WO2023284721A1 - 一种免疫细胞的培养方法及其用途 - Google Patents

一种免疫细胞的培养方法及其用途 Download PDF

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WO2023284721A1
WO2023284721A1 PCT/CN2022/105137 CN2022105137W WO2023284721A1 WO 2023284721 A1 WO2023284721 A1 WO 2023284721A1 CN 2022105137 W CN2022105137 W CN 2022105137W WO 2023284721 A1 WO2023284721 A1 WO 2023284721A1
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cells
cell
yeats
present application
immune cells
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PCT/CN2022/105137
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English (en)
French (fr)
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刘雅容
赵佩佩
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苏州沙砾生物科技有限公司
上海沙砾生物科技有限公司
珠海拓域生物科技有限公司
珠海沙砾生物科技有限公司
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Priority to CN202280007366.4A priority Critical patent/CN116406421A/zh
Publication of WO2023284721A1 publication Critical patent/WO2023284721A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464488—NY-ESO
    • 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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte

Definitions

  • the present application relates to the field of biomedicine, in particular to a method for culturing immune cells and its use.
  • immunotherapy is an effective approach to treat patients with poor prognosis.
  • the immune cells used in immunotherapy have the problem of low cell activity or weak proliferation ability.
  • the present application provides a method for cultivating immune cells, which may have one or more effects selected from the following group: improved cell proliferation ability, increased live cell ratio, improved cell subset ratio, increased cytokine Secretory ability and enhanced tumor cell killing ability.
  • the present application provides a method for culturing immune cells, reducing the expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragments in the immune cells.
  • the present application provides an immune cell obtained through the method of the present application.
  • the present application provides a composition comprising the immune cells of the present application.
  • the present application provides a pharmaceutical composition, which comprises the immune cells of the present application and/or the composition of the present application, and optionally a pharmaceutically acceptable carrier.
  • the present application provides a method for affecting cell growth, comprising administering the immune cells of the present application, the composition of the present application and/or the pharmaceutical composition of the present application.
  • the present application provides the application of the immune cells of the present application, the composition of the present application and/or the pharmaceutical composition of the present application in the preparation of medicines for preventing and/or treating diseases and/or symptoms.
  • Figure 1 shows the proliferation results of the experimental group added with YEATS inhibitor.
  • Figures 2A-2B show the results of cell viability in the experimental group added with YEATS inhibitor for different donors.
  • Figure 3 shows the proportion of CD8 + cells in the experimental group added with YEATS inhibitor.
  • Figures 4A-4C show the proportion of CD28 + cells in the experimental group with YEATS inhibitor added for different donors.
  • Figures 5A-5E show the proportion of CD25 + cells in the experimental group with YEATS inhibitor added for different donors.
  • Figures 6A-6D show the proportion of 41BB + cells in the experimental group added with YEATS inhibitor for different donors.
  • Figures 7A-7K show the proportion of PD1 + cells in the experimental group added with YEATS inhibitor for different donors.
  • Figures 8A-8F show the proportion of LAG3 + cells in the experimental group with YEATS inhibitor added for different donors.
  • Figures 9A-9C show the proportion of TIM3 + cells in the experimental group with YEATS inhibitor added for different donors.
  • Figures 10A-10B show the proportion of CD39 + cells in the experimental group added with YEATS inhibitor for different donors.
  • Figure 11 shows the ratio of Treg cells (regulatory T cells) in the test group added with YEATS inhibitor.
  • Figures 12A-12C show the proportion of apoptotic cells in the experimental group added with YEATS inhibitor for different donors.
  • Figures 13A-13C show, for different donors, the proportion of stem cells in the test group added with YEATS inhibitor.
  • Figures 15A-15B show the proportion of CD107A-secreting cells in the experimental group supplemented with YEATS inhibitor for different donors.
  • Figures 16A-16D show the proportion of IFN ⁇ -secreting cells in the test group supplemented with YEATS inhibitor for different donors.
  • Fig. 17 shows the ratio of TNF-secreting cells in the test group to which the YEATS inhibitor was added.
  • Figures 18A-18D show the proportion of GZMB-secreting cells in the test group added with YEATS inhibitor for different donors.
  • Figure 19 shows the transfection efficiency of T cells cultured with YEATS inhibitor for different donors.
  • Figure 20 shows the proportion of stemness-associated CD69 - CD39 - cells cultured with YEATS inhibitor for different donors.
  • Figures 21-22 show the proportion of depletion-associated CD39 + or PD1 + cells cultured with YEATS inhibitor for different donors.
  • Figures 23-24 show the proportion of activation-related 41BB + cells or CD25 + cells for different donors cultured with YEATS inhibitor.
  • Figure 25 shows the results of cell proliferation for different donors cultured with YEATS inhibitor. The results showed that YEATS inhibitors can improve the proliferation ability of cells.
  • Figures 26-30 show the results of secretion of IL-2, GZMB, TNF- ⁇ , IFN- ⁇ and IL-6 for different donors cultured with YEATS inhibitor.
  • Figure 31 shows that the release of cytokines (such as IL-2, TNF- ⁇ and IFN- ⁇ ) from NK cells is significantly increased after YEATS inhibitor treatment.
  • cytokines such as IL-2, TNF- ⁇ and IFN- ⁇
  • Figure 32 shows the proportion of activation-associated CD25 + cells cultured with YEATS inhibitor for different donors. The results showed that YEATS inhibitors can increase the proportion of activation-associated cells.
  • Figures 33-34 show the proportion of depletion-associated PD1 + cells or LAG3 + cells cultured with YEATS inhibitor for different donors. The results showed that YEATS inhibitors could reduce the proportion of exhaustion-associated cells.
  • Figures 35-36 show the proportion of stemness-associated TCF1 + or CD69 ⁇ CD39 ⁇ cells cultured with YEATS inhibitor for different donors. The results showed that YEATS inhibitors can increase the proportion of stemness-related cells.
  • the term "immune cell” generally refers to cells involved in carrying out innate and adaptive immune responses.
  • lymphocytes such as T cells (including thymocytes) and B cells
  • natural killer (NK) cells such as T cells (including thymocytes) and B cells
  • NK natural killer
  • NKT cells NKT cells
  • macrophages monocytes, eosinophils, basophils cells, neutrophils, dendritic cells, and mast cells.
  • the modified immune effector cells are T cells, such as CD4 + T cells, CD8 + T cells (also known as cytotoxic T cells or CTLs), regulatory T cells (Treg), Th1 cells, Th2 cells , Th17 cells ⁇ T cells and/or ⁇ T cells.
  • a CAR generally refers to an engineered antigen receptor.
  • a CAR may comprise an extracellular antigen-binding domain fused via a hinge and transmembrane domain to a cytoplasmic domain comprising a signaling domain.
  • the CAR extracellular domain can bind to an antigen expressed by a target cell in an MHC-independent manner, resulting in activation and proliferation of the cell.
  • the extracellular domain of the CAR can recognize a tag fused to an antibody or antigen-binding fragment thereof.
  • a single CAR construct can be made to target multiple different antigens by substituting one antibody for another.
  • the extracellular domain of the CAR may comprise an antigen-binding fragment derived from an antibody.
  • Antigen binding domains useful in the present disclosure may include, for example, scFvs, antibodies, antigen binding regions of antibodies, variable regions of heavy/light chains, and/or single chain antibodies.
  • T cell receptor generally refers to an engineered antigen receptor.
  • a TCR may comprise a TCR alpha and/or TCR beta chain that has been isolated and cloned from a population of T cells that recognize a particular target antigen.
  • the TCR ⁇ and/or TCR ⁇ genes i.e., TRAC and TRBC
  • TRAC and TRBC can be derived from T cell populations isolated from individuals with specific malignancies or from T cells that have been isolated from humanized mice immunized with specific tumor antigens or tumor cells cloned from the population.
  • Engineered TCRs can recognize antigens by the same mechanism as their endogenous counterparts (e.g., by recognizing their cognate antigens presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of target cells), thereby It can lead to the activation and proliferation of TCR engineered cells.
  • MHC major histocompatibility complex
  • YEATS generally refers to family members having domains that recognize modified proteins.
  • the YEATS domain can recognize histone modifications, such as histone acetylation.
  • proteins of family members comprising a YEATS domain can comprise ENL (UniProt No Q03111), AF9 (UniProt No P42568), and YEATS2 (UniProt No Q9ULM3) and GAS41 (UniProt No O95619).
  • YEATS inhibitor generally refers to a substance capable of affecting the activity and/or function of YEATS protein.
  • Suitable inhibitor molecules may include antagonist antibodies or antibody fragments, fragments or derivatives of small molecules, peptides, antisense oligonucleotides, small organic molecules, and the like.
  • a method of identifying an inhibitor of the present application comprises contacting a cell expressing a molecule inhibited by the present application with a candidate inhibitor molecule, detecting a detectable change in one or more biological activities associated with the molecule inhibited by the present application, YEATS inhibition
  • the agent may comprise a substance capable of reducing the expression and/or activity of a nucleic acid molecule encoding a protein comprising a YEATS domain.
  • a YEATS inhibitor can inhibit the function of YEATS by binding to the histone-binding domain of the YEATS protein.
  • the YEATS inhibitor may include SGC-iMLLT (CAS NO.: 2255338-25-9) and any known YEATS inhibitor, as well as derivatives of the above compounds.
  • a YEATS inhibitor can be specific, having higher binding activity for one or more proteins that comprise members of the YEATS domain family.
  • alkyl is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso Butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc.
  • “Lower alkyl” is an alkyl group containing one to six carbon atoms.
  • alkenyl is a hydrocarbon group of 2 to 24 carbon atoms, and the formula contains at least one carbon-carbon double bond.
  • alkynyl is a hydrocarbon group of 2 to 24 carbon atoms, and the formula contains at least one carbon-carbon triple bond.
  • aryl is any carbon-based aromatic group including, but not limited to, benzene, naphthalene, and the like.
  • aryl also includes “heteroaryl” or “heteroaryl”, which are defined as aromatic groups having at least one heteroatom incorporated into the ring of the aryl group, while “heteroaryl” or “heteroaryl group” has 5 to 9 ring atoms ("C 5-9 heterocyclic aryl”), for example, 5 or 6 ring atoms (“C 5 or C 6 heterocyclic aryl”), selected from carbon atoms and heterocyclic atom.
  • heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus.
  • Aryl is substituted or unsubstituted aryl.
  • Aryl groups may be substituted with one or more groups including, but not limited to: alkyl, alkynyl, alkenyl, aryl, halogen, nitro, amino, ester, ketone, aldehyde, hydroxyl, carboxylic acid, or alkoxy .
  • cyclic oligomer is an oligomer whose termini, side chains or a combination thereof are covalently bonded to form a ring structure.
  • the term "effective amount" of a compound refers to a non-toxic amount of the compound which is sufficient to provide the desired result.
  • the exact amount required will vary from patient to patient, depending on the type, age, and general condition of the patient, the severity of the condition being treated, the particular compound being used, its mode of administration, and the like, as described below. Accordingly, an exact “effective amount” cannot be specified. However, an appropriate effective amount can be determined by one of ordinary skill in the art using no more than routine experimentation.
  • esters is represented by the formula -C(O)OA, where A can be alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkane as described above group, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl.
  • thioester refers to a functional group characterized by a sulfur atom flanked by a carbonyl and a carbon of any hybridization.
  • sulfonate refers to a salt or ester of sulfonic acid.
  • peptide refers to a class of compounds consisting of amino acids chemically bonded together. Typically, the amino acids are chemically joined together by an amide linker (CONH); however, the amino acids can be joined together by other chemical bonds known in the art. For example, amino acids can be attached via amine linkers.
  • peptides include oligomers of amino acids and small and large peptides, including polypeptides.
  • ⁇ - ⁇ - ⁇ stacking ⁇ - ⁇ stacking
  • ⁇ - ⁇ stacking ⁇ - ⁇ stacking
  • ⁇ -stacking ⁇ -stacking
  • conjugated and delocalized refer to delocalized ⁇ -electrons (may also be referred to as conjugated ⁇ -electrons).
  • Conjugated ⁇ -electrons or conjugated systems refer to systems of p-orbitals (bridging intermediate sigma bonds) overlapping with delocalized electrons that exist in compounds with alternating single and multiple bonds (usually double bonds).
  • pharmacological activity refers to an inherent physical property of a peptide or polypeptide. These properties include, but are not limited to, half-life, solubility and stability, and other pharmacokinetic properties.
  • the term "pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., it can be administered to a patient together with a nucleic acid or carrier without causing any adverse biological effects or Interact in a harmful manner with any other ingredient of the contained pharmaceutical composition.
  • the carrier will naturally be chosen to minimize any degradation of the active ingredient, and to minimize any adverse side effects in the patient.
  • the material can be a solution, a suspension (eg, incorporated into microparticles, liposomes, or cells).
  • ring refers to a ring of atoms and bonds in a molecule, or to a group of connected atoms and bonds, each of which is a member of a ring.
  • “Monocyclic” generally consists of one ring structure; “bicyclic” has two ring structures; “tricyclic” has three ring structures; “tetracyclic” has four ring structures, and so on.
  • selective targeting refers to the ability to specifically target within a specific region.
  • permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, but are not limited to: halogen, hydroxyl, or any other organic group containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally in chain, branched, or cyclic structural forms including One or more heteroatoms, such as oxygen, sulfur or nitrogen groups.
  • substituents include: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, Substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio radical, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl , amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphono, substituted
  • 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.
  • CD4 + cells generally refers to CD4 positive cells, such as T cells.
  • CD4 + cells CD4 positive 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.
  • 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.
  • 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 "specific inhibitor” generally refers to an inhibitor that acts specifically on a molecule of the present application.
  • the YEATS selective inhibitor can inhibit one or more subtypes in YEATS.
  • 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.
  • 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 immune cells go through in vitro. increase process.
  • each stage can be divided by changes in the number of immune cells. For example, when the number of immune cells increases by at least about 1-fold, it can be considered that the immune cells have entered the next stage of in vitro expansion.
  • the number of immune cells when the number of immune 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, or at least about 50 times, it can be considered that the immune cells have entered the next stage of in vitro expansion. For example, each stage can also be divided by the conditions of immune cell culture.
  • the immune cells when cell activators and/or cell growth factors are added or supplemented to the cell culture medium, it can be considered that the immune cells enter the next stage of in vitro expansion. For example, after the immune cells have been centrifuged and/or washed, the immune cells can be considered to enter the next stage of in vitro expansion. For example, each stage can also be divided by the number of days of immune cell culture.
  • the immune cells when the immune cells are cultured in vitro for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days , about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, about 50 days or about After 100 days, the immune cells can be considered to enter the next stage of in vitro expansion.
  • first-stage in vitro expansion generally refers to the stage of expansion using 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, pleural effusion and peritoneal 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 in vivo amplification by autologous or allogeneic, 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.
  • 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 times). , 60, 70, 80 or 90 fold), or for example the number of cells can be increased by at least about 100 fold.
  • the culture conditions of the second-stage in vitro expansion may be different from those of the first-stage in vitro expansion, for example, the culture substances added may be different.
  • the second stage of in vitro expansion of the present application may also be referred to as the REP (Rapid Expansion) stage.
  • in vivo generally refers to an event that occurs in 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 subject's body through surgery or therapy.
  • secretion generally refers to the transfer of an expressed polypeptide or protein by a cell to the extracellular environment.
  • 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, alpha rays, beta rays, or gamma rays, for example.
  • 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.
  • an engineered cell can be an immune cell that has been genetically modified to express a cell activator and/or a cell growth factor 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 in the present application can, for example, be through direct contact, ie, where cells of one population are in direct physical contact with cells of another population.
  • indirect contact can be mediated, for example, 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.
  • the term "contacting" generally means that two or more substances of different types are brought into contact together in any order, in any manner, and for any length of time.
  • one or more feeder cells, cell activators and/or cell growth factors can be added to the culture medium of immune cells, for example, one or more feeder cells, cell activators and/or cell growth factors can be added or cell growth factor medium to add and/or replace immune cell culture medium, for example, a medium comprising one or more feeder cells, cell activators and/or cell growth factors may be used for the cultivation of immune cells;
  • through indirect contact for example, metabolites produced and released by feeder cells can be used to cultivate immune 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), for example, 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, for example, the number of cells can 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.
  • polymer can 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.
  • polymers may include, for example, polysaccharides, dextran, hydrogels, polyethylene glycols, or poloxamers.
  • 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 six CDRs is provided 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.
  • IL-2 or "IL2” generally refers to the cellular growth factor known as interleukin 2 and includes all forms of IL-2, which may include, for example, human and mammalian forms, conservative amino acid substitutions , glycoform modifications or variants, or active fragments thereof.
  • the GeneID of the gene encoding IL-2 may be 3558.
  • antigen presenting cell 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.
  • antigen presenting cells may include those selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.
  • the term "improved immune cell characteristics" generally refers to the immune cell characteristics that appear after the cells are expanded and/or cultured. Improved immune cell properties may include changes in the number and/or ratio of cells, changes in secretion capacity, changes in killing capacity or expression capacity, or any combination thereof. According to different evaluation criteria, the improvement in the present application can be an increase or a decrease, such as an increase in killing ability, and a decrease in the level of cell exhaustion.
  • 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
  • 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
  • CD58 the gene GeneID encoding it can be 965
  • 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 an immune cell. 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.
  • immune 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 grows in vitro and secretes at least one factor into the medium and can be used to support the growth of another cell of interest in culture.
  • feeder cells can 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 usually refers to the separation of cells from tumor tissue, pleural effusion and/or peritoneal effusion isolated from the patient, after one or more expansions, and finally obtaining The complete course of administration of cells to a subject.
  • cell culture medium generally refers to a nutrient solution in which cells, such as mammalian cells, are grown.
  • the formulation of cell culture media is well known in the art.
  • 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 cells generally refers to cells that have long-term memory and are capable of receiving antigen restimulation.
  • Central memory cells may have a phenotype of CD45RA ⁇ CCR7 + or CD45RO + CD62L + , for example, central memory cells may be identified by CD45RA ⁇ and CCR7 + or CD45RO + and CD62L + .
  • Central memory cells may have a stronger ability to resist tumor growth than normal cells.
  • regulatory cells generally refers to a subpopulation of cells that control autoimmune reactivity in the body. Regulatory cells may have a phenotype of CD4 + CD25 + Foxp3 + , for example, regulatory cells may be identified by CD4 + , CD25 + and Foxp3 + . Regulatory cells may have the ability to suppress the anti-tumor growth of cells.
  • activated cells generally refers to cells that have been activated to have the ability to resist tumor growth.
  • the activated cells may have the phenotype of CD28 + , CD25 + , or 41BB + , for example, activated cells may be identified by CD28 + , CD25 + , or 41BB + .
  • Activated cells may have the ability to resist tumor growth.
  • exhausted cells generally refers to cells in which immune cells are continuously stimulated by antigens and gradually lose their effector functions.
  • the function of an exhausted cell can be reversible, or partially reversible.
  • the exhausted cells can have a phenotype of PD1 + , LAG3 + , TIM3 + , or CD39 + , for example, the exhausted cells can be identified by PD1 + , LAG3 + , TIM3 + , or CD39 + .
  • Exhausted cells can be characterized by reduced immune function.
  • apoptotic cells generally refers to immune cells undergoing programmed death.
  • Apoptotic cells may have the phenotype of CD95 + caspass3 + cells and/or CD95 + DR5 + , for example, apoptotic cells may be identified by CD95 + caspass3 + cells and/or CD95 + DR5 + .
  • Apoptotic cells can cause a decrease in cell number.
  • tumor-specific cells generally refers to cells that can specifically resist tumor growth.
  • the tumor-specific cells may have a phenotype of CD103 + CD39 + , for example, tumor-specific cells may be identified by CD103 + and CD39 + .
  • Tumor-specific cells may have a more specific ability to resist tumor growth than normal cells.
  • stem cell-like cell generally refers to a type of cell that may have the potential of self-proliferation and/or differentiation (stemness, stemness).
  • Stem cell-like cells may have a phenotype of CD69 - CD39 - or TCF1 + , for example, stem cell-like cells may be identified by CD69 - CD39 - or TCF1 + .
  • Tumor-specific cells may have a stronger and/or longer-term ability to resist tumor growth than normal 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.
  • cell activator generally refers to a substance that binds to a corresponding binding receptor on a cell and mediates a costimulatory response of the cell.
  • Cell activators can be substances other than antigen receptors that are required for cells to mount an effective immune response.
  • a cell activator may refer to a cell co-stimulatory molecule.
  • the cell activator of the present application may comprise its variant, homologue or any substance comprising its functionally active fragment.
  • 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 of encoding it can be 151888), Toll ligand receptor, OX40 (the gene GeneID of encoding it can be 7293), CD2 (the gene GeneID of encoding it can be 914), CD7 (the gene GeneID of encoding it can be 914), CD7 (the gene GeneID of 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 term "cell growth factor” generally refers to a biologically active polypeptide or small molecule compound that causes cell proliferation.
  • the cell growth factor of the present application may comprise its variant, homologue or any substance comprising its functionally active fragment.
  • the 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-7 (the gene GeneID encoding it can be 3565), IL-7 (encoding Its gene GeneID can be 3574), IL-10 (the gene GeneID encoding it can be 3586), IL-12 (the gene GeneID encoding it can be 3592 or 3593), IL-15 (the gene GeneID encoding it can be 3592 or 3593), IL-15 (the gene GeneID encoding it can be 3600), and gamma interferon (the gene GeneID encoding it may be 3458).
  • IL-2 the gene GeneID encoding it can be 35
  • substantially simultaneously usually means that the immune cells can be in contact with two or more substances at the same time during a period of time during the contact process, but it is not limited to the fact that the immune cells are always in contact with two or more substances during the entire contact process. Substances come into contact at the same time.
  • substantially simultaneously can mean that the immune cells can interact with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% for a period of time. % of two or more substances of each substance in simultaneous contact.
  • 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 incorporate one or more cell activating agents.
  • the solid-phase medium of the present application may refer to a medium in which CD28 antibody or its antigen-binding fragment and CD3 antibody or its antigen-binding fragment are bound in the medium and/or on the surface of the medium through covalent binding and/or non-covalent binding. Material.
  • the solid phase medium of the present application may be microspheres comprising the OKT3 antibody and the 15E8 antibody with a diameter of about 500 nanometers to about 10 micrometers.
  • the solid phase medium of the present application may be a polymeric material.
  • the solid phase media of the present application can be microspheres having a diameter of at least about 500 nanometers.
  • the solid phase medium of the present application may be a nanomatrix.
  • the solid phase medium of the present application may be a nanomatrix comprising the OKT3 antibody and the 15E8 antibody with a diameter of about 1 nanometer to about 500 nanometers.
  • the term "nanomatrix" generally refers to a material with a diameter ranging from about 1 nanometer to about 500 nanometers.
  • the nanomatrix can have a binding function, for example, the nanomatrix of the present application can be combined with one or more cell activating agents.
  • 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 cells in vitro and can elicit primary cellular responses both 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 immune cells, such as detecting the ability of immune cells to release cytokines, the operation steps performed on immune cells (such as adding a One or more substances to detect the ability of immune 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-21, and/or IL-12.
  • ILs interleukins
  • cytokines can include CD107A (UniProt No P11279), IFN ⁇ (UniProt No P01579), TNF (UniProt No P01375) or GZMB (UniProt No P10144).
  • CD107A UniProt No P11279
  • IFN ⁇ UniProt No P01579
  • TNF UniProt No P01375
  • GZMB UniProt No P10144
  • 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.
  • cell subgroup ratio generally refers to the ratio of different cell subgroups to immune cells or immune cell populations.
  • different cell subpopulations of the present application have different immunological activity and/or differentiation capacity.
  • cell subpopulations of the present application can be distinguished based on cell surface markers.
  • the term "number of immune cells” generally refers to the number of cells in the immune cells of the present application.
  • the number of immune cells may refer to the number of cells in the immune cell population obtained at any stage of this application.
  • the number of immune cells can refer to the number of cells of the first immune cell population derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion.
  • the number of immune 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 immune cells may refer to the number of immune cells finally obtained by any of the cultivation methods of the present application.
  • the number of immune cells can be measured by methods commonly used in the art, such as 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 numerical values.
  • the present application provides a method for culturing immune cells, which reduces the expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragments in the immune cells.
  • said reducing the activity of the protein comprising the YEATS domain and/or its functionally active fragment comprises inhibiting the binding ability of the YEATS domain to histone.
  • said reducing the activity of the protein comprising the YEATS domain and/or its functionally active fragment may comprise inhibiting the binding ability of the YEATS domain to histone.
  • a substance capable of inhibiting the binding of the YEATS domain to histone may be added during the cultivation of immune cells.
  • a substance capable of inducing the inactivation of a protein containing a YEATS domain can be added during the cultivation of immune cells.
  • substances capable of binding to the YEATS domain can be added during the cultivation of immune cells.
  • the methods of the present application comprise: contacting the immune cells with one or more YEATS inhibitors.
  • the corresponding immune cells that have not been in contact with the YEATS inhibitor may refer to immune cells that are derived from the same donor and have not been in contact with the YEATS inhibitor of the present application.
  • immune cells that have not been in contact with the YEATS inhibitor of the present application can be contacted with other target inhibitors (for example, AKT inhibitors (AKT inhibitor VIII, AKTi-1/2, CAS accession number: 612847-09-3), Immune cells exposed to a PI3K inhibitor (Idelalisib, CAS accession number: 870281-82-6).
  • the corresponding immune cells that have not been in contact with the YEATS inhibitor of the present application may refer to immune cells that have been isolated in the same way from the same donor and have not been in contact with the YEATS inhibitor of the present application.
  • the corresponding immune cells that have not been in contact with the YEATS inhibitor of the present application may refer to immune cells from the same tumor origin of the same donor and that have not been in contact with the YEATS inhibitor of the present application.
  • the corresponding immune cells that have not been in contact with the YEATS inhibitor of the present application may refer to immune cells that have been isolated in the same way from the same tumor source of the same donor and have not been in contact with the YEATS inhibitor of the present application.
  • the corresponding immune cells that have never been in contact with the YEATS inhibitor of the present application can refer to dividing the immune cells from the same donor into two groups, wherein a group of immune cells that have never been in contact with the YEATS inhibitor of the present application can be those that have not been in contact with the YEATS inhibitor of the present application. Apply YEATS inhibitor to the corresponding immune cells contacted.
  • the corresponding immune cells that have not been in contact with the YEATS inhibitor of the present application can refer to dividing the immune cells from the same donor in the same way into two groups, one of which is the immune cells that have not been in contact with the YEATS inhibitor of the present application It may be corresponding immune cells that have not been in contact with the YEATS inhibitor of the present application.
  • the corresponding immune cells that have not been in contact with the YEATS inhibitor of the present application can refer to dividing the immune cells of the same tumor origin from the same donor into two groups, wherein a group of immune cells that have not been in contact with the YEATS inhibitor of the present application can be Corresponding immune cells that have never been in contact with the YEATS inhibitor of the present application.
  • the corresponding immune cells that have not been in contact with the YEATS inhibitor of the present application may refer to dividing the immune cells from the same tumor source from the same donor into two groups, one of which has not been contacted with the YEATS inhibitor of the present application.
  • the contacted immune cells may be corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application.
  • the immune cells comprise phagocytes, lymphocytes, neutrophils, eosinophils and/or basophils.
  • the immune cells comprise monocytes, macrophages and/or dendritic cells.
  • the immune cells comprise B cells, T cells, natural killer cells and/or natural killer-like T cells.
  • an "unmodified immune cell” or “unmodified immune cell” can refer to one in which the genome has not been modified and contains no gene regulation system or contains a control gene regulation system (e.g., empty vector control, non-targeting gRNA, plus scrambling siRNA, etc.) cells or cell populations.
  • TIL cells exposure of TIL cells to YEATS inhibitors during culture resulted in increased cell proliferation, increased cell viability, increased proportion of CD8 + cells, increased proportion of central memory cells, decreased proportion of regulatory cells, increased increased proportion of activated cells, increased proportion of tumor-specific cells, and/or increased proportion of stem-like cells; for example, T cells, TCR-T cells, and/or CAR-T cells exposed to a YEATS inhibitor during culture can achieving increased cell proliferation capacity, increased cell viability, increased proportion of CD8 + cells, increased proportion of central memory cells, decreased proportion of regulatory cells, increased proportion of activated cells, increased proportion of tumor-specific cells, and and/or increased proportion of stem-like cells; for example, NK cells exposed to YEATS inhibitors during culture can achieve increased cell proliferation, increased cell viability, increased proportion of CD8 + cells, increased proportion of central memory cells , a decreased proportion of regulatory cells, an increased proportion of activated cells, an increased proportion of tumor-specific cells, and/or an increased proportion of stem-like cells.
  • the immune cells comprise ⁇ T cells and/or ⁇ T cells.
  • the immune cells comprise tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • the TILs are TILs derived from fragments of tumor tissue, pleural effusion and/or peritoneal effusion and/or TILs derived from thawed after cryopreservation.
  • the TILs of the present application may be TILs derived from fragments of tumor tissue, pleural effusion and/or peritoneal effusion and/or TILs derived from resuscitation after cryopreservation.
  • TILs of the present application can be obtained by processing tumor tissue into tumor fragments.
  • the tumor fragments of the present application have a volume of about 1-27 cubic millimeters.
  • the tumor fragments of the present application have a volume of about 1 cubic millimeter, about 2 cubic millimeters, about 3 cubic millimeters, about 4 cubic millimeters, about 5 cubic millimeters, about 6 cubic millimeters, about 7 cubic millimeters, about 8 cubic millimeters , about 9 cubic millimeters, about 10 cubic millimeters, about 11 cubic millimeters, about 12 cubic millimeters, about 13 cubic millimeters, about 15 cubic millimeters, about 17 cubic millimeters, about 19 cubic millimeters, about 20 cubic millimeters, about 21 cubic millimeters , about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters, or about 27 cubic millimeters.
  • the immune cells comprise engineered immune receptors displayed on the cell surface.
  • the engineered immune receptor specifically binds to an antigen expressed on a target cell.
  • the immune cells comprise chimeric antigen receptors and/or T cell receptors.
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: reducing the expression and/or activity of a protein containing a YEATS domain and/or a functionally active fragment thereof in the TIL.
  • TIL tumor-infiltrating lymphocytes
  • TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro can be subjected to at least one stage of in vitro expansion, wherein, in at least one stage of the in vitro expansion, Decreasing the expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragment in the TIL.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage in vitro expansion and the second stage in vitro expansion, and in the first stage of the present application
  • the expression and/or activity of the protein containing the YEATS domain in the TIL and/or its functionally active fragment can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage in vitro expansion and the second stage in vitro expansion, and in the first stage of the present application
  • the expression and/or activity of the protein containing the YEATS domain in the TIL and/or its functionally active fragment can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage in vitro expansion and the second stage in vitro expansion, and in the first stage of the present application
  • the expression and/or activity of the protein containing the YEATS domain in the TIL and/or its functionally active fragment can be reduced, and in the second-stage in vitro expansion of the present application, all The expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragment in the TIL is reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the first stage of in vitro amplification of the present application, the expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragments in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the second stage of in vitro amplification of the present application, the expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragment in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the third stage of in vitro amplification of the present application, the expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragment in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the first stage of in vitro amplification of the application, the expression and/or activity of the protein containing the YEATS domain in the TIL and/or its functionally active fragment can be reduced, and in the second stage of the application in vitro During amplification, the expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragment in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the first stage of in vitro amplification of the present application, the expression and/or activity of the protein containing the YEATS domain in the TIL and/or its functionally active fragments can be reduced, and in the third stage of the present application in vitro During amplification, the expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragment in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the second stage of in vitro amplification of the application, the expression and/or activity of the protein containing the YEATS domain in the TIL and/or its functionally active fragment can be reduced, and in the third stage of the application in vitro During amplification, the expression and/or activity of the protein containing the YEATS domain and/or its functionally active fragment in the TIL can be reduced.
  • the TIL derived from tumor tissue, pleural effusion and/or peritoneal effusion of the present application and not expanded in vitro can be subjected to the first stage of in vitro expansion, the second stage of in vitro expansion and the third stage of in vitro expansion , and in the first stage of in vitro amplification of the application, the expression and/or activity of the protein containing the YEATS domain in the TIL and/or its functionally active fragment can be reduced, and in the second stage of the application in vitro During the expansion, the expression and/or activity of the protein containing the YEATS domain in the TIL and/or its functionally active fragment can be reduced, and in the third stage of in vitro expansion of the application, the expression and/or activity of the protein in the TIL can be reduced.
  • the expression and/or activity of a YEATS domain-containing protein and/or a functionally active fragment thereof is reduced.
  • each stage of in vitro expansion can be divided by the change in the number of TIL cells.
  • the number of TIL cells increases by at least about 1-fold, it can be considered that the TIL cells have entered the next stage of in vitro expansion.
  • the number of TIL cells is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times, or at least about 1000 times, TIL cells can be considered to enter the next stage of in vitro expansion.
  • each stage of in vitro expansion can also be divided by changing the conditions of TIL cell culture.
  • TIL cells can be considered to enter the next stage of in vitro expansion when 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 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.
  • TIL cells can be considered to enter the next stage of in vitro expansion.
  • each stage can also be divided by the days of TIL cell culture.
  • TIL cells when TIL cells are cultured in vitro for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days , about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, about 50 days or about After 100 days, TIL cells can be considered to enter the next stage of in vitro expansion.
  • the immune cells that have been contacted with the YEATS inhibitor exhibit improved cellular properties compared to immune cells that have not been contacted with the YEATS inhibitor.
  • the improved cell properties comprise one or more selected from the group consisting of improved cell proliferation, increased proportion of viable cells, improved proportion of cell subsets, increased cytokine secretion and increased tumor cell Lethality.
  • the improved proportion of cell subpopulations comprises one or more selected from the group consisting of increased proportion of activated cells, decreased proportion of regulatory cells, decreased proportion of exhausted cells, increased proportion of central memory cells, decreased The proportion of apoptotic cells and the proportion of stem cell-like cells increased.
  • the number of immune cells improved in the present application refers to the corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application or contacted with other inhibitors in the in vitro expansion stage.
  • the number of cells of the immune cells of the present invention contacted by the YEATS inhibitor can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, At least about 40 times, or at least about 50 times.
  • an increased proportion of viable cells can be manifested as an increase in immune cell viability.
  • the proportion of viable cells of the present application can refer to the corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application or contacted with other inhibitors in the in vitro expansion stage, and in at least one in vitro expansion stage.
  • the proportion of living cells of the immune cells of the present application contacted by the YEATS inhibitor of the present application can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the improved cytokine secretion ability of the present application may refer to the increase of cytokine secretion ability of immune cells selected from the following group: IL-2, IL-6, CD107a, GZMB, TNF and IFN ⁇ .
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of cytokine-secreting cells in immune cells of the present application can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of cytokine-secreting cells in immune cells of the present application can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, At least about 1%, at least about
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of cells secreting CD107A among immune cells of the present application can be increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least About 40 times, or at least about 50 times.
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of cells secreting CD107A among immune cells of the present application can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least About 1%, at least about 0.5%
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of IL-2-secreting cells among immune cells of the present application can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold , at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times , at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of cells secreting IL-2 among immune cells of the present application can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40% , at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12% , at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2% , at least about 1%, at least
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of IL-6 secreting cells in the immune cells of the present application can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times , at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times , at least about 40 times, or at least about 50 times.
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of cells secreting IL-6 among immune cells of the present application can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40% , at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12% , at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2% , at least about 1%, at least
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the ratio of IFN ⁇ -secreting cells among immune cells of the present application can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 7-fold, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least About 40 times, or at least about 50 times.
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of IFN ⁇ -secreting cells in the immune cells of the present application can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least About 1%, at least about
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of TNF-secreting cells among immune cells of the present application can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least About 40 times, or at least about 50 times.
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of TNF-secreting cells in the immune cells of the present application can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least About 1%, at least about 0.
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of GZMB-secreting cells in immune cells of the present application can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least About 40 times, or at least about 50 times.
  • the increased cytokine secretion capacity of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the proportion of GZMB-secreting cells among immune cells of the present application can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least About 1%, at least about 0.
  • the determination of the cytokine secreting ability of the immune cells of the present application may be by measuring the cytokine expressing ability of the immune cells.
  • the cytokine secreting ability of the immune cells of the present application can be determined by the method of cell flow cytometry.
  • the cytokine secreting ability of the immune cells of the present application is determined by measuring the cytokine releasing ability of the immune cells.
  • the cytokine secretion ability of the immune cells of the present application is measured by the CBA method (Cytometric Bead Array).
  • the improved tumor cell killing ability of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with the corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the tumor cell killing rate of the immune cells of the present application can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times.
  • the improved tumor cell killing ability of the present application may refer to contact with the YEATS inhibitor of the present application in at least one in vitro expansion stage compared with the corresponding immune cells that have not been contacted with the YEATS inhibitor of the present application in the in vitro expansion stage
  • the tumor cell killing rate of immune cells of the present application can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%,
  • the improved cell subset ratio of the present application may comprise one or more selected from the following group: increased CD8 + cell ratio, increased central memory cell ratio, decreased regulatory cell ratio, increased activated cell ratio proportion, increased proportion of tumor-specific cells, and increased proportion of stem-like cells.
  • the increased proportion of CD8 + cells of the present application may be an increase in the proportion of CD8 positive cells among immune cells.
  • the proportion of CD8 + cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the increased proportion of activated cells of the present application may be an increase in the proportion of CD28 + , CD25 + , and/or 41BB + cells among immune cells.
  • the proportion of activated cells in immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% , at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% , at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1% , at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or may be increased
  • the proportion of CD28 + cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the proportion of CD25 + cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the proportion of 41BB + cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or may be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the reduced proportion of exhausted cells of the present application can be an increase in the proportion of PD1 + , LAG3 + , TIM3 + , and/or CD39 + cells among immune cells.
  • the proportion of exhausted cells in immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% , at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% , at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1% , at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0. 0.
  • the proportion of PD1 + cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11 %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the proportion of LAG3 + cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the proportion of TIM3 + cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the proportion of CD39 + cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold , at least about 5 times, at least about 6 times, at least about
  • the reduced proportion of regulatory cells of the present application may be a reduction in the proportion of CD4 + CD25 + Foxp3 + cells among immune cells.
  • the proportion of regulatory cells among immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the reduced proportion of apoptotic cells of the present application may be a decrease in the proportion of CD95 + caspass3 + cells and/or CD95 + DR5 + cells in immune cells.
  • the proportion of apoptotic cells in immune cells can be reduced by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%,
  • the increased proportion of stem cells of the present application may be an increase in the proportion of CD69 ⁇ CD39 ⁇ cells and/or TCF1 + cells among immune cells.
  • the proportion of cells with stemness among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least About 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • the increased proportion of central memory cells of the present application may be an increase in the proportion of CD45RA ⁇ CCR7 + or CD45RO + CD62L + cells among immune cells.
  • the proportion of central memory cells among immune cells can be increased by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30% %, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11% %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, at least about 1 %, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%.
  • it comprises subjecting said immune cells to at least one stage of in vitro expansion, wherein during at least one stage of said in vitro expansion, said immune cells are contacted with said YEATS inhibitor.
  • the TILs derived from tumor tissue, pleural effusion and/or peritoneal effusion and not expanded in vitro are subjected to a first stage of in vitro expansion and a second stage of in vitro expansion, and in the second stage In the one-stage in vitro expansion, the TILs expanded in vitro by the first stage are contacted with the YEATS inhibitor.
  • the first stage of in vitro expansion is performed for at least about 7 days.
  • the second stage of in vitro expansion is performed for at least about 7 days.
  • the YEATS inhibitor of the present application may comprise a substance capable of inhibiting the interaction between the YEATS domain and histone.
  • a YEATS inhibitor of the present application may bind to a substance of the histone binding domain of YEATS.
  • the YEATS inhibitors of the present application can bind to substances that are modified from the histone binding domain of YEATS.
  • the YEATS inhibitors of the present application can bind to substances in the histone binding domain of acetylated YEATS.
  • the YEATS inhibitor of the present application may comprise a substance capable of inhibiting the interaction between the YEATS domain and histone.
  • the YEATS inhibitor may comprise a substance capable of inhibiting the binding of the YEATS domain to histone.
  • the YEATS inhibitor may comprise a substance capable of inhibiting the binding of the YEATS domain to histone with an IC 50 value of about 200 ⁇ M or less.
  • the YEATS inhibitor can comprise a compound capable of producing at about 190 ⁇ M or less, about 180 ⁇ M or less, about 170 ⁇ M or less, about 160 ⁇ M or less, about 150 ⁇ M or less, about 140 ⁇ M or less, about 130 ⁇ M or Lower, about 120 ⁇ M or lower, about 110 ⁇ M or lower, about 100 ⁇ M or lower, about 90 ⁇ M or lower, about 70 ⁇ M or lower, about 50 ⁇ M or lower, about 20 ⁇ M or lower, about 10 ⁇ M or lower Agents that inhibit the binding of the YEATS domain to histones with an IC50 value of low, about 5 ⁇ M or less, about 2 ⁇ M or less, about 1 ⁇ M, about 0.5 ⁇ M, about 0.3 ⁇ M or less.
  • the YEATS inhibitors of the present application may inhibit YEATS by at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, compared to the inhibition of non-YEATS targets. %, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14% %, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4% %, at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be increased by at least about 1-fold , at least about 2 times, at least about 3 times, at least about
  • the YEATS inhibitor can comprise a substance capable of binding a YEATS domain with a KD value of about 0.13 ⁇ M or less.
  • the YEATS inhibitor can comprise a compound capable of producing at about 9 ⁇ M or less, about 8 ⁇ M or less, about 7 ⁇ M or less, about 6 ⁇ M or less, about 5 ⁇ M or less, about 4 ⁇ M or less, about 3 ⁇ M or Lower, about 2 ⁇ M or lower, about 1 ⁇ M or lower, about 0.9 ⁇ M or lower, about 0.8 ⁇ M or lower, about 0.7 ⁇ M or lower, about 0.6 ⁇ M or lower, about 0.5 ⁇ M or lower , about 0.4 ⁇ M or less, about 0.3 ⁇ M or less, about 0.2 ⁇ M or less, or about 0.1 ⁇ M or less of a KD value that binds to a YEATS domain.
  • the YEATS inhibitor may comprise one or more selected from the group consisting of nucleic acid molecules, polypeptides and small molecule compounds.
  • the YEATS inhibitor can comprise DNA, and/or RNA; for example, the YEATS inhibitor can comprise an antibody and/or an antigen-binding fragment thereof; for example, the YEATS inhibitor can comprise a small molecule compound, such as a small molecule Inhibitors.
  • the YEATS inhibitor does not substantially bind one or more targets selected from the group consisting of: Akt, and PI3K.
  • the YEATS inhibitor does not substantially affect the activity of Akt, and/or PI3K.
  • adding the YEATS inhibitor of the present application can only reduce the activity of Akt and/or PI3K by about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less, or about 0.01% or less.
  • the activity of Akt and/or PI3K can be measured by methods commonly used in the art.
  • the YEATS inhibitors of the present application may have at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60% higher inhibition of YEATS than the inhibition of the Akt target , at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14% , at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4% , at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be increased by at
  • the YEATS inhibitors of the present application may have at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60% greater inhibition of YEATS than the inhibition of the PI3K target , at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14% , at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4% , at least about 3%, at least about 2%, at least about 1%, at least about 0.5%, at least about 0.4%, at least about 0.3%, at least about 0.2%, or at least about 0.1%, or can be increased by at least about 1-fold, At least about 2 times, at least about 3 times, at least about 4 times, at least about
  • the YEATS inhibitor can comprise a YEATS specific inhibitor, eg, SGC-iMLLT (CAS Accession No.: 2255338-25-9).
  • a YEATS-specific inhibitor may comprise only substances that directly and specifically inhibit the YEATS protein.
  • a YEATS-specific inhibitor may not include substances that affect YEATS activity by affecting YEATS transcription.
  • the YEATS inhibitor can comprise a substance capable of reducing the expression and/or activity of a nucleic acid molecule encoding YEATS, for example, can reduce the maturation process of transcription, translation and/or post-translational modification of a nucleic acid molecule encoding YEATS.
  • the YEATS inhibitor comprises a substance capable of reducing the expression of YEATS protein.
  • the YEATS inhibitor comprises a substance capable of affecting the activity of the YEATS protein.
  • the initial concentration of each of the YEATS inhibitors in the cell culture medium of the immune cells is at least about 1 ⁇ M.
  • the initial concentration of SGC-iMLLT in the cell culture medium of the immune cells can be at least about 1 ⁇ M.
  • the initial concentration of each of the YEATS inhibitors in the cell culture medium of the immune cells is at least about 1 ⁇ M.
  • the initial concentration of each of the YEATS inhibitors in the cell culture medium of the immune cells can be at least about 0.01 ⁇ M, at least about 0.02 ⁇ M, at least about 0.03 ⁇ M, at least about 0.05 ⁇ M, at least about 0.07 ⁇ M, at least about 0.1 ⁇ M, at least about 0.2 ⁇ M, at least about 0.3 ⁇ M, at least about 0.4 ⁇ M, at least about 0.5 ⁇ M, at least about 0.6 ⁇ M, at least about 0.7 ⁇ M, at least about 0.8 ⁇ M, at least about 0.9 ⁇ M, at least about 1 ⁇ M, at least about 1.5 ⁇ M, at least about 1.6 ⁇ M, at least about 1.7 ⁇ M, at least about 1.8 ⁇ M, at least about 1.9 ⁇ M, at least about 2 ⁇ M, at least about 2.5 ⁇ M, at least about 3 ⁇ M, at least about 3.5 ⁇ M, at least about 4 ⁇ M, at least About 4.5 ⁇ M,
  • the initial concentration of each of the YEATS inhibitors in the cell culture medium of the immune cells may independently of each other be from about 1 ⁇ M to about 200 ⁇ M, from about 5 ⁇ M to about 200 ⁇ M, from about 10 ⁇ M to about 200 ⁇ M, from about 50 ⁇ M to about 200 ⁇ M, about 100 ⁇ M to about 200 ⁇ M, about 150 ⁇ M to about 200 ⁇ M, about 1 ⁇ M to about 150 ⁇ M, about 5 ⁇ M to about 150 ⁇ M, about 10 ⁇ M to about 150 ⁇ M, about 50 ⁇ M to about 150 ⁇ M, about 100 ⁇ M to about 150 ⁇ M, about 1 ⁇ M to about 100 ⁇ M, About 5 ⁇ M to about 100 ⁇ M, about 10 ⁇ M to about 100 ⁇ M, about 50 ⁇ M to about 100 ⁇ M, about 1 ⁇ M to about 50 ⁇ M, about 2 ⁇ M to about 50 ⁇ M, about 5 ⁇ M to about 50 ⁇ M, about 10 ⁇
  • the YEATS inhibitor comprises a compound as defined in formula (I) or a pharmaceutically acceptable salt thereof,
  • A is CR 2 or N
  • each B 1 and B 2 is an independently selected divalent linking unit, wherein the divalent linking unit passes through amide, ester, thioester, sulfamide, imidate, imide, sulfonate or sulfonate Amides are linked head-to-tail;
  • D is H or unsubstituted or substituted hydrocarbyl, carbocyclyl or heterocyclyl ;
  • D 2 is H, NH 2 , NHR 15 , NR 16 R 17 , OH, OR 18 , or unsubstituted or substituted hydrocarbon, carbocyclyl or heterocyclyl;
  • X is unsubstituted or substituted C 1-10 alkyl, C 1-10 alkenyl, C 1-10 alkynyl, C 1-10 carbocyclyl, C 1-10 heteroalkyl, C 1-10 10 heteroalkenyl, C 1-10 heteroalkynyl or C 1-10 heterocyclyl;
  • Y is NR 3 , O or S
  • Z is -CO-, -CS-, -CNR 4 -, -SO- or -SO 2 -;
  • R 2 , R 3 and R 4 are independently H or C 1-10 hydrocarbon group, C 1-10 carbocyclyl or C 1-10 heterocyclyl;
  • R is a conjugated/delocalized group, for example, an unsubstituted or substituted heterocyclic or carbocyclic aromatic ring (monocyclic, bicyclic, tricyclic, tetracyclic), or unsubstituted or substituted alkenyl or alkynyl;
  • R 15 , R 16 , R 17 and R 18 can be H or alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, alkoxy, alkylamino, di Alkylamino, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted alkoxy, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, C 3 -C 20 ring, substituted C 3 -C 20 ring, heterocycle or substituted heterocycle; and
  • n and n are each independently an integer of 0 to 10, wherein at least one of m or n is not 0.
  • A could be CR 2 or N.
  • R can be H or alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, alkoxy, alkylamino, dialkylamino, hydroxy, Aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted alkoxy, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, C 3 -C 20 ring , a substituted C 3 -C 20 ring, a heterocycle or a substituted heterocyclic group.
  • A can be CR 2 and R 2 can be H.
  • B 1 may be -NH-, -O-, -S- or -(CH 2 ) p- , wherein p is an integer from 1 to 6, eg, 1, 2, 3, 4, 5, and 6.
  • D 2 can be H, NH 2 , NHR 15 , NR 16 R 17 , OH, OR 18 or unsubstituted or substituted hydrocarbyl, carbocyclyl or heterocyclyl, wherein R 15 , R 16 , R 17 and R 18 can be H or alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, alkoxy, alkylamino, dialkylamino, hydroxy, aryl radical, substituted aryl, heteroaryl, substituted heteroaryl, substituted alkoxy, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, C 3 -C 20 ring, Substituted C3 - C20 ring, heterocycle or substituted heterocycle.
  • a divalent unit could be
  • W can be CR 2
  • R 11 can be a side chain
  • R 12 and R 13 can be such as amide linking group, ester linking group, thioester linking group, thiamine linking group, imide ester linking group, imide Part of linkers, sulfonate linkers, and sulfonamide linkers.
  • Different divalent units can be attached to the same or different types of linkers, the attached R12 and R13 groups being compatible components of the target linker.
  • R 11 may consist of an existing side chain or part of a normal side chain of an oligomer monomer.
  • the chain base oligomer is a peptide
  • one of the side chains of the amino acids in the peptide may comprise a portion of R 11 , or may be substituted by R 11 .
  • compositions of this form include at least one R 11 terminating in R 1 (wherein R 1 is as defined herein).
  • compositions of this form include at least one R 11 defined according to the following structure:
  • dashed lines indicate points of attachment to the structures above, where R 1 can be a conjugated/delocalized group and X, Y, and Z are as defined herein.
  • the divalent units are all independently any ⁇ -amino acid.
  • the divalent units are each an ⁇ -amino acid independently selected from Lys, Gln, Thr, Ala, Arg, Ser, and Gly.
  • D can be H or unsubstituted or substituted hydrocarbyl, carbocyclyl or heterocyclyl.
  • D can be alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, alkoxy, alkylamino, dialkylamino, hydroxy, aryl , substituted aryl, heteroaryl, substituted heteroaryl, substituted alkoxy, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, C 3 -C 20 ring, substituted C 3 -C 20 ring, heterocycle or substituted heterocycle group.
  • D 1 can be H.
  • D 1 can be carboxybenzyl (Cbz). In some forms, D can be acetyl. In some forms, D can be benzylcarbonyl. In some forms, D can be phenethylcarbonyl. In some forms, D can be phenylpropylcarbonyl. In some forms, D can be naphthylethylcarbonyl.
  • D 2 can be H, -NH 2 , -NHR 15 , -NR 16 R 17 , -OH, -OR 18 or unsubstituted or substituted hydrocarbyl, carbocyclyl or heterocyclyl, wherein R 15 , R 16 , R 17 and R 18 can be H or alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, alkoxy, alkylamino, dialkylamino, hydroxy, Aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted alkoxy, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, C 3 -C 20 ring , a substituted C 3 -C 20 ring, a heterocycle or a substituted heterocycle.
  • D2 can be H or -NH2 .
  • D can be alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, alkoxy, alkylamino, dialkylamino, hydroxy, aryl , substituted aryl, heteroaryl, substituted heteroaryl, substituted alkoxy, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, C 3 -C 20 ring, substituted C 3 -C 20 ring, heterocycle or substituted heterocycle group.
  • D2 can be H.
  • D2 can be -NH2 .
  • D 2 can be OH or OR 18 .
  • D2 can be OH.
  • X can be unsubstituted or substituted C 1-10 alkyl, C 1-10 alkenyl, C 1-10 alkynyl, C 1-10 carbocyclyl, C 1-10 heteroalkyl, C 1 -10 heteroalkenyl, C 1-10 heteroalkynyl or C 1-10 heterocyclyl.
  • X can be alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, alkoxy, alkylamino, dialkylamino, hydroxy, aryl, Substituted aryl, heteroaryl, substituted heteroaryl, substituted alkoxy, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, C 3 -C 20 ring, substituted C 3 -C 20 ring, heterocycle or substituted heterocycle group.
  • X can be -(CH 2 ) p -, where p is an integer from 1 to 6.
  • X can be -(CH 2 ) p -, where p is an integer from 2 to 5. In some forms, X can be -(CH 2 ) p- where p is 3 or 4. In some forms, X can be -(CH 2 ) p - where p is 4.
  • Y can be -NR 3 , -O- or -S-.
  • R 3 can be H or C 1-10 hydrocarbyl, C 1-10 carbocyclyl, or C 1-10 heterocyclyl.
  • R can be H or alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, alkoxy, alkylamino, dialkylamino, hydroxy, Aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted alkoxy, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, C 3 -C 20 ring , a substituted C 3 -C 20 ring, a heterocycle or a substituted heterocyclic group.
  • Y can be NR3 , and R3 can be H.
  • Z may be -CO-, -CS-, -CNR4- , -SO- or -SO2- .
  • Z can be -CO-.
  • R 4 can be H or C 1-10 alkyl, C 1-10 carbocyclyl, or C 1-10 heterocyclyl.
  • R can be H or alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, alkoxy, alkylamino, dialkylamino, hydroxy, Aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted alkoxy, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, C 3 -C 20 ring , a substituted C 3 -C 20 ring, a heterocycle or a substituted heterocyclic group.
  • the conjugated/delocalized group can be an unsubstituted or substituted heterocyclic or carbocyclic aromatic ring, or an unsubstituted or substituted alkenyl or alkynyl group, wherein the conjugated/delocalized Domain groups include p-orbitals that allow the delocalization of ⁇ electrons.
  • the conjugated/delocalized group can be an unsubstituted or substituted aromatic ring.
  • the conjugated/delocalized group can be unsubstituted or substituted aromatic monocyclic, bicyclic, tricyclic or tetracyclic.
  • the conjugated/delocalized group can be an unsubstituted or substituted alkenyl or alkynyl group.
  • R can be an unsubstituted or substituted heterocyclic or carbocyclic aromatic ring, or an unsubstituted or substituted alkenyl or alkynyl group, wherein the conjugated/delocalized group includes Delocalized p-orbitals.
  • R 1 can be an unsubstituted or substituted aromatic ring.
  • R can be unsubstituted or substituted aromatic monocyclic, bicyclic, tricyclic or tetracyclic.
  • R can be unsubstituted or substituted alkenyl or alkynyl.
  • R can be:
  • dashed lines indicate points of attachment of the aforementioned groups.
  • an unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 2-position and oxygen at the 3-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 2-position and oxygen at the 4-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 2-position and oxygen at the 5-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 2-position and oxygen at the 7-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 2-position and oxygen at the 8-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 2-position and oxygen at the 9-position.
  • an unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 3-position and oxygen at the 4-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 3-position and oxygen at the 5-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 3-position and oxygen at the 7-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 3-position and oxygen at the 8-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 3 position and oxygen at the 9 position.
  • an unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 4-position and oxygen at the 5-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 4-position and oxygen at the 7-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 4-position and oxygen at the 8-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 4-position and oxygen at the 9-position.
  • an unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 5-position and oxygen at the 7-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 5-position and oxygen at the 8-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 5-position and oxygen at the 9-position.
  • an unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 6-position and oxygen at the 7-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 6-position and oxygen at the 8-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 6-position and oxygen at the 9-position.
  • an unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 7-position and oxygen at the 8-position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 7 position and oxygen at the 9 position.
  • the unsubstituted C9 bicyclic heterocyclic aromatic ring can have nitrogen at the 8-position and oxygen at the 9-position.
  • m is an integer of 0 to 10.
  • m can be 0 to 10, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 0 to 9, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 0 to 8, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 0 to 7, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 0 to 6, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 0 to 5, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 0 to 4, 1 to 4, 2 to 4, Integers of 3 to 4, 0 to 3, 1 to 3, 2 to 3, 0 to 2, 1 to 2, and 0 to 1.
  • m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • n is an integer of 0 to 10.
  • n can be 0 to 10, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 0 to 9, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 0 to 8, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 0 to 7, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 0 to 6, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 0 to 5, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 0 to 4, 1 to 4, 2 to 4, Integers of 3 to 4, 0 to 3, 1 to 3, 2 to 3, 0 to 2, 1 to 2, and 0 to 1.
  • n can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • m+n may be an integer of 0 to 20.
  • the YEATS inhibitor comprises a compound as defined by formula (II) or a pharmaceutically acceptable salt thereof,
  • each J1 and J2 is independently any ⁇ - amino acid ;
  • R is a conjugated/delocalized group, for example, an unsubstituted or substituted heterocyclic or carbocyclic aromatic ring (monocyclic, bicyclic, tricyclic, tetracyclic), or unsubstituted or substituted alkenyl or alkynyl; and
  • n and n are each independently an integer of 0 to 10, wherein at least one of m or n is not 0.
  • the YEATS inhibitor comprises a compound as defined by formula (III) or a pharmaceutically acceptable salt thereof,
  • each J1 and J2 is independently any ⁇ - amino acid ;
  • X is unsubstituted or substituted C 1-10 alkyl, C 1-10 alkenyl, C 1-10 alkynyl, C 1-10 carbocyclyl, C 1-10 heteroalkyl, C 1-10 10 heteroalkenyl, C 1-10 heteroalkynyl or C 1-10 heterocyclyl;
  • Y is NR 3 , O or S
  • Z is -CO-, -CS-, -CNR 4 -, -SO- and -SO 2 -;
  • R 3 and R 4 are independently H, C 1-10 hydrocarbon group, C 1-10 carbocyclyl or C 1-10 heterocyclyl;
  • R is a conjugated/delocalized group, for example, an unsubstituted or substituted heterocyclic or carbocyclic aromatic ring (monocyclic, bicyclic, tricyclic, tetracyclic), or unsubstituted or substituted alkenyl or alkynyl; and
  • n and n are each independently an integer of 0 to 10, wherein at least one of m or n is not 0.
  • the YEATS inhibitor comprises a compound as defined by formula (IV) or a pharmaceutically acceptable salt thereof,
  • each E 1 and E 2 are independently unsubstituted or substituted C 1-10 alkyl, C 1-10 alkenyl, C 1-10 alkynyl, C 1-10 carbocyclyl, C 1- 10 heteroalkyl, C 1-10 heteroalkenyl, C 1-10 heteroalkynyl or C 1-10 heterocyclyl, or O, S or NR 5 ;
  • X is unsubstituted or substituted C 1-10 alkyl, C 1-10 alkenyl, C 1-10 alkynyl, C 1-10 carbocyclyl, C 1-10 heteroalkyl, C 1-10 10 heteroalkenyl, C 1-10 heteroalkynyl or C 1-10 heterocyclyl;
  • Y is NR 3 , O or S
  • Z is -CO-, -CS-, -CNR 4 -, -SO- or -SO 2 -;
  • R 3 , R 4 and R 5 are independently H, C 1-10 hydrocarbon group, C 1-10 carbocyclyl or C 1-10 heterocyclyl;
  • R is a conjugated/delocalized group, for example, an unsubstituted or substituted heterocyclic or carbocyclic aromatic ring (monocyclic, bicyclic, tricyclic, tetracyclic), or unsubstituted or substituted alkenyl or alkynyl; and
  • n and n are each independently an integer of 0 to 10, wherein at least one of m or n is not 0.
  • At least two of the Bi linking units comprise side chains, wherein the two side chains of the Bi linking units are covalently linked to each other to form a cyclic oligomer.
  • the YEATS inhibitor comprises a compound as defined by formula (V) or a pharmaceutically acceptable salt thereof,
  • R is a conjugated/delocalized group, including an unsubstituted or substituted aromatic ring (monocyclic, bicyclic, tricyclic, tetracyclic), unsubstituted or substituted alkenyl or alkynyl;
  • R 6 , R 7 , R 8 , R 9 and R 10 are independently H or C 1-10 hydrocarbon group, C 1-10 carbocyclyl or C 1-10 heterocyclyl;
  • each of G 1 , G 2 and G 3 is unsubstituted or substituted C 1-10 alkyl, C 1-10 alkenyl, C 1-10 alkynyl, C 1-10 carbocyclyl, C 1 -10 heteroalkyl, C 1-10 heteroalkenyl, C 1-10 heteroalkynyl, C 1-10 heterocyclyl or 5 to 9 membered heteroaryl; and
  • i 1 , i 2 , j 1 , j 2 , k 1 , and k 2 are each independently an integer of 0 to 10.
  • B2 linking units comprise side chains, wherein the two side chains of the B2 linking units are covalently linked to each other to form a cyclic oligomer.
  • the compound can inhibit ⁇ - ⁇ - ⁇ stacking interactions.
  • the compound selectively targets the YEATS protein domain.
  • the initial concentration of each of the YEATS inhibitors in the cell culture medium of the immune cells is about 1 ⁇ M or greater.
  • the initial concentration of each of the YEATS inhibitors in the cell culture medium of the immune cells is about 1 ⁇ M to about 200 ⁇ M.
  • the immune cells may be contacted with the one or more cell activators and the YEATS domain-containing protein and/or its function in the immune cells may be in a single stage of the in vitro expansion of the present application Expression and/or activity of the active fragment is reduced.
  • the cell activator may comprise an agonist for one or more targets selected from the group consisting of: CD3, CD28, HVEM, CD40L, OX40, and 4-1BB.
  • immune cells of the present application are contacted with a YEATS inhibitor of the present application and with one or more cell activators of the present application.
  • the TILs of the present application can be contacted with the YEATS inhibitor of the present application and contacted with one or more cell activators of the present application.
  • the TILs of the present application can be contacted with the YEATS inhibitor of the present application and contacted with one or more cell activators of the present application.
  • the TILs of the present application can be contacted with the YEATS inhibitor of the present application and contacted with one or more cell activators of the present application.
  • the immune cells of the present application can be contacted with the YEATS inhibitor of the present application and one or more cell activators of the present application substantially simultaneously.
  • the immune cells of the present application in the in vitro expansion of the present application in a single stage, can be contacted with the YEATS inhibitor of the present application 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 cell activators of the present application.
  • the immune cells of the present application can be contacted with one or more cell activators of the present application first, for example, 2 hours in advance, 4 hours in advance, and 8 hours in advance , 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with the YEATS inhibitor of the present application.
  • the TILs of the present application can be contacted with the YEATS inhibitor of the present application and one or more cell activators of the present application substantially simultaneously.
  • the TILs of the present application in the second stage of in vitro expansion of the present application, can be contacted with the YEATS inhibitor of the present application and one or more cell activators of the present application substantially simultaneously.
  • the TILs of the present application in the third stage of in vitro expansion of the present application, can be contacted with the YEATS inhibitor of the present application and one or more cell activators of the present application substantially simultaneously.
  • the second stage in vitro expansion of the present application is performed for at least about 9 days.
  • the second stage in vitro expansion of the present application can be performed for at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the second stage of in vitro expansion of the present application can be carried out for about 9 days to about 14 days
  • the second stage of in vitro expansion of the present application can be carried out for about 9 days to about 14 days, about 10 days to about 14 days, About 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days days to about 13 days, about 9 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the second stage of in vitro expansion in the present application can be considered as the REP (rapid expansion protocol) stage.
  • the first stage of in vitro expansion of the present application can be considered the preREP stage.
  • the number of days for the second stage of in vitro expansion in the present application can be calculated from the start of the second stage of in vitro expansion. For example, when the second-stage in vitro expansion starts, it can be considered that the second-stage in vitro expansion has been performed for about 0 days. For example, if the second-stage in vitro expansion proceeds for about 24 hours, it can be considered that the second-stage in vitro expansion has been performed for about one day. For example, the day when the second-stage in vitro expansion starts can be considered as about 0 days after the second-stage in vitro expansion.
  • the number of days for the second stage of in vitro expansion in the present application can be calculated based on the number of days for the second stage of in vitro expansion. For example, on the second day after the start of the second-stage in vitro expansion, it can be considered that the second-stage in vitro expansion has been performed for about one day.
  • the cell activator of the present application may comprise one or more selected from the following group: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, and their functional activities fragment.
  • the cell activator of the present application may comprise an agonist of one or more targets selected from the following group: CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • the cell activator of the present application may comprise antibodies and antigen-binding fragments thereof selected from the group consisting of CD3, CD28, HVEM, CD40L, OX40 and 4-1BB.
  • a cell activator of the present application may comprise a CD3 agonist.
  • the cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, such as OKT3 from Miltenyi Biotech, or SP34 from BD.
  • a cell activator of the present application may comprise a CD28 agonist.
  • the cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, such as Merck's 15E8.
  • the cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain VL and heavy chain VH of OKT3 of Miltenyi Biotech, may comprise the light chain VL and heavy chain of SP34 of BD VH.
  • a cell activator of the present application may comprise a CD28 agonist.
  • the cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, for example may comprise Merck's 15E8 light chain VL and heavy chain VH.
  • the cell activator of the present application may comprise an anti-CD3 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of Miltenyi Biotech's OKT3, and may comprise the light chain of BD's SP34 LCDR1-3 and heavy chain HCDR1-3, the anti-CD3 antibody and/or antigen-binding fragment thereof of the present application may have CD3-binding ability.
  • a cell activator of the present application may comprise a CD28 agonist.
  • the cell activator of the present application may comprise an anti-CD28 antibody and/or an antigen-binding fragment thereof, for example, may comprise the light chain LCDR1-3 and heavy chain HCDR1-3 of Merck's 15E8, the anti-CD28 antibody of the present application and/or Or an antigen-binding fragment thereof may have CD28-binding ability.
  • the antibody of the present application or its antigen-binding protein comprises at least one CDR in the variable region VH of the heavy chain of the antibody.
  • the CDRs of this application may be defined according to IMGT nomenclature, the CDRs of this application may be defined according to Chothia, or the CDRs of this application may be defined according to Kabat.
  • contacting the immune cells of the present application with one or more cell activators of the present application may include one or more methods selected from the following groups: (1) adding the cell activators of the present application to the cell activators of the present application In the cell culture medium of immune cells; (2) Add the engineered cells expressing the cell activator of the present application to the cell culture medium of the immune cells of the present application; (3) Add the solid phase containing the cell activator of the present application The medium is added to the cell culture medium of the immune cells of the application.
  • contacting the immune cells of the present application with one or more cell activators of the present application may comprise adding a solid phase medium comprising the cell activators of the present application to the cell culture medium of the immune cells of the present application.
  • contacting the immune cells of the present application with one or more cell activators of the present application may comprise adding the solid phase medium comprising the CD28 antibody and the CD3 antibody of the present application to the cell culture medium of the immune cells of the present application.
  • the initial concentration of the cell activator in the cell culture medium of the immune cells of the present application may be at least about 30 ng/mL.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the immune cells of the present application can be at least about 30 ng/mL;
  • the initial concentration of the CD3 antibody of the present application in the cell culture medium of the immune cells of the present application can be At least about 30 ng/mL.
  • the selection of the initial concentration of the CD28 antibody of the present application can be independent of the selection of the initial concentration of the CD3 antibody of the present application; for example, the initial concentration of the CD28 antibody of the present application and the CD3 antibody of the present application in the cell culture medium of the immune cells of the present application Concentrations can be combined arbitrarily.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the immune cells of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL.
  • the initial concentration of the CD3 antibody of the present application in the cell culture medium of the immune cells of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL.
  • the initial concentration of the CD28 antibody of the present application in the cell culture medium of the immune cells of the present application can be arbitrarily selected from about 30 ng/mL to about 300 ng/mL, and the CD3 antibody of the present application in the cell culture medium of the immune cells of the present application
  • the initial concentration of can be arbitrarily selected from about 30ng/mL to about 300ng/mL, and the selection of the initial concentration of the CD28 antibody of the present application can be independent of the selection of the initial concentration of the CD3 antibody of the present application.
  • the diameter of the solid phase media of the present application can be from about 500 nanometers to about 10 micrometers.
  • the diameter of the solid phase medium of the present application can be measured by a transmission electron microscope.
  • the diameter of the solid phase media of the present application can be from about 1 nanometer to about 500 nanometers.
  • the diameter of the solid phase media of the present application can be from about 100 nanometers to about 500 nanometers.
  • the diameter of the solid phase media of the present application can be from about 200 nanometers to about 500 nanometers.
  • the diameter of the solid phase medium of the present application can be measured by a transmission electron microscope.
  • the solid phase medium of the present application may comprise a polymer.
  • the solid phase media of the present application may comprise dextran.
  • the solid phase medium of the present application contains at least about 25 ⁇ g of the cell activator of the present application per mg.
  • the solid phase medium containing the cell activator of the present application is added to the cell culture medium of the immune cells of the present application at a ratio of the solid phase medium of the present application to the immune cells of the present application of about 1:100-about 1:2000.
  • the solid phase medium containing the cell activator of the present application is added to the cell culture medium of the immune cells of the present application at a ratio of about 2:1 to about 1:2 of the solid phase medium of the present application to the immune cells of the present application.
  • the ratio of the solid phase medium of the present application to the immune cells of the present application can be about 2:1 to about 1:2.
  • the solid phase medium of the cell activator is added to the cell culture medium of the immune cells of the present application.
  • the diameter of the solid phase medium of the present application when the diameter of the solid phase medium of the present application is about 100 nanometers to about 500 nanometers, it can be about 2:1-about 1:2, about 2:1-about 1:1, or about 1:1 -
  • the ratio of the solid medium of the present application to the immune cells of the present application is about 1:2
  • the solid phase medium comprising the cell activator of the present application such as CD3 agonist and/or CD28 agonist, is added to the cell culture of the immune cells of the present application Base.
  • the ratio of the solid phase medium of the present application to the immune cells of the present application can be about 1:100-about 1:2000.
  • the solid phase medium of the cell activator is added to the cell culture medium of the immune cells of the present application.
  • the diameter of the solid phase medium of the present application when the diameter of the solid phase medium of the present application is about 100 nanometers to about 500 nanometers, it can be about 1:100-about 1:2000, about 1:200-about 1:2000, about 1:300- About 1:2000, about 1:400-about 1:2000, about 1:500-about 1:2000, about 1:600-about 1:2000, about 1:700-about 1:2000, about About 1:800-about 1:2000, about 1:900-about 1:2000, about 1:1000-about 1:2000, about 1:1200-about 1:2000, about 1:1400-about 1 :2000, about 1:1600-about 1:2000, or about 1:1800-about 1:2000 of the ratio of the solid phase medium of the application to the immune cells of the application, for example, the CD28 agonist of the application and the CD3
  • the solid phase medium of the agonist is added to the cell culture medium of the immune cells of the present application.
  • the method of the present application may further comprise: contacting the immune cells of the present application with one or more cell growth factors during at least one stage of the in vitro expansion of the present application.
  • immune cells of the present application can be contacted with a cell activator of the present application and contacted with one or more cellular growth factors of the present application.
  • the TIL of the present application in the first stage of in vitro expansion of the present application, can be contacted with the cell activator of the present application and contacted with one or more cell growth factors of the present application.
  • the TILs of the present application in the second stage of in vitro expansion of the present application, can be contacted with the cell activator of the present application and contacted with one or more cell growth factors of the present application.
  • the TIL of the present application in the third stage of in vitro expansion of the present application, can be contacted with the cell activator of the present application and contacted with one or more cell growth factors of the present application.
  • the immune cells of the present application are contacted with the cell activating agent of the present application and one or more cell growth factors of the present application substantially simultaneously.
  • immune cells of the present application can be contacted with one or more cellular growth factors of the present application and one or more cellular activators of the present application substantially simultaneously.
  • the immune cells of the present application can be first contacted with one or more cell growth factors of the present application, for example, 2 hours in advance, 4 hours in advance, or 8 hours in advance , 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more cell activators of the present application.
  • one or more cell growth factors of the present application for example, 2 hours in advance, 4 hours in advance, or 8 hours in advance , 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc.
  • the immune cells of the present application can be contacted with one or more cell activators of the present application first, for example, 2 hours in advance, 4 hours in advance, and 8 hours in advance , 12 hours in advance, 24 hours in advance, or 48 hours in advance, etc., and then contact with one or more cell growth factors of the present application.
  • the immune cells of the present application can be contacted with the cell activator of the present application and one or more cell growth factors of the present application substantially simultaneously.
  • the TILs of the present application can be contacted with the cell activator of the present application and one or more cell growth factors of the present application substantially simultaneously.
  • the TILs of the present application can be contacted with the cell activator of the present application and one or more cell growth factors of the present application substantially simultaneously.
  • the cell growth factor of the present application can be selected from one or more of the following groups: IL-2, IL-7, IL-12, IL-15, IL-21, gamma interferon, and their functionally active fragments .
  • the cell growth factors of the present application may comprise IL-2 and/or functionally active fragments thereof.
  • a functionally active fragment of IL-2 may comprise a fragment of IL-2 known in the art that binds to the IL-2 receptor of a cell.
  • contacting the immune cells of the present application with one or more cell growth factors of the present application may comprise adding the cell growth factors of the present application to the cell culture medium of the immune cells of the present application.
  • the initial concentration of the cell growth factors of the present application in the cell culture medium of the immune cells of the present application can be at least about 300 IU/mL.
  • the initial concentration of IL-2 of the present application in the cell culture medium of the immune cells of the present application can be at least about 350IU/mL, at least about 400IU/mL, at least about 500IU/mL, at least about 600IU/mL, at least about 700IU/mL mL, at least about 800 IU/mL, at least about 900 IU/mL, at least about 1000 IU/mL, at least about 1100 IU/mL, at least about 1200 IU/mL, at least about 1300 IU/mL, at least about 1400 IU/mL, at least about 1500 IU/mL, At least about 2000 IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL, at least about 2700 IU/mL, at least about 2800 IU/mL, at least about 2900 IU/mL, at least about 3000 IU/mL, at least about 3100 IU/mL, at least about 3200
  • the method of the present application may further comprise: in at least one stage of the in vitro expansion of the present application, the immune cells of the present application may be co-cultured with the feeder cells.
  • the immune cells of the present application can be contacted with one or more cell activators and/or one or more cell growth factors and co-cultured with the feeder cells of the present application, e.g.
  • the in vitro amplification of the present application at a single stage may refer to the in vitro amplification of the present application at the same stage, for example, the same in the first stage of in vitro amplification of the present application, the same in the second stage of in vitro amplification of the present application, Or the third stage of in vitro amplification in the present application.
  • the TILs of the present application can be contacted with one or more cell activators and/or one or more cell growth factors and co-cultured with the feeder cells of the present application.
  • the TIL of the present application in the second stage of in vitro expansion of the present application, can be contacted with one or more cell activators and/or one or more cell growth factors of the present application and with the feeder cells of the present application. Co-culture.
  • the TIL of the present application in the third stage of in vitro expansion of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application and with the feeder cells of the present application. Co-culture.
  • the immune cells of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then contacted with the present application.
  • Feeder cell co-culture For example, in the first stage of in vitro expansion of the present application, the TILs of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then fed with the present application. Cell co-culture.
  • the TILs of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then fed with the present application.
  • Cell co-culture For example, in the third stage of in vitro expansion of the present application, TILs of the present application can be brought into contact with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then fed with the present application. Cell co-culture.
  • the immune cells of the present application can be contacted with one or more cell activators and/or one or more cell growth factors of the present application for a certain period of time, and then contacted with the present application.
  • Feeder cell co-culture For example, the certain period of time for this application may be at least about 2 hours.
  • the certain period of time of the present application can be at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least About 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, or at least about 72 hours.
  • a certain period of time for the present application may be from about 2 hours to about 72 hours.
  • the certain period of time of the present application can be about 6 hours to about 7 hours, about 6 hours to about 8 hours, about 6 hours to about 9 hours, about 6 hours to about 10 hours, about 6 hours to about 11 hours, about 6 hours to about 12 hours, about 6 hours to about 13 hours, about 6 hours to about 14 hours, about 6 hours to about 15 hours, about 6 hours to about 16 hours, about 6 hours to about 17 hours, about 6 hours to about 18 hours, about 6 hours to about 19 hours, about 6 hours to about 20 hours, about 6 hours to about 21 hours, about 6 hours to about 22 hours, about 6 hours to about 23 hours, about 6 hours to about 24 hours, about 6 hours to about 36 hours, about 6 hours to about 48 hours, about 6 hours to about 60 hours, or about 6 hours to about 72 hours.
  • the certain period of time of the present application can be about 12 hours to about 13 hours, about 12 hours to about 14 hours, about 12 hours to about 15 hours, about 12 hours to about 16 hours, about 12 hours to about 17 hours, about 12 hours to about 18 hours, about 12 hours to about 19 hours, about 12 hours to about 20 hours, about 12 hours to about 21 hours, about 12 hours to about 22 hours, about 12 hours to about 23 hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, or about 12 hours to about 72 hours.
  • the certain period of time in the present application can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours , about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours.
  • feeder cells of the present application may comprise antigen presenting cells.
  • the feeder cells of the present application may comprise one or more selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen-presenting cells.
  • the feeder cells of the present application may be peripheral mononuclear cells.
  • the feeder cells of the present application can be irradiated feeder cells.
  • the feeder cells of the present application can be isolated artificial antigen-presenting cells (aAPC), and the artificial antigen-presenting cells of the present application can comprise cells expressing HLA-A/B/C, CD64, CD80, ICOS-L and/or CD58 , and can be modified to express more than one cell activator of the present application.
  • the feeder cells of the present application can be irradiated, eg, can be gamma irradiated, or can be X-ray irradiated.
  • the co-cultivation of the immune cells of the present application and the feeder cells of the present application may include contacting the surface of the feeder cells of the present application with the surface of the immune cells of the present application.
  • the co-culture of the immune cells of the present application and the feeder cells of the present application comprises adding the feeder cells of the present application to the cell culture medium of the immune cells of the present application.
  • the present application can add the feeder cells of the present application to the cell culture medium of the immune cells of the present application at a ratio of about 40:1 to about 400:1 of the feeder cells of the present application to the immune cells of the present application.
  • the present application can be about 40:1 to about 400:1, about 40:1 to about 300:1, about 40:1 to about 200:1, about 40:1 to about 100:1, and About 40:1-about 90:1, about 40:1-about 80:1, about 40:1-about 70:1, about 40:1-about 60:1, about 40:1-about 50:1, about 50:1-about 400:1, about 60:1-about 400:1, about 70:1-about 400:1, about 80:1-about 400:1, about 90:1 to about 400:1, about 100:1 to about 400:1, about 200:1 to about 400:1, or about 300:1 to about 400:1 of the feeder cells of the present application and the present application
  • the feeder cells of the present application are added to the cell culture medium
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) making tumor-infiltrating lymphocytes derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion The first TIL population is contacted with one or more cell growth factors; wherein, the second TIL population is obtained through the step (A); (B) inhibiting the second TIL population with the one or more YEATS agent contact; wherein, the third TIL group is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may include: (A) making tumor-infiltrating lymphocytes derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion The increased first TIL population is contacted with one or more cell growth factors; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population is contacted with SGC-iMLLT, and SGC-
  • the initial concentration of iMLLT in the cell culture medium of the TIL may be at least about 1 ⁇ M to about 200 ⁇ M such that the TIL is contacted with one or more cell activators and with SGC-iMLLT substantially simultaneously; wherein, via the The above step (B) obtains the third TIL group.
  • the first-stage in vitro expansion of the present application can be optionally used interchangeably with step (A) in the method of the above aspects.
  • the second-stage in vitro expansion of the present application can be used in any substitution with step (B) in the method of the above aspect.
  • the TILs expanded in vitro in the first stage of the present application can be used in any substitution with the second population of TILs obtained through step (A) of the method of the above aspects.
  • the second-stage in vitro expanded TILs of the present application can be used in any substitution with the third TIL population obtained through step (B) of the method of the above aspects.
  • the third-stage in vitro amplification of the present application can be used in any replacement with any added step (C) in the method of the above aspects.
  • the third-stage in vitro expanded TILs of the present application can be used in any replacement with the fourth TIL population obtained through any additional step (C) in the method of the above aspects .
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion The first TIL population is contacted with various cell growth factors; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with various cell growth factors, and with various Contacting the cell activator, contacting with various YEATS inhibitors of the present application, and co-cultivating the TILs with the feeder cells; wherein, the third TIL population is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion The first TIL population is contacted with the cell growth factor; wherein, the second TIL population is obtained through the step (A); (B) the second TIL population can be contacted with the cell growth factor, with the cell activator, with the One or more YEATS inhibitors of the present application are contacted, and the TILs are co-cultured with feeder cells, and the initial concentration of the YEATS inhibitors in the cell culture medium of the TILs can be at least about 1 ⁇ M to about 200 ⁇ M; wherein , the third TIL group is obtained through the step (B).
  • TIL tumor-infiltrating lymphocytes
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion
  • the first TIL group is 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, and a cell activator (for example, can be A nanomatrix containing CD3 antibody and CD28 antibody, CD3 antibody or a mixture of CD3 antibody and CD28 antibody) is in contact with one or more YEATS inhibitors of the present application, such as SGC-iMLLT (commercial source: MCE (HY-112804) ), YDi-1( The synthesis method can be found in Example XL-13m) of WO WO2019101195A1, or YDi-2 ( The synthesis method can be found in Example XL-13n) of WO WO2019101195A1, and
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion
  • the first TIL group is 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, and contains CD3 antibody and CD28 antibody
  • YEATS inhibitors of the present application such as SGC-iMLLT (commercial source: MCE (HY-112804)), YDi-1
  • SGC-iMLLT commercial source: MCE (HY-112804)
  • YDi-1 YEATS inhibitors of the present application
  • the synthesis method can be found in Example XL-13m) of WO WO2019101195A1, or YDi-2
  • the synthesis method can be found in Example XL-13n) of WO WO2019101195A1, and the TIL is co
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion
  • the first TIL group is 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, and contains CD3 antibody and CD28 antibody
  • YEATS inhibitors of the present application such as SGC-iMLLT (commercial source: MCE (HY-112804)), YDi-1
  • SGC-iMLLT commercial source: MCE (HY-112804)
  • YDi-1 The synthesis method can be found in Example XL-13m) of WO WO2019101195A1, or YDi-2 ( The method of synthesis can be found in Example XL-13n) of WO WO2019101195A1, and the TILs are co-cultured with feeder cells, the diameter of the
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion
  • the first TIL population is contacted with IL-2, and the initial concentration of said IL-2 in the cell culture medium of said TIL can be at least about 300IU/mL; wherein, the second TIL population is obtained through said step (A)
  • B) the second TIL population can be contacted with IL-2, with a nanomatrix comprising CD3 antibody and CD28 antibody, with one or more YEATS inhibitors of the present application, such as SGC-iMLLT (commercial source :MCE(HY-112804)), YDi-1(
  • SGC-iMLLT commercial source :MCE(HY-112804)
  • YDi-1 The synthesis method can be found in Example XL-13m) of WO WO2019101195A1, or YDi-2 (
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion
  • the first TIL population is contacted with IL-2, and the initial concentration of said IL-2 in the cell culture medium of said TIL can be at least about 300IU/mL; wherein, the second TIL population is obtained through said step (A)
  • B) the second TIL population can be contacted with IL-2, with a nanomatrix comprising CD3 antibody and CD28 antibody, with one or more YEATS inhibitors of the present application, such as SGC-iMLLT (commercial source :MCE(HY-112804)), YDi-1(
  • SGC-iMLLT commercial source :MCE(HY-112804)
  • YDi-1 The synthesis method can be found in Example XL-13m) of WO WO2019101195A1, or YDi-2 (
  • the ratio of the nanomatrix to the TIL is added to the cell culture medium of the TIL, the feeder cells may comprise peripheral mononuclear cells, and the feeder cells to the TIL may be in a ratio of about 40:1 to about 400:1
  • the feeder cells are added to the cell culture medium of the TIL, and the initial concentration of the YEATS inhibitor in the cell culture medium of the TIL can be at least about 1 ⁇ M to about 200 ⁇ M; wherein, after the step (B) Obtaining the third TIL population.
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion
  • the first TIL population is contacted with IL-2, and the initial concentration of said IL-2 in the cell culture medium of said TIL can be at least about 300IU/mL; wherein, the second TIL population is obtained through said step (A)
  • B) the second TIL population can be contacted with IL-2, with a nanomatrix comprising CD3 antibody and CD28 antibody, with one or more YEATS inhibitors of the present application, such as SGC-iMLLT (commercial source :MCE(HY-112804)), YDi-1(
  • SGC-iMLLT commercial source :MCE(HY-112804)
  • YDi-1 The synthesis method can be found in Example XL-13m) of WO WO2019101195A1, or YDi-2 (
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TIL), which may comprise: (A) can be derived from tumor tissue, pleural effusion and/or peritoneal effusion without in vitro expansion
  • the first TIL population is contacted with IL-2, and the initial concentration of said IL-2 in the cell culture medium of said TIL can be at least about 300IU/mL; wherein, the second TIL population is obtained through said step (A)
  • B) the second TIL population can be contacted with IL-2, with a nanomatrix comprising CD3 antibody and CD28 antibody, with one or more YEATS inhibitors of the present application, such as SGC-iMLLT (commercial source :MCE(HY-112804)), YDi-1(
  • SGC-iMLLT commercial source :MCE(HY-112804)
  • YDi-1 The synthesis method can be found in Example XL-13m) of WO WO2019101195A1, or YDi-2 (
  • the present application provides a method of culturing tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • the method of obtaining TIL cells from a tissue sample of a subject can be obtained by surgically obtaining an orthotopic tumor sample or a metastatic tumor sample, the weight of which can be at least about 1 g, or multiple pieces of tissue can be combined.
  • Tumor tissue, pleural effusion and/or peritoneal effusion should be transported at about 2-8 degrees in sample transport fluid, such as commonly used commercial tumor tissue transport fluid, tumor tissue preservation fluid or tumor tissue transport fluid, and processed within 48 hours .
  • the tissue pieces can be mechanically broken to about 1-27 cubic millimeters in size, transferred into a gas-permeable culture bag or Grex, and added with a cell serum-free medium and a concentration of 300-9000IU/mL (for example, it can be 1000-9000IU/mL, such as Can be 6000IU/mL) IL-2 culture for about 3-14 days.
  • Cells in the culture medium are collected and transferred into a gas-permeable culture bag, or Grex, or Xuri equipment, and the serum-free medium of the cells can be added with the CD28 antibody, CD3 antibody and CD28 antibody of the application, magnetic beads comprising CD3 antibody and CD28 antibody (such as Dynabeads ) and/or a nanomatrix (such as transACT) comprising CD3 antibody and CD28 antibody, IL-2 with a concentration of 300-9000IU/mL (such as 1000-9000IU/mL, such as 6000IU/mL) and an initial concentration of At least about 1 ⁇ M of YEATS inhibitor (such as SGC-iMLLT (commercial source: MCE (HY-112804)), YDi-1 ( The synthesis method can be found in Example XL-13m) of WO WO2019101195A1, or YDi-2 ( The synthesis method can be found in Example XL-13n)) of WO WO2019101195A1.
  • YEATS inhibitor such as S
  • PBMC PBMC
  • the ratio of TIL to PBMC is about 1:40-about 1:400
  • the expansion culture is about 3 -14 days.
  • Cells in the culture medium can be collected using a cell processing system, washed and frozen, and detected.
  • the CD3 ratio of the final product can be greater than 80%, the cell viability can be greater than 50%, and the cells greater than 80% can be memory effector cells and effector cells.
  • IFN ⁇ can be secreted after stimulation, and/or can be characterized by an up-regulation of the proportion of activated cells.
  • the present application provides an immune cell, and the immune cell of the present application can be cultivated according to the cultivation method of the present application.
  • the immune cells provided in the present application may comprise one or a batch of immune cells cultured by the cultivation method of the present application.
  • the immune cells provided in the present application may comprise multiple or multiple batches of immune cells cultured by the cultivation method of the present application and combined in any proportion.
  • immune cells expanded using the methods of the present application can be administered to a patient as a pharmaceutical composition.
  • the pharmaceutical composition may be a suspension of immune cells in a sterile buffer.
  • Immune cells expanded using the PBMCs of the present application can be administered by any suitable route known in the art.
  • the cells can be administered as a single intra-arterial or intravenous infusion, which can last for about 30 to 60 minutes.
  • Other suitable routes of administration may include intraperitoneal, intrathecal and intralymphatic administration.
  • any suitable dose of immune cells can be administered.
  • about 2.3 x 10 9 to about 13.7 x 10 10 immune cells may be administered.
  • about 1 x 109 to about 12 x 1010 immune cells may be administered.
  • about 1.2 x 1010 to about 4.3 x 1010 immune cells may be administered.
  • about 3 x 1010 to about 12 x 1010 immune cells may be administered.
  • about 4 x 1010 to about 10 x 1010 immune cells may be administered.
  • about 5 x 1010 to about 8 x 1010 immune cells may be administered.
  • the therapeutically effective dose may be from about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 . In some embodiments, the therapeutically effective dose may be from about 1 ⁇ 10 9 to about 12 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 3 ⁇ 10 10 to about 12 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 4 ⁇ 10 10 to about 10 ⁇ 10 10 immune cells.
  • the therapeutically effective dose may be from about 5 ⁇ 10 10 to about 8 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 6 ⁇ 10 10 to about 8 ⁇ 10 10 immune cells. In some embodiments, the therapeutically effective dose may be from about 7 ⁇ 10 10 to about 8 ⁇ 10 10 immune cells.
  • the number of immune cells provided in the composition of the present application may be about 1 ⁇ 10 6 , about 2 ⁇ 10 6 , about 3 ⁇ 10 6 , about 4 ⁇ 10 6 , about 5 ⁇ 10 6 , About 6 ⁇ 10 6 , About 7 ⁇ 10 6 , About 8 ⁇ 10 6 , About 9 ⁇ 10 6 , About 1 ⁇ 10 7 , About 2 ⁇ 10 7 , About 3 ⁇ 10 7 , About 4 ⁇ 10 7 , About 5 ⁇ 10 7 , about 6 ⁇ 10 7 , about 7 ⁇ 10 7 , about 8 ⁇ 10 7 , about 9 ⁇ 10 7 , about 1 ⁇ 10 8 , about 2 ⁇ 10 8 , about 3 ⁇ 10 8 , about 4 ⁇ 10 8 , about 5 ⁇ 10 8 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 , about 8 ⁇ 10 8 , about 9 ⁇ 10 8 , about 1 ⁇ 10 9 , about 2 ⁇ 10 9 , about 3 ⁇ 10 9 , About 4 ⁇ 10 9 , about 5 ⁇ 10 8 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 ,
  • the number of immune cells provided in the composition of the present application may range from about 1 ⁇ 10 6 to 5 ⁇ 10 6 , about 5 ⁇ 10 6 to 1 ⁇ 10 7 , about 1 ⁇ 10 7 to 5 ⁇ 10 7 , about 5 ⁇ 10 7 to 1 ⁇ 10 8 , about 1 ⁇ 10 8 to 5 ⁇ 10 8 , about 5 ⁇ 10 8 to 1 ⁇ 10 9 , about 1 ⁇ 10 9 to 5 ⁇ 10 9 , about 5 ⁇ 10 9 to 1 ⁇ 10 10 , about 1 ⁇ 10 10 to 5 ⁇ 10 10 , about 5 ⁇ 10 10 to 1 ⁇ 10 11 , about 5 ⁇ 10 11 to 1 ⁇ 10 12 , about 1 ⁇ 10 12 to 5 ⁇ 10 12 , or about 5 ⁇ 10 12 to 1 ⁇ 10 13 .
  • the concentration of immune cells provided in the compositions of the present application may be less than, for example, about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40% of the composition. %, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, About 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2 %, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%, about 0.009%, about 0.008%, About 0.007%, about 0.006%, about 0.005%, about 0.004%, about 0.003%, about 0.002%, about 0.001%, about 0.0009%, about 0.0009%, about
  • the concentration of immune cells provided in the composition of the present application can be greater than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30% of the composition , about 20%, about 19.75%, about 19.50%, about 19.25%, about 19%, about 18.75%, about 18.50%, about 18.25%, about 18%, about 17.75%, about 17.50%, about 17.25%, about 17%, about 16.75%, about 16.50%, about 16.25%, about 16%, about 15.75%, about 15.50%, about 15.25%, about 15%, about 14.75%, about 14.50%, about 14.25%, about 14% , about 13.75%, about 13.50%, about 13.25%, about 13%, about 12.75%, about 12.50%, about 12.25%, about 12%, about 11.75%, about 11.50%, about 11.25%, about 11%, about 10.75%, about 10.50%, about 10.25%, about 10%, about 9.75%, about 9.50%, about 9.25%,
  • the concentration of immune cells provided in the composition of the present application can range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% of the composition. % to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to About 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17% %, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v/v.
  • the immune cells provided in the composition of the present application may be present in a concentration range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% of the composition % to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to About 1%, or about 0.1% to about 0.9% w/w, w/v or v/v.
  • the amount of immune cells provided in the composition of the present application can be equal to or less than about 10 g, about 9.5 g, about 9.0 g, about 8.5 g, about 8.0 g, about 7.5 g, about 7.0 g, about 6.5g, about 6.0g, about 5.5g, about 5.0g, about 4.5g, about 4.0g, about 3.5g, about 3.0g, about 2.5g, about 2.0g, about 1.5g, about 1.0g, about 0.95g , about 0.9g, about 0.85g, about 0.8g, about 0.75g, about 0.7g, about 0.65g, about 0.6g, about 0.55g, about 0.5g, about 0.45g, about 0.4g, about 0.35g, about 0.3g, about 0.25g, about 0.2g, about 0.15g, about 0.1g, about 0.09g, about 0.08g, about 0.07g, about 0.06g, about 0.05g, about 0.04g, about 0.03g, about 0.02g ,
  • the amount of immune cells provided in the compositions of the present application may be greater than about 0.0001 g, about 0.0002 g, about 0.0003 g, about 0.0004 g, about 0.0005 g, about 0.0006 g, about 0.0007 g, about 0.0008 g, about 0.0009g, about 0.001g, about 0.0015g, about 0.002g, about 0.0025g, about 0.003g, about 0.0035g, about 0.004g, about 0.0045g, about 0.005g, about 0.0055g, about 0.006g, About 0.0065g, about 0.007g, about 0.0075g, about 0.008g, about 0.0085g, about 0.009g, about 0.0095g, about 0.01g, about 0.015g, about 0.02g, about 0.025g, about 0.03g, about 0.035 g, about 0.04g, about 0.045g, about 0.05g, about 0.055g, about 0.06g, about
  • the immune cells can be administered in a single dose. Such administration may be by injection, for example intravenously. In some embodiments, immune cells can be administered in multiple doses. Doses may be once, twice, three, four, five, six or more than six times per year. Dosage can be monthly, biweekly, weekly, or every 2 days. In some embodiments, the administration of immune cells can be administered sequentially.
  • the present application provides a pharmaceutical composition.
  • it may comprise the immune cells of the present application and/or the composition of the present application, and a pharmaceutically acceptable carrier.
  • the present application provides a kit.
  • the kit of the present application may include the cell activator, cell growth factor and/or feeder cells of the method for culturing immune cells of the present application, and instructions describing the steps of the method of culturing immune cells of the present application.
  • the present application provides a kit, which may include the immune cells of the present application and/or the pharmaceutical composition of the present application.
  • the present application provides a method for affecting the growth of cells, such as tumor cells, which may include administering the immune cells of the present application and/or the pharmaceutical composition of the present application to a subject.
  • affecting tumor growth can comprise reducing the volume of the tumor to, for example, about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40% of its pre-administration volume.
  • the present application provides the application of the immune cells of the present application and/or the pharmaceutical composition of the present application in the preparation of medicines, and the medicines of the present application can be used to prevent and/or treat diseases and/or symptoms.
  • the diseases and/or symptoms of the present application may include tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • the present application provides a method for preventing and/or treating diseases and/or symptoms, which may include administering the immune cells of the present application and/or the pharmaceutical composition of the present application to a subject.
  • the diseases and/or symptoms of the present application may include tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • the present application provides a TIL of the present application and/or the pharmaceutical composition of the present application, which can be used to prevent and/or treat diseases and/or symptoms.
  • the diseases and/or symptoms of the present application may include tumors.
  • the tumor of the present application is selected from solid tumors.
  • the tumor of the present application can be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, tumor rectal cancer, and kidney cancer.
  • 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 and a culture bag with a gas-permeable surface, such as a culture bag (Origen), and add 300mL of rewarmed complete medium.
  • a culture bag with a gas-permeable surface, such as a culture bag (Origen)
  • the complete medium can be arbitrarily 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.
  • preREP stage The first stage of in vitro amplification and harvesting
  • 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 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 with a concentration of 300-9000IU/mL (for example, 1000-9000IU/mL, for example, 6000IU/mL), such as 6000IU/mL IL-2.
  • 3-14 days of the first phase of in vitro expansion for example, samples can be taken and counted at the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th or 14th day, if the cell number is at 5 ⁇ 10 5 When it reaches 5 ⁇ 10 8 , it enters the harvesting step of the first stage of in vitro amplification described below.
  • Collect the cells after the first stage of in vitro expansion centrifuge, discard the medium, wash the cells once with PBS or saline, obtain the TILs (second TIL population) that have undergone the first stage of in vitro expansion, and take a sample and count about 5 From ⁇ 10 5 to 2 ⁇ 10 8 cells enter the following first-stage in vitro expansion step; about 5 ⁇ 10 5 cells can be taken for quality control testing; the rest of the cells are added to cryopreservation medium and frozen.
  • 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 culture medium, and can add essential amino acids and antibiotics, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 cells/mL, in a suspension 24-well culture plate , 1 mL/well, add IL-2 at a concentration of 300-9000 IU/mL (eg, 1000-9000 IU/mL, eg, 6000 IU/mL).
  • T cell medium such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium
  • essential amino acids and antibiotics adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 cells/mL, in a suspension 24-well culture plate , 1 mL/well, add IL-2 at a concentration of 300-9000 IU/mL (eg, 1000-9000 IU/mL, eg, 6000 IU/mL).
  • CD28 agonists can be added, such as adding an antibody mixture containing CD3 antibody and CD28 antibody, adding magnetic beads containing CD3 antibody and CD28 antibody, and ⁇ or adding CD3 antibody and CD28 antibody Antibody for transACT (approximately 100 to 500 nm in diameter, Miltenyi).
  • YEATS inhibitor test group such as SGC-iMLLT (commodity source: MCE (HY-112804)), YDi-1 ( The synthesis method can be found in Example XL-13m) of WO WO2019101195A1, YDi-2( The synthesis method can be found in Example XL-13n) of WO WO2019101195A1.
  • the initial concentration of YEATS inhibitor in the cell culture medium of TIL can be about 1 ⁇ M or more, for example about 1 ⁇ M to about 200 ⁇ M, for example 20 ⁇ M, other inhibitor test group, add other inhibitors, for example AKT inhibitor VIII (commercial source: MCE (HY-10355)), or PI3K inhibitor Idelalisib (commercial source: MCE (HY-13026)).
  • AKT inhibitor VIII commercial source: MCE (HY-10355)
  • PI3K inhibitor Idelalisib commercial source: MCE (HY-13026)
  • Tn can be taken from 0 hours to 14 days, such as 24 hours or 48 hours, such as 48 hours
  • Tn can be taken from 0 hours to 14 days, such as 24 hours or 48 hours, such as 48 hours
  • revive the feeder cells mixed with 1-5 donors transfer the activated TIL cells and feeder cells into G-Rex100 culture flasks or air-permeable bags, supplement complete medium, take samples and count every 1-3 days, and
  • Add liquid or change half of the liquid until the total number of cells is greater than 1 ⁇ 10 9 or the second stage of in vitro expansion culture reaches 13 days, then terminate the culture.
  • the YEATS inhibitor test group can be added with the YEATS inhibitor throughout the whole process, and the TIL function test is performed after the 7th, 9th or 14th day of the second stage of in vitro expansion culture;
  • TILs amplified in vitro in the second stage Take the cells amplified in vitro in the second stage, centrifuge, discard the supernatant of the medium, and wash three times with PBS or saline or compound electrolyte solution to obtain TILs amplified in vitro in the second stage (the third TIL population). 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.
  • Figure 1 shows the proliferation results of the experimental group added with YEATS inhibitor.
  • the numerical value of the ordinate in Fig. 1 indicates that the third TIL population obtained by the second stage in vitro expansion of each test group is compared with the second TIL population before the second stage in vitro expansion, and the expansion of the number of TIL cells to multiple.
  • the results showed that the addition of YEATS inhibitors (for example, SGC-iMLLT) in the second stage of in vitro expansion resulted in increased proliferative capacity of TILs obtained.
  • YEATS inhibitors for example, SGC-iMLLT
  • Figures 2A-2B show the results of cell viability in the experimental group added with YEATS inhibitor for different donors.
  • the numerical value of the ordinate in Fig. 1 represents the proportion of living cells of the third TIL population obtained from the second-stage in vitro expansion of each test group.
  • the results showed that the addition of YEATS inhibitors (for example, SGC-iMLLT) in the second stage of in vitro expansion resulted in increased cell viability of TILs obtained.
  • 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 ⁇ 105 to 5 ⁇ 105 cell samples per 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 250 ⁇ L/well, discard supernatant. Add the prepared antibody working solution for cell surface staining, the concentration of antibody (BD or Biolegend) is 1:100 to 1:200, and the activity detection dye is 1:10000. Flow tube 100 ⁇ L/tube, 96-well plate 50 ⁇ L/well for staining, incubate at 2-8°C for 30 minutes in the dark.
  • Cell fixation and membrane rupture Sufficiently resuspend the cells, add an appropriate amount (96-well plate 100 ⁇ L/well, flow tube 1mL/tube) 1 ⁇ working solution A to fix and rupture the membrane, and incubate at 2-8°C for 40-50 minutes in the dark. After fixation and membrane rupture, add 1 ⁇ working solution B to wash the cells (250 ⁇ 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 (250 ⁇ 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 (96-well plate 250 ⁇ L/time, flow tube 2 mL/time), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice. After surface staining, cells were washed once with PBS (250 ⁇ L/time for 96-well plate, 1 mL/time for flow tube), centrifuged at 600 g for 3 minutes at room temperature, and the supernatant was discarded after centrifugation. Use 100-500 ⁇ L PBS to resuspend the cells for flow cytometry detection.
  • Figure 3 shows the proportion of CD8 + cells in the experimental group added with YEATS inhibitor.
  • YEATS inhibitors eg, SGC-iMLLT
  • Figures 4A-4C show the proportion of CD28 + cells in the experimental group with YEATS inhibitor added for different donors.
  • YEATS inhibitor for example, SGC-iMLLT
  • TILs TILs with an increased ratio of CD8 + cells and/or CD28 + activated cells among CD4 + cells.
  • Figures 5A-5E show the proportion of CD25 + cells in the experimental group with YEATS inhibitor added for different donors.
  • the results showed that the addition of YEATS inhibitors (for example, SGC-iMLLT) to the second stage of in vitro expansion resulted in TILs with increased CD8 + cells and/or CD4 + cells in the proportion of CD25 + activated cells, and the addition of YEATS inhibitors to the activation of cells The effect is better than other inhibitors.
  • YEATS inhibitors for example, SGC-iMLLT
  • Figures 6A-6D show the proportion of 41BB + cells in the experimental group added with YEATS inhibitor for different donors.
  • YEATS inhibitor e.g., SGC-iMLLT
  • TILs TILs with an increased proportion of 41BB + activated cells in CD8 + cells and/or CD4 + cells
  • YEATS inhibitors to the activation of cells The effect is better than other inhibitors.
  • Figures 7A-7K show the proportion of PD1 + cells in the experimental group added with YEATS inhibitor for different donors.
  • Figures 7A-7D, 7F-7G, and 7I-7J show the results of detection of TIL cells from different donors on the 7th day of the second stage in vitro expansion culture;
  • Figure 7E, 7H, and 7K show the results of TIL cells from different donors. The results of the detection of human-derived TIL cells on the 14th day of the second stage of in vitro expansion culture.
  • YEATS inhibitor e.g., SGC-iMLLT
  • TILs TILs with reduced proportion of CD8 + cells and/or CD4 + cells in PD1 + depleted cells
  • anti-cells with YEATS inhibitor added The depletion effect is superior to other inhibitors.
  • Figures 8A-8F show the proportion of LAG3 + cells in the experimental group with YEATS inhibitor added for different donors.
  • YEATS inhibitors e.g., SGC-iMLLT
  • TILs TILs with reduced proportions of LAG3 + depleted cells among CD8 + cells and/or CD4 + cells, and anti-cells with YEATS inhibitors added
  • the depletion effect is superior to other inhibitors.
  • Figures 9A-9C show the proportion of TIM3 + cells in the experimental group with YEATS inhibitor added for different donors.
  • Figures 9A-9B show the detection results of TIL cells from different donors on the 7th day of the second-stage in vitro expansion culture;
  • Figure 9C shows the second-stage in vitro expansion of TIL cells from different donors The results were detected on the 14th day of culture.
  • the results showed that the addition of a YEATS inhibitor (eg, SGC-iMLLT) to the second stage of in vitro expansion yielded TILs with a reduced proportion of TIM3 + depleted cells among CD8 + cells and/or CD4 + cells.
  • a YEATS inhibitor eg, SGC-iMLLT
  • Figures 10A-10B show the proportion of CD39 + cells in the experimental group added with YEATS inhibitor for different donors.
  • the results showed that the addition of a YEATS inhibitor (eg, SGC-iMLLT) to the second stage of in vitro expansion resulted in TILs with a reduced proportion of CD39 + depleted cells among CD4 + cells.
  • a YEATS inhibitor eg, SGC-iMLLT
  • Figure 11 shows the ratio of Treg cells (regulatory T cells) in the test group added with YEATS inhibitor.
  • YEATS inhibitors eg, SGC-iMLLT
  • Figures 12A-12C show the proportion of apoptotic cells in the experimental group added with YEATS inhibitor for different donors.
  • YEATS inhibitors eg, SGC-iMLLT
  • TILs TILs with reduced proportions of CD95 + caspass3 + cells and/or CD95 + DR5 + apoptotic cells among CD4 + cells.
  • Figures 13A-13C show, for different donors, the proportion of stem cells in the test group added with YEATS inhibitor.
  • Figures 13A and 13C show the results of TIL cells from different donors on the 7th day of in vitro expansion culture in the second stage;
  • Figure 13B shows the TIL cells from different donors in the second stage of in vitro expansion The results were detected on the 14th day of culture.
  • the results showed that the addition of YEATS inhibitors (for example, SGC-iMLLT) to the second stage of in vitro expansion resulted in TILs with increased proportions of CD69 ⁇ CD39 ⁇ cells and/or TCF1 + stem cells among CD8 + cells.
  • YEATS inhibitors for example, SGC-iMLLT
  • Figures 14A-14H show the proportion of central memory T cells (Tcm, such as CD45RA - CCR7 + in CD4 + cells or CD45RA - CCR7 + in CD8 + cells) of the experimental group added with YEATS inhibitor for different donors .
  • Figures 14A-14F show the results of TIL cells from different donors on the 7th day of in vitro expansion culture in the second stage;
  • Figures 14G-14H show the TIL cells from different donors in the second stage in vitro The results of detection were carried out on the 14th day of the expansion culture.
  • TILs from each test group were incubated overnight in a 96-well plate, and flowed through BD Cytofix/CytopermTM Plus Fixation/permeabilization kit BD GolgiStopTM (Cat. No.: BD 554715) and Fixation/Permeabilization Solution kit BD GolgiPlugTM (Cat. No.: BD 555028).
  • the formula detects the proportion of cells secreting cytokines.
  • Figures 15A-15B show the proportion of CD107A-secreting cells in the experimental group supplemented with YEATS inhibitor for different donors.
  • YEATS inhibitors for example, SGC-iMLLT
  • SGC-iMLLT SGC-iMLLT
  • Figures 16A-16D show the proportion of IFN ⁇ -secreting cells in the test group supplemented with YEATS inhibitor for different donors.
  • YEATS inhibitors for example, SGC-iMLLT
  • SGC-iMLLT SGC-iMLLT
  • Fig. 17 shows the ratio of TNF-secreting cells in the test group to which the YEATS inhibitor was added.
  • YEATS inhibitors for example, SGC-iMLLT
  • SGC-iMLLT SGC-iMLLT
  • Figures 18A-18D show the proportion of GZMB-secreting cells in the test group added with YEATS inhibitor for different donors.
  • YEATS inhibitors for example, SGC-iMLLT
  • SGC-iMLLT SGC-iMLLT
  • T cells from the donor add antibodies to activate, and add 10 ⁇ M YEATS inhibitor, such as YEATS SGC (commodity source: MCE (HY-112804), CAS number: 2255338-25-9), and culture for 72 hours.
  • YEATS SGC modifiedity source: MCE (HY-112804), CAS number: 2255338-25-9
  • TCR-T cells were obtained by transfecting NYESO1TCR (HLA-A*0201NY-ESO-1), and CAR-T cells were obtained by transfecting CD19 CAR (FMC63).
  • NT indicates unmodified T cells
  • CAR-T indicates T cells transfected with CAR
  • TCR-T indicates T cells transfected with TCR.
  • the cells continued to be cultured for 5-8 days, and 10 ⁇ M of YEATS inhibitor was added during the culture, and detected after the culture was over.
  • 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 250 ⁇ 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.
  • Figure 19 shows the transfection efficiency of T cells cultured with YEATS inhibitor for different donors. The results showed that YEATS inhibitor can increase the transfection efficiency of T cells.
  • the cultured NT, TCR-T, and CAR-T were collected for flow detection.
  • 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 250 ⁇ 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 (250 ⁇ 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 (250 ⁇ 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 (96-well plate 250 ⁇ L/time, flow tube 2 mL/time), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice. After surface staining, cells were washed once with PBS (250 ⁇ L/time for 96-well plate, 1 mL/time for flow tube), centrifuged at 600 g for 3 minutes at room temperature, and the supernatant was discarded after centrifugation. Use 100-500 ⁇ L PBS to resuspend the cells for flow cytometry detection.
  • Figure 20 shows the proportion of stemness-associated CD69 - CD39 - cells cultured with YEATS inhibitor for different donors. The results showed that YEATS inhibitors can increase the proportion of stemness-related cells.
  • Figures 21-22 show the proportion of depletion-associated CD39 + or PD1 + cells cultured with YEATS inhibitor for different donors. The results showed that YEATS inhibitors could reduce the proportion of exhaustion-associated cells.
  • Figures 23-24 show the proportion of activation-related 41BB + cells or CD25 + cells for different donors cultured with YEATS inhibitor. The results showed that YEATS inhibitors can increase the proportion of activation-associated cells.
  • NT, TCR-T, and CAR-T cells after culture were taken, and their proliferation ability was detected after being stimulated with no antibody or CD3 antibody for 72 hours.
  • Figure 25 shows the results of cell proliferation for different donors cultured with YEATS inhibitor. The results showed that YEATS inhibitors can improve the proliferation ability of cells.
  • NT, TCR-T, and CAR-T cells after culture were taken, and the secretion of cytokines was detected after being stimulated without adding antibodies or adding CD3 antibodies for 24 hours.
  • the kit is made of soluble protein master buffer kit (BD), reconstituted with 2mL Assay Diluent (BD) (the concentrations of IL-2, TNF- ⁇ , IFN- ⁇ and IL-6 cytokines in the standard solution are all 2500pg/mL , GZMB cytokine concentration is 10000pg/mL) and in order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256 gradient dilution, followed by Mix Capture Beads (BD), incubate for 2 hours at room temperature in the dark, then add PE Detection Reagent (BD) and mix, incubate for 1 hour in the dark at room temperature, transfer to a 15mL conical bottom centrifuge tube, labeled as "standard tube”. Take 1 tube containing Assay Diluent dilution only as a negative control.
  • BD Assay Diluent
  • Figures 26-30 show the results of secretion of IL-2, GZMB, TNF- ⁇ , IFN- ⁇ and IL-6 for different donors cultured with YEATS inhibitor. The results showed that YEATS inhibitors can improve the ability of cells to secrete cytokines.
  • PBMC Peripheral blood mononuclear cells
  • NK cells were cultured in 6-well plates at a concentration of 1.5E6 cells/mL, and 500 IU/mL IL-2 was added.
  • YEATS inhibitor treatment group 10 ⁇ M of YEATS inhibitor (such as SGC (commodity source: MCE (HY-112804)) was added; the control group did not add YEATS inhibitor. After culturing in the cell culture box for 5 days, the supernatant was collected and Perform Cytometric Bead Array (CBA) detection.
  • CBA Cytometric Bead Array
  • Figure 31 shows that the release of cytokines (such as IL-2, TNF- ⁇ and IFN- ⁇ ) from NK cells is significantly increased after YEATS inhibitor treatment.
  • cytokines such as IL-2, TNF- ⁇ and IFN- ⁇
  • the results show that YEATS inhibitors can enhance the activation effect and/or killing ability of NK cells.
  • YEATS inhibitors such as SGC-iMLLT (commercial source: MCE (HY-112804)
  • SGC-iMLLT commercial source: MCE (HY-112804)
  • YDi-1 The synthesis method can be found in Example XL-13m) of WO WO2019101195A1, YDi-2( The synthesis method can be found in Example XL-13n) of WO WO2019101195A1.
  • the initial concentration of the YEATS inhibitor in the cell culture medium of the TIL may be about 1 ⁇ M or more, eg about 1 ⁇ M to about 200 ⁇ M, eg 20 ⁇ M.
  • 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 250 ⁇ 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 (250 ⁇ 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 (250 ⁇ 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 (96-well plate 250 ⁇ L/time, flow tube 2 mL/time), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice. After surface staining, cells were washed once with PBS (250 ⁇ L/time for 96-well plate, 1 mL/time for flow tube), centrifuged at 600 g for 3 minutes at room temperature, and the supernatant was discarded after centrifugation. Use 100-500 ⁇ L PBS to resuspend the cells for flow cytometry detection.
  • Figure 32 shows the proportion of activation-associated CD25 + cells cultured with YEATS inhibitor for different donors. The results showed that YEATS inhibitors can increase the proportion of activation-associated cells.
  • Figures 33-34 show the proportion of depletion-associated PD1 + cells or LAG3 + cells cultured with YEATS inhibitor for different donors. The results showed that YEATS inhibitors could reduce the proportion of exhaustion-associated cells.
  • Figures 35-36 show the proportion of stemness-associated TCF1 + or CD69 ⁇ CD39 ⁇ cells cultured with YEATS inhibitor for different donors. The results showed that YEATS inhibitors can increase the proportion of stemness-related cells.

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Abstract

提供了一种免疫细胞的培养方法及其用途，具体包含使免疫细胞中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。还提供了使用所述细胞预防和/或治疗肿瘤的方法。

Description

一种免疫细胞的培养方法及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种免疫细胞的培养方法及其用途。
背景技术
目前，免疫治疗是一种治疗预后不良患者的有效方法。但是免疫治疗中使用的免疫细胞存在细胞活性不强或增殖能力弱的问题。
因此如何提供一种稳健可靠的免疫细胞的培养方法是亟待解决的问题。
发明内容
本申请提供了一种培养免疫细胞的方法，可以具有选自以下组的一种或多种的效果：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力和提高的肿瘤细胞杀伤能力。
一方面，本申请提供了一种培养免疫细胞的方法，使所述免疫细胞中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
一方面，本申请提供了一种免疫细胞，所述免疫细胞经过本申请的方法获得。
一方面，本申请提供了一种组合物，其包含本申请的免疫细胞。
一方面，本申请提供了一种药物组合物，其包含本申请的免疫细胞和/或本申请的组合物，以及任选地药学上可接受的载体。
一方面，本申请提供了一种影响细胞生长的方法，包含施用本申请的免疫细胞、本申请的组合物和/或本申请的药物组合物。
一方面，本申请提供了本申请的免疫细胞、本申请的组合物和/或本申请的药物组合物在制备药物中的应用，所述药物用于预防和/或治疗疾病和/或症状。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是，添加YEATS抑制剂的试验组的增殖结果。
图2A-2B显示的是，对于不同供者，添加YEATS抑制剂的试验组的细胞活率结果。
图3显示的是，添加YEATS抑制剂的试验组的CD8 +细胞比例。
图4A-4C显示的是，对于不同供者，添加YEATS抑制剂的试验组的CD28 +细胞比例。
图5A-5E显示的是，对于不同供者，添加YEATS抑制剂的试验组的CD25 +细胞比例。
图6A-6D显示的是，对于不同供者，添加YEATS抑制剂的试验组的41BB +细胞比例。
图7A-7K显示的是，对于不同供者，添加YEATS抑制剂的试验组的PD1 +细胞比例。
图8A-8F显示的是，对于不同供者，添加YEATS抑制剂的试验组的LAG3 +细胞比例。
图9A-9C显示的是，对于不同供者，添加YEATS抑制剂的试验组的TIM3 +细胞比例。
图10A-10B显示的是，对于不同供者，添加YEATS抑制剂的试验组的CD39 +细胞比例。
图11显示的是，添加YEATS抑制剂的试验组的Treg细胞(调节T细胞)比例。
图12A-12C显示的是，对于不同供者，添加YEATS抑制剂的试验组的凋亡细胞比例。
图13A-13C显示的是，对于不同供者，添加YEATS抑制剂的试验组的具有干性细胞比例。
图14A-14H显示的是，对于不同供者，添加YEATS抑制剂的试验组的中心记忆T细胞(Tcm)比例。
图15A-15B显示的是，对于不同供者，添加YEATS抑制剂的试验组的CD107A分泌细胞的比例。
图16A-16D显示的是，对于不同供者，添加YEATS抑制剂的试验组的IFNγ分泌细胞的比例。
图17显示的是，添加YEATS抑制剂的试验组的TNF分泌细胞的比例。
图18A-18D显示的是，对于不同供者，添加YEATS抑制剂的试验组的GZMB分泌细胞的比例。
图19显示的是，对于不同供者通过YEATS抑制剂培养，T细胞的转染效率。
图20显示的是，对于不同供者通过YEATS抑制剂培养，干性相关CD69 -CD39 -细胞比例。
图21-22显示的是，对于不同供者通过YEATS抑制剂培养，耗竭相关CD39 +细胞或PD1 +细胞比例。
图23-24显示的是，对于不同供者通过YEATS抑制剂培养，活化相关41BB +细胞或CD25 +细胞比例。
图25显示的是，对于不同供者通过YEATS抑制剂培养，细胞增殖结果。结果显示，YEATS抑制剂可以提高细胞的增殖能力。
图26-30显示的是，对于不同供者通过YEATS抑制剂培养，IL-2、GZMB、TNF-α、IFN-γ和IL-6的分泌结果。
图31显示的是在YEATS抑制剂处理后，NK细胞的细胞因子(例如IL-2、TNF-α和IFN-γ)释放显著增加。
图32显示的是，对于不同供者通过YEATS抑制剂培养，活化相关CD25 +细胞比例。结果显示，YEATS抑制剂可以提高活化相关细胞的比例。
图33-34显示的是，对于不同供者通过YEATS抑制剂培养，耗竭相关PD1 +细胞或LAG3 +细胞比例。结果显示，YEATS抑制剂可以降低耗竭相关细胞的比例。
图35-36显示的是，对于不同供者通过YEATS抑制剂培养，干性相关TCF1 +或CD69 -CD39 -细胞比例。结果显示，YEATS抑制剂可以提高干性相关细胞的比例。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“免疫细胞”通常是指参与进行先天性和适应性免疫应答的细胞。例如，可以包括但不限于淋巴细胞(诸如T细胞(包括胸腺细胞)和B细胞)、自然杀伤(NK)细胞、NKT细胞、巨噬细胞、单核细胞、嗜酸性粒细胞、嗜碱性粒细胞、嗜中性粒细胞、树突状细胞和肥大细胞。在一些实施方案中，修饰的免疫效应细胞是T细胞，诸如CD4 +T细胞、CD8 +T细胞(也称为细胞毒性T细胞或CTL)、调控性T细胞(Treg)、Th1细胞、Th2细胞、Th17细胞αβT细胞和/或γδT细胞。
在本申请中，术语“嵌合抗原受体”通常是指一种工程化抗原受体。例如，CAR可以包含经由铰链和跨膜结构域与包含信号传导结构域的细胞质结构域融合的细胞外抗原结合结构域。在一些实施方案中，CAR细胞外结构域可以以MHC非依赖性方式与由靶细胞表达的抗原结 合，从而导致细胞的活化和增殖。在一些实施方案中，CAR的细胞外结构域可以识别与抗体或其抗原结合片段融合的标签。例如，可以使得单个CAR构建体可以通过用一种抗体取代另一种抗体来靶向多种不同的抗原。在一些实施方案中，CAR的细胞外结构域可以包含来源于抗体的抗原结合片段。可用于本公开的抗原结合结构域可以包括例如scFv、抗体、抗体的抗原结合区、重链/轻链的可变区、和/或单链抗体。
在本申请中，术语“T细胞受体”通常是指一种工程化抗原受体。例如，TCR可以包含已从识别特定靶抗原的T细胞群体中分离并克隆的TCRα和/或TCRβ链。例如，TCRα和/或TCRβ基因(即TRAC和TRBC)可以从分离自患有特定恶性肿瘤的个体的T细胞群体或已分离自用特异性肿瘤抗原或肿瘤细胞免疫的人源化小鼠的T细胞群体中克隆而来。工程化TCR可以通过与其内源对应物相同的机制识别抗原(例如，通过识别在靶细胞表面上表达的主要组织相容性复合物(MHC)蛋白的背景下呈递的其同源抗原)，从而可以导致TCR工程化细胞的活化和增殖。
在本申请中，术语“YEATS”通常是指具有识别修饰蛋白的结构域的家族成员。例如，YEATS结构域可以识别组蛋白的修饰，例如组蛋白的乙酰化修饰。例如，包含YEATS结构域的家族成员的蛋白可以包含ENL(UniProt No Q03111)、AF9(UniProt No P42568)、和YEATS2(UniProt No Q9ULM3)和GAS41(UniProt No O95619)。
在本申请中，术语“YEATS抑制剂”通常是指能够影响YEATS蛋白的活性和/或功能的物质。合适的抑制剂分子可以包括拮抗剂抗体或抗体片段、小分子的片段或衍生物、肽、反义寡核苷酸、小的有机分子等。鉴定本申请的抑制剂的方法包括使表达本申请所抑制的分子的细胞与候选抑制剂分子接触，检测本申请所抑制的分子相关的一种或多种生物学活性可检测的变化，YEATS抑制剂可以包含能够降低编码包含YEATS结构域的蛋白的核酸分子的表达和/或活性的物质。例如，YEATS抑制剂可以通过结合YEATS蛋白的用于结合组蛋白的结构域，实现抑制YEATS功能的效果。例如，YEATS抑制剂可以包含SGC-iMLLT(CAS NO.：2255338-25-9)以及已知的任意YEATS抑制剂，以及上述化合物的衍生物。例如，YEATS抑制剂可以具有特异性，对于包含YEATS结构域的家族成员的一种或多种蛋白具有更高的结合活性。
在本申请中，术语“烷基”是1至24个碳原子的支链或无支链的饱和烃基团，例如，甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基、己基、庚基、辛基、癸基、十四烷基、十六烷基、二十烷基、二十四烷基，等等。“低级烷基”是含有一个至六个碳原子的烷基。
在本申请中，术语“烯基”是2至24个碳原子的烃基团，并且结构式含有至少一个碳-碳 双键。非对称的结构，例如，(AB)C＝C(CD)，包括E和Z两种异构体。这可以在本文中存在不对称烯烃的结构式中推定，也可以用键符号C明确表示。
在本申请中，术语“炔基”是2至24个碳原子的烃基团，并且结构式含有至少一个碳-碳叁键。
在本申请中，术语“芳基”是任何基于碳的芳香基，包括但不限于：苯、萘，等等。术语“芳香基”还包括“杂芳基”或“杂环芳基”，其定义为具有至少一个杂原子并入芳香基的环内的芳香基，而“杂芳基”或“杂环芳基”有5至9个环原子(“C 5-9杂环芳基”)，例如，5或6个环原子(“C 5或C 6杂环芳基”)，选自碳原子和杂原子。杂原子的例子包括但不局限于：氮、氧、硫和磷。芳基是取代的或未取代的芳基。芳基可以被一个或多个基团取代，包括但不限于：烷基、炔基、烯基、芳基、卤素、硝基、氨基、酯、酮、醛、羟基、羧酸或烷氧基。
在本申请中，术语“环状低聚物”是一种低聚物，其末端、两侧链或其组合共价结合，形成环状结构。
在本申请中，术语化合物的“有效量”是指无毒但足以提供所需结果的化合物的量。如下文所述，所需的确切数量因患者而异，取决于患者的种类、年龄和一般情况、正在治疗的疾病的严重程度、使用的具体化合物、其给药方式，等等。因此，无法具体说明确切的“有效量”。然而，本领域普通技术人员只使用常规实验，就可以确定合适的有效量。
在本申请中，术语“酯”由式-C(O)OA表示，其中，A可以是上文所述的烷基、卤代烷基、烯基、炔基、芳基、杂芳基、环烷基、环烯基、杂环烷基或杂环烯基。
本文所使用的术语“硫酯”是指以硫原子为特征的官能团，其两侧为任何杂化的一个羰基和一个碳。
在本申请中，术语“磺酸盐/酯”是指磺酸的盐或酯。
在本申请中，术语“肽”是指由化学结合在一起的氨基酸组成的一类化合物。通常，氨基酸通过酰胺连接基(CONH)化学结合在一起；然而，氨基酸可以通过本领域已知的其它化学键结合在一起。例如，氨基酸可以通过胺连接基结合。本文使用的肽包括氨基酸的低聚物和小肽和大肽，包括多肽。
在本申请中，术语“π-π-π堆积”、“π-π堆积”或“π-堆积”或“pi-堆积”可互换使用，指的是含有π-键的芳香环之间的吸引性非共价相互作用。
在本申请中，术语“共轭”和“离域”指离域π-电子(也可称为共轭π电子)。共轭π-电子或共轭系统是指在具有交替的单键和多键(通常是双键)的化合物中存在的与离域电子重叠的p轨道(桥接中间sigma键)系统。
在本申请中，术语“药理学活性”是指肽或多肽的固有物理性质。这些性能包括但不局限于：半衰期、溶解性和稳定性及其它药物动力学性能。
在本申请中，术语“可药用”是指不是在生物学上或其它方面不合乎需要的材料，即，该材料可与核酸或载体一起给予患者，不会造成任何不良的生物效应或以有害方式与所含药物组合物的任何其它成分相互作用。如本领域技术人员所熟知的，将自然地选择所述载体，使所述活性成分的任何降解最小化，并使所述患者中的任何不良副作用最小化。所述材料可以是溶液、悬浮液(例如，结合进微粒、脂质体或细胞中)。
在本申请中，术语“环”是指分子中原子和键的环，或是指一组相连的原子和键，其中每个原子和键都是环的成员。“单环”通常由一个环结构组成；“双环”有两个环结构；“三环”有三个环结构；“四环”有四个环结构，等等。
在本申请中，术语“选择性靶向”是指在一个特定区域内特定导向目标的能力。
在本申请中，术语“取代”是指本文所述化合物的所有允许的取代基。从最广泛的意义上讲，允许的取代基包括有机化合物的非环形和环形的、支链和无支链的、碳环和杂环的、芳香和非芳香的取代基。说明性取代基包括但不限于：卤素、羟基或含有任意数量碳原子(优选1-14个碳原子)的任何其它有机基团，并且任选在链状、支链或环状结构形式中包括一个或多个杂原子，例如氧、硫或氮基团。代表性的取代基包括：烷基、取代的烷基、烯基、取代的烯基、炔基、取代炔基、苯基、取代的苯基、芳基、取代的芳基、杂芳基、取代的杂芳基、卤素、羟基、烷氧基、取代的烷氧基、苯氧基、取代的苯氧基、芳氧基、取代的芳氧基、烷基硫基、取代的烷基硫基，苯基硫基、取代的苯基硫基、芳基硫基、取代的芳基硫基、氰基、异氰基、取代的异氰基、羰基、取代的羰基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、磺酰基、取代的磺酰基、磺酸、磷酰基、取代的磷酰基、膦酰基、取代的膦酰基，聚芳基、取代的聚芳基、C 3-C 20环、取代的C 3-C 20环、杂环、取代的杂环、氨基酸、肽和多肽基团。
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，“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。
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“特异性抑制剂”通常是指特异性地对本申请的分子起作用的抑制剂。例如，主要针对YEATS有抑制作用，而对于YEATS之外的其他分子基本没有抑制作用。例如，特异性抑制剂可以不排除其对于其他分子有抑制作用的可能。其中YEATS选择性抑制剂可以对YEATS中的一种或多种亚型有抑制作用。
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“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”通常是指免疫细胞在体外经过的一段扩增过程。例如，每一个阶段之间可以是通过免疫细胞数量的变化来划分的，例如，当免疫细胞的数量增加至少约1倍时，可以认为免疫细胞进入了下一个阶段的体外扩增。在一些实施方式中，当免疫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， 可以认为免疫细胞进入了下一个阶段的体外扩增。例如，每一个阶段之间也可以是通过免疫细胞培养的条件来划分的。例如，当细胞培养基中添加了或补充添加了细胞激活剂和/或细胞生长因子后，可以认为免疫细胞进入了下一个阶段的体外扩增。例如，当免疫细胞进行了离心和/或细胞洗涤后，可以认为免疫细胞进入了下一个阶段的体外扩增。例如，每一个阶段之间也可以是通过免疫细胞培养的天数来划分的。例如，当免疫细胞体外培养约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，可以认为免疫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，使用细胞生长因子进行扩增的阶段。例如，本申请的组织可以选自以下组：肿瘤组织、胸腔积液和腹腔积液，本申请的胸腔积液可以是有转移癌的患者的胸腔积液。例如，本申请的扩增可以是自体或者异体进行的体内扩增，或者可以是体外扩增。本申请的第一阶段体外扩增也可以称为preREP(快速扩增前)阶段。
在本申请中，术语“第二阶段体外扩增”通常是指从受试者体内取出的组织并进行扩增后，再次进行扩增的阶段。例如，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)，或者例如细胞的数量可以增加至少约100倍。例如，第二阶段体外扩增可以与第一阶段体外扩增的培养条件不同，例如加入的培养物质可以不同。本申请的第二阶段体外扩增也可以称为REP(快速扩增)阶段。
在本申请中，术语“体内”通常是指发生在受试者体内的事件。
在本申请中，术语“体外”通常是指在受试者体外发生的事件。
在本申请中，术语“离体”通常是指涉及对已从受试者体内移除的细胞、组织和/或器官进行治疗或进行手术的事件。例如，该细胞、组织和/或器官可以通过手术或治疗方法返回到受试者的身体。
在本申请中，术语“分泌”通常是指细胞将表达的多肽或蛋白转移到细胞外环境。
在本申请中，术语“分泌能力”通常是指细胞表达多肽或蛋白并将本申请的多肽或蛋白转移到细胞外环境的能力。
在本申请中，术语“辐照”通常是指通过射线对物质进行的处理。例如，例如，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。例如，工程化细胞可以经过基因修饰以表达本申请的细胞激活剂和/或细胞生长因子的免疫细胞。
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”，例如可以通过直接接触，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。或者例如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。例如可以通过直接接触，例如可以将一种或多种饲养细胞、细胞激活剂和/或细胞生长因子加入免疫细胞的培养基，例如可以将包含一种或多种饲养细胞、细胞激活剂和/或细胞生长因子的培养基加入和/或替换免疫细胞的培养基，例如，可以将包含一种或多种饲养细胞、细胞激活剂和/或细胞生长因子的培养基用于免疫细胞的培养；例如可以通过间接接触，例如可以将饲养细胞产生和释放的代谢产物，用于培养免疫细胞。
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。例如细胞的数量可以增加至少约3倍(或4、5、6、7、8或9倍)，例如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)，或者例如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。例如，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。例如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的细胞生长因子，并包括所有形式的IL-2，可以包括例如人和哺乳动物形式、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。例如，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“改善的免疫细胞特性”通常是指细胞经过扩增和/或培养后出现的免疫细胞特性。改善的免疫细胞特性可以包括，细胞的数量和/或比例变化，分泌能力变化，杀伤能力变化或表达能力的变化，或它们的任何组合。根据评价标准的不同，本申请的改善可以是提高或者降低，例如杀伤能力的提高，细胞耗竭水平的降低。
在本申请中，术语“纳米颗粒”通常是指至少一个尺寸小于100nm的微观颗粒。通常，纳米颗粒具有50nm至500nm(即0.05μm至0.5μm)范围内的直径；在生理环境中结构稳定；且可以容纳更小的分子(如药物或其他生物活性剂)，然后可以将该分子递送至希望的部位。例如，本申请的纳米颗粒可以包含CD28抗体或其抗原结合片段。例如，本申请的纳米颗粒可以包含CD28抗体或其抗原结合片段以及CD3抗体或其抗原结合片段。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免疫细胞。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，包含本申请的免疫细胞。
在本申请中，术语“受试者”通常是指细胞或动物，可以是哺乳动物，诸如人、非人灵长类动物(猿、长臂猿、大猩猩、黑猩猩、猩猩、猕猴)、家畜(狗和猫)、农场动物(家禽如鸡和鸭、马、牛、山羊、绵羊、猪)和实验动物(小鼠、大鼠、兔、豚鼠)。人受试者包括胎儿、新生儿、婴儿、青少年和成人受试者。受试者包括动物疾病模型，例如肿瘤动物模型，和本领域技术人员已知的其它动物模型。
在本申请中，术语“饲养细胞(feeder)”通常是指体外生长和分泌至少一种因子至培养基并且可以用于支持培养另一种所关注的细胞生长的培养细胞。例如，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、胸腔积液和/或腹腔积液中分离开始，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。例如，本申请的TIL可以是指TIL群。
在本申请中，术语“中心记忆细胞”通常是指具有长期记忆性的，并能够接受抗原再刺激的细胞。中心记忆细胞可以具有CD45RA -CCR7 +或者CD45RO +CD62L +的表型，例如可以是通过CD45RA -和CCR7 +或者CD45RO +和CD62L +来鉴定中心记忆细胞。中心记忆细胞可以相比普通细胞具有更强的抗肿瘤生长的能力。
在本申请中，术语“调节性细胞”通常是指一类控制体内自身免疫反应性的细胞亚群。调节性细胞可以具有CD4 +CD25 +Foxp3 +的表型，例如可以是通过CD4 +、CD25 +和Foxp3 +来鉴定调节性细胞。调节性细胞可以具有抑制细胞的抗肿瘤生长的能力。
在本申请中，术语“活化细胞”通常是指经过活化而可以具有抗肿瘤生长的能力的细胞。活化细胞可以具有CD28 +、CD25 +、或41BB +的表型，例如可以是通过CD28 +、CD25 +、或41BB +来鉴定活化细胞。活化细胞可以具有抗肿瘤生长的能力。
在本申请中，术语“耗竭细胞”通常是指免疫细胞受到抗原的持续刺激，逐渐失去效应功能的细胞。例如，耗竭细胞的功能可以是可逆转的，或者部分可逆转的。耗竭细胞可以具有PD1 +、LAG3 +、TIM3 +、或CD39 +的表型，例如可以是通过PD1 +、LAG3 +、TIM3 +、或CD39 +来鉴定耗竭细胞。耗竭细胞可以具有免疫功能降低的特征。
在本申请中，术语“凋亡细胞”通常是指正在经历程序性死亡的免疫细胞。凋亡细胞可以 具有CD95 +caspass3 +细胞和/或CD95 +DR5 +的表型，例如可以是通过CD95 +caspass3 +细胞和/或CD95 +DR5 +来鉴定凋亡细胞。凋亡细胞可以造成细胞数量的降低。
在本申请中，术语“肿瘤特异性细胞”通常是指可以特异性抗肿瘤生长的细胞。肿瘤特异性细胞可以具有CD103 +CD39 +的表型，例如，可以是通过CD103 +和CD39 +来鉴定肿瘤特异性细胞。肿瘤特异性细胞可以相比普通细胞具有更特异性的抗肿瘤生长的能力。
在本申请中，术语“干细胞样细胞”通常是指可以具有自我增殖和/或分化的潜能(干性，stemness)的一类细胞。干细胞样细胞可以具有CD69 -CD39 -或TCF1 +的表型，例如可以是通过CD69 -CD39 -或TCF1 +来鉴定干细胞样细胞。肿瘤特异性细胞可以相比普通细胞具有更强和/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“细胞激活剂”通常是指与细胞上的相应结合受体结合，并介导细胞共刺激反应的物质。细胞激活剂可以是细胞产生有效免疫应答所需的除抗原受体之外的物质。 细胞激活剂可以是指细胞共刺激分子。例如，本申请的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。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)、CD80(编码其的基因GeneID可以为941)、CD86(编码其的基因GeneID可以为942)、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“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。例如，细胞生长因子可以选自以下组的一种或多种：IL-2(编码其的基因GeneID可以为3558)、IL-4(编码其的基因GeneID可以为3565)、IL-7(编码其的基因GeneID可以为3574)、IL-10(编码其的基因GeneID可以为3586)、IL-12(编码其的基因GeneID可以为3592或3593)、IL-15(编码其的基因GeneID可以为3600)、和γ干扰素(编码其的基因GeneID可以为3458)。
在本申请中，术语“基本上同时”通常是指接触过程的一段时间内免疫细胞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免疫细胞总是与两种以上的物质同时接触。例如，基本上同时可以是指一段时间内免疫细胞可以与至少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细胞的抗原特异性分化，并且能够在体外和体内引发原代细胞应答。
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；为了检测免疫细胞的功能，例如检测免疫细胞释放细胞因子能力，而对免疫细胞进行的操作步骤(例如向免疫细胞的培养基中加入一种或一种以上物质以检测免疫细胞释放细胞因子能力)，可以不属于本申请的体外扩增。
在本申请中，术语“外周单个核细胞”或“外周血单个核细胞”通常是指外周血中具有单个核的细胞。例如，在本申请中，本申请的外周血单个核细胞可以包括淋巴细胞、单核细胞和/或树突状细胞。
在本申请中，术语“细胞因子”通常是指由一个细胞群释放的对另一个细胞起细胞间调节剂作用的蛋白。本申请的细胞因子可以是淋巴细胞因子(lymphokines)、单核细胞因子(monokines)和多肽激素。本申请的细胞因子可以包括白细胞介素(ILs)如IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-21、和/或IL-12。例如，细胞因 子可以包括CD107A(UniProt No P11279)、IFNγ(UniProt No P01579)、TNF(UniProt No P01375)或GZMB(UniProt No P10144)。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“细胞亚群比例”通常是指根据不同细胞亚群占免疫细胞或免疫细胞群中的比例。例如，本申请不同的细胞亚群具有不同的免疫活性和/或分化能力。例如，本申请的细胞亚群可以根据细胞表面标志物进行区分。
在本申请中，术语“免疫细胞数量”通常是指本申请的免疫细胞中细胞数量。在本申请中，免疫细胞数量可以是指本申请任一阶段获得的免疫细胞群中的细胞数量。例如，免疫细胞数量可以是指源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一免疫细胞群的细胞数量。例如，免疫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，免疫细胞数量可以是指本申请任意一种培养方法最终获得的免疫细胞的细胞。在本申请中，免疫细胞数量可以通过本领域常用的方法测量，例如可以包括但不限于细胞计数板手动细胞计数和/或自动细胞计数器计数。
在本申请中，术语“约”和“大约”通常是指在统计上有意义的数值范围内。这样的范围可以在给定值或范围的一个数量级内，可以包括在50％内，可以包括在20％内，可以包括在10％内，可以包括在5％内。术语“约”或“大约”所包含的可允许变化可以取决于所研究的特定系统，并且本领域普通技术人员可以容易地理解。术语“以上”、“以下”、“至多”和“至少”可以包括本 数。
发明详述
一方面，本申请提供一种培养免疫细胞的方法，使所述免疫细胞中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，所述使包含YEATS结构域的蛋白和/或其功能活性片段的活性降低包含抑制所述YEATS结构域与组蛋白的结合能力。
例如，所述使包含YEATS结构域的蛋白和/或其功能活性片段的活性降低可以包含抑制所述YEATS结构域与组蛋白的结合能力。例如，可以在免疫细胞的培养过程中加入能够抑制所述YEATS结构域与组蛋白的结合的物质。例如，可以在免疫细胞的培养过程中加入能够诱导包含YEATS结构域的蛋白失活的物质。例如，可以在免疫细胞的培养过程中加入能够结合所述YEATS结构域的物质。
例如，本申请方法包含：使所述免疫细胞与一种或多种YEATS抑制剂接触。
例如，未曾与YEATS抑制剂接触的相应免疫细胞可以是指源自同一供体的且未曾与本申请YEATS抑制剂接触的免疫细胞。例如，未曾与本申请YEATS抑制剂接触的免疫细胞可以是与其它靶点抑制剂(例如，AKT抑制剂(AKT抑制剂VIII，AKTi-1/2，CAS登录号：612847-09-3)、PI3K抑制剂(Idelalisib，CAS登录号：870281-82-6))接触的免疫细胞。例如，未曾与本申请YEATS抑制剂接触的相应免疫细胞可以是指源自同一供体的经过同样方式分离的且未曾与本申请YEATS抑制剂接触的免疫细胞。例如，未曾与本申请YEATS抑制剂接触的相应免疫细胞可以是指源自同一供体的同一肿瘤来源的且未曾与本申请YEATS抑制剂接触的免疫细胞。例如，未曾与本申请YEATS抑制剂接触的相应免疫细胞可以是指源自同一供体的同一肿瘤来源的经过同样方式分离的且未曾与本申请YEATS抑制剂接触的免疫细胞。例如，未曾与本申请YEATS抑制剂接触的相应免疫细胞可以是指将源自同一供体的免疫细胞分为两组，其中一组未曾与本申请YEATS抑制剂接触的免疫细胞可以为未曾与本申请YEATS抑制剂接触的相应免疫细胞。例如，未曾与本申请YEATS抑制剂接触的相应免疫细胞可以是指将源自同一供体的经过同样方式分离的免疫细胞分为两组，其中一组未曾与本申请YEATS抑制剂接触的免疫细胞可以为未曾与本申请YEATS抑制剂接触的相应免疫细胞。例如，未曾与本申请YEATS抑制剂接触的相应免疫细胞可以是指将源自同一供体的同一肿瘤来源的免疫细胞分为两组，其中一组未曾与本申请YEATS抑制剂接触的免疫细胞可以为未曾与本申请YEATS抑制剂接触的相应免疫细胞。例如，未曾与本申请YEATS抑制剂接触的相应免疫细胞可以是指将源自同一供体的同一肿瘤来源的经过同样方式分离的免疫细胞分 为两组，其中一组未曾与本申请YEATS抑制剂接触的免疫细胞可以为未曾与本申请YEATS抑制剂接触的相应免疫细胞。
例如，所述免疫细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
例如，所述免疫细胞包含单核细胞、巨噬细胞和/或树突状细胞。
例如，所述免疫细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞。例如，“未修饰的免疫细胞”或“未改造的免疫细胞”可以是指其中基因组未被修饰并且不包含基因调控系统或包含对照基因调控系统(例如，空载体对照、非靶向gRNA、加扰siRNA等)的细胞或细胞群体。例如，TIL细胞培养过程中与YEATS抑制剂接触，可以实现提高的细胞增殖能力，提高的细胞活率，增加的CD8 +细胞比例，增加的中心记忆细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例，和/或增加的干细胞样细胞比例；例如，T细胞、TCR-T细胞和/或CAR-T细胞在培养过程中与YEATS抑制剂接触，可以实现提高的细胞增殖能力，提高的细胞活率，增加的CD8 +细胞比例，增加的中心记忆细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例，和/或增加的干细胞样细胞比例；例如，NK细胞在培养过程中与YEATS抑制剂接触，可以实现提高的细胞增殖能力，提高的细胞活率，增加的CD8 +细胞比例，增加的中心记忆细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例，和/或增加的干细胞样细胞比例。
例如，所述免疫细胞包含αβT细胞和/或γδT细胞。
例如，所述免疫细胞包含肿瘤浸润淋巴细胞(TIL)。
例如，所述TIL为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
例如，本申请的TIL可以为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。例如，可以通过将肿瘤组织处理成肿瘤碎片获得本申请的TIL。例如，本申请的肿瘤碎片的体积约为1-27立方毫米。例如，本申请的肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方毫米、约6立方毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约15立方毫米、约17立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或约27立方毫米。
例如，所述免疫细胞包含展示在细胞表面上的工程化免疫受体。
例如，所述工程化免疫受体与靶细胞上表达的抗原特异性结合。
例如，所述免疫细胞包含嵌合抗原受体和/或T细胞受体。
一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，可以包含：使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本申请的第二阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低，且在本申请的第二阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第二阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第三阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性 降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低，且在本申请的第二阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低，且在本申请的第三阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第二阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低，且在本申请的第三阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，可以使本申请的源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增、第二阶段体外扩增和第三阶段体外扩增，且在本申请的第一阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低，且在本申请的第二阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低，且在本申请的第三阶段体外扩增中，可以使所述TIL中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
例如，每一个阶段体外扩增之间可以是通过TIL细胞数量的变化来划分的，例如，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、至少约50倍、至少约100倍、至少约200倍、至少约500倍、或者至少约1000倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，每一个阶段的体外扩增之间也可以是通过TIL细胞培养的条件的变化来划分的。例如，当细胞培养基中添加了或补充添 加了细胞激活剂和/或细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了IL-2后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了一种或多种抑制剂后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了饲养细胞后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当TIL细胞进行了离心和/或细胞洗涤的操作后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。例如，当TIL细胞体外培养约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天、约20天、约30天、约40天、约50天或约100天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
例如，与未曾与所述YEATS抑制剂接触的免疫细胞相比，与所述YEATS抑制剂接触过的所述免疫细胞显示出改善的细胞特性。
例如，所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力和提高的肿瘤细胞杀伤能力。
例如，所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
例如，本申请的改善的免疫细胞数量是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触或与其它抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞的细胞数量可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，增加的活细胞比例可以表现为免疫细胞活率的增加。例如，本申请的增加的活细胞比例可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的或与其它抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞的活细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、 至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指免疫细胞的选自以下组的细胞因子分泌能力提高：IL-2、IL-6、CD107a、GZMB、TNF和IFNγ。例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌细胞因子的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌细胞因子的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌CD107A的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌CD107A的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、 至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌IL-2的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌IL-2的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌IL-6的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌IL-6的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请 YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌IFNγ的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌IFNγ的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌TNF的细胞比例可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌TNF的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本申请的TNF可以为TNF-α。
例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌GZMB的细胞比例可以增加至少约1倍、至少约2倍、至 少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的细胞因子分泌能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞中分泌GZMB的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本申请的免疫细胞的细胞因子分泌能力的测定可以是通过测量免疫细胞的细胞因子表达能力。例如，本申请的免疫细胞的细胞因子分泌能力可以通过细胞流式检测的方法测定。例如，本申请的免疫细胞的细胞因子分泌能力通过测量免疫细胞的细胞因子释放能力测定。例如，本申请的免疫细胞的细胞因子分泌能力是通过CBA法(Cytometric Bead Array)测定。
例如，本申请的提高的肿瘤细胞杀伤能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞的肿瘤细胞杀伤率可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。例如，本申请的提高的肿瘤细胞杀伤能力可以是指与在体外扩增阶段未曾与本申请YEATS抑制剂接触的相应免疫细胞相比，在至少一个体外扩增阶段中与本申请YEATS抑制剂接触过的本申请免疫细胞的肿瘤细胞杀伤率可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。例如，本申请的免疫细胞的肿瘤细胞杀伤率可以通过CFSE和DAPI染色法测量。例如，本申请的免疫细胞的肿瘤细胞杀伤可以是指免疫细胞杀伤实体瘤细胞的能力。
例如，本申请的改善的细胞亚群比例可以包含选自以下组的一种或多种：增加的CD8 +细胞比例，增加的中心记忆细胞比例，降低的调节性细胞的比例，增加的活化细胞比例，增加的肿瘤特异性细胞比例，和增加的干细胞样细胞比例。
例如，本申请的增加的CD8 +细胞比例可以是免疫细胞中CD8阳性细胞的比例的增加。例如，在免疫细胞中CD8 +细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的增加的活化细胞比例可以是免疫细胞中CD28 +、CD25 +、和/或41BB +细胞的比例的增加。例如，在免疫细胞中活化细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中CD28 +细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中CD25 +细胞比例可以增加至少约100％、至少约90％、至少约80％、 至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中41BB +细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本申请的降低的耗竭细胞比例可以是免疫细胞中PD1 +、LAG3 +、TIM3 +、和/或CD39 +细胞的比例的增加。例如，在免疫细胞中耗竭细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中PD1 +细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、 至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中LAG3 +细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中TIM3 +细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，在免疫细胞中CD39 +细胞比例可以降低至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％，或可以降低至少约1倍、至少约2倍、至少约 3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
例如，本申请的减少的调节性细胞的比例可以是免疫细胞中CD4 +CD25 +Foxp3 +细胞的比例的减少。例如，在免疫细胞中调节性细胞比例可以减少至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的减少的凋亡细胞的比例可以是免疫细胞中CD95 +caspass3 +细胞和/或CD95 +DR5 +细胞的比例的减少。例如，在免疫细胞中凋亡细胞比例可以减少至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的增加的具有干性的细胞的比例可以是免疫细胞中CD69 -CD39 -细胞和/或TCF1 +细胞的比例的增加。例如，在免疫细胞中具有干性的细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，本申请的增加的中心记忆细胞比例可以是免疫细胞中CD45RA -CCR7 +或者CD45RO +CD62L +细胞的比例的增加。例如，在免疫细胞中中心记忆细胞比例可以增加至少约100％、至少约90％、至少约80％、至少约70％、至少约60％、至少约50％、至少约40％、至少约30％、至少约20％、至少约19％、至少约18％、至少约17％、至少约16％、至少约15％、至少约14％、至少约13％、至少约12％、至少约11％、至少约10％、至少约9％、至少约8％、至少约7％、至少约6％、至少约5％、至少约4％、至少约3％、至少约2％、 至少约1％、至少约0.5％、至少约0.4％、至少约0.3％、至少约0.2％、或至少约0.1％。
例如，其包含：使所述免疫细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述免疫细胞与所述YEATS抑制剂接触。
例如，其中，使所述源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使经所述第一阶段体外扩增的TIL与所述YEATS抑制剂接触。
例如，所述第一阶段体外扩增进行至少约7天。
例如，所述第二阶段体外扩增进行至少约7天。
例如，本申请的YEATS抑制剂可以包含能够抑制YEATS结构域与组蛋白相互作用的物质。例如，本申请的YEATS抑制剂可以结合YEATS的组蛋白结合结构域的物质。例如，本申请的YEATS抑制剂可以结合修饰的YEATS的组蛋白结合结构域的物质。例如，本申请的YEATS抑制剂可以结合乙酰化修饰的YEATS的组蛋白结合结构域的物质。
例如，本申请的YEATS抑制剂可以包含能够抑制YEATS结构域与组蛋白相互作用的物质。例如，所述YEATS抑制剂可以包含能够抑制YEATS结构域与组蛋白结合的物质。例如，所述YEATS抑制剂可以包含能够以约200μM或更低的IC 50值抑制YEATS结构域与组蛋白结合的物质。例如，所述YEATS抑制剂可以包含能够以约190μM或更低、约180μM或更低、约170μM或更低、约160μM或更低、约150μM或更低、约140μM或更低、约130μM或更低、约120μM或更低、约110μM或更低、约100μM或更低、约90μM或更低、约70μM或更低、约50μM或更低、约20μM或更低的、约10μM或更低、约5μM或更低、约2μM或更低、约1μM、约0.5μM、约0.3μM或更低的IC 50值抑制YEATS结构域与组蛋白结合的物质。例如，与对于非YEATS靶点的抑制作用相比，本申请的YEATS抑制剂对于YEATS的抑制作用可以高至少约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，所述YEATS抑制剂可以包含能够以约0.13μM或更低的K D值结合YEATS结构域的物质。例如，所述YEATS抑制剂可以包含能够以约9μM或更低、约8μM或更低、约7μM或更低、约6μM或更低、约5μM或更低、约4μM或更低、约3μM或更低、约2μM或更低、约1μM或更低、约0.9μM或更低、约0.8μM或更低、约0.7μM或更低、约0.6μM或更低、约0.5μM或更低的、约0.4μM或更低、约0.3μM或更低、约0.2μM或更低、或者约0.1μM或更低的K D值结合YEATS结构域的物质。
例如，所述YEATS抑制剂可以包含选自以下组的一种或多种：核酸分子、多肽和小分子化合物。例如，所述YEATS抑制剂可以包含DNA、和/或RNA；例如，所述YEATS抑制剂可以包含抗体和/或其抗原结合片段；例如，所述YEATS抑制剂可以包含小分子化合物，例如小分子抑制剂。
例如，所述YEATS抑制剂基本上不结合选自以下组的一种或多种靶点：Akt、和PI3K。例如，所述YEATS抑制剂基本上不影响Akt、和/或PI3K的活性。例如，相比于不加入YEATS抑制剂，加入本申请的YEATS抑制剂可以仅降低Akt、和/或PI3K的活性约50％以下、约40％以下、约30％以下、约20％以下、约10％以下、约9％以下、约8％以下、约7％以下、约6％以下、约5％以下、约4％以下、约3％以下、约2％以下、约1％以下、约0.5％以下、约0.1％以下、或约0.01％以下。例如，Akt、和/或PI3K的活性可以通过本领域通用的方法测量。例如，与对于Akt靶点的抑制作用相比，本申请的YEATS抑制剂对于YEATS的抑制作用可以高至少约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，与对于PI3K靶点的抑制作用相比，本申请的YEATS抑制剂对于YEATS的抑制作用可以高至少约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，所述YEATS抑制剂可以包含YEATS特异性抑制剂，例如，SGC-iMLLT(CAS登录号：2255338-25-9)。例如，YEATS特异性抑制剂可以仅包含直接且特异性抑制YEATS蛋白的物质。例如，YEATS特异性抑制剂可以不包含通过影响YEATS转录，从而影响YEATS活性的物质。
例如，所述YEATS抑制剂可以包含能够降低编码YEATS的核酸分子的表达和/或活性的物质，例如，可以降低编码YEATS的核酸分子的转录、翻译和/或翻译后修饰的成熟过程。例如，所述YEATS抑制剂包含能够降低YEATS蛋白表达的物质。例如，所述YEATS抑制剂包含能够影响YEATS蛋白活性的物质。
例如，每一种所述YEATS抑制剂在所述免疫细胞的细胞培养基中的初始浓度为至少约1μM。例如，SGC-iMLLT在所述免疫细胞的细胞培养基中的初始浓度可以为至少约1μM。例如，每一种所述YEATS抑制剂在所述免疫细胞的细胞培养基中的初始浓度为至少约1μM。例如，每一种所述YEATS抑制剂在所述免疫细胞的细胞培养基中的初始浓度可以相互独立地为至少约0.01μM、至少约0.02μM、至少约0.03μM、至少约0.05μM、至少约0.07μM、至少约0.1μM、至少约0.2μM、至少约0.3μM、至少约0.4μM、至少约0.5μM、至少约0.6μM、至少约0.7μM、至少约0.8μM、至少约0.9μM、至少约1μM、至少约1.5μM、至少约1.6μM、至少约1.7μM、至少约1.8μM、至少约1.9μM、至少约2μM、至少约2.5μM、至少约3μM、至少约3.5μM、至少约4μM、至少约4.5μM、至少约5μM、至少约10μM、至少约20μM、至少约30μM、至少约40μM、至少约50μM、至少约60μM、至少约70μM、至少约80μM、至少约90μM、至少约100μM、至少约150μM、或至少约200μM。
例如，每一种所述YEATS抑制剂在所述免疫细胞的细胞培养基中的初始浓度可以相互独立地为约1μM至约200μM、约5μM至约200μM、约10μM至约200μM、约50μM至约200μM、约100μM至约200μM、约150μM至约200μM、约1μM至约150μM、约5μM至约150μM、约10μM至约150μM、约50μM至约150μM、约100μM至约150μM、约1μM至约100μM、约5μM至约100μM、约10μM至约100μM、约50μM至约100μM、约1μM至约50μM、约2μM至约50μM、约5μM至约50μM、约10μM至约50μM、约 20μM至约50μM、约30μM至约50μM、约40μM至约50μM、约45μM至约50μM、约1μM至约40μM、约2μM至约40μM、约5μM至约40μM、约10μM至约40μM、约20μM至约40μM、约30μM至约40μM、约1μM至约30μM、约2μM至约30μM、约5μM至约30μM、约10μM至约30μM、约20μM至约30μM、约1μM至约20μM、约2μM至约20μM、约5μM至约20μM、约10μM至约20μM、约1μM至约10μM、约2μM至约10μM、约5μM至约10μM、约1μM至约5μM、约2μM至约5μM、或约1μM至约2μM。
例如，所述YEATS抑制剂包含如式(I)定义的化合物或其可药用盐，
Figure PCTCN2022105137-appb-000001
其中，A是CR 2或N；
其中，每个B 1和B 2是独立选择的二价连接单元，其中，所述二价连接单元通过酰胺、酯、硫酯、硫酰胺、亚胺酯、酰亚胺、磺酸酯或磺酰胺以首尾连接方式连接；
其中，D 1是H或未取代的或取代的烃基、碳环基或杂环基；
其中，D 2是H、NH 2、NHR 15、NR 16R 17、OH、OR 18，或未取代的或取代的烃基、碳环基或杂环基；
其中，X是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基或C 1-10杂环基；
其中，Y是NR 3、O或S；
其中，Z是-CO-、-CS-、-CNR 4-、-SO-或-SO 2-；
其中，R 2、R 3和R 4独立地是H或C 1-10烃基、C 1-10碳环基或C 1-10杂环基；
其中，R 1是共轭/离域基团，例如，未取代的或取代的杂环的或碳环的芳香环(单环、双环、三环、四环)，或未取代的或取代的烯基或炔基；
其中，R 15、R 16、R 17和R 18可以是H或烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环；且
其中，m和n各自独立地是0至10的整数，其中，m或n中的至少一个不是0。
例如，A可以是CR 2或N。在一些形式中，R 2可以是H或烷基、烯基、炔基、取代的烷 基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环基团。在一些形式中，A可以是CR 2，且R 2可以是H。
例如，B 1可以是-NH-、-O-、-S-或-(CH 2) p-、其中，p是1至6的整数，例如，1、2、3、4、5和6。在一些形式中，端部B 2可以是-C(＝O)-、-C(＝NH)-、-C(＝S)-、-S(＝O) 2-或-(代表B 2上的末端键)。在一些形式中，D 2可以是H、NH 2、NHR 15、NR 16R 17、OH、OR 18或未取代的或取代的烃基、碳环基或杂环基，其中，R 15、R 16、R 17和R 18可以是H或烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环。
例如，二价单元可以是
Figure PCTCN2022105137-appb-000002
例如，W可以是CR 2，R 11可以是侧链，R 12和R 13可以是诸如酰胺连接基、酯连接基、硫酯连接基、硫胺连接基、亚胺酯连接基、酰亚胺连接基、磺酸酯连接基和磺酰胺连接基的组成部分。不同的二价单元可以与相同或不同类型的连接基连接，连接的R 12和R 13基团是目标连接基的相容的组成部分。在一些形式中，R 12可以是-C(＝O)-R 19、-C(＝NH)-R 19、-C(＝S)-R 19或-S(＝O) 2-R 19。在一些形式中，R 19可以是N(R 14)-、O-、S-或N(R 14)-C(＝O)-，其中，R 14可以是H或烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环。
例如，R 11可以由低聚物单体的现有侧链或正常侧链的一部分组成。因此，例如，在链状基础低聚物是肽的组合物中，肽中的氨基酸的侧链之一可包含R 11的一部分，或可被R 11取代。通常，这种形式的组合物包括至少一个终止于R 1的R 11(其中R 1如本文所定义)。在一些形式中，这种形式的组合物包括至少一个根据以下结构定义的R 11：
Figure PCTCN2022105137-appb-000003
例如，虚线表示上述结构的连接点，其中，R 1可以是共轭/离域基团，X、Y和Z如本文所定义。
例如，二价单元全部独立地是任何α-氨基酸。在一些形式中，二价单元各自是α-氨基酸，独立地选自Lys、Gln、Thr、Ala、Arg、Ser和Gly。
例如，D 1可以是H或未取代的或取代的烃基、碳环基或杂环基。在一些形式中，D 1可以是烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环基团。在一些形式中，D 1可以是H。在一些形式中，D 1可以是羧基苄基(Cbz)。在一些形式中，D 1可以是乙酰基。在一些形式中，D 1可以是苯甲基羰基。在一些形式中，D 1可以是苯乙基羰基。在一些形式中，D 1可以是苯丙基羰基。在一些形式中，D 1可以是萘乙基羰基。
例如，D 2可以是H、-NH 2、-NHR 15、-NR 16R 17、-OH、-OR 18或未取代的或取代的烃基、碳环基或杂环基，其中R 15、R 16、R 17和R 18可以是H或烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环。在一些形式中，D 2可以是H或-NH 2。在一些形式中，D 2可以是烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环基团。在一些形式中，D 2可以是H。在一些形式中，D 2可以是-NH 2。在一些形式中，D 2可以是OH或OR 18。在一些形式中，D 2可以是OH。
例如，X可以是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基或C 1-10杂环基。在一些形式中，X可以是烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环基团。在一些 形式中，X可以是-(CH 2) p-，其中，p是1至6的整数。在一些形式中，X可以是-(CH 2) p-，其中，p是2至5的整数。在一些形式中，X可以是-(CH 2) p-，其中，p是3或4。在一些形式中，X可以是-(CH 2) p-，其中，p是4。
例如，Y可以是-NR 3、-O-或-S-。在一些形式中，R 3可以是H或C 1-10烃基、C 1-10碳环基或C 1-10杂环基。在一些形式中，R 3可以是H或烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环基团。在一些形式中，Y可以是NR 3，且R 3可以是H。
例如，Z可以是-CO-、-CS-、-CNR 4-、-SO-或-SO 2-。在一些形式中，Z可以是-CO-。在一些形式中，R 4可以是H或C 1-10烃基、C 1-10碳环基或C 1-10杂环基。在一些形式中，R 4可以是H或烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环基团。
例如，所述共轭/离域基团可以是未取代的或取代的杂环的或碳环的芳香环，或未取代的或取代的烯基或炔基，其中，所述共轭/离域基团包括允许π电子离域的p-轨道。在一些形式中，共轭/离域基团可以是未取代的或取代的芳香环。在一些形式中，共轭/离域基团可以是未取代的或取代的芳香单环、双环、三环或四环。在一些形式中，共轭/离域基团可以是未取代的或取代的烯基或炔基。
例如，R 1可以是未取代的或取代的杂环的或碳环的芳香环，或未取代的或取代的烯基或炔基，其中，所述共轭/离域基团包括允许π电子离域的p-轨道。在一些形式中，R 1可以是未取代的或取代的芳香环。在一些形式中，R 1可以是未取代的或取代的芳香单环、双环、三环或四环。在一些形式中，R 1可以是未取代的或取代的烯基或炔基。在一些形式中，R 1可以是∶
Figure PCTCN2022105137-appb-000004
例如，虚线表示上述基团的连接点。
例如，未取代的C 9双环杂环芳香环可以在2位具有氮，且在3位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在2位具有氮，且在4位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在2位具有氮，且在5位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在2位具有氮，且在7位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在2位具有氮，且在8位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在2位具有氮，且在9位具有氧。
例如，未取代的C 9双环杂环芳香环可以在3位具有氮，且在4位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在3位具有氮，且在5位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在3位具有氮，且在7位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在3位具有氮，且在8位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在3位具有氮，且在9位具有氧。
例如，未取代的C 9双环杂环芳香环可以在4位具有氮，且在5位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在4位具有氮，且在7位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在4位具有氮，且在8位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在4位具有氮，且在9位具有氧。
例如，未取代的C 9双环杂环芳香环可以在5位具有氮，且在7位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在5位具有氮，且在8位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在5位具有氮，且在9位具有氧。
例如，未取代的C 9双环杂环芳香环可以在6位具有氮，且在7位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在6位具有氮，且在8位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在6位具有氮，且在9位具有氧。
例如，未取代的C 9双环杂环芳香环可以在7位具有氮，且在8位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在7位具有氮，且在9位具有氧。在一些形式中，未取代的C 9双环杂环芳香环可以在8位具有氮，且在9位具有氧。
例如，m是0至10的整数。在一些形式中，m可以是0至10、1至10、2至10、3至10、4至10、5至10、6至10、7至10、8至10、9至10、0至9、1至9、2至9、3至9、4至9、5至9、6至9、7至9、8至9、0至8、1至8、2至8、3至8、4至8、5至8、6至8、7至8、0至7、1至7、2至7、3至7、4至7、5至7、6至7、0至6、1至6、2至6、3至6、4至6、5至6、0至5、1至5、2至5、3至5、4至5、0至4、1至4、2至4、3至4、0至3、1至3、2至3、0至2、1至2和0至1的整数。在一些形式中，m可以是0、1、 2、3、4、5、6、7、8、9或10。
例如，n是0至10的整数。在一些形式中，n可以是0至10、1至10、2至10、3至10、4至10、5至10、6至10、7至10、8至10、9至10、0至9、1至9、2至9、3至9、4至9、5至9、6至9、7至9、8至9、0至8、1至8、2至8、3至8、4至8、5至8、6至8、7至8、0至7、1至7、2至7、3至7、4至7、5至7、6至7、0至6、1至6、2至6、3至6、4至6、5至6、0至5、1至5、2至5、3至5、4至5、0至4、1至4、2至4、3至4、0至3、1至3、2至3、0至2、1至2和0至1的整数。在一些形式中，n可以是0、1、2、3、4、5、6、7、8、9或10。
例如，m+n可以是0至20的整数。例如，0至20、1至20、2至20、3至20、4至20、5至20、6至20、7至20、8至20、9至20、10至20、11至20、12至20、13至20、14至20、15至20、16至20、17至20、18至20、19至20、0至19、1至19、2至19、3至19、4至19、5至19、6至19、7至19、8至19、9至19、10至19、11至19、12至19、13至19、14至19、15至19、16至19、17至19、18至19、0至18、1至18、2至18、3至18、4至18、5至18、6至18、7至18、8至18、9至18、10至18、11至18、12至18、13至18、14至18、15至18、16至18、17至18、0至17、1至17、2至17、3至17、4至17、5至17、6至17、7至17、8至17、9至17、10至17、11至17、12至17、13至17、14至17、15至17、16至17、0至16、1至16、2至16、3至16、4至16、5至16、6至16、7至16、8至16、9至16、10至16、11至16、12至16、13至16、14至16、15至16、0至15、1至15、2至15、3至15、4至15、5至15、6至15、7至15、8至15、9至15、10至15、11至15、12至15、13至15、14至15、0至14、1至14、2至14、3至14、4至14、5至14、6至14、7至14、8至14、9至14、10至14、11至14、12至14、13至14、0至13、1至13、2至13、3至13、4至13、5至13、6至13、7至13、8至13、9至13、10至13、11至13、12至13、0至12、1至12、2至12、3至12、4至12、5至12、6至12、7至12、8至12、9至12、10至12、11至12、0至11、1至11、2至11、3至11、4至11、5至11、6至11、7至11、8至11、9至11、10至11、0至10、1至10、2至10、3至10、4至10、5至10、6至10、7至10、8至10、9至10、0至9、1至9、2至9、3至9、4至9、5至9、6至9、7至9、8至9、0至8、1至8、2至8、3至8、4至8、5至8、6至8、7至8、0至7、1至7、2至7、3至7、4至7、5至7、6至7、0至6、1至6、2至6、3至6、4至6、5至6、0至5、1至5、2至5、3至5、4至5、0至4、1至4、2至4、3至4、0至3、1至3、2至3、0至2、1至2和0至1。例如，m+n可以是0、1、2、3、4、5、6、7、8、9、10、 11、12、13、14、15、16、17、18、19或20。
例如，所述YEATS抑制剂包含如式(II)定义的化合物或其可药用盐，
Figure PCTCN2022105137-appb-000005
其中，每个J 1和J 2独立地是任何α-氨基酸；
其中，R 1是共轭/离域基团，例如，未取代的或取代的杂环的或碳环的芳香环(单环、双环、三环、四环)，或未取代的或取代的烯基或炔基；且
其中，m和n各自独立地是0至10的整数，其中，m或n中的至少一个不是0。
例如，所述YEATS抑制剂包含如式(III)定义的化合物或其可药用盐，
Figure PCTCN2022105137-appb-000006
其中，每个J 1和J 2独立地是任何α-氨基酸；
其中，X是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基或C 1-10杂环基；
其中，Y是NR 3、O或S；
其中，Z是-CO-、-CS-、-CNR 4-、-SO-和-SO 2-；
其中，R 3和R 4独立地是H、C 1-10烃基、C 1-10碳环基或C 1-10杂环基；
其中，R 1是共轭/离域基团，例如，未取代的或取代的杂环的或碳环的芳香环(单环、双环、三环、四环)，或未取代的或取代的烯基或炔基；且
其中，m和n各自独立地是0至10的整数，其中，m或n中的至少一个不是0。
例如，所述YEATS抑制剂包含如式(IV)定义的化合物或其可药用盐，
Figure PCTCN2022105137-appb-000007
其中，每个E 1和E 2独立地是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基或C 1-10杂环基，或O、S或NR 5；
其中，X是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基或C 1-10杂环基；
其中，Y是NR 3、O或S；
其中，Z是-CO-、-CS-、-CNR 4-、-SO-或-SO 2-；
其中，R 3、R 4和R 5独立地是H、C 1-10烃基、C 1-10碳环基或C 1-10杂环基；
其中，R 1是共轭/离域基团，例如，未取代的或取代的杂环的或碳环的芳香环(单环、双环、三环、四环)，或未取代的或取代的烯基或炔基；且
其中，m和n各自独立地是0至10的整数，其中，m或n中的至少一个不是0。
例如，至少两个B 1连接单元包含侧链，其中，B 1连接单元的两个侧链彼此共价地连接，形成环状低聚物。
例如，所述YEATS抑制剂包含如式(V)定义的化合物或其可药用盐，
Figure PCTCN2022105137-appb-000008
其中，R 1是共轭/离域基团，包括未取代的或取代的芳香环(单环、双环、三环、四环)、未取代的或取代的烯基或炔基；
其中，R 6、R 7、R 8、R 9和R 10独立地是H或C 1-10烃基、C 1-10碳环基或C 1-10杂环基；
其中，每个G 1、G 2和G 3是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳 环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基、C 1-10杂环基或5至9元杂芳基；且
其中，i 1、i 2、j 1、j 2、k 1、k 2各自独立地是0至10的整数。
例如，其中，至少两个B 2连接单元包含侧链，其中，B 2连接单元的两个侧链彼此共价地连接，形成环状低聚物。
例如，其中，所述化合物可以抑制π-π-π堆积相互作用。
例如，其中，所述化合物选择性地靶向YEATS蛋白结构域。
例如，每一种所述YEATS抑制剂在所述免疫细胞的细胞培养基中的初始浓度为约1μM或以上。
例如，每一种所述YEATS抑制剂在所述免疫细胞的细胞培养基中的初始浓度为约1μM至约200μM。
例如，可以在单个阶段的本申请的体外扩增中，使所述免疫细胞与所述一种或多种细胞激活剂接触以及使所述免疫细胞中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。例如，细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，在单个阶段的所述体外扩增中，使本申请的免疫细胞与本申请的YEATS抑制剂接触且与本申请的一种或多种细胞激活剂接触。例如，在本申请第一阶段体外扩增中，可以使本申请的TIL与本申请的YEATS抑制剂接触且与本申请的一种或多种细胞激活剂接触。例如，在本申请第二阶段体外扩增中，可以使本申请的TIL与本申请的YEATS抑制剂接触且与本申请的一种或多种细胞激活剂接触。例如，在本申请第三阶段体外扩增中，可以使本申请的TIL与本申请的YEATS抑制剂接触且与本申请的一种或多种细胞激活剂接触。
例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞基本上同时与本申请的YEATS抑制剂以及本申请的一种或多种细胞激活剂接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞先与本申请的YEATS抑制剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本申请的一种或多种细胞激活剂接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞先与本申请的一种或多种细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本申请的YEATS抑制剂接触。
例如，在本申请第一阶段体外扩增中，可以使本申请的TIL基本上同时与本申请的YEATS抑制剂以及本申请的一种或多种细胞激活剂接触。例如，在本申请第二阶段体外扩增中，可 以使本申请的TIL基本上同时与本申请的YEATS抑制剂以及本申请的一种或多种细胞激活剂接触。例如，在本申请第三阶段体外扩增中，可以使本申请的TIL基本上同时与本申请的YEATS抑制剂以及本申请的一种或多种细胞激活剂接触。
例如，本申请的第二阶段体外扩增进行至少约9天。例如，本申请的第二阶段体外扩增可以进行至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本申请的第二阶段体外扩增可以进行约9天至约14天，例如，本申请的第二阶段体外扩增可以进行约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。例如，本申请的第二阶段体外扩增可以认为是REP(rapid expansion protocol)阶段。例如，本申请的第一阶段体外扩增可以认为是preREP阶段。
例如，本申请第二阶段体外扩增进行的天数可以是从第二阶段体外扩增的开始时刻进行计算。例如，第二阶段体外扩增开始的当时，可以认为是第二阶段体外扩增进行了约0天。例如，第二阶段体外扩增开始后进行了约24小时，可以认为是第二阶段体外扩增进行了约1天。例如，第二阶段体外扩增开始的当天，可以认为是第二阶段体外扩增进行了约0天。例如，本申请第二阶段体外扩增进行的天数可以是通过第二阶段体外扩增进行的天数进行计算。例如，第二阶段体外扩增开始后的第二天，可以认为是第二阶段体外扩增进行了约1天。
例如，本申请的细胞激活剂可以包含选自以下组的一种或多种：CD80、CD86、B7-H3、4-1BBL、CD27、CD30、CD134、B7h、CD40、LIGHT、以及它们的功能活性片段。例如，本申请的细胞激活剂可以包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、HVEM、CD40L、OX40和4-1BB。例如，本申请的细胞激活剂可以包含选自以下组：CD3、CD28、HVEM、CD40L、OX40和4-1BB的抗体以及它们的抗原结合片段。例如，本申请的细胞激活剂可以包含CD3激动剂。例如，本申请的细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以是Miltenyi Biotech的OKT3，可以是BD的SP34。例如，本申请的细胞激活剂可以包含CD28激动剂。例如，本申请的细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以是Merck的15E8。
例如，本申请的细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链VL和重链VH，可以包含BD的SP34的轻链VL和重链VH。例如，本申请的细胞激活剂可以包含CD28激动剂。例如，本申请的细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链VL和重链 VH。例如，本申请的细胞激活剂可以包含抗CD3的抗体和/或其抗原结合片段，例如可以包含Miltenyi Biotech的OKT3的轻链LCDR1-3和重链HCDR1-3，可以包含BD的SP34的轻链LCDR1-3和重链HCDR1-3，本申请的抗CD3的抗体和/或其抗原结合片段可以具有CD3结合能力。例如，本申请的细胞激活剂可以包含CD28激动剂。例如，本申请的细胞激活剂可以包含抗CD28的抗体和/或其抗原结合片段，例如可以包含Merck的15E8的轻链LCDR1-3和重链HCDR1-3，本申请的抗CD28的抗体和/或其抗原结合片段可以具有CD28结合能力。在本申请中，本申请抗体或其抗原结合蛋白包含抗体重链可变区VH中的至少一个CDR。本申请CDR可以是根据IMGT命名法定义的，本申请CDR可以是根据Chothia定义的，或本申请CDR可以是根据Kabat定义的。
例如，使本申请的免疫细胞与本申请的一种或多种细胞激活剂接触可以包含选自以下组的一种或多种方式：(1)将本申请的细胞激活剂添加至本申请的免疫细胞的细胞培养基中；(2)将表达本申请的细胞激活剂的工程化细胞添加至本申请的免疫细胞的细胞培养基中；(3)将包含本申请的细胞激活剂的固相介质添加至本申请的免疫细胞的细胞培养基中。例如，使本申请的免疫细胞与本申请的一种或多种细胞激活剂接触可以包含将包含本申请的细胞激活剂的固相介质添加至本申请的免疫细胞的细胞培养基中。例如，使本申请的免疫细胞与本申请的一种或多种细胞激活剂接触可以包含将包含本申请的CD28抗体与CD3抗体的固相介质添加至本申请的免疫细胞的细胞培养基中。
例如，所述细胞激活剂在本申请免疫细胞的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本申请的CD28抗体在本申请免疫细胞的细胞培养基中的初始浓度可以为至少约30ng/mL；例如，本申请的CD3抗体在本申请免疫细胞的细胞培养基中的初始浓度可以为至少约30ng/mL。例如，本申请的CD28抗体初始浓度的选择可以与本申请的CD3抗体初始浓度的选择相互独立；例如，本申请的CD28抗体与本申请的CD3抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意组合。例如，本申请的CD28抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL。例如，本申请的CD3抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL。例如，本申请的CD28抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL，且本申请的CD3抗体在本申请免疫细胞的细胞培养基中的初始浓度可以任意选自约30ng/mL-约300ng/mL，本申请的CD28抗体初始浓度的选择可以与本申请的CD3抗体初始浓度的选择相互独立。例如，本申请的固相介质的直径可以为约500纳米至约10微米。例如，本申请的固相介质的直径可以通过透射电子显微镜测量。例如，本申请的固相介 质的直径可以为约1纳米至约500纳米。例如，本申请的固相介质的直径可以为约100纳米至约500纳米。例如，本申请的固相介质的直径可以为约200纳米至约500纳米。例如，本申请的固相介质的直径可以通过透射电子显微镜测量。
例如，本申请的固相介质可以包含聚合物。例如，本申请的固相介质可以包含葡聚糖。
例如，每mg本申请的固相介质包含至少约25μg的本申请的细胞激活剂。
例如，以约1:100-约1:2000的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂的固相介质添加至本申请免疫细胞的细胞培养基中。例如，以约2:1-约1:2的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂的固相介质添加至本申请免疫细胞的细胞培养基中。
例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约2:1-约1:2的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂的固相介质添加至本申请免疫细胞的细胞培养基中。例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约2:1-约1:2、以约2:1-约1:1、或以约1:1-约1:2的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂，例如CD3激动剂和/或CD28激动剂的固相介质添加至本申请免疫细胞的细胞培养基中。
例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000的本申请固相介质与本申请免疫细胞的比例，将包含本申请细胞激活剂的固相介质添加至本申请免疫细胞的细胞培养基中。例如，当本申请的固相介质的直径为约100纳米至约500纳米时，可以以约1:100-约1:2000、以约1:200-约1:2000、以约1:300-约1:2000、以约1:400-约1:2000、以约1:500-约1:2000、以约1:600-约1:2000、以约1:700-约1:2000、以约1:800-约1:2000、以约1:900-约1:2000、以约1:1000-约1:2000、以约1:1200-约1:2000以约1:1400-约1:2000、以约1:1600-约1:2000、或以约1:1800-约1:2000的本申请固相介质与本申请免疫细胞的比例，例如可以将包含本申请CD28激动剂和CD3激动剂的固相介质添加至本申请免疫细胞的细胞培养基中。
例如，本申请的方法还可以包含：在至少一个阶段的本申请体外扩增中，使本申请免疫细胞与一种或多种细胞生长因子接触。
例如，在单个阶段的本申请体外扩增中，可以使本申请的免疫细胞与本申请的细胞激活剂接触且与本申请的一种或多种细胞生长因子接触。例如，在本申请第一阶段体外扩增中，可以使本申请的TIL与本申请的细胞激活剂接触且与本申请的一种或多种细胞生长因子接触。例如，在本申请第二阶段体外扩增中，可以使本申请的TIL与本申请的细胞激活剂接触且与 本申请的一种或多种细胞生长因子接触。例如，在本申请第三阶段体外扩增中，可以使本申请的TIL与本申请的细胞激活剂接触且与本申请的一种或多种细胞生长因子接触。
例如，在单个阶段的本申请体外扩增中，使本申请免疫细胞基本上同时与本申请细胞激活剂以及本申请一种或多种细胞生长因子接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞基本上同时与本申请的一种或多种细胞生长因子以及本申请的一种或多种细胞激活剂接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞先与本申请的一种或多种细胞生长因子接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本申请的一种或多种细胞激活剂接触。例如，在单个阶段的本申请的体外扩增中，可以使本申请的免疫细胞先与本申请的一种或多种细胞激活剂接触，例如，可以提前2小时、提前4小时、提前8小时、提前12小时、提前24小时、或提前48小时等，再与本申请的一种或多种细胞生长因子接触。
例如，在本申请第一阶段体外扩增中，可以使本申请免疫细胞基本上同时与本申请细胞激活剂以及本申请一种或多种细胞生长因子接触。例如，在本申请第二阶段体外扩增中，可以使本申请TIL基本上同时与本申请细胞激活剂以及本申请一种或多种细胞生长因子接触。例如，在本申请第三阶段体外扩增中，可以使本申请TIL基本上同时与本申请细胞激活剂以及本申请一种或多种细胞生长因子接触。
例如，本申请的细胞生长因子可以选自以下组的一种或多种：IL-2、IL-7、IL-12、IL-15、IL-21、γ干扰素、以及它们的功能活性片段。例如，本申请的细胞生长因子可以包含IL-2和/或其功能活性片段。例如，IL-2的功能活性片段可以包含本领域已知的可以与细胞的IL-2受体结合的IL-2的片段。
例如，本申请的免疫细胞与本申请一种或多种细胞生长因子接触可以包含将本申请细胞生长因子添加至本申请免疫细胞的细胞培养基中。例如，本申请的细胞生长因子在本申请免疫细胞的细胞培养基中的初始浓度可以为至少约300IU/mL。例如，本申请IL-2在本申请免疫细胞的细胞培养基中的初始浓度可以为至少约350IU/mL、至少约400IU/mL、至少约500IU/mL、至少约600IU/mL、至少约700IU/mL、至少约800IU/mL、至少约900IU/mL、至少约1000IU/mL、至少约1100IU/mL、至少约1200IU/mL、至少约1300IU/mL、至少约1400IU/mL、至少约1500IU/mL、至少约2000IU/mL、至少约2500IU/mL、至少约2600IU/mL、至少约2700IU/mL、至少约2800IU/mL、至少约2900IU/mL、至少约3000IU/mL、至少约3100IU/mL、至少约3200IU/mL、至少约3300IU/mL、至少约3400IU/mL、至少约3500IU/mL、至少约4000IU/mL、至少约4500IU/mL、至少约5000IU/mL、至少约5500IU/mL、至少约 6000IU/mL、至少约6500IU/mL、至少约7000IU/mL、至少约7500IU/mL、至少约8000IU/mL、至少约8500IU/mL、或至少约9000IU/mL。
例如，本申请的方法还可以包含：在至少一个阶段的本申请体外扩增中，可以使本申请免疫细胞与饲养细胞共培养。
例如，在单个阶段的本申请体外扩增中，可以使本申请免疫细胞与一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本申请饲养细胞共培养，例如，单个阶段的本申请体外扩增可以指在同一个阶段的本申请的体外扩增，例如，可以同在本申请的第一阶段体外扩增、同在本申请的第二阶段体外扩增、或同在本申请的第三阶段体外扩增等。
例如，本申请的第一阶段体外扩增中，可以使本申请TIL与一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本申请饲养细胞共培养。例如，本申请的在本申请第二阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本申请饲养细胞共培养。例如，本申请的在本申请第三阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触且与本申请饲养细胞共培养。
例如，在单个阶段的本申请体外扩增中，可以使本申请免疫细胞与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。例如，在本申请第一阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。例如，在本申请第二阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。例如，在本申请第三阶段体外扩增中，可以使本申请TIL与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。
例如，在单个阶段的本申请体外扩增中，可以使本申请免疫细胞与本申请一种或多种细胞激活剂和/或一种或多种细胞生长因子接触一定时间之后，再与本申请饲养细胞共培养。例如，本申请的一定时间可以为至少约2小时。例如，本申请的一定时间可以为至少约1小时、至少约2小时、至少约3小时、至少约4小时、至少约5小时、至少约6小时、至少约7小时、至少约8小时、至少约9小时、至少约10小时、至少约11小时、至少约12小时、至少约13小时、至少约14小时、至少约15小时、至少约16小时、至少约17小时、至少约18小时、至少约19小时、至少约20小时、至少约21小时、至少约22小时、至少约23小时、至少约24小时、至少约36小时、至少约48小时、至少约60小时或至少约72小时。例如， 本申请的一定时间可以为约2小时至约72小时。例如，本申请的一定时间可以为约6小时到约7小时、约6小时到约8小时、约6小时到约9小时、约6小时到约10小时、约6小时到约11小时、约6小时到约12小时、约6小时到约13小时、约6小时到约14小时、约6小时到约15小时、约6小时到约16小时、约6小时到约17小时、约6小时到约18小时、约6小时到约19小时、约6小时到约20小时、约6小时到约21小时、约6小时到约22小时、约6小时到约23小时、约6小时到约24小时、约6小时到约36小时、约6小时到约48小时、约6小时到约60小时或约6小时到约72小时。例如，本申请的一定时间可以为约12小时到约13小时、约12小时到约14小时、约12小时到约15小时、约12小时到约16小时、约12小时到约17小时、约12小时到约18小时、约12小时到约19小时、约12小时到约20小时、约12小时到约21小时、约12小时到约22小时、约12小时到约23小时、约12小时到约24小时、约12小时到约36小时、约12小时到约48小时、约12小时到约60小时或约12小时到约72小时。例如，本申请的一定时间可以为约1小时、约2小时、约3小时、约4小时、约5小时、约6小时、约7小时、约8小时、约9小时、约10小时、约11小时、约12小时、约13小时、约14小时、约15小时、约16小时、约17小时、约18小时、约19小时、约20小时、约21小时、约22小时、约23小时、约24小时、约36小时、约48小时、约60小时或约72小时。
例如，本申请的饲养细胞可以包含抗原呈递细胞。例如，本申请的饲养细胞可以包含选自以下组的一种或多种：外周单个核细胞，树突状细胞，和人工抗原呈递细胞。例如，本申请的饲养细胞可以为外周单个核细胞。例如，本申请的饲养细胞可以为经过辐照的饲养细胞。例如，本申请的饲养细胞可以为分离的人工抗原呈递细胞(aAPC)，本申请的人工抗原呈递细胞可以包含表达HLA-A/B/C、CD64、CD80、ICOS-L和/或CD58的细胞，并可以被修饰以表达一种以上本申请的细胞激活剂。例如，本申请的饲养细胞可以经过辐照，例如，可以经过伽马射线辐照，或可以经过X射线辐照。
例如，本申请的免疫细胞与本申请的饲养细胞共培养可以包含使本申请的饲养细胞的表面与本申请的免疫细胞的表面相接触。例如，本申请的免疫细胞与本申请的饲养细胞共培养包含将本申请的饲养细胞添加至本申请的免疫细胞的细胞培养基中。
例如，本申请可以以约40:1-约400:1的本申请饲养细胞与本申请免疫细胞的比例，将本申请饲养细胞添加至本申请免疫细胞的细胞培养基中。例如，本申请可以以约40:1-约400:1、以约40:1-约300:1、以约40:1-约200:1、以约40:1-约100:1、以约40:1-约90:1、以约40:1-约80:1、以约40:1-约70:1、以约40:1-约60:1、以约40:1-约50:1、以约50:1-约400:1、以约60:1- 约400:1、以约70:1-约400:1、以约80:1-约400:1、以约90:1-约400:1、以约100:1-约400:1、以约200:1-约400:1、或以约300:1-约400:1的本申请饲养细胞与本申请免疫细胞的比例，将本申请饲养细胞添加至本申请免疫细胞的细胞培养基中。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群与所述一种或多种YEATS抑制剂接触；其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与一种或多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)使所述第二TIL群与SGC-iMLLT接触，SGC-iMLLT在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM，使所述TIL基本上同时与一种或多种细胞激活剂以及与SGC-iMLLT接触；其中，经所述步骤(B)得到第三TIL群。
在一种实施方式中的术语中，本申请的第一阶段体外扩增可以与以上方面的方法中的步骤(A)任意替换使用。在一种实施方式中的术语中，本申请的第二阶段体外扩增可以与以上方面的方法中的步骤(B)任意替换使用。在一种实施方式中的术语中，本申请的经第一阶段体外扩增的TIL可以与经以上方面的方法中步骤(A)得到的第二TIL群任意替换使用。在一种实施方式中的术语中，本申请的经第二阶段体外扩增的TIL可以与经以上方面的方法中步骤(B)得到的第三TIL群任意替换使用。在一种实施方式中的术语中，如有需要，本申请的第三阶段体外扩增可以与以上方面的方法中任意增加的步骤(C)任意替换使用。在一种实施方式中的术语中，如有需要，本申请的经第三阶段体外扩增的TIL可以与经以上方面的方法中任意增加的步骤(C)得到的第四TIL群任意替换使用。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与多种细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与多种细胞生长因子接触、与多种细胞激活剂接触、与本申请的多种YEATS抑制剂接触，且使所述TIL与饲养细胞共培养；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与细胞生长因子接触；其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与细胞生长因 子接触、与细胞激活剂接触、与本申请的一种或多种YEATS抑制剂接触，且使所述TIL与饲养细胞共培养，YEATS抑制剂在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与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、与细胞激活剂(例如可以包含CD3抗体以及CD28抗体的纳米基质、CD3抗体或CD3抗体和CD28抗体的混合物)接触、与本申请的一种或多种YEATS抑制剂，例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000009
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，或YDi-2(
Figure PCTCN2022105137-appb-000010
合成方法可以见于WO WO2019101195A1的实施例XL-13n)接触，且使所述TIL与饲养细胞共培养，YEATS抑制剂在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与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、与本申请的一种或多种YEATS抑制剂，例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000011
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，或YDi-2(
Figure PCTCN2022105137-appb-000012
合成方法可以见于WO WO2019101195A1的实施例XL-13n)接触，且使所述TIL与饲养细 胞共培养，YEATS抑制剂在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与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、与本申请的一种或多种YEATS抑制剂，例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000013
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，或YDi-2(
Figure PCTCN2022105137-appb-000014
合成方法可以见于WO WO2019101195A1的实施例XL-13n)接触，且使所述TIL与饲养细胞共培养，所述纳米基质的直径可以为约1纳米至约500纳米，所述饲养细胞可以包含外周单个核细胞，YEATS抑制剂在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与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、与包含CD3抗体以及CD28抗体的纳米基质接触、与本申请的一种或多种YEATS抑制剂，例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000015
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，或YDi-2(
Figure PCTCN2022105137-appb-000016
合成方法可以见于WO WO2019101195A1的实施例XL-13n)接触，且使所述TIL与饲养细胞共培养， 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，所述纳米基质的直径可以为约1纳米至约500纳米，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，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，YEATS抑制剂在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与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、与包含CD3抗体以及CD28抗体的纳米基质接触、与本申请的一种或多种YEATS抑制剂，例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000017
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，或YDi-2(
Figure PCTCN2022105137-appb-000018
合成方法可以见于WO WO2019101195A1的实施例XL-13n)接触，且使所述TIL与饲养细胞共培养，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，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，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，YEATS抑制剂在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与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、与包含CD3抗体以及CD28 抗体的纳米基质接触、与本申请的一种或多种YEATS抑制剂，例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000019
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，或YDi-2(
Figure PCTCN2022105137-appb-000020
合成方法可以见于WO WO2019101195A1的实施例XL-13n)接触，且在步骤(B)进行至少约2小时之后使所述TIL与饲养细胞共培养，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，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，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，YEATS抑制剂在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其可以包含：(A)可以使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的第一TIL群与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、与包含CD3抗体以及CD28抗体的纳米基质接触、与本申请的一种或多种YEATS抑制剂，例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000021
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，或YDi-2(
Figure PCTCN2022105137-appb-000022
合成方法可以见于WO WO2019101195A1的实施例XL-13n)接触，且在步骤(B)进行至少约2小时之后使所述TIL与饲养细胞共培养，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，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，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，YEATS抑制剂在所述TIL的细胞培养基中的初始浓度可以为至少为约1μM至约200μM；其中，经所述步骤(B)得到第三TIL群。
在另一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织、胸腔积液和/或腹腔积液在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液，内约2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入细胞无血清培养基和浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2培养约3-14天。收集培养基中细胞，转移入透气培养袋、或Grex、或Xuri设备，细胞无血清培养基可以添加本申请的CD28抗体、CD3抗体以及CD28抗体、包含CD3抗体以及CD28抗体的磁珠(例如Dynabeads)和/或包含CD3抗体以及CD28抗体的纳米基质(例如transACT)、浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2以及初始浓度为至少约1μM的YEATS抑制剂(例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000023
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，或YDi-2(
Figure PCTCN2022105137-appb-000024
合成方法可以见于WO WO2019101195A1的实施例XL-13n))，活化本申请的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于50％，大于80％的细胞可以为记忆效应细胞和效应细胞。经刺激后可以分泌IFNγ，和/或可以具有活化细胞比例上调的特征。
一方面，本申请提供一种免疫细胞，本申请的免疫细胞可以根据本申请的培养方法培养 得到。在一种实施方式中，本申请提供的免疫细胞可以包含一种或一个批次的本申请的培养方法培养得到免疫细胞。在一种实施方式中，本申请提供的免疫细胞可以包含多种或多个批次的本申请的培养方法培养得到并以任意比例组合的免疫细胞。
在一些实施方式中，可以将使用本申请方法扩增的免疫细胞作为药物组合物施用于患者。在一些实施方式中，药物组合物可以是免疫细胞在无菌缓冲液中的悬液。使用本申请的PBMC扩增的免疫细胞可以通过本领域已知的任何合适途径施用。在一些实施方式中，细胞可以以单次动脉内或静脉内输注施用，输注可以持续约30至60分钟。其他合适的施用途径可以包括腹膜内、鞘内和淋巴管内施用。
在一些实施方式中，可以施用任何合适剂量的免疫细胞。在一些实施方式中，例如当肿瘤是黑色素瘤时，可以施用约2.3×10 9至约13.7×10 10个免疫细胞。在一些实施方式中，可以施用约1×10 9至约12×10 10个免疫细胞。在一些实施方式中，可以施用约1.2×10 10至约4.3×10 10个免疫细胞。在一些实施方式中，可以施用约3×10 10至约12×10 10个免疫细胞。在一些实施方式中，可以施用约4×10 10至约10×10 10个免疫细胞。在一些实施方式中，可以施用约5×10 10至约8×10 10个免疫细胞。在一些实施方式中，可以施用约6×10 10至约8×10 10个免疫细胞。在一些实施方式中，可以施用约7×10 10至约8×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约2.3×10 9至约13.7×10 10。在一些实施方式中，治疗有效剂量可以为约1×10 9至约12×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约1.2×10 10至约4.3×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约3×10 10至约12×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约4×10 10至约10×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约5×10 10至约8×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约6×10 10至约8×10 10个免疫细胞。在一些实施方式中，治疗有效剂量可以为约7×10 10至约8×10 10个免疫细胞。
在一些实施方式中，本申请的组合物中提供的免疫细胞的数量可以为约1×10 6、约2×10 6、约3×10 6、约4×10 6、约5×10 6、约6×10 6、约7×10 6、约8×10 6、约9×10 6、约1×10 7、约2×10 7、约3×10 7、约4×10 7、约5×10 7、约6×10 7、约7×10 7、约8×10 7、约9×10 7、约1×10 8、约2×10 8、约3×10 8、约4×10 8、约5×10 8、约6×10 8、约7×10 8、约8×10 8、约9×10 8、约1×10 9、约2×10 9、约3×10 9、约4×10 9、约5×10 9、约6×10 9、约7×10 9、约8×10 9、约9×10 9、约1×10 10、约2×10 10、约3×10 10、约4×10 10、约5×10 10、约6×10 10、约7×10 10、约8×10 10、约9×10 10、约1×10 11、约2×10 11、约3×10 11、约4×10 11、约5×10 11、约6×10 11、约7×10 11、约8×10 11、约9×10 11、约1×10 12、约2×10 12、约3×10 12、约4×10 12、约5×10 12、约6×10 12、约7×10 12、约8×10 12、约 9×10 12、约1×10 13、约2×10 13、约3×10 13、约4×10 13、约5×10 13、约6×10 13、约7×10 13、约8×10 13，或约9×10 13。在一些实施方式中，本申请的组合物中提供的免疫细胞数量的范围可以为约1×10 6至5×10 6、约5×10 6至1×10 7、约1×10 7至5×10 7、约5×10 7至1×10 8、约1×10 8至5×10 8、约5×10 8至1×10 9、约1×10 9至5×10 9、约5×10 9至1×10 10、约1×10 10至5×10 10、约5×10 10至1×10 11、约5×10 11至1×10 12、约1×10 12至5×10 12，或约5×10 12至1×10 13。
在一些实施方式中，本申请的组合物中提供的免疫细胞的浓度可以小于组合物的例如约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约0.0002％，或约0.0001％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的免疫细胞的浓度可以大于组合物的约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19.75％、约19.50％、约19.25％、约19％、约18.75％、约18.50％、约18.25％、约18％、约17.75％、约17.50％、约17.25％、约17％、约16.75％、约16.50％、约16.25％、约16％、约15.75％、约15.50％、约15.25％、约15％、约14.75％、约14.50％、约14.25％、约14％、约13.75％、约13.50％、约13.25％、约13％、约12.75％、约12.50％、约12.25％、约12％、约11.75％、约11.50％、约11.25％、约11％、约10.75％、约10.50％、约10.25％、约10％、约9.75％、约9.50％、约9.25％、约9％、约8.75％、约8.50％、约8.25％、约8％、约7.75％、约7.50％、约7.25％、约7％、约6.75％、约6.50％、约6.25％、约6％、约5.75％、约5.50％、约5.25％、约5％、约4.75％、约4.50％、约4.25％、约4％、约3.75％、约3.50％、约3.25％、约3％、约2.75％、约2.50％、约2.25％、约2％、约1.75％、约1.50％、约125％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约或0.0002％，或者约0.0001％w/w、w/v或v/v。
在一些实施方式中，本申请的组合物中提供的免疫细胞的浓度范围可以为组合物的约0.0001％至约50％、约0.001％至约40％、约0.01％至约30％、约0.02％至约29％、约0.03％至约28％、约0.04％至约27％、约0.05％至约26％、约0.06％至约25％、约0.07％至约24％、约0.08％至约23％、约0.09％至约22％、约0.1％至约21％、约0.2％至约20％、约0.3％至约19％、约0.4％至约18％、约0.5％至约17％、约0.6％至约16％、约0.7％至约15％、约0.8％至约14％、约0.9％至约12％，或约1％至约10％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的免疫细胞的浓度范围可以为组合物的约0.001％至约10％、约0.01％至约5％、约0.02％至约4.5％、约0.03％至约4％、约0.04％至约3.5％、约0.05％至约3％、约0.06％至约2.5％、约0.07％至约2％、约0.08％至约1.5％、约0.09％至约1％、或约0.1％至约0.9％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的免疫细胞的量可以等于或小于约10g、约9.5g、约9.0g、约8.5g、约8.0g、约7.5g、约7.0g、约6.5g、约6.0g、约5.5g、约5.0g、约4.5g、约4.0g、约3.5g、约3.0g、约2.5g、约2.0g、约1.5g、约1.0g、约0.95g、约0.9g、约0.85g、约0.8g、约0.75g、约0.7g、约0.65g、约0.6g、约0.55g、约0.5g、约0.45g、约0.4g、约0.35g、约0.3g、约0.25g、约0.2g、约0.15g、约0.1g、约0.09g、约0.08g、约0.07g、约0.06g、约0.05g、约0.04g、约0.03g、约0.02g、约0.01g、约0.009g、约0.008g、约0.007g、约0.006g、约0.005g、约0.004g、约0.003g、约0.002g、约0.001g、约0.0009g、约0.0008g、约0.0007g、约0.0006g、约0.0005g、约0.0004g、约0.0003g、约0.0002g，或者约0.0001g。
在一些实施方式中，本申请的组合物中提供的免疫细胞的量可以大于约0.0001g、约0.0002g、约0.0003g、约0.0004g、约0.0005g、约0.0006g、约0.0007g、约0.0008g、约0.0009g、约0.001g、约0.0015g、约0.002g、约0.0025g、约0.003g、约0.0035g、约0.004g、约0.0045g、约0.005g、约0.0055g、约0.006g、约0.0065g、约0.007g、约0.0075g、约0.008g、约0.0085g、约0.009g、约0.0095g、约0.01g、约0.015g、约0.02g、约0.025g、约0.03g、约0.035g、约0.04g、约0.045g、约0.05g、约0.055g、约0.06g、约0.065g、约0.07g、约0.075g、约0.08g、约0.085g、约0.09g、约0.095g、约0.1g、约0.15g、约0.2g、约0.25g、约0.3g、约0.35g、约0.4g、约0.45g、约0.5g、约0.55g、约0.6g、约0.65g、约0.7g、约0.75g、约0.8g、约0.85g、约0.9g、约0.95g、约1g、约1.5g、约2g、约2.5g、约3g、约3.5g、约4g、约4.5g、约5g、约5.5g、约6g、约6.5g、约7g、约7.5g、约8g、约8.5g、约9g、约9.5g，或者约10g。
在一些实施方式中，免疫细胞可以单剂量施用。此种施用可以通过注射，例如可以静脉内注射。在一些实施方式中，免疫细胞可以多剂量施用。剂量可以是每年一次、两次、三次、 四次、五次、六次或超过六次。剂量可以是每月一次、每两周一次、每周一次或每2天一次。在一些实施方式中，免疫细胞的施用可以连续施用。
一方面，本申请提供一种药物组合物。在一些实施方式中，其可以包含本申请的免疫细胞和/或本申请的组合物，与药学上可接受的载体。
一方面，本申请提供一种试剂盒，本申请的试剂盒可以包含本申请培养免疫细胞方法的细胞激活剂、细胞生长因子和/或饲养细胞与记载本申请培养免疫细胞方法的步骤的说明书。一方面，本申请提供一种试剂盒，本申请试剂盒可以包含本申请的免疫细胞和/或本申请的药物组合物。
一方面，本申请提供一种影响细胞，例如肿瘤细胞，生长的方法，可以包括向受试者施用本申请的免疫细胞和/或本申请的药物组合物。在一些实施方式中，影响肿瘤生长可以包含肿瘤的体积减少到施用前的例如约99％、约95％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％或约0.1％。
一方面，本申请提供本申请的免疫细胞和/或本申请的药物组合物在制备药物中的应用，本申请的药物可以用于预防和/或治疗疾病和/或症状。例如，本申请的疾病和/或症状可以包含肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
一方面，本申请提供一种预防和/或治疗疾病和/或症状的方法，可以包括向受试者施用本申请的免疫细胞和/或本申请的药物组合物。例如，本申请的疾病和/或症状可以包含肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
一方面，本申请提供一种本申请的TIL和/或本申请的药物组合物，其可以用于预防和/或治疗疾病和/或症状。例如，本申请的疾病和/或症状可以包含肿瘤。在一些实施方式中，本申请的肿瘤选自实体瘤。在一些实施方式中，本申请的肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
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方法和用途等，而不用 于限制本申请发明的范围。
实施例
实施例1
TIL(肿瘤浸润淋巴细胞)的培养结果
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和培养袋，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第一阶段体外扩增及收获(preREP阶段)
1.3.1第一阶段体外扩增
根据细胞生长状态，每3-7天补液或半量换液，保证细胞营养。使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，并添加浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2，例如6000IU/mL 的IL-2。第一阶段体外扩增的3-14天，例如可以第3、4、5、6、7、8、9、10、11、12、13或14天时取样计数，若细胞数目处于5×10 5至5×10 8之间时进入下述第一阶段体外扩增的收获步骤。
1.3.2第一阶段体外扩增的收获
收集第一阶段体外扩增结束细胞，离心，弃去培养基，使用PBS或生理盐水洗涤细胞一次，获得经第一阶段体外扩增的TIL(第二TIL群)，并取样计数留取约5×10 5至2×10 8个细胞进入下述第一阶段体外扩增步骤；取约5×10 5个细胞可以进行质量控制检测；其余细胞加入冻存液冻存。
1.4第二阶段体外扩增(REP阶段)
1.4.1第二阶段体外扩增的TIL活化
取5×10 5至2×10 8的第一阶段体外扩增的细胞量，使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，调整细胞密度为5×10 5至2×10 6个细胞/mL，于悬浮24孔培养板内，1mL/孔，添加浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2。各个试验组加入IL-2的同时，可以添加对应的CD28激动剂，例如加入添加CD3抗体和CD28抗体的抗体混合物、添加包含CD3抗体和CD28抗体的磁珠、和\或添加包含CD3抗体和CD28抗体的transACT(直径约100至500nm，Miltenyi)。
同时，YEATS抑制剂试验组加入对应的YEATS抑制剂，例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000025
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，YDi-2(
Figure PCTCN2022105137-appb-000026
合成方法可以见于WO WO2019101195A1的实施例XL-13n)。YEATS抑制剂在TIL的细胞培养基中的初始浓度可以为约1μM或以上，例如约1μM至约200μM，例如20μM，其它抑制剂试验组，加入其它抑制剂，例如AKT抑制剂VIII(商品来源：MCE(HY-10355))、或PI3K抑制剂Idelalisib(商品来源：MCE(HY-13026))。对照组不加入抑制剂或仅加入阴性对照PBS。
1.4.2第二阶段体外扩增的扩大培养
以上各个试验组，在第二阶段体外扩增加入IL-2与不同形式的T细胞激活剂后的若干时间T n以后(T n可以取0小时到14天，例如24小时或48小时，例如48小时)，复苏1-5名供者混合的饲养细胞；将活化的TIL细胞，饲养细胞转入G-Rex100培养瓶或者透气袋内，补充完全培养基，每1-3天取样计数，并根据细胞状态补液或半量换液直至细胞总数大于1×10 9或第二阶段体外扩增培养达13天，终止培养。在培养过程中，YEATS抑制剂试验组可以全程添加YEATS抑制剂，第二阶段体外扩增培养的第7天、9天或14天后进行TIL功能检测；
1.4.3肿瘤浸润淋巴细胞的收获
取第二阶段体外扩增的细胞，离心后弃去培养基上清，并使用PBS或生理盐水或复方电解质溶液清洗三次，获得经第二阶段体外扩增的TIL(第三TIL群)，第三次清洗时取样计数，根据计数结果，最后一次离心后弃上清，取3×10 6细胞送质量控制检测；其余全部细胞加入冻存液，调整细胞密度1-3×10 8个细胞/mL冻存。
TIL增殖能力和细胞活率检测
对于本申请中各个试验组第二阶段体外扩增培养获得的第三TIL群进行细胞计数。
图1显示的是，添加YEATS抑制剂的试验组的增殖结果。图1中纵坐标的数值表示，各个试验组第二阶段体外扩增获得的第三TIL群相比于第二阶段体外扩增开始前的第二TIL群，TIL细胞数量扩增至的扩增倍数。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的增殖能力。
图2A-2B显示的是，对于不同供者，添加YEATS抑制剂的试验组的细胞活率结果。图1中纵坐标的数值表示，各个试验组第二阶段体外扩增获得的第三TIL群的活细胞所占的比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的细胞活率。
TIL流式检测
对于本申请中各个试验组第二阶段体外扩增培养获得的第三TIL群进行流式检测。
TIL流式检测试验材料的来源
转录因子缓冲组(Transcription Factor Buffer Set)，厂家BD，货号562574；V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×105至5×105个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板250μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend) 浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板250μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板250μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板250μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。表面染色结束后，PBS清洗细胞一次(96孔板250μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
图3显示的是，添加YEATS抑制剂的试验组的CD8 +细胞比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的CD8 +细胞比例。
图4A-4C显示的是，对于不同供者，添加YEATS抑制剂的试验组的CD28 +细胞比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的CD8 +细胞和/或CD4 +细胞中CD28 +活化细胞比例。
图5A-5E显示的是，对于不同供者，添加YEATS抑制剂的试验组的CD25 +细胞比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的CD8 +细胞和/或CD4 +细胞中CD25 +活化细胞比例，添加YEATS抑制剂的细胞活化效果优于其它抑制剂。
图6A-6D显示的是，对于不同供者，添加YEATS抑制剂的试验组的41BB +细胞比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的CD8 +细胞和/或CD4 +细胞中41BB +活化细胞比例，添加YEATS抑制剂的细胞活化效果优于其它抑制剂。
图7A-7K显示的是，对于不同供者，添加YEATS抑制剂的试验组的PD1 +细胞比例。图7A-7D、7F-7G、7I-7J显示的是不同供者来源的TIL细胞在第二阶段体外扩增培养的第7天进行检测的结果；图7E、7H、7K显示的是不同供者来源的TIL细胞在第二阶段体外扩增培养的第14天进行检测的结果。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC- iMLLT)，获得的TIL具有降低的CD8 +细胞和/或CD4 +细胞中PD1 +耗竭细胞比例，添加YEATS抑制剂的抗细胞耗竭效果优于其它抑制剂。
图8A-8F显示的是，对于不同供者，添加YEATS抑制剂的试验组的LAG3 +细胞比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有降低的CD8 +细胞和/或CD4 +细胞中LAG3 +耗竭细胞比例，添加YEATS抑制剂的抗细胞耗竭效果优于其它抑制剂。
图9A-9C显示的是，对于不同供者，添加YEATS抑制剂的试验组的TIM3 +细胞比例。图9A-9B显示的是不同供者来源的TIL细胞在第二阶段体外扩增培养的第7天进行检测的结果；图9C显示的是不同供者来源的TIL细胞在第二阶段体外扩增培养的第14天进行检测的结果。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有降低的CD8 +细胞和/或CD4 +细胞中TIM3 +耗竭细胞比例。
图10A-10B显示的是，对于不同供者，添加YEATS抑制剂的试验组的CD39 +细胞比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有降低的CD4 +细胞中CD39 +耗竭细胞比例。
图11显示的是，添加YEATS抑制剂的试验组的Treg细胞(调节T细胞)比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有降低的Treg细胞比例。
图12A-12C显示的是，对于不同供者，添加YEATS抑制剂的试验组的凋亡细胞比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有降低的CD4 +细胞中CD95 +caspass3 +细胞和/或CD95 +DR5 +凋亡细胞比例。
图13A-13C显示的是，对于不同供者，添加YEATS抑制剂的试验组的具有干性细胞比例。图13A和13C显示的是不同供者来源的TIL细胞在第二阶段体外扩增培养的第7天进行检测的结果；图13B显示的是不同供者来源的TIL细胞在第二阶段体外扩增培养的第14天进行检测的结果。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的CD8 +细胞中CD69 -CD39 -细胞和/或TCF1 +干性细胞比例。
图14A-14H显示的是，对于不同供者，添加YEATS抑制剂的试验组的中心记忆T细胞(Tcm，例如CD4 +细胞中的CD45RA -CCR7 +或CD8 +细胞中的CD45RA -CCR7 +)比例。图14A-14F显示的是不同供者来源的TIL细胞在第二阶段体外扩增培养的第7天进行检测的结果；图14G-14H显示的是不同供者来源的TIL细胞在第二阶段体外扩增培养的第14天进行检测的结果。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL 具有提高的中心记忆T细胞比例，添加YEATS抑制剂的提高中心记忆T细胞比例效果优于其它抑制剂。
TIL细胞因子分泌检测
对于本申请中各个试验组第二阶段体外扩增培养获得的第三TIL群进行细胞因子分泌检测。
取各个试验组的TIL在96孔板内孵育过夜，通过BD Cytofix/CytopermTM Plus Fixation/permeabilization试剂盒BD GolgiStopTM(货号：BD 554715)以及Fixation/Permeabilization Solution试剂盒BD GolgiPlug TM(货号：BD 555028)流式检测分泌细胞因子的细胞的比例。
图15A-15B显示的是，对于不同供者，添加YEATS抑制剂的试验组的CD107A分泌细胞的比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的分泌CD107A的细胞比例，添加YEATS抑制剂的刺激细胞因子分泌效果优于其它抑制剂。
图16A-16D显示的是，对于不同供者，添加YEATS抑制剂的试验组的IFNγ分泌细胞的比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的分泌IFNγ的细胞比例，添加YEATS抑制剂的刺激细胞因子分泌效果优于其它抑制剂。
图17显示的是，添加YEATS抑制剂的试验组的TNF分泌细胞的比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的分泌TNF的细胞比例，添加YEATS抑制剂的刺激细胞因子分泌效果优于其它抑制剂。
图18A-18D显示的是，对于不同供者，添加YEATS抑制剂的试验组的GZMB分泌细胞的比例。结果显示，第二阶段体外扩增添加YEATS抑制剂(例如，SGC-iMLLT)，获得的TIL具有提高的分泌GZMB的细胞比例，添加YEATS抑制剂的刺激细胞因子分泌效果优于其它抑制剂。
实施例2
T细胞培养
取来自供者的T细胞，添加抗体活化，并加入10μM的YEATS抑制剂，例如YEATS SGC(商品来源：MCE(HY-112804)，CAS号：2255338-25-9)，培养72小时。通过转染NYESO1TCR(HLA-A*0201NY-ESO-1)获得TCR-T细胞，通过转染CD19 CAR(FMC63)获得CAR-T细胞。NT表示未改造的T细胞，CAR-T表示转染CAR的T细胞，TCR-T表示转染TCR 的T细胞。转染后细胞继续培养5-8天，培养过程中添加10μM的YEATS抑制剂，培养结束后进行检测。
转染效率测试
培养结束的细胞使用流式方法检测转染效率。本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板250μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。表面染色结束后，PBS清洗细胞一次(96孔板250μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
图19显示的是，对于不同供者通过YEATS抑制剂培养，T细胞的转染效率。结果显示，YEATS抑制剂可以提高T细胞的转染效率。
细胞流式分析
取培养结束的NT、TCR-T、CAR-T进行流式检测。
转录因子缓冲组(Transcription Factor Buffer Set)，厂家BD，货号562574；V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板250μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板250μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板250μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板250μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。表面染色结束 后，PBS清洗细胞一次(96孔板250μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
图20显示的是，对于不同供者通过YEATS抑制剂培养，干性相关CD69 -CD39 -细胞比例。结果显示，YEATS抑制剂可以提高干性相关细胞的比例。
图21-22显示的是，对于不同供者通过YEATS抑制剂培养，耗竭相关CD39 +细胞或PD1 +细胞比例。结果显示，YEATS抑制剂可以降低耗竭相关细胞的比例。
图23-24显示的是，对于不同供者通过YEATS抑制剂培养，活化相关41BB +细胞或CD25 +细胞比例。结果显示，YEATS抑制剂可以提高活化相关细胞的比例。
增殖能力检测
取培养结束的NT、TCR-T、CAR-T细胞，通过未添加抗体刺激或添加CD3抗体刺激72h后检测其增殖能力。
图25显示的是，对于不同供者通过YEATS抑制剂培养，细胞增殖结果。结果显示，YEATS抑制剂可以提高细胞的增殖能力。
细胞因子分泌检测
取培养结束的NT、TCR-T、CAR-T细胞，通过未添加抗体刺激或添加CD3抗体刺激24h后检测细胞因子分泌结果。
标准品准备
试剂盒选用soluble protein master buffer kit(BD)，使用2mL Assay Diluent稀释液(BD)复溶(标准品原液IL-2、TNF-α、IFN-γ和IL-6细胞因子浓度均为2500pg/mL，GZMB细胞因子浓度为10000pg/mL)并按顺序：1:2，1:4，1:8，1:16，1:32，1:64，1:128，1:256梯度稀释，随后与Capture Beads(BD)混合，室温下避光孵育2h，后添加PE Detection Reagent检测试剂(BD)混合，室温下避光孵育1h，转移至15mL锥底离心管，标记为“标准品管”。取1管仅含有Assay Diluent稀释液作为阴性对照。
检测步骤
取各组细胞培养结束上清，按照10μL/孔加入孔板，随后按照0.2μL/Beads/孔使用beads diluent稀释后10μL/孔与样品混合，室温下避光孵育2h后，按照10μL/孔添加PE Detection Reagent检测试剂(BD)，室温下避光孵育1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26-30显示的是，对于不同供者通过YEATS抑制剂培养，IL-2、GZMB、TNF-α、IFN- γ和IL-6的分泌结果。结果显示，YEATS抑制剂可以提高细胞分泌细胞因子的能力。
NK细胞培养
取来自供者的外周血单个核细胞PBMC。通过在PBMC中加入阴性磁珠，以去除NK细胞以外的细胞，最终分选分离得到NK细胞。之后将NK细胞以1.5E6个/mL的浓度培养于6孔板中，并加入500IU/mL IL-2。在YEATS抑制剂处理组中，加入10μM的YEATS抑制剂(例如SGC(商品来源：MCE(HY-112804))；对照组不加YEATS抑制剂。于细胞培养箱中培养5天后，收集上清并进行Cytometric Bead Array(CBA)检测。
图31显示的是在YEATS抑制剂处理后，NK细胞的细胞因子(例如IL-2、TNF-α和IFN-γ)释放显著增加。结果显示YEATS抑制剂可以提高NK细胞的激活效果和/或杀伤能力。
实施例3
不同种类YEATS抑制剂的培养效果
对于TIL细胞采用YEATS抑制剂进行培养，例如SGC-iMLLT(商品来源：MCE(HY-112804))，YDi-1(
Figure PCTCN2022105137-appb-000027
合成方法可以见于WO WO2019101195A1的实施例XL-13m)，YDi-2(
Figure PCTCN2022105137-appb-000028
合成方法可以见于WO WO2019101195A1的实施例XL-13n)。YEATS抑制剂在TIL的细胞培养基中的初始浓度可以为约1μM或以上，例如约1μM至约200μM，例如20μM。
细胞流式分析
转录因子缓冲组(Transcription Factor Buffer Set)，厂家BD，货号562574；V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×10 5至5×10 5个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板250μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD， Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板250μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板250μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板250μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。表面染色结束后，PBS清洗细胞一次(96孔板250μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
图32显示的是，对于不同供者通过YEATS抑制剂培养，活化相关CD25 +细胞比例。结果显示，YEATS抑制剂可以提高活化相关细胞的比例。
图33-34显示的是，对于不同供者通过YEATS抑制剂培养，耗竭相关PD1 +细胞或LAG3 +细胞比例。结果显示，YEATS抑制剂可以降低耗竭相关细胞的比例。
图35-36显示的是，对于不同供者通过YEATS抑制剂培养，干性相关TCF1 +或CD69 -CD39 -细胞比例。结果显示，YEATS抑制剂可以提高干性相关细胞的比例。
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 (91)

  1. 一种培养免疫细胞的方法，使所述免疫细胞中包含YEATS结构域的蛋白和/或其功能活性片段的表达和/或活性降低。
  2. 根据权利要求1所述的方法，所述使包含YEATS结构域的蛋白和/或其功能活性片段的活性降低包含抑制所述YEATS结构域与组蛋白的结合能力。
  3. 根据权利要求1-2中任一项所述的方法，其包含：使所述免疫细胞与一种或多种YEATS抑制剂接触。
  4. 根据权利要求1-3中任一项所述的方法，所述免疫细胞包含吞噬细胞、淋巴细胞、中性粒细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
  5. 根据权利要求1-4中任一项所述的方法，所述免疫细胞包含单核细胞、巨噬细胞和/或树突状细胞。
  6. 根据权利要求1-5中任一项所述的方法，所述免疫细胞包含B细胞、T细胞、自然杀伤细胞和/或自然杀伤样T细胞。
  7. 根据权利要求1-6中任一项所述的方法，所述免疫细胞包含αβT细胞和/或γδT细胞。
  8. 根据权利要求1-7中任一项所述的方法，所述免疫细胞包含肿瘤浸润淋巴细胞(TIL)。
  9. 根据权利要求8所述的方法，所述TIL为源自肿瘤组织的碎片、胸腔积液和/或腹腔积液的TIL和/或源自冷冻保存后复苏的TIL。
  10. 根据权利要求9所述的方法，所述碎片的体积为约1立方毫米至约27立方毫米。
  11. 根据权利要求1-10中任一项所述的方法，所述免疫细胞包含展示在细胞表面上的工程化免疫受体。
  12. 根据权利要求11所述的方法，所述工程化免疫受体与靶细胞上表达的抗原特异性结合。
  13. 根据权利要求11-12中任一项所述的方法，所述免疫细胞包含嵌合抗原受体和/或T细胞受体。
  14. 根据权利要求1-13中任一项所述的方法，与未曾与所述YEATS抑制剂接触的免疫细胞相比，与所述YEATS抑制剂接触过的所述免疫细胞显示出改善的细胞特性。
  15. 根据权利要求14所述的方法，所述改善的细胞特性包含选自以下组的一种或多种：改善的细胞增殖能力、增加的活细胞比例、改善的细胞亚群比例、提高的细胞因子分泌能力和提高的肿瘤细胞杀伤能力。
  16. 根据权利要求15所述的方法，所述改善的细胞亚群比例包含选自以下组的一种或多种：增加的活化细胞比例、降低的调节性细胞比例、降低的耗竭细胞的比例、增加的中心记忆细胞比例、降低的凋亡细胞的比例和增加的干细胞样细胞比例。
  17. 根据权利要求1-16中任一项所述的方法，其包含：使所述免疫细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述免疫细胞与所述YEATS抑制剂接触。
  18. 根据权利要求17所述的方法，其中，使所述源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使经所述第一阶段体外扩增的TIL与所述YEATS抑制剂接触。
  19. 根据权利要求18所述的方法，所述第一阶段体外扩增进行至少约7天。
  20. 根据权利要求18-19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。
  21. 根据权利要求17-20中任一项所述的方法，所述YEATS抑制剂包含能够抑制YEATS结构域与组蛋白相互作用的物质。
  22. 根据权利要求17-21中任一项所述的方法，所述YEATS抑制剂包含能够结合YEATS的组蛋白结合结构域的物质。
  23. 根据权利要求17-22中任一项所述的方法，所述YEATS抑制剂能够以约0.3μM或更低的IC 50值抑制YEATS结构域与组蛋白结合。
  24. 根据权利要求17-23中任一项所述的方法，所述YEATS抑制剂能够以约0.13μM或更低的K D值结合YEATS结构域。
  25. 根据权利要求17-24中任一项所述的方法，所述YEATS抑制剂选自以下组的一种或多种：核酸分子、多肽和小分子化合物。
  26. 根据权利要求17-25中任一项所述的方法，所述YEATS抑制剂包含能够降低包含YEATS结构域的蛋白表达的物质。
  27. 根据权利要求17-26中任一项所述的方法，所述YEATS抑制剂包含能够影响YEATS蛋白活性的物质。
  28. 根据权利要求17-27中任一项所述的方法，所述YEATS抑制剂包含YEATS特异性抑制剂。
  29. 根据权利要求17-28中任一项所述的方法，所述YEATS抑制剂包含SGC-iMLLT。
  30. 根据权利要求17-29中任一项所述的方法，所述YEATS抑制剂包含如式(I)定义的化合物或其可药用盐，
    Figure PCTCN2022105137-appb-100001
    其中，A是CR 2或N；
    其中，每个B 1和B 2是独立选择的二价连接单元，其中，所述二价连接单元通过酰胺、酯、硫酯、硫酰胺、亚胺酯、酰亚胺、磺酸酯或磺酰胺以首尾连接方式连接；
    其中，D 1是H或未取代的或取代的烃基、碳环基或杂环基；
    其中，D 2是H、NH 2、NHR 15、NR 16R 17、OH、OR 18，或未取代的或取代的烃基、碳环基或杂环基；
    其中，X是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基或C 1-10杂环基；
    其中，Y是NR 3、O或S；
    其中，Z是-CO-、-CS-、-CNR 4-、-SO-或-SO 2-；
    其中，R 2、R 3和R 4独立地是H或C 1-10烃基、C 1-10碳环基或C 1-10杂环基；
    其中，R 1是共轭/离域基团，例如，未取代的或取代的杂环的或碳环的芳香环(单环、双环、三环、四环)，或未取代的或取代的烯基或炔基；
    其中，R 15、R 16、R 17和R 18可以是H或烷基、烯基、炔基、取代的烷基、取代的烯基、取代的炔基、烷氧基、烷基氨基、二烷基氨基、羟基、芳基、取代的芳基、杂芳基、取代的杂芳基、取代的烷氧基、羧基、取代的羧基、氨基、取代的氨基、酰胺基、取代的酰胺基、C 3-C 20环、取代的C 3-C 20环、杂环或取代的杂环；且
    其中，m和n各自独立地是0至10的整数，其中，m或n中的至少一个不是0。
  31. 根据权利要求17-30中任一项所述的方法，所述YEATS抑制剂包含如式(II)定义的化合物或其可药用盐，
    Figure PCTCN2022105137-appb-100002
    其中，每个J 1和J 2独立地是任何α-氨基酸；
    其中，R 1是共轭/离域基团，例如，未取代的或取代的杂环的或碳环的芳香环(单环、双环、三环、四环)，或未取代的或取代的烯基或炔基；且
    其中，m和n各自独立地是0至10的整数，其中，m或n中的至少一个不是0。
  32. 根据权利要求17-31中任一项所述的方法，所述YEATS抑制剂包含如式(III)定义的化合物或其可药用盐，
    Figure PCTCN2022105137-appb-100003
    其中，每个J 1和J 2独立地是任何α-氨基酸；
    其中，X是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基或C 1-10杂环基；
    其中，Y是NR 3、O或S；
    其中，Z是-CO-、-CS-、-CNR 4-、-SO-和-SO 2-；
    其中，R 3和R 4独立地是H、C 1-10烃基、C 1-10碳环基或C 1-10杂环基；
    其中，R 1是共轭/离域基团，例如，未取代的或取代的杂环的或碳环的芳香环(单环、双环、三环、四环)，或未取代的或取代的烯基或炔基；且
    其中，m和n各自独立地是0至10的整数，其中，m或n中的至少一个不是0。
  33. 根据权利要求17-32中任一项所述的方法，所述YEATS抑制剂包含如式(IV)定义的化合物或其可药用盐，
    Figure PCTCN2022105137-appb-100004
    其中，每个E 1和E 2独立地是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基或C 1-10杂环基，或O、S或NR 5；
    其中，X是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基或C 1-10杂环基；
    其中，Y是NR 3、O或S；
    其中，Z是-CO-、-CS-、-CNR 4-、-SO-或-SO 2-；
    其中，R 3、R 4和R 5独立地是H、C 1-10烃基、C 1-10碳环基或C 1-10杂环基；
    其中，R 1是共轭/离域基团，例如，未取代的或取代的杂环的或碳环的芳香环(单环、双环、三环、四环)，或未取代的或取代的烯基或炔基；且
    其中，m和n各自独立地是0至10的整数，其中，m或n中的至少一个不是0。
  34. 根据权利要求30-33中任一项所述的方法，至少两个B 1连接单元包含侧链，其中，B 1连接单元的两个侧链彼此共价地连接，形成环状低聚物。
  35. 根据权利要求17-34中任一项所述的方法，所述YEATS抑制剂包含如式(V)定义的化合物或其可药用盐，
    Figure PCTCN2022105137-appb-100005
    其中，R 1是共轭/离域基团，包括未取代的或取代的芳香环(单环、双环、三环、四环)、未取代的或取代的烯基或炔基；
    其中，R 6、R 7、R 8、R 9和R 10独立地是H或C 1-10烃基、C 1-10碳环基或C 1-10杂环基；
    其中，每个G 1、G 2和G 3是未取代的或取代的C 1-10烷基、C 1-10烯基、C 1-10炔基、C 1-10碳环基、C 1-10杂烷基、C 1-10杂烯基、C 1-10杂炔基、C 1-10杂环基或5至9元杂芳基；且
    其中，i 1、i 2、j 1、j 2、k 1、k 2各自独立地是0至10的整数。
  36. 根据权利要求30-35中任一项所述的方法，其中，至少两个B 2连接单元包含侧链，其中，B 2连接单元的两个侧链彼此共价地连接，形成环状低聚物。
  37. 根据权利要求30-36中任一项所述的方法，其中，所述化合物可以抑制π-π-π堆积相互作用。
  38. 根据权利要求30-37中任一项所述的方法，其中，所述化合物选择性地靶向YEATS蛋白结构域。
  39. 根据权利要求17-38中任一项所述的方法，每一种所述YEATS抑制剂在所述免疫细胞的细胞培养基中的初始浓度为约1μM或以上。
  40. 根据权利要求17-39中任一项所述的方法，每一种所述YEATS抑制剂在所述免疫细胞的细胞培养基中的初始浓度为约1μM至约200μM。
  41. 根据权利要求1-40中任一项所述的方法，其还包含：使所述免疫细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述免疫细胞与一种或多种细胞激活剂接触。
  42. 根据权利要求41所述的方法，使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过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与所述细胞激活剂接触。
  43. 根据权利要求41-42中任一项所述的方法，其中，在单个阶段的所述体外扩增中，使所述免疫细胞与所述细胞激活剂以及所述YEATS抑制剂接触。
  44. 根据权利要求41-43中任一项所述的方法，其中，在单个阶段的所述体外扩增中，使所述免疫细胞基本上同时与所述细胞激活剂以及所述YEATS抑制剂接触。
  45. 根据权利要求41-44中任一项所述的方法，所述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。
  46. 根据权利要求41-45中任一项所述的方法，所述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。
  47. 根据权利要求41-46中任一项所述的方法，所述细胞激活剂包含CD3激动剂和/或CD28激动剂。
  48. 根据权利要求41-47中任一项所述的方法，所述细胞激活剂包含CD3激动剂。
  49. 根据权利要求41-48中任一项所述的方法，所述细胞激活剂包含抗CD3的抗体和/或其抗原结合片段。
  50. 根据权利要求41-49中任一项所述的方法，所述细胞激活剂包含CD28激动剂。
  51. 根据权利要求41-50中任一项所述的方法，所述细胞激活剂包含抗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。
  52. 根据权利要求41-51中任一项所述的方法，所述使免疫细胞与所述细胞激活剂接触包含选自以下组的一种或多种方式：(1)将所述细胞激活剂添加至所述免疫细胞的细胞培养基中；(2)将表达所述细胞激活剂的工程化细胞添加至所述免疫细胞的细胞培养基中；(3)将包含所述细胞激活剂的固相介质添加至所述免疫细胞的细胞培养基中。
  53. 根据权利要求52所述的方法，每一种所述细胞激活剂在所述免疫细胞的细胞培养基中的初始浓度为至少约30ng/mL。
  54. 根据权利要求52-53中任一项所述的方法，每一种所述细胞激活剂在所述免疫细胞的细胞培养基中的初始浓度为约30ng/mL至约300ng/mL。
  55. 根据权利要求52-54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。
  56. 根据权利要求52-55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。
  57. 根据权利要求55-56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。
  58. 根据权利要求52-57中任一项所述的方法，所述固相介质包含聚合物。
  59. 根据权利要求52-58中任一项所述的方法，每mg所述固相介质包含每一种所述细胞激活剂至少约25μg。
  60. 根据权利要求52-59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免疫细胞的比例，将包含所述细胞激活剂的固相介质添加至所述免疫细胞的细胞培养基中。
  61. 根据权利要求52-60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免疫细胞的比例，将包含所述细胞激活剂的固相介质添加至所述免疫细胞的细胞培养基中。
  62. 根据权利要求1-61中任一项所述的方法，其还包含：使免疫细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述免疫细胞与一种或多种细胞生长因子接触。
  63. 根据权利要求62所述的方法，使源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过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与所述细胞生长因子接触。
  64. 根据权利要求62-63中任一项所述的方法，其中，在所述单个阶段的所述体外扩增中，使所述免疫细胞与所述细胞激活剂且与所述细胞生长因子接触。
  65. 根据权利要求62-64中任一项所述的方法，其中，在所述单个阶段的所述体外扩增中，使所述免疫细胞基本上同时与所述细胞激活剂以及所述细胞生长因子接触。
  66. 根据权利要求62-65中任一项所述的方法，所述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。
  67. 根据权利要求62-66中任一项所述的方法，所述细胞生长因子包含IL-2和/或其功能活性片段。
  68. 根据权利要求62-67中任一项所述的方法，所述免疫细胞与所述细胞生长因子接触包含将所述细胞生长因子添加至所述免疫细胞的细胞培养基中。
  69. 根据权利要求62-68中任一项所述的方法，每一种所述细胞生长因子在所述免疫细胞的细胞培养基中的初始浓度为至少约300IU/mL。
  70. 根据权利要求1-69中任一项所述的方法，其还包含：使免疫细胞经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述免疫细胞与饲养细胞共培养。
  71. 根据权利要求70所述的方法，使所述源自肿瘤组织、胸腔积液和/或腹腔积液且未经体外扩增的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使经所述第一阶段体外扩增的TIL与所述饲养细胞共培养。
  72. 根据权利要求70-71中任一项所述的方法，其中，在单个阶段的所述体外扩增中，使所述免疫细胞与所述细胞激活剂和/或所述细胞生长因子接触且与所述饲养细胞共培养。
  73. 根据权利要求70-72中任一项所述的方法，其中，在单个阶段的所述体外扩增中，使所述免疫细胞与所述细胞激活剂和/或细胞生长因子接触一定时间之后，再与所述饲养细胞共培养。
  74. 根据权利要求73所述的方法，使所述免疫细胞在与所述细胞激活剂接触至少约2小时之后与所述饲养细胞共培养。
  75. 根据权利要求73-74中任一项所述的方法，使所述免疫细胞在与所述细胞激活剂接触约2小时至约72小时之后与所述饲养细胞共培养。
  76. 根据权利要求73-75中任一项所述的方法，使所述免疫细胞在与所述细胞激活剂接触约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。
  77. 根据权利要求70-76中任一项所述的方法，所述饲养细胞包含抗原呈递细胞。
  78. 根据权利要求70-77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。
  79. 根据权利要求70-78中任一项所述的方法，所述饲养细胞为外周单个核细胞。
  80. 根据权利要求70-79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。
  81. 根据权利要求70-80中任一项所述的方法，所述免疫细胞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免疫细胞的表面相接触。
  82. 根据权利要求70-81中任一项所述的方法，所述免疫细胞与所述饲养细胞共培养包含将所述饲养细胞添加至所述免疫细胞的细胞培养基中。
  83. 根据权利要求71-82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免疫细胞的比例，将所述饲养细胞添加至所述免疫细胞的细胞培养基中。
  84. 一种免疫细胞，所述免疫细胞经过权利要求1-83中任一项所述的方法获得。
  85. 一种组合物，其包含权利要求84所述的免疫细胞。
  86. 一种药物组合物，其包含权利要求84所述的免疫细胞和/或权利要求85所述的组合物，以及任选地药学上可接受的载体。
  87. 一种影响细胞生长的方法，包含施用权利要求84所述的免疫细胞、权利要求85所述的组合物和/或权利要求86所述的药物组合物。
  88. 权利要求84所述的免疫细胞、权利要求85所述的组合物和/或权利要求86所述的药物组合物在制备药物中的应用，所述药物用于预防和/或治疗疾病和/或症状。
  89. 根据权利要求88所述的应用，所述疾病和/或症状包含肿瘤。
  90. 根据权利要求88-89中任一项所述的应用，所述疾病和/或症状包含实体瘤。
  91. 根据权利要求88-90中任一项所述的应用，所述疾病和/或症状包含选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌和肾癌。
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