can include simultaneous administration in different compositions, administration in different compositions at different times, or administration in a composition in which two or more active pharmaceutical ingredients are present.
  • “simultaneous contacting” in this application may generally refer to substantially simultaneous contacting.
  • the term “expansion” generally refers to a several-fold increase in the number of cells over a period of time.
  • the number of cells can be increased by at least about 3-fold (or 4, 5, 6, 7, 8 or 9-fold), in one embodiment the number of cells can be increased by at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80 or 90 fold), or in one embodiment the number of cells may be increased by at least about 100 fold.
  • the term “expanded” generally means that the cells of the present application have undergone one or more expansions as described above.
  • polymer generally refers to a molecule consisting of individual chemical moieties linked together, the polymer moieties herein being the same or different.
  • the term “polymer” may refer to individual chemical moieties joined end to end to form a linear molecule, as well as individual chemical moieties linked together in branched (eg, "multi-armed” or "star") structures .
  • the polymer may include, for example, a polysaccharide, dextran, hydrogel, polyethylene glycol, or poloxamer.
  • Poloxamers are nonionic triblock copolymers having a polyoxypropylene (poly(propylene oxide)) central hydrophobic chain flanked by two polyoxyethylene (poly(ethylene oxide)) hydrophilic chains.
  • the materials encompassed herein may be formulated with, or administered with, any polymer described herein or known in the art.
  • chimeric antibody generally refers to an antibody in which the variable region of a murine antibody is fused with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • a chimeric antibody you can establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and clone the constant region gene of the human antibody as needed, and combine the mouse variable region gene with the Human constant region genes are connected into chimeric genes and then inserted into expression vectors to express chimeric antibody molecules in eukaryotic or prokaryotic systems.
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) usually refers to the antibody variable region framework grafted to the human antibody CDR sequence, that is, different Types of antibodies produced in the framework sequences of human germline antibodies. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database.
  • the term "fully human antibody”, “fully human antibody” or “fully human antibody”, also known as “fully human monoclonal antibody”, the variable region and constant region of the antibody can be human Source, remove immunogenicity and toxic side effects.
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the antibody or ligand described in this application may be a fully human monoclonal antibody.
  • the relevant technologies for the preparation of fully human antibodies can be: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.
  • CDR generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • One of the most commonly used definitions of the 6 CDRs can be given by Kabat E.A. et al., Chothia et al. and MacCallum et al.
  • the Kabat definition of CDR can be applied to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and the heavy chain variable domain CDR1, CDR2 and CDR3 (CDR H1, CDR H2, CDR H3 or H1, H2, H3).
  • anti-CD3 antibody generally refers to CD3-targeting antibodies or variants thereof, such as monoclonal antibodies, including human, humanized, chimeric or murine antibodies, which are directed against mature T-cell T cells CD3 receptor among antigen receptors.
  • Anti-CD3 antibodies can include OKT-3.
  • Anti-CD3 antibodies can include SP34.
  • Anti-CD3 antibodies can also include other anti-CD3 antibodies including, for example, otelixizumab, teplizumab, and visilizumab in one embodiment.
  • IL-2 or "IL2” generally refers to the T-cell growth factor known as interleukin 2, and includes all forms of IL-2, which can include in one embodiment humans and mammals forms, conservative amino acid substitutions, glycoform modifications or variants, or active fragments thereof.
  • the GeneID of the gene encoding IL-2 may be 3558.
  • the term "antigen presenting cell”, “antigen presenting cell”, or “APC” generally refers to an immune cell displaying on its surface exogenous antigen complexed with major histocompatibility complex (MHC).
  • Systemic cells such as accessory cells (eg, B cells, dendritic cells, etc.).
  • T cells can recognize these complexes using their T cell receptor (TCR).
  • APCs can process antigens and present them to T cells.
  • the antigen presenting cells may comprise a group selected from peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.
  • TIL characteristics generally refers to the characteristics of TIL cells obtained through the cultivation method of this application. Changes in TIL properties can include: increased number of TIL cells, increased proportion of viable cells, increased viability, improved proportion of T cell subsets, increased cytokine secretion, increased tumor cell killing capacity, increased T cell Receptor (TCR) clonal diversity and increased number of TIL cells in tissue, or any combination thereof. The changes in this application can be increased or decreased.
  • an increase in the viability of TIL cells can refer to an increase in the time that TIL cells exist in vivo.
  • increased viability can refer to an increase in the time a cell persists in a tissue of a subject, such as a tumor, spleen, bone marrow, lung tissue, and blood.
  • nanoparticle generally refers to microscopic particles having at least one dimension smaller than 100 nm.
  • nanoparticles have a diameter in the range of 50 nm to 500 nm (ie, 0.05 ⁇ m to 0.5 ⁇ m); are structurally stable in physiological environments; and can accommodate smaller molecules (such as drugs or other bioactive agents), which can then be Deliver to desired site.
  • a nanoparticle of the present application may comprise a CD28 antibody or an antigen-binding fragment thereof.
  • a nanoparticle of the present application may comprise a CD28 antibody or an antigen-binding fragment thereof and a CD3 antibody or an antigen-binding fragment thereof.
  • an anti-CD3 antibody can include OKT3.
  • an anti-CD28 antibody can include 15E8.
  • artificial antigen-presenting cell generally refers to the artificially constructed immune cells used to present foreign antigens, for example, the way of presenting foreign antigens can be that the surface of artificial antigen-presenting cells contains foreign Histocompatibility complex (MHC) complex.
  • MHC foreign Histocompatibility complex
  • isolated artificial antigen-presenting cells may be included, which may contain genes expressing HLA-A/B/C (the gene GeneID encoding it may be 3105, 3106, or 3107), CD64 (the gene encoding it GeneID can be 2209), CD80 (the gene GeneID encoding it can be 941), ICOS-L (the gene GeneID encoding it can be 23308) and CD58 (the gene GeneID encoding it can be 965), and can be modified To express more than one T cell activator, the above of the present application may contain this number.
  • fusion protein generally refers to an amino acid sequence comprising a first polypeptide or protein or a fragment, analog or derivative thereof and a heterologous polypeptide or protein (i.e., different from the first polypeptide or protein or the amino acid sequence of a second polypeptide or protein, or a fragment, analog or derivative thereof, or generally not part of the first polypeptide or protein, or a fragment, analog or derivative thereof) of a fragment, analog or derivative thereof peptide or protein.
  • a fusion protein may comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide.
  • the heterologous protein, polypeptide or peptide of the present application may or may not be different types of preventive or therapeutic drugs.
  • two different proteins, polypeptides or peptides with immunomodulatory activity can be fused together to form a fusion protein.
  • the fusion protein may retain or increase activity compared to the activity of the original polypeptide or protein prior to fusion of the heterologous protein, polypeptide or protein.
  • the fusion protein of the present application may be a fusion protein fused with a CD28 antibody or an antigen-binding fragment thereof and a CD3 antibody or an antigen-binding fragment thereof.
  • the term "killing ability" generally means that the target cells are killed by contacting the cells of the present application with an effective amount of substances.
  • the substance of the present application may be TIL cells. Killing in the present application may include killing cells by itself or promoting CDC, apoptosis, ADCC, and/or phagocytosis of other cells or substances, or by a combination of two or more of these mechanisms.
  • administering generally refers to delivering a substance to a subject in need thereof by any route known in the art.
  • Pharmaceutically acceptable carriers and formulations or compositions are also well known in the art. Routes of administration may include: intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral and/or mucosal.
  • kit generally refers to two or more components packaged together in a container, receptacle or other container, one of which corresponds to the substance of the present application.
  • TIL cells of the present application are included.
  • the term "subject” generally refers to a cell or an animal, which can be a mammal such as a human, a non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), a domestic animal (dog and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs) and laboratory animals (mice, rats, rabbits, guinea pigs).
  • Human subjects include fetal, neonatal, infant, adolescent and adult subjects.
  • Subjects include animal disease models, such as tumor animal models, and other animal models known to those skilled in the art.
  • feeder generally refers to a cultured cell that can be used to support the growth of another cell of interest in culture. For example, by growing in vitro and secreting at least one factor into the culture medium.
  • feeder cells may include antigen presenting cells.
  • the term "specific binding” generally refers to a binding substance that recognizes a specific target substance, but does not substantially recognize or bind to other molecules in a sample.
  • a binding substance can specifically bind the specific target substance of the application from one species
  • the binding substance of the present application can also specifically bind the target substance of the application from one or more other species or homologous target substances. This cross-species reactivity by itself may not alter the classification of the binding substance as specific.
  • a binding substance that specifically binds to a target substance may also bind to a different allelic form of the target substance.
  • complete culture process generally refers to the complete process of separating cells from tumor tissues isolated from patients, expanding them once or more, and finally obtaining cells that can be administered to subjects .
  • cell culture medium generally refers to a nutrient solution in which cells, such as mammalian cells, are grown.
  • the formulation of cell culture media is well known in the art.
  • cell culture media include buffers, salts, carbohydrates, amino acids, vitamins and necessary trace elements.
  • Cell culture media may or may not contain serum, peptone, and/or protein.
  • Cell culture media can be supplemented with additional components or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, etc., depending on the cells to be cultured requirements and/or desired cell culture parameters.
  • the term "pharmaceutical composition” or “pharmaceutical preparation” generally refers to a preparation, which may allow the biological activity of the active ingredient to be effective, and may not contain any substances that are harmful to the subject to which the preparation will be administered.
  • the tester was unacceptably toxic for additional components.
  • Such preparations are sterile.
  • “Pharmaceutically acceptable” excipients carriers, additives are those which can reasonably be administered to a subject mammal to provide an effective dosage of the active ingredient employed.
  • TIL tumor infiltrating lymphocytes
  • TILs may include, but are not limited to, CD8 + cytotoxic T cells (lymphocytes), Th1 and Th17CD4 + T cells, natural killer cells, dendritic cells, and M1 macrophages.
  • TILs can include primary TILs and secondary TILs.
  • Primary TILs can be those TIL cells obtained from a tissue sample of a subject, and "secondary TILs" can be any population of TILs that have been or have been expanded in this application.
  • the tumor-infiltrating lymphocytes of the present application may not be isolated and purified, or may infiltrate with tumor cells.
  • TIL in the present application may refer to a group of TILs.
  • central memory T cells generally refers to T cells that have long-term memory and are capable of receiving antigen restimulation.
  • Central memory T cells may have the phenotype of CD45RA - CCR7 + or CD45RO + CD62L + , for example, central memory T cells may be identified by CD45RA - and CCR7 + , or central memory T cells may be identified by CD45RO + and CD62L + .
  • Central memory T cells can have a stronger ability to resist tumor growth than ordinary T cells.
  • regulatory T cells generally refers to a subset of T cells that control autoimmune reactivity in vivo. Regulatory T cells may have a phenotype of CD4 + CD25 + Foxp3 + , for example, regulatory T cells may be identified by CD4 + , CD25 + and Foxp3 + . Regulatory T cells may have the ability to suppress the anti-tumor growth of T cells.
  • activated T cells generally refers to T cells that have been activated to have the ability to resist tumor growth.
  • Activated T cells may have a phenotype of PD1 + , LAG3 + or CD28 + , for example, activated T cells may be identified by PD1 + , LAG3 + or CD28 + .
  • Activated T cells may have the ability to fight tumor growth.
  • tumor-specific T cells generally refers to T cells that can specifically fight tumor growth.
  • Tumor-specific T cells may have a phenotype of CD103 + CD39 + , for example, tumor-specific T cells may be identified by CD103 + and CD39 + .
  • Tumor-specific T cells can have a more specific ability to resist tumor growth than ordinary T cells.
  • stem cell-like T cell generally refers to a type of T cell that may have the potential of self-proliferation and/or differentiation.
  • Stem cell-like T cells may have a TCF1 + phenotype, for example, stem cell-like T cells may be identified by TCF1 + .
  • Tumor-specific T cells may have stronger and/or longer-term anti-tumor growth capabilities than ordinary T cells.
  • tumor fragments generally refers to tumor fragments that can be formed by mechanical disruption, enzymatic hydrolysis and/or other disruption methods after the tumor tissue is removed from the subject.
  • composition or “pharmaceutical composition” generally refers to at least one cell and at least one and optionally more than one other pharmaceutically acceptable chemical components such as carrier, stabilizer , diluents, dispersants, suspending agents, thickeners and/or mixtures of excipients.
  • the term "pharmaceutically acceptable carrier” generally refers to one or more non-toxic materials that do not interfere with the active ingredient.
  • a pharmaceutically acceptable carrier may not interfere with the biological activity of the active ingredient; for example, a pharmaceutically acceptable carrier may not interfere with the effectiveness of the biological activity possessed by the active ingredient.
  • Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for human administration.
  • contemplated carriers, excipients, and/or additives may include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids , protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), etc.
  • pharmaceutically acceptable carrier carrier
  • carrier can be understood as a carrier (vector) that does not include the nucleic acid form used in genetic engineering.
  • the term "functionally active fragment” generally refers to a fragment that has a partial region of a full-length protein or nucleic acid, but retains or partially retains the biological activity or function of the full-length protein or nucleic acid.
  • a functionally active fragment may retain or partially retain the ability of the full-length protein to bind another molecule.
  • the functionally active fragment of the growth factor IL-2 may retain or partially retain the biologically active function of the full-length IL-2 to cause cell proliferation.
  • T cell activator generally refers to a substance that binds to the corresponding binding receptor on T cells and mediates costimulatory responses of T cells.
  • T cell activators may be substances other than antigen receptors that are required for T cells to mount an effective immune response.
  • a T cell activator may refer to a T cell co-stimulatory molecule.
  • the T cell activator of the present application may comprise its variant, homologue or any substance comprising its functionally active fragment.
  • T cell activators may include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), NK cell activating receptors, BTLA (the gene GeneID encoding it can be 151888), Toll ligand receptor, OX40 (the gene GeneID encoding it can be 7293), CD2 (the gene GeneID encoding it can be 914), CD7 (the gene GeneID encoding it can be 914), CD7 (the gene GeneID encoding it can be 924), CD27 (the gene GeneID encoding it can be 939), CD28 (the gene GeneID encoding it can be 940), CD30 (the gene GeneID encoding it can be 943), CD40 (the gene GeneID encoding it can be 958 ), CDS, ICAM-1 (the gene GeneID encoding it can be 3383), LFA-1 (CD11a/CD18) (the gene
  • T cell growth factor generally refers to a biologically active polypeptide or small molecule compound that causes cell proliferation.
  • the T cell growth factor of the present application may comprise its variant, homologue or any substance comprising its functionally active fragment.
  • the T cell growth factor can be selected from one or more of the following groups: IL-2 (the gene GeneID encoding it can be 3558), IL-4 (the gene GeneID encoding it can be 3565) , IL-6 (the gene GeneID encoding it can be 3569), IL-7 (the gene GeneID encoding it can be 3574), IL-10 (the gene GeneID encoding it can be 3586), IL-12 (the gene GeneID encoding it can be 3586), IL-12 (the gene GeneID encoding it can be 3586), Gene GeneID can be 3592 or 3593), IL-15 (the gene GeneID encoding it can be 3600), IL-21 (the gene GeneID encoding it can be 59067), TNF- ⁇ (the gene GeneID encoding it can be 100137091) , gamma interferon (the gene GeneID encoding it can be 3458) and the like.
  • IL-2 the gene GeneID encoding it can be 3558
  • IL-4 the gene
  • substantially simultaneously usually means that TIL can be contacted with two or more substances at the same time during a period of time in the contact process, but it is not limited to that TIL is always in contact with two or more substances at the same time during the entire contact process. touch.
  • substantially simultaneously can mean that TIL can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% of each of the two or more substances are in contact simultaneously.
  • solid phase medium generally refers to a solid phase material having a binding function.
  • a solid phase medium in this application may refer to a material that binds one or more substances in the medium and/or on the surface of the medium through covalent bonding and/or non-covalent bonding.
  • the solid phase media of the present application can be combined with one or more T cell activators.
  • the solid-phase medium of the present application may refer to a medium in which CD28 antibody or its antigen-binding fragment and CD3 antibody or its antigen-binding fragment are bound in the medium and/or on the surface of the medium through covalent binding and/or non-covalent binding. Material.
  • the solid phase medium of the present application may be microspheres comprising the OKT3 antibody and the 15E8 antibody with a diameter of about 500 nanometers to about 10 micrometers.
  • the solid phase medium of the present application may be a polymeric material.
  • the solid phase media of the present application can be microspheres having a diameter of at least about 500 nanometers.
  • the solid phase medium of the present application may be a nanomatrix.
  • the solid phase medium of the present application may be a nanomatrix comprising the OKT3 antibody and the 15E8 antibody with a diameter of about 1 nanometer to about 500 nanometers.
  • the term "nanomatrix" generally refers to a material with a diameter ranging from about 1 nanometer to about 500 nanometers.
  • the nanomatrix 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.
  • dendritic cell generally refers to an antigen-presenting cell present in vivo, in vitro, ex vivo or within a host or subject, or which may be derived from hematopoietic stem cells or monocytes.
  • Dendritic cells and their precursors can be isolated from various lymphoid organs such as spleen, lymph nodes as well as bone marrow and peripheral blood.
  • the dendritic cells of the present application may have characteristic morphology such as thin layers (lamellipodia) extending in multiple directions from the dendritic cell body.
  • dendritic cells can express high levels of MHC and co-stimulatory (eg, B7-1 and B7-2) molecules.
  • Dendritic cells can induce antigen-specific differentiation of T cells in vitro and can elicit primary T cell responses in vitro and in vivo.
  • in vitro expansion generally refers to cultured to produce changes in the number of cells, expanded cells may also produce changes in the number and/or ratio of cells, changes in secretion capacity, changes in killing capacity or expression of Changes in capabilities, or any combination of them.
  • the changes in this application can be increased or decreased.
  • in vitro expansion may be for the purpose of expansion; in order to detect the function of TIL cells, such as detecting the ability of TIL cells to release cytokines, the operation steps performed on TIL cells (such as adding a One or more substances to detect the ability of TIL cells to release cytokines), may not belong to the in vitro expansion of this application.
  • peripheral mononuclear cells or “peripheral blood mononuclear cells” generally refer to cells in peripheral blood having a single nucleus.
  • the peripheral blood mononuclear cells of the present application may include lymphocytes, monocytes and/or dendritic cells.
  • cytokine generally refers to a protein released by a population of cells that acts as an intercellular regulator of another cell.
  • the cytokines of the present application may be lymphokines, monokines and polypeptide hormones.
  • the cytokines of the present application may include interleukins (ILs) such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-15, IL-21, and/or IL-12.
  • ILs interleukins
  • the term cytokine may include proteins from natural sources or from recombinant cell culture, biologically active equivalents of native sequence cytokines, and functionally active fragments thereof.
  • the term “diameter” generally refers to the diameter of a cross-section of a substance of the present application.
  • the term “diameter” generally refers to the maximum diameter and/or average diameter of the largest cross-section of the material of the present application.
  • the method for determining the diameter of a substance may be a method commonly used in the art, such as transmission electron microscopy.
  • the term "neoplastic” generally refers to any new pathological growth of tissue.
  • the tumors of this application may be benign or malignant.
  • the tumors of this application may be solid or hematological.
  • the term “tumor” may be selected from one or more of the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer .
  • tumor tissue generally refers to a sample of any tissue from a tumor in a subject, including any solid tumor and/or non-solid tumor in a subject.
  • CD28 agonist generally refers to a compound that binds to the cell surface CD28 protein and elicits a response in the cell.
  • a CD28 agonist of the present application may be a small molecule agent that binds CD28.
  • the CD28 agonist of the present application can be an antibody or antigen-binding fragment thereof that binds CD28.
  • the term "proportion of T cell subsets" generally refers to the proportion of different T cell subsets in TIL cells or TIL populations.
  • different T cell subsets of the present application have different immunological activity and/or differentiation ability.
  • T cell subsets of the present application can be differentiated based on T cell surface markers.
  • central memory T cells can have a CD45RA - CCR7 + phenotype.
  • regulatory T cells can have a phenotype of CD4 + CD25 + Foxp3 + .
  • activated T cells can have a CD25 + , CD28 + , PD1 + , or 41BB + phenotype.
  • tumor-specific T cells can have a CD103 + CD39 + phenotype.
  • stem-like T cells can have a TCF1 + phenotype.
  • the term "number of TIL cells” generally refers to the number of cells in the TIL cells of the present application.
  • the number of TIL cells may refer to the number of cells in the TIL population obtained at any stage of the present application.
  • the number of TIL cells can refer to the number of cells of the first TIL population derived from tumor tissue and not expanded in vitro.
  • the number of TIL cells can refer to the number of cells of the second TIL population expanded in vitro by the first stage.
  • the number of TIL cells can refer to the number of cells of the third TIL population expanded in vitro by the second stage.
  • the number of TIL cells may refer to the TIL cells finally obtained by any of the culturing methods in the present application.
  • the number of TIL cells can be measured by methods commonly used in the art, for example, including but not limited to manual cell counting on a cell counting board and/or automatic cell counter counting.
  • the terms “about” and “approximately” generally mean within a statistically meaningful range of values. Such ranges may be within an order of magnitude of a given value or range, may be within 50%, may be within 20%, may be within 10%, may be within 5%.
  • the permissible variations encompassed by the term “about” or “approximately” may depend on the particular system under study and are readily understood by those of ordinary skill in the art.
  • the terms “above”, “below”, “at most” and “at least” may be inclusive of numbers.
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), the method may include: reducing the expression and/or weakening the activity of at least one target gene of the TIL, and making the TIL in Co-cultivation with feeder cells followed by exposure to T cell activators and/or T cell growth factors for a certain period of time.
  • TIL tumor-infiltrating lymphocytes
  • the method may comprise: reducing the expression and/or attenuating the activity of at least one target gene of the TIL after co-cultivating the TIL with the feeder cells.
  • the method may comprise: co-cultivating the TIL with the feeder cells after reducing the expression and/or attenuating the activity of at least one target gene of the TIL.
  • the method can comprise: after contacting the TIL with the T cell activator and/or the T cell growth factor and before the TIL is co-cultivated with the feeder cells, at least one of the TILs The expression and/or activity of a target gene is reduced.
  • the method may comprise reducing expression and/or attenuating the activity of at least one target gene of the TIL substantially simultaneously with contacting the TIL with the T cell activator and/or the T cell growth factor .
  • the method may comprise: reducing the expression and/or attenuating the activity of at least one target gene of the TIL substantially simultaneously with the co-cultivation of the TIL with the feeder cells.
  • the present application provides a method for culturing tumor infiltrating lymphocytes (TIL), the method may comprise: reducing the expression and/or weakening the activity of at least one target gene of the TIL.
  • TIL tumor infiltrating lymphocytes
  • the present application also provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: reducing the expression and/or weakening the activity of at least one target gene of the TIL, wherein the TIL Contains TIL obtained from co-culture with feeder cells after exposure to T cell activators and/or T cell growth factors for a certain period of time.
  • TIL tumor-infiltrating lymphocytes
  • the present application also provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: contacting the TIL with a T cell activator and/or a T cell growth factor for a certain period of time and then contacting with a feeder Cell co-cultivation, wherein the TIL comprises TIL obtained by reducing the expression and/or weakening the activity of at least one target gene of the TIL.
  • TIL tumor-infiltrating lymphocytes
  • the present application also provides a method for culturing tumor-infiltrating lymphocytes (TIL), the method may include: reducing the expression and/or weakening the activity of at least one target gene of the TIL, and making the TILs were exposed to CD28 agonists.
  • TIL tumor-infiltrating lymphocytes
  • the present application also provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: reducing the expression and/or weakening the activity of at least one target gene of the TIL, wherein the TIL Contains TILs obtained by exposure to a CD28 agonist.
  • TIL tumor-infiltrating lymphocytes
  • the present application also provides a method for culturing tumor infiltrating lymphocytes (TIL), the method may comprise: contacting the TIL with a CD28 agonist, wherein the TIL comprises at least one of the TIL Reduced expression and/or attenuated activity of the target gene in TILs obtained.
  • TIL tumor infiltrating lymphocytes
  • the CD28 agonist comprises an anti-CD28 antibody and/or its antigen-binding fragment, CD80 and/or its functionally active fragment and/or CD86 and/or its functionally active fragment, and recombinant proteins of the above substances.
  • TILs obtained by reducing the expression and/or activity of at least one target gene of said TIL may exhibit improved TIL properties compared to TILs with unchanged expression and/or activity of the target gene.
  • the TIL whose expression and/or activity of the target gene is unchanged may refer to a TIL derived from the same donor and which has not reduced the expression and/or activity of at least one target gene of said TIL cell.
  • the TIL whose expression and/or activity of the target gene has not been changed may refer to genes other than the target gene of the TIL derived from the same donor (for example, knocking out the other gene, for TIL cells with reduced expression and/or attenuated activity that have essentially no effect on cell function.
  • the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL may refer to a TIL that has been isolated in the same way from the same donor and that has not made the TIL TIL cells with reduced expression and/or reduced activity of at least one gene of interest.
  • the corresponding TIL that has not reduced the expression and/or attenuated the activity of at least one target gene of said TIL may refer to the same tumor origin from the same donor and has not reduced at least one target gene of said TIL.
  • a TIL cell with reduced expression and/or reduced activity of a gene of interest may refer to a TIL that has been isolated in the same way from the same donor and that has not made the TIL TIL cells with reduced expression and/or reduced activity of at least one gene of interest.
  • the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL may refer to the same tumor source from the same donor that has been isolated in the same way and that has not been subjected to the same method. TIL cells with reduced expression and/or reduced activity of at least one target gene of TIL.
  • the corresponding TIL that has not reduced the expression and/or attenuated the activity of at least one target gene of said TIL may refer to dividing TIL cells derived from the same donor into two groups, one of which has not
  • the TIL cells that reduce the expression and/or attenuate the activity of the at least one target gene of the TIL may be corresponding TILs that have not reduced the expression and/or attenuation of the at least one target gene of the TIL.
  • the corresponding TIL that has not reduced the expression and/or attenuated the activity of at least one target gene of said TIL may refer to dividing TIL cells derived from the same donor in the same way into two groups , wherein a group of TIL cells that have not reduced the expression and/or weakened activity of at least one target gene of said TIL may be corresponding TILs that have not reduced the expression and/or weakened activity of at least one target gene of said TIL .
  • the corresponding TIL that has not reduced the expression and/or attenuated the activity of at least one target gene of said TIL may refer to dividing the TIL cells of the same tumor origin from the same donor into two groups, One group of TIL cells that have not reduced the expression and/or weakened the activity of the at least one target gene of the TILs may be the corresponding TILs that have not reduced the expression and/or weakened the activity of the at least one target gene of the TILs. In one embodiment, the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL may refer to the TIL cells isolated in the same way from the same tumor source from the same donor.
  • a group of TIL cells that have not reduced the expression and/or activity of the at least one target gene of the TIL may be those that have not reduced the expression and/or activity of the at least one target gene of the TIL Corresponding TIL attenuated.
  • the reduced expression and/or weakened activity of at least one target gene may mean that the target gene of the natural cell is in a certain degree of expression state, and after the treatment of the present application, the expression level of the target gene of the cell can be reduced. Decreasing, that is, reducing the expression level of the target gene can be to make the natural cell change from expressing the target gene to substantially not expressing the target gene or expressing a decrease in the amount of the target gene.
  • the improved TIL properties of the present application comprise one or more selected from the following group: increased TIL cell number and expansion capacity, increased live cell ratio, increased viability, improved TIL Proportions of cell subpopulations, increased cytokine secretion, increased tumor cell killing, increased anti-apoptosis, and increased T cell receptor (TCR) clonal diversity.
  • the TIL prepared by the method of the present application can improve the viability of the TIL.
  • the TIL obtained in the present application may have increased long-term persistence under culture conditions in which IL-2 is removed.
  • the concentration of IL-2 in the culture conditions of the TILs obtained in the present application can be reduced by 100%, at least 90%, at least 80%, at least 70%, at least 60%. %, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, or at least 1%, the TIL obtained in the present application can continue to expand.
  • the improved TIL cell number and expansion ability of the present application refers to the expression of at least one target gene of the TIL that has not been reduced in the in vitro expansion stage and/or or the corresponding TIL with weakened activity
  • the cell number of the TIL of the present application whose expression of at least one target gene of said TIL is reduced and/or the activity is weakened in at least one in vitro expansion stage can be increased by at least about 1-fold, 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.
  • improved expansion capacity of TILs can be manifested as an increase in the fold proliferation of TILs.
  • improved TIL cell populations can be manifested as increased TIL cell viability.
  • the increased number of TIL cells in the present application may refer to the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL in the in vitro expansion stage.
  • the number of cells of the TIL of the present application that reduces the expression and/or activity of at least one target gene of the TIL during at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least About 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • an improved number of TIL cells can be manifested as a reduction in the proportion of TIL cells that are apoptotic.
  • the reduced TIL cell apoptosis ratio of the present application may refer to the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL in the in vitro expansion stage
  • the apoptotic ratio of the TIL of the present application that reduces the expression and/or weakens the activity of at least one target gene of the TIL in at least one stage of in vitro expansion 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
  • the increased cytokine secretion ability of the present application may refer to the increased cytokine secretion ability of TIL cells selected from the group consisting of CD107a, GZMB, IL-2, TNF- ⁇ and IFN- ⁇ .
  • the increased cytokine secretion ability of the present application may refer to the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL during the in vitro expansion stage, In at least one in vitro expansion stage, the cytokine secretion ability of the TILs of the present application whose expression and/or activity of at least one target gene of the TILs is reduced can be increased by at least about 1 times, at least about 2 times, at least about 2 times.
  • the increased cytokine secretion ability of the present application may refer to the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL during the in vitro expansion stage,
  • the cytokine secretion ability of the TILs of the present application whose expression and/or activity of at least one target gene of the TILs has been reduced 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%
  • the determination of the cytokine secretion ability of TILs of the present application may be by measuring the cytokine expression ability of TIL cells. In one embodiment, the cytokine secreting ability of TILs of the present application is determined by measuring the cytokine releasing ability of TIL cells. In one embodiment, the cytokine secretion ability of the TIL of the present application is determined by the CBA method (Cytometric Bead Array).
  • the increased proportion of NK cells of the present application may be an increase in the proportion of NK cells among TIL cells.
  • the proportion of NK 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%.
  • the improved tumor cell killing ability of the present application may refer to the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL in the in vitro expansion stage,
  • the tumor cell killing rate of the TIL of the present application that reduces the expression and/or weakens the activity of at least one target gene of the TIL can be increased by at least about 1 times, at least about 2 times, at least about 2 times.
  • the improved tumor cell killing ability of the present application may refer to the corresponding TIL that has not reduced the expression and/or weakened the activity of at least one target gene of the TIL in the in vitro expansion stage
  • the tumor cell killing rate of the TIL of the present application whose expression of at least one target gene of the TIL is reduced and/or the activity is weakened in at least one in vitro expansion stage can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about
  • the tumor cell killing rate of the TIL of the present application can be measured by CFSE and DAPI staining. In one embodiment, the tumor cell killing rate of the TIL of the present application can be measured by measuring Caspase-3/7 activity using the IncuCyte system. In one embodiment, the tumor cell killing of TIL in the present application may refer to the ability of TIL to kill solid tumor cells. In one embodiment, the tumor cell killing of TIL in the present application may refer to the ability of TIL to kill melanoma cells.
  • the improved T cell receptor (TCR) clonal diversity of the present application can be included in the process of long-term culture, and the expression of at least one target gene of said TIL has not been made in the in vitro expansion stage. Compared with the corresponding TIL with reduced and/or weakened activity, the expression of at least one target gene of said TIL is reduced and/or the activity is weakened in at least one stage of in vitro expansion.
  • the TCR expressed in the TIL cell population of the present application More kinds, for example, can increase 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 improved T cell subset ratio of the present application may comprise one or more selected from the following group: increased CD4 + cell ratio, decreased CD8 + cell ratio, increased central memory T cell proportion, decreased proportion of regulatory T cells, increased proportion of activated T cells, increased proportion of tumor-specific T cells, and increased proportion of stem-like T cells.
  • the increased proportion of CD4 + cells of the present application may be an increase in the proportion of CD4 positive cells among TIL cells.
  • the proportion of CD4 + 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%.
  • the reduced proportion of CD8 + cells of the present application may be a reduction in the proportion of CD8 positive cells among TIL cells.
  • the proportion of CD8 + 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 central memory T cells of the present application may be an increase in the proportion of CD45RA ⁇ CCR7 + cells among TIL cells.
  • the proportion of central memory T cells among 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%.
  • the reduced ratio of regulatory T cells of the present application may be a reduction in the ratio of CD4 + CD25 + Foxp3 + cells among TIL cells.
  • the proportion of regulatory T cells among 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 among TIL cells.
  • the proportion of activated T cells among 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
  • 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 may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the proportion of 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 may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the proportion of 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 may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, or may
  • 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 may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • reducing the expression and/or weakening the activity of at least one target gene of TIL in the method of the present application may comprise introducing a gene regulation system into the TIL cells.
  • the gene regulation system can disrupt the target gene at the DNA level.
  • the gene regulation system can disrupt the region of the target gene or a fragment thereof in the genome of the TIL cell.
  • the DNA region where the target gene is located or its fragment in TIL cells is cleaved and the expression ability of the target gene is reduced or the activity of the target gene is inhibited.
  • the editing effect of the gene regulation system on the target gene can be long-term and sustained.
  • the gene regulation system may comprise a guide nucleic acid molecule and an enzyme protein.
  • the enzyme protein may have nucleic acid scissor activity, and the guide nucleic acid molecule may guide the enzyme protein to specifically cut the region where the target gene is located or a fragment thereof.
  • the guide nucleic acid molecule and the enzyme protein may exist in the form of ribonucleoprotein complexes (RNP), or each independently.
  • the enzyme protein may comprise a Cas protein.
  • reducing the expression and/or weakening the activity of at least one target gene of TIL in the present application may comprise: introducing a ribonucleoprotein complex (RNP) comprising the guide nucleic acid molecule and the enzyme protein into the TIL.
  • RNP ribonucleoprotein complex
  • the enzyme protein may comprise a Cas protein, a Cas protein homologue, or a functionally active fragment thereof.
  • the guide nucleic acid molecule can comprise a guide RNA (gRNA).
  • the gRNA can be used to bind to the sequence of the target gene.
  • the combination of the gRNA and the sequence of the target gene may be fully complementary, partially complementary, or hybridized to the sequence of the target gene under moderate stringency or stringent conditions.
  • the combination of the gRNA and the sequence of the target gene can allow the CRISPR system of the gRNA to specifically cut the target gene.
  • the target genes of the present application may include genes encoding NF- ⁇ B pathway inhibitory molecules.
  • the NF- ⁇ B pathway inhibitory molecule of the present application can ubiquitinate a protein selected from the group consisting of: receptor interacting protein 1 (RIP1), mucosa-associated lymphoid tissue lymphoma translocator protein 1 (MALT1), receptor interacting Interacting protein 2 (RIP2) and tumor necrosis factor receptor-associated factor 6 (TRAF6).
  • the NF- ⁇ B pathway inhibitory molecules of the present application can ubiquitinate receptor interacting protein 1 (RIP1).
  • the NF- ⁇ B pathway inhibitory molecule of the present application may comprise tumor necrosis factor alpha-inducible protein (TNFAIP), such as tumor necrosis factor alpha-inducible protein 3 (TNFAIP3).
  • TNFAIP tumor necrosis factor alpha-inducible protein
  • the guide nucleic acid molecule described in the present application can be combined with the region where exon 2 and/or exon 7 of TNFAIP3 gene is located or a fragment thereof.
  • the guide nucleic acid molecules described in the present application can be combined with regions or fragments thereof selected from the following groups: SEQ ID NO: 34 to 47.
  • PAM protospacer adjacent motif
  • the PAM region of the target gene when the PAM region of the target gene is determined, those skilled in the art can easily determine the target sequence consisting of about 15 to about 25 nucleotides upstream of the 5' end of the PAM of the target gene, and at the same time design suitable gRNA for the target sequence.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by GGG in the DNA where the TNFAIP3 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr6:137876102-137876121 or chr6:137874868-137874887.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by TGG in the DNA where the TNFAIP3 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr6:137871485-137871504 or chr6:137871486-137871504.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by CGG in the DNA where the TNFAIP3 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, About 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to about 19, about 17 to about 19, or about 15 to about 17 nucleotides in the target sequence.
  • the target sequence may be derived from human chr6:137871475-137871494, chr6:137871501-137871520 or chr6:137871502-137871520.
  • the guide nucleic acid molecule may comprise a target sequence capable of binding to the first about 10 to about 30 nucleotides of the PAM region indicated by AGG in the DNA where the TNFAIP3 gene is located.
  • the guide nucleic acid molecule may comprise about 15 to about 25, about 17 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 23 to about 25, about 15 to about 23, about 17 to about 23, about 19 to about 23, about 20 to about 23, about 21 to about 23, about 15 to about 21, about 17 to about 21, about 19 to about 21, about 20 to about 21, about 15 to about 20, about 17 to about 20, about 19 to about 21, about 15 to A target sequence of about 19, about 17 to about 19, or about 15 to about 17 nucleotides.
  • the guide nucleic acid molecule can comprise a sequence as shown in any one of SEQ ID NO: 48 to 61.
  • the proportion of cells expressing the product of the target gene in the TIL obtained by reducing the expression and/or weakening the activity of at least one target gene of the TIL compared with the TIL in which the expression and/or activity of the target gene are not changed The expression level of said gene of interest in a single cell can be reduced and/or can be reduced.
  • the target gene is expressed in the TIL obtained by reducing the expression and/or weakening of the activity of at least one target gene of the TIL
  • the cell fraction of the product is reduced by at least about 5%.
  • the proportion of cells expressing the product of the TNFAIP3 gene is reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the proportion of cells expressing the product of the TNFAIP3 gene can range from an observable proportion of cells to 0%.
  • the proportion of cells expressing the product of the TNFAIP3 gene can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, At least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the proportion of cells expressing the product of the TNFAIP3 gene can be detected by flow cytometry.
  • the proportion of cells expressing the product of the TNFAIP3 gene in the TIL obtained by reducing the expression and/or weakening the activity of at least one target gene of the TIL in the method of the present application may be at most about 95%.
  • the proportion of cells expressing the product of the TNFAIP3 gene can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40% %, up to about 30%, up to about 20%, up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15%, up to about 14%, up to about 13%, up to about 12% %, up to about 11%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, or up to about 5%.
  • the proportion of cells expressing the product of the TNFAIP3 gene can be detected by flow cytometry.
  • the TIL obtained by reducing the expression and/or activity of at least one target gene of the TIL is described in a single cell.
  • the expression level of the gene of interest may be reduced by at least about 5%.
  • the expression of the gene of interest in a single cell can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least About 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%.
  • the expression level of the target gene in a single cell can range from an observable amount to 0%.
  • the expression level of the gene of interest in a single cell can be reduced to at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40% , at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12% , at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, or at least about 1%.
  • the expression level of the target gene in a single cell in the TIL obtained by reducing the expression and/or activity of at least one target gene of the TIL in the method of the present application can be the expression and/or activity of the target gene Up to about 95% of TIL is unchanged.
  • the expression of TNFAIP3 in a single cell in TIL can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most About 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15%, up to About 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%.
  • the method of the present application may further comprise: subjecting TILs derived from tumor tissue and not expanded in vitro to undergo at least one stage of in vitro expansion, wherein, in at least one stage of in vitro expansion of the present application , TILs of the present application can be co-cultured with feeder cells.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and/or the activity can be weakened and co-cultured with the feeder cells of the present application
  • the in vitro amplification of the present application in a single stage may refer to the in vitro amplification of the present application in the same stage, for example, the same in the first stage in vitro expansion of the present application, and the same in the second stage in vitro expansion of the present application. increase, or in vitro expansion in the third stage of this application, etc.
  • the expression and/or activity of at least one target gene of the TIL in the first stage of in vitro expansion of the present application, can be reduced and co-cultured with the feeder cells of the present application. In one embodiment, in the second stage of in vitro expansion of the present application, the expression and/or activity of at least one target gene of the TIL can be reduced and co-cultured with the feeder cells of the present application. In one embodiment, in the third stage of in vitro expansion of the present application, the expression and/or activity of at least one target gene of the TIL can be reduced and co-cultured with the feeder cells of the present application.
  • each stage of in vitro expansion can be divided by the change in the number of TIL cells.
  • TIL cells when the number of TIL cells increases by at least about 1-fold, TIL cells can be considered Into the next stage of in vitro expansion.
  • the number of TIL cells when the number of TIL cells is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times, or at least about 1000 times, TIL cells can be considered to enter the next stage of in vitro expansion.
  • each stage of in vitro expansion can also be divided by changing the conditions of TIL cell culture.
  • T cell activators and/or T cell growth factors are added or supplemented to the cell culture medium, it can be considered that the TIL cells enter the next stage of in vitro expansion.
  • TIL cells can be considered to enter the next stage of in vitro expansion when IL-2 is added or supplemented to the cell culture medium.
  • IL-2 is added or supplemented to the cell culture medium.
  • TIL cells can be considered to enter the next stage of in vitro expansion when feeder cells are added or supplemented to the cell culture medium.
  • each stage can also be divided by the days of TIL cell culture.
  • TIL cells when the TIL cells are cultured in vitro for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, After about 50 days or about 100 days, TIL cells can be considered to enter the next stage of in vitro expansion.
  • the second stage of in vitro expansion can be performed for at least about 7 days.
  • the second stage of in vitro expansion can be performed for at least about 9 days.
  • the second stage of in vitro expansion can be performed for up to about 14 days.
  • the second stage of in vitro expansion can be performed for up to about 13 days.
  • the second stage of in vitro expansion can be performed for about 7 days to about 14 days, about 9 days to about 14 days, about 7 days to about 13 days, or about 9 days to about 13 days.
  • the second stage in vitro expansion of the present application can be performed for at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the second stage of in vitro expansion of the present application can be carried out for about 9 days to about 14 days
  • the second stage of in vitro expansion of the present application can be carried out for about 9 days to about 14 days, about 10 days to about 14 days, About 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days days to about 13 days, about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the second stage of in vitro expansion in the present application can be considered as the REP (rapid expansion protocol) stage.
  • the first stage of in vitro expansion can be performed for at least about 7 days.
  • the first stage of in vitro expansion can be performed for about 7 days to about 14 days.
  • the first stage of in vitro expansion of the present application can be performed for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least About 14 days.
  • the first stage of in vitro expansion of the present application can be performed for about 7 days to about 14 days, about 8 days to about 14 days, about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days to about 13 days , about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the first stage of in vitro expansion of the present application can be considered the preREP stage.
  • the number of days for the second-stage in vitro expansion of the present application can be calculated from the start of the second-stage in vitro expansion. For example, when the second-stage in vitro expansion starts, it can be considered that the second-stage in vitro expansion has been performed for about 0 days. For example, if the second-stage in vitro expansion proceeds for about 24 hours, it can be considered that the second-stage in vitro expansion has been performed for about one day. For example, the day when the second-stage in vitro expansion starts can be considered as about 0 days after the second-stage in vitro expansion.
  • the number of days for the second stage of in vitro expansion in the present application can be calculated based on the number of days for the second stage of in vitro expansion. For example, on the second day after the start of the second-stage in vitro expansion, it can be considered that the second-stage in vitro expansion has been performed for about one day.
  • the cultivation method of the present application can be divided into two steps.
  • the first TIL population derived from tumor tissue and not expanded in vitro can be contacted with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • the TIL population can be made The expression of at least one target gene of the second TIL population is reduced and/or the activity is weakened, and the second TIL population is co-cultured with feeder cells after being contacted with a T cell activator and/or a T cell growth factor for a certain period of time , wherein, the third TIL group is obtained through the step (B).
  • step (A) may be performed for about 7 days to about 14 days.
  • step (B) may be performed for about 7 days to about 14 days.
  • the cultivation method of the present application can be divided into three steps.
  • (A) the first TIL population derived from tumor tissue and not expanded in vitro can be contacted with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • (B) the TIL population can be made The expression of at least one target gene of the second TIL population is reduced and/or the activity is weakened, and the second TIL population can be contacted with a T cell activator and/or a T cell growth factor, wherein, after the step ( B) Obtaining the third TIL population;
  • (C) The third TIL population can be co-cultured with feeder cells, wherein the fourth TIL population is obtained through the step (C).
  • step (A) may be performed for about 7 days to about 14 days.
  • step (B) can be performed for about 0 days to about 8 days.
  • step (C) may be performed for about 5 days to about 14 days.
  • the cultivation method of the present application can be divided into four steps.
  • the first TIL population derived from tumor tissue and not expanded in vitro can be contacted with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • the TIL population can be made The second TIL population is contacted with a T cell activator and/or a T cell growth factor, wherein a third TIL population is obtained through the step (B);
  • at least one target of the third TIL population can be made The expression of the gene is reduced and/or the activity is weakened, wherein, the fourth TIL group is obtained through the step (C);
  • the fourth TIL group can be co-cultured with feeder cells, wherein, after the step (D) ) to obtain the fifth TIL group.
  • step (A) may be performed for about 7 days to about 14 days.
  • step (B) can be performed for about 0 days to about 4 days.
  • step (C) can be performed for about 0 days to about 4 days.
  • step (D) may be performed for about 5 days to about 14 days.
  • step (A) of the culturing method of the present application is to obtain a second TIL population from the recovered and/or continued culture of the in vitro TIL population.
  • the TIL population in vitro may comprise a TIL population obtained by in vitro expansion of a first TIL population derived from tumor tissue and not expanded in vitro.
  • the in vitro TIL population can comprise a TIL population obtained by contacting the first TIL population with a T cell growth factor.
  • said in vitro TIL population can comprise a TIL population obtained by cryopreservation of said first TIL population.
  • the in vitro TIL population may comprise a TIL population obtained by contacting the first TIL population with a T cell growth factor and cryopreserving.
  • the step (A) of the present application is to obtain the second TIL population from the recovered and/or continued culture of the in vitro TIL population, the step (A) can be carried out for about 2 hours to about 4 days.
  • the TILs of the present application in a single stage of the in vitro expansion of the present application, can be contacted with one or more T cell activators and/or one or more T cell growth factors of the present application for a certain period of time Afterwards, co-cultivate with the feeder cells of the present application.
  • the certain period of time of the application may be at least about 2 hours.
  • the certain period of time of the present application can be at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours or at least About 72 hours.
  • the certain period of time for this application may be from about 6 hours to about 72 hours.
  • the certain period of time of the present application can be about 6 hours to about 7 hours, about 6 hours to about 8 hours, about 6 hours to about 9 hours, about 6 hours to about 10 hours, about 6 hours to about About 11 hours, about 6 hours to about 12 hours, about 6 hours to about 13 hours, about 6 hours to about 14 hours, about 6 hours to about 15 hours, about 6 hours to about 16 hours, about 6 hours to about 17 hours hours hours, about 6 hours to about 18 hours, about 6 hours to about 19 hours, about 6 hours to about 20 hours, about 6 hours to about 21 hours, about 6 hours to about 22 hours, about 6 hours to about 23 hours, From about 6 hours to about 24 hours, from about 6 hours to about 36 hours, from about 6 hours to about 48 hours, from about 6 hours to about 60 hours, or from about 6 hours to about 72 hours.
  • the certain period of time of the present application can be about 12 hours to about 13 hours, about 12 hours to about 14 hours, about 12 hours to about 15 hours, about 12 hours to about 16 hours, about 12 hours to About 17 hours, about 12 hours to about 18 hours, about 12 hours to about 19 hours, about 12 hours to about 20 hours, about 12 hours to about 21 hours, about 12 hours to about 22 hours, about 12 hours to about 23 hours hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, or about 12 hours to about 72 hours.
  • the certain time of the present application can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, About 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours hours, about 23 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours.
  • the feeder cells of the present application may comprise antigen presenting cells.
  • the feeder cells of the present application may comprise one or more selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen-presenting cells.
  • the feeder cells of the present application may be peripheral mononuclear cells.
  • the feeder cells of the present application may be irradiated feeder cells.
  • the feeder cells of the present application can be isolated artificial antigen-presenting cells (aAPC), and the artificial antigen-presenting cells of the present application can comprise cells expressing HLA-A/B/C, CD64, CD80, ICOS-L and/or CD58 , and can be modified to express more than one T cell activator of the present application.
  • the feeder cells of the present application can be irradiated, for example, can be gamma irradiated, or can be X-ray irradiated.
  • the co-cultivation of the TIL of the present application and the feeder cell of the present application may comprise contacting the surface of the feeder cell of the present application with the surface of the TIL of the present application. In one embodiment, the co-cultivation of the TIL of the present application and the feeder cell of the present application comprises adding the feeder cell of the present application to the cell culture medium of the TIL of the present application.
  • the present application can add the feeder cells of the present application to the cell culture medium of the TIL of the present application at a ratio of about 40:1 to about 400:1 of the feeder cells of the present application to the TIL of the present application.
  • the present application can be about 40:1 to about 400:1, about 40:1 to about 300:1, about 40:1 to about 200:1, about 40:1 to about 100:1, about 40:1-about 90:1, about 40:1-about 80:1, about 40:1-about 70:1, about 40:1-about 60:1, about 40:1-about 50:1, about 50:1-about 400:1, about 60:1-about 400:1, about 70:1-about 400:1, about 80:1-about 400 :1, about 90:1-about 400:1, about 100:1-about 400:1, about 200:1-about 400:1, or about 300:1-about 400:1 of the present application
  • the ratio of the feeder cells to the TIL of the present application, the feeder cells of the present application are added to the cell
  • the method of the present application may further comprise: subjecting TILs derived from tumor tissue and not expanded in vitro to undergo at least one stage of in vitro expansion, wherein, in at least one stage of in vitro expansion of the present application , contacting TILs of the present application with one or more T cell activators.
  • said TILs are contacted with said one or more T cell activators during a single stage of in vitro expansion of the application.
  • the T cell activator may comprise an agonist for one or more targets selected from the group consisting of: CD3, CD28, HVEM, CD40L, OX40, and 4-1BB.
  • the expression and/or activity of at least one target gene of the TIL is reduced and combined with one or more T cell activators of the present application touch.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and can be combined with one or more T cell activation of the application. agent contact.
  • the expression and/or activity of at least one target gene of the TIL in the second stage of in vitro expansion of the application, can be reduced and can be combined with one or more T cell activation of the application. agent contact.
  • the expression and/or activity of at least one target gene of the TIL in the third stage of in vitro expansion of the present application, can be reduced and can be combined with the activation of one or more T cells of the present application. agent contact.
  • the expression and/or activity of at least one target gene of the TIL in a single stage of the in vitro expansion of the present application, can be reduced and/or the activity of the TIL can be substantially simultaneously reduced and the TIL can be combined with one of the present application. or multiple T cell activators.
  • the expression and/or activity of at least one target gene of the TIL in a single stage of the in vitro amplification of the present application, can be reduced first, for example, 2 hours in advance, 4 hours in advance , 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more T cell activators of the present application.
  • the TILs of the present application in the in vitro expansion of the present application in a single stage, can be contacted with one or more T cell activators of the present application first, for example, 2 hours in advance, 4 hours in advance. hours, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then reduce the expression and/or activity of at least one target gene of the TIL.
  • the expression and/or activity of at least one target gene of the TIL in the first stage of in vitro expansion of the present application, can be reduced and/or the activity of the TIL can be substantially simultaneously reduced and the TIL can be contacted with one or more of the present application. Exposure to multiple T cell activators. In one embodiment, in the second stage of in vitro expansion of the present application, the expression and/or activity of at least one target gene of the TIL can be reduced and/or the activity of the TIL can be substantially simultaneously reduced and the TIL can be contacted with one or more of the present application. Exposure to multiple T cell activators.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and/or the activity of at least one target gene of the TIL can be substantially simultaneously reduced and the TIL can be contacted with one or more of the present application. Exposure to multiple T cell activators.
  • the T cell activator of the present application may comprise one or more selected from the following group: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT , and their functionally active fragments.
  • the T cell activator of the present application may comprise an agonist of one or more targets selected from the following group: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the T cell activator of the present application may comprise antibodies selected from the group consisting of CD3, CD28, HVEM, CD40L, OX40 and 4-1BB and their antigen-binding fragments.
  • the T cell activator of the present application may comprise a CD3 agonist.
  • the T cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, it may be OKT3 from Miltenyi Biotech, or it may be SP34 from BD.
  • the T cell activator of the present application may comprise a CD28 agonist.
  • the T cell activator of the present application may comprise an anti-CD28 antibody and/or its antigen-binding fragment, such as Merck's 15E8, and the T cell activator of the present application may comprise CD80 and/or its function Active fragments and/or CD86 and/or functionally active fragments thereof, as well as recombinant proteins of the above substances.
  • the T cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain VL and heavy chain VH of Miltenyi Biotech's OKT3, may comprise the SP34 of BD Light chain VL and heavy chain VH.
  • the T cell activator of the present application may comprise a CD28 agonist.
  • the T cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, for example may comprise the light chain VL and heavy chain VH of Merck's 15E8.
  • the T cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of Miltenyi Biotech's OKT3, may comprise The light chain LCDR1-3 and heavy chain HCDR1-3 of SP34 of BD, the anti-CD3 antibody and/or antigen-binding fragment thereof of the present application may have CD3-binding ability.
  • the T cell activator of the present application may comprise a CD28 agonist.
  • the T cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, for example may comprise Merck's 15E8 light chain LCDR1-3 and heavy chain HCDR1-3, the present application's Anti-CD28 antibodies and/or antigen-binding fragments thereof may have CD28-binding ability.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VH of the heavy chain of the antibody.
  • the CDRs of this application may be defined according to IMGT nomenclature, the CDRs of this application may be defined according to Chothia, or the CDRs of this application may be defined according to Kabat.
  • the CD3 agonist of the present application may be a CD3 antibody or an antigen-binding protein thereof.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VH of the heavy chain of the antibody.
  • the CDRs of this application may be defined according to IMGT nomenclature, the CDRs of this application may be defined according to Chothia, or the CDRs of this application may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1, and the HCDR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 2 and 12;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR2, and the HCDR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 3 and 13;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR3, and the HCDR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 4 and 14;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise HCDR1-3, wherein the HCDR1 of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 2 and 12, and the HCDR2 of the present application can comprise SEQ ID NO: 3 and the amino acid sequence shown in any one of 13, and the HCDR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 4 and 14;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDR of the present application It may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same HCDR1-3 as OKT3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 3 amino acid sequence, and the HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 4; the CDR of the present application may be defined according to the IMGT nomenclature; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same HCDR1-3 as SP34, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 13
  • the amino acid sequence of the present application, and the HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 14; the CDR of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VL of the light chain of the antibody.
  • the CDRs of the present application may be defined according to the IMGT nomenclature, or the CDRs of the present application may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1, and the LCDR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 5 and 15;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR2, and the LCDR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 6 (DTS) and 16;
  • the CDR of the present application may be according to the IMGT nomenclature Defined;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR3, and the LCDR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 7 and 17;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1-3, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 5 and 15, and the LCDR2 of the present application may comprise SEQ ID NO: 6 (DTS) and the amino acid sequence shown in any one of 16, and the application LCDR3 can comprise the amino acid sequence shown in any one of SEQ ID NO:7 and 17;
  • the application CDR can be defined according to the IMGT nomenclature;
  • the CDRs of the present application may be defined according to Kabat; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same LCDR1-3 as OKT3, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 5, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 6 (DTS ), and the application LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 7; the CDR of the application may be defined according to the IMGT nomenclature; for example, the antigen binding protein of the application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same LCDR1-3 as SP34, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 15, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 16
  • the amino acid sequence of the present application, and the LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 17; the CDR of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise HCDR1-3 and LCDR1-3, wherein the HCDR1 of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 2 and 12, and the HCDR2 of the present application can comprise SEQ ID NO:
  • the amino acid sequence shown in any one of ID NO: 3 and 13 can comprise the amino acid sequence shown in any one of SEQ ID NO: 4 and 14, and the LCDR1 of this application can comprise SEQ ID NO: 5 and 15
  • the amino acid sequence shown in any one, the application LCDR2 can comprise the amino acid sequence shown in any one of SEQ ID NO: 6 (DTS) and 16, and the application LCDR3 can comprise SEQ ID NO: 7 and 17 in any One of the amino acid sequences shown;
  • the CDRs of this application can be defined according to IMGT nomenclature; the CDRs of this application can be defined according to Kabat; for example, the antigen binding protein of this application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1-3 and LCDR1-3 identical to OKT3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR2 of the present application may comprise SEQ ID NO :
  • the amino acid sequence shown in 3 the HCDR3 of the present application may contain the amino acid sequence shown in SEQ ID NO: 4
  • the LCDR1 of the present application may contain the amino acid sequence shown in SEQ ID NO: 5
  • the LCDR2 of the present application may contain the amino acid sequence shown in SEQ ID NO: 6
  • the amino acid sequence shown in (DTS), and the application LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 7;
  • the application CDR can be defined according to IMGT nomenclature;
  • the antigen binding protein of the application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same HCDR1-3 as SP34, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 13 HCDR3 of this application may include the amino acid sequence shown in SEQ ID NO: 14, LCDR1 of this application may include the amino acid sequence shown in SEQ ID NO: 15, and LCDR2 of this application may include the amino acid sequence shown in SEQ ID NO: 16 Sequence, and the LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 17; the CDR of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain variable region VH, and the VH of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 8 and 18; for example, The antigen-binding protein of the present application may have CD3-binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH as OKT3, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 8; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH as SP34, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 18; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a light chain variable region VL, and the VL of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 9 and 19; for example, The antigen-binding protein of the present application may have CD3-binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same VL as OKT3, and the VL of the present application can comprise the amino acid sequence shown in SEQ ID NO: 9; for example, the antigen-binding protein of the present application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VL as SP34, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 19; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, and the VH of the present application may comprise any one of SEQ ID NO: 8 and 18.
  • the amino acid sequence shown, the VL of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 9 and 19; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH and VL as OKT3, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 8, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 9 Amino acid sequence; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH and VL as SP34, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 18, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 19 Amino acid sequence; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise a heavy chain, and the heavy chain of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 10 and 20;
  • the present application The antigen binding protein may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain as OKT3, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 10;
  • the antigen-binding protein of the present application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain as SP34, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 20;
  • the antigen-binding protein of the present application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise a light chain, and the light chain of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 11 and 21;
  • the present application The antigen binding protein may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same light chain as OKT3, and the light chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 11;
  • the antigen-binding protein of the present application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same light chain as SP34, and the light chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 21;
  • the antigen-binding protein of the present application can have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain and a light chain
  • the heavy chain of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 10 and 20
  • the present application The light chain may comprise the amino acid sequence shown in any one of SEQ ID NO: 11 and 21; for example, the antigen binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain and light chain as OKT3, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 10, and the light chain of the present application can comprise SEQ ID NO : the amino acid sequence shown in 11; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain and light chain as SP34, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 20, and the light chain of the present application can comprise SEQ ID NO : the amino acid sequence shown in 21; for example, the antigen-binding protein of the present application may have CD3 binding ability.
  • the CD28 agonist of the present application may be a CD28 antibody or an antigen-binding protein thereof.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VH of the heavy chain of the antibody.
  • the CDRs of the present application may be defined according to the IMGT nomenclature, or the CDRs of the present application may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1, and the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR2, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 23; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR3, and the HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 24; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1-3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 23, And the HCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 24;
  • the CDR of the present application may be defined according to the IMGT nomenclature;
  • the CDR of the present application may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may contain the same HCDR1-3 as 15E8, wherein the HCDR1 of the present application may contain the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 of the present application may contain the amino acid sequence shown in SEQ ID NO: 23
  • the amino acid sequence of the HCDR3 of the present application may include the amino acid sequence shown in SEQ ID NO: 24; the CDR of the present application may be defined according to the IMGT nomenclature; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VL of the light chain of the antibody.
  • the CDRs of the present application may be defined according to the IMGT nomenclature, or the CDRs of the present application may be defined according to Kabat.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1, and the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 25; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR2, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 26 (AAS); the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application It may be defined according to Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • AAS amino acid sequence shown in SEQ ID NO: 26
  • the CDR of the present application may be defined according to the IMGT nomenclature
  • the CDR of the present application It may be defined according to Kabat
  • the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR3, and the LCDR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 27; the CDR of the present application may be defined according to the IMGT nomenclature; the CDR of the present application may be defined according to As defined by Kabat; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise LCDR1-3, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 26 (AAS).
  • Amino acid sequence, and the application LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27; the application CDR may be defined according to the IMGT nomenclature; the application CDR may be defined according to Kabat; for example, the antigen binding protein of the application May have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same LCDR1-3 as 15E8, wherein the LCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 26 (AAS ), and the application LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 27; the CDR of the application may be defined according to the IMGT nomenclature; for example, the antigen binding protein of the application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1-3 and LCDR1-3, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 23 HCDR3 of this application may include the amino acid sequence shown in SEQ ID NO: 24, LCDR1 of this application may include the amino acid sequence shown in SEQ ID NO: 25, and LCDR2 of this application may include the amino acid sequence shown in SEQ ID NO: 30 sequence, and the application LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26 (AAS); the application CDR may be defined according to the IMGT nomenclature; the application CDR may be defined according to Kabat; for example, the antigen of the application A binding protein may have CD28 binding ability.
  • the application CDR may be defined according to the IMGT nomenclature
  • the application CDR may be defined according to Kabat
  • the antigen of the application A binding protein
  • the antibody of the present application or its antigen-binding protein may comprise HCDR1-3 and LCDR1-3 identical to 15E8, wherein the HCDR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 of the present application may comprise SEQ ID NO : the amino acid sequence shown in 23, the HCDR3 of the present application may contain the amino acid sequence shown in SEQ ID NO: 24, the LCDR1 of the present application may contain the amino acid sequence shown in SEQ ID NO: 25, and the LCDR2 of the present application may contain the amino acid sequence shown in SEQ ID NO: 26
  • the amino acid sequence shown in (AAS), and the application LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 27;
  • the application CDR can be defined according to IMGT nomenclature;
  • the antigen binding protein of the application can have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain variable region VH, and the VH of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 28 and 29; for example, The antigen-binding protein of the present application may have CD28-binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a VH identical to a 15E8, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 28; for example, the antigen-binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same VH as another 15E8, and the VH of the present application can comprise the amino acid sequence shown in SEQ ID NO: 29; for example, the antigen-binding protein of the present application can have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a light chain variable region VL, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 30; for example, the antigen-binding protein of the present application May have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VL as 15E8, and the VL of the present application may comprise the amino acid sequence shown in SEQ ID NO: 30; for example, the antigen-binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, and the VH of the present application may comprise any one of SEQ ID NO:28 and 29.
  • the amino acid sequence shown in the present application VL may comprise the amino acid sequence shown in SEQ ID NO: 30; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH and VL as a 15E8, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 28, and the VL of the present application may comprise SEQ ID NO: 30
  • the amino acid sequence shown; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same VH and VL as another 15E8, and the VH of the present application may comprise the amino acid sequence shown in SEQ ID NO: 29, and the VL of the present application may comprise SEQ ID NO: The amino acid sequence shown in 30; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise a heavy chain, and the heavy chain of the present application can comprise the amino acid sequence shown in any one of SEQ ID NO: 31 and 32;
  • the present application The antigen binding protein may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain identical to a 15E8, and the heavy chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 31; for example, the antigen-binding protein of the present application may have CD28 binding capacity.
  • the antibody of the present application or its antigen-binding protein can comprise the same heavy chain as another 15E8, and the heavy chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 32; for example, the antigen-binding protein of the present application can be Has CD28 binding ability.
  • the antibody of the present application or its antigen binding protein can comprise a light chain, and the light chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 33;
  • the antigen binding protein of the present application can have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein can comprise the same light chain as 15E8, and the light chain of the present application can comprise the amino acid sequence shown in SEQ ID NO: 33;
  • the antigen-binding protein of the present application can have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise a heavy chain and a light chain
  • the heavy chain of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 31 and 32
  • the present application The light chain may comprise the amino acid sequence shown in SEQ ID NO: 33; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same heavy chain and light chain as a 15E8, and the heavy chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 31, and the light chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 31.
  • the amino acid sequence shown in ID NO: 33; for example, the antigen-binding protein of the present application may have CD28 binding ability.
  • the antibody of the present application or its antigen-binding protein may comprise the same heavy chain and light chain as another 15E8, and the heavy chain of the present application may comprise the amino acid sequence shown in SEQ ID NO: 32, and the light chain of the present application may comprise The amino acid sequence shown in SEQ ID NO: 33; for example, the antigen binding protein of the present application may have CD28 binding ability.
  • contacting the TIL of the present application with one or more T cell activators of the present application may comprise one or more methods selected from the following group: (1) activating the T cell of the present application (2) adding engineered cells expressing the T cell activator of the present application to the cell culture medium of TIL of the present application; (3) adding the T cell culture medium containing the TIL of the present application The solid phase medium of the cell activator was added to the cell culture medium of the TIL of the present application.
  • contacting the TILs of the present application with one or more T cell activators of the present application may comprise adding a solid phase medium comprising the T cell activators of the present application to the cell culture of the TILs of the present application. Base.
  • contacting the TILs of the present application with one or more T cell activators of the present application may comprise adding a solid phase medium comprising the CD28 antibody and the CD3 antibody of the present application to the cells of the TILs of the present application medium.
  • the initial concentration of the T cell activator in the TIL cell culture medium of the present application may be at least about 30 ng/mL.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of TIL of the present application can be at least about 30 ng/mL;
  • the initial concentration of the CD3 antibody of the present application in the cell culture medium of TIL of the present application can be at least about 30 ng/mL; 30ng/mL.
  • the selection of the initial concentration of the CD28 antibody of the present application can be independent of the selection of the initial concentration of the CD3 antibody of the present application; for example, the initial concentration of the CD28 antibody of the present application and the CD3 antibody of the present application in the cell culture medium of the TIL of the present application Any combination is possible.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the TIL of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL.
  • the initial concentration of the CD3 antibody of the present application in the cell culture medium of the TIL of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the TIL of the present application can be arbitrarily selected from about 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 30ng/mL to about 300ng/mL, and the selection of the initial concentration of the CD28 antibody of the present application can be independent of the selection of the initial concentration of the CD3 antibody of the present application.
  • the solid phase media of the present application may have a diameter of about 500 nanometers to about 10 microns. In one embodiment, the diameter of the solid phase medium of the present application can be measured by a transmission electron microscope.
  • the diameter of the solid phase medium of the present application may be from about 1 nanometer to about 500 nanometers. In one embodiment, the diameter of the solid phase medium of the present application may be from about 100 nanometers to about 500 nanometers. In one embodiment, the diameter of the solid phase medium of the present application may be from about 200 nanometers to about 500 nanometers. In one embodiment, the diameter of the solid phase medium of the present application can be measured by a transmission electron microscope.
  • the solid phase medium of the present application may comprise a polymer. In one embodiment, the solid phase medium of the present application may comprise dextran.
  • each mg of the solid medium of the present application comprises at least about 25 ⁇ g of the T cell activator of the present application.
  • the solid phase medium comprising the T cell activator of the present application is added to the cell culture of the TIL of the present application at a ratio of the solid phase medium of the present application to the TIL of the present application of about 1:100-about 1:2000 Base. In one embodiment, the solid phase medium comprising the T cell activator of the present application is added to the cell culture of the TIL of the present application at a ratio of the solid phase medium of the present application to the TIL of the present application of about 2:1 to about 1:2 Base.
  • the ratio of the solid phase medium of the present application to the TIL of the present application can be about 2:1 to about 1:2.
  • the solid phase medium of cell activator was added to the cell culture medium of TIL in this application.
  • the diameter of the solid phase medium of the present application when the diameter of the solid phase medium of the present application is about 100 nanometers to about 500 nanometers, it can be about 2:1-about 1:2, about 2:1-about 1:1, or about 1:1 -The ratio of the solid phase medium of the present application to the TIL of the present application is about 1:2, the solid phase medium comprising the T cell activator of the present application, such as CD3 agonist and/or CD28 agonist is added to the cell culture medium of the TIL of the present application middle.
  • the solid phase medium comprising the T cell activator of the present application such as CD3 agonist and/or CD28 agonist
  • the ratio of the solid phase medium of the present application to the TIL of the present application can be about 1:100-about 1:2000.
  • the solid phase medium of cell activator was added to the cell culture medium of TIL in this application.
  • the diameter of the solid phase medium of the present application when the diameter of the solid phase medium of the present application is about 100 nanometers to about 500 nanometers, it can be about 1:100-about 1:2000, about 1:200-about 1:2000, about 1:300- About 1:2000, about 1:400-about 1:2000, about 1:500-about 1:2000, about 1:600-about 1:2000, about 1:700-about 1:2000, about About 1:800-about 1:2000, about 1:900-about 1:2000, about 1:1000-about 1:2000, about 1:1200-about 1:2000, about 1:1400-about 1 :2000, about 1:1600-about 1:2000, or about 1:1800-about 1:2000 of the ratio of the solid phase medium of the present application to the TIL of the present application, for example, the CD28 agonist and CD3 agonist of the present application can be included
  • the solid medium of the agent was added to the cell culture medium of TIL in this application.
  • the method of the present application may further comprise: subjecting TILs derived from tumor tissue and not expanded in vitro to undergo at least one stage of in vitro expansion, wherein, in at least one stage of in vitro expansion of the present application , contacting TILs of the present application with one or more T cell growth factors.
  • TILs of the present application may be contacted with a T cell activator of the present application and contacted with one or more T cell growth factors of the present application in a single stage of in vitro expansion of the present application.
  • the TIL of the present application in the first stage of in vitro expansion of the present application, can be contacted with the T cell activator of the present application and contacted with one or more T cell growth factors of the present application.
  • the TIL of the present application in the second stage of in vitro expansion of the present application, can be contacted with the T cell activator of the present application and contacted with one or more T cell growth factors of the present application.
  • the TILs of the present application in the third stage of in vitro expansion of the present application, can be contacted with the T cell activator of the present application and contacted with one or more T cell growth factors of the present application.
  • the expression and/or activity of at least one target gene of the TIL is reduced and the TIL is contacted with a T cell growth factor substantially simultaneously. In one embodiment, in a single stage of the in vitro expansion of the present application, the expression and/or activity of at least one target gene of said TIL is reduced and the TIL is contacted with a T cell growth factor substantially simultaneously.
  • the expression and/or activity of at least one target gene of the TIL can be reduced first, for example, 2 hours in advance, 4 hours in advance , 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact the TIL of the present application with one or more T cell growth factors of the present application.
  • the TIL of the present application in the in vitro expansion of the present application in a single stage, can be contacted with one or more T cell growth factors of the present application first, for example, 2 hours in advance, 4 hours in advance hours, 8 hours in advance, 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then reduce the expression and/or activity of at least one target gene of the TIL.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and the TIL can be contacted with T cell growth factors substantially at the same time.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and the TIL can be contacted with T cell growth factors substantially simultaneously.
  • the expression and/or activity of at least one target gene of the TIL can be reduced and the TIL can be contacted with T cell growth factors substantially at the same time.
  • the T cell growth factor of the present application can be selected from one or more of the following groups: IL-2, IL-7, IL-12, IL-15, IL-21, interferon gamma, and their functionally active fragments.
  • the T cell growth factor of the present application may comprise IL-2 and/or a functionally active fragment thereof.
  • a functionally active fragment of IL-2 may comprise a fragment of IL-2 known in the art that binds to the IL-2 receptor of a T cell.
  • contacting the TIL of the present application with one or more T cell growth factors of the present application may comprise adding the T cell growth factor of the present application to the cell culture medium of the TIL of the present application.
  • the initial concentration of the T cell growth factor of the present application in the cell culture medium of the TIL of the present application can be at least about 300 IU/mL.
  • the initial concentration of IL-2 of the present application in the cell culture medium of TIL of the present application can be at least about 350IU/mL, at least about 400IU/mL, at least about 500IU/mL, at least about 600IU/mL, At least about 700 IU/mL, at least about 800 IU/mL, at least about 900 IU/mL, at least about 1000 IU/mL, at least about 1100 IU/mL, at least about 1200 IU/mL, at least about 1300 IU/mL, at least about 1400 IU/mL, at least about 1500 IU/mL, at least about 2000 IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL, at least about 2700 IU/mL, at least about 2800 IU/mL, at least about 2900 IU/mL, at least about 3000 IU/mL, at least about 3100 IU/mL mL, at least about 3200
  • the TIL of the present application may be the TIL derived from the fragments of the tumor tissue of the present application. In one embodiment, the TIL of the present application can be obtained by processing tumor tissue into tumor fragments. In one embodiment, the tumor fragments of the present application have a volume of about 1-27 cubic millimeters.
  • the tumor fragments of the present application have a volume of about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters , about 8 cubic millimeters, about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 15 cubic millimeters, about 17 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters , about 21 cubic millimeters, about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters, or about 27 cubic millimeters.
  • the TILs may be selected from the group consisting of TILs derived from fragments of tumor tissue, TILs derived from fragments of lymphatic metastases, TILs derived from pleural effusions, TILs derived from peritoneal effusions, and TILs derived from cryopreserved After resuscitating the TIL.
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor , wherein, the second TIL population is obtained through the step (A); (B) reducing the expression and/or activity of at least one target gene of the TIL, and making the second TIL population interact with T cells The activator and/or the T cell growth factor are contacted for a certain period of time and then co-cultured with the feeder cells, wherein the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: (A) resuscitating and/or continuing to cultivate an in vitro TIL population to obtain a second TIL population, wherein the in vitro TIL population comprising a TIL population obtained by in vitro expansion of a first TIL population, the first TIL population is a TIL population derived from tumor tissue and not amplified in vitro; (B) expressing at least one target gene of the TIL reduced and/or weakened in activity, and the second TIL population is co-cultured with feeder cells after being in contact with T cell activators and/or T cell growth factors for a certain period of time, wherein the third TIL population is obtained through the step (B) TIL group.
  • TIL tumor-infiltrating lymphocytes
  • TIL populations derived from tumor tissue and not expanded in vitro can be contacted with T cell growth factors at a certain time and/or at a certain location to obtain TIL populations in vitro.
  • the TIL populations in vitro can be continuously cultivated , to carry out step (B), on the other hand, the in vitro TIL population can be cryopreserved first, and the in vitro TIL population can be revived when necessary, and then step (B) can be carried out.
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor , wherein, the second TIL group is obtained through the step (A); (B) the expression and/or activity of the TNFAIP3 gene of the TIL is reduced and/or the activity is weakened, and the second TIL group is combined with a T cell activator and /or co-cultivate with feeder cells after being exposed to T cell growth factors for a certain period of time, wherein the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) contacting a first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor , wherein, the second TIL population is obtained through the step (A); (B) the proportion of cells expressing the TNFAIP3 gene in the TIL is about 95% or less, and the second TIL population is compared with T The cell activator and/or the T cell growth factor are contacted for a certain period of time and then co-cultured with the feeder cells, wherein the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the first-stage in vitro expansion of the present application can be optionally used interchangeably with step (A) in the method of the above aspect.
  • the second-stage in vitro expansion of the present application can be used in any substitution with step (B) in the method of the above aspect.
  • the TILs expanded in vitro in the first stage of the present application can be used in any substitution with the second population of TILs obtained through step (A) of the method of the above aspects.
  • the second-stage in vitro expanded TILs of the present application can be used in any substitution with the third TIL population obtained through step (B) of the method of the above aspects.
  • the third-stage in vitro amplification of the present application can be used in any replacement with any added step (C) in the method of the above aspects.
  • the third-stage in vitro expanded TILs of the present application can be used in any replacement with the fourth TIL population obtained through any additional step (C) in the method of the above aspects .
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The expression and/or activity of at least one target gene of the TILs is reduced and/or the activity is weakened, and the TILs are co-cultured with the feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The expression and/or activity of the TNFAIP3 gene of the TILs is reduced and/or the activity is weakened, and the TILs are co-cultured with the feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The proportion of cells expressing the TNFAIP3 gene in the TIL is about 95% or less, and the TIL is co-cultured with feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The proportion of cells expressing the TNFAIP3 gene in the TIL is about 95% or less, and the TIL is co-cultured with the feeder cells after at least 2 hours; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The proportion of cells expressing the TNFAIP3 gene in the TIL is about 95% or less, and after at least 2 hours, the TIL is co-cultured with feeder cells, which may comprise peripheral mononuclear cells, and the feeder cells added to the cell culture medium of the TIL; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) combining the first TIL population derived from tumor tissue and not expanded in vitro with a variety of T cells growth factor contact; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with a variety of T cell growth factors, with a variety of T cell activators, and the The proportion of cells expressing the TNFAIP3 gene in the TIL is about 95% or less, and after at least 2 hours, the TIL is co-cultured with feeder cells, which may include peripheral mononuclear cells, and may be at about 40: The ratio of the feeder cells to the TILs is about 1-400:1, the feeder cells are added to the cell culture medium of the TILs; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 ; Wherein, the second TIL group is obtained through the step (A); (B) the second TIL group can be contacted with IL-2, contacted with various T cell activators, and expressed in the TIL
  • the proportion of cells with the TNFAIP3 gene is about 95% or less, and after at least 2 hours, the TILs are co-cultured with feeder cells, which may comprise peripheral mononuclear cells, and may be in a ratio of about 40:1 to about 400:1
  • the ratio of the feeder cells to the TIL, the feeder cells are added to the TIL cell culture medium; wherein, the third TIL population is obtained through the step (B).
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 , the initial concentration of the IL-2 in the cell culture medium of the TIL can be at least about 300IU/mL; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be made
  • the TIL population is contacted with IL-2, which may have an initial concentration of at least about 300 IU/mL in the cell culture medium of the TILs, with a CD3 antibody, the CD3 antibody in the cell culture medium of the TILs an initial concentration of at least about 30 ng/mL, such that the proportion of cells expressing the TNFAIP3 gene in the TIL is about 95% or less, and after at least 2 hours, the TIL is co-cultured with feeder cells, which may comprise Peripheral mononuclear cells, the feeder cells can be
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 ; Wherein, the second TIL group is obtained through the step (A); (B) the second TIL group can be contacted with IL-2, contacted with a nano-matrix comprising CD3 antibody and CD28 antibody, and made in the TIL The proportion of cells expressing the TNFAIP3 gene is about 95% or less, and the TILs are co-cultured with the feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 ; Wherein, the second TIL group is obtained through the step (A); (B) the second TIL group can be contacted with IL-2, with the nano-matrix comprising CD3 antibody and CD28 antibody, and the nano-matrix of the nano-matrix The diameter can be about 1 nanometer to about 500 nanometers, and each mg of the nanomatrix can comprise about 25 ⁇ g of CD3 antibody and CD28 antibody, so that the proportion of cells expressing the TNFAIP3 gene in the TIL is about 95% or less, And the TILs are co-cultured with the feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor infiltrating lymphocytes
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 ; Wherein, the second TIL group is obtained through the step (A); (B) the second TIL group can be contacted with IL-2, with the nano-matrix comprising CD3 antibody and CD28 antibody, and the nano-matrix of the nano-matrix
  • the diameter can be about 1 nanometer to about 500 nanometers, and each mg of the nanomatrix can contain about 25 ⁇ g of the CD3 antibody and the CD28 antibody, and the nanomatrix and the TIL can be in a ratio of about 1:100 to about 1:2000
  • the proportion of adding to the cell culture medium of the TIL so that the proportion of cells expressing the TNFAIP3 gene in the TIL is about 95% or less, and the TIL is co-cultured with the feeder cells; wherein, after the step (B)
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TIL), which may comprise: (A) a first TIL population derived from tumor tissue and not expanded in vitro may be contacted with IL-2 , the initial concentration of the IL-2 in the cell culture medium of the TIL can be at least about 300IU/mL; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be made
  • the population of TILs is contacted with IL-2, which may have an initial concentration of at least about 300 IU/mL in a cell culture medium of the TILs, with a nanomatrix comprising an antibody to CD3 and an antibody to CD28, the nanomatrix having The diameter can be about 1 nanometer to about 500 nanometers, and each mg of the nanomatrix can contain about 25 ⁇ g of the CD3 antibody and the CD28 antibody, and the nanomatrix and the TIL can be in a ratio of about 1:100 to about 1:2000 The ratio of
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • the method of obtaining TIL cells from a tissue sample of a subject can be obtained by surgically obtaining an orthotopic tumor sample or a metastatic tumor sample, the weight of which can be at least about 1 g, or multiple pieces of tissue can be combined.
  • the tumor tissue is transported at about 2-8 degrees in the sample transport solution, for example, a commonly used commercial tumor tissue transport solution, tumor tissue preservation solution or tumor tissue transfer solution, and processed within 48 hours.
  • the tissue pieces can be mechanically broken to a size of about 1-27 cubic millimeters per piece, transferred into a gas-permeable culture bag or Grex, and T cell serum-free medium is added with a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/mL, For example, 6000 IU/mL) of IL-2 can be cultured for about 3-14 days.
  • T cell serum-free medium is added with a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/mL, For example, 6000 IU/mL) of IL-2 can be cultured for about 3-14 days.
  • the harvested TIL cells can be frozen and resuscitated, or the cells in the culture medium can be directly collected and transferred into a gas-permeable culture bag, or Grex, or Xuri equipment, and the CD28 antibody and CD3 antibody of this application can be added to the T cell serum-free medium And CD28 antibody, magnetic beads comprising CD3 antibody and CD28 antibody (such as Dynabeads) and/or nano-matrix (such as transACT) comprising CD3 antibody and CD28 antibody, the concentration is 300-9000IU/mL (for example, it can be 1000-9000IU/mL , such as IL-2 that can be 6000IU/mL), and the ribonucleoprotein complex (RNP) that carries the gRNA shown in any one of SEQ ID NO: 48 to 61 and Cas protein is transduced to make the The proportion of cells expressing the TNFAIP3 gene in the TIL is about 95% or less.
  • CD28 antibody and CD3 antibody of this application can be added to the T cell serum
  • TIL and PBMC irradiated PBMC
  • PBMC PBMC
  • the CD3 ratio of the final product can be greater than 80%, the cell viability can be greater than 50%, and the T cells greater than 80% can be memory effector T cells and effector T cells.
  • IFN- ⁇ can be secreted after stimulation, and/or can be characterized by an up-regulated proportion of activated T cells.
  • the present application provides a tumor infiltrating lymphocyte (TIL), and the TIL of the present application can be cultured according to the cultivation method of the present application.
  • TILs provided in the present application may comprise one or a batch of TILs cultured by the cultivation method of the present application.
  • the TIL provided in the present application may comprise multiple or multiple batches of TIL cultured by the cultivation method of the present application and combined in any ratio.
  • TILs expanded using the methods of the present application can be administered to a patient as a pharmaceutical composition.
  • the pharmaceutical composition may be a suspension of TIL in a sterile buffer.
  • TILs expanded using the PBMCs of the present application can be administered by any suitable route known in the art.
  • T cells can be administered as a single intra-arterial or intravenous infusion, which can last for about 30 to 60 minutes. Other suitable routes of administration may include intraperitoneal, intrathecal and intralymphatic administration.
  • any suitable dose of TIL can be administered.
  • about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 TILs may be administered.
  • about 1 ⁇ 10 9 to about 12 ⁇ 10 10 TILs may be administered.
  • about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 TILs may be administered.
  • about 3 ⁇ 10 10 to about 12 ⁇ 10 10 TILs may be administered.
  • about 4 ⁇ 10 10 to about 10 ⁇ 10 10 TILs may be administered.
  • about 5 ⁇ 10 10 to about 8 ⁇ 10 10 TILs may be administered.
  • about 6 ⁇ 10 10 to about 8 ⁇ 10 10 TILs may be administered.
  • the therapeutically effective dose may be from about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 . In some embodiments, the therapeutically effective dose may be from about 1 ⁇ 10 9 to about 12 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 3 ⁇ 10 10 to about 12 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 4 ⁇ 10 10 to about 10 ⁇ 10 10 TILs.
  • a therapeutically effective dose may be from about 5 ⁇ 10 10 to about 8 ⁇ 10 10 TILs. In some embodiments, a therapeutically effective dose may be from about 6 ⁇ 10 10 to about 8 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 7 ⁇ 10 10 to about 8 ⁇ 10 10 TILs.
  • the amount of TIL provided in the composition of the present application can be about 1 ⁇ 10 6 , about 2 ⁇ 10 6 , about 3 ⁇ 10 6 , about 4 ⁇ 10 6 , about 5 ⁇ 10 6 , about 6 ⁇ 10 6 , about 7 ⁇ 10 6 , about 8 ⁇ 10 6 , about 9 ⁇ 10 6 , about 1 ⁇ 10 7 , about 2 ⁇ 10 7 , about 3 ⁇ 10 7 , about 4 ⁇ 10 7 , about 5 ⁇ 10 7 , about 6 ⁇ 10 7 , about 7 ⁇ 10 7 , about 8 ⁇ 10 7 , about 9 ⁇ 10 7 , about 1 ⁇ 10 8 , about 2 ⁇ 10 8 , about 3 ⁇ 10 8 , about 4 ⁇ 10 8 , about 5 ⁇ 10 8 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 , about 8 ⁇ 10 8 , about 9 ⁇ 10 8 , about 1 ⁇ 10 9 , about 2 ⁇ 10 9 , about 3 ⁇ 10 9 , about 4 ⁇ 10 9 , about 5 ⁇ 10 9 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 ,
  • the amount of TIL provided in the compositions of the present application may range from about 1 ⁇ 10 6 to 5 ⁇ 10 6 , about 5 ⁇ 10 6 to 1 ⁇ 10 7 , about 1 ⁇ 10 7 to 5 ⁇ 10 7 , about 5 ⁇ 10 7 to 1 ⁇ 10 8 , about 1 ⁇ 10 8 to 5 ⁇ 10 8 , about 5 ⁇ 10 8 to 1 ⁇ 10 9 , about 1 ⁇ 10 9 to 5 ⁇ 10 9 , about 5 ⁇ 10 9 to 1 ⁇ 10 10 , about 1 ⁇ 10 10 to 5 ⁇ 10 10 , about 5 ⁇ 10 10 to 1 ⁇ 10 11 , about 5 ⁇ 10 11 to 1 ⁇ 10 12 , about 1 ⁇ 10 12 to 5 ⁇ 10 12 , or about 5 ⁇ 10 12 to 1 ⁇ 10 13 .
  • the concentration of TIL provided in the composition of the present application can be less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40% of the composition , about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2% , about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%, about 0.009%, about 0.008%, about 0.007%, about 0.006%, about 0.005%, about 0.004%, about 0.003%, about 0.002%, about 0.001%, about 0.0009%, about 0.0008%, about 0.0007%, about
  • the concentration of TIL provided in the composition of the present application can be greater than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, About 20%, about 19.75%, about 19.50%, about 19.25%, about 19%, about 18.75%, about 18.50%, about 18.25%, about 18%, about 17.75%, about 17.50%, about 17.25%, about 17 %, about 16.75%, about 16.50%, about 16.25%, about 16%, about 15.75%, about 15.50%, about 15.25%, about 15%, about 14.75%, about 14.50%, about 14.25%, about 14%, About 13.75%, about 13.50%, about 13.25%, about 13%, about 12.75%, about 12.50%, about 12.25%, about 12%, about 11.75%, about 11.50%, about 11.25%, about 11%, about 10.75 %, about 10.50%, about 10.25%, about 10%, about 9.75%, about 9.50%, about 9.25%, about 9%
  • the concentration of TIL provided in the composition of the present application can range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% of the composition to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17% , about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v /v.
  • the concentration of TIL provided in the compositions of the present application may range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% of the composition to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w/w, w/v or v/v.
  • the amount of TIL provided in the compositions of the present application may be equal to or less than about 10 g, about 9.5 g, about 9.0 g, about 8.5 g, about 8.0 g, about 7.5 g, about 7.0 g, about 6.5 g, about 6.0g, about 5.5g, about 5.0g, about 4.5g, about 4.0g, about 3.5g, about 3.0g, about 2.5g, about 2.0g, about 1.5g, about 1.0g, about 0.95g, About 0.9g, about 0.85g, about 0.8g, about 0.75g, about 0.7g, about 0.65g, about 0.6g, about 0.55g, about 0.5g, about 0.45g, about 0.4g, about 0.35g, about 0.3 g, about 0.25g, about 0.2g, about 0.15g, about 0.1g, about 0.09g, about 0.08g, about 0.07g, about 0.06g, about 0.05g, about 0.04g, about 0.03g, about 0.02g
  • the amount of TIL provided in the compositions of the present application may be greater than about 0.0001 g, about 0.0002 g, about 0.0003 g, about 0.0004 g, about 0.0005 g, about 0.0006 g, about 0.0007 g, about 0.0008 g , about 0.0009g, about 0.001g, about 0.0015g, about 0.002g, about 0.0025g, about 0.003g, about 0.0035g, about 0.004g, about 0.0045g, about 0.005g, about 0.0055g, about 0.006g, about 0.0065g, about 0.007g, about 0.0075g, about 0.008g, about 0.0085g, about 0.009g, about 0.0095g, about 0.01g, about 0.015g, about 0.02g, about 0.025g, about 0.03g, about 0.035g , about 0.04g, about 0.045g, about 0.05g, about 0.055g, about 0.06g,
  • TILs can be administered in a single dose. Such administration may be by injection, for example intravenously. In some embodiments, TILs can be administered in multiple doses. Doses may be once, twice, three, four, five, six or more than six times per year. Dosage can be monthly, biweekly, weekly, or every 2 days. In some embodiments, the administration of TILs can be administered continuously.
  • the present application provides a pharmaceutical composition.
  • it may comprise the TIL of the present application and/or the composition of the present application, and a pharmaceutically acceptable carrier.
  • the present application provides a kit
  • the kit of the present application may comprise the T cell activator, T cell growth factor and/or feeder cells of the method for culturing tumor infiltrating lymphocytes (TIL) of the present application and the method for culturing tumors described in the present application Description of the steps of the infiltrating lymphocyte (TIL) method.
  • the present application provides a kit, which may comprise the TIL of the present application and/or the pharmaceutical composition of the present application.
  • the present application provides a method for affecting the growth of tumor cells, which may include administering the TIL of the present application and/or the pharmaceutical composition of the present application to a subject.
  • affecting tumor growth can comprise reducing the volume of the tumor to, for example, about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40% of its pre-administration volume.
  • the present application provides the application of the TIL of the present application and/or the pharmaceutical composition of the present application in the preparation of medicines, and the medicines of the present application can be used to prevent and/or treat tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • the present application provides a method for preventing and/or treating tumors, which may include administering the TIL of the present application and/or the pharmaceutical composition of the present application to a subject.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • the present application provides a TIL of the present application and/or the pharmaceutical composition of the present application, which can be used for preventing and/or treating tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • PBMC peripheral blood mononuclear cells
  • red blood cells If there are many red blood cells, you can split the red blood cells after centrifugation. Add the red blood cell lysate according to the volume of the cell pellet and the red blood cell lysate at a ratio of 1:2 to 1:3, mix well, and lyse at room temperature for 10 minutes. Mix gently in the middle of the centrifuge tube 2- 3 times to ensure the lysis effect. After the lysis is completed, add PBS or saline to wash the cells. After cleavage, the cells were washed twice, centrifuged at 400g for 6 minutes, and samples were taken and counted before the last centrifugation.
  • Discard the supernatant resuspend the cells in the basal medium, adjust the cell density to about 2-3 ⁇ 107 cells/mL, the liquid level may not exceed 1 cm, and the volume in each T225 culture bottle may be less than 200 mL; , X-ray irradiation 50Gy.
  • the supernatant was discarded by centrifugation, and the cells were frozen according to the counting results, about 1-2 ⁇ 108 cells/mL, 1-2 mL/vessel; the cells were placed in a programmed cooling box and transferred to a -80°C refrigerator for freezing.
  • cpro separation kit Aseptically connect the tubing of the blood bag to the input end of the cpro separation kit (Cytiva). If the blood volume is greater than 120 mL, a pre-concentration step is performed to concentrate the blood volume to less than 120 mL.
  • the neatcell program can be used to separate and wash PBMCs.
  • the washing liquid is physiological saline, with an intermediate volume of 20 mL; the resuspension liquid is the basal medium, and 80 mL/batch is added.
  • each donor’s PBMC is a bag of 100mL. In the flat state, the height of the liquid level can not exceed 1 cm, and the X-ray irradiation is 50Gy.
  • the washing solution is normal saline; set the intermediate volume and final volume so that every 1 ⁇ 10 9 cells are not less than 2 mL; add an equal amount to 2 times for freezing Mix well. Adjust the cell density from 1 ⁇ 10 7 cells/mL to 2 ⁇ 10 8 cells/mL with 1-times cryopreservation solution, aliquot 20 mL/bag, freeze in a programmed cooling apparatus, and store in liquid nitrogen.
  • PBMC cells in blood samples were separated and frozen according to the above PBMC manual separation and cryopreservation procedures. Take a culture bottle or culture bag with a gas-permeable surface, such as a culture bag (Origen), and add 300 mL of rewarmed complete medium.
  • a culture bag with a gas-permeable surface, such as a culture bag (Origen)
  • the complete medium can be optionally selected from X-vivo15 medium or other commercial T cell culture medium , such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and can add essential amino acids and antibiotics, and the added concentration is 300-9000IU/mL (for example, it can be 1000-9000IU/mL, for example, it can be 6000 IU/mL) of IL-2.
  • T cell culture medium such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium
  • the added concentration is 300-9000IU/mL (for example, it can be 1000-9000IU/mL, for example, it can be 6000 IU/mL) of IL-2.
  • ophthalmic scissors and ophthalmic tweezers to make preliminary cuts to remove fat tissue and necrotic tissue, and continue to cut each tissue block to a size of about 27 cubic millimeters.
  • take the non-suspended tumor tissue block use a 20mL syringe to remove the internal piston, connect it to the culture bag, and use a pipette to transfer about 1g of the tissue block into the culture bag through the syringe. Put the culture bag into the carbon dioxide incubator for cultivation.
  • the scissors and tweezers were cleaned, and after initial disinfection with 75% alcohol, ultrasonic cleaning and sterilization were performed to obtain the first TIL group.
  • the liquid should be replaced every 3-7 days or half of the liquid should be replaced to ensure the nutrition of the cells.
  • Use complete medium complete medium can choose X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium, and can add necessary Amino acids and antibiotics, and IL-2 (Double Heron) with a concentration of 300-9000IU/mL (for example, 1000-9000IU/mL, for example, 6000IU/mL) was added.
  • T cell medium such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium
  • IL-2 Double Heron
  • samples can be taken and counted on the 13th or 14th day. If the number of cells is between 5 ⁇ 10 5 and 5 ⁇ 10 8 , enter the harvesting step of step (A).
  • step (A) Collect the cells after step (A) in vitro expansion, centrifuge, discard the medium, wash the cells once with PBS or normal saline, obtain the TILs amplified in vitro (the second TIL population) in step (A), and take a sample for counting About 5 ⁇ 10 5 to 2 ⁇ 10 8 cells enter the subsequent in vitro expansion step; about 5 ⁇ 10 5 cells can be taken for quality control testing; the rest of the cells are added to the cryopreservation solution and stored as cryopreserved preREP TIL in vitro cells .
  • step (A) Continue to culture the TILs (second TIL population) amplified in step (A) in vitro, or perform cell recovery on the frozen preREP TILs in vitro to activate TILs in step (B).
  • complete medium can choose X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium, and can add necessary For amino acids and antibiotics, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 cells/mL, place in a suspended 24-well culture plate, 1 mL/well, and add a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/ mL, for example may be 6000 IU/mL or 6000 IU/mL) of IL-2.
  • T cell medium such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium
  • T cell activators such as CD3 agonists and/or CD28 agonists
  • CD3 antibody Miltenyi Biotech, OKT3
  • CD28 Antibody Merck, 15E8
  • magnetic beads diameter about 1 to 10 ⁇ m Dynabeads, Thermo Fisher
  • the ratio of transACT (approximately 100 to 500 nm in diameter, Miltenyi) to TIL was added to transACT. After culturing for about 0-4 days, the third TIL population was obtained.
  • the gRNA of the present application (sequence shown in any one of SEQ ID NO: 48 to 61) was thawed and added with nuclease-free water to a concentration of about 100 ⁇ M.
  • the above RNP was electroporated with about 1 ⁇ 10 6 cells of the third TIL population by a Lonza electroporator.
  • the electroporation procedure can be human T cell stim (EO115).
  • the fourth TIL population was obtained by culturing for about 0-4 days after gene editing by electroporation.
  • Tn for each test group can be taken from 0 hours to 12 days, such as 24 hours or 48 hours).
  • step (D) First resuscitate the feeder cells mixed with 1-5 donors; mix the activated TIL cells and feeder cells according to the ratio of TIL cells:feeder cells about 1:200, transfer them into G-Rex100 culture flasks or air-permeable bags, and replenish completely Culture medium, sampling and counting every 1-3 days, and rehydration or half-quantity replacement according to the cell state until the total number of cells is greater than 1 ⁇ 10 9 or step (D) in vitro expansion and culture for about 5 days to about 14 days, and step (D) is terminated Culture for in vitro expansion.
  • step (D) Take the cells amplified in step (D), discard the medium supernatant after centrifugation, and wash three times with PBS or physiological saline or compound electrolyte solution to obtain TILs amplified in step (D) (the fifth TIL group), the first Sampling and counting during the three washes, according to the counting results, discard the supernatant after the last centrifugation, take 3 ⁇ 10 6 cells and send them to quality control testing; add all the remaining cells to the cryopreservation solution, adjust the cell density to 1-3 ⁇ 10 8 cells/ mL frozen.
  • Tn can range from 0 hour to 14 days
  • Tn is selected from 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 5 days, 7 days, and 9 days to obtain TIL cultured at different addition times of feeder cells, and compare the cell counts test.
  • FIG. 1 shows the analysis results of the proliferation ability of TIL cultured with feeder cells at different addition times.
  • the values on the vertical axis in each group of graphs in which TIL was cultured with feeder cells at different addition times indicate the expansion factor of the number of TIL cells after the end of in vitro expansion compared with before the start of in vitro expansion.
  • the proliferation results of TILs from 4 donors showed that the TILs cultured with feeder cells added 0 hours after the addition of OKT3 and IL-2 (that is, at the same time), the proliferation ability was weaker than that of 24 hours or 48 hours after the addition of OKT3 and IL-2 Then add feeder cell cultured TIL.
  • T n can range from 0 hour to 14 days
  • Tn is selected from 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 5 days, 7 days, and 9 days to obtain TIL cultured at different addition times of feeder cells, and perform flow detection Comparative Test.
  • Transcription Factor Buffer Set manufacturer BD, product number 562574; V-bottom 96-well plate, manufacturer Corning, product number 3894; flow tube, manufacturer Corning, product number 352052.
  • the flow cytometry antibodies in this example were purchased from BD or Biolegend. Add 1 ⁇ 10 5 to 5 ⁇ 10 5 cell samples in each group into flow tubes or V-bottom 96-well plates. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, flow tube 1mL/tube, 96-well plate 200 ⁇ L/well, discard supernatant. Add the prepared antibody working solution for cell surface staining, the antibody (BD or Biolegend) concentration is 1:100 to 1:200, and the activity detection dye is 1:10000. Flow tube 100 ⁇ L/tube, 96-well plate 50 ⁇ L/well for staining, incubate at 2-8°C for 30 minutes in the dark.
  • BD Transcription Factor Buffer Set
  • BD Fixation/Permeabilization
  • BD Fixation/Permeabilization
  • BD Fixation/Permeabilization
  • BD Perm/Wash Buffer
  • Cell fixation and membrane rupture Sufficiently resuspend the cells, add an appropriate amount (96-well plate 100 ⁇ L/well, flow tube 1mL/tube) 1 ⁇ working solution A to fix and rupture the membrane, and incubate at 2-8°C for 40-50 minutes in the dark. After fixation and membrane rupture, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B to prepare intracellular antibody, the antibody concentration is 1:100 to 1:200, 50 ⁇ L/well of 96-well plate, 100 ⁇ L/tube of flow tube, and stain for 30 minutes at 2-8°C in the dark. After staining, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice. . Use 100-500 ⁇ L PBS to resuspend the cells for flow cytometry detection.
  • Figure 38 to Figure 44 show the flow cytometric analysis of TIL cultured with feeder cells at different addition times.
  • FIGS. 2 and Figure 3 show the proportion of CD45RA - CCR7 + central memory T cells (Tcm) in TIL cells cultured after adding OKT3 and IL-2 0 hours, 24 hours or 48 hours after adding feeder cells. .
  • the results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of central memory T cells than TILs cultured with feeders at the same time.
  • FIG. 4 shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells obtained by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a lower proportion of regulatory T cells than TILs cultured with feeder cells at the same time.
  • Figures 5 and 6 show the proportion of activated T cells in TIL cells cultured by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TIL supplemented with feeder cells after 24 hours or 48 hours had a higher proportion of activated T cells, such as a higher proportion of PD1 + , LAG3 + and/or CD28 + cells, than TILs cultured with feeder cells at the same time. high.
  • Figure 7 shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells cultured by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of tumor-specific T cells than TILs cultured with feeder cells at the same time.
  • Figure 8 shows the ratio of TCF1 + stem cell-like T cells in TIL cells obtained by adding feeder cells cultured TILs 0 hours, 24 hours or 48 hours after the addition of OKT3 and IL-2. The results showed that the TIL cultured with feeder cells after 24 hours or 48 hours had a higher proportion of stem cell-like T cells than the TILs cultured with feeder cells at the same time.
  • Figure 9 shows the analysis results of the proliferation ability of the test group and the control group added with different forms of CD28 agonists.
  • the value on the ordinate in the figure indicates the expansion factor to which the number of TIL cells is amplified to when the TIL population obtained from in vitro expansion of each test group is compared with the TIL population before in vitro expansion.
  • the results showed that the in vitro expansion of step (B) in the four-step division method added CD28 antibody, and the proliferation ability of TIL obtained was stronger than that of TIL cultured in the control group (without adding CD28 antibody).
  • FIG 10 shows the ratio of T cell subsets of TIL cells cultured in the mixed antibody group and the control group.
  • step (B) in the four-step division method was expanded in vitro and CD28 antibody was added, and compared with the control group (without adding CD28 antibody), the TIL obtained had an improved ratio of T cell subsets.
  • higher proportion of activated T cells CD28 + or 41BB +
  • lower proportion of regulatory T cells Treg, e.g. CD4 + CD25 + Foxp3 +
  • TCF1 + stem cell-like T cells
  • Tcm central memory T cells
  • FIG 11 shows the ratio of T cell subsets of TIL cells cultured in the mixed antibody group and the control group.
  • step (B) in the four-step division method was expanded in vitro and CD28 antibody was added, and compared with the control group (without adding CD28 antibody), the TIL obtained had an improved ratio of T cell subsets.
  • a higher proportion of tumor-specific T cells CD103 + CD39 +
  • a higher proportion of activated T cells CD25 +
  • Reg regulatory T cells
  • FIG 12 shows the ratio of T cell subsets of TIL cells cultured in the magnetic bead group and the control group.
  • step (B) in the four-step division method was expanded in vitro and added with CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had improved Proportions of T cell subsets.
  • CD28 antibody for example, adding magnetic beads containing CD3 antibody and CD28 antibody
  • TCF1 + stem cell-like T cells
  • Tcm central memory T cells
  • Figure 13 shows the ratio of T cell subsets of TIL cells cultured in the magnetic bead group and the control group.
  • step (B) in the four-step division method was expanded in vitro and added with CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had improved Proportions of T cell subsets.
  • TCF1 + stem cell-like T cells
  • 41BB + activated T cells
  • Tcm central memory T cells
  • Reg Proportion of regulatory T cells
  • CD33 + CD39 + tumor-specific T cells
  • FIG 14 shows the ratio of T cell subsets of TIL cells cultured in the nano-matrix group and the control group.
  • step (B) in the four-step division method was expanded in vitro and added CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had improved T Proportions of cell subpopulations.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • the obtained TIL had improved T Proportions of cell subpopulations.
  • a higher proportion of tumor-specific T cells CD103 + CD39 +
  • a higher proportion of activated T cells CD25 + or PD1 +
  • Tcm central memory T cells
  • target cells such as Hela tumor cells
  • CFSE (6(6)-Carboxyfluorescein diacetate N-succinimidyl ester, Sigma, 21888-25MG-F): wash tumor cells with PBS, resuspend tumor cells in 500 ⁇ L of PBS; add CFSE to 500 ⁇ L of PBS , mixed with 500 ⁇ L of tumor cell resuspension in PBS, to a final concentration of CFSE of 0.5 ⁇ mol/L. After incubating at 37°C for 6 minutes, add medium containing 10% FBS to wash, centrifuge at 600g for 5 minutes, use X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi, etc.
  • TIL cells Resuspend tumor cells in branded T cell medium at a concentration of 5 x 105 cells/mL.
  • the TIL cells in each test group were centrifuged at 600g for 5 minutes, and the TIL cells were resuspended according to the effect-to-target ratio (ratio of TIL cells to tumor cells) of 3:1 (that is, the concentration of resuspended TIL cells was 1.5 ⁇ 10 6 cells/mL).
  • ratio of TIL cells to tumor cells was 1.5 ⁇ 10 6 cells/mL.
  • a group of control groups containing only tumor cells was set up, and different reagents were added according to different groups of experiments. Centrifuge the plate at 200g for 1 minute, and incubate at 37°C for 4 hours to overnight.
  • Killing rate% Dapi + CFSE + cell number/total CFSE + ⁇ 100%.
  • Figure 15 shows the cell killing ability of TIL cells cultured in the nano-matrix group and the control group.
  • step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher cell killing ability.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • Figure 16 shows the detection results of intracellular factor expression in TIL cells cultured in the mixed antibody group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added with CD28 antibody.
  • the TIL obtained had a higher ability to express intracellular factors. For example, higher CD107a expression capacity.
  • Figure 17 shows the detection results of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher expression of intracellular factors. For example, higher CD107a expression capacity.
  • Figure 18 shows the detection results of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher expression of intracellular factors. For example, higher CD107a expression capacity.
  • Figure 19 shows the detection results of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher expression of intracellular factors. For example, higher CD107a expression capacity.
  • Figure 20 shows the detection results of intracellular factor expression in TIL cells cultured in the magnetic bead group and the control group.
  • the results showed that the step (B) in the four-step division method was amplified in vitro and added CD28 antibody (for example, adding magnetic beads containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher expression of intracellular factors. For example, higher CD107a expression capacity.
  • Figure 21 shows the detection results of intracellular factor expression in TIL cells cultured in the nano-matrix group and the control group.
  • the results showed that the step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher Intracellular factor expression ability.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • the control group without adding CD28 antibody
  • Figure 22 shows the results of cytokine secretion detection of TIL cells cultured in the nano-matrix group and the control group.
  • the results showed that the step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher Cytokine secretion capacity.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • control group without adding CD28 antibody
  • the TIL obtained from each test group was incubated with the tumor cells overnight, and after the incubation, the supernatant was taken to detect cytokine secretion according to the detection steps in this example.
  • Fig. 23 shows the cytokine secretion detection results after co-incubating TIL cells and tumor cells obtained from the nano-matrix group and the control group.
  • the results showed that the step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), compared with the control group (without adding CD28 antibody), the obtained TIL had higher Cytokine secretion capacity.
  • CD28 antibody for example, adding transACT containing CD3 antibody and CD28 antibody
  • TIL cells of each test group in Example 1 were expanded and cultured in vitro for 48 hours in step (B) of the four-step division method, and the gene knockout efficiency was detected.
  • Nuclease-free water (commercial source: Shanghai Youfan Biotechnology Co., Ltd.; RT121-02) was used to prepare sgRNA (sequence shown in SEQ ID NO: 1, GGAGAATGACGAGTGGACCC), and the concentration was adjusted to 50 ⁇ mol/L. Take 2 ⁇ L of gRNA and add it to a PCR tube, Nuclease-free water is used as a negative control, incubate in a PCR instrument at 95°C for 2 minutes, then cool at room temperature for 10 minutes.
  • P3Buffer commercial source: Lonza; V4XP-3032
  • 61.7 ⁇ mol/L Cas9 nuclease commercial source: Suzhou Kerui Gene Biotechnology Co., Ltd.; C01-2019-11-001
  • T cell medium at 1 mL/well and place in a CO 2 incubator to preheat.
  • an electroporation apparatus Lionza
  • feeder cells irradiated PBMC cells
  • the TIL cells of each test group after the end of the culture were taken for cell counting, and 2 ⁇ 10 5 cells were taken from each test group, centrifuged at 500g for 3 minutes, and the supernatant was discarded after centrifugation.
  • Fixable Viability Dye eFluor 780 (commercial source: eBioscience; 65-0865-18) and dilute it 10000 times in PBS; take 100 ⁇ L of diluted Fixable Viability Dye eFluor 780 in PBS solution, add 1 ⁇ L TCR- ⁇ -APC (commercial source: eBioscience; 17-9986-42), 1 ⁇ L of BB515 Mouse Anti-Hu CD8 (commercial source: BD Pharmingen; 564526), 1 ⁇ L of PE-Cy7Mouse Anti-Hu CD4 (commercial source: BD Pharmingen; 557852), mix well.
  • Figure 24 shows the gene knockout efficiency results of TIL cells cultured in the nano-matrix group and the control group.
  • step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), and compared with the control group (without adding CD28 antibody), the resulting TILs had improved genes.
  • Knockout efficiency capability For example, improved TCR ⁇ gene knockout efficiency.
  • Figure 25 shows the results of gene knockout efficiency of TIL cells cultured in the nano-matrix group and the control group.
  • step (B) in the four-step division method was expanded in vitro by adding CD28 antibody (for example, adding transACT containing CD3 antibody and CD28 antibody), and compared with the control group (without adding CD28 antibody), the resulting TILs had improved genes.
  • Knockout efficiency capability For example, improved TCR ⁇ gene knockout efficiency.
  • the first TIL population derived from tumor tissue and not expanded in vitro was obtained, and the first TIL population was subjected to steps (A), step (B), and step (C) in the four-step division method in the same manner and step (D) to obtain a fifth population of TILs.
  • the fifth TIL population was randomly divided into 3 groups, and IL-2 was added to the T cell culture medium of each test group, while the blank group was not added any T cell activator, and the CD3 antibody was added to the CD28 agonist group at about 30 ng/mL
  • a CD3 agonist and a CD28 agonist were added, for example, transACT was added at a ratio of transACT to TIL of about 1:100-1:2000.
  • the TIL (terminal stimulatory cell population) obtained after culturing for 3 days was used to detect the proliferation ability of TIL cells by using the CellTiter-Glo kit (commercial source: Promega) by the cell viability detection method.
  • Figure 26, Figure 27 and Figure 28 respectively show the proliferative ability analysis results of the test groups that were expanded in vitro in different ways at the terminal stimulation stage for TILs from different donors.
  • the fluorescence values on the ordinate in the figure reflect the proliferation ability of TIL cells subjected to terminal stimulation in different ways in each test group.
  • the results showed that the addition of a CD28 agonist to the terminal stimulation had similar proliferative capacity of TILs compared to the terminal stimulation without the addition of a CD28 agonist.
  • DNA extraction solution QuickExtract DNA extraction solution, Lucigen, QE09050
  • nuclease-free water RNase/DNase free water, Tiangen
  • EDTA Etggong, 0.5M
  • Genomic DNA extraction About 4 days after TIL cell knockout, take about 1 ⁇ 105 to about 2 ⁇ 105 cells, add the prepared nuclease mixture (DNase I), and incubate at 37°C for 5 minutes . Add 2.5 ⁇ L of 0.5M EDTA to the sample and incubate at 80°C for 10 minutes. After centrifugation, add 50 ⁇ L of DNA extraction solution, run the following program after brief centrifugation: 75°C-10 minutes; 95°C-5 minutes; 4°C-maintain. DNA sample concentration can be detected using a spectrophotometer (NanoDrop TM ).
  • PCR primers can be designed in a region of about 100 to about 200 nucleotides upstream and downstream of the PAM site. Design the PCR reaction system as follows:
  • Reagent volume 2 ⁇ PCR Premix Buffer 25 ⁇ L DNA template About 100-500ng Forward primer (10 ⁇ M) 1 ⁇ L Reverse primer (10 ⁇ M) 1 ⁇ L double distilled water Replenish to 50 ⁇ L DMSO 1.5 ⁇ L
  • the PCR products were subjected to Sanger sequencing analysis.
  • TILs can be derived from different tumor patients.
  • Donors A and B are patients with skin malignant melanoma
  • donors C are patients with pancreatic cancer
  • donors D and E are patients with non-small cell lung cancer.
  • the sgRNA indicated by (TATGCCATGAGTGCTCAGAG), namely TP4 has a knockout efficiency of about 59.9% for donor A, about 34% for donor B, and about 60.4% for donor C, The knockout efficiency for donor D was about 33.8%.
  • Various gene editing methods of the present application can achieve a certain percentage of knockout efficiency.
  • TIL cells in each group were re-plated with the same total number of cells, and cultured in a cell culture medium that did not contain IL-2, that is, IL-2 was removed for culture. After 3 days, the expansion efficiency of TIL cells was analyzed with CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega).
  • Figure 29, Figure 30, Figure 31 and Figure 32 show, for TIL cells derived from different donors, the amount of fluorescence after expansion of TIL cells in each group. The results show that the gene-edited TIL cells of the present application can still have significant expansion ability even if cultured in a cell culture medium that does not contain IL-2.
  • the 96-well plate was pretreated with 30ng/mL CD3 antibody (Miltenyi Biotech, OKT3) and incubated overnight at 4°C; Cell culture medium without IL-2. After 3 days, the expansion efficiency of TIL cells was analyzed with CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega).
  • Figure 33 and Figure 34 show, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group.
  • NT represents the negative control group, TIL that has not undergone gene editing
  • ST3 represents the irrelevant knockout group, TIL that is knocked out in a region that does not affect cell function
  • Knockout TILs TP4 represents TILs knocked out by sgRNA shown in SEQ ID NO:58. * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, *** indicates p ⁇ 0.001, **** indicates p ⁇ 0.0001.
  • TIL cells:tumor cells, E:T effect-to-target ratio
  • the instructions of the apoptosis detection reagent (Incucyte Caspase-3/7 Green Dye for Apoptosis, Sartorius), add the apoptosis detection reagent at 0.2 ⁇ L/well, and add the medium to dilute Caspase 3/7 Green Dye at 25 ⁇ L/well .
  • the incucyte recorder (Sartorius) was used to record the activity of Caspase 3/7 to analyze the killing ability of TIL on tumor cells, and recorded every 3 hours, and the total recording time was about 5 days.
  • Figure 35 shows the test results of the killing ability of TIL cells derived from donor B co-cultured with tumor cells at an effect-to-target ratio of 1:1.
  • Figure 36 and Figure 37 show the killing ability test results of TIL cells derived from donor C co-cultured with tumor cells at an effect-to-target ratio of 1:1 and an effect-to-target ratio of 1:3. The results showed that gene-edited TIL cells can have more significant tumor cell killing ability.
  • IL-2 is an important factor that regulates the growth of T cells. This example tests whether the gene-edited TIL cells of the present application can survive and/or expand independently of IL-2.
  • TIL cells in each group were re-plated with the same total number of cells, and cultured in a cell culture medium that did not contain IL-2, that is, IL-2 was removed for culture.
  • the expansion efficiency and viability of TIL cells were analyzed with a cell counter every 2 to 4 days.
  • the amplification efficiency is expressed by the multiplication factor: the total number of cells on the day of IL-2 removal (the 0th day) is taken as 1, and the proliferation factor on the nth day is expressed as the total number of cells on the nth day/the total number of cells on the 0th day; Indicates the percentage of the number of surviving cells to the total number of cells.
  • Figure 38 and Figure 39 show, for TIL cells derived from different donors, the proliferation factor of TIL cells in each group.
  • Figure 40 and Figure 41 show the viability of TIL cells in each group for TIL cells from different donors. The results show that even if IL-2 is withdrawn, the gene-edited TIL cells of the present application can still have significant expansion ability and maintain a high cell viability.
  • TIL populations obtained on the 8th day after gene editing in each test group in Example 1 were detected by flow cytometry.
  • V-bottom 96-well plate manufacturer Corning, product number 3894; flow tube, manufacturer Corning, product number 352052.
  • the flow cytometry antibodies in this example were purchased from BD or Biolegend. Add 1 ⁇ 10 5 to 5 ⁇ 10 5 cell samples in each group into flow tubes or V-bottom 96-well plates. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, flow tube 1mL/tube, 96-well plate 200 ⁇ L/well, discard supernatant. Add the prepared antibody working solution for cell surface staining, the antibody (BD or Biolegend) concentration is 1:100 to 1:200, and the activity detection dye is 1:10000. Flow tube 100 ⁇ L/tube, 96-well plate 50 ⁇ L/well for staining, incubate at 2-8°C for 30 minutes in the dark.
  • Figure 42 and Figure 43 show, for TIL cells derived from donor C, the ratio of TIM-3 positive and CD101 positive cells in TIL cells of each group. The results showed that the gene-edited TIL cells had a lower proportion of exhausted cells.
  • FIG 44 shows, for TIL cells derived from donor C, the ratio of CD45RA-negative CCR7-positive memory T cells (Tcm) among TIL cells in each group. The results showed that gene-edited TIL cells had a higher proportion of central memory T cells.
  • TIL populations obtained on the 7th or 8th day after the gene editing in each test group in Example 1 the expression of cytokines was detected by flow cytometry.
  • CD3 antibody group 30 ng/ml CD3 antibody (Miltenyi Biotech, OKT3) was used to coat flat-bottomed 96-well plates 1 day in advance, and coated overnight at 4°C; on the day of plating in the PMA group, phorbol-myristate- Acetate (PMA, 25ng/ml) and ionomycin (Ionomycin, 1 ⁇ g/ml) are sufficient.
  • PMA phorbol-myristate- Acetate
  • Ionomycin 1 ⁇ g/ml
  • Cell fixation and membrane rupture Sufficiently resuspend the cells, add an appropriate amount (96-well plate 100 ⁇ L/well, flow tube 1mL/tube) 1 ⁇ working solution A to fix and rupture the membrane, and incubate at 2-8°C for 40-50 minutes in the dark. After fixation and membrane rupture, add 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B 1 ⁇ working solution B to wash the cells (200 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • FIG. 49, Fig. 50, Fig. 51 and Fig. 52 show is, under the condition that CD3 antibody (Miltenyi Biotech, OKT3, CD3 antibody is pretreated with 30ng/mL 96-well plates) stimulated overnight, for TIL derived from different donors Cells, the proportion of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group.
  • CD3 antibody Miltenyi Biotech, OKT3, CD3 antibody is pretreated with 30ng/mL 96-well plates
  • Fig. 53, Fig. 54, Fig. 55 and Fig. 56 show the conditions under which phorbol-myristate-acetate (PMA, 25ng/ml) and ionomycin (Ionomycin, 1 ⁇ g/ml) stimulated overnight Bottom, for TIL cells from different donors, the proportion of CD107a, GZMB, TNF- ⁇ and IFN- ⁇ expressing cells in TIL cells of each group.
  • PMA phorbol-myristate-acetate
  • Ionomycin Ionomycin
  • TIL cells in each group were re-plated with the same total number of cells, and cultured in a cell culture medium that did not contain IL-2, that is, IL-2 was removed for culture.
  • T cell receptor (TCR) clonal diversity in TILs was analyzed on days 8 and 18 after gene editing.
  • TCR pedigree kit Beta Mark TCR V ⁇ Repertoire Kit, Beckman Coulter
  • 1 ⁇ L of PerCP-cy5.5-labeled CD8 antibody BD
  • PE-cy7-labeled CD4 antibody BD
  • 0.01 ⁇ L of eFluor 780-labeled dead cell dye eBioscience
  • TCR diversity of TIL cells was identified according to the instructions of the TCR lineage kit (Beta Mark TCR V ⁇ Repertoire Kit, Beckman Coulter). Table 1 shows the correspondence between the fluorescence from tube A to tube H and the TCR V ⁇ clone. Where the TCR V ⁇ clone is other, it may mean that the T cells under this ratio do not have the TCR V ⁇ that can be identified by the TCR lineage kit. Tables 2 to 5 show the diversity of TCR V ⁇ clones of CD4 + T cells and CD8 + T cells on day 8 and day 18 after gene editing of TIL cells.
  • Table 2 shows the diversity of TCR V ⁇ clones in CD4 + T cells on day 8 after TIL cell gene editing.
  • Table 3 shows the diversity of TCR V ⁇ clones in CD4 + T cells on day 18 after gene editing in TIL cells.
  • Table 4 shows the diversity of TCR V ⁇ clones in CD8 + T cells on day 8 after gene editing in TIL cells.
  • Table 5 shows the diversity of TCR V ⁇ clones in CD8 + T cells on day 18 after TIL cell gene editing.
  • Figure 57 and Figure 58 show the Shannon diversity index of TCR V ⁇ clones of CD4 + T cells and CD8 + T cells on day 8 and day 18 after gene editing of TIL cells.
  • the Shannon diversity index reflects the diversity of TCR V ⁇ clones, and the results show that gene-edited TIL cells can maintain a higher diversity of ⁇ -chain subtypes at day 18, indicating that gene-edited TIL cells can significantly maintain TCR for a long time
  • the diversity of clones can help to achieve stronger antigen recognition and immune response to tumor cells.
  • TIL activation of the second-stage expansion in 1.4 of Example 1 take the amount of cells expanded in the first stage, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, and place them in a suspended 24-well culture plate , 1 mL/well, add CD3 antibody, eg OKT3 about 30 ng/mL, add IL-2 at a concentration of about 1000-9000 IU/mL, eg 3000 or 6000 IU/mL IL-2.
  • feeder cells were added into the culture environment of tumor infiltrating lymphocytes.
  • TIL and feeder cells can be added at a ratio of 1:40-1:400, and all the cells are collected after about 9-14 days of expansion and culture in the second stage, and the results of TIL obtained from the culture are detected and counted.
  • TILs derived from tumors from different donors were regarded as different batches; the data of each batch of the test group in which OKT3 and IL-2 were added to feeder cells at the same time (0h group) were used as benchmark 1, and the data of other time points in the same batch were tested The data of each group were standardized, and the relative proliferative ability of each test group in the second stage of amplification relative to the 0h group was counted.
  • Fig. 59A shows the results of cell proliferation ability of TIL cells obtained by adding feeder cell-cultured TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. Compared with the TILs cultured with feeder cells added 0 hours after the addition of OKT3 and IL-2 (that is, at the same time), the proliferation ability of TILs cultured with feeder cells 24 hours or 48 hours after the addition of OKT3 and IL-2 was significantly enhanced.
  • Flow cytometric detection was performed on the TIL population obtained by culturing the above-mentioned feeder cells at different addition times.
  • TILs derived from tumors from different donors were regarded as different batches; the data of each batch of the test group in which OKT3 and IL-2 were added to feeder cells at the same time (0h group) were used as benchmark 1, and the data of other time points in the same batch were tested The data of each group were standardized, and the cell composition ratio of each experimental group expanded in the second stage relative to the 0h group was counted.
  • test procedure of flow detection refer to the content of Example 3 of the present application.
  • FIG 59B shows the results of CD45RA - CCR7 + central memory T cell (Tcm) ratio of TIL cells obtained by adding feeder cell-cultured TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Tcm central memory T cell
  • Figure 59C shows the ratio of TCF1 + stem cell-like T cells in TIL cells cultured after adding OKT3 and IL-2 0 hours, 24 hours or 48 hours after adding feeder cells cultured TILs. The results showed that TIL cultured with feeder cells added after 24 hours or 48 hours had a higher proportion of stem cell-like T cells in CD8 + than TILs cultured with feeder cells added at the same time.
  • Figure 59D shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells obtained by adding feeder cell cultured TILs after adding OKT3 and IL-2 for 0 hours, 24 hours or 48 hours. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a lower proportion of regulatory T cells than TILs cultured with feeder cells at the same time.
  • Figure 59E shows the proportion of activated T cells (PD-1 + ) in TIL cells cultured after adding OKT3 and IL-2 0 hours, 24 hours or 48 hours after adding feeder cell-cultured TILs.
  • the results showed that TIL cultured with feeder cells supplemented after 24 hours or 48 hours had a higher proportion of activated T cells, such as PD-1 in CD8 + and/or CD4 + , than TILs cultured with feeder cells at the same time + Higher percentage of cells.
  • Figure 59F shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours after adding feeder cell-cultured TILs.
  • the results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of tumor-specific T cells in CD8 + and/or CD4 + than TILs cultured with feeder cells at the same time.
  • FIG. 59G shows the proportion of activated T cells (CD28 + ) in TIL cells obtained by adding feeder cells cultured TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of activated T cells, such as a higher proportion of CD8 + CD28 + cells, than TILs cultured with feeder cells at the same time.
  • Figure 59H shows the proportion of activated T cells (41BB + ) in TIL cells cultured by adding feeder cell-cultured TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TILs cultured with feeder cells supplemented after 24 hours or 48 hours had a higher proportion of activated T cells, such as 41BB + cells in CD8 + and/or CD4 + cells, than TILs cultured with feeder cells at the same time The proportion is higher.
  • Figure 59I shows the percentage of activated T cells (CD25 + ) in the cultured TIL cells obtained by adding feeder cell-cultured TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TILs cultured with feeder cells supplemented after 24 hours or 48 hours had a higher proportion of activated T cells, such as CD25+ cells in CD8 + and/or CD4 + cells, than TILs cultured with feeder cells at the same time The proportion is higher.
  • Figure 59J shows the detection results of intracellular factor expression in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours after adding feeder cell-cultured TIL.
  • the results showed that the TIL cultured with feeder cells added after 24 hours or 48 hours had a higher ability to express intracellular factors than the TILs cultured with feeder cells added at the same time. For example, higher CD107a expression capacity in CD3 + , CD8 + and/or CD4 + .
  • the cytokine secretion detection method can refer to the instructions of the cytokine detection kit (BD), reconstitute the human Th1/Th2/Th17 cytokine standard freeze-dried powder (BD) with 2mL Assay Diluent diluent (BD) (Standard stock solution The concentration of each cytokine is 5000pg/mL) and in order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 serial dilution, marked as "standard tube”. Take 1 tube containing Assay Diluent dilution only as a reference.
  • BD cytokine detection kit
  • BD Assay Diluent diluent
  • Figure 59K shows the cytokine secretion detection results of TIL cells cultured by adding feeder cell-cultured TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • the results showed that TIL cultured with feeder cells added after 24 hours or 48 hours had higher cytokine secretion ability than TILs cultured with feeder cells added at the same time. For example, higher TNF- ⁇ secretion capacity, or higher IFN- ⁇ secretion capacity.
  • TIL activation of the second-stage expansion in 1.4 of Example 1 take the amount of cells expanded in the first stage, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, and place them in a suspended 24-well culture plate , 1 mL/well, add CD3 antibody, such as OKT3 about 30 ng/mL, add IL-2 at a concentration of about 1000-9000 IU/mL, such as 3000 or 6000 IU/mL IL-2.
  • feeder cells were added to the culture environment of tumor infiltrating lymphocytes.
  • TIL and feeder cells can be added at a ratio of 1:40-1:400, such as 1:200, and all cells are collected after about 9-14 days of second-stage expansion culture, and the results of cultured TIL are detected and counted.
  • Figure 59L shows that the TIL cultured by feeder cells was added after 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding OKT3 and IL-2, and the cell proliferation ability of the TIL cells obtained by culturing Result graph. Compared with the TILs cultured with feeder cells added 0 hours after the addition of OKT3 and IL-2 (i.e. at the same time), the proliferation ability of TILs cultured with feeder cells after 12 hours or more after the addition of OKT3 and IL-2 was significantly enhanced .
  • Flow cytometric detection was performed on the TIL population obtained by culturing the above-mentioned feeder cells at different addition times.
  • TILs derived from tumors from different donors were regarded as different batches; the data of each batch of the test group in which OKT3 and IL-2 were added to feeder cells at the same time (0h group) were used as benchmark 1, and the data of other time points in the same batch were tested The data of each group were standardized, and the cell composition ratio of each experimental group expanded in the second stage relative to the 0h group was counted.
  • test procedure of flow detection refer to the content of Example 3 of the present application.
  • Figure 59M shows the CD8 + T of TIL cells cultured after adding OKT3 and IL-2 at 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder-cultured TILs. cell ratio. The results showed that TILs cultured with feeder cells added 12 hours or more after the addition of OKT3 and IL-2 had a higher proportion of CD8 + T cells than TILs cultured with feeder cells at the same time.
  • Figure 59N shows the CD45RO + CD62L of TIL cells cultured after adding OKT3 and IL-2 at 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells. + T cell ratio.
  • the results showed that TILs cultured with feeder cells added 12 hours or more after addition of OKT3 and IL-2 had higher memory T cells (Tcm, CD45RO + CD62L + ) compared with TILs cultured with feeder cells at the same time. proportion.
  • Figure 59O shows the addition of feeder cell-cultured TILs at 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after the addition of OKT3 and IL-2, and the NK T cells of the resulting TIL cells. Proportion. The results showed that TILs cultured with feeder cells added 12 hours or more after the addition of OKT3 and IL-2 had a higher proportion of NK T cells than TILs cultured with feeder cells at the same time.
  • Figure 59P shows the CD4 + CD25 CD4 + CD25 + Foxp3 + regulatory T cell (Treg) ratio. The results showed that TILs cultured with feeder cells added 12 hours or more after the addition of OKT3 and IL-2 had a lower proportion of regulatory T cells than TILs cultured with feeder cells at the same time.
  • TIL activation in the second stage of expansion in 1.4 of Example 1 take the amount of cells expanded in the first stage, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, and place them in a suspended 24-well culture plate , 1 mL/well, add CD3 antibody, such as OKT3 about 30 ng/mL, add IL-2 at a concentration of about 1000-9000 IU/mL, such as 3000 or 6000 IU/mL IL-2. 12 hours to 14 days after the addition of the above OKT3 and IL-2, for example 48 hours later, the feeder cells are added into the culture environment of tumor infiltrating lymphocytes. Among them, TIL and feeder cells can be added at a ratio of 1:40-1:400, and all cells are collected after about 9-14 days of expansion and culture in the second stage, and the cell killing ability of TIL obtained from culture is detected and counted.
  • CD3 antibody such as OKT3 about 30 ng/mL
  • IL-2 at a concentration of about 1000-9
  • TILs obtained from each test group for detection and target cells for co-culture such as A375 melanoma cells and/or Hela cervical cancer cells.
  • CFSE (6(6)-Carboxyfluorescein diacetate N-succinimidyl ester, Sigma, 21888-25MG-F): wash tumor cells with PBS, resuspend tumor cells in 500 ⁇ L of PBS; add CFSE to 500 ⁇ L of PBS , mixed with 500 ⁇ L of tumor cell resuspension in PBS, to a final concentration of CFSE of 0.5 ⁇ mol/L. After incubating at 37°C for 6 minutes, add medium containing 10% FBS to wash, centrifuge at 600g for 5 minutes, use X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi, etc.
  • TIL cells Resuspend tumor cells in branded T cell medium at a concentration of 1 x 106 cells/mL.
  • the TIL population of each test group was centrifuged at 600g for 5 minutes, and the TIL cells were resuspended according to the effect-to-target ratio (the ratio of TIL cells to tumor cells) of 3:1 (that is, the concentration of resuspended TIL cells was 3 ⁇ 10 6 cells/mL).
  • TIL and tumor cells are co-cultured, substances that activate TIL cells can be omitted as the non-activation group, or transACT (Miltenyi, a nano-matrix material containing CD3 antibody and CD28 antibody) can be added as the activation group.
  • transACT Miltenyi, a nano-matrix material containing CD3 antibody and CD28 antibody
  • Killing rate% Dapi + CFSE + cell number/total CFSE + ⁇ 100%, or the killing rate can be expressed by Dapi + cell number/total tumor cell number.
  • Figure 59Q shows the results of the cell killing ability of TIL cells cultured by adding feeder cells cultured TILs 48 hours after the addition of OKT3 and IL-2. The results showed that 48 hours after the addition of OKT3 and IL-2, the TIL cultured with feeder cells had significant tumor cell killing ability, such as melanoma and/or cervical tumor.
  • the knockout efficiency detection refers to the cell knockout efficiency detection method of the present application, and the results are shown in the table below.
  • D903 is from a patient with cervical cancer
  • D904 is from a patient with non-small cell lung cancer
  • D107 is from a patient with non-small cell lung cancer
  • D812 is from a patient with skin malignant melanoma
  • D003 is from a patient with skin cancer.
  • TILs obtained from the sgRNA knockout group or control group (NT-no treatment) provided in this application are spread on a 96-well plate at 1.5e5/well 7 days after electroporation.
  • the CTG kit was used to analyze the cell expansion after 3 days.
  • Figure 60A shows, for TIL cells from different donors, the amount of fluorescence after expansion of TIL cells in each group under the non-stimulation detection condition.
  • anti-CD3 antibody for example, OKT3, 30ng/ml
  • Fig. 60B shows, for TIL cells derived from different donors, the amount of fluorescence after expansion of TIL cells in each group under the stimulation detection condition of anti-CD3 antibody. The results show that the gene-edited TIL cells of the present application can have significant expansion ability.
  • This application also detects the long-term proliferation/survival ability of the gene-edited TIL cells after culture without IL-2.
  • the TIL obtained from the sgRNA knockout group or control group (NT-no treatment) provided in this application was expanded in vitro on the 7th day, and the culture medium did not contain IL-2 (as D0 day). After the cells were re-plated, the total number of cells was counted every 2-4 days and normalized.
  • Figure 60C shows the results of long-term proliferation/persistence of TIL cells after withdrawal of IL-2.
  • the results show that the gene-edited TIL cells of this application can have significant long-term proliferation/survival ability after culture without IL-2.
  • the matching killing test is carried out.
  • the tumor target cell line (matched cell line) that is the same/similar to the tumor type of the donor is plated on a 96-well plate and cultured overnight; Compared with co-cultured with matching cell lines, IncuCyte was used to analyze the killing of tumor cells by TIL by detecting the activity of Caspase 3/7 in real time.
  • Figure 60D shows the killing effect of the gene-edited TIL cells of the present application on the tumor cell line matched with the TIL donor tumor type.
  • Figure 60E shows the killing effect of the gene-edited TIL cells of the present application on a tumor cell line unmatched with the tumor type of the TIL donor.
  • the TILs obtained from the sgRNA knockout group or control group (NT-no treatment) provided by the application were tested by flow cytometry 8 days after electroporation.
  • the expression of CD107a, GZMB, TNF- ⁇ and IFN- ⁇ was detected.
  • Fig. 60F shows, for TIL cells from different donors, the percentages of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group under the condition of no stimulation test.
  • cytokine expression flow detection method of this application under the condition of anti-CD3 antibody stimulation detection, add anti-CD3 to the TIL obtained from the sgRNA knockout group or control group (NT-no treatment) provided by this application, 7 days after electroporation Antibody (for example, OKT3, 30ng/ml) was stimulated overnight, and the expression of CD107a, GZMB, TNF- ⁇ and IFN- ⁇ was detected by flow cytometry the next day.
  • electroporation Antibody for example, OKT3, 30ng/ml
  • Figure 60G shows the proportion of cells expressing CD107a, GZMB, TNF- ⁇ and IFN- ⁇ in TIL cells of each group under the condition of anti-CD3 antibody stimulation overnight, for TIL cells from different donors.
  • the TIL population obtained from the sgRNA knockout group or the control group (NT-no treatment) provided by the present application is subjected to flow cytometry detection.
  • Figure 60H shows, for TIL cells derived from different donors, the ratio of expression of exhaustion marker molecules in TIL cells of each group. The results showed that gene-edited TIL cells had a lower ratio of expression of exhaustion marker molecules. For example, the TIL population had lower expression of CD38, CD101, LAG-3, TIM-3 and PD-1.
  • Figure 60I shows, for TIL cells derived from different donors, the proportion of central memory cells (CD45RO positive CD62L positive) among TIL cells in each group. The results showed that gene-edited TIL cells had a higher proportion of central memory cells (CD45RO positive CD62L positive).
  • TILs obtained from the sgRNA knockout group or control group (NT-no treatment) provided in this application apoptosis detection was performed 7 days after gene editing.
  • TILs from the gene knockout group or the control group (NT-no treatment) were used to detect the apoptosis level of T cells with a cell apoptosis detection kit (BD 559763 Annexin V PE Apoptosis kit).
  • Figure 60J shows the results of the apoptosis detection of TIL cells derived from donor 709. The results showed that gene-edited TIL cells could have more significant anti-apoptotic ability.
  • the following table 6 shows the human TNFAIP3 genome coordinates corresponding to the gRNA provided by this application

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Abstract

提供一种修饰的肿瘤浸润淋巴细胞及其用途，也提供一种培养肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte，TIL)的方法，其包含使所述TIL的NF-κB通路抑制分子的表达降低和/或活性减弱。还提供使用肿瘤浸润淋巴细胞预防和/或治疗肿瘤的方法。

Description

一种修饰的肿瘤浸润淋巴细胞及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种修饰的肿瘤浸润淋巴细胞及其用途。
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，而且目前来自患者肿瘤的肿瘤浸润淋巴细胞在体内的存续和扩增能力弱，杀伤靶细胞的能力不强，同时受到来自肿瘤微环境的多种抑制而功能受限。
因此如何提供一种稳健可靠的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
一方面，本申请提供了一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
在一种实施方式中，所述方法包含：使所述TIL与所述饲养细胞共培养之前，使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
在一种实施方式中，所述方法包含：在所述TIL与所述T细胞激活剂和/或所述T细胞生长因子接触之后且在所述TIL与所述饲养细胞共培养之前使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，其中所述TIL包含与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养获得的TIL。
一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中所述TIL包含使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL。
在一种实施方式中，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少 一种目标基因的表达降低和/或活性减弱获得的TIL显示出改善的TIL特性。
在一种实施方式中，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的抗凋亡能力，和提高的T细胞受体(TCR)克隆多样性。
在一种实施方式中，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，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含将基因调控系统引入所述TIL细胞中。
在一种实施方式中，所述基因调控系统能够在DNA水平破坏所述目标基因。
在一种实施方式中，所述基因调控系统包含指导核酸分子和酶蛋白。
在一种实施方式中，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。
在一种实施方式中，所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
在一种实施方式中，所述指导核酸分子包含指导RNA(gRNA)。
在一种实施方式中，所述指导核酸分子能够与所述目标基因的序列结合。
在一种实施方式中，所述目标基因包含编码NF-κB通路抑制分子的基因。
在一种实施方式中，所述NF-κB通路抑制分子能够使选自以下组的蛋白泛素化：受体相互作用蛋白1(RIP1)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1)、受体相互作用蛋白2(RIP2)和肿瘤坏死因子受体相关因子6(TRAF6)。
在一种实施方式中，所述NF-κB通路抑制分子包含肿瘤坏死因子α诱导蛋白3(TNFAIP3)。
在一种实施方式中，所述指导核酸分子能够与TNFAIP3基因的第2外显子和/或第7外显子所在的区域或其片段结合。
在一种实施方式中，所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：34至47。
在一种实施方式中，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：GGG、TGG、CGG和AGG。
在一种实施方式中，所述指导核酸分子包含如SEQ ID NO：48至61中任一项所示的序列。
在一种实施方式中，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例降低和/或单个细胞的表达量下降。
在一种实施方式中，所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中，表达所述目的基因的细胞比例为约95％或以下。
在一种实施方式中，所述方法还包含：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与所述饲养细胞共培养。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL与所述饲养细胞共培养。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL的至少一种目标基因的表达降低和/或活性减弱且使所述TIL与所述饲养细胞共培养。
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，使所述TIL与所述饲养细胞共培养。
在一种实施方式中，所述第一阶段体外扩增进行至少约7天。
在一种实施方式中，所述第一阶段体外扩增进行约7天至约14天。
在一种实施方式中，所述第二阶段体外扩增进行至少约7天。
在一种实施方式中，所述第二阶段体外扩增进行约7天至约14天。
在一种实施方式中，使所述TIL在与T细胞激活剂和/或T细胞生长因子接触至少约2小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时至约72小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约12小时至约48小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时、约12小时、约24小时、约48小时或约72小时之后与所述饲养细胞共培养。
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，所述方法还包含：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与所述T细胞激活剂接触。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞激活剂接触。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL的至少一种目标基因的表达降低和/或活性减弱且使所述TIL与所述T细胞激活剂接触。
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，使所述TIL与所述T细胞激活剂接触。
在一种实施方式中，所述T细胞激活剂包含选自以下组的一种或多种：分化簇80(CD80)、CD86、CD276、4-1BB配体(4-1BBL)、CD27、CD30、CD134、CD275、CD40、CD258、以及它们的功能活性片段。
在一种实施方式中，所述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、CD28、疱疹病毒进入介质(HVEM)、CD40L、OX40和4-1BB。
在一种实施方式中，所述T细胞激活剂包含CD3激动剂和/或CD28激动剂。
在一种实施方式中，所述T细胞激活剂包含CD3激动剂。
在一种实施方式中，所述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T细胞激活剂包含CD28激动剂。
在一种实施方式中，所述T细胞激活剂包含抗CD28的抗体和/或其抗原结合片段、CD80和/或其功能活性片段和/或CD86和/或其功能活性片段。
在一种实施方式中，所述使TIL与所述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；和(3)将包含所述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，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为至少约30ng/mL。
在一种实施方式中，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为约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所述固相介质中包含的每一种所述T细胞激活剂的量各自独立地至少为约25μg。
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，所述方法还包含：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与所述T细胞生长因子接触。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞生长因子接触。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞激活剂以及所述T细胞生长因子接触。
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，使所述TIL与T细胞生长因子接触。
在一种实施方式中，使所述TIL基本上同时与所述T细胞激活剂以及所述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，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
在一种实施方式中，所述TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
在一种实施方式中，所述碎片的体积为约1立方毫米至约27立方毫米。
另一方面，本申请还提供了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T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
(B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
(A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
(B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中，所述体外TIL群包含由所述第一TIL群接触T细胞生长因子获得的TIL群。
在一种实施方式中，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
在一种实施方式中，所述步骤(A)进行约7天至约14天。
在一种实施方式中，所述步骤(B)进行约7天至约14天。
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T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
(B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
(C)使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。
一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
(A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源 自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
(B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
(C)使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。
在一种实施方式中，所述体外TIL群包含由所述第一TIL群接触T细胞生长因子获得的TIL群。
在一种实施方式中，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
在一种实施方式中，所述步骤(A)进行约7天至约14天。
在一种实施方式中，所述步骤(B)进行约0天至约8天。
在一种实施方式中，所述步骤(C)进行约5天至约14天。
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T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
(B)使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
(C)使所述第三TIL群的至少一种目标基因的表达降低和/或活性减弱，其中，经所述步骤(C)得到第四TIL群；
(D)使所述第四TIL群与饲养细胞共培养，其中，经所述步骤(D)得到第五TIL群。
一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
(A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
(B)使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
(C)使所述第三TIL群的至少一种目标基因的表达降低和/或活性减弱，其中，经所述步骤(C)得到第四TIL群；
(D)使所述第四TIL群与饲养细胞共培养，其中，经所述步骤(D)得到第五TIL群。
在一种实施方式中，所述体外TIL群包含由所述第一TIL群接触T细胞生长因子获得的TIL群。
在一种实施方式中，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
在一种实施方式中，所述步骤(A)进行约7天至约14天。
在一种实施方式中，所述步骤(B)进行约0天至约4天。
在一种实施方式中，所述步骤(C)进行约0天至约4天。
在一种实施方式中，所述步骤(D)进行约5天至约14天。
在一种实施方式中，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL显示出改善的TIL特性。
在一种实施方式中，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的抗凋亡能力，和提高的T细胞受体(TCR)克隆多样性。
在一种实施方式中，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，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含将基因调控系统引入所述TIL细胞中。
在一种实施方式中，所述基因调控系统能够在DNA水平破坏所述目标基因。
在一种实施方式中，所述基因调控系统包含指导核酸分子和酶蛋白。
在一种实施方式中，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。
在一种实施方式中，所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
在一种实施方式中，所述指导核酸分子包含指导RNA(gRNA)。
在一种实施方式中，所述指导核酸分子能够与所述目标基因的序列结合。
在一种实施方式中，所述目标基因包含编码NF-κB通路抑制分子的基因。
在一种实施方式中，所述NF-κB通路抑制分子能够使选自以下组的蛋白泛素化：受体相互作用蛋白1(RIP1)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1)、受体相互作用蛋白2(RIP2)和肿瘤坏死因子受体相关因子6(TRAF6)。
在一种实施方式中，所述NF-κB通路抑制分子包含肿瘤坏死因子α诱导蛋白3(TNFAIP3)。
在一种实施方式中，所述指导核酸分子能够与TNFAIP3基因的第2外显子和/或第7外显子所在的区域或其片段结合。
在一种实施方式中，所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：34至47。
在一种实施方式中，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：GGG、TGG、CGG和AGG。
在一种实施方式中，所述指导核酸分子包含如SEQ ID NO：48至61中任一项所示的序列。
在一种实施方式中，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例降低和/或单个细胞的表达量下降。
在一种实施方式中，所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中，表达所述目的基因的细胞比例为约95％或以下。
在一种实施方式中，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触至少约2小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时至约72小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约12小时至约48小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时、约12小时、约24小时、约48小时或约72小时之后与所述饲养细胞共培养。
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，所述T细胞激活剂包含选自以下组的一种或多种：分化簇80(CD80)、CD86、CD276、4-1BB配体(4-1BBL)、CD27、CD30、CD134、CD275、CD40、CD258、以 及它们的功能活性片段。
在一种实施方式中，所述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、CD28、疱疹病毒进入介质(HVEM)、CD40L、OX40和4-1BB。
在一种实施方式中，所述T细胞激活剂包含CD3激动剂和/或CD28激动剂。
在一种实施方式中，所述T细胞激活剂包含CD3激动剂。
在一种实施方式中，所述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T细胞激活剂包含CD28激动剂。
在一种实施方式中，所述T细胞激活剂包含抗CD28的抗体和/或其抗原结合片段、CD80和/或其功能活性片段和/或CD86和/或其功能活性片段。
在一种实施方式中，所述使TIL与所述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；和(3)将包含所述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，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为至少约30ng/mL。
在一种实施方式中，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为约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所述固相介质中包含的每一种所述T细胞激活剂的量各自独立地至少为约25μg。
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，使所述TIL基本上同时与所述T细胞激活剂以及所述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，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
在一种实施方式中，所述TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
在一种实施方式中，所述碎片的体积为约1立方毫米至约27立方毫米。
另一方面，本申请还提供了一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL与CD28激动剂接触。
在一种实施方式中，所述方法包含：使所述TIL与CD28激动剂接触之后，使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，其中所述TIL包含与CD28激动剂接触获得的TIL。
一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL与CD28激动剂接触，其中所述TIL包含使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL。
在一种实施方式中，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL显示出改善的TIL特性。
在一种实施方式中，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，和提高的T细胞受体(TCR)克隆多样性。
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，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含将基因调控系统引入所述TIL细胞中。
在一种实施方式中，所述基因调控系统能够在DNA水平破坏所述目标基因。
在一种实施方式中，所述基因调控系统包含指导核酸分子和酶蛋白。
在一种实施方式中，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。
在一种实施方式中，所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
在一种实施方式中，所述指导核酸分子包含指导RNA(gRNA)。
在一种实施方式中，所述指导核酸分子能够与所述目标基因的序列结合。
在一种实施方式中，所述目标基因包含编码NF-κB通路抑制分子的基因。
在一种实施方式中，所述NF-κB通路抑制分子能够使选自以下组的蛋白泛素化：受体相互作用蛋白1(RIP1)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1)、受体相互作用蛋白2(RIP2)和肿瘤坏死因子受体相关因子6(TRAF6)。
在一种实施方式中，所述NF-κB通路抑制分子包含肿瘤坏死因子α诱导蛋白3(TNFAIP3)。
在一种实施方式中，所述指导核酸分子能够与TNFAIP3基因的第2外显子和/或第7外显子所在的区域或其片段结合。
在一种实施方式中，所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：34至47。
在一种实施方式中，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：GGG、TGG、CGG和AGG。
在一种实施方式中，所述指导核酸分子包含如SEQ ID NO：48至61中任一项所示的序列。
在一种实施方式中，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例降低和/或单个细胞的表达量下降。
在一种实施方式中，所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中，表达所述目的基因的细胞比例为约95％或以下。
在一种实施方式中，其中，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，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。
在一种实施方式中，所述第一阶段体外扩增进行至少约7天。
在一种实施方式中，所述第一阶段体外扩增进行约7天至约14天。
在一种实施方式中，所述第二阶段体外扩增进行至少约7天。
在一种实施方式中，所述第二阶段体外扩增进行约7天至约14天。
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、CD80和/或其功能活性片段、和/或CD86和/或其功能活性片段。
在一种实施方式中，所述方法还包含：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与所述CD28激动剂之外的其它T细胞激活剂接触。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL与所述其它T细胞激活剂接触。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL的至少一种目标基因的表达降低和/或活性减弱且使所述TIL与所述其它T细胞激活剂接触。
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，使所述TIL与所述其它T细胞激活剂接触。
在一种实施方式中，使所述TIL基本上同时与所述CD28激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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，所述方法还包含：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在与CD28激动剂接触一定时间之后与饲养细胞共培养。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL与所述饲养细胞共培养。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL与CD28激动剂接触且使所述TIL与所述饲养细胞共培养。
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，使所述TIL与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述CD28激动剂接触至少约2小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述CD28激动剂接触约6小时至约72小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述CD28激动剂接触约12小时至约48小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述CD28激动剂接触约6小时、约12小时、约24小时、约48小时或约72小时之后与所述饲养细胞共培养。
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，所述方法还包含：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T细胞生长因子接触。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞生长因子接触。
在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂以及所述T细胞生长因子接触。
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，使所述TIL与T细胞生长因子接触。
在一种实施方式中，使所述TIL基本上同时与所述CD28激动剂以及所述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，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
在一种实施方式中，所述TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移 灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
在一种实施方式中，所述碎片的体积为约1立方毫米至约27立方毫米。
另一方面，本申请还提供了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T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
(B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL与CD28激动剂接触，其中，经所述步骤(B)得到第三TIL群。
一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
(A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
(B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL与CD28激动剂接触，其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中，所述体外TIL群包含由所述第一TIL群接触T细胞生长因子获得的TIL群。
在一种实施方式中，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
在一种实施方式中，所述步骤(A)进行约7天至约14天。
在一种实施方式中，所述步骤(B)进行约7天至约14天。
在一种实施方式中，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL显示出改善的TIL特性。
在一种实施方式中，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，和提高的T细胞受体(TCR)克隆多样性。
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，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱 包含将基因调控系统引入所述TIL细胞中。
在一种实施方式中，所述基因调控系统能够在DNA水平破坏所述目标基因。
在一种实施方式中，所述基因调控系统包含指导核酸分子和酶蛋白。
在一种实施方式中，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。
在一种实施方式中，所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
在一种实施方式中，所述指导核酸分子包含指导RNA(gRNA)。
在一种实施方式中，所述指导核酸分子能够与所述目标基因的序列结合。
在一种实施方式中，所述目标基因包含编码NF-κB通路抑制分子的基因。
在一种实施方式中，所述NF-κB通路抑制分子能够使选自以下组的蛋白泛素化：受体相互作用蛋白1(RIP1)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1)、受体相互作用蛋白2(RIP2)和肿瘤坏死因子受体相关因子6(TRAF6)。
在一种实施方式中，所述NF-κB通路抑制分子包含肿瘤坏死因子α诱导蛋白3(TNFAIP3)。
在一种实施方式中，所述指导核酸分子能够与TNFAIP3基因的第2外显子和/或第7外显子所在的区域或其片段结合。
在一种实施方式中，所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：34至47。
在一种实施方式中，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：GGG、TGG、CGG和AGG。
在一种实施方式中，所述指导核酸分子包含如SEQ ID NO：48至61中任一项所示的序列。
在一种实施方式中，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例降低和/或单个细胞的表达量下降。
在一种实施方式中，所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中，表达所述目的基因的细胞比例为约95％或以下。
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、CD80和/或其功能活性片段、和/或CD86和/或其功能活性片段。
在一种实施方式中，使所述TIL基本上同时与所述CD28激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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，使所述TIL在与所述CD28激动剂接触至少约2小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述CD28激动剂接触约6小时至约72小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述CD28激动剂接触约12小时至约48小时之后与所述饲养细胞共培养。
在一种实施方式中，使所述TIL在与所述CD28激动剂接触约6小时、约12小时、约24 小时、约48小时或约72小时之后与所述饲养细胞共培养。
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，使所述TIL基本上同时与所述CD28激动剂以及所述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，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
在一种实施方式中，所述TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
在一种实施方式中，所述碎片的体积为约1立方毫米至约27立方毫米。
另一方面，本申请还提供了一种肿瘤浸润淋巴细胞(TIL)，所述TIL经过本申请的方法获得。
另一方面，本申请还提供了一种组合物，其包含本申请的TIL。
另一方面，本申请还提供了一种药物组合物，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。
另一方面，本申请还提供了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、本申请的组合物和/或本申请的药物组合物。
另一方面，本申请还提供了本申请的TIL、本申请的组合物和/或本申请的药物组合物在制备药物中的应用，所述药物用于预防和/或治疗肿瘤。
在一种实施方式中，其中，所述肿瘤为实体瘤。
在一种实施方式中，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。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是，饲养细胞不同添加时间培养的TIL的增殖能力分析结果。
图2和图3显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA -CCR7 +中心记忆T细胞(Tcm)比例。
图4显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。
图5和图6显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞比例。
图7显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103 +CD39 +肿瘤特异性T细胞比例。
图8显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1 +干细胞样T细胞比例。
图9显示的是，添加不同形式的CD28激动剂的试验组以及对照组的增殖能力分析结果。
图10和图11分别显示的是，对于不同供者来源的TIL，混合抗体组与对照组培养所得的TIL细胞的T细胞亚群比例。
图12和图13分别显示的是，对于不同供者来源的TIL，磁珠组与对照组培养所得的TIL细胞的T细胞亚群比例。
图14显示的是，纳米基质组与对照组培养所得的TIL细胞的T细胞亚群比例。
图15显示的是，纳米基质组与对照组培养所得的TIL细胞的细胞杀伤能力。
图16显示的是，混合抗体组与对照组培养所得的TIL细胞的胞内因子表达检测结果。
图17、图18、图19和图20分别显示的是，对于不同供者来源的TIL，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。
图21显示的是，纳米基质组与对照组培养所得的TIL细胞的胞内因子表达检测结果。
图22显示的是，纳米基质组与对照组培养所得的TIL细胞的细胞因子分泌检测结果。
图23显示的是，纳米基质组与对照组培养所得的TIL细胞与肿瘤细胞共同孵育后的细胞因子分泌检测结果。
图24和图25分别显示的是，对于不同供者来源的TIL，纳米基质组与对照组培养所得的TIL细胞的基因敲除效率结果。
图26、图27和图28分别显示的是，对于不同供者来源的TIL，在终末刺激阶段中，以不同方式进行体外扩增的试验组的增殖能力分析结果。
图29、图30、图31和图32显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞在没有额外刺激条件下扩增后的荧光量。
图33和图34显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞在anti-CD3抗体(Miltenyi Biotech，OKT3)刺激下扩增后的荧光量。
图35显示的是，对于来源于供者B的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。
图36和图37显示的是，对于来源于供者C的TIL细胞，以效靶比1:1和效靶比1:3与肿瘤细胞共培养的杀伤能力检测结果。
图38和图39显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞在撤去IL-2后长期培养的增殖倍数。
图40和图41显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞在撤去IL-2后长期培养的活率。
图42和图43显示的是，对于来源于供者C的TIL细胞，各组TIL细胞中TIM-3阳性和CD101阳性细胞的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
图44显示的是，对于来源于供者C的TIL细胞，各组TIL细胞中CD45RA阴性CCR7 阳性记忆T细胞(Tcm)的比例。
图45、图46、图47和图48显示的是，在没有刺激的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图49、图50、图51和图52显示的是，在CD3抗体(Miltenyi Biotech，OKT3，CD3抗体以30ng/mL预处理96孔板)刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图53、图54、图55和图56显示的是，在佛波醇-十四酸酯-乙酸酯(PMA，25ng/ml)和离子霉素(Ionomycin，1μg/ml)刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图57和图58显示的是，TIL细胞基因编辑后的第8天和第18天的CD4 +T细胞和CD8 +T细胞TCR Vβ克隆的香农多样性指数。
图59A显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。
图59B显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA -CCR7 +中心记忆T细胞(Tcm)比例结果图。
图59C显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1 +干细胞样T细胞比例。
图59D显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。
图59E显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(PD-1 +)比例。
图59F显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103 +CD39 +肿瘤特异性T细胞比例。
图59G显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD28 +)比例。
图59H显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(41BB +)比例。
图59I显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD25 +)比例。
图59J显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的 TIL，培养所得的TIL细胞的胞内因子表达检测结果。
图59K显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞因子分泌检测结果。
图59L显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。
图59M显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD8 +T细胞比例。
图59N显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RO +CD62L +T细胞比例。
图59O显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的NK T细胞比例。
图59P显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。
图59Q显示的是加入OKT3和IL-2的48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞杀伤能力结果。
图60A显示的是，对于来源于不同供者的TIL细胞，无刺激检测条件下各组TIL细胞的扩增后的荧光量。
图60B显示的是，对于来源于不同供者的TIL细胞，抗CD3抗体刺激检测条件下各组TIL细胞的扩增后的荧光量。
图60C显示的是，TIL细胞撤去IL-2后长期增殖/存续的增殖倍数结果。
图60D显示的是，本申请的基因编辑的TIL细胞对与该TIL的供者瘤种配型的肿瘤细胞系的杀伤效果。
图60E显示的是，本申请的基因编辑的TIL细胞对与该TIL的供者瘤种未配型的肿瘤细胞系的杀伤效果。
图60F显示的是，在无刺激检测条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图60G显示的是，在抗CD3抗体刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图60H显示的是，对于不同供者来源的TIL细胞，各组TIL细胞中不同耗竭标识分子表达的比例。
图60I显示的是，对于不同供者来源的TIL细胞，各组TIL细胞中中心记忆细胞(CD45RO阳性CD62L阳性)的比例。
图60J显示的是，对于来源于供者709的TIL细胞的凋亡检测结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“NF-κB通路抑制分子”通常是指一种信号通路的抑制分子。例如，NF-κB通路抑制分子可以通过使目标蛋白泛素化，实现抑制信号通路传递的功能。例如，NF-κB通路抑制分子可以使以下蛋白泛素化：受体相互作用蛋白1(RIP1，UniProt登录号Q13546)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1，UniProt登录号Q9UDY8)、受体相互作用蛋白2(RIP2，UniProt登录号O43353)和肿瘤坏死因子受体相关因子6(TRAF6，UniProt登录号Q9Y4K3)。
在本申请中，术语“肿瘤坏死因子α诱导蛋白3(TNFAIP3)”通常是指一种信号通路的抑制分子。例如，TNFAIP3可以使得NF-κB通路的信号转导物质泛素化。例如，TNFAIP3的UniProt登录号可以是P21580。本申请中，TNFAIP3可以涵盖未加工的TNFAIP3、任何形式加工的TNFAIP3、TNFAIP3的变体或包含TNFAIP3的功能活性片段的物质。
在本申请中，术语“基因调控系统”通常是指调节目标基因表达或活性的系统。例如，基因调控系统可以包含基因调控分子。例如，基因调控系统可以调节基因的表达或活性，如使该基因处于失活或激活的状态、使该基因的数量增加或降低、使该基因处于转录量提高或降低的状态、和/或使该基因的转录产物处于失活或激活的状态；例如，基因调控系统可以调节基因的表达或活性，如使单个细胞中该基因的表达产物的量提高或降低、和/或使表达该基因的表达产物的细胞的数量提高或降低。
在本申请中，术语“指导核酸分子”通常是指一种可以用于基因编辑的核酸分子。例如，指导核酸分子可以提供核苷酸插入或删除的信息，指导编辑过程。例如，指导核酸分子可以是指导RNA或向导RNA(guide RNA，gRNA)。例如，“gRNA”可以是指结合到Cas蛋白并 使Cas蛋白靶向靶DNA内特定位置的RNA分子。例如，其中gRNA与DNA靶向序列之间的杂交促进CRISPR复合物的形成，可以不必要求完全互补性，例如只要存在引起杂交并促进CRISPR复合物形成的充分互补性即可。
在本申请中，术语“酶蛋白”通常是指一种具有酶活性的蛋白。例如，酶蛋白可以是指Cas蛋白。例如，Cas蛋白可以包含至少一个RNA识别或结合结构域，该结构域可以与gRNA相互作用。Cas蛋白还可以包含核酸酶结构域(例如，DNA酶或RNA酶结构域)、DNA结合结构域、解旋酶结构域、蛋白-蛋白相互作用结构域、二聚化结构域和/或其他结构域。核酸酶结构域可以具有用于核酸切割的催化活性。切割可以包括核酸分子共价键的断裂。Cas蛋白可以为野生型蛋白(即，自然界存在的蛋白)、经修饰的Cas蛋白(即，Cas蛋白变体)或者野生型或经修饰的Cas蛋白的片段。Cas蛋白还可以是野生型或经修饰的Cas蛋白的活性变体或片段。本申请中，Cas蛋白可以涵盖未加工的Cas蛋白、任何形式加工的Cas蛋白、Cas蛋白的变体或包含Cas蛋白的功能活性片段的物质。
在本申请中，术语“核糖核蛋白复合物”通常是指一种蛋白与核酸形成的复合体。例如，核糖核蛋白复合物中的蛋白可以具有核酸酶活性。例如，核糖核蛋白复合物可以在其中核酸的指导下，对目标序列进行切割。例如，核糖核蛋白复合物可以是Cas蛋白与gRNA形成的复合物。
在本申请中，术语“外显子”通常是指基因上可以被表达为蛋白质的部分。例如，外显子可以是指在蛋白质生物合成过程中具有被表达为蛋白质的能力。例如，剪切目标基因的外显子序列可以降低目标基因的活性或功能。
在本申请中，术语“原型间隔序列毗邻基序(PAM)”通常是指靶序列后的短序列。例如，Cas9对靶DNA的位点特异性切割时，PAM序列可以用于决定切割的位置。例如，确定了PAM的区域，本领域技术人员可以容易确定合适的靶序列位置，以及可以容易设计出用于切割目标序列的gRNA序列。
在本申请中，术语“表达降低”通常是指产物或其基因的表达量的降低和/或能够表达所述产物的细胞比例下降。例如，可以是细胞该基因表达的产物的量降低或包含该基因表达的产物的细胞比例降低，或者分泌该基因表达的产物的细胞比例降低。例如，可以通过检测细胞的基因组中该基因的敲除量，间接表示该基因的表达降低。例如，可以通过检测一个细胞群中该基因被敲除的细胞所占的比例，间接表示该基因的表达降低。
在本申请中，术语“活性”通常是指物质的生物学功能。例如，基因的活性可以是指该基因的转录和/或翻译状态。例如，基因的活性减弱可以是指该基因的转录功能减弱、该基因无法被正常转录或该基因转录产物的功能被抑制。
在本申请中，术语“CD80”通常是指一种细胞刺激分子。例如，CD80可以是CD28的配体。例如，CD80可以见于GenBank登记号P33681。本申请的CD80蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含CD80的功能活性片段的物质。例如，本申请的CD80可以包含CD80的功能活性片段以及其它任意的结构域。
在本申请中，术语“CD86”通常是指一种细胞刺激分子。例如，CD86可以是CD28的配体。例如，CD86可以见于GenBank登记号P42081。本申请的CD86蛋白还可以涵盖其功能活性片段，不限于在细胞中发生的加工和/或修饰后产生的包含CD86的功能活性片段的物质。例如，本申请的CD86可以包含CD86的功能活性片段以及其它任意的结构域。
在本申请中，术语“分泌”通常是指一种物质可以定位于细胞的胞外。例如，分泌的物质可以在细胞内合成之后，被运送到细胞的胞外空间。例如，可以通过酶联免疫吸附剂测定或其它检测方法检测一种物质是否是分泌的物质。
在本申请中，术语“T细胞受体”或“TCR”通常是指响应于抗原的呈递参与T细胞的活化的膜蛋白的复合体。TCR可以负责识别结合至主要组织相容性复合体分子的抗原。TCR可以由alpha(α)和beta(β)链的异二聚体组成，或由gamma和delta(γ/δ)链构成。TCR可以以α/β和γ/δ形式存在，其是结构上相似的，但是具有独特的解剖学位置和功能。例如，TCR可以是在表达TCR的任何细胞上被修饰的TCR。例如，TCR的种类可以通过TCR亚型分析试剂进行分析。
在本申请中，术语“克隆多样性”通常是指某种物质具有多种克隆型。例如，TCR的克隆多样性可以是指TCR可以具有不同序列结构和/或抗原识别能力。例如，TCR具有的多样性常用β链亚型来区分，可以包括Vβ23、Vβ7.2、Vβ5.2、Vβ11、Vβ16、Vβ3等，当一个T细胞群具有更多的β链亚型时，可以认为该T细胞群具有更高的克隆多样性。
在本申请中，“CD4 +细胞”通常是指CD4阳性的细胞，例如可以是T细胞。术语“CD4 +细胞”，“CD4阳性细胞”可以同义使用。这些细胞可通过本领域知道的方法来鉴定，例如通过用荧光标记的针对CD4的抗体对细胞染色和使用荧光激活细胞分选。例如，已有的数据可以证明，CD4 +细胞比例的提高可以使得细胞群分泌IFN-γ和/或TNF的能力提高，并可以提高T细胞群的促进肿瘤抑制的效果。例如，请见Tay,R.E.,Richardson,E.K.等人(2020). Cancer Gene Therapy,1-13.但是，本领域缺少一种提高CD4 +细胞比例的方法，本申请可以提供一种影响CD4 +细胞比例的方法。
在本申请中，“CD8 +细胞”通常是指CD8阳性的细胞，例如可以是T细胞。术语“CD8 +细胞”，“CD8阳性细胞”可以同义使用。这些细胞可通过本领域知道的方法来鉴定，例如通过用荧光标记的针对CD8的抗体对细胞染色和使用荧光激活细胞分选。
在本申请中，术语“IC 50值”或“IC50值”通常是指目标物获得生物学过程50％抑制需要的浓度。可以使用Cheng-Prusoff方程(Biochem.Pharmacol.(1973)22:3099)将IC50值换算成绝对抑制常数(Ki)。
在本申请中，术语“K D值”或“KD值”通常是指解离常数，其可通过表面等离子体共振进行测定。通常，表面等离子体共振分析使用BIAcore系统(Pharmacia Biosensor，Piscataway，NJ)，通过表面等离子体共振(SPR)，测量配体(固定化于生物传感器基质上的物质)和分析物(溶液中的物质)之间的实时结合相互作用。也可以通过固定化分析物(生物传感器基质上的物质)和呈递配体，进行表面等离子体分析。
在本申请中，术语“编码”通常是指能够根据基本上确定的规则，由一种分子的结构或组成信息，直接或间接推断出与其相关的另一类分子的结构或组成信息。例如，可以根据氨基酸的序列推断出其核苷酸序列，例如根据脱氧核糖核酸转录互补核酸的特性，包括能翻译成多肽的核酸。例如，脱氧核糖核酸可编码从脱氧核糖核酸转录的RNA。脱氧核糖核酸可类似地编码从脱氧核糖核酸所转录的RNA翻译的多肽。
在本申请中，术语“小分子化合物”通常是指肽、肽模拟物、氨基酸、氨基酸类似物、多核苷酸、多核苷酸类似物、核苷酸、核苷酸类似物、分子量小于约10,000克/摩尔的有机或无机物(即包括异源有机物和有机金属化合物)、分子量小于约5,000克/摩尔的有机或无机物、分子量小于约1,000克/摩尔的有机或无机物、分子量小于约500克/摩尔的有机或无机物，以及这类药物的盐、酯和其它药学上可接受的形式。
在本申请中，术语“NK细胞”也称为“自然杀伤细胞”，通常是指一种细胞质中具有大颗粒的细胞。NK细胞由骨髓淋巴样干细胞发育而成，可以依赖于骨髓或胸腺微环境分化、发育。在本申请中，TIL细胞中的NK细胞的比例可以通过本申请的方法加以改变。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本申请 的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab')2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合CD3)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。根据重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。目前存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。
在本申请中，术语“抗原结合片段”通常指具有特异结合抗原(例如，CD3)能力的一个或多个多肽片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv和/或dAb。
在本申请中，术语“固相介质”或“介质”通常是指结合功能的固相材料。例如，本申请固相介质可以是指通过共价结合和/或非共价结合的作用，将一种或一种以上的物质结合在介质内和/或介质表面的材料。例如，本申请的固相介质可以是指通过共价结合和/或非共价结合的作用将CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段结合在介质内和/或介质表面的材料。例如，本申请的固相介质可以是聚合物材料。
在本申请中，术语“表达”通常是指编码目标多肽的基因在细胞内发生的转录和/或翻译过程。可以通过测量存在于细胞中的相应mRNA的量来确定宿主细胞中编码目标多肽的基因的转录水平。例如，可通过PCR或通过RNA杂交对编码目标多肽的基因转录的mRNA进行定量测量。可以通过多种方法测量编码目标多肽的基因的翻译水平，例如通过ELISA，通过多肽生物活性测试，或通过蛋白质印迹或放射免疫测试法。在本申请中，术语“表达”通常也可以是指产物发生的转录和/或翻译过程。例如，细胞因子的表达可以是细胞转录和/或 翻译该细胞因子的过程。例如，细胞因子的表达可以通过检测存在于细胞中的相应mRNA的量或检测通过细胞生产的该细胞因子的量，或两者进行确定。
在本申请中，术语“一个阶段的体外扩增”、“单个阶段的体外扩增”、或“第一阶段体外扩增”等中的“阶段”通常是指TIL在体外经过的一段扩增过程。在一种实施方式中，每一个阶段之间可以是通过TIL细胞数量的变化来划分的，在一种实施方式中，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的条件来划分的。在一种实施方式中，当细胞培养基中添加了或补充添加了T细胞激活剂和/或T细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，当TIL细胞进行了离心和/或细胞洗涤后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。在一种实施方式中，当TIL细胞体外培养约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天、约20天、约30天、约40天、约50天或约100天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
在本申请中，术语“第一阶段体外扩增”通常是指从组织中获得初级TIL后，使用T细胞生长因子进行扩增的阶段。在一种实施方式中，本申请的组织可以选自以下组：肿瘤组织和胸腔积液，本申请的胸腔积液可以是有转移癌的患者的胸腔积液。在一种实施方式中，本申请的扩增可以是自体或者异体进行的体内扩增，或者可以是体外扩增。本申请的第一阶段体外扩增也可以称为preREP(快速扩增前)阶段。例如，源自肿瘤组织且未经体外扩增的TIL可以称为第一TIL群。例如，在两步骤法划分的本申请的培养方式中经过第一阶段体外扩增获得的TIL可以称为第二TIL群。
在本申请中，术语“第二阶段体外扩增”通常是指从受试者体内取出的组织并进行扩增后，再次进行扩增的阶段。在一种实施方式中，与经第一阶段体外扩增的TIL相比，本申请的经第二阶段体外扩增的TIL细胞数量增加，例如，可以增加至少约10倍(或至少约20、30、40、50、60、70、80或90倍)，或者在一种实施方式中细胞的数量可以增加至少约100倍。在一种实施方式中，第二阶段体外扩增可以与第一阶段体外扩增的培养条件不同，例如加入的培 养物质可以不同。例如，在两步骤法划分的本申请的培养方式中第二阶段体外扩增也可以称为REP(快速扩增)阶段。例如，在两步骤法划分的本申请的培养方式中经过第二阶段体外扩增获得的TIL可以称为第三TIL群。
在本申请中，术语“体内”通常是指发生在受试者体内的事件。
在本申请中，术语“体外”通常是指在受试者体外发生的事件。
在本申请中，术语“离体”通常是指涉及对已从受试者体内移除的细胞、组织和/或器官进行治疗或进行手术的事件。在一种实施方式中，该细胞、组织和/或器官可以通过手术或治疗方法返回到受试者的身体。
在本申请中，术语“分泌能力”通常是指细胞表达多肽或蛋白并将本申请的多肽或蛋白转移到细胞外环境的能力。
在本申请中，术语“辐照”通常是指通过射线对物质进行的处理。例如，在一种实施方式中，辐照可以是指通过X射线、α射线、β射线或γ射线对物质进行辐照。
在本申请中，术语“工程化细胞”通常是指将DNA或RNA形式的额外遗传物质加入细胞的总遗传物质而被基因修饰的细胞。在一种实施方式中，工程化细胞可以经过基因修饰以表达本申请的T细胞激活剂和/或T细胞生长因子的TIL。
在本申请中，术语“共培养”通常是指将两个或更多个不同群体的细胞在它们之间有一定程度的接触的情况下培养。本申请的两个或更多个不同群体的细胞的“接触”，在一种实施方式中可以通过直接接触，即其中一个群体的细胞与另一个群体的细胞直接物理接触。或者在一种实施方式中可以通过共用培养基所介导的间接接触。本申请的共用的培养基可以含有由共培养细胞的至少一个群体所产生和释放的代谢产物，并用于培养另一个群体的细胞。
在本申请中，术语“接触”通常是指两个或更多个不同类型的物质以任何顺序、任何方式以及任何时长接触在一起。在一种实施方式中可以通过直接接触，例如可以将一种或多种饲养细胞、T细胞激活剂和/或T细胞生长因子加入TIL细胞的培养基，例如可以将包含一种或多种饲养细胞、T细胞激活剂和/或T细胞生长因子的培养基加入和/或替换TIL细胞的培养基，例如，可以将包含一种或多种饲养细胞、T细胞激活剂和/或T细胞生长因子的培养基用于TIL细胞的培养；在一种实施方式中可以通过间接接触，例如可以将饲养细胞产生和释放的代谢产物，用于培养TIL细胞。
在本申请中，术语“混合物”通常是指两个或更多个不同物质的组合。例如，本申请的CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段可以在混合后作为混合物加入细胞培养基。
在本申请中，术语“同时接触”、“共同接触”、“与...接触同时”、“同时”和“共同”通常是指向受试者和/或细胞施用两种以上物质，使得物质同时存在于受试者和/或细胞培养的环境中。同时接触可以包括以不同的组合物同时施用、以不同的组合物在不同时间施用，或以其中存在两种以上活性药物成分的组合物施用。例如，本申请中“同时接触”通常可以是指基本上同时接触。
在本申请中，术语“扩增”通常是指在一段时间内细胞的数量增加若干倍。在一种实施方式中细胞的数量可以增加至少约3倍(或4、5、6、7、8或9倍)，在一种实施方式中细胞的数量可以增加至少约10倍(或20、30、40、50、60、70、80或90倍)，或者在一种实施方式中细胞的数量可以增加至少约100倍。在本申请中，术语“经扩增”通常是指本申请的细胞经过上述一种或多种扩增。
在本申请中，术语“聚合物”通常是指由连接在一起的单独化学部分组成的分子，本申请的聚合物部分可相同或不同。在一种实施方式中，术语“聚合物”可以指尾尾相连而形成线性分子的单独化学部分，以及以分支(如“多臂”或“星型”)结构形式连接在一起的单独化学部分。在一种实施方式中聚合物可以包括例如多糖、葡聚糖、水凝胶、聚乙二醇、或泊洛沙姆。泊洛沙姆是非离子三嵌段共聚物，其具有聚氧丙烯(聚(环氧丙烷))中央疏水链，侧连两条聚氧乙烯(聚(环氧乙烷))亲水链。本申请包含的物质可以与本文所描述的或本领域已知的任何聚合物一起配制，或与它们一起给予。
在本申请中，术语“嵌合抗体(chimeric antibody)”通常是指鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，可以建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，可以根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，可以在真核系统或原核系统中表达嵌合抗体分子。
在本申请中，术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，通常是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库。
在本申请中，术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区可以都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和 全人源单克隆抗体。本申请所述抗体或配体可以为全人源单克隆抗体。全人抗体制备的相关技术可以为：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
在本申请中，术语“CDR”通常是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一可以由Kabat E.A.等人，Chothia等人和MacCallum等人提供。如本申请中使用的，CDR的Kabat定义可以应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR1、CDR2和CDR3(CDR H1、CDR H2、CDR H3或H1、H2、H3)。
在本申请中，术语“抗CD3抗体”通常是指靶向CD3的抗体或其变体，例如单克隆抗体，包括人、人源化、嵌合或鼠抗体，其针对成熟T细胞的T细胞抗原受体中的CD3受体。抗CD3抗体可以包括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“TIL特性”通常是指TIL细胞经过本申请培养方法获得的特性。TIL特性的变化可以包含：增加的TIL细胞数量，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的T细胞受体(TCR)克隆多样性和提高的组织中TIL细胞数量，或它们的任何组合。本申请的变化可以是提高或者降低。
在本申请中，术语“存续”通常是指细胞在受试者体内的存在。例如，TIL细胞存续能力的增加，可以是指TIL细胞在体内存在的时间增加。例如，存续能力增加可以是指细胞在受试者组织内，例如肿瘤、脾脏、骨髓、肺组织及血液中存在的时间的增加。
在本申请中，术语“纳米颗粒”通常是指至少一个尺寸小于100nm的微观颗粒。通常，纳米颗粒具有50nm至500nm(即0.05μm至0.5μm)范围内的直径；在生理环境中结构稳定； 且可以容纳更小的分子(如药物或其他生物活性剂)，然后可以将该分子递送至希望的部位。例如，本申请的纳米颗粒可以包含CD28抗体或其抗原结合片段。例如，本申请的纳米颗粒可以包含CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段。例如，抗CD3抗体可以包括OKT3。例如，抗CD28抗体可以包括15E8。
在本申请中，术语“人工抗原呈递细胞”通常是指人工构建的用于呈递外源抗原的免疫细胞，例如，呈递外源抗原的方式可以是人工抗原呈递细胞的表面包含外源抗原与主要组织相容性复合物(MHC)的复合物。在一个实施方案中，可以包括分离的人工抗原呈递细胞(aAPC)，其可以包含表达HLA-A/B/C(编码其的基因GeneID可以为3105、3106或3107)、CD64(编码其的基因GeneID可以为2209)、CD80(编码其的基因GeneID可以为941)、ICOS-L(编码其的基因GeneID可以为23308)和CD58(编码其的基因GeneID可以为965)的细胞，并可以被修饰以表达一种以上T细胞激活剂，本申请的以上可以包含本数。
在本申请中，术语“融合蛋白”通常是指含有第一多肽或蛋白质或其片段、类似物或衍生物的氨基酸序列和异源多肽或蛋白质(即，不同于第一多肽或蛋白质或其片段、类似物或衍生物的第二多肽或蛋白质或其片段、类似物或衍生物，或者通常不是第一多肽或蛋白质或其片段、类似物或衍生物的一部分)的氨基酸序列的多肽或蛋白质。在某些情形中，融合蛋白可包含与异源蛋白、多肽或肽融合的预防性或治疗性药物。其中，本申请的异源蛋白、多肽或肽可以是或不是不同类型的预防性或治疗性药物。例如，可将具有免疫调节活性的两种不同蛋白质、多肽或肽融合到一起形成融合蛋白。在某些情形中，与异源蛋白、多肽或蛋白质融合前的初始多肽或蛋白质的活性相比，融合蛋白可以保留或提高了活性。例如，本申请的融合蛋白可以是融合了CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段的融合蛋白。
在本申请中，术语“杀伤能力”通常是指通过使本申请的细胞接触有效量的物质从而杀伤靶细胞来实现。在一个实施方案中，本申请的物质可以是TIL细胞。本申请的杀伤可以包括通过自身或者促进其它细胞或物质的CDC、凋亡、ADCC、和/或吞噬作用，或通过两种或更多种这些机制的组合以杀伤细胞。
在本申请中，术语“施用”通常是指通过本领域已知的任意途径，将物质递送给有此需要的受试者。药用载体和制剂或组合物也是本领域众所周知的。给药途径可以包括：静脉内的、肌肉内的、真皮内的、皮下的、透皮的、粘膜的、瘤内的和/或粘膜的。
在本申请中，术语“试剂盒”通常是指一起被包装在容器、接受器或其它容器中的两种或更多种组分，其中一种对应于本申请的物质。例如，包含本申请的TIL细胞。
在本申请中，术语“受试者”通常是指细胞或动物，可以是哺乳动物，诸如人、非人灵长类动物(猿、长臂猿、大猩猩、黑猩猩、猩猩、猕猴)、家畜(狗和猫)、农场动物(家禽如鸡和鸭、马、牛、山羊、绵羊、猪)和实验动物(小鼠、大鼠、兔、豚鼠)。人受试者包括胎儿、新生儿、婴儿、青少年和成人受试者。受试者包括动物疾病模型，例如肿瘤动物模型，和本领域技术人员已知的其它动物模型。
在本申请中，术语“饲养细胞(feeder)”通常是指可以用于支持培养另一种所关注的细胞生长的培养细胞。例如，可以通过体外生长和分泌至少一种因子至培养基。在一种实施方式中，饲养细胞可以包括抗原呈递细胞。
在本申请中，术语“特异性结合”通常是指识别特异性靶点物质，但是基本不识别或结合样品中其它分子的结合物质。例如，如果一种结合物质可以特异性结合来自一个物种的本申请的特异性靶点物质，则本申请的结合物质还可以特异性结合来自其它的一个或多个物种的本申请的靶点物质或同源靶点物质。这种种间反应性本身可以不会改变结合物质作为特异性的分类。在某些情形中，特异性结合至靶点物质的结合物质还可以结合至靶点物质的不同等位形式。
在本申请中，术语“完整的培养过程”通常是指将细胞从患者体内分离的肿瘤组织中分离开始，经过一次或一次以上的扩增，最终获得可以施用于受试者的细胞的完整过程。
在本申请中，术语“细胞培养基”通常是指细胞例如哺乳动物细胞在其中生长的营养液。细胞培养基的配制在本领域中是熟知的。典型地，细胞培养基包括缓冲液、盐、碳水化合物、氨基酸、维生素以及必要的微量元素。细胞培养基可以含有或不含有血清、蛋白胨、和/或蛋白质。细胞培养基可以补充有另外的组分或浓度增加的组分，如氨基酸、盐、糖、维生素、激素、生长因子、缓冲液、抗生素、脂质、微量元素等，这取决于有待培养的细胞的要求和/或所希望的细胞培养参数。
在本申请中，术语“药物组合物”或“药物制剂”通常是指一种制备物，本申请的制备物可以允许有效成分的生物活性有效，并且可以不含有对于将会施用该制剂的受试者不可接受地有毒的额外组分。这类制剂是无菌的。“可药用的”赋形剂(载体、添加物)是可以合理地施用至受试哺乳动物以提供有效剂量的所用有效成分的那些赋形剂。
在本申请中，术语“肿瘤浸润淋巴细胞”或“TIL”通常是指最初作为白细胞获得的细胞群，本申请的细胞已经离开受试者的血流并迁移到肿瘤中。TIL可以包括但不限于CD8 +细胞毒性T细胞(淋巴细胞)、Th1和Th17CD4 +T细胞、天然杀伤细胞、树突细胞和M1巨噬细胞。TIL可以包括初级TIL和次级TIL。“初级TIL”可以是从受试者组织样品获得的那些TIL细胞， “次级TIL”可以是本申请中已扩增或经扩增的任何TIL群。在一些实施方式中，本申请的肿瘤浸润淋巴细胞可以是未经分离纯化的，或者可以是与肿瘤细胞相互浸润的。在一种实施方式中，本申请的TIL可以是指TIL群。
在本申请中，术语“中心记忆T细胞”通常是指具有长期记忆性的，并能够接受抗原再刺激的T细胞。中心记忆T细胞可以具有CD45RA -CCR7 +或者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，也可以通过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。本申请中，“药学上可接受的载体(carrier)”可以理解为不包含基因工程用到的核酸形式的载体(vector)。
在本申请中，术语“功能活性片段”通常是指具有全长蛋白质或核酸的部分区域，但保留或部分保留全长蛋白质或核酸的生物活性或功能的片段。例如，功能活性片段可以保留或部分保留全长蛋白质结合另一种分子的能力。例如，生长因子IL-2的功能活性片段，可以保留或部分保留全长IL-2的引起细胞增殖的生物活性功能。
在本申请中，术语“T细胞激活剂”通常是指与T细胞上的相应结合受体结合，并介导T细胞共刺激反应的物质。T细胞激活剂可以是T细胞产生有效免疫应答所需的除抗原受体之外的物质。T细胞激活剂可以是指T细胞共刺激分子。例如，本申请的T细胞激活剂可以包含其变体、同源物或包含其功能活性片段的任何物质。T细胞激活剂可以包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号淋巴细胞活化分子(SLAM蛋白)、NK细胞活化受体、BTLA(编码其的基因GeneID可以为151888)、Toll配体受体、OX40(编码其的基因GeneID可以为7293)、CD2(编码其的基因GeneID可以为914)、CD7(编码其的基因GeneID可以为924)、CD27(编码其的基因GeneID可以为939)、CD28(编码其的基因GeneID可以为940)、CD30(编码其的基因GeneID可以为943)、CD40(编码其的基因GeneID可以为958)、CDS、ICAM-1(编码其的基因GeneID可以为3383)、LFA-1(CD11a/CD18)(编码其的基因GeneID可以为3689)、4-1BB(CD137)(编码其的基因GeneID可以为3604)、B7-H3(编码其的基因GeneID可以为80381)、ICOS(CD278)(编码其的基因GeneID可以为29851)、GITR(编码其的基因GeneID可以为8784)、BAFFR(编码其的基因GeneID可以为115650)、LIGHT(编码其的基因GeneID可以为8740)、HVEM(LIGHTR)(编码其的基因GeneID可以为8764)、KIRDS2、SLAMF7(编码其的基因GeneID可以为57823)、NKp80(KLRF1)(编码其的基因GeneID可以为51348)、NKp44(编码其的基因GeneID可以为9436)、NKp30(编码其的基因GeneID可以为259197)、NKp46(编码其的基因GeneID可以为9437)、CD19(编码其的基因GeneID可以为930)、CD4(编码其的基因GeneID可以为920)、CD8α(编码其的基因GeneID可以为925)、CD8β(编码其的基因GeneID可以为926)、IL-2Rβ、IL-2Rγ、IL7Rα(编码其的基因GeneID可以为)、ITGA4(编码其的基因GeneID可以为3676)、VLA1(编码其的基因GeneID可以为3672)、CD49a(编码其的基因GeneID可以为3672)、IA4(编 码其的基因GeneID可以为3732)、CD49D(编码其的基因GeneID可以为3676)、ITGA6(编码其的基因GeneID可以为3655)、VLA-6(编码其的基因GeneID可以为3655)、CD49f(编码其的基因GeneID可以为3655)、ITGAD(编码其的基因GeneID可以为3681)、CD11d(编码其的基因GeneID可以为3681)、ITGAE(编码其的基因GeneID可以为3682)、CD103(编码其的基因GeneID可以为3682)、ITGAL(编码其的基因GeneID可以为3683)、CD11a(编码其的基因GeneID可以为3683)、LFA-1(编码其的基因GeneID可以为3683)、ITGAM(编码其的基因GeneID可以为3684)、CD11b(编码其的基因GeneID可以为3684)、ITGAX(编码其的基因GeneID可以为3687)、CD11c(编码其的基因GeneID可以为3687)、ITGB1(编码其的基因GeneID可以为3688)、CD29(编码其的基因GeneID可以为3688)、ITGB2(编码其的基因GeneID可以为3689)、CD18(编码其的基因GeneID可以为3689)、LFA-1(编码其的基因GeneID可以为3689)、ITGB7(编码其的基因GeneID可以为3695)、NKG2D(编码其的基因GeneID可以为22914)、NKG2C(编码其的基因GeneID可以为3822)、TNFR2(编码其的基因GeneID可以为7133)、TRANCE/RANKL(编码其的基因GeneID可以为8600)、DNAM1(CD226)(编码其的基因GeneID可以为10666)、SLAMF4(CD244、2B4)(编码其的基因GeneID可以为51744)、CD84(编码其的基因GeneID可以为8832)、CD96(Tactile)(编码其的基因GeneID可以为10225)、CEACAM1(编码其的基因GeneID可以为634)、CRTAM(编码其的基因GeneID可以为56253)、Ly9(CD229)(编码其的基因GeneID可以为4063)、CD160(BY55)(编码其的基因GeneID可以为11126)、PSGL1(编码其的基因GeneID可以为6404)、CD100(SEMA4D)(编码其的基因GeneID可以为10507)、CD69(编码其的基因GeneID可以为969)、SLAMF6(NTB-A、Ly108)(编码其的基因GeneID可以为114836)、SLAM(SLAMF1、CD150、IPO-3)(编码其的基因GeneID可以为6504)、BLAME(SLAMF8)(编码其的基因GeneID可以为56833)、SELPLG(CD162)(编码其的基因GeneID可以为6404)、LTBR(编码其的基因GeneID可以为4055)、LAT(编码其的基因GeneID可以为27040)、GADS(编码其的基因GeneID可以为9402)、SLP-76(编码其的基因GeneID可以为3937)、PAG/Cbp(编码其的基因GeneID可以为55824)、CD19a、和特异性结合CD3的配体、特异性结合CD28的配体、特异性结合HVEM的配体、特异性结合CD40L的配体、特异性结合OX40的配体、和特异性结合4-1BB的配体。共刺激胞内信号传导结构域可以是指T细胞激活剂的胞内部分。胞内信号传导结构域可以包含从中衍生的分子的完整胞内部分或完整天然胞内信号传导结构域或其功能性片段。
在本申请中，术语“T细胞生长因子”通常是指引起细胞增殖的生物活性多肽或小分子化合物。例如，本申请的T细胞生长因子可以包含其变体、同源物或包含其功能活性片段的任 何物质。在一种实施方式中，T细胞生长因子可以选自以下组的一种或多种：IL-2(编码其的基因GeneID可以为3558)、IL-4(编码其的基因GeneID可以为3565)、IL-6(编码其的基因GeneID可以为3569)、IL-7(编码其的基因GeneID可以为3574)、IL-10(编码其的基因GeneID可以为3586)、IL-12(编码其的基因GeneID可以为3592或3593)、IL-15(编码其的基因GeneID可以为3600)、IL-21(编码其的基因GeneID可以为59067)、TNF-α(编码其的基因GeneID可以为100137091)、γ干扰素(编码其的基因GeneID可以为3458)等等。
在本申请中，术语“基本上同时”通常是指接触过程的一段时间内TIL可以与两种以上的物质同时接触，但是可以不限于在整个接触过程中TIL总是与两种以上的物质同时接触。在一种实施方式中，基本上同时可以是指一段时间内TIL可以与至少10％、20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％的两种以上的物质的每种物质同时接触。
在本申请中，术语“固相介质”或“介质”通常是指具有结合功能的固相材料。例如，本申请固相介质可以是指通过共价结合和/或非共价结合的作用，将一种或一种以上的物质结合在介质内和/或介质表面的材料。例如，本申请的固相介质可以结合一种或一种以上的T细胞激活剂。例如，本申请的固相介质可以是指通过共价结合和/或非共价结合的作用将CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段结合在介质内和/或介质表面的材料。例如，本申请的固相介质可以是包含OKT3抗体和15E8抗体的直径为约500纳米至约10微米的微球。例如，本申请的固相介质可以是聚合物材料。例如，本申请的固相介质可以是直径至少约500纳米的微球。例如，本申请的固相介质可以是纳米基质。例如，本申请的固相介质可以是包含OKT3抗体和15E8抗体的直径为约1纳米至约500纳米的纳米基质。
在本申请中，术语“纳米基质”通常是指一种直径在约1纳米到约500纳米的材料。在本申请中，纳米基质可以具有结合功能，例如，本申请的纳米基质可以结合一种或一种以上的T细胞激活剂。在本申请中，纳米基质可以包含聚合物，例如，本申请的纳米基质可以包含可降解聚合物。在本申请中，纳米基质可以包含多糖、和/或葡聚糖。
在本申请中，术语“树突状细胞”通常是指存在于体内、体外、离体或宿主或受试者内的或可衍生自造血干细胞或单核细胞的抗原递呈细胞。树突状细胞及其前体可以从各种淋巴器官例如脾脏、淋巴结以及骨髓和外周血分离。本申请的树突状细胞可以具有特征形态，例如在树突细胞体的多个方向上延伸的薄层(板状伪足)。通常，树突细胞可以表达高水平的MHC和共刺激(例如B7-1和B7-2)分子。树突状细胞可以在体外诱导T细胞的抗原特异性分化，并且能够在体外和体内引发原代T细胞应答。
在本申请中，术语“体外扩增”通常是指经过培养以产生细胞的数量的变化，经扩增的细 胞也可以产生细胞的数量和/或比例变化，分泌能力变化，杀伤能力变化或表达能力的变化，或它们的任何组合。本申请的变化可以是提高或者降低。在本申请中，体外扩增可以是为了扩增目的；为了检测TIL细胞的功能，例如检测TIL细胞释放细胞因子能力，而对TIL细胞进行的操作步骤(例如向TIL细胞的培养基中加入一种或一种以上物质以检测TIL细胞释放细胞因子能力)，可以不属于本申请的体外扩增。
在本申请中，术语“外周单个核细胞”或“外周血单个核细胞”通常是指外周血中具有单个核的细胞。例如，在本申请中，本申请的外周血单个核细胞可以包括淋巴细胞、单核细胞和/或树突状细胞。
在本申请中，术语“细胞因子”通常是指由一个细胞群释放的对另一个细胞起细胞间调节剂作用的蛋白。本申请的细胞因子可以是淋巴细胞因子(lymphokines)、单核细胞因子(monokines)和多肽激素。本申请的细胞因子可以包括白细胞介素(ILs)如IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-15、IL-21、和/或IL-12。在本申请中，术语细胞因子可以包括来自天然来源或来自重组细胞培养物的蛋白，天然序列细胞因子的生物活性等价物，以及其功能活性片段。
在本申请中，术语“直径”通常是指本申请物质的截面的直径。例如，当本申请的物质不是球形时，则术语“直径”通常是指本申请物质的最大截面的最大直径和/或平均直径。确定物质的直径的方法可以是本领域通用的方法，例如透射电子显微镜。
在本申请中，术语“肿瘤”通常是指任何新的病理性的组织增生。本申请的肿瘤可能是良性的，也可能是恶性的。本申请的肿瘤可能是实体的，也可能是血液的。术语“肿瘤”可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
在本申请中，术语“肿瘤组织”通常是指来自对象中的肿瘤，包括对象中的任何实体肿瘤和/或非实体肿瘤的任何组织的样品。
在本申请中，术语“CD28激动剂”通常是指结合细胞表面CD28蛋白并且在细胞中引发应答的化合物。例如，本申请的CD28激动剂可以是结合CD28的小分子制剂。例如，本申请的CD28激动剂可以是结合CD28的抗体或其抗原结合片段。
在本申请中，术语“T细胞亚群比例”通常是指根据不同T细胞亚群占TIL细胞或TIL群中的比例。例如，本申请不同的T细胞亚群具有不同的免疫活性和/或分化能力。例如，本申请的T细胞亚群可以根据T细胞表面标志物进行区分。例如，中心记忆T细胞可以具有CD45RA -CCR7 +的表型。例如，调节性T细胞可以具有CD4 +CD25 +Foxp3 +的表型。例如，活 化T细胞可以具有CD25 +、CD28 +、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，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
例如，所述方法可以包含：使所述TIL与所述饲养细胞共培养之后，使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
例如，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱之后，使所述TIL与所述饲养细胞共培养。
例如，所述方法可以包含：在所述TIL与所述T细胞激活剂和/或所述T细胞生长因子接触之后且在所述TIL与所述饲养细胞共培养之前使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
例如，所述方法可以包含：在所述TIL与所述T细胞激活剂和/或所述T细胞生长因子接触基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
例如，所述方法可以包含：在所述TIL与所述饲养细胞共培养基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使 所述TIL的至少一种目标基因的表达降低和/或活性减弱。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，其中所述TIL包含与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养获得的TIL。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中所述TIL包含使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL与CD28激动剂接触。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，其中所述TIL包含与CD28激动剂接触获得的TIL。
另一方面，本申请还提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法可以包含：使所述TIL与CD28激动剂接触，其中所述TIL包含使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL。
例如，所述CD28激动剂包含抗CD28的抗体和/或其抗原结合片段、CD80和/或其功能活性片段和/或CD86和/或其功能活性片段，以及上述物质的重组蛋白。
例如，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL可以显示出改善的TIL特性。在一种实施方式中，目标基因的表达和/或活性未改变的TIL可以是指源自同一供体的且未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞。在一种实施方式中，目标基因的表达和/或活性未改变的TIL可以是指源自同一供体的且未曾使所述TIL的目标基因以外的其它基因(例如敲除该其它基因，对细胞功能基本没有影响)的表达降低和/或活性减弱的TIL细胞。
在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指源自同一供体的经过同样方式分离的且未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指源自同一供体的同一肿瘤来源的且未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指源自同一供 体的同一肿瘤来源的经过同样方式分离的且未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指将源自同一供体的TIL细胞分为两组，其中一组未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞可以为未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指将源自同一供体的经过同样方式分离的TIL细胞分为两组，其中一组未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞可以为未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指将源自同一供体的同一肿瘤来源的TIL细胞分为两组，其中一组未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞可以为未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL。在一种实施方式中，未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL可以是指将源自同一供体的同一肿瘤来源的经过同样方式分离的TIL细胞分为两组，其中一组未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的TIL细胞可以为未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL。例如，至少一种目标基因的表达降低和/或活性减弱可以是指天然的细胞的该目标基因的处于一定程度的表达状态，经过本申请的处理，可以使得该细胞的该目标基因的表达量降低，即该目标基因的表达量降低可以是使天然细胞从表达该目标基因转变为基本不表达该目标基因或表达该目标基因的量降低。
在一种实施方式中，本申请的改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的抗凋亡能力，和提高的T细胞受体(TCR)克隆多样性。例如，本申请的方法制备的TIL可以提高TIL的存续能力。例如，本申请获得的TIL可以在撤去IL-2的培养条件下，可以具有增加的长期存续能力。例如，相对于未经过本申请培养方法获得的TIL的培养条件，本申请获得的TIL的培养条件中IL-2的浓度可以降低100％、至少90％、至少80％、至少70％、至少60％、至少50％、至少40％、至少30％、至少20％、至少10％、至少5％、或至少1％时，所述本申请获得的TIL可以持续扩增。
在一种实施方式中，本申请的改善的TIL细胞数量和扩增能力，提高的抗凋亡能力是指与在体外扩增阶段未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL 相比，在至少一个体外扩增阶段中使所述TIL的至少一种目标基因的表达降低和/或活性减弱的本申请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。例如，改善的TIL的扩增能力可以表现为TIL增殖倍率的增加。例如，改善的TIL细胞数量可以表现为TIL细胞活率的增加。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使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL相比，在至少一个体外扩增阶段中使所述TIL的至少一种目标基因的表达降低和/或活性减弱的本申请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％。例如，改善的TIL细胞数量可以表现为TIL细胞凋亡比例的减少。在一种实施方式中，本申请的减少的TIL细胞凋亡比例可以是指与在体外扩增阶段未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL相比，在至少一个体外扩增阶段中使所述TIL的至少一种目标基因的表达降低和/或活性减弱的本申请TIL的细胞凋亡比例可以减少至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，凋亡细胞比例可以通过本领域已知的凋亡试剂盒进行检测。
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-α和IFN-γ。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使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL相比，在至少一个体外扩增阶段中使所述TIL的至少一种目标基因的表达降低和/或活性减弱过的本申请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使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL相比，在至少一个体外扩增阶段中使所述TIL的至少一种目标基因的表达降低和/或活性减弱过的本申请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，本申请的增加的NK细胞比例可以是TIL细胞中NK细胞的比例的增加。例如，在TIL细胞中NK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使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL相比，在至少一个体外扩增阶段中使所述TIL的至少一种目标基因的表达降低和/或活性减弱过的本申请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使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL相比，在至少一个体外扩增阶段中使所述TIL的至少一种目标基因的表达降低和/或活性减弱过的本申 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的肿瘤细胞杀伤率可以通过使用IncuCyte系统测量Caspase-3/7活性测量。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杀伤黑色素瘤细胞的能力。
在一种实施方式中，本申请的提高的T细胞受体(TCR)克隆多样性可以包含在长期培养的过程中，与在体外扩增阶段未曾使所述TIL的至少一种目标基因的表达降低和/或活性减弱的相应TIL相比，在至少一个体外扩增阶段中使所述TIL的至少一种目标基因的表达降低和/或活性减弱过的本申请TIL细胞群中表达的TCR的种类更多，例如，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，本申请的改善的T细胞亚群比例可以包含选自以下组的一种或多种：增加的CD4 +细胞比例，降低的CD8 +细胞比例，增加的中心记忆T细胞比例，降低的调节性T细胞的比例，增加的活化T细胞比例，增加的肿瘤特异性T细胞比例，和增加的干细胞样T细胞比例。
在一种实施方式中，本申请的增加的CD4 +细胞比例可以是TIL细胞中CD4阳性细胞的比例的增加。例如，在TIL细胞中CD4 +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，本申请的降低的CD8 +细胞比例可以是TIL细胞中CD8阳性细胞的比例的降低。例如，在TIL细胞中CD8 +细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
在一种实施方式中，本申请的增加的中心记忆T细胞比例可以是TIL细胞中CD45RA -CCR7 +细胞的比例的增加。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 +细胞的比例的减少。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。
例如，本申请的方法中使TIL的至少一种目标基因的表达降低和/或活性减弱可以包含将基因调控系统引入所述TIL细胞中。
例如，所述基因调控系统可以在DNA水平破坏所述目标基因。例如，所述基因调控系统可以破坏所述TIL细胞的基因组中的所述目标基因的区域或其片段。例如，使用所述基因调控系统后，TIL细胞中的所述目标基因所在的DNA区域或其片段被剪切而该目标基因的表达能力降低或该目标基因的活性被抑制。例如，所述基因调控系统对目标基因的编辑效果可以是长期的、持续的。
例如，所述基因调控系统可以包含指导核酸分子和酶蛋白。例如，所述酶蛋白可以具有核酸剪切酶活性，所述指导核酸分子可以指导所述酶蛋白特异性剪切目标基因所在的区域或其片段。例如，指导核酸分子和酶蛋白可以以核糖核蛋白复合物(RNP)形式存在、或各自独立地单独存在。例如，所述酶蛋白可以包含Cas蛋白。
例如，本申请使TIL的至少一种目标基因的表达降低和/或活性减弱可以包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。例如，所述酶蛋白可以包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。例如，所述指导核酸分子可以包含指导RNA(gRNA)。
例如，所述gRNA可以用于与所述目标基因的序列结合。例如，所述gRNA与所述目标基因的序列的结合可以是完全互补、可以是部分互补、也可以是中等严紧或严紧条件杂交于所述目标基因的序列。例如，所述gRNA与所述目标基因的序列的结合可以使得gRNA的CRISPR系统特异性剪切所述目标基因。
例如，本申请的目标基因可以包含编码NF-κB通路抑制分子的基因。例如，本申请的NF-κB通路抑制分子可以使选自以下组的蛋白泛素化：受体相互作用蛋白1(RIP1)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1)、受体相互作用蛋白2(RIP2)和肿瘤坏死因子受体相关因子6(TRAF6)。例如，本申请的NF-κB通路抑制分子可以使受体相互作用蛋白1(RIP1)泛素化。例如，本申请的NF-κB通路抑制分子可以包含肿瘤坏死因子α诱导蛋白(TNFAIP)， 例如肿瘤坏死因子α诱导蛋白3(TNFAIP3)。
例如，本申请所述指导核酸分子可以与TNFAIP3基因的第2外显子和/或第7外显子所在的区域或其片段结合。例如，本申请所述指导核酸分子可以与选自以下组所示的区域或其片段结合：SEQ ID NO：34至47。例如，本申请的指导核酸分子靶向的区域下游可以有原型间隔序列毗邻基序(PAM)，所述原型间隔序列毗邻基序(PAM)可以是GGG、TGG、CGG或AGG。例如，当确定了目标基因的PAM区域，本领域人员可以容易确定目标基因PAM的5′端上游约15至约25个核苷酸组成的靶序列，同时可以针对该靶序列设计合适的gRNA。
例如，所述指导核酸分子可以包含能够结合TNFAIP3基因所在DNA中GGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合TNFAIP3基因所在DNA中CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr6:137876102-137876121或chr6:137874868-137874887。
例如，所述指导核酸分子可以包含能够结合TNFAIP3基因所在DNA中TGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合TNFAIP3基因所在DNA中CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr6:137871485-137871504或chr6:137871486-137871504。
例如，所述指导核酸分子可以包含能够结合TNFAIP3基因所在DNA中CGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合TNFAIP3基因所在DNA中CGG所示PAM区前约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例 如，靶序列可以是源自于人chr6:137871475-137871494、chr6:137871501-137871520或chr6:137871502-137871520。
例如，所述指导核酸分子可以包含能够结合TNFAIP3基因所在DNA中AGG所示PAM区前约10至约30个核苷酸组成的靶序列。例如，所述指导核酸分子可以包含能够结合TNFAIP3基因所在DNA中AGG所示PAM区约15至约25个、约17至约25个、约19至约25个、约20至约25个、约21至约25个、约23至约25个、约15至约23个、约17至约23个、约19至约23个、约20至约23个、约21至约23个、约15至约21个、约17至约21个、约19至约21个、约20至约21个、约15至约20个、约17至约20个、约19至约21个、约15至约19个、约17至约19个、或约15至约17个核苷酸组成的靶序列。例如，靶序列可以是源自于人chr6:137879020-137879039、chr6:137874833-137874852、chr6:137877195-137877214、chr6:137878652-137878672、chr6:137878653-137878672或chr6:137874842-137874860。
例如，所述指导核酸分子可以包含如SEQ ID NO：48至61中任一项所示的序列。
例如，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例可以降低和/或单个细胞中所述目的基因的表达量可以下降。
例如，本申请的方法中与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例降低至少约5％。例如，表达所述TNFAIP3基因的产物的细胞比例降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，表达所述TNFAIP3基因的产物的细胞比例可以从可以观测的细胞比例到0％。例如，表达所述TNFAIP3基因的产物的细胞比例可以降低到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。例如，表达所述TNFAIP3基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本申请的方法中所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的 TIL中表达所述TNFAIP3基因的产物的细胞比例可以为至多约95％。例如，表达所述TNFAIP3基因的产物的细胞比例的细胞比例可以为至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。例如，表达所述TNFAIP3基因的产物的细胞比例可以通过细胞流式仪进行检测。
例如，本申请的方法中与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中单个细胞中所述目的基因的表达量可以下降至少约5％。例如，单个细胞中所述目的基因的表达量可以下降至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、或至少约5％。例如，单个细胞中所述目的基因的表达量可以从可以观测的量到0％。例如，，单个细胞中所述目的基因的表达量可以下降到至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、或至少约1％。
例如，本申请的方法中所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中单个细胞中所述目的基因的表达量可以为所述目标基因的表达和/或活性未改变的TIL的至多约95％。例如，TIL中单个细胞中所述TNFAIP3的表达量可以为所述TNFAIP3的表达和/或活性未改变的TIL的至多约95％、至多约90％、至多约80％、至多约70％、至多约60％、至多约50％、至多约40％、至多约30％、至多约20％、至多约19％、至多约18％、至多约17％、至多约16％、至多约15％、至多约14％、至多约13％、至多约12％、至多约11％、至多约10％、至多约9％、至多约8％、至多约7％、至多约6％、或至多约5％。
在一种实施方式中，本申请的方法还可以包含：使源自肿瘤组织且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的本申请体外扩增中，可以使本申请TIL与饲养细胞共培养。
在一种实施方式中，在单个阶段的本申请体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请饲养细胞共培养，在一种实施方式中，单个阶段的 本申请体外扩增可以指在同一个阶段的本申请的体外扩增，例如，可以同在本申请的第一阶段体外扩增、同在本申请的第二阶段体外扩增、或同在本申请的第三阶段体外扩增等。
在一种实施方式中，本申请的第一阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请饲养细胞共培养。在一种实施方式中，本申请的在本申请第二阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请饲养细胞共培养。在一种实施方式中，本申请的在本申请第三阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请饲养细胞共培养。
在一种实施方式中，每一个阶段体外扩增之间可以是通过TIL细胞数量的变化来划分的，在一种实施方式中，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、至少约50倍、至少约100倍、至少约200倍、至少约500倍、或者至少约1000倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段的体外扩增之间也可以是通过TIL细胞培养的条件的变化来划分的。在一种实施方式中，当细胞培养基中添加了或补充添加了T细胞激活剂和/或T细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了IL-2后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，使所述TIL的至少一种目标基因的表达降低和/或活性减弱，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了饲养细胞后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，当TIL细胞进行了离心和/或细胞洗涤的操作后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。在一种实施方式中，当TIL细胞体外培养约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天、约20天、约30天、约40天、约50天或约100天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
例如，所述第二阶段体外扩增可以进行至少约7天。例如，所述第二阶段体外扩增可以进行至少约9天。例如，所述第二阶段体外扩增可以进行至多约14天。例如，所述第二阶段体外扩增可以进行至多约13天。例如，所述第二阶段体外扩增可以进行约7天至约14天、约9天至约14天、约7天至约13天或约9天至约13天。例如，本申请的第二阶段体外扩增 可以进行至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本申请的第二阶段体外扩增可以进行约9天至约14天，例如，本申请的第二阶段体外扩增可以进行约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。例如，本申请的第二阶段体外扩增可以认为是REP(rapid expansion protocol)阶段。
例如，所述第一阶段体外扩增可以进行至少约7天。例如，所述第一阶段体外扩增可以进行约7天至约14天。例如，本申请的第一阶段体外扩增可以进行至少约7天、至少约8天、至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本申请的第一阶段体外扩增可以进行约7天至约14天、约8天至约14天、约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。例如，本申请的第一阶段体外扩增可以认为是preREP阶段。
在一种实施方式中，本申请第二阶段体外扩增进行的天数可以是从第二阶段体外扩增的开始时刻进行计算。例如，第二阶段体外扩增开始的当时，可以认为是第二阶段体外扩增进行了约0天。例如，第二阶段体外扩增开始后进行了约24小时，可以认为是第二阶段体外扩增进行了约1天。例如，第二阶段体外扩增开始的当天，可以认为是第二阶段体外扩增进行了约0天。在一种实施方式中，本申请第二阶段体外扩增进行的天数可以是通过第二阶段体外扩增进行的天数进行计算。例如，第二阶段体外扩增开始后的第二天，可以认为是第二阶段体外扩增进行了约1天。
在一种实施方式中，本申请培养方法可以按照两步骤划分方式进行划分。例如，(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。例如，所述步骤(A)可以进行约7天至约14天。例如，所述步骤(B)可以进行约7天至约14天。
在一种实施方式中，本申请培养方法可以按照三步骤划分方式进行划分。例如，(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤 (A)得到第二TIL群；(B)可以使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且可以使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；(C)可以使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。例如，所述步骤(A)可以进行约7天至约14天。例如，所述步骤(B)可以进行约0天至约8天。例如，所述步骤(C)可以进行约5天至约14天。
在一种实施方式中，本申请培养方法可以按照四步骤划分方式进行划分。例如，(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；(C)可以使所述第三TIL群的至少一种目标基因的表达降低和/或活性减弱，其中，经所述步骤(C)得到第四TIL群；(D)可以使所述第四TIL群与饲养细胞共培养，其中，经所述步骤(D)得到第五TIL群。例如，所述步骤(A)可以进行约7天至约14天。例如，所述步骤(B)可以进行约0天至约4天。例如，所述步骤(C)可以进行约0天至约4天。例如，所述步骤(D)可以进行约5天至约14天。
在一种实施方式中，本申请的培养方式的步骤(A)是从复苏和/或继续培养体外TIL群得到第二TIL群。例如，所述体外TIL群可以包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群。例如，所述体外TIL群可以包含由所述第一TIL群接触T细胞生长因子获得的TIL群。例如，所述体外TIL群可以包含由所述第一TIL群冷冻保存获得的TIL群。例如，所述体外TIL群可以包含由所述第一TIL群接触T细胞生长因子且冷冻保存获得的TIL群。例如，当本申请的步骤(A)为是从复苏和/或继续培养体外TIL群得到第二TIL群时，此时步骤(A)可以进行约2小时至约4天。
在一种实施方式中，在单个阶段的本申请体外扩增中，可以使本申请TIL与本申请一种或多种T细胞激活剂和/或一种或多种T细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。在一种实施方式中，本申请的一定时间可以为至少约2小时。在一种实施方式中，本申请的一定时间可以为至少约1小时、至少约2小时、至少约3小时、至少约4小时、至少约5小时、至少约6小时、至少约7小时、至少约8小时、至少约9小时、至少约10小时、至少约11小时、至少约12小时、至少约13小时、至少约14小时、至少约15小时、至少约16小时、至少约17小时、至少约18小时、至少约19小时、至少约20小时、至少约21小时、至少约22小时、至少约23小时、至少约24小时、至少约36小时、至少约48小时、至少约60小时或至少约72小时。在一种实施方式中，本申请的一定时间可以为约6小时至约72小时。在一种实施方式中，本申请的一定时间可以为约6小时到约7小时、约6小时到 约8小时、约6小时到约9小时、约6小时到约10小时、约6小时到约11小时、约6小时到约12小时、约6小时到约13小时、约6小时到约14小时、约6小时到约15小时、约6小时到约16小时、约6小时到约17小时、约6小时到约18小时、约6小时到约19小时、约6小时到约20小时、约6小时到约21小时、约6小时到约22小时、约6小时到约23小时、约6小时到约24小时、约6小时到约36小时、约6小时到约48小时、约6小时到约60小时或约6小时到约72小时。在一种实施方式中，本申请的一定时间可以为约12小时到约13小时、约12小时到约14小时、约12小时到约15小时、约12小时到约16小时、约12小时到约17小时、约12小时到约18小时、约12小时到约19小时、约12小时到约20小时、约12小时到约21小时、约12小时到约22小时、约12小时到约23小时、约12小时到约24小时、约12小时到约36小时、约12小时到约48小时、约12小时到约60小时或约12小时到约72小时。在一种实施方式中，本申请的一定时间可以为约1小时、约2小时、约3小时、约4小时、约5小时、约6小时、约7小时、约8小时、约9小时、约10小时、约11小时、约12小时、约13小时、约14小时、约15小时、约16小时、约17小时、约18小时、约19小时、约20小时、约21小时、约22小时、约23小时、约24小时、约36小时、约48小时、约60小时或约72小时。
在一种实施方式中，本申请的饲养细胞可以包含抗原呈递细胞。在一种实施方式中，本申请的饲养细胞可以包含选自以下组的一种或多种：外周单个核细胞，树突状细胞，和人工抗原呈递细胞。在一种实施方式中，本申请的饲养细胞可以为外周单个核细胞。在一种实施方式中，本申请的饲养细胞可以为经过辐照的饲养细胞。例如，本申请的饲养细胞可以为分离的人工抗原呈递细胞(aAPC)，本申请的人工抗原呈递细胞可以包含表达HLA-A/B/C、CD64、CD80、ICOS-L和/或CD58的细胞，并可以被修饰以表达一种以上本申请的T细胞激活剂。在一种实施方式中，本申请的饲养细胞可以经过辐照，例如，可以经过伽马射线辐照，或可以经过X射线辐照。
在一种实施方式中，本申请的TIL与本申请的饲养细胞共培养可以包含使本申请的饲养细胞的表面与本申请的TIL的表面相接触。在一种实施方式中，本申请的TIL与本申请的饲养细胞共培养包含将本申请的饲养细胞添加至本申请的TIL的细胞培养基中。
在一种实施方式中，本申请可以以约40:1-约400:1的本申请饲养细胞与本申请TIL的比例，将本申请饲养细胞添加至本申请TIL的细胞培养基中。在一种实施方式中，本申请可以以约40:1-约400:1、以约40:1-约300:1、以约40:1-约200:1、以约40:1-约100:1、以约40:1-约90:1、以约40:1-约80:1、以约40:1-约70:1、以约40:1-约60:1、以约40:1-约50:1、以约 50:1-约400:1、以约60:1-约400:1、以约70:1-约400:1、以约80:1-约400:1、以约90:1-约400:1、以约100:1-约400:1、以约200:1-约400:1、或以约300:1-约400:1的本申请饲养细胞与本申请TIL的比例，将本申请饲养细胞添加至本申请TIL的细胞培养基中。
在一种实施方式中，本申请的方法还可以包含：使源自肿瘤组织且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的本申请体外扩增中，使本申请TIL与一种或多种T细胞激活剂接触。
在一种实施方式中，在单个阶段的本申请的体外扩增中，使所述TIL与所述一种或多种T细胞激活剂接触。例如，T细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。在一种实施方式中，在单个阶段的所述体外扩增中，使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本申请第一阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本申请第二阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本申请第三阶段体外扩增中，可以使所述TIL的至少一种目标基因的表达降低和/或活性减弱且与本申请的一种或多种T细胞激活剂接触。
在一种实施方式中，在单个阶段的本申请的体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以先使所述TIL的至少一种目标基因的表达降低和/或活性减弱，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以使本申请的TIL先与本申请的一种或多种T细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
在一种实施方式中，在本申请第一阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL接触本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本申请第二阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL接触本申请的一种或多种T细胞激活剂接触。在一种实施方式中，在本申请第三阶段体外扩增中，可以基本上同时使所述TIL的 至少一种目标基因的表达降低和/或活性减弱以及使TIL接触本申请的一种或多种T细胞激活剂接触。
在一种实施方式中，本申请的T细胞激活剂可以包含选自以下组的一种或多种：CD80、CD86、B7-H3、4-1BBL、CD27、CD30、CD134、B7h、CD40、LIGHT、以及它们的功能活性片段。在一种实施方式中，本申请的T细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。在一种实施方式中，本申请的T细胞激活剂可以包含选自以下组：CD3、CD28、HVEM、CD40L、OX40和4-1BB的抗体以及它们的抗原结合片段。在一种实施方式中，本申请的T细胞激活剂可以包含CD3激动剂。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以是Miltenyi Biotech的OKT3，可以是BD的SP34。在一种实施方式中，本申请的T细胞激活剂可以包含CD28激动剂。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以是Merck的15E8，本申请的T细胞激活剂可以包含CD80和/或其功能活性片段和/或CD86和/或其功能活性片段，以及上述物质的重组蛋白。
在一种实施方式中，本申请的T细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链VL和重链VH，可以包含BD的SP34的轻链VL和重链VH。在一种实施方式中，本申请的T细胞激活剂可以包含CD28激动剂。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链VL和重链VH。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链LCDR1-3和重链HCDR1-3，可以包含BD的SP34的轻链LCDR1-3和重链HCDR1-3，本申请的抗CD3的抗体和/或其抗原结合片段可以具有CD3结合能力。在一种实施方式中，本申请的T细胞激活剂可以包含CD28激动剂。在一种实施方式中，本申请的T细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链LCDR1-3和重链HCDR1-3，本申请的抗CD28的抗体和/或其抗原结合片段可以具有CD28结合能力。在本申请中，本申请抗体或其抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，本申请CDR可以是根据Chothia定义的，或本申请CDR可以是根据Kabat定义的。
在一种实施方式中，本申请的CD3激动剂可以为CD3抗体或其抗原结合蛋白。
在本申请中，本申请抗体或其抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR。 本申请CDR可以是根据IMGT命名法定义的，本申请CDR可以是根据Chothia定义的，或本申请CDR可以是根据Kabat定义的。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1，且本申请HCDR1可以包含SEQ ID NO：2和12中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR2，且本申请HCDR2可以包含SEQ ID NO：3和13中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR3，且本申请HCDR3可以包含SEQ ID NO：4和14中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：2和12中任一项所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：3和13中任一项所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：4和14中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：2所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：3所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：4所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：12所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：13所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：14所示的氨基酸序列；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在本申请中，本申请抗体或其抗原结合蛋白包含抗体轻链可变区VL中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，或本申请CDR可以是根据Kabat定义的。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR1，且本申请LCDR1可以包含SEQ ID NO：5和15中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR2，且本申请LCDR2可以包含SEQ ID NO：6(DTS)和16中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR3，且本申请LCDR3可以包含SEQ ID NO：7和17中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：5和15中任一项所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：6(DTS)和16中任一项所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：7和17中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：5所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：6(DTS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：7所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：15所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：16所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：17所示的氨基酸序列；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1-3和LCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：2和12中任一项所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：3和13中任一项所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：4和14中任一项所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：5和15中任一项 所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：6(DTS)和16中任一项所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：7和17中任一项所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的HCDR1-3和LCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：2所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：3所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：4所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：5所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：6(DTS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：7所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：12所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：13所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：14所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：15所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：16所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：17所示的氨基酸序列；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链可变区VH，且本申请VH可包含SEQ ID NO：8和18中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的VH，且本申请VH可包含SEQ ID NO：8所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的VH，且本申请VH可包含SEQ ID NO：18所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含轻链可变区VL，且本申请VL可包含SEQ ID NO：9和19中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的VL，且本申请VL可包 含SEQ ID NO：9所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的VL，且本申请VL可包含SEQ ID NO：19所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链可变区VH和轻链可变区VL，且本申请VH可包含SEQ ID NO：8和18中任一项所示的氨基酸序列，本申请VL可包含SEQ ID NO：9和19中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的VH和VL，且本申请VH可包含SEQ ID NO：8所示的氨基酸序列，本申请VL可包含SEQ ID NO：9所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的VH和VL，且本申请VH可包含SEQ ID NO：18所示的氨基酸序列，本申请VL可包含SEQ ID NO：19所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链，且本申请重链可包含SEQ ID NO：10和20中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的重链，且本申请重链可包含SEQ ID NO：10所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的重链，且本申请重链可包含SEQ ID NO：20所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含轻链，且本申请轻链可包含SEQ ID NO：11和21中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的轻链，且本申请轻链可包含SEQ ID NO：11所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的轻链，且本申请轻链可包 含SEQ ID NO：21所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链和轻链，且本申请重链可包含SEQ ID NO：10和20中任一项所示的氨基酸序列，本申请轻链可包含SEQ ID NO：11和21中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与OKT3相同的重链和轻链，且本申请重链可包含SEQ ID NO：10所示的氨基酸序列，本申请轻链可包含SEQ ID NO：11所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与SP34相同的重链和轻链，且本申请重链可包含SEQ ID NO：20所示的氨基酸序列，本申请轻链可包含SEQ ID NO：21所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD3结合能力。
在一种实施方式中，本申请的CD28激动剂可以为CD28抗体或其抗原结合蛋白。
在本申请中，本申请抗体或其抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，或本申请CDR可以是根据Kabat定义的。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1，且本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR2，且本申请HCDR2可以包含SEQ ID NO：23所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR3，且本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：23所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的HCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO： 23所示的氨基酸序列，且本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在本申请中，本申请抗体或其抗原结合蛋白包含抗体轻链可变区VL中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，或本申请CDR可以是根据Kabat定义的。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR1，且本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR2，且本申请LCDR2可以包含SEQ ID NO：26(AAS)所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR3，且本申请LCDR3可以包含SEQ ID NO：27所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：26(AAS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：27所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的LCDR1-3，其中本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：26(AAS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：27所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含HCDR1-3和LCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：23所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：30所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：26(AAS)所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；本申请CDR可以是根据Kabat定义的； 例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的HCDR1-3和LCDR1-3，其中本申请HCDR1可以包含SEQ ID NO：22所示的氨基酸序列，本申请HCDR2可以包含SEQ ID NO：23所示的氨基酸序列，本申请HCDR3可以包含SEQ ID NO：24所示的氨基酸序列，本申请LCDR1可以包含SEQ ID NO：25所示的氨基酸序列，本申请LCDR2可以包含SEQ ID NO：26(AAS)所示的氨基酸序列，且本申请LCDR3可以包含SEQ ID NO：27所示的氨基酸序列；本申请CDR可以是根据IMGT命名法定义的；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链可变区VH，且本申请VH可包含SEQ ID NO：28和29中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与一种15E8相同的VH，且本申请VH可包含SEQ ID NO：28所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与另一种15E8相同的VH，且本申请VH可包含SEQ ID NO：29所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含轻链可变区VL，且本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的VL，且本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链可变区VH和轻链可变区VL，且本申请VH可包含SEQ ID NO：28和29中任一项所示的氨基酸序列，本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与一种15E8相同的VH和VL，且本申请VH可包含SEQ ID NO：28所示的氨基酸序列，本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与另一种15E8相同的VH和VL，且本申请VH可包含SEQ ID NO：29所示的氨基酸序列，本申请VL可包含SEQ ID NO：30所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链，且本申请重链可包含SEQ ID NO：31和32中任一项所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与一种15E8相同的重链，且本申请重链可包含SEQ ID NO：31所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与另一种15E8相同的重链，且本申请重链可包含SEQ ID NO：32所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含轻链，且本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与15E8相同的轻链，且本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，本申请的抗体或其抗原结合蛋白可以包含重链和轻链，且本申请重链可包含SEQ ID NO：31和32中任一项所示的氨基酸序列，本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与一种15E8相同的重链和轻链，且本申请重链可包含SEQ ID NO：31所示的氨基酸序列，本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
例如，本申请的抗体或其抗原结合蛋白可以包含与另一种15E8相同的重链和轻链，且本申请重链可包含SEQ ID NO：32所示的氨基酸序列，本申请轻链可包含SEQ ID NO：33所示的氨基酸序列；例如，本申请的抗原结合蛋白可以具有CD28结合能力。
在一种实施方式中，使本申请的TIL与本申请的一种或多种T细胞激活剂接触可以包含选自以下组的一种或多种方式：(1)将本申请的T细胞激活剂添加至本申请的TIL的细胞培养基中；(2)将表达本申请的T细胞激活剂的工程化细胞添加至本申请的TIL的细胞培养基 中；(3)将包含本申请的T细胞激活剂的固相介质添加至本申请的TIL的细胞培养基中。在一种实施方式中，使本申请的TIL与本申请的一种或多种T细胞激活剂接触可以包含将包含本申请的T细胞激活剂的固相介质添加至本申请的TIL的细胞培养基中。在一种实施方式中，使本申请的TIL与本申请的一种或多种T细胞激活剂接触可以包含将包含本申请的CD28抗体与CD3抗体的固相介质添加至本申请的TIL的细胞培养基中。
在一种实施方式中，所述T细胞激活剂在本申请TIL的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本申请的CD28抗体在本申请TIL的细胞培养基中的初始浓度可以为至少约30ng/mL；例如，本申请的CD3抗体在本申请TIL的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本申请的CD28抗体初始浓度的选择可以与本申请的CD3抗体初始浓度的选择相互独立；例如，本申请的CD28抗体与本申请的CD3抗体在本申请TIL的细胞培养基中的初始浓度可以任意组合。例如，本申请的CD28抗体在本申请TIL的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL。例如，本申请的CD3抗体在本申请TIL的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL。例如，本申请的CD28抗体在本申请TIL的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL，且本申请的CD3抗体在本申请TIL的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL，本申请的CD28抗体初始浓度的选择可以与本申请的CD3抗体初始浓度的选择相互独立。在一种实施方式中，本申请的固相介质的直径可以为约500纳米至约10微米。在一种实施方式中，本申请的固相介质的直径可以通过透射电子显微镜测量。在一种实施方式中，本申请的固相介质的直径可以为约1纳米至约500纳米。在一种实施方式中，本申请的固相介质的直径可以为约100纳米至约500纳米。在一种实施方式中，本申请的固相介质的直径可以为约200纳米至约500纳米。在一种实施方式中，本申请的固相介质的直径可以通过透射电子显微镜测量。
在一种实施方式中，本申请的固相介质可以包含聚合物。在一种实施方式中，本申请的固相介质可以包含葡聚糖。
在一种实施方式中，每mg本申请的固相介质包含至少约25μg的本申请的T细胞激活剂。
在一种实施方式中，以约1:100-约1:2000的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂的固相介质添加至本申请TIL的细胞培养基中。在一种实施方式中，以约2:1-约1:2的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂的固相介质添加至本申请TIL的细胞培养基中。
例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约2:1-约1:2的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂的固相介质添加至本申请TIL的细胞培养基中。例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约2:1-约1:2、以约2:1-约1:1、或以约1:1-约1:2的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂，例如CD3激动剂和/或CD28激动剂的固相介质添加至本申请TIL的细胞培养基中。
例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000的本申请固相介质与本申请TIL的比例，将包含本申请T细胞激活剂的固相介质添加至本申请TIL的细胞培养基中。例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000、以约1:200-约1:2000、以约1:300-约1:2000、以约1:400-约1:2000、以约1:500-约1:2000、以约1:600-约1:2000、以约1:700-约1:2000、以约1:800-约1:2000、以约1:900-约1:2000、以约1:1000-约1:2000、以约1:1200-约1:2000以约1:1400-约1:2000、以约1:1600-约1:2000、或以约1:1800-约1:2000的本申请固相介质与本申请TIL的比例，例如可以将包含本申请CD28激动剂和CD3激动剂的固相介质添加至本申请TIL的细胞培养基中。
在一种实施方式中，本申请的方法还可以包含：使源自肿瘤组织且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的本申请体外扩增中，使本申请TIL与一种或多种T细胞生长因子接触。
在一种实施方式中，在单个阶段的本申请体外扩增中，可以使本申请的TIL与本申请的T细胞激活剂接触且与本申请的一种或多种T细胞生长因子接触。例如，在本申请第一阶段体外扩增中，可以使本申请的TIL与本申请的T细胞激活剂接触且与本申请的一种或多种T细胞生长因子接触。例如，在本申请第二阶段体外扩增中，可以使本申请的TIL与本申请的T细胞激活剂接触且与本申请的一种或多种T细胞生长因子接触。例如，在本申请第三阶段体外扩增中，可以使本申请的TIL与本申请的T细胞激活剂接触且与本申请的一种或多种T细胞生长因子接触。
在一种实施方式中，在单个阶段的本申请体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以先使所述TIL的至少一种目标基因的表达降低和/或活性减弱， 例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使本申请的TIL与本申请的一种或多种T细胞生长因子接触。在一种实施方式中，在单个阶段的本申请的体外扩增中，可以使本申请的TIL先与本申请的一种或多种T细胞生长因子接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
例如，在本申请第一阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。例如，在本申请第二阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。例如，在本申请第三阶段体外扩增中，可以基本上同时使所述TIL的至少一种目标基因的表达降低和/或活性减弱以及使TIL与T细胞生长因子接触。
在一种实施方式中，本申请的T细胞生长因子可以选自以下组的一种或多种：IL-2、IL-7、IL-12、IL-15、IL-21、γ干扰素、以及它们的功能活性片段。在一种实施方式中，本申请的T细胞生长因子可以包含IL-2和/或其功能活性片段。例如，IL-2的功能活性片段可以包含本领域已知的可以与T细胞的IL-2受体结合的IL-2的片段。
在一种实施方式中，本申请的TIL与本申请一种或多种T细胞生长因子接触可以包含将本申请T细胞生长因子添加至本申请TIL的细胞培养基中。在一种实施方式中，本申请的T细胞生长因子在本申请TIL的细胞培养基中的初始浓度可以为至少约300IU/mL。在一种实施方式中，本申请IL-2在本申请TIL的细胞培养基中的初始浓度可以为至少约350IU/mL、至少约400IU/mL、至少约500IU/mL、至少约600IU/mL、至少约700IU/mL、至少约800IU/mL、至少约900IU/mL、至少约1000IU/mL、至少约1100IU/mL、至少约1200IU/mL、至少约1300IU/mL、至少约1400IU/mL、至少约1500IU/mL、至少约2000IU/mL、至少约2500IU/mL、至少约2600IU/mL、至少约2700IU/mL、至少约2800IU/mL、至少约2900IU/mL、至少约3000IU/mL、至少约3100IU/mL、至少约3200IU/mL、至少约3300IU/mL、至少约3400IU/mL、至少约3500IU/mL、至少约4000IU/mL、至少约4500IU/mL、至少约5000IU/mL、至少约5500IU/mL、至少约6000IU/mL、至少约6500IU/mL、至少约7000IU/mL、至少约7500IU/mL、至少约8000IU/mL、至少约8500IU/mL、或至少约9000IU/mL。
在一种实施方式中，本申请的TIL可以为源自本申请的肿瘤组织的碎片的TIL。在一种实施方式中，可以通过将肿瘤组织处理成肿瘤碎片获得本申请的TIL。在一种实施方式中，本申请的肿瘤碎片的体积约为1-27立方毫米。在一种实施方式中，本申请的肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方毫米、约6立方 毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约15立方毫米、约17立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或约27立方毫米。例如，所述TIL可以选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由第一TIL群体外扩增获得的TIL群，所述第一TIL群为源自肿瘤组织且未经体外扩增的TIL群；(B)使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
例如，可以先将在一定的时间和/或一定位置的源自肿瘤组织且未经体外扩增的TIL群与T细胞生长因子接触，得到体外TIL群，一方面可以继续培养所述体外TIL群，进行步骤(B)，另一方面可以先冷冻保存所述体外TIL群，在有需要的时候复苏所述体外TIL群，进行步骤(B)。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)使所述TIL的TNFAIP3基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞 共培养，其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中的术语中，本申请的第一阶段体外扩增可以与以上方面的方法中的步骤(A)任意替换使用。在一种实施方式中的术语中，本申请的第二阶段体外扩增可以与以上方面的方法中的步骤(B)任意替换使用。在一种实施方式中的术语中，本申请的经第一阶段体外扩增的TIL可以与经以上方面的方法中步骤(A)得到的第二TIL群任意替换使用。在一种实施方式中的术语中，本申请的经第二阶段体外扩增的TIL可以与经以上方面的方法中步骤(B)得到的第三TIL群任意替换使用。在一种实施方式中的术语中，如有需要，本申请的第三阶段体外扩增可以与以上方面的方法中任意增加的步骤(C)任意替换使用。在一种实施方式中的术语中，如有需要，本申请的经第三阶段体外扩增的TIL可以与经以上方面的方法中任意增加的步骤(C)得到的第四TIL群任意替换使用。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL的TNFAIP3基因的表达降低和/或活性减弱，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与多种T细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种T细胞生长因子接触、与多种T细胞激活剂接触、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，可以以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触、与多种T细胞激活剂接触、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，可以以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为至少约300IU/mL；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为至少约300IU/mL、与CD3抗体接触，CD3抗体在所述TIL的细胞培养基中的初始浓度至少约30ng/mL、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且在至少2小时后使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，可以以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A) 可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触、与包含CD3抗体以及CD28抗体的纳米基质接触、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触、与包含CD3抗体以及CD28抗体的纳米基质接触，所述纳米基质的直径可以为约1纳米至约500纳米，每mg所述纳米基质可以分别包含CD3抗体以及CD28抗体各为约25μg、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触、与包含CD3抗体以及CD28抗体的纳米基质接触，所述纳米基质的直径可以为约1纳米至约500纳米，每mg所述纳米基质可以分别包含CD3抗体以及CD28抗体各为约25μg，可以以约1:100-约1:2000的所述纳米基质与所述TIL的比例添加至所述TIL的细胞培养基中、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与IL-2接触，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为至少约300IU/mL；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与IL-2接触，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为至少约300IU/mL、与包含CD3抗体以及CD28抗体的纳米基质接触，所述纳米基质的直径可以为约1纳米至约500纳米，每mg所述纳米基质可以分别包含CD3抗体以及CD28抗体各为约25μg，可以以约1:100-约1:2000的所述纳米基质与所述TIL的比例添加至所述TIL的细胞培养基中、使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，且使所述TIL与饲养细胞共培养，所述饲养细胞可以包含外周单个核细胞，可以以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液，内约2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入T细胞无血清培养基和浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2培养约3-14天。可以将收获的TIL细胞冻存后再复苏，也可以直接收集培养基中细胞，转移入透气培养袋、或Grex、或Xuri设备，T细胞无血清培养基可以添加本申请的CD28抗体、CD3抗体以及CD28抗体、包含CD3抗体以及CD28抗体的磁珠(例如Dynabeads)和/或包含CD3抗体以及CD28抗体的纳米基质(例如transACT)、浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2，以及用携带包含SEQ ID NO：48至61中任一项所示的gRNA与Cas蛋白形成的核糖核蛋白复合物(RNP)进行转导使所述TIL中表达所述TNFAIP3基因的细胞比例为约95％或以下，活化本申请的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于50％，大于80％的T细胞可以为记忆效应T细胞和效应T细胞。经刺激后可以分泌IFN-γ，和/或可以具有活化T细胞比例上调的特征。
一方面，本申请提供一种肿瘤浸润淋巴细胞(TIL)，本申请的TIL可以根据本申请的培养方法培养得到。在一种实施方式中，本申请提供的TIL可以包含一种或一个批次的本申请的培养方法培养得到TIL。在一种实施方式中，本申请提供的TIL可以包含多种或多个批次的本申请的培养方法培养得到并以任意比例组合的TIL。
在一些实施方式中，可以将使用本申请方法扩增的TIL作为药物组合物施用于患者。在一些实施方式中，药物组合物可以是TIL在无菌缓冲液中的悬液。使用本申请的PBMC扩增的TIL可以通过本领域已知的任何合适途径施用。在一些实施方式中，T细胞可以以单次动脉内或静脉内输注施用，输注可以持续约30至60分钟。其他合适的施用途径可以包括腹膜内、鞘内和淋巴管内施用。
在一些实施方式中，可以施用任何合适剂量的TIL。在一些实施方式中，例如当肿瘤是黑色素瘤时，可以施用约2.3×10 9至约13.7×10 10个TIL。在一些实施方式中，可以施用约1×10 9至约12×10 10个TIL。在一些实施方式中，可以施用约1.2×10 10至约4.3×10 10个TIL。在一些实施方式中，可以施用约3×10 10至约12×10 10个TIL。在一些实施方式中，可以施用约4×10 10 至约10×10 10个TIL。在一些实施方式中，可以施用约5×10 10至约8×10 10个TIL。在一些实施方式中，可以施用约6×10 10至约8×10 10个TIL。在一些实施方式中，可以施用约7×10 10至约8×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约2.3×10 9至约13.7×10 10。在一些实施方式中，治疗有效剂量可以为约1×10 9至约12×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约1.2×10 10至约4.3×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约3×10 10至约12×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约4×10 10至约10×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约5×10 10至约8×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约6×10 10至约8×10 10个TIL。在一些实施方式中，治疗有效剂量可以为约7×10 10至约8×10 10个TIL。
在一些实施方式中，本申请的组合物中提供的TIL的数量可以为约1×10 6、约2×10 6、约3×10 6、约4×10 6、约5×10 6、约6×10 6、约7×10 6、约8×10 6、约9×10 6、约1×10 7、约2×10 7、约3×10 7、约4×10 7、约5×10 7、约6×10 7、约7×10 7、约8×10 7、约9×10 7、约1×10 8、约2×10 8、约3×10 8、约4×10 8、约5×10 8、约6×10 8、约7×10 8、约8×10 8、约9×10 8、约1×10 9、约2×10 9、约3×10 9、约4×10 9、约5×10 9、约6×10 9、约7×10 9、约8×10 9、约9×10 9、约1×10 10、约2×10 10、约3×10 10、约4×10 10、约5×10 10、约6×10 10、约7×10 10、约8×10 10、约9×10 10、约1×10 11、约2×10 11、约3×10 11、约4×10 11、约5×10 11、约6×10 11、约7×10 11、约8×10 11、约9×10 11、约1×10 12、约2×10 12、约3×10 12、约4×10 12、约5×10 12、约6×10 12、约7×10 12、约8×10 12、约9×10 12、约1×10 13、约2×10 13、约3×10 13、约4×10 13、约5×10 13、约6×10 13、约7×10 13、约8×10 13，或约9×10 13。在一些实施方式中，本申请的组合物中提供的TIL数量的范围可以为约1×10 6至5×10 6、约5×10 6至1×10 7、约1×10 7至5×10 7、约5×10 7至1×10 8、约1×10 8至5×10 8、约5×10 8至1×10 9、约1×10 9至5×10 9、约5×10 9至1×10 10、约1×10 10至5×10 10、约5×10 10至1×10 11、约5×10 11至1×10 12、约1×10 12至5×10 12，或约5×10 12至1×10 13。
在一些实施方式中，本申请的组合物中提供的TIL的浓度可以小于组合物的例如约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约0.0002％，或约0.0001％w/w、w/v 或者v/v。
在一些实施方式中，本申请的组合物中提供的TIL的浓度可以大于组合物的约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19.75％、约19.50％、约19.25％、约19％、约18.75％、约18.50％、约18.25％、约18％、约17.75％、约17.50％、约17.25％、约17％、约16.75％、约16.50％、约16.25％、约16％、约15.75％、约15.50％、约15.25％、约15％、约14.75％、约14.50％、约14.25％、约14％、约13.75％、约13.50％、约13.25％、约13％、约12.75％、约12.50％、约12.25％、约12％、约11.75％、约11.50％、约11.25％、约11％、约10.75％、约10.50％、约10.25％、约10％、约9.75％、约9.50％、约9.25％、约9％、约8.75％、约8.50％、约8.25％、约8％、约7.75％、约7.50％、约7.25％、约7％、约6.75％、约6.50％、约6.25％、约6％、约5.75％、约5.50％、约5.25％、约5％、约4.75％、约4.50％、约4.25％、约4％、约3.75％、约3.50％、约3.25％、约3％、约2.75％、约2.50％、约2.25％、约2％、约1.75％、约1.50％、约125％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约或0.0002％，或者约0.0001％w/w、w/v或v/v。
在一些实施方式中，本申请的组合物中提供的TIL的浓度范围可以为组合物的约0.0001％至约50％、约0.001％至约40％、约0.01％至约30％、约0.02％至约29％、约0.03％至约28％、约0.04％至约27％、约0.05％至约26％、约0.06％至约25％、约0.07％至约24％、约0.08％至约23％、约0.09％至约22％、约0.1％至约21％、约0.2％至约20％、约0.3％至约19％、约0.4％至约18％、约0.5％至约17％、约0.6％至约16％、约0.7％至约15％、约0.8％至约14％、约0.9％至约12％，或约1％至约10％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的TIL的浓度范围可以为组合物的约0.001％至约10％、约0.01％至约5％、约0.02％至约4.5％、约0.03％至约4％、约0.04％至约3.5％、约0.05％至约3％、约0.06％至约2.5％、约0.07％至约2％、约0.08％至约1.5％、约0.09％至约1％、或约0.1％至约0.9％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的TIL的量可以等于或小于约10g、约9.5g、约9.0g、约8.5g、约8.0g、约7.5g、约7.0g、约6.5g、约6.0g、约5.5g、约5.0g、约4.5g、约4.0g、约3.5g、约3.0g、约2.5g、约2.0g、约1.5g、约1.0g、约0.95g、约0.9g、约0.85g、 约0.8g、约0.75g、约0.7g、约0.65g、约0.6g、约0.55g、约0.5g、约0.45g、约0.4g、约0.35g、约0.3g、约0.25g、约0.2g、约0.15g、约0.1g、约0.09g、约0.08g、约0.07g、约0.06g、约0.05g、约0.04g、约0.03g、约0.02g、约0.01g、约0.009g、约0.008g、约0.007g、约0.006g、约0.005g、约0.004g、约0.003g、约0.002g、约0.001g、约0.0009g、约0.0008g、约0.0007g、约0.0006g、约0.0005g、约0.0004g、约0.0003g、约0.0002g，或者约0.0001g。
在一些实施方式中，本申请的组合物中提供的TIL的量可以大于约0.0001g、约0.0002g、约0.0003g、约0.0004g、约0.0005g、约0.0006g、约0.0007g、约0.0008g、约0.0009g、约0.001g、约0.0015g、约0.002g、约0.0025g、约0.003g、约0.0035g、约0.004g、约0.0045g、约0.005g、约0.0055g、约0.006g、约0.0065g、约0.007g、约0.0075g、约0.008g、约0.0085g、约0.009g、约0.0095g、约0.01g、约0.015g、约0.02g、约0.025g、约0.03g、约0.035g、约0.04g、约0.045g、约0.05g、约0.055g、约0.06g、约0.065g、约0.07g、约0.075g、约0.08g、约0.085g、约0.09g、约0.095g、约0.1g、约0.15g、约0.2g、约0.25g、约0.3g、约0.35g、约0.4g、约0.45g、约0.5g、约0.55g、约0.6g、约0.65g、约0.7g、约0.75g、约0.8g、约0.85g、约0.9g、约0.95g、约1g、约1.5g、约2g、约2.5g、约3g、约3.5g、约4g、约4.5g、约5g、约5.5g、约6g、约6.5g、约7g、约7.5g、约8g、约8.5g、约9g、约9.5g，或者约10g。
在一些实施方式中，TIL可以单剂量施用。此种施用可以通过注射，例如可以静脉内注射。在一些实施方式中，TIL可以多剂量施用。剂量可以是每年一次、两次、三次、四次、五次、六次或超过六次。剂量可以是每月一次、每两周一次、每周一次或每2天一次。在一些实施方式中，TIL的施用可以连续施用。
一方面，本申请提供一种药物组合物。在一些实施方式中，其可以包含本申请的TIL和/或本申请的组合物，与药学上可接受的载体。
一方面，本申请提供一种试剂盒，本申请的试剂盒可以包含本申请培养肿瘤浸润淋巴细胞(TIL)方法的T细胞激活剂、T细胞生长因子和/或饲养细胞与记载本申请培养肿瘤浸润淋巴细胞(TIL)方法的步骤的说明书。一方面，本申请提供一种试剂盒，本申请试剂盒可以包含本申请的TIL和/或本申请的药物组合物。
一方面，本申请提供一种影响肿瘤细胞生长的方法，可以包括向受试者施用本申请的TIL和/或本申请的药物组合物。在一些实施方式中，影响肿瘤生长可以包含肿瘤的体积减少到施用前的例如约99％、约95％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约 1％、约0.5％、约0.4％、约0.3％、约0.2％或约0.1％。
一方面，本申请提供本申请的TIL和/或本申请的药物组合物在制备药物中的应用，本申请的药物可以用于预防和/或治疗肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
一方面，本申请提供一种预防和/或治疗肿瘤的方法，可以包括向受试者施用本申请的TIL和/或本申请的药物组合物。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
一方面，本申请提供一种本申请的TIL和/或本申请的药物组合物，其可以用于预防和/或治疗肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的产品、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1
1.1饲养细胞接收及制备
1.1.1单采血接收
记录单采血信息，批号及体积，并复温至室温。
1.1.2 PBMC(外周血单个核细胞)手动分离及冻存
使用75％酒精消毒血袋，转移至生物安全柜内。使用无菌剪刀剪开血袋后，将单采血转移至50mL离心管内，使用20mL注射器注入20mL PBS或生理盐水清洗血袋，将洗涤液一并转入50mL离心管内。每个50mL离心管内液体体积可以不超过30mL。将单采血3000g离心10分钟。离心过程中准备6-8支50mL离心管，加入已复温的淋巴细胞分离液(天津灏洋Ficoll)，20mL/支。离心结束后，弃掉上层血浆，使用PBS或生理盐水稀释细胞沉淀，将稀释后的血细胞混合液缓慢滴加上淋巴细胞分离液上层，可以不破坏界面，每管约加25mL样品，可以不超过28mL。
离心使用水平转子，500-600g离心15-30分钟，温度18-22℃，离心结束后得到的白膜 层将处于生理盐水及淋巴细胞分离液Ficoll的分界面处。吸弃上层血浆及生理盐水，用移液管吸取中间白膜层至另一干净的50mL离心管内。使用PBS或生理盐水稀释收集到的白膜层，600g离心10分钟，室温。离心结束后弃上清，PBS或生理盐水清洗细胞一次，500g离心5分钟，室温。
如红细胞较多，离心结束后可以进行裂红，按照细胞沉淀体积与红细胞裂解液1:2至1:3加入红细胞裂解液，混匀，室温裂解10分钟中，中间轻柔混匀离心管2-3次，保证裂解效果，裂解完成后加入PBS或生理盐水清洗细胞。裂红后清洗细胞两次，400g离心6分钟，最后一次离心前取样计数。
弃上清，基础培养基重悬细胞，调整细胞密度约2-3×10 7个细胞/mL，液面高度可以不超过1厘米，每T225培养瓶中体积可以低于200mL；平铺状态下，X射线辐照50Gy。离心弃上清，根据计数结果冻存细胞，约1-2×10 8个细胞/mL，1-2mL/支；将细胞放入程序降温盒内转移至﹣80℃冰箱内冻存。
1.1.3 PBMC自动分离及冻存
将血袋的管路与cpro分离套件(Cytiva)输入端无菌接管。若血量大于120mL，进行预浓缩步骤，可以将血液体积浓缩至120mL以内。可以使用neatcell程序进行PBMC分离及洗涤，洗涤液为生理盐水，中间体积20mL；重悬液为基础培养基，添加80mL/批。分离后每供者PBMC为一袋100mL，在平铺状态下，液面高度可以不超过1厘米，X射线辐照50Gy。辐照后取样计数，使用culture wash程序收集细胞并洗涤三次，洗涤液为生理盐水；设置中间体积及终体积，使得每1×10 9个细胞不少于2mL；加入等量至2倍冻存液混匀。使用1倍冻存液调整细胞密度约为1×10 7个细胞/mL至2×10 8个细胞/mL，分装20mL/袋，程序降温仪内冻存，液氮保存。
1.2肿瘤组织接收及处理
1.2.1组织接收
接收供者的肿瘤组织及血样，核对样品信息并记录，打印相应样品标签。
1.2.2组织处理及培养
使用75％酒精消毒样品管及采血管，转移至生物安全柜内。根据上述PBMC手动分离及冻存操作程序分离血样中PBMC细胞并进行冻存。取一种具有透气表面的培养瓶或培养袋，例如培养袋(Origen)，加入300mL已复温的完全培养基，完全培养基可以任意地选用X-vivo15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，并添加浓度为300-9000IU/mL(例如可以是 1000-9000IU/mL，例如可以是6000IU/mL)的IL-2。取数个10厘米培养皿，加入适量培养基，使用无菌眼科镊从样品管中取出肿瘤组织于10厘米培养皿中，培养基量以刚没过肿瘤组织为准，观察组织形态并记录。洗涤组织并更换培养皿。使用眼科剪及眼科镊将进行初步剪切，去除脂肪组织及坏死组织，每块组织块继续剪碎至约27立方毫米大小。取非悬浮肿瘤组织块，使用20mL注射器去除内部活塞后，与培养袋连接，使用移液管将约1g组织块通过注射器转入培养袋内。将培养袋放入二氧化碳培养箱内进行培养。清理剪刀及镊子，并用75％酒精进行初步消毒后，超声清洗后进行灭菌，得到第一TIL群。
1.3步骤(A)体外扩增及收获
1.3.1步骤(A)体外扩增
根据细胞生长状态，每3-7天补液或半量换液，保证细胞营养。使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，并添加浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2(双鹭)。步骤(A)的3-14天，例如可以第13或14天时取样计数，若细胞数目处于5×10 5至5×10 8之间时进入步骤(A)的收获步骤。
1.3.2步骤(A)的收获
收集步骤(A)体外扩增结束细胞，离心，弃去培养基，使用PBS或生理盐水洗涤细胞一次，获得经步骤(A)体外扩增的TIL(第二TIL群)，并取样计数留取约5×10 5至2×10 8个细胞进入后续体外扩增步骤；取约5×10 5个细胞可以进行质量控制检测；其余细胞加入冻存液冻存，作为冻存的preREP TIL体外细胞。
1.4步骤(B)TIL活化
继续培养经步骤(A)体外扩增的TIL(第二TIL群)，或者对冻存的preREP TIL体外细胞进行细胞复苏，进行步骤(B)的TIL活化。
使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，调整细胞密度为5×10 5至2×10 6个细胞/mL，于悬浮24孔培养板内，1mL/孔，添加浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL或6000IU/mL)的IL-2。各TIL细胞群的培养基中同时可以添加T细胞激活剂，例如添加CD3激动剂和/或CD28激动剂，例如，约30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)、约30ng/mL的CD28抗体(Merck，15E8)、以约1:2-2:1的磁珠与TIL的比例加入磁珠(直径约1 至10μm Dynabeads，Thermo Fisher)和/或以约1:100-1:2000的transACT(直径约100至500nm，Miltenyi)与TIL的比例加入transACT。培养约0-4天，获得第三TIL群。
1.5步骤(C)TIL细胞基因编辑
将本申请的gRNA(序列如SEQ ID NO：48至61任一项所示)，解冻并加入无核酸酶水，配至浓度为约100μM。将约2μL的gRNA(50μM)在95℃孵育2分钟退火后加入到P3缓冲液，并加入0.3-1μL的Cas9(克睿，10mg/mL)，25℃孵育10分钟以形成核糖核蛋白复合物(RNP)。在P3缓冲液(Lonza)中，通过Lonza电转仪将上述RNP与第三TIL群的约1×10 6个细胞进行电转。例如，电转程序可以是human T cell stim(EO115)。电转的基因编辑后培养约0-4天，获得第四TIL群。
1.6步骤(D)TIL细胞基因编辑后培养
在第四TIL细胞群中加入饲养细胞进行培养。TIL与饲养细胞接触的时间需要在步骤(B)的TIL与IL-2以及T细胞激活剂接触后的若干时间T n以后(各个试验组的T n可以取0小时到12天，例如24小时或48小时)。首先复苏1-5名供者混合的饲养细胞；将活化的TIL细胞、饲养细胞按照TIL细胞：饲养细胞约为1:200的比例混合，转入G-Rex100培养瓶或者透气袋内，补充完全培养基，每1-3天取样计数，并根据细胞状态补液或半量换液直至细胞总数大于1×10 9或步骤(D)体外扩增培养约5天至约14天，终止步骤(D)体外扩增的培养。
1.7肿瘤浸润淋巴细胞的收获
取步骤(D)扩增的细胞，离心后弃去培养基上清，并使用PBS或生理盐水或复方电解质溶液清洗三次，获得经步骤(D)扩增的TIL(第五TIL群)，第三次清洗时取样计数，根据计数结果，最后一次离心后弃上清，取3×10 6细胞送质量控制检测；其余全部细胞加入冻存液，调整细胞密度1-3×10 8个细胞/mL冻存。
实施例2 饲养细胞不同添加时间培养的TIL增殖能力对比
实施例1的加入IL-2与不同形式的T细胞激活剂后的若干时间T n以后(T n可以取0小时到14天)，将饲养细胞与肿瘤浸润淋巴细胞共培养。本实施例中T n选取0小时、6小时、12小时、24小时、48小时、72小时、5天、7天、和9天获得饲养细胞不同添加时间培养的TIL，并进行细胞计数的对比试验。
图1显示的是，饲养细胞不同添加时间培养的TIL的增殖能力分析结果。饲养细胞不同添加时间培养TIL的各组图中纵坐标的数值表示，体外扩增结束后相比于体外扩增开始前，TIL细胞数量扩增至的扩增倍数。4名供者来源的TIL增殖结果显示，加入OKT3和IL-2后的0小时后(即同时)添加饲养细胞培养的TIL，增殖能力弱于加入OKT3和IL-2后的24小 时或48小时后加饲养细胞培养的TIL。
实施例3 饲养细胞不同添加时间培养的TIL流式检测对比
在实施例1的加入IL-2与不同形式的T细胞激活剂后的若干时间T n以后(T n可以取0小时到14天)，将饲养细胞与肿瘤浸润淋巴细胞共培养。本实施例中T n选取0小时、6小时、12小时、24小时、48小时、72小时、5天、7天、和9天获得饲养细胞不同添加时间培养的TIL，并进行流式检测的对比试验。
TIL流式检测试验材料的来源
转录因子缓冲组(Transcription Factor Buffer Set)，厂家BD，货号562574；V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。。使用100-500μL PBS重悬细胞，进行流式上机检测。
饲养细胞不同添加时间培养的TIL的流式结果分析如图38到图44所示。
图2和图3显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA -CCR7 +中心记忆T细胞(Tcm)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高中心记忆T细胞的比例。
图4显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的 TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更少的调节性T细胞的比例。
图5和图6显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如PD1 +、LAG3 +和/或CD28 +细胞比例更高。
图7显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103 +CD39 +肿瘤特异性T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的肿瘤特异性T细胞的比例。
图8显示的是，加入OKT3和IL-2后0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1 +干细胞样T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的干细胞样T细胞的比例。
实施例4 加入CD28激动剂刺激的TIL增殖能力检测
对于实施例1中各个试验组培养获得的TIL群进行细胞计数。
图9显示的是，添加不同形式的CD28激动剂的试验组以及对照组的增殖能力分析结果。图中纵坐标的数值表示，各个试验组体外扩增获得的TIL群相比于体外扩增开始前的TIL群，TIL细胞数量扩增至的扩增倍数。结果显示，四步骤划分法中的步骤(B)的体外扩增添加CD28抗体，获得的TIL增殖能力强于对照组(不添加CD28抗体)培养的TIL。
实施例5 加入CD28激动剂刺激的TIL流式检测
对于实施例1中各个试验组体外扩增培养获得的TIL群进行流式检测。
添加不同形式的CD28激动剂的TIL的流式结果分析如图46到图50所示。
图10显示的是，混合抗体组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如，更高的活化T细胞(CD28 +或41BB +)的比例，更低的调节性T细胞(Treg，例如CD4 +CD25 +Foxp3 +)比例，更高的干细胞样T细胞(TCF1 +)比例，和/或更高的中心记忆T细胞(Tcm，例如CD45RA -CCR7 +)的比例。
图11显示的是，混合抗体组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如，更高的肿瘤特异性T细胞(CD103 +CD39 +)比例，更高的活化T细胞(CD25 +)的比例，和/或更低的调节性T细胞(Treg，例如CD4 +CD25 +Foxp3 +)比例。
图12显示的是，磁珠组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如，更高的活化T细胞(CD28 +、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13显示的是，磁珠组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如，更高的干细胞样T细胞(TCF1 +)比例，更高的活化T细胞(41BB +)的比例，更高的中心记忆T细胞(Tcm，例如CD45RA -CCR7 +)的比例，更低的调节性T细胞(Treg，例如CD4 +CD25 +Foxp3 +)比例，和/或更高的肿瘤特异性T细胞(CD103 +CD39 +)比例。
图14显示的是，纳米基质组与对照组培养所得的TIL细胞的T细胞亚群比例。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的T细胞亚群比例。例如，更高的肿瘤特异性T细胞(CD103 +CD39 +)比例，更高的活化T细胞(CD25 +或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6 加入CD28激动剂刺激的TIL细胞杀伤能力检测
对于实施例1中各个试验组四步骤划分法中的步骤(B)体外扩增培养获得的TIL群进行细胞杀伤能力检测。
细胞准备
准备用于检测的各个试验组获得的TIL和用于共培养的靶细胞(例如Hela肿瘤细胞)。
检测步骤
用CFSE(5(6)-Carboxyfluorescein diacetate N-succinimidyl ester，Sigma，21888-25MG-F)标记肿瘤细胞：用PBS清洗肿瘤细胞，重悬肿瘤细胞于500μL的PBS中；将CFSE加入500μL的PBS中，与500μL的肿瘤细胞PBS重悬液混合，至CFSE的终浓度为0.5μmol/L。37℃ 孵育6分钟后，加含10％FBS的培养基清洗，600g离心5分钟，用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基重悬肿瘤细胞浓度为5×10 5个细胞/mL。对各个试验组的TIL细胞600g离心5分钟，按照效靶比(TIL细胞与肿瘤细胞的比例)3:1重悬TIL细胞(即重悬TIL细胞浓度为1.5×10 6个细胞/mL)。于U底96孔板(Corning)中加入肿瘤细胞和TIL细胞各100μL，每组设置三个复孔。同时设置一组只包含肿瘤细胞的对照组并按照实验不同分组加入不同试剂。将孔板200g离心1分钟，置于37℃孵育4小时至过夜。
孵育完成后，600g离心3分钟，弃上清，每孔加入20μL胰酶，37℃培养箱内孵育3-5分钟消化肿瘤细胞，消化完成后加入180μL含10％FBS的培养基终止消化。将Dapi(碧云天，C0060)用1:100稀释，然后每孔加入20μL稀释后的Dapi。进行流式上机检测。
杀伤率％＝Dapi +CFSE +细胞数/总CFSE +×100％。
图15显示的是，纳米基质组与对照组培养所得的TIL细胞的细胞杀伤能力。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的细胞杀伤能力。
实施例7 加入CD28激动剂刺激的TIL胞内因子表达检测
对于实施例1中各个试验组四步骤划分法中的步骤(B)体外扩增培养获得的TIL群进行胞内因子表达检测。
试验准备
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比1:500添加CD107a抗体(BD)。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，100μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，50μL/组染色，2-8℃避光孵育30分钟。染色结束后清洗细胞，使用PBS重悬，进行流式上机检测。
图16显示的是，混合抗体组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图17显示的是，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显 示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图18显示的是，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图19显示的是，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图20显示的是，磁珠组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的磁珠)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力。
图21显示的是，纳米基质组与对照组培养所得的TIL细胞的胞内因子表达检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力或更高的GZMB表达能力。
实施例8 加入CD28激动剂刺激的TIL细胞因子分泌检测
对于实施例1中各个试验组四步骤划分法中的步骤(B)体外扩增培养获得的TIL群进行细胞因子分泌检测。
图22显示的是，纳米基质组与对照组培养所得的TIL细胞的细胞因子分泌检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的细胞因子分泌能力。例如，更高的IL-2分泌能力、更高的TNF分泌能力、或更高的IFN-γ分泌能力。
取各个试验组获得的TIL与肿瘤细胞过夜孵育，孵育结束后取上清液参照本实施例检测步骤进行细胞因子分泌检测。
图23显示的是，纳米基质组与对照组培养所得的TIL细胞与肿瘤细胞共同孵育后的细胞因子分泌检测结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有更高的细胞因子分泌能力。例如，更高的IL-2分泌能力、更高的TNF分泌能力、或更高的IFN-γ分泌能力。
实施例9 加入CD28激动剂刺激的TIL基因敲除效率检测
取实施例1中各个试验组在四步骤划分法中的步骤(B)体外扩增培养48小时后的TIL细胞，进行基因敲除效率检测。
用Nuclease-free water(商业来源：上海右凡生物科技有限公司；RT121-02)配制sgRNA(序列如SEQ ID NO:1所示，GGAGAATGACGAGTGGACCC)，调节浓度至50μmol/L。取2μL gRNA加入PCR管中，Nuclease-free water作为阴性对照，在PCR仪中95℃孵育2分钟后，室温冷却10分钟。
按照体积比sgRNA:P3Buffer:Cas9核酸酶＝2:2:1，在含有sgRNA的PCR管中依次加入P3Buffer(商业来源：Lonza；V4XP-3032)及61.7μmol/L Cas9核酸酶(商业来源：苏州克睿基因生物科技有限公司；C01-2019-11-001)，将PCR管放入PCR仪，25℃孵育10分钟以形成RNP，放入4℃备用。
按照1mL/孔加入T细胞培养基，放置于CO 2培养箱中预热。取实施例1中各个试验组在四步骤划分法中的步骤(B)体外扩增培养48小时后的TIL细胞，混匀后进行计数，每个试验组样品取5×10 5个细胞加入P3Buffer(20μL)，混匀细胞；将细胞加入新的PCR管中，与5μL的RNP混合；将细胞与RNP的混合物加入电转条板中，在电转仪(Lonza)中进行电转(human T cell stimulated(E0115))。电转程序结束后，立即加入预热的180μL的T细胞培养基，将全部体积转移到24孔悬浮板中，放置于CO 2培养箱中进行培养。24小时后进行细胞计数，按照TIL：饲养细胞＝1:200比例加入饲养细胞(辐照后的PBMC细胞)，放置于CO 2培养箱中继续培养72小时。取培养结束后的各个试验组的TIL细胞进行细胞计数，每个试验组取2×10 5个细胞，500g离心3分钟，离心后吸弃上清。
配制流式检测混合抗体：取Fixable Viability Dye eFluor 780(商业来源：eBioscience；65-0865-18)在PBS中稀释10000倍；取100μL稀释后的Fixable Viability Dye eFluor 780的PBS溶液，分别加入1μL的TCR-αβ-APC(商业来源：eBioscience；17-9986-42)、1μL的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24显示的是，纳米基质组与对照组培养所得的TIL细胞的基因敲除效率结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的基因敲除效率能力。例如，提高的TCRβ基因敲除效率。
图25显示的是，纳米基质组与对照组培养所得的TIL细胞的基因敲除效率结果。结果显示，四步骤划分法中的步骤(B)体外扩增添加CD28抗体(例如添加包含CD3抗体和CD28抗体的transACT)，相比对照组(不加入CD28抗体)，所得的TIL具有改善的基因敲除效率能力。例如，提高的TCRβ基因敲除效率。
实施例10 REP阶段结束后添加CD28激动剂的TIL增殖能力检测
参考实施例1得到源自肿瘤组织且未经体外扩增的第一TIL群，将第一TIL群经过相同方式的四步骤划分法中的步骤(A)、步骤(B)、步骤(C)和步骤(D)得到第五TIL群。将第五TIL群随机分为3组，各个试验组的T细胞培养基加入IL-2的同时，其中空白组不添加任何T细胞激活剂，未添加CD28激动剂组添加CD3抗体约30ng/mL，添加CD28激动剂组添加CD3激动剂和CD28激动剂，例如以约1:100-1:2000的transACT与TIL的比例加入transACT。培养3天得到的TIL(终末刺激细胞群)，通过细胞活力检测方法使用CellTiter-Glo试剂盒(商业来源：Promega)的进行TIL细胞增殖能力的检测。
图26、图27和图28分别显示的是，对于不同供者来源的TIL，终末刺激阶段以不同方式进行体外扩增的试验组的增殖能力分析结果。图中纵坐标荧光值反映了各个试验组以不同方式进行终末刺激的TIL细胞的增殖能力。结果显示，终末刺激添加CD28激动剂，与终末刺激不添加CD28激动剂相比，具有类似的TIL增殖能力。
实施例11 TIL细胞的敲除效率检测
试剂和材料：DNA提取液(QuickExtract DNA extraction solution，Lucigen，QE09050)、无核酸酶水(RNase/DNase free water，天根)、EDTA(生工，0.5M)。
提取基因组DNA：在TIL细胞敲除后的约4天，取约1×10 5个至约2×10 5个细胞，并加入配置的核酸酶混合液(DNase I)，在37℃孵育5分钟。样品中加入2.5μL 0.5M的EDTA，并80℃孵育10分钟。离心后，加入50μL DNA提取液，短暂离心后运行以下程序：75℃- 10分钟；95℃-5分钟；4℃-维持。可以利用分光光度计(NanoDrop TM)检测DNA样品浓度。
测序：可以在PAM位点上下游约100至约200个核苷酸的区域设计PCR引物。按照以下设计PCR反应体系：
试剂 体积
2×PCR Premix Buffer 25μL
DNA模板 约100-500ng
正向引物(10μM) 1μL
反向引物(10μM) 1μL
双蒸水 补充到50μL
DMSO 1.5μL
并按照以下PCR程序扩增：
Figure PCTCN2022109577-appb-000001
将PCR产物进行Sanger测序分析。
分析Crispr Cas9敲除效率
利用Tracking of Indels by DEcomposition(Tide)方法根据Sager测序数据，分析Crispr Cas9敲除效率，具体方法可以参见(Brinkman et al,Nucl.Acids Res.(2014)或shinyapps.datacurators.nl/tide/)。通过输入本申请相应的sgRNA序列、敲除前对照序列、Crispr Cas9敲除后的测试序列，P-value阈值设置为0.001，进行敲除效率分析。
不同的TIL可以是来源于不同的肿瘤患者，供者A、供者B为皮肤恶性黑色素瘤患者，供者C为胰腺癌患者，供者D、供者E为非小细胞肺癌患者。
结果显示，SEQ ID NO：57(CTTGTGGCGCTGAAAACGAA)所示的sgRNA，即TP2，对于供者B的敲除效率为约28％，对于供者C的敲除效率为约47.7％；SEQ ID NO：58(TATGCCATGAGTGCTCAGAG)所示的sgRNA，即TP4，对于供者A的敲除效率为约59.9％， 对于供者B的敲除效率为约34％，对于供者C的敲除效率为约60.4％，对于供者D的敲除效率为约33.8％。本申请的各种基因编辑方式均可以实现一定比例的敲除效率。
实施例12 TIL细胞的扩增情况的检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板，并换用不含有IL-2的细胞培养基培养，即撤去IL-2进行培养。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图29、图30、图31和图32显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。结果表明，即使不含有IL-2的细胞培养基培养，本申请的基因编辑的TIL细胞仍然可以具有显著的扩增能力。
基因编辑后的第6天开始，用30ng/mL的CD3抗体(Miltenyi Biotech，OKT3)预处理96孔板，4℃过夜孵育；次日各组TIL细胞以相同的细胞总数重新铺板，并换用不含有IL-2的细胞培养基培养。3天后用CTG试剂盒(CellTiter-Glo Luminescent Cell Viability Assay，Promega)分析TIL细胞的扩增效率。
图33和图34显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的扩增后的荧光量。
本申请中，NT表示阴性对照组，未经过基因编辑的TIL；ST3表示无关敲除组，在不影响细胞功能的区域进行敲除的TIL；TP2表示通过SEQ ID NO：57所示的sgRNA进行敲除的TIL；TP4表示通过SEQ ID NO：58所示的sgRNA进行敲除的TIL。*表示p<0.05，**表示p<0.01，***表示p<0.001，****表示p<0.0001。
实施例13 TIL细胞杀伤能力检测
基因编辑后的第6天开始，将A375肿瘤细胞系铺于96孔板中。次日，将各组TIL细胞以效靶比(TIL细胞：肿瘤细胞，E：T)为1:1或1:3的比例与上述A375细胞共培养。
根据细胞凋亡检测试剂(Incucyte Caspase-3/7 Green Dye for Apoptosis，Sartorius)的说明书，按照0.2μL/孔加入细胞凋亡检测试剂，并按照25μL/孔加入培养基稀释Caspase 3/7 Green Dye。使用Incucyte记录仪(Sartorius)记录Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤能力，每3小时记录1次，总记录时长约5天。
图35显示的是，对于来源于供者B的TIL细胞，以效靶比1:1与肿瘤细胞共培养的杀伤能力检测结果。
图36和图37显示的是，对于来源于供者C的TIL细胞，以效靶比1:1和效靶比1:3与 肿瘤细胞共培养的杀伤能力检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的肿瘤细胞杀伤能力。
实施例14 TIL细胞的扩增效率以及活率的检测
IL-2是调节T细胞生长的重要因子，本实施例检测本申请基因编辑的TIL细胞是否可以不依赖于IL-2而依然可以存活和/或扩增。
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板，并换用不含有IL-2的细胞培养基培养，即撤去IL-2进行培养。每隔2至4天用细胞计数仪分析TIL细胞的扩增效率及活率。其中扩增效率以增殖倍数表示：将撤去IL-2当天(第0天)的细胞总数作为1，第n天的增殖倍数表示为第n天的细胞总数/第0天的细胞总数；活率表示存活细胞数量占总细胞数量的百分比。
图38和图39显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的增殖倍数。
图40和图41显示的是，对于来源于不同供者的TIL细胞，各组TIL细胞的活率。结果表明，即使撤去IL-2，本申请的基因编辑的TIL细胞仍然可以具有显著的扩增能力，以及可以保持较高的细胞活率。
实施例15 TIL流式检测
对于实施例1中各个试验组基因编辑后的第8天获得的TIL群进行流式细胞仪检测。
TIL流式检测试验材料的来源
V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板200μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板200μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
图42和图43显示的是，对于来源于供者C的TIL细胞，各组TIL细胞中TIM-3阳性和CD101阳性细胞的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭细胞的比例。
图44显示的是，对于来源于供者C的TIL细胞，各组TIL细胞中CD45RA阴性CCR7阳性记忆T细胞(Tcm)的比例。结果显示，基因编辑后的TIL细胞具有更高中心记忆T细 胞的比例。
实施例16 TIL细胞因子表达流式检测
对于实施例1中各个试验组基因编辑后的第7天或第8天获得的TIL群进行流式细胞仪检测细胞因子表达情况。
试验准备
CD3抗体组提前1天使用30ng/ml的CD3抗体(Miltenyi Biotech，OKT3)包被平底96孔板，4℃包被过夜；PMA组铺板当天在培养基中添加佛波醇-十四酸酯-乙酸酯(PMA，25ng/ml)和离子霉素(Ionomycin，1μg/ml)即可。
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比添加：Golgistop 0.7:1000，Golgiplug 1：1000，CD107a抗体1:500即2μL/mL。不添加白介素。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，200μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，96孔板50μL/孔，流式管100μL/管染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板200μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体(CD107a，GZMB，TNF-α和IFN-γ，BD/BioLegend)，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板200μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用100-500μL PBS重悬细胞，进行流式上机检测。图45、图46、图47和图48显示的是，在没有刺激的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图49、图50、图51和图52显示的是，在CD3抗体(Miltenyi Biotech，OKT3，CD3抗 体以30ng/mL预处理96孔板)刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
图53、图54、图55和图56显示的是，在佛波醇-十四酸酯-乙酸酯(PMA，25ng/ml)和离子霉素(Ionomycin，1μg/ml)刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
结果显示，基因编辑后的TIL细胞具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力、更高的TNF-α表达能力或更高的GZMB表达能力。
实施例17 TIL细胞的TCR克隆多样性检测
基因编辑后的第7天开始，各组TIL细胞以相同的细胞总数重新铺板，并换用不含有IL-2的细胞培养基培养，即撤去IL-2进行培养。
在基因编辑后的第8天和第18天对TIL中T细胞受体(TCR)克隆多样性进行分析。根据TCR谱系试剂盒(Beta Mark TCR Vβ Repertoire Kit，Beckman Coulter)的说明书，在A管至H管的8个管中分别加入4μL的Beta Mark TCR Vβ Repertoire Kit Tube A-H抗体，并在每个管中加入1μL的BV510标记的CD3抗体(BD)、1μL的PerCP-cy5.5标记的CD8抗体(BD)、1μL的PE-cy7标记的CD4抗体(BD)和0.01μL的eFluor 780标记的死活细胞染料(eBioscience)。将待测细胞分为8份，分别加入A管至H管，混合均匀，4℃孵育30分钟。染色结束，离心并弃去上清，使用PBS重悬洗涤1次，用于流式细胞仪检测。
按照TCR谱系试剂盒(Beta Mark TCR Vβ Repertoire Kit，Beckman Coulter)的说明书对TIL细胞的TCR多样性进行鉴定。表1显示的是，A管至H管的荧光与TCR Vβ克隆的对应关系。其中TCR Vβ克隆为其它，可以是指该比例下的T细胞没有TCR谱系试剂盒可以鉴定的TCR Vβ。表2至表5显示的是，TIL细胞基因编辑后的第8天和第18天的CD4 +T细胞和CD8 +T细胞的TCR Vβ克隆的多样性。
表1 A管至H管的荧光与TCR Vβ克隆的对应关系
Figure PCTCN2022109577-appb-000002
Figure PCTCN2022109577-appb-000003
表2 显示的是，TIL细胞基因编辑后的第8天的CD4 +T细胞TCR Vβ克隆的多样性。
Figure PCTCN2022109577-appb-000004
Figure PCTCN2022109577-appb-000005
表3 显示的是，TIL细胞基因编辑后的第18天的CD4 +T细胞TCR Vβ克隆的多样性。
Figure PCTCN2022109577-appb-000006
Figure PCTCN2022109577-appb-000007
表4 显示的是，TIL细胞基因编辑后的第8天的CD8 +T细胞TCR Vβ克隆的多样性。
Figure PCTCN2022109577-appb-000008
Figure PCTCN2022109577-appb-000009
表5 显示的是，TIL细胞基因编辑后的第18天的CD8 +T细胞TCR Vβ克隆的多样性。
Figure PCTCN2022109577-appb-000010
Figure PCTCN2022109577-appb-000011
图57和图58显示的是，TIL细胞基因编辑后的第8天和第18天的CD4 +T细胞和CD8 +T细胞TCR Vβ克隆的香农多样性指数。香农多样性指数反应了TCR Vβ克隆的多样性，结果表明，基因编辑的TIL细胞在第18天时，可以保持更高的β链亚型多样性，说明基因编辑的TIL细胞可以显著地长期保持TCR克隆的多样性，从而可以有助于实现对于肿瘤细胞更强的抗原识别和免疫应答能力。
实施例18
饲养细胞不同添加时间培养的TIL的结果统计
在实施例1的1.4的第二阶段扩增的TIL活化中，取第一阶段扩增的细胞量，调整细胞密度为5×10 5至2×10 6/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3 约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如3000或6000IU/mL的IL-2。加入上述OKT3和IL-2后的0小时、24小时、48小时以后，将饲养细胞加入肿瘤浸润淋巴细胞的培养环境中。其中，TIL与饲养细胞可以按照比率1:40-1:400加入，第二阶段扩增培养约9-14天后收集全部细胞，检测和统计培养所得TIL的结果。
增殖能力检测
对于上述饲养细胞不同添加时间培养获得的TIL进行细胞计数。
不同供者肿瘤来源的TIL作为各自不同的批次；将各个批次的加入OKT3和IL-2同时(0h组)加入饲养细胞的试验组的数据作为基准1，将同批次其它时间点试验组的数据进行标准化处理，统计各试验组在第二阶段扩增相对于0h组的相对增殖能力。
图59A显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。相比于加入OKT3和IL-2后的0小时后(即同时)添加饲养细胞培养的TIL，加入OKT3和IL-2后的24小时或48小时后加饲养细胞培养的TIL增殖能力显著增强。
流式检测TIL细胞组成
对于上述饲养细胞不同添加时间培养获得的TIL群进行流式检测。
不同供者肿瘤来源的TIL作为各自不同的批次；将各个批次的加入OKT3和IL-2同时(0h组)加入饲养细胞的试验组的数据作为基准1，将同批次其它时间点试验组的数据进行标准化处理，统计各试验组在第二阶段扩增相对于0h组的细胞组成比例。
流式检测的试验流程可以参照本申请实施例3的内容。
图59B显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA -CCR7 +中心记忆T细胞(Tcm)比例结果图。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高CD8 +中和/或CD4 +中的中心记忆T细胞的比例。
图59C显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1 +干细胞样T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高CD8 +中的干细胞样T细胞的比例。
图59D显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更少 的调节性T细胞的比例。
图59E显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(PD-1 +)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8 +中和/或CD4 +中的PD-1 +细胞比例更高。
图59F显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103 +CD39 +肿瘤特异性T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的CD8 +中和/或CD4 +中的肿瘤特异性T细胞的比例。
图59G显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD28 +)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8 +CD28 +细胞比例更高。
图59H显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(41BB +)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8 +中和/或CD4 +中的41BB +细胞比例更高。
图59I显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD25 +)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8 +中和/或CD4 +中的CD25 +细胞比例更高。
胞内因子表达检测
试验准备
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比1:500添加CD107a抗体(BD)。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×10 6个细胞/mL，加入96孔板内，100μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，50μL/组 染色，2-8℃避光孵育30分钟。染色结束后清洗细胞，使用PBS重悬，进行流式上机检测。
图59J显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的胞内因子表达检测结果。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的胞内因子表达能力。例如，更高的CD3 +中、CD8 +中和/或CD4 +中的CD107a表达能力。
细胞因子分泌检测
细胞因子分泌检测方法可以参照细胞因子检测试剂盒(BD)的说明书，将人Th1/Th2/Th17细胞因子标准品冻干粉(BD)使用2mL Assay Diluent稀释液(BD)复溶(标准品原液各细胞因子浓度均为5000pg/mL)并按顺序：1:2，1:4，1:8，1:16，1:32，1:64，1:128，1:256，1:512，1:1024梯度稀释，标记为“标准品管”。取1管仅含有Assay Diluent稀释液作为参照。按照2μL/Beads/孔加入每种Capture Beads(BD)，然后按照10μL/孔加入PE Detection Reagent检测试剂(BD)并混合配制为混合物(mix)，按照22μL/孔加入V底96孔板内，随后按照10μL/孔加入各标准品和试验组的上清并混合，室温下避光孵育3小时。
孵育结束，每孔加入200μL Wash Buffer(BD)，500g离心3分钟。离心结束，每孔加入100μL Wash Buffer(BD)重悬，进行流式分析。
图59K显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞因子分泌检测结果。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的细胞因子分泌能力。例如，更高的TNF-α分泌能力、或更高的IFN-γ分泌能力。
饲养细胞不同添加时间培养的TIL的结果统计
在实施例1的1.4的第二阶段扩增的TIL活化中，取第一阶段扩增的细胞量，调整细胞密度为5×10 5至2×10 6/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如3000或6000IU/mL的IL-2。加入上述OKT3和IL-2后的0小时、6小时、12小时、24小时、48小时、72小时、或5天以后，将饲养细胞加入肿瘤浸润淋巴细胞的培养环境中。其中，TIL与饲养细胞可以按照比率1:40-1:400加入，例如1:200，第二阶段扩增培养约9-14天后收集全部细胞，检测和统计培养所得TIL的结果。
增殖能力检测
对于上述饲养细胞不同添加时间培养获得的TIL进行细胞计数。
图59L显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、 72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。相比于加入OKT3和IL-2后的0小时后(即同时)添加饲养细胞培养的TIL，加入OKT3和IL-2后的12小时或更多时间后加饲养细胞培养的TIL增殖能力显著增强。
流式检测TIL细胞组成
对于上述饲养细胞不同添加时间培养获得的TIL群进行流式检测。
不同供者肿瘤来源的TIL作为各自不同的批次；将各个批次的加入OKT3和IL-2同时(0h组)加入饲养细胞的试验组的数据作为基准1，将同批次其它时间点试验组的数据进行标准化处理，统计各试验组在第二阶段扩增相对于0h组的细胞组成比例。
流式检测的试验流程可以参照本申请实施例3的内容。
图59M显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD8 +T细胞比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的CD8 +T细胞的比例。
图59N显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RO +CD62L +T细胞比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的记忆T细胞(Tcm，CD45RO +CD62L +)的比例。
图59O显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的NK T细胞比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的NK T细胞的比例。
图59P显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4 +CD25 +Foxp3 +调节性T细胞(Treg)比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更少的调节性T细胞的比例。
本申请培养的TIL的杀伤能力检测
对于实施例1的1.4的第二阶段扩增的TIL活化中，取第一阶段扩增的细胞量，调整细胞密度为5×10 5至2×10 6/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如3000或6000IU/mL的IL-2。加 入上述OKT3和IL-2后的12小时至14天后，例如48小时以后，将饲养细胞加入肿瘤浸润淋巴细胞的培养环境中。其中，TIL与饲养细胞可以按照比率1:40-1:400加入，第二阶段扩增培养约9-14天后收集全部细胞，检测和统计培养所得TIL的细胞杀伤能力检测。
细胞准备
准备用于检测的各个试验组获得的TIL和用于共培养的靶细胞(例如A375黑色素瘤细胞和/或Hela宫颈癌细胞)。
检测步骤
用CFSE(5(6)-Carboxyfluorescein diacetate N-succinimidyl ester，Sigma，21888-25MG-F)标记肿瘤细胞：用PBS清洗肿瘤细胞，重悬肿瘤细胞于500μL的PBS中；将CFSE加入500μL的PBS中，与500μL的肿瘤细胞PBS重悬液混合，至CFSE的终浓度为0.5μmol/L。37℃孵育6分钟后，加含10％FBS的培养基清洗，600g离心5分钟，用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基重悬肿瘤细胞浓度为1×10 6个细胞/mL。对各个试验组的TIL群600g离心5分钟，按照效靶比(TIL细胞与肿瘤细胞的比例)3:1重悬TIL细胞(即重悬TIL细胞浓度为3×10 6个细胞/mL)。于U底96孔板(Corning)中加入肿瘤细胞和TIL细胞各100μL，每组设置三个复孔。同时设置一组只包含肿瘤细胞的对照组。将孔板200g离心1分钟，置于37℃孵育4小时至过夜。其中，TIL与肿瘤细胞共培养时，可以不加激活TIL细胞的物质作为无激活组，或者加入transACT(Miltenyi，包含CD3抗体和CD28抗体的纳米基质材料)作为激活组。
孵育完成后，600g离心3分钟，弃上清，每孔加入20μL胰酶，37℃培养箱内孵育3-5分钟消化肿瘤细胞，消化完成后加入180μL含10％FBS的培养基终止消化。将Dapi(碧云天，C0060)用1：100稀释，然后每孔加入20μL稀释后的Dapi。进行流式上机检测。
杀伤率％＝Dapi +CFSE +细胞数/总CFSE +×100％，或杀伤率可以通过Dapi +细胞数/总肿瘤细胞数表示。
图59Q显示的是，加入OKT3和IL-2的48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞杀伤能力结果。结果显示，加入OKT3和IL-2后的48小时后添加饲养细胞培养的TIL均具有显著的肿瘤细胞杀伤能力，例如黑色素瘤和/或宫颈肿瘤。
实施例19
TNFAIP3各个亚区域的编辑效果
敲除效率检测参照本申请的细胞的敲除效率检测方法，结果如下表所示。
Figure PCTCN2022109577-appb-000012
Figure PCTCN2022109577-appb-000013
  TP-15 TP-17 TP2 TP4 TP-4 TP-5 TP-6
gRNA序列(SEQ ID NO) 55 56 57 58 59 60 61
D903 66 87.5 85.1 89.6 - - -
D107 78.5 97.3 91.8 97.2 - - -
D812 - 98.1 92.5 97 - - -
D904 - 98.3 89.3 - - - -
D003 - - 24.8 22.7 21.6 16.7 16.6
D607 - - 74.6 98.7 - 74 55.7
D105 - - 83.7 97.2 - 86.3 58.2
D713 - - 86.9 98.2 - 87.2 58.7
D313 - - 65.6 83.6 - - -
D316 - - - 79.5 - - -
D709 - - - 98.6 - - -
其中，各个TIL细胞编号与供者来源的瘤种对应关系为：D903来自宫颈癌患者、D904来自非小细胞肺癌患者、D107来自非小细胞肺癌患者、D812来自皮肤恶性黑色素瘤患者、D003来自皮肤恶性黑色素瘤、D105来自胰腺癌患者、D607来自卵巢癌、D713来自宫颈癌患者、D313来自皮肤恶性黑色素瘤、D316来自皮肤恶性黑色素瘤、以及D709来自宫颈癌患者。
细胞增殖效果
参照本申请的细胞扩增情况检测方法，例如将本申请提供的sgRNA敲除组或对照组(NT-no treatment)获得的TIL，在电转后7天以1.5e5/孔铺于96孔板。
无刺激检测条件下，不加额外刺激，3天后用CTG试剂盒分析细胞扩增情况。
图60A显示的是，对于来源于不同供者的TIL细胞，无刺激检测条件下各组TIL细胞的扩增后的荧光量。
抗CD3抗体刺激检测条件下，加抗CD3抗体(例如，OKT3，30ng/ml)刺激，3天后用CTG试剂盒分析细胞扩增情况。
图60B显示的是，对于来源于不同供者的TIL细胞，抗CD3抗体刺激检测条件下各组 TIL细胞的扩增后的荧光量。结果表明，本申请的基因编辑的TIL细胞可以具有显著的扩增能力。
长期增殖能力
本申请同时检测得到的基因编辑TIL细胞撤去IL-2培养后长期增殖/存续能力。将本申请提供的sgRNA敲除组或对照组(NT-no treatment)获得的TIL在体外扩增的第7天，培养基中不包含IL-2(作为D0天)。细胞重新铺板后，每隔约2-4天统计细胞总数并进行归一化分析。
图60C显示的是，TIL细胞撤去IL-2后长期增殖/存续的增殖倍数结果。结果表明，本申请的基因编辑的TIL细胞在撤去IL-2培养后，可以具有显著的长期增殖/存续能力。
配型杀伤
配型杀伤的试验方法根据与TIL的供者瘤种配型的肿瘤细胞系，进行配型杀伤测试。例如，将与供者瘤种相同/相近的肿瘤靶细胞系(配型细胞系)铺于96孔板，过夜培养；第二天将本申请靶点敲除或对照组TIL以相应的效靶比与配型细胞系共培养，用IncuCyte通过实时检测Caspase 3/7的活性来分析TIL对肿瘤细胞的杀伤。
图60D显示的是，本申请的基因编辑的TIL细胞对与该TIL的供者瘤种配型的肿瘤细胞系的杀伤效果。
图60E显示的是，本申请的基因编辑的TIL细胞对与该TIL的供者瘤种未配型的肿瘤细胞系的杀伤效果。
细胞因子表达
参照本申请的细胞因子表达流式检测方法，无刺激检测条件下，将本申请提供的sgRNA敲除组或对照组(NT-no treatment)获得的TIL，在电转后8天用流式细胞仪检测CD107a,GZMB,TNF-α和IFN-γ的表达。
图60F显示的是，在无刺激检测条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
参照本申请的细胞因子表达流式检测方法，抗CD3抗体刺激检测条件下，将本申请提供的sgRNA敲除组或对照组(NT-no treatment)获得的TIL，在电转后7天加抗CD3抗体(例如，OKT3，30ng/ml)刺激过夜，第二天用流式细胞仪检测CD107a,GZMB,TNF-α和IFN-γ的表达。
图60G显示的是，在抗CD3抗体刺激过夜的条件下，对于来源于不同供者的TIL细胞，各组TIL细胞中CD107a，GZMB，TNF-α和IFN-γ的表达细胞的比例。
结果显示，基因编辑后的TIL细胞具有更高的胞内因子表达能力。例如，更高的CD107a表达能力、更高的IFN-γ表达能力、更高的TNF-α表达能力或更高的GZMB表达能力。
细胞流式检测
参照本申请的细胞流式检测方法，对获得的本申请提供的sgRNA敲除组或对照组(NT-no treatment)获得的TIL群进行流式细胞仪检测。
图60H显示的是，对于不同供者来源的TIL细胞，各组TIL细胞中耗竭标识分子表达的比例。结果显示，基因编辑后的TIL细胞具有更低的耗竭标识分子表达的比例。例如，TIL群具有更低的CD38、CD101、LAG-3、TIM-3和PD-1表达。
图60I显示的是，对于不同供者来源的TIL细胞，各组TIL细胞中中心记忆细胞(CD45RO阳性CD62L阳性)的比例。结果显示，基因编辑后的TIL细胞具有更高的中心记忆细胞(CD45RO阳性CD62L阳性)的比例。
降低TIL细胞凋亡
对于本申请提供的sgRNA敲除组或对照组(NT-no treatment)获得的TIL，在基因编辑后7天进行凋亡检测。将基因敲除组或对照组(NT-no treatment)组的TIL用细胞凋亡检测试剂盒(BD 559763 Annexin V PE Apoptosis kit)检测T细胞凋亡水平。
图60J显示的是，对于来源于供者709的TIL细胞的凋亡检测结果。结果表明，基因编辑的TIL细胞可以具有更显著的抗凋亡能力。
下表6显示的是本申请提供的gRNA对应的人TNFAIP3基因组坐标
gRNA编号 SEQ ID NO 染色体坐标
TP-N8 48 chr6:137876102-137876121
TP-N10 49 chr6:137874868-137874887
TP-N14 50 chr6:137879020-137879039
TP-N15 51 chr6:137874833-137874852
TP-N16 52 chr6:137877195-137877214
TP-N19 53 chr6:137871485-137871504
TP-N20 54 chr6:137871475-137871494
TP-15 55 chr6:137878543-137878562
TP-17 56 chr6:137878652-137878672
TP2 57 chr6:137871501-137871520
TP4 58 chr6:137878653-137878672
TP-4 59 chr6:137871502-137871520
TP-5 60 chr6:137871486-137871504
TP-6 61 chr6:137874842-137874860
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 (293)

  1. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
  2. 根据权利要求1所述的方法，所述方法包含：使所述TIL与所述饲养细胞共培养之前，使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
  3. 根据权利要求1-2中任一项所述的方法，所述方法包含：在所述TIL与所述T细胞激活剂和/或所述T细胞生长因子接触之后且在所述TIL与所述饲养细胞共培养之前使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
  4. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，其中所述TIL包含与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养获得的TIL。
  5. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中所述TIL包含使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL。
  6. 根据权利要求1-5中任一项所述的方法，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL显示出改善的TIL特性。
  7. 根据权利要求6所述的方法，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的抗凋亡能力，和提高的T细胞受体(TCR)克隆多样性。
  8. 根据权利要求7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。
  9. 根据权利要求1-8中任一项所述的方法，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含将基因调控系统引入所述TIL细胞中。
  10. 根据权利要求9所述的方法，所述基因调控系统能够在DNA水平破坏所述目标基因。
  11. 根据权利要求9-10中任一项所述的方法，所述基因调控系统包含指导核酸分子和酶蛋白。
  12. 根据权利要求11所述的方法，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。
  13. 根据权利要求11-12中任一项所述的方法，所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
  14. 根据权利要求11-13中任一项所述的方法，所述指导核酸分子包含指导RNA(gRNA)。
  15. 根据权利要求11-14中任一项所述的方法，所述指导核酸分子能够与所述目标基因的序列结合。
  16. 根据权利要求1-15中任一项所述的方法，所述目标基因包含编码NF-κB通路抑制分子的基因。
  17. 根据权利要求16所述的方法，所述NF-κB通路抑制分子能够使选自以下组的蛋白泛素化：受体相互作用蛋白1(RIP1)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1)、受体相互作用蛋白2(RIP2)和肿瘤坏死因子受体相关因子6(TRAF6)。
  18. 根据权利要求17所述的方法，所述NF-κB通路抑制分子包含肿瘤坏死因子α诱导蛋白3(TNFAIP3)。
  19. 根据权利要求18所述的方法，所述指导核酸分子能够与TNFAIP3基因的第2外显子和/或第7外显子所在的区域或其片段结合。
  20. 根据权利要求11-19中任一项所述的方法，所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：34至47。
  21. 根据权利要求11-20中任一项所述的方法，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：GGG、TGG、CGG和AGG。
  22. 根据权利要求11-21中任一项所述的方法，所述指导核酸分子包含如SEQ ID NO：48至61中任一项所示的序列。
  23. 根据权利要求1-22中任一项所述的方法，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例降低和/或单个细胞的表达量下降。
  24. 根据权利要求1-23中任一项所述的方法，所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中，表达所述目的基因的细胞比例为约95％或以下。
  25. 根据权利要求1-24中任一项所述的方法，所述方法还包含：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与所述饲养细胞共培养。
  26. 根据权利要求25所述的方法，在单个阶段的所述体外扩增中，使所述TIL与所述饲养细胞共培养。
  27. 根据权利要求25-26中任一项所述的方法，在单个阶段的所述体外扩增中，使所述TIL的至少一种目标基因的表达降低和/或活性减弱且使所述TIL与所述饲养细胞共培养。
  28. 根据权利要求25-27中任一项所述的方法，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，使所述TIL与所述饲养细胞共培养。
  29. 根据权利要求28所述的方法，所述第一阶段体外扩增进行至少约7天。
  30. 根据权利要求28-29中任一项所述的方法，所述第一阶段体外扩增进行约7天至约14天。
  31. 根据权利要求28-30中任一项所述的方法，所述第二阶段体外扩增进行至少约7天。
  32. 根据权利要求28-31中任一项所述的方法，所述第二阶段体外扩增进行约7天至约14天。
  33. 根据权利要求1-32中任一项所述的方法，使所述TIL在与T细胞激活剂和/或T细胞生长因子接触至少约2小时之后与所述饲养细胞共培养。
  34. 根据权利要求1-33中任一项所述的方法，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时至约72小时之后与所述饲养细胞共培养。
  35. 根据权利要求1-34中任一项所述的方法，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约12小时至约48小时之后与所述饲养细胞共培养。
  36. 根据权利要求1-35中任一项所述的方法，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时、约12小时、约24小时、约48小时或约72小时之后与所述饲养细胞共培养。
  37. 根据权利要求1-36中任一项所述的方法，所述饲养细胞包含抗原呈递细胞。
  38. 根据权利要求1-37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。
  39. 根据权利要求1-38中任一项所述的方法，所述饲养细胞为外周单个核细胞。
  40. 根据权利要求1-39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。
  41. 根据权利要求1-40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。
  42. 根据权利要求1-41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。
  43. 根据权利要求1-42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。
  44. 根据权利要求1-43中任一项所述的方法，所述方法还包含：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与所述T细胞激活剂接触。
  45. 根据权利要求44所述的方法，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞激活剂接触。
  46. 根据权利要求44-45中任一项所述的方法，在单个阶段的所述体外扩增中，使所述TIL的至少一种目标基因的表达降低和/或活性减弱且使所述TIL与所述T细胞激活剂接触。
  47. 根据权利要求44-46中任一项所述的方法，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，使所述TIL与所述T细胞激活剂接触。
  48. 根据权利要求1-47中任一项所述的方法，所述T细胞激活剂包含选自以下组的一种或多种：分化簇80(CD80)、CD86、CD276、4-1BB配体(4-1BBL)、CD27、CD30、CD134、CD275、CD40、CD258、以及它们的功能活性片段。
  49. 根据权利要求1-48中任一项所述的方法，所述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、CD28、疱疹病毒进入介质(HVEM)、CD40L、OX40和4-1BB。
  50. 根据权利要求1-49中任一项所述的方法，所述T细胞激活剂包含CD3激动剂和/或CD28激动剂。
  51. 根据权利要求1-50中任一项所述的方法，所述T细胞激活剂包含CD3激动剂。
  52. 根据权利要求1-51中任一项所述的方法，所述T细胞激活剂包含抗CD3的抗体和/或其抗原结合片段。
  53. 根据权利要求1-52中任一项所述的方法，所述T细胞激活剂包含CD28激动剂。
  54. 根据权利要求1-53中任一项所述的方法，所述T细胞激活剂包含抗CD28的抗体和/或其抗原结合片段、CD80和/或其功能活性片段和/或CD86和/或其功能活性片段。
  55. 根据权利要求1-54中任一项所述的方法，所述使TIL与所述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；和(3)将包含所述T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  56. 根据权利要求55所述的方法，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为至少约30ng/mL。
  57. 根据权利要求55-56中任一项所述的方法，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为约30ng/mL-约300ng/mL。
  58. 根据权利要求55-57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。
  59. 根据权利要求55-58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。
  60. 根据权利要求58-59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。
  61. 根据权利要求55-60中任一项所述的方法，所述固相介质包含聚合物。
  62. 根据权利要求55-61中任一项所述的方法，每mg所述固相介质中包含的每一种所述T细胞激活剂的量各自独立地至少为约25μg。
  63. 根据权利要求55-62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T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  64. 根据权利要求55-63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T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  65. 根据权利要求1-64中任一项所述的方法，所述方法还包含：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与所述T细胞生长因子接触。
  66. 根据权利要求65所述的方法，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞生长因子接触。
  67. 根据权利要求65-66中任一项所述的方法，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞激活剂以及所述T细胞生长因子接触。
  68. 根据权利要求65-67中任一项所述的方法，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，使所述TIL与T细胞生长因子接触。
  69. 根据权利要求65-68中任一项所述的方法，使所述TIL基本上同时与所述T细胞激活剂以及所述T细胞生长因子接触。
  70. 根据权利要求1-69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。
  71. 根据权利要求1-70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。
  72. 根据权利要求1-71中任一项所述的方法，所述TIL与所述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。
  73. 根据权利要求1-72中任一项所述的方法，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
  74. 根据权利要求1-73中任一项所述的方法，所述TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
  75. 根据权利要求74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。
  76. 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T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
    (B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
  77. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
    (A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
    (B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
  78. 根据权利要求77所述的方法，所述体外TIL群包含由所述第一TIL群接触T细胞 生长因子获得的TIL群。
  79. 根据权利要求77-78中任一项所述的方法，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
  80. 根据权利要求76-79中任一项所述的方法，所述步骤(A)进行约7天至约14天。
  81. 根据权利要求76-80中任一项所述的方法，所述步骤(B)进行约7天至约14天。
  82. 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T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
    (B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
    (C)使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。
  83. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
    (A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
    (B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
    (C)使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。
  84. 根据权利要求83所述的方法，所述体外TIL群包含由所述第一TIL群接触T细胞生长因子获得的TIL群。
  85. 根据权利要求83-84中任一项所述的方法，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
  86. 根据权利要求82-85中任一项所述的方法，所述步骤(A)进行约7天至约14天。
  87. 根据权利要求82-86中任一项所述的方法，所述步骤(B)进行约0天至约8天。
  88. 根据权利要求82-87中任一项所述的方法，所述步骤(C)进行约5天至约14天。
  89. 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T细胞生长因子接触，其中，经 所述步骤(A)得到第二TIL群；
    (B)使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
    (C)使所述第三TIL群的至少一种目标基因的表达降低和/或活性减弱，其中，经所述步骤(C)得到第四TIL群；
    (D)使所述第四TIL群与饲养细胞共培养，其中，经所述步骤(D)得到第五TIL群。
  90. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
    (A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
    (B)使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
    (C)使所述第三TIL群的至少一种目标基因的表达降低和/或活性减弱，其中，经所述步骤(C)得到第四TIL群；
    (D)使所述第四TIL群与饲养细胞共培养，其中，经所述步骤(D)得到第五TIL群。
  91. 根据权利要求90所述的方法，所述体外TIL群包含由所述第一TIL群接触T细胞生长因子获得的TIL群。
  92. 根据权利要求90-91中任一项所述的方法，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
  93. 根据权利要求89-92中任一项所述的方法，所述步骤(A)进行约7天至约14天。
  94. 根据权利要求89-93中任一项所述的方法，所述步骤(B)进行约0天至约4天。
  95. 根据权利要求89-94中任一项所述的方法，所述步骤(C)进行约0天至约4天。
  96. 根据权利要求89-95中任一项所述的方法，所述步骤(D)进行约5天至约14天。
  97. 根据权利要求76-96中任一项所述的方法，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL显示出改善的TIL特性。
  98. 根据权利要求97所述的方法，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的抗凋亡能力，和提高 的T细胞受体(TCR)克隆多样性。
  99. 根据权利要求98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。
  100. 根据权利要求76-99中任一项所述的方法，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含将基因调控系统引入所述TIL细胞中。
  101. 根据权利要求100-101中任一项所述的方法，所述基因调控系统能够在DNA水平破坏所述目标基因。
  102. 根据权利要求100-101中任一项所述的方法，所述基因调控系统包含指导核酸分子和酶蛋白。
  103. 根据权利要求102所述的方法，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。
  104. 根据权利要求102-103中任一项所述的方法，所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
  105. 根据权利要求102-104中任一项所述的方法，所述指导核酸分子包含指导RNA(gRNA)。
  106. 根据权利要求102-105中任一项所述的方法，所述指导核酸分子能够与所述目标基因的序列结合。
  107. 根据权利要求76-106中任一项所述的方法，所述目标基因包含编码NF-κB通路抑制分子的基因。
  108. 根据权利要求107所述的方法，所述NF-κB通路抑制分子能够使选自以下组的蛋白泛素化：受体相互作用蛋白1(RIP1)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1)、受体相互作用蛋白2(RIP2)和肿瘤坏死因子受体相关因子6(TRAF6)。
  109. 根据权利要求108所述的方法，所述NF-κB通路抑制分子包含肿瘤坏死因子α诱导蛋白3(TNFAIP3)。
  110. 根据权利要求109所述的方法，所述指导核酸分子能够与TNFAIP3基因的第2外显子和/或第7外显子所在的区域或其片段结合。
  111. 根据权利要求102-110中任一项所述的方法，所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：34至47。
  112. 根据权利要求102-111中任一项所述的方法，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：GGG、TGG、CGG和AGG。
  113. 根据权利要求102-112中任一项所述的方法，所述指导核酸分子包含如SEQ ID NO：48至61中任一项所示的序列。
  114. 根据权利要求76-113中任一项所述的方法，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例降低和/或单个细胞的表达量下降。
  115. 根据权利要求76-114中任一项所述的方法，所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中，表达所述目的基因的细胞比例为约95％或以下。
  116. 根据权利要求76-115中任一项所述的方法，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触至少约2小时之后与所述饲养细胞共培养。
  117. 根据权利要求76-116中任一项所述的方法，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时至约72小时之后与所述饲养细胞共培养。
  118. 根据权利要求76-117中任一项所述的方法，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约12小时至约48小时之后与所述饲养细胞共培养。
  119. 根据权利要求76-118中任一项所述的方法，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时、约12小时、约24小时、约48小时或约72小时之后与所述饲养细胞共培养。
  120. 根据权利要求76-119中任一项所述的方法，所述饲养细胞包含抗原呈递细胞。
  121. 根据权利要求76-120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。
  122. 根据权利要求76-121中任一项所述的方法，所述饲养细胞为外周单个核细胞。
  123. 根据权利要求76-122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。
  124. 根据权利要求76-123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。
  125. 根据权利要求76-124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。
  126. 根据权利要求76-125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。
  127. 根据权利要求76-126中任一项所述的方法，所述T细胞激活剂包含选自以下组的一种或多种：分化簇80(CD80)、CD86、CD276、4-1BB配体(4-1BBL)、CD27、CD30、CD134、CD275、CD40、CD258、以及它们的功能活性片段。
  128. 根据权利要求76-127中任一项所述的方法，所述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、CD28、疱疹病毒进入介质(HVEM)、CD40L、OX40和4-1BB。
  129. 根据权利要求76-128中任一项所述的方法，所述T细胞激活剂包含CD3激动剂和/或CD28激动剂。
  130. 根据权利要求76-129中任一项所述的方法，所述T细胞激活剂包含CD3激动剂。
  131. 根据权利要求76-130中任一项所述的方法，所述T细胞激活剂包含抗CD3的抗体和/或其抗原结合片段。
  132. 根据权利要求76-131中任一项所述的方法，所述T细胞激活剂包含CD28激动剂。
  133. 根据权利要求76-132中任一项所述的方法，所述T细胞激活剂包含抗CD28的抗体和/或其抗原结合片段、CD80和/或其功能活性片段和/或CD86和/或其功能活性片段。
  134. 根据权利要求76-133中任一项所述的方法，所述使TIL与所述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；和(3)将包含所述T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  135. 根据权利要求134所述的方法，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为至少约30ng/mL。
  136. 根据权利要求134-135中任一项所述的方法，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为约30ng/mL-约300ng/mL。
  137. 根据权利要求134-136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。
  138. 根据权利要求134-137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。
  139. 根据权利要求137-138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。
  140. 根据权利要求134-139中任一项所述的方法，所述固相介质包含聚合物。
  141. 根据权利要求134-140中任一项所述的方法，每mg所述固相介质中包含的每一种所述T细胞激活剂的量各自独立地至少为约25μg。
  142. 根据权利要求134-141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T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  143. 根据权利要求134-142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T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  144. 根据权利要求76-143中任一项所述的方法，使所述TIL基本上同时与所述T细胞激活剂以及所述T细胞生长因子接触。
  145. 根据权利要求76-144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。
  146. 根据权利要求76-145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。
  147. 根据权利要求76-146中任一项所述的方法，所述TIL与所述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。
  148. 根据权利要求76-147中任一项所述的方法，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
  149. 根据权利要求76-148中任一项所述的方法，所述TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
  150. 根据权利要求149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。
  151. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL与CD28激动剂接触。
  152. 根据权利要求151所述的方法，所述方法包含：使所述TIL与CD28激动剂接触之后，使所述TIL的至少一种目标基因的表达降低和/或活性减弱。
  153. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL的至少 一种目标基因的表达降低和/或活性减弱，其中所述TIL包含与CD28激动剂接触获得的TIL。
  154. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，所述方法包含：使所述TIL与CD28激动剂接触，其中所述TIL包含使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL。
  155. 根据权利要求151-154中任一项所述的方法，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL显示出改善的TIL特性。
  156. 根据权利要求155所述的方法，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的抗凋亡能力，和提高的T细胞受体(TCR)克隆多样性。
  157. 根据权利要求156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。
  158. 根据权利要求151-157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。
  159. 根据权利要求158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。
  160. 根据权利要求151-159中任一项所述的方法，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含将基因调控系统引入所述TIL细胞中。
  161. 根据权利要求160所述的方法，所述基因调控系统能够在DNA水平破坏所述目标基因。
  162. 根据权利要求160-161中任一项所述的方法，所述基因调控系统包含指导核酸分子和酶蛋白。
  163. 根据权利要求162所述的方法，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。
  164. 根据权利要求162-163中任一项所述的方法，所述酶蛋白包含Cas蛋白、Cas蛋 白同系物，或其功能活性片段。
  165. 根据权利要求162-164中任一项所述的方法，所述指导核酸分子包含指导RNA(gRNA)。
  166. 根据权利要求162-165中任一项所述的方法，所述指导核酸分子能够与所述目标基因的序列结合。
  167. 根据权利要求151-166中任一项所述的方法，所述目标基因包含编码NF-κB通路抑制分子的基因。
  168. 根据权利要求167所述的方法，所述NF-κB通路抑制分子能够使选自以下组的蛋白泛素化：受体相互作用蛋白1(RIP1)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1)、受体相互作用蛋白2(RIP2)和肿瘤坏死因子受体相关因子6(TRAF6)。
  169. 根据权利要求168所述的方法，所述NF-κB通路抑制分子包含肿瘤坏死因子α诱导蛋白3(TNFAIP3)。
  170. 根据权利要求169所述的方法，所述指导核酸分子能够与TNFAIP3基因的第2外显子和/或第7外显子所在的区域或其片段结合。
  171. 根据权利要求162-170中任一项所述的方法，所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：34至47。
  172. 根据权利要求162-171中任一项所述的方法，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：GGG、TGG、CGG和AGG。
  173. 根据权利要求162-172中任一项所述的方法，所述指导核酸分子包含如SEQ ID NO：48至61中任一项所示的序列。
  174. 根据权利要求151-173中任一项所述的方法，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例降低和/或单个细胞的表达量下降。
  175. 根据权利要求151-174中任一项所述的方法，所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中，表达所述目的基因的细胞比例为约95％或以下。
  176. 根据权利要求151-175中任一项所述的方法，其中，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。
  177. 根据权利要求176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。
  178. 根据权利要求177所述的方法，所述第一阶段体外扩增进行至少约7天。
  179. 根据权利要求177-178中任一项所述的方法，所述第一阶段体外扩增进行约7天至约14天。
  180. 根据权利要求177-179中任一项所述的方法，所述第二阶段体外扩增进行至少约7天。
  181. 根据权利要求177-180中任一项所述的方法，所述第二阶段体外扩增进行约7天至约14天。
  182. 根据权利要求151-181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、CD80和/或其功能活性片段、和/或CD86和/或其功能活性片段。
  183. 根据权利要求151-182中任一项所述的方法，所述方法还包含：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与所述CD28激动剂之外的其它T细胞激活剂接触。
  184. 根据权利要求183所述的方法，在单个阶段的所述体外扩增中，使所述TIL与所述其它T细胞激活剂接触。
  185. 根据权利要求183-184中任一项所述的方法，在单个阶段的所述体外扩增中，使所述TIL的至少一种目标基因的表达降低和/或活性减弱且使所述TIL与所述其它T细胞激活剂接触。
  186. 根据权利要求183-185中任一项所述的方法，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，使所述TIL与所述其它T细胞激活剂接触。
  187. 根据权利要求183-186中任一项所述的方法，使所述TIL基本上同时与所述CD28激动剂以及所述其它T细胞激活剂接触。
  188. 根据权利要求183-187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。
  189. 根据权利要求183-188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。
  190. 根据权利要求183-189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。
  191. 根据权利要求183-190中任一项所述的方法，所述使TIL与所述CD28激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  192. 根据权利要求191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。
  193. 根据权利要求191-192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。
  194. 根据权利要求191-193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。
  195. 根据权利要求191-194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。
  196. 根据权利要求194-195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。
  197. 根据权利要求191-196中任一项所述的方法，所述固相介质包含聚合物。
  198. 根据权利要求191-197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激动剂以及所述其它T细胞激活剂。
  199. 根据权利要求191-198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激动剂以及所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  200. 根据权利要求191-199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激动剂以及所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  201. 根据权利要求151-200中任一项所述的方法，所述方法还包含：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在与CD28激动剂接触一定时间之后与饲养细胞共培养。
  202. 根据权利要求201所述的方法，在单个阶段的所述体外扩增中，使所述TIL与 所述饲养细胞共培养。
  203. 根据权利要求201-202中任一项所述的方法，在单个阶段的所述体外扩增中，使所述TIL与CD28激动剂接触且使所述TIL与所述饲养细胞共培养。
  204. 根据权利要求201-203中任一项所述的方法，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，使所述TIL与所述饲养细胞共培养。
  205. 根据权利要求201-204中任一项所述的方法，使所述TIL在与所述CD28激动剂接触至少约2小时之后与所述饲养细胞共培养。
  206. 根据权利要求201-205中任一项所述的方法，使所述TIL在与所述CD28激动剂接触约6小时至约72小时之后与所述饲养细胞共培养。
  207. 根据权利要求201-206中任一项所述的方法，使所述TIL在与所述CD28激动剂接触约12小时至约48小时之后与所述饲养细胞共培养。
  208. 根据权利要求201-207中任一项所述的方法，使所述TIL在与所述CD28激动剂接触约6小时、约12小时、约24小时、约48小时或约72小时之后与所述饲养细胞共培养。
  209. 根据权利要求201-208中任一项所述的方法，所述饲养细胞包含抗原呈递细胞。
  210. 根据权利要求201-209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。
  211. 根据权利要求201-210中任一项所述的方法，所述饲养细胞为外周单个核细胞。
  212. 根据权利要求201-211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。
  213. 根据权利要求201-212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。
  214. 根据权利要求201-213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。
  215. 根据权利要求201-214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。
  216. 根据权利要求151-215中任一项所述的方法，所述方法还包含：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T细胞生长因子接触。
  217. 根据权利要求216所述的方法，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞生长因子接触。
  218. 根据权利要求216-217中任一项所述的方法，在单个阶段的所述体外扩增中，使所述TIL与所述CD28激动剂以及所述T细胞生长因子接触。
  219. 根据权利要求216-218中任一项所述的方法，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，使所述TIL与T细胞生长因子接触。
  220. 根据权利要求216-219中任一项所述的方法，使所述TIL基本上同时与所述CD28激动剂以及所述T细胞生长因子接触。
  221. 根据权利要求216-220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。
  222. 根据权利要求216-221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。
  223. 根据权利要求216-222中任一项所述的方法，所述TIL与所述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。
  224. 根据权利要求216-223中任一项所述的方法，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
  225. 根据权利要求151-224中任一项所述的方法，所述TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
  226. 根据权利要求225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。
  227. 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T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
    (B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL与CD28激动剂接触，其中，经所述步骤(B)得到第三TIL群。
  228. 一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包含：
    (A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
    (B)使所述第二TIL群的至少一种目标基因的表达降低和/或活性减弱，且使所述TIL与CD28激动剂接触，其中，经所述步骤(B)得到第三TIL群。
  229. 根据权利要求228所述的方法，所述体外TIL群包含由所述第一TIL群接触T细胞生长因子获得的TIL群。
  230. 根据权利要求228-229中任一项所述的方法，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
  231. 根据权利要求227-230中任一项所述的方法，所述步骤(A)进行约7天至约14天。
  232. 根据权利要求227-231中任一项所述的方法，所述步骤(B)进行约7天至约14天。
  233. 根据权利要求227-232中任一项所述的方法，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL显示出改善的TIL特性。
  234. 根据权利要求233所述的方法，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的抗凋亡能力，和提高的T细胞受体(TCR)克隆多样性。
  235. 根据权利要求234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。
  236. 根据权利要求227-235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。
  237. 根据权利要求236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。
  238. 根据权利要求227-237中任一项所述的方法，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含将基因调控系统引入所述TIL细胞中。
  239. 根据权利要求238所述的方法，所述基因调控系统能够在DNA水平破坏所述目标基因。
  240. 根据权利要求238-239中任一项所述的方法，所述基因调控系统包含指导核酸分 子和酶蛋白。
  241. 根据权利要求240所述的方法，其中所述使所述TIL的至少一种目标基因的表达降低和/或活性减弱包含：将包含所述指导核酸分子和所述酶蛋白的核糖核蛋白复合物(RNP)引入所述TIL中。
  242. 根据权利要求240-241中任一项所述的方法，所述酶蛋白包含Cas蛋白、Cas蛋白同系物，或其功能活性片段。
  243. 根据权利要求240-242中任一项所述的方法，所述指导核酸分子包含指导RNA(gRNA)。
  244. 根据权利要求240-243中任一项所述的方法，所述指导核酸分子能够与所述目标基因的序列结合。
  245. 根据权利要求227-244中任一项所述的方法，所述目标基因包含编码NF-κB通路抑制分子的基因。
  246. 根据权利要求245所述的方法，所述NF-κB通路抑制分子能够使选自以下组的蛋白泛素化：受体相互作用蛋白1(RIP1)、粘膜相关淋巴组织淋巴瘤易位蛋白1(MALT1)、受体相互作用蛋白2(RIP2)和肿瘤坏死因子受体相关因子6(TRAF6)。
  247. 根据权利要求246所述的方法，所述NF-κB通路抑制分子包含肿瘤坏死因子α诱导蛋白3(TNFAIP3)。
  248. 根据权利要求247所述的方法，所述指导核酸分子能够与TNFAIP3基因的第2外显子和/或第7外显子所在的区域或其片段结合。
  249. 根据权利要求240-248中任一项所述的方法，所述指导核酸分子能够与选自以下组所示的区域或其片段结合：SEQ ID NO：34至47。
  250. 根据权利要求240-249中任一项所述的方法，所述指导核酸分子能够与选自以下组所示的原型间隔序列毗邻基序(PAM)5′端上游约15至约25个核苷酸组成的序列结合：GGG、TGG、CGG和AGG。
  251. 根据权利要求240-250中任一项所述的方法，所述指导核酸分子包含如SEQ ID NO：48至61中任一项所示的序列。
  252. 根据权利要求227-251中任一项所述的方法，与目标基因的表达和/或活性未改变的TIL相比，使所述TIL的至少一种目标基因的表达降低和/或活性减弱获得的TIL中表达所述目的基因的产物的细胞比例降低和/或单个细胞的表达量下降。
  253. 根据权利要求227-252中任一项所述的方法，所述使所述TIL的至少一种目标基 因的表达降低和/或活性减弱获得的TIL中，表达所述目的基因的细胞比例为约95％或以下。
  254. 根据权利要求227-253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、CD80和/或其功能活性片段、和/或CD86和/或其功能活性片段。
  255. 根据权利要求227-254中任一项所述的方法，使所述TIL基本上同时与所述CD28激动剂以及所述其它T细胞激活剂接触。
  256. 根据权利要求255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。
  257. 根据权利要求255-256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。
  258. 根据权利要求255-257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。
  259. 根据权利要求255-258中任一项所述的方法，所述使TIL与所述CD28激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它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激动剂以及所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  260. 根据权利要求259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。
  261. 根据权利要求259-260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。
  262. 根据权利要求259-261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。
  263. 根据权利要求259-262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。
  264. 根据权利要求262-263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。
  265. 根据权利要求259-264中任一项所述的方法，所述固相介质包含聚合物。
  266. 根据权利要求259-265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激动剂以及所述其它T细胞激活剂。
  267. 根据权利要求259-266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激动剂以及所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  268. 根据权利要求259-267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激动剂以及所述其它T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  269. 根据权利要求227-268中任一项所述的方法，使所述TIL在与所述CD28激动剂接触至少约2小时之后与所述饲养细胞共培养。
  270. 根据权利要求269所述的方法，使所述TIL在与所述CD28激动剂接触约6小时至约72小时之后与所述饲养细胞共培养。
  271. 根据权利要求269-270中任一项所述的方法，使所述TIL在与所述CD28激动剂接触约12小时至约48小时之后与所述饲养细胞共培养。
  272. 根据权利要求269-271中任一项所述的方法，使所述TIL在与所述CD28激动剂接触约6小时、约12小时、约24小时、约48小时或约72小时之后与所述饲养细胞共培养。
  273. 根据权利要求269-272中任一项所述的方法，所述饲养细胞包含抗原呈递细胞。
  274. 根据权利要求269-273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。
  275. 根据权利要求269-274中任一项所述的方法，所述饲养细胞为外周单个核细胞。
  276. 根据权利要求269-275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。
  277. 根据权利要求269-276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。
  278. 根据权利要求269-277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。
  279. 根据权利要求269-278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。
  280. 根据权利要求227-279中任一项所述的方法，使所述TIL基本上同时与所述CD28激动剂以及所述T细胞生长因子接触。
  281. 根据权利要求227-280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。
  282. 根据权利要求227-281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。
  283. 根据权利要求227-282中任一项所述的方法，所述TIL与所述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。
  284. 根据权利要求227-283中任一项所述的方法，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
  285. 根据权利要求227-284中任一项所述的方法，所述TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL和源自冷冻保存后复苏的TIL。
  286. 根据权利要求285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。
  287. 一种肿瘤浸润淋巴细胞(TIL)，所述TIL经过权利要求1-286中任一项所述的方法获得。
  288. 一种组合物，其包含权利要求287所述的TIL。
  289. 一种药物组合物，其包含权利要求287所述的TIL和/或权利要求288所述的组合物，以及任选地药学上可接受的载体。
  290. 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287所述的TIL、权利要求288所述的组合物和/或权利要求289所述的药物组合物。
  291. 权利要求287所述的TIL、权利要求288所述的组合物和/或权利要求289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。
  292. 根据权利要求291所述的应用，其中，所述肿瘤为实体瘤。
  293. 根据权利要求291-292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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